Fermentation for the Future

Microorganisms-bacteria and yeast-have been widely used for the production of genetically engineered (recombinant) biopharmaceuticals. Typical examples include the prokaryotic bacteria Escherichia coli (E. coli) and the eukaryotic yeasts Saccharomyces cerevisiae, Hansenula polymorpha, and Pichia pastoris (P. pastoris). Recombinant technology is used on these microbes to produce large quantities of desired substances, including peptides, proteins, and nucleic acids. Newer drug substances, such as single-domain antibodies, peptibodies, or antibody fragments that contain the effective domains, have recently been produced using microbes.

Microbial expression is often preferred over cell culture for the production of smaller peptides and proteins that do not require glycosylation because the desired substances may be produced in much shorter times, according to Daniela Reinisch, a scientist responsible for fermentation technology with Boehringer Ingelheim RCV (BI). It is also useful for the production of cytokines, growth factors, and plasmid DNA. Reinisch also notes that media costs for microbial fermentation are lower, although media costs for cell culture have been declining. Drivers of new technology development in microbial fermentation are similar to those for cell-culture processes and include reducing costs, increasing efficiency/productivity, and enhancing quality, she continues. As a consequence, significant effort has been invested in addressing certain key issues associated with microbial fermentation, including soluble protein expression in bacteria rather than production in inclusion bodies (IBs) and the engineering of yeast strains and processes to enable efficient expression and human-like glycosylation.

Soluble expression
One of the drawbacks of many bacterial expression systems, particularly E. coli, is the formation of insoluble aggregates of the overexpressed recombinant protein, referred to as IBs. In most cases, the IBs consist largely of the protein of interest, but may also include other proteins, such as co-precipitates. “These inclusion bodies are isolated during bioprocessing. Subsequently, the IBs are solubilized, and in a refolding step, the protein is renatured and afterwards purified. Therefore, production of recombinant proteins via inclusion body expression requires additional processing steps. Given that there is a strong dependence between protein structure and performance of protein-based drugs, extensive refolding know-how and high-throughput screening capabilities are key to achieving the intended product quality,” Reinisch explains.

On the other hand, with bacterial systems that promote soluble expression and can secrete the protein into the periplasm or media, the product can likely be purified more rapidly at lower cost and with the greatest chance for obtaining the pharmaceutical substance with its native conformation. “From a process perspective, soluble expression is also beneficial for establishing automated bioprocessing with cultivation and purification closely linked and supported by timely analysis,” Reinisch illustrates. Ideally, the product is accessible without cell disruption (i.e., can be analyzed with minimal effort in a relatively pure matrix). “Clearly, soluble expression addresses all three major drivers for biopharmaceutical developmentcost, efficiency, and quality--and it is therefore evident that companies are seeking to engineer bacterial strains to fully benefit from the advantages of soluble expression,” says Reinisch.

Recent developments at Boehringer Ingelheim
Boehringer Ingelheim has focused on establishing automated high-throughput systems for design of experiment-based, rapid bioprocess development. The company is currently implementing an automated microbial mini-fermentation system integrated in a robotic platform. The 15-mL bioreactor is based on single-use technology and allows up to 48 parallel fermentations. It also integrates with BI’s modular vector technology for optimized cloning and genomic expression. “This technology will help us to gain early insight into process performance and product quality according to BI’s quality culture concept,” Reinisch says. For soluble proteins and microbial expression, BI provides an integrated approach that combines strain development, automated upstream- and downstream-processing, and analytical assessment.

Alternative microbial expression
Some companies are also developing alternative bacterial strains for efficient fermentation. Pfenex, for example, has large libraries of genetically engineered Pseudomonas fluorescens bacterial expression strains that it can screen using high-throughput technology to identify the optimal choice for a given therapeutic protein. These strains do not form IBs and produce the protein in the periplasm, from which it is much easier to recover. According to the company, decisions on suitable expression systems for proteins, even those difficult to express, can be made much more rapidly with its Pfenex Expression Technology than with conventional development programs. In addition, the production strains will be optimized for rapid cycle times, high cell densities, and low-cost media.

Optimized yeast expression systemP. pastoris yeast expression systems have been shown to be effective for the production of drug substances because of high production levels and the ability for use in high cell-density fermentation protocols. Researchers at VTU Technology developed a proprietary P. pastoris production platform based on first- and second-generation aldehyde oxidase promoter libraries that allows for the fine-tuning of gene expression by carefully matching promoters and target genes. The company also offers a modular plasmid system, platform host strains and a set of helper proteins designed to work with the platform. In addition, VTU can produce up to 25,000 clones per week in 96-deep well plates, which reduces the time required to develop customized, high-yielding expression strains.

Post-translational modification
The other major limitations of microbial fermentation are the production of small proteins and the inability to achieve post-translational glycosylation. Both academic and industry researchers are working to overcome these issues and have made notable advances in the development of engineered yeast strains.

Much of the work was accomplished in P. pastoris. In yeast, N-glycosylation does not occur in the same manner as it does in humans, which can affect the efficacy of the therapeutic protein. Therefore, genetic engineering of P. pastoris was pursued to eliminate the undesired glycosylation pathways and introduce human-like genes that direct appropriate glycosylation patterns. Such glycoengineered P. pastoris strains were developed by Research Corporation Technologies (the Pichia GlycoSwitch system) and provide the advantages of microbial fermentation combined with the ability to produce proteins with uniform and customized glycosylation patterns.

There are, however, some issues with glycoengineered P. pastoris expression systems. The intellectual property around the technology is quite extensive and provides a barrier to further development. P. pastoris strains can also lack an effective unfolded protein response to manage the production of unfolded or misfiled proteins.

Moving toward synthetic processes
An entirely different approach to the production of biopharmaceuticals involves elimination of the use of cells or microbes. One example of a biochemical synthetic strategy was developed by Sutro, which offers the Xpress CF platform. The company extracts all of the required components for the production of proteins, including the biochemical components necessary for energy production, transcription, and translation. The components are then used for cell-free biochemical protein synthesis following the addition of a DNA sequence for the desired protein. Notably, the technology can be used to produce proteins incorporating non-natural amino acids and for the production of challenging biologic substances, such as difficult-to-fold proteins. In addition, the system is not limited to the production of small proteins and peptides, as is the case with microbial fermentation, and it is also readily scalable, according to Sutro.

About the AuthorCynthia A. Challener, PhD, is a contributing editor to BioPharm International.

Article DetailsBioPharm International, Vol. 28, Issue 1
Pages: 36-39
Citation: When referring to this article, please cite it as: C. Challener, "Fermentation for the Future," BioPharm International, Vol 28 (1) 2015.