Assessment of susceptible chemical modification sites of trastuzumab and endogenous human immunoglobulins at physiological conditions

Abstract

The quality control testing of chemical degradations in the bio-pharmaceutical industry is currently under controversial debate. Here we have systematically applied in vitro and in vivo stress conditions to investigate the influence of protein degradation on structure-function. Extensive purification and characterization enabled identification and functional assessment of the physiological degradation of chemical modification sites in the variable complementarity-determining regions (CDRs) and conserved region of trastuzumab. We demonstrate that the degradation of the solvent-accessible residues located in the CDR and the conserved fragment crystallizable region (Fc) occurs faster in vivo (within days) compared to the levels observed for bio-process and real-time storage conditions. These results hence question the rationality of extreme monitoring of low level alterations in such chemical modifications as critical patient safety parameters in product quality control testing, given that these modifications merely mirror the natural/physiological aging process of endogenous antibodies.

Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes

“…3 and ESM Tables S7 and S8). Levels are also lowered when compared to previously reported data generated using long digestion times in basic buffer [28].…”

Assessing Particle Formation of Biotherapeutics in Biological Fluids

“…17 As an alternative to serum, several studies used PBS due to its buffering capacity at pH 7.4, similar ionic strength as blood, and significantly less complex matrix to circumvent analytical challenges encountered with biological fluids. 2,5,18 While certain chemical modifications showed similar results in PBS and serum, few studies have compared physical stability in PBS to serum. 6,18 Our results provide further evidence that mAbs are less stable in serum compared to PBS in regards to protein aggregation.…”

“…2,5,18 While certain chemical modifications showed similar results in PBS and serum, few studies have compared physical stability in PBS to serum. 6,18 Our results provide further evidence that mAbs are less stable in serum compared to PBS in regards to protein aggregation. Thus, PBSmay not be considered a suitable in vitro model to assess protein liabilities as the stability may not be impacted to an extent that enables to benchmark molecule candidates.…”

The Impact of Immunoglobulin G1 Fc Sialylation on Backbone Amide H/D Exchange

“…Liquid chromatography-mass spectrometry (LC-MS/MS) peptide mapping and the quantification of relevant amino acid modifications were principally conducted as previously described [52,53]. In brief, all samples were denatured with 8 M Gua-HCl (pH 6.0) and reduced with 10 µL (c = 0.1 g/mL) dithiothreitol (DTT) at 50 • C for 1 h. Samples were buffer-exchanged (0.02 M His-HCl, pH 6.0) and further digested with 10 µL (c = 0.25 mg/mL) trypsin (Roche Diagnostics GmbH) at 37 • C for 18 h. Peptide separation (BEH C 18 1.7 µm, 2.1 × 150 mm) was performed on a Waters ACQUITY UPLC system.…”