Rapid Assessment of Molecular Similarity between a Candidate Biosimilar and an Innovator Monoclonal Antibody Using Complementary LC–MS Methods - Intact protein LC–MS detected a mass varian

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Rapid Assessment of Molecular Similarity between a Candidate Biosimilar and an Innovator Monoclonal Antibody Using Complementary LC–MS Methods
Intact protein LC–MS detected a mass variance of 62 Da and peptide mapping located a difference of two amino acids.


BioPharm International Supplements


Application of Peptide Mapping to Locate Sequence Variants


Figure 3. UHPLC–MS tryptic peptide maps of innovator and biosimilar MAbs presented as total ion chromatograms.
Tryptic digests of both samples were analyzed using the data-independent acquisition UHPLC–MS approach ("UPLC–MSE "). It has been shown that data-independent acquisition LC–MS with alternate low and elevated collision energy scanning provides significant benefits in terms of completeness and speed of peptide mapping.8 In contrast to data-dependent acquisition (DDA) LC–MS–MS, which requires a list of precursors (targeted DDA) or relies on intensity-biased precursor selection on the fly (often resulting in omission of minor peptides), data-independent acquisition MS allows for sequencing of all peptides above the limit of detection and for accurate quantification by MS signals in a single analysis.


Figure 4. UHPLC–MS data of peptide maps shown as deisotoped and charge reduced chromatograms (all charge states and isotopes were deconvoluted into singly charged ion "stick"). Selected part of chromatogram between 3.5 and 7.5 min shows one unique peptide detected in innovator and biosimilar MAb digests.
Figure 3 shows a mirror plot of UHPLC–MS peptide maps of the biosimilar and innovator MAbs generated by BiopharmaLynx software. The detected tryptic peptide masses were matched against the theoretical values using a published trastuzumab sequence.12 Although the total ion chromatogram traces do not show significant differences between samples (Figure 3), the compare function in the software visualized chromatogram regions for each of the MAbs that differed. Besides differences in glycosylation, the software indicated additional differences, made apparent by displaying charge-reduced and isotope-deconvoluted MS "stick chromatogram" plots, as shown in Figure 4. The mirror plot of the selected region of the chromatogram highlights that the innovator and biosimilar MAbs each contain a unique peptide without a corresponding signal in the other sample. The peptide HT35 (heavy chain, tryptic peak T35) with the amino acid sequence EEMTK found in the innovator product was not present in the biosimilar candidate MAb. Instead, a peak with a mass of 605.32 Da was observed. The calculated difference in mass between this unknown peptide and EEMTK is ~32 Da, which correlates with our previous results for the differences in mass in the heavy chain (~32 Da) and intact MAb (~64 Da).


Figure 5. Spectra of unique peptides, obtained using data-independent acquisition MS, confirm the primary sequence of HCT35 peptides for innovator antibody. As expected, it is EEMTK. Biosimilar drug candidate sequence of the HCT35 peptide is DELTK, which is a known MAb allotype.
The MS data suggest that the innovator and biosimilar MAbs have a local inconsistency in the primary amino acid sequence located in the HT35 peptide. Because the UHPLC–MS data contain fragmentation data for all peptides (including the unknown and unidentified ones) it was possible to investigate the unknown peak in BiopharmaLynx, as well as in the PepSeq programs, with the latter capable of assisting de novo sequencing. This task was simplified by the availability of information about alternative MAb allotypes in the DrugBank.13,14 An alternative sequence matched the HT35 peptide mass and the fragmentation pattern matched the DELTK sequence, with a difference of two amino acids from the innovator MAb EEMTK peptide. Both sequences were confirmed using data-independent acquisition MS (Figure 5).


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