Analysis of ADCs by Native Mass Spectrometry

International audienceMass spectrometry performed in nondenaturing conditions (native MS) has proven its utility for the quantitative and qualitative analysis of antibody-drug conjugates (ADCs), especially when ADCs’ subunits involve noncovalent interactions (i.e., cysteine-conjugated ADCs). Its hyphenation to ion mobility spectrometry (IM-MS) allows differentiation of gas-phase ions based on their rotationally averaged collision cross section providing an additional dimension of conformational characterization of ADCs. More recently, size exclusion chromatography (SEC) appeared as an interesting technique to perform online buffer exchange in an automated way prior to native MS/IM-MS analysis. Online SEC-native MS/IM-MS allows the global structural characterization of ADCs and the assessment of some critical quality attributes (CQAs) required for ADC release on the market, such as drug load distribution (DLD), drug-to-antibody ratio (DAR), the average DAR (DARav), and the relative amount of unconjugated mAb

Asparagine-linked Oligosaccharides Present on a Non-consensus Amino Acid Sequence in the CH1 Domain of Human Antibodies

We report that N-linked oligosaccharide structures can be present on an asparagine residue not adhering to the consensus site motif NX(S/T), where X is not proline, described in the literature. We have observed oligosaccharides on a non-consensus asparaginyl residue in the CH1 constant domain of IgG1 and IgG2 antibodies. The initial findings were obtained from characterization of charge variant populations evident in a recombinant human antibody of the IgG2 subclass. HPLC-MS results indicated that cation-exchange chromatography acidic variant populations were enriched in antibody with a second glycosylation site, in addition to the well documented canonical glycosylation site located in the CH2 domain. Subsequent tryptic and chymotryptic peptide map data indicated that the second glycosylation site was associated with the amino acid sequence TVSWN162SGAL in the CH1 domain of the antibody. This highly atypical modification is present at levels of 0.5–2.0% on most of the recombinant antibodies that have been tested and has also been observed in IgG1 antibodies derived from human donors. Site-directed mutagenesis of the CH1 domain sequence in a recombinant-human IgG1 antibody resulted in an increase in non-consensus glycosylation to 3.15%, a greater than 4-fold increase over the level observed in the wild type, by changing the −1 and +1 amino acids relative to the asparagine residue at position 162. We believe that further understanding of the phenomenon of non-consensus glycosylation can be used to gain fundamental insights into the fidelity of the cellular glycosylation machinery