Production of F(ab′)2 from Monoclonal and Polyclonal Antibodies

Abstract

Antibodies are widely used in therapeutic, diagnostic, and research applications, and antibody derivatives such as F(ab′)2 fragments are used when only a particular antibody region is required. F(ab′)2 can be produced through antibody engineering, but some applications require F(ab′)2 produced from an original formulated antibody or directly from a polyclonal antibody pool. The cysteine protease immunoglobulin-degrading enzyme (IdeS) from Streptococcus pyogenes digests immunoglobulin G (IgG) specifically and efficiently to produce F(ab′)2. Here we detail the production and purification of recombinant IdeS; its utilization to digest monoclonal or polyclonal antibodies to F(ab′)2 fragments; and F(ab′)2 purification through consecutive affinity chromatography steps. The resultant F(ab′)2 exhibit high purity, retain antigen-binding functionality, and are readily utilizable in various downstream applications. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Production and purification of F(ab′)2 fragments from monoclonal and polyclonal antibodies using IdeS. Alternate Protocol: Purification of polyclonal antigen–specific F(ab′)2 fragments from human serum or secretions. Support Protocol: Production and purification of IdeS.