The Use of Freeze-Drying Microscopy (FDM) to Determine Critical Parameters

of Materials Prior to Lyophilisation

Dr Kevin R. WardDirector of Research & Development, BTLWinnall Valley Road, Winchester SO23 0LD

T: 01962 841092E: kward@biopharma.co.ukW: www.btl-solutions.net

What is Freeze-Drying?

• Freeze-drying (lyophilisation) is the removal of frozen solvent by sublimation under vacuum, and unfrozen solvent by desorption

• Materials should be characterised and freeze-dried below their intrinsic critical temperature (Tc / Teu)

• A wide range of materials across the pharmaceutical, diagnostics and food sectors are freeze-dried

Examples of freeze-dried products

Vials of freeze-dried product

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Good OK PoorPoor

The product in the “Poor” vials has become soft and dense during freeze-drying, because it has become warmer than its “Critical Temperature”

“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

(although in some cases, it is possible to dry above this)

• We can analyse the critical temperature of a formulation before freeze-drying it, using, for example:

– Freeze-Drying Microscopy (FDM)

– Impedance (Zsinφ) and Thermal Analysis5

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-60s• Now manufactured commercially• Our original Lyostat instrument

was developed with Oxford Instruments (1997)

• Lyostat2 was developed jointly with Linkam (2001)

• Now Lyostat3 (also with Linkam)• Allows for precise temperature

control and programmed ramp / hold steps

• Vacuum tight system

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Sample Preparation in Lyostat3 FDM

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Sample holder Side

Door

Block

• Sample loading takes about 60 seconds.• Routine analysis usually takes 20 - 30 minutes, or up to 60

minutes if heat annealing is used.

Sample Format in Lyostat3 FDM

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Temperature-Controlled Block

Light Source (from below)

Aperture

Quartz cover slip (16 mm dia.)

Glass cover slip (13 mm dia.)

2µl of sample

Objective Lens (usually 10 x)

Metal Spacer (70µm thick)

• 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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FDM image 1

Frozen Sample

Sublimation Front

Dried Sample

Temperature / Time Profile

Real-time Chart

• 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

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FDM image 2

Collapsed Material

Frozen Sample

Sublimation Front

• Sample structure lost when collapse temperature was exceeded.

• Structure regained as sample was re-cooled to below its collapse temperature.

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FDM image 3

Regained Structure

Frozen Sample

Sublimation Front

Collapsed Sample

• Sample temperature was again increased to above its collapse temperature, causing the sample to collapse.

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FDM Image 4

Frozen sample

Collapsing again on reheating

Sublimation front

Dried structure

So, what else can FDM tell us?

• Eutectic melting temperature

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NaCl Below Eutectic Temperature

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Frozen

Dry

NaCl Above Eutectic Temperature

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Note changes in appearance of frozen structure

Eutectic liquid

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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Layer of concentrated solute at edge of sample

Drying only occurs through ruptures in the skin / crust

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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

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Sample cooled to -40°C, then warmed to -10°C

Same sample after a further 15 minutes at -10°C

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.

FDM setup with polarised light

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Polariser

Analyser

Sample

Camera

Effect of annealing on solute behaviour:FDM with basic plane polarised light function

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Sample quench cooled below -40°C

No sign of crystals (no light rotation)

Drying at -18°C

Polariser shows presence of crystals (white areas)

Frozen Mannitol Solution, annealed to -5oC

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Significant crystallisation, moving in from edge of sample

Crystal Formation at Controlled Temperatures (1)

Crop Protection Molecule in MeOHprior to solvent evaporation

Crystal Formation at Controlled Temperatures (2)

Same Molecule after MeOHevaporated at 20°C

Crystal Formation at Controlled Temperatures (3)

Same Molecule after MeOHevaporated below -80°C

Crystal Formation at Controlled Temperatures (4)

Same Molecule upon crystallisation from THF(evaporated at 25°C)

Crystal Formation at Controlled Temperatures (5)

Same Molecule upon crystallisation from THF(evaporated slowly at reduced temperature)

Sheep RBC in suspension

Sheep RBC upon initial freezing...

…and 1 minute later…

…and after rapid thawing…

Was it the freezing or the thawing that caused lysis?

It’s difficult to tell using conventional methods, because the cells may have been damaged during freezing, yet fixed in position, thereby making damage impossible to identify…

Issues with RBC lyophilisation

• Freezing damage:– from ice crystal growth– from pH gradients– from freeze-concentration / osmotic effects

• Drying damage:– Physical action of ice removal on membrane– Dehydration causing deformation of RBC

• Rehydration damage:– Concentration and pH effects (e.g. localised hypotonic / acidic /

alkaline regions, causing lysis)– Wetting issues exacerbating the above effects

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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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– Behaviour of cells and other structures as well as simple solutions

during freezing and drying

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All the above information can be useful for formulation & freeze-drying cycle development

Acknowledgements• Linkam Scientific Instruments (for

development of Lyostat3 FDM):– Ian Pearce– Vince Kamp– Peter Grocutt

• Members of the BTL scientific team:– Isobel Cook– Tom Peacock– Marc Townell

Thank You for your attention!

T: 01962 841092E: kward@biopharma.co.ukW: www.btl-solutions.net

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Presented during “Emerging Technologies in Freeze

Drying”, Cambridge, 11th May 2011. Event organised by BPS

and BTL, www.biopharma.co.uk

www.btl-solutions.net