Anouk Donners

31 Tutorial on LC-MS/MS methods quantifying mAbs Generally, these published methods share similarities as they all include steps depicted in Figure 3. The major differences between these methods are the selection of internal standard, sample processing and digestion conditions (Table 1). The merits and drawbacks of various options in each steps of method development will be discussed in next sections. Figure 3. General workflow to develop quantitative LC-MS/MS method to measure therapeutic mAbs. Signature peptide selection The first step in method development is selection of unique signature peptides to serve as surrogate for quantification. The peptide sequence of the therapeutic mAb is essential for this step. Sequences of approved therapeutic mAb can be found in the Immunogenetics Information system® ( or in Drugbank ( For the quantification of chimeric mAbs in human serum, tryptic peptides from the entire variable region can be chosen and targeted. However, for human or humanized mAbs the choice is limited to the complementarity-determining regions (CDRs) of which there are six in the variable light and heavy chains. In silico tryptic digestion can be performed manually or by using an online tool ‘Protein Prospector’ (http://prospector. The peptides generated can be screened online with protein Blast® software ( The peptide sequence is compared against the target organism (biological matrix of the analyte) from an appropriate database such as UniProtKB/Swiss-prot. Any peptide scoring under 100% for either the query cover or the positive identification, represents a unique peptide and as such a potential quantifier. Thereafter, a list of potential signature peptides can be screened for amino acid stability. Amino acids such as cysteine and methionine are susceptible to oxidation leading to mass shifts of +16, +32, +48 Da depending on the number of oxidation reactions. Asparagine followed by a smaller amino acid such as glycine can readily be deamidated into aspartic acid, isoaspartic acid and succinimide during tryptic digestion, causing a mass shift of +0.98, +0.98, and -17.03 Da respectively. Also N terminal glutamine cyclization can occur at alleviated pH and prolonged digestion time. Here, the loss of ammonia leads to a mass shift of -17.03 Da [87]. Preferably, these amino acids should be avoided. However, when stable alternatives are unavailable, these peptides can still be used if a stable isotopically labeled (SIL) internal standard is included. For example, infliximab, somatropin and nivolumab quantification with signature peptides containing a methionine were previously reported [47, 66, 68]. Also, endogenous insulin-like growth factor 1 was successfully quantified in serum with a signature peptide containing two cysteines that were protected from oxidation by attaching a carboxymethyl group on the thiol moiety through iodoacetamide alkylation and acid hydrolysis following disulfide bond 2