Structural characterization of expressed monoclonal antibodies by single sample mass spectral analysis after IdeS proteolysis

Abstract

Simple and rapid methods for analysis of monoclonal antibody structure and post-translational modifications are increasingly needed due to the explosion of therapeutic monoclonal antibodies and monoclonal antibody applications. Mass spectral analysis is a powerful method for characterizing monoclonal antibodies. Recent discovery and commercialization of the Immunoglobulin G-degrading enzyme of Streptococcus pyogene (IdeS protease) has facilitated and improved the generation of antibody fragments of suitable size to allow characterization of the structure of the entire antibody molecule via analysis of just a few fragments. In this study, we coupled IdeS fragmentation and simultaneous reduction and alkylation of the resultant fragments using tributylphosphine and iodoacetamide to prepare samples in about 2 hours. Following simple introduction of a single, unseparated mixture of alkylated fragments into a mass spectrometer, detailed structural information is obtained, covering the entire antibody molecule. The large majority of the glycoforms present on the single, conserved N-linked glycosylation site of the heavy chain is elucidated, although some of the very low abundance glycoforms are not determined by this protocol. The ease, simplicity, speed, and power of this method make it attractive for analysis of the developmental stages and production batches of therapeutic monoclonal antibodies.

Characterization of a recombinant humanized anti-cocaine monoclonal antibody produced from multiple clones for the selection of a master cell bank candidate

“…The selection of a single clone is a critical step in establishing this cell bank [10]. It was shown, using the same material as in the current study, that post-translational modifications (glycosylation) can vary between h2E2 produced by different clones (cell lines) with different production yields [11]. The effects of these differences in glycosylation on the pharmacokinetics of monoclonal antibodies are unclear, since contradictory results have been obtained in previous studies [11, 12].…”

Selective disulfide reduction for labeling and enhancement of Fab antibody fragments

“…To confirm and further investigate the selectivity of reduction of the single Fab disulfide bond, the selectively reduced Fab was alkylated with IAM, following our established procedures for quantitative alkylation and preparation of the sample for mass spectral analyses [8]. Two independent samples were analyzed on different days, sample 1 was reduced for 24 h, and had 2.7 cys/Fab, while sample 2 was reduced for 17 h and had 1.9 cys/Fab fragment prior to alkylation.…”

“…Light chain (LC) = 22,748.6 (for the 215 amino acids) − 17.03 (for the N -terminal Gln to pyroGlu [8]) − 4.04 Da (4 × 1.01 (for 2 intact internal disulfides)) + 57.05 (from one IAM) = 22,784.6 Da (compared to the observed mass of the main peak seen in Fig. 3 of 22,785.9 Da, they differ by + 1.3 Da).…”

“…To separate iodoacetamide from the Fab fragment after reduction and alkylation for mass spectral analysis, Sephadex G-25-80 size exclusion beads were from Sigma, and disposable columns were from BioRad (Econo-Pac 14 cm–high 1.5 × 12 cm polypropylene columns, cat. # 7321010EDU), equilibrated with 0.1% formic acid just before use, as previously described [8]. Tris base, NaCl, EDTA, and other buffer components, as well as sequencing grade concentrated formic acid, were from Fisher Scientific.…”

“…The IAM alkylated Fab samples were analyzed by electrospray ionization time-of-flight mass spectrometry (ESI-Tof MS), as described previously [8]. Briefly, samples were diluted by transferring 1 µL of the concentrated sample in 0.1% formic acid into 19 µL of 50% methanol/0.1% formic acid.…”