For pharmaceutical proteins, it is a requirement that the protein N-terminals should be analyzed in the ICH Q6B Guideline. And the information should be used throughout all stages of drug development, to demonstrate comparability and consistency of lots during manufacturing.
▶ Edman sequencing
Developed by Pehr Edman more than 60 years ago, the Edman sequencing reaction is a cyclic procedure where the N-terminal amino acid residue is cleaved off and identified by chromatography. It is by far the most important method in N-terminal sequence analysis.
It involves stepwise degradation of peptides with phenyl isothiocyanate, starting at the N-terminus of the polypeptide. In the first step, N-terminal amino acid is converted to phenylthiocarbamoyl derivative by phenylisothiocyanate under mild alkaline conditions. Under acidic conditions, this derivative of the N-terminal amino acid is then cleaved as a thiazolinone derivative. Finally, the the thiazolinone amino acid is converted to the more stable phenylthiohydantoin amino acid derivative after extraction, which can be identified with using chromatography. A standard mixture of 19 PTH-amino acids is also injected onto the column for separation as the first cycle of the sequencing run, which offers standard retention times of the amino acids for comparison with each Edman degradation cycle chromatogram.
▶ N/C-terminal sequencing (LC-MS/MS)
Edman sequencing has its own disadvantages which cannot deliver sequence information from blocked N-termini and C-terminus which is essential for complete characterizatiom. This drawback can be overcomed by mass spectrometry analysis.
ICH Q6B guidelines suggest “new analytical technology and modifications to existing technology are continually being developed and should be utilized when appropriate”. So APTBIO can also offer N/C-terminal sequencing with LC-MS/MS platform.