A study of vial headspace moisture in an entire freeze-dried batch and the factors affecting moisture content variability

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. 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
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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
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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
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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)
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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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.
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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