Glu-C – an orthogonal and alternative enzyme for protein quantitation by LC-MS/MS

To date, almost all protein LC-MS/MS based bioanalysis has involved the use of trypsin to cleave biopharmaceuticals into manageable peptides for quantitation. In some cases, where trypsin does not generate useful peptide sequences, a different cleavage agent is required. Glu-C is an enzyme that is frequently used for protein characterisation, and cuts proteins on the C-terminal side of the aspartic and glutamic acid residues. These two molecules are acidic amino acids, whereas the ubiquitous trypsin cleaves at the basic residues (lysine and arginine), therefore making this an orthogonal cleavage enzyme.

Here, we present a Glu-C digestive enzyme method for detecting and determining the amount of a human or humanized antibody of interest in pre-clinical animal biological samples including tissue, plasma, serum or cells.  Application of Glu-C enzyme for quantitative LC-MS/MS analysis was demonstrated with an IgG1 monoclonal antibody (mAb) biopharmaceutical, where human framework peptide sequences were used to monitor mAb levels in rat and mouse plasma.  Linearity, accuracy and precision were assessed, a heavy chain framework peptide (LLGGPSVFLFPPKPKDTLMISRTPE) providing satisfactory performance with %RE < 9% and %CV < 7%.

In monkey serum, a more complex bispecific antibody was assessed.  Here, two light chain framework peptides, one for kappa (AKVQWKVDNALQSGNSQE) and one for lambda (TTTPSKQSNNKYAASSYLSLTPE) light chains, were monitored, achieving %RE < 6% and %CV < 17% in accuracy and precision assessments. A LLOQ of 1 µg/mL was achieved, a performance comparable to a trypsin digestion approach. In summary, Glu-C digestion provides an efficient alternative means for quantification of biopharmaceuticals in biological samples.

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