(T1230-07-37) Using Enzymes to Accelerate PS80 Degradation and Elucidate Mechanistic Impacts of PS80 Degradation on Biologics Drug Product Quality

Purpose: Polysorbate 80 (PS80) is commonly added to biologics formulations for protection against agitation stresses. However, PS80 can degrade through various pathways that result in different combinations of degradants that impact the stability of the biologic. Recent studies indicate an impact of individual PS80 degradants on protein stability, but the combined effects of all the degradants that result from a particular PS80 degradation pathway is not well understood. This work demonstrates a method to enable mechanistic understanding on the correlation between specific PS80 degradation patterns on biologic product stability, specifically focusing on enzymatic PS80 hydrolysis.

Methods: 32 different esterases, lipases, and proteases immobilized on beads were screened for PS80 hydrolysis activity by incubating the enzymes with 2% PS80 in formulation buffer for 24 h at 30 °C on a 200 RPM shaker. PS80 degradation profiles were determined by using high performance liquid chromatography with a charged aerosol detector. Three enzymes were selected for unique degradation profiles. The enzymes were removed from solution via membrane filtration and the resulting hydrolyzed PS80 was used to formulate MAb1 samples. These MAb1 samples were stressed by agitation for up to 14 days and thermally stressed at 40 °C for up to 4 weeks and analyzed for subvisible particles using backgrounded membrane imaging, fragmentation and aggregation using size exclusion chromatography (SEC) and charged variants using ion exchange chromatography (IEX).

Results: PS80 degraded by Candida Antarctica lipase B (CALB), Rhizomucor miehei lipase (RML), and Bacillus subtilis protease (BSP) were selected to formulate MAb1 samples as they exhibited distinguishable PS80 degradation profiles (Table 1). To accelerate MAb1 degradation induced by the resulting PS80 degradants, MAb1 samples were either agitated at room temperature for up to 14 days or thermally stressed at 40 °C for up to 4 weeks. Of the samples containing neat or degraded PS80, CALB-degraded PS80 induced the highest level of particulate counts after both agitation and thermal stress (Fig. 1). However, it did not result in any noticeable deviations in MAb1 degradation by UPSEC and IEX compared to the control sample. During incubation at 40 °C, RML-degraded PS80 induced an appreciable increase in subvisible particulates compared to the control sample with intact PS80. However, this was not the case during agitation at room temperature, when the samples with RML-degraded PS80 and intact PS80 exhibited comparable numbers of subvisible particles. Lastly, BSP-degraded PS80 did not yield an increase in subvisible particles during agitation or thermal stress, but instead resulted in different MAb1 degradation profiles. Despite exhibiting similar fractions of monomer and main peaks as all other samples, BSP-degraded PS80 induced increased levels of fragmentation and basic variants and lower levels of aggregation (Fig. 2).

Conclusion: These results indicate that different PS80 degradation profiles can have with unique mechanistic impacts on protein stability. Overall, this work demonstrates a method to investigate the impacts of combinations of PS80 enzymatic degradants on the stability of a biologic. This tool will enable a better understanding of the relationship between PS80 degradation and biologics stability, and more robust therapeutic products.

References: Larson, Nicholas R., et al. "Comparison of polysorbate 80 hydrolysis and oxidation on the aggregation of a monoclonal antibody." Journal of Pharmaceutical Sciences 109.1 (2020): 633-639.

Zhang, Jingtao, Jiayi He, and Katelyn J. Smith. "Fatty Acids Can Induce the Formation of Proteinaceous Particles in Monoclonal Antibody Formulations." Journal of Pharmaceutical Sciences 111.3 (2022): 655-662.

Table 1: Summary of Enzymatic PS80 hydrolysis patterns after 24 h



Figure 1: Of the samples containing intact or degraded PS80, CALB-degraded PS80 induced the highest levels of subvisible particles under thermal stress.



Figure 2: BSP-degraded PS80 leads to higher levels of MAb1 fragmentation compared to all other samples evaluated.