Tuning Relative Polypeptide Expression to Optimize Assembly, Yield and Downstream Processing of Bispecific Antibodies

Abstract

Bispecific antibodies (bsAbs) are often composed of several polypeptide chains that have to be expressed adequately to enable optimal assembly and yield of the bsAb. κλ bodies are a bispecific format with a native IgG structure, composed of two different light chains that pair with a common heavy chain. Introduction of non-optimal codons into the sequence of a particular polypeptide is an effective strategy for down modulating its expression. Here we applied this strategy but restricted the modification of the codon content to the constant domain of one light chain. This approach facilitates parallel optimization of several bsAbs by using the same modified constant domains. Partial sequence de-optimization reduced expression of the targeted polypeptide. Stable cell pools could be isolated displaying increased bispecific antibody titers as well as changes in the abundance of undesired by-products that require elimination during downstream processing. Thus, modulating the relative expression of polypeptides can have a significant impact on bsAb titer and product related impurities; which are important factors for large scale manufacturing for clinical supply.

The Confluence of Innovation in Therapeutics and Regulation: Recent CMC Considerations

“…For example, for hetero-IgG-based BsAb constructs formed from 2 distinctive heavy chain subunits and 2 distinctive light chain subunits, random assembly during synthesis can result in 16 unique combinations, in which only two represent the desired product. 4,5 Additionally, purification, analytics, and characterization of BsAb molecules presents additional challenges. 6 Therefore, manufacturing of bispecific antibodies requires both sophisticated protein engineering and formulation for the creation of high quality and consistent products.…”

Vector design for enhancing expression level and assembly of knob-into-hole based FabscFv-Fc bispecific antibodies in CHO cells

“…Chinese hamster ovary (CHO) cells are the dominant host cells for producing therapeutic antibodies due to their advantages of robust cell growth and capability of performing proper post-translational modifications [ 11 ]. Studies to optimize the relative expressions of multiple chains for enhancing titre and assembly of BsAbs have been done in CHO cells in transient transfections [ 9 , 10 , 12 , 13 ]. Co-expression of multiple chains is often achieved by co-transfecting CHO cells with multiple plasmids, with one plasmid expressing one chain.…”

Expression liabilities in a four‐chain bispecific molecule

“…The four heteroIgG chains are often engineered to promote correct heavy‐heavy chain and heavy‐light chain pairing through electrostatic or steric steering (Atwell et al, 1997; Gunasekaran et al, 2010; Liu et al, 2015; Ridgway et al, 1996; Strop et al, 2012). However, imperfect steering and expression level differences between each polypeptide chain can result in undesired product‐related contaminants such as half antibodies (one HC and one LC paired, either correctly or incorrectly), free light chain monomer or dimer, and up to nine incorrect four‐chain species (Gomez et al, 2018; Magistrelli et al, 2018). For instance, excess LC1/HC1 over LC2/HC2 can produce either homodimer (LC1/HC1‐HC1/LC1) or half antibody (LC1/HC1).…”

“…The ideal ratio to support high productivity and product quality may not necessarily be stoichiometric. Factors that should be considered in determining the ideal chain ratio include chain–chain interactions as well as chain‐specific synthetic kinetics, folding/assembly kinetics, secretion rates, and degradation (Davies et al, 2011; Magistrelli et al, 2018; Schlatter et al, 2005). For monoclonal antibodies (mAbs), it is well accepted that expression of excess light chain over heavy chain improves IgG secretion (Bhoskar et al, 2013; Magistrelli et al, 2018; Schlatter et al, 2005).…”

“…Factors that should be considered in determining the ideal chain ratio include chain–chain interactions as well as chain‐specific synthetic kinetics, folding/assembly kinetics, secretion rates, and degradation (Davies et al, 2011; Magistrelli et al, 2018; Schlatter et al, 2005). For monoclonal antibodies (mAbs), it is well accepted that expression of excess light chain over heavy chain improves IgG secretion (Bhoskar et al, 2013; Magistrelli et al, 2018; Schlatter et al, 2005). Antibody polypeptide chains, synthesized separately by ribosomes, translocate to the endoplasmic reticulum (ER) for posttranslational modification, folding, and maturation (Pobre et al, 2019; M. Wang & Kaufman, 2016).…”