Platform technologies facilitate development and accelerate commercialization of protein therapeutics.
By Cynthia A. Challener
Speed to market is essential in the biopharmaceutical industry today. Manufacturers are seeking mechanisms for increasing efficiencies and reducing costs without compromising the safety and efficacy of their drug products. In addition to exploring novel production methods such as continuous processes and disposable production technology, manufacturers also heavily invest in platform technologies for protein expression that will not only facilitate the development of biologic drug candidates, but also increase yield and speed to market.
A platform of benefits
Platform technologies for protein expression that have been successfully used to produce biologic drug substances (i.e., substances that have made it to late-stage clinical trials and commercialization after being subjected to regulatory scrutiny) can thus be considered safe, according to Menzo Havenga, CEO at Batavia Biosciences. In addition, any technologies underlying these platforms have at that stage been well-studied, making them predictable, and predictability creates manufacturing comfort. They are also expected to speed up the development of novel specific molecules based on the use of chemistry, manufacturing, and controls (CMC) standards and by leveraging capabilities and capacities, notes Beate Mueller-Tiemann, head of business integration and innovation at Sanofi. “Consequently, platform expression technologies may lead to substantial benefits in terms of speed to market, assuming CMC aspects are on the critical path, and reduced cost of goods manufactured (COGM),” she states.
In addition, new technologies for genomic engineering of cells for the production of therapeutic proteins are “opening up a new world of possibilities to tailor-make protein-based drugs with appropriate post-translational modifications that meet the needs of the pharmaceutical industry, physicians, and ultimately patients in need of novel medications,” notes Bjørn Voldborg, director of CHO cell-line development at the Novo Nordisk Foundation Center for Biosustainability.
Improving efficiency, yield, and functionality
There are a large number of factors to be taken into account in the development of new platform technologies for protein expression ranging from the choice of cell line, expression plasmid design, cultivation medium, growth conditions, equipment, scalability, stability, and matching purification capability, to name but a few. Primary drivers to the development of a new therapeutic protein platform are the desire to improve efficiency and productivity, the need to increase the agility of manufacturing, and the fact that complex non-natural proteins are not well-expressed in the current systems such as Chinese hamster ovary (CHO) cell lines, according to Mueller-Tiemann.
Improving yield has always been and will continue to be one of the key drivers for a technology platform to be successful, explains Havenga, although clearly agility, product quality, and regulatory acceptance are highly significant. Next to yield, the stability of protein-producing cell clones remains an issue to be studied as well. Furthermore, Havenga points out that the use of novel platform technologies for protein expression has not yet resulted in significant reductions in consumer prices.
“The goal beyond making some of these new entities viable is achieving a significant reduction in COGM, thus increasing the affordability of biotherapeutics and improving overall healthcare economics,” agrees Mueller-Tiemann.
With respect to the impact of technology, the main driver for recent advances in the development of protein expression platforms is the discovery of CRISPR-Cas9-based genome editing, according to Voldborg. “This technology has made it possible to specifically engineer genes in existing hosts to improve their properties and performance and to add completely new functionality,” he observes. There is one hurdle yet to overcome, however; the intellectual property landscape surrounding the CRISPR-Cas9 approach is unresolved, which makes it less attractive for use in industrial settings.
Voldborg expects that most therapeutic proteins will continue to be produced using well-known platforms (e.g., CHO cells and Escherichia coli [E. coli ] bacteria), but with the addition of engineered versions that may overcome the drawbacks of earlier non-engineered versions of these platforms. “I have to say, however, that the possibility to specifically delete or insert genes by demand using CRISPR-Cas9 technology is a game-changer in the field.” He adds that combining genetic engineering tools like CRISPR/CAS9 with bioinformatics to identify optimum sequences using a bio-mimetic approach could make a difference. Tools enabling modification of metabolic pathways for glycosylation in algae and yeast glycosylation are also noteworthy. “These approaches offer avenues for designing better fit-for-purpose molecules not only in terms of their activity and safety, but also with respect to desirable CMC attributes,” says Voldborg.
The availability of several molecular biology tools developed in the recent past by the scientific community in diverse applications beyond biopharmaceuticals is starting to make a difference, agrees Mueller-Tiemann. She notes that there are several platform approaches based on diverse host systems that seem promising for the expression of therapeutic proteins due to their increased productivity and ability to be genetically engineered toward specific molecule characteristics and levels of expression.
In addition, according to Mueller-Tiemann, the simplicity of the culture media required for their growth presents opportunities from a cost-reduction perspective. Use of chemically defined media also makes the risk of exposure to zoonotic adventitious agents extremely unlikely. “High-quality, defined, animal-free reagents and cell-culture products help biomanufacturers eliminate the risk of contamination that has been associated with animal- and human-serum-derived media,” agrees Scott Deeter, president and CEO of Ventria Bioscience. Cell-free systems, which are currently at the earliest stage of development, could be important in the long term, according to Mueller-Tiemann.
Advances in process equipment are also expected to have an impact, according to Havenga. He points to the use of high-cell-density, fixed-bed bioreactors for adherent cell culture as an exciting new development. “With these bioreactors, it is possible to increase cell densities from 10-20 million cells per milliliter in typical bioreactors to an impressive 100-200 million cells per milliliter, resulting in significant savings in facility costs due to the reduction in required floor space,” he explains.
Overcoming cellular complexity
While a lack of suitable genome engineering tools is no longer a bottleneck for improvement of protein expression platforms, biologic drug manufacturers still face many challenges. The perfect system is not around the corner. Understanding and tools for better engineering the overall metabolism of host cells with the capability to balance protein generation in several different phases with cell growth and survival will remain a key research focus, according to Mueller-Tiemann. “Significant progress has been made, but a lot has yet to be discovered,” she asserts.
Adds Voldborg: “Cellular expression systems are highly complex, and we still lack knowledge of the cellular machinery that is used to transcribe, translate, fold, modulate, and finally secrete desired protein products.” He does note, however, that the use of computer-based models and big data analysis are being used to improve this knowledge.
For Havenga, continuing issues with the stability of expression cell lines and clonality associated with gene amplification technologies, such as dihydrofolate reductase (DHFR) selection, remain a concern. “The problem here is loss of expression, and [the use of] technologies that do not involve gene amplification, such as the STEP technology developed by Batavia Biosciences, is one approach to addressing this problem,” he says.
Engineering CHO cells
The platform technology developed at The Novo Nordisk Foundation Center for Biosustainability at the Technical University of Denmark is focused on engineering cell lines according to the needs of the pharmaceutical industry working with protein-based therapeutics, says Voldborg. “We have been able to engineer cell lines that solve a lot of the problems and challenges experienced by the industry,” he says. As one example, Voldborg points to engineered CHO cell lines that cannot produce lactate, thereby nearly eliminating the need for pH adjustment via base addition during cell culture. “With these expression systems, it will be possible to conduct much longer fed-batch runs and significantly increase the amount of product that can be made from each production run.” The center has also developed cell lines engineered to produce proteins with tailor-made, highly homogenous glycoprofiles, highly homogenous cell lines that exhibit reduced host-cell protein secretion, and cell lines resistant to certain virus infections.
At Sanofi, establishing differentiating CHO expression systems is a clear goal for the company, says Mueller-Tiemann. Sanofi is also pursuing process intensification for high-throughput, semi-continuous manufacturing in the short term.
Batavia Biosciences’ plasmid-based STEP technology increases protein expression in CHO cells by at least 10-fold, taking just 12 weeks to generate stable cell clones, according to Havenga. A cytomegalovirus (CMV) promoter drives the transcription of one mRNA from which two proteins are translated (i.e., the protein of interest [product] and a functionally impaired Zeocin selection marker). As the impaired Zeocin selection marker needs to be expressed in a CHO cell to high levels for the cell to survive the antibiotic pressure, that cell per definition also makes high levels of the desired product.
The platform rapidly achieves high protein-expression levels without the need for gene amplification. In addition, cell clones developed thus far with STEP (n=12) have all proven to be stable in the expression of the desired product for more than 60 passages in the absence of selection pressure, according to Havenga. “In the rapidly growing market of recombinant proteins and antibodies, our STEP technology provides a tool to complete preclinical phases at higher speed with reduced costs and with a higher success rate,” he asserts. Regulatory approval of the STEP technology is expected in 2018 at the latest, according to Havenga, with first products on the market using STEP in 2024.
Ventria Bioscience’s proprietary ExpressTec technology is a plant-based expression system. Recombinant proteins, peptides, multi-subunit molecules, monoclonal antibodies, fusion proteins, and enzymes are manufactured within a growing plant using sunlight, soil, water, and air as raw materials, according to Deeter. He adds that products manufactured using this expression platform are cost-effective and free of animal, human, and bacterial contaminants, which is an important safety factor.
“ExpressTec also delivers meaningful advantages by making molecules that would be difficult to produce in other systems, enabling new product opportunities that were not previously available. Ventria is in the process of doubling our production capacity with the platform to meet growing needs in developing new biotherapeutics, cell-culture media reagents for biomanufacturing, and industrial and animal nutrition enzymes,” says Deeter.