April 1, 2013 (Vol. 33, No. 7)

Many cell culture experts will argue that improvements in media and feed have been more responsible than any other factor in raising production titers for therapeutic proteins. While the “nature vs. nurture” argument is far from settled, culture media is at worst the partner of cell-line development in the quest for productivity, safety, and efficacy.

Media selection supports every stage of development and manufacturing, from clone selection to process development, scale-up, and commercial manufacturing. Absent the optimization of cell culture media, meeting biomanufacturing challenges such as scalability, process robustness, time to market, supply chain reliability, and cost of goods becomes impossible.

According to Achim Quandt, cell culture media and consumables business leader at GE Healthcare, scalability, especially for monoclonal antibody production, has improved through platform processes that allow using the same host cell line and culture medium for whole families of products.

“A robust process provides manufacturers with a relatively large design space where product quality remains unaltered. Once validated, platform processes provide huge advantages during scale-up, as wider acceptable ranges for the process parameters can be used.”

One company’s platform may differ significantly from another’s, particularly with respect to media and feed. Yet media manufacturers need to stay ahead of the curve(s). “For media manufacturers, the challenge is to offer a wide portfolio that reflects the needs of the most common cell culture platforms,” notes Pascal Lefebvre, media product manager at Sartorius Stedim Biotech.

Supply chain reliability is a key factor in modern bioprocessing that employs specialized equipment and raw material from a multitude of vendors. “Manufacturers strive for improved control of their supply situation by careful selection of vendors, conducting of supplier audits, and requirements for safety stocks where applicable,” says Quandt.

Cell culture media and media development are significant contributors to cost of goods, but when executed perfectly can greatly shorten time-to-market, Lefebvre notes. “As competition becomes more intense, fast-tracking to the commercial phase becomes critical.”

Related manufacturing issues include process robustness, reproducibility for both upstream and downstream processing, “quick-and-clean” processing, technical and regulatory support, and matching titers for optimal upstream and downstream operations. “You can’t run purification identically for 1 g/L and 6 g/L processes.”

The bioprocessing landscape is not quite settled in terms of culture type: fed-batch (including variants) or perfusion. “We’re seeing hybrid approaches—quasi-continuous processing—employing feeding strategies that do not involve complete media exchanges as you’d see with perfusion,” notes Shawn Smith, senior director, upstream bioprocess technologies at Pall Life Sciences.

The trend toward continuous manufacturing as a cost-saving measure will create additional challenges to media manufacturers and end-users. Right now this is an open-ended area for vendor and customer alike, since broad experience in this manufacturing mode does not yet exist. Top media vendors tell us, however, that they will be prepared when the dust settles.

Media Definition

All the experts interviewed for this article agree that end-users are looking for high-quality media that provides reproducible results and robust yields. As the industry moves toward more chemically defined media products, Stephen W. Brown, Ph.D., CTO at Vivalis, notes that “chemically defined media don’t always provide what you need in terms of yield. That means you have to go back, in some cases, to somewhat more complex media.” That is why poor characterization of media components causes problems.

The adoption of chemically defined media has been slow but steady—a “city on the hill”—for both bioprocessors and media manufacturers. Each step along the way, from serum-based to serum-sparing, serum-free, animal-derived component (ADC)-free, and chemically defined presents new challenges in terms of media optimization and the addition of trace ingredients. Jon Wannlund, R&D director for BD Biosciences, advises companies transitioning between media types to focus on media optimization as early in development as possible.

Paradoxically, as users and vendors alike strive for better definition, uniformity, and simplicity, the process of designing and optimizing cell culture media has taken on greater complexity, demanding a level of formulation sophistication that was hitherto unknown—not to mention a higher level of attention toward media optimization. End-users optimize media by evaluating spent components, then selectively replenishing them through a process Pall’s Smith describes as “an input/output problem that is almost like live biofeedback control, with the bioreactors behaving like a living organism.”

Ken Ludwig, business director, bioprocess at Corning Life Sciences, estimates that approximately 20% of today’s mammalian cell cultures for biotherapeutics are ADC-free, with a slightly lower percentage for vaccine cultures. For vaccines the figure is about 50% serum-free, while therapeutic protein processes are now virtually serum-free, and up to 40% ADC-free. But up to 90% of cultures for cell therapy still employ serum. “They would like to reach the same level of serum-free culture as biotherapy, but the challenges in growing stem cells suggest the transition will take longer.”

The majority of new processes, according to Roberta Morris, cell culture business director at Thermo Fisher Scientific, are attempting to go serum-free, with many attempting the chemically defined route as well. Cell therapy developers try to avoid serum as well, “but this is a very early-stage market that lacks standardization. Everyone is rushing to enter clinical stages. These cells, particularly human embryonic stem cells, are extremely sensitive to culture conditions, including media. But the closer they get to market, the more likely developers will attempt to optimize processes away from serum.”

Stem cell commercialization is further challenged by the fact that subpopulations exist even within distinct lineages, many with their own specific media/feed requirements. “Optimizing these media involves quite a bit of trial and error,” Morris says.

The intrinsic inconsistency of ill-defined media components, along with poor control over culture conditions, is one of the great sources of variability in cell culture, says Suzanne Hector, Ph.D., market development specialist at PromoCell.

“The total absence of human- or animal-derived ingredients, compared with traditional media systems, promotes a more controlled and standardized culture environment with minimal variation, particularly for sensitive cells such as stem cells and immune system cells.”

Defined media enhance culture performance by eliminating the deleterious effects of poorly defined growth supplements. Additionally, the risk of contamination from mammalian pathogens is reliably eliminated, meeting the demands of customers and regulators.

A researcher prepares freezing media using Thermo Scientific Nalgene filtration unit for cryopreservation of cell samples.

A huge benefit of well-characterized media and feeds is the potential for a simplified fed-batch regimen that incorporates as many ingredients as possible in the main feed.

“Batch-to-batch consistency, a major industry trend, is more than just a buzzword,” comments Jörg von Hagen, head of process development at Merck Millipore. Characterization of ingredients is a big deal at Merck Millipore. The company uses a “toolbox” approach involving several orthogonal analytic techniques to provide customers with a principal component analysis which, when applied properly, confers a “full perspective,” according to von Hagen, of media performance.

The switch to chemically defined and ADC-free media, and traceability of media components, are the most significant trends in today’s cell culture media. “While abandoning animal-derived components is driven by regulatory issues, chemically defined media provides direct advantages to the bioprocessor,” notes Quandt of GE.

ADC-free media originally contained peptones and hydrolysates of plant origins consisting of undefined amino acids, peptides, and other components. With increasing understanding of cell metabolism, media manufacturers began substituting undefined ingredients, leading to significantly improved batch-to-batch consistency for media, and therefore better reproducibility of cell culture processes.

Raw material quality is critical in both chemically defined and ADC-free media, requiring thorough screening of raw suppliers, tight control over incoming goods, and thorough raw material traceability. This has brought about more sensitive, sophisticated ingredient milling technologies such as temperature-controlled impact mills, says Quandt.

Search for Consistency

A key issue for media suppliers, says BD’s Wannlund, is assuring product and process consistency. “We’ve seen issues related to consistency that go back to the consistency of raw materials.” Media companies must pay special attention to low-level components, particularly metal ions. In some instances, and within certain concentrations, some “impurities” promote cell culture consistency. It works the other way around as well, with some impurities inhibiting cell growth and productivity. “There are inconsistencies even in chemically defined media,” Wannlund adds.

According to Gautam Choudhary, director of Gibco Cell Culture at Life Technologies, the main challenges facing cell culture process developers are the desires to increase process titer without modifying product quality and to obtain batch-to-batch consistency.

“Manufacturers are looking to incorporate commonly used raw materials to achieve greater predictability,” he said. “Consistency may be influenced by factors such as clone stability to media and feeds, as well as other add-backs.”

Media performance often depends on the presence of trace elements that exist at well-defined concentrations. Merck Millipore quantifies these ingredients, and makes sure they are added in precise quantities. “Media suppliers must assure that they do not introduce more of these trace ingredients than necessary through the addition of standard raw materials that may contain them,” says von Hagen. “You have to have a grip on the quality and composition of all individual components, not just the trace elements.”

Media optimization is iterative and often proprietary because sponsors are unwilling to share details of their process with anyone, including a preferred media supplier. Merck Millipore often sends a range of media to customers to try out and optimize with their cell lines, and provides a powdered medium based on the results. From medium batch to batch, the principal component analysis provides assurance that the customer is receiving exactly the same product, with component concentrations as identical as possible.

Industry-wide, media manufacturers are demanding more-complete transparency from their suppliers, while providing the same level of pellucidity to end-users. BD has set up secure data sites that customers access under confidentiality agreements that provide full analytical details on media makeup.

Re-Thinking Plastics and Viruses

Improvements in culture and cell productivity have paved the way for smaller processes employing single-use equipment. This has pushed demand upward for prepared, ready-to-use liquid media vs. media powders, says Bruce Lehr, director of development at SAFC. “This requires us to support customers logistically, or maintain manufacturing sites that are better aligned with where products are used.”

As discrete components are added to the media or through feeding, bioprocessors must be conscious of not just their concentrations and accurate delivery, but increasingly, their interaction with plastic films from which single-use equipment is fabricated.

As a member of PDA’s single-use taskforce, Stephen Brown of Aptalis has observed an increasing tendency for media suppliers to provide fully constituted, ready-made media in disposable bags. “Too often the data package arriving with that type of media product is not sufficiently thorough, particularly with respect to the interaction between media and bag materials.”

The media itself may be well-characterized, but not the container or its potential chemical contribution. For example, leachables and extractables can interfere with critical cell functions, while the bags themselves may sequester essential lipophilic media components. Thus, due diligence is at least twice as complex with bagged liquid media than for powdered media.

The packaging of liquid media in plastic bags has also changed how bioprocessors view virus safety, particularly with respect to early upstream operations. With a greater emphasis on elimination of upstream contaminants, producers and suppliers are looking further upstream than ever before, to unit ingredient sources. Part of this strategy involves the use of ADC-free components when appropriate, and sourcing only from highly trusted countries and suppliers. Future ingredient suppliers will therefore need to be completely transparent with regard to the sources of their raw materials, and the manner in which they prepare them.

Users are employing (or asking suppliers to apply) such techniques as virus filtration for media, as well as gamma irradiation and high-temperature-short-time, in addition to virus filtration of finished or near-finished product. The point is the elimination of viruses as early in the process—and as far from the patient—as possible.

Life Technologies’ Gautam Choudhary notes a greater emphasis on process safety as biomanufacturers explore ways to reduce risk. In addition to the conventional approaches such as media filtration and pasteurization, “suppliers are scrutinizing raw materials closely, and investing in completely ADC-free manufacturing,” he explained.

Manufacturers also are employing rapid qPCR methods to detect contaminants like mycoplasma and viruses.

Want more on cell culture? Be sure to check out the Expert Tips “Best Practices to Optimize Your Cell-Based Assays“.

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