Improving Protein Expression with Novel Systems

Published on: 
BioPharm International, BioPharm International-05-01-2014, Volume 27, Issue 5

New human and plant-based expressions systems can enable faster product development and improve quality at potentially lower costs.

Biopharmaceuticals are generally manufactured in established cell lines that are dominated by mammalian cells, particularly those based on Chinese hamster ovary (CHO) cells, but that also include E. coli (bacterial) and S. cerevisiae (yeast) cells. While these traditional cell lines are productive, their development can be slow and costly, and animal-derived systems provide non-human glycosylation patterns. Despite these difficulties, these traditional expression systems are generally used because they are approved by FDA, they are familiar, and there are established downstream purification processes for removing contaminants. Alternative expression systems, on the other hand, are being designed to avoid the performance issues associated with traditional cell lines. They offer biopharmaceutical manufacturers the chance to establish a stronger intellectual property position and improve their processes. New animal (including human), yeast, and plant-based expression systems have been shown to be effective for the production of biologics.

Human cells ideal for expressing human proteins
Human cells present the proper background to express human proteins. They can add the correct posttranslational modifications to these proteins, according to Nicole Faust, senior vice-president of development and services with CEVEC Pharmaceuticals. “Having the correct posttranslational modifications is of particular importance when expressing complex glycoproteins; human cells only add modifications normally found in humans, while with rodent cells, foreign structures can be added to the proteins that are potentially immunogenic in humans, such as N-glycolylneuraminic acid,” she explains.

The use of human cells is also advantageous because any potential host-cell proteins (HCPs) present in the final preparation of the therapeutic protein will be of human origin and therefore present a far lower immunogenic risk than host-cell proteins derived from nonhuman cells. Such an immune response to contaminating HCPs has been observed with rhFIX produced by CHO cells, according to Faust. “Some human host-cell expression systems can also be used to propagate a variety of different human-pathogenic viruses, which extends the versatility of the expression host from recombinant proteins per se to relevant vaccines and viruses,” she adds.


Plant-based expression offers speed and simplicity
With plant-based expressions systems, such as the transient expression system in whole green plants (Nicotiana benthamiana) from iBio, there is no requirement to identify, amplify, and adapt high-expressing cell clones to large-scale culture, saving a year to 18 months in product development time, according to Terence E. Ryan, iBio’s senior vice-president and chief scientific officer. With iBio’s system, production of protein can take place within 21 days of knowing a gene sequence, and this speed has attracted a lot of attention in the area of vaccine antigen production for pandemic diseases, particularly for influenza, according to Ryan.  

The process is also simple, with plant biomass grown over five weeks prior to vector infiltration. The plants are grown in hydroponic medium in a soil-free substrate under controlled conditions. Once the plants reach an appropriate size, the vectors are introduced by vacuum, delivering the vectors to all of the leaf cells at the same time. Once gene expression reaches its peak, the plants are ground and homogenized, allowing the desired protein to be purified by standard chromatography. “No bioreactors or staff skilled in aseptic cell culture are required, and thus this technology is highly attractive in areas of the world where capital and a highly skilled biopharmaceutical work force are in short supply,” Ryan says.  In addition, he notes that because the process is virtually the same regardless of the product (only the purification chromatography varies significantly), a single factory design can support multiple products, allowing for “campaign-style” production in one site.  

The speed and flexibility offers tremendous advantages, particularly at the early stages of development, by allowing product optimization and product risk mitigation in much less time and at much lower cost, according to Gene Garrard Olinger, a principal science advisor with MRIGlobal. He adds that in comparison with CHO, plant systems, specifically the rapid antibody manufacturing platform (RAMP) developed by Icon Genetics and Kentucky BioProcessing, offer the ability to control specific glycosylation patterns on the protein (1). “For antibodies, this specificity in glycosylation yields products with superior antibody-dependent, cell-mediated cytotoxicity (ADCC) profiles,” he observes. Ultimately, Olinger believes that with scale, the method should offer a significant cost advantage compared to cell-culture-based production.

Meaningful scientific advantages

Plant-based expression systems are also valuable because plants are capable of producing just about every type of biopharmaceutical product, including vaccine antigens, enzymes, replacement proteins, monoclonal antibodies, and others, according to Ryan.  In addition, because no animal products are used in iBio’s technology, there is no risk of contamination by animal viruses or other adventitious agents.

Meanwhile, the human amniocyte CAP cells developed by CEVEC, which are not derived from cancer cells or aborted embryos, are designed to efficiently produce complex proteins and glycoproteins at commercial scale, and therefore high product titers can be obtained even for difficult-to-express proteins, according to Faust. “We have found that in some cases, CAP cells have yielded 5- to 10-fold more protein than CHO cells. In addition, less or no proteolytic degradation of sensitive proteins was observed with the CAP system,” Faust observes. She adds that the cells are robust and easy to handle, and unlike traditional expression systems, the product quality is unaffected by small variations in the upstream process, making production runs highly reproducible. CEVEC has also developed a highly efficient transient protein expression platform (CAP-T cells) that is based on CAP cells and produces proteins with nearly identical post-translational modifications. “As a result, the transition from transient protein production in CAP-T cells at early project stages to stable production in CAP cells at later stages is very easy,” Faust says.

Further advances and demonstration of utility
Going forward, Faust believes that an increasing number of genetically engineered cell lines that have a bias towards certain post-translational modifications will be developed. She also expects further improvements in expression levels of CEVEC’s human expression systems through optimization of vectors, the use of cells with altered metabolisms, and further improvements in chemically defined media.

With respect to plant-based expression systems, Larry Zeitlin, president of Mapp Biopharmaceutical, expects rapid increases in yield to be realized. Zeitlin is hopeful that existing facilities such as the one at Kentucky BioProcessing, which can produce 10 kg of GMP protein per year, will be expanded to produce 100s of kg/year. “We also hope that the reduced capital costs to build plant manufacturing facilities will enable local production in developing nations to address their unique public health needs,” he says. Olinger adds that there is advancing work in tobacco with vaccines that use production of protein subunits to fully-formed, virus-like particles (VLPs), and he anticipates that with FDA approval of elelyso, which is derived from a carrot-based expression system, more plant-derived pharmaceuticals will be commercialized over the short and long term.

Efforts at iBio are directed at demonstrating the utility of its expression systems in new biotherapeutic classes, which often requires the coexpression of heterologous proteins within the same cell to provide helper functions or enzymatic cleavages not normally accomplished by plant cells. “Many interesting biotherapeutics are naturally expressed as ‘pre-pro’ proteins whose active form is produced as a result of post-translational cleavage or other activation,” he explains. iBio’s long-term focus is on the recapitulation of the complete human glycosylation machinery so that plant proteins are identical at the glycoform level to human products, according to Ryan.

Collaborating to advance novel expression technologies
One area that iBio is particularly proud of is the use of its technology to develop a next-generation yellow fever vaccine in partnership with the Fraunhofer Center for Molecular Biotechnology, the Oswaldo Cruz Institute (Fio-Cruz), and Bio-Maguinhos, with the latter two entities from Brazil, according to Ryan. “Bio-Maguinhos is the largest manufacturer of yellow fever vaccines in the world, and the perceived safety advantages of a recombinant subunit vaccine over the current (but 70-year old) live attenuated virus vaccine has spurred the development of the new vaccine,” he notes. In fact, the design for a manufacturing facility in northeastern Brazil capable of processing 1000 kg of plant material per week is in the second of three planning stages under sponsorship by the Brazilian government. “Through this project, we have received major endorsement of our technology and been able to demonstrate how quickly it can be recognized, adapted, and brought into manufacture,” Ryan concludes.
iBio is also collaborating with Caliber Biotherapeutics (College Station, TX) to establish a turn-key plant-based biopharmaceutical development capability, from the earliest stage of product selection and optimization through large-scale production.

For CEVEC, the growing market for complex, glycosylated molecules is reflected in the increasing number of worldwide licensees using its CAP technology, according to Faust. In addition to several top pharmaceutical customers, the company recently signed a licensing deal with Yuhan Pharma, one of the larger Korean drug manufacturers. A clinical Phase I study with CAP cell-derived human alkaline phosphatase was successfully completed in Q4 of 2013 in the Netherlands, and several customer molecules are nearing clinical trials. CEVEC also achieved two major milestones by showing excellent viral yields for influenza and RSV in addition to licensing its CAP cell line for the exclusive production of a cytomegalovirus vaccine based on dense bodies.

1. Zeitlin et al. PNAS 108 (51), 18030-18035 (2011).

About the Author
Cynthia A. Challener is a contributing editor to BioPharm International.