April 15, 2016 (Vol. 36, No. 8)

A Productive Cell Line Needs More Than a Good Pedigree, It Needs Proper Care and Feeding

Developing a highly productive cell line is a balancing act. Due weight must be given to various factors, particulary clone selection and media feed. And yet more finesse will be required if bioprocessors are to get the most out of the newer cell lines, many of which are bound to embody the most subtle shifts gene-editing technologies such as CRISPR can allow.

To help our readers maintain their equilibrium, GEN has consulted with the steadiest cell-line experts available. They explain how to keep cell-line productivity high—and stable.

GEN: If the goal of CHO cell-line development is to improve productivity and/or protein quality, which factor, clone selection or media feed, has been most responsible for these improvements and why?

Dr. Pankiewicz Development of CHO stable cell lines expressing high amounts of high-quality recombinant protein is the first step in efficient production of therapeutic proteins. Historically, the main focus in cell-line development has been on generating and finding highly expressing clones through optimizing selection systems, expression vectors, and screening.

Lately, development of chemically defined media and feeds combined with accessibility to high-throughput analytical tools allow better understanding of nutrient influence on the quality of expressed protein. I believe that including feeds and protein quality testing early in clone screening is beneficial for selection of clones with higher protein quality.

Dr. Racher Both contribute to product quality and productivity; it depends upon your objective. Current process productivities are a combination of an effective protein expression system, a good host cell line, selection of a suitable product variant, appropriate media feeds, and process operating conditions.

The importance of clone selection depends on whether you need “good enough” productivity or require a high-producing outlier to meet projected demand; this is more important if a specific quality attribute is desired. A selected clone gets you close to the target. Then you can use modifications to media feeds and process to optimize to the specific target attribute.

Ms. Holroyd Both factors are equally important. The large degree of genomic “plasticity” by the CHO genome has always been exploited by developers when selecting clonal cell lines. Selection of better parental cell line pedigrees has improved the clonal productivity efficiencies. The engineering of the parental cell to create improved selection systems (for example, GS/ using zinc finger nuclease technology) now provides the ability to select for a particular phenotype (productivity and/or protein quality) by screening fewer clones giving one a high statistical probability of finding clones with the desired traits.

In conjunction with the cell line, the development of optimized media and feeds plays an integral role in a platform approach across multiple clones and molecules. The increasing focus on biosimilar drug development, media, and feed optimization will be major effectors on modulation of protein quality attributes with the goal of matching the innovator drug profiles.

Dr. Fisch To rapidly select a stable and high-producing cell clone in a defined media and feed, Selexis has taken an approach that has impacted protein productivity and quality from its CHO-K1 cell line. First, Selexis identified an epigenetic DNA element (SGE) that increases the transcription rate at the site of integration by favoring a unique recombination mode. Each copy of the integrated transgene is in active transcriptional mode.

Our approach for speed, productivity, and quality includes a) codon optimization of the gene of interest (GOI), b) promoter sequence adapted to CHO cells, c) epigenetic DNA element (SGE), d) transfection procedure, e) selection with ClonePix, f) serum-free media, and g) animal-derived component-free feed.

Dr. Powers The goal in our ExpiCHO system was to improve protein yield, as this was the primary barrier to the use of transient CHO systems for screening of lead molecules. The approach that we took in the development of this product was to optimize every aspect of this system (cells, media, feeds, enhancers, transfection reagents, protocols), and it was only by this systems-based approach that we were able to achieve the results that we did: orders of magnitude higher titers (up to 3 g/L) than what is achievable with existing transient CHO-based systems.

Historically, the main focus in cell-line development has been on generating and finding highly expressing clones through optimizing selection systems, expression vectors, and screening. [iStock/Reptile8488]

GEN: What, if anything, will the application of CRISPR/Cas9 or related gene-editing techniques have on future cell-line development?

Dr. Tedesco CRISPR is the only available gene-editing approach effective enough to enable knockout studies even without clonal cell selection. For example, a pool of cells transduced with a lentiviral CRISPR construct can often harbor knockout cells at high enough frequency to assess gene-knockout effects on drug sensitivity. These “quick and dirty” assays aren’t possible with any other genome-editing methods.

However, to make reliable cell models for more sophisticated studies, the targeted genetic change is just the first step. Downstream characterization of cells to assess secondary or off-target effects that might have been introduced is essential and requires much time and effort.

Dr. Pankiewicz The CRISPR/Cas9 technology is a powerful genome-engineering tool that is winning popularity due to its efficiency, specificity, and relative simplicity compared to other technologies. This system was successfully used to mutate genes in diverse organisms and cell lines including CHO cells.

I believe that CRISPR/Cas9 technology will be further used to engineer CHO cells to express proteins with desired quality, higher  specific productivity, and better stability of expression; to improve clone selection systems; and to develop efficient targeted transgene integration systems.

Dr. Alberts CRISPR/Cas9 can have a tremendous impact on cell-line development. Using CRISPR, you can easily, quickly, and precisely insert your specific gene and promoter stably into the genome. Furthermore, the utility of CRISPR allows the additional modification and customization of protein production cell lines.

For example, glycosylation patterns of expressed therapeutic antibodies can significantly impact the pharmacokinetics of monoclonal antibodies and antibody-dependent cellular cytotoxicity (ADCC) activities, making them an attractive knockout target. The ability to customize your cell line by knockout of glycosylation pathways or other factors affecting the efficacy or effectiveness of the desired gene product is a potential game-changer.

Ms. Holroyd CRISPR/Cas9 technology allows for a cost-effective high-throughput screening method (for example, pooled CHO CRISPR libraries) to identify novel targets by allowing a deeper and broader screening approach. Therefore, CRISPRs can serve as a tool for target identification as well as target validation.

Orthogonal gene-editing technologies such as zinc finger nuclease technology can then be employed to precisely modify the validated sites to generate a final bioproduction cell line. Such precise modifications will lead to better understanding of cellular metabolism, and this better understanding will in turn translate to even higher efficiencies in productivity and protein quality.

Dr. Fisch The combination of data from next-generation sequencing and bioinformatics along with CRISPR/Cas9 will have a profound impact on the development and manufacturing of recombinant protein drugs. Selexis has developed bioinformatics tools to assemble the genome of its CHO-K1 cell line.

The direct outcome from this effort? Selexis can now fully characterize, fully trace, and determine the clonality and integrity of a transgene sequence, or any other genes, that may play a role in the stability of its research cell banks. This work also allows the identification of viral sequences (adventitious agents) present in Selexis’ CHO cell.

Technologies such as CRISPR/Cas9 will allow Selexis to create a novel, viral-free CHO cell line that can mitigate the risks associated with the presence of viral particles in today’s manufacturing process.

Dr. Powers The key element that gene-editing technologies (such as CRISPR/Cas9 and transcription activator-like effector nucleases) bring to the future of cell-line development is well captured in the phrase “precision genome engineering.” The ability of these technologies to precisely target specific genes for creation of knockouts and knockins, the correction of mutations, and the introduction of other modifications has enabled the creation of cell lines that would have previously been considered virtually impossible.

We are already seeing this have a dramatic effect in the creation of cellular models of disease. And the approach is starting to emerge as an important element in the development of future cell-based therapies.

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