Peptide Mapping for Biotherapeutics

Advanced Peptide Mapping Approaches for Biotherapeutic Characterization

Význam tématu
Peptide mapping is a cornerstone of biotherapeutic analysis, enabling protein identification, primary structure confirmation and monitoring of critical quality attributes (CQAs) such as post-translational modifications (PTMs). As regulatory and research demands intensify, there is a need for faster, more reproducible and high-throughput workflows that generate clean data with minimal sample preparation artifacts.

Cíle a přehled studie / článku
This collection of application studies presents novel strategies to streamline peptide mapping workflows: automation of sample preparation, accelerated digestions using engineered trypsins, improved chromatographic performance to reduce adsorption effects, enhanced PTM discovery, spatial proteomic profiling and optimized LC-UV-MS detection. Together, these advances address common bottlenecks in speed, robustness and data quality.

Použitá metodika a instrumentace

• Sample preparation and digestion: Waters PeptideWorks Tryptic Digestion Kits with homogeneously methylated RapiZyme™ Trypsin; manual and automated protocols on Andrew+ Pipetting Robot; one-pot diluted protocols; rapid 30-minute and overnight digestions with varied enzyme-to-protein ratios.
• Chromatography: ACQUITY UPLC I-Class PLUS and H-Class PLUS systems; ACQUITY Premier Peptide CSH C18 Columns featuring MaxPeak™ HPS Technology for reduced metal-ion mediated adsorption; RapiZyme digestion compatible columns.
• Mass spectrometry and detection: ACQUITY RDa™, QDa, BioAccord™ LC-MS System, SYNAPT™ XS and UV detection at 219 nm; waters_connect and UNIFI software for data acquisition, peptide mapping and MAM analysis.

Hlavní výsledky a diskuse

• PeptideWorks kit enables automatable preparation of 24 mAb digests in <2.5 h, reducing missed cleavages by over 90 % and minimizing autolysis, with consistent manual and robotic results.
• High-throughput Andrew+ workflow digests mAbs from microbioreactor samples (30 µg) in under 4 h, achieving reproducible CQA peptide quantification with <20 % RSD.
• ACQUITY Premier Columns with MaxPeak HPS eliminate non-specific adsorption of acidic peptides, improving recovery by 94 %, reducing tailing by 80 % and enhancing MS signal reproducibility.
• New insights into non-canonical PTMs and spatial proteomics highlight emerging methods for urea- and guanidine-resistant enrichment, antibody-based detection challenges and single-cell localization techniques.
• RapiZyme Trypsin accelerates traditional 3 h digestions (1:20 E:P) to 1 h (1:20 E:P) or 30 min (1:5 E:P) without autolysis; supports one-pot protocols in 0.6 M guanidine; and yields clean, high-coverage peptide maps.
• IonHance™ DFA as mobile phase additive provides adduct-free MS spectra, enhances UV sensitivity and retention of low-abundance peptides, and delivers stable baselines at 219 nm for QC-ready analyses.

Přínosy a praktické využití metody
These innovations reduce sample preparation time by up to 75 %, improve data clarity by eliminating unmatched peaks and metal-induced adsorption artifacts, and support both manual and automated high-throughput pipelines. They are directly applicable to QC release testing, multi-attribute monitoring and research in biopharma development.

Budoucí trendy a možnosti využití
Ongoing trends include integration of spatial proteomics with single-cell omics, AI-driven PTM prediction and database expansion, further miniaturization of workflows, and the convergence of imaging-based and LC-MS techniques for comprehensive protein localization and modification profiling. As column and enzyme technologies continue to evolve, streamlined MAM methods and real-time process monitoring will become standard in regulated environments.

Závěr
The combined use of RapiZyme Trypsin, IonHance DFA and advanced chromatographic hardware significantly accelerates and simplifies peptide mapping workflows without compromising data quality. Robust automation, rapid digestions and improved peak shapes meet the demands of modern biotherapeutic analysis and set new standards for reproducibility, sensitivity and throughput.

Reference
1. Mouchahoir T., Schiel J.E. Anal Bioanal Chem. 2018;410:2111–2126.
2. Ippoliti S. et al. Waters Application Note. 2023;720007840.
3. Finny A.S. et al. Waters Application Note. 2023;720007864.
4. Birdsall R. et al. Waters White Paper. 2020;720006930EN.
5. Yang H. et al. Waters Application Note. 2023;720007885.