Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry. Issue 1 (2nd January 2016)

• Record Type: Journal Article
• Authors: Schaefer, Wolfgang; Völger, Hans R; Lorenz, Stefan; Imhof-Jung, Sabine; Regula, Jörg T; Klein, Christian; Mølhøj, Michael
• Is Part Of: MAbs. Volume 8:Issue 1(2016)
• Page Start: 49
• Page End: 55
• Publication Date: 2016-01-02
• Subjects: bispecific antibody -- CrossMab -- chain pairing -- ESI-QTOF mass spectrometry -- knobs-into-holes -- quadroma; Monoclonal antibodies -- Therapeutic use -- Periodicals; Monoclonal antibodies -- Periodicals; Antibodies, Monoclonal -- Periodicals; 616.0798
• DOI: 10.1080/19420862.2015.1111498
• Languages: English
• ISSNs: 1942-0862

Abstract

Abstract : The quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.