October 1, 2017 (Vol. 37, No. 17)

Technologies That Increase Protein Expression, Enhance Protein Folding, and Expedite Clone Development Are Bridging the Lab-Clinic Gap

The pharmaceutical industry has changed. While chemically synthesized medicines continue to line store shelves, biologically produced drugs have stormed the market—filling the void that their predecessors could not by providing new, targeted approaches for a growing list of diseases like cancer and autoimmune disorders.

While these therapies offer hope to patients with these conditions, coercing cells to produce them has challenged biopharmaceutical manufacturers and driven drug prices skyward. In addition, as increasingly complex, difficult-to-express molecules enter the pipeline, biopharmaceutical manufacturers will need to come up with innovative solutions to increase product titers. Otherwise, cost constraints will strangle these therapies before they ever reach patients.

The “Optimizing Cell Line Development” program at Cambridge HealthTech’s Bioprocessing Summit, held in Boston in late August 2017, provided an opportunity for biomanufacturers to discuss the challenges facing the industry, and to share their solutions for designing stable, high-titer, production cell lines that could reduce manufacturing costs.

High-Quality Clones in Three Weeks

According to Anton Bauer, Ph.D., COO at The Antibody Lab, one of the major challenges in generating a stable production cell line arises from the host cell’s rebellion against incorporation of the foreign DNA that drives the production of recombinant proteins. The host cell battles this foreign invasion using chromatin regulatory elements that effectively shut down foreign genes and halt protein production. The most commonly used approaches attempt to strong-arm chromatin regulation by introducing simple chromatin-modifying elements.

Looking to the mouse genetics community for answers, The Antibody Lab engineered a vector capable of transporting large pieces of DNA—even a whole gene locus—to the host cell’s chromatin. Stable integration of multiple copies of a modified ROSA26 locus, which has enough of its own regulatory elements to maintain an open configuration that allows constitutive expression, creates a veritable army that shields the vector from sieges by the host cell chromatin that tries to block expression.

To identify clones with the strongest battlements, The Antibody Lab first reveals positive clones by using high selective pressure that forces protein expression over a short time period. High-quality clones will continue to produce the protein of interest at high levels after releasing the selective pressure for a two-week period before screening. The Antibody Lab used conventional culturing methods for 70 days or more to confirm that their process enables the selection of stable, high-yield, production cell lines in a fraction of the time typically required.

“The idea that you can get these clones in three weeks is really amazing,” said The Antibody Lab’s CSO, Gottfried Himmler, Ph.D., “because the industry standard to get a stable clone and cell line is 6–9 months. This closes the gap in the timeline between transient and stable expression and makes the generation of stable cell lines feasible at the research stage.”

In addition to shortening the timeline for cell-line development, The Antibody Lab’s BESTcell cloning system can produce cell lines with higher titers than classical plasmid-based technologies. A study published in Nucleic Acids Research in 2015 showed a significant increase in the expression of a difficult-to-express HIV-1 glycoprotein when researchers delivered the ROSA26 locus to the host-cell chromosome using BAC (Bacterial Artificial Chromosome) vectors. Dr. Bauer stated that the authors, “were surprised that they could achieve so much by applying this [expression] technology,” which “contributed about 20-fold improvement [in protein expression].”

Vaccine Production Program

The Vaccine Production Program (VPP), a division of the National Institutes of Health’s Vaccine Research Center (VRC), has also increased protein production by modifying their expression vectors. The VPP, which develops cell lines and processes for manufacturing clinical candidates developed by the Vaccine Research Center, is studying how introducing different combinations of elements—known from the literature to improve expression—affect protein yield and quality (Figure 1).

Elizabeth Scheideman, Ph.D., director of cell-line development at VPP, said her group has managed to increase protein expression by adding matrix-associated regions and amplification-promoting sequences to their vectors, but reaching the high product titers set by monoclonal antibodies remains a challenge for many of the molecules they work with, which often have unusual glycosylation patterns that affect therapeutic activity.

The VPP is also developing an innovative FACS-based system that can establish cell clonality and help identify the most productive cell clones. “We really started looking at the FACS-based platform because you can have clear traceability that you have started from a single cell,” explained Dr. Scheideman.

Their FACS-based system includes a module that images plates after sorting and allows cell line developers to track a single cell as it divides and forms a clonal cell line. “You can really tell if something is not clonal,” said Dr. Scheideman, who explained that the lack of clonality could cause variation in product quality and homogeneity.

Another advantage of using a FACS-based system is its potential to detect and seed only the highest-producing cell clones. Fluorescently tagging the protein of interest with an antibody can provide a direct measurement of protein production, but secreted proteins will diffuse away from the cell and make it difficult to determine their origin. Thus, VPP has started developing fluorescent reporter systems to indirectly measure protein production by linking the expression of the reporter with the gene of interest or the selection marker.

The VPP still has challenges to tackle, including optimizing cell-sorting conditions and assessing the effect of the reporter construct on protein expression, but the benefits of clonal traceability and more improved clone screening may make their conquest worth the effort.

Figure 1. Researchers participating in the NIH’s Vaccine Production Program are refining clone selection through the development of a FACS-based platform for single-cell cloning. This platform optimizes sorting conditions, medium development, and reporter-based systems for sorting protein targets. The researchers hope to define product quality metrics that can be incorporated earlier into the clone-selection process.

Protein Folding

While both VPP and The Antibody Lab have engineered expression vectors and clone-selection methods that emphasize increased protein expression, Sola Biosciences has focused slightly downstream on protein folding. Protein therapeutics require proper folding to achieve the three-dimensional conformation that confers their functionality. Not only can misfolding inactivate proteins or alter their function—turning an origami crane into a platypus—but it can also reduce overall productivity by slowing down secretion, destabilizing proteins, and increasing cellular toxicity.

Akinori Hishiya and Keizo Koya recently described Sola Biosciences’ new technology, TapBoost®, in Nature’s Scientific Reports. In addition to increasing protein yields for manufacturing, they also reported on its potential as a therapeutic for diseases caused by protein misfolding and aggregation. The proprietary protein has two functional domains, the effector (or TapBoost) sequence and the target protein-binding domain, which work together to enhance the effectiveness of protein folding and cell quality-control systems.

The TapBoost sequence, which is based on a known chaperone protein, supervises protein folding, while the targeting domain binds to the protein of interest, temporarily associating the TapBoost sequence with the protein during the folding process. Sola Biosciences identified the TapBoost sequence after an intensive search for a specific amino acid sequence that enhanced folding and secretion of bound proteins within the endoplasmic reticulum.

To meet the demands of pharmaceutical companies, Sola Biosciences developed a second proprietary technology that dissociates the TapBoost protein from the target protein before it’s secreted into cell culture media. Since the TapBoost protein remains inside the cell after protein secretion, no additional downstream purification is required.

“This technology is unique,” commented Akinori Hishiya, Ph.D., principal scientist and cofounder of Sola Biosciences, “because it exploits the protein folding systems.”

In addition to increasing product yields for difficult-to-express proteins, whose complicated structures can make them susceptible to protein folding issues, the technology can also work in tandem with other protein-expression technologies that enhance translation, stabilize RNA, or increase cell-line stability.

Sola Biosciences has developed TapBoost, a technology that can facilitate protein folding and enhance protein production. Notice that the TapBoost protein consists of two functional domains, the effector sequence (for enhancing folding and secretion of bound proteins within the endoplasmic reticulum) and the target protein binding domain (for protein targeting), According to the company, TapBoost improves the production of proteins such as antibodies and Fc-fusion proteins.

In Silico Optimization

Instead of giving the protein a chaperone to oversee proper folding, Boehringer Ingelheim has designed an in silico optimization tool that analyzes the protein’s amino acid sequence and identifies potential troublemakers before they can crash the party. Originally developed to reduce aggregation in the final drug product, the optimization tool searches for liabilities in the amino acid sequence and suggests substitutions. According to Simon Fischer, Ph.D., head of cell-line development at Boehringer Ingelheim, the optimized sequences also resulted in enhanced protein folding, secretion, and expression in CHO cells. “Product titers were enhanced in a fed-batch process by at least four-fold” when they tested the technology on two ‘very difficult-to-express’ proteins,” said Dr. Fischer.

Boehringer Ingelheim’s in silico molecular optimization tool is one of multiple technologies that the research-driven pharmaceutical company has applied to cell-line development. Genome-editing and microRNA technologies have also given the company new tools for engineering cell lines.

The cell-line development group used zinc finger nuclease technology to knock out the glutamine synthetase (GS) metabolic pathway in CHO-K1 cells and establish a new cell line with a built-in selection system. Since GS-deficient cells cannot generate glutamine on their own, only cells that stably integrate the genes carried by the plasmid—which encode glutamine synthetase and the protein of interest—will survive.

 Dr. Fischer noted that previous studies have shown that the GS-selection system results in higher expression stability and productivity than the more commonly used DHFR (dihydrofolate reductase) systems.

While not as prominent, noncoding microRNAs can also have a profound effect on cell productivity. Discovered 15 years ago due to their role in cancer development, microRNAs post-transcriptionally regulate gene expression. A single microRNA can regulate an entire pathway without adding a translational burden to the cell—making them an attractive tool for cell-line development. After screening hundreds of microRNA, Boehringer Ingelheim has identified several microRNA that enhance cell growth and productivity.

Perhaps the biggest challenge looming on the horizon for biopharma is the increasing number of new-molecule formats in the development pipeline. Dr. Fischer believes that the increasingly artificial biologics marching towards the market—multispecific molecules, fusion proteins, and enzymes—will require innovative solutions to obtain product titers that rival that of naturally occurring monoclonal antibodies.

New technologies that increase protein expression, enhance protein folding, and shorten clone-development timelines will all have an important role in carrying these new drugs from the science bench to the frontlines of medicine.

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