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

Transcription

1 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.

2 Vials of freeze-dried product Poor Good OK Poor The product in the Poor vials has become soft and dense during freeze-drying, because it has become warmer than its Critical Temperature! 2

3 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 (Zsinφ) and Thermal Analysis 3

4 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 4

5 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 5

6 FDM set-up with LN2 cooling system BTL Lyostat2 Liquid nitrogen pump Freeze-Drying Stage Camera Compound Microscope Temperature controller To vacuum pump Vacuum gauge Liquid nitrogen Dewar 6

7 Sample Preparation for FDM Sample loading takes about 60 seconds. Routine analysis usually takes minutes, or up to 60 minutes if heat annealing is used. Block Sample holder Side Door 7

8 Sample Format in Lyostat2 Objective Lens (usually 10 x) Glass cover slip (13 mm dia.) Metal Spacer (70µm thick) 2µl of sample Aperture Quartz cover slip (16 mm dia.) Temperature-Controlled Block Light Source (from below) 8

9 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!) 9

10 Sublimation front Temp/time table INITIAL FDM IMAGE When sample reaches the holding temperature and has been observed to freeze, vacuum pump is switched on and drying begins. Dried sample Frozen sample On-line plot Sublimation interface can be seen moving through the frozen sample. 10

11 INTERPRETATION OF EVENTS Collapsed material Increasing or decreasing the temperature of the sample allows you to view its freeze-drying characteristics. By examining the freezedried structure behind the interface, the collapse temperature of the material can be determined. Sublimation front Frozen sample The temperature may be cycled in order to evaluate Tc more closely 11

12 INTERPRETATION OF EVENTS Regained structure Frozen sample Sample structure lost when collapse temperature was exceeded. Structure regained as sample was re-cooled to below its collapse temperature. Collapsed sample Sublimation front 12

13 INTERPRETATION OF EVENTS Sublimation front Frozen sample 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 13

14 Micro-collapse (see e.g. Wang, 2004) Macroscopically similar but is it: Wetter? Less stable? More difficult to reconstitute? Below Tc of amorphous phase Above Tc of amorphous phase 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) 14

15 FDM of 2% Mannitol + 1% Glucose Frozen material (Drying front) Regions of (micro) collapse. Just glucose? Regions with good dried structure. Just mannitol? -41 o C, around Tc for glucose. Micro-collapse or just poor solute mixing? 15

16 So, what else can FDM tell us? Eutectic melting temperature 16

17 NaCl Below Eutectic Temperature Dry Frozen 17

18 NaCl Above Eutectic Temperature Note changes in appearance of frozen structure Eutectic liquid 18

19 So, what else can FDM tell us? Eutectic melting temperature May give some indication of skin (crust) formation potential of a formulation 19

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22 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 22

23 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. 23

24 FDM setup with polarised light Camera Analyser Sample Polariser 24

25 Effect of annealing on solute behaviour: FDM with polarised light function KCl solution quench cooled below -40 C No sign of crystals (no light rotation) Drying at -18 C Polariser shows presence of crystals (white areas) 25

26 Effect of annealing on solute behaviour: FDM with polarised light function Start of Eutectic Melt at -11 C Eutectic Melt complete but ice still present. Crystals were indeed 26 due to eutectic KCl

27 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,

28 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 28

29 Mannitol solution at 20 o C (liquid) 29

30 Mannitol solution cooled to -30 o C Amorphous solid plus small layer of excluded liquid at edge 30

31 Warming to -25 o C (note changes in appearance already) 31

32 Annealing to -5 o C Significant crystallisation, moving in from edge of sample 32

33 CONCLUSIONS FDM can provide a visual indication of: Collapse temperature (T c ) Eutectic temperature (T eu ) 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 33