Peptide digestion analysis of a monoclonal therapeutic antibody
28th Mar 2022
MS of a Trastuzumab biosimilar digestion using an IDA MSMS methodology
The amino acid sequence of proteins is essential to its biological and physiological properties. When expressing a therapeutic protein its primary sequence is a critical quality attribute (CQA) that needs to be monitored to ensure its efficacy. In addition, there are a number of post-translational modifications such as deamidation and oxidation which are indicative of degradation. The use of tryptic digestion allows these CQAs to be monitored when they may present as too small a mass shift and to be done on the intact protein level.
Here we outline an analysis strategy to confirm the primary amino acid sequence of a trastuzumab biosimilar after a Trypsin/Lys-C (Promega) digestion. Mass spectrometry conditions were optimised for maximum peptide intensity. All data was collected on a SCIEX X500B Mass Spectrometer with an EXION HPLC. The chromatography was performed with a bioZen XB-C18 Column (1.7 µm, 150 x 2.1 mm). See diagram here
The sample was separated on a 70-minute gradient and analysed using BioPharmaView Flex. Figure 1 shows all peptides which were matched with MSMS data above a set of tolerances. Most peptides were observed in multiple charge states and the digestion was not run to completion resulting in a variety of missed cleavages.
Figure 2 shows MSMS data of an example peptide VVSVLTVLHQDWLNGK. All assigned peptides were required to have sequence coverage greater than 40% to exclude poor matches. In addition, a scoring algorithm was applied with an acceptance criteria of 2% false discovery rate. Several of the methionine residues were observed in an oxidised state ranging between 0.2 and 1.6%.
Figure 1: Sequence coverage of trastuzumab with peptides assigned based upon MSMS coverage with an FDR of less than 2% and a peptide coverage of >40%. Based upon IDA MSMS an overall 99.1% of the sequence was confirmed. Individual peptides are shown in green with any modifications such as carbamidomethylation, or oxidation shown in red. See more here.
Figure 2: MSMS data from 3+ peptide VVSVLTVLHQDWLNGK. Eluting at 59.27 minutes in the 2 and 3+ charge states, Fragmentation provides 100% peptide sequence coverage. See more here.
