Modulation of the high concentration viscosity of IgG1 antibodies using clinically validated Fc mutations.

The self-association of therapeutic antibodies can result in elevated viscosity and create problems in manufacturing and formulation, as well as limit delivery by subcutaneous injection. The high concentration viscosity of some antibodies has been reduced by variable domain mutations or by the addition of formulation excipients. In contrast, the impact of Fc mutations on antibody viscosity has been minimally explored. Here, we studied the effect of a panel of common and clinically validated Fc mutations on the viscosity of two closely related humanized IgG1, κ antibodies, omalizumab (anti-IgE) and trastuzumab (anti-HER2). Data presented here suggest that both Fab-Fab and Fab-Fc interactions contribute to the high viscosity of omalizumab, in a four-contact model of self-association. Most strikingly, the high viscosity of omalizumab (176 cP) was reduced 10.7- and 2.2-fold by Fc modifications for half-life extension (M252Y:S254T:T256E) and aglycosylation (N297G), respectively. Related single mutations (S254T and T256E) each reduced the viscosity of omalizumab by ~6-fold. An alternative half-life extension Fc mutant (M428L:N434S) had the opposite effect in increasing the viscosity of omalizumab by 1.5-fold. The low viscosity of trastuzumab (8.6 cP) was unchanged or increased by ≤2-fold by the different Fc variants. Molecular dynamics simulations provided mechanistic insight into the impact of Fc mutations in modulating electrostatic and hydrophobic surface properties as well as conformational stability of the Fc. This study demonstrates that high viscosity of some IgG1 antibodies can be mitigated by Fc mutations, and thereby offers an additional tool to help design future antibody therapeutics potentially suitable for subcutaneous delivery.

Characterization of Recombinantly-Expressed Hydrolytic Enzymes from Chinese Hamster Ovary Cells: Identification of Host Cell Proteins that Degrade Polysorbate.

Enzymatic hydrolysis of polysorbate in drug products is a major challenge for the biopharmaceutical industry. Polysorbate hydrolysis caused by host cell proteins (HCPs) co-purified during bioprocessing can reduce the protective effects of the surfactant for the active pharmaceutical ingredient and cause the accumulation of low-solubility degradation products over the long-term storage. The identities of such HCPs are elusive due to their extremely low concentrations after the efficient purification processes of most biopharmaceuticals. In this work, 20 enzymes-selected for their known or putative hydrolytic activity and potential to degrade polysorbate-were recombinantly expressed, purified, and characterized via orthogonal methods. First, these recombinant HCPs were assessed for hydrolytic activity against a fluorogenic esterase substrate in a recently-developed, high-throughput assay. Second, these HCPs were screened for hydrolytic activity against polysorbate in a representative mAb formulation. Third, HCPs that displayed hydrolytic activities in the first two assays were subjected to more detailed characterization of their enzyme kinetics against polysorbates. Finally, these HCPs were evaluated for substrate specificity towards different sub-species of polysorbates. This work provides critical new insights for targeted LC-MS/MS approaches for identification of relevant polysorbate-degrading enzymes and supports improvements to remove such HCPs, including knockouts or targeted removal during purification.