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The use of bioengineering offers practical tools for the evolution of host cells.
Bioengineering has benefitted biomanufacturing by creating cells with enhanced productivity and robustness. However, using a cell-line engineering approach has its challenges, among them the capability to develop clonal production cell lines and cell culture media formulations that are scalable while at the same time maintaining robust titers and the desired critical quality attributes (CQAs) needed for molecules to be successful in both the clinical and commercial setting, says Ademola Kassim, product specialist, Cell Line Development, Sartorius.
“While developing an appropriate cell line is important, a media formulation that complements a host-cell line is just as critical as it helps improve scalability and the success of the molecule,” Kassim says.
Overall, innovations in bioprocessing, including equipment and technology, aim to make scale-up of upstream processing easier. Process intensification, for instance, has been useful in improving overall upstream processing outcomes, Kassim points out.
Process intensification consists of several technologies, including single-use systems, perfusion media/feeds, scale-down models (e.g., Ambr 15/250, Sartorius), process control, and data analytics. “Overall, process intensification helps maximize the productivity of unit operations by improving drug development timelines, increasing titers, lowering costs, and reducing the manufacturing footprint. As the manufacturing footprint is reduced, this allows ‘multi-product facilities,’ thus potentially improving cost efficiencies and drug development timelines,” Kassim explains.
There remains industry focus on ways to better develop cell lines to optimize production yields at the start. Bioengineering has increasingly been focused to develop efficient cell lines for bioproduction. As an example, Kassim points out, targeted integration has been a big interest for production cell lines such as Chinese hamster ovary (CHO) cells.
“The goal of targeted integration is to engineer a cell line in which the transgene always integrates into a specific site or ‘hot spot’ of the genome. This ‘hot spot’ is a region within the genome that allows for consistently high expression and genetic stability of the biotherapeutic,” Kassim states.
Kassim also explains that, additionally, there is an increasing need to knock-out specific genes within the CHO genome by using gene-editing tools such as clustered regularly interspaced short palindromic repeats-cas9 (CRISPR/Cas9). “At times, customers see their biotherapeutic co-purifying with a specific host-cell protein (HCP) during downstream processing. If an HCP co-purifies with a therapeutic, this not only causes process-related impurity issues to arise, but also may increase immunogenicity issues,” he states.
One way that process-related impurity issues are being remedied is through the engineering of a host cell line in which potentially problematic HCPs have been knocked-out. “Once those HCPs have been knocked out, the host cell line can now be used to express the gene of interest,” says Kassim.
Engineering the cell line to knock out HCPs has several potential advantages, according to Kassim: it allows cellular resources to focus on expressing the gene of interest; it potentially reduces immunogenicity risk; and it helps for easier and cheaper downstream processing. “Other ways bioengineering can help improve productivity include engineering cells to achieve specific product quality profiles, such as afucosylation, and engineering cells to remove growth inhibitory or toxic byproducts, such as lactate,” he adds.
Kassim also explains how research has shown that the elimination of HCPs in CHO cells in some instances allows for improvements in expression levels, cell viability, and viable cell densities; however, the improvements seen may not warrant a significant reduction in bioreactor size.
“The removal of HCPs may not necessarily change the way cell culture media or feeds are formulated. However, the improvements we see in growth parameters may require a different feed regimen during a fed-batch process. Removing HCPs tends to have a bigger effect on downstream processing versus upstream processing,” Kassim says.
Other benefits to the removal of HCPs includes improved efficiencies and cost reductions during filtration and chromatography steps because fewer resources (e.g., resins, etc.) are used. “We also see scenarios where the removal of genes that produce toxic by products such as lactate occurs. In some instances, reduced production of lactate and other toxic metabolites has been found to improve growth parameters and production yield in CHO cells,” according to Kassim.
Recent research examined the effects of engineering CHO cells. In Budge et al. (1), for instance, researchers noted that, while CHO cell expression systems have been specialized to the point where they are able to generate high concentrations of efficacious, multi-domain proteins with “human-like post translational modifications” with the appropriate product quality attributes, there nevertheless remains a need for the development of new CHO cell expression systems that can produce more challenging recombinant biotherapeutics at higher yield. Furthermore, these new expression systems are also required to produce challenging biotherapeutics with improved product quality attributes.
The Budge et al. researchers decided to investigate the results of engineering the lipid metabolism in CHO cells to enhance product quality attributes, an angle that had not been investigated before. Lipid metabolism became the target for this investigation because the biosynthesis of recombinant proteins is controlled in part by cellular process that are highly dependent on lipid metabolism. The researchers were able to show that, to different degrees, genes involved in lipid biosynthesis can be overexpressed in CHO cells, and they were thus able to increase the expression of a number of model secretory biopharmaceuticals by 1.5- to nine-fold. Budge et al. concluded that manipulation of lipid metabolism in CHO cells using genetic engineering can be a new approach to enhance these cells’ ability to produce a range of different types of recombinant protein products (1).
In another study, Mistry et al. studied the effect of engineering CHO cells to resist hydrogen peroxide and found that hydrogen peroxide-evolved CHO cells showed improved product expression. In particular, the manufacture of bispecific antibodies using CHO cells was known to have lower yields compared to monoclonal antibodies, and it was recently shown that “reactive oxygen species” of CHO cells negatively impacted antibody production. In comparison, boosting the cells’ antioxidant capacity had a beneficial effect on the expression of recombinant proteins. Mistry et al. used directed host cell evolution to generate novel hydrogen peroxide-evolved host cells and demonstrated that the new host has a hydrogen peroxide resistance that was heritable in future cell generations (2). The new host cells also demonstrated other beneficial characteristics, such as enhanced antioxidant capacity via gene activity and improved glutathione content (which plays a role in the antioxidant defense mechanism).
The Mistry et al. researchers were further able to demonstrate that their new evolved host cells have significantly improved recovery times following transfection, show improved growth and viability properties in a fed-batch production process, and had elevated expression of two difficult-to-express bispecific antibodies, compared to unevolved CHO host cells (used as the control). Their reported findings suggest that host cell evolution can be an important methodology for improving specific host cell characteristics, which in turn can benefit the expression of difficult-to-express biotherapeutics. The team’s data also offer insights into the role that cellular antioxidants play in the production of cell lines, supporting a growing body of research being conducted to control cellular redox and thus boost the production of recombinant proteins (2).
In yet another study, Eisenhut et al. demonstrated the ability to better “tune” protein translation in cells using a synthetic RNA structure. By tuning the protein translation mechanism in mammalian cells, both the yield and quality of complex proteins can conceivably be improved. The researchers developed an easy-to-implement toolbox based on the use of synthetic 5’-untranslated region (UTR) RNA structures that would facilitate the regulation of protein expression in mammalian cells. The specific benefit here is that protein expression can be regulated in a fast, reliable, and predictable manner, according to the study. The toolbox works by introducing defined RNA hairpins, which have been termed “regulation elements (RgE)”, in the 5-UTR with the aim of impacting protein translation efficiency (3).
The Eisenhut et al. study also showed that RgEs can be used to characterize and optimize the recombinant production characteristics of CHO cells. What’s more, this can be done by intentionally regulating the expression of protein subunits, or by regulating the expression of a required helper factor. “Together, these elements represent an easy-to-implement and reliable toolbox for future mammalian cell line engineering applications where precise control of protein expression levels is desired to improve the cellular phenotype, but they can also be employed in the investigation of basic biological processes,” Eisenhut et al. reported in the study.
With such intense focus on ways to engineer cells to develop optimal cell line hosts, what does this mean for an approach that uses a cell-free method? How much progress has been made so far in moving toward a cell-free cell culture process, and does bioengineering influence a shift in that direction?
Kassim observes that, over the past few years, cell-free expression technologies have improved significantly. He notes that yields have been increased and some cell-free systems and that yields can now reach 1–2 g/L of recombinant protein. “This was mainly achieved by optimization of lysates and energy generation systems. The generation of cell-free lysates has been simplified and optimized, resulting in more potent and robust lysates with reduced batch-to-batch variability,” Kassim says.
“Bioengineering has been and will be very important for improvements in cell-free manufacturing,” he emphasizes. “Genetic engineering of host strains is essential to generate lysates with higher performance and to enable post-translational modifications.” Overall, Kassim says that improved lysate generation has decreased manufacturing costs and increased yields of cell-free expression systems, but he also notes that costs in cell-free systems are still significantly higher in comparison with cell-based manufacturing.
1. J.D. Budge, et al., Metab Eng. 57, 203–216 (January 2020).
2. R.K. Mistry, et al., Biotechnol Bioeng. 118 (6) 2326–2337 (2021).
3. P. Eisenhut, et al., Nucleic Acids Research 48 (20) e119 (2020).
Feliza Mirasol is the science editor for BioPharm International.
Vol. 35, No. 3
When referring to this article, please cite it as F. Mirasol, “Outlining Cell Lines’ Future with Engineering Approaches,” BioPharm International 35 (3) 10–13 (2022).