A few basic steps are generally followed to make a host cell become a high producer of the desired product. First, a transgene coding for the product protein is typically introduced to the host cell using a plasmid. In addition to the transgene, the plasmid carries a gene that confers a selectable trait, such as antibiotic resistance, so that after transfection, a selective pressure can be applied to enrich for those cells which have internalized the plasmid. The plasmid does not replicate in mammalian cells and would otherwise be gradually lost as cells multiply. By applying selective pressure over an extended period, all of the cells that are selected for would have the plasmid integrated into the chromosome. After a stable cell population is obtained, preliminary in vitro screening is then performed to screen for clones with the highest production levels. This is often fulfilled by assaying the product concentration in the supernatant of a 96-well plate. Another common practice is to grow cell clones as small colonies on soft agar and then perform immunoassays in situ to identify those with a larger immunoprecipitation zone around the colony. The next step, the amplification of transgene copy number, is practiced in some instances (such as when CHO cells are used), but not all (such as when myeloma cells are used). To do this, the stable cells are subjected to a high concentration of an inhibitor
Steps in Cell Line Creation
• Transfection • Selection • Amplification • Single cell cloning • Screening
to the amplification marker, which the cells require for survival. This high concentration of inhibitor kills the vast majority of cells, except those that have multiple copies of the marker and resistance genes. As the amplification marker multiplies, the adjacent integrated transgene(s) are also co-replicated. The number of copies may increase dramatically in different regions of the genome, thereby giving rise to high levels of transcription and translation. Through this process, in some high-producing cells, the transcript level of recombinant IgG heavy chain becomes the most highly expressed transcript in the cell. An excessive expression of protein can overwhelm cell’s protein folding capacity and lead to an unfolded protein response in the endoplasmic reticulum and, thereby, induce apoptosis. Consequently, cells which have not developed appropriate machinery to handle the increased production may not survive. After amplification, the selected cells not only have multiple copies of the transgene and a high level expression of resistance and product genes, but they also have developed the secretory capacity to allow for enhanced protein secretion. These clones have a high propensity to become high producers, but not all of them do. Hyper-productivity also requires many other traits, such as the capability to quickly grow to a high cell density and the capability to sustain a high viability over a long duration in the stationary phase. Subsequently, single-cell cloning is performed on those surviving cells, typically by sorting single cells into culture wells by flow cytometry. Cloning can also be performed by dispensing cells (approximately 0.2 cell per well) into multi-welled culture plates, so that the probability of having more than one cell in each well is very low. Thus, all cells that arise from a given well all originated from the same cell. Single cell cloning is necessary because a pool of stable cells would have vastly different genetic backgrounds, perhaps in the loci of exogenous gene integration, and potentially have different mutations caused by those integration and other unknown aberrations. Such a mixture of cells of genetic heterogeneity is called a “cell pool”.
Fig. 5.2: Typical steps in introducing a transgene for generating high producing cell lines for manufacturing.
After single cell cloning, the productivity of each clone is then assessed and those with high productivity are isolated. The selected clones are further expanded for stock preparation, growth characterization and product quality assessment. In some cases, the producing cells are then adapted to growth conditions more amenable to manufacturing conditions. Single cell cloning is considered critical for establishing a production cell line, as the cells arising from a single cell are genetically homogenous. It is practiced regardless of whether there is an amplification step or not. Although the host cells used for establishing production lines are all aneuploid and can be prone to further genome reorganization and epigenetic reprograming, a culture of cloned cells are much more homogenous than cell pools. From a single cell at the beginning of single cell clone to the end of a production run, the production cell may have gone through more than 60 doublings. If that is extended to the entire production lifetime, the number of cell doublings may be greater than 80 doublings. It is important to minimize the outgrowth of mutated cells in the original pool, during the course of cell expansion, by single cell cloning during the generation of production cell lines. A number of different methods are commonly used to introduce expression vectors into host cells. The choice of method is dependent on cell type, the available quantity of cells and plasmids, and the experience of the lab practicing it. Although different methods depend on different mechanisms of plasmid uptake by cells, all methods require the cytoplasmic membrane to first become permeable to plasmids. Plasmid delivery by calcium phosphate precipitation, cationic polymers, and liposomes relies on direct interactions of the particles, or lipid vesicles containing plasmid DNA, with the cellular membrane through an endocytosis- like of mechanism. In electroporation and microinjection, physical force is used to introduce openings in the cell membrane for DNA entry. The “Methods of Gene Transfer” table lists
DNA-Calcium Phosphate Co-Precipitation
DNA and calcium chloride is added dropwise into a HEPES buffer with sodium phosphate (1 mM). A fine precipitate forms in 5-30 min, and is added directly to the cells (~2- 40μg/106) .
Electroporation
Expose cells to a high-voltage electropulse in the presence of DNA solution. This introduces pores in the plasma membrane, allowing entry of DNA. Duration of pulse and strength of electric field varies with cell type .
Lipofection/Lipid Mediated Gene Transfer
A mixture of DNA with amphipathic compound (DOTMA, DOPE, etc), that simultaneously interacts with DNA and hydrophobic portions of the membrane, allowing passage of DNA into the cell .
commonly used methods of DNA introduction. All methods use a very high plasmid-to-cell ratio, but only a moderate DNA concentration. While up to a thousand copies of the plasmid can enter cells, only a small proportion actually translocate to the nucleus and are transcribed to allow for the expression of selectable marker resistance gene.
DNA-Calcium Phosphate Co-Precipitation
DNA and calcium chloride is added dropwise into a HEPES buffer with sodium phosphate (1 mM). A fine precipitate forms in 5 - 30 min, and is added directly to the cells (~2- 40μg/106) .
Electroporation
Expose cells to a high-voltage electropulse in the presence of DNA solution. This introduces pores in the plasma membrane, allowing entry of DNA. Duration of pulse and strength of electric field varies with cell type .
Fig. 5.3: A typical vector for introducing transgene into host cells for recombinant protein production.