“Critical temperatures for intelligent freeze-

drying”

Nektaria Servi – BTL Instrument Product Manager

Agenda

• History of BTL

• How to determine the Critical temperatures

• Lyostat4 LED Fourth Generation Freeze-Drying

Microscope

• Lyotherm2 – DTA & Impedance Analysis

• Consulting and R&D Services Overview

• Case Studies

• Conclusions

• Questions

History of BTL• Biopharma Technology Ltd (BTL) was founded in 1997

• Sister company of Biopharma Process Systems (BPS)

• To date, BTL has worked with over 1000 products, from

small molecules to proteins, organisms, whole cells,

collagen products, blood and archaeological samples

• BTL has expertise in the formulation of products,

accounting for the lyophilisation steps involved in the

development and/or production process. Acting at early

stage minimises issues otherwise encountered later in

scale-up and downstream process

Why are Critical Temperatures

important in freeze-drying?

• Freeze-drying above the

product critical

temperature can lead to:

– Loss of physical structure

– Incomplete drying (high

moisture content)

– Decreased solubility

– Reduced activity and/or

stability

• Freeze-drying too far

below the product critical

temperature can lead to:

– Poor efficiency

– High running costs

– Longer cycles than

necessary

Critical Temperatures for freeze-

drying

• Collapse Temperature (Tc)

- This is the temperature at which the material softens to

the point of not being able to support its own structure

• Eutectic Temperature (Teu)

- This is the temperature at which the solute material

melts, preventing any structure from forming after the

solvent has been removed

• Glass Transition (Tg)

- This is the temperature at which the glass first exhibits

a change in viscosity from a brittle solid into a soft solid.

Effect on formulation components on

critical temperature

• Higher molecular weight components such as polymers

tend to have higher critical temperatures

• Lower molecular weight components such as salts and

small sugars tend to have lower critical temperatures

• Additionally, crystalline/amorphous mix can have a major

impact on critical temperature:

– Lactose + NaCl (1:1) Tc ≈ -30°C

– Lactose + NaCl (1:0.3) Tc ≈ -45°C

Critical Temperature

Determination

• Using extensive knowledge and experience in

the freeze-drying industry, BTL has developed

two unique analytical instruments. These

bring scientific understanding and a rational

approach to freeze-drying cycle development:

Lyostat4

Freeze-drying microscope

Lyotherm2

DTA & Impedance Analyser

Lyostat4 LED Fourth Generation

Freeze-Drying Microscope

• Lyostat was developed by BTL to provide a rational scientific basis for freeze-drying cycle development. Combined with BTL’s extensive experience in the freeze-drying industry, Lyostat provides a powerful tool for freeze-drying R&D Scientists

• BTL has used Lyostat to analyse over 1000 different formulations, from small drug molecules to large complex biomolecules for biopharmaceutical companies worldwide

Lyostat4 – Freeze-drying

microscope

• Enables real-time observation of the behaviour of your

formulation during freeze-drying

• Enables temperature control between -196°C and

125°C to an accuracy of 0.1°C

• By observing the sample structure during drying as the

temperature is raised, the exact point of collapse or

eutectic melt can be observed under the microscope.

Freeze-drying microscopy

• Enables real-time observation of the behaviour

of your formulation during freeze-drying

• Obtains vital information on the critical

temperatures of your product relevant to freeze-

drying

• Enables you to develop a safe and efficient

freeze-drying cycle specific your product

Benefits of using Lyostat4

• Help to improve old and develop new formulations

• Provide essential data to develop cycles on a rational scientific basis

• Save valuable development time and money

• Enables rapid determination of collapse, melting and eutectic event temperatures

• Archives data and image capture for future reference

• Only 2µl of sample material is required to determine the key parameters

Lyostat4 System Components

System Components

• Lyostat4 Freeze-Drying Cryostage

– Temperature range -196°C to +125°C

– Up to 130°C per min heating/cooling rates

– Silver heating block for high thermal conductivity

• Innovative New Imaging Station with LED lamp

– Purpose Built

– Small benchtop footprint

– Higher resolution than standard compound microscopes

– Tilting lens and camera for easy loading of samples

System Components

• Temperature Control System

– Temperature is controlled by carefully balancing heat

input from a heating element and the precise control

of liquid nitrogen through the silver block

– Temperature is measured to 0.01°C by a Pt100

resistor mounted close to the sample and controlled

to 0.1°C even down to pressures as low as 10-3mBar

System Components

• High Performance Digital Firewire Camera

• Dedicated Digital Image Capture Software

– Displays real-time temperature and pressure information and

gives the user full control heating rate, limit and hold time of up

to 100 ramps within the temperature profile.

– Image capture is integrated with temperature programmer data

capture.

– Images can be loaded into a gallery of either 2,8 or 32 images,

for closer inspection.

• Vacuum Pump

• Vacuum Gauge

• Optional Motorised Vacuum Control System

– Software-controlled motorized valve controls chamber pressure

– Enables close investigation of the effects of pressure on sample

collapse

– Chamber pressure displayed in Torr, microns, mBar and Pa

– Pressure plotted with temperature against time and can be

saved for later analysis.

System Components

Lyostat Key Components

LinkSys

Workstation

Software &

Control

Create MovieView Pictures

Data Overview

This file contains all the

information from the analytical

run

From here you can view the

pictures recorded in a gallery

or put them together to create

a movie (.avi format)

The data chart shows temperature and pressure information throughout the

procedure

Results Data

Data OverviewEach point on the purple image line

represents a single image viewed by

selecting it with the cursor

The single image view enables the user to

draw lines and a size bar to measure

between points of interest on the image

(e.g. ice crystal size)

Date, time, vacuum, temperature and ramp

information are displayed under the image

Results Data

Image Gallery

The gallery view enables

fast sorting of images,

showing how the drying

progresses across a longer

timeframe

Gallery Navigation

Zoom

Image Number

For larger images the gallery can be viewed showing less images – 32, 8 and 2

are available

Results Data

Crust Formation

The black line around the sample

edge is an area of higher solute

concentration that impedes vapour

flow and inhibits drying

Image of a solution showing evidence of crust formation

When the crust ruptures the sample will

dry from the site of the break. In this

case the temperature had been raised

above the sample Tc and so shows

evidence of collapse

Eutectic temperature of a

crystalline material

Lyostat FDM

Screenshots of Lyostat analysis, showing frozen product (1), dried with good structure (2)

and collapse (3)

1

2

3

Lyostat provides you with essential information on the critical

temperatures of your formulation, enabling you to get the best results

from your product

Determination of critical

temperatures

‘Trial and Error’ Cycle

resulting in loss of

product or costly

inefficient cycle

New cycle based

on actual

parameters

Problem Solved

This method is sensitive to endothermic

and exothermic processes

Exotherms

•Crystal transitions

•Crystallization

Endotherms

•Melting

•Transitions in the frozen state

Measures the difference in temperature

between a sample and a reference as heat

is applied or removed from the system.

Differential Thermal Analysis

Lyotherm2 is a unique cutting edge technology pioneeredby Biopharma Technology Ltd with the help of ProfessorLouis Rey as a solution to some of the most difficultchallenges faced by freeze drying R&D scientists

Biopharma Technology Ltd has used Lyotherm2 to analyseover 1000 different formulations, from small drug molecules tolarge complex biomolecules for biopharmaceutical companiesworldwide

Lyotherm2

Impedance analysis is fixed frequency

dielectric analysis

It can provide an indication

of the mobility of a sample in

its frozen state, as

demonstrated in a study

carried out by Professor

Louis Rey*

*Freeze-Drying/ Lyophilization of pharmaceutical and biological products. Vol 137, “Drugs and the

pharmaceutical sciences” series. 2nd edition, Eds L. Rey, J.C. May, Marcel Dekker Inc., New York

Impedance Analysis (ZSinφ)

Benefits of Using Lyotherm2

• Help to improve old and develop new formulations

• Provide essential data to develop cycles on a rational scientific basis

• Save valuable development time and money

• Enables rapid determination of frozen state behaviour

• Archives data for future reference

• Not limited to freeze-drying research but to all frozen state applications

AdvantagesLyotherm2 performs a similar function to DSC (differential scanning

calorimetry) as it has a built in differential thermal analyser which

identifies significant thermal events.

The advantage of Lyotherm2 is that it combines DTA with an Impedance

Analysis (ZSinφ) function, which can provide an indication of the

mobility of a sample in its frozen state. Thus giving a more complete

picture of the events occurring within your product than thermal analysis

alone.

Lyotherm2 System Components

Aluminium heating block

designed to distribute heat

evenly, minimising variation

between samples

DTA Probes (Sample and

Reference)

Impedance probe

(ZSinφ)

Dewar for rapid

cooling by Liquid

Nitrogen

Lyotherm2 Control Box

System Components

Graphical plots of both the ZSinϕ

and DTA values are available

tracking the raw data.

Near-linear cooling rates are

provided by the thermally

distributing design of the sample

holding block.

Data Recording

Real-time analysis of both

ZSinϕ and DTA give results

during the analysis,

updated every 5 seconds.

This provides usable data

before the analysis is

finished.

Data Analysis

The software package

allows you to export

your data directly to

Microsoft Excel for easy

data interpretation and

graph manipulation

Temperature (°°°°C)

Ele

ctr

ica

l Im

pe

da

nc

e Z

(k

Oh

ms

)

De

lta

-T (°° °°

C)

(En

do

Do

wn

)

Data Analysis

Case StudymDSC & Lyotherm 2 Study

Case StudymDSC & Lyotherm 2 Study

Case StudymDSC & Lyotherm 2 Study

Lyotherm2 provides you with essential information on the

critical temperatures of your formulation, enabling you to get

the best results from your product

Determination of critical

temperatures and / or annealing

step requirement

‘Trial and Error’

Cycle resulting in

loss of product

New cycle based

on actual

parameters

Problem Solved

Consulting and R&D

Services

• Formulation Development

• Cycle Development

• Product Characterisation

• Production

• Scale-up: Technology and Cycle Transfer

• New Facility for Cytotoxic Compounds

Products Handled

• Small Molecules (Traditional drugs, small

peptides, crop protection agents

• Large Molecules (Large peptides, proteins, DNA

based reagents)

• Structured Materials (Collagen matrices,

drug/vaccine delivery systems)

• Cellular/Organisms (Bacteria, tissue samples)

Case Study 1 – Cycle Development using FDM,

DTA and impedance analysis

Customer Issue• A customer approached BTL with a product that was being freeze-

dried. Their current cycle duration was 64 hours, which they wanted

to be reduced to increase throughput. FDM, DTA and impedance

analysis was proposed to ascertain the lyophilization and frozen

state behavior of the product, which could then be used to improve

the cycle.

• FDM analysis using the BTL Lyostat and DTA and impedance

analysis using the BTL Lyotherm was carried out on the sample

supplied.

• Once the critical temperatures had been established, three

developmental cycles were undertaken to aim for a cycle with a

duration of no more than 32 hours.

• Following the FDM, DTA and impedance analysis, BTL

recommended that the 25 hour cycle was used to enable a higher

throughput of 5 cycles per week.

Conclusions

• BTL can offer a complete service including

- supply of state-of-the-art high quality systems for FDM and DTA &

Impedance

• BTL Consulting offers

- formulation development

- temperature cycle optimization

- product characterization

Advantages:

• Financial: Formulation of a product that aligns with the most cost

effective lyophilisation process

• Operational: Robust, time-efficient procedure

• Regulatory: Characterised product that mitigates risks

Questions

• Thank you for attending the webinar today

• Please email any questions to:

nservi@biopharma.co.uk

Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK

+44 (0)1962 841092 intelligentfreezedrying.com