A cell line development strategy to improve a bispecific antibody expression purity in CHO cells

Among antibody-based therapies, bispecific antibodies (BsAbs) have gained momentum in preclinical and clinical investigations following the regulatory approvals of the T-cell engaging BsAb blinatunomab and the FVIII cofactor mimicking BsAb emicizumab. Production of BsAbs with high purity is one of the challenges in commercializing BsAb therapeutics. Therefore, several protein engineering approaches have been developed to increase the correct assembly of the desired target product. However, few reports investigated the cell line development strategies in further improving the expression purity of BsAbs. In this paper, we investigated the plasmid transfection ratio and pool selection approach to enhance the purity of an IgG-like BsAb containing a heavy chain (H), a light chain (L), and a ScFv-Fc polypeptide (ScFv-Fc). Two plasmids were used to express HL and ScFv-Fc, respectively. A two-stage pool selection method to favor ScFv-Fc expression increased the expression purity of the BsAb from 44.5%–88.6%. The single clones isolated from the best pool after the second stage of pool selection could produce the BsAb with tripled titer and similar purity to its parental pool.