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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: [email protected]: 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!