Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry

Abstract

Charge variant analysis is a widely used tool to monitor changes in product quality during the manufacturing process of monoclonal antibodies (mAbs). Although it is a powerful technique for revealing mAb heterogeneity, an unexpected outcome, for example the appearance of previously undetected isoforms, requires further, time-consuming analysis. The process of identifying these unknowns can also result in unwanted changes to the molecule that are not attributable to the manufacturing process. To overcome this, we recently reported a method combining highly selective cation exchange chromatography-based charge variant analysis with on-line mass spectrometric (MS) detection. We further explored and adapted the chromatographic buffer system to expand the application range. Moreover, we observed no salt adducts on the native protein, also supported by the optimal choice of MS parameters, resulting in increased data quality and mass accuracy. Here, we demonstrate the utility of this improved method by performing an in-depth analysis of adalimumab before and after forced degradation. By combining molecular mass and retention time information, we were able to identify multiple modifications on adalimumab, including lysine truncation, glycation, deamidation, succinimide formation, isomerisation, N-terminal aspartic acid loss or C-terminal proline amidation and fragmentation along with the N-glycan distribution of each of these identified proteoforms. Host cell protein (HCP) analysis was performed using liquid chromatography-mass spectrometry that verified the presence of the protease Cathepsin L. Based on the presence of trace HCPs with catalytic activity, it can be questioned if fragmentation is solely driven by spontaneous hydrolysis or possibly also by enzymatic degradation.

Hyphenation of strong cation exchange chromatography to native mass spectrometry for high throughput online characterization of charge heterogeneity of therapeutic monoclonal antibodies

“…The broad applications of this column as reported in the literature (charge variant characterizations using MS compatible and MS incompatible buffers) also make it a suitable tool for comparison of the new SCX salt-pH gradient method we developed with historical results from established buffer systems. 17,[23][24][25] Antibodies may undergo modifications or degradation under high or low pH. The instability of protein modifications (e.g., hydrolysis of succinimide under alkaline pH) has also been reported by different groups.…”

Are Biotransformation Studies of Therapeutic Proteins Needed? Scientific Considerations and Technical Challenges

“…To date, nonvolatile salts in the buffers limited the use of IEX with MS. Recently, successful approaches to make CVA compatible with MS have been reported, in which volatile ammonium salts have been used (Füssl et al, 2018(Füssl et al, , 2019. The challenge that the authors faced was the poor buffering capacity of ammonium salts at pH around the isoelectric point of the analytes.…”

Middle-up characterization of monoclonal antibodies by online reduction liquid chromatography-mass spectrometry

“…A key step in the structural characterization of mAbs and variants thereof, is the measurement of the intact molecule using LC-MS under denaturing or native conditions. Historically, reversed phase liquid chromatography (RPLC) has been preferred for the up-front separations while more recently techniques like sizeexclusion chromatography (SEC) and cation exchange chromatography (CEX) are also implemented to directly introduce size or charge variants in the MS [9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28] . Intact measurements provide an initial confirmation of the gene-derived protein sequence, reveals the structural integrity and highlights post-translational modifications, however, for large molecules such as mAbs, modifications resulting in minor mass differences might be missed and highly heterogeneous antibodies might generate convoluted spectra [9][10][11][12] .…”