Hybridoma vs Phage Display for Monoclonal Antibody Production: Which Technique for Which Purpose?

What is the ideal antibody affinity you need? What is the final application of your project? Do you have any timeline or budget constraints? These are some of the parameters you may have to consider if you are hesitating between hybridoma development and phage display for a monoclonal antibody production project. This short guide aims to highlight the key advantages and drawbacks of both techniques, in order to help you determine the best method for your needs.

Hybridoma 

Hybridoma generation was first developed in 1975 by George Köhler and César Milstein. It relies on the fusion of mouse immunized B spleen cells with myeloma cells. The immortal B cells producing the antibody of interest are then selected and the best clones are screened in order to obtain monoclonal antibodies with the desired antigen affinity.

Hybridoma development is one of the more traditional methods used to generate monoclonal antibodies, as it allows you to produce highly sensitive binders at an affordable price. Their high specificity makes them particularly adapted for assay development. It is also important to note that the mammalian origin of the cells integrates in vivo post translational modifications, which decreases the risk of aggregation or recognition failures.

These characteristics suggest that hybridomas may also be well suited for therapeutic antibody development, however, there are some significant drawbacks to using hybridomas for this purpose that are worth noting. Firstly, the hybridoma development process is very long: it takes on average between 6 and 8 months to obtain a reasonable amount of monoclonal antibodies (against a few weeks for phage display). Secondly, the murine origin of the antibodies implies further humanization for a therapeutic purpose, which importantly induces an extra cost. For these reasons, hybridoma development is being progressively replaced by faster, more appropriate techniques for biotherapeutic development.

Phage display

Monoclonal antibody production based on phage display represents as one alternative to hybridoma technology for drug development.

The antibody phage display method was attributed to Smith in 1985. A gene sequence coding for a particular antibody is integrated into the DNA sequence of a filamentous bacteriophage, which allows its expression on the surface of the bacteriophage capsid. This peculiarity establishes a link between the genotype and the phenotype. The phage infects Escherichia coli and uses its inner replication system to continuously display new phage, without killing the host cell. This enables fast production of antibodies, in large numbers. A library of naïve or immune phage (up to 10¹⁰ phage) is therefore constituted and can be used to detect an antigen-antibody interaction of interest thanks to screening methods. Libraries can be generated from any animal – even humans, which makes it possible to directly screen human antibodies. As the genotype is linked to the phenotype, it is also easy to obtain direct access to the sequence, which facilitates further engineering or recombinant protein production.

Unlike hybridoma development techniques, when a naïve library is already available, this process can be very fast and generally lasts only a few weeks, it also allows you to screen a greater diversity of antibodies. Phage display is commonly used for toxicology and antivenom research due to its ability to facilitate work with both toxic and non-immunogenic antigens.

In comparison to hybridoma, phage display is more expensive, and there is a chance that the binders may result in a lower affinity when panning naïve libraries.

To establish which display technique is most appropriate for your needs, you should weigh the pros and the cons of each method, summarized in the table below:

	Hybridoma	Phage Display
Advantages	Large scale production High antibody yield High specificity High antibody sensitivity Lower cost	Large scale production Fast process Great control over the selection process Easy to screen a large diversity of clones Possible to directly screen human libraries Possible to screen toxic antigens No immunogenicity issue (for naïve libraries)  No clone viability issues Direct access to sequence No animal use (for naïve libraries)
Drawbacks	Long generation time Incomplete epitope identification Often requires humanization	More expensive Binders may have lower affinity Technically more difficult