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. Nov-Dec 2018;10(8):1236-1247.
doi: 10.1080/19420862.2018.1505398. Epub 2018 Sep 20.

Characterization and Analysis of scFv-IgG Bispecific Antibody Size Variants

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Free PMC article

Characterization and Analysis of scFv-IgG Bispecific Antibody Size Variants

Mingyan Cao et al. MAbs. .
Free PMC article

Abstract

Bispecific antibodies are an emergent class of biologics that is of increasing interest for therapeutic applications. In one bispecific antibody format, single-chain variable fragments (scFv) are linked to or inserted in different locations of an intact immunoglobulin G (IgG) molecule to confer dual epitope binding. To improve biochemical stability, cysteine residues are often engineered on the heavy- and light-chain regions of the scFv to form an intrachain disulfide bond. Although this disulfide bond often improves stability, it can also introduce unexpected challenges to manufacturing or development. We report size variants that were observed for an appended scFv-IgG bispecific antibody. Structural characterization studies showed that the size variants resulted from the engineered disulfide bond on the scFv, whereby the engineered disulfide was found to be either open or unable to form an intrachain disulfide bond due to cysteinylation or glutathionylation of the cysteines. Furthermore, the scFv engineered cysteines also formed intermolecular disulfide bonds, leading to the formation of highly stable dimers and aggregates. Because both the monomer variants and dimers showed lower bioactivity, they were considered to be product-related impurities that must be monitored and controlled. To this end, we developed and optimized a robust, precise, and accurate high-resolution size-exclusion chromatographic method, using a statistical design-of-experiments methodology.

Keywords: appended scFv-IgG bispecific antibody; bispecific antibody; cysteinylation; glutathionylation; size variant.

Figures

Figure 1.
Figure 1.
SEC chromatograms of Bis-A on an Aquity BEH SEC-200 column. (A) SEC of Bis-A affinity purified Protein A product pool. (B) SEC overlay of enriched fractions 1 (black), 2 (red), and 3 (blue).
Figure 2.
Figure 2.
Intact and reduced mass analysis of Bis-A enriched fractions. (A1) Mass spectrum of intact fraction 1; (A2) mass spectrum of intact fraction 2; (A3) mass spectrum of intact fraction 3; (B1) intact mass of fraction 1; (B2) intact mass of fraction 2; (B3) intact mass of fraction 3 (B3); (C1) ion chromatogram of reduced fraction 1; (C2) ion chromatogram of fraction 2; (C3) ion chromatogram of fraction 3.
Figure 3.
Figure 3.
IdeS digestion scheme and masses of (Fab′)2 and Fc/2 after IdeS digestion. (A1) (Fab′)2 of fraction 1; (A2) (Fab′)2 of fraction 2; (A3) (Fab′)2 of fraction 3; (B1) Fc/2 of fraction 1 after IdeS digestion; (B2) Fc/2 of fraction 2 after IdeS digestion; (B3) Fc/2 of fraction 3 after IdeS digestion; (C1) Fc/2 of fraction 1 after IdeS/PNGase F/CpB digestion; (C2) Fc/2 of fraction 2 after IdeS/PNGase F/CpB digestion; (C3) Fc/2 of fraction 3 after IdeS/PNGase F/CpB digestion.
Figure 4.
Figure 4.
UV chromatograms of nonreduced peptide mapping. (A) Fraction 1; (B) fraction 2; (C) fraction 3.
Figure 5.
Figure 5.
Mass spectra of peptides with cysteinylation on Cys442 at 29.5 min and cysteinylation on Cys640 at 41.9 min in fraction 2. (A) MS spectrum of peak at 29.5 min with in-source fragments; (B) MS2 spectra of parent ion 597.79(+ 2) at 29.5 min; (C) MS spectrum of peak at 41.9 min with in-source fragments; (D) MS3 spectrum from fragment ion Y9 of peak at 41.9 min.
Figure 6.
Figure 6.
Total ion chromatograms of the three fractions and deconvoluted masses of the Fc/2 dimer in fraction 3 after IdeS/PNGase F/CpB digestion. (A) Ion chromatograph of fraction 1; (B) ion chromatograph of fraction 2; (C) ion chromatograph of fraction 3; (D) deconvoluted mass of Fc/2 dimer in fraction 3.
Figure 7.
Figure 7.
Putative head-to-tail structure of dimer molecule.
Figure 8.
Figure 8.
SEC separation of Bis-A under different conditions on an Aquity BEH SEC-200 column. Shown are SEC separations on (A) 150 × 4.6–mm column with150 mM sodium phosphate, pH 6.8, mobile phase; (B) 150 × 4.6–mm column with 750 mM NaCl, 150 mM sodium phosphate, pH 6.8, mobile phase; and (C) 300 × 4.6–mm column with 750 mM NaCl, 150 mM sodium phosphate, pH 6.8, mobile phase.

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Grant support

This study was supported by MedImmune, the global biologics R&D arm of AstraZeneca.
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