Insights in Lyophilization

Science Tour India 2016

Freeze Dry Microscopy (FDM) A Key Tool to

Determine the Collapse Temperature of a
Formulation

Dr. Margit Gieseler

Friedrich-Bergius-Ring 15 E-Mail: info@gilyos.com
D-97076 Wrzburg Web: www.gilyos.com
 Insights in Lyophilization
Science Tour India 2016

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 FDM
General Introduction, 1

Conservative
drying conditions
to prevent
collapse /
impairment of
product
appearance.

Increase of TP by 1C
can shorten primary
drying time up to 13%.
High product
temperature (TP)
to keep drying Pikal M.J.; Use of Laboratory Data in Freeze Drying
time short. Process Design: Heat and Mass Transfer Coefficients
and the Computer Simulation of Freeze Drying. J.
Parent. Sci. Technol. 1985, 39(3), 115-138.

Page 3
 FDM
General Introduction, 2

Knowledge about the

Conservative
drying conditions critical formulation
to prevent temperature of utmost
collapse / importance!
impairment of
product
appearance.
Only an optimized
TP
cycle can satisfy both
requirements!

High product Dependent on the

temperature to
keep drying time formulation composition
short. must be determined for
every formulation!

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 FDM
Product Defects, 1

Classification of product defects (selection)

Meltback:
Meltback = product temperature exceeds Teut (crystalline materials).
Meltback = ice meltback due to improper endpoint detection (primary drying).
Shrinkage / Microcollapse (A) or full collapse (B).

A B

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 FDM
Product Defects, 2

Microcollapse
Collapse only on -scale level.
Not directly detectable during visual inspection.

50 mg/mL sucrose 50 mg/mL sucrose

Product temperature during primary drying below Toc. Product temperature during primary drying above Toc.

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 FDM
Definitions

Eutectic temperature (Teut)

Critical formulation temperature of crystalline systems.
If product temperature during primary drying > Teut melt.
Analytical method: Differential Scanning Calorimetry (DSC), Freeze Dry Microscopy
(FDM).

Glass transition temperature (Tg')

Temperature of maximally freeze-concentrated solute of amorphous systems.
If product temperature during primary drying >> Tg' shrinkage / collapse.
Analytical method: Differential Scanning Calorimetry (DSC).

Collapse temperature (Tc)

Temperature, at which the first structural changes in the dried product matrix
become visible in amorphous systems.
Increased mobility due to viscous flow.
If product temperature during primary drying > Tc collapse.
By definition: Tc Toc (onset of collapse).
Analytical method: Freeze Dry Microscopy (FDM).
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 FDM
Technology - Background

Glass transition
During the DSC measurement, the sample is in constant contact with ice, i.e. it is a
STATIC measurement.
Water acts as a plasticizer.

Collapse
DYNAMIC process, starting in the pore walls of the dried product matrix due to a
temperature induced increase in molecular mobility.
Already during primary drying, secondary drying effect leads to diffusion /
desorption of water and thus (additional) water removal from the (dry) product.

Pikal M.J.; Shah S. The collapse temperature in freeze drying:

Dependence on measurement methodology and rate of water removal
from the glassy phase. Int. J. Pharm. 62:165-186, 1990

Page 8
 FDM
Comparison FDM - DSC

Comparison DSC - FDM

-4
-6
-8 Toc
-10
Tfc
-12
Temperature [C]

-14 Tg'
-16
-18
-20
-22
-24
-26
-28
-30
-32
-34
-36
5) 0) 5) 0) 0) 5) 0) 5) 0) 0)
5 /3 0 /5 5 /6 0 /8 0/9 5/3 0/5 5/6 0/8 0/9
(6 (5 (3 (2 (1 (6 (5 (3 (2 (1
/s /s /s /s /s A/t A /t A /t A /t A /t
A A A A A
BS BS BS BS BS BS BS BS BS BS

Meister E.; Gieseler H. Freeze-Dry Microscopy of Protein/Sugar Mixtures: Drying Behavior, Interpretation of Collapse
Temperatures and a Comparison to Corresponding Glass Transition Data. J. Pharm. Sci., 98(9):3072-3087, 2009.

Page 9
 FDM
Equipment and Sample Preparation

A 6
1 Light microscope
2 Freeze drying stage
3 Vacuum pump
9
8 4 Electronic valve
5 Pirani gauge
1 6 Digital camera
7 Dewar with LN2
5
8 Controller (pressure, LN2 feed, temperature)
4 9 Computer with control and recording
2
3 software

B C small cover slip

spacer (H:25m)
sample(~2L)
largecover slip
silicone oil

silver block
PT100

Lightsource

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 FDM
Example Amorphous Systems, 1

Sucrose-based
formulation, approx.
50 mg/mL total solids.
- Cooling rate 1C/min.
- Heating rate 1C/min.

Temperature: -33.4C.

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 FDM
Example Amorphous Systems, 2

Sucrose-based
formulation, approx.
50 mg/mL total solids.
- Cooling rate 1C/min.
- Heating rate 1C/min.

Temperature: -33.3C.

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 FDM
Example Amorphous Systems, 3

Sucrose-based
formulation, approx.
50 mg/mL total solids.
- Cooling rate 1C/min.
- Heating rate 1C/min.

Toc: -33.3C.

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 FDM
Example Amorphous Systems, 4

Sucrose-based
formulation, approx.
50 mg/mL total solids.
- Cooling rate 1C/min.
- Heating rate 1C/min.

Tfc (full collapse): -30.5C.

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 FDM
Example Crystalline Systems

Mannitol 100 mg/mL

Heating rate: 1C/min.

Cooling rate: Quench-cooled (ca. 70C/min). Cooling rate: Quench-cooled (approx. 70C/min) +
T = -30.0C. annealing (30 min @ -15C).
T = -30.0C.

Page 15
 FDM
Example Partially Crystalline Systems

Mannitol / Trehalose 50 mg/mL

Cooling / heating rate: 1C/min.

Mixing ratio 50:50 Mixing ratio 90:10

T = -34.9C T = -35.0C

Page 16
 FDM
Methodology - Important Factors

Cooling rate
Influences size, structure and homogeneity of ice crystals.
A partially crystalline material can remain amorphous due to a high cooling rate. Its
Toc contributes to the overall Toc, shifting it to lower temperatures.
The FDM result might not be representative if a different cooling rate is used or an
annealing step is implemented in a subsequent freeze drying cycle.

Heating rate
High heating rates (5 - 10C/min): time saving, BUT: detection of Toc impaired.

Sample layer thickness / sample volume

Differences in viscosity lead to variations in spreading behavior Toc might be
detected too late (thick layer) or too early (thin layer) or is not detectable at all (very
thin layer).

Concentration of formulation
High total solid concentrations (> 10%): dark pictures, Toc difficult to detect shift
to higher Toc.

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