LYOPHILIZATION

Introduction to Freeze-Drying
Second of Four Lectures
Physical Properties and Characterization of Materials

J. Jeff Schwegman, Ph.D.
AB BioTechnologies
P.O. Box 1430
Bloomington, Indiana, 47402
812-327-6898
jjschwegman@gmail.com
AB|BioTechnologies
AB Biotechnologies has
a new website
www.ab-biotech.com
Experts in Formulation, Lyophilization Cycle
Design/Optimization, Thermal
Characterization, and Education and Training
in the Development of Injectable Drug
Products and Diagnostics
 Definitions
A eutectic mixture is an intimate mixture of
2 (or more) crystalline species that are in
such close contact that they melt like a
single, pure substance.

A glass is an amorphous species (randomly

oriented molecules) which forms a solid,
non flowing mass below its Tg’

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 Freezing of Aqueous Systems
Solution
Solute crystallizes?
Ice Nucleation
Ice crystal growth, freeze
concentration

Lyotropic
Solute Crystallizes, Liquid Crystal
eutectic formation

Stable Glass
Metastable
Glass
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 Thermal Transition Terms Important
in Freeze-Drying
Eutectic temperature (Te)
Refers to crystalline systems and measured by
thermal or thermoelectric analyses
Glass transition temperature (Tg’ and Tg)
Refers to amorphous systems and also measured by
thermal or thermoelectric analyses
Collapse temperature (Tc)
Essentially the same as Tg’, but measure by freeze-dry
microscopy.

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 Eutectics and Glasses

Why are These Important?

The eutectic or glass transition
temperatures determine maximum
temperature that the product can
withstand during primary drying
without loss of structure
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 Unfrozen Water

The amorphous solute, when in

equilibrium with ice contains large
amounts (10-50%) of unfrozen water
which is dissolved in the solute phase
The crystalline solute contains no
unfrozen water except surface adsorbed
water on crystal surface or occluded water
in crystals (<1%)
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 Eutectics and Glasses

These two different species dry very

differently both in primary and
secondary drying.
Cycle development conditions must
take this into consideration

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 Eutectics and Glasses

In primary drying, must stay below Tg’

or Te or complete structural loss or the
product will occur (Tg’ typically much
lower than Te)
Water embedded in glass while
adsorbed to the surface of crystals

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 Eutectics and Glasses
Interstitial space must be rigid enough to support itself
after water (acting as the scaffolding) sublimes away

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 Eutectics and Glasses

In secondary drying:
•Water must diffuse to surface of glass
before vaporization (very slow)
•Water easily vaporized off of the crystal
surface (very fast)

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 Collapse

Complete Collapse of
the Product

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 Melt Back

Melt Back
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 Cake Shrinkage – Partial and Complete
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 Characterization

Critical Temperatures in Freeze-

Drying
The eutectic or glass transition
temperature determines maximum
temperature that the product can
withstand during primary drying
without loss of structure

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 Characterization

How?
• Differential Scanning Calorimetry (DSC)
or Modulated DSC
• Dielectric Analysis (DEA)
• Differential Thermal Analysis (DTA)
• Thermoelectric Analysis (TEA)
• Freeze-Dry Microscopy
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 Thermal Analysis

Physical or chemical changes occurring in a

material with changes in temperature are
generally accompanied by the absorption or
release of energy in the form of heat.
The methods for characterizing these
alterations upon heating or cooling a sample
are referred to as “Thermal Analysis”.
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 DSC

In DSC, the sample and a reference are

subjected to a carefully controlled temperature
program each with their own furnace.
When a transition occurs that results in a
change in sample temperature, heat is added or
taken away from the sample so that both cells
remain at the same temperature.
This heat is equal to the energy of the
transition.
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 DSC

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 DSC

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 DSC

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 DSC

When using DSC for conducting thermal

analysis studies for lyophilization cycle
development, discard the cooling curve and
only focus on the heating curve.
The sample has the potential for
“supercooling” and gives misleading data.

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 DSC

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 DSC Thermal Transitions

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 DSC

A shift in the baseline toward a higher heat

capacity is indicative of a glass transition which
denotes a decrease in order within the system.
Endotherms generally indicate physical changes
rather than chemical changes (sharp endotherms
are characteristic of melting of pure compounds).
Exothermic behavior is associated with increased
molecular order within the system such as
crystallization.
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 DSC

What if I can’t detect my low temperature

glass transition?
Increasing both the heating rate and sample
concentration increases the sensitivity of the
DSC making low temperature transitions
visible.
PE Diamond DSC has “Hyper DSC” capable
of accurate heating rates of up to 250°C/min.
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 DSC

20 W /g
Endotherm Up
Heat Flow, W/g

0.1 W /g

-50 -48 -46 -44 -42 -40 -38 -36 -34 -32 -30

-60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25
Tem perature, °C

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 DSC

20 W /g
Endotherm Up
Heat Flow, W/g

0.1 W /g

-50 -48 -46 -44 -42 -40 -38 -36 -34 -32 -30

-60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25
Tem perature, °C

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 DSC

What if I know I have a eutectic and ice but

only see one peak?
Decreasing the heating rate increases the
resolution of the DSC making overlapping
over lapping thermal events visible.
Can warm through transitions at 0.1°C/min
to resolve over lapping thermal events.
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 DSC Thermogram of Glycine vs.
Heating Rate

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 DSC

DSC Rule of Thumb

Increase heating rate and or sample
concentration to see low energy transitions
(glass transitions) which may not be detected at
lower heating rates.
Decrease heating rate to resolve overlapping
thermal events (Ice and eutectic melt thermal
transitions)
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 DSC Technique (Liquids)

• 7 to 10 µL are hermetically sealed into aluminum sample

pans (pre-filter samples to remove particles)
• Sample cell and reference cell (empty pan) are placed in the
sample and reference furnaces
• The sample is then cooled to a predetermined Temp (-60°C)
• The sample is then warmed through all of the thermal events
(25°C)
• Discard cooling curve and focus on warming curve
• Look for thermal transitions (low energy and overlapping)
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 DSC Technique (Solids)

• Prepare amorphous or partially amorphous samples in a dry

box.
• Add enough solid material to cover the bottom of the pan.
• Compress lightly to approximately 1 mm thickness.
• Sample cell and reference cell (empty pan) are placed in the
sample and reference furnaces
• The sample is then warmed through all of the thermal events
(be aware of exploding/leaking pans at high temps)
• Look for thermal transitions (low energy and overlapping)
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 Freeze-Dry Microcopy

Direct examination of freezing and

freeze-drying via a special microscope
and thermal stage

Compliments and supports the

information gained from the DSC

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 Equipment

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 Equipment

Necessary equipment includes:

• Polarized Light Microscope
• Liquid nitrogen cooled thermal stage
• Vacuum pump
• Complete systems available at McCrone
Microscopes in Chicago, IL
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 Thermal Stage
Linkam FDCS 196 Freeze-Dry Microscopy Stage

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 Thermal Stage
Linkam FDCS 196 Freeze-Dry Microscopy Stage

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 Technique

1. Insert the sample manipulator into the chamber

2. Place a small amount of silicone oil on the silver
block (aids in sample movement and heat transfer)
3. Place a 13 mm window on the block within the
sample manipulator
4. Pipet 4-7µL of sample onto the top, center of the
window
5. Lightly place a small cover slip on top of the sample
being careful not to let fluid wick underneath the
lower window
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 Technique

6. Seal the chamber and begin cooling

7. Bathe the upper and lower exterior windows with
dry nitrogen to prevent condensation
8. Freeze the sample to below the eutectic or glass
transition temperature (Determined from DSC)
9. Collect images of the frozen state
10.Set the vacuum to the desired level and collect
images below the critical temperature to show an
intact dried layer

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 Technique

11.Slowly warm the sample and collect images of the

various thermal events
12.Repeat experiment if necessary to determine the
effect of annealing

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 Types of Freeze-Drying Behavior:
Eutectic Melting

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 What have we learned?

Thermal analysis studies (DSC/Freeze-Dry

microscopy have given us several key pieces of
information:
Tells us if the system is amorphous, crystalline, or
partially crystalline
Tells us our critical temperatures (Tg’, Te)
Tells us if we need to anneal the system and
approximately what those conditions are

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 What have we learned?

Thermal analysis studies allow us to take

an empirical approach to lyophilization
cycle development as opposed to using
trial-and-error which has been the
dominant means of development in the
past. These techniques also allow us to
better troubleshoot problem cycles and
formulations
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 Thank you.
The next lecture in this series is
April 18th at 6:30 am & 10 am (NY time)
Experts in Formulation, Lyophilization Cycle Design/Optimization,
Thermal Characterization, and Education and Training in the
Development of Injectable Drug Products and Diagnostics

J. Jeff Schwegman, Ph.D.

jjschwegman@gmail.com
AB Biotechnologies has
a new website
www.ab-biotech.com
AB|BioTechnologies 52