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Jan 1, 2006 (Vol. 26, No. 1)

Cell Culture Advances Enable Better Biology

Customized Formulations Prove to be the Key to Media Optimization

  • Two main forces are driving the cell culture marketplace, according to some industry observers.

    "Improving the user's experience and creating better biology," is how Martin Naley, director of the business segment for cell culture research products at Invitrogen (www.invitrogen.com), puts it.

    The first goal involves making it easier for users to find, buy, and use cell culture products. To achieve this, Invitrogen sent observers to 57 cell culture laboratories to learn how materials are delivered and used, with the intent of using this information to guide the development of new product formats (e.g., novel dispensing mechanisms to facilitate the transfer of media from its packaging to the cells).

    On the biology side, Naley points to customer demands for more predictive cell-based models for use in assays to screen compound libraries and to study cell function; i.e., cell culture systems that "have higher fidelity to in vivo conditions."

    Although many different types of cells and cell lines may grow in a standard media formulation, cell growth is no longer a sufficient criteria for cell culture researchers. They are looking for more specialized media that support the growth of healthy cells with normal morphology and activity, such as protein expression; i.e., media tailored to a specific cell line and application.

    For biomanufacturing, "the biggest trend we see is the drive toward animal-component free, serum-free media," says Brandon Pence, bioproduction market manager at HyClone (www.hyclone.com).

    Lisa Masterson, product manager at SAFC Biosciences, a division of Sigma-Aldrich (www.sigmaaldrich.com), concurs.

    "Serum-free conditions are desirable for all areas of cell culture. The necessity for serum-free environments is not as intense in the research area, but as more and more research labs are seeking licenses and patents, serum-free conditions add value to their IP," she explains, especially in the areas of cell therapeutics and regenerative medicine.

    Non-animal product options have expanded to include synthetic and recombinant media components, such as amino acids and cytokines.

    There is still strong demand for serum for established therapeutic products and processes, but, "I would be surprised if you would see any major therapeutics coming through in the future that would contain serum," says Archie Cullen, vp, sales and marketing at SAFC Biosciences.

    Sigma-Aldrich acquired JRH Biosciences in February, and the JRH Biosciences business transferred into the SAFC Biosciences brand during September.

    Driving demand for animal-product free formulations are concerns about safety and, specifically, transmissible spongiform encephalitis. How far to go in developing an animal component-free product and how far back in the process to drive this change are issues to be determined by individual therapeutics producers, as the regulatory agencies have not made specific recommendations or established standards or guidelines.

  • Move to Serum Free

    For production of a therapeutic, "clearly the elimination of serum from media used in bioprocessing reduces regulatory risks, which ultimately saves time and money," says Wendy Bray, senior marketing manager for biotherapeutic media and sera at Cambrex BioScience Walkersville (www. cambrex.com).

    "The research community and those in process development are attracted to serum-free media for the increased consistency of performance, reduction of experimental variables, and easier downstream purification," adds William Siegel, product manager at Cambrex BioScience Walkersville.

    Therefore, Bray notes, new products introduced under the BioWhittaker brand are all serum-free formulations, including cell culture media and support products, such as freeze media and growth factors."Serum-free media, protein-free media, and chemically defined media all have applications in both the research and biotherapeutic arena, but biotherapeutic/industrial demand will out-pace research demand," predicts Siegel.

    Protein-free media are of primary importance for research applications involving purification of recombinant proteins in the research and industrial market. Chemically defined media, a subset of serum-free media, "is important to the research community because it gives added control over experimental variables and allows better interpretation of results," says Siegel.

    The demand for animal product-free components in chemically defined media is most relevant to the biotherapeutic market segment because of the demands increasingly introduced by global regulatory authorities.

    According to Pence, HyClone's clients are taking a full systems approach and not only focusing on the media formulation itself but they also want to know how the formulation is manufactured.

    Pence attributes this to the current regulatory environment for cell culture media, which continues to evolve from traditional serum-containing media to animal-component free, serum-free media and to an emerging focus on controlling the manufacturing process used to produce cell culture media.

    In response, HyClone invested in technology that enabled the company to use more disposable production materials and to establish dedicated facilities for producing animal-component free formulations.

  • Optimizing Formulations

    Another advantage of serum-free formulations is that they give researchers more flexibility to optimize the media, tweaking the additives and components to meet their individual needs.

    A large percentage of Mediatech's (www.cellgro.com) business is custom-formulated media, a market sector that is showing strong growth. Mediatech emphasizes manufacturing efficiencies to reduce media production costs and patented a new piece of manufacturing equipment that currently produces approximately two million bottles of media per year and will ultimately generate more than five million bottles each year, according to Karen Evans, marketing manager.

    Evan cites two key benefits of this equipment: it yields a substantial decrease in overhead, lowering the cost/bottle and improving production efficiency and it reduces the number of workers that handle the material during production, thereby increasing sterility assurance.

    "In research, standard formulations are the rule as they are economical, readily available, and often cited in the scientific literature," says Masterson.

    For biodevelopment and biomanufacturing applications, proprietary media (modified versions of off-the-shelf formulations) are more often used for R&D applications, and custom media optimized for an individual end-user's cell culture platform are more the norm.

    SAFC's new LookOut line of kits for mycoplasma detection, elimination, and clean up is scheduled for release in 2006.

    CHO and NS0 cell lines dominate the cell culture arena, with growing interest in other platforms, such as PER.C6 cells. Cambrex recently introduced its chemically defined, non-animal origin PowerCHO 1, 2, and 3 media serum-free applications using CHO cells in suspension and it plans to launch a new serum-free media for NS0 cells in 2006.

    SAFC is collaborating with Vivalis (www.vivalis.com) to optimize growth of its EBx cell lines that the company plans to use to develop a cell culture-based manufacturing process for producing viral vaccines.

    "Recent worldwide concerns about avian flu have sparked increases in funding for vaccines," observes Siegel.

    Flu vaccine development efforts are shifting away from the time- and labor-intensive, egg-based production methods to novel cell culture-based systems. "We anticipate increased demand for our ProVero 1 serum-free, protein-free media in response to this," says Siegel, as Vero cells are a good host for the flu virus.

    Pence echoes the trend toward a greater desire for media optimization in biopharmaceutical development. As the biotech market grows, and companies develop their own internal research capabilities and begin to move products into clinical trials, "they start to look at scale-up and want to maximize performance of their cell line.

  • Formulation Modifications

    Simple modifications of a standard medium formulation can improve performance 2- to 3-fold. Even for well established CHO, hybridoma, and NS0 cells, for example, each individual clone may have unique nutritional requirements, and media optimization designed to meet those requirements can boost growth and protein/antibody expression.

    HyClone has expanded its line of animal-component free, chemically defined media to include HyQ CDM4NS0, HyQ CDM4CHO, and HyQ CDM4MAb for therapeutic protein/Mab production in NS0, CHO, and hybridoma cells, respectively.

    To facilitate cell culture optimization and scale-up, HyClone focused new product initiatives on improving process performance. In October the company launched HyQSphere microcarriers for adherent cell lines, aimed at optimizing scale-up for such cell lines as Vero and MDCK cells used in viral vaccine production.

    Developed in collaboration with Baxter Biosciences (www. baxter.com), HyClone's Single-Use Bioreactor, designed for scalable mammalian cell culture in working volumes of 50 L to 1000 L will come to the market in April.

    Invitrogen's line of Advanced Medium contains 10-fold less sera and includes Advanced RPMI, MEM, and D-MEM Medium products. Earlier this year, the company also introduced a new, freezing medium for mammalian cell cultures, called Recovery, aimed at improving workflows and results by enabling higher yields of recovered cells after storage in liquid nitrogen.

    Efforts to lower the cost of cell culture are focusing, in large part, on a switch to disposable containers and devices for materials storage and transfer and cell culture processing. Invitrogen introduced its Universal Bag format this year, providing media in 1-L to 1000-L bags with four connector ports.

    Media manufactured in HyClone's dedicated animal-component-free powder facility can be hydrated using the company's HyNetics module, a single-use, disposable system for automated production of liquid formulations. The production tanks contain large, sealed plastic bags, where the plastic film used to make the bags contains no animal components, which are filled through a closed transfer system. "Everything that comes into contact with the liquid product manufactured in HyNetics is disposable," says Pence.

    Bray points to growing demand among Cambrex customers for disposable products throughout the process stream; i.e., products that can improve efficiency by eliminating the time and cost for cleaning validation and reduce the risk of contamination.

    Cost-conserving measures in media production include "automation and optimal batch sizing for custom products" and "a more efficient supply chain throughout the operation, starting with receipt of raw materials through inventory and shipping of finished goods," says Bray.

    "New technology for tracking materials, planning, and scheduling and testing have opened the door for operational improvements." Masterson emphasizes that cost savings "should not be limited to the product price component alone. Organizations now have the ability to utilize a single supplier for the whole of their media and related components across the continuum from bench-size quantity research all the way to full industrial production.

    "Cost savings at this level comes in the form of multi-vendor consolidation, streamlined scale-up procedures, and reduction of qualification and validation testing requirements," she says.

    At the research level, the growing use and diverse applications of stem cells are having a significant impact on the media market, as companies work to understand how best to optimize their growth.

    For stem cell applications, "serum-free media that lack growth factors, cytokines, or artificial stimulators of proliferation will play an increasingly important role," says Siegel, pointing to the Cambrex' BioWhittaker X-VIVO serum-free media, which has been used in published studies to support the growth of CD34+ cells.

    SAFC added two products to its Stemline media platform, T-Cell Expansion Medium and Mesenchymal Expansion Medium, and will launch Stemline Dendritic Maturation Medium in early 2006.

    StemCell Technologies' (www.stemcell. com) product line includes StemSpan Serum-Free Expansion Medium (SFEM), StemSpan H3000, a chemically defined, animal-free medium that contains only human-derived or recombinant human proteins, and the StemSpan line of cytokine cocktails.

  • Tailored Feeding Strategies

    Another emerging trend focuses on custom feeds and tailored feeding strategies, according to Tom Fletcher, director of R&D for industrial cell culture at Irvine Scientific (www.irvinesci.com).

    "The business continues to be dominated by CHO cell applications," says Fletcher, and although a good selection of serum-free, animal protein-free, and chemically defined starting media are already available, he points to the growing importance of customizing the feed to the metabolic requirements of the culture to replace the nutrients being depleted.

    This focus on refining culture conditions in real-time ties in with the need for enhanced online process monitoring capabilities. Irvine has an ongoing collaboration with a manufacturer of chemistry analyzers and is expanding its use of online monitoring technology for the development of customized feeds.

    With regard to feeding strategies, Fletcher describes two key variables being evaluated to enhance the overall health and productivity of cells grown in fed-batch cultures: the timing of feedingwhether to rely on one or multiple feeding events; and the concentration of feed solutionsmoving to more concentrated feed solutions to minimize dilution of the culture.

    Sandy Parten, senior product manager for industrial cell culture at Irvine, sees an increasing interest in protein hydrolysate supplementation. She notes that in many cases the greatly improved performance achieved with hydrolysates more than compensates for the risks associated with their use, these being mainly lot-to-lot inconsistencies.

    The three hydrolysates recently introduced by Irvineone derived from soy, one from yeast, and one a hydrolysate mixture optimized for CHO cellsare all ultrafiltered to reduce endotoxin and minimize the risk of inconsistency, according to Parten.

    Irvine offers a CHO media optimization kit to customers that participate in the company's prototype small-batch media development program, in which Irvine helps optimize media in small, non-GMP samples with a one to two week turnaround time.

    The media optimization kit contains 1-L samples of six chemically defined media and a hydrolysate supplement, allowing customers to evaluate the growth and performance of their cell cultures under 12 different media conditions.



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