The Use of Freeze-Drying Microscopy (FDM) to Determine Critical Parameters of Materials Prior to Lyophilisation Dr Kevin R...
What is Freeze-Drying? <ul><li>Freeze-drying (lyophilisation) is the removal of frozen solvent by sublimation under vacuum...
Examples of freeze-dried products
Vials of freeze-dried product Good OK Poor Poor The product in the “Poor” vials has become soft and dense during freeze-dr...
“ How do we know what the Critical Temperature is for our product?” <ul><li>The “Critical Temperature” will be: </li></ul>...
Freeze-drying microscopy (FDM) <ul><li>FDM is the study of freeze-drying at the microscopic level </li></ul><ul><li>FDM al...
What is a Freeze-Drying Microscope? <ul><li>Effectively a ‘micro freeze-dryer’ where the freeze-drying of a small sample m...
Sample Preparation in  Lyostat3  FDM Sample holder Side Door Block <ul><li>Sample loading takes about 60 seconds. </li></u...
Sample Format in  Lyostat3  FDM Temperature-Controlled Block Light Source (from below) Aperture Quartz cover slip (16 mm d...
<ul><li>When sample reaches the holding temperature and has been observed to freeze ,  vacuum pump is switched on and dryi...
<ul><li>Increasing or decreasing the temperature of the sample allows you to view its freeze-drying characteristics. </li>...
<ul><li>Sample structure lost when collapse temperature was exceeded. </li></ul><ul><li>Structure regained as sample was r...
<ul><li>Sample temperature was again increased to above its collapse temperature, causing the sample to collapse.  </li></...
So, what else can FDM tell us? <ul><li>Eutectic melting temperature </li></ul>
NaCl Below Eutectic Temperature Frozen Dry
NaCl Above Eutectic Temperature Note changes in appearance of frozen structure Eutectic liquid
So, what else can FDM tell us? <ul><li>Eutectic melting temperature </li></ul><ul><li>May give some indication of skin (cr...
Layer of concentrated solute at edge of sample Drying only occurs through ruptures in the skin / crust
So, what else can FDM tell us? <ul><li>Eutectic melting temperature </li></ul><ul><li>May give some indication of skin (cr...
Effect of annealing on ice crystal size Sample cooled to -40 ° C, then warmed to -10 ° C Same sample after a further 15 mi...
FDM setup with polarised light Polariser Analyser Sample Camera
Effect of annealing on solute behaviour: FDM with basic plane polarised light function Sample quench cooled below -40 ° C ...
Frozen Mannitol Solution, annealed to -5 o C Significant crystallisation, moving in from edge of sample
Crystal Formation at Controlled Temperatures (1) Crop Protection Molecule in MeOH prior to solvent evaporation
Crystal Formation at Controlled Temperatures (2) Same Molecule after MeOH evaporated at 20°C
Crystal Formation at Controlled Temperatures (3) Same Molecule after MeOH evaporated 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 reduc...
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...
Issues with RBC lyophilisation <ul><li>Freezing damage: </li></ul><ul><ul><li>from ice crystal growth </li></ul></ul><ul><...
Further applications of FDM <ul><li>It is possible to examine differences in relative drying rates: </li></ul><ul><ul><li>...
CONCLUSIONS <ul><li>FDM can provide a  visual  indication of: </li></ul><ul><ul><li>Collapse temperature (T c )  </li></ul...
Acknowledgements <ul><li>Linkam Scientific Instruments (for development of  Lyostat3  FDM): </li></ul><ul><ul><li>Ian Pear...
Thank You for your attention! T: 01962 841092 E:  [email_address] W:  www.btl-solutions.net
Presented during  “Emerging Technologies in Freeze Drying”,  Cambridge, 11 th  May 2011. Event organised by BPS and BTL,  ...
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Use Of Freeze Drying Microscopy To Determine Critical Parameters

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Materials for freeze drying should be characterised in order to ensure that freeze-drying takes place below their intrinsic critical temperature (Tc / Teu). This presentation evaluates freeze drying microscopy, an established technique for formulation analysis.

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  • K: Formulation Characterisation 1
  • K: Formulation Characterisation 1
  • K: Formulation Characterisation 1
  • K: Formulation Characterisation 1
  • K: Formulation Characterisation 1
  • K: Formulation Characterisation 1
  • K: Formulation Characterisation 1
  • K: Formulation Characterisation 1
  • Use Of Freeze Drying Microscopy To Determine Critical Parameters

    1. 1. The Use of Freeze-Drying Microscopy (FDM) to Determine Critical Parameters of Materials Prior to Lyophilisation Dr Kevin R. Ward Director of Research & Development, BTL Winnall Valley Road, Winchester SO23 0LD T: 01962 841092 E: [email_address] W: www.btl-solutions.net
    2. 2. What is Freeze-Drying? <ul><li>Freeze-drying (lyophilisation) is the removal of frozen solvent by sublimation under vacuum, and unfrozen solvent by desorption </li></ul><ul><li>Materials should be characterised and freeze-dried below their intrinsic critical temperature (Tc / Teu) </li></ul><ul><li>A wide range of materials across the pharmaceutical, diagnostics and food sectors are freeze-dried </li></ul>
    3. 3. Examples of freeze-dried products
    4. 4. 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”
    5. 5. “ How do we know what the Critical Temperature is for our product?” <ul><li>The “Critical Temperature” will be: </li></ul><ul><ul><li>The eutectic temperature (Teu) for crystalline materials </li></ul></ul><ul><ul><li>The collapse temperature (Tc) for amorphous materials (somewhere at or above the glass transition temperature) </li></ul></ul><ul><ul><li>The lower of the above temperatures for mixed systems (although in some cases, it is possible to dry above this) </li></ul></ul><ul><li>We can analyse the critical temperature of a formulation before freeze-drying it, using, for example: </li></ul><ul><ul><li>Freeze-Drying Microscopy (FDM) </li></ul></ul><ul><ul><li>Impedance (Zsin φ ) and Thermal Analysis </li></ul></ul>
    6. 6. Freeze-drying microscopy (FDM) <ul><li>FDM is the study of freeze-drying at the microscopic level </li></ul><ul><li>FDM allows determination of collapse, melting and “qualitative phenomena” such as skin formation </li></ul>
    7. 7. What is a Freeze-Drying Microscope? <ul><li>Effectively a ‘micro freeze-dryer’ where the freeze-drying of a small sample may be observed </li></ul><ul><li>First designs in the mid-60s </li></ul><ul><li>Now manufactured commercially </li></ul><ul><li>Our original Lyostat instrument was developed with Oxford Instruments (1997) </li></ul><ul><li>Lyostat2 was developed jointly with Linkam (2001) </li></ul><ul><li>Now Lyostat3 (also with Linkam) </li></ul><ul><li>Allows for precise temperature control and programmed ramp / hold steps </li></ul><ul><li>Vacuum tight system </li></ul>
    8. 8. Sample Preparation in Lyostat3 FDM Sample holder Side Door Block <ul><li>Sample loading takes about 60 seconds. </li></ul><ul><li>Routine analysis usually takes 20 - 30 minutes, or up to 60 minutes if heat annealing is used. </li></ul>
    9. 9. Sample Format in Lyostat3 FDM Temperature-Controlled Block Light Source (from below) Aperture Quartz cover slip (16 mm dia.) Glass cover slip (13 mm dia.) 2 µl of sample Objective Lens (usually 10 x) Metal Spacer (70 µm thick)
    10. 10. <ul><li>When sample reaches the holding temperature and has been observed to freeze , vacuum pump is switched on and drying begins. </li></ul><ul><li>Sublimation interface can be seen moving through the frozen sample. </li></ul>FDM image 1 Frozen Sample Sublimation Front Dried Sample Temperature / Time Profile Real-time Chart
    11. 11. <ul><li>Increasing or decreasing the temperature of the sample allows you to view its freeze-drying characteristics. </li></ul><ul><li>By examining the freeze-dried structure behind the interface, the collapse temperature of the material can be determined. </li></ul><ul><li>The temperature may be cycled in order to evaluate Tc more closely </li></ul>FDM image 2 Collapsed Material Frozen Sample Sublimation Front
    12. 12. <ul><li>Sample structure lost when collapse temperature was exceeded. </li></ul><ul><li>Structure regained as sample was re-cooled to below its collapse temperature. </li></ul>FDM image 3 Regained Structure Frozen Sample Sublimation Front Collapsed Sample
    13. 13. <ul><li>Sample temperature was again increased to above its collapse temperature, causing the sample to collapse. </li></ul>FDM Image 4 Frozen sample Collapsing again on reheating Sublimation front Dried structure
    14. 14. So, what else can FDM tell us? <ul><li>Eutectic melting temperature </li></ul>
    15. 15. NaCl Below Eutectic Temperature Frozen Dry
    16. 16. NaCl Above Eutectic Temperature Note changes in appearance of frozen structure Eutectic liquid
    17. 17. So, what else can FDM tell us? <ul><li>Eutectic melting temperature </li></ul><ul><li>May give some indication of skin (crust) formation potential of a formulation </li></ul>
    18. 18. Layer of concentrated solute at edge of sample Drying only occurs through ruptures in the skin / crust
    19. 19.
    20. 20. So, what else can FDM tell us? <ul><li>Eutectic melting temperature </li></ul><ul><li>May give some indication of skin (crust) formation potential of a formulation </li></ul><ul><li>Whether heat-annealing may be of benefit </li></ul><ul><ul><li>To increase ice crystal size – and what conditions are required for this (above Tg’?) </li></ul></ul><ul><ul><li>To encourage some components to crystallise </li></ul></ul>
    21. 21. Effect of annealing on ice crystal size 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.
    22. 22. FDM setup with polarised light Polariser Analyser Sample Camera
    23. 23. Effect of annealing on solute behaviour: FDM with basic plane polarised light function Sample quench cooled below -40 ° C No sign of crystals (no light rotation) Drying at -18 ° C Polariser shows presence of crystals (white areas)
    24. 24. Frozen Mannitol Solution, annealed to -5 o C Significant crystallisation, moving in from edge of sample
    25. 25. Crystal Formation at Controlled Temperatures (1) Crop Protection Molecule in MeOH prior to solvent evaporation
    26. 26. Crystal Formation at Controlled Temperatures (2) Same Molecule after MeOH evaporated at 20°C
    27. 27. Crystal Formation at Controlled Temperatures (3) Same Molecule after MeOH evaporated below -80°C
    28. 28. Crystal Formation at Controlled Temperatures (4) Same Molecule upon crystallisation from THF (evaporated at 25°C)
    29. 29. Crystal Formation at Controlled Temperatures (5) Same Molecule upon crystallisation from THF (evaporated slowly at reduced temperature)
    30. 30. Sheep RBC in suspension
    31. 31. Sheep RBC upon initial freezing...
    32. 32. … and 1 minute later…
    33. 33. … 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…
    34. 34. Issues with RBC lyophilisation <ul><li>Freezing damage: </li></ul><ul><ul><li>from ice crystal growth </li></ul></ul><ul><ul><li>from pH gradients </li></ul></ul><ul><ul><li>from freeze-concentration / osmotic effects </li></ul></ul><ul><li>Drying damage: </li></ul><ul><ul><li>Physical action of ice removal on membrane </li></ul></ul><ul><ul><li>Dehydration causing deformation of RBC </li></ul></ul><ul><li>Rehydration damage: </li></ul><ul><ul><li>Concentration and pH effects ( e.g. localised hypotonic / acidic / alkaline regions, causing lysis) </li></ul></ul><ul><ul><li>Wetting issues exacerbating the above effects </li></ul></ul>
    35. 35. Further applications of FDM <ul><li>It is possible to examine differences in relative drying rates: </li></ul><ul><ul><li>For different formulations </li></ul></ul><ul><ul><li>For a specific formulation at different temperatures </li></ul></ul><ul><li>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 </li></ul>
    36. 36. CONCLUSIONS <ul><li>FDM can provide a visual indication of: </li></ul><ul><ul><li>Collapse temperature (T c ) </li></ul></ul><ul><ul><li>Eutectic temperature (T eu ) </li></ul></ul><ul><ul><li>Skin formation potential </li></ul></ul><ul><ul><li>Annealing effects: on ice structure, solute crystallisation, critical temperature </li></ul></ul><ul><ul><li>Relative rates of drying for different formulations, or for the same formulation at different temperatures </li></ul></ul><ul><ul><li>Behaviour of cells and other structures as well as simple solutions during freezing and drying </li></ul></ul><ul><ul><li>All the above information can be useful for formulation & freeze-drying cycle development </li></ul></ul>
    37. 37. Acknowledgements <ul><li>Linkam Scientific Instruments (for development of Lyostat3 FDM): </li></ul><ul><ul><li>Ian Pearce </li></ul></ul><ul><ul><li>Vince Kamp </li></ul></ul><ul><ul><li>Peter Grocutt </li></ul></ul><ul><li>Members of the BTL scientific team: </li></ul><ul><ul><li>Isobel Cook </li></ul></ul><ul><ul><li>Tom Peacock </li></ul></ul><ul><ul><li>Marc Townell </li></ul></ul>
    38. 38. Thank You for your attention! T: 01962 841092 E: [email_address] W: www.btl-solutions.net
    39. 39. Presented during “Emerging Technologies in Freeze Drying”, Cambridge, 11 th May 2011. Event organised by BPS and BTL, www.biopharma.co.uk www.btl-solutions.net

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