PROCESS VALIDATION OF LYOPHILIZATION PROCESS A...
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PROCESS VALIDATION OF LYOPHILIZATION PROCESS A
REVIEW
Aditya Mishra*, Dr. T. R. Sainia and Dr. V. K. Maurya
b
*,aDepartment of Pharmacy Shri G. S. Institute of Technology and Science, Indore, Madhya
Pradesh 452003.
bGovt. College of Pharmacy, Aurangabad, Aurangabad, Maharashtra 431005.
ABSTRACT
Lyophilization mainly considered as one of best method to promote the
long-term stability of many pharmaceutical drug product.
Lyophilization, more generally known as “freeze-drying,” is a means
of dehydration process (a process that remove water molecules/
desiccation) used in the foods like meat industries, many chemicals
industries, pharmaceutical, and biotechnology industries. In each of
cases, lyophilization is used to promote the long term stability of a
decomposable product and /or making the product easier to transport
or/ and store in different words the degradative or less stable in an
aqueous medium of these systems forms a real barrier against the
clinical use of many pharmaceuticals. In the biotechnology industry
and most other pharmaceutical industries, lyophilization /freeze drying is used as a last
processing step for purified active pharmaceutical ingredients (APIs) or/ and drug
formulation to stabilize the protein and other pharmaceuticals for long-term storage. This
article reviews the state of the art of lyophilization and process validation considerations of
freeze dried product with parameter of PAT and QbD tools in each stage. This review
discusses about the most important parameters that promote the success of lyophilization of
these frangible systems, and gives an overview of freeze-drying process and formulation
strategies which is focused on the impact of formulation and process on particle stability
point of view.
KEYWORDS: Process validation, Lyophilization, QbD, Primary Drying, Secondary Drying,
Freezing, Quality, Sublimation, PAT, Freeze- Drying.
WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES
SJIF Impact Factor 6.647
Volume 7, Issue 1, 365-397 Review Article ISSN 2278 – 4357
*Corresponding Author
Aditya Mishra
Department of Pharmacy
Shri G. S. Institute of
Technology and Science,
Indore, Madhya Pradesh
452003.
Article Received on
11 Nov. 2017,
Revised on 30 Nov. 2017,
Accepted on 21 Dec. 2017
DOI: 10.20959/wjpps20181-10766
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1. Lyophilization
Lyophilization is the most general technique for formulating Parenterals products when
stability in aqueous solution is an issue. It is central to the protection of materials, which
require low moisture content (less than 1%) in order to ensure stability and require a sterile
and gentle preservation process. Lyophilization produces excellent quality products, both
foodstuff and pharmaceuticals, due to the moderate temperatures at which the process takes
place, contributing to the formation of highly porous solids that retain aroma, colour, and
flavour.[1,2]
Vacuum lyophilization takes place at very low pressures so that the operation
occurs below the triple point of water, leading to high investment and operating costs.[2,3]
Lyophilization is a technology, method, process by a product is frozen (converting all mater
to solid state) and then water removed by sublimation (primary drying) of the freezed water
molecule (solid particles) i. e. Ice. The complete process of freeze drying requires steps;
freezing of the molecule in witch water is there by nucleation and many other method; after
that second step is main drying (MD) by this sublimation of the ice molecules happens; then
secondary drying (SD) in this step desorption of the water molecule which is bounded to the
solid particle or particles, and in the last packing should be done in the vails/ containers to
prohibit reabsorption of water and/or oxygen from the atmosphere. With the help of freeze-
drying a product unstable/ less stable / decomposable/ degradative in the presence of moisture
is converted into a dried and stable formulation.[4]
Development of this technique to comply four demands on the Rinsed product: its volume
should remain in the frozen state, the structure and the biological activity of the dried solid
and the original substance should be same as far as possible, the dried product remains stable
during storage in room temperature. It is possible that the product can be stable at
temperatures up to 40 degree C during storage for up to 2 y;; and When water is added then
the lyophilized product is quickly reconstituted.[5]
See Fig. 1.
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Fig 1: Steps in freeze drying of herbal material.
Pre-freezing
In lyophilization method and final temperature affect ability to successfully freeze dry the
material. In lyophilization freezing step mainly affected by cooling rate. Rapid cooling rate
mainly used for preserving stature to be examined in the microscopically but product is more
difficult to lyophilized. Slow cooling rate can use to bigger ice crystal but in the case of
human or plant cell larger crystal can rupture and product is less restriction channels in the
matrix to freeze dry.[6]
Product can be freeze by two ways. Product consist of primarily of
water, solvent material dissolved/ material suspended in the water or solute. Most sample are
eutectics which freeze at lower temperature than surrounding water. In pre freezing step on
cooling pocket are formed, ice contain pockets in which solute is present and have lower
freezing temperature than water. Product looks as frozen but it is not frozen completely till
solute in the suspension is completely frozen. Only when all of mixture (eutectic mixture) is
frozen then only product fitly frozen. This temperature is called the eutectic temperature. Pre
freezing of the formulation is performed below this temperature (eutectic temperature) before
freezing step is performed because small pockets of unfrozen suspension leftover in the
product amplify and degrade the structure stability of the lyophilized product. In other way,
formulation that bear glass formation during freeze drying process. The complete suspension
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as the temperature lower the formulation becomes more viscous. At last the product freezes at
the point forming called as glass transition point when the formulation becomes a viscous
solid. This type of product is very difficult to lyophilized.[7–12]
1.1. Freezing
While in simple terms, the freezing step is first step in processing and apparently the most
complicated step in freeze drying process. A phase stated the “freeze-concentrate” when
water freezes the soluble particles in the product remain in the remaining liquid. At which
point ice formation became maximal, that make up the lattice when the freeze concentrate
solidifies between the ice crystals. At the stage of primary drying, ice is formed during
freezing is which is the crystalline and it‟s removed by sublimation. That‟s why, the vapor
pressure of the chamber is decreased below the ice vapor pressure, and the temperature of
shelf is increased to supply the heat removed by sublimation of ice.[13]
Fig 2: Phase diagram of water - Ice- Vapor system
As per Fig. 2 (phase diagram of water)[14]
, most product are frozen well below their
eutectic/glass transition point (A). Temperature is decreased to just below critical temperature
(B). No matter what type of freeze drying system is used condition must be crated to
encourage the free flow of water molecule. Therefore vacuum pump is an essential
component of a freeze drying system and is used to lower the pressure (C). the molecule have
a natural affinity to move towards the collector chamber because it‟s vapour pressure is lower
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than that of the product. Therefore the collector temperature (D) must be significantly lower
than the product temperature.
So freezing can be define as it is a process when ice crystallization occurs from super cold
water. In simple, in freezing process first Colling of solution that will lead to “nucleation”
(nucleation is a process in which small nuclei is formed in solution or saturated solution). The
degree of supercooling is mainly responsible for the rate of ice growth, number of ice nuclei
formed, and the ice crystal‟s size.[16]
That will lead to growing of Ice crystals at a defined
rate, freeze concentration of the solution will be formed, a process that can result in
crystalline and amorphous solids, or in mixtures of amorphous & crystalline.[17,18]
The
freezing rate of a formulation is not necessarily related to its cooling rate.[18]
Because the
cooling rate is defined “as the rate at which a solution is cooled & the freezing rate is the rate
of post nucleation ice crystal growth, which is largely determined by the amount of
supercooling prior to nucleation”.[17–19]
Ice nuclei getting more time to grow when providing slow rate of freezing, the ice will grow
and form the ice crystals and solution which is present in between the crystal becomes more
concentrated. But in the case rapid freezing will leads to grow small and the remaining
solution will become so much vicious. As the viscosity increased, water molecule becomes a
part of concentrated liquid (glass) in between ice crystals and not able to diffuse.[17]
The cryoprotective effect of four carbohydrates (glucose, fructose, mannose and maltose) on
paradodecanoyl-calixarene based SLNs (solid lipid nanoparticles) has been investigated by
PCS (photon correlation spectroscopy) and these four carbohydrates have been shown to act
as good cryoprotectants, allowing reconstitution of the suspensions after the freeze-drying
process.[6,20]
If other solid/ dissolved substances are present the freezing behaviour of water
changes differently, e.g. cryoprotective agents (CPAs). They all protect quality of the product
to be lyophilized in one way or another, they can be used in alone or in combination.
Lyophilization of liposomes without special protectants, cryoprotectors, causes them to
coalesce and aggregate. The included water-soluble drug can also leak out.[6]
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The most commonly used cryoprotectants material can be classified in Fig 3.[21]
Fig 3: commonly used cryoprotectants material.
1.1.1. Freezing method
There are several freezing method are describe[6,22]
Shelf-ramped freezing
Pre-cooled shelf method
Annealing
Quench freezing Quench
Directional freezing
Ice fog technique
Electro-freezing
Ultrasound-controlled ice nucleation
High-pressure shift freezing or depressurization technique
Vacuum-induced surface freezing
Non-aqueous co-solvents
Addition of ice nucleating agents
Others
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1.2. Primary drying
Primary drying is also known as main drying because in this phase of lyophilisation
sublimation occurs. Sublimation occurs when a frozen solvent passes to gaseous phase
without passing through liquid phase. The crystals of ice by using a special freezing method
will grow extremely uniformly. The ice sublimes and the remaining solids show their original
structure after freezing. In the process of sublimation the ice temperature at the sublimation
front (Tice) should be done at well below the collapse temperature (Tc).[17]
The end of the
sublimation phase corresponds to the decrease in the moisture sensor signal down to a low
constant value.[23]
The vapour transportation of vapour and supply of heat to the condenser
which is shown Fig. 4 are most important parameter during primary drying. That‟s why the
operation pressure is a very mandatory tool to control Tice, if the temperature of shelf is
conserved constant and the temperature of condenser is always down to a maximum, which
depends on the design of the plant and water vapour pressure in the chamber. Sublimation is
a process when matter directly convert from solid state to vapour state without melting (liquid
Phase). Sublimation occurs at a controlled environment for a particular substances at define
range of pressures and temperatures.[24]
The phase diagram of pure water shows that
sublimation of water ice can be done when the temperature and vapor pressure are below the
triple point of water - i.e., below 0.010C and 611.73 Pa, accordingly.
Fig 4: The collecting system which acts as a cold trap to collect the vapors.
In the end of sublimation process most of the water which is present in the form of moisture
removed from formulation.[17]
In lyophilizer, vapour is formed after sublimation process in
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lyophilization chamber goes to condenser. Condenser continuously remove it.[25]
When main
drying MD completed the most of the ice is sublimated. And the standard deviation of Tice
above the measured Tice decreases during MD. This parameter can be utilized to change
continuously from second stage to third stage that is main drying MD to secondary drying
(SD), e.g. if the average becomes 2-30C above than measured Tice the during main drying
(MD).[17]
1.3. Secondary drying
After primary drying all ice will be sublimated but moisture will be present in form of bound
to the solid particle pf formulation. The formulation seems to be dried but it‟s not because of
bound residual moisture can be present as high as 7-8%, so it is mandatory to drying at higher
temperature. This will leads to reduce the moisture. When the bound water is desorbed from
formulation then it‟s called as Isothermal Desorption. Secondary drying carried out at high
vacuum and moderate temperature (20–60°C). The dryer loses shelf control for 30 mins
during secondary drying as a result of a brief power outage. This results in the shelf
temperature being maintained at 5°C cooler than the set point of 25°C. In the freeze drying
process the third stage secondary drying is used for desorption of water which is present in
the bound form until target residual water content is achieved. At this this stage, the drying
temperature is more important determinant of the amount of moisture with drying time.[26]
1.4. Sealing Lyophilized Products
A freeze dried product should be closed within its container before removing it from the
ultra-dry atmosphere present at the end of the freeze drying process. The formulation which
has been go through this cycle most of the time it contains less than 1 % of moisture, so when
it will come contact with moisture containing environment, product will try to take moisture
as its capacity. The quality of the product will be degraded immediately. Enhanced chemical
performance, increased shelf life, and rapid reconstitution properties required by lyophilized
product after freeze drying, will be compromised. If moisture is again taken by product then
loss of product, false result, product failure and product recalls will be consequences. To use
of packages that can‟t be sealed inside the lyophilizer before to re-pressurization that is the
most common mistake by companies. For example, the manufacturing process for some
diagnostic products can require lyophilizing the product inside a large number of screw-top
tubes. Sealing of these tubes inside of a lyophilizer before terminating the batch, there is no
practical way, that‟s why the company will gather a large production staff to apply manually
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to a room which is incompatible with freeze drying process. Recently, stable chemistry will
be endangered by the risk of unacceptable high and variable moisture levels during manual
sealing process. So exposing lyophilized material to atmospheric moisture in this way may
result in an unstable product.[27]
For the successful performance of any lyophilization process all four stages of the
lyophilization process (freezing, primary drying, secondary drying and sealing) carrying
equally importance , So it can produce a dried and stabilized product for storage at long-term.
Subsequent steps, final moisture level, or quality of overall product can largely affected by
any change in one step of any stage of lyophilization cycle. The basic principles of
lyophilization must be understand and then it should apply to lyophilization process and
individual product. To ensure proper “validation proper process qualification and continuous
process monitoring” should be performed.
For scrutinizing the process is under control or not, process validation of lyophilization
process should include the validating process variables of formulation. This process should
take consideration of critical variables a. e. Final mixing, filtration, filling, partial stoppering
of three batch after that stoppering sealing sand packing.
2. PROCESS ANALYTICAL TECHNOLOGY (PAT)
PAT is defined as „a system for designing, analyzing and controlling manufacturing through
timely measurements (in and after processing) of critical quality and performance attributes
of raw and in-process materials and processes with the goal of ensuring final product quality‟.
PAT is a unique approach for process validation. It combines techniques, procedures and
tools like quality by design (QbD) and real time release (RTR) that enable online and offline
verification of key process parameters. PAT creates a robust control strategy for the ongoing
process monitoring. PAT is thus, increasingly explored and adopted by pharmaceutical and
biotechnological set ups for enhanced process understanding and risk management. In the
PAT Table 1 shows the type of measurement for characteristics with method is used and
Table 2 explain how the PAT can be for lyophilization process with the help of stages,
physical parameter. In this process most of the control parameter is mentioned in Table 3.
PAT from an implementation perspective is visualized as a three-step process.
Design
Analysis
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Control
Table 1: Process analysis measurement.
Type of PAT
measurements Method Characteristics
In-line No removal of the sample
Quick and quality result
obtained. on-line
Sample is diverted from the
main process, analyzed and
may be returned
at-line Sample is removed and
analyzed closed to the process. More time consuming.
off-line
Sample is removed and
analyzed away from the
process.
Difference between at-line
and off-line measurements
in lab scale processing is
tough to define.
Table 2: PAT for Freeze-Drying Process Development.
Stages Physical parameter Tools of Process analytical
technology (PAT)
Potential product
quality product
Freezing Ice nucleation
temperature
Ice fog
Nucleation-technique Controlled-ice
Reconstitution
time
Residual water
Physical stability
Cake appearance
Primary drying
Product temperature,
Sublimation rate
Drying time
Gas flow velocity
Vail heat transfer
Product resistances
coefficient
Manometric temperature
measurement (MTM)
Freeze drying
Control system
Wireless temperature monitoring
Pirani gauge
Spectroscopic-technique
residual gas analyzer (RGA)
Secondary
drying
Residual water
Product temperature
Drying time
Tuneable diode laser absorption
spectroscopy (TDLAS)
Table 3: Process Control Parameters.
S. No. Parameters Source Source
1 Product Final Mixing
Testing End Product
1.1 Description of formulation
1.2 pH after and before reconstituted
1.3 % Assay of API
2 Filtration
2.1 Sterility Test of the final formulation
3 Filling of Bulk
3.1 Filled Volume of Bulk
3.2 % Assay at Bulk
4 Lyophilization
4.1 Water Content in %
5 Sealing of Vials (Full Stoppered)
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5.1 Leak Test of vails
6 Description
Testing In Process
7 Content of Water
8 Average Weight of Cake
9 Dosage form Uniformity
10 Constituted solution
11 Description of solution
12 Time take to Reconstitution
13 Particulate matter in the solution
14 pH of the bulk
15 Bacterial Endotoxins limit
16 Sterility
17 % Assay
18 Relative substances
3. QUALIY BY DESIGN (QbD)
Pharmaceutical Quality by Design (QbD) is a systemic, scientific, risk-based, holistic and
proactive approach to pharmaceutical development that begins with predefined objectives and
emphases product and processes understanding and process control based on sound science
and quality risk management. The relationships between formulation and manufacturing
process variables which must include excipient attributes, drug substances, and process
parameter and product characteristics are established then Source of quality variation
identified. Because of this knowledge and experience used to regulate a robust and flexible
process of manufacturing than can be adapted and produce a consistent quality product.
Process understanding is a major goal of a QbD program.[28,29]
QbD include following
element.[15]
Define target product quality profile
Design and develop product and manufacturing process
Identify critical quality attributes, process parameter, and sources of variability
Control manufacturing process to produce consistent quality over time.
The variables from the first three buckets directly impacts the process performance and
imposes boundaries on the “Design Space” while the variables from the fourth bucket not so
much influence the process but impacts the product quality attributes. The next step is to
design multivariate experiments supported with the stability studies to determine the degrees
of impact each parameter has on the CQAs. This evaluation could be based on statistical
significance in the experiments and, process parameters that significantly impact CQAs will
be categorized as Critical Process Parameters (CPP) and those that are not but are important
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for consistency of the process performance or other business related factors are categorized as
Key Process Parameters (KPP). The parameters or material attributes that are demonstrated to
be influential and critical to the CQAs need to be further studied using DOEs involving
multivariate combinations and interactions with other parameters to define the operating
boundaries around the target within the design space. These studies can be performed using a
scale-up model that mimics and represents the commercial scale freeze dryer conditions. The
validity of the model and the operating space within the design space can be confirmed and
demonstrated with a verification experiments at full scale. In order to have the understanding
and data to support the non-conformances during commercial manufacturing, univariate
studies can be performed and proven acceptable limits can be established but these do not
constitute the design space.[30]
The most challenging part of the freeze drying process is the construction of the “Design
Space,” which is one of the key elements of QbD and requires thorough “Process
Understanding” which means the CPPs controlling the variability in the CQAs be identified
and understood during process development so that they can be measured and controlled in
real-time during manufacturing process. One way of doing this is by using Prior-knowledge
and risk based assessment to list all the parameters that have the potential to influence the
process performance and product quality attributes. They typically fall into four buckets.
Freeze drying process operating parameters (shelf temperature, chamber pressure, ramp
rates and hold-times).
Product parameters (protein concentration, excipients and their concentrations, vial
configuration, stoppers, fill volume).
Equipment (capabilities and limitations, batch load/size, scale effects).
Components preparation and Devices.
4. PROCESS VALIDATION
The basic principle of the process of validation that should be used to produce a drug that is
suitable for its designed use. USFDA‟s Guidance for Industry on “Process Validation:
General Principles and Practices” states that “Process validation is 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”. Definition „„Process Validation is establishing documented evidence which
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provides a high degree of assurance that a specific process will consistently produce a
product meeting its predetermined specifications and quality characteristics‟‟.[25]
Welfare of human beings and betterment of humanity is the main one aim of science and
technology. This can be achieved by many means. It may be useful to his mental functioning
human beings so that it can be obtained from the diet and nutrition. Other limitations that
address human's intellectual and emotional needs are less clear but no less important. There is
health care in different areas of much more importance because it is directly related to
maintaining and improving the quality of human life.[25]
With the focus on verification requirements, the development of a new freeze dried product is
easy to integrate into the production environment, while attempts are made in the production
along with further development studies. In designing the freeze drying process, it is advisable
to complete the process study on the limits of the process parameter limit at time stability
studies are made. From such an approach, the selection of parameters and the more robust
process results in greater safety and efficiency.[25]
4.1. Process Validation program Contents:
Process Validation Program should be include the following points...
History of the development and product details (If available, the development report
would be useful)
Manufacturing process with a manufacturing procedure and flowchart
Equipment list mandatory for process in production
Critical production stages list for product quality
Process Validation test procedures schedule
All test procedures must contains detailed description, which include:
Acceptance criteria
Test procedure
Evaluation procedure
Sampling procedure
Intermediate and finished products Specification
Labelling of the samples
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History and origin of process validation can be explained by Table 4[25,31]
Table 4: History and origin of process validation[25],[31]
Problem identified Enactments for problem
Year Problem Year Enactments
Early 1900s plants were revealed as unclean
conditions in meat packaging 1906
“Meat Inspection Act and
the Food and Drug Act”
1937
more than 100 people died after
taking an elixir of sulphanilamide
which was formulated using a
toxic substance
1938 “Food, Drug and Cosmetics
Act”
late 1950s
Thousands of babies were born in
Europe with birth defects.
Investigations carried out revealed
in the early 1960s that these
babies were born to mothers who
had taken the drug Thalidomide
1962
“Kefauver–Harris
amendments to the Food,
Drug and Cosmetics Act”
early to
mid-1970s
Several people died after injection
of a drug formulation that turned
out to be severely non-
conforming.
1970s
“QC approach alone is not
sufficient, but a QA
approach must be applied as
well”
early to
mid-1970s
Several people died after injection
of a drug formulation that turned
out to be severely non-
conforming.
1970s
1978
“This is regarded by many
as the true start of the
application of process
validation in industry, and
indeed the FDA in the 1970s
required and enforced
validation, especially on
sterile processes.”
“The FDA required the
process to be validated, but
the term „process validation‟
was not defined.”
1987
“FDA issued its guideline
document”
1996
“FDA issued a set of
proposed rules modifying
some of the points in the
cGMP that related to
validation”
4.2. Types of process validation
Process validation can be done by four type of method.[25,31]
4.2.1. Prospective validation
This type of process validation approach is generally main choice when the new formula
should be validated before the regular pharmaceutical production begins. Process validation
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method normally guide to transfer development function to production manufacturing
process. Production process must be divided to many steps during development of
formulation. Each product must be evaluated important parameters on the basis of experience
or theoretical considerations to determine, which can affect the finished product quality.
4.2.2. Retrospective validation
Retrospective validation is an approach of process validation which used for operating
processes, process controls and facilities, which are not formally documented. Validation of
these facilities, processes, and process controls is can be perform by using historical data
which can give the mandatory documentary evidence so that this process is doing what is
supposed to do So that‟s why retrospective validation is can be perform whenever process is
well established. But in case change control occurs like change in composition of product,
change in the operating procedure, change in process or change in part/ parts of equipment or
equipment itself then this approach is not suitable. In recent trends this type of validation
approach is not common because of prospective validation process is perform from
development stage. Nowadays this approach mainly used in validation process audit.
List of some of the mandatory elements are.
Number of lots released per annum
An analysis of trends including those for quality related complaints.
Batches processed for a defined period (atleast10 last consecutive batches)
Manufacturer / Strength / Period / Batch Size / Year
List of process corrective and preventive action
Current specifications for active pharmaceutical ingredients/ excipient/ finished products.
. Master manufacturing / packaging documents
Data for stability testing for several batches.
4.2.3. Concurrent validation
It includes a review of the original validation attempt or repeat part of it and examining
current performance data. It can also be said in the form of re-qualification, revalidation or
re-verification of a continuous process in response to a significant change in product
components. Examples of these can be.
When process is previously validated and it going to transfer to another manufacturing
site or third party as contract manufacturer.
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Whenever product having same ratio of inactive/ active ingredient and they are validated
then for product of different strength this approach can be a choice.
4.2.4. Revalidation
Re-validation, the name itself explain that its repeatability of validation of a part of process
or/ and whole process and which should be include reviewing of existed data of performance.
Revalidation can be understand by some example like.
Major change in the manufacturing process by which quality of the formulation can
affected.
Change in the manufacturing location.
Change in the specification and / or change in the source of Active Pharmaceutical
Ingredient.
Change in Primary Packaging material.
Changed in the batch size.
Change in the batch formula.
Modification / Change in equipment used which is expected to affect the quality of
pharmaceutical product.
4.3. Regulatory requirements for process validation
Process validation for drugs (finished pharmaceuticals and components) is a legally
enforceable requirement under section 501(a) (2) (B) of the Act (21 U.S.C. 351(a) (2) (B)).
FDA regulations describing current good manufacturing practice (CGMP) for finished
pharmaceuticals are provided in 21 CFR parts 210 and 211. To fulfilment of requirement of
FDA, all record should be available of all data of lyophilization cycle. International Society
of Pharmaceutical Engineers (ISPE)‟s Good Automated Manufacturing Practice (GAMP)
requirements will apply when data recorders are used, which should add the stipulation that
any recording equipment must be verified and validated for use in pharmaceutical processes.
Recorder which having the facility of Ethernet connectivity can store historical record and
alarm and trail of audit information is to be obtained automatically in a central database
where archiving and analysis can be done if it is necessary. “The Guide to Inspections of
Lyophilization of Parenterals, published by the US Food and Drug Administration, July 1993,
contains among others the chapters „Lyophilization Cycle and Controls‟, „Cycle Validation‟
and „Lyophilizer Sterilization/ Design‟”.[25]
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4.4. Process validation of lyophilization
After development of freeze dried products will go to next stage scale up of manufacturing
process in this time more often validation of product and process is performed. Realization of
many benefits and pressure of regulatory requirement all the activities are underway with
respect to development pathway. There may be parameter (condition) for what type
validation will be mandatory for commercial products which are already existing, because of
compliance of requirement of regulation and if any change control or change in the process
performed. It is mainly integral part of new product development. Required application of
principal should be applied in the case change control process or in the case of revalidation.
4.4.1. Stage 1- Process design
4.4.1.1. Composing validation protocol and sops
For composing validation protocol of lyophilizer need to carry out its qualification test and
decide acceptance criteria. So during selection of size shape and other prospective need to
have one sole document. Comparing different activities of the protocol into small parts makes
communication between individuals and departments more convenient. During design
qualification as per meet the applicable user‟s requirements. During the IQ, the reviewing and
verification of utility connections, piping of the refrigeration and heat transfer system,
reconnecting the vacuum system, rewiring of the control system, start-up and testing may be
organized into distinct documents for each activity. This “modular” approach becomes more
effective and efficient as the complexity of the procedures and equipment increases. The
document should provide sufficient detail to develop the validation master plan that will
describe the approach, justification and rationale for moving to Process performance
Qualification. The theoretical results of in-process product temperature, primary drying time,
and moisture content mapping and history are consistent with the experimental results,
suggesting the theoretical model should be useful in process development and "trouble-
shooting" applications.[32]
Fig. 5 shows the documents required in different stages of the
process of process design.
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Fig 5: Process design.
4.4.1.2. Responsibilities
Responsibility of different departments[33]
Head Quality Assurance responsible for “preparation and evaluation of the validation data as
well as deviations during execution of process validation protocol.”
Head Quality Control responsible for “analysis and evaluation of the analytical results for in-
process and finished product samples as per validation sampling plan.”
Head Production responsible for “qualification and calibration of all the processing
equipment/ instrument/ utilities and maintain its efficacy during the manufacturing.”
Engineering responsible for “qualification and calibration of all the processing
equipment/instrument/utilities and maintain its efficacy during the manufacturing.”
Research & Development responsible for “providing necessary support to the verification and
validation activity.”
Establishing acceptance criteria: - The selection of acceptance criteria is dependent on the
circumstances under which validation is being undertaken and requires judicious
consideration. If new equipment is under the qualification test then perform limitation test
challenge and based on its purpose to warrant the acceptance criteria will be decided.
Sometime OQ made to common performance capabilities of equipment.[25]
Table 5 describe
the critical parameter in freeze drying process. Table 6, 7, & 8 describe technique used for
Temp measurement, % residual moisture during late primary and early secondary drying,
miscellaneous monitors respectively.
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Table 5: Critical parameter of lyophilization process.
STEPS Critical parameters of
PROCESS PRODUCT
STEP-1
FREEZING
RAMP
Freezing temperature and time
Annealing
Morphology
Crestline
Amorphous
STEP-2
PRIMARY DRYING
RAMP
Target product temperature
Self-temperature
Primary drying end point
Chamber pressure
Glass transition
temperature.
TRANSITION PHASE
pH
Target product temperature
Chamber pressure
Phase transition
temperature
STEP-3
SECONDARY DRYING
Heating rate
Chamber pressure
Self- temperature
Residual moisture
FINAL PRODUCT Physical appearance
Residual moisture
Physical appearance a.e.
colour & clarity after
reconstitution.
Residual moisture
Table 6: Temperature measurement during the FD process.
Technique Characteristics
Temperature Remote Interrogation
System. (TEMPRIS)
Wireless temperature sensors to observe
temperature profiles[33]
Manometric temperature
measurement.(MTM)
A valid measurement of product temperature
during primary drying even at temperatures
as low as - 45°C. It measure dry-layer
resistance and vial heat transfer coefficients
and also used for measurement of motor flux
of water[34–38]
Freeze-drying microscope (FDM) based
on time-domain Optical Coherence
Tomography (OCT).
Measure Tc of product formulations in
standard pharmaceutical vials without loss of
product quality, Provides quantitative
justification for FD above Tc and provides
an upper limit to the temperature at which a
FD cycle may run without macroscopic
product collapse. It helps to reduce the time
for primary drying and increase process
efficiency for FD products with more
accuracy than light transmission or
differential scanning calorimetry; other
benefits are product microstructure
visualization[39]
Differential scanning calorimetry (DSC). The glass transition of maximally freeze
concentrated amorphous phase (Tg‟)[40]
Freeze dry microscopy Used to determine the temperature at which
lyophile collapse occurs[41]
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Table 7: Determine % residual moisture during late primary and early secondary
drying.
Technique Characteristics
Karl Fischer technique Not suitable for small samples[42–44]
Gravimetric method. Accuracy depends upon hygroscopic nature of sample[42]
Gas chromatography. Based on the adsorption of water from the sample using organic solvents[44]
Multipoint NIR spectroscopy In-line quantification of moisture content and hence used for drying end point
determination of a residual moisture level[45]
Multipoint NIR spectroscopy. Detection of unequal sublimation rates within a freeze-dryer shelf[23,46,47]
Phenolphthalein Colour changes of phenolphthalein show a high sensitivity to different forms
of water, which makes it possible to implement a well control over the whole
freeze-drying process.[48]
Table 8: Miscellaneous Monitors. Technique Characteristics
Raman spectroscopy (in-line) and
NIR spectroscopy and X-ray
powder diffractometry (XRPD)
(at-line)
Real time monitoring of FD processes in combination with experimental designs. Both
techniques not only complement each other, but they also provide mutual confirmation
of specific conclusions[39,40,43,49,50]
Raman spectroscopy Useful technique to monitor physical changes during FD[50–52]
Capacitive manometer Using for measuring the total pressure inside the vacuum chamber[23]
Cold plasma ionization device Plasma tool, a relevant method for monitoring FD processes
The Smart Soft Sensor The coefficient of heat transfer between the shelf and the product and the resistance of
the dried cake to vapour flow, as well as Uses the product temperature, a mathematical
model of the process, and the Kalman filter algorithm to estimate the residual amount
of ice in the vial as a function of time.[53]
XRD technique Characterize the phase transitions during FD and useful in developing a mechanistic
understanding of the solid state alterations during FD of complex, multi-component,
pharmaceutical systems[54]
scanning electron microscope (SEM) Morphology of the freeze-drying HA fibres was characterized[55–57]
The transmission electron
microscopic (TEM, Hitachi H-
7500,Tokyo, Japan)
Technique was used for this study to observe whether any change has occurred on the
morphology of final optimized nanopolymersomes during lyophilization process and
prove the conservation of vesicular integrity[58]
Frequency Modulation
Spectroscopy (FMS)
Helps to demonstrate the uniformity and create a map of headspace moisture (HSM)
for vials which allow inspection and play an important part in process validation and
quality assurance.[59]
Used to test vacuum seal integrity of lyophilized protein pharmaceuticals in glass
vials.[60]
Tunable diode laser absorption
spectroscopy (TDLAS) technique
Motor flux of water[35]
The Optical Fiber Sensors (OFS) It helps to obtain three-dimensional temperature profiles with an Optical Fiber Sensors
helix configuration which enables non-invasive, automatic loading compatible
monitoring of FD process. Allow easy handling and positioning along with detection
of excipient crystallization events.[13]
Vial impedance spectroscopy
(off-line)
Useful in the concurrent product formulation development and FD cycle without any
uncertainty introduced so as to define the critical process parameters[61]
Brunauer–Emmett–Teller (BET) by
direct flow gas absorption analysis
Specific surface area (SSA) measurement of the freeze dried samples
Temperature profile and X-ray
technique
Used to detected Sublimation during lyophilization[43]
Flow meter Motor flux of inert gas[35]
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4.4.2. Stage 2- Process Qualification
4.4.2.1. Equipment validation
Complete process validation will include the validation of all process chemicals and raw
materials used in each unit operation, validation of all supporting facilities and utilities
necessary for the manufacturing process, qualification and validation of all process
equipment, validation of each individual unit operation, and validation of the entire process
as it is intended to be operated at commercial scale. Model based tools allow optimizing the
freeze-drying of a pharmaceutical formulation in a specific freeze-dryer, thus minimizing the
duration of the process, besides maintaining product temperature below a limit value to
preserve product quality.[62]
Equipment validation (stage of equipment validation) normally
broken down into four phase as sown in the Fig.6.[25,31]
Fig 6: Stage of equipment validation.
4.4.2.2. Construction of lyophilizer
The instrument of lyophilizer or freeze dryer is consist of following essential parts[25]
Vacuum pump
Shelf tray dryer
Control and monitoring system
Tray dryer
Shelf with controlled temperature system
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Stop ring facility for vials
Condenser or collector chamber
Controlling and monitoring system
4.4.2.3. Equipment performance test
Separate test should be used for each function. For testing of performance of equipment
should include major system like heat transfer system, condenser & vacuum system.[25]
Testing of lyophilizer Qualification can be performed.
Condenser capacity
Shelf heating rate
Shelf cooling rate
Condenser cooling
Pressure control
Rate of Sublimation
Shelf temperature control
Leak test
System evacuation rate
4.4.2.3.1. Heat transfer system
Determination of heat transfer and temperature profile during freezing step is fundamental to
predict the final structure of a lyophilized product. Heat transfer system is used to provide a
specific temperature. Cooling is required for freezing the product and heating is required for
rate of sublimation. So cooling and heating rate along with control at set point and
temperature uniformity must be tested.[25]
In heat transfer uniformity of self-temperature
transmission plays no important role. When the guardrail was absent and Styrofoam was
replaced by the steel band. So metal band can be used as a thermal shield but some heat can
be convicts by many principle like radiation and conduction from shelf of lyophilizer at that
circumstances higher sublimation rates were observed. Radiation seem to be major effect in
heat transfer from the chamber wall to formulation.[35]
The guardrail is not much responsible
for heat transfer by conduction However for higher sublimation rates for vials located at the
side and front of an array radiation heat transfer is contributes.[63]
A significant increase in
product resistance was produced by a decrease in nucleation temperature. Self-temperature
uniformity across any one self and all of selves should be in acceptable range. The ice fog
technique was refined to successfully control the ice nucleation temperature of solutions
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within 10C.[34]
The stated capability for self-temperature uniformity by many of lyophilizer
vendor is ± 1 degree C at the study state condition. The effect of the curvature of the vial
bottom, the heat accumulation in the glass vial and the heat transfer to the sidewalls of vials
in the corner of the shelf all contributed to a significant radial influence on the heat
transfer.[64]
Fig. 7 shows the effect of the heat transfer in shelf of lyophilizer.
Fig 7: Heat Transfer in Shelf of Lyophilizer.
Vials which present at edge get experience of heat transfer by radiation. So heat transfer rate
influenced by warmer surface. This phenomenon can be avoided by using appropriate
radiation shields.[63]
No systematic variation was observed in residual moisture an vapour
composition as load decreased.[35]
That means load on shelf decreases lead to shorter the
primary drying process. Suggesting by use of the measurements emitted on various freeze
dryer that vials present in front in a manufacturing lyophilizer getting 1.8 times lesser than
vials present in front in a laboratory freeze dryer.[65]
That‟s why, the front vials in the
laboratory are very unusual in the manufacturing sector compared to the front vials. Heat
transfer can be done different methods like Heat transfer by gas conduction, Heat transfer by
radiation.
4.4.2.3.2. Condenser
The refrigeration unit parameter like shape type, size numbers affect the shelf cooling rate on
the system. Temperature of condenser is mainly depend upon on the which solvent system
can be used aqueous or organic, for example aqueous solvent system having the commonly
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maximum allowance temperature is -50 degree Celsius, but in the case of organic solvent like
butyl alcohol must be cooled at not much colder than water, and in the case of ethanol vapour
should be condensed at well below – 115 degree Celsius. Sublimation – condensation test can
elaborate load capacity of ice and the rate of condensation. The condensation rate can be used
to development of freeze drying cycle by setting the limit to the parameter of process, and it
can be indicated by kg of ice /h;. So the limit of product batch size can be decided be ice
capacity test.[25]
4.4.2.3.3. Freeze dry rates
Freeze dry rates mainly depend on three factor
Surface area and the thickness of the sample
The condenser temperature and vacuum obtained
The eutectic point and solute concentration of the sample.
It is important to remember these three factors when trying to obtain efficient utilization of
freeze dry system. Surface area of sample is directly proportional to the rate of freeze drying.
And as rate of freeze dry decreasing vapour pressure also decreasing. Vapour pressure
depend upon both eutectic temperature and solute concentration of the sample.
4.4.2.3.4. Vacuum
Vacuum should be enforced during the freeze drying process to remove the solvent at a
correct time. Two-stage rotary vacuum pump is used to achieve desired vacuum range which
is generally such low vacuum 50 to 100µ bar. If chamber is larger than multiple pump may
be a choice. For large chambers, multiple pumps may be used. So vacuum is to very
necessary to observed and monitor.[25]
4.4.2.4. Process testing
Process testing uses a module product to test different functions in the preceding stages of
OQ studies. In such a study, the integrity control capabilities and the capabilities of each
component of the system are challenged to implement the actual processing parameters for a
completely lyophilization cycle. So it is done after operational qualification in equipment
validation. In this studies we can locate area of monitoring or sampling. And perform the test
like product uniformity testing.[25]
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4.4.2.5. Product qualification
Applying these validation steps to freeze drying processes and products, appropriate
recognition in the importance of development activities and growth is evident. Product
qualification can become a tool to identify the source to ensure the process is which are using
good enough or not.
4.4.2.5.1. Preformulation study
Product qualification as part of Preformulation activities, investigations, including physical
and chemical properties, optimum pH, purity, stability, solubility and density studies.[66,67]
Beta-lactam antibiotics can be solidify to a crystalline or amorphous morphology.[25]
Each
different form displays different physical properties, such as solubility and stability. When
substance undergoes phase transition then pH can be an effective factor. By choosing of some
excipients can also change the morphology of the many active drug substance.[68,69]
Development studies become critical parts of validation plan by summarize within a separate
report on development studies, drug substance characteristics, prepared product, and physical
educational aspects characteristics.[25]
4.4.2.5.2. Drug substances
The physicochemical character of the excipients and the physicochemical character of the
active ingredient govern design of product development. For example, if drugs tend to create
an amorphous or crystalline phase, then character freezing method and materials should be
evaluated during development. On the basis of theoretical point of view, when material
solidified during freezing then with compare to amorphous form a crystalline form is more
thermodynamically stable.[25,70]
4.4.2.5.3. Excipients
Freeze drying is a generally used method for product development of drug particle which are
not stable in the presence of moisture and/or unstable in high temperature. Freeze drying of
active pharmaceutical ingredient only, yet having un-doubtful product development
challenges, which can be overcome by using of many excipients (e.g. collapse temperature
modifiers, buffering agents, preservatives, wetting agent, cosolvents, tonicifying agent and
bulking agents) in the final formula. For covenants selection of excipients in the formulation
for freeze drying A needful access should be executed, because of that the formulation should
maintain an optimal functionality a. e. simple and easier processing.
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4.4.2.5.4. Finished Product Formulation
As they are intended for parenteral administration, excipients used to formulate the freeze
dried cake must have regulatory acceptance by all means. For lyophilization of small
molecule should take account to selection criteria a, e. the list of approved excipients with
their maximum limit for finished product formulation.[25]
4.4.2.5.5. Determining Thermal Characteristics
Determination of this critical temperature is very necessary parameter for developing an
optimized freeze drying cycle. While performing primary drying of formulation, drying
temperature should below the critical temperature, which otherwise give problems i. e.
„collapse‟ or „meltback‟ phenomenon in case of amorphous substance or crystalline
respectively. Before doing experiment on lyophilization thermal parameter/ critical
temperature like Tg & Tc should be known. Eutectic temperature can be explained if in the
formulation the solute separates out in crystalline form. Besides, if it is separate out in an
amorphous form then temperature is referred to as the glass transition temperature (Tg ‟).[18]
To achieve the shelf temperature important to totally solidify the product during first step
freezing, the temperature which is required it‟s mandatory to establish complete solidification
of water and solute is scrutinized with the help of thermal analysis early in formulation
development. Apart from that optimal processing parameter should be defined in case
crystallization of product occurs at freezing.[25]
4.4.2.5.6. Finished Product Attributes
Lyophilization technique is one of the outstanding methods used for drying and producing
end products of highest quality as scrutinizing too many other drying methods. As well as the
moisture content of products is become low, the processing temperature is decreased and
most of the degradation & microbiological reactions are also ended. So that‟s why, both
preservation and value food are produced by using this lyophilization technology. If
lyophilization method the retain the physiochemical parameter of the starting formulation
solution and, rather, detention of the structure performed during first step “freezing” then the
results will be successful and effective. And assay of the constituted solution will assures the
preservation of the desired activity in the starting material which is present in it. Assay of
multiple samples of dried material is used to demonstrate content.[25]
To decide finished
product attributes should take account.
Physical Appearances
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Residual Moisture
Reconstitution
Assay
5. CONCLUSION
Development of a successful lyophilized product can be a time- and energy consuming
process. In general, high concentration formulations are usually more resistant to both
freezing and lyophilization. Given the complexity of lyophilization process and formulation
design, attainment of a high concentration lyophilized formulation with a fast reconstitution
time requires optimization of individual process parameters and proper understanding of the
different freezing and drying stresses. This review provided an insight into the different
stresses, their impact and approaches to mitigate them for achieving the highest drug quality
at the least cost.
6. ACKNOWLEDGMENTS
This review was partially supported by ICPA HEALTH PRODUCTS LTD., GIDC
ANKLESHWAR GUJRAT. I am thankful to Mangesh Kumar Padme and my colleagues who
provided expertise that greatly assisted the research, although they may not agree with all of
the interpretations provided in this paper.
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