Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

Abstract

2020) Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis, mAbs, 12:1, 1739825, ABSTRACT Recently, cation exchange chromatography (CEX) using aqueous volatile buffers was directly coupled with mass spectrometry (MS) and applied for intact analysis of therapeutic proteins and antibodies. In our study, chemical modifications responsible for charge variants were identified by CEX-UV-MS for a monoclonal antibody (mAb), a bispecific antibody, and an Fc-fusion protein. We also report post-CEX column addition of organic solvent and acid followed by mixing at elevated temperatures, which unfolded proteins, increased ion intensity (sensitivity) and facilitated top-down analysis. mAb stressed by hydrogen peroxide oxidation was used as a model system, which produced additional CEX peaks. The on-line CEX-UV-MS top-down analysis produced gas-phase fragments containing one or two methionine residues. Oxidation of some methionine residues contributed to earlier (acidic), some to later (basic) eluting peaks, while oxidation of other residues did not change CEX elution. The abundance of the oxidized and non-oxidized fragment ions also allowed estimation of the oxidation percentage of different methionine residues in stressed mAb. CEX-UV-MS measurement revealed a new intact antibody proteoform at 5% that eluted as a basic peak and included paired modifications: high-mannose glycosylation and remaining C-terminal lysine residue (M5/M5 + K). This finding was confirmed by peptide mapping and on-column disulfide reduction coupled with reversed-phase liquid chromatographytop-down MS analysis of the collected basic peak. Overall, our results demonstrate the utility of the on-line method in providing site-specific structural information of charge modifications without fraction collection and laborious peptide mapping. ARTICLE HISTORY

Identification of critical chemical modifications by size exclusion chromatography of stressed antibody-target complexes with competitive binding

“… 22 We have also performed peptide mapping and characterized the main proteoform in each CEX peak. 29 The potency measurements of the collected CEX fractions showed that the main CEX peak had a potency of 140%, while the CEX peak characterized as HC D102 isomerization only had a potency of 12–30%. 22 A potency of 98% was found for the second acidic peak on the CEX profile of antibody that mainly contains LC N30 deamidation.…”

“…CEX-UV was performed as described previously. 29 For peptide mapping, the SEC fractions were collected into an Eppendorf tube with the presence of 200 µL denature solution containing 6 M guanidine hydrochloride, 0.2 M Tris, 2 mM EDTA, and 20 mM methionine (pH 3.5) to minimize artificial oxidation caused by collection. SEC runs and fraction collection were carried out in triplicate.…”

“…Detailed digestion and LC-MS/MS analysis procedures have been described elsewhere. 29 , 61 Corresponding fractions from each run were transferred onto a 500 µL Microcon filter (30 kDa MWCO, Sigma-Aldrich, St. Louis, MO) and were dried by spinning for 15 min at 14,000 × g. Then, each fraction was dissolved in the denature solution with 10 mM dithiothreitol for reduction at 37°C for 30 min, followed by alkylation (10 mM iodoacetamide) in dark at room temperature for 30 min. The filter of each fraction was further washed with digestion solution (0.1 M Tris, 20 mM methionine, and 5% acetonitrile (ACN)) three times to remove the residual denature solution.…”

“…As an example, trastuzumab and its target, human epidermal growth factor receptor-2 (HER2), were studied as a model system using competitive binding SEC followed by peptide mapping. Prior collection and study of CEX fractions 22 , 29 and also online CEX-mass spectrometry (MS) studies 28 revealed the presence of three main attributes in trastuzumab: light chain (LC) N30 deamidation, heavy chain (HC) N55 deamidation, and HC D102 isomerization. According to the sequence analysis and available crystal structure, 30 all three residues are located in the complementarity-determining regions (CDRs) of trastuzumab and within close distance to HER2.…”

Toward Drug-Like Multispecific Antibodies by Design

“…Fortunately, a combined top-down fragmentation and cation-exchange chromatography method coupled with mass spectrometry has been developed for the site-specific identification of chemical variants that result in charge differences, specifically methionine oxidation. Furthermore, this technology was applied to the analysis of a bispecific antibody [ 129 ]. One notable advantage of the top-down methodology is the reduced need for peptide mapping, which is a major drawback of previous strategies.…”