October 15, 2006 (Vol. 26, No. 18)

Helping Biologists Turn Ideas into Productive Cell Lines

Because it covers such a broad end-use range from single-cell assays through mega-processing, one could say that as cell culture goes, so goes biotechnology. Among the emerging, perennial trends in cell culture are automation, disposable systems, the ongoing evolution of cell culture media, and increased interest in cell culture for vaccine production.

The Automation Partnership (TAP; www.automationpartnership.com) focuses on automating the research end of the business, which is rapidly growing due to interest in cell-based assays and cell line engineering. TAP’s SelecT™ and CompacT SelecT instruments streamline manual culture of multiple cell lines in an assay-ready plate format. TAP’s Cello™ provides automated clone selection from multiple cell lines cultured in parallel, while Piccolo™ optimizes protein production from microbial or insect cell lines. Moving up a notch, the company’s Cellmate offers larger-volume cultured cells for roller bottles and T-flasks.

According to Tim Ward, director of cell culture, TAP’s goal is to improve the efficiency with which biologists turn ideas into productive cell lines. Traditionally, researchers perform experiments sequentially. Cello, Piccolo, and the SelecT line help optimize cells through parallel culture of tens of thousands of clones.

“By applying automation, scientists do not need to make deductions from the first experiment,” says Ward. Another automated, parallel instrument is in the works for testing media.

Heidi McIntosh, product manager for cell culture at Nalge Nunc International (www.nalgenunc.com), sees a lot of specialty cells cultured for cell-based assays and corresponding interest in specially coated roller bottles and consumables (e.g., media, buffers, reagents).

Nalge Nunc’s line of flasks, roller bottles, and microtiter plates include products coated with poly D-lysine (PDL), a poly amino acid that enhances cell adherence. The company also offers a chemical mimic of PDL, CC3™, which unlike PDL is sterilizable and has a five-year shelf life (as opposed to three for PDL). Nunc also offers low-binding coatings suitable for stem cell research.

Rising protein titers and cell densities continue to blur the distinction between large- and small-scale cell culture, to the point where small-scale equipment is increasingly used for manufacturing. For medium-sized batches of vaccines, antibodies, or therapeutic proteins, Nalge Nunc’s Nunclon™ Cell Factory uses stackable trays with working volumes of 200 to 8,000 mL. Up to four 8-liter cell factory units can be daisy-chained.

According to McIntosh, demand for small- to mid-sized cell culture equipment is rising as customers seek inexpensive alternatives to bioreactors during early product development. “These are not competing with 10,000-liter stainless steel bioreactors,” she says, “but they provide a way to research a process at a low capital investment.”

New Brunswick Scientific (www.nbsc.com) just introduced a benchtop bioreactor for R&D through small-scale production. The CelliGen® 310 bioreactor with advanced touchscreen controller provides built-in control of up to 32 process parameters per vessel and the ability to control up to four reactor vessels, or over 120 control loops, simultaneously.

Features include the ability to choose up to five thermal mass flow controllers for sparge and gas overlay and connect over 10 external devices, including sensors, scales, gas analyzers, and more, for optimized process control. A variety of specialized vessels and impellers are offered, including the packed-bed basket.

SAFC Biosciences (www.sigmaaldrich.com), a supplier of equipment and raw materials to companies producing proteins through cell culture, offers buffers, reagents, liquid and powdered media (serum-based and -free), single-use bags, pre-assembled tubing and filter sets, filters, sera and serum-free media, and cell culture development services. Among the company’s latest products are ExCell™ 302 serum-free media for CHO cells and the BIOEAZE™ lines of polyethylene and ethyl vinyl acetate storage bags.

According to Denise DeTommaso, market manager for single-use products, interest in disposable process equipment continues. The economic and logistic advantages of single-use equipment remain compelling at all scales, she says, but handling 2,000-L bags presents certain ergonomic issues. “How do you move such a large bag around after it’s filled, how do you get it into the rigid container, and how do you fill it?” Although hardly insurmountable, these problems demand novel facility and equipment solutions. Luckily, processes continue to shrink through productivity improvements.

Along with continuing growth in disposable storage and cell culture bags look for more disposable downstream process equipment such as process-scale filtration devices. Vijay Singh, Ph.D., president of Wave Biotech(www.wavebiotech.com), expects disposable chromatography will be “widespread in the near future. With these developments, purification will evolve to a more closed system with the benefits of lower cost and higher quality.”

In the cell culture arena, Dr. Singh sees disposables as central to patient-specific (personalized) therapies, particularly for gene therapy vectors, cell therapy, stem cell culture, and ex vivo applications. “Many of these technologies are already in advanced clinical trials using our single-use Wave bioreactors.”

Disposables will also gain from smaller cell culture batches (thanks to titer improvements), as well as for microbial applications. Wave recently introduced the WavePOD instrument module, which integrates a bioreactor’s analytical instrumentation for dissolved oxygen, pH, and gas composition controls in a single, portable device.

Experts increasingly credit rising protein titers to superior media composition, to the point where time and investment on media development can reap hundred- or thousand-fold returns by eliminating the need for plant expansions and new construction.

The Media’s the Thing

Not too long ago bioprocessors lamented the switch to serum-free media, claiming that some cells only grew well in that mysterious mixture of cow blood and other ingredients. Brandon Pence, bioproduction market manager at Hyclone (www.hyclone.com), does not believe that serum-free runs counter to increased productivity. “On the contrary, the serum-free trend has been driving higher protein titers because serum, which contains everything, masked the unique nutrient requirements of particular cell lines,” he explains.

Chemically defined media enhances the ability to optimize cell culture because processors know the precise composition and can adjust individual components up or down to maximize productivity. Chemically defined media provides greater confidence in scaleup as well, since scaling a defined media recipe is straightforward. “You know it will behave the same at 10,000 liters as 10 liters,” Pence adds.

Hyclone has been active in custom media development through its Rapid Response Production™ (RRP) service. RRP provides liquid media in sizes up to 200 L and powdered lots up to 10 kg within seven days of request to support cell culture in hybridoma, NS0, insect, CHO, PER.C6™, HEK 293, MDCK, MDBK, and Vero cell lines. In addition, RRP manufactures custom-made buffers. Pence describes growth of the RRP service as phenomenal.

Anyone working in the food or beverage industries (think soft drink syrups) would find it incredible that at one time, most cell culture media shipped in fully constituted liquid form. There were actually good reasons for this. Some media were proprietary, so vendors maintained control by distributing only the final product. Plus many media contained animal-derived components, such as serum and salts and other solids that either did not dissolve readily or did not lend themselves to powdered formats. The trend toward chemically defined media and custom formulations has changed the face of media and media development, although hydration and formulation difficulties remain.

Invitrogen (www.invitrogen.com), through its Gibco brand, offers chemically defined dry form media made using AGT (advanced granulation technology). Where conventional powdered media require addition of multiple components and pH adjustments, AGT-based media are reconstituted in one step—adding high-purity water (typically water-for-injection grade). The key is AGT’s granular formulation, which hydrates quickly.

According to Bob Burrier, Ph.D., vp of research and development, customers are turning to dry-form media earlier to eliminate the transition from liquid at the research stage to dry-form formulations at pilot and manufacturing scale. “We have removed that unknown from the equation,” he says.

Chuck Velnoskey, bionutrients market development manager at BD (www.bd.com), believes there is still quite a bit of life remaining in media based on peptones (protein hydrosylates). BD’s protein hydrosylates are derived from nonanimal sources such as soy or yeast, out of concern over prion diseases. Velnoskey describes these products as “productivity enhancers” with a long history of boosting cell culture performance at a competitive price. “Peptones can achieve better performance than chemically defined media,” he says, “and at much lower cost.”

BD is in the process of characterizing peptone media ingredients to identify components critical for cell growth, with the idea of eventually creating a peptone-based media that is fully characterized for amino acid concentrations.

Cell Culture Solutions (www.cellculturesolutions.com) focuses on services for cell culture design of experiment (DOE), a quality tool borrowed from Six Sigma. MKC Consulting, a Netherlands-based six sigma consultancy, defines DOE as “a process using planning and statistical tools for gathering the maximum amount of useful information from an experimental program.”

According to Brigitte Van der Hagen, M.D., Ph.D., president of Cell Culture Solutions, cell culture processes used to be designed one variable at a time, “by trial and error. If twenty factors were deemed important, they would optimize by holding nineteen constant and changing one.” DOE applies statistical tools to multivariate analysis of cell culture, thereby streamlining the optimization process. “DOE quickly eliminates irrelevant factors.”

Despite its appeal, pharma/biotech companies are resisting DOE due to its unfamiliarity. But with powerful desktop computing and readily available software, the real hurdles to adopting it are gone, says Dr. Van der Hagen. “You don’t need to be a statistician, so there is no excuse for not adopting it from the very beginning of a cell culture project.”

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