Laboratory Testing Blog

Peptide Mapping - An Essential Analytical Development Technique

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Developing new drugs, especially monoclonal antibody drugs can be a very complex and time-consuming process.  It's no wonder that the failure rate of these types of drugs is higher than conventional compounds.

Peptide mapping is an essential technique for studying the primary structure of proteins.  For recombinant proteins, peptide mapping is used for the initial ‘proof of structure’ characterization; for example: to confirm the expression of the desired amino acid sequence and to characterize any post-translational modifications.  Furthermore, peptide mapping is employed for subsequent lot-to-lot identity testing or ‘finger-printing’ in support of bio-process development and clinical trials.  Peptide mapping is also the current method of choice for monitoring the ‘genetic stability’ of recombinant cell lines.  

Normally, amino acid sequence analysis by peptide mapping at the protein level includes complex instrumentation and procedures

The simples approach is that a mAb is digested with various endoproteases and these peptide fragments are analyzed by UPLC-UV/MS.  This is a regular amino acid sequence confirmation procedure for the known entity when the sequence is clearly identified and you want to confirm the primary structure of the mAb.  So, it's best if the sequence is first identified and understood with the digest procedures documented.  Once that is accomplished, the peptide mapping (separation) method can be developed and confirm the amino acid sequence coverage.

However, you want to establish a validated peptide mapping method for identity testing and process monitoring, then it can become quite complicated and needs a much more complex approach.

For instance:

• Reproducible Tryptic and Lys-C digestion procedures and stability must be established    

• Develop an optimal peptide mapping method - can be UPLC-UV

• Amino acid analysis by ACQUITY UPLC, AccQ.Tag per column derivatization method and Florescence or UV detection

• SDS-PAGE light and heavy chain purity

• LC-ESI-MS or UPLC-MS/MS LC:ESI-MS is very valuable in that it gives a large amount of m/z information across the entire map from a single instrument run.  However, sifting through all this MS data can be laborious and identification by MS alone can sometimes be difficult or ambiguous without some other independent data

• Reverse-phase HPLC-UV separation (purification) carboxymethylated light chain and heavy chain species  

• MALDI-TOF MS of the isolated peaks gcan provide the expected m/z values for the light and heavy chain species  

• N-terminal sequencing (Edman degradation) of isolated peaks

• If identification of a modified protein is required, then work will becomes even more complicated.

  So, there is a difficulty to estimate the time line and amount of work that is required.  It can be very unpredictable and may not be easy to achieve.