Antibody library generation for phage display

Phage Display form

    Whether you need ready-made naïve antibody libraries or a partner to develop high-diversity immune libraries for antibody phage display, ProteoGenix offers the market’s most extensive guarantees. Drawing from our experience in creating over 60 high diversity libraries (109 to 1010), our library generation process is designed to recover and optimize antibody repertoires from any host species (mouse, rabbit, camelid, human, etc.) and in any format (scFv, Fab, VHH) maximizing library diversity.

    Our solutions for antibody library generation

    Use our naïve antibody libraries

    Naïve antibody libraries

    Use our naïve antibody libraries to quickly develop functional antibodies for therapy (3 fully human libraries) and analytical applications (rabbit library), as well as next-generation VHH antibodies in less than 7 weeks

    Build your immune antibody library

    Immune antibody libraries

    Develop functional and high-diversity immune antibody libraries (109-1010 different clones) even against the most challenging of antigens in only 14-19 weeks thanks to our vast expertise in antibody library generation processes.

    Why choose ProteoGenix for your antibody library development projects?

    Best high diversity libraries for any antigen and application
    Optimized libraries for any
    antigen and application

    Our optimized library generation protocols allow the construction of high-quality repertoires from any format, origin, or species for maximal clonal diversity (1010) and functionality adapted to any antigen and application

    IP free antibody libraries for phage display
    IP free

    You get full ownership over your custom-made immune antibody libraries and binders obtained from our highly diverse naïve antibody libraries

    Ready-to-use antibody libraries for phage display
    Ready-to-use libraries

    Partner with us for your phage display projects or buy the libraries (immune or naïve) with all essential components to perform your own efficient panning campaigns

    Experts in antibody library generation for phage display
    Experts in creating antibody libraries from any species

    Drawing from our experience in library creation from any host species (mouse, sheep, llama, rabbit, etc.), we have developed optimal immunization and library generation protocols that prime your projects for success.

    scFv, Fab, and VHH antibody libraries for phage display
    scFv, Fab, or VHH antibodies

    Wide range of antibody formats to fit all types of applications

    Animal free antibody phage display using naïve libraries

    Minimize animal use in your antibody discovery projects by choosing our premium naïve libraries built for the highest possible quality and diversity

    Our antibody phage display service process

    Antigen design and production for antibody library generation
    Antigen design & production
    • Choice of an adequate antigen format (protein, peptide, DNA, small molecules, or cells)
    • Antigen production
    • Conjugation to carriers (only for peptides or small molecules)
    Host selection and immunization for antibody library generation
    Host selection & immunization*
    • Choice of the most suitable host species and antibody format (scFv, Fab, or VHH)
    • Design of an efficient immunization strategy

    *only for immune library generation

    Cell harvesting for antibody library generation
    Cell harvesting
    • Harvesting and cell separation
    • Isolation of spleen, bone marrow, and peripheral blood mononuclear cells (PBMCs)
    Antibody repertoire harvesting for library generation
    Antibody repertoire harvesting
    • mRNA isolation
    • cDNA synthesis
    • Amplification of antibody encoding genes with specific primers (Fv, Fab, or VHH regions)
    Phagemid library construction
    Phagemid library construction
    • Randomized heavy and light chain pairing (only for Fab and scFv libraries)
    • Cloning of the antibody fragments (fused to M13’s minor coat protein G3P) into the phagemid vectors
    • Library QC

    14-19 weeks

    Antibody library purchase for phage display
    Library purchase

    Buy our naïve or immune libraries to carry out phage display campaigns at your facilities

    Phage display using naïve or immune antibody libraries
    Phage display

    Choose to perform your phage display panning campaigns with us

    Naïve or immune antibody phage display libraries?

    Antibody libraries can be generated from naïve hosts who have never been challenged with a specific immunogen or from immune hosts who have developed a strong and specific humoral response against the desired target. In comparison to other antibody generation approaches, antibody libraries have considerable advantages including:

    • Elimination of the need for animal immunization (naïve libraries only)
    • Elimination of the need for antibody humanization (fully human antibody libraries only)
    • Compatibility with nonimmunogenic or toxic antigens (it is unnecessary to generate an immune response)
    • Faster turnaround times (a few weeks instead of months)

    Naïve antibody libraries typically comprise the vast naïve (unchallenged) antibody repertoire of many individual hosts to maximize diversity and functionality. In contrast, immune antibody libraries are built from a reduced number of hosts and maximize the affinity of the antibody repertoire against a specific immunogen (require animal immunization).

    Antibodies generated from naïve libraries may, at times, present lower affinity towards a specific target in comparison to antibodies generated from immune libraries. But the choice between the two library types can be a complex one. It depends on several factors including the desired timeline, available budget, and project’s goals.

    Naïve libraries are uniquely adapted to projects requiring fast turnarounds or projects focused on developing antibodies against different antigens (i.e. development of antibody cocktail therapies, antivenom treatments, etc.). Moreover, naïve library construction is generally considered more time-consuming and labor-intensive, these libraries can serve a wide diversity of projects in comparison to immune libraries, which are restricted to a single antigen. However, for projects that prize antibody affinity, the development of immune antibody libraries should be preferred.

    Naïve libraries Immune libraries
    Species Human, rabbit, camelids (camel, llama, alpaca) No limitation except humans
    Formats Fab, scFv, VHH Fab, scFv, VHH
    Number of binders ++ +++
    Affinity of binders ++ +++
    Potential immunogenicity issues No Yes
    Animal use No Yes
    Timelines 6-7 weeks 14-19 weeks + 6-7 weeks
    Price + ++

    How are phagemid antibody libraries generated?

    Antibody libraries are built by harvesting peripheral blood mononuclear cells (PBMCs), spleen cells, and bone marrow cells from challenged (immunized) or unchallenged (naïve) hosts. These cells are subsequently used for mRNA isolation, cDNA synthesis, amplification of antibody encoding genes, the randomized pairing of antibody’s heavy and light chains, and cloning into phage or phagemid vectors.

    Phagemid vectors have become the most commonly used approach for antibody library generation. These vectors are typically derived from filamentous phage M13, a single-stranded DNA (ssDNA) phage with a circular genome capable of infecting strains of Escherichia coli harboring the F (fertility) pili. This phage carries all genes necessary for infection, replication, assembly, as well as all structural proteins. In contrast, phagemid vectors encode only a “minimal” version of the M13 phage containing key elements such as:

    •  a selection marker (i.e. antibiotic resistance gene)
    •  genes encoding for specific antibody fragments fused to one of the phage’s coat proteins
    •  the phage’s origin of replication – crucial for phage DNA replication

    Antibody-encoding genes in the form of scFv (single-chain fragment variable regions), Fab, or VHH are typically fused to one of M13’s coat proteins – G3P, also known as pIII. About 4-5 copies of G3P are expressed on the tip of fully assembled M13 phages and these copies are responsible for binding to the F pili in E. coli.

    However, fused copies of G3P cannot interact with E. coli’s F pili. Because, when all copies of the G3P protein are coupled to antibody fragments, the phage is unable to infect E. coli and, thus, no replication can take place.

    The most common approach to circumventing this issue is the use phagemid vectors in conjugation with helper phages. Helpers contain all genes of the M13 phage essential for capsid production, assembly, replication, and budding, including the wild-type unfused GP3

    In E. coli, helper phage vectors compete with the phagemid encoding the G3P-antibody fusion protein. As a result, the majority of phage particles either have no antibody fragment displayed on its surface or display a single G3P-antibody fusion protein alongside wild-type G3P. The mixed phenotype allows the reconciliation of the phage’s role in antibody display with its ability to infect and replicate in E. coli.

    Wild type G3P versus G3P-antibody fusion in M13 phage

    Adapted from Ledsgaard, L. et al. Basics of Antibody Phage Display Technology. Toxins. 2018; 10:236. doi:10.3390/toxins10060236

    What are the advantages of the phagemid system for antibody library construction?

    Antibody libraries based on phagemid vectors have a critical advantage over full phage libraries – a higher transformation efficiency. Due to its “minimal” size, E. coli is more easily transformed by phagemid than by phage vectors. Moreover, since most resulting phage particles derived from phagemid libraries have only a single antibody fragment on its surface (monovalent), the selection of high-affinity antibodies is more efficient than when multivalent formats are used (phage vectors). In other words, the monovalent format ensures that antibodies with weak affinity can be easily washed off during panning campaigns.

    However, because phagemid libraries need to be amplified by direct competition with a helper phage with wild-type G3P, the vast majority of the phages will be devoid of antibody fragments (“bald” phages”) but still retain their infectivity.

    To circumvent this issue and reduce the abundance of “bald” phages, a trypsin cleavage site is often introduced into the wild-type G3P. This modification ensures that, after panning and enrichment of high-affinity antibody fragments, trypsin treatment can be performed to render “bald” phages non-infective. The same trypsin cleavage site is also added to the linker region between the antibody fragment and G3P, so that antibody fragments can be cleaved from positive phages to allow them to infect E. coli.

    Trypsin cleavage of antibody phage display libraries

    Adapted from Ledsgaard, L. et al. Basics of Antibody Phage Display Technology. Toxins. 2018; 10:236. doi:10.3390/toxins10060236