AMINO ACID SEQUENCE AND PEPTIDE MAPPING
For a mAb, the EMA guideline and ICH Q6B request a deduction of the amino acid sequence from the DNA sequence and confirmation
of the DNA-derived sequence experimentally by appropriate methods (e.g., peptide mapping, amino-acid sequencing, mass spectrometry
analysis). In addition, the variability of the N-terminal amino acid sequences (whether a free amino acid or a pyroglutamic acid is present) and the C-terminal amino-acid sequences (e.g. the presence or absence of C-terminal lysine(s) on the heavy chain) should be analyzed.
Peptide mapping (i.e., analysis of specific protease digests of the mAb followed by analysis of the products using on-line
RP-HPLC with ultraviolet [UV] and electrospray mass spectrometric detection [LC/ES–MS]) provides molecular weight information
for the peptides released from the mAb by the protease of choice. The data obtained are able to provide mapping confirmation
(or otherwise) of the DNA-derived sequence but does not provide confirmation of the sequence of the light and heavy chains
of the mAb. To provide confirmation at the primary amino acid sequence level, a number of protease digests combined with on-line
RP–HPLC with tandem MS/MS (LC/ES–MS/MS) analysis are required. ICH Q6B does not actually request sequencing at the primary
amino acid level. There is a move, however, initiated by some of the regulatory bodies, to provide confirmation of the primary
protein sequence for new mAb products and also provide data showing comparability of the the primary amino acid sequence of
mAb biosimilar products to a reference product.
As mass spectrometric-based sequencing is unable (in most cases) to differentiate between isoleucine and leucine (these amino
acids have the same molecular weight), automated N-terminal sequencing of purified peptides is required for unambiguous assignment of these two amino acids, particularly within
the variable regions of the light and heavy chains.
TERMINAL AMINO ACID SEQUENCE
Terminal amino acid analysis is performed to identify the amino- and carboxy-terminal amino acid sequence(s) of the light
and heavy chains of a mAb. If the product exhibits more than one terminal amino acid sequence, the relative amounts of the
termini should be determined.
Automated N-terminal sequencing following separation of the light and heavy chains using sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE) and blotting onto polyvinylidene fluoride (PVDF) membrane is used routinely for analysis of the N-termini of mAb light and heavy chains. Automated N-terminal sequencing uses Edman chemistry which requires a free amino functionality at the N-terminus of a protein for labeling prior to cleavage of the N-terminal and subsequent amino acids. This means that for a number of mAbs (which have a pyroglutamic acid at the N-terminus and therefore no free amino group), N-terminal sequencing using this method will not provide sequence information. In this case pyroglutaminase can be used to
enzymatically remove the N-terminal pyroglutamic acid residue before N-terminal sequencing.
There is no fully reliable method analogous to Edman chemistry for determining the C-terminal amino acid sequence for a biological product. Information relating to the C-terminal sequence of a peptide or protein can be obtained using carboxypeptidase digestion and/or mass-spectrometric mapping
strategies. 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 and intact molecular weight
analysis of the product.
SULFHYDRYL GROUP(S) AND DISULFIDE BRIDGES
During a peptide mapping/sequencing analysis, free sulfhydryl groups and disulfide bridges should also be considered. Peptide
mapping post-digestion with a specific protease, followed by analysis using on-line LC/ES–MS or LC/ES–MS/MS prior to/and following
reduction, provides the data necessary for a full assessment of disulfide bridges and free thiols within the mAb. Care must
be taken when digesting proteins containing free thiols at basic pH as the free thiol can initiate scrambling within the digest,
and the result obtained may therefore not be consistent with the true disulfide bridge/free thiol pattern within the product.