Drug development and production rely on stringent quality controls, reproducibility, and verification. Biopharmaceuticals, including protein-based therapies, require extensive characterization, standardization, and batch validation to ensure safety and efficacy. Peptide mapping provides a precise and reliable means to verify and analyze the structure of protein biopharmaceuticals.
What is Peptide Mapping?
Protein mapping is a high-resolution analytical technique that enables the identification of the amino acid sequence of a protein, along with any chemical modifications to those amino acids. A typical peptide mapping workflow includes:
- Enzymatic digestion - The target protein is digested into peptide fragments by proteolytic enzymes, commonly trypsin. The protein is broken at specific sites to generate predictable and reproducible patterns of peptide fragments.
- Separation by chromatography - Peptide fragments are then separated, often using liquid chromatography (LC), most commonly reverse-phase LC, enabling high resolution and detection of individual peptides.
- Analysis by mass spectrometry (MS) - Separated peptides are analyzed by MS to determine their masses/spectra. Data are compared to a proteomic database to confirm or characterize their identity.
Together, these steps provide a detailed molecular fingerprint of the protein, allowing precise characterization of its structure and modifications.
Quality Control in Drug Development
Peptide mapping is an indispensable tool for quality control in drug development and production. Protein therapeutics represent the largest class of biopharmaceuticals, and therapies using monoclonal antibodies (mAbs) or antibody-drug conjugates (ADCs) have emerged as leaders in new approvals and sales. Due to the biological nature of antibody production, they are inherently heterogeneous and pose challenges to reproducibility and storage.
Regulatory agencies require extensive analytical characterization at each stage of the drug development and throughout the manufacturing pipeline to ensure consistency, stability, and safety. Therefore, peptide mapping plays an important role during protein-based pharmaceutical R&D. It is used to verify the amino acid sequence to ensure the protein matches its intended composition and structure. Peptide mapping can also identify potential impurities, enabling the monitoring of lot-to-lot consistency and contamination in antibody production processes.
Importantly, peptide mapping also identifies post-translational modifications, which can significantly impact therapeutic function. Common modifications include:
- Glycolysation - The addition of sugar moieties influences protein folding, solubility, and immunogenicity.
- Methionine or tryptophan oxidation - Oxidative alterations can affect binding affinity and shelf life
- Asparagine deamination - The conversion of asparagine to aspartate or isoaspartate can lead to charge variants that significantly change antibody efficacy and stability
- C-terminal clipping - Loss of lysine residues at the C-terminus can similarly lead to charge variants with altered binding affinity and stability.
These post-translational modifications can all significantly influence protein folding, antigen binding, immunogenicity, and shelf stability. Subtle changes in post-translational modifications can disrupt function and alter pharmacokinetics. As such, peptide mapping supports robust structure-function correlation.
Read about how peptide mapping was incorporated into a high-throughput antibody purification process here.
Incorporating peptide mapping into high-throughput purification and production pipelines is essential for efficient biopharmaceutical development. Advanced platforms now enable automation and faster turnaround, making it feasible to routinely implement peptide mapping during early-stage development and scale-up for manufacturing.
Cytiva is a global supplier of chromatography resins and provides extensive support for scientists at every stage of the drug discovery pipeline. Explore the full range of available products to see how you can boost your liquid chromatography capabilities for peptide mapping and achieve more precise, insightful results in your workflows.
