A Novel Redox Method for Rapid Production of Functional Bi-Specific Antibodies For Use in Early Pilot Studies

Abstract

We demonstrate here a rapid alternative method for the production of functional bi-specific antibodies using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA). Following reduction of a mixture of two monoclonal antibodies with MESNA to break inter heavy chain bonds, this solution is dialysed under oxidising conditions and antibodies are allowed to reform. During this reaction a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. Following purification, bi-specificity of antibodies is determined in enzyme-linked immunosorbent assays and by flow cytometry. Using this redox method we have been successful in producing hybrid and same-species bi-specific antibodies in a time frame of 6–10 working days, making this production method a time saving alternative to the time-consuming traditional heterohybridoma technology for the production of bi-specific antibodies for use in early pilot studies. The use of both rat and mouse IgG antibodies forming a rat/mouse bi-specific antibody as well as producing a pure mouse bi-specific antibody and a pure rat bi-specific antibody demonstrates the flexibility of this production method.

A Murine, Bispecific Monoclonal Antibody Simultaneously Recognizing β-Glucan and MP65 Determinants in Candida Species

“…The construction of the MP65/bglu mAb relied upon a rapid method based on reduction-oxidation of the parental IgGs [ 19 ]. As illustrated in Fig 1 , the method exploits the mild reducing agent 2-mercaptoethanesulfonic acid (MESNA) to break the inter-heavy chain bonds of the parental IgGs and to reduce them to monovalent mAbs (mvAbs).…”

“…The 2G8/4C8 bsmAb was prepared by adapting the redox method described by Carling et al [ 19 ]. To reduce the parental mAbs to monovalent antibody (mvAb), 2-mercaptoethanesulfonic acid sodium salt (Sigma-Aldrich, USA) was diluted in H 2 O and added to 1 mg of mAb 2G8 or to 1 mg of mAb 4C8, both diluted in PBS, pH 7.2, at the final concentration of 10 mM (mAb 2G8) or 30 mM (mAb 4C8).…”

A detergent-based procedure for the preparation of IgG-like bispecific antibodies in high yield

“…Nonetheless, efforts to refine the chemical procedures to obtain BsAbs continue, including those in combination with recombinant procedures29. More recently a simple procedure for the production of BsAbs from a mixture of monoclonal antibodies by reduction of inter-heavy chain disulfides followed by their reoxidation was described16. The procedure subsequently employed to generate BsAbs both from monoclonal and polyclonal antibodies5 however gives poor yields.…”

“…Chemical conjugation procedures are more efficient in producing bispecifics formed from antigen binding fragments like the Fab’s, rather than in the generation of IgG like BsAbs, because of the presence of strong interactions between the Fc regions of the two heavy chains that interfere with the dissociation of the two half molecules and consequently in the formation of the bifunctionals15. More recently a redox procedure has been described by Carlring et al 16. in which a mixture of two different antibody molecules is subjected to reduction under the conditions causing the selective disruption of inter-heavy chain thiols followed by reoxidation of the thiols, to generate BsAbs in moderate yield.…”

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

“…19,20 Similarly, full-length bispecific IgG antibodies can be generated from two IgG antibodies by reduction/oxidation followed by affinity chromatography using the respective antigens. 19,20 However, these approaches do not allow the large scale supply of bispecific antibodies in qualities required for clinical trials; thus, ideally, the desired bispecific heterodimeric IgG antibody is produced in one cell line. Figure 1 illustrates the basic challenge in generating bispecific heterodimeric IgG antibodies from 4 antibody chains (2 different heavy and 2 different light chains) in one expression cell line, the so-called chain association issue.…”