Application of Peptide Mapping to Locate Sequence Variants
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
Figure 3. UHPLC–MS tryptic peptide maps of innovator and biosimilar MAbs presented as total ion chromatograms.
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 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.
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).
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.