Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry.
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.
a Roche Pharma Research and Early Development; Large Molecule Research, Roche Innovation Center Penzberg ; Penzberg , Germany.
Wolfgang
W
Schaefer
a Roche Pharma Research and Early Development; Large Molecule Research, Roche Innovation Center Penzberg ; Penzberg , Germany.
Hans R
HR
Völger
a Roche Pharma Research and Early Development; Large Molecule Research, Roche Innovation Center Penzberg ; Penzberg , Germany.
Stefan
S
Lorenz
a Roche Pharma Research and Early Development; Large Molecule Research, Roche Innovation Center Penzberg ; Penzberg , Germany.
Sabine
S
Imhof-Jung
a Roche Pharma Research and Early Development; Large Molecule Research, Roche Innovation Center Penzberg ; Penzberg , Germany.
Jörg T
JT
Regula
b Oncology Discovery & Translational Area, Roche Innovation Center Zurich ; Schlieren , Switzerland.
Christian
C
Klein
a Roche Pharma Research and Early Development; Large Molecule Research, Roche Innovation Center Penzberg ; Penzberg , Germany.
Michael
M
Mølhøj
D018033:Antibodies, Bispecific / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D057809:HEK293 Cells; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000276:immunology; D013058:Mass Spectrometry; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000276:immunology
D018033:Antibodies, Bispecific; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D011994:Recombinant Proteins
CrossMab; ESI-QTOF mass spectrometry; bispecific antibody; chain pairing; knobs-into-holes; quadroma