Full validation of therapeutic antibody sequences by middle-up mass measurements and middle-down protein sequencing

Abstract

The regulatory bodies request full sequence data assessment both for innovator and biosimilar monoclonal antibodies (mAbs). Full sequence coverage is typically used to verify the integrity of the analytical data obtained following the combination of multiple LC-MS/MS datasets from orthogonal protease digests (so called “bottom-up” approaches). Top-down or middle-down mass spectrometric approaches have the potential to minimize artifacts, reduce overall analysis time and provide orthogonality to this traditional approach. In this work we report a new combined approach involving middle-up LC-QTOF and middle-down LC-MALDI in-source decay (ISD) mass spectrometry. This was applied to cetuximab, panitumumab and natalizumab, selected as representative US Food and Drug Administration- and European Medicines Agency-approved mAbs. The goal was to unambiguously confirm their reference sequences and examine the general applicability of this approach. Furthermore, a new measure for assessing the integrity and validity of results from middle-down approaches is introduced – the “Sequence Validation Percentage.” Full sequence data assessment of the 3 antibodies was achieved enabling all 3 sequences to be fully validated by a combination of middle-up molecular weight determination and middle-down protein sequencing. Three errors in the reference amino acid sequence of natalizumab, causing a cumulative mass shift of only −2 Da in the natalizumab Fd domain, were corrected as a result of this work.

Combination of intact, middle-up and bottom-up levels to characterize 7 therapeutic monoclonal antibodies by capillary electrophoresis – Mass spectrometry

“…An increasingly number of reports from worldwide drug agencies such as the FDA and the EMA highlight the importance of multi-level characterization of mAbs [25][26][27] Separations have been performed in less than 11 minutes for each mAbs (Fig. 1) (RSD < 3% on migration times (n=3)).…”

In-Source Decay and Pseudo-MS3of Peptide and Protein Ions Using Liquid AP-MALDI

“…The lower mass range of the AP-MALDI source limits ISD and pseudo-MS 3 to small proteins, but both analyses benefit from the accurate mass and high mass resolution of the Orbitrap mass analyzer. Conversely, the much wider mass range of a vacuum MALDI TOF instrument can be used to acquire ISD spectra from large proteins, including antibodies [27], and pseudo-MS 3 spectra from smaller ISD fragments, albeit with significantly lower mass resolution and mass accuracy. It should be noted that the liquid matrix is less applicable to vacuum MALDI and intermediate pressure MALDI systems that require an extraction field in the MALDI source because the liquid matrix droplet distorts the electric field lines.…”

LC–MS bioanalysis of intact proteins and peptides

“…The advantage of middle‐down approach compared with top‐down is ascribed to the reduced size of the analyte, which enables better resolution and sensitivity. This is particularly useful for the detection of modified species with small mass shift, such as oxidation, acetylation and even deamidation, which is detected by ultra‐high‐resolution QTOF (Resemann et al, ), as well as biotransformation of payloads on ADCs (Kellie, Kehler, Mencken, et al, ; Su et al, ). Regarding assay sensitivity, bottom‐up approach is certainly the best among the three approaches.…”