Multiple Expression Systems
Diosynth Biotechnology (www.diosynthbiotechnology.com) signed a long-term commercial supply agreement with Dendreon (www.dendreon.com) to produce a bulk ancillary component for its Provenge® product. Diosynth’s experience with the baculovirus expression vector system (BEVS) production technology played a role in that agreement. Currently, the company is manufacturing in the insect cell system at the 2,000-L scale.
According to Frank Tielens, president of Diosynth Biotechnology, “We have been rewarded with projects that take advantage of our expertise in the newer systems such as baculovirus-based and Pichia pastoris expression.”
George Koch, Ph.D., CSO at Diosynth, adds, “It is likely that our ability to scale-up, BEVS in particular, as well as our international capabilities and infrastructure, influenced Dendreon’s decision to go with Diosynth.”
In addition, the company has a history working with expression systems that are standard in the industry, such as E. coli and mammalian cell culture. Diosynth manufactures both clinical material and commercially approved products. “We are manufacturing difficult-to-produce macromolecules in evolving expression systems as well as the current state-of-the-art systems, whether the drugs are therapeutic enzymes, Mabs, or other recombinant proteins,” Tielens adds.
The pharmaceutical industry has added regulatory constraints, so contract manufacturers must be extremely cautious regarding the use of new technologies. “Within these constraints, technology advances that enhance processing capabilities are used for the benefit of customer projects,” Dr. Koch says.
Diosynth increased the cooling capacity in its production fermenters and development units for optimized production processes. The company is using disposable reactors in cGMP production as part of a seed train toward the larger-scale bioreactors to save time and costs with regards to sterilization, cleaning, and validation.
The company has experience with fed-batch processes as well as perfusion processes. “Perfusion is well-suited for the production of complex glycoproteins requiring a low residence time in the bioreactor,” Jan Visser, director of small-scale cell culture and development at Diosynth, states.
A perfusion mode of operation is characterized by the continuous addition of fresh medium and the withdrawal of an equal volume of used medium while retaining the cells in the bioreactor. “We currently have several cell culture perfusion processes under development in addition to commercially producing Organon’s (www.organon.com) Puregon® perfusion-based product,” says Visser.
At Boehringer Ingelheim (BI; www.boehringeringelheim.com), high-throughput screening of microbial hosts, high-yield expression systems, and alternatives to chromatography are three strategic areas for new technologies. Roman Necina, Ph.D., head of biopharmaceutical production, says, “Efficient technology and quality are key to the success of the project, so our approach is to develop competitive technology rather than building capacity.”
The quick identification of an optimum expression system and the best-suited host or plasmid combination, in terms of quality and yield, are critical for efficient biomanufacturing. BI Austria established a high-throughput-screening approach that allows the screening of a high number of bacterial and yeast hosts in parallel. Both qualitative and quantitative parameters are evaluated.
“The expression systems we evaluate include E. coli, based on periplasmic and cytosolic production (preferred as insoluble inclusion bodies), and product-secreting yeast systems,” Dr. Necina says. “After a screening round, the host with the highest productivity and best quality is selected.”
Increasing the volumetric yield of microbial expression systems is required to achieve low cost-of-goods-sold figures. “Our new Autoprotease Platform technology based on E. coli provides high-yield expression of finicky and large proteins,” Dr. Necina explains. “The fusion partner tends to aggregate and guides the fusion partner into inclusion bodies, providing yields up to 15 g/L. Subsequent refolding is mediated by the fusion partner, which cleaves itself off after correct refolding.”
As a different example, expression yields of pDNA, as high as 2 g/L, are based on BI’s E. coli host and a high cell density fermentation process. “A closed semi-continuous lysis system and purification process are used to manufacture large quantities of pDNA with high ccc content in a single downstream batch.”
For the future of biopharmaceutical contract manufacturing, Dr. Necina sees a demand for increased flexibility at the plant and reduced lead time, which means an increasing use of disposables and mobile equipment. “High-yield, high cell density fermentations will become more important in the future, challenging plant design and processes for primary recovery.”
“BI Austria is developing alternative downstream technologies such as crystallization and extraction. These technologies allow fast and cost-efficient processing of large amounts of biopharmaceuticals without investing in large vessels, expensive chromatographic resins, and chromatographic hardware. These processes can be operated as closed or continuous systems.”