Freeze-drying microscopy (FDM) allows observation of materials during freeze-drying at the microscopic level. FDM can determine critical parameters like collapse temperature and eutectic temperature prior to lyophilization. It does this by observing changes in sample structure as the temperature is varied, such as when the sample collapses or melts. FDM provides visual information on various freeze-drying phenomena that can be useful for formulation and process development.
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Use Of Freeze Drying Microscopy To Determine Critical Parameters
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
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”
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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)
16. NaCl Above Eutectic Temperature Note changes in appearance of frozen structure Eutectic liquid
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18. Layer of concentrated solute at edge of sample Drying only occurs through ruptures in the skin / crust
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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. FDM setup with polarised light Polariser Analyser Sample Camera
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. Frozen Mannitol Solution, annealed to -5 o C Significant crystallisation, moving in from edge of sample
25. Crystal Formation at Controlled Temperatures (1) Crop Protection Molecule in MeOH prior to solvent evaporation
26. Crystal Formation at Controlled Temperatures (2) Same Molecule after MeOH evaporated at 20°C
27. Crystal Formation at Controlled Temperatures (3) Same Molecule after MeOH evaporated below -80°C
28. Crystal Formation at Controlled Temperatures (4) Same Molecule upon crystallisation from THF (evaporated at 25°C)
29. Crystal Formation at Controlled Temperatures (5) Same Molecule upon crystallisation from THF (evaporated slowly at reduced temperature)
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…
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38. Thank You for your attention! T: 01962 841092 E: [email_address] W: www.btl-solutions.net
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
![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](https://image.slidesharecdn.com/useoffreezedryingmicroscopytodeterminecriticalparameters-13061449661042-phpapp01-110523052207-phpapp01/85/Use-Of-Freeze-Drying-Microscopy-To-Determine-Critical-Parameters-1-320.jpg)
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