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Characterization and Quantification of Lyophilized Product Appearance

and Structure

Dr Kevin R. Ward B.Sc. Ph.D. MRSCDirector of Research & DevelopmentBiopharma Technology Ltd.Winchester SO23 0LD, UKTel: +44 (0)1962 841092E-mail: kward@biopharma.co.ukWeb: www.btl-solutions.net

ISL-FD Conference, Bologna, Italy, March 2012

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Synopsis of Presentation

• Subjectivity of product appearance assessment– Macroscopic features of lyophiles– Descriptors that might be used– Microscopic features

• What other features related to appearance and structure can be quantified?– Porosity– Specific surface area– Density variations within a cake– Stress / strain testing for mechanical properties

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Acknowledgements

Dr Daryl WilliamsSharmila Devi

My team at BTL:

• Isobel Cook• Tom Peacock• Mervyn Middleton• Nick White

for sponsoring Imperial College studentship

Dr Andrew InghamEdmond Ekenlebie

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Lyophile Characterization

• Appearance• Residual moisture (KF, LoD, NIR, FMS…)• Container seal integrity (pressure)• Oxygen levels in vials / ampoules• Molecular integrity assays• Activity and Stability assays• Crystal / polymorphic forms / hydrates• Thermal properties – Tg, relaxation times• Reconstitution properties

Appearance seems to be the only parameter that is not usually quantified!

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Product Appearance

• Difficult to quantify by eyesight alone!• We can tell total collapse and partial collapse

from something that is not collapsed…

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Product Appearance

• Or a product that has undergone severe eutectic melting…

• Or a friable one that became powdery…

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Product Appearance• Or where there almost seem to be several

different products in one vial…!

Disc on top (due to crust formation or early collapse)

Powder underneath

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Product Appearance

• But of the ones that appear to look “good”, there are still differences…

Uniform; adhered to vial Uniform; not adhered to vial Does the peak matter?!

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Some Features / Adjectives

Texture: rough, smooth, porous, “grainy”…

Surface features (peak, sheen, skin, crust…) Adhesion to vial

Cake height (vs. fill height)

Shape

Cohesive or powdery?

Colour (or shade of white?!)

Uniformity (homogeneous / heterogeneous?)

Collapsed or melted

…but what about the microscopic differences we can’t see?

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Electron microscopySEM, TEM• Can be used to look at porosity, microstructure,

possible microcollapse

But remember, the image may depend on sampling technique:• Is the sample representative of the whole cake?• Has the sampling process changed it in any way?

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SEM analysis and annealingFor mannitol + sucrose mixture

Annealed sample:

Mannitol crystallised and/or larger ice crystals, giving ‘rougher’ structure

Non-annealed sample:

Mannitol amorphous and/or ice crystals smaller, giving ‘smoother’structure

However, this method is still not quantitative!

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Methods of quantifyingappearance and structure

• Gas adsorption methods– Specific surface area– Mean pore diameter

• Micro-CT scanning– Porosity– Heterogeneity

• Stress / strain testing– Mechanical properties

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Surface Area & Porosityby gas adsorption methods

• N2 sorption based on Brunauer-Emmett-Teller (BET) and Barrett-Joyner-Halenda (BJH) / Kelvin equations

• BET and BJH equations allow determination of specific surface area and pore size distribution

• We carried out a simple study for mannitol:– Nitrogen adsorption and desorption isotherms were

measured at -195.8°C, using an ASAP Tristar 3000 (Micromeritics Instrument Corporation, USA) volumetric adsorption system

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• Similar isotherms but hysteresis is wider for the LN2 quench‐cooled mannitol 

• This indicates that the energy needed for evaporation from the pores is distinctly different from the energy associated with condensation within it

• This in turn implies that desorption (evaporation) is inhibited due to constriction, thereby suggesting pores are smaller

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BET adsorption analysisFor surface area of cake

Spe

cific

sur

face

are

a, m

2 /gra

m

0

1

2

3

4

5

6

7

8

Slow cool Fast cool LN2 cool

SA ads

SA des

SA BET

Data for mannitol lyophilized using different initial cooling rates (BTL / ICL)

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BJH adsorption analysisFor mean pore diameter

0

100

200

300

400

500

600

Slow cool Fast cool LN2 cool

ads  pore diam

des  pore diam

Mea

n po

re d

iam

eter

, Ang

stro

ms

Data for mannitol lyophilized using different initial cooling rates (BTL / ICL)

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Structural assessment:Micro-CT scanning (tomography)

Dr Andrew Parker (Molecular Profiles) gave an excellent presentation on this technique at Visiongain’s 2010 Lyo conference in London

Micro-CT scanning enables a 3D picture to be built up of a freeze-dried product, showing pore structure and cake uniformity

Ref: “Determination of the influence of primary drying rates on the microscale structural attributes and physicochemical properties of protein containing lyophilized products”, A. Parker et al, J. Pharm. Sci.(published online 28/6/10). DOI: 10.1002/jps22185

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Three-dimensional cross-sectionalX-ray CT into packed powders

Scale bar: 1.25 mmFreeze-dried mannitol powder Fluid bed dried mannitol powder

Images courtesy of Dr Andrew Ingham & Edmond Ekenlebie, Aston University, UK.

Taken from their publication “Short Cycle Times for Cost-Efficient Processing in Lyophilized Formulations”, American Pharmaceutical Review (2011)

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Mechanical Properties:Stress / strain measurement

• In collaboration with Imperial College London, we are currently developing a miniature load cell to measure stress and strain in a lyophilized cake while it is still in the vial

• Measuring the stress (σ) and strain (ε) can also give us elasticity, Young’s modulus (E):

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• Stable Microsystems Texture analyser and Lloyds EZ50.• Loaded at speed of 1mm/sec, tested at 0.01mm/sec to a 

depth of 3mm (start at 1g force)• Samples tested in the vial• Samples:  Fast, slow and LN2 quench cooled mannitol 

Stress / Strain Testing

AIM:To compare the strength of the cakes

•Compression testing 

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Data for mannitol

E modulus (N/m2) Mean

Fast Cooled60863

45428.529994

LN2 Quench Cooled12213

86925171

Slow Cooled 3303

23281353

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• Fast cooled mannitol strongest, slow cooled mannitol has lowest strength • Could be due to morphology or fundamental material properties• Morphology : 

• Slow cooled has larger pores therefore the support may not be strong• From the SEM pictures, small holes are visible in the LN2‐ and slow‐ cooled 

samples; holes are the weakest points for the cakes to crack/break easily• Material property: Strength/physical stability of the polymorphic form but not clear

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Summary

• Subjectivity of product appearance assessment– Macroscopic features of lyophiles– Descriptors that might be used– Microscopic features

• Other features related to appearance and structure that can be quantified– Porosity– Specific surface area– Density variations within a cake– Stress / strain testing for mechanical properties

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Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK

Tel: +44 (0)1962 841092 Web: www.btl-solutions.net

Thank You for your attention!