A study of vial headspace moisture in
 an entire freeze dried batch and the
  factors affecting moisture content
         ...
Contents
• Freeze drying basics
• Moisture in the freeze dried product
• Frequency Modulated Spectroscopy (FMS)
• FMS and ...
The freeze drying basics
Why? - Freeze drying can increase product shelf life from a few days to months/years.

Freeze dry...
Moisture in the freeze dried product
The moisture content within a freeze dried material has a direct effect on the glass
...
Frequency Modulated Spectroscopy (FMS)
FMS enables the measurement of water and pressure within the headspace of the vial....
FMS and Karl Fischer moisture correlations
Water may be present in a variety of “forms”/locations – free, adsorbed, chemic...
Vial heat transfer in a freeze dryer
  Heat transfer occurs by various means but is not uniform throughout the
  batch.

 ...
Factors affecting moisture variation
                                         Moisture content of samples in a tray
Heat t...
Frozen structure of Mannitol
Annealing involves cooling and re-warming of the frozen structure
• Encourages crystallisatio...
Moisture mapping variations for 2% Mannitol
• Freeze drying cycle involved an annealing step to encourage crystallisation
...
Moisture mapping variations for 2% Mannitol
• Freeze drying cycle involved an annealing step to encourage crystallisation
...
Further investigations
Freeze dried mannitol can exist in several forms:
• Amorphous mannitol
• Crystalline hydrate(s) of ...
FMS ratio variations over time

Direct shelf contact batch
• Higher ratio change
• Random spread
  (highest ratio change
 ...
FMS ratio variations over time

No direct contact (tray)
 batch
• Lower ratio change
• Random spread
  (Higher ratio chang...
Summary
Headspace moisture analysis can be used as a non-destructive method to further our
understanding of the factors in...
For more on FMS, vial headspace
 analysis, or freeze drying product and
         process analysis, visit
        www.btl-s...
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A study of vial headspace moisture

  1. 1. A study of vial headspace moisture in an entire freeze dried batch and the factors affecting moisture content variability Isobel Cook Principal Scientist BTL– specialists in freeze drying research and development www.btl- www.btl-solutions.net
  2. 2. Contents • Freeze drying basics • Moisture in the freeze dried product • Frequency Modulated Spectroscopy (FMS) • FMS and Karl Fischer moisture correlations • Heat transfer within a freeze dryer • Factors affecting moisture variation • Frozen structure of mannitol • Moisture mapping variations for 2% mannitol • Further investigations • FMS ratio variations over time • Summary www.btl- www.btl-solutions.net
  3. 3. The freeze drying basics Why? - Freeze drying can increase product shelf life from a few days to months/years. Freeze drying involves removing the water from the material which involves three stages: • Freezing - freeze the product to below its critical temperature • Primary drying - remove the free ice by sublimation under vacuum • Secondary drying - remove any remaining unfrozen water, the temperature is typically raised above 0°C and the vacuum increased For successful freeze drying: • Choose suitable components Freeze for the formulation dried • Establish the critical temperatures for the product Liquid fill Good cake Collapsed cake www.btl- www.btl-solutions.net
  4. 4. Moisture in the freeze dried product The moisture content within a freeze dried material has a direct effect on the glass transition (Tg) of the material. The Tg is the point at which a material can be observed to undergo structural change, this has a direct effect on the • Long term stability • Storage temperature Establishing moisture content uniformity is an important quality control tool Moisture is commonly measured by Karl Fischer (KF) analysis and is often considered the industry standard • Measures the total water but • Labour intensive • Destroys the sample • Uses toxic reagents www.btl- www.btl-solutions.net
  5. 5. Frequency Modulated Spectroscopy (FMS) FMS enables the measurement of water and pressure within the headspace of the vial. • The laser light is tuned to match the internal water absorption frequency at 1400nm • The amount of laser light absorbed is proportional to the water vapour concentration Analysis time ~ 5 seconds per vial • Non-destructive (monitoring same vial over time) • 100% inspection Stopper Sensor Near infrared laser measures light absorption Vial headspace Freeze dried material FMS-1400 (Lighthouse Instruments) www.btl- www.btl-solutions.net
  6. 6. FMS and Karl Fischer moisture correlations Water may be present in a variety of “forms”/locations – free, adsorbed, chemically bound, hydration shells (e.g. of proteins), water of crystallisation Water can have different association/affinity within the freeze dried material which varies with the formulation Sucrose KF vs FMS Mannitol KF vs FMS 3.50 7.00 y = 1.0274x + 0.796 y = 0.4242x - 0.3634 3.00 R2 = 0.9894 6.00 R2 = 0.939 2.50 5.00 KF % water 2.00 4.00 % KF 1.50 3.00 1.00 2.00 0.50 1.00 0.00 0.00 0.00 0.50 1.00 1.50 2.00 2.50 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 FMS Torr FMS Torr Intercept and gradient vary with the formulation based on intrinsic properties of excipients and active www.btl- www.btl-solutions.net
  7. 7. Vial heat transfer in a freeze dryer Heat transfer occurs by various means but is not uniform throughout the batch. Vapour escapes through gap in stopper Heat transfer by radiation from side walls of the freeze Top layer dries first dryer Heat transfer by Central vials have greater direct conduction shielding from side wall from the shelf to radiation the vial and product Heat transfer by gaseous Freeze dryer shelf convection www.btl- www.btl-solutions.net
  8. 8. Factors affecting moisture variation Moisture content of samples in a tray Heat transfer by • Conduction • Gaseous convection Degree of shelf contact • Tray • Direct shelf contact • Sample container Moisture content of samples with direct shelf contact Radiative heating from freeze dryer • Freeze dryer door • Freeze dryer walls • Extent of shielding Cycle/processing conditions responsible for observed differences www.btl- www.btl-solutions.net
  9. 9. Frozen structure of Mannitol Annealing involves cooling and re-warming of the frozen structure • Encourages crystallisation • Encourages the growth of larger ice crystals (slower cooling larger ice crystals) It is easier for vapour to Annealing escape Energy input required More ordered reduced open structure Structure reduces the impact of heat transfer variation due to shelf contact Gaseous convection not observed as open structure allows for efficient drying Material structure and treatment can have large impact on the observed moisture www.btl- www.btl-solutions.net
  10. 10. Moisture mapping variations for 2% Mannitol • Freeze drying cycle involved an annealing step to encourage crystallisation • Primary drying conducted at -5°C, Vacuum set at 1000 mtorr Mannitol-Headspace Moisture (Direct) Mannitol-Headspace Moisture (Tray) 20 20 18 18 16 16 14 14 Moisture (Torr) Moisture (Torr) 12 12 10 10 8 8 6 6 4 4 2 2 0 0 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 1 12 23 34 45 56 67 78 89 100 111 122 133 144 155 166 177 188 199 210 221 232 243 Vial number Vial number • Direct shelf contact • No direct shelf contact (Tray) • Average torr 5.24 / ~ 2 % KF • Average torr 4.72 / ~ 2 % KF • Standard deviation 2.31 • Std deviation 1.30 Sample sets have a similar moisture content – gaseous convection plays a role! Significant variation within each sample set Tray samples have a lower standard deviation – Related to slower cooling rate www.btl- www.btl-solutions.net
  11. 11. Moisture mapping variations for 2% Mannitol • Freeze drying cycle involved an annealing step to encourage crystallisation • Primary drying at -40°C, Vacuum set at 50 mtorr, shortened secondary drying Mannitol-Headspace Moisture (Direct) Mannitol-Headspace Moisture (Tray) 20 20 18 18 16 16 14 14 Moisture (Torr) Moisture (Torr) 12 12 10 10 8 8 6 6 4 4 2 2 0 0 1 12 23 34 45 56 67 78 89 100 111 122 133 144 155 166 177 188 199 210 221 232 243 1 12 23 34 45 56 67 78 89 100 111 122 133 144 155 166 177 188 199 210 221 232 243 Vial number Vial number • Direct shelf contact • No direct shelf contact (Tray) • Average torr 10.31 • Average torr 9.69 • Standard deviation 2.79 • Standard deviation 2.12 Sample sets have a similar moisture content – gaseous convection not a factor! Higher moisture results for both sets due to shortened secondary drying step Significant variation within each tray www.btl- www.btl-solutions.net
  12. 12. Further investigations Freeze dried mannitol can exist in several forms: • Amorphous mannitol • Crystalline hydrate(s) of mannitol • Anhydrous crystalline mannitol - Alpha (α), beta (β) and delta (δ) Mannitol KF vs FMS 7.00 y = 0.3191x - 0.0673 Further analysis and closer 6.00 R2 = 0.6003 inspection of the KF/FMS 5.00 KF % water 4.00 correlation revealed a 3.00 deviation and lack of 2.00 correlation for some samples 1.00 0.00 0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 FMS (torr) This variation appeared to be related to a change in the mannitol form (observed by comparing FMS data over several days) www.btl- www.btl-solutions.net
  13. 13. FMS ratio variations over time Direct shelf contact batch • Higher ratio change • Random spread (highest ratio change towards front half of tray) Ratio change= Day 3 / Day 1 Green = no / small change Mannitol-Headspace Moisture (Direct) 20 High level of variation observed for 18 16 headspace moisture Moisture (Torr) 14 12 Standard deviation 2.31 10 8 6 4 Indicate headspace moisture variation 2 within a shelf related to the different 0 1 11 21 31 41 51 61 71 81 91 101 111 121 131 141 151 161 171 181 191 201 211 221 231 proportion of mannitol crystalline forms Vial number www.btl- www.btl-solutions.net
  14. 14. FMS ratio variations over time No direct contact (tray) batch • Lower ratio change • Random spread (Higher ratio changes towards tray edge) Ratio change= Day 3 / Day 1 Green = no / small change Mannitol-Headspace Moisture (Tray) 20 18 Lower level of variation observed for 16 headspace moisture 14 Moisture (Torr) 12 Standard deviation 1.30 10 8 6 4 2 Indicate slower heat transfer results in 0 1 12 23 34 45 56 67 78 89 100 111 122 133 144 155 166 177 188 199 210 221 232 243 more controlled crystallisation and a Vial number smaller variation in mannitol form www.btl- www.btl-solutions.net
  15. 15. Summary Headspace moisture analysis can be used as a non-destructive method to further our understanding of the factors involved in obtaining a uniform moisture content Processing factors • Efficiency of heat transfer – conduction and convection, container material • Degree of shelf contact e.g. tray/no tray, container shape • Radiative heating – larger shelves = fewer vials exposed to side wall radiation • Annealed or non-annealed - ice crystal size, pathways for vapour to escape • Cooling and re-warming rates Excipients/active material • Material type/structure e.g. amorphous or crystalline, material complexity It is important to fully understand reasons for moisture variation due to processing and material choices. This understanding can assist in validation and in assessing any changes made. www.btl- www.btl-solutions.net
  16. 16. For more on FMS, vial headspace analysis, or freeze drying product and process analysis, visit www.btl-solutions.net Isobel Cook Principal Scientist BTL- specialists in freeze drying research and development www.btl- www.btl-solutions.net

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