Why Fetal Bovine Serum (FBS) Is Problematic for Modern Cell Therapy

1. Ethical Concerns

• Animal Welfare Issues: FBS is collected from fetuses removed from pregnant cows during slaughter, raising concerns about the suffering of the animals. The process often involves no anesthetics, and the fetus is alive when blood is extracted, leading to ethical dilemmas.

• Lack of Regulation: Many countries lack strict regulations on fetal blood collection, meaning procedures may vary and animal welfare standards can be inconsistent.

• Connection to the Meat Industry: Since FBS is a byproduct of the meat industry, its production is tied to factory farming and unsustainable agricultural practices, further raising ethical questions.

• Environmental Impact: The meat industry, which produces FBS, is a significant contributor to environmental harm, including high water usage, greenhouse gas emissions, and land degradation.

2. Batch-to-Batch Variability and Challenges for Scalability

FBS, being a complex medium supplement, carries inherent batch-to-batch variability. The subtle variations in its composition can significantly impact cell culture conditions, which, in turn, can lead to inconsistencies in cell properties and even alter the final therapeutic product. This becomes particularly problematic as cells are highly sensitive to their culture environment, and even minor changes can affect their functionality and performance.

The factors contributing to this variability include:

• The genetic diversity of the source herds.
• Variations in the animal diet.
• The serum manufacturing process itself.
• The sheer complexity of serum composition, which includes around 1800 proteins and 4000 metabolites.
• The broad acceptable range of serum specifications for release

As the cell therapy industry matures and clinical trials progress into pivotal phase 3 studies, there will be an increasing demand for scale-up, process validation, and raw material quality assurance. This growing need will likely drive changes in culturing technologies, raw material sourcing, and testing methods.

Because cells are highly sensitive to their culture conditions, any change in the culture environment can alter cellular physiology and performance characteristics, potentially affecting the product’s critical quality attributes. Therefore, uncontrolled scale-up can lead to variations in the final product’s activity, making it difficult to achieve consistency between smaller-scale and larger-scale production.

Additionally, there are significant challenges in ensuring a consistent supply of high-quality raw materials in the required quantities. The difficulty in scaling raw material production to meet demand while maintaining quality poses a substantial challenge to the commercialization of cell therapies.

3. Safety Concerns with Human-Derived Components

• Serum-free formulations based on human blood components carry additional safety risks due to the pooled nature of the material.
• Ensuring safety involves thorough donor screening procedures, safety testing of viral infections, and viral inactivation during the manufacturing process
• Complex Regulatory Requirements for Blood-Derived Products:
  Different regulatory agencies have specific safety and testing requirements for blood-derived components, which adds complexity and may limit material availability.
  • Plasma donor criteria (e.g., CJD and vCJD transmission risk).
  • Pathogen testing, testing methodology, and source material traceability.
  • Licensing of plasma collection facilities and necessary certifications (e.g., FDA, EMA).
  • Viral testing
  • Viral inactivation and removal steps in manufacturing.

4. Limitations in Scaling Supply and Commercialization Risks

• Global Supply Sources: Approximately 90% of the fetal bovine serum (FBS) used in the cell therapy industry is sourced from three countries: the United States, Australia, and New Zealand.
• Given that FBS is harvested as a byproduct of the meat industry, the industry has no incentive to scale up cattle farming exclusively for serum.  
• The reliance on the meat industry’s byproduct limits the ability to significantly expand serum production.
• Any significant increase in FBS demand cannot easily be met by simply raising cattle for this purpose, making supply inherently constrained.

Given the combination of limited supply, ethical scrutiny, and rising costs, dependence on FBS could become a major bottleneck for the future scalability and commercialization of cell-based medicines.

The industry is increasingly motivated to seek serum-free or xeno-free alternatives to ensure manufacturing consistency, regulatory compliance, and long-term sustainability.

How Fetal Bovine Serum (FBS) Is Made: Step-by-Step Overview

1. FBS production begins at abattoirs, where the fetus is removed from pregnant cows that have been slaughtered for meat.
2. Blood is collected directly from the fetal heart to minimize microbial contamination.
3. The collected blood is centrifuged to separate the serum from blood clots and blood cells.
4. Serum undergoes filtration through a 0.1-micron filter, removing bacteria, fungi, and mycoplasma, effectively sterilizing the product.
5. Some batches also receive gamma irradiation to eliminate viruses and very small pathogens that could pass through filtration.
6. To preserve the integrity of proteins and biological components, the serum is kept frozen throughout the manufacturing steps.
7. After pooling and processing, representative samples from serum batches are tested for sterility and other quality parameters before release.

Top Alternatives to Fetal Bovine Serum in Cell Culture Media

Human Platelet Lysate (HPL)

• Role in Regenerative Medicine: Platelets are crucial for wound healing and tissue repair, making their derivatives ideal for regenerative medicine.
• Growth Factors and Cytokines: Platelet granules store abundant growth factors and cytokines, which can be released naturally by thrombin activation or artificially through freeze/thaw, sonication, or chemical treatment.
• Application in Cell Culture: HPL has become a viable alternative to FBS in animal serum-free cell cultures, supporting efficient human cell propagation for advanced somatic cell therapy and tissue engineering.
• Historical Use: In the 1980s, HPL was first used to culture fibroblasts, endothelial cells, and tumor cell lines.
• Source of HPL: Approximately 15-20% of whole blood donations are used to create platelet concentrates, and 5-20% of those expired concentrates can be used to produce HPL. With 100 million whole blood donations annually, around 100,000–250,000 L of HPL could be sourced per year from outdated platelet concentrates.

Human serum albumin

• Potential Alternative: Human AB serum (HABS) has emerged as a promising alternative to FBS. It is routinely tested for viral contamination and supports the growth of human osteoblasts, chondrocytes, bone marrow cells, and various cancer cell lines like glioma and melanoma.
• Limitations: The main challenges with HABS include limited collection capacity and the risks associated with human blood products, including potential exposure to new or unknown pathogens.

Serum-Free, Xeno-Free, and Chemically Defined Media

• Industry and Academic Efforts: Both academia and industry have focused on developing serum replacements like serum-free, xeno-free, and chemically defined media. These alternatives aim to provide controlled and consistent environments for cell culture.
• Terminology Confusion: The terminology surrounding these media has evolved over time, creating confusion in the industry. Efforts are being made to clarify and standardize the definitions of these alternatives for better understanding and application.
• Common misconceptions (xeno-free ≠ serum-free)

Key terms used in cell culture media explained:

Transitioning from FBS to Serum-Free Media in CAR T-Cell Manufacturing

Challenges and Ethical Considerations in FBS Use for CAR T-Cell Production

• FBS-containing media are still the most commonly used in GMP core facilities worldwide
• However, FBS composition differs significantly from human serum and does not adequately replicate the human environment encountered by CAR T-cells post-administration
• Due to ethical and environmental considerations, An increasing number of GMP laboratories are optimizing HS-containing media formulations for CAR T-cell production

Variability and Safety Concerns in Serum-Based Media for GMP Manufacturing

• Serum requires extensive testing prior to GMP manufacturing, leading to increased costs.
• Human serum provides nutrients and growth factors that more closely mirror the human microenvironment.
• Both human serum and FBS exhibit donor-dependent variability, leading to product-to-product inconsistency
• The risk of contamination is a critical concern:
  • FBS: Potential transmission of bovine spongiform encephalopathy (BSE) and viral pathogens.
  • HS: Risk of viral contamination
• Serum requires extensive testing prior to GMP manufacturing, leading to increased costs.

 Shift Toward Defined and Serum-Free Media

• An increasing trend is emerging towards the use of defined media formulations, which may include or exclude chemically defined xeno-free components, in CAR T-cell production.
• Serum-free media, while excluding serum, can incorporate purified or synthetic components.

Supporting Evidence for Serum-Free Media

Smith et al. demonstrated:

• Comparable T-cell expansion rates in xeno-free media versus HS- or FBS-supplemented media.
• A higher percentage of central memory T-cells in xeno-free media.
• Similar growth of lentivirus-transduced T-cells in both media types.
• Coeshott et al. successfully expanded T-cells at large scale in a functionally closed, automated bioreactor system using xeno-free media, showing increased central memory T-cell populations after expansion.

In this context, Kohjin Bio’s serum-free Media for T-cell expansion and Activation and Expansion Media for T-cells can further enhance CAR T-cell production. Specifically formulated to promote both activation and expansion, this media supports high-efficiency T-cell activation and robust cell growth. Kohjin Bio’s media formulations help streamline the production process, improving scalability while maintaining high-quality T-cell populations essential for therapeutic applications.

Regulatory Standards for Serum-Free Media in Cell Therapy

• The term “cGMP-grade” serum is often misleading.
• In the European Union (EU), regulators emphasize that raw materials are not manufactured under full cGMP (current Good Manufacturing Practices) guidelines.
• The EU only grants cGMP licenses to facilities producing active pharmaceutical ingredients and final drug products, not raw materials like serum.

Why a Robust Quality System Is Essential in Serum-Free Media Manufacturing

• Even though raw materials aren’t produced under cGMP, having a robust quality system is crucial.
• A proper quality system ensures:
• Thorough documentation of the manufacturing process.
• Mechanisms for change control to track any adjustments in procedures.

The Hidden Limitations of Quality Systems in Cell Therapy Supply Chains

• Having a quality system does not automatically guarantee the quality of the final raw material.
• Ultimate responsibility for the safety and efficacy of the finished cell therapy product lies with the cell therapy manufacturer.
• Therefore, manufacturers must audit and approve the raw material suppliers’ quality systems themselves.

Key Process Design Strategies to Guarantee Consistent Serum-Free Media Quality

• Manufacturers should evaluate whether the supplier’s process design includes:
• Viral reduction or elimination steps, if necessary.
• Strict temperature control during production.
• In-process monitoring and control measures.
• Up-to-date process validation studies to ensure consistency.

Why Supplier QC Alone Isn’t Enough for Cell Therapy Compliance

• Suppliers’ QC testing might not meet all the needs of a cell therapy developer.
• Typical supplier QC tests include:
• Total protein content.
• Immunoglobulin levels.
• Sterility testing.
• pH measurement.
• Osmolality testing.
• However, these basic tests might be insufficient for clinical applications.

Supplemental Testing: A Critical Step Beyond Supplier QC

• Developers often need to perform additional functional assays, such as:
• Cell growth performance tests.
• Marker expression analysis.
• Activity profiling, depending on the application.

How Gamma Irradiation Affects Serum Quality and Cell Therapy Performance

• QC testing is typically performed before gamma irradiation.
• Since irradiation can alter serum performance, a post-irradiation quality assurance strategy is essential to verify that the serum still meets required specifications.

Certification & Documentation Must-Haves for Regulatory-Ready Serum-Free Media

• Besides having a quality system and QC validation, suppliers should also provide:
• A Certificate of Suitability from the European Directorate for the Quality of Medicines and Healthcare (EDQM) regarding the risk of transmissible spongiform encephalopathies (TSE).
• A certificate of origin detailing the source of the raw material.
• Different countries may impose additional testing requirements and specifications.
• It is critical to identify the intended market for the product and ensure compliance with local health authority regulations.

In line with FDA expectations for cell therapy manufacturing (21 CFR 1271, cGMP, BLA submissions), developers should also address common challenges, including:

• Selecting an appropriate serum-free medium for the target cell type.
• Adjusting factors to modulate cell growth rates and doubling times.
• Using attachment factors to support initial cell adherence after seeding.
• Aligning serum-free media supplements and protocols with the culture surface and cell type.
• Optimizing cell seeding densities.
• Managing changes in cellular characteristics such as secretome, differentiation profile, cell size, or immune-related properties.
• Monitoring shifts in surface marker expression or distribution.
• Developing adaptation strategies for transitioning cells to serum-free conditions.
• Establishing primary isolation methods in serum-free media.
• Ensuring proper media shelf-life and storage (e.g., temperature, light sensitivity).
• Accounting for the impact of different culture systems on cell adherence and shear stress.

Conclusion

Shifting from FBS to xeno-free and serum-free media in cell therapy addresses ethical, safety, and regulatory concerns but presents challenges in scalability and consistency. Continued innovation is essential to improve these media’s commercial viability and support the growth of sustainable, safe cell therapies.

References

1. Smith C et al (2015). Ex vivo expansion of human T cells for adoptive immunotherapy using the novel xeno-free CTS immune cell serum replacement. Clin Transl Immunology. Jan 16;4(1):e31.
2. Karnieli O et al (2017). A consensus introduction to serum replacements and serum-free media for cellular therapies. Cytotherapy. Feb;19(2):155-169.
3. Coeshott C et al. (2019) Large-scale expansion and characterization of CD3+ T-cells in the quantum® cell expansion system. J Transl Med. Aug 7;17(1):258.
4. Alnabhan R et al (2018) Media evaluation for production and expansion of anti-CD19 chimeric antigen receptor T cells. Cytotherapy. 20:941–951.
5. Versteegen RJ, Murray J, Doelger S. (2021) Animal welfare and ethics in the collection of fetal blood for the production of fetal bovine serum. ALTEX.