Monday, 20 January 2020

Cell Line Development

Cell Line Development

Host Cells and Transfection

Many commonly used host cells for the generation of industrial production cell lines were derived from rodents. For example, Chinese hamster ovary (CHO) cells were originally from hamsters, and NS0 cells were from mouse myeloma cells.

A transgene (a gene of interest, i.e., the product gene) coding for the product protein is typically introduced into the host cell using a plasmid. The transgene is driven by a strong promoter and is followed by a polyadenylation signal for proper mRNA formation. In addition to the transgene, the plasmid carries a gene that confers a selectable trait, such as antibiotic resistance. After transfection, selective pressure can be applied to enrich for those cells that have internalized the plasmid.

 cell line development

Figure 1. A vector for the expression of a transgene using a DHFR amplification system.

The plasmid also consists of elements that are necessary for it to be propagated in bacteria, for generating many copies of itself for transfection into the host cell. The plasmid does not replicate in mammalian cells and would otherwise be lost as cells multiply. By applying selective pressure over an extended period, the surviving cells are likely to have the plasmid integrated into a chromosome and be stable transfectants.

Amplification

To achieve high productivity, the producing cell must have a high level of the mRNA for the product protein. In general, the amount of mRNA for a gene accumulated in a cell increases with the promoter strength that drives the transcription and the number of copies of the gene. To increase the transcript level, the number of transgenes in a cell is often “amplified” to tens or even hundreds of copies. This is done by including an amplification marker near the transgene in the plasmid. After the plasmid is integrated into the chromosome, the transfected cells are subjected to a high concentration of an inhibitor to the amplification marker. The high concentration of inhibitor kills the vast majority of cells, sparing only those that have multiple copies of the resistance genes.

Cell Line Development

Figure 2. Schematic of cell line development for the production of recombinant proteins.

A commonly used amplification system is the dihydrofolate reductase (DHFR) system. The enzyme DHFR is required for the synthesis of nucleoside hypoxanthine and is inhibited by methotrexate (MTX). By dosing cells with a very high concentration of MTX, only cells with multiple copies of DHFR will survive. As the amplification marker multiplies, the adjacent integrated transgene(s) will also be co-amplified, thus giving rise to high levels of transgene transcription and translation.

Through this process, some cells develop a very high capacity for product synthesis. Typically, single-cell cloning is performed on the population of surviving cells after selection and amplification(at the least, after amplification).Thesurvivingcellsmakeup a very heterogeneous population. Single-cell cloning, with the entire population, started from a single cell (thus, a clone), generates a more homogeneous population. This is typically done by sorting a single cell into each culture well using flow cytometry.

Cloning can also be performed by dispensing cells (approximately 0.2 cells per well) into multiwalled culture plates. At an average of 0.2 cells/well, the Poisson distribution predicts that the chance of a well having two cells is low. Therefore, one can assume that all cells arising from a given well all originated from the same cell and area clonal population. Without single-cell cloning, the population of the producing cells is heterogeneous and poses a high risk of a population drift, which occurs when subpopulation outgrows other cells in the original population overtime. This may lead to changes in productivity or even product quality, and it is a risk that must be minimized if not eliminated.

After single-cell cloning, the productivity of each clone is then assessed. Those with high productivity are isolated. To establish the producing cell line, the product quality is further assessed in two respects:
 (1) protein sequence (lest a mutation in the product gene may have occurred in the course of cell line development), and 
(2) structure and glycosylation pattern. The cell line is also evaluated for its long-term stability. 

To generate a sufficient amount of cells for manufacturing over many years, the single cell that was first isolated must undergo a large number of cell divisions. Long-term cultivation thus Carried out to ensure that the probability of major alteration in the cell's production characteristics is stable. This occurs before the selected producing cell line is expanded for the establishment of a cell bank for manufacturing.

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