ADVANCED USES OF FREEZEDRYING MICROSCOPY FDM FOR PRODUCT FORMULATION AND LYOCYCLE DEVELOPMENT

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ADVANCED USES OF FREEZE-DRYING MICROSCOPY (FDM)
FOR PRODUCT FORMULATION AND LYO-CYCLE DEVELOPMENT

• Kevin R. Ward Ph.D. MRSC
• Director of Research and Development,
• Biopharma Technology Ltd.
• Winchester, UK.

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Vials of freeze-dried product
Good
OK
Poor
Poor
The product in the Poor vials has become soft
and dense during freeze-drying, because it has
become warmer than its Critical Temperature!
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How do we know what the Critical Temperature is
for our product?

• The Critical Temperature will be
• The eutectic temperature (Teu) for crystalline
  materials
• The collapse temperature (Tc) for amorphous
  materials (somewhere at or above the glass
  transition temperature)
• The lower of the above temperatures for mixed
  systems (depending on whether micro-collapse is
  acceptable)
• We can analyse the critical temperature of a
  formulation before freeze-drying it, using, for
  example
• Freeze-Drying Microscopy (FDM)
• Impedance (Zsinf) and Thermal Analysis

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Freeze-drying microscopy (FDM)

• FDM is the study of freeze-drying at the
  microscopic level
• FDM allows determination of collapse, melting and
  qualitative phenomena such as skin formation

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What is a Freeze-Drying Microscope?

• Effectively a micro freeze-dryer where the
  freeze-drying of a small sample may be observed
• First designs in the mid- 1960s
• Now manufactured commercially

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• FDM set-up with LN2 cooling system BTL Lyostat2

Freeze-Drying Stage
Camera
Compound Microscope
Liquid nitrogen pump
Temperature controller
Liquid nitrogen Dewar
To vacuum pump
Vacuum gauge
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Sample Preparation for FDM

• Sample loading takes about 60 seconds.
• Routine analysis usually takes 20 - 30 minutes,
  or up to 60 minutes if heat annealing is used.

Block
Sample holder
Side Door
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Sample Format in Lyostat2
Objective Lens (usually 10 x)
Glass cover slip (13 mm dia.)
Metal Spacer (70µm thick)
2µl of sample
Quartz cover slip (16 mm dia.)
Aperture
Temperature-Controlled Block
Light Source (from below)
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Sample Loading and Cooling

• Ideally the raw formulation is used
• Sometimes necessary to use samples that have
  previously been frozen or lyophilised
• After loading the sample, the Lyostat2 is set to
  cool to the desired temperature
• The sample is allowed to cool and freeze (Note
  for eutectic materials, there will be more than
  one freezing event!)

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INITIAL FDM IMAGE

• When sample reaches the holding temperature and
  has been observed to freeze, vacuum pump is
  switched on and drying begins.
• Sublimation interface can be seen moving through
  the frozen sample.

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INTERPRETATION OF EVENTS

• Increasing or decreasing the temperature of the
  sample allows you to view its freeze-drying
  characteristics.
• By examining the freeze-dried structure behind
  the interface, the collapse temperature of the
  material can be determined.
• The temperature may be cycled in order to
  evaluate Tc more closely

Collapsed material
Sublimation front
Frozen sample
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INTERPRETATION OF EVENTS
Frozen sample
Regained structure

• Sample structure lost when collapse temperature
  was exceeded.
• Structure regained as sample was re-cooled to
  below its collapse temperature.

Collapsed sample
Sublimation front
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INTERPRETATION OF EVENTS
Frozen sample
Sublimation front

• 100 structure has been regained by lowering the
  sample temperature.
• Sample temperature was again increased to above
  its collapse temperature, causing the sample to
  collapse.

Dried sample with structure
Collapsing again on reheating
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Micro-collapse (see e.g. Wang, 2004)
Macroscopically similar but is it Wetter? Less
stable? More difficult to reconstitute?
A similar effect may also be observed due to the
melting of crystalline component(s) onto a rigid
amorphous structure (depending on which has the
lower critical temperature)
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FDM of 2 Mannitol 1 Glucose
Frozen material
(Drying front)
Regions of (micro) collapse. Just glucose?
Regions with good dried structure. Just mannitol?
-41oC, around Tc for glucose. Micro-collapse or
just poor solute mixing?
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So, what else can FDM tell us?

• Eutectic melting temperature

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NaCl Below Eutectic Temperature
Dry
Frozen
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NaCl Above Eutectic Temperature
Note changes in appearance of frozen structure
Eutectic liquid
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So, what else can FDM tell us?

• Eutectic melting temperature
• May give some indication of skin (crust)
  formation potential of a formulation

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So, what else can FDM tell us?

• Eutectic melting temperature
• May give some indication of skin (crust)
  formation potential of a formulation
• Whether heat-annealing may be of benefit
• To increase ice crystal size and what
  conditions are required for this (above Tg?)
• To encourage some components to crystallise

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Effect of annealing on ice crystal size
Sample cooled to -40C, then warmed to -10C
Same sample after a further 15 minutes at -10C
Experiments can be carried out to compare rates
of change at different temperatures, in order to
establish what annealing temperature might be
most efficient to use in the freeze-dryer.
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FDM setup with polarised light
Camera
Analyser
Sample
Polariser
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Effect of annealing on solute behaviourFDM with
polarised light function
KCl solution quench cooled below -40C No sign of
crystals (no light rotation)
Drying at -18C Polariser shows presence of
crystals (white areas)
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Effect of annealing on solute behaviourFDM with
polarised light function
Start of Eutectic Melt at -11C
Eutectic Melt complete but ice still
present. ?Crystals were indeed due to eutectic KCl
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Further applications of FDM

• It is possible to examine differences in relative
  drying rates
• For different formulations
• For a specific formulation at different
  temperatures
• Ref Zhai, S., Taylor, R., Sanches, R. and N.K.H.
  Slater (2003). Measurement of Lyophilisation
  primary drying rates by freeze-drying microscopy.
  Chem. Eng. Sci. 58, 2313-2323

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Use of Phase Contrast / DIC

• Phase Contrast may be used to artificially
  colour different parts of a sample
• The following sequence of slides shows a solution
  of mannitol
• In the liquid state
• After initial freezing
• Following annealing

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Mannitol solution at 20oC (liquid)
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Mannitol solution cooled to -30oC
Amorphous solid plus small layer of excluded
liquid at edge
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Warming to -25oC
(note changes in appearance already)
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Annealing to -5oC
Significant crystallisation, moving in from edge
of sample
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CONCLUSIONS

• FDM can provide a visual indication of
• Collapse temperature (Tc)
• Eutectic temperature (Teu)
• Skin formation potential
• Annealing effects on ice structure, solute
  crystallisation, critical temperature
• Relative rates of drying for different
  formulations, or for the same formulation at
  different temperatures
• All the above information can be useful for
  formulation cycle development