Key factors on the installation and validation of a production line … · Key factors on the...

Key factors on the installation and validation of aproduction line for infusion bags

Ricardo Jorge Dias Leandro

Thesis to obtain the Master of Science Degree in

Biological Engineering

Supervisors: Prof. José Monteiro Cardoso de MenezesEng. Samuel Mendes Geraldes Camocho

Examination Committee:

Chairperson: Prof. Arsénio do Carmo Sales Mendes FialhoSupervisor: Eng. Samuel Mendes Geraldes Camocho

Member of the Committee: Prof. Frederico Castelo Alves Ferreira

November 2015

"It is the nature of man to rise to greatness if greatness is expected of him."John Steinbeck

Acknowledgments

For contributing for this work, at different levels, there are some people that I would like to dedicate

a special acknowledgment. Firstly, I would like to thank my supervisor, Eng. Samuel Camocho, not

only for the enthusiasm and daily support that he gave me throughout my internship but also for all

the patience, important advices and commitment that he had towards me and this work. I also have to

thank him for allowing me to be the first Masters student to enter Hikma as an intern and contributing

decisively for the launch of my hopefully successful career.

On this note, I also have to thank Prof. José Cardoso Menezes for the confidence that he deposited

in me and for his inputs since the first day. Furthermore, a special gratitude note has to be issued to

Técnico Lisboa, and to all the people that make it the best Engineering University in Portugal.

Within Hikma, there are several people that I would like to give a special thanks to. Inês Graça and

Omar Murad, for all the help they gave me throughout my stay at Hikma, as well as to all the personnel

from Lines 1 and 4, the Validation department and the ladies from the Compliance department, for all

the great moments throughout my internship and their constant support.

On a more personal level, I have to save some thank you notes for my friends. Thank you Alexan-

dre, Ana Maria, Beatriz, Eddie e Rita for putting up with me in this crazy semester, for all the support,

encouragement and patience that you had and also for all the laughter and fun moments that also

helped shaping this thesis.

Last, but certainly not least, I would like to express my heart-felt gratitude to my family. This

dissertation is dedicated to them because none of this would be possible without their constant love,

concern, support and strength throughout these years. I would like to express my heart-felt gratitude

to my family. For making me the man that I am today, Mom, Dad, little sister and Artur, thanks for

everything!

iii

Abstract

This main goal of this work is to identify and discuss the main focus points on the instalation

and validation of a new commercial pharmaceutical line for the manufacture of terminally sterilized

infusion bags. This was achieved through a close monitoring and participation in the initial installation

and qualification stages carried out by the engineering, validation, quality control and production

departments.

A practical and functional-orientated assessment of the main factors that influence the outcome

of the new manufacturing department’s validation stage was developed. An integrated description

of the main focal points that contribute to the overall prevention of contamination development in the

department and to the safety of the final product is provided. Specifically, the main steps involved in

the installation stage, with a tight focus on the structure, equipment and utilities installed to support

the department, as well as in the qualification stage and all the test runs and validation protocols

implemented are explained and their relation and connection with the overall success of the validation

is demonstrated.

With the completion of this work, conditions have been created for an efficient transition to the

routine production stage, in compliance with the regulators’ requirements, allowing for the continuous

monitoring and improvement of the process throughout its lifecycle.

Keywords

Validation - Installation - Qualification - Quality - Sterility - Safety

v

Resumo

O principal objectivo deste trabalho consiste na identificação e discussão dos pontos chave en-

volvidos na instalação e validação de uma nova linha farmacêutica de produção de sacos de infusão

com esterilização terminal. Isto foi conseguido com base numa monitorização e participação nas

fases de instalação e qualificação iniciais levadas a cabo pelos departamentos de engenharia, vali-

dação, controlo de qualidade e produção.

Foi efectuada uma avaliação prática dos principais factores que influenciam a etapa de validação

do novo departamento de produção. Com base nesta, foi possível descrever de forma integrada os

factores chave que contribuem para a prevenção generalizada de contaminações no departamento

e para a segurança do produto final. Especificamente, foram explicados os principais passos en-

volvidos na fase de instalação, com um foco preferencial na estrutura, equipamentos e utilidades

instaladas para apoiar o departmento. Na fase de qualificação, foram explicados todos os ensaios de

teste e protocolos de validação implementados e foi demonstrada a sua interligação e contribuição

para o sucesso global da validação.

Com a conclusão deste trabalho, as condições para uma transição eficiente para a etapa de pro-

dução em rotina foram criadas, em conformidade com os requerimentos das entidades reguladores,

o que permite a contínua monitorização e melhoria do processo ao longo do seu ciclo-de-vida.

Palavras Chave

Validação - Instalação - Qualificação - Qualidade - Esterilidade - Segurança

vii

Contents

1 Introduction 1

1.1 Key focal points of the work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.2 Chronological Overview on Process Validation . . . . . . . . . . . . . . . . . . . . . . . 4

1.3 Process Validation Throughout Process Lifecycle . . . . . . . . . . . . . . . . . . . . . . 9

1.3.1 Process design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.3.2 Process Qualification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

1.3.3 Continued Process Verification (CPV) . . . . . . . . . . . . . . . . . . . . . . . . 16

1.3.4 EMA’s 2014 guideline on process validation - a new approach . . . . . . . . . . 18

1.4 Validating a new manufacturing line of parenterals . . . . . . . . . . . . . . . . . . . . . 20

1.4.1 Parenteral preparations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

1.4.2 Hikma’s approach to validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

1.4.3 Components of the department . . . . . . . . . . . . . . . . . . . . . . . . . . . . 23

2 Contamination prevention 25

2.1 Contaminations in the pharmaceutical environment . . . . . . . . . . . . . . . . . . . . . 26

2.1.1 Sources of contamination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

2.2 Contamination control in the department . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2.2.1 Facilities and environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2.2.1.A Surface materials and construction . . . . . . . . . . . . . . . . . . . . 32

2.2.2 HVAC, air handling and airflow patterns . . . . . . . . . . . . . . . . . . . . . . . 35

2.2.3 Equipment and utilities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

2.2.4 Personnel and material flows to and from the cleanrooms . . . . . . . . . . . . . 45

2.2.5 Cleaning and disinfection plans . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

ix

3 Product safety 49

3.1 Assessing product safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50

3.1.1 Product and container/closure compatibility . . . . . . . . . . . . . . . . . . . . . 50

3.2 Terminal sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

3.3 Filtration for bioburden reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

4 Results: Validation and qualification of the installed line 59

4.1 Validation of the department’s components . . . . . . . . . . . . . . . . . . . . . . . . . 60

4.1.1 Installation and qualification tests . . . . . . . . . . . . . . . . . . . . . . . . . . . 60

4.1.2 Assessment and control of air quality . . . . . . . . . . . . . . . . . . . . . . . . 61

4.1.3 Monitoring and validation of the environmental conditions . . . . . . . . . . . . . 64

4.1.4 Monitoring and validation of the water quality . . . . . . . . . . . . . . . . . . . . 68

4.2 Validation of cleaning and sterilization methods . . . . . . . . . . . . . . . . . . . . . . . 73

A – Cleaning and disinfection methods’ validation . . . . . . . . . . 73

B – Validation of the terminal sterilization . . . . . . . . . . . . . . 74

C – Validation of the filling machine’s SIP cycle . . . . . . . . . . . 77

D – Validation of the filtration step to reduce the microbial load . . 79

5 Discussion and conclusions 81

5.1 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

6 Future work 85

6.1 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

Bibliography 86

Appendix A Material and Personnel flows of the department A-1

Appendix B Rationale behind the choice of products for leachable studies B-1

Appendix C Sampling locations included in the Environmental Monitoring Plan C-1

Appendix D Trends of viable particles for environmental monitoring D-1

Appendix E Detail of the SIP course in the filling machine E-1

x

Appendix F Example of a supporting protocol of the new department F-1

xi

List of Figures

1.1 Process salidation sequence throughout the process lifecycle. . . . . . . . . . . . . . . . 2

1.2 Chronological evolution of Process and Product Validation Guidelines. Source: Institute

of Validation Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

1.3 Main principles of FDA’s Guideline for Industry on Process Validation, 2011. Source:

Biopharm International . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

1.4 Standard set of utilities found in Hikma plants. Source: Applying Process Systems

Engineering for Continuous Improvement in Pharmaceutical Production, 2013 . . . . . . 14

2.1 Sources of contamination in the pharmaceutical environment. . . . . . . . . . . . . . . . 27

2.2 Cause and effect diagram with main parameters to be controlled to avoid contamination

in the manufacturing process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2.3 Typical control layout for clean areas. Source: World Health Organization . . . . . . . . 31

2.4 Cleanroom classification from USA, EU and Japanese regulatory agencies. Source:

Manufacturing Sterile Products to Meet EU and FDA Guidelines, FDA News . . . . . . . 32

2.5 Manufacturing operations to perform according with the cleanroom grade for termi-

nally sterilized products. Source: Manufacturing Sterile Products to Meet EU and FDA

Guidelines, FDA News . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

2.6 Airflow patterns implemented in the department. Source: ISO 14644-4 (2001) Clean-

rooms and associated controlled environments - Part 4: Design, construction and start-up 38

2.7 Water pre-treatment system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

2.8 Basic principle of reverse osmosis. Source: Puretec - Industrial Water . . . . . . . . . . 42

2.9 Water treatment system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

2.10 Water for injection distribution loops. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

xiii

3.1 Survival curve of Bacillus stearothermophilus spores with a standard sterilization method

(A). Thermal survival curve of Bacillus stearothermophilus spores with a standard ster-

ilization method (B). Source: Manufacturing Sterile Products to Meet EU and FDA

Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

4.1 Scheme illustrating the EM process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

4.2 Trend of non-viable particles equal or greater than 0,5 µm over the 5 days of initial

qualification in Grade C rooms. The several series represent the different Grade C

rooms in the department, whereas the top series corresponds to the acceptance criteria

defined. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69


qualification in Grade D rooms. The several series represent the different Grade D


defined. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70


qualification in Grade C rooms. The several series represent the different Grade C


defined. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70


qualification in Grade D rooms. The several series represent the different Grade D


defined. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

4.6 Trend of viable particles sampled through air sampling for the 5 days of initial qualifica-

tion in Grade C rooms. The several series represent the different Grade C rooms in the

department. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4.7 Trend of viable particles sampled through settling plates for the 5 days of initial qualifi-

cation in Grade C rooms. The several series represent the different Grade C rooms in

the department. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4.8 Trend of viable particles sampled through contact plates for the 5 days of initial qual-

ification in Grade C. The several series represent the different Grade C rooms in the

department. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

4.9 Schematic representation of the SIP cycle for the terminal sterilization process. . . . . . 75

A.1 Material flow of the infusion bags department. . . . . . . . . . . . . . . . . . . . . . . . . A-2

A.2 Personnel flow of the infusion bags department. . . . . . . . . . . . . . . . . . . . . . . A-3

B.1 Rational for the choice of products to be considered for the leachables’ study. . . . . . . B-2

xiv

C.1 Locations for the air sampling included in the EM plan. . . . . . . . . . . . . . . . . . . . C-2

C.2 Locations for the settling plates sampling included in the EM plan. . . . . . . . . . . . . C-3

C.3 Locations for the RODAC sampling included in the EM plan. . . . . . . . . . . . . . . . . C-4

C.4 Locations for the non-viable particle sampling included in the EM plan. . . . . . . . . . . C-5

D.1 Trend of viable particles sampled through air sampling for the 5 days of initial qualifica-

tion in Grade B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

D.2 Trend of viable particles sampled through air sampling for the 5 days of initial qualifica-

tion in Grade D rooms. The several series represent the different Grade D rooms in the

department. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

D.3 Trend of viable particles sampled through settling plates for the 5 days of initial qualifi-

cation in Grade D rooms. The several series represent the different Grade D rooms in

the department. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-2

D.4 Trend of viable particles sampled through contact plates for the 5 days of initial qualifi-

cation in Grade B. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3

D.5 Trend of viable particles sampled through contact plates for the 5 days of initial qual-

ification in Grade D. The several series represent the different Grade C rooms in the

department. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D-3

E.1 Detail of the SIP course of the filling machine. . . . . . . . . . . . . . . . . . . . . . . . . E-2

F.1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-2

F.2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-3

F.3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-4

F.4 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F-5

xv

List of Tables

1.1 Summary of the current recommended approach to process validation. . . . . . . . . . 18

2.1 Acceptance criteria for the number of viable particles within clean areas. . . . . . . . . . 36

4.1 Rationale for the choice of the non-viable, air sampling and settling plates’ sampling

locations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

4.2 Rationale for the choice of the RODAC surface plates’ sampling locations. . . . . . . . . 69

4.3 Action levels for viable particle monitoring. . . . . . . . . . . . . . . . . . . . . . . . . . . 69

4.4 Set of microorganisms to be used for disinfectant efficacy tests . . . . . . . . . . . . . . 74

4.5 Sequence of the SIP cycle of the filling machine. . . . . . . . . . . . . . . . . . . . . . . 78

4.6 SIP cycle parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78

4.7 Biological Indicators characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

4.8 Results of the test runs for qualification of the SIP of the filling line. . . . . . . . . . . . . 79

xvi

Abbreviations

API - Active Pharmaceutical Ingredient

CCP - Critical Control Parameters

cGMP - Current Good Manufacturing Practices

CPP - Critical Process Parameter

CPV -Continued Process Verification

CQA - Critical Quality Attribute

DS - Design Space

DQ - Design Qualification

EM - Environmental Monitoring

EMA - European Medicines Agency

FDA - U. S. Food and Drug Administration

ICH - International Conference on Harmonization

IPC - In Process Control

IQ - Installation Qualification

NOR - Normal Operating Range

OOS - Out of Specifications

OQ - Operational Qualification

PAT - Process Analytical Technology

PQ - Performance Qualification

PS - Pure Steam

PPQ - Process Performance Qualification

PV - Process Validation

QbD - Quality by Design

QRM - Quality Risk Management

QTPP - Quality Target Product Profile

RTRT - Real-time Release Testing

SOP - Standard Operating Procedure

UFH - Unidirectional Flow Hood

USP - United States Pharmacopeia

WFI - Water for Injectables

xvii

1Introduction

Contents1.1 Key focal points of the work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.2 Chronological Overview on Process Validation . . . . . . . . . . . . . . . . . . . . 41.3 Process Validation Throughout Process Lifecycle . . . . . . . . . . . . . . . . . . 91.4 Validating a new manufacturing line of parenterals . . . . . . . . . . . . . . . . . . 20

1

1.1 Key focal points of the work

Validation is an integral part of quality assurance. It involves the systematic study of systems,

facilities and processes with the goal of determining whether they perform their intended functions

in an adequate and consist manner. A validated operation is one which has been demonstrated to

provide a high degree of assurance that uniform batches that meet the required specifications will be

produced and has, therefore, been formally approved.

Unlike many other requirements of GMP, validation in itself does not improve processes. It can

only confirm (or not, as the case may be) that the process has been properly developed and is

under control. Good validation practices require the close collaboration of departments such as those

concerned with design, production, engineering, quality assurance and control. Well established initial

validation stages are essential for a controlled routine full-scale production. [1].

Adequate validation may be beneficial for the manufacturer in many ways: [2]

• It deepens the understanding of processes, decreases the risks of processing problems, and

thus assures the smooth running of the process;

• It decreases the risks of defect costs;

• It decreases the risks of regulatory non-compliance;

• A fully validated process may require less in-process control and end-product testing.

The aim of this thesis is to define and analyze the main focal points, in terms of the installation

and validation, of a new commercial manufacturing line to produce terminally sterilized infusion bags.

Considering the process validation sequence throughout the process lifecycle, illustrated in Figure 1.1

the scope of this work is focused on the installation and qualification stages, that precede the phase

of routine production in a commercial scale.

Figure 1.1: Process salidation sequence throughout the process lifecycle.

By the end of the installation process and throughout the validation cycle, the safety of the product

and the efficacy of the production processes has to be demonstrated, in order to guarantee that all

bags that are produced will consistently conform with the required compliance parameters and that

the consumers’ safety is safeguarded.

2

To fully understand the approach of this thesis, it is important to know the background behind

this work. The internship that led to the work here presented represented a new effort from the

company in creating a position that acted as link between the production, validation, quality control

and engineering departments. The goal was that the intern would be exposed to the initial stages

of installation and qualification and would be a part of both the decision-making processes and the

implementation of procedures that would act as the foundation for an active contribution in the routine

operations of the product line or an equivalent one.

Furthermore, the training and the experience gained with the internship would allow for the com-

pliant application of the knowledge acquired in a second stage, after the installation and initial qualifi-

cation works were complete. This stage would include, as an example, the scaling-up of the different

products that will be transfered to new line, the preparation and filling of submission batches for the

new products, the validation of specific elements of the department (namely autoclave loads and rou-

tine environmental monitoring) and the training of the production team that will be operating within the

new department.

With this said, it is relevant to mention that all the specific validation tests and projects that are

described in the course of this thesis did not result of the author’s individual work. Instead, they

reflect the work of the multidisciplinary teams (in which the author was included) that contributed for

the installation, initial qualification and overall validation of the department and its elements. Still, the

discussion, comments and conclusions that are presented throughout this work result of the author’s

analysis of the process and reflect the knowledge and experience that were gained during the course

of the internship.

In what concerns the regulators’ requirements, there are several cGMP considerations that have

to be addressed throughout the design phase and that will move on to impact the project lifecycle.

Common elements that concern the authorities when it comes to establishing a new production line

or alter an existing one include: [3]

• An effective quality assurance system is established and maintained;

• The processes are in a state of control;

• Personnel are suitably qualified, trained and supervised;

• Processes and equipment are designed, installed, operated and maintained to suit the intended

operations;

• Detailed records of all aspects of the process are generated, analyzed and archived, in order to

allow the identifications and tracing of root causes for potential problems that might arise;

• A generalized contamination prevention plan is established in the facility

Besides this, there are also different sets of specific requirements that have to be thought since

the design phase (and, posteriorly, maintained during the process activity) that are specific to the

3

type of product to be produced, the processes underlined with the production process and the type of

strategy used to assure its sterility.

Considering this, and having in mind that the outcome of this thesis must be within the scope of

the Biological Engineering Masters, two main areas have been outlined as critical to be addressed

during the validation of the manufacturing line: contamination prevention and product safety.

Securing a contaminants-free product is crucial in the manufacturing of parenteral preparations.

Given their route of administration, the patient’s health would be seriously compromised if any sort of

microbial, chemical or other type of contaminant was present in the formulation of the drug that would

be injected.

While manufacturing terminally sterilized products, the final step of sterilization is responsible for

the elimination of all contaminants that may be on the preparation or in the container/closure pair.

However, and even though the processing and the filling of the infusion bags is not performed in

totally aseptic conditions (that is, within a sterile room following the FDA/EMA specification for aseptic

filling and manufacturing conditions), it is still of major relevance to implement protocols intended to

minimize the risks of microbial or particulate contamination of the intermediates and guarantee the

success of the subsequent sterilization.

In chapter 2, there is a preliminary analysis of the main contaminants that appear within the phar-

maceutical environment. Following this, a detailed description of the systems and the installation

protocols of the different components of the department are detailed, with a tighter focus on the mea-

sures taken to avoid and control potential contamination ingress in the department.

On the other hand, in chapter 3 the focus will be on the critical steps taken to assure the safety

of the product to the patients that will use it. Part of this chapter will be dedicated to the process

of terminal sterilization, but further comments will be made regarding the interactions between the

solution and the container/closure pair, as well as additional steps that were implemented in the

manufacturing process to safeguard the quality and the safety of the products.

1.2 Chronological Overview on Process Validation

The introduction of the Process Validation concept occurred in 1987, when FDA (United States

Food and Drug Administration) issued the Guideline on General Principles of Process Validation, as

a way to push pharmaceutical manufacturers to better their practices regarding process validation

(Figure 1.2). [4]

On this guidance, process validation was defined as "establishing documented evidence which

provides a high degree of assurance that a specific process will consistently produce a product meet-

ing its predetermined specifications and quality attributes." [4]

With this guidance, several types of process validation were defined, based on the time within

the product lifecycle on which they were applied: prospective validation, retrospective validation and

4

Figure 1.2: Chronological evolution of Process and Product Validation Guidelines. Source: Institute of ValidationTechnology

revalidation.

Prospective process validation (also called premarket validation) was defined as the establishment

of documented evidence, prior to process implementation, that a system does what it proposed to do

based on preplanned protocols. This approach to validation was normally undertaken whenever the

process for a new formula (or within a new facility) had to be validated before routine pharmaceutical

production commences. In fact, validation of a process by this approach often led to transfer of the

manufacturing process from the development function to production. [2]

Under this perspective, validation was faced as a multiple tests demonstrations, that intended to

prove consistency in the new process, by submitting it to a series of sequential steps that challenge

it in worst-case scenarios. Based on the results obtained, the equipment and supporting processes

should be designed and/or selected so that the facility is qualified. This should be done with the partic-

ipation of all appropriate groups that are concerned with assuring a quality product, e.g., engineering

design, production operations, and quality assurance personnel. [5]

There were two main elements associated with prospective validation: installation qualification

(IQ) and performance qualification (PQ).

Installation qualification studies establish confidence that the process equipment and ancillary sys-

tems are capable of consistently operating within established limits and tolerances. [5] After process

equipment was designed or selected, it should be evaluated and tested to verify that it was capable

of operating satisfactorily within the operating limits required by the process. This phase of validation

includes:

• Examination of equipment design;

5

• Determination of calibration, maintenance and adjustment requirements;

• Identification of critical equipment features that could affect the process and product

Information obtained from these studies should be used to establish written procedures covering

equipment calibration, maintenance, monitoring, and control. Furthermore, based on the variability of

the results from the worst-case scenario testing done with the equipment, the total number of trials

selected for the subsequent stage of performance qualification studies may be determined. [6]

On the performance qualification stage, the goal was to demonstrate the effectiveness and repro-

ducibility of the process based on rigorous testing. When this stage started, the process specifications

should be established and described with sufficient detail to allow for the challenge of the critical parts

that may affect the quality of the product. The test runs are designed to simulate those that would be

encountered during routine production, including worst-case scenarios. [6]

By the end of the two stages, the consistency and reproducibility of the specific manufacturing

processes had to be proven in order to guarantee a successful validation step.

Retrospective validation was, on the other hand, a concept that involved solely the examination

of past experience of production with the assumption that composition, procedures, and equipments

remained unchanged. Such experience and the results of in-process and final control tests were

then evaluated. Recorded difficulties and failures in production were analyzed to determine the limits

of process parameters and a trend analysis would be conducted to determine the extent to which

the process parameters were within the permissible range. [7] Retrospective validation is obviously

not a quality assurance measure in itself, and should never be applied to new processes or products;

however, it is frequently used as a tool to establish priorities in the development of validation programs

when such requirements are first introduced in a company.

Batches selected for retrospective validation should be representative of all batches made during

the review period, including any batches that failed to meet specifications, and should be sufficient in

number to demonstrate process consistency. Additional testing of retained samples may be needed

to obtain the necessary amount or type of data to retrospectively validate the process. Generally, data

from ten to thirty consecutive batches should be examined to assess process consistency, but fewer

batches may be examined if justified. [7]

Finally, the 1987 guideline pushed forward the concept of revalidation of processes when changes

to a process are introduced, or when process variation is detected. A quality assurance system

should be in place in order to assure the implementation of a revalidation protocol whenever there

were changes in packaging, formulation, raw-materials, equipments or processes which could impact

on the product’s effectiveness and characteristics. [4]

The quality assurance procedures should establish the circumstances under which revalidation is

required. These may be based upon equipment, process, and product performance observed during

the initial validation challenge studies. The extent of revalidation will depend upon the nature of the

6

changes and how they impact upon different aspects of production that had previously been validated.

It may not be necessary to revalidate a process from scratch merely because a given circumstance

has changed. However, it is important to carefully assess the nature of the change to determine

potential ripple effects and what needs to be considered as part of revalidation.

Summing up, the main principles introduced by the 1987 Guidance on General Principles of Pro-

cess Validation can be stated as follows:

• The process equipment operates consistently within required pre-specified limits;

• The ancillary equipment and materials, such as measuring and monitoring equipments are ca-

pable of operating in conformity with the process equipment;

• A validated process is guaranteed by assuring consistent performance within a certain opera-

tional range for a series of test batches, that translate in a product that complies with the required

specifications for quality and function;

• Changes to the process, the equipment or other relevant production parameters are addressed

through an analysis of the impact of that change on the process and product’s characteristics.

Requalification and revalidation are introduced whenever needed.

Regarding the view of EMA on process validation, the first guideline concerning this subject was

released on 2001, as Annex 15 of its EU Guidelines for Good Manufacturing Practice for Medicinal

Products for Human and Veterinary Use. This Annex contained several of the concepts initially pre-

sented on FDA’s guideline and starting framing the methodology for process validation within the EU

area. [8]

However, the issuance of the most recent FDA and EMA guidelines have been changing the orig-

inal paradigm initially proposed. The complexity of the manufacturing processes, derived from the

countless variables to it associated (where can be stated equipments, process strategy, process con-

trol, raw-materials, operating units, amongst others), along with the process variability that arises over

time led to the issuance of a new set of guidances, that reflect a modern take on process validation.

The Guidance for Industry - Process Validation: General Principles and Practices [9], issued by

FDA on 2011, reflects a closer link between process validation and drug quality. For purposes of this

guidance, process validation is now defined as the collection and evaluation of data, from the process

design stage through commercial production, which establishes scientific evidence that a process is

capable of consistently delivering quality product. [9] This new definition encompasses a broader look

on the process itself, pushing the idea that process validation should be continuous throughout the

process and product’s lifecycles.

With this guidance, the concepts of retrospective validation and worst-case scenario testing were

removed and the concept of revalidation was altered with the introduction of the stage of Continuous

Process Verification. Validation is described as an on-going process, that can be approached in three

7

stages (detailed in section 1.3.) The main ideas introduced with this new guideline are mentioned

next:

• Quality, safety, and efficacy are designed or built into the product - Quality by Design [10];

• Detailed information on the process and product design and development are key to a successful

validation program;

• The introduction of a Quality Risk Management approach throughout the process lifecycle is

described as an essential requirement; [11]

• Establishing a robust and well defined Quality System throughout the product’s lifecycle through-

out the product lifecycle should facilitate innovation and continual improvement and strengthen

the link between pharmaceutical development and manufacturing activities. [12]

• Objective measures, statistical tools and a better control of process variability are strongly rec-

ommended to assure that the finished product meets all design characteristics and quality at-

tributes including specifications.

Following the release of this new guidance, EMA reviewed its previous guideline and released

draft versions for both its Guideline on the Process Validation for finished products and Annex 15,

whose final versions became effective last year (2014). [13]

These documents comply with the latest developments on Product Validation, being in sync with

FDA’s Guideline for Industry, as well as ICH’s guidelines. Together, they facilitate companies to use

detailed process knowledge and understanding, coupled with risk management when working in the

process validation quality management system.

Both the guideline and the new revision of the Annex 15 promote a lifecycle approach to validation

linking product and process development, restating the three stages of process lifecycle already men-

tioned in FDA’s guideline: process design, process qualification and continued process verification.

The validation of commercial manufacturing process and maintenance of the process in a state of

control during routine production are also emphasized. Besides this, they allow flexibility between us-

ing the traditional approach, a continuous process verification approach or a hybrid approach, where

either may be used for different steps in a manufacturing process. Companies can also choose to

migrate from the traditional approach to the continuous verification mode when revalidating commer-

cialized products as part of a process change or in support of continuous improvement.

The principles and implications of the current guidelines and annexes from both FDA and EMA will

be detailed in section 1.3.

8

1.3 Process Validation Throughout Process Lifecycle

For manufacturing processes to be truly validated, each of its stages must be addressed and

integrated. This integration of development work, process conformance, and continued verification

provides assurance that the product or process will consistently remain in control throughout the

entire product lifecycle. [14]

Process validation must not be considered a one-time event or a focused one-time task performed

just prior to commercial launch that emphasizes only the manufacture of three conformance lots.

Acceptable manufacture of three conformance batches must not be interpreted as completion of val-

idation, as defended in the traditional approach to validation. These batches cannot truly represent

the future manufacturing process, with all the variability to it associated. Conformance lots are often

inadvertently biased (i.e., they may utilize well-characterized and controlled active pharmaceutical in-

gredients (API) and excipients, be manufactured under well controlled conditions, be monitored by

expert individuals, and performed by most experienced or well trained personnel - all "best-case" con-

ditions). It is highly unrealistic to contend that the manufacture of three conformance batches under

"best-case" conditions conclusively predicts successful manufacturing over the product lifetime. True

process validation must be a process that is never completed and is always ongoing. [14]

The approach to process validation stated in the 2011 FDA’s Guidance for Industry on Process

Validation clearly emphasizes contemporary concepts and expectations for pharmaceutical manufac-

turing. The manufacturers should have great confidence that the performance of the process will

consistently produce APIs and drug products meeting expected attributes.

This guidance describes process validation activities in three stages (see Figure 1.3):

• Stage 1: Process Design - The commercial manufacturing process is defined during this stage

based on knowledge gained through development and scale-up activities;

• Stage 2: Process Qualification - During this stage, the process design is evaluated to de-

termine if the process is capable of reproducible commercial manufacturing. Besides this, the

facility, equipment and support utilities are also evaluated and validated to confirm their compli-

ance with the processes’ and the regulators’ requirements;

• Stage 3: Continued Process Verification - Ongoing assurance is gained during routine pro-

duction that the process remains in a state of control.

These sections of the 2011 guidance clearly identify the key difference between the current

paradigm of FDA towards validation and the one presented with the 1987 guideline. The 2011

lifecycle approach to process validation encompasses product and process activities beginning in

development and continuing throughout the commercial life of the product. The 1987 definition and

subsequent discussion in the guidance placed major emphasis on the validation protocol, testing,

results, and documentation (what is now considered to be the process qualification stage in the lifecy-

9

Figure 1.3: Main principles of FDA’s Guideline for Industry on Process Validation, 2011. Source: BiopharmInternational

cle approach).[14] Moreover, this guidance aligns process validation activities with a product lifecycle

concept and with the International Conference on Harmonisation (ICH) Guidances for Industry: Phar-

maceutical Development (Q8) [10], Quality Risk Management (Q9) [11] and Pharmaceutical Quality

System (Q10) [12].

1.3.1 Process design

The commercial manufacturing process is defined during this stage. Its main goal is to "design

a process suitable for routine commercial manufacturing that can consistently deliver a product that

meets its quality attributes" [9]. This stage is critical for the success of validation, since the design

choices made at this stage will influence the outcome of the process and the overall quality of the final

product. Furthermore, at this stage studies are also commonly conducted to develop and characterize

both the product and its production process, as well as to decide on what may be the best layout and

structural options for the manufacturing department and the support systems to be installed within it.

Process design can be approached either from an empirical perspective or a more systematic one,

or even a combination of both. At a minimum, the approach followed regarding process design must

always include the following elements:

• Selection of the appropriate manufacturing process, as well as the basic layout of the manufac-

turing department and the support equipment and utilities;

• Definition of the Quality Target Product Profile (QTPP), since it relates to quality, safety and

efficacy, and includes considerations on e.g., the route of administration, dosage form, bioavail-

10

ability, strength and stability; [10]

• Identifying Potential Critical Quality Attributes (CQA) of both the product and the manufacturing

process, so that those characteristics having an impact on product quality can be studied and

controlled; [10]

A more systematic approach to development (also defined as Quality by Design (QbD)) can in-

clude, for example, incorporation of prior knowledge, results of studies using design of experiments,

use of Quality Risk Management (QRM) and use of knowledge management [12] throughout the life-

cycle of the product. Such a systematic approach can facilitate the achievement of the desired quality

of the product and help the regulators to better understand a company’s strategy. Product and pro-

cess understanding can be updated with the knowledge gained over the product lifecycle. [10] This

enchanced approach would also include:

• A systematic evaluation, understanding and refining of the formulation and manufacturing pro-

cess, including;

– Identifying, through e.g., prior knowledge, experimentation, and risk assessment, the ma-

terial attributes and process parameters that can have an effect on product CQAs;

– Determining the functional relationships that link material attributes and process parame-

ters to product CQAs.

• Using the enhanced product and process understanding in combination with quality risk man-

agement to establish an appropriate control strategy which can, for example, include a proposal

for a design space (DS) and/or real-time release testing (RTRT).

The basis for design of a pharmaceutical product is the quality target product profile (QTPP),

defined as "a prospective summary of the quality characteristics of a drug product that ideally will be

achieved to ensure the desired quality, taking into account safety and efficacy of the drug product"

[15]. The QTPP forms the basis of design for development of the product, the process used to

manufacture it and the infrastructural system that supports its implementation and should be one of

the first documents to be placed in a product specification or design-history file, since it includes both

the expected drug product quality attributes and the general manufacturing pathway.

Designing an efficient process with an effective process control approach is dependent on process

knowledge and understanding obtained. Therefore, product and process characterization activities

are crucial. There are several strategies that can be implemented for this purpose, namely design of

experiments (DOE), risk assessments, laboratory or pilot-scale experiments and computer modeling.

[7]

More advanced strategies, which may involve the use of Process Analytical Technology (PAT),

can include timely analysis and control loops to adjust the processing conditions so that the output

remains constant. [9]

11

Regardless of the approach taken, once the process design stage is completed and all desired

variations were tested, the knowledge over the process undergoing validation should be enough to

assure its full understanding and a proper control strategy must be in place to be applied during the

following stages of validation and, later on, during the commercial stage.

1.3.2 Process Qualification

During the process qualification (PQ) stage of process validation, the process design is evaluated

to determine if it is capable of reproducible commercial manufacture.

Throughout this stage, cGMP-compliant procedures must be followed. This is relevant because a

successful completion of the process qualification is necessary before commercial distribution can be

initiated. This stage has two elements: design of the facility and qualification of the equipment and

utilities and process performance qualification (PPQ).

- Design of the Facility and Qualification of Utilities and Equipment

Proper design of a manufacturing facility is a GMP requirement and is essential for the success

of the production processes on it developed. It is essential that all activities performed to assure

proper facility design and commissioning precede PPQ. Here, the term qualification refers to activities

undertaken to demonstrate that utilities and equipment are suitable for their intended use and perform

properly. These activities necessarily precede manufacturing products at the commercial scale.

For a new or upgraded facility, commissioning and facility validation is the foundation for assuring

success in further manufacturing process validation. Before you begin validating a manufacturing

process, an acceptable facility and the utilities and equipment to support manufacturing operations

must be in place and fully validated.

Facility qualification (a part of validation that proves and documents that equipment or ancillary

systems are properly installed, work correctly and actually lead to the expected results) and validation

(establishing documented evidence that provides a high degree of assurance that the manufacturing

processes, including buildings, systems and equipment consistently produce the desired results, ac-

cording to predetermined specifications and quality attributes) activities are commonly described in

four stages:

• A Design Qualification (DQ) stage, used to verify that the premises, supporting utilities, equip-

ment and processes have been designed in accordance with the requirements of GMP;

• An Installation Qualification (IQ) stage, where it is assessed if the premises, supporting utilities

and equipment have been built and installed in compliance with their design specifications;

• An Operational Qualification (OQ) stage, where it is verified that the facilities, supporting utilities

and equipment operate in accordance with their design specifications

• The Equipment Performance Qualification (PQ), designed to confirm that the facilities, utilities,

12

or equipment that can affect product quality perform as intended, meeting predetermined ac-

ceptance criteria.

For the particular case of manufacturing areas, as defined in the regulations, separate or de-

fined areas of operation within the pharmaceutical manufacturing environment should be maintained

and controlled during production. The design of a given area involves satisfying microbiological and

particle criteria, as defined by the equipment, components and products exposed, as well as the op-

erational activities conducted in the area. Clean area control parameters should be supported by both

viable and non-viable particulate data obtained during qualification studies. [9],

The initial qualification of pharmaceutical controlled areas includes, in part, an assessment of air

quality under as-built and static conditions, but has a main focus on the data generated under dynamic

conditions, i.e., simulating the normal operation routine conditions. Besides this, an adequate plan for

the monitoring of the environmental conditions of the controlled areas is also essential to assess the

conformance with the regulated clean area classification specified for the manufacture of parenteral

products.

Qualification of utilities and equipment are approach under a different perspective, generally in-

cluding the following activities: [9]

• Selection of utilities and equipment construction materials, operating principles, and perfor-

mance characteristics based on whether they are appropriate for their specific uses;

• Verification that utility systems and equipment are built and installed in compliance with the

design specifications (e.g., built as designed with proper materials, capacity, and functions, and

properly connected and calibrated);

• Verification that utility systems and equipment operate in accordance with the process require-

ments in all anticipated operating ranges. This should include challenging the equipment or

system functions while under load comparable to that expected during routine production. It

should also include the performance of interventions, stoppage, and start-up as is expected

during routine production. Operating ranges should be shown capable of being held as long as

would be necessary during routine production.

The standard set of utilities that can be found in Hikma plants, supplying its production lines are

summed in Figure 1.4.

Qualification of utilities and equipment can be covered under individual plans or as part of an

overall project plan. The plan should consider the requirements of use and can incorporate risk man-

agement to prioritize certain activities and to identify a level of effort in both the performance and

documentation of qualification activities. The plan should identify all the tests performed, along with

the criteria used to assess outcomes, the timings of the qualification activities and the procedures for

documenting and approving by the responsible departments. By the end of this stage, the installation

13

Figure 1.4: Standard set of utilities found in Hikma plants. Source: Applying Process Systems Engineering forContinuous Improvement in Pharmaceutical Production, 2013

and operational qualifications of all the equipments and utility sets that will be a part of the manufac-

turing process must be complete.

- Process Performance Qualification (PPQ)

The process performance qualification combines the actual facility, utilities, equipment (each now

qualified) and the trained personnel with the commercial manufacturing process, control procedures,

and components to produce commercial batches. A successful PPQ will confirm the process design

and demonstrate that the commercial manufacturing process performs as expected and will consis-

tently deliver quality products.

The approach to PPQ should be based on sound science and the manufacturer’s overall level

of product and process understanding and demonstrable control. Accumulated experience is highly

valuable and the introduction of inputs that have proven successful in previous similar approaches

and products is usually a valuable insight at this point. The cumulative data from all relevant studies

(e.g., designed experiments laboratory, pilot, and commercial batches) should be used to establish

the manufacturing conditions in the PPQ.

In most cases, PPQ will have a higher level of sampling, additional testing, and greater scrutiny of

process performance than would be typical of routine commercial production. The increased level of

scrutiny, testing, and sampling should continue through the process verification stage as appropriate,

to establish levels and frequency of routine sampling and monitoring for the particular product and

process.

This stage is also critical to assess the robustness of the process regarding several variables that

occur during production. One of the most relevant concerns the process’ capacity to control the level

14

of potential contaminations. Prevention of the presence of bioburden, endotoxins or foreign contam-

inants in the aseptic process streams must be demonstrated in PPQ batches. This demonstration

can be accomplished through a combination of process controls such as raw material specifications

and testing, equipment cleaning and sanitization, facility/environmental requirements and controls,

operational controls and in-process monitoring during the production of the drug product.

Associated with the PPQ stage, there are two main sets of documentation that must be created:

the PPQ protocol and the PPQ report. Both documents are essential to support the validation program

that is applied to processes and products, since they contain all the information regarding the way that

the validation activities were performed and the results they originated.

The Process Performance Qualification Protocol is a written plan stating how validation will be

conducted, including test parameters, product characteristics, production equipment, and decision

points on what constitutes acceptable test results. Thus, this plan should document a complete list

of process validation studies required for product/process approval, including the appropriate level

of effort and timing of process validation activities. A typical PPQ protocol discusses the following

elements:

• The manufacturing conditions, including operating parameters, processing limits, and compo-

nent inputs;

• The data to be collected, as well as the timings and procedures for its evaluation;

• Tests to be performed (in-process, release, characterization) and acceptance criteria for each

significant processing step;

• The sampling plan, including sampling points, number of samples, and the frequency of sam-

pling for each unit operation and attribute. The number of samples should be adequate to

provide sufficient statistical confidence of quality both within a batch and between batches. The

confidence level selected can be based on risk analysis as it relates to the particular attribute

under examination. Sampling during this stage should be more extensive than is typical during

routine production;

• Criteria and process performance indicators that allow for a science- and risk-based decision

about the ability of the process to consistently produce quality products. The criteria should

include:

– A description of the statistical methods to be used in analyzing all collected data (e.g.,

statistical metrics defining both intra-batch and inter-batch variability);

– Provision for addressing deviations from expected conditions and handling of nonconform-

ing data. Data should not be excluded from further consideration in terms of PPQ without

a documented, science-based justification.

15

• Design of facilities and the qualification of utilities and equipment, personnel training and qual-

ification, and verification of material sources (components and container/closures), if not previ-

ously accomplished;

• Status of the validation of analytical methods used in measuring the process, in-process mate-

rials, and the product;

Once the PPQ protocol is executed, a Process Performance Validation Report documenting and

assessing adherence to the written PPQ protocol should be prepared. On this report, there should

be:

• Discussion and cross-referencing of all aspects of the PPQ protocol;

• Summary of the collected data and respective analysis, as specified in the protocol;

• Evaluation of any additional observations and data that wasn’t specified on the protocol;

• Summary and discussion of all manufacturing non-conformances, such as deviations, aberrant

test results or information that conflicts with the validity of the process;

• Detailed description of any corrective actions or changes that should be made to any procedure

or control undergoing validation;

• Statement of a clear set of conclusions stating whether the data indicates the process met the

conditions established in the protocol and whether the process is considered to be in a state of

control. If not, the report should state what should be accomplished before such a conclusion

can be reached.

The conclusions stated in the report should be based on a documented justification for the ap-

proval of the process, successful release of lots produced by it to the market in consideration of the

entire compilation of knowledge and information gained from the design stage through the process

qualification stage.

1.3.3 Continued Process Verification (CPV)

The main goal of the Continued Process Verification stage is to guarantee that the process re-

mains in a state of control (the validated state) during commercial manufacture. To accomplish it, the

integration of a system or systems to detect process variability or other changes to the original design

is essential. Specifically, the collection and evaluation of information and data about the performance

of the process will allow the detection of undesired process variability. Evaluating the performance of

the process identifies problems and determines whether actions must be taken to correct, anticipate,

and prevent problems, so that the process remains in control.

CPV can either be applied to a new process/product or to legacy products, i.e., existing validated

products in commercial manufacturing.

16

The application of CPV to a new product should be started immediately after they successfully

complete their Process Performance Qualification stage. In these cases, the usual approach consists

on applying a consistent program of monitoring and sampling of process parameters and quality

attributes at the level established during the process qualification stage until sufficient data is available

to generate significant variability estimates. These estimates can provide the basis for establishing

levels and frequency of routine sampling and monitoring for the particular product and process.

On the other hand, when it comes to legacy products, the attributes and parameters to be included

in the CPV plan should be defined and evaluated based on historical and routine manufacturing data.

Besides this, the initial evaluations may also determine the need to return to some of the activities

described in the first two stages of the process validation program, which should be used to improve

and enhance the monitoring policies implemented in the manufacturing process.

Regardless of the type of product to be evaluated, the implementation of a successful CPV plan

usually encompasses the following points:

• Implementation of systems to detect unplanned divergence from the process design, which may

include deviations / non-conformances, out-of-specification results (OOS), out-of-trend results

(OOT), erroneous batch records, defect complaints, adverse event reports, process yield varia-

tion, among others;

• Ongoing program to collect and analyze product and process data that relate to product quality

(process trends, in-process controls (IPC), finished product testing against specifications);

• Ongoing process monitoring of critical process parameters and quality attributes (CPPs and

CQAs);

• Statistical trending (reviewed by trained personnel);

• Annual Product Quality Reviews (PQR);

• Improvement initiatives through process experience, taking into account not only management

experience reviews but also production staff feedback and experience from executed batch

records;

• Ongoing risk management policies;

• Corrective Actions and Preventive Actions (CAPAs) to address process malfunctions or devia-

tions

The systems to collect and analyze product and process data that relate to product quality are the

most important part in the CPV programs. The process capability must be evaluated and the data

collected should include relevant process trends and quality of incoming materials or components,

facilities, in-process materials and finished products. The data should be statistically trended and

17

reviewed by trained personnel, in order to verify that the quality attributes are being appropriately

controlled throughout the process.

On this stage, a more detailed assessment of the quality of the previous validation stages can

also be accomplished. Good process design and development should anticipate significant sources

of variability and establish appropriate detection, control, and/or mitigation strategies, as well as ap-

propriate alert and action limits. However, a process is likely to encounter sources of variation that

were not previously detected or to which the process was not previously exposed. Many tools and

techniques, some statistical and others more qualitative, can be used to

Variation can also be detected by the timely assessment of defect complaints, out-of-specification

findings, process deviation reports, process yield variations, batch records, incoming raw material

records, and adverse event reports.

Regarding process improvement, the analysis of the data collected during the initial phases of this

stage may also provide different ways to improve and/or optimize the process by altering some aspect

of the process or product, such as the operating conditions (ranges and set-points), process controls,

components or in-process material characteristics.

Maintenance of the facility, utilities and equipment is another important aspect of ensuring that a

process remains in control. Once established, qualification status must be maintained through routine

monitoring, maintenance, and calibration procedures and schedules, whose frequency should be

adjusted based on the data collected during these activities. The equipment and facility qualification

data should be assessed periodically to determine whether re-qualification should be performed and,

if so, to what extent.

In Table 1.1, a summary of the current recommended approach to validation suggested by FDA is

presented.

Table 1.1: Summary of the current recommended approach to process validation.

Stage Objective Characteristic elements

Process Design

To define the commercial process on knowledge gainedthrough development and scale-up activities. The outcomeis the design of a process suitable for routine manufacturethat will consistently deliver product that meets its critical

quality attributes.

Quality by design;Product developments activities;

Design of experiments;Risk assessments.

Performance Qualification To confirm the process designas capable of reproducible commercial manufacture.

Facility design;Equipment and utilities qualification;Process performance qualification.

Continuous ProcessVerification

To provide ongoing assurance that the process remains ina state of control during routine production through quality

procedures and continuous improving initiatives.

Trend analysis;Statistical data review;

Periodic review of maintenance andcalibration;

Improvements through process experience.

1.3.4 EMA’s 2014 guideline on process validation - a new approach

In June of 2014, the new guideline from EMA on process validation for finished products came

into effect and with it, a new approach to process validation was introduced. In this guideline (which

18

complies with the ICH’s Q8, Q9 and Q10 documents), the European agency introduces a new take

on process validation, which is described as a hybrid approach. EMA describes three possible ways

that manufacturers can follow when establishing the validation of a process: the traditional approach,

continuous process verification and the hybrid approach. [13]

The traditional process validation is normally performed when the pharmaceutical development

and/or process development is concluded, after scale-up to production scale and prior to marketing of

the finished product. On this approach, conformity with the regulators’ requirements is proven through

a series of test batches, that must show that the process consistently delivers outputs with the desired

level of quality.

The number of batches should be based on the variability of the process, the complexity of the pro-

cess/product, process knowledge gained during development, supportive data at commercial scale

during technology transfer and the overall experience of the manufacturer. Data on a minimum of 3

production scale batches should be submitted unless otherwise justified. Data on 1 or 2 production

scale batches may suffice where these are supported by pilot scale batches and production scale

validation data at the time of regulatory submission is provided. [13]

On the other hand, continuous process verification is an alternative approach to traditional process

validation in which manufacturing process performance is continuously monitored and evaluated (ICH

Q8). Continuous process verification can be used in addition to, or instead of, traditional process

validation.

This approach shares many common points with the FDA’s 2011 Guideline on Process Validation,

defending the take on validation should be a science and risk-based real-time approach to verify and

demonstrate that a process that operates within the predefined specified parameters consistently pro-

duces material which meets all its critical quality attributes (CQAs) and control strategy requirements

thorughout its lifecycle. Relevance to the application of Quality Risk Management, Process Analytic

Technology and statistical-based methods is highlighted in this perspective. [13]

The novelty from EMA comes with the concept of the hybrid approach. EMA recognizes that it

may be necessary to use either the traditional process validation or the continuous process verifi-

cation approach for different steps within the manufacturing process. The validation requirements in

terms of batch size and number of batches would depend on the extent to which continuous process

verification has been used. For situations where continuous process verification does not address the

critical unit operation(s), the process validation requirements highlighted in the traditional approach

should be applied. [13]

This idea allows manufacturers to adapt the strategies on process validation with the specification

of the process and the product they are validation, which potentially leads to an improvement on the

results of the validation protocols.

19

1.4 Validating a new manufacturing line of parenterals

1.4.1 Parenteral preparations

Parenteral preparations are sterile preparations containing one or more active ingredients intended

for administration by injection, infusion or implantation into the body. They are packaged in either

single-dose or multi-dose containers. [16]. A parenteral drug is defined as one intended for injection

through the skin or other external boundary tissue, rather than through the alimentary canal, so that

active substances they contain are administered, using gravity or force, directly into a blood vessel,

organ, tissue, or lesion. [17] They are infused when administered intravenously, or injected when

administered intramuscularly, or subcutaneously into the human body. A large volume parenteral

(LVP) is a unit dose container of greater than 100 ml that is terminally sterilized by heat. Small volume

parenteral (SVP) is a "catch-all" for all non-LVP parenterals products except biologicals. A small

volume parenteral is a unit dose container of less than 100 ml. [18]

The preparation of parenteral medicines always to be performed in compliance with the cGMP

requirements. Nevertheless, according with the product specifications and the customer’s needs, the

sterility of the final product is assured in one of two ways: through aseptic manufacture or through

terminal sterilization. In the manufacturing line studied on this thesis, the infusion bags produced are

terminally sterilized and this process is discussed in further detail on section 3.2.

Parenteral preparations can be presented in different ways, according with the physical state of the

product or the clinical use they are intended for. Sterile solutions, suspensions, emulsions or solids

can be formulated as parenteral preparations and presented under different categories, which include

injections, intravenous infusions, powders for injections and implants.

A - Manufacture:

The manufacturing process should meet the requirements of good manufacturing practices (GMP).

The quality and grade of starting materials, the design and maintenance of the equipment and the

method of manufacture must ensure the stability of the active substance and the final product, as well

as the sterility of the final product, guaranteeing that it is free of pyrogens and particulate matter.

Parenteral preparations may require the use of excipients such as solvents, substances to en-

hance solubility, suspending agents, buffering agents, substances to make the preparation isotonic

with blood, stabilizers or antimicrobial preservatives. The addition of excipients is kept to a minimum

and incompatibilities between any of the components of the dosage form must be avoided completely.

Besides this, when excipients are used they do not adversely affect the stability, bioavailability, safety

or efficacy of the active ingredient(s), cause toxicity or undue local irritation.

Water for injections (WFI) is used as the vehicle for aqueous injections. Sterilization at this stage

may be omitted, provided that the preparation is subjected to terminal sterilization. For non-aqueous

injections, fixed oils of vegetable origin are used as vehicles. Unless otherwise specified in the individ-

ual monograph, sodium chloride or other suitable substance(s) may be added to an aqueous solution

20

for injection in order to render the preparation isotonic. When an individual monograph defines a

particular parenteral preparation simply as a solution, emulsion or suspension in WFI, this does not

preclude the inclusion of such substances, where necessary, for this purpose.[16]

Throughout manufacturing, certain procedures should be validated and monitored by carrying

out appropriate in-process controls. These should be designed to guarantee the effectiveness of

each stage of production. In-process controls during manufacture of parenteral preparations should

include monitoring of environmental conditions (especially with respect to particulate and microbial

contamination), bacterial endotoxins, pH and clarity of solution, freedom from particulate matter and

integrity of the container-closure system (absence of leakage, etc.). For powders for injections con-

trols should also include uniformity of mass, moisture content and the ease of reconstitution of a

solution or suspension. The validation of the manufacturing process and the in-process controls have

to be documented.

B - Containers and closures:

Parenteral preparations are usually supplied in glass ampoules, vials, plastic bottles or bags or in

prefilled syringes. In case of light-sensitive substances, the container should protect the contents (for

example, by the use of coloured glass).

Containers are made, as far as possible, from material that is sufficiently transparent to permit

the visual inspection of the contents, except for implants and in other justified and authorized cases.

They do not adversely affect the quality of the preparation, allow diffusion of any kind into or across

the material of the container or yield foreign substances into the preparation.

Regarding the closures for parenteral preparation containers, they are typically equipped with a

firm seal to prevent entry of microorganisms and other contaminants while permitting the withdrawal

of a part or the whole of the contents without removal of the closure. They are not made of com-

ponents that react with the contents or that allow foreign substances to diffuse into the preparation.

The closure, composed of plastic materials or elastomers, is sufficiently firm and elastic to allow the

passage of a needle with the least possible shedding of particles. Closures for multi-dose containers

are sufficiently elastic to allow the puncture to reseal when the needle is withdrawn and protect the

contents from airborne contamination. A tamper-evident container is fitted with a device that reveals

clearly whether it has ever been opened. [16]

C - Visual inspection and contamination:

The containment and closure of the parenteral preparations must be performed in a way that

ensures that a visual inspection of the solutions, whether they are liquid or reconstituted, grants

them to be clear and free from visible particulate matter. Any trace of a defected unit during visual

inspection, such as a phase separation in emulsions or discoloration/precipitation of solid matter, must

result in the rejection of said unit.

Visual inspection is typically complemented with a series of tests to detect any sort of contamina-

21

tion in the preparations. Regarding this subject, there are several sources of contamination that have

to be analyzed, namely particulate contamination, bacterial endotoxins contamination and pyrogens

contamination.

Regarding particulate contamination, parenterals may be tested to detect visible particles, are

commonly detected using a double-faced lightbox. or subvisible particles, using one of two main

procedures: light obscuration particle count test and microscopic particle count test.

1.4.2 Hikma’s approach to validation

The company manufactures sterile injectable products for parenteral use, including liquid dosage

forms (LVP and SVPs) and lyophilized powders. Regardless of the type of product being manufac-

tured, several quality assessment mechanisms are in place in the company to ensure that all products

are fit for their intended use, in compliance with the requirements of the regulative authorities and the

marketing authorizations, and that they do not place patients at risk due to inadequate safety, quality

or efficacy.

Regarding the validation policy applied by the company, compliance with both the FDA’s Guideline

on process validation and EMA’s Guideline on process validation for finished products is maintained

and preserved throughout the lifecycles of the processes and products. The company applies a hybrid

approach to validation, as suggested in EMA’s latest guideline, combining both the lifecycle approach

of the three stages of process validation with some of the traditional validation practices, which include

a series of test-run batches to evaluate the process performance.

During a process validation step, three consecutive batches are manufactured, and all in-process

and finished product specifications are verified as per a specific pre-approved protocol. Critical pa-

rameters are set at different ranges during the manufacturing process to verify that the product con-

sistently meets its predetermined quality specifications over the range of critical parameter settings

(acceptance criteria).

This practical methodology is complemented with the information that the company generates by

applying its risk management and assessment program to all processes and products undergoing

validation. This approach allows the company to assess critical parameters and priorities within the

process and controlling the associated risk factors to an appropriate and acceptable level.

The past experience of the company with similar products and processes is also a key element in

the definition of new validation plans, since it can provide a clearer insight on the approaches to follow

regarding the different steps of the validation plan.

There is an integrated Quality System in place in the company, that is in sync with the recommen-

dations issued by the authorities (namely ICH’s Q10) [12], whose purpose is to assure the quality

of all products that are released into the market, as well as the quality of their respective production

processes and the overall procedures followed within the factory. All validation plans initiated in the

22

factory are closely monitored and evaluated by the quality department.

1.4.3 Components of the department

The focal point of this work is to describe the main focal points on the installation and validation of

a new commercial manufacturing line, for the production of terminally sterilized infusion bags.

This new line will be installed in Hikma Pharmaceuticals’ production facilities in Sintra, Portugal,

being the second manufacturing line to commercially produce terminally sterilized infusion bags in

the company. The new line will be integrated with the pre-existing one in the new infusion bags

department, that will host all the production of terminally sterilized infusion bags in the company.

In total, and after the installation and validation of the new line is complete and the transfer of the

existing line is performed, the new department will include two filling sections, each with its preparation

and compounding section, a section for material preparation, as well as an inspection section and

several support rooms. A layout of the department can be found on Appendix A.

Regarding the new line, which is the aim of study of this work, the following elements will be

installed in and for the support of the classified areas in the department:

• Clean room construction elements, which include walls, ceilings, doors and floors;

• One fully automatic bag making, filling and sealing machine for polypropylene materials, as well

as four laminar flow units to support the machine;

• Two stainless steel tanks, with a capacity of 8000 l, for the compounding of the parenteral

preparations and support the filling machine;

• An HVAC system, which includes one air handling unit (AHU) to provide the necessary environ-

mental conditions and air changes to the department.

• A new loop for the distribution of Water for injection (WFI) and pure steam (PS)

This elements will be complemented with a secondary area, that is not classified as a "lint-free"

area, where the inspection to the manufactured bags, the terminal sterilization step and the packaging

will be performed.

The central point of the department is the bags’ filling machine. The machine installed in the

department is fully automatic, securing the fabrication, filling and closure of flexible single chamber

bags under pharmaceutical conditions. It is in compliance with actual cGMP and FDA standards. The

individual working stations can process 4 bags in one cycle and are mounted in the machine frame.

The machine framework consists of stainless steel AISI 304 quality or comparable. All components

in touch with the product are made of stainless steel AISI 316 quality or comparable. As port system

one or two flexible tubes could be sealed per bag, and the solution is filled through the tube(s). After

23

the filling process, the tube is sealed with one of different stoppering systems, according with the

product specifications, limiting the contact between the preparation and the air.

After production and inspection, the bags will be terminally sterilized in a steam autoclave, in order

to guarantee that the parenteral preparations will be sterile and will not pose any health hazard to the

patients it will be administered to.

Further information on the department’s construction process and materials, the auxiliary struc-

tures installed and the bags sterilization process will be provided and discussed in the following chap-

ters of this work.

24

2Contamination prevention

Contents2.1 Contaminations in the pharmaceutical environment . . . . . . . . . . . . . . . . . 262.2 Contamination control in the department . . . . . . . . . . . . . . . . . . . . . . . 29

25

2.1 Contaminations in the pharmaceutical environment

The development of a contamination control plan is critical to the success of aseptic, terminal

sterilization and non-sterile manufacturing facilities. This is most obvious in the aseptic case, where

the FDA has issued clear regulatory guidance on the need for control of contamination at all stages

of the process. Nevertheless, in aseptic manufacturing conditions, the sterility of the various product

containers may not be dependent solely on the terminal sterilization step.

Therefore, the preceding production steps must satisfy special requirements: starting materials

(such as active ingredients and excipients), primary containers, and the components of the container

closure system, intermediates, and finished products must be treated at every processing level in a

manner that ensures that they are protected against contamination. [19]

In terms of contaminants of parenteral preparations, different classes of contaminants can be

distinguished, including microorganisms (general bioburden, pyrogens, bacterial toxins) chemicals

and particles (physical contamination). [20]

The control and reduction of the presence of microorganisms within pharmaceutical environments

is fundamental when parenteral preparations are manufactured. High levels of bioburden pose a

serious threat to the safety of the manufactured drugs, since they can not only directly contaminate

the product but also produce toxins that may pose serious risks to a patient’s health.

There are two general kinds of bacterial toxins. Exotoxins are produced during the growth phase

of certain kinds of bacteria and are liberated into the medium or tissue. They are protein in nature

and their reactions are specific. For example, Clostridium botulinum produces an exotoxin of unusual

potency which affects only neurological tissue. Other well-known examples of exotoxins are tetanus

toxin, shiga toxin, and diphtheria toxin. [21]

Endotoxins are another kind of toxin that can be extracted from a wide variety of gram-negative

bacteria. The term "endotoxin" is usually interchangeable with the term "pyrogen", although not all

pyrogens are endotoxins and pyrogen testing alone cannot be used entirely for detection and charac-

terization of microbial endotoxins. Higher doses of endotoxin are required to produce a lethal effect in

the experimental animal when compared to exotoxins.

The effects produced by endotoxins on the host are systemic such as fever and general body

reactions, rather than strictly neurological effects, as is the case with most exotoxins. Endotoxins are

found in the gram-negative bacteria mostly, and are obtained subsequent to the death and autolysis

of the cells. The endotoxins are extracted from and associated with the cell structure (cell wall). Good

examples of pyrogen producing bacteria are S. typhosa, E. coli, and Ps. aeruginosa. [22]

On a different level, chemical contamination is also a threat in parenteral preparations. Contamina-

tion may arise from metals deliberately added as catalysts or reagents. Natural occurrence in source

materials (e.g., in minerals or herbals) or processing equipment like vessels, pipes or metal connec-

tions to tubes or hoses may be further causes for metal residues. As contaminants, these metals may

26

exert toxicological effects and therefore they should be excluded or limited to an acceptable threshold.

[23]

The parenteral preparations may also be contaminated with visible or sub-visible particles, as a

result of the interactions of the solution with the surfaces that enclose it or due to a direct exposure to

a particle-releasing environment.

2.1.1 Sources of contamination

Given the complexity of the manufacturing process and the several variables that introduce in-

puts in the process, the sources of contamination within the pharmaceutical environment are diverse

and influence differently the quality and safety of the processes and products. The most commonly

addressed sources of contamination to pharmaceuticals are featured in Figure 2.1.

Figure 2.1: Sources of contamination in the pharmaceutical environment.

Personnel are among the more critical sources of contamination. Unlike the other sources already

mentioned, human actions and behaviors are not measurable and, as a result, it is more difficult to

control and monitor their impact on the process and the product’s quality.

The potential for contamination coming from personnel can result from different factors, including:

[24]

• Deficient training, which results in a lack of understanding of the process and the procedures to

follow;

27

• Poor hygiene habits;

• Incorrect behaviour in production (e.g. moving a contaminated object from a less clean area to

a cleaner one without proper disinfection;)

• Incorrect gowning procedure.

Besides this, factory personnel, whether they are part of the operations, maintenance or quality

teams, can make mistakes while performing their designated tasks and may end up affecting inadver-

tently, directly or indirectly, the quality of the product.

Water and air are featured as sources of contamination since both act as vectors for different

contaminants and, in the case of water, act as a growth media for many microorganisms. [25]

In the case of water, piping system defects and faults on the production of pharmaceutical class

water may cause contamination of clean incoming water. Seasonal variations in temperature and

growth of flora may also cause fluctuations in microbial content of source water. [26]

The materials, and in particular the surface materials, also represent possible sources of contami-

nation. First of all, parts that come into contact with the product must be considered to be particularly

critical for product contamination, as organisms can be directly transferred from the surface to the

product.

There are also issues that have to see with the interaction of the materials chosen and the way

their are installed. In particular, if a facility presents, for instance, unsealed ceilings and windows

or poorly equipped airlocks, the potential for contamination increases. Defects on the materials,

like scratches and cracks in the wall or floor covering give organisms a good chance of surviving

disinfection measures and grow on those locations.

Still regarding the facilities, the disposal of waste and the flows of materials are also a possible

cause for contamination. Inappropriately designed material flows may lead to contamination of or from

those materials into others or into the facility. On a different level, a poor system of waste removal

may lead to the accumulation of microbial and particulate generators like bacteria and others, which

may compromise the sterility level of the plant.

The inclusion of the equipment as a source of contamination can correlate with the personnel-

based contamination. Improperly cleaned or sanitized equipment, as well as incorrect assembly

may lead to the accumulation and/or generation of contaminants in the equipment. Other concerns

may include equipment with irregular surfaces, that can lead to the growth of microbial organisms or

to contaminants retention, as well as equipments that tend to generate particles. The retention of

particles in pieces of equipment that are used in consecutive batches may be extremely problematic

and lead to cases of product cross-contamination.

Cross-contamination is a particular case of contamination, in which a starting material, interme-

diate product or finished product is contaminated with another starting material or a product. This

risk of accidental cross-contamination arises from the uncontrolled release of dust, gases, particles,

28

vapours, sprays or organisms from materials and products in process, from residues on equipment,

from intruding insects, and from operators’ clothing, skin, etc. The significance of this risk varies with

the type of contaminant and of the product being contaminated. Among the most hazardous con-

taminants are highly sensitizing materials, biological preparations such as living organisms, certain

hormones, cytotoxic substances, and other highly active materials. Products in which contamination

is likely to be most significant are those administered by injection or applied to open wounds and

those given in large doses and/or over a long time.[27]

2.2 Contamination control in the department

It is state of the art that parenteral products must be produced under controlled conditions. Taking

into account the regulations and the requirements set by the authorities regarding parenteral prepa-

rations and bearing in mind the different sources of contamination cited on the previous section, it is

clear that the existence of a multidisciplinary and broad plan for contamination prevention is key. That

plan must cover all the areas that end up affecting the production process and/or the final product

and, with it, the measures to be taken to prevent contamination must be discriminated.

That plan must be implemented together with an action plan for potential contamination and a

sampling and monitoring plan to evaluate the quality of the processes and the success of the contam-

ination prevention program.

In Figure 2.2 is presented a cause-effect diagram that sums up the most critical areas to be

addressed in order to prevent contamination ingress in the production department.

Figure 2.2: Cause and effect diagram with main parameters to be controlled to avoid contamination in themanufacturing process.

2.2.1 Facilities and environment

The buildings that contain the production processes and the environment surrounding their loca-

tion are one of the fundamental aspects to look after when addressing process and product sterility.

The location of the facility and the way that the buildings are distributed and constructed can critically

29

influence both the outcome of the processes to be developed on that site and the quality of the product

that will be manufactured.

When it concerns the overall manufacturing facility, there are some general considerations that

can be discussed, which include: [3]

• local environmental conditions(including pollution and security);

• suitability/acceptability of physical segregation of processes for manufacture and holding prod-

ucts (this includes the potential segregation of production stages of the same, similar and differ-

ent products and the use of dedicated or shared facilities);

• overall layout of the facility (including the process cores, the position of technical and other

non-production areas with respect to processing areas);

• general layout of the production processes, with concerns over the logical flow through the

facility to reduce to a minimum the crossover of processing streams and different process flows;

• generalized access to the facilities, both pedestrian and by vehicles;

• pest and contamination controls.

Most of this considerations are addressed during the stage of the facility design and, posteriorly,

during its construction and installation. On the particular case of this thesis, since the new line will be

installed in a facility already functional and running other validated and approved commercial man-

ufacturing lines, all these points were already considered and evaluated in the best way to fulfill the

needs and requirements of the processes performed in the plant.

On a more specific level, for each of the areas that affect or interact in any way with the manufac-

turing process, there is a set of requirements that has to be followed based on the type of process

implemented and, obviously, the type of product being manufactured.

Firstly, it is important to distinguish the different areas that can be found within a manufacturing

pharmaceutical plant. All areas in which a specific task or process will be performed that, directly or

indirectly, affects the final quality of the product have to be designed as cleanrooms. On the other

hand, if that area is only planned to act as a passage or holding space, or if the activities happening

there do not influence the final quality of the product, its level of containment can be different and

there may be differences on the overall construction and procedures to follow within it.

Cleanrooms and associated controlled environments are essential to ensure the control of air-

borne particulate contamination to levels appropriate for accomplishing contamination-sensitive ac-

tivities, as well as to reduce to a minimum the risk of microbiological and pyrogen contamination.

[28]

A cleanroom is a room or a specific area with a defined environment control of particulate and

microbial contamination, constructed and used in such a way as to reduce the introduction, generation

30

and retention of contaminants within the area. [29] Within a cleanroom, environmental conditions such

as temperature, humidity and pressure are controlled and monitored.

For economic, technical and operational reasons, clean zones are often enclosed or surrounded by

further zones of lower cleanliness classification. This can allow the zones with the highest cleanliness

demands to be reduced to the minimum size. Movement of material and personnel between adjacent

clean zones gives rise to the risk of contamination transfer, therefore special attention should be paid

to the detailed layout and management of material and personnel flow. [30] A typical example of the

layout of the different rooms inside a clean area environment is displayed in Figure 2.3.

Figure 2.3: Typical control layout for clean areas. Source: World Health Organization

The main goal of the cleanrooms is, as previously stated, to reduce to a minimum the occurrence

of contamination. The sources of contamination are diverse and the design and planning of the clean-

room has to be performed in order to reduce to a minimum the impact of the potential contaminations

in the product’s final quality.

Clean areas for the manufacture of sterile products are classified according to the required char-

acteristics of the environment. Each manufacturing operation requires an appropriate level of envi-

ronmental cleanliness in the operational state to minimize the risks of particulate or microbial contam-

ination of the product or materials being handled.

For the manufacture of sterile pharmaceutical preparations, four grades of clean areas are distin-

guished as follows (see Figure 2.4): [31]

• Grade A: The local zone for high-risk operations, e.g. filling zones, placement of stopper bowls,

open containers or making aseptic connections. Normally such conditions are achieved by

using a unidirectional airflow workstation. Unidirectional airflow systems should provide a ho-

mogeneous air speed of 0.36 - 0.54 m/s (guidance value) at a defined test position 15 - 30

cm below the terminal filter or air distributor system. The velocity at working level should not

31

be less than 0.36 m/s. The uniformity and effectiveness of the unidirectional airflow should be

demonstrated by undertaking airflow visualization tests;

• Grade B: In aseptic preparation and filling, this is the background environment for the Grade A

zone;

• Grades C and D: Clean areas for carrying out less critical stages in the manufacture of sterile

products or carrying out activities during which the product is not directly exposed (i.e. aseptic

connection with aseptic connectors and operations in a closed system).

Figure 2.4: Cleanroom classification from USA, EU and Japanese regulatory agencies. Source: ManufacturingSterile Products to Meet EU and FDA Guidelines, FDA News

When manufacturing terminally sterilized products, the design of the manufacturing line implies

the establishment of the cleanroom grades. This is a critical step, since it will have to fit the tasks that

are intended to be performed in each room, as well as the procedures that will have to be followed

within in. The classifications will have to harmonize with the flows established for the process, making

it so that the sequence of events and the flows of both materials and personnel occur efficiently and

in a way that ensures the minimum risk of contamination for the area and the final product.

The cleanliness grades with the corresponding environmental conditions for the individual manu-

facturing operations are determined in Annex 1 of the EU GMP Guide (2009). [32] This conditions

comply with the recommendations from FDA on the matter and are presented in Figure 2.5

2.2.1.A Surface materials and construction

Special considerations have to be made when selecting and installing the cleanrooms. Not only

the materials chosen must be adequate to the tasks to be performed and chosen based on the

32

Figure 2.5: Manufacturing operations to perform according with the cleanroom grade for terminally sterilizedproducts. Source: Manufacturing Sterile Products to Meet EU and FDA Guidelines, FDA News

assumption that the cleanliness of the area will be maintained as high as possible, but the layout of

the different cleanrooms and the connections between them must also contribute to maintain the area

free from contaminants.

Considering that the manufacturing line produces terminally sterilized products, following the

guidelines from the EMA and FDA (see Figure 2.5), the department’s cleanrooms are all classified as

being grade C, with two exceptions: the rooms used to access the department (which include the per-

sonnel’s gowning rooms and the materials entrance/exit airlock) are classified as grade D cleanroom,

whereas the inside of the filling machine is under a grade A air supply (under static conditions), being

considered a grade B environment for viable environmental monitoring.

All areas that are used to access the department are airlocks, since they separate rooms with

different cleanliness requirements. Air locks for personnel and material are intended to ensure that

the quality of clean room conditions is not impaired by people and material entering and leaving the

rooms.

The doors of the airlocks are electronically locked to assure that they cannot be opened simultane-

ously. Besides this, the locks are adequately ventilated (according with their grade) and are designed

to allow for the personnel to change into gowning that fits the grade of the room they will enter (see

more on section 2.2.4).

There is a pressure cascade that is maintained between the airlocks and their adjoining rooms

(varies from 10 to 15 Pa) which helps guaranteeing that any contaminants flow towards the areas with

the lower grades.

33

Regarding the materials used for the walls, floors and ceilings, their suitability for the intended

operation and the way they are installed has to be carefully selected, in order to prevent the appear-

ance of defected fittings and sealings. Besides this, it is also important to consider the provision and

location of support material and utilities, as well as the overall cleanability and easiness to disinfect of

all the materials and the room.

The design and selection of all the materials to include in the cleanrooms of the manufacturing

line was done according with the current cGMP regulations.

For all the lint free and sterile areas, the laminated internal and external surfaces of walls, floors,

doors and ceilings have smooth and cleanable finishes, being free from cracks and cavities. In ad-

dition to this, they are impervious to water, as well as to cleaning and sanitizing solutions, and they

don’t shed or accumulate particulate matter, which reduces the risk of contaminations occurring on

these areas.

Resistance to degradation is another important feature that must be present within said environ-

ments. On this case, all the materials resist chipping, flaking and oxidizing; besides this, they exhibit

resistance to thermal expansion and contraction, as well as to changes in the environmental condi-

tions.

As for the ceilings, their installation was made so that they were sealed to prevent contamination

from the space above, including the light fixtures. The windows are also sealed, being also non-

opening and flush fitted to prevent the ingress of contamination. On the matter of contamination

ingress and egress prevention, it is important to mention that the joints between the various wall

panels are sealed flush to provide a totally airtight room. This is complemented with the pressurization

of all the rooms, which are electronically interlocked, and the HVAC system and air handling protocols

implemented, which will be discussed ahead.

All additional elements that are integrated within the cleanroom and they may affect the outcome

of the process and the quality of the final product have to be designed and installed in a way that

facilitates the cleaning and disinfection of said element and that reduces its impact within the area

to a minimum. In particular, the installation of communication systems to and from the cleanroom

and the passage of pipes, ducts and other utilities was made so that recesses, unsealed openings or

surfaces that might be hard to clean were not created.

Other essential features, such as power take-off points, switchboards, taps and connections were

designed and installed in a way that facilitates regular cleaning and avoids the build-up of contamina-

tion in or behind blanking covers.

An important side note to be made on this stage relates to the contamination levels that typically

arise in a construction and installation phase of a new manufacturing line. Many tasks involved in

construction and assembly intrinsically generate contamination. With this in mind, the construction

and installation protocol was developed and enforced to satisfy and achieve specified contamination

control objectives, that were established to reduce as much as possible the impact that the new

34

incomplete line would have on the factory environment.

Throughout the earlier stages of the installation, the entire area that encompassed the future line

was sealed from the rest of the factory, so that the ingress of potential contamination to the pharma-

ceutical area was avoided. Besides this, the scheduling of the different tasks to be performed was

constructed so that the tasks which were the greatest sources of contamination were accomplished

before tasks which were lesser sources of contamination or more contamination-sensitive.

Following the completion of the department’s construction, the area of the line was pressurized,

so that the potential contamination that might be generated was contained within the department.

2.2.2 HVAC, air handling and airflow patterns

Heating, Ventilation, and Air Conditioning (HVAC) can be a critical system that affects the ability

of a pharmaceutical facility to meet its objective of providing safe and effective product to the patient.

Environmental control systems that are appropriately designed, built, commissioned, operated, and

maintained can help ensure the quality of product manufactured in a facility, improve reliability, and

reduce both initial costs and ongoing operating costs for a facility. [33] The design of HVAC systems

for the pharmaceutical industry requires additional considerations, particularly with regard to providing

a clean and safe space environment. With high running costs (energy associated with the movement,

cooling and heating of air) and the potential to impact upon safety and product quality, getting them

right is important for business, safety and good manufacturing practice (GMP) criticality. [34]

The design of the HVAC system must be based upon the clean room suite that it serves, and will

be affected by factors such as the number of rooms served, the layout of the rooms, the equipment

within the rooms and, most critically from a qualification perspective, the environmental conditions

that the rooms must achieve. [34]

A properly designed HVAC system has to ensure that the manufactures can monitor and control, at

least, three fundamental parameters: air cleanliness, by promoting the containment and elimination of

airborne particles; the temperature of the air, which may influence the product’s temperature and the

relative humidity of the air, which may influence the product’s moisture content. [35]By fulfilling these

requirements, the HVAC will inherently be controlling the impact of the environment on the finished

product, securing the quality of the product.

Other HVAC variables, such as the rooms’ relative pressure, the integrity of the air filters, airflow

patterns and airflow volume, can affect one or more of the above parameters.

A - Air handling and airflow patterns

Pharmaceutical HVAC should control airborne contamination and needs to help to ensure the

"...purity, identity and quality..." of the product. [36]. The level of air cleanliness (level of airborne

particles) depends on: [35]

35

• internal activities (control of particle generation into the space from people and processes);

• particles entering from outside the space, and the ability to keep these external contaminants

out of the space;

• airflow patterns in the unidirectional flow space;

• general room air patterns;

• the quantity of dilution (supply) airflow;

• cleanliness (quality) of the air being introduced to the space.

The majority of airborne particles are non-viable. A fraction (< 1%) of airborne particles are viable,

e.g., bacteria and viruses; however, these can multiply. Viable particles travel with non-viable parti-

cles; therefore, controlling the total number of airborne particles also controls the number of viable

particles. [33]

The concentration of total airborne particles and microbial contamination within the space is a

key measurement of room environmental conditions for pharmaceutical operations. The different

regulatory agencies differ on the specifications regarding the concentration limits of airborne particles

within clean environments, namely when it comes to the size of particles considered, the state of the

cleanroom (if it is operating or "at rest") and the establishment of cleanliness levels. In Table 2.1, the

acceptance criteria used for the assessment of the level of non-viable particles within clean areas is

shown. This criteria was established based on the limits imposed by USP, FDA and the EU GMP

guide[37], [31]

Table 2.1: Acceptance criteria for the number of viable particles within clean areas.

Roomclassification

In staticconditions

In dynamicconditions

In staticconditions

In dynamicconditions

Maximum number ofparticles / ft3 equal or above:

Maximum number ofparticles / m3 equal or above:

0.5 µm 5.0 µm 0.5 µm 5.0 µm 0.5 µm 5.0 µm 0.5 µm 5.0 µm

A 100 1 100 1 3520 20 3520 20B 100 1 100 82 3520 29 352000 2900C 9968 82 99675 821 352000 2900 3520000 20000D 99675 821 Not defined Not defined 3520000 29000 Not defined 0

In Table 2.1, the static state is the condition where the installation is complete with equipment

installed and operating in a manner agreed upon by the customer and supplier, but with no personnel

present. On the other hand, the dynamic state is the condition where the installation is functioning in

the defined operating mode and the specified number of personnel is present. The areas and their

associated environmental control systems should be designed to comply with the acceptance criteria

for both the static and the dynamic conditions.

Particles generated from internal activities may be controlled to a limited extent by local exhaust

(for particle generating processes), by local airflow patterns or by gowning (for people). On the other

36

hand, particles generated outside the clean areas should be kept out of the critical zone, especially

viable particles from operators in the surrounding area. Particles generated outside the room are

commonly kept out of the room by airflow created by room pressurization.[33]

The ingress of particles and other contaminants from the outside to the interior of the clean areas

is contained through air filtration. The air that supplies the department undergoes different filtration

steps based on the room it will enter. All the air destined for the filling room passes through a pre-

filter, an intermediate filter installed in the AHU and terminal high efficiency particulate air (HEPA)

filters installed at the entrance of the room. The latter are designed to remove from the air that passes

through it a minimum of 99.97% of particles with a size down to 0,3 µm. [38]

For the remaining rooms, where the cleanliness requirements are not as strict, the air passes

though a HEPA filter with primary and intermediate filters before entering the room.

The control and management of the particles generated within the process is more troublesome.

Overall, there are three main elements that are designed and implemented to access this situation:

air change rates, airflow patters and pressurization cascades.

Cleanrooms can be divided in four types, according to the type of airflow pattern: [37]

• Conventional (or non-unidirectional flow);

• Unidirectional flow;

• Mixed flow;

• Isolators, Restrited Access Barrier Systems (RABS) or microenvironments.

A RABS provides a level of separation between operator and product that affords product protec-

tion superior to the other systems. The typical characteristics of these systems include the existence

of a rigid wall enclosure that provides full physical separation of the aseptic processing operations, a

unidirectional airflow system specific for the critical area covered by the RABS and the use of glove

ports, halfsuits or automation to access all areas of the enclosure. [39]

In the department analyzed on this thesis, RABS are not implemented. Instead, a combination of

unidirectional and conventional flow rooms is used to reduce the impact of airborne contamination.

Within the department, only the area of the filling machine is considered a unidirectional flow area

(is classified as a Grade A area, under the EU GMP standards). The machine core, where the bags’

making, filling and sealing procedures take place, is completely covered by a unidirectional (laminar)

flow of clean HEPA-filtered air. The air flows perpendicularly to the working plane, assuring that the

positions immediately adjacent to the clean air supply offer optimal contamination control conditions.

The rest of the department is supplied with a conventional flow of air. The type, position and

number of inlets and outlets depends on the room, but overall the air suppliers are located in the

higher parts of the rooms, generating a downward motion of the introduced air from the ceiling to

37

the floor, where the exhausts are located. This sort of airflow pattern does not allow for a uniform

motion of air (and, consequently, a uniform motion of the airborne particles to remove) but the flows

are aligned to flow from the direction of the higher pressurized room to the lower pressurized room

and away from the product line, which allows for the containment of possible contaminations.

The different airflow patterns employed in the department are schematically represented in Fig-

ure 2.6.

Figure 2.6: Airflow patterns implemented in the department. Source: ISO 14644-4 (2001) Cleanrooms andassociated controlled environments - Part 4: Design, construction and start-up

Typically, in non-sterile rooms (Grade C and below), dilution of airborne particles using high room

airflow rates is common, relying on adequate mixing of room air with clean air to minimize local areas

of high particle concentration. The flow of make-up air introduced in the room is directly related with

the particle generation rate of the process, the operations and the equipment present in the clean-

room. [33] Therefore, knowing the particle generation potential within the process is key to calculate

and adjust the amount of necessary make-up air to dilute the airborne particles concentration and

allow for their continuous removal from the critical areas.

The values for air change rates in the filling room are around the 60 changes per hour, with the

remaining department having about 25 air changes per hour per room.

As it was already mentioned, the department is also pressurized and the pressure cascade imple-

mented between the different cleanrooms helps providing an additional method to remove airborne

contamination from product exposed areas. The filling room is designed to have a positive pressure

in comparison with the remaining rooms, which promotes the flow of air from this room to the adjacent

rooms, with lower pressure and less critical in terms of containment. The pressure cascade is main-

tained throughout the department, promoting airflow to move into the room with lower pressurization.

To complement this, the department also has airlocks, both for the materials and the personnel

(gownings). The primary role of these airlocks is to provide an effective obstacle to airborne contami-

nation. These rooms control traffic into and out of the department through a series of doors and also

provide a location for gowning/de-gowning, decontamination and cleaning procedures.

38

The airlocks installed in the department are cascade airlocks. On these airlocks, the pressure

inside them is somewhere in between the two areas that it connects. This prevents the pressure

differentials between rooms with different air classes from dropping to zero when one of the doors of

the airlock is opened. [29]

B - Temperature and humidity

These two parameters can have a great impact on the quality of the final product and the cleanli-

ness of the production process. Comfortable personnel produce fewer environmental contaminants:

a typical worker will discharge 100,000 particles (sized 0,3 µm and larger) a minute doing relatively

sedentary work. Higher room temperature may affect the comfort of operators in the room, causing

them to release more viable particles through perspiration and respiration. [33]. A high degree of

protective gowning may minimize contamination from operators, but cooler temperature and lower

relative humidity may be needed to keep operators comfortable with more gowning.

Avoiding rooms with high humidity levels is also important, since a high moisture content may

lead to mold and microbial growth in some areas or, on a different level, may increase the chance of

corrosion of metallic materials.

The temperature and humidity of the air are managed on the air handling unit (AHU) that supports

the department. Cooling and heating coils are located within the AHU, increasing or decreasing the

air temperature to ensure that the room temperatures remains within specifications (typical operating

range is around 22 ◦C ± 2). Relative humidity is set to never surpass the 60%.

The HVAC system designed for the manufacturing line studied took into consideration a set of

factors with the goal of guaranteeing that the product quality was not compromised due to poor air

conditioning but also that the personnel and the product safety were maintained. With the imple-

mented system, it is possible to assure that:

1. Cross contamination between the different areas is avoided by proper zoning and pressurization,

air locks, and having a flow of air flooding from the cleanest to the less clean space (until

exhausted), at predetermined points, with a periodic monitoring of the process;

2. Temperature and humidity are carefully monitored and controlled, avoiding disturbances in the

production operation;

3. Records of the pressure, temperature, humidity and filter cleanliness are checked and trended

periodically, to assure the right functioning of the system;

2.2.3 Equipment and utilities

Water is one of the major commodities used by the pharmaceutical industry. It may be present

as an excipient, or used for reconstitution of products, during synthesis, during production of the

39

finished product, as a sterilizing agent (in the form of steam) or as a cleaning agent for rinsing vessels,

equipment, primary packaging materials, etc. [40].

In the production of terminally sterilized parenteral products, there are two main utilities that are

critical for the quality and the safety of the manufactured product: Water for Injection (WFI) and Pure

Steam (PS).

WFI is water for the preparation of medicines for parenteral administration when water is used

as a vehicle (WFI in bulk) and for dissolving or diluting substances or preparations for parenteral

administration before use (sterilized water for injections). [40] WFI is of mandatory use due to its

sterility - the absence of pyrogens, endotoxins or other contaminants is essential. Its use applies to

the formulation of products, as well as to the final washing of components and equipment used in their

manufacture.

Distillation and Reverse Osmosis (RO) filtration are the only acceptable methods listed in the USP

for producing WFI. In the company, the system of production and treatment of WFI that supplies the

needs of the department is split in two phases: a pretreatment stage where the feedwater is pro-

cessed through several equipments until compliance with WFI requirements is met, and a treatment

step where the WFI is maintained, heated and distributed and where the WFI is directed for the pro-

duction of PS.

A - Pretreatment stage

Pretreatment of feedwater is recommended by most manufacturers of distillation equipment and

is definitely required for RO units. WFI in bulk is obtained from feedwater which, in this case, is

water that is intended for human consumption and that is monitored and evaluated by the competent

authority that oversees its distribution. [41].

The pretreatment system used in the company to produce WFI is illustrated in Figure 2.7. This

system is composed by a sand filter (with anthracite incorporated), a pair of softeners, a cement

holding tank of 70 m3, a microfilter and two reverse osmosis apparatus. To complement this system,

there are also an intermediate water hardness analyzer, as well as two stages of addition of chlorine

and bissulfite.

The initial step of the pretreatment stage is a filtration step in a sand and anthracite filter. The pur-

pose of this first filtration step is to remove solid contaminants from the incoming source water supply

and protect the downstream system components from particulates that can inhibit the equipment’s

performance. The usage of a dual filter media with anthracite over sand permits more penetration of

the suspended matter into the filter bed, thus resulting in more efficient filtration and longer runs be-

tween cleaning. During operation, influent water to be filtered enters at the top of the filter, percolates

through the filter bed, and is drawn off through the collector system at the bottom, which directs it to

the softeners.

Periodically, the filter is backwashed and rinsed to carry away the deposited matter. Before a back-

40

Figure 2.7: Water pre-treatment system.

washed filter is placed back into service, it is rinsed to drain until the filtrate meets the specification.

After the filtration, the water enters one of the two available softeners. There are two softeners

to assure that the flow of water is continuous (when one of them is being used, the other one is

undergoing a regeneration step). They utilize sodium-based cation-exchange resins to remove water

hardness ions, such as calcium and magnesium, that could possibly foul or interfere with some of the

downstream processes (like the reverse osmosis step).

The softeners’ operation depends on the flow of water that is supplied and on the hardness of

that water. The water hardness must be provided as inputs to the machine, so that it can calculate

the amount of water that can be treated before a new regeneration step must be initiated. The

regeneration of the resin beds is done with concentrated sodium chloride solutions (brine). Following

the decalcification process, the water is stored in the 70 m3 cement holding tank.

In the holding tank, the water is analyzed to confirm that all the hardness was removed. If the

test indicates that the hardness removal was not effective, the valve that is placed at the outlet of the

working decalcifier will be closed, preventing the entrance of any more hard water into the tank. When

this happens, the water will be re-treated until it complies with the requirements.

Within the tank, the soft water will undergo a chlorination step, which aims to prevent any sort

of microbiological growth in the water. FDA’s regulations state that chlorination of potable water is

an effective treatment if minimum levels of 0.2mg/liter of free chlorine are attained. [26]. On this

case, levels of free chlorine are maintained between 0.3 and 0.7 mg/liter, to assure the efficacy of the

treatment.

41

Before being directed to the reverse osmosis processes, the water is first passed through a set of

microfilters of 5 µm operated in parallel, to provide an additional step of particles that may still be in

the water. From here, the water is then directed to the reverse osmosis systems.

When entering both the first and the second osmosis systems, the chlorine content in the water

is measured. Based on the value, bissulfite is added to the feed water of the osmolizers to prevent

the entrance of chlorine to the membranes. A set of 1 µm microfilters is also incorporated in the

osmolizers to protect the membranes.

There are two reverse osmosis systems, of different capacities, that operate in parallel. In both

systems, water recirculation is used to direct a significant amount of the rejected back to the line

were the water is fed to the osmolizers. In Figure 2.8 is a diagram outlining the process of RO.

When pressure is applied to the concentrated solution, the water molecules are forced through the

semi-permeable membrane and the contaminants are not allowed through.

Figure 2.8: Basic principle of reverse osmosis. Source: Puretec - Industrial Water

An RO membrane rejects contaminants based on their size and charge, being capable of removing

up to 99% of the dissolved salts (ions), particles, colloids, organics, bacteria and pyrogens from the

feed water. For the production of WFI, this step is complemented with the distillation step, so that the

entire load of the feedwater in bacteria and viruses is removed.

In the smaller system, the normal permeate values vary from 800 to 1100 l/h, with a recovery rate

of 80%. In this system, the pressure at the entrance of the membranes goes from 6 to 9 bar, with

a pressure differential in the system of 1.9 to 2.2 bar. Regarding the second system, the permeate

values processes vary from 3500 to 5000 l/h and the entrance pressure values range from 10 to 18

bar, with the recovery rates being similar to the first system.

From the osmosis steps, the water is now directed to a stainless steel (SS) holding tank, with a

2000 l capacity, to go through the treatment stage. This holding tank has a vent filter - hydrophobic

sterilizing-grade filter - used as an air vent. The purpose of the tank vent filter is twofold: maintain

near ambient pressure in the tank while ensuring sterility in the tank. The tank vent filter removes

42

viruses and microorganisms from the air as it flows into or out of the tank.

B - Treatment stage

The outline of the treatment stage, as well as the two loops used for WFI distribution are presented

in Figure 2.9 and Figure 2.10, respectively.

Figure 2.9: Water treatment system.

This is the stage that guarantees the supply of the WFI either to the distiller or to the pure steam

generator. Besides the SS holding tank, the treatment system also includes a heat exchanger, a

microfilter and a UV radiation system.

After leaving the reverse osmosis systems and passing to the SS holding tank, the treated water is

passed through a heat exchanger. The main objective of this equipment is to heat the water up to 80◦C to perform the sanitization of the loop of treated water, an essential step to assure the sterility of

43

Figure 2.10: Water for injection distribution loops.

the treated water. After the sanitization is performed, the cooling of the treated water is also performed

by this heat exchanger.

The water that passes through the treatment system is continuously filtered through the 1 µm filter

installed after the heat exchanger. Alongside the filtration step, the treated water is also sterilized to

remove any microbial load that may remain in it at this stage by a UV irradiation system.

Besides the equipment mentioned above, the treatment loop has two additional elements: a pres-

sure control valve and a conductivity reader. The pressure control valve is located near the holding

tank and is responsible for maintaining a constant pressure in the system, that enables the right

functioning of both the distiller and the PS generator. On the other hand, the conductivity reader is

present to monitor the quality of the circulating water and allows the operator to assess the efficacy

of the treatment system on-line.

44

Additional tests are performed to the water throughout the process, but they will be discussed in

section 4.1.4.

The distiller is the final stage on the WFI production process. The distiller installed has a production

capacity of 3000 l/h of WFI and supplies the two loops that distribute the WFI to the manufacturing

departments.

In each loop there is a SS holding tank, with a capacity of 8000 l. The water is stored at 85 ◦C and

is kept in motion around the loops, and this two factors contribute to reduce the risk of contaminations.

The choice to store the WFI at this temperature is based on the fact that that hot systems (typically

ranging from 60 to 85 ◦C) are self sanitizing, which contributes to the maintenance of the quality of

the WFI. In addition, the fact that the water is kept in constant motion makes it less liable to have high

levels of contaminants.

In both loops, heat exchangers are installed to allow the users to cool down the water, if need be.

In the IBD, which is supplied by loop 1, there are five use points of the WFI: two points supply the

compounding and preparation rooms, two points are used to supply the compounding tanks and a

fifth point is destined to be used for the tanks’ clean in place CIP operations.

In parallel with the steps taken to assure the low bioburden of the water produced, the design

of the production system and the distribution loops was also made considering the impact of the

installation, the materials and the components used in the WFI. It is clear that the piping, valves and

other elements selected have not only to withstand the sterilization processes used to sanitize the

water but also to have virtually no impact on the water’s characteristics. Besides this, their installation

and placement has to be done to reduce to a minimum the risk of ingress of contamination in the

water systems.

Regarding the quality of the materials that compose the treatment systems and the distribution

loops, all product contact steel parts are of stainless steel AISI 316L or comparable. All other product

contact parts such a flexible hoses and gaskets are not toxic, do not establish any sort of chemical

reactions with the water, can stand sterilization temperatures up to 125 ◦C, do not release particles

and are wear resisting, anti-aging and non-deformable.

All the welds that exist in the system are stainless steel, due to their internal smoothness and

resistance to corrosion. Besides this, a special care was taken throughout the system’s design to

make sure that gravity drainage is promoted whenever possible, so as to avoid any stopped water for

staying in the system.

2.2.4 Personnel and material flows to and from the cleanrooms

In order to produce an acceptable sterile product, the design of personnel and material flows

should minimize or prevent the introduction of contaminants to the clean area. Fulfilling this latter

objective is particularly significant in systems where containers, closures and product are exposed

45

and activity is conducted in the immediately adjacent environment.

In the case of the manufacturing line analyzed on this work, the area was designed in a unidirec-

tional manner, which allows for raw materials to enter at one end of the department and for finished

product to flow out the opposite way. The design of the personnel flows was made in order to guar-

antee that the routes that must be followed are clearly defined, with smooth transitions for gowning

zones from the facility entrance, offices, general plant, and operational areas.

The overall establishment of both the personnel and the material flows was thought having in mind

the details of the department, namely the grades of the cleanrooms, the layout of the department and

the logical flow of the process. With this in mind, some general considerations regarding both flows

in the department were issued and include:

• The flows aim to cross contamination and environmental contamination, forcing both personnel

and material to pass through primary disinfection or gowning areas before entering cleaner

areas;

• The use of airlocks as a central part of the flows guarantees the maintenance of the rooms

cleanliness conditions. Furthermore, the access to the filling room is obligatorily made through

a specific airlock, allowing for an additional steps of gowning or disinfection of personnel and

material, respectively, to comply with the requirements of the filling room environment.

• Separate entry and exit routes for personnel and materials were designed, to prevent contami-

nation;

• The number of interventions on the critical areas were designed to be maintained at a minimum.

Elements like communication means between the areas and processing instructions were de-

signed to comply with this premise.

2.2.5 Cleaning and disinfection plans

Cleaning and disinfection of surfaces are essential steps for maintaining the cleanliness of phar-

maceutical manufacturing operations. The establishment of any production process must include

dedicated cleaning and disinfection programs to be applied on a routine basis. This programs must

be presented as written procedures (SOPs) and identify the sequence of the cleaning and disinfection,

the detergents and disinfectants to use, the frequency of procedures and the appropriate techniques

to use. The cleaning programs are constructed with different levels, that separate the number and

type of procedures that should be established based on the needs for said procedures (daily, weekly

and monthly cleaning and disinfection requirements are differentiated and the requirements for each

of these cleaning procedures are specified).

There is also an additional cleaning and disinfection plan for the department that states the proce-

dures and materials to use when the normal environmental conditions of the department are affected.

46

The total cleaning and disinfection plan represents the worst-case scenario for the cleaning and dis-

infection procedures and its implementation guarantees that, after the environmental conditions are

reset, the department returns to the normal cleanliness settings.

The cleaning and sanitization program should achieve specified cleanliness standards, control mi-

crobial contamination of products, and be designed to prevent the chemical contamination of pharma-

ceutical ingredients, product-contact surfaces and/or equipment, packaging materials and ultimately

the drug products. [42]

The construction of the cleaning and validation protocols implies the selection of appropriate deter-

gents and disinfectants, as well as of the cleaning materials and require a solid validation program of

all the choices made, that establishes the operating conditions and parameters that should be applied

when operating.

A disinfectant’s effectiveness depends on its intrinsic biocidal activity, the concentration of the

disinfectant, the contact time, the nature of the surface disinfected, the hardness of water used to

dilute the disinfectant, the amount of organic materials present on the surface, and the type and the

number of microorganisms present. [42]

When choosing the detergents and disinfectants to use, their mode of activity and effectiveness

against microorganisms must be considered. Preferentially, the disinfectants should have a wide

spectrum of activity. The spectrum of activity refers to the properties of a disinfectant being effec-

tive against a wide range of vegetative microorganisms, including Gram-negative and Gram-positive

bacteria. Generally, the actuation of the disinfectant on microbial cells varies from case to case. Dis-

infectants may act on the cell wall, the cytoplasmic membrane (where the matrix of phospholipids and

enzymes provide various targets) or the cytoplasm. Understanding the distinction between different

disinfectants is important when selecting them between. [43]

Two major categories of disinfectants can be distinguished: the non-oxidizing disinfectants, which

include alcohols, aldehydes, amphoterics, biguanide, phenolics, and quaternary ammonium com-

pounds, and the oxidizing disinfectants, also known as sporicidal disinfectants, which include halo-

gens and oxidizing agents like peracetic acid and chlorine hypochlorite. [42]

There are a number of factors which affect how well disinfectants work in practical situations, and

it is important to understand these in order for the cleaning program to be effective. The choice of

detergents and disinfectants to use on the department was based on the validation program applied

to those disinfectants, further detailed on section 4.2. Based on the results of the tests performed, the

effectiveness of the disinfectants against a series of microorganisms was evaluated and parameters

like the concentration and the action time were determined for all the disinfectants selected.

The cleaning and disinfection techniques are extremely important for a successful sanitization

since if detergents and disinfectants are not applied in the correct way, areas will not be cleaned

effectively and unduly high levels of microbial contamination will remain as the disinfectant will not

eliminate all the contaminants. [44] In addition, the cleaning materials used to apply disinfectants and

47

detergents have to be appropriate for the task. The materials must be able to apply an even layer

of each agent. For disinfectants and detergents used for floors, surfaces, and walls in sterile manu-

facturing areas, these must be applied using materials which are cleanroom certified and nonparticle

shedding (non-woven and lint-free).

To begin the execution of the cleaning and disinfection plan, a primary step of vacuum cleaning

can be used, when applicable, to remove larger particles and debris that may be in the area to clean.

The cleaning protocols designed for the department plan for the use of sterilized mops to perform the

cleaning and disinfection protocols. The mops must be used to clean ceilings, walls and floor, on this

order, in a straight motion that avoid overlap of the mop with already clean areas.

The areas must be firstly cleaned with a detergent solution and, posteriorly, disinfected with an

appropriate disinfectant solution. The contact time established for the disinfectants must be respected,

since that is the only way to ensure that the validated conditions for microbial elimination are fulfilled.

Disinfectants for use on surfaces (walls, floors) are applied using the double or triple-bucket system

to avoid cross contamination. Both of these techniques involve using a bucket of disinfectant and a

bucket of water. In the "two-bucket" technique there is a wringer (for the mop) over the bucket of water.

In the "three-bucket" technique there is a third bucket, empty except for having a wringer mounted over

it.

All the disinfectants that are validated to be used in the department are included in a disinfectant

rotation plan, to satisfy cGMP regulations. [42] Because it is theoretically possible that the selective

pressure of the continuous use of a single disinfectant could result in the presence of disinfectant-

resistant microorganisms in a manufacturing area, quarterly rotation of the disinfectants used is im-

plemented in the department.

Parallel to the cleaning and disinfection program for the department, it is also necessary to con-

sider and develop adequate plans to clean and sanitize all the pieces of equipment that are in the

department, as well as the materials that are introduced into the clean area. When it concerns the

materials, before entering the department all carton or styrofoam covers are removed, since they are

particle-generator materials. After they are removed, a disinfection step using either 70 % Isopropanol

or a sporicidal disinfectant is performed.

Regarding the equipment, and in particular the filling machine and the compounding tanks, both

elements have integrated clean-in-place (CIP) and sterilize-in-place (SIP) that are used to perform

the cleaning and disinfection of said equipment. The outer parts are cleaned following the general

protocols for the cleaning of the department.

The qualification of both the CIP and the SIP cycles of the filling machine are a critical step within

the overall validation plan of the department, and further detail regarding the tests conducted to

validate the SIP cycle and the outcome of that validation step will be detailed in section 4.2.

48

3Product safety

Contents3.1 Assessing product safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 503.2 Terminal sterilization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 533.3 Filtration for bioburden reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57

49

3.1 Assessing product safety

The prevention of contamination ingress into the clean areas is not sufficient to guarantee that, by

the end of the process, the product will be safe for use and risk-free for the patient’s health. Aside

from contaminants, that have to be actively prevented and dealt with through sterilization processes,

there are other issues that can compromise the quality and safety of the final product.

Some of these issues were already discussed and include the quality of the water used for the

product’s formulation and the terminal sterilization process used to eliminate contaminants in the

product. In addition to these topics, and considering the specificity of the product being manufactured,

additional references to the interactions between the container and closures used with the product and

the additional process implemented to reduce the microbial load of the product prior to the bags’ filling

were considered pertinent and will be discussed next.

3.1.1 Product and container/closure compatibility

The US Food and Drug Administration (FDA) Guidance for Industry: Container Closure Systems

(CCS) for Packaging Human Drugs and Biologics establishes that, "Every proposed packaging sys-

tem should be shown to be suitable for its intended use: it should adequately protect the dosage

form; it should be compatible with the dosage form; and it should be composed of materials that are

considered safe for use with the dosage form and the route of administration." [45]

Failure to meet any of these criteria can cause a drug product to be contaminated. Examples of

failures include: [46]

• Uneffective protection - allowing contaminants to enter the container and contact with the

product;

• Incompatibility - loss of safety and drug potency due to a leachable from the container/closure;

• Failure to achieve safety - introduction of microorganisms or endotoxins into the drug product

These contaminants can enter the product due to several reasons, including inappropriate materi-

als of construction, improper closure preparation processes, compromised container closure integrity

or degradation of closures and leaching of compounds from the closures.

Issues related with the potential microbial contamination of the product and the actions taken to

prevent it were already discussed in chapter 2. On this section, the focus is on the risks to patients’

health posed by possible incompatibilities between a product and the containers and closures used

to contain it, namely when it concerns the potential leaching of chemical contaminants from the con-

tainers or closures to the product.

Leachables are chemical entities, both organic and inorganic, that migrate from components of

a container closure system or device into a drug product over the course of its shelf-life. Usually

50

they can be found in drug product matrices as complex mixtures at trace levels relative to the active

pharmaceutical ingredient (API). [47]

Management of leachables is important to pharmaceutical and biotechnology/biologic product

manufacturers and regulatory authorities because certain leachables above specific concentrations

can present safety concerns for patients and/or compatibility issues for drug product formulations.

[48]

The choice of the materials to be used as new bag system was done based on the bag system in

use on the currently installed manufacturing line for infusion bags’ production and on a detailed study

on the general information of those and other potential materials, which included the analysis of their

general composition, pharmaceutical conformance, process of manufacturing and physical, chemical

and toxicological specifications, in compliance with the requirements from USP and EMA.

Based on the results, the company chose to use a Polyolefin Multilayer film, a Polyolefin Tube and

a PP twist-off port as the components for the new bag system. The bag is available in a 100 ml and

200 ml nominal volume and will be filled with different filling volumes, specific for each application

(ranging from 50 to 250 ml). The bags are terminally sterilized by a water cascade sterilizer and

subsequently stored in an overpouch for a maximum of 3 years.

In order to assess the toxicological safety of these materials (which is a requirement of the reg-

ulatory agencies), an analytical study was set-up to determine if, and to what extent, the polymer

material of the container bags system will release chemical compounds during its contact with the

current infusion products (active substances, diluents and solvents).

The study was set in two phases,the first being the execution of extractables studies to identify

potential leachables compounds. Extractables are chemical entities, both organic and inorganic, that

will extract from components of a container closure system or device into solvents under controlled

conditions. They are used to identify and quantify potential leachables. [47] With this test, it became

possible to determine a worst case migration profile of the bag system and select target compounds

to be monitored in the leachables study.

For this purpose, three different extraction solvents were stored in the bag system at worst case

extraction conditions. The following extraction solvents were selected to represent a worst case for

the solutions to be stored in the bag system:

1. Acidified UltraPure Water (UPW), pH 2 (using HCl);

2. Alkaline UltraPure Water (UPW), pH 9 (using NaOH);

3. 20% Ethanol in UPW.

These extraction solvents cover the pH range of the different drug products as well as the organic

content (all drug products are aqueous based with an organic content well below 20%, the ethanol

represents the organic content of the drug products).

51

The solutions were screened by a series of analytical methods, including GC/MS screening to

identify volatile organic compounds, High Resolution Accurate Mass (HRAM) UPLC/MS screening to

identify non-volatile organic compounds and ICP Optical Emission Spectrometry to identify elements,

to determine the composition of the materials that were extracted from the containers/closures to

the solutions. The detected substances included both semi and non-volatile organic compounds and

metallic elements, including magnesium, zinc and mercury.

Based on the results of this study and on preliminary interaction studies carried out by the the

plastic film, the plastic tube and the closures’ manufacturers, the target compounds for the leachables

studies were selected.

After the target compounds to be screened with the leachables tests were identified, the products

to be tested had to be chosen. The criteria to bracket Hikma products concerning their impact on

extractables profile were: pH, storage temperature, surface contact area and organic content.

Based on this criteria, one product with acidic pH, another with neutral pH and another with ba-

sic pH were chosen for this evaluation. Also the more concentrated products on NaCl and dextrose

diluents were chosen. For these products, test batches were performed using the new bag system

configurations to test and the bags produced were placed under stability at different storage condi-

tions, so that the leachables assessment was done in three stages: immediately after production; at

accelerated conditions storage (40 ± 2 ◦C / < 25 % RH) at 6 months and during long term conditions

storage (25 ± 2 ◦C / 40 % ± 5 % RH) at 24 months.

The rational for the choice of the manufactured products that were analyzed, with information

relative to their formulation and the parameters that influence the study can be found in Appendix B.

The results obtained will be used to develop drug product leachables specifications and the prod-

uct’s acceptance criteria for the leachables, based on the qualitative/quantitative results of the leach-

ables study, manufacturing process capability considerations and, most importantly, the potential

safety, compatibility or impact on product quality of the leachables.

When dealing with high-risk dosage forms (such as parenterals), it may be meaningful, useful,

and at times required to routinely monitor finished drug products for leachables. Under such circum-

stances, leachables specifications and acceptance criteria must be established.

It is important to note that leachables specifications should be applicable to a product during all

stages of its shelf-life, including release and at end of shelf-life. This is necessary because leachables

accumulate over the entire shelf-life of a drug product. [48] Acceptance criteria can be both qualita-

tive and quantitative for both known and unspecified leachables. For example, a typical leachables

specification could include:

• Quantitative end of shelf-life limits for target leachables, which apply over the shelf-life of the

drug product;

• A quantitative limit for previously unidentified and uncorrelated leachables

52

3.2 Terminal sterilization

The main requirement for medicinal products that are sterilized in the final container is sterility,

which is achieved by means of the terminal sterilization step and is proven by sterility testing according

to the pharmacopeia method. [49]

Sterilization is necessary for the complete destruction or removal of all microorganisms (including

spore-forming and non-spore-forming bacteria, viruses, fungi and protozoa) that could contaminate

the product and that would, consequently, constitute a health hazard. Since the achievement of the

absolute state of sterility cannot be demonstrated, the sterility of a pharmaceutical preparation can be

defined only in terms of probability. The efficacy of any sterilization process will depend on the nature

of the product, the extent and type of any contamination and the conditions under which the final

product has been prepared. The requirements for cGMP should be observed throughout all stages of

manufacture and sterilization. [50]

The death of a homogeneous culture of microorganisms exposed to a constant lethal stress has

been shown empirically to follow a first-order kinetics, known as the survivor curve. The rate of the

microbial death is a function of the thermal resistance of the microorganism and the lethal stress and

is independent of the number of microorganisms in the challenge.

If microorganisms are subjected to moist heat (at a constant temperature), the microbial count

decreases in relation to time. The D value is the decimal mortality rate in minutes at a given tempera-

ture, or the time in minutes that is required to kill 90 percent (a one log reduction) of the population of

microorganisms udes as a biological indicator under specified lethal conditions. The D value always

refers to one temperature and one microbial species. [31]

The z value indicates the change in temperature in degrees Celsius that causes the D value to

change by a power of 10. The z value is also defined as the relative resistance of a given microor-

ganism against different temperatures. [31]

An example is shown in Figure 3.1 of the relationship of the D value to the killing kinetic when the

microbial count is reduced by a power of 10 (A) and of the relationship of the z value to the D value in

a thermal survival curve. n is the number of powers of 10 by which the initial microbial count should

be reduced.

In Figure 3.1 (B), there is a reference to the F value of the process and equivalent values for

sterilization methods at different temperatures. The F value is a measurement of sterilization effec-

tiveness, i.e., of the lethality of the process. F(T,z) is defined as the equivalent time at temperature T

delivered to a container or unit of product for the purpose of sterilization, calculated using a specific

value of z. There is a particular case of F value which is used as a reference to compare the efficacy

of sterilization processes that use different parameters. The reference exposure time, or F0, is defined

as the number of equivalent minutes of steam sterilization at the reference temperature of 121.1 ◦C

delivered to a container or unit of product calculated, using a z-value of 10 ◦C.

53

Figure 3.1: Survival curve of Bacillus stearothermophilus spores with a standard sterilization method (A). Ther-mal survival curve of Bacillus stearothermophilus spores with a standard sterilization method (B). Source: Man-ufacturing Sterile Products to Meet EU and FDA Guidelines

The value of F0 for a certain sterilization process can be calculated using the formula on Equa-

tion 3.1, where N0 represents the actual microbial count, D121 is the D-value for a certain microor-

ganism at 121 ◦C and N is the microbial count desired after sterilization. The lethality factor can

also be determined using the formula on Equation 3.2, where T is the temperature within the item

being heated, z corresponds to the z-value of the challenge organism and ∆T is the exposure time,

in minutes, at the sterilization temperature.

F0 = (logN0 − logN)×D121 (3.1)

F0 = ∆T∑

10T−121

z (3.2)

Each sterilization method has a particular biological performance capability, that is, a proper ca-

pability to kill viable organisms. In the case of terminal sterilized products, the sterilization method

selected must achieve a sterility assurance level (SAL) of, at least, 10−6, that is, a 6 log reduction

(each log reduction [10−1] represents a 90 % reduction in the microbial population).

The SAL is the probability of a single unit being non-sterile after it has been subjected to steriliza-

tion. In microbiology, it is impossible to prove that all organisms have been destroyed as the likelihood

of survival of an individual microorganism is never zero, so SAL is used to express the probability of

the survival. The minimum value of SAL acceptable for parenteral drugs is 10−6, which means that

the chance to find a non-sterile unit is only 1 in 1.000.000. [51]

The design of the sterilization cycles to be employed to sterilize the bags’ loads in the department

was done following the so-called overkill approach. The overkill method relies upon the selection of a

lethality level known to be adequate to ensure sterilization without routine control over bioburden.

The overkill approach stems from the concept that the sterilization process will inactivate a high

54

micro-biological challenge with an additional safety factor. The microbiological challenge will consist

of a biological indicator with a specific number of microorganisms (usually 106), and a worst-case

assumption is made that the heat resistance of the bioburden is equivalent to that of the biological in-

dicator. Therefore, the cycle conditions established are more severe than those re-quired to inactivate

the real product bioburden, and a theoretical spore reduction of 1012 is expected to prove the overkill

assurance.[52]

The basis for this level is that if the bioburden on an article was one million and all of that bioburden

consisted of resistant spores with a D121 value of 1 min, then a 10−6 probability of a nonsterile unit

(PNSU) would be consistently attained. Obviously, this reflects worst-case assumptions regarding

both the bioburden level and resistance, which would in every instance be lower in the real-world

condition.

Classical sterilization techniques using saturated steam under pressure or hot air are the most

reliable and also so most commonly used amongst manufacturers. Other sterilization methods include

filtration, ionizing radiation (gamma and electron-beam radiation) and gas sterilization (ethylene oxide,

formaldehyde). [50]

For each new sterilization process that is introduced, the EU GMP Guide [28] requires that the

process be appropriate for the product. Evidence should, if required, be provided by physical mea-

surements and biological indicators. The validity of the process should be tested at regular intervals,

and at least once a year. The process should also be checked after major changes are made to

the equipment. Validated load patterns should be defined for each sterilization process. The FDA’s

Guidance for Industry: Sterile Drug Products Produced by Aseptic Processing [53] describes this in

a very similar way. The effectiveness of a sterilization cycle should be proven by validation studies.

Requalification studies should be conducted regularly and, in addition, the load patterns used and the

placement of bioindicators and temperature sensors should be determined and documented in the

validation studies in advance.

According to the standards of both authorities, the validation process always consists of the ele-

ments’ installation, operational and performance qualification. In the course of installation qualification

(IQ) and operational qualification (OQ) it should be demonstrated that the sterilizer and the room in

which it is installed comply with the specified requirements. For the purpose of operational qualifica-

tion, a check is made to see whether the instruments that are used for control, display and recording

are calibrated within the specified limits. Performance qualification (PQ) documents that the defined

sterilization conditions are maintained constantly and everywhere in the sterilizer load. It includes the

interaction between the sterilizer, process, items to be sterilized and packaging.

The standard process according to the European Pharmacopeia [54] envisage sterilization using

saturated steam for 15 minutes at 121 ◦C. When these parameters are put in relation to the F value

(specified in minutes), it follows that the initial microbial count may not exceed 104 germs/ml. [31] On

the basis of this conclusion, it is clear that the introduction of germ-reducing measures throughout the

55

production process should explicitly be implemented, validating the approach taken in the develop-

ment of the contamination control plan for the department and the introduction of the additional step

of filtration to reduce the bioburben.

Whichever method of sterilization is chosen, the procedure must be validated for each type of

product or material, both with respect to the assurance of sterility and to ensure that no adverse

change has taken place within the product. Failure to follow precisely a defined, validated process

could result in a non-sterile or deteriorated product.

A typical validation program for steam or dry-heat sterilization requires the correlation of tempera-

ture measurements, made with sensory devices to demonstrate heat penetration and heat distribution,

with the destruction of the used biological indicators, i.e. preparations of specific microorganisms,

known to have high resistance to the particular sterilization process. Biological indicators are also

used to validate other sterilization methods and sometimes for routine control of individual cycles.

The difference in microbial resistance is critical to sterilization validation. The microbial genera

Geobacilli, Bacilli, and Clostridia, having substantial resistance to the sterilization process, are com-

monly chosen as BIs to provide an appropriate evaluation of the process. These BI organisms are

stipulated to be spore populations that have much higher resistance to sterilization processes than

the vegetative cells that predominate in the normal microflora found in pharmaceutical production en-

vironments. Using these spores as indicator organisms creates a process challenge that is inherently

worst-case. In the case of moist heat in which sterilization conditions are very well defined and under-

stood, BIs are best used to establish that there is sufficient correlation between physically measured

lethality, generally in the form of thermometric data, and biological lethality measured using calibrated

BIs. [55]

For the case of overkill systems, the role of the BI would be to prove that there is a strong cor-

relation between a physically determined F0 of 12 min and biological lethality at the location of the

indicator. A good correlation between biological and physical lethality ensures that an efficient and

well-designed cycle with suitable steam penetration and air removal (where necessary) exists.

The description of the plan of initial qualification of the terminal sterilization cycles to be imple-

mented in the department is detailed in section 4.2

A - Superheated water sterilization:

Sterilization with recirculating superheated water (sometimes referred to as water cascade or rain-

ing process) is the method of choice for the sterilization of the infusion bags.

The water-cascade sterilizer installed in the department is primarily intended for large volumes of

liquids in sealed glass or plastic containers. The sterilization process is very straightforward: first,

the chamber containing the item to be sterilized is filled to a pre-defined level (below the load being

sterilized) with water (for sterilization purposes in the new department, only WFI will be used in the

autoclaves). The WFI then circulates through a steam-heated heat exchanger and cascades over the

load being sterilized at a continuously rising temperature.

56

The bags are placed in perforated stainless steel trays, which permits the water to circulate through

the trays and contact directly with the bags, increasing the efficiency of the process.

The advantage of the hot water cascade system lies in its very short cycle times, which are

achieved through a high circulation rate and cascade density in combination with short heating up and

cooling down times. Throughout the process, temperature-controlled supporting pressure generated

by sterile-filtered compressed air prevents the tightly closed receptacle from bursting or deforming.

[56]

The sterilization process, like all processes within the department, must be validated before being

used in commercial manufacturing. The parameters of the cycle have to comply with the requirements

of the regulators and be adequate to the materials used and the type of product to sterilize. [16]

Further details on the validation of the sterilization procedures are presented on section 4.2.

3.3 Filtration for bioburden reduction

Filtration is the process of removing the microorganisms and particles that are present in liquids

and gases with the help of suitable filter materials. When dealing with the aseptic manufacturing

of sterile pharmaceutical products, sterile filtration is key to assure the sterility of the final product,

since in most cases they cannot be terminally sterilized. However, filtration can also be employed in

the manufacturing of terminally sterilized products as a way to reduce contaminants in the product,

increasing as a result the chances of a successful traditional sterilization step. [31]

To guarantee that the reduction of bioburden is substantial using filtration, the selection of a suit-

able filtration system is very important. This includes the filter device and the associated filter medium.

A number of details play a significant role in making the right decisions, including:

• Compatibility of the solution to be filtered with the envisaged filtration system;

• Batch volume;

• Total particles in the solution to be filtered;

• Number of organisms in the solution to be filtered;

• Requirements regarding the pyrogen content.

The company’s experience with the filtration train already in use in its running infusion bags manu-

facturing line played a major role in the selection of the appropriate train system to employ in the new

department. Considering that the majority of the products being produced will be transfered for the

new department, a worst-case scenario condition was establish based on the formulation that pre-

sented the greater challenge for the filtration system. The major difference was in the batch volume,

since the amount of solution to be filtered could, in some cases, increase about 20 times (going up to

8000 l).

57

The selection of the filtration system and the type of filter to use was based on a conjoint study

performed between the company and the filter’s manufacturer. In a lab-scale model, the conditions

of the filtration process in the worst-case scenario were simulated and the operational parameters to

be tested during validation, as well as the most adequate types of filter (and pre-filter) to use were

chosen.

Membrane filters will be used to perform the filtration steps. This filters are made of polymer

starting materials such as polyvinyl chloride and are mounted in a housing, that is installed directly

on the product line piping. The filter has a sterilizing retention grade of 0,2 µm, while the pre-filter’s

is 1,2 µm. Considering that, for the case studied, most of the process parameters were already well

defined (including the product’s density, viscosity and pH, as well as the expected bioburden and

particle level), the focus was on establishing what were the optimal conditions in terms of pressure

differential and product flow rate for the different filter system settings testes.

Ideally, it would be beneficial is the pressure differential was kept as low as possible, to increase the

probability of microorganisms and particles’ segregation and that the residence time of the particles

in the pores was as long as possible to increase the reliability of the retention in the filter. [31]

The filtration system was chosen bearing in mind that the filtration step will be applied in the

manufacturing step immediately before the filling step. The filtration system is designed to act in two

different areas: the pre-filter is installed at the exit of the compounding tanks, retaining the larger

impurities and preventing the clogging of the sterilizing filter.

The filters to be used in the department will be previously sterilized in line before the product pas-

sage is initiated. This increases the sterility of the entire filling process and prevents the contamination

of the product with any viable particles that may have been in the piping or the filter.

Before and after the filters are used, they are subjected to a filter integrity test, to confirm the

quality of the filter used and the success of the microbial load reduction process.[57]. The integrity of

the filter is tested by performing a bubble point test. In this test, the pressure at which the transition

of the diffusive gas flow to the free flow via pores in the filter material that are no longer wet takes

place is determined. With this process, the gas pressure is increased continuously or in stages on the

non-sterile side, and a check is made to see when the pressure decreases disproportionately. [31].

58

4Results: Validation and qualification

of the installed line

Contents4.1 Validation of the department’s components . . . . . . . . . . . . . . . . . . . . . . 604.2 Validation of cleaning and sterilization methods . . . . . . . . . . . . . . . . . . . 73

59

4.1 Validation of the department’s components

4.1.1 Installation and qualification tests

Once the department is installed and the ancillary equipment that is fixed and used in production

is already in place (including the filling machine, the compounding tanks, etc.), the next step on the

process validation plan is to qualify the installation of said equipments and guarantee that they are ca-

pable of performing consistently the tasks they were designed to perform. All pieces of equipment that

will be used or installed in the line have to be previously validated before being used in a production

context.

The qualification of the filling machine was performed in two separate stages: prior to the instal-

lation, a factory acceptance test (FAT) was conducted to determine if the machine was capable to

comply with the requirements for production established in the project design phase. This test was

performed in the manufacturer’s facility with a similar machine that was fully functional and operating.

A specific protocol was designed for this stage and in it, the parameters that were going to be

evaluated were described, as well as the acceptance conditions for the several elements analyzed.

The protocol was divided in two major sections: the functional inspection phase and the bags’ quality

stage.

The functional inspection stage was performed with the goal to evaluate if the different stations of

the machine (which include the bag making station, the filling station and the bags’ sealing station,

among others) are working like described in the contract of the machine. The different components

that are activated in each station were challenged regarding their functioning and capability to perform

their designated roles and the outcome was recorded in the protocol.

When it concerns the bags’ quality verification, a series of tests was conducted to evaluate not

only the visual appearance of the product but also the conformity with the regulators’ guidelines. The

bags were verified to assess their general design and appearance, as well as the quality of the printing

of the batch’s information on their surface, the outline welding of both the bag and the tube and the

insertion of the closures. Besides this, compressive and drop tests were also performed on sample

bags, according with ISO’s regulation on plastic containers for intravenous injections [58] to guarantee

their compliance with the requirements on containers’ integrity.

With the approval on the FAT, the second stage of the machine’s validation is the site acceptance

test (SAT), performed after the machine was installed in the department. The SAT is a more strict

test, that aims to challenge the performance of the machine and to evaluate the optimal operating

conditions and parameters that should be used to develop the operating protocols and the production

recipes.

Just like in the case of FAT, a protocol was issued for this stage of the validation process, and

the combined results of both protocols were documented as a report that certifies the results ob-

tained in the initial qualification stage and that will be used as the baseline for the routine validation

60

interventions that will have to be performed throughout the process lifecycle.

Similarly to what happened to the filling machine, the remaining pieces of equipment installed

have to undergo a process qualification step, followed by the general process performance qualifica-

tion stage before being validated as fit to perform in routine manufacturing procedures. All the pieces

of equipment to be validated as covered either by the validation master plan established for the de-

partment or an individual validation plan, that will specify all the conditions that must be met and all

the challenges that the equipment must pass in order to become apt for use.

The standard process qualification protocols applied in the department include a first stage of

installation qualification, where the equipment is evaluated and its installation is conducted and certi-

fied, followed by an operational qualification stage where the functioning of the equipment under the

desired settings of operation must be demonstrated.

4.1.2 Assessment and control of air quality

Following the installation of the HVAC system and the HEPA filters that handle and sterilize the air

that enters the department it is necessary, both for validation and for routine qualification purposes,

to establish the monitoring protocols used to evaluate the state of those elements.

There is a series of specific tests that have to be performed to validate the air handling units,

including: calculation of the air changes per hour, measurements of the air velocity, airflow and air

patters, measurement of the differential pressure between rooms and, for the HEPA filters, measure-

ment of the pressure drop of the filter and leak tests.

A - Air patterns determination

A major part in the contamination prevention plan, which is inherently connected with the assur-

ance of the product’s sterility, is the control of the airflow patterns within the cleanrooms. At it was

previously mentioned, the area of the filling machine is considered as a grade A area regarding air

supply, being under a unidirectional flow hood, while the air supply to the rest of the department is

performed through air inlets that provide a non-unidirectional air supply.

Based on this, the validation of the air handling unit must include a qualification of the airflow pat-

terns within the cleanroom, as a way of not only guaranteeing that an adequate unidirectional airflow

is maintained in the critical areas but also that the airflow between adjoining rooms within the depart-

ment and between the airlocks and the pharmaceutical grade areas outside the department is directed

from the most critical to the less critical areas (grade C to grade D and grade D to pharmaceutical

areas).

The determination of the airflow patterns is made through smoke tests. These tests must be per-

formed both under static and under dynamic conditions and are a part of both the initial qualification

of the department and the lifecycle-based validation plan.

When assessing the airflow patterns under the laminar flow hood of the filling machine, the gen-

61

erated smoke should be unidirectional, showing uniform patterns with minimum turbulence and no

back flow. There is also a second acceptance criteria that has to be verified, that is the flow of the

smoke towards potential sources of contamination and away from the the product path. In this case,

however, since the area of the machine which includes the unidirectional flow hood (UFH) is delimited

by a series of Plexiglas doors, and the velocity of the air flowing from the unidirectional flow to the

outside of the delimited area is greater than the velocity of the airflow in the rest of the cleanroom,

there will be no movement of air from outside the UFH into the machine area.

In the case of the non-unidirectional airflow, the acceptance criteria defined states that the smoke

should flow from the higher pressurized rooms to the lower pressurized rooms, which assures that

possible contaminations in the air are directed to less critical areas.

The acceptance criteria must be met in both cases before the testing under dynamic conditions

is performed. For this case, the purpose is to evaluate how the normal operating conditions and the

personnel movement affect the airflow patterns and the expected movement of the air inside and

in-between the cleanrooms.

No back flow due to the turbulence can be verified. Slight turbulence due to equipment config-

uration is accepted but if the air returns to critical areas then the system must be adjusted. The

aerodynamic patterns must be changed if the turbulence cannot be stopped, in order to prevent the

flow towards more critical areas and to allow for the system to become qualified.

B - HEPA filters integrity and performance tests

Leak testing have to be performed at installation to detect integrity breaches around the sealing

gaskets, through the frames, or through various points on the filter media. Thereafter, leak tests will

be performed every six months for the unidirectional grade A air supply flow of the filling machine

and once per year on the filters installed in the remaining cleanrooms of the department. Additional

testing may be appropriate when air quality is found to be unacceptable, facility renovations might

be the cause of disturbances to ceiling or wall structures or as part of an investigation into product

sterility failure. [53]

The purpose of performing regularly scheduled leak tests is to detect leaks from the filter media,

filter frame or seal. The test method applied for the testing of the installed filters is the standard

aerosol photometer test method described in ISO’s 14464 Part 3 guideline on Test methods. [59] This

is commonly known as the DOP test and uses the aerosol photometer as the measuring device and

an aerosol generator to produce an aerosol challenge.

The challenge involves use of a polydispersed aerosol composed of particles with a light-scattering

mean droplet diameter in the submicron size range, including a sufficient number of particles at ap-

proximately 0,3 µm. The aerosol photometer uses a near forward scattered light chamber and a

photomultiplier tube as its detection method. The forward scattered light is directly proportional to the

aerosol mass concentration. The instrument is a continuous real-time detector and usually allows a

62

pre-set alarm point to be set for easy detection of leaks.

Performing a leak test without introducing a sufficient upstream challenge of particles of known

size upstream of the filter is ineffective for detecting leaks. It is important to introduce an aerosol

upstream of the filter in a concentration that is appropriate for the accuracy of the aerosol photometer.

The leak test should be done in place, and the filter face scanned on the downstream side with an

appropriate photometer probe, at a sampling rate of, at least, 1 ft3 per minute.

The downstream leakage measured by the probe should then be calculated as a percent of the

upstream challenge. The procedure to perform the leak test is described in a SOP, indicating that the

scan should be conducted on the entire filter face and frame, at a position about 5 cm from the face

of the filter. The maximum permissible leak is set at 0.01%, and a single probe reading that fails to

meet the requirement would be considered as indicative of a significant leak.

When a leak is found, the replacement of the HEPA filter has to be done or, when appropriate, the

limited area of the leak may be repaired. A subsequent confirmatory retest should be performed in

the area of any repair.

The success of the leak tests alone is not enough to validate a HEPA filter. Additionally, measure-

ments of the air velocity downstream of the filter and of teh pressure drop across the surface of the

filter must be performed.

Usually, the results of these tests are interpreted together, and are use to evaluate the performance

of the HEPA filters and, simultaneously, of the air distribution system to the inside of the cleanroom

(this applies both to unidirectional flow hoods and to standard air entrances).

The maximum pressure drop admitted in all ceiling mounted HEPA filters in the department, where

they are part of a unidirectional flow hood (UFH) or not, should be 500 Pa, while the air velocity

downstream of the filter must be between 0,36 and 0,45 m/s under UFH and 0,25 and 0,56 for the

non-unidirectional flows.

The values for the air velocity were established based on the "as built" specifications, that is,

the velocity estimated as the optimum velocity to assure the room’s requirements (in terms of air

changes and air supply to the room) in the situation where the installation is complete with all services

connected and functioning but with no production equipment, materials or personnel present.

Situations in where the acceptance criteria for the pressure drop across the filter is not met and the

air velocity measured is below to the established limits should lead to the replacement of the filter. The

monitoring of this parameters is part of the validation protocol of the HVAC system and the successful

validation of the filters installed in the system can only be achieved if the acceptance criteria are met

and the leak test is negative for flaws on the filter.

63

4.1.3 Monitoring and validation of the environmental conditions

As it was emphasized throughout this work, the maintenance of the environmental conditions re-

quired within the cleanrooms has a major relevance in avoiding the contamination of the product,

therefore allowing for the maintenance of a state of control of the manufacturing process that con-

tributes to guaranteeing the quality of the product.

After the installation of the cleanrooms is complete and the ancillary systems that help containing

the ingress of contamination are functioning (namely, the HVAC system), the result of said installation

and the impact of the routine operations on the sterility levels that has to be assessed.

Monitoring cannot identify and quantify all microbial contaminants present. Furthermore, micro-

biological monitoring of a cleanroom is technically a semi-quantitative exercise, given the limitations

in sampling equipment and the lack of precision of counting methods and limited sample volumes

mean that environment monitoring is incapable of providing quantitative information regarding sterility

assurance. [60]

However, the real value of a microbiological monitoring program lies in its ability to confirm con-

sistent, high quality environmental conditions at all times. Monitoring programs can detect changes

in the contamination recovery rate, that may be indicative of changes in the state of control within the

environment, making it possible to act in order to reestablish the normal conditions.

The evaluation of the environmental conditions of the department for the purpose of its initial

qualification was performed following a written procedure for environmental monitoring (EM). In this

procedure, all the knowledge regarding the layout of the area, the air handling within the department

and the operations performed was used to establish a general microbiological sampling plan, that

includes the type of sampling to perform, the sampling points and number of measures to perform

during the validation of the department and throughout the routine operation. The several stages

that go from the design of the EM plan until the trending of the obtained results are summarized in

Figure 4.1

The rationale behind the choice of the number of sampling locations is dependent on the room

size, as per the International Standard EN ISO 14644-1. [61] The minimum number of sampling

locations is determined based on the following equation:

NL =√A (4.1)

In Equation 4.1, NL is the minimum number of samplings locations (rounded up to a whole num-

ber) and A is the area of the clean room or clean zone (in square meters).

Once the number of locations per room is calculated, the distribution of those sampling points

within the area has to be done and is usually based on the following criteria:

It is important that locations posing the most microbiological risk to the product be a key part of

64

Figure 4.1: Scheme illustrating the EM process.

the program. It is especially important to monitor the microbiological quality of the areas that have a

direct impact on the quality and the steility assurance level (SAL) of the product, to determine whether

or not the desired environmental conditions are maintained throughout the activities taking place in

those areas. Air and surface samples should be taken at the locations where significant activity or

product exposure occurs during production. Critical surfaces that come in contact with the sterile

product should remain sterile throughout the operation. When identifying critical sites to be sampled,

consideration should be given to the points of contamination risk in a process, including factors such

as difficulty of setup, length of processing time and the impact of interventions. [62]

The choice of the sampling locations considered not only the criticality of the operations performed

at each location, but also the results of the smoke tests to determine airflow patters, the number and

the location of the HEPA filters and the existence of high traffic areas in the department (entrance/exit

areas for personnel and materials).

For the purpose of the initial qualification, the sampling procedures of both viable and non-viable

particles were designed to be performed in static and dynamic conditions, following the Environmental

Monitoring (EM) plan designed for the initial qualification of the department. For static monitoring,

a room must have no activity other than the highly trained sampling operators for generally three

hours or greater to be considered in a static condition. A dynamic room is expected to be operating

according to routine manufacturing operations and personnel in order to have a representative viable

and non-viable particulate reading. [63]

The static monitoring is performed to establish the baseline count of particles in a room, that will

represent the optimal conditions and will be used as a reference to determine the decontamination

time of the room. On the other hand, monitoring under dynamic conditions is used to routinely monitor

points of critical exposure of the product and other reference points in the room, with the presence of

the operators and the normal functioning of the production process.

65

During the initial qualification stage, the monitoring of both viable and non-viable particles was

performed during 5 consecutive days in static conditions. Following this stage, viable particles were

monitored during 5 more consecutive days under dynamic conditions, whereas non-viable particles

were monitored during 3 consecutive days under the latter conditions. Once the dynamic studies

are completed, the monitoring of the area will be performed on a weekly basis until all the results

from the initial qualification stage are available and the routine sampling procedures and locations are

established.

The environmental monitoring plan also defined the type of samples to be gathered, in order for the

area monitored to be qualified. Overall, there are two main types of samples that can be considered:

air samples and surface samples. In addition, sterile water lines, product ingredients and finished

products are randomly selected for sampling. [64]

Regarding the monitoring of airborne particles, there are different protocols used for the monitor-

ing of viable and non-viable particles. For non-viable particles, the sampling is performed using an

airborne particle counter, that traces the existence of particulates such as dust, skin and other con-

taminates suspended in the room air. The volume of air sampled to perform the particle count was

different whether initial qualification or routine monitoring was being performed: for initial qualification,

1 m3 of air was sampled in the locations classified as Grade B and Grade A air supply (in the depart-

ment, only the inside of the filling machine has this classification), whereas for grades C and D, the

volume to collect will be 1 ft3. In all cases, the sampling results are the average of three consecutive

reads. The acceptance criteria varies based on the room grade, according with Table 2.1.

For the monitoring of viable particles, the distinction between sampling for airborne particles and

surface contaminant particles can be made. There are two main procedures that are implemented to

perform the sampling of airborne viable particles: active air sampling using air samplers and passive

air sampling using settling plates.

Air samplers draw in predetermined volumes of air. The air is drawn over a sterile media plate,

which is later incubated to reveal the number of viable organisms per volume of collected air. The

sampler holds the media plate under a perforated lid and draws in a known amount of air that passes

to the agar plate, allowing for the accurate measure of the amount of viable bacteria in the air. [65]

Distinctively, settling plates are essentially Petri dishes containing sterile growth media, that are

exposed to the environment for a specific period of time (usually between 30-60 minutes but can be

exposed up to four hours before compromising the integrity of the media itself). Viable microorganisms

which settle onto the media surface will grow after the plates are incubated. However, passive air

sampling is tending to be phased out because it does not reflect microbial contamination with an

accurately measured volume of air.

Both procedures are implemented within the department and were performed in the sampling

locations that were established in the EM plan for the purpose of initial qualification.

On the case of surface particle monitoring, two cases can be considered. To determine the envi-

66

ronmental conditions of the department regarding surface contamination - this is a measurement of

the efficacy of the cleaning and disinfection procedures - RODAC or surface plates are used. This

plates contain a sterile growth media (usually Tryptone Soy agar enriched with lecithin and tweet

80 is used, when the goal is to determine the total amount of microorganisms [general hygienic

state]). When ready to use, the lid is removed and the plated is gently pushed onto the surface of

area/equipment that needs to be sampled. Afterwards, the plated is sealed and incubated.

RODAC plates are also the sampling method used when personnel gowning has to be monitored.

There is a standard protocol for sampling, regarding the sampling locations and order of sampling, that

must be followed to access that the gowning procedures are well performed and that no contamination

is being introduced in the area via gowning.

There is also another protocol of surface sampling that can be used to access mainly the effec-

tiveness of the cleaning protocols on equipment and other materials, which is swabbing. To sample,

the swabs’ heads are wet on a proper solvent and the swab is passed in the area to sample according

with the swabbing protocol. Afterwards, a solvent extraction is used to extract any particles that were

present in the swab’s head and an analytical technique (usually HPLC) is used to trace and identify

the extracted particles.

The overall rationale for the choice of the sampling locations for both viable and non-viable condi-

tions for the filling room is presented in Table 4.1 and Table 4.2. Furthermore, the sampling locations

for both the non-viable and the viable sampling procedures are displayed in Appendix C.

The limits for viable particles that define the acceptance criteria of the tests were established in-

house, based on the FDA and EEC guidelines on the subject. [66], [57]. The limits used are more

strict than the levels suggested in both guidelines, as shown in Table 4.3.

Regarding the static monitoring of non-viable particles, the results of the reads inside the filling

machine (that corresponds to a Grade A air supply zone) indicate a total absence of particles. For the

remaining rooms, the results obtained over the 5 days of initial qualification were trended, according

with the room classification. In Figure 4.2 and Figure 4.3, the trends of non-viable particles equal or

above 0,5 µm are presented, while in Figure 4.4 and Figure 4.5, the trends shown are for particles

equal or greater than 5,0 µm. The series represent the different rooms of the department, and they

were obtained by averaging the results of the several sampling positions within the room for each of

the 5 days of monitoring.

For the case of viable particles, the static monitoring results were already trended for air sampling,

settling plates and contact samples. The trends were made based on the grade of the room and the

type of sample. For each room, the several sampling locations were averaged and the results were

trended for the 5 days of monitoring.

In Figure 4.6, Figure 4.7 and Figure 4.8 are represented the trends obtained by air sampling,

settling plates and contact plates in grade C rooms during the initial qualification stage. The trends

for the remaining rooms sampled are presented in Appendix D. The results of the dynamic monitoring

67

Table 4.1: Rationale for the choice of the non-viable, air sampling and settling plates’ sampling locations.

Area (Room #) /Equipment /

Grade

LocationIdentification

Rational for choosing criticality of the location chosen Does the locationimpact the quality

and SAL of the product?Interventions Other Rationale for choosing critically(add detailed information on how the location was chosen)

IB01 - Line 7Filling Machine(Under Grade A

Air supply)

Description: locationsdistributed within the machinetaking into consideration thenumber of HEPA filters (12);

NL Machine: 12

NV (6): L7-1, L7-2, L7-3, L7-4,L7-5, L7-6, L7-8, L7-9, L7-10,

L7-11, L7-12

Air samplers (6)AS: AS1 - In the tube feeding

area inside the machine; AS2 -In the filling area inside the

machine; AS3 - In thestoppering area inside the

machine; AS4 - In the formingarea inside the machine; AS8 -

In the exiting grippers areainside the machine; AS9 - Inthe bags transfer area inside

the machine

Settings (6)S: S3 - inside the machine

cabinet near the filling needles;S5 - inside the machine cabinet

near the exit belt; S6 - insidethe machine cabinet near the

stoppering area; S7 - inside themachine cabinet near the bags

transfer; S8 - inside themachine cabinet near the tubeinfeed; S9 - inside the machinecabinet near the forming/film

transfer area

- Filling andstoppering area- Filling machine

assembling- Interventions during

filling/stoppering- Tube Loading /

Unloading- Film Loading /

Unloading- Stoppers Loading

/Unloading

This Machine is Minor since product is exposed to airunder UFH before being stoppered, and will beterminally sterilized in the bag after being filled.

Due to criticality of the operations to assure the SALof the product, this area has a total of 12 HEPA filters

(12 UFHs).

As per NL a minimum of 12 locations is required forqualification of the machine for both NV and AS/S;

NV: Considering the number of HEPAs it was decidedto have 12 locations in the area;

AS: locations (6) were distributed to cover all thecritical operations.

S: These locations (6) were chosen taking intoconsideration the criticality of the operations thatoccur and the direct impact to the quality of the

product.

Yes(Direct Impact)

IB01 - Line 7Filling Room

(Grade C)

Description: locationsdistributed within the room

taking into consideration thenumber of HEPA filters (13);

NL Room: 12

NV (6): L7-14, L7-15, L7-16, L7-18,L7-19, L7-20, L7-21, L7-22, L7-23 &

L7-24

Air samplers (6)AS: AS5 - In the Room near the

stopper bowls I; AS6 - in theRoom in front if the manualstations; AS7 - In the Roomnear the stopper bowls II &

Blowing Station; AS10 - In theRoom at the right end corner;AS11 - In the Room at the left

end corner;AS12 - In theRoom behind the tube rolls.

Settings (6)S: S1 - on the wall, near thefilm station; S2 - on the wall

near the speaker to thecorridor; S4 - on the wall nearthe stoppering bowls; S10 - on

the wall near the speaker tothe compounding; S11 - on thewall near the exit belt; S12 - onthe wall near the material exit

door.

- Filling machineassembling

- Interventions duringfilling/stoppering- Tube Loading /


Unloading- Stoppers Loading /

Unloading

The Criticality of Operations taking place in this roomare considered Minor since product is exposed to

air under UFH before being stoppered.

Due to criticality of the operations to assure the SALof the product, this area has a total of 13 HEPA filters.

As per NL a minimum of 12 locations is required forqualification of the machine for both NV and AS/S;

NV: Considering the number of HEPAs it was decidedto have 12 locations in the area;

AS: locations (6) were distributed to cover all thecritical operations.

S: These locations (6) were chosen taking intoconsideration the criticality of the operations thatoccur and the direct impact to the quality of the

product.

Yes(Direct Impact)

were unavailable at the date of submission of this work.

4.1.4 Monitoring and validation of the water quality

As it was said before, to comply with the regulatory requirements, it is expected that the water for

injection (WFI) used to produce any sort of parenteral preparation is essentially sterile.

68

Table 4.2: Rationale for the choice of the RODAC surface plates’ sampling locations.

Area (Room #) /Equipment /

Grade

LocationIdentification

Rational for choosing criticality of the location chosen Does the locationimpact the quality

and SAL of the product?Interventions Other Rationale for choosing critically(add detailed information on how the location was chosen)

IB01 - Line 7Filling Machine

/ Under Grade AAir supply

Description: locations underunidirectional flow hood and

room.

R: R1 - Inside the machine,near to the forming area

R2 - Inside the machine, nearto tube feeding station

R3 - Internal surface of thePlexiglas, near to the manual

feeding stationR4 - Inside the machine, onthe stoppering infeed tracksR5 - Internal surface of the

Plexiglas, near to the filling areaR6 - Inside the machine, on

the output conveyor

- Filling andstoppering area- Filling machine

assembling- Interventionsduring filling/stoppering

- Tube Loading /Unloading

- Film Loading /Unloading

- Stoppers Loading/Unloading

This Machine is Minor since product is exposed to airunder UFH before being stoppered, and will beterminally sterilized in the bag after being filled.

Due to criticality of the operations to assure the SALof the product, this area has a total of 12 HEPA filters

(UFHs).

To properly evaluate the cleaning and disinfectionprocedure, in critical areas such as the filling machine

were evaluated.Also the general cleaningless of the room, such as the

doors and floor.

Yes(Direct Impact)

IB01 - Line 7Filling Room

/ Grade C

R: R7 - Middle of the room onthe floor

R8 - On the wall, right corner ofthe room

R9 - On the wall, near to themelaphone to the

compounding roomR10 - In the door push area to

enter room IB06R11 - External surface of thePlexiglas, near to the filling

areaR12 - External surface of thePlexiglas belt cover, near to

output belt

- Filling machineassembling

- Interventions duringfilling/stoppering- Tube Loading /


Unloading- Stoppers Loading /

Unloading

The Criticality of Operations taking place in this roomare considered Minor since product is exposed to

air under UFH before being stoppered.

Due to criticality of the operations to assure the SAL ofthe product, this area has a total of 13 Ceiling Mounted

HEPA filters.

To properly evaluate the cleaning and disinfectionprocedure, in critical areas such as the filling machine

were evaluated.Also the general cleaningless of the room such as the

doors and floor.

Yes(Direct Impact)

Table 4.3: Action levels for viable particle monitoring.

Action level

Airborne SurfaceGrade Air sampler (CFU/m3) Settling plates (CFU/4 hours) RODAC (CFU/plate)

A <1 <1 <1B 7 3 5

C The action levels for these rooms are established after the execution ofthe performance qualification protocol which, for the case of the department,

will consist on five consecutive days of environmental monitoring indynamic conditions.D

Figure 4.2: Trend of non-viable particles equal or greater than 0,5 µm over the 5 days of initial qualification inGrade C rooms. The several series represent the different Grade C rooms in the department, whereas the topseries corresponds to the acceptance criteria defined.

69

Figure 4.3: Trend of non-viable particles equal or greater than 0,5 µm over the 5 days of initial qualification inGrade D rooms. The several series represent the different Grade D rooms in the department, whereas the topseries corresponds to the acceptance criteria defined.

Figure 4.4: Trend of non-viable particles equal or greater than 5,0 µm over the 5 days of initial qualification inGrade C rooms. The several series represent the different Grade C rooms in the department, whereas the topseries corresponds to the acceptance criteria defined.

Figure 4.5: Trend of non-viable particles equal or greater than 5,0 µm over the 5 days of initial qualification inGrade D rooms. The several series represent the different Grade D rooms in the department, whereas the topseries corresponds to the acceptance criteria defined.

After all the equipment and piping has been verified as installed correctly and working as spec-

ified, the initial phase of the water system validation can begin. During this phase, the operational

70

Figure 4.6: Trend of viable particles sampled through air sampling for the 5 days of initial qualification in GradeC rooms. The several series represent the different Grade C rooms in the department.

Figure 4.7: Trend of viable particles sampled through settling plates for the 5 days of initial qualification in GradeC rooms. The several series represent the different Grade C rooms in the department.

Figure 4.8: Trend of viable particles sampled through contact plates for the 5 days of initial qualification in GradeC. The several series represent the different Grade C rooms in the department.

parameters and the cleaning and sanitization procedures and frequency were developed. Although

the manufacturing line studied in this thesis is not yet cleared for production, the utilities that will

supply the department, namely WFI and PS, were sampled daily after each step in the purification

71

process and at each point of use for three weeks.

The SOPs for the operation of the water system were already developed, since the main system

was already being used to support the running production lines. There was the need to review it in

order to include the parameter specifications required for the new department and the settings for the

cleaning and disinfection of the loop that distributes the WFI to the department.

The sampling procedure for the sampling in the multiple points of use was established at this

stage, with a special concern to assure that the sampling protocol reflected how the water will be

drawn during routine operations (e.g. if a hose is usually attached to the use point, the sample should

be taken at the end of the hose).

The second phase of the system validation consisted on the demonstration that the system will

consistently produce the desired water quality when operated in conformance with the SOPs. The

sampling was performed as in the initial phase and for the same time period. At the end of this phase

the data should demonstrate that the system will consistently produce the desired quality of water.

The third phase of validation fits the lifecycle approach to validation, being designed to demon-

strate that when the water system is operated in accordance with the SOPs over a long period of time

and that it will consistently produce water of the desired quality. Considering that the main system for

the production of WFI has been fully functioning for over two years, typical variations in the quality of

the feedwater that could affect the operation and ultimately the water quality are already identified and

measures to correct them were, for most cases, already implemented. Nevertheless, there is still an

on-going monitoring plan that includes not only the main system but also the new loop that supplies

the department, built to assure the compliance of the WFI production conditions with the required

specifications.

All the samples that are gathered from the WFI production system and the several use points that

supply the department have to be analyzed to determine the quality of the water being produced.

The regulatory requirements for WFI require that the water is free from microorganisms and py-

rogens, presents no particulate matter traces and complies with the restrictions regarding chemicals

composition.

In order to comply with the regulators’ requirements, the WFI cannot include more than 10 CFU/100

ml. This is defined as the action limit for contamination in the water, meaning that an investigation

must be issued to assess the impact of the microbial contamination on products manufactured with

the water whenever this value is passed and corrective actions must be taken to fix the identified

problems. [67]

An additional compliance is related with the quantity of endotoxins in the water. FDA and USP

regulations admit a maximum of 0,25 USP units of endotoxins (equivalent to the IU units) per ml.

The determination of the endotoxin concentration is performed using the LAL test. The limulous

amoebocyte lysate test (LAL test) is based in the biology of the horseshoe crab (Limulous). These

animals produce LAL enzymes in blood cells (amoebocytes) to bind and inactivate endotoxin from

72

invading bacteria. The LAL test exploits the action of this enzyme, by adding LAL reagent to the

tested product, and assaying for clot formation.

All the laboratory tests performed to assess the quality of the water for injection used in the de-

partment are complemented with a preventive maintenance program that was designed to ensure that

the water system remains in a state of control.

This program includes, besides the before mentioned monitoring and sampling programs, the

following elements:

• Detailed procedures describing how to operate the water system, what are the points to analyze

during routine maintenance and how to perform if any corrective action is to be implemented;

• Description of the timings to initiate a maintenance program, the frequency of that work and how

the work is to be documented;

• Assessment of the operating parameters and other quality attributes, through a combination of

on-line sensors and process trends, that allows the constant evaluation of the process’ status

and the recalculation of the needs for a sanitization step or any other preventive maintenance

actions.

4.2 Validation of cleaning and sterilization methods

A – Cleaning and disinfection methods’ validation The cleaning validation involves a series

of stages over the lifecycle of the product and cleaning process: cleaning process design, cleaning

process qualification and continued cleaning process verification.

Cleaning process design intends to design, develop and understand the cleaning process residues

and to establish the strategy for the cleaning process control. The main activities in this stage include

the evaluation of the chemical and physical properties of the residues, as well as the integration of

the information on process sequence and the area layout that should culminate with the elaboration

of the cleaning and disinfection protocols.

In parallel with this stage, the disinfectants that will be used were also validated. To demonstrate

the efficacy of a disinfectant within a pharmaceutical manufacturing environment, the validation pro-

tocol implemented on the factory assumes the realization of the following tests:

1. Use-dilution tests (screening disinfectants for their efficacy at various concentrations and con-

tact times against a wide range of standard test organisms and environmental isolates);

2. Surface challenge tests (using standard test microorganisms and microorganisms that are typ-

ical environmental isolates, applying disinfectants to surfaces at the selected use concentration

with a specified contact time, and determining the log reduction of the challenge microorgan-

isms);

73

3. A statistical comparison of the frequency of isolation and numbers of microorganisms isolated

prior to and after the implementation of a new disinfectant.

The microorganisms used to perform the disinfectant validation, represented in Table 4.4 include

a set of representative organisms of major different classes of microbes, as well as isolates that are

part of the natural flora of the facility’s location and that should be eliminated with the disinfection.

Table 4.4: Set of microorganisms to be used for disinfectant efficacy tests

Challenge microorganism ATCC n◦

Gram negative rod: Escherichia coli 8793Gram negative rod: Pseudomonas aeruginosa 9027Geam positive cocci: Staphylococcus aureus 6538

Gram positive spore-forming rod: Bacillus subtilits 6633Yeast: Candida albicans 10231

Mold: Aspergillus brasiliensis 16404In-house isolate: Micrococcus luteus N/A

With the tests above mentioned, it was made not only the selection of the disinfectants to use in the

department but also the definition of the contact times per disinfectant and the optimal concentration.

The information of the running lines was an important insight on this stage, since any of the

residues that will be found on the department are similar to the ones already found in the infusion

bags manufacturing line already running. Additional information regarding the residues generated by

new materials and equipment were collected and analyzed at this stage. In addition, the results of

the cleaning and disinfection plans already implemented were used to create the plans for the new

department, with information regarding the most successful techniques and procedures to be used to

optimize the new protocols.

Afterwards, the cleaning process qualification can be performed, aiming to demonstrate that the

cleaning procedure works as expected. Some of the activities performed on this stage include the

qualification of clean in place (CIP) systems of the equipment in the department (namely the CIP

systems of the filling machine and the compounding tanks), cleaning operational parameters (e.g.

temperature, flow rates, pressure, etc.), as well as the training of the operators.

Once the qualification is complete, similarly to what happens with the other elements to validate,

it should be demonstrated that the cleaning process remains in control throughout the product lifecy-

cle. This is achieved mainly through the monitoring program of the environmental conditions already

mentioned, that can assess the conditions of the department and provide an insight on the success

of the cleaning and disinfection protocols.

B – Validation of the terminal sterilization The validation plan of a terminal sterilization stage

occurs in multiple steps. Firstly, the autoclave used to run the sterilization cycles has to be fully

validated to confirm its ability to properly sterilize the loads. In parallel, there is also a need to validate

the loads that you intend to sterilize, namely the disposition of the load and the operating parameters,

74

in order to assess the optimal functioning conditions and to assure the final sterility of the product.

The qualification of a sterilization cycle is achieved through heat distribution and heat penetra-

tion studies, that demonstrate the uniformity, reproducibility and conformance to specifications of the

production sterilization cycle for a specific autoclave.

The studies were performed in two phases: firstly, empty chamber studies were conducted, in

order to measure the temperature distribution profile within the autoclave, so that hot and cold spots

in the sterilizer were detected and the temperature uniformity was verified. Following this study, addi-

tional runs with both maximum and minimum loads should be performed, to demonstrate the effects

of loading on thermal input to the product. The basic cycle followed to conduct the heat distribution

studies is displayed in Figure 4.9.

Figure 4.9: Schematic representation of the SIP cycle for the terminal sterilization process.

For the empty chamber studies, a series of thermocouples were placed within the autoclave, in

specific positions, and a series of three runs were performed in order to evaluate the performance of

the sterilizer, according with the following acceptance criteria:

• All thermocouples should attain a temperature of 121.1 ± 1 ◦C during the sterilization phase;

• The maximum temperature disparity between the hottest and the coldest spots should not ex-

ceed 1 ◦C during the steady phase of the cycle.

By the end of the three runs, the values of the temperature disparity between the thermocouples

during the sterilization period of the study were below the acceptance criteria of 1 ◦.

75

The next stage will be the heat penetration studies with both maximum and minimum loads, de-

signed to ensure that the most difficult items to be sterilized are exposed to sufficient heat dose to

assure sterilization.

Heat penetration studies should be conducted with the maximum and minimum loading configura-

tions for each sterilization cycle using the sterilization parameters specified for the normal production

cycles.

During heat penetration studies, sensors will be placed in the containers at their slowest heating

point. The majority of these containers will be located at the slowest heating point in the loading

pattern, as determined by the heat distribution studies. The amount of heat delivered to the slowest

heating unit of the load will be monitored and this data will be employed to compute the minimum

lethality (F0) of the process. Once the slowest heating units of the load have been identified, three

replicate runs will be performed to verify that the desired minimum process lethality factor can be

achieved reproducibly throughout the load. The process is considered acceptable once such consis-

tency in lethality has been adequately established.

For the heat penetration studies, the acceptance criteria already take into consideration not only

the lethality factor of the cycle but also the actual capability of the cycle to eliminate bioburden. The

requirements to be satisfied are the following:

• The minimum F0 time is 15 minutes. This value may be updated if any additional information

on the process or the type of load is already available but is serves as a reference for initial

qualification stages;

• All biological indicators must give negative growth result, except for the positive BI, which must

give a positive growth result;

• The minimum exposure time in order to achieve a 6-log reduction is calculated as 6D+2 minutes,

according with the BI strip certificate D-value.

In order to set-up the heat penetration studies, the thermocouples and the biological indicators will

be placed in pre-defined locations, which have into account the results of the initial empty chamber

studies, and the joint data of the two will be used to validate the sterilization cycle for that load.

For each run, the value of the lethality factor F0 will be calculated per position, based on the

average temperature registered by the thermocouple, according with Equation 4.2

F0 = ∆T∑

10T−121

z (4.2)

In the end, the results for the lethality factors for each of the thermocouples’ position will be de-

termined and matched with the data obtained with the incubation of the biological indicators placed

in the same position. The cycle will only be considered as qualified if all the acceptance criteria are

met, proving that that cycle has the capacity to properly sterilize the tested load.

76

Once all the elements regarding heat distribution and heat penetration studies to validate the ster-

ilization cycles are complete, the next stage will be load optimization. This will consist on conducting

a series of runs varying the size, distribution and operational parameters used (always within the re-

quirements imposed by the authorities) in order to assess the optimal conditions for sterilization for

each type of load to sterilize.

All the tests conducted will have to be annually repeated, as part of the requalification program

effective in the company towards sterilization cycles and sterilizers. On this scenarios, the loads that

have been determined to be the worst-case will be the ones assessed and the results of the tests will

have to remain in agreement with the ones obtained during the initial qualification stages.

The loads to be sterilized coming from the new department will be validated according with a

CBE30 supplement procedure. By definition, a CBE30 is a filing with the FDA to gain approval of

a moderate change, i.e., a change that has a moderate potential to have an adverse effect on the

identity, strength, quality, purity, or potency of the drug product as these factors may relate to the

safety or effectiveness of the drug product. FDA has 30 days to respond prior to implementation of

the change. If the company receives no word from FDA in 30 days, it is assumed that the change was

approved and that it can be considered effective. [68]

For this case, the idea behind the CBE30 is to explore the similarities between the loads produced

in the existing department for the production of infusion bags and the new department studied in this

work. The filling to be presented to the FDA uses as an argument the fact that the data regarding

the type of autoclave to use, all the operational parameters and the distribution and overall charac-

teristics of the loads currently being sterilized (produced in the existent department) and the ones to

be sterilized coming from the new department will be the same. Therefore, it will be assumed that if

all this variables are kept constant and if the original runs of sterilization were already validated and

were demonstrated to comply with the regulatory requirements, then it is likely that the new loads will

present the same behaviour.

C – Validation of the filling machine’s SIP cycle As a validation requirement, both a heat distri-

bution test and a heat penetration test & bacterial challenge (Heat penetration study) were performed

to validate the sterilization in place (SIP) in the filling machine located in the new department.

The SIP system is designed for automatic sterilization without major disassembly and assembly

work of the parts in the machine were is installed. The SIP temperature is measured by one tem-

perature probe located at the coldest position in the system, which is inside the collector pipe (to the

drain), before the steam trap. The main product valve will act as a regulator for the temperature inside

the filling system: the valve will close at a temperature of 127,5 ◦C and reopen when the temperature

drops to 125,5 ◦C. If the temperature falls under the desired set point, the SIP process will restart

automatically; the system is design to restart for three times and then abort the process.

The purpose of this study is to measure the temperature distribution profile, in order to locate hot

77

or cold spots in the system and verify the temperature uniformity. With this, the parameters of the

system may be optimized to assure that the most difficult locations to be sterilized are exposed to

sufficient heat to achieve the sterilization requirements.

The validation study included three runs with 8 calibrated thermocouples for the SIP-Filling Line

Cycle and three runs with 8 Calibrated Thermocouples for the SIP-Vacuum Line Cycle. The load

consists on assembling the 8 thermocouples inside the pipes of the system, alongside 8 strips with

Geobacillus stearothermophilus, the biological indicator (BI) chosen for the qualification runs, inside

the SIP system. Once a run was completed, the thermocouples’ results were analyzed and the BIs

were subjected to a microbiological analysis to assess their condition.

The system configuration with the location of the thermocouples and BI’s is described in Appendix

D.

In tables Table 4.5, Table 4.6 and Table 4.7, a description of the automatic SIP process, as well

as the operational parameters and the characteristics of the BI used are detailed are presented,

respectively.

Table 4.5: Sequence of the SIP cycle of the filling machine.

SIP sterilization cycle

Start up SIP

Select CIP/SIPManual Fitting of the CIP/SIP drain barAll parts are mounted in a good wayLowering the Filling StationSelecting the Desired CycleInitiating the SIP of the Filling Machine from theTanks Control PanelStarting the Cycle on the Filling Machine

SIP: Heating and Sterilization(Automatic Procedure)

Main Product/SIP/CIP valve opensMain Drain opens to remove condensate untilPre-temperature is reachedSwitch valves to Drain only through the Steam TrapHeat up Machine to Set point TemperatureSterilization Starts after Set point is Reached

End Sterilization Ends after the Set time has elapsedMain Drain Opens again and Main Valve Closes

Table 4.6: SIP cycle parameters

Parameter Value Unit

Pre-Temperature 100Temperature 121

Filling Line SIP 20 minVacuum Line SIP 20 min

The acceptance criteria defined for the qualification of the SIP cycle assume that, by the ends of

the sterilization cycle, a 6-log reduction must obligatorily be obtained. The following criteria have to

be verified:

78

Table 4.7: Biological Indicators characteristics

Characteristics

Supplier SPORDEXLot. No. 1014A

Expiry date 11/10/2015Mean Population 2.1x106 CFUD121 (certificate) 2.2 min

Organism Geobacillus stearothermophilus

1. The minimum F0 time allowed is 20,0 minutes;

2. All biological indicators must give negative growth result, except for the positive BI, which must

give a positive growth result.

3. The minimum exposure time in order to achieve a 6-log reduction is 15,2 minutes, calculated

according with the BI strip certificate D-value.

The values of the lethality factor were calculated based on the D-value for the biological indicator

used and the exposure time for each thermocouple (using Equation 4.2) and the biological indicators

were incubated after the runs were completed. The success of the run is established by crossing the

information from the physical and biological data obtained.

The results obtained after the three runs of SIP performed in the filling line are shown in Table 4.8.

Table 4.8: Results of the test runs for qualification of the SIP of the filling line.

Location of the thermocouple Min. F0 Value Min. ExposureTime

Results of the BI incubation(for the three runs)

L # Description of the location Run # Minutes Channel # Run # Minutes Channel #

1 Inside the Left endof the Distributor Pipe 3 141.16 8 1, 3 00:21:00 8 Negative

2 Inside the Right endof the Distributor Pipe 3 160.80 12 3 00:22:30 12 Negative

3 Inside the Pipe before thefilling Valve of needle A 3 141.87 7 1, 3 00:21:00 7 Negative

4 Inside the Pipe before thefilling Valve of needle B 3 145.13 3 3 00:21:00 3 Negative

5 Inside the Pipe before thefilling Valve of needle C 2 86.99 17 1, 3 00:21:00 17 Negative

6 Inside the Pipe before thefilling Valve of needle D 3 162.36 4 3 00:22:30 4 Negative

7 Inside the Drain next tothe Temperature Probe 2 71.95 10 2, 3 00:20:00 10 Negative

8 Inside the Drain next tothe Conductivity Probe 1 101.32 19 3 00:20:00 19 Negative

D – Validation of the filtration step to reduce the microbial load Aside from the validation of

the terminal sterilization step, the filtration step for the reduction of the microbial load that is employed

prior to the filling must also be validated to assure that it complies with the regulations and that the

outcome of that filtration corresponds to the expectations regarding microbiological load reduction.

The tests performed to assess the quality and the functionality of the filters used are an important

contribution to the overall validation process. Among the critical steps in the validation of the filtration

79

methods, prove that the filters used are capable of removing bacteria from the stream and that the

outcome of the microbial load reduction process does not adversely affect the product stream are

essential.

There are also other considerations that have to be verified when validating this process, including

proofs that:

• The filter’s bacterial retention capability remains unaltered throughout the process (meaning that

the integrity of the filter has to be guaranteed);

• The filter does not adversely affect the process stream, which means that not only will the filter

not remove any components from the process stream but will also not contaminate the flow with

compounds that migrate from the filter structure and that may be question the final quality of

product;

The validation of the filters’ characteristics is usually based on the performance tests done by the

manufacturers of filters, which have to comply with the requirements imposed by both the FDA and

EMA. During routine production, the overall integrity of the filters will be tested, and the results of the

integrity tests will be used to attest that the filters used comply with the conditions required.

80

5Discussion and conclusions

Contents5.1 Discussion and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

81

5.1 Discussion and conclusions

Overall, the main objectives outlined for this work were achieved. A deep insight on the more

important parameters that have a direct impact on the quality of the final product was presented and

the information detailed on this thesis acts as an important tool to describe, qualify and integrate the

different aspects that make the global validation plan of the department.

On a more specific level, the company ended up training and developing someone that fits into

an existent gap in the organization, and that is able to not only integrate and apply knowledge from

areas like microbiology, transfer phenomena and general engineering that are needed during process

validation but that can also make the connection between the several departments that contribute to

the department’s validation.

Regarding the current status of the validation program, the stages of design qualification have

been successfully completed for all the different elements of the department. By the date of sub-

mission of this work, the final process qualification and performance qualification stages are being

completed and, following that, it will be possible to begin the first batches of routine-like operation,

to start the continuous verification stage of validation, that will allow for the beginning of the normal

manufacturing conditions.

In terms of area, utilities and equipment, most of the performance qualification stages are already

completed. The installation protocols to be followed for the qualification of these elements specified

the requirements that had to be met in order to achieve qualification and those requirements were

met for all the elements installed in the department, namely: the materials used comply with the

normative specifications, all valves, gauges and piping are sealed and appropriately installed, the

air handling units are fully functional and met their design specifications and all the utility sets that

support the department are qualified and readily available. Therefore, it can be stated that all the

elements installed in the department are currently validated and consistently perform according with

their design specifications.

Currently, there are some results still pending related with the application of the environmental

monitoring plan, as well as with the validation of the sterilization cycles, the final characterization of

the products and the overall procedures to be implemented within the department.

Regarding the data obtained under static conditions for the non-viable particle determination, it

is clear that the acceptance criteria defined as the limits for the differently classified rooms was re-

spected, with to record of excursions outside the defined levels. Since non-viable particles essentially

act as transport vehicles for microorganisms (bacteria and other living organisms can attach them-

selves to non-viable particles and be carried by available air currents), the fact that the number of

non-viable particles in the department is low contributes to reduce the bioburden within the clean

area.

In what concerns the viable particle sampling, an excursion (deviation from the established limits)

82

was detected for air sampling in one of the days of implementation of the EM plan. The fact that the

excursion happened under a unidirectional laminar flow makes it a major issue. Since the excursion

happened during the monitoring under static conditions, there will be a reevaluation of the locations

in which the deviation occurred under dynamic conditions.

Considering that the excursion happened in a phase where the cleaning and disinfection protocols

of the machine are still being applied and the training of the operators that perform them is still in an

early stage, if the results under dynamic conditions comply with the criteria established, the excursion

can be considered an isolated event and the validation of the environmental conditions may not be

affected.

The data from the EM plan under dynamic conditions was, as mentioned above, still unavailable at

the time this work was submitted. There are specific acceptance criteria defined for the different types

of samples obtained and the success of the EM plan will be judged per comparison with that criteria.

Besides this, and since the plan is being used for the initial qualification of the department, all the

microorganisms that grow during the incubation period will be fully identified, so that a characterization

of the department’s typical flora can be made.

Once the results are obtained, if any deviations from the acceptance criteria are verified, an inves-

tigation will have to be issued to assess the root cause associated with that excursion. Typically, the

most common causes for deviations are poorly trained personnel, inefficient cleaning and disinfection

protocols or, in some cases, failures in the control or the design of the area or the ancillary equipment

which affect the control of the environmental conditions. Corrective actions will necessarily be initi-

ated to fully restore the accepted conditions and to address the causes of the deviation, so that the

conditions that led to the excursion are eliminated.

For the results from Grades C and D, since there were no limits imposed, the goal of both the

static and the dynamic conditions is to assess the trends and the average number of CFU present in

each room, so that the action limits for the department may be set. Analyzing the results obtained,

and comparing them with the average limits established for other departments of the factory (limits for

Grade C are typically around 25 CFU/plate and for Grade D revolve around 50 CFU/plate), the fact that

this limits are being respected and that the overall trends per room are similar leads to the conclusion

that there are no major deviations from the typical environmental conditions for these areas and, as

a result, there should be no need to change the current procedures of cleaning and disinfection and

the parameters of the ancillary equipment, namely the air handling units.

However, once the results from the dynamic monitoring are available, there will be a re-evaluation

of the sampling positions (namely in terms of number and location) to define the procedure to be

followed under routine production, so that the monitoring continues to be effected during production

and the overall environmental condition and the efficacy of the cleaning and disinfection protocols

continues to be verified.

In what concerns the results of the sterilization cycles’ qualification, any sort of failure will com-

83

promise the entire project, since the safety and quality of the product will be severely compromised.

Regarding the terminal sterilization validation step, a distinction will have to be made whether the

deviation to the acceptance criteria defined occurs in the empty chamber studies or during a heat

penetration test (with loads already included in the study). The empty chamber studies conducted in

the autoclave used to sterilize the bags were successful, since not only was it possible to determine

the position of the cold spots of the autoclave, but it was also demonstrated that there is a uniform

temperature distribution inside the autoclave that matches the acceptance criteria.

On the case of the heat penetration studies, the possibilities for potential deviations increase,

since the number of variables being evaluated is greater. Variation in the results is expected and quite

common, especially for products which are non-homogeneous or exhibit complex heating behavior.

Variability is generally evaluated based on plots of the heating and cooling curves and/or lethality

calculations and will be considered when identifying the slowest heating behavior of a process and

during the optimization of the operational parameters for specific loads.

If the supplement for CBE30 is approved and the steps of validation of the sterilization-in-place

cycle of the filling machine are successful, the main sterility premises will be verified and, when

normal production routines are in place, there will be an assurance that the final product will be safe

and have the predetermined quality attributes desired.

In what concerns the validation of the SIP of the filling line of the bags’ filling machine, the results

demonstrated that the parameters tested for the sterilization cycle are able to assure a sterility level

on the filling line. Both the physical data, obtained by the thermocouples placed in the line, and the

biological data, resulting from the incubation of the biological indicators shown compliance with the

acceptance criteria defined for the cycle.

Overall, the lethality factor achieved is highly greater than the minimum required, which guarantees

the efficacy of the process as a sterilizing cycle. Nevertheless, it is important to discuss the data

obtained by two of the positions within the line, since the lethality factor calculated is smaller than the

average value for the filling line. In those positions, which correspond to the thermocouples placed

inside the drain, there is an accumulation of condensates that are generated during the sterilization

process and, as a result, the temperature measured by the thermocouples will be lower compared

with the probes placed at the edges of the piping and at the valves. However, since the biological

indicators placed at those positions matches their acceptance criteria and the lethality factor achieved

was greater than the minimum accepted, the effects of condensate accumulation at the drain sites do

not compromise the sterilization process.

The lower lethality factor obtained in position 5, which corresponded to the filling valve of needle C,

was reduced compared to the others due to a temporary stoppage of the reading of the thermocouple

used. Still, since the exposure time and the lethality factor obtained were above the acceptance

criteria, the issue is not considered critical and the outcome of the measurement is not affected.

84

6Future work

Contents6.1 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

85

6.1 Future work

The current approach to validation recommended by the regulatory authorities, combined with the

fact that the ongoing validation program implemented in the department is still not complete makes

it fairly clear that, in the near future, there will be a dedicated effort to finish all the qualification and

validation stages that are still remaining, so that the department can begin to operate under routine

conditions as soon as possible.

Once that is done, the transition to the continuous process verification phase will be made, as

a part of the overall validation master plan, reflecting the ideas and criteria established during the

initial qualification stages. From the experience of the company with other departments and similar

situations, the general monitoring program to be implemented will be capable of detecting gradual or

unplanned departures from the process as designed.

On a personnel level, the fact that the internship granted me exposure to multiple areas of exper-

tise, allowing me to participate in the installation and qualification stages in an active and relevant way

was extremely beneficial. With the knowledge gathered during this period, I now have the capability of

actively contributing to assure an efficient and compliant transition into the commercial manufacturing

stage.

Focusing more specifically on the continuous monitoring plan to be implemented in the depart-

ment, amongst the tools used for continuous verifications, product stability programs, change control

processes and the Annual Product Review Process are vehicles used for monitoring and assessing

process stability. As a complement to these tools, an approach to sampling with a focus on looking at

intra and inter-batch variation of the critical quality attributes (CQA) of the product of the product will

also be used to monitor the commercial process stability. The data obtained during production and

revalidation efforts will be trended and analyzed employing statistical process control and process

control charts. With it, there will be a constant re-evaluation of the alerts and action limits established

during the initial qualification stages, since the commercial process will display variation coming from

the raw material, process and testing methods.

In medium and long-term perspectives, the introduction of computational and advances strate-

gies for process status evaluation and manufacturing risk analysis may be the next improvement in

the lifecycle approach of the process validation. The limits of the design spaces and other process

parameters can be challenged using computational simulations and specific software applications,

that contribute to the constant acquisition of data that may lead to direct update and evaluation of

specific product parameters. In addition, the establishment of new technologies for the evaluation of

the lifecycle conditions of the process may allow for the immediate correlation between upstream and

downstream parameters, which could result in an automatic assessment of the risk factors that may

interfere with the process outcomes and contribute to the reduction of the overall variability and the

improvement of the process knowledge and control.

86

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1

AMaterial and Personnel flows of the

department

A-1

Figure A.1: Material flow of the infusion bags department.

A-2

Figure A.2: Personnel flow of the infusion bags department.

A-3

BRationale behind the choice ofproducts for leachable studies

B-1

Figure B.1: Rational for the choice of products to be considered for the leachables’ study.

B-2

CSampling locations included in the

Environmental Monitoring Plan

C-1

Figure C.1: Locations for the air sampling included in the EM plan.

C-2

Figure C.2: Locations for the settling plates sampling included in the EM plan.

C-3

Figure C.3: Locations for the RODAC sampling included in the EM plan.

C-4

Figure C.4: Locations for the non-viable particle sampling included in the EM plan.

C-5

DTrends of viable particles for

environmental monitoring

D-1

Figure D.1: Trend of viable particles sampled through air sampling for the 5 days of initial qualification in GradeB.

Figure D.2: Trend of viable particles sampled through air sampling for the 5 days of initial qualification in GradeD rooms. The several series represent the different Grade D rooms in the department.

Figure D.3: Trend of viable particles sampled through settling plates for the 5 days of initial qualification in GradeD rooms. The several series represent the different Grade D rooms in the department.

D-2

Figure D.4: Trend of viable particles sampled through contact plates for the 5 days of initial qualification in GradeB.

Figure D.5: Trend of viable particles sampled through contact plates for the 5 days of initial qualification in GradeD. The several series represent the different Grade C rooms in the department.

D-3

EDetail of the SIP course in the filling

machine

E-1

Figure E.1: Detail of the SIP course of the filling machine.

E-2

FExample of a supporting protocol of

the new department

F-1

Figure F.1

F-2

Figure F.2

F-3

Figure F.3

F-4

Figure F.4

F-5

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