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".