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Feature Articles : Oct 15, 2011 (Vol. 31, No. 18)

Tracking Key Biomanufacturing Trends

Researchers Seek to Produce Safer and Highly Effective Products in More Economical Manner

Cell culture media, novel resin technologies, and single-use systems are among the most active areas of R&D in the biomanufacturing sector. GEN recently interviewed several scheduled speakers for the upcoming “Bioprocess International Conference” in Long Beach, CA, to get a better sense of how this market is evolving and to see what types of new products are available.

Until the late 1980s, manufacturers of biopharmaceutical products cultured their cells in fetal bovine serum, despite lot-to-lot inconsistency, contamination, and supply issues. With the discovery of bovine spongiform encephalopathy in the late 1980s, and with resulting increases in regulations, manufacturers began to aggressively explore alternatives to serum-based media.

The upshot has been a growing interest in supplements in which all components are entirely chemically defined, animal-free, and protein-free.

In response to this demand, said Samuel Denby, Eng.D., scientific manager for technical applications at BD Biosciences, the company is now marketing BD Recharge™, a chemically defined cell culture media supplement. The product is the first in a series that BD is developing.

“We use proprietary fractionation processes combining analytical methods and bioassays to identify the key components in a yeast extract peptone,” he said. “From these we then selected the optimal chemical constituents for cell nutrition. Although our initial focus has been CHO cell lines, we have several customers evaluating other lines.”

Dr. Denby claimed the product offers equivalent or better yield as compared with traditional peptone supplementation, a reduction in variability while retaining protein quality, and the potential for an easier regulatory filing in biopharmaceutical manufacture.

Supplements need to be evaluated not only for their growth potential but also for their productivity, continued Dr. Denby.

“We typically start with small batch cultures and move up to confirm our results in small-scale bioreactors. In terms of developing our next-generation supplements, pulling the peptones apart is challenging, but putting a chemically defined alternative back together again is equally challenging.

“We use a cross disciplinary team, as well as alpha site evaluations with multiple biopharmaceutical manufacturers who tested the product with production cell lines.”

Medium Suitability

Among the many factors that determine the suitability of a cell culture medium, two are paramount, noted Tom Fletcher, director of R&D at Irvine Scientific.

“The first is the metabolic behavior of the chosen cell line and the second is the content of the initial growth medium,” he explained.

The company has been rapidly expanding its media production facility. Beginning in the early 1970s Irvine began producing media and researching improvements that resulted in the chemically defined, serum-free products now available.

Because of its many favorable attributes, CHO has become the premier cell line for biopharmaceutical production.

“Most of our work has been with CHO lines, and we have been engaged in a number of collaborative research projects with companies to develop optimal media for specific CHO clones,” Fletcher added. “This has been a boon for us since we find that different CHO clones have undergone significant metabolic alterations and their nutritional requirements may be radically altered.”

According to Fletcher, significant changes have taken place with the opening of a sister facility in Japan, near Tokyo. Located in Saitama prefecture, the plant has the capacity to manufacture animal-component-free cell culture media in volume requirements ranging from research use to industrial scale.

The facility has been commissioned and validated as animal-component-free. The new site is designed to replicate the production and processes used at the company’s Santa Ana, CA, manufacturing facility and is outfitted with the same formulation and packaging equipment.

Risk Mitigation

Wade Nudson, a technical manager at SAFC, discussed his company’s efforts to design effective risk-mitigation strategies for cell culture reagents.

Over the last 20 years, media evaluation has become much more demanding and complex as the number of reagents and media options grows and as more and more production volume shifts to biopharmaceutical uses. Quality control is one of the most pressing concerns.

“We deal with approximately 400 different raw materials in our production schedule,” said Nudson. “We put an enhanced program in place over the last two years that evaluates them analytically and biologically, especially when chemical characterization may not be sufficient.

“We establish dose-response growth curves that are precise and allow accurate, repeatable characterization of our products. We are constantly refining our evaluation procedures, doing a lot more mass spectroscopy, HPLC, and similar studies.”

While the company’s raw materials are purchased only from approved, qualified vendors, quality is not the only issue. There is also availability of the materials used in cell culture.

For example, single-source components like specific plant-derived hydrolysates have experienced supply issues in the past. Any changes in raw materials are carefully planned in advance. Change control and change notification are extremely important in this industry.

“If you lose a bioreactor run in a drug production process because of medium or other raw material failure it could cost the client $50 million in final product or more,” according to Nudson. “Therefore, we make no changes in major components without an extended consultation with the client.”

For single chemicals, it is usually relatively straightforward to check identity and to certify that they are sufficiently pure. For other more complex components, it can be much more difficult. This is a particular problem for undefined components, such as hydrolysates, whose composition is naturally variable, even between batches from the same supplier.

A rule of thumb employed within the industry is that 10–15% biological variability from batch to batch is to be anticipated, and could represent the lower limit of acceptability. To deal with this issue, Nudson describes a chemically defined hydrolysate option offered by SAFC in which the components (such as peptides, amino acids, vitamins, and trace elements) are known and reproducibly formulated.

Investigation of growth parameters has established that this defined medium option supports growth as well or better than the previous undefined product, noted Nudson. This represents a long-term trend in the industry aimed at the eventual elimination of all undefined components in cell culture media formulations.

“That’s what we’re striving for,” Nudson stated, “as this would lessen our dependence on biological assays for quality assurance.”

Novel Resin Technology

Layered beads is a novel resin design with the potential to provide molecular functionalities that can take on the challenge of complex separation problems, according to Klas Allmér, Ph.D., director of R&D at GE Healthcare.

The approach is essentially an onion design with differing functional layers building out from an agarose bead core.

“By designing the beads with different ligands in the inner and outer layers, new properties and features of the chromatography resins can be realized,” explained Dr. Allmér. “We feel the layered beads technology will solve difficult separation problems while at the same time offering significant economic benefits.”

Current downstream vaccine processes use centrifugation with sucrose gradients or size exclusion chromatography, a strategy providing quite limited throughput. One possible configuration of the layered bead technology is using an inert shell and a functionalized core.

This bead design can eliminate host cell proteins and DNA fragments in a single chromatographic step, using multimodal ligands in combination with size exclusion. If the ligand is located in the core of the bead, large entities, such as whole virus particles, pass through the column for collection, while smaller contaminating proteins are retained.

“An additional advantage is the scalable nature of the layered beads technology that makes it applicable to small lab-scale operation or industrial procedures,” said Dr. Allmér.

Single-Use Systems

In light of the increasing popularity of single-use systems, Thermo Fisher Scientific is rapidly expanding its technology base in this area, according to Justin Hutchinson, product manager, single-use technology. Hutchinson will be involved in a strategy discussion forum at the Long Beach meeting.

He pointed out that when Thermo Fisher Scientific first began building single-use containers they were just simple bags.

“But over the years we’ve created a new generation of delivery systems,” he said. “Now we’re developing complex options that are more than just static units for holding the culture medium. We have a vision to turn the entire bioprocess operation cycle into a single-use function, and we’ll be looking to both enhance our current product lines and move into complete, integrated bioprocessing platform solutions.”

Noting that single-use containers are, by definition, disposable, Hutchinson explained that Thermo Fisher Scientific has invested substantial effort into creating recycling technology for the materials after they have been used. There are a number of cost-effective ways that polyethylene and other components can be used as fuel sources for the operation of water-purification systems and other energy-intensive tasks.

Recycling can be carried out in-house or at large facilities that are able to deal with waste that may consist of several types of plastic and even some stainless steel and electronic components mixed in.

“The challenge of pure recycling is significant since these devices are laminated and difficult to disassemble,” said Hutchinson. “Yet, in the final analysis, we note a wealth of data establishing the green credentials of single-use devices as being much more energy efficient compared to reusable hardware.”