Microorganisms play a vital role in modern life — with applications ranging from wine fermentation to biofuel production to solutions for complex mathematical problems (1). During the past decade, microbial fermentation for protein production reached a higher level of sophistication and wider adoption. When BPI was first published in 2003, the physical and biological characteristics of many microbial cells and the attributes of their fermentation processes were well known. Nonetheless, the economic environment at that time created immense pressure on the industry to drive innovation and emphasize manufacturing efficiency (2).
BPI’s Protein Expressions supplement in 2004 reviewed microbial fermentation, its advantages over mammalian cell expression (e.g., lower generation time, growth time, media costs, robustness), and its shortfalls (e.g., for most systems, glycosylation and posttranslational modifications) (3). Our 2008 coverage of microbial expressions confirmed that companies continued to use those same systems, typically prokaryotes such as bacteria (e.g., Escherichia coli), eukaryotes such as filamentous fungi (e.g., Aspergillus spp) and yeast (e.g. Saccharomyces cerevisiae and Pichia pastoris) (4). At that time, of the expression systems used for producing recombinant proteins in the United States and Europe, 55% were expressed using microbes — 40% bacteria (39% of which as a form of Escherichia coli) and 15% yeast (5). Also at that time, however, a few other promising microbial systems had begun to gain attention along with studies involving novel applications, “platform” technologies, and improved analytical and montitoring techniques.Escherichia coli
The most prominent expression system after CHO cells is E. coli. Especially robust and economical for the production of antibodies and recombinant proteins, it continues to be the top microbial host (of more than 30) (6, 7). Studies have demonstrated the ability of E. coli to make milligram-quantities of glycosylated proteins, thereby broadening its potential applications (8).
During the past decade, new E. coliplatform technologies have been developed. For example, in 2009, Mücke et al. demonstrated a successful technology based on E. coli secretion and high-cell-density fermentation for producing high yields of human antibody fragments (Fabs). (A separate study on Fabs expressed using Pichia pastoris resulted in mixed results when compared with expression from a CHO platform (9)). Also in 2009, Pattnaik et al. described the application of membrane technology in the production of inclusion-body proteins from E. coli(10). They used a “highly productive fermentation process and high-throughput purification” to successfully produce (and scale) therapeutic proteins from E. coli.
Industrial applications of E. coli host systems began in the 1980s and continue to be core business strengths for many companies. Boehringer Ingelheim Austria GmbH, for example, claims to have “pioneered the microbial fermentation and purification of therapeutic proteins from E. coli since 1982.” (11). Sandoz was also an early (1980) developer of the recombinant protein interferon alpha at an industrial-scale fermentation.
In 2008, more than 70% of Lonza’s fermentation projects were on an E. coli platform (with Pichia pastoris gaining preference) (12). Since then, the company has developed its XS Microbial Expression Technologies platform and expanded its E. coli and yeast expression technologies into developing and manufacturing plasmid DNA, antibody fragments, and protein scaffolds. Other major companies providing microbial fermentation capabilities include SAFC, Life Technologies, and Biocon.Upcoming Microbial Host Systems
The past decade saw the introduction and growing interest of other expression technologies. Here are two we are keeping an eye on during our next 10 years.
Pseudomonas fluorescens: In 2009, Dow announced the formation of a new company (Pfenex) based on its Pfēnex Expression Technology system. The Pseudomonas fluorescens-based platform uses “high throughput, parallel processing methodologies for optimized protein production” (13). Since then, the product pipeline already contains five products, including biosimilars, biodefense molecules, and vaccine antigens. The company published cost comparisons with other microbial systems using “a computer aided approach to process economics” to show costs associated with the production of an aglycosylated protein (14).
Lactoccocus latis: This system has shown potential as “a viable choice for membrane proteins” (8). Traditionally used in food fermentation, L. latis is a gram-positive lactic bacterium that is “now used widely for large-scale overproduction of heterologously expressed proteins. Recombinant membrane proteins can be produced with affinity tags for efficient detection and purification from crude membrane protein extracts” (15).Fermentation Monitoring Systems
Advances in monitoring systems and enumerating technologies have also taken place this past decade. BD, for example, developed its FACSMicroCount system, and scientists from Hamburg University of Applied Sciences have a developed biomass monitoring system based on radiofrequency impedance (16). Single-use bioreactors for both mammalian cells and microbial fermentation have been developed that integrate sensors and provide feedback through various display units (e.g., Cell-tainer from CELL-ution Biotech BV).
Maribel Rios is managing editor of BioProcess International;
HP. 2008. An Emerging Star for Therapeutic and Catalytic Protein Production. BioProcess Int 6:S10-S21.