Characterization and Quantification of Lyophilized Product Appearance and Structure

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1 Characterization and Quantification of Lyophilized Product Appearance and Structure Dr Kevin R. Ward B.Sc. Ph.D. MRSC Director of Research & Development Biopharma Technology Ltd. Winchester SO23 0LD, UK Tel: +44 (0) Web: ISL-FD Conference, Bologna, Italy, March

2 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 2

3 Acknowledgements My team at BTL: Dr Daryl Williams Sharmila Devi Isobel Cook Tom Peacock Mervyn Middleton Nick White for sponsoring Imperial College studentship Dr Andrew Ingham Edmond Ekenlebie 3

4 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! 4

5 Product Appearance Difficult to quantify by eyesight alone! We can tell total collapse and partial collapse from something that is not collapsed 5

6 Product Appearance Or a product that has undergone severe eutectic melting Or a friable one that became powdery 6

7 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 7

8 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?! 8

9 Some Features / Adjectives Colour (or shade of white?!) Shape Texture: rough, smooth, porous, grainy Surface features (peak, sheen, skin, crust ) Adhesion to vial Uniformity (homogeneous / heterogeneous?) Cohesive or powdery? Cake height (vs. fill height) Collapsed or melted but what about the microscopic differences we can t see? 9

10 Electron microscopy SEM, 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? 10

11 SEM analysis and annealing For mannitol + sucrose mixture Annealed sample: Non-annealed sample: Mannitol crystallised and/or larger ice crystals, giving rougher structure Mannitol amorphous and/or ice crystals smaller, giving smoother structure 11 However, this method is still not quantitative!

12 Methods of quantifying appearance and structure Gas adsorption methods Specific surface area Mean pore diameter Micro-CT scanning Porosity Heterogeneity Stress / strain testing Mechanical properties 12

13 Surface Area & Porosity by gas adsorption methods N 2 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 C, using an ASAP Tristar 3000 (Micromeritics Instrument Corporation, USA) volumetric adsorption system 13

14 Similar isotherms but hysteresis is wider for the LN 2 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 14

15 BET adsorption analysis For surface area of cake 8 Specific surface area, m 2 /gram Slow cool Fast cool LN2 cool SA ads SA des SA BET Data for mannitol lyophilized using different initial cooling rates (BTL / ICL) 15

16 BJH adsorption analysis For mean pore diameter Mean pore diameter, Angstroms Slow cool Fast cool LN2 cool ads pore diam des pore diam Data for mannitol lyophilized using different initial cooling rates (BTL / ICL) 16

17 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: /jps

18 Three-dimensional cross-sectional X-ray CT into packed powders Freeze-dried mannitol powder Fluid bed dried mannitol powder Scale bar: 1.25 mm 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) 18

19 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): 19

20 Stress / Strain Testing AIM: To compare the strength of the cakes Compression testing 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 LN 2 quench cooled mannitol 20

21 Data for mannitol Fast Cooled LN2 Quench Cooled Slow Cooled E modulus (N/m2) Mean

22 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 22

23 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 23

24 Thank You for your attention! Biopharma House, Winnall Valley Road, Winchester SO23 0LD, UK Tel: +44 (0) Web: 24

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