Standard-Free Absolute Quantitation of Antibody Deamidation Degradation and Host Cell Proteins by Coulometric Mass Spectrometry

Abstract

Proteomic absolute quantitation strategies mainly rely on the use of synthetic stable isotope-labeled peptides or proteins as internal standards, which are highly costly and time-consuming to synthesize. To circumvent this limitation, we recently developed a coulometric mass spectrometry (CMS) approach for absolute quantitation of proteins without the use of standards, based on the electrochemical oxidation of oxidizable surrogate peptides, followed by mass spectrometry measurement of the peptide oxidation yield. Previously, CMS was only applied for single-protein quantitation. In this study, first, we demonstrated absolute quantitation of multiple proteins in a mixture (e.g., β-lactoglobulin B, α-lactalbumin, and carbonic anhydrase) by CMS in one run, without using any standards. The CMS quantitation result was validated with a traditional isotope dilution method. Second, CMS can be used for absolute quantitation of a low-level target protein in a mixture; for instance, 500 ppm of PLBL2, a problematic host cell protein (HCP), in the presence of a highly abundant monoclonal antibody (mAb) was successfully quantified by CMS with no use of standards. Third, taking one step further, this study demonstrated the unprecedented quantitative analysis of deamidated peptide products arising from the mAb heavy chain deamidation reaction. In particular, absolute quantitation of the deamidation succinimide intermediate which had not been performed before due to the lack of standard was conducted by CMS, for the first time. Overall, our data suggest that CMS has potential utilities for quantitative proteomics and biotherapeutic drug discovery.

Figure 1

(a) Sequences of β-lactoglobulin B and α-lactalbumin (the chosen surrogate peptides VLVLDTDYK and LDQWLEK for CMS quantitation are highlighted in red); b) electric current diagrams were collected from oxidation of blank solvent (inset) and the digested protein sample after LC separation; MS spectra of LDQWLEK (c) without oxidation and (d) with oxidation (applied potential: +1.05 V). MS spectra of VLVLDTDYK (e) without oxidation and (f) with oxidation (applied potential: + 1.05 V). EICs of LDQWLEK are shown in (g) without oxidation and (h) with oxidation (applied potential: + 1.05V); EICs of VLVLDTDYK were acquired (i) without oxidation and (j) with oxidation (applied potential: + 1.05V).

Figure 2.

(a) Sequence of PLBL2 (the chosen surrogate peptide NPALWK is highlighted in red). MS spectra of NPALWK from the digested sample (mAb:PLBL2=200:1) (b) when the cell was off and (c) when the cell was turned on (applied potential: +1.05 V). The oxidation product of NPALWK was detected at m/z 371.70, m/z 372.71, and m/z 380.70. EICs of NPALWK were acquired (d) when the cell was off and (e) when the cell was turned on (applied potential: +1.05 V). Electric current diagrams were collected from oxidation of (f) a blank solvent and (g) NPALWK.

Figure 3.

(a) Sequence of NIST 8671 light chain and heavy chain (the chosen N318 surrogate peptide VVSVLTVLHQDWLN₃₁₈GK from HC is highlighted in red). EICs of (b) unmodified peptide VVSVLTVLHQDWLN₃₁₈GK, (c) deamidated peptides VVSVLTVLHQDWLisoD₃₁₈GK and VVSVLTVLHQDWLD₃₁₈GK, and (d) succinimide intermediate VVSVLTVLHQDWLSuc₃₁₈GK. Electric oxidation current diagrams are shown due to the oxidation of (e) a solvent blank and (f) the mAb digest. MS spectra of succinimide intermediate VVSVLTVLHQDWLSuc₃₁₈GK was recorded (g) without oxidation and (h) with oxidation (applied potential: +1.05 V).

Figure 4.

Comparison of quantitation results for the deamidated products (a) and the succinimide intermediate (b) as measured based on EIC peak areas and CMS absolute quantitation

Scheme 1.

Mechanism of asparagine deamidation to aspartic acids via a succinimide intermediate

Scheme 2.

Workflows showing absolute quantitation for a) multiple proteins in a protein mixture, b) HCPs in mAb , and c) mAb deamidation by CMS.