Abstract

In pharmaceutical companies, the production of glycoproteins is achieved in cell transient or stable gene expression systems. When fast delivery and economical cost are required, transient expression is still the first choice for protein production, which skips the lengthy screening step and simply introduces the plasmid containing the target gene into the cell. It is faster and more efficiently. The rate of protein synthesis depends on many factors, such as the efficiency of transfection, the cytotoxicity of the transfection reagent, and the feeding strategy during culture. Therefore, although the transient turnover is not stable, the protein expression level is high. In fact, since the plasmid is transiently outside the chromosome, cell division is prone to loss plasmid, which limits protein production. So far, gene therapy by viral plasmids has mainly been completed by transients. A stable gene expression system is a better choice when performing large-scale glycoprotein production. In order to improve protein yield, process tolerance and reduce cell culture time, various efforts and optimizations have been made in these systems.



Screening system
In order to improve the rate of protein synthesis and screening efficiency, many screening systems have been developed in recent years. In the stable expression system, when the target cDNA is integrated into the plasmid, a gene marker is often added. The copy number of the plasmid and the loci integrated into the host gene are the key factors in stabilizing the gene expression system. There are two common screening markers in biopharmaceuticals, the glutamine synthetase (GS) and the dihydrofolatereductase (DHFR). The GS screening system was originally developed by Celltech (Lonza) and was obtained by a glutamine auxotrophic complement strain constructed from recombinant GS genes. Since NS0 and Sp2/0 only express a small amount of endogenous GS, it is only necessary to remove glutamine from the culture medium to meet the screening conditions. In the CHO cell line, in order to increase the screening pressure of the GS screening system, it is necessary to add methionine sulfoximine (MSX), a glutamic acid analog to the culture medium, and MSX can inhibit endogenous glutamine activity. To increase the performance of this screening system, Eli Lilly has developed a glutamine synthetase knockout cell line (KO) in the CHO cell line. Similarly, DHFR-deficient strains were also constructed in CHO cells to establish a DHFR screening system. The recombinant DHFR gene is integrated into a plasmid, and the constructed cell strain is cultured in a medium free of nucleotides and a DHFR enzyme activity inhibitor (methotrexate), and cultured with the concentration of methotrexate. By this, the copy number of the target gene is increasing. In addition to the two screening systems mentioned above, there are other screening systems, such as OSCAR, but so far only DHFR and GS screening systems have been used in large scale production.

Gene expression
The screening system mentioned above can achieve our goal, but the plasmid containing the target gene is randomly integrated into the host gene. This random integration not only causes inconsistency in cell population, but also leads to different level of protein expression by various cell lines. This transgene is likely to be introduced into the heterochromatin region, resulting in low levels of gene expression. It is a tedious task to screen cell lines that integrate plasmids into the nuclear chromatin region from a large number of cell pools. So, a proper gene editing tool will greatly increase efficiency. Currently, a variety of molecular and cellular biological tools have been integrated for specific gene loci, which will help biopharmaceutical companies reduce the randomness of gene integration and prediction of highly expressed cell lines. Although it takes more work to isolate high-yielding and stable cell lines than random integration strategies, this is still a more attractive approach because it can control and predict the expression levels of progeny clones. When the target gene and the selection marker are integrated into the nuclear chromatin region on the same plasmid, the probability of increased expression of the target gene and the marker increases.

The first generation of gene tools, using specific recombinases (Cre/Lox and Flp/FRT) to specifically knockout target DNA. Recombinase-mediated cassette exchange technology (RMCE) is favored by companies because it can target gene inserts. RMCE utilizes recombinase to recognize differences in DNA recognition site mutants through two relatively independent recombination reaction. RMCE improves the construction success rate of CHO cells and shortens the cell construction time.

Among the second generation of gene tools, different types of endonucleases, such as zinc finger ribonuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9, are employed. These endonucleases can induce double-strand breaks at specific locus in the host DNA, thereby facilitating integration or homologous direct repair of the plasmid by non-homologous end joining. Both ZFN and TALEN technologies can add specific DNA sequences in the nuclease effect region, so they can develop a DNA recombination sequence with high specificity and homology. Since the gene sequence contains numerous repeats and highly homologous DNA sequences, a protein-based gene editing technique has been applied to increase endonuclease specificity and reduce off-target effects, which is CRISPR/Cas9. It is composed of Cas9 protein, tracrRNA and crRNA, and is an RNA-directed gene editing technology that has been used in CHO cells to reduce protein yield differences between different clones. ZFN, TALEN and CRISPR/Cas9 technologies can be used to specific gene knockouts, such as GS and DHFR genes, and ZFN is one of the most successful techniques. Although these techniques have significant advantages in the specific gene knockouts, there are still challenging, for example, the confirmation of stability and high expression hotspots in host genes. It should also be noted that a single knockout does not solve all the problems, and other quality adjustments may be required for certain proteins, such as folding degree and glycosylation, which can also be achieved by gene editing.

There are also a number of less-used gene tools, such as mammalian artificial chromosome expression technology (ACE). This minigenome can be expressed in a self-controlled manner in the cell, its gene sequence can be customized and expressed as needed, and it can also be appreciably expressed in CHO. Comparison of ACE and random plasmid integration systems in fed-batch cultures of CHO cells, the results revealed comparable cell performance and stability. In addition, the transposon enzyme system can easily introduce the target gene into the host gene, such as the transposon PiggyBac purified from Lepidoptera, which has been shown to increase yield in CHO cells.

Certain cis-acting epigenetic regulatory components can also be used to increase cellular protein production and stability, and these components stabilize the transcriptional activity of the target gene by remodeling the nuclear chromatin environment. One of the most widely used cis-acting components is MAR. It consists of a non-histone fibrin and a large molecular weight RNA to form a three-dimensional network system. Studies have shown that the presence of the MAR component in the recombinant protein expression system can significantly improve the expression level, but some scholars have pointed out that MAR has a requirement for the sequence, and only plays a role in a specific sequence. Other genetic regulatory components include UCOEs and STAR.