Methods used in antibody and antibody mimetic production

Polyclonal antibodies and monoclonal antibodies from hybridomas

Polyclonal antibodies are generated by repeatedly immunising animals, such as rabbits, goats, or chickens, with an antigen. Blood is collected, and the serum, which contains a mixture of antibodies produced by different B cells targeting multiple epitopes on the antigen, is separated and purified. While polyclonal antibodies can recognise multiple epitopes, they suffer from several drawbacks:

  • High variability: Batch-to-batch differences can compromise reproducibility.
  • Limited specificity: Off-target binding can occur.
  • Unknown sequences: Without a defined DNA sequence, long-term reproducibility is not possible.

Monoclonal antibodies differ from polyclonal antibodies in that they are identical antibodies produced by a single B cell clone, designed to bind a specific epitope. These are typically made by fusing B cells (harvested from immunised animals) with immortal myeloma cells, creating hybridomas. These hybridomas are screened to identify clones producing antibodies with the desired specificity and affinity. Monoclonal antibodies are then produced by culturing hybridomas in vitro or in vivo (e.g., in mouse ascites fluid). Following this, the antibodies are purified. Drawbacks associated with monoclonal antibodies include:

  • Genetic drift: Hybridoma instability can affect antibody consistency.
  • Unknown sequences: Without a sequenced antibody, reproducibility is limited.
  • Ethical concerns: The ascites method is banned in some countries due to animal welfare issues.

Recombinant antibodies

Recombinant antibodies are monoclonal antibodies created using recombinant DNA technology, ensuring precise and reproducible antibody sequences. This method can bypass the need for using animals and involves two key stages – discovery & production.

The generation of antibody libraries involves creating a diverse repertoire of antibody fragments (e.g., single-chain variable fragments [scFv] or Fab fragments), created from:

Naïve B cells: These libraries are derived from B cells isolated from pooled blood of human or non-human donors who have not been intentionally immunised with a specific antigen. Antibody-encoding gene segments from isolated B cells are amplified using PCR with primers targeting the variable (V) regions, then cloned into vectors to construct a recombinant antibody library. Naïve B cells inherently reflect the broad natural immune repertoire, with each donor contributing up to 10 million unique antibody gene sequences due to hypervariable regions. When pooled, the resulting diversity greatly enhances the likelihood of identifying an antibody targeting the antigen of interest during in vitro selection. The copied sequences are expressed using display methods (described below). Although billions of antibodies may be theoretically generated, not all are stable, which can make synthetic methods preferable in certain cases.

Framework Sequences: Derived from conserved antibody gene regions; these frameworks serve as stable scaffolds for creating synthetic or naïve libraries. Complementarity-determining regions (CDRs), which dictate antigen specificity, are inserted into the variable domains of the antibody’s heavy chain (VH) and light chain (VL), regions critical for the antibody’s ability to bind specifically to its target antigen. This enables a high degree of diversity. Potentially problematic (“liability”) sequences can be removed during the design phase. The high level of control afforded by gene synthesis, as opposed to relying on natural B cell diversity, ensures consistency and stability. Frameworks are often engineered to improve solubility, stability, and expression in host systems (e.g., bacteria, yeast, or mammalian cells). For therapeutic applications, human frameworks are typically employed to minimise immune responses.

  • Synthetic Framework Sequences: Computationally designed and optimised using combinatorial mutagenesis, rather than being directly derived from natural repertoires
  • Natural Framework Sequences: Sequences are sourced from various species (e.g., human, mouse, rabbit, camel, or shark) and are available in databases, published research, expired patents, or genome sequencing projects, or may be consensus sequences from multiple natural frameworks. Their use does not require new animal immunisation or tissue harvesting.

Immunised animals: Immunised animals, such as mice, rabbits, or camelids, produce diverse antibody repertoires specific to a target antigen (e.g., a protein or peptide). B cells are harvested from the spleen, lymph nodes, or blood, and their mRNA is extracted, reverse-transcribed into cDNA, and amplified to construct recombinant libraries. While immunisation remains part of some discovery pipelines, particularly for generating libraries from immunised animals, this method does not align with the May 2020 ECVAM recommendations and is increasingly being replaced by synthetic and naïve approaches while maintaining the diversity and specificity necessary for high-quality antibodies.

Display systems facilitate the selection and production of recombinant antibodies from libraries. Antibody fragments are physically linked to display platforms (e.g., bacteriophage, yeast cells, or ribosome complexes), ensuring their presentation on the host’s surface alongside the genetic information encoding them. For example, natural or synthetic gene fragments can be cloned into a phagemid vector and fused with a minor coat protein (e.g., protein III). This enables antibodies to be displayed on the surface of phage particles, where they are exposed for antigen binding. Each phage in the library expresses an antibody fragment specific to a single epitope. Display systems allow researchers to screen billions of antibody variants in a single experiment, offering scalability and efficiency.

This method also supports flexibility in generating different antibody formats, including single-chain variable fragments (scFv), Fab fragments, or full-length IgG. The theoretical diversity achievable in this process can reach up to 1011 independent clones, effectively equivalent to a lifetime supply of antibodies derived from immunised animals.

Selected antibodies are subsequently mass produced using bacterial, yeast, or mammalian systems. The selected antibody genes are cloned into suitable expression vectors equipped with the necessary regulatory sequences for transcription and translation in host cells. The choice of host system depends on the desired antibody format and properties and may include bacteria (e.g., E. coli), yeast, or mammalian cells (e.g., CHO or HEK293). In yeast or mammalian cells, recombinant antibodies are typically secreted into the culture medium, while in bacteria, they are often retained within the cells.

Use of Animal-Derived Biomaterials (ADBs)

Animal-derived biomaterials (ADBs) are sometimes used in antibody production or final formulations, raising concerns regarding reproducibility, contamination, and ethics. Alternatives to these materials are increasingly available, with the market for animal-free biomaterials rapidly growing. Transitioning to animal-free biomaterials in antibody production is an essential step toward more ethical and scientifically robust practices.

Commonly used in cell culture media, FCS is derived from bovine foetuses during slaughter. Variability in composition and the risk of contamination make it less desirable compared to animal-free alternatives. Serum-free media options are now widely available, alongside many protocols to humanise common cell lines. Researchers can explore serum-free media compositions for hundreds of commonly used cell lines using the Fetal Calf Serum-free Database.

Another option is the use of human serum which has advantages over animal serum that include direct translatability into human-specific, clinical applications, and no risk of external pathogen transmission. As human serum is harvested from volunteer donors, a consistent supply is ensured. However, as it is collected from multiple sources batch-to-batch variation is an issue and due to its limited supply, costs tend to be higher.

Derived from bovine blood, BSA is used as a stabiliser, blocking agent, carrier or buffer additive in various processes. While each purification method produces BSA that meets standard specifications, there are several attributes which are often overlooked such as fatty acid and hormone profiles, contaminants and the presence of IgG, transferrin, and other peptides. These factors can significantly impact the performance of BSA in certain applications. This contributes to batch-to-batch and supplier variability in the BSA market. Synthetic or plant-based stabilisers are available as replacements.

KLH is used to conjugate small, immunogenic molecules (haptens) for antibody production. Synthetic Carriers made from materials like polyethylene glycol (PEG) are available.

Often derived from porcine pancreas, trypsin is used for detaching adherent cells during cell culture. Issues around this ADB include contamination, impurities, and the spread of porcine viruses. Alternatives to trypsin are commercially available, including TrypZean solution, a recombinant animal-free dissociation reagent expressed in corn.

Derived from casein or meat, these serve as nutrients in cell cultures. Alternatives include chemically defined media composed of precise amounts of pure chemicals, ensuring consistency and eliminating animal-derived components; plant-derived hydrolysates; synthetic peptides custom-designed to mimic the nutrient profile of enzymatic digests; yeast extracts and algal-based hydrolysates.

Extracted from animal connective tissues, these are occasionally used as stabilisers or in cell culture media. Animal-free and synthetic alternatives include those sourced from plant-based materials, fibrin derived from human plasma, alginate sourced from brown algae, and synthetic polymers.

Antibody mimetics

What are Antibody Mimetics?

Antibody mimetics are engineered proteins designed to replicate the binding capabilities of antibodies, often providing advantages in stability, specificity, and ease of production. They are increasingly favoured in biotechnology and bioanalysis due to their potential for high binding affinity, enhanced cellular and tumour penetration, suitability for mass production, and resistance to environmental challenges like temperature and pH fluctuations.

Design and Development of Antibody Mimetics

The development of antibody mimetics typically begins with selecting a naturally occurring scaffold. These are stable protein structures that serve as a framework or template for engineering high affinity and high specificity ligand binding functions, typically by introducing or modifying amino acid sequences within it. Some scaffolds naturally possess cavities, loops, or pockets that can be engineered into functional binding sites and some are chosen for their stable structure, allowing them to withstand changes in temperature, pH, and other environmental conditions.

This scaffold is then subjected to processes like optimisation and selection to enhance its binding characteristics. Library generation involves creating a diverse collection of protein variants which increases the likelihood of identifying variants with desired traits, such as high affinity for a particular target or increased stability under specific conditions.

Once the library is generated, techniques like phage display, yeast display, or ribosome display are often used to present these protein variants and test them for binding to a specific target. Through several rounds of selection, the variants with the best binding properties are isolated and further optimised if needed.

Read more about the advantages of recombinant antibodies and mimetics

Types of Antibody Mimetics

Each type of antibody mimetic offers distinct advantages in terms of size, stability, diversity of scaffolds, and specific binding capabilities, making them valuable tools in diagnostics, therapeutics, and research. In addition to those listed below, emerging antibody mimetics include Atrimers, Knottins, Kunitz Domains, Fynomers, β-Hairpin Mimetics, nanoCLAMPs, and Optimers.

  • Source: Derived from the B-domain of staphylococcal protein A.
  • Characteristics: Small, cost-effective proteins with high solubility and thermal stability. Known for high binding affinity.
  • Applications: Commonly used in diagnostics and therapeutic targeting.

  • Source: Based on the extracellular domain of human fibronectin III.
  • Characteristics: Exhibit antibody-like specificity and affinity.
  • Applications: Widely used in therapeutic targeting and biomolecular research.

  • Source: Derived from the DNA-binding protein Sac7d from Sulfolobus acidocaldarius, a thermophilic bacterium.
  • Characteristics: Extremely heat-resistant due to thermophilic origin.
  • Applications: Suited for use in high-temperature environments and industrial applications.

  • Source: Developed from human lipocalins, naturally occurring binding proteins.
  • Characteristics: Useful for binding small molecules with their cup-shaped binding sites.
  • Applications: Frequently used in diagnostics and small-molecule binding studies.

  • Source: Based on an ankyrin repeat scaffold.
  • Characteristics: Small, highly stable single-domain proteins.
  • Applications: Employed in various research applications due to high stability.

  • Source: Modular proteins designed for multi-target binding.
  • Characteristics: Able to bind multiple targets, beneficial for complex therapeutic applications.
  • Applications: Utilised in multi-target therapies and complex molecular interactions.

  • Source: Derived from natural ankyrins, consisting of at least three ankyrin repeat motifs.
  • Characteristics: Known for stability, ease of production, and high affinity.
  • Applications: Used in therapeutic targeting and structural biology.

  • Source: Nucleic acid molecules that mimic antibodies by forming complex 3D shapes.
  • Characteristics: Easily generated, low cost, low immunogenicity, reversible folding, and minimal batch-to-batch variability.
  • Applications: Diagnostic applications and molecular interaction studies.

  • Source: Engineered from the cystatin protein scaffold or other scaffolds.
  • Characteristics: Robust and stable, easily produced in bacterial systems, and cost-effective.
  • Applications: Commonly used in diagnostic assays and as research reagents.

References for Recombinant Antibodies

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Reasons for using Recombinant Antibodies

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Antibodies – Accio Biobank Online – Tissue for Research

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Replacing animal-derived antibodies with animal-free affinity reagents | NC3Rs

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Animal-free alternatives and the antibody iceberg | Nature Biotechnology

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Guide to Understanding the Benefits and Uses of Recombinant Antibodies

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How animal-derived antibodies contribute an estimated loss of $800 million p.a. to biomedical research

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Generating recombinant antibodies to the complete human proteome

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EURL ECVAM recommendation on non-animal-derived antibodies

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An approach to identifying quality research antibodies

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A worldwide survey on the use of animal-derived materials and reagents in scientific experimentation

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Animal-Friendly Affinity Reagents: Replacing the Needless in the Haystack

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Animal free antibody webinar series by Afability Animal Friendly Affinity Reagents

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The case for replacement

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Reproducibility: Standardise antibodies used in research

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Reproducibility crisis: Blame it on the antibodies

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Can antibodies be “vegan”? A guide through the maze of today’s antibody generation methods

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Achieving an animal free IHC

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Making the Switch to Non-Animal Derived Antibodies

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Generation of synthetic nanobodies against delicate proteins

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Increasing the use of animal-free recombinant antibodies

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Advances in Animal-Free Monoclonal Antibody Production

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Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display

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Everything You Need to Know About Recombinant Antibodies

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Antibody essentials blog series

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Fetal Calf Serum-free Database

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