September 15, 2009 (Vol. 29, No. 16)

Vicki Glaser Writer GEN

Fears Surrounding Pandemic Influenza Have Energized Companies with Enabling Technologies

In today’s highly competitive and rapidly evolving global vaccines market, companies are looking for techniques, technologies, and tools to optimize processes, accelerate the timeline for moving candidate vaccines into the clinic, and build flexibility into vaccine production. All of these initiatives seek to enable rapid and reliable scale-up, expedite process validation and regulatory review, and facilitate the transfer of manufacturing technology from site to site and country to country.

The 21st century may be the “new Golden Age of vaccines,” says Catorina Flyborg, Ph.D., leader of vaccine initiatives at GE Healthcare, who will speak about critical process parameters to maximize vaccine yield and quality at the “World Vaccine Congress” next month in Lyon, France.

Dr. Flyborg will compare traditional and disposable manufacturing platforms and describe how to identify the threshold at which it is economical and prudent—based on parameters of quality and quantity—to switch to disposable systems. As vaccines tend to be produced in smaller quantities than most other biopharmaceuticals, disposable production systems offer particular advantages. Dr. Flyborg expects the industry to continue to use a combination of stainless steel and disposable platforms.

Process optimization in vaccine production has many different facets and tends to be more complicated than for monoclonal antibodies (mAbs), for example, due to the greater complexity of the products. A key area for improvement early in the developmental pipeline is boosting expression levels in a cell line of interest to enable smaller volume, more efficient bioprocesses. Scale-up of mammalian cell culture is inherently more difficult due mainly to the need for higher oxygen concentrations. A generic goal for process optimization in vaccine production focuses on reducing water consumption and overall energy use.

Philip Ball, Ph.D., technical director at Eden Biodesign, will talk about end-to-end process development and scalable manufacturing at next month’s meeting. Capacity and scalability are key buzzwords in the industry today, as current vaccine capacity is insufficient, especially for influenza, despite continuing increases in global biomanufacturing capacity, according to Dr. Ball.

“We need transferrable and scalable production platforms,” similar to what has been achieved for mAbs. “I think we can learn from the antibody industry,” he says, in terms of rapid development of robust, transferrable processes for localized production, for which disposable technologies available up to about 2,000 liter scale are ideal. Disposable options are also rapidly expanding beyond the bioreactor and fermentor space and, for example, into purification processes, with growing interest in prefilled chromatography columns and single-use sterile filtration products.

When Dr. Ball speaks of end-to-end process development, he advises vaccine manufacturers to consider process development as part of, and not separate from, the product-development cycle. “Begin with the end in mind,” is Eden’s philosophy, and he encourages clients to understand the end-points of their processes, consider scale-up needs early on, and not look at a single process step in isolation.  

Eden strives to apply enabling technologies to maximize flexibility and efficiency. Examples include adapting adherent cell lines to grow in suspension for enhanced scalability in stirred tank reactors. The company offers expertise in a range of expression technologies and culture systems, including microbial, viral, baculovirus, and mammalian.

Novel purification strategies include the use of monolithic membrane absorbers with high binding capacity for both product capture and polishing steps, and a move away from centrifugation to more scalable and transferrable chromatographic techniques. Dr. Ball also describes the adoption of analytical methods and tools developed for the mAb industry, which are improving process characterization and product analysis, including novel HPLC- and mass spectrometry-based methods.

Medicago’s transient expression manufacturing platform has been developed and optimized for use with Nicotiana benthamiana, a relative of the tobacco plant.

Vaccines Go Green

Pandemic influenza has grabbed the media spotlight in recent months. Medicago reports that it has overcome many of the challenges associated with traditional egg-based influenza vaccine production, and now offers an alternative to cell culture methods. The company’s transient expression system produces  recombinant vaccine antigens in the cells of Nicotiana benthamiana.

The cells are transfected with the genes for key viral proteins, but unlike with viral vector-based transfection, there is no infection process and the transfected DNA does not integrate into the plant genome. Once the genetic sequence of a pandemic flu strain is identified, Medicago can reportedly deliver a vaccine for testing within about a month’s time. Rapid scale-up to produce large volumes of antigens can be achieved in dedicated greenhouses and processing facilities, which would ideally be built on-site in regions where vaccine need is most urgent.

At the “World Vaccine Congress Asia,”  held in June, Andy Sheldon, president and CEO of Medicago, gave a presentation describing the application of its Proficia™ manufacturing technology to produce H5N1 avian influenza virus-like particles (VLPs). The VLPs are the same size and shape as actual virus and contain hemagglutinin antigen on their surface, but do not contain any viral DNA and cannot reproduce. They induce both an antibody and a cell-based immune response.

Sheldon also reviewed the company’s R&D efforts related to an H1N1 swine flu vaccine candidate and described Medicago’s ability to generate H1 antigen within 14 days of receiving the DNA sequence of the H1N1 swine flu strain.

Medicago’s technology involves introducing a DNA sequence of interest into Agrobacterium tumefaciens, a bacterium present in soil that is a convenient vector and readily taken up by plants. The company grows Nicotiana benthamiana plants from seed—it belongs to the tobacco family and has large leaves and grows rapidly in the company’s computer-controlled greenhouses. The plants are grown in a mossy substrate, and each production lot yields about 25 kg of biomass.

Once the plants have reached a suitable size, they are transferred from the greenhouse to an adjacent processing facility where the plants are dipped in inoculants containing the engineered Agrobacterium. They are then placed in a vacuum container to optimize uptake of the liquid inoculant into the leaves.

“We trick the plant to produce the protein,” says Brigitte Barbeau, vp of manufacturing at Medicago, and they synthesize the VLPs as though they were a regular component of plant proteins.

The transfected plants are incubated for six days to allow for maximum antigen production. The VLPs bud between the cell wall and the plasma membrane. The leaves are harvested, and an extraction process grinds the leaves, breaking open the cells. The liquid produced is filtered and clarified to remove the plant proteins and other bioburden, and a chromatographic purification process captures the virus-like particles (90–95% pure), which are diluted in a saline buffer with no preservative.

As no actual virus is produced, there is no need for an inactivation step. Barbeau says that stability programs for the diluted doses ready for inoculation are prepared to cover a 12-month period—the target shelf-life for influenza vaccines. 

A High-Throughput Approach

The Pfenex Expression Technology™ platform from Dow Chemical exploits the advantages of Pseudomonas fluorescens, including its ability to achieve high optical densities in both small- and large-scale fermentation, with high levels of specific protein expression and a high success rate relative to traditional E. coli and yeast-based fermentation systems. The scalable Pfenex platform reportedly enables the evaluation of more than 1,000 host strain/plasmid combinations within five weeks in parallel through the use of robotics to identify those strains producing the highest titer of soluble, properly folded, aglycosylated, active protein.

The platform is especially valuable for the rapid evaluation of “numerous putative antigens in late-stage discovery and/or preclinical studies in vaccine development,” says Patrick Lucy, global business development leader at Pfenex. He describes the toolbox of Pseudomonas-derived components Pfenex has developed for selecting production strain characteristics and fermentation conditions that optimize protein expression and folding.

These include a two-plasmid system—with one carrying the gene for the target protein and the other carrying genes coding for helper proteins, each under separate control (a complementation strategy that eliminates the need for antibiotics), a selection of ribosomal RNA binding sites to effect the rate of translation initiation, more than two dozen, well-characterized secretion leaders, and more than 150 unique protease deletion strains to address proteolysis.

In these engineered strains the protein of interest typically accumulates in the  periplasm, where cleavage of the secretion leader and in vivo protein folding take place.

“We can rapidly screen over 1,000 strains in 96-well arrays using high-throughput screening methods,” says Lucy. The company recently implemented automated postexpression processing, including sonication to release the protein and subsequent sample handling and analysis of the antigens, which are ready for testing in animals. The platform has an “85% success rate for soluble protein expression for proteins that previously failed to express in other systems, and can produce milligram quantities of soluble antigen in eight to ten weeks,” Lucy adds.

Pfenex platform reportedly enables the evaluation of more than 1,000 host strain/plasmid combinations within five weeks in parallel through the use of robotics to identify those strains.
(Dow Pharma)

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