Peptide Mapping

Biopharmaceutical peptide mapping

Peptide mapping is a critical workflow in biotherapeutic protein characterization and is essential for elucidating the primary amino acid structure of proteins. For recombinant protein pharmaceuticals, such as monoclonal antibodies (mAbs) and antibody-drug conjugates (ADCs), peptide mapping is used for proof of identity, primary structural characterization and quality assurance/quality control (QA/QC). Global regulatory agencies, including US Food and Drug Administration (US FDA) and European Medicines Agency (EMA), look to harmonized guidelines from the International Council for Harmonisation (ICH). ICH Q6B  covers the test procedures and acceptance criteria for biologic drug products, and specifies the use of peptide mapping as a critical quality test procedure for drug characterization used to confirm desired product structure for lot release purposes.

Comprehensive peptide maps, simplified

In order to generate a peptide map, the therapeutic protein must first be digested into its constituent peptides via a chemical or enzymatic reaction. Robust separation and identification of the resultant peptides then provides insight into a protein’s full sequence information; displaying each amino acid component and the surrounding amino acid microenvironment, including disulfide linkage information. Structural characterization at this level highlights post translational modifications (PTMs) such as site-specific glycosylation, amino acid substitutions (sequence variants) and/or truncations which may result from erroneous transcription of complementary DNA. Within a bioproduction environment, peptide mapping is necessary for manufacturing process monitoring and QC. It facilitates product comparability testing, which is necessary to identify any product-related impurities, such as deamidation and/or oxidation following any formulation, manufacturing process or storage change.

Due to its complexity and inherent variability, peptide mapping is generally performed in a comparative manner; for example, biosimilars would be compared to a reference or control substance, such as the innovator biologic, in a side-by-side experiment. An in-depth analysis is then required to identify minor and even isobaric differences in protein primary structure. The modern biopharmaceutical and protein research laboratory is tasked with providing high quality analytical results, often in high-throughput, regulated environments. Some technologies currently employed for biopharmaceutical peptide mapping are subject to high levels of irreproducibility, poor sensitivity, and high levels of time-consuming manual work—with protracted methodologies that are not amenable to automation and often require 24 hours to achieve full protein digestion.

This variability impacts data confidence. Moreover, it increases potential for introduction of non-product related artifacts during manual sample handling.

The most recent advances in protein sample preparation chemistries, ultra-high performance liquid chromatography (UHPLC), mass spectrometry (MS) hardware and intuitive software facilitate the generation of comprehensive, confident peptide maps.

Peptide mapping products and workflows ›

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AppsLabAppsLab Peptide Mapping eWorkflows
Application NotePeptide Mapping of Challenging Monoclonal Antibodies
Application BriefHigh Resolution, High-Precision Peptide Mapping of a Monoclonal Antibody Digest
Application NoteComparison of Alternative Approaches to Trypsin Protein Digestion for Reproducible and Efficient Peptide Mapping Analysis of Monoclonal Antibodies
Application NoteHigh-throughput Peptide Mapping of Trastuzumab using a Tandem LC-MS Workflow
Application NoteAutomated Biopharmaceutical Protein Digestion Optimization for Peptide Mapping Method Development
Application NoteHigh-precision, automated peptide mapping of proteins
Application NoteFast Digestion Method Optimization Using Innovative Trypsin Technology
Application NoteHigh-Throughput Peptide Mapping with the Vanquish UHPLC System and the Q Exactive HF Mass Spectrometer
Application NoteImpact on Tryptic Digestion when Detergents and Chaotropes are Essential: A Case Study with Ribonuclease A
Application NoteImprovement in Speed and Reproducibility of Protein Digestion and Peptide Quantitation, Utilizing Novel Sample Preparation Technology in a Full Solution Workflow
Application NoteIncreased Efficiency of Biomolecule Identification by Optimization of Trypsin Digestion Buffers
Application NoteIncreased Long-term Stability of Peptide Mapping Using the Vanquish UHPLC System
Application NoteLC-UV-MS Peptide Mapping Development for Easy Transfer to LC-UV QA/QC
Application NoteIncreasing Sensitivity in HCP Analysis: from Simplified Sample Preparation to Reliable Results
Application NoteReliable Results in Peptide Mapping Using the Vanquish Flex UHPLC System
Application NoteSelecting Buffers to Remove Uncertainty in Tryptic Digestion
Application NoteHILIC – an alternative separation technique for glycopeptides
Application NoteInvestigating Process-Related Post-Translational Modifications in NISTmAb RM 8671 Using High-Throughput Peptide Mapping Analysis
ArticleDevelopments in Peptide Mapping Technology for the Biopharmaceutical Industry
ArticleColumn Temperature Control in Peptide Mapping. Maximizing Peak Capacity by Keeping Your Column Hot the Right Way.
ArticleHigh-Throughput, High-Resolution Peptide Maps
BlogAdmiring the Architecture of the Building Blocks of Life
BlogAhoy! Do I Need Satellite Navigation for my Peptide Map?
BlogFinding HOPE Using Peptide Mapping?
BlogNot All Who Wander Are Lost
BlogWhat’s faster? Digesting a protein, or reading this blog?
BlogBioPharMoore: Unravelling Biopharmaceutical Protein Digestion
BrochurePeptide Mapping Workflows: Upgrade Your Maps
BrochureVanquish Flex UHPLC System: Built for BioPharma
BrochureBioPharma Finder Software - Brochure
BrochureQ Exactive BioPharma
eBookPutting Peptides on the Map
eBookSimpler, Faster and More Reproducible Protein Digestion
Online ToolSMART Digest Kits - Digest Time Calculator
PosterDifferentiate Minor Difference of Protein Structure in Biosimilar and Reference Products Using High-Resolution Orbitrap LC-MS/MS
Poster NoteCharacterization of Monoclonal Antibodies and ADCs using a Benchtop Orbitrap Mass Spectrometer
Poster NoteComprehensive Sequence and Post-translational Modifications Analysis of Monoclonal Antibody by Flash Digest and LC-High Resolution MS
Poster NoteDegradation Profiling of a Monoclonal Antibody Using Multiple Fragmentation Techniques and a Novel Peptide Mapping Software
Poster NoteDifferentiate Minor Difference of Protein Structure in Biosimilar and Reference Products Using High-Resolution Orbitrap LC-MS/MS
Poster NoteDisulfide Bond Analysis on Orbitrap Mass Spectrometry
Poster NoteRapid Peptide Mapping via Automated Integration of On-line Digestion, Separation and Mass Spectrometry for the Analysis of Therapeutic Proteins
Poster NoteComplete Characterization of a Cysteine linked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass Range
Poster NoteOrbitrap Based Mass Spectrometric Characterization of Antibody Drug Conjugates Engineered through Antibody Glycans
Poster NoteComplete Characterization of a Cysteinelinked Antibody-Drug Conjugate Performed on a Hybrid Quadrupole-Orbitrap Mass Spectrometer with High Mass Range
Application NoteRobust and reproducible peptide mapping and intact mass analysis workflows on a single instrument platform
Poster NoteCharacterization of Monoclonal Antibodies and ADCs using a Benchtop Orbitrap Mass Spectrometer
Poster NoteFull Characterization of Heterogeneous Antibody Samples Under Denaturing and Native Conditions on a Hybrid Quadrupole-Orbitrap Mass Spectrometer
Poster VideoConvenient High pH Peptide Fractionation
Poster VideoHigh throughput Peptide Mapping by UHPLC-MS
Poster VideoImproved Protein Identification using SubCellular Localization
Poster VideoOrbitrap Based Mass Spectrometric Antibody Drug Conjugate Characterization
PresentationNew, rapid and reproducible peptide mapping workflows
Product SpotlightSMART Digest Kit Q&A
Product SpotlightSMART Digest Soluble Trypsin Kit Smarter protein digestion
Technical NoteHigh Resolution Peptide Mapping for Biopharmaceutical Analysis
Web PageBuilt For Biopharma Hypersite - Peptide Mapping
Web PagePeptide Mapping Webpage
WebinarImproved Characterization of Biotherapeutics with Better Peptide Mapping
Web PageBioPharma Finder Software - Mass Spectrometry - Orbitrap
Web PageBioPharma Finder Software - Product page