Production of Functional Proteins
Elena Kovaleva, Ph.D., head of R&D at Chesapeake PERL, discussed her company’s program of large-scale production of proteins by various lepidopterin species including the cabbage looper. The process involves growing a large number of larvae, infecting them with baculovirus, followed by processing and protein purification.
Cultivation is on a massive scale, generating greater than 10 kg of biomass per hour to a capacity of 200 kg per week for the facility. The company has years of experience with the process, so stability and reproducibility can be assured, Dr. Kovaleva said. By the same token, the baculovirus expression vector system is well-established with custom made vectors based on PERXpress protein production in whole larvae. The work is done with the preoccluded form of the virus, which is infectious when ingested but noninfectious when initiated into cultures in vitro.
Dr. Kovaleva asserted that the heterogeneity of cell types present in the larva allows for a higher fidelity of protein production than that realized with homogeneous cell cultures. Larvae can participate in propeptide and c-terminal cleavage, dimerization, tetramerization, and association with ancillary molecules. Examples of proteins successfully expressed in the PERL platform include fluorescent proteins, viral antigens, virus–like particles, and a range of different enzymes.
A colorful example of the the PERL expression technology is the fluorescent protein DsRed, cloned from the coral Discosoma. Highly stable and bright red, the protein serves as an excellent marker for tracking protein behavior. In order to produce kilogram quantities of protein, infected larvae from the second passage are stored away for later preparation for the next round, thereby overcoming one of the main impediments to large-scale protein production in insect bioreactors.
Baculovirus makes a convenient system for vaccine development, especially given that subunit vaccines are composed of antigen subunits rather than a live virus.
“We are able to continually improve the process, based on host characterization and our broad base of technical experience,” said Dr. Kovaleva. “This places us in a strong position for reaching unlimited production of identical proteins.”
Producing Recombinant Proteins
“In an alternative approach to the use of baculovirus, we have used Spodoptera frugiperda insect cell lines stably transformed to express secreted alkaline phosphatase,” said Satya Prakash, Ph.D., director of the biomedical technology and cell therapy research laboratory at McGill University.
Dr. Prakash said his system offers a number of important features. Recombinant proteins generated in insect cells are glycosylated and biologically active, and large-scale, high cell densities cultures are achievable while maintaining specific productivity. Moreover, stably transformed lines don’t have the problem of protease interferences observed in the lytic system, and the proteins produced are homogenous and consistent from batch to batch. Perfusion and fed-batch modes are easily scalable for insect cell culture, he added.
“We have also generated a potential therapeutic glycoprotein, human Interleukin-7,” he said. This protein is critical for cellular proliferation during B-cell maturation and for T cell and natural killer cell survival, development, and homeostasis. It is an essential growth factor for the immune T cell.
The investigation included a comparison of protein expression using the Baculovirus system with the stably transfected Sf9 insect cell cultures in a conventional stirred-tank bioreactor or the disposable Wave bioreactor system (GE Healthcare), using batch and fed-batch strategies.
The protein synthesized was more homogeneous in the stably transformed cell lines than in the baculovirus-infected cells. Both systems are nonapoptotic and perfect candidates for perfusion and fedbatch systems, he concluded.
Algae excels as a recombinant protein production platform due to its scalability, cost, and containment. In analyzing the economic factor, Dr. Mayfield cited the astronomical yearly costs to patients of a laundry list of protein therapeutics: Avastin $50–100,000; Herceptin $40,000; Erbitux $60,000; and finally Ceredase weighing in at a staggering $500,000. While a number of factors (including corporate marketing decisions) factor into these figures, the estimated financial burden per gram of raw material ranges from $150 for protein produced in mammalian cells down to a minuscule $0.05 per gram for transgenic plants. So it appears that a plant-based protein production platform could only affect consumer prices in a positive fashion.
According to Dr. Mayfield, given selectable markers, transforming plant cells is a relatively easy task, proceeding by homologous recombination. These transformed lines can be built up to commercial scale, at a level of thousands of liters of cell mixtures, in a matter of three to four months. This includes the optimization of properties and the screening and evaluation that accompanies the process. Algal species are GRAS organisms (generally regarded as safe), and although not included in the supermarket gourmet section, they are indeed edible, thus lending themselves to use in vaccine technology.
The organism has another important advantage; it is endowed with chloroplasts that can be easily and conveniently manipulated genetically. Engineering of the chloroplast genome in Chlamydomonas is accomplished through well-described use of promoters and expression elements, and recombinant proteins can be driven to accumulate at high levels. Dr. Mayfield highlighted the use of his team’s technology for codon optimization using GFP markers.
As a proof-of-principle, Dr. Mayfield’s team has engineered strains of Chlamydomonas to produce the bovine serum amyloid protein A3, as well as seven human proteins. This effort included a full-length human monoclonal antibody, which can be shown to accumulate in the chloroplasts.
The use of micro-algae as a biotechnology platform lags behind other organisms, especially bacteria and yeast. This is somewhat surprising given the ability of algae to be grown at large scale in a cost-effective manner. Eukaryotic algae offer tremendous potential for the large-scale and cost-effective production of recombinant proteins.
Fabien Walas, Ph.D., a project leader at ERA Biotech, presented data at the summit concerning the in vivo encapsulation of recombinant proteins in the development of a universal production platform. The technology is based on the zeins or prolaminins, major storage proteins in plants. Specifically, g-zein has the unique property of being soluble in aqueous solution, yet forming aggregates in cells. This process involves a specific, sequential interaction between the various zein species leading to protein body biogenesis. Maize endosperm is especially rich in these large entities.
The Zera® self-assembly peptide was engineered by scientists at ERA Biotech as a fusion protein into which a target of interest was incorporated. Recombinant cells package fusion protein into storage organelles. The Zera fusion peptides interact with the endoplasmic reticulum membrane, inducing the formation of StorPro® organelles, which are insulated from proteolysis. The sequestered proteins are well folded and protected from proteolytic degradation. The platform allows accumulation of protected biomass in a rapid time frame, Dr. Walas added.
The beauty of the system is that the product is protected from the cell, while at the same time the cell is protected from the product and stabilized for long-term storage. This means simpler downstream processing including washing and removal of contaminants.
According to David Wood, Ph.D., a member of the department of chemical and biochemical engineering at Ohio State University, current developments in the bioprocessing industry are driving the need for constant reinvention. These include the completion of the human genome project, the expiration of patents on various biologicals, a massive increase in the yield of recombinant proteins, and a streamlining of the validation process. These changes ensure that there will be a continuing demand on companies and academic investigators to bring forth updated versions of their favorite bioprocessing technologies.