Serum-free media: ask the experts

Written by RegMedNet

In this ‘Ask the experts’ feature, we have brought together a panel of experts from across the industry to share their current perspectives on the challenges and benefits of serum-free media and animal-free reagents in cell and gene therapy. For example, what considerations should users bear in mind when selecting and upscaling serum-free media for research purposes? Does serum-free media influence culture conditions? How might serum-free and animal-free reagents evolve and impact future production of therapeutics? Discover more on the topic with our panel of thought leaders, featuring Jason Mills (Century Therapeutics; PA, USA), Marlin Frechette (FUJIFILM Irvine Scientific; CA, USA), Yas Heidari (Bio-Techne; MN, USA), James J. Hickman (Hesperos, Inc.; FL, USA) and Ulla Schultz (Sartorius CellGenix; Freiburg, Germany).


Please introduce yourself and your institution.

Yas Heidari (YH): My name is Yas Heidari, Product Manager for the Cell and Protein biology portfolio of Bio-Techne (MN, USA) where I oversee a range of our reagents and technologies for research and beyond. My role is focused on helping support our customers’ applications through innovative product introductions and partnerships with scientific networks, academia and industry.

Marlin Frenchette (MF): My name is Marlin Frechette and I am the Chief Quality & Compliance Officer (CQO) at FUJIFILM Irvine Scientific (CA, USA).

FUJIFILM Irvine Scientific is a medical device organization offering a diverse portfolio of advanced cell culture media solutions for biopharmaceutical, vaccine, and cell and gene therapy manufacturers, and complete workflow solutions for assisted reproductive technologies. As the CQO, I bring more than 30 years of industry experience to my role and am responsible for the company’s global compliance within quality systems, global regulatory, corporate compliance, and environmental health & safety.

Ulla Schultz (US): My name is Ulla Schultz and I am Manager of R&D Cell Culture Systems at Sartorius CellGenix (Freiburg, Germany). I was trained in virology and immunology before I joined CellGenix GmbH, a leading global supplier of high-quality raw materials for the expanding cell and gene therapy and regenerative medicine space. At Sartorius CellGenix, we develop, manufacture and market human cytokines and growth factors in preclinical and good manufacturing practice (GMP) quality in addition to GMP serum-free media for further manufacturing of advanced therapy medicinal products.

Given our over 25 years of experience, we are experts in GMP manufacturing of raw materials for the cell and gene therapy space. As a former advanced therapy medicinal product developer and manufacturer, we gained in-depth cell culture knowledge and superior regulatory expertise. Based on this unique background, we very well understand the challenges our customers face during product development and the high requirements of the regulatory approval process. By offering expert technical and regulatory support, we can help simplify raw material qualification and validation efforts.

Jason Mills (JAM): My name is Jason ‘Jay’ Mills, I am a Director of Process Development at Century Therapeutics (PA, USA). Century Therapeutics is an allogeneic iPSC-derived cell therapy company integrating cutting edge gene editing, protein engineering, technical development and manufacturing capabilities to generate iPSC-derived natural killer and T cell therapies for hematological and solid tumor malignancies.

James J. Hickman (JH): Hello, I am James J. Hickman, co-founder and Chief Scientist at Hesperos, Inc. (FL, USA) as well as Professor of Nanoscience Technology, Chemistry, Biomolecular Science and Electrical Engineering and Head of the Hybrid Systems Laboratory at the University of Central Florida. Our company focuses on human-on-a-chip applications for rare diseases as well as efforts to reduce and replace animal use in drug discovery, cosmetic evaluations and for general toxicity. I created the first serum-free defined culture system in 1995 and I have pioneered the use of serum-free media in many organ systems as well as interconnected multi-organ systems.


Why would someone choose a serum-free media or animal-free reagents, and what are the benefits?

YH: Serum- and animal-free reagents are increasingly sought after by researchers, both basic and clinical, to bridge the gap between the two realms. There are a number of different reasons ranging from scientific, manufacturing and ethical considerations. One theme that straddles all is maintaining that all-important reproducibility in data gained. The latter is critical for correct interpretations of results that could impact the lives of patients.

MF: Using serum-free media minimizes the risk of introducing adventitious agents, provides lot-to-lot consistency and simplifies regulatory submissions. Because serum contains various materials, it is undefined, variable and requires extensive screening to minimize the risk of infectious disease transmission. The screening process of serum can be very costly and time-consuming.

US: Pharma companies and everybody engaged in clinical applications are aware that regulatory requirements will become more and more stringent within the next few years, which will call for more defined culture conditions to ensure safe and potent cellular therapeutics. Undefined raw materials, in particular complex mixtures of many different components, such as cell culture media, or those of biological origin, such as serum, may introduce variability and bear safety concerns. Serum may contain adventitious agents and slight differences in impurity profiles, such as trace elements content. Variations in serum composition or biological activity may lead to unwanted effects such as altered cell surface marker profiles and lower expansion rates. This may ultimately cause decreased potency of the final cell product. Another major advantage of using serum-free media is the possibility to select for specific cell types by adding the appropriate cytokine combinations.

JAM: The field of cell and gene therapy is rapidly growing and the FDA regulatory guidelines are requiring more control of raw materials to maintain safe and reliable manufacture of drug products. The use of serum-free media provides an opportunity to increase the definition, produce more reproducible formulations and less batch-to-batch variability. When coupled with animal-free reagents, this provides a material that has a better safety profile and may limit the amount of adventitious agent testing and risk of transmissible spongiform encephalopathies (TSE) contamination when manufactured appropriately.

JH: The primary benefit for using serum-free media is that the components are defined, reproducible and remove an important variable, serum, in cell culture and other assays as well as in expansion and differentiation of stem cells. Animal sera can also induce epigenetic changes that will increase variability of stem cell use in assay development or if being considered for therapeutic utilization.


Does serum-free media recapitulate the characteristics of serum-containing media?

YH: Serum-free formulations have come a long way from their early days. Nowadays they can not only replicate the characteristics of serum-free media but are in many instances superior due to their defined composition; animal-derived sera can lead to lack of reproducibility and varying results sets. Sera is a complex undefined mixture of factors that may affect long term viability of cells, with unforeseen toxicity effects.

MF: The development and design of FUJIFILM Irvine Scientific serum-free media was intended to recreate the same media without serum, but with the natural characteristics of serum-containing products. One replacement component is recombinant human albumin, which enhances serum-free cell culture media performance because of its favorable lipid profile. Cell types such as T cells, mesenchymal stem cells, and natural killer cells can all successfully be maintained in these enriched serum-free media.

US: It is still common practice to supplement media with serum as it contains various growth factors, extracellular matrix proteins and hormones that stimulate cell activation and promote expansion. However, serum contains unidentified components that can have unknown effects on the functionality of the cell product. The variability in serum composition and performance has driven the cell therapy field to search for serum- and xeno-free media alternatives. Such alternatives provide improved safety profiles, increased consistency and allow for simplified regulatory procedures. Serum-free media contain a finite number of well-defined, traceable ingredients reducing the risk of inconsistencies. Supplementing serum-free media with animal component-free cytokines and growth factors manufactured according to GMP guidelines allows for greater control of your cell culture process and precise evaluation of cellular function.

JAM: The first step in adopting serum-free media is to identify if the composition of the media contains the required nutrients, such as proteins, lipids, carbohydrates, micro- and macro-nutrients, that are amenable to grow, expand, and/or differentiate your cells to desired final cellular product. There are many commercially available products that have been tailored to a variety of defined cell types. However, in many cases, additional recombinant proteins or small molecules need to be included to serum-free media to meet the cellular characteristics that serum-containing medias may provide. Having a thorough and robust understanding of the cell type(s) of interest can provide a roadmap on how to reliably and reproducibly maintain, expand or generate cells with characteristics of serum containing medias.

JH: Serum-free medias have been shown to be as good as, and in most cases better than, serum-based systems due to their reproducibility as well as the ability to tailor them for specific applications. It also removes many of the undesirable components contained in serum that cause de-differentiation of primary cells and interferences in other assay systems.


Does serum-free media influence culture conditions and how may this impact cell growth in culture?

YH: As long as the medium used is a robust rich formulation at physiological pH and with good buffering capacity, it will enable the cells or cell lines in question to thrive in culture. It is important to bear in mind that besides the media used to culture cells, other important factors include supplements added to the media, such as growth factors and small molecules, and the extracellular matrix or substrate on which the cells are cultured. The exact supplement cocktail incorporated into the media is dependent on the needs and characteristics of the cultured cells.

MF: Serum-free media allows researchers to grow a specific cell type or perform a specific application without serum; however, almost every cell type has its own media supplement requirements, which is why a universal serum-free cell culture medium would need to be user-qualified for the specific application. For commercial-scale production, serum-free media significantly reduces batch-to-batch variations, increases lot-to-lot consistency and does not require lengthy or rigorous additional screening. The technical documentation of serum-free media also supports easier reviews during regulatory submissions.

US: Given the central functional role of serum in the current production processes, inconsistencies in growth factor and hormone concentrations can greatly affect the overall media performance. When switching to serum-free media, growth factor and cytokine concentrations may need to be adapted to ensure optimal proliferation and cellular function. Adherent cells may also require precoating of cell culture plasticware with defined extracellular matrix proteins for optimal adherence and survival when serum-free media are used.

JAM: The use of serum- and animal-free reagents have shown to enhance the growth, viability and expansion of cells. Due to the chemically defined formulation, the cultures have a more consistent performance and provide better control over evaluations of the physiologic responsiveness, phenotype uniformity and cellular functions. However, before considering switching over to serum-free media immediately, there should be a cell adaption protocol to ensure optimized results.

JH: Serum-free media has been shown to substitute well in almost every culture system in which the effort has been expended to remove serum as a variable. The majority of applications that still use serum are simply due to manufacturers and practitioners not wanting to change protocols that have been approved by the FDA or simply due to inertia in many labs and businesses to change.


What considerations should users bear in mind when selecting serum-free media for research purposes, and subsequently upscaling production?

YH: One key consideration is that the media supports your cells to maintain a healthy status for the entirety of the period in which they need to remain in culture. For upscaling, it is important that the defined medium in question is from a reliable supplier. In certain cases, it may be best to opt for a custom formulation and hence it is beneficial to work with a manufacturer with custom capabilities both on media production, analytical and other bio-assays. At Bio-Techne, we endeavor to be a partner that can offer you flexibility and a seamless transition from research to translation.

MF: Different cell types have diverse receptors involved in cell survival, growth and differentiation, releasing various factors into their environment. Each cell type also has requirements concerning media supplements. Therefore, manufacturers should select a serum-free medium that fulfills the cells’ requirements used in their experiments. Additionally, serum-free media reduces the need for extensive quality assurance testing of raw materials, including media for their process.

US: When deciding on a cell culture media, functionality of the media under specific culture conditions is of utmost importance: do the cells proliferate, do they have the desired phenotype and do they meet the criteria for viability and functionality?

The need for cell culture media to abide to international regulatory guidelines can arise in later steps of the development process, for example during commercial manufacturing and clinical stages. Cell culture media should be chosen not only based on performance, but also on safety and quality aspects. Individual countries tend to have local requirements; therefore, it is important to ensure that the media is compliant with both international regulatory guidelines and local requirements. We recommend choosing a supplier that can provide customized regulatory support.

JAM: Selection of serum-free media can be a considerable decision; there are hundreds of different media available through various suppliers. Even at the early research stage of any program, the selection of media should consider a supplier with a reliable supply chain track record, short lead times and lot sizes large enough to satisfy the consumers requirements for research to upscaling production. Exploring multiple serum-free media suppliers with comparable products should be considered, as formulations or backorders of material can delay or eliminate the potentiality of media sources. The use of ‘boutique’ media supplies should be explored, as these vendors can create custom formulations where components and concentrations of formulation are provided as proprietary media to the consumer. This can reduce the dependence of single supplier media sources. Additional analytical methodologies should be developed to identify plans for a comparability assessment between media or reagents that are being explored for research to later stage development, or upscaling purposes.

JH: There are many different variations of serum-free medias that are validated in the literature or have extensive data sheets from manufacturers and suppliers. In many cases a quick review of this literature or a conversation with the supplier will lay out the parameters that are necessary to adapt or utilize serum-free medias for their procedures or products.


How can manufacturers optimize their manufacturing processes when moving to serum-free or animal-free reagents?

YH: The manufacturing processes have to be set up so that the reagents produced are reliable, consistent from lot to lot, and meet their safety and other QC specifications. Again, that flexibility has to be built in for upscaling and potential custom requirements. For animal-free formulations, origin of raw materials is important, provided in relevant documentation in order to meet quality and regulatory standards.

MF: Optimizing a cell-based manufacturing process is much more straightforward when serum-free media are used. Customers have essentially eliminated the media’s most variable and troublesome portion by removing the serum. Removing serum opens up the opportunity to conduct optimization experiments in a design space that is better defined as the extrinsic and variable influences of sera have been significantly reduced. Therefore, efforts can be focused on identifying and optimizing the factors that can be reliably controlled. Removing serum helps produce higher confidence levels in the conclusions drawn from process experiments.

US: To enable seamless transition from research to preclinical development and further on to the clinical stage it is highly recommended to identify the appropriate raw materials, such as media and cytokines, already during early preclinical research. In addition, to meet global regulatory requirements, as well as for commercial considerations, it is advantageous to omit human serum because of supply limitations, safety issues and high lot-to-lot variation. The lot-to-lot variation requires costly and often time-consuming pretesting. Using serum-free media and animal-free reagents manufactured to all applicable GMP guidelines will provide documented evidence of safety, purity, potency, consistency and stability.

JAM: Serum-free or animal-free reagents should come from a vendor that has a manufacturing process that is robust, reproducible and reliable to ensure that products are safe, consistent and of superior quality. Manufacturers should consider the market demands and prepare to generate batch sizes that support current scale and potential for upscaling efforts. These upscaling efforts could be through larger volume media or reagents, or custom fill options. For complex media formulations, the manufacturer should ensure solid documentation that details the source and origin of raw materials and provide post-production analytical testing. This generally is provided through certificates of origins and certificates of analysis documents provided by the supplier. Implementing more robust analytical testing, such as adventitious agents, mycoplasma, endotoxin, and sterility, on media or reagents would ensure compliance with regulations and provision of cell therapy products that are intended for patient delivery. A robust quality and audit management systems aid in the coordination and directs a supplier’s activities to meet customers’ needs and align with documentation and regulatory requirements.

JH: Actually, the optimization of their processes will be made easier as the components are defined and the inherent batch-to-batch variability present when utilizing animal sera will not be present with serum-free formulations, making it a better alternative from the start. However, just as with any process supply chain, factors should be considered in development of the manufacturing schedule and establishment of reagent availability from multiple vendors would be important.


At what stage in the development process should the decision about media be made?

YH: If you mean at which stage the decision should be made to go serum-free, or animal-free for cultured cells, then as early as possible is good practice. It means that any troubleshooting and optimization that may be necessary for the process can be done upfront, circumventing any potential delays. Furthermore, if the process needs to be fully animal-free, then that needs to be set up as such from the outset.

MF: Selection and qualification of serum-free media should occur early in process development to avoid delays during scale-up. Considerations should include the ability of the medium to support consistent cell expansion and function, availability of a GMP form of the components of the chosen medium, and a reliable and cooperative supplier with an established supply chain that meets the cell and gene therapy manufacturer’s quality and regulatory requirements. For the end user, the qualification and regulatory process is costly, which is why we recommend they use serum-free media early: to begin process development.

US: The appropriate serum-free GMP culture media in cell therapy should be considered before clinical development, with global regulatory guidelines in mind. It is also important to factor in the suitability of the production of cell culture media in different cell culture devices.

Envision the future cell culture workflow: is it upscale or scale out, are you using static or agitated bioreactors? Is the cell culture media available globally to supply all your manufacturing plants? Is there a secured supply chain available? Will you receive change notifications prior to relevant changes on the cell culture media?

Conducting these considerations early on in your development process will prevent the need for expensive and laborious clinical comparability studies later on to proof process adaptations or cell culture media changes. Changes in the manufacturing process can trigger a round of regulatory re-review and approval because the change might alter the safety and efficacy of the manufactured cells.

JAM: As early as the research and discovery stage, media and other raw materials should be assessed with forward looking approaches to move toward development and eventually GMP manufacturing.

JH: Given the ethical and regulatory considerations that are moving government, industry and academia away from animal and animal product usage, many difficulties can be avoided by establishing serum-free media as the basis of the system from the beginning of the process. Switching from serum-containing formulations at the development stage to serum-free will require much time and effort in scaling up for production.


How might serum-free and animal-free reagents, and their applications, evolve and impact future production of therapeutics?

YH: Cell culture is constantly evolving – its gradual evolution may mean that we perhaps forget that it is still happening. We are moving away from sera, to xeno-free, to eventually animal-free. The big driver is to tackle the reproducibility and ethical issues on the research side. However, on the translational side there is an absolute requirement for production and eventually therapeutics to be animal-free for safety, toxicity and reliability purposes. Quality and compliance procedures thus need to be in place to meet the evolving guidelines, regulations and standardizations. As cell culture evolves to be more animal-free, it should positively impact the future production of therapies.

MF: The development of new technologies to produce recombinant molecules will result in newer, more efficient serum-free cell culture media. As a result, many cell types will be maintained successfully in these enriched serum-free media and the number of cell-specific media will grow steadily.

US: The high cost of cell therapies remains a major obstacle to their widespread use, particularly as first-line or second-line therapies; for example, CAR-T therapies like Kymriah and Yescarta are approved for third-line only at this time. Reducing the costs of the manufacturing process is therefore of utmost importance not only for the sake of the patients and the healthcare system but also for cell therapy manufacturers, who will have to cope with the pressure on the market price. The establishment of automated manufacturing platforms, and novel AI-based tools and technologies, bear the opportunity for consistent and cost-efficient manufacturing. Using serum-free and animal-free reagents procured from safe and traceable sources will significantly reduce qualification and validation efforts and will increase speed-to-market.

JAM: While off-the-shelf media can provide a relatively turnkey approach for expedited and efficient path forward in early discovery, there needs to be added considerations when upscaling the process for development, clinical or commercial manufacturing. In these cases, having a vendor that understands the regulatory landscape of advanced therapy medicinal products will make the path to patients more straight forward. This usually involves a solid quality management system, audit control, quality assurances and compliance with USP, EP, and ISO technical standards to ensure manufacturing is using high quality raw materials. Standardization of the quality requirements of serum-free material or animal-free reagents will help at harmonize the industry and provide more clarity of acceptable guidelines, rather than non-supervised recommendations.

JH: I expect that serum-containing media will gradually be phased out of all processes within 10 years, which will make it more difficult for regulatory approval and customer acceptance with continued use of serum-containing products or products that utilize serum during their development or manufacture. As the information concerning epigenetics and the changes that are induced in cells during culture become more apparent, undefined systems, such as those containing animal sera will be harder to justify, especially for therapeutic applications.


Meet the experts

Yas Heidari | Product Manager, Protein & Cell biology, Europe and Emerging Markets, Bio-Techne 

Yas Heidari obtained her Ph.D. in 2003 from King’s College London (UK), on identification and characterization of a novel isoform of the NADPH oxidase enzyme complex, involved in immune defense and cell signaling. She received a Wellcome Trust fellowship grant to pursue her post-doctoral studies at Centre for Inflammatory Diseases, Imperial College London (UK), where she studied genes that may be implicated in the onset of the autoimmune disease systemic lupus erythematosus (SLE). After leaving research, Yas spent 8 years at Thermo Fisher Scientific in various commercial roles and departments gaining experience of the biotech sector. Yas joined Bio-Techne in 2019 as Product Manager for the Cell and Protein biology portfolio, overseeing a range of reagents and technologies for Europe and emerging markets. Her role is focused on helping the team to support our customers’ applications, where she aspires to use her experience and skills to help advance regenerative medicine through innovative product introductions and partnerships with stem cell and organoid networks, academia and industry. 

Marlin Frechette | Chief Quality & Compliance Officer (CQO), FUJIFILM Irvine Scientific

Marlin Frechette has over 30 years of experience in the Medical Device industry, servicing Pharmaceutical and Biopharmaceutical customers. As Chief Quality & Compliance Officer at FUJIFILM Irvine Scientific, she holds global responsibility for all FUJIFILM Irvine Scientific sites and affiliates for Compliance, Quality Systems, Global Regulatory and EHS.  She holds a Bachelor’s of Science with a major in Business Administration & Personnel Management. 

Ulla Schultz | Head of R&D Cell Culture Systems, Sartorius CellGenix

Ulla Schultz studied biology and veterinary medicine and graduated in Virology at the University of Giessen (Germany). As a research fellow at The Scripps Research Institute in La Jolla (CA USA) she conducted research in the field of avian immunology. In 1998 Ulla moved to the University of Freiburg (Germany) as a senior scientist before joining Sartorius CellGenix in 2002.  She has been Head of R&D Cell Culture Systems at Sartorius CellGenix since 2010.

Jason ‘Jay’ Mills | Director of Process Development, Technical Development and Manufacturing, Century Therapeutics

Jay Mills is the Director of Process Development in the Technical Development and Manufacturing organization at Century Therapeutics.  In his role, Jay oversees the early platform development strategies for clinical manufacturing of novel iPSC-derived allogeneic cell therapies (NK and T-cells).  Through his work there, Jay and his colleagues have developed scalable platforms that will enable CenturyTX to advance multiple therapeutic programs from PhI-III at their GMP manufacturing facility in Branchburg, NJ. 

James J. Hickman | Co-Founder and Chief Scientist, Hesperos Inc. 

James J. Hickman is a founder and Chief Scientist at Hesperos, Inc., a biotechnology CRO leveraging its’ patented, Human-on-a-Chip multi-organ platform to accelerate drug discovery by providing safety and efficacy testing for novel therapeutics. He is also the Founding Director of the NanoScience Technology Center, a Professor of Nanoscience Technology, and Electrical Engineering at the University of Central Florida. Previously, he was the Hunter Endowed Chair in the Bioengineering Department at Clemson University. Dr. Hickman has a Ph.D. from MIT in Chemistry. For the past thirty years, he has been studying the interaction of biological species with modified surfaces, first in industry and later in academia. He has worked at NSF and DARPA in the area of biological computation. He has 162 publications and 20 book chapters, in addition to 30 issued patents out of 49 total patent applications. He is a Fellow of the American Institute of Medical and Biomedical Engineers (2004), the American Vacuum Society (2007) International Academy of Nanobiotechnology (2019) and the National Academy of Inventors (2020). 

 

Disclaimers

The opinions expressed in this interview are those of the interviewees and do not necessarily reflect the views of RegMedNet or Future Science Group.

This feature was produced in association with Sartorius CellGenix, FUJIFILM Irvine Scientific and Bio-Techne.