Lyostat4 freeze drying microscope provides brighter, clearer images for easy analysis of formulations for freeze drying. Freeze drying microscopy is used to identify the collapse temperature of a product, which is necessary for product and process development.

1. Lyostat 4 freeze drying microscope

2. Contents
• History of Biopharma Technology
• How to determine the critical temperatures
• Lyostat LED Fourth Generation Freeze-Drying
Microscope
• Lyotherm – Frozen-State Analysis
• Consulting and R&D Services Overview
• Case Studies
• Conclusions
• Questions

3. History of Biopharma Technology
• Biopharma Technology Ltd (BTL) was founded in
1997
• Part of the Biopharma Group
• To date, BTL has worked with over 1800 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 lyophilization (freeze drying) steps
involved in the development and/or production
process. Acting at early stage minimizes 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 frozen 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. Tc vs Tg’
• Many cycles use Tg’ as the critical temperature
for freeze drying cycle development
• However Tg’ can be significantly lower than Tc
• Lower primary drying temperatures mean slower
processing and more expensive cycles
• Although Tg’ is significant, many formulations
can be dried safely above this point
• Knowledge of Tc is strongly recommended for
formulation and cycle development

8. • BTL has used Lyostat to analyze over 1800 different
formulations, from small drug molecules to large
complex biomolecules
Lyostat 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

9. Freeze Drying Microscopy
• Real-time observation of the behavior of your
formulation during freeze drying
• By observing the sample structure the exact
point of collapse or eutectic melt can be
observed

10. Benefits of Using Lyostat
• 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

11. Lyostat System Components

12. System Components
• Lyostat 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

13. 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

14. 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.

15. • Vacuum Pump
• Vacuum Gauge
• Optional Motorized 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

16. Lyostat Software
LinkSys
Workstation
Software &
Control

17. Assay Procedure
• Unscrew lid and side door
• Place sample holder on
block and close door
• Place drop of silicone oil
on sample block
• Place quartz cover slip in
the sample holder
• Place spacer ring on
quartz cover slip
• Pipette 2 l of sample
onto quartz cover slip
• Place a smaller cover slip
on top of the spacer ring
Sample
Block
Sample
Holder
Stage Lid

18. Temperature
Controlled Block
Light Source
(from below)
Aperture
Quartz cover slip (16 mm
dia.)
Glass cover slip (13 mm dia.)
2 l of sample
Objective Lens (usually 10x)
Metal Spacer (70 m thick)
Sample Holder
Assay Procedure
Sample Loading
Drop of silicone oil for low
temperature lubrication

19. Assay Procedure
• Once sample is loaded
onto stage it can be
viewed in real-time on the
computer screen
• Program your freezing
temperature and initiate
the pump with one click
• When your freezing
temperature is attained it
can be held to ensure
complete freezing
• Turn on the vacuum
pump to begin drying the
sample
Freezing

20. Assay Procedure
Drying – Amorphous Product
When drying, the sublimation
front will move from one side
of the sample to the other. In
front of it will be the frozen
material and behind will be
the dried material
The collapse temperature (Tc) is obtained by gradually increasing the temperature
until product collapse is observed
Sublimation
Front
Movement of
sublimation Front
Dried
Amorphous
Material
Frozen
Amorphous
Material

21. Assay Procedure
Drying – Amorphous Product
Raising the temperature above the
Tc of the sample will cause it to
undergo collapse
A precise Tc can be found by going
back and viewing the images
recorded during the analytical run
For crystalline products the same method is applied but a eutectic temperature
(Teu) is obtained instead
Dried
Amorphous
Material
Collapsed
Material
Frozen
Amorphous
Material

22. Assay Procedure
Drying - Crystalline Product
Frozen
Crystalline
Material
Dried
Crystalline
Material
Sublimation
Front
Eutectic melt of material at
interface
Change in gross morphology of
frozen phase to give more liquid
appearance
Increasing
Temperature

23. 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

24. 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

25. 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

26. Crust Formation
The black line around the sample
edge is an area of higher solute
concentration that impedes vapor
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

27. Eutectic Temperature of a
Crystalline Material

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

29. Case Study 1- Using FDM to design
a more efficient freeze drying cycle
Customer Issue
BTL was asked to reduce the freeze drying time for a product that
currently had a cycle duration of 61 hours. The formulation was
analysed using BTL’s freeze drying microscope Lyostat. The FDM
analysis identified a Tc range of -18.6°C to -17.4°C. Differential
thermal analysis and impedance analysis was carried out using
Lyotherm2. This identified a softening event in the impedance at -
25°C. A freeze drying cycle was carried out that was designed to
maintain the product temperature below -30°C, allowing a 5.0°C
safety margin below the lowest critical temperature identified. This new
cycle, designed by BTL, was 45 hours long. This was 16 hours shorter
than the original cycle, resulting in a significant time and energy saving
for the manufacturer.

30. Case Study 2 – Using FDM to
improve cycles
Customer Issue
A customer approached BTL with a product that was being freeze-dried using a
generic cycle (which was applied to a number of other products). It was failing
to meet the required moisture threshold (no more than 2%) and each batch was
therefore rejected. FDM analysis was proposed to ascertain the lyophilisation
behaviour of the product and to see if any improvements could be made on the
existing cycle.
FDM analysis using the BTL Lyostat was carried out on the sample supplied.
Once the critical temperatures had been established a critical review of the
existing cycle was carried out to see what changes could be made to prevent
future batch failure.
Following the FDM analysis and the critical review of the existing cycle, BTL
recommended that the temperature used in primary drying was kept constant
about 5.0°C below Tc. This was implemented by the customer and it
immediately led to a reduced moisture content and thus no more batch failures.

31. Case Study 3 – Formulation
Development
Customer Issue
• The customer wanted to develop a freeze drying cycle for a product.
However freeze drying microscopy had identified a low collapse
temperature of –42°C.
• Low collapse temperatures require cycles that are slow and lengthy and
energy expensive.
• BTL were able to reformulate to replace some of the sucrose, which has a
low collapse temperature, with dextran. Dextran can affect biological
products but the small amount used ensured there were no detrimental
effects to the active ingredient.
• The final formulation had an overall collapse temperature of -23°C, a
significant improvement over the original.

32. 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

33. Other Products and
Services from BTL
• Lyotherm frozen-state analyzer
– Provides detailed information about behavior of the
product in the frozen state, including Tg’,
crystallizations, melts, molecular mobility
• Training courses in freeze drying technology
– Regular scheduled courses held worldwide
– Customizable courses held at any preferred location
• Product and process development
– Experience with over 1800 different products
– Standalone facility for cytotoxic / potent products

34. intelligentfreezedrying.com