Educational guide
Speeding Up Peptide Mapping
In manufacturing biopharmaceuticals, companies must characterize the molecules to ensure efficacy and safety. For proteins, that characterization can be done with peptide mapping, which can be used to confirm the sequence of amino acids and reveal any post-tra
This guide cannot diagnose a condition or recommend a personal treatment plan. Discuss medical questions with a qualified professional.
In manufacturing biopharmaceuticals, companies must characterize the molecules to ensure efficacy and safety. For proteins, that characterization can be done with peptide mapping, which can be used to confirm the sequence of amino acids and reveal any post-translational modifications (PTMs).
To do this, companies take a time-consuming approach to digest the proteins and then analyze the resulting peptides with reversed-phase chromatography, but Jonathan Bones, PhD, an analytical chemist at the National Institute for Bioprocessing Research and Training in Dublin, Ireland, and his colleagues described a faster method, which automates the digestion by using magnetic beads.
Three members of the Bones lab—Silvia Millán-Martín, Pharm D, Sara Carillo, PhD, and Craig Jakes—teamed up to field questions about this work. They note: “Peptide mapping analysis offers the advantage of providing site-specific information regarding post-translational and chemical modifications that may arise during production, processing, or storage.”
As these scientists point out, some “PTMs can negatively impact potency, immunogenicity, and stability.” The most challenging PTMs, they say, “are asparagine deamidation and aspartic acid isomerization and glycosylation, which may potentially cause a decrease in antigen binding affinity, resulting in loss of potency or impact immunogenicity.” The scientists add that, “oxidation occurs frequently with methionine and tryptophan residues and results in decreased thermal stability, increased aggregation potential, and thus an increased immunogenicity risk.”
These researchers showed that a simplified combination of automated sample digestion followed by analysis with LC-MS—specifically, high-resolution mass-accurate mass spectrometry, or HRAM MS—can track multiple PTMs and confirm the amino-acid sequence. Plus, they even tested the workflow in four independent labs to confirm that other scientists can implement this method consistently in the real world.