Bioproduction and characterisation of recombinant anti-CRP antibody

Abstract

C-reactive protein (CRP) is an acute-phase protein mainly synthesised and secreted by hepatocytes in response to inflammation. Serum CRP level is a sensitive marker for systemic inflammation and is widely used to distinguish between bacterial and viral infection and to evaluate the risk of suffering from cardiovascular diseases. The wide application of the CRP test creates a large demand for high-quality immunoassays. As one of the key materials for immunoassays, anti-CRP antibodies with a high production yield as well as high batch-to-batch consistency is well needed. Traditionally, monoclonal anti-CRP antibodies are produced by hybridomas. The yield of hybridoma culture is usually below 40 mg/L and the accumulated mutation will result in loss of binding affinity or decline in yield. Thus, it is of significance to set up a platform to produce anti-CRP antibodies with high yield, and more importantly, high batch-to-batch consistency. In this study, a platform producing recombinant anti-CRP antibodies was developed. Using the complementary DNA (cDNA) from a previously established anti-CRP antibody hybridoma cell line 7E12 as the template, a pair of sequences from the heavy variable domain and light variable domain were obtained through cloning and amplicon next-generation sequencing. Sequences were identified by the international ImMunoGeneTics information system (IMGT) database. The recombinant anti-CRP antibody is designed with the mouse IgG1 Fc region and the mouse Igκ constant region as the heavy chain and light chain constant region separately. The anti-CRP antibody expression level was compared by different cistron and leading sequences arrangements. Co-expressing heavy chain and light chain by monocistronic vectors which is led by Igκ signal sequence showed the highest anti-CRP antibody expression level. ExpiCHO transient expression system, which was supplied by ThermoFisher Scientific company, was introduced for anti-CRP antibody production, with a highly improved yield to over 90 mg/L after affinity purification using protein G sepharose. The binding affinity of the recombinant anti-CRP antibody was measured and compared with the hybridoma-derived antibody by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) measurement. ELISA suggested that the recombinant anti-CRP antibody and hybridoma-derived anti-CRP antibody showed similar sensitivity within the range of 0-50 ng/ml. The SPR measurement revealed that the dissociation constants (KD) values of the recombinant anti-CRP antibody and the hybridoma-derived anti-CRP antibody were 1.08×10-8 M and 1.30×10-8 M, respectively. Hence, the binding affinity of the recombinant anti-CRP antibody is similar to that of the hybridoma-derived anti-CRP antibody. Meanwhile, the two antibodies exhibited similar performance in western blotting when used as primary antibodies at concentrations of 0.2 μg/ml and 1.0 μg/ml. The recombinant anti-CRP antibody was further applied to high-sensitivity CRP turbidimetric immunoassay (ITA). Using world health organization (WHO) international standard for human CRP (85/506) as the testing sample, the assay showed accurate readouts. To sum up, the platform established in the study can produce a 2 to 3 times higher yield of antibodies than hybridomas. The produced recombinant antibody shows similar binding affinity and function in immunoassays with the hybridoma-derived one. Hence, it is a suitable approach for in-house large-scale production.