Moisture mapping using headspace moisture analysis

Isobel Cook BSc MRSCPrincipal ScientistBiopharma Technology Limited – specialists in freeze drying research and development

www.btl-solutions.net

Importance of residual moistureThe 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

Incorrect drying /moisture too high

Moisture in the freeze dried product

Establishing moisture content uniformity is an important quality

control tool

Moisture is commonly measured by Karl Fischer (KF), other methods

include, TGA, NIR, though KF analysis is often considered the industry

standard

• Measure the total water!

• Labour intensive

• Destroy the sample

• Toxic reagents

Sampling product for moisture analysis

• Sample from different positions on shelf….and from different shelves

• Useful to sample vials with different dried product appearance (esp. for R&D)

• Obviously any method that could allow 100%inspection has many advantages

Frequency Modulation Spectroscopy (FMS)

FMS-1400 (Lighthouse Instruments)

Measures water and pressure within the headspace of the vial

• The laser light tuned to match internal water absorption frequency

• Laser light absorbed is proportional to the water vapour concentration

Mahajan et al, J. Pharmaceutical Technology, Data and Review, 83, (2005)

FMS / KF moisture correlationWater can have different association/affinity within the freeze dried material which varies with the formulation

For amorphous sucrose the intercept is at 1% w/w by KF

Indicate moisture retained in cake when none observed in headspace.

This is not unsurprising given the hygroscopicity of sucrose

Moisture mapping for Sucrose

Direct shelf contact

• Uniform headspace moisture (HSM)• Lowest 0.2 torr / 1% water KF• Highest 1.0 torr / 1.8% water KF• Average 0.49 torr / 1.3% KF• Standard deviation 0.15

Sucrose-Headspace Moisture

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• Primary drying conducted at -40°C, Vacuum set at 50 mtorr

Shelf front

Moisture mapping for Sucrose

Sucrose-Headspace Moisture

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No direct shelf contact (tray)

• High variation in HSM• Lowest 0.4 torr / 1.2% water KF• Highest 3.2 torr / 4.0% water KF• Average 1.05 torr / 1.9% KF• Standard deviation 0.49

• Primary drying conducted at -40°C, Vacuum set at 50 mtorr

Shelf front

Vial heat transfer in a freeze dryer

Vapour escapes through gap in stopper

Top layer dries first

Freeze dryer shelf

Heat transfer by direct conduction from the shelf to the vial and product Heat transfer by gaseous

convection

Heat transfer by radiation from side Walls of the freeze dryer

Central vials have greater shielding from side wall radiation

This observation can be explained by radiation and shielding effects Edge vial effects bigger at lower temperatures

Factors affecting moisture variationHeat transfer by• Conduction• Gaseous convection

Degree of shelf contact• Tray• Direct shelf contact• Sample container

Radiative heating• Freeze dryer door• Freeze dryer walls• Extent of shielding

• Cycle/processing conditions responsible for observed differences

Samples in a tray

Direct shelf contact

FMS and Karl Fischer moisture correlations

Water may be present in a variety of “forms”/locations – free, adsorbed, chemically bound, hydration shells (e.g. proteins), water of crystallisation

Intercept and gradient vary with the formulation based on intrinsic properties of excipients and active

Moisture mapping variations for Mannitol

Primary drying conducted at -5°C after annealing, vacuum set at 1000 mTorr

• Direct shelf contact• Average Torr 5.24 / ~ 2 % KF• Standard deviation 2.31

• No direct shelf contact (Tray)• Average Torr 4.72 / ~ 2 % KF• Std deviation 1.30

Significant variation within each sample setTray samples have a lower standard deviation – slower cooling rate!Similar moisture content – gaseous convection plays a role!

Moisture mapping variations for Mannitol

Mannitol-Headspace Moisture (Direct)

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Mannitol-Headspace Moisture (Tray)

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Primary drying at -40°C after annealing, vacuum set at 50 mTorr, shortened secondary drying

• Direct shelf contact• Average Torr 10.31 ~ 5%KF• Standard deviation 2.79

• No direct shelf contact (Tray)• Average Torr 9.69 ~5%KF• Standard deviation 2.12

Sample sets have similar moisture content – gaseous convection not a factor

Significant variation within each tray

Mannitol – Frozen structure• Annealing – involves cooling and re-warming of the frozen structure• Encourages crystallisation and growth of larger ice crystals

(slower cooling larger ice crystals)

Structure reduces 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 moisture

Mannitol – further investigationsFreeze dried mannitol can exist in several forms

• Amorphous mannitol • Crystalline hydrate(s) of mannitol • Anhydrous crystalline mannitol - Alpha (α), beta (β) and delta (δ)

Further analysis and closer inspection of the KF/FMS correlation revealed a deviation and lack of correlation for some samples

This variation appeared to be related to a change in the mannitol form (observed by comparing FMS data over several days)

Mannitol KF vs FMS

y = 0.3191x - 0.0673

R2 = 0.6003

0.00

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FMS (torr)

KF

% w

ater

Mannitol – FMS ratio variations

• Direct shelf contact• Higher ratio change• Random spread

(highest ratio change

towards front half of

tray)

• High level of variation observed• Standard deviation 2.31

• Indicate headspace moisture variation within a shelf could be related to the different proportion

of mannitol crystalline forms

Ratio change= Day 3 / Day 1

Mannitol – FMS ratio variations• No direct contact• Lower ratio change• Random spread

(Higher ratio changes

towards tray edge)

• Lower level of variation observed• Standard deviation 1.30

• Indicate slower heat transfer results in more controlled crystallisation / smaller variation in mannitol form

Ratio change= Day 3 / Day 1

Factors affecting moisture variation

Excipients/active material• Excipients play a critical role in dynamics of water exchange

Processing factors

• Heat transfer efficiency– conduction, convection, container

• Degree of shelf contact e.g. tray/no tray, container shape

• Radiative heating – larger shelves = fewer vials exposed

• Annealed or non-annealed - ice crystal size, pathways for vapour to escape

• Cooling and re-warming rates

• Assess stopper type and treatment (autoclave, sterilisation, drying times)

FMS summary

• Important to fully understand what the moisture result is telling you

• Headspace moisture analysis is a non-destructive method allowing long term monitoring

• Understand moisture variation due to

processing and material choices.

• 100% inspection can assist in

validation and scale up/transfer

Thank you

Any questions?

Isobel CookPrincipal Scientist BSc MRSCBiopharma Technology Limited – specialists in freeze drying research and development

www.btl-solutions.net

Presented during “Emerging Technologies in Freeze Drying”, Cambridge, 11th May 2011. Event

organised by BPS and BTL, www.biopharma.co.uk

www.btl-solutions.net