April 15, 2015 (Vol. 35, No. 8)

Chemically Defined Media Achieve Consistency, not Universality—Not without Supplements

Cell culture preferences have slowly shifted from serum-based to hydrolysate-supplemented to chemically defined. Dominant drivers for this trend have been a desire for consistency in product titer and quality, and a greater appreciation for process understanding.

“The unexpected problem,” says Jon Wannlund, Ph.D., director of R&D for advanced processing at BD Biosciences, “is the level of variability that still exists in media products.”

For example, Genentech recently reported the effect of manganese impurities in a ferrous sulfate media component. As a cofactor for enzymes involved in glycosylation, fluctuating manganese levels caused unpredictable glycosylation patterns and glycan composition. Taken alone, these effects were not necessarily bad, but the resulting inconsistency was unacceptable.

Genentech’s solution was to raise the manganese concentration in their culture media to levels that swamped the affected enzymes and eliminated variability.

Impurities may arise from any ingredient, including those prepared through biological processes. Again, their effects may not harm the process, but they contribute to unpredictability.

“Solving the impurity carryover problem can be quite an issue, even for chemically defined media,” Dr. Wannlund adds. “It would be nice if trace metals would exist at constant levels, but they do not.”

As many as 80 components exist in chemically defined media. Controlling each concentration precisely is relatively easy. But each component may carry several contaminants or trace chemicals, which is problematic. Analyzing each bulk ingredient’s carry-overs is something some media manufacturers do, but even when precise concentrations are known, the effect on the process may be unpredictable.

BD Biosciences’ BD Select CD1000™ is a chem-ically defined platform medium optimized for hydrolysate or CD feed supplementation.

Universal, Chemically Defined? Unlikely

Dr. Wannlund is skeptical of the notion of developing a universal culture medium, even with strict chemical definition. Expression systems behave uniquely, but even one cell line, e.g. CHO, exhibits a great deal of diversity and genetic instability.

“When optimizing media for a specific cell line, customers sometimes send us sister clones of the main candidate,” according to Dr. Wannlund. He has found that nutritional requirements for two different lines from the same cloning can be significantly different. “The DNA inserts into different locations, so the cells may be metabolically different. In addition, there are process parameters that drive different nutritional requirements.”

The closest chemically defined media can achieve in terms of a “universal medium” is a basal formulation that promotes cell growth. But as with serum-based and complex peptone-based media, process developers must still experiment with media components and feeds.

Uma Lakshmipathy, Ph.D., principal scientist at Thermo Fisher Scientific, concurs: “Basal media contain the very basic components necessary for most cells to grow. But it is unrealistic to expect a universal medium that covers all cell types.”

Something approaching a defined medium might be possible. Dr. Lakshmipathy cites her company’s Essential 8™ basal medium for pluripotent stem cells. As the name implies, the product provides supplementation with eight essential nutrients for basal media used to grow pluripotent stem cells. Supplement packages would differ for other cell types. But even at this level of universality, Dr. Lakshmipathy is skeptical: “Would this type of product be available for every cell type out there? I doubt it.”

Developers and experimenters should select the point on the serum-to-defined continuum that their project requires, Dr. Lakshmipathy says. “Chemically defined media is most desirable, but it’s not trivial to develop such a system and confirm that cells that are traditionally cultured in fetal bovine serum-containing media will grow and function as well in a xeno-free or chemically defined system.”

The critical question users should ask is: How are the cells and products to be used? If it is to serve as a research-only reagent, a medium has only one function: keep cells healthy and relatively productive. The closer a project gets to the clinic, however, the more seriously developers must consider chemically defined media, and the cGMP implications of how their media are manufactured. The additional advantage of working with chemically defined media is less variability from different lots of the media and, consesquently, greater reproducibility.

According to Steven Richardson, senior scientist at SAFC, perfusion cell-culture processes are eager to switch from hydrolysates to chemically defined media, but current commercial products fall short. “The inherent variability with hydrolysates can wreak havoc on perfusion cultures,” notes Richardson, who compares the variability of hydrolysates to that of a wine. Peptone-based media vary significantly depending on weather, season, and locations where their raw materials are sourced. This variability directly affects processes and products.

Why are chemically defined media for perfusion cultures not widely available? Richardson offers that companies are still on the fence regarding the appropriateness of perfusion cultures.

“Biopharmaceutical companies are still reconciling the operational and economic benefits and the robustness of perfusion technology,” he says. “Moreover, realizing high-throughput small-scale perfusion systems that mimic production scales has been challenging.”

SAFC and other companies have developed their own such systems, but this hurdle has hindered efforts to model large perfusion cultures reliably.

The human induced pluripotent stem cells shown here were cultured in Thermo Fisher Scientific’s Essential 8® medium on a vitro-nectin-coated tissue culture dish.

Several vendors of culture media design those products for specific cell lines, e.g. CHO GS or CHO S. “We’ve taken on the challenge to create an off-the-shelf chemically defined medium that works with all mammalian cells,” Richardson says.

A recently released SAFC medium, Ex-Cell® Advanced™, serves as a platform medium for all CHO lineages. The product arose from a customer program to replace a hydrolysate-based medium for a perfusion process with a defined medium. “Having one platform medium throughout a large biomanufacturing organization reduces supply chain risk,” points out Richardson.

Even such “universal” media have their limitations. Users still must optimize supplementation and feeds for their specific cell line and process.

“Our philosophy is a medium should support cell line development, cloning, and high growth while not harming the overall process at scale,” Richardson explains.

Vendors are beginning to understand this idea, with the introduction of supplement packages for specific processes. SAFC, for example, has introduced a protein quality supplement, Ex-Cell® Glycosylation Adjust (Gal+), which when used with a platform media helps to direct glycosylation.

The recently launched Ex-Cell® Advanced™ medium from SAFC has been specifically developed to support growth and productivity across a diverse set of CHO cells. The formulation was developed using multivariate analysis of 10,000+ data points that included performance, physical, regulatory, and safety design specifications.