Research

Cell line development

In order to develop therapeutic protein producing cell lines, the gene-of-interest (GOI), which encodes therapeutic proteins, should be integrated in the genome of host cells by transfection. The transfected cells are screened by selection process, such as DHFR or GS system to ensure the expression of GOI. By applying the selective pressure, the GOI can be co-amplified with selection marker gene, dhfr or gs gene, which is located near the GOI. After the pool selection, clonal cell lines showing desired growth profile and productivity are selected by limiting dilution. However, the conventional method of cell line development has some drawbacks, such as clonal variation and laborious selection process. Therefore, it is required to establish more efficient platform of cell line development.

rAAV production

Recombinant adeno-associated virus (rAAV) is a leading vector for gene therapy, yet its manufacture still relies on transient triple-plasmid transfection of HEK293 cells — a process limited by low yield and a low proportion of full capsids. Our laboratory quantifies this process stage by stage, from polyplex formation through cellular uptake, nuclear entry, viral gene expression and genome packaging, to identify where productivity is actually lost. This work has shown that the three plasmids are neither delivered nor expressed uniformly and that vector production is concentrated in a small subpopulation of cells; we translate these findings into design-of-experiment optimization of plasmid ratio and transfection conditions that raise both titer and full-capsid ratio. In parallel, we apply genome-wide CRISPR knockout screening to engineer host cells with reduced plasmid-associated cytotoxicity and improved transfection efficiency. By combining mechanistic understanding of the process with host-cell engineering, we aim to establish an rAAV production platform that delivers both quantity and vector quality.

Process optimization 

In biopharmaceutical production processes, the yield and quality of recombinant protein products are affected by various culture process conditions, such as temperature, culture pH, osmolality, pCO2, DO and by-products (ammonia and lactic acid) accumulation. Therefore, it is necessary to find the suitable culture conditions that allow the rCHO cells to grow well and to produce recombinant protein products properly. Among these factors, the temperature can be easily controlled. Culture pH can affect intracellular pH, cell metabolism, and protein glycosylation. Osmolality also plays an important role in regulating cell size and intracellular amino acid transport. Ammonia and lactic acid adversely affect cell growth and product quality. In this laboratory, we are trying to find the optimal culture process conditions for the high productivity of the recombinant proteins while keeping a good quality in the bioreactor.

Apoptosis and autophagy

The programmed cell death (PCD), such as apoptosis and autophagy, is considered as an significant issue for production cell lines when it comes to improving the productivity and quality of therapeutic proteins. Apoptosis has been investigated in Chinese hamster ovary (CHO) cells to extend the culture longevity thereby increase the overall productivity. Autophagy, another type of PCD, has been extensively studied in cell biology related to the diseases and senescence. It has been known that autophagy induces cell survival and the inhibition of autophagy increases the specific productivity of recombinant CHO cell lines. Elucidation of the mechanism by which autophagy enhances specific productivity will be enable to control the culture process more elaborately.

Proteomics

During the production of recombinant protein products, manufacturers must demonstrate clearance of host cell impurities and contaminants, such as host cell proteins (HCPs), to appropriate levels prior to use in the clinic. Also, HCPs, secreted from viable cells and released from dead cells, accumulate extracellularly during the culture process, potentially impairing product quality. Therefore, it is important to characterize the HCPs in the culture process to ensure safety and optimal product quality. Our group is identifying and quantifying the HCPs of rCHO in the culture process and searching for specific targets for cell engineering.