Every freeze-drying formulation has a critical temperature. During primary drying the formulation must be kept below this temperature in order to prevent processing defects or complete collapse. Improperly dried products will exhibit reduced levels of activity, decreased solubility, and poor stability - even if they do not look any different. Freeze drying microscopy enables critical temperatures such as collapse, melting and freezing point to be quickly and easily identified with as little 2ml of the formulation. With this information freeze drying cycles can be designed that are efficient, robust and safe for your product.

1. Lyostat3
Third Generation
Freeze-Drying Microscope

2. Lyostat3
• Lyostat3 was developed by Biopharma Technology
Ltd to provide a rational scientific basis for freeze-
drying cycle development. The Lyostat3 combined
with BTL’s extensive experience in the freeze-drying
industry provides a powerful tool for freeze-drying
R&D Scientists
• Biopharma Technology Ltd has used Lyostat3 to
analyse over 700 different formulations, from small
drug molecules to large complex biomolecules for
biopharmaceutical companies worldwide

3. 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

4. Benefits of using Lyostat3
• It can help improve old and develop new
formulations
• It can provide essential data to develop cycles on a
rational scientific basis
• It can 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

5. Lyostat3 System Components

6. System Components
• Lyostat3 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
– Purpose Built
– Small benchtop footprint
– Higher resolution than standard compound microscopes
– Tilting lens and camera for easy loading of samples

7. 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

8. 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.

9. System Components
• 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.

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

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

12. Assay Procedure
Freezing
• 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

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

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

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

16. Crust Formation
Image of a solution showing evidence of crust formation
The black line around the sample When the crust ruptures the sample will
edge is an area of higher solute dry from the site of the break. In this
concentration that impedes vapour case the temperature had been raised
flow and inhibits drying above the sample Tc and so shows
evidence of collapse

17. Micro-collapse
Image of a 2% Mannitol + 1% Glucose solution drying under vacuum at -41.8°C,
around the Tc of Glucose
Frozen material
Movement of
sublimation front
Glucose Mannitol
collapses retains
within mannitol structure
structure
Dried material

18. Results Data
Data Overview
View Pictures Create Movie
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

19. Results Data
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

20. Results Data
Gallery Navigation
Image Gallery Zoom
Image Number
The gallery view enables
fast sorting of images,
showing how the drying
progresses across a longer
timeframe
For larger images the gallery can be viewed showing less images – 32, 8 and 2
are available

21. Problem Solved
‘Trial and Error’ Cycle
New cycle based
resulting in loss of Determination of critical
on actual
product or costly temperatures
parameters
inefficient cycle
Lyostat3 provides you with essential information on the critical
temperatures of your formulation, enabling you to get the best results
from your product

22. Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK
Tel: +44 (0)1962 841092 Web: www.btl-solutions.net