For decades commercial and academic scientists have sought reliable serum-free media. As far back as the 1960s a number of options were available for the cultivation of some cell lines. Now, after years of effort, many of the shortcomings of these early formulations have been resolved, and researchers have a number of highly successful alternatives available. GEN spoke with several experts on current cell culture media trends:

Andrew Bulpin, PhD, Head of Process Solutions, MilliporeSigma
Nicole Wellens, Global Product Manager, BioResearch and Bioprocess Media, Lonza
Maureen Bunger, PhD, Senior Product Manager, ADME/Tox Solutions, Lonza
Tobias Schenk, PhD, Product Manager, Bioprocess Solutions, Sartorius-Stedim Biotech
Randy Alfano, PhD, Vice President, Product Development, InVitria.

GEN: There are a number of media options available for in vitro protein production; is there a particular formulation that you favor, and what are its main benefits?

Bulpin: For CHO bioproduction in a fed-batch process, there are two media and feed systems that we recommend. The first, the EX-CELL^® Advanced CHO Fed Batch System, is optimized for fed-batch processes. The second, Cellvento^® 4CHO medium and 4Feed, are unique in that the feed contains a modified form of tyrosine, which allows high concentrations of this amino acid at neutral pH.

Both systems contain only chemically defined components and are animal- component free. These two media systems display high titers, protein quality, and scalability when operating in a fed-batch mode. However, if the bioproduction process is perfusion, then the nutritional needs of the cell line will be different and a medium optimized for that mode of production is essential.

Wellens: When generating proteins, we recommend the choice of serum-free medium, as this will simplify the downstream processing step. Besides being serum free, the use of chemically defined medium is an advantage, as we have found that it reduces batch-to-batch variability.

Alfano: Primary cells have shown high variance in metabolic activity due to an assortment of factors including donor-to-donor variability, but also due to variance derived from different culture methods. The goal for larger scale clinical manufacturing is to identify sources of inconsistency to streamline the manufacturing process and reduce the contribution of this variability to the final product. This is the goal of our research and development at InVitria. We are designing systems to eliminate variability emanating from culturing methods by implementing a blood-free and chemically defined media within a defined culture system to reduce or eliminate sources of variability coming from serum use or undefined components in cell culture media.

GEN: Dong et al. (Cytotechnology. 2008 Jul; 57[3]: 251–261) observed that “experiments on primary human hepatocyte cultures showed high variance in metabolic activities in cells from different individuals, making determination of optimal levels of factors more difficult.” Does this natural variance make it impossible to design a standard medium for evaluating primary hepatocytes, or are there strategies that could smooth over this natural variance in order to provide reliable, comparative data?

Bulpin: No, the individual variability in enzyme activities has not precluded the development of high-quality media for primary human hepatocyte (PHH) cultures. Media were initially optimized for animal hepatocyte cultures (i.e., mouse, rat) and then modified for PHH. The variability in cells isolated from animals is much less than in those isolated from humans. Also, improvements in media formulations can be tested within a given lot of PHH in order to optimize the formulation. Then, it can be tested in additional lots to validate the improved performance. The critical components for PHH culture and robust activity are now well-known. Of course, additional improvements are always being sought for specific aspects of PHH culture.

Bunger: This is a challenge because the donor variability may be due not only to inherent differences between donors, but also because of the tissue health and robustness of the processing method. We can’t solve the former but can mitigate the latter. For processing, the media that are used are highly specialized and quality controlled to reduce processing variability as much as possible. For culturing hepatocytes, the hepatocyte is highly metabolic and therefore uses up nutrients very fast, so highly supplemented medium and good buffering against pH change is really important.

GEN: Cell culture media design is pursued vigorously in both the academic and commercial sectors, but there is a great deal of repetition in the industry due to privacy concerns, failure to research older literature, and lack of effective communication. How might this situation be improved in order to avoid costly repetition? Or are conditions in different laboratories so complex and multifaceted that it would be difficult or impossible to faithfully replicate them?

Bulpin: The privacy protection of complex commercial media formulations is important. However, there are still opportunities to collaborate with academic partners to facilitate the advancement of the cell culture media technology for the industry as well as our understanding of the cellular mechanisms that are driving the performance. MilliporeSigma is an active member in AMBIC and NIIMBL, where we are collaborating with academic partners to improve the speed of developing new formulations, as well as to improve our understanding of CHO cell metabolism.

Wellens: Development of a medium in an academic (or even commercial) setting is not a guarantee the medium can be produced on large scale. Development of in-house media might also be used to gain a benefit over competition resulting in proprietary formulations. Biotech companies will strive for optimal results, and many companies have set their own release criteria even for commercially available media.

Alfano: Although it has been a slow movement, the cell culture media industry has advanced over the years from one reliant on serum and classical media combinations to one where most clinical products—now in cell therapy, gene therapy, tissue processing, and recombinant protein manufacturing—are optimized without serum. Some have even removed all undefined media components due to the intense need to eliminate the supply chain risk represented by serum and undefined components for the manufacture of clinical drug products.

The research community has been even slower to adopt chemically defined and blood-free cell culture systems. This is partly due to an unwillingness to change long-established research protocols or because there is little repetitive use of the same protocol. To that end, highly individualized processes will drive media diversification and create the need for further optimization around a particular process or protocol.

However, as the industry continues to shift towards blood-free and defined systems, the research field that these cell and gene therapy products stem from will also make that shift and we are already beginning to see this happen among many groups.

GEN: Raw materials (cell culture media) represent a significant source of variability to biopharmaceutical manufacturing processes that can detrimentally affect cellular growth, viability, and specific productivity or alter the quality profile of the expressed therapeutic protein (McGilicutty et al, Biotechnol Lett. 2018 Jan; 40[1]: 5-21). Is this a significant problem for the industry? If so, how does MilliporeSigma propose to deal with it?

Bulpin: Variability in the biopharmaceutical manufacturing process is a key concern, and understanding the individual, raw material components that make up a cell culture medium is crucial to reducing the risk of variation. Historically, much of this variation was introduced in complex and undefined components, such as hydrolysates. However, as we move toward chemically defined formulations, there are still components that can introduce variation in performance.

MilliporeSigma takes a proactive approach to raw material characterization. Each component is assigned a risk score based upon complexity, vendor, sourcing, and historical knowledge of variations in the product. For the components that are more likely to vary, we implement a second tier of testing. Importantly, this second tier of testing takes an orthogonal approach—first, to analyze the material for impurities and composition and second, to design a cell-based assay to test for biological response. By implementing these second-tier tests, we can reduce the risk of variability in the performance of the final media formulation.

Wellens: Biopharmaceutical manufacturing companies have moved away from serum and moved to serum-free media and chemically defined media, to assure reproducibility of results. The media manufacturing industry has realized biomanufacturing often starts with medium in liquid form; but when moving to the clinical phase studies, one often makes the change to powder. To create liquid from powder several components are combined. Optimal standardization would mean one powder is used to produce the liquid, providing consistency.

Schenk: Raw materials will have an effect on media performance; that’s why it is important to have a good quality control in place and experience with a media producer and its raw material sources. Having a second media source on file is crucial to ensure continuous supply with high batch-to-batch consistency.

Alfano: It is a significant problem for the industry as the appropriate control of the risk of raw materials is absolutely essential to ensure safety of the final product. Simplification of cell culture media to incorporate the minimal number and concentration of the key components can help to reduce variability of components that may not be necessary. We have experienced that the more effort invested in optimization of the media and manufacturing system, the larger the benefit and the likelihood that manufacturing objectives can be achieved.

GEN: Animal serum is still used to support growth of many cell lines, although its critical constituents have been identified. Is it possible or desirable to completely eliminate serum from cell culture protein production protocols?

Bulpin: Animal serum can introduce variability and cost, as well as the risk of contaminants. We strongly believe that it is possible and desirable to remove serum from recombinant biotherapeutic production processes. Beyond these animal-containing components, it is further desirable to move towards a completely chemically defined process. We routinely help our customers move away from protein hydrolysates and into chemically defined media, either through consultations to help them choose the right medium, or by employing our in-house high-throughput methodologies to develop a custom formulation.

Wellens: It is indeed desirable to produce proteins in a serum-free environment. It will help reduce risks of virus contamination and it will ease the difficulties encountered in the downstream processing step.

Schenk: The demand for serum-free media applications is increasing and more media are being developed to support this need. While serum-free cell culture protein production is already possible, there is a critical need to improve it to maximize protein titers.

Alfano: For protein production, the industry has been able to eliminate undefined components, including serum, while also improving productivity. There are some legacy products that may use animal or blood-derived components, but newer products have already made the conversion. However, in cell and gene therapies, vaccines, tissue preservation, and tissue processing, there is still a need for innovation in cell culture media systems to achieve the goal of chemically defined and high-performance media. However, strides have been made in the most recent years, proving that there are multiple advantages that can come from a fully defined and serum-free system.

GEN: Important improvements have been made in cell culture media over the decades. Has the end point been reached? Or, will improvements as defined above continue into the future with no limit?

Bulpin: In the past decade, the industry has made massive gains in terms of improving the growth and titer produced in biotherapeutic manufacturing. However, there are still significant improvements that can be achieved beyond enhancing these performance characteristics of the cells—the end point has not yet been reached. Novel, hard-to-express therapeutics are becoming more prevalent and may require more specific optimization. As the analysis of the protein quality becomes more detailed, tight control of the protein quality characteristics is becoming more and more important. In addition, we see a drive to drastically reduce the cost of goods sold (COGS) of bioproduction, whether through reduction of cell culture media cost or by easing the process.

Finally, we see a trend towards perfusion processes as a means to increase flexibility, reduce manufacturing footprints, and reduce costs. We are exploring new cell culture media formulations that can drive these improvements.

Wellens: There will still be development on protein production media to be more efficient and economic.

Schenk: I believe that future developments will focus in the direction of dedicated media rather than on generalized, all-purpose media. Such custom-designed media will be targeted to a defined purpose to maximize precise needs, including maximizing protein titer and cell growth. Since serum-free proliferation has not reached a theoretical maximum, there is a lot of room for further improvement.

Alfano: Today’s cell culture systems are becoming more complex as we see the launch of gene therapies, CAR-T cell therapies, and advanced tissue regeneration systems, in addition to advancements in protein production. Moreover, there is a budding industry in alternatives to in vivo meat that rely on cell culture methods. As our industry expands, our cell culture systems need to be improved in scale, performance, and cost effectiveness. Our future medicines and even the protein we select for dinner will be made possible, in part, by advancements in cell biology and cell culture systems. We have an exciting future ahead if we are to realize what these innovations promise. But we will need a long road of discovery to achieve long term, lasting success.