Culture Media Purpose-Fit for New Therapies

To Generate New Biologics, You Need Specialty Media—Defined Media, Animal-Component-Free Media, and Other Wonders of Formulation

Hunger for cell culture media is greatest among developers of traditional biopharmaceuticals. Contributing less to current demand—but demonstrating a growing appetite—are developers of drugs based on antibody and protein variants. Also, developers of nonprotein biological therapeutics are acquiring a taste for cell culture media.

If developers of traditional therapeutics keep working in established fields, while developers of alternative therapeutics start opening new vistas, demand for basal and specialty media can only increase. Also, demand is bound to rise as products of all types transition from discovery and development to large-scale production.

“Cell culture–based vaccines and stem cell therapeutics are also on the rise,” says Margarita Hunter-Panzica, senior product marketing manager, bioprocess cell culture, GE Healthcare Life Sciences. “Media development and manufacturing have become highly sophisticated sectors that are greatly affecting the overall bioproduction efficiency.”

The shift from liquid to powdered media continues, particularly in more mature European and U.S. markets, reflecting a growing appreciation for powdered media’s longer shelf life and lower transportation and storage costs. The uptake is already 60–70% of the total market by revenue. Liquid media still captures about half the market in the Asia-Pacific region, “but we are expecting a similar development there in the next few years,” Ms. Hunter-Panzica adds.

As bioprocesses mature, they demonstrate greater consistency, efficiency, and total output—improvements largely attributable to advances in culture media. Yet media components, which may number 100 or more, can be the greatest sources of bioprocess variability. “We see biopharmas turning more often to their suppliers, requesting detailed information about critical raw materials, including genealogy and property profiles,” confides Ms. Hunter-Panzica. “Biopharma companies hope that this information will help them improve biomanufacturing outcomes.”

Old-Fashioned Cell Cultures

With renewed interest in primary cell culture for research and discovery, keeping cultures alive and physiologically relevant for extended periods has been a key goal. In late 2017, Thermo Fisher Scientific launched a media system, the Gibco B-27 Plus Neuronal Culture System, to solve a thorny problem related to cultured primary neuronal cells. The new system, which consists of the company’s B-27 Plus Supplement and Neurobasal Plus Medium, extends survival of neuronal cell models by 50% without the need to change workflows.

The Gibco B-27 Supplement and Neurobasal Medium is cited in the materials and methods sections of more than 11,000 scientific publications. The enhancements represent a next-generation formulation and upgraded production and quality control.

Among primary cells, neurons are particularly fragile, to the point where obtaining viable cultures can be difficult. For neuronal cultures derived from rodent or human pluripotent stem cells, Thermo Fisher offers the CultureOne Supplement in addition to the Neuronal Culture System.

“Neurons are very sensitive cells,” emphasizes Laura Requena, Ph.D., director of product management, primary and stem cell systems, Thermo Fisher Scientific. “Since observing neuronal network-wide activity takes weeks to months, ensuring survival of over time is a major objective. Scientists are seeking to maintain higher neuron density over longer time periods than was possible with our classic B-27 supplements and Neurobasal media.”

Thermo Fisher optimized the B-27 and Neurobasal formulation with no special additives or growth factors to avoid inhibiting downstream processes or affecting the cells’ biology. “You just get more neurons,” Dr. Requena explains. “We also implemented more stringent requirements for acceptance of raw materials for better lot-to-lot consistency.”

Scientists are showing renewed interest in primary cells, like neurons, in large part due to the availability of tools to maintain the cells in healthy, physiologically relevant states. According to Dr. Requena, this is particularly true for primary neuronal cultures.

Perfusion Media

Last year, MilliporeSigma launched EX-CELL® Advanced™ HD Perfusion Medium, the first off-the-shelf, high-density cell culture medium to support perfusion cell culture. The medium processes at low perfusion rates, thus increasing production yield for a range of perfusion cultures.

In a perfusion medium, nutrients are balanced to sustain very high cell densities with the lowest possible perfusion rates—depending on medium depth, cellular requirements, and the product’s inherent characteristics, according to Andrew Bulpin, Ph.D., head of process solutions, MilliporeSigma. “The medium,” he explains, “should provide the nutrients in balanced amounts, while maintaining all other components at the lowest possible concentrations to provide optimum metabolic conditions while minimizing buildup of byproducts.”

So, while critical components are the same in both media types, their concentrations may differ significantly.

Since launching EX-CELL Advanced, MilliporeSigma has noted an uptick in demand for perfusion media. “Customers are interested in evaluating it for suitability testing on their cell lines and processes,” informs Dr. Bulpin. “The initial clients who evaluated it are experienced in perfusion cultures. We are receiving very good feedback and expect that to continue as our clients fine-tune and scale up their processes.” Users inexperienced with perfusion culture have also been evaluating the new medium.

“Cell culture media should support optimal performance and ease of use at minimum cost,” Dr. Bulpin continues. “This starts with media hydration, which should involve a simple reconstitution protocol with short mixing times.

“More highly concentrated formulations reduce the volume of media or feeds needed in the cell culture media and are designed to extend culture duration and, ultimately, support more efficient processes. However, concentrated solutions contain components that are difficult to solubilize, and are generally difficult to be maintain in solution. There is a need and the potential for novel components that enable higher concentrations and improved solubility to increase the overall performance in modern processes.”

Animal-Free Still Trending

In November 2017, Ventria subsidiary InVitria launched a media-formulation service. InVitria generates reagents and supplements by using its parent company’s ExpressTec technology, a plant-based platform for expressing peptides, proteins, and other pharmacologically significant molecules that are completely free of animal-derived components (Figure 1).

Figure 1. A scientist at InVitria tests components for inclusion in an animal-component-free media formulation. InVitria’s product line includes albumin, transferrin, lactoferrin, lysozyme, and insulin generated by ExpressTec, a plant-based expression platform. InVitria products work with varied cell lines, including those consisting of CHO, hybridoma, Vero and MDCK, and stem and primary cells.

Regulators discourage the use media and feeds containing animal-derived components (ADCs), which risk the transfer of pathogens to processes and products. By harnessing the power of ExpressTec, InVitria generates the components required to formulate completely ADC-free media for cGMP production.

InVitria manufactures several crucial components, including Cellastim (recombinant albumin), Optiferrin (transferrin), LIF (recombinant leukemia inhibitory factor), and ITS/ITSE (insulin and transferrin supplements that include ADC-free recombinant insulin).

The ExpressTec host is a monocot plant. Such plants, which include rice, barley, and sorghum, express target proteins in their seeds. InVitria’s manufacturing facility, which holds ISO 9001 certification and follows cGMPs, is animal-free at the tertiary level, meaning that all raw materials, processing equipment, and packaging materials are certified to be free of animal components.

“InVitria’s vision is to offer a portfolio of the critical animal-free components to improve performance, consistency, quality, and safety of cell culture media, not just for therapeutic proteins, but also for chimeric antigen receptor T-cell therapies, other cell therapies, vaccines, and regenerative medicine,” says Randy Alfano, Ph.D., vice president of product development. “Realistically, there are hundreds of proteins in fetal bovine serum, and InVitria has developed many of the critical factors, but not all.”

InVitria is not alone in this vision. For example, CellNest is a very attractive recombinant collagen-like product from FujiFilm, and PeproTech has a portfolio of cytokines and growth factors. Combined, there are animal-free products available today that provide cell culture scientists the tools they need to optimize animal-free media and eliminate serum without sacrificing performance.

Dr. Alfano notes that media for research differs significantly from production media. Customization is less prevalent, and serum is widely used. In research, experimental integrity depends on the robustness and reproducibility of the components incorporated into the experimental system. Since serum protein components can vary by an order of magnitude in different lots of serum, this introduces an unacceptable degree of inconsistency that can result in unreproducible results and erroneous conclusions.

“So, there is an advantage for researchers to move to more defined and blood-free cell culture media,” asserts Dr. Alfano. “This is starting to happen with the first movers to animal-free media being those thought leaders who seek consistency.”

Keep Your Powder Dry

Figure 2. Irvine Scientific added a new large-scale powder blender as part of the recent expansion of the company’s animal-component-free manufacturing facility in Santa Ana, CA. The expansion provides tumble blending capacity of 12,000 L total dry volume. This additional capacity enables the company to provide customers with homogeneous powder media in batch sizes of up to 7,000 kg.

Irvine Scientific recently expanded production of dry powder culture media at its Santa Ana, CA, facility. The expansion dedicates more capacity to Irvine’s animal-component-free dry powder production, particularly to the company’s impact milling, blending, and cleaning capabilities. With the expansion, Irvine now accommodates production of powder batch sizes of up to 7,000 kg (Figure 2).

A great deal of development work related to this expansion focused on assuring consistency and quality for media products across the range of batch sizes.

Irvine has demonstrated blend homogeneity for powdered media, with relative standard deviations of key component concentrations at less than 6%, even for trace metals.

Due to weight, sterility, and storage considerations, powder is by far the preferred format for shipping production-scale quantities of culture media. Reconstitution involves dissolving powders in injection-grade water, adding various ingredients, and carrying out sterile filtration.

Concentrated liquid media and granulated formulations are additional possibilities. With the former, reconstitution occurs either in batches or, less frequently, through inline dilution. Either way, users pay a premium for sterility assurance and for logistics associated with shipping and delivery of a weighty product.

But in general, these formulations are less common. “Beyond early-phase development,” maintains Tom Fletcher, scientific director, Irvine Scientific, “a customer specifying liquid media for large-scale usage is the exception.” Irvine is working on a system that would allow inline dilution of powdered media, which according to Fletcher, would be an industry first.

To ensure blend uniformity of its powdered media, Irvine follows the USP monograph relating to blend uniformity for pharmaceutical actives, which specifies that inhomogeneities should be no greater than 6%. “We chose that number because it’s a benchmark for drugs,” remarks Fletcher. Note that batch-to-batch uniformity of final formulated is much more tightly controlled.

Specialty Media

Advances in cell therapy and stem cells have created the need for equipment and specialty media to support associated research, development, and clinical applications. Corning Life Sciences has long been active on the equipment side with its varied cultureware for both adherent and attachment-free cells. Through its Mediatech cell culture subsidiary, Corning now offers NutriStem® hPSC, a xeno-free human pluripotent stem cell medium for stem cell production, in addition to culture bottles, flasks, and the CellCube system for adherent cells.

NutriStem was developed in a collaboration between two of Corning’s commercial partners, the media specialty firm Biological Industries and Israel’s Technion Institute of Technology. The medium is being manufactured by Biological Industries.

“Many types of media developed today are formulated based on the cell-line requirements, if known, or on the application in which they will be used,” explains Mark Rothenberg, Ph.D., product line manager for media, Corning.

For these cultures, particularly those destined for clinical applications, defined components are the prevailing trend. “Media developed for stem cell use,” Dr. Rothenberg points out, “require even more clarity and definition as to the components added to the formulation, compared to those for CHO, VERO, and other bioproduction lines.”

Stem cells require unique media formulations to maintain viability and the pluripotent state of the line during expansion. Such components may include ROCK (Rho-associated protein kinase) inhibitors. Targeting ROCK1 increases stem cell survival and cloning efficiency without affecting pluripotency.

“Many ingredients in these unique media formulations are similar, but they vary in terms of growth factors types, chemical components, added proteins, glutamine, and other supplements,” details Dr. Rothenberg. “The compositions may also include unique additives that provide an enhanced characteristic for cell line maintenance or differentiation.”