Non-Animal-Derived Antibodies  

Recommendation on Non-Animal-Derived Antibodies

In 2020, the EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) recommendation on the use of animal-free antibodies was published, which states that animals should no longer be used for the development and production of antibodies for research, regulatory, diagnostic as well as therapeutic purposes. In the EU, the provisions of Directive 2010/63/EU should be respected and EU countries should no longer allow the development and production of antibodies by animal immunization unless there is a robust, scientifically legitimate justification. 

Phagedisplay and Antibody-Libraries

Bacteriophages, yeasts or mammalian cells and even ribosomes allow the formation of so-called genetic packages, where a recombinant protein such as an antibody or antibody fragment is expressed on its surface, while the coding genetic information is contained inside the package. This direct link between genotype and phenotype allows the creation of large and diverse libraries of antibody fragments, where all clones differ in the region that contacts the antigen.  Antibody fragment libraries can be enriched and selected for clones that bind to a specific antigen, mimicking immune selection. Selected clones can be isolated and further processed, resulting in human antibodies with high affinity and specificity, which can then be expressed and purified on a large scale from cell cultures in vitro and used clinically for therapy in humans.

Find more information in the presentation of Univ. Prof. DI Dr. Florian Rüker:

"Circumventing immunization by in vitro directed evolution and selection of human antibodies from large surface display libraries"

Yeast Based Production of Antibody Molecules

Nowadays, yeasts are not only used for the production of alcohol, but also for the production of industrially relevant substances. Whether yeasts are a good cellular factory for the production of monoclonal antibodies (mAbs) depends on what the mAbs are produced for. mAbs for the treatment of human diseases are still preferentially produced in mammalian cells, while mAbs for protein-based diagnostic tests are already produced in yeast. As early as 2020, the first mAb produced in yeast was approved for the treatment of migraine in humans. However, yeasts have some disadvantages to be effective cellular factories for mAbs production. mAbs carry sugar residues (glycans) that affect their binding properties and they contain multiple disulfide bridges for their stability. In recombinant systems such as yeast, cells must first be taught how to properly implement these changes to the mAb, as these factors differ between yeast and human cells. Yeast can be humanized by genetic modification of glycans or chemo-enzymatic glycosylation. Through these procedures, it is now possible to produce mAbs with uniform N-glycans. During the production of the recombinant protein, disulfide bridging is a strong limiting factor that greatly hinders the production of full-length mAbs. Overexpression of biocatalysts can enhance the formation of disulfide bridges in Fab fragments and their secretion. Since the secretion of mAbs from the cell is very complex and limited in capacity, large amounts of the recombinant protein can remain in the cell, affecting production. However, by using certain factors to inhibit vacuolar protein-sorting complexes and simultaneously pulling them into and pushing them out of the endoplasmic reticulum, secretion of mAbs fragments can be enhanced. Current results therefore suggest that yeasts will be the system of choice in the future especially for the production of mAbs fragments and nanobodies.

For further details on this topic, please refer to the presentation by Assoc. Prof. Dipl.-Ing. Dr. Brigitte Gasser:

“Strategies to improve yeast-based production of antibody molecules”

EARA/EFPIA Statement on the EURL ECVAM Recommendation on Non-Animal-Derived Antibodies

After EURL ECVAM (European Union Reference Laboratory for Alternatives to Animal Testing) issued a recommendation for the development and production of exclusively animal-free antibodies in 2020, the European Animal Research Association (EARA) together with the European Federation of Pharmaceutical Industries and Associations (EFPIA) commented on this publication in the same year. Overall, EARA/EFPIA believe such a recommendation is premature, particularly in the area of vaccines and therapeutics, although the scientific community recognizes the forward-looking approach. However, there are many unanswered questions that need to be addressed in order to fully utilize animal-free antibody development methods in all areas. The research community is calling for additional support to diversify production capacity, investment in lowering the cost of new production methods, and adequate support for project and application evaluation. EARA/EFPIA's key recommendations are: - A thorough evaluation of the feasibility of therapeutic applications using animal-free antibody development. - Regulatory support and legal certainty in line with EU industrial and pharmaceutical strategies - Use of existing or new platforms for a more comprehensive exchange between scientists, industry, regulators and civil society on the production and use of animal-free antibodies - Establish a permanent stakeholder platform that includes a balanced representation of all relevant stakeholders and whose goal would be to identify areas where alignment is possible - Support projects that explore and develop robust technologies for the development and production of animal-free antibodies and conduct high-quality research in this area, thereby replacing, reducing, and improving the use of animals In addition, the statement details the advantages and disadvantages of animal-free and animal-developed antibodies. It then discusses the use of the two types of antibodies as research tools, therapeutics, and for the production of in vitro diagnostics. Also addressed is the aspect of animal welfare and the socio-economic impact of a switch to purely animal-free antibodies. The statement ends with an outlook on a world where there are no more antibodies developed in vivo and warns that such a restriction would prevent access to the best available medicines while jeopardizing Europe's position as a global center for innovation and research & development in the life sciences.

Follow the link to the statement