Intact molecular weight analysis of the product can also be used as an orthogonal procedure to support conclusions regarding the structures of the N- and C-termini.
This includes the generation of confirmatory peptide fragment ions to confirm the nature of the C-terminal peptide or peptides. In the latter approach, intactness of a protein C-terminus or the presence of ragged ends can be assessed using data obtained from a peptide map. For an assessment of the C-termini, BioPharmaSpec scientists use carboxypeptidase digestion and/or mass-spectrometric mapping strategies. There is no fully analogous method akin to Edman chemistry for determining the C-terminal sequencing of a biopharmaceutical. Leucine and isoleucine derivatives have unique chromatographic elution positions and can thus be categorically identified. In these cases, peptides containing Leucine or Isoleucine can be purified and subjected to N-terminal sequencing using Edman chemistry as described above. However, since Leucine and Isoleucine are isomers (they both have the same mass), they cannot be categorically identified by mass. Mass spectrometry can provide much useful structural information as part of an investigation of this nature by generating amino acid sequence information from chromatographically separated peptides. Techniques must be used that can unambiguously define the sequence. As mentioned above, it is a regulatory requirement for the protein sequence to be determined (see Q6B section 6.1.1 a)). This last use of N-terminal sequencing is very important.
Unambiguously defining the Isoleucine and Leucine residues within the protein sequence.Demonstrating batch-to-batch consistency.Showing that the N-terminus of your protein is intact and as expected.N-terminal sequence analysis has a number of different applications in drug development such as:
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