Peptide Mapping & PTM Analysis | ICH Q6B Compliant

As part of our protein characterization services, BioPharmaSpec provides a peptide mapping service enabling you to meet the structural characterization requirements of the ICH Q6B guidelines, understand the primary structure of your protein, assess post translational modifications (PTMs) and make key development decisions based on this knowledge.

Examples of post-translation modifications (PTMs) to protein primary structure

Peptide Mapping of Biosimilars

Peptide mapping is an essential fingerprint tool during initial characterization of your biosimilar and subsequent biosimilar-to-innovator comparability studies.

Data obtained from on line LC ES/MS analysis of a protease digest of a Monoclonal Antibody Reference Product and three batches of Biosimilar

In the above biosimilar to reference product comparability study that BioPharmaSpec performed for a client, it can be seen that Biosimilar batches 2 and 3 differ from the Reference Product around the 12-minute mark. The mass spectrometric data generated from the peptide map was used to investigate the structure of these components and orthogonal tests such as icIEF were then be performed to corroborate the reasons for differences in the peptide mapping data.

Peptide Mapping Service

Peptide mapping analysis procedures at BioPharmaSpec are tailored to your molecule, but follow the basic steps outlined below:

1. Reduction and alkylation of the protein 
   • Reduction of S-S bridges and blocking of the generated free thiol opens the protein structure and allows proteolytic digestion to be more efficient. In the case of multi-chain proteins which have disulfide bridges linking the chains (e.g. monoclonal antibodies) this step will also break the bridges between the chains.
2. Digestion of the protein using specific enzymes
   • This step breaks the protein down into peptides. Enzymes  are chosen based on the theoretical sequence so this informed choice of enzymes(s) results in the best peptide mapping analysis.

1. Analysis of the digested peptides using on-line RP-HPLC with UV and LC/ES-MS.

• • LC separates the peptides based on polarity (reverse phase-LC).
  • The Mass Spec generates mass information for each peptide as it elutes from the LC column. Fragment ion information is also generated, allowing the primary sequence to be determined which serves to confirm peptide identity. The mass spectrometers at BioPharmaSpec are also capable of generating fragmentation information from the peptides as they enter the MS source from the LC column. This is either selective (MS/MS) or non-selective (MSe), depending on the requirements of the study and can be considered as a form of tandem mass spectrometry.

Multiple Applications of Peptide Mapping

Our scientists will apply peptide mapping mass spec techniques to your product to  provide detailed information on the primary structure of the protein including:

Our Global Laboratory Locations

Wherever you are, BioPharmaSpec offers access to high-quality analytical services through our labs in the United States, United Kingdom, Germany, Italy, and Lithuania.

Need peptide mapping advice?

Peptide mapping is at the core of our comprehensive protein mass spectrometry services. Talk to one of our scientists today about the best peptide mapping strategies for your molecule.