Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

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.

Keywords: CrossMab; ESI-QTOF mass spectrometry; bispecific antibody; chain pairing; knobs-into-holes; quadroma.

Figure 1.

Quadroma and knobs-into-holes antibodies. Theoretical combinations and statistical distribution of quadroma and knobs-into-holes antibodies consisting of heavy chains A (blue) and B (red) and light chains a (cyan) and b (orange). The framed structures aABb (intended bispecific antibody, yellow background) and bABa are isobaric.

Figure 2.

ESI-QTOF MS of the quadroma antibody. Deconvoluted spectrum of (A) the deglycosylated, intact quadroma antibody demonstrating the presence of 9 different masses representing the 10 antibodies theoretically possible by combinations of 2 different heavy chains A (blue) and B (red), and 2 different light chains a (cyan) and b (orange). The intended bispecific antibody aABb (yellow background) and bABa are isobaric and thus cannot be distinguished by mass spectrometry of the intact antibody. (B) Deconvoluted spectrum of the deglycosylated, TCEP-reduced quadroma showing the presence of the 2 different heavy chains and 2 different light chains. Expected and determined average masses are listed in the upper right corners. *Without C-terminal Gly.

Figure 3.

ESI-QTOF MS of the knobs-into-holes antibody. Deconvoluted spectrum of (A) the deglycosylated, intact knobs-into-holes antibody demonstrating the presence of 3 different masses representing the 4 antibodies theoretically possible by combinations of the knob heavy chain A (blue), the hole heavy chain B (red), and 2 different light chains a (cyan) and b (orange). The intended bispecific antibody aABb (yellow background) and bABa are isobaric and thus cannot be distinguished by mass spectrometry of the intact antibody. (B) Deconvoluted spectrum of the deglycosylated, TCEP-reduced knobs-into-holes antibody showing the presence of the knob and hole heavy chains and 2 different light chains. Expected and determined average masses are listed in the upper right corners. *Without C-terminal Gly.

Figure 4.

ESI-QTOF MS of the CrossMab^Fabantibody. Deconvoluted spectrum of (A) the deglycosylated, intact CrossMab^Fab demonstrating the presence of the intended product aABb consisting of the knob heavy chain A (blue), the hole CL-VL “heavy chain” B (red-orange), the wt light chain a (cyan) and the CH1-VH “light chain” b (red), and hole-hole and knob-hole heavy chain dimer side-products. (B) Deconvoluted spectrum of the deglycosylated, TCEP-reduced CrossMab^Fab showing the presence of the 4 different chains. Expected and determined average masses are listed. *Without C-terminal Gly. ^#Phosphate adduct.