February 1, 2015 (Vol. 35, No. 3)

GEN Interviews Specialists Who Can Help You Harmonize Novel Formulations and Rigorous Processes

Fed-batch processing employing classical media plus periodic nutrient supplementation remains the most prevalent mammalian cell culture mode. But perfusion cell culture, with its own requirements for media and feeding, is gaining. The first phase in fed-batch processes is biomass expansion. Once cells reach desired density the emphasis is on productivity. With longer-duration perfusion cultures, cells are removed and media replenished periodically.

“Perfusion cultures must strike a finer balance between maintaining cell viability and productivity over time,” says Marin Parenty, vp, cell culture media, Sartorius Stedim Biotech. Achieving this balance demands more exquisitely tuned media and nutritional supplementation.

In virus vaccine production, manufacturers are moving away from egg-based production to mammalian cell culture, which drives the need for specialized or dedicated media for commercial cell lines and nonproprietary cells, such as MDCK and Vero, that support both cell growth and virus infection.
Some vaccine-worthy cell lines are adherent, which requires specialized media and culture conditions to wean them from attachment dependence. Processing virus production in suspension cells is easier than for adherent cells, according to Parenty.

Customers are demanding increasingly stringent media release tests to reduce the impact of media batch-to-batch variability. Parenty mentions near-infrared techniques (for solid media ingredients) and Raman spectroscopy (for liquids) as emerging techniques for stricter quality control. End users also ask suppliers to develop stricter analytics for raw materials used in production to test for any contaminant that may inhibit cell growth or productivity. “This raises requirements for analytics to sensitivity levels that were previously unseen.”

Concerns over virus and prion contamination push media manufacturers to segregate production lines employing animal component-derived products from animal component-free materials. In the same vein, virus contamination risk forces customers to ensure virus reduction in media through UV irradiation, filtration, or heat inactivation.

According to Nikolai Stankiewicz, Ph.D., who heads the Technology Transfer Laboratory for Cell Culture Media at Merck in Darmstadt, Germany, the goal of continuous processing has been the impetus for perfusion cell culture: “More and more of our customers request it.” Because perfusion cultures result in longer process time enhancing the productivity, suppliers must find new ways to formulate culture media that support these processes.

In particular, the low solubilities of cystine and tyrosine pose challenges. “We needed to come up with new formulation procedures for these single ingredients,” says Dr. Stankiewicz.

The group led by Dr. Stankiewicz arrived at a solution that involves mixed crystals of poorly soluble amino acids and counterions to enhance solubility. Another approach is to employ chemically modified amino acids that are converted to the unmodified ingredient such as phosphotyrosine, which is highly soluble at neutral pH. Phosphatases released from dead cells cleave this molecule to tyrosine and phosphate.

In a published account that appeared last July, Dr. Stankiewicz’ group  described how it applied its approach in CHO fed-batch cultures. Phosphotyrosine is thus equivalent to an on-demand form of this essential amino acid. “We had to demonstrate, of course, that the modified tyrosine does not get into the cell and is not incorporated into the final protein product,” Dr. Stankiewicz adds.

High-throughput screening of novel cell culture media components at Merck KGaA, Darmstadt, Germany.

Media Optimization

Development and optimization strategies of cell culture media widely vary and sometimes result in overcomplicated formulations with 80-plus components. The Media Development team at SAFC has been using advanced statistical tools to better understand critical media components that are cell-line specific, to help remove legacy nonessential components.

“Fewer components translate to reduced variability due to raw material impurities, simplified supply chains, and overall improved reliability in long-term media manufacture and supply,” says Delia Lyons, a senior scientist at the company. “It is this reliability of supply that ultimately translates to reduced direct and indirect end-user costs.”

Bioprocessors have shown concern over trace element impurities in composite media. Such impurities, which can be traced back to starting materials, may adversely affect glycosylation and protein quality. SAFC’s Trace Element Initiative works with customers to understand these impurities with the objective of troubleshooting variability in drug manufacturing. Once the variability is identified and understood, appropriate sourcing and procurement strategies can be put in place to mitigate potential impact further downstream.

While reduction of media components is attractive for obvious reasons, it should not sacrifice productivity or product quality. Biopharmaceutical manufacturers therefore rely on fed-batch and continuous fed-batch production using highly concentrated feeds. One of the significant limitations of these types of feeds, however, is feed stability in solution. An understanding of component solubility, dissolution, and the complex chemistries occurring during manufacture is essential to building better feeds and supplements.

Much can be learned and applied using advanced statistical tools in media development. For example, EX-CELL® Glycosylation Adjust (Gal+) (recently introduced by SAFC) provides for functionally relevant shifts in N-linked glycosylation with titration into bioreactors resulting in a two- to fourfold increase of relative G1F and G2F distribution.

Media development will continue to evolve, driven by end-user requirements (high titers and protein quality) and emerging formulation technologies. “As defined components replace undefined components, the complexity of formulations has increased,” Lyons explains. “Reducing that complexity in favor of more efficient media products, using methods such as advanced statistical analysis, is becoming increasingly important to sourcing strategies, manufacturing processes, and final product.”

Diffuse Supply

Biosimilars and biobetters, combined with rapid globalization, are key trends affecting biomanufacturing, notes William Whitford, senior manager, cell culture, GE Healthcare Life Sciences: “Dedicated in-country, for-country initiatives for vaccines and other critical products are shaping the future.”

To meet these needs, suppliers are increasing the number of cell culture media facilities worldwide. This, with advances in packaging and shipping technologies, has led to a reconsideration of whether liquid or powder media best serves cGMP manufacturing.

Large volumes of ready-to-use liquid can now be delivered safely and efficiently as needed, thus saving end-users time, storage space, and effort. “In addition, the water quality is kept under tight control by the cell culture media supplier,” notes Whitford, “minimizing the risk for variability in the process and decreasing the need for cleaning and validation.”

Fed-batch production continues to be the workhorse mode for mammalian biopharmaceutical processes, to the point where fed-batch titers are no longer bottlenecks. This, Whitford argues, arises from developments in cell culture media and novel expression systems.

“These technologies address bottlenecks in translation and transcription for the product gene and help ascertain [whether] the final construct [has been inserted] at productive chromosomal locations using a landing pad or site-directed integration,” explains Whitford. “Stable, high-producing clones are developed in less than 10 weeks compared with approximately 40 weeks using traditional technology.”

The challenge now is to find flexible, serum-free media formulations as starting points for multiple clones in the early stages of process development. Such platform approaches facilitate clone selection and speeds the overall media optimization effort.

“Interest in new process formats is still growing, especially for niche applications and emerging production systems,” Whitford asserts. “This in turn drives the development of new cell culture media such as the avian and human platforms EB66™ and Per.C6™ [respectively] that require tailored cell culture media formulations to fully realize their potential.”

Whitford’s final observation involves increased demand for security of supply and raw-material quality. Initiatives such as the pharma industry’s Rx-360 address quality and standardization in cell culture media, and performance standards support industry best practices. “Essential quality metrics are also emerging from the process analytical technology (PAT) and quality by design (QbD) approaches to operational excellence,” Whitford adds.

User operating the recently launched GE Healthcare Life Sciences’ ReadyToProcess WAVE™ 25 bioreactor system, which is capable of either single- or dual-mode functionality. When dual mode is used, two cultivations can be run using disposable Cellbag™ culture chambers of different sizes and/or working volumes. Each chamber can be controlled independently for parameters such as temperature, pH, dissolved oxygen, and pump speed.

An Interesting Coalescence

Consolidation among suppliers has begun to affect how biomanufacturers do business. “The commingling of medium and single-use systems is going to shake up the field,” says Alexis Bossie, Ph.D., director, media R&D, Lonza Biologics.

She refers to the acquisition of Life Technologies by Thermo Fisher Scientific, Merck KGaA’s acquisition of Sigma-Aldrich, and her own company’s alliance with Sartorius Stedim Biotech. The commingling of media and plastic makes sense in one significant way that equivalent deals between stainless bioreactor companies and media vendors would not. As disposable bioprocessing takes hold, every single-use bioreactor needs medium and vice versa. “Every bag customer is a potential medium customer,” Dr. Bossie explains.

And those customers, she says, are looking for ease of use and one-stop shopping—one source of expertise for the entire process. “Our users want assurance that there will be no issue hooking up media to bioreactors,” notes Dr. Bossie. “Filters will be included, and downstream processing expertise is there as well if they require it.” Vertically integrated suppliers lower the entry barriers as well, particularly for small, development-stage companies or biotechs in emerging markets.

Related is the renewed interest in medium optimization. Off-the-shelf media have their place, according to Dr. Bossie: “Everyone has accepted that. But when you are investing heavily in a process for producing protein, media optimization is absolutely necessary because every cell line is unique.”

At the same time, companies may lack the specific expertise demanded of medium development, or the bandwidth to conduct a prolonged search for the right culture medium. And in all cases, companies are wary of providing vendors with cells or revealing details about processes and products. For them, Lonza (through its affiliation with Sartorius Stedim) can provide on-site media development expertise that includes thorough scale-down modeling and testing.

“They may have the cell culture expertise, but they don’t want to divulge proprietary information or become indebted to a vendor,” adds Dr. Bossie. “What they need is guidance and the tools to do it quickly.”

The commingling of medium and single-use systems, as exemplified by the alliance between Lonza Biologics and Sartorius Stedim Biotech, may affect how biomanufacturers do business. For example, many customers may look for ease of use and one-stop shopping—one source of expertise for the entire process. Together, Lonza and Sartorius Stedim Biotech can provide on-site media development expertise that includes thorough scale-down modeling and testing.

Mosaic of Trends

Ramin Cyrus, marketing director for BD Advanced Bioprocessing, notes that there is a “mosaic” of trends that in one way or another escapes notice: “More and more biological drugs are entering development and coming to market. But a bifurcation exists between the up-front R&D folks trying to develop biopharmaceuticals and process development [specialists] who have to bring the molecule into production.”

Although the buzz these days surrounds chemically defined media and supplements, very few late-stage or commercial products actually use them. Instead, many processes employ animal-free peptones and hydrolysates. “Although the longer-term trend is toward chemically defined media, it does not provide the titers and consistency that is needed during production,” Cyrus explains. “That is why after early development, process development teams tend to reformulate toward more conventional media and animal-free peptones and hydrolysates.”

Cyrus, who is responsible at BD for identifying trends in media usage and acting on them, bases his position on filings for clinical trials that range from Phase I to Phase III. While chemically defined processes do exist among these projects, they are outweighed by hydrolysates and animal-origin media.

“Companies are no doubt experimenting with chemically defined media at the research stage, but they’re simply not getting the results they would in serum-based media,” Cyrus tells GEN. He refers to chemically defined and serum-based media as the “bookends” of the medium spectrum. “The industry grew up on animal origin products, and chemically defined is the future. But today the best compromise are peptones and hydrolysates.”

“We have seen several significant technology trends driving advances in cell culture media, which drive how we work with customers and partners to meet their research and bioproduction needs,” says Jason T. Walsh, business director, media products, Corning Life Sciences. The significant supply constraints and resulting price increases for fetal bovine serum have renewed customer interest in serum substitutes, as well as reduced-serum or serum-free media. “We expect these supply and cost pressures to continue to drive the trend toward alternatives,” notes Walsh.

In addition, the groundbreaking research and emerging therapeutic use of cell therapies is spurring new media development critical to this work.

Finally, with its long history of supporting tissue processing and engineered tissue production for regenerative medicine, Corning expects robust growth in these applications and in the emerging technology of 3D bioprinting as well. “These developing areas,” Walsh points out, “will need strong media formulations and media performance to support their continued advancement.”

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