Rapid comparison of a candidate biosimilar to an innovator monoclonal antibody with advanced liquid chromatography and mass spectrometry technologies

Abstract

This study shows that state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) can be used for rapid verification of identity and characterization of sequence variants and posttranslational modifications (PTMs) for antibody products. A candidate biosimilar IgG1 monoclonal antibody (mAb) was compared in detail to a commercially available innovator product. Intact protein mass, primary sequence, PTMs, and the micro-differences between the two mAbs were identified and quantified simultaneously. Although very similar in terms of sequences and modifications, a mass difference observed by LC-MS intact mass measurements indicated that they were not identical. Peptide mapping, performed with data independent acquisition LC-MS using an alternating low and elevated collision energy scan mode (LC-MS(E)), located the mass difference between the biosimilar and the innovator to a two amino acid residue variance in the heavy chain sequences. The peptide mapping technique was also used to comprehensively catalogue and compare the differences in PTMs of the biosimilar and innovator mAbs. Comprehensive glycosylation profiling confirmed that the proportion of individual glycans was different between the biosimilar and the innovator, although the number and identity of glycans were the same. These results demonstrate that the combination of accurate intact mass measurement, released glycan profiling, and LC-MS(E) peptide mapping provides a set of routine tools that can be used to comprehensively compare a candidate biosimilar and an innovator mAb.

Versatile characterization of glycosylation modification in CTLA4-Ig fusion proteins by liquid chromatography-mass spectrometry

“…To expedite the development of biosimilars in Europe, the European Medicines Agency (EMA) has established a series of guidelines. 5 Although biosimilar products such as human growth hormone, granulocyte colony-stimulating factor and epoetin have already been approved by regulators, including the EMA, only one biosimilar antibody has been approved because of the complexity of the molecule. Compared to other biosimilar therapeutics, mAb and antibody-derivative biosimilars guidance has experienced delays.…”

The glycosylation aspects of biosimilarity

“…Their results confirmed that the ratio of individual glycans (quantified by integrated peak area) differed between the biosimilar and the reference product [72]. In another case study, Sanchez-De Meloa et al analyzed the glycan profile of trastuzumab and its biosimilar candidates by normal-phase HPLC and MALDI-TOF-MS [73].…”

Top-down proteomic analysis of protein pharmaceuticals, mixtures of protein complexes, and ribosomal protein extracts by capillary zone electrophoresis-mass spectrometry

“…The difference between the theoretical and experimental molecular masses of the dimer was 34.48 Da, possibly due to strong coordination of one water molecule or replacement of a proton with an ammonium ion per monomer. It is worth noting that DrugBank (www.drugbank.ca/drugs/DB00072) has a different sequence for the heavy chain (HC) of the innovator version of Trastuzumab (Genentech), including DELTK for residues 360-364 of the HC instead of the EEMTK, and an extra P in the sequence with VEPPK instead of VEPK at residues 218-222 of the HC, as also reported by others [338,339]. Additionally, the absence of the C-terminal K is yet another difference between the DrugBank sequence and the one reported in [338], which also agrees with our experimental findings.…”