Transient and stable CHO expression, purification and characterization of novel hetero‐dimeric bispecific IgG antibodies

Abstract

IgG bispecific antibodies (BsAbs) represent one of the preferred formats for bispecific antibody therapeutics due to their native-like IgG properties and their monovalent binding to each target. Most reported studies utilized transient expression in HEK293 cells to produce BsAbs. However, the expression of biotherapeutic molecules using stable CHO cell lines is commonly used for biopharmaceutical manufacturing. Unfortunately, limited information is available in the scientific literature on the expression of BsAbs in CHO cell lines. In this study we describe an alternative approach to express the multiple components of IgG BsAbs using a single plasmid vector (quad vector). This single plasmid vector contains both heavy chain genes and both light chain genes required for the expression and assembly of the IgG BsAb, along with a selectable marker. We expressed, purified, and characterized four different IgG BsAbs or "hetero-mAbs" using transient CHO expression and stable CHO minipools. Transient CHO titers ranged from 90 to 160 mg/L. Stable CHO titers ranged from 0.4 to 2.3 g/L. Following a simple Protein A purification step, the percentage of correctly paired BsAbs ranged from 74% to 98% as determined by mass spectrometry. We also found that information generated from transient CHO expression was similar to information generated using stable CHO minipools. In conclusion, the quad vector approach represents a simple, but effective, alternative approach for the generation of IgG BsAbs in both transient CHO and stable CHO expression systems. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:469-477, 2017.

Design and Production of Bispecific Antibodies

“…The CHO cell pools transiently transfected with two plasmid vectors of which one heavy chain and a light-chain pairing of the bispecific antibody were harbored on individual plasmids yielded 0.09–0.15 g/L with 73–92% correctly paired bispecific antibody as determined by mass spectrometry. The cell lines transiently transfected with a single plasmid vector containing all the components for bispecific antibody generated similar yields of correctly paired bispecific antibody [183]. However, a stable CHO pool with a single plasmid expression resulted in a higher titer ranging from 0.6 up to 2.2 g/L while the percentage of correct pairing ranged from 74 to 98% [183].…”

“…The cell lines transiently transfected with a single plasmid vector containing all the components for bispecific antibody generated similar yields of correctly paired bispecific antibody [183]. However, a stable CHO pool with a single plasmid expression resulted in a higher titer ranging from 0.6 up to 2.2 g/L while the percentage of correct pairing ranged from 74 to 98% [183]. Their results indicated that a single plasmid system could be comparable to multiple plasmid system in terms of titers and it may facilitate the generation of stable CHO cells [183].…”

Computational stabilization of T cell receptors allows pairing with antibodies to form bispecifics

“…Protein expression and purification. α/β constructs were co-transfected for transient expression in either 24 well plates (1 mL scale) or individual flasks (100-200 mL scale) as described previously 43 . Briefly, plasmids were transfected in CHO K1SV KO cells maintained in a DMEM-based medium with 8-12 mM L-Glutamine (LM-Growth, SAFC Cat#59202C-100).…”

Computational design of a specific heavy chain/κ light chain interface for expressing fully IgG bispecific antibodies

“…For IgG BsAb production, four plasmids individually harboring each of the 2 HC and 2 LC DNA inserts were transfected using 1:3 HC:LC plasmid ratios in HEK293F cells and using 1:1 HC:LC plasmid ratios for CHO as described previously . For both HEK293 and CHO, secreted protein material was harvested by centrifugation at 5 K rpm for 5 min at the end of the culture period.…”

“…Ideally, fully IgG BsAbs could be generated from two naturally occurring IgGs within a single mammalian cell line, which is the current standard for mAb manufacturing . This would dramatically reduce the complexity and cost of their production.…”