Why Cell Line Construction Important in Recombinant Antibody Production?

In general, polyclonal antibodies are made by collecting blood from an immunized animal stimulated with a target antigen, which can be provided all the time as long as the animal is still alive, while monoclonal antibodies are produced continuously by immunizing the host animal with the target protein and then extracting B cells that recognize and respond to the antigen and fuse them with myeloma cells to become permanent culturable cells, widely used in laboratory research, diagnostic products, and immunotherapy.

Compared to polyclonal antibodies, monoclonal antibodies have multiple advantages, featured by strong specificity for a single epitope, little or no variability, and the easy operation to modify and customize in terms of different needs, thereby turning out to be the most suitable candidate for recombinant antibodies (rAbs). The rAbs are obtained through sequencing of the animal's immune cells and inserting the gene into a suitable cell strain, such as bacterium, yeast, and mammalian cells. Even if the original cell strain dies or is mutated, the desired cell strain can be generated by gene insertion since the antibody is definitive.

The recombinant antibody production firstly requires the high-quality construction of production cell line. Current expression levels of engineering cell lines used for recombinant antibody production internationally can reach 20-70pcd and a variety of animal cells have been applied into the antibody production according to their distinct characteristics. In recent years, the development of engineering cell lines mainly focused on cell engineering techniques that improve the growth and expression ability of the host, site-directed integration techniques that overcome the position effects during the random vector integration, and flow cytometry and high-throughput screening that enhance the screening throughput and efficiency of recombinant cells.

The complex antibody structure of tetrameric glycoprotein and the multi-step extracellular expression both pose a potential challenge to a perfect antibody production process. Only after translation, folding, assembly and glycosylation of both light and heavy chains, can the antibody be equipped with biological activity. It has been proved that the post-translational modification of recombinant antibodies by host cells directly affects the clinical efficacy and immunogenicity of antibody drugs.

In the commonly used heterologous protein expression systems, E. coli can only express scFv or antibody fragments instead of a whole antibody molecule; yeast cells, insect cells, and transgenic plants, etc., due to the lack of post-translational modification ability, present significantly difference compared to human antibody. Hence, only mammalian cells are suitable for heterologous expression of recombinant antibodies, in which CHO, myeloma cells, hybridoma cells, PerC.6, HEK293 and others have been practically used for recombinant antibody production.

There are a series of requirements on the engineered cell line used for antibody drug production, such as good growth characteristics, high density of serum-free suspension culture, strong expression ability of heterologous protein with correct post-translational modification ability, clear genetic background and stable phenotype of host and recombinant cell line, in line with relevant regulatory requirements.

At present, DHFR and GS systems are two most common screening systems in the construction of cell lines in the industry, in which after transfection of plasmids carrying target genes and screening markers (Dhfr, GS, or other resistance genes neo, hygro) into host cells, recombinant cells are selected using corresponding media. To expand the expression level of recombinant cells, multiple rounds of selective pressure can be used to increase the target gene copy number. After suspension and serum-free acclimatization, the growth ability, expression level and product quality of the candidate clones are comprehensively evaluated in the reactor. Finally, the master cell bank and working cell bank are established in compliance with cGMP requirements.

Based on a comprehensive cell line, the recombinant antibodies produced in an animal-free system can be widely used in medical research, as well as the preparation of pharmaceutical compounds to treat a variety of diseases, even cancers.