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Laboratory Production Methods

Detailed below are opportunities to end the use of animal-derived products for scientific or medical purposes and to reduce significantly the use of animals for the production of drugs and vaccines.

Antibody Production

Affinity reagents such as antibodies are essential tools used in research to bind to a molecule to identify or influence its activity. Every year, millions of animals are injected with viruses, bacteria, or other foreign substances and then killed for the antibodies that their bodies produce in response. Animals used in antibody production are subjected to many invasive and painful procedures, including antigen injection and repeated blood or ascites collection, before being killed. In the ascites method of antibody production, animals have been reported to be unable to eat, walk, or breathe properly. Several countries, including Australia, Canada, Germany, the Netherlands, Switzerland, and the U.K., have restricted or banned the production of antibodies obtained via the ascites method because of animal welfare concerns.12

Growing concern about the lack of quality and reproducibility of animal-derived antibodies, which often show poor specificity or fail to recognize their targets, is also evident in the literature. In a 2015 Nature commentary, 111 academic and industry scientists called for an international shift to recombinant antibodies for increased reliability and reduced batch-to-batch variability in affinity reagents.3 In addition, a 2015 Nature news feature reported that antibodies might be the laboratory tool most commonly contributing to the “reproducibility crisis.”4 In fact, poorly characterized and ill-defined antibodies were considered a primary cause of irreproducible research in a survey of preclinical studies that found that 47 out of 53 studies could not be replicated. Furthermore, a systematic analysis of 185 commercially available hybridoma monoclonal antibodies found that one-third were not reliably monospecific, and the authors recommended replacing the use of animal-derived monoclonal antibodies with sequence-defined recombinant antibodies as a straightforward and cost-effective solution to this serious problem.5 This issue is not limited to monoclonal antibodies. Polyclonal antibodies, which depend on the animal used to produce the antibodies and, by definition, vary in their composition, cannot be consistently reproduced, leading to calls within the scientific community to phase them out of research completely.3

In addition to the lack of scientific reliability and animal welfare concerns, there are significant economic issues related to using animal-derived antibodies. An estimated $800 million is wasted annually worldwide on unreliable antibodies.4 Thus, there are potential cost savings associated with the more reproducible research that would result from using higher-quality affinity reagents.

Non-animal affinity reagents, such as recombinant antibodies, can be used in all traditional antibody applications, including in basic research, regulatory testing, and clinical applications. They are commercially available and, with appropriate resources, can be developed by researchers in their own laboratories.56 The numerous scientific advantages of non-animal affinity reagents over animal-derived antibodies include high affinity and specificity, shorter generation time, reduced immunogenicity, the ability to control selection conditions, and the ability to be generated against unstable, toxic, immunosuppressant, and non-immunogenic antigens.78

International efforts have highlighted the importance of a large-scale transition from animal-derived antibodies to animal-free affinity reagents.

In its 2020 Recommendation on Non-Animal-Derived Antibodies, EURL ECVAM stated the following:

EURL ECVAM recommends that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic and therapeutic applications. … EU countries should no longer authorise the development and production of antibodies through animal immunisation, where robust, legitimate scientific justification is lacking.9

In the U.S., experts and organizations, including NICEATM and PETA Science Consortium International, are working to increase access to animal-free affinity reagents. In December 2019, both organizations convened a meeting to outline a pathway to improve the quality and reproducibility of research and testing by accelerating their production and use. The subsequent meeting report describes steps to overcome hurdles to a comprehensive shift from animal-derived to animal-free, sequence-defined affinity reagents.10 More information on sources of animal-free affinity reagents, webinars, publications, and details of the scientific, economic, and ethical advantages of replacing animal-derived antibodies with animal-free options are available at ThePSCI.eu/our-work/antibodies.

Governments have the opportunity to advance science by committing to developing, producing, and importing animal-free antibodies and banning monoclonal antibodies produced via the ascites method. In 2022, the Recombinant Antibody Challenge was launched by PETA Science Consortium International, the Physicians Committee for Responsible Medicine, and the Alternatives Research and Development Foundation, offering grants for free catalog recombinant antibodies for use in research and testing (ThePSCI.eu/funding/recombinant-antibody-challenge). In order to further expedite the replacement of animal-derived antibodies, we recommend providing additional grant opportunities for the generation and use of non-animal affinity reagents.

Biological Drugs

Many vaccines and other biological drugs are produced or tested for quality, identity, safety, and efficacy in experiments that require large numbers of animals. These procedures often cause severe suffering before the animals die or are killed. Methods to produce and test these drugs without using animals are increasingly available, but experience has shown that validation and regulatory acceptance of these methods have not guaranteed their use.1112131415 Activities intended to phase out the use of animals in this context must ensure that regulatory authorities and the industry commit to (1) transitioning to non-animal biological drug production platforms; (2) developing non-animal replacements for quality, identity, safety, and efficacy tests for all biological drugs; and (3) ensuring that non-animal methods are consistently used in place of animal-based tests whenever they are available.

Production platforms are available that replace animal-derived substances with recombinant, cell-based equivalents. Antitoxins, for example, have been produced historically by hyper-immunizing horses and other large mammals and isolating the resulting immunoglobulins from their blood. These animal-derived immunoglobulins have disadvantages intrinsic to their animal origin, including the risk of adverse human immune response, high batch-to-batch variability, and the potential to transmit pathogens between species. Animal-derived antitoxins can be replaced with recombinant human antitoxins expressed in cell culture. Several recombinant antibodies have been marketed,1617 and more are in development,18 including candidate therapeutic human recombinant antibodies created with funding from PETA Science Consortium International.1920

With adequate funding and support from regulators, all biological drugs of animal origin, including antibodies (described above), can and should be replaced to resolve issues inherent in using antibodies derived from animals.

Non-animal tests for assessing quality are available, but no formal mechanism exists to ensure that barriers to their implementation are resolved in a timely manner.21 In some instances, manufacturers report difficulty meeting the technical criteria for using validated non-animal methods (as with the in vitro Leptospira vaccine potency tests).22 In other instances, international regulators have yet to agree on technical criteria for using non-animal methods (as with the in vitro rabies vaccine potency test).23 Without formal oversight of the implementation process, these barriers are left to be resolved informally through workshops and decentralized problem-solving by consortia of interested parties, which is prohibitively expensive and slow. As a consequence, the industry’s adoption of non-animal methods remains limited.24 Additional barriers to the implementation of currently available alternative tests have been discussed at length in workshops and the literature for many human and veterinary biological drugs.252627 Accelerating and standardizing processes that facilitate the use of these existing replacement methods is crucial.

Leadership from regulators that ensures coordination among international regulatory bodies and the industry on best practices will remove these barriers. Authorities must establish harmonized manufacturing consistency requirements, as tightly controlled manufacturing consistency policies are the foundation of many animal replacement strategies.2829

Fetal Bovine Serum

Fetal bovine serum (FBS) is a supplement for cell culture media that provides an undefined mixture of macromolecules that maintain cell viability and facilitate cell metabolism, growth, proliferation, and spreading in culture. When pregnant cows are slaughtered, a large-gauge needle is used to draw the blood from the beating heart of the fetus.3031 Because the unborn calves are not anesthetized at the time of blood collection, they likely experience pain. In 2007, it was estimated that 600,000 liters of FBS were produced globally each year, which translates to the use of up to 1.8 million bovine fetuses for this purpose.32 Given the significant increase in the use of cell culture for research and testing, the number of fetuses used is expected to increase substantially.

Several scientific concerns are associated with the use of FBS: (1) batch variation leads to reproducibility issues for in vitro studies using FBS (or other undefined animal-derived products such as bovine pituitary extract); (2) the unknown composition of the serum may complicate the analysis of data obtained from cultured cells and reduce human relevance, leading to potentially unexpected or undesirable outcomes; and (3) the risk of contamination by animal proteins or pathogens is especially problematic in the manufacture of biological drugs for human therapies.

Chemically defined, serum-free media or human platelet lysates can replace FBS in cell culture media. For optimal definition and reproducibility, a chemically defined, animal-free medium that avoids all animal-derived supplements should be used. Scientists have published workshop proceedings for more than 20 years calling for the transition from FBS to animal component–free and chemically defined media.3334353637 Animal-free and chemically defined serum-free media are available for some cell types. For others, researchers may still need to optimize the concentration of supplement to replace FBS. Medium providers can assist researchers in finding the right animal component–free medium. Researchers are also working to develop animal component–free media that can work across cell types.38 Information on replacing FBS in cell culture media and developing serum-free media and listings of companies offering FBS-free products are available on PETA Science Consortium International’s website (ThePSCI.eu/fbs) and in the Fetal Calf Serum-Free Database (https://fcs-free.org). PETA Science Consortium International has funded the transition of commonly used lung cell lines to cell culture media without animal-derived products.39

Government and regulatory agencies should move expediently to restrict the production and use of FBS and prioritize the development and use of non-animal media and supplements. Funding organizations should also provide funding for the transition of cells to available non-animal media and for developing and optimizing non-animal, serum-free media when needed. In addition, any research project proposal application should include a section on whether animal-derived products (including serum) will be used and, if animal-derived products are used, details on the search for non–animal derived products and an explanation for why an adequate substitute couldn’t be found for that project.

References
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  2. Research Councils UK, National Centre for Replacement, Refinement and Reduction of Animals in Research. Animal welfare standards expected of suppliers of antibodies to Research Council establishments. NC3Rs. Accessed March 26, 2025. https://www.nc3rs.org.uk/sites/default/files/documents/Funding/Antibody%20supplier%20policy%20-%20will%20be%20updated.pdf ↩︎
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