Applications Of Headspace Moisture Analysis

Applications of headspace moisture

analysis for investigating the water

dynamics within a sealed vial

containing freeze dried material

Isobel Cook BSc MRSC

Principal Scientist

Biopharma Technology Limited – specialists in freeze drying

research and development

www.btl-solutions.net

Contents

• Importance of moisture

• Factors contributing to moisture variation

• Introduction to headspace moisture analysis

• What headspace moisture can tell us

• Moisture mapping

• Summary

Importance of residual moisture

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

Incorrect drying /moisture too high

Factors contributing to

variability in moisture content

• Container Geometry (tray, vial, stopper…)

• Differences in container-shelf contact (e.g. warped of trays, vial base)

• The nature of the formulation itself

• Drying rate variations due to product temperature differences

– Shelf temperature variation (ramp, hold),

– Radiative heating effects

– „Flat spots‟ in chamber pressure (e.g. insufficient shelf spacing)

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

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

Frequency Modulation

Spectroscopy (FMS)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

Analysis time ~ 5 seconds per vial

• Non-destructive (monitor same vial over time)

• 100% inspection

Stopper water in the headspace

Headspace moisture (HSM) analysis allowed a comparison of stopper

treatments and storage conditions and the effect this had on HSM levels

Empty stoppered vial with no freeze dried material were analysed

Data indicates that the stopper/headspace moisture equilibrium is highly

sensitive to temperature

Lower HSM at lower

temperature

Higher HSM at higher

temperature

Stopper water in the headspace

Stoppers were dried (3hr at 105°C) to minimise residual moisture in the

headspace and compared with un-dried stoppers.

Dried stoppers revealed an average of 4.25 Torr higher partial pressure of

moisture within the headspace

This demonstrates the increased level of moisture ingress from the stopper

when stoppers are not oven dried prior to freeze drying

0

1

2

3

4

5

6

7

Dried stopper (n=15) Un-dried stopper (n=15)

Stopper water in the headspace

Moisture ingress will also occur directly after sealing and removal from

the freeze drier

This moisture increase is predominantly due to the desorption of

residual water vapour from the stopper

PEG (10kMW)

Excipient effect on HSMIncrease in the HSM is evident by FMS for excipients in vials with stoppers

that were not oven dried compared to those that were oven dried

Amorphous sucrose and crystalline mannitol in the vials display different

dynamics of interaction with water, resulting in a marked difference in the

observed increase in headspace moisture

Significance of stopper moisture!

A difference of 4.25 headspace moisture between the dried and un-dried

stoppers results in 64.8µg/0.0648mg of extra water available to the cake

A solution with 4% solids and a 2 ml fill , gives a theoretical cake weight

of 80mg, the extra headspace moisture from stopper is 0.08%

A solution with 2% solids and a 1 ml fill , gives a theoretical cake weight

of 20mg, the extra headspace moisture from stopper 0.3%

Long term monitoring

Monitoring the same selection of vials over time may allow us to

observe if any changes occur, signifying a change in product form

Mannitol is crystalline and

relatively non-hydroscopic

Pressure moisture ratios

indicate that headspace

moisture stays constant.

The freeze drying cycle for mannitol produced a stable crystalline form of

mannitol which has little interaction with the headspace moisture

Long term monitoring

The degree and speed of the observed change is related to excipient

effect, its affinity for water and time taken to reach equilibrium

Sucrose is amorphous and

relatively hydroscopic

Pressure moisture (P/M)

ratios indicate that HSM

takes 2 weeks to level off

Sucrose takes water into the headspace, therefore initially the P/M ratio is

very high. The P/M ratio is higher than mannitol as more is absorbed in

the cake

FMS / KF moisture correlation

Water 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 hydroscopicity 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

0

0.5

1

1.5

2

2.5

3

3.5

1

12

23

34

45

56

67

78

89

100

111

122

133

144

155

166

177

188

199

210

221

232

Vial number

Mo

istu

re (

To

rr)

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

Shelf

front

Moisture mapping for Sucrose

Sucrose-Headspace Moisture

0

0.5

1

1.5

2

2.5

3

3.5

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

Mo

istu

re (

To

rr)

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

Factors affecting moisture variation

Heat 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

MannitolPrimary 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 set

Tray 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)

0

2

4

6

8

10

12

14

16

18

20

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

Mo

istu

re (

To

rr)

Mannitol-Headspace Moisture (Tray)

0

2

4

6

8

10

12

14

16

18

20

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

Mo

istu

re (

To

rr)

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

1.00

2.00

3.00

4.00

5.00

6.00

7.00

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00

FMS (torr)

KF

% w

ate

r

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

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 Cook

Principal Scientist BSc MRSC

Biopharma Technology Limited – specialists in freeze drying

research and development

www.btl-solutions.net