Generation of Bispecific Antibodies Using FAST-Ig™

Robust generation of bispecific antibodies (BsAbs) in single-cell expression systems is essential for basic research and industrial manufacturing. However, the low yield of BsAbs and complicated downstream processing caused by the random assembly of heavy chains (HCs) and light chains (LCs) have limited the use of BsAbs in clinical and preclinical studies. Major technological improvements have been established to address the issues of HC homodimerization and noncognate HC and LC pairing. Nevertheless, current existing methods need to determine common light chains, extensively engineer each Fv portion or perform labor-intensive biochemical processing making downstream processing highly challenging. Here, we describe an engineering technology for preferential cognate HC/LC and HC/HC pairing called four-chain assembly by electrostatic steering technology-immunoglobulin (FAST-Ig^TM). When co-expressed in single host cells, parental monoclonal antibodies incorporating these interfaces can preferentially assemble into BsAbs. The remaining trace amount of mispairing antibodies can be easily removed via ion exchange chromatography. In this chapter, we provide a detailed protocol for the systematic generation of IgG-like BsAb designs based on the FAST-Ig™ technology in combination with knobs-into-holes technology to obtain highly purified BsAbs. Additionally, we present a detailed protocol for the analysis and characterization of target BsAbs.