BTL "Lyostat" and "Lyotherm" instruments are designed to provide information on "critical temperatures" that are necessary for successful freeze drying product and process development. This presentation gives a brief overview of critical temperatures and explains what the instruments are and how they function.

1. “Critical temperatures for intelligent freeze-
drying”
Nektaria Servi – BTL Instrument Product Manager

2. 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

3. 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

4. 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

5. 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.

6. 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

7. 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

8. 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

9. 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.

10. 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

11. 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 2 l of sample material is required to determine
the key parameters

12. Lyostat4 System Components

13. 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

14. 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

15. 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.

16. • 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

17. Lyostat Key Components
LinkSys
Workstation
Software &
Control

18. 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

19. Data Overview
Each 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

20. 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

21. 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

22. Eutectic temperature of a
crystalline material

23. Lyostat FDM
Screenshots of Lyostat analysis, showing frozen product (1), dried with good structure (2)
and collapse (3)
1
2
3

24. 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

25. 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

26. Lyotherm2 is a unique cutting edge technology pioneered
by Biopharma Technology Ltd with the help of Professor
Louis Rey as a solution to some of the most difficult
challenges faced by freeze drying R&D scientists
Biopharma Technology Ltd has used Lyotherm2 to analyse
over 1000 different formulations, from small drug molecules to
large complex biomolecules for biopharmaceutical companies
worldwide
Lyotherm2

27. 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)

28. 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

29. Advantages
Lyotherm2 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.

30. Lyotherm2 System Components

31. 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

32. 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

33. 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

34. The software package
allows you to export
your data directly to
Microsoft Excel for easy
data interpretation and
graph manipulation
Temperature (°°°°C)
ElectricalImpedanceZ(kOhms)
Delta-T(°°°°C)(EndoDown)
Data Analysis

35. Case Study
mDSC & Lyotherm 2 Study

36. Case Study
mDSC & Lyotherm 2 Study

37. Case Study
mDSC & Lyotherm 2 Study

38. 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

39. Consulting and R&D
Services
• Formulation Development
• Cycle Development
• Product Characterisation
• Production
• Scale-up: Technology and Cycle Transfer
• New Facility for Cytotoxic Compounds

40. 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)

41. 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.

42. 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

43. Questions
• Thank you for attending the webinar today
• Please email any questions to:
nservi@biopharma.co.uk

44. Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK
+44 (0)1962 841092 intelligentfreezedrying.com