Definitions & Background: What Are Hybridoma Technology & Phage Display?

Hybridoma technology, developed by Köhler & Milstein in1975, fuses antibody-producing B cells from an immunized animal with myeloma cells to create immortal hybridomas that secrete monoclonal antibodies. The output is typically full-length IgG amenable to direct functional testing. However, central tolerance can eliminate B cells against conserved human proteins; fusion efficiency, immunogenicity of the antigen, and screening throughput limit accessible diversity. Phage display (mid-1980s onward) expresses antibody fragments (like scFv, Fab, V_HH) on the surface of bacteriophage, linking genotype to phenotype. Libraries—naïve, immune, synthetic, or semi-synthetic—can reach billions of unique variants. Iterative panning enriches binders with desired properties (affinity, specificity, kinetics, stability) under precisely controlled conditions. Creative Biolabs routinely deploys both modalities, often in hybrid strategies to accelerate hit discovery, optimize affinity/epitope, and de-risk developability.

• Phage display maximizes diversity, control, and speed, especially for difficult epitopes and multi-constraint property windows.
• Hybridoma delivers in vivo-matured IgGs ideal for function-first programs with immunogenic antigens.

Phage Display: The In Vitro Revolution

Phage display technology uncouples antibody selection from the constraints of the immune system. It leverages the simple biology of bacteriophages to create a direct, physical link between a protein (the antibody fragment, or "phenotype") and the gene that encodes it (the "genotype").

Fig.1 Phage display workflow steps for mAb generation.

1. Library Construction: A vast collection of genes encoding antibody fragments—typically single-chain variable fragments (scFv) or antigen-binding fragments (Fab)—is cloned into a phagemid vector. Each gene is fused to a phage coat protein gene (pIII or pVIII).
2. Phage Display: The library of phagemids is transformed into E. coli. The bacteria are then co-infected with a helper phage, which provides the necessary proteins for phage assembly. The resulting phage particles incorporate the fusion protein, "displaying" the antibody fragment on their surface while carrying the corresponding gene inside the phage particle. A single library can contain from billions to trillions (10⁹ to >10¹¹) of unique antibody variants.
3. Biopanning: Biopanning is the iterative selection workflow in which a phage library is first incubated with the target antigen immobilized on a solid surface, followed by washes that progressively increase in stringency to remove non-specific or weak binders; specifically bound phages are then eluted—commonly via pH shift or competitive ligand—and the recovered pool is amplified in fresh E. coli to generate enriched input for the next round, steadily concentrating high-affinity, target-specific clones.
4. Screening and Characterization: After 3-5 rounds of biopanning, the enriched phage population is used to infect E. coli for soluble expression of the antibody fragments. These fragments are then screened to identify the best candidates, which are subsequently sequenced and reformatted into full-length IgGs for further characterization.

Advantages of Phage Display

• Fast campaign turnaround and high-throughput automation
• Fully human antibodies with no humanization and reduced immunogenicity risk
• Immune-system independent selection for toxic, self, and non-immunogenic antigens
• Ultra-high library diversity (10¹¹ scale) and increased rare-binder probability
• Precise selection control and rapid in vitro affinity maturation

Limitations of Phage Display

• Fragment hits require IgG reformatting and risk affinity or stability loss
• E. coli display lacks mammalian post-translational modifications
• Lower initial affinity from naïve or synthetic libraries requiring in vitro maturation

Hybridoma Technology: The Classic Gold Standard

Hybridoma technology harnesses the animal's own immune system to create specific antibodies. The process is a masterpiece of cell biology, leveraging B cell biology and cell fusion to create immortal antibody-producing cell lines.

The Hybridoma Workflow

The generation of mAbs via hybridoma technology is a multi-step in vivo process:

Fig.2 Hybridoma steps for mAb generation.

1. Immunization: A host animal, typically a mouse or rat, is injected with the target antigen. The animal's immune system responds by producing a polyclonal mixture of B cells, each secreting antibodies against different epitopes of the antigen. This process involves a series of boosts over several weeks to stimulate a strong memory B cell response and drive affinity maturation.
2. Cell Fusion: The animal's spleen is harvested, and the antibody-secreting B cells (splenocytes) are isolated. These mortal cells are then fused with immortal myeloma (cancerous B cell) partner cells that cannot produce their own antibodies. This fusion is typically induced by PEG.
3. Selection: The resulting mixture of fused and unfused cells is cultured in a selective HAT (Hypoxanthine-Aminopterin-Thymidine) medium. Unfused B cells naturally die off after a few days. Unfused myeloma cells, which have a genetic defect in a salvage pathway for nucleotide synthesis, are killed by aminopterin. Only the successfully fused hybridoma cells—possessing the longevity of myeloma cells and the antibody-producing machinery of B cells—can survive and proliferate.
4. Screening and Cloning: The surviving hybridoma pools are then screened, often by ELISA, to identify wells containing cells that produce the antibody with the desired specificity. Positive clones are then sub-cloned via limiting dilution to ensure each colony originates from a single parent cell, guaranteeing the monoclonality and stability of the antibody production.

Advantages of Hybridoma Technology

• Full-length IgG with natural heavy–light pairing and in vivo matured affinity
• Correct mammalian folding and glycosylation ensuring function and stability
• Proven and reliable workflows with decades of rodent antibody experience

Limitations of Hybridoma Technology

• Long timelines and labor intensive with 4–6 months to stable clones
• Dependent on animal immunity and unsuitable for toxic, non-immunogenic, or self-like antigens
• Rodent origin requiring costly humanization to reduce anti-drug antibody risk
• Limited repertoire diversity relative to the available sequence space
• Low throughput and difficult to automate at scale

Phage Display & Hybridoma Comparative Snapshot: When to Use Which?

The Creative Biolabs Advantage: Harnessing Next-Generation Phage Display

While both technologies are valuable, the future of therapeutic antibody discovery clearly favors in vitro display technologies. At Creative Biolabs, we have pushed the boundaries of this platform. Our cutting-edge phage display services include:

• Integrated Library Construction Suite（Peptide | Antibody | Scaffold | cDNA）

From day one we unify peptide-library build, antibody-library build, scaffold-library build, and cDNA-library build into a single, high-diversity workflow for engineered for rapid hit discovery and smooth downstream optimization. Custom phage display library constructions for different species are also available at Creative Biolabs.

• Phage Display System Construction

We provide system builds spanning M13 system build, Lambda system build, T4 system build, T7 system build, Fab-display build, scFv-display build, hyperphage display, and dual-genome phagemid/helper setup, aligning display format and valency with your target class.

• Phage Display Library Screening and Biopanning

Our selection toolbox covers naïve-library screen, immune-library screen, and peptide-library screen, enabling fit-for-purpose enrichment paths from early binders to prioritized leads.

• Other Services Based on Phage Display

Engagement options include phage-vaccine design, epitope mapping and mimetics, protein–protein interaction profiling, recombinant antibody production, directed protein evolution, and in-vitro diagnostic assay support.

Phage display and hybridoma technology are two pivotal methods in monoclonal antibody discovery, each revolutionizing the landscape in different ways. Phage display delivers speed, control, and unmatched diversity for difficult or highly specified targets, while hybridoma provides in vivo–matured, full-length IgGs ideal for function-first screening. The most resilient path often combines both—leveraging immune experience with display-based steering and rapid maturation to reach developable leads faster. Partner with Creative Biolabs. We provide end-to-end, research-use solutions spanning next-generation phage display (peptide, antibody, scaffold, cDNA libraries; M13, λ, T4, T7, Fab, scFv, Hyperphage, dual-genome systems), library screening and biopanning (naïve, immune, peptide), hybridoma generation, IgG reformatting and humanization, affinity maturation, epitope binning, and developability profiling—delivered through a single, integrated workflow. Ready to accelerate your program? Contact us to request a tailored proposal now!

FAQs

Q: Are antibodies discovered via phage display functional in vivo?

A: Absolutely. Once the selected scFv or Fab fragments are reformatted into full-length human IgG formats (like IgG1 or IgG4) and produced in a mammalian cell line, they behave just like human antibodies. Many of the world's best-selling antibody drugs were discovered using phage display technology.

Q: How do I discover binders to a glycosylated or phosphorylated site?

A: Use phage display with precisely modified antigen and counter-select against unmodified forms. Competitive elution with the natural ligand or unmodified peptide sharpens specificity.

Q: Can I start with hybridoma and then use phage display?

A: Absolutely. Sequence the hybridoma VH/VL, transfer to display, and run maturation to enhance affinity, reduce polyspecificity, or engineer pH-dependent binding.

Q: Can you generate antibodies against self-antigens using these methods?

A: It is extremely difficult with hybridoma technology because the animal's immune system is tolerant to its own proteins (self-antigens). Phage display, however, completely bypasses this limitation. Since the selection is done in vitro, it is an excellent method for discovering antibodies against self-antigens, which is crucial for many autoimmune and cancer targets.

Q: How does the affinity of antibodies from phage display compare to hybridoma?

A: Antibodies from a well-executed hybridoma project often have very high (picomolar to low nanomolar) affinity due to the natural in vivo maturation process. Initial hits from a phage display naïve library may have more moderate affinity (nanomolar range). However, these candidates can be readily optimized using in vitro affinity maturation techniques, often achieving affinities that are equal to or greater than those from hybridomas.

Q: What is "humanization" and why is it so important?

A: Humanization is a genetic engineering process that modifies a non-human (e.g., murine) antibody to make it more similar to human antibodies. This involves grafting the murine CDRs onto a human antibody framework. It is critical because the human immune system can recognize a murine antibody as foreign and mount an immune response against it (an ADA response), which can neutralize the drug and cause adverse effects.

Q: Can I get fully human antibodies without human donors?

A: Yes. Synthetic phage display libraries built on human frameworks with designed CDRs deliver fully human sequences suitable for research applications without relying on human donors.

Related Services

• Phage Display Library Construction
• Phage Display System Construction
• Phage Display Library Screening and Biopanning
• Custom Services Based on Phage Display
• Applications of Phage Display Platform

Please kindly note that our services can only be used to support research purposes (Not for clinical use).