Standardized Kit-Based Sample Preparation for Multi-Attribute Method Analysis of Biotherapeutics

Background/Objectives: LC–MS/MS-based Multi-Attribute Method (MAM) workflows are increasingly used to characterize biotherapeutic proteins using peptide mapping and site-specific monitoring of critical quality attributes (CQAs). Sample preparation can impact foulant carryover, digestion efficiency, host cell protein (HCP) detection, and can induce artifactual chemical modifications. Herein, we compared a published National Institute for Bioprocessing Research and Training (NIBRT) MAM workflow with the S-Trap™ MAM Kit using NISTmAb RM 8671 with and without foulants. Methods: Both bottom-up workflows used tryptic digestion, performed in solution for NIBRT and on-trap for the S-Trap™ MAM Kit. Matched samples were prepared with or without added foulants from a panel of 42 bioprocess-relevant contaminants including salts, buffers, surfactants, stabilizers, and excipients. LC–MS/MS analysis compared peptide-mapping performance, foulant carryover, sequence coverage, HCP detection and background signals, as well as preparation-induced modifications, in particular deamidation. Protein recovery and complementary Tryp-N™ digestion were also evaluated. Results: Compared with the NIBRT workflow, the S-Trap™ MAM Kit had higher NISTmAb protein recovery (92.3% versus 48.4%); both workflows yielded full sequence coverage given matched peptide input. The S-Trap™ workflow produced greater total LC–MS signal from non-product proteins like HCPs. Under the conditions evaluated, signals corresponding to polysorbate-80, Brij-35, PEG-8000 and other contaminants were not detected following S-Trap™ preparation. Lower measured deamidation at the evaluated NISTmAb sites was observed using the kit and Tryp-N™ provided complementary peptide-mapping coverage. Conclusions: The S-Trap™ MAM Kit offers several advantages for NISTmAb sample preparation: higher recovery, greater LC-MS signal from non-product proteins like HCPs, lower overall deamidation, and no detectable signals from the examined surfactant and polymer foulants under the conditions evaluated. The standardized, plate-based workflow offers a promising approach for biotherapeutic MAM sample preparation. Broader applicability and suitability for use in quality-control require further product-specific qualification or validation. Data are available via ProteomeXchange with identifier PXD086642.