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

Schaefer, W.; Völger, Hans Rainer; Lorenz, Stefan; Imhof-Jung, Sabine; Regula, Jörg T.; Klein, Christian; Mølhøj, Michael

doi:10.1080/19420862.2015.1111498

Cited by 31 publications

(31 citation statements)

References 38 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Second, we assumed that the BsIgG should have a higher or equal percentage than the light chain-scrambled IgG. This second assumption is consistent with previous reports that some coexpressed antibody pairs show significant preference for pairing of cognate heavy and light chains, 16 and other antibody pairs have little (if any) preference for cognate chain pairing 24,33 . There are no reports (to our knowledge) of co-expressed antibodies exhibiting a preference for non-cognate heavy and light chain pairing.…”

Section: Resultssupporting

confidence: 78%

Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

Yin¹,

Han²,

Zhou³

et al. 2016

mAbs

View full text Add to dashboard Cite

Bispecific IgG are heterotetramers comprising 2 pairs of heavy and light chains. Co-expression of the 4 component chains in a single host cell typically yields the desired bispecific IgG plus up to 9 additional incorrect chain pairings. Several protein engineering strategies have been reported to facilitate the heterodimerization of antibody heavy chains or cognate pairing of antibody heavy and light chains. These technologies have been used to direct the efficient assembly of bispecific IgG in single host cells and minimize unwanted chain pairings. When purifying bispecific IgGs, the identification and quantification of low levels of closely related IgG contaminants are substantial analytical challenges. Here we have developed a robust high-throughput method for quantitative analysis of bispecific IgG preparations using novel online liquid chromatography in conjunction with an extended mass range Orbitrap-based high-resolution mass spectrometer. A mathematical method was developed to estimate the yields of the 2 isobaric species, namely the desired bispecific IgG and the light chain-scrambled IgG. The analytical methods described herein are anticipated to be broadly applicable to the development of bispecific IgG as drugs and potentially to other complex next-generation biotherapeutics.

show abstract

Section: Resultssupporting

confidence: 78%

Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

Yin¹,

Han²,

Zhou³

et al. 2016

mAbs

View full text Add to dashboard Cite

show abstract

“…For some control BsIgG such as anti-HER2/CD3 and anti-VEGFA/ANG2, the yield of BsIgG was 22-24%, 21 consistent with random pairing of HC and LC as previously observed by others. 23 In contrast, high BsIgG yield, arbitrarily defined here as >65%, was observed for other control bispecifics such as anti-EGFR/MET and anti-IL-4/IL-13 BsIgG, consistent with a cognate HC/LC pairing preference. 21 Examples of elevated BsIgG yield without Fab mutations have also been observed by others.…”

Section: Introductionmentioning

confidence: 81%

Elucidating heavy/light chain pairing preferences to facilitate the assembly of bispecific IgG in single cells

Joshi¹,

Phung²,

Han³

et al. 2019

mAbs

View full text Add to dashboard Cite

Multiple strategies have been developed to facilitate the efficient production of bispecific IgG (BsIgG) in single host cells. For example, we previously demonstrated near quantitative (≥90%) formation of BsIgG of different species and isotypes by combining 'knob-into-hole' mutations for heavy chain heterodimerization with engineered antigen-binding fragments (Fabs) for preferential cognate heavy/light chain pairing. Surprisingly, in this study we found high yield (>65%) of BsIgG 1 without Fab engineering to be a common occurrence, i.e., observed for 33 of the 99 different antibody pairs evaluated. Installing charge mutations at both C H 1/C L interfaces was sufficient for near quantitative yield (>90%) of BsIgG 1 for most (9 of 11) antibody pairs tested with this inherent cognate chain pairing preference. Mechanistically, we demonstrate that a strong cognate pairing preference in one Fab arm can be sufficient for high BsIgG 1 yield. These observed chain pairing preferences are apparently driven by variable domain sequences and can result from a few specific residues in the complementarity-determining region (CDR) L3 and H3. Transfer of these CDR residues into other antibodies increased BsIgG 1 yield in most cases. Mutational analysis revealed that the disulfide bond between heavy and light chains did not affect the yield of BsIgG 1. This study provides some mechanistic understanding of factors contributing to antibody heavy/light chain pairing preference and subsequently contributes to the efficient production of BsIgG in single host cells.

show abstract

“…Historically, the fundamental issue in the generation of bispecific heterodimeric/asymmetric IgG antibodies has been the random association of heavy and light chains. 1,2 While the correct heavy chain heterodimerization was enabled early on using the knobs-into-holes (KiH) approach, 3 the correct association of generic light chains has remained a problem for decades. 2 Since 2011, when we described the CrossMAb technology as a method to enforce correct light chain association in bispecific heterodimeric IgG antibodies, 4 this technology has proven to be one of the most versatile antibody engineering technologies, allowing the generation of various bispecific antibody formats, including bi- (1+1), tri- (2+1) and tetra-(2+2) valent bispecific antibodies, as well as non-Fc tandem antigen-binding fragment (Fab)-based antibodies.…”

Section: Introduction To Crossmab Technologymentioning

confidence: 99%

The use of CrossMAb technology for the generation of bi- and multispecific antibodies

Klein

Schaefer

Regula

2016

mAbs

Self Cite

150

112

View full text Add to dashboard Cite

The major challenge in the generation of bispecific IgG antibodies is enforcement of the correct heavy and light chain association. The correct association of generic light chains can be enabled using immunoglobulin domain crossover, known as CrossMAb technology, which can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology or electrostatic steering. Since its development, this technology has proven to be very versatile, allowing the generation of various bispecific antibody formats, not only heterodimeric/asymmetric bivalent 1+1 CrossMAbs, but also tri- (2+1), tetravalent (2+2) bispecific and multispecific antibodies. Numerous CrossMAbs have been evaluated in preclinical studies, and, so far, 4 different tailor-made bispecific antibodies based on the CrossMAb technology have entered clinical studies. Here, we review the properties and activities of bispecific CrossMAbs and give an overview of the variety of CrossMAb-enabled antibody formats that differ from heterodimeric 1+1 bispecific IgG antibodies.

show abstract

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

Cited by 31 publications

References 38 publications

Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

Precise quantification of mixtures of bispecific IgG produced in single host cells by liquid chromatography-Orbitrap high-resolution mass spectrometry

Elucidating heavy/light chain pairing preferences to facilitate the assembly of bispecific IgG in single cells

The use of CrossMAb technology for the generation of bi- and multispecific antibodies

Contact Info

Product

Resources

About