Polysorbate 80 Hydrolysis and Considerations for Control Strategy Development

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1 Polysorbate 80 Hydrolysis and Considerations for Control Strategy Development

2 Outline 1. Background 2. Considerations for control strategy development Development experience Impact to product quality Drug product testing 3. Conclusions

3 Evolving Understanding of Polysorbate Degradation Pathways Kishore RK, et. al Degradation of polysorbate 20 and 80: Studies on thermal autoxidation and hydrolysis. J Pharm Sci Kishore RK, et. al The degradation of polysorbates 20 and 80 and its potential impact on the stability of biotherapeutics. Pharm Res Labrenz SR Ester hydrolysis of polysorbate 80 in mab drug product: Evidence in support of the hypothesized risk after the observation of visible particulate in mab formulations. J Pharm Sci Cao X, et. al Free fatty acid particles in protein formulations, Part 1: Microspectroscopic identification. J Pharm Sci Siska CC, et. al Free fatty acid particles in protein formulations, Part 2: Contribution of polysorbate raw material. J Pharm Sci Doshi N, et. al Understanding particle formation: Solubility of free fatty acids as polysorbate 20 degradation byproducts in therapeutic monoclonal antibody formulations. Mol Pharm Tomlinson A, et al Polysorbate 20 degradation in biopharmaceutical formulations: Quantification of free fatty acids, characterization of particulates, and insights into the degradation mechanism. Mol Pharm Dixit N, et. al Residual host cell protein promotes polysorbate 20 degradation in a sulfatase drug product leading to free fatty acid particles. J Pharm Sci Hall T. et al Polysorbate 20 and 80 degradation by group XV lysosomal phospholipase A 2 isomer X1 in monoclonal antibody formulations. J Pharm Sci

4 Acknowledgements Analytical Andy Carr Michael DeFelippis Melody Gossage Lihua Huang Rick Meyer Dawn Norris Carissa Susalla Tewelde Tesfai William Weiss Shou Song Zhang Formulation Shermeen Abbas Patrick Donovan Jose Flores Nagarajan Thyagarajapuram NMR Spectroscopy Scott Bradley Lisa Zollars Purification Bill Holmes Brian Hotovec Statistics Eric Adamec Surface Science Group Xia Dong Craig Kemp

5 Background Polysorbate 80 heterogeneity as a raw material Monoclonal antibody > 50 mg/ml Changing API storage from frozen in polycarbonate containers to refrigerated in LDPE bags PS80 Analytical Method Sorbitan headgroup MW = 1310 Oleic Acid PS80 lacks a chromophore for standard HPLC-UV detection High protein concentrations causes chromatographic interference and/or incompatibility with analysis of PS80 Selected the hydrolysis method for detection of oleic acid relative to PS80

6 Polysorbate 80 Content Results for API Stored in PC Bottles and LDPE Bags Temperature Dependent Loss of PS80 (oleic acid) in LDPE Bags

7 Container Material of Construction Stability Study 25ºC Polysorbate 80 loss dependent on temperature and container material of construction

8 NMR Spectra of the Solutions Sorbitan Headgroup Signal Signal for the CH 2 s in oleic acid PS80 Standard PC Bottle (no loss) LDPE Bag (80% loss) PETG Bottle (no loss) HDPE Bottle (75% loss) NMR data suggests a loss of the oleic acid portion, and not a decrease in the sorbitan headgroup

9 ToF-SIMS of Bag Interior Surfaces BN TDC - Ions 400µm cts LA426 unuse 15 Negative Ion Spectra Counts Unused Bag Mass [m/z] m/z 281 Free oleic acid anion Counts BN TDC - Ions 400µm cts LA426 placebo bag Bag Exposed to Placebo normal PS80 assay Mass [m/z] ToF-SIMS indicates oleic acid sorption to the surface of the bag. Counts BN TDC - Ions 400µm cts Lot A 281 Bag Exposed to Product low PS80 assay Mass [m/z]

10 Further Evidence that Material of Construction Influences Hydrolysis Monoester Diester Triester Polysorbate 80 control Prefilled syringe 9 months 5C Prefilled syringe 9 months 25C LDPE bag with polysorbate 80 depleted PS80 Monoester depleted with higher substituted PS80 remaining in prefilled syringe but depleted in LDPE based on LC/MS (Tetraester not shown)

11 Hydrolysis Assay Oleic Acid Sorbitan Headgroup Can we differentiate between oleic acid free in solution prior to sample preparation and the oleic acid freed during the base hydrolysis? Internal standard: Cis-10-Nonadecenoic acid

12 Oleic Acid Internal Standard Hydrolysis Assay (with & without base hydrolysis sample preparation) PC without hydrolysis PC with hydrolysis LDPE without hydrolysis LDPE with hydrolysis Cleavage of PS80 occurs in all containers Sorption of oleic acid is dependent on container material of construction

13 Influence of Container Material of Construction on Hydrolysis PC Bottle vs LDPE Bag Based on TOA Glass Syringe Based on TOA and FOA Three Lots of Drug Product at 5 and 25C TOA: Total Oleic Acid (after sample hydrolysis) FOA: Free Oleic Acid (NO sample hydrolysis) Containers that reduce oleic acid by sorption accelerate the rate and extent of PS80 loss Surface area to volume ratio influences sorption (stability simulators)

14 Impact of Polysorbate 80 Hydrolysis on Product Quality Analytical Control Strategy: In this case Hydrolysis Method Three Lots of Drug Product Syringes at 5 & 25C TOA FOA = Polysorbate 80 Level

15 Impact of Polysorbate 80 Hydrolysis on Product Quality (continued) Determine the Minimum Effective Polysorbate 80 Level Agitation Study Assessing PS80 Level Three Lots of Syringes at 5 & 25C Studies that contribute to setting minimum effective level: Freeze thaw, agitation, shipping, stability (include aged material) Include a safety margin to address product and method variability

16 Impact of Polysorbate 80 Hydrolysis on Product Quality (continued) Product Quality: Three Lots of Drug Product Syringes at 5 & 25C Studies that contribute to justifying product quality: Freeze thaw, agitation, shipping, stability

17 Impact of Polysorbate 80 Hydrolysis on Product Quality (continued) 3. Product Quality: Three Lots of Drug Product Syringes at 5 & 25C Studies that contribute to justifying product quality: Freeze thaw, agitation, shipping, stability

18 Drug Product Testing Timing of assay versus degradation rate Minimum Effective PS80 Level Potential timing of importation testing

19 Drug Product Testing (continued) Batch Number Polysorbate 80, % Modeling of data indicates that at least 0.020% PS80 is needed at release to ensure PS80 remains above lower effective level A B C D E F G H I J K L M N O Adjusted TImepoint, Month

20 Conclusions 1. The reduction of PS80 resulted from hydrolysis beyond the rate expected from hydrolytic reactions 2. Container material of construction can influence the rate and extent of hydrolysis Selection of stability simulator Drug substance storage at -70C essentially elements hydrolysis 3. The PS80 remaining in the drug product maintained product quality over the shelf-life 4. Considerations for developing the drug product testing strategy Rate of degradation In-process assay versus release End of shelf life relative to minimum level of PS80