Identification of Hetero-aggregates in Antibody Co-formulations by Multi-dimensional Liquid Chromatography Coupled to Mass Spectrometry.

Antibody combination therapies have become viable therapeutic treatment options for certain severe diseases such as cancer. The co-formulation production approach is intrinsically associated with more complex drug product variant profiles and creates more challenges for analytical control of drug product quality. In addition to various individual quality attributes, those arising from the interactions between the antibodies also potentially emerge through co-formulation. In this study, we describe the development of a widely applicable multi-dimensional liquid chromatography coupled to tandem mass spectrometry method for antibody homo- versus hetero-aggregate characterization. The co-formulation of trastuzumab and pertuzumab was used, a challenging model system, comprising two monoclonal antibodies with very similar physicochemical properties. The data presented demonstrate the high stability of the co-formulation, where only minor aggregate formation is observed upon product storage and accelerated temperature or light-stress conditions. The results also show that the homo- and hetero-aggregates, formed in low and comparable proportions, are only marginally impacted by the formulation and product storage conditions. No preferential formation of hetero-aggregates, in comparison to the already existing pertuzumab and trastuzumab homo-aggregates, was observed.

Multiattribute Monitoring of Antibody Charge Variants by Cation-Exchange Chromatography Coupled to Native Mass Spectrometry.

The aim of this study was to characterize the product variants of a therapeutic T-cell bispecific humanized monoclonal antibody (TCB Mab, ∼200 kDa, asymmetric) and to develop an online cation-exchange chromatography native electrospray mass spectrometry method (CEC-UV-MS) for direct TCB Mab charge variant monitoring during bioprocess and formulation development. For the identification and functional evaluation of the diverse and complex TCB Mab charge variants, offline fractionation combined with comprehensive analytical testing was applied. The offline fractionation of abundant product variant peaks enabled identification of coeluting acid charge variants such as asparagine deamidation, primary and secondary Fab glycosylation (with and without sialic acid), and the presence of O-glycosylation in the G4S-linker region. Consequently, a new nonconsensus N-glycosylation motif (N-338-FG) in the heavy chain CDR region was discovered. Functional evaluation by cell-based potency testing demonstrated a clear and negative impact of both asparagine deamidations, whereas the O-glycosylation did not affect the TCB Mab biological activity. We established an online native CEC-UV-MS method, with an ammonium acetate buffer and pH gradient, to directly monitor the TCB Mab charge variants. All abundant chemical degradations and post-translational amino acid modifications already identified by offline fraction experiments and liquid chromatography mass spectrometry peptide mapping could also be monitored by the online CEC-UV-MS method. The herein reported online native CEC-UV-MS methodology represents a complementary or even alternative approach for multiattribute monitoring of biologics, offering multiple benefits, including increased throughput and reduced sample handling and intact protein information in the near-native state.

Analytical protein a chromatography as a quantitative tool for the screening of methionine oxidation in monoclonal antibodies.

The presence of oxidized methionine residues in therapeutic monoclonal antibodies can potentially impact drug efficacy, safety, as well as antibody half-life in vivo. Therefore, methionine oxidation of antibodies is a strong focus during pharmaceutical development and a well-known degradation pathway. The monitoring of methionine oxidation is currently routinely performed by peptide mapping/liquid chromatography-mass spectrometry techniques, which are laborious and time consuming. We have established analytical protein A chromatography as a method of choice for fast and quantitative screening of total Fc methionine oxidation during formulation and process development. The principle of this method relies on the lower binding affinity of protein A for immunoglobulin G-Fc domains containing oxidized methionines, compared with nonoxidized Fc domains. Our data reveal that highly conserved Fc methionines situated close to the binding site to protein A can serve as marker for the oxidation of other surface-exposed methionine residues. In case of poor separation of oxidized species by protein A chromatography, analytical protein G chromatography is proposed as alternative. We demonstrate that analytical protein A chromatography, and alternatively protein G chromatography, is a valuable tool for the screening of methionine oxidation in therapeutic antibodies during formulation and process development.