Construction, expression, and purification of single-chain antibodies (scFv-Fc) antibodies

Introduction

The clinical development of bispecific antibodies (BsAb) as therapeutic drugs hinders the clinical development of drugs due to the difficulty of preparing sufficient quantity and quality materials by traditional methods. Here, we describe an effective method for producing new bispecific antibody fragments by genetically fusing single-stranded Fv (scFv) to light-chain C-end syltos or fab fragments bound with different antigens. Bispecific Fab-scFv fragments are expressed in a single E. coli host and purified to homogenization through a step affinity chromatography. Two tyrosine kinase receptors of the endothelial growth factor of the blood vessel were constructed with two different versions of the bispecific Fab-scFv fragment. These bispecific antibody fragments not only retain the antigen binding ability of each parent antibody, but also bind to both targets at the same time, as shown in cross-linked ELISA. In addition, bispecific antibodies are as effective as their parent antibodies in blocking ligands from binding to receptors and inhibiting biological activity caused by ligands.

Our service

We have developed a technical platform for mammalian cells to express and produce antibody Fab fragments. The recombinant antibody Fab fragments produced by our technology have superior quality over the antibody Fab fragments produced by full antibody enzyme lysis.

ScFv (Single Chain Fragment Variable) antibody expression and production is a routine effort to support antibody library antibody discovery. ScFv protein can be easily expressed in microbial expression systems such as E. coli. It can also be expressed in mammalian cells through transient transfection. The Chinese organism is able to produce ScFv single-chain antibody fragments using any production system according to the customer's preferences.

Process

Service procedures	Specification	Timeline	Deliverables
Gene synthesis and codon optimization (optional)		1-2 weeks	• Purified antibody • SDS-PAGE data • CoA
Vector construction	• Expression vector construction • Plasmid sequencing • Large scale plasmid production	1-2 weeks
Expression & purification	• Transfection of HEK293 / CHO cells or E.coli. fermentation • Purify the antibody • Polishing if needed	1-2 weeks
QC analysis	• Analysis by SDS-PAGE and UV • Purity analysis by HPLC-SEC and endotoxin level analysis (optional)	3 days