Modifications like asparagine deamidation, aspartate isomerization, methionine oxidation, and lysine glycation are typical degradations for recombinant antibodies. For the identification and functional evaluation of antibody critical quality attributes (CQAs) derived from chemical modifications in the complementary-determining regions (CDRs) and the conserved regions, an approach employing specific stress conditions, elevated temperatures, pH, oxidizing agents, and forced glycation with glucose incubation, was applied. The application of the specific stress conditions combined with ion exchange chromatography, proteolytic peptide mapping, quantitative liquid chromatography mass spectrometry, and functional evaluation by surface plasmon resonance analysis was adequate to identify and functionally assess chemical modification sites in the CDRs of a recombinant IgG1. LC-Met-4, LC-Asn-30/31, LC-Asn-92, HC-Met-100c, and HC Lys-33 were identified as potential CQAs. However, none of the assessed degradation products led to a complete loss of functionality if only one light or heavy chain of the native antibody was affected.

https://www.tandfonline.com/doi/pdf/10.4161/mabs.27876

Assessment of chemical modifications of sites in the CDRs of recombinant antibodies: Susceptibility vs. functionality of critical quality attributes

Preserving the chemical and structural integrity of therapeutic antibodies during manufacturing and storage is a major challenge during pharmaceutical development. Oxidation of Fc methionines Met252 and Met428 is frequently observed, which leads to reduced affinity to FcRn and faster plasma clearance if present at high levels. Because oxidation occurs in both positions simultaneously, their individual contribution to the concomitant changes in pharmacokinetic properties has not been clearly established. A novel pH-gradient FcRn affinity chromatography method was applied to isolate three antibody oxidation variants from an oxidized IgG1 preparation based on their FcRn binding properties. Physico-chemical characterization revealed that the three oxidation variants differed predominantly in the number of oxMet252 per IgG (0, 1, or 2), but not significantly in the content of oxMet428. Corresponding to the increase in oxMet252 content, stepwise reduction of FcRn affinity in vitro, as well as faster clearance and shorter terminal half-life, in huFcRn-transgenic mice were observed. A single Met252 oxidation per antibody had no significant effect on pharmacokinetics (PK) compared with unmodified IgG. Importantly, only molecules with both heavy chains oxidized at Met252 exhibited significantly faster clearance. In contrast, Met428 oxidation had no apparent negative effect on PK and even led to somewhat improved FcRn binding and slower clearance. This minor effect, however, seemed to be abrogated by the dominant effect of Met252 oxidation. The novel approach of functional chromatographic separation of IgG oxidation variants followed by physico-chemical and biological characterization has yielded the first experimentally-backed explanation for the unaltered PK properties of antibody preparations containing relatively high Met252 and Met428 oxidation levels.

A novel approach to investigate the effect of methionine oxidation on pharmacokinetic properties of therapeutic antibodies

https://www.onlinelibrary.wiley.com/doi/pdf/10.1002/jps.24509

Selective Oxidation of Methionine and Tryptophan Residues in a Therapeutic IgG1 Molecule

https://onlinelibrary.wiley.com/doi/pdf/10.1002/jps.24136

Effect of Individual Fc Methionine Oxidation on FcRn Binding: Met252 Oxidation Impairs FcRn Binding More Profoundly than Met428 Oxidation

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.

/pdf/assessment-of-susceptible-chemical-modification-sites-of-2sxaepebib.pdf

Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions.

http://www.protocol-online.org/forums/uploads/monthly_10_2012/msg-23183-0-70615700-1349324203.ipb

Analytical Protein A Chromatography as a Quantitative Tool for the Screening of Methionine Oxidation in Monoclonal Antibodies

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

https://pubs.acs.org/doi/pdf/10.1021/acs.analchem.2c03099

Jürgen Fichtl

Papers

Analytical Protein A Chromatography as a Quantitative Tool for the Screening of Methionine Oxidation in Monoclonal Antibodies

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

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