Peptide Mapping

SMART Digest Mag Beads (Part no. 60109-101-MB)

• Peptide mapping is a regulatory expectation to verify sequence and monitor post-translational modifications (PTMs)

• Proteolytic digestion remains labor-intensive and challenging to accurately reproduce

• SMART Digest provides a fast, reproducible, automated protocol for protease digestion incorporated into an optimized peptide mapping workflow

• This protocol is suitable for use with multi-attribute method (MAM) in drug development, production and QC laboratories.

“The sample preparation workflow is simple, ideally suited to inexperienced operators and has been extensively studied to show global applicability and robustness for mAbs by performing sample digestion and LC-MS analysis at four independent sites in Europe.” (Anal Bioanal Chem. 2020 Oct;412(25):6833-6848.)

 Example protocol

• Samples diluted to 2 mg/mL in water

• 15uL SMART Digest Mag Beads (Part no. 60109-101-MB), 150uL SMART Digest Buffer

• Incubated for 45 minutes at 70 °C then cooled (on KingFisher)

• Disulfide bond reduction performed with 10 mM DTT for 30 minutes at 57 °C then alkylation with 20 mM IA in darkness for 30 minutes

• The reaction was quenched with 15.45μL of 100 mM DTT followed by 15.64μL 10% TFA (final concentration 11 mM DTT and 1% TFA)

• Samples were then injected immediately into the LC-MS

 
Select Publications

• Millán-Martín, et al. (2020). Inter-laboratory study of an optimised peptide mapping workflow using automated trypsin digestion for monitoring monoclonal antibody product quality attributes. Analytical and Bioanalytical Chemistry, 412(25), 6833–6848.

• Carillo, S., et al. (2019). Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies. Journal of Pharmaceutical Analysis, 10(1), 23–34.

• Füssl, F., et al. (2018). Comprehensive characterisation of the heterogeneity of adalimumab via charge variant analysis hyphenated on-line to native high resolution Orbitrap mass spectrometry. mAbs, 11(1), 116–128.

• Carillo, S., et al. (2019b). Comparing different domains of analysis for the characterisation of N-glycans on monoclonal antibodies. Journal of Pharmaceutical Analysis, 10(1), 23–34.

• Torrente-López, A., et al. (2023). Comprehensive physicochemical and functional analysis of pembrolizumab based on controlled degradation studies: Impact on antigen–antibody binding. European Journal of Pharmaceutics and Biopharmaceutics, 194, 131–147.

• Rauniyar, N., et al. (2024) Comparative analysis of Herceptin N-Linked glycosylation by HILIC-FLD and LC-MS/MS methods. Journal of Pharmaceutical and Biomedical Analysis, 244

• Torres-García, A., et al. (2024). Comprehensive analysis of Cetuximab Critical quality attributes: Impact of handling on Antigen-Antibody binding. Pharmaceutics, 16(9), 1222.