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.

Charge sensitive separation methods such as ion exchange chromatography (CEX) and capillary electrophoresis (CE) have recently been coupled to mass spectrometry to facilitate high resolution profiling of proteoforms present within the charge variant profile of complex biopharmaceuticals. Here we apply pH gradient cation exchange chromatography and microfluidic capillary electrophoresis using the ZipChip platform for comparative characterization of the monoclonal antibody Cetuximab. Cetuximab harbors four glycans per molecule, two on each heavy chain, of which the Fab glycans have been reported to be complex and multiply sialylated. The presence of these extra glycosylation sites in the variable region of the molecule causes significant charge variant and glycan heterogeneity, which makes comprehensive analysis on the intact protein level challenging. Both pH gradient CEX-MS and CE-MS were found to be powerful for the separation of Cetuximab charge variants with eight major peaks being baseline resolved using both separation platforms. Informative native-like mass spectra were collected for each charge variant peak using both platforms that facilitated deconvolution and further analysis. The total proteoform coverage was exceptionally high with >100 isoforms identified and relatively quantified with CEX-MS, in case of CE-MS the proteoform coverage was >200. A deep insight into the heterogeneity of Cetuximab was unveiled, the high level of sensitivity achievable enables the implementation of the presented technologies even at early stages of the biopharmaceutical development platform, such as in developability assessment, process development and also for monitoring process consistency.

RationaleMutations in the cystic fibrosis transmembrane regulator (CFTR) gene form the basis of cystic fibrosis (CF). There remains an important knowledge gap in CF as to how diminished CFTR activity leads to the dominant inflammatory response within CF airways.ObjectivesTo investigate if extracellular vesicles (EVs) contribute to inflammatory signalling in CF.MethodsEVs released from CFBE41o-, CuFi-5, 16HBE14o- and NuLi-1 cells were characterised by nanoparticle tracking analysis (NTA). EVs isolated from bronchoalveolar lavage fluid (BALF) from 30 people with CF (PWCF) were analysed by NTA and mass spectrometry and compared with controls. Neutrophils were isolated from the blood of 8 PWCF to examine neutrophil migration in the presence of CFBE41o- EVs.ResultsA significantly higher level of EVs were released from CFBE41o- (p<0.0001) and CuFi-5 (p=0.0209) relative to control cell lines. A significantly higher level of EVs were detected in BALF of PWCF, in three different age groups relative to controls (p=0.01, 0.001, 0.002). A significantly lower level of EVs were released from CFBE41o- (p<0.001) and CuFi-5 (p=0.0002) cell lines treated with CFTR modulators. Significant changes in the protein expression of 126 unique proteins was determined in EVs obtained from the BALF of PWCF of different age groups (p<0.001–0.05). A significant increase in chemotaxis of neutrophils derived from PWCF was observed in the presence of CFBE41o EVs (p=0.0024) compared with controls.ConclusionThis study demonstrates that EVs are produced in CF airway cells, have differential protein expression at different ages and drive neutrophil recruitment in CF.