Are you striving to create potent antibody therapies? Our process of hybridoma production allies the cost-efficiency of hybridomas with the market’s most diverse offer of downstream services to bring you antibodies with enhanced affinity, epitope specificity, and stability. With over 30 antibodies undergoing preclinical and clinical trials and 3 others already on the market, ProteoGenix can help you develop efficient immunotherapies even against the most challenging antigens like membrane-bound receptors and large protein complexes.

Our process of hybridoma production
for therapeutic applications

Antigen Design

  • Definition of the most relevant immunization strategy.
    Antigen design: peptide synthesis, gene synthesis & protein production in 2 systems or DNA Immunization.


  • Immunization of 5 mice with our optimized proprietary protocol

Cell Fusion

  • Collection of the splenocytes from 2 mice for 2 fusions with a myeloma cell line

Hybridoma Selection And Screening (polyclonal Stage)

  • Hybrid cells selection (HAT selection) Culture supernatant screening vs. target antigen (ELISA screening)

Isolation And Selection Of The Best Monoclones

  • Isolation of monoclones by limiting dilution.
  • Expansion and screening of the monoclones by ELISA or in target application.

Why do hybridoma-generated antibodies have better
biophysical properties than their in vitro generated counterparts?

Antibodies are produced by B cells in higher eukaryotes in response to invading pathogens or toxins, thus, acting as key players of the humoral immune response (adaptive immune system).

B cells, as well as T cells (important for producing long-lasting immunity), carry immense antibody repertoires that are constantly evolving in response to the selective pressure (i.e. pathogens, toxins, etc.) a person experiences throughout his/her life.

Antigens trigger the immune response initiating a process of recombination which endows T and B cells with the ability to quickly generate high-affinity antibodies against new antigens. The governing mechanisms of this process of sequence diversification are:

  • Combinatorial diversification consisting of the rearrangement of the different genes that compose the variable regions – variable (V), diversity (D), and joining (J) genes
  • The imprecise joining of the different gene fragments in these variable regions
  • Somatic hypermutations (SHM – insertions, deletions, single-point mutations) which further diversify the sequences in the variable regions

The combination of these processes drives the selection of antibodies able to recognize gradually decreasing concentrations of a specific antigen and it can be regarded as the organism’s way to fine-tune its already immense repertoire for optimal affinity as efficiently as possible.

Why is hybridoma production still
relevant for therapeutic applications?

The knowledge that B cells carry these massive repertoires (up to 1011 antibodies in humans) and that they adapt to different antigens rather quickly through extensive recombination, prompted the development of the hybridoma technology.

Hybridomas remain one of the most cost-effective solutions for the production of therapeutic antibodies. They consist of fusing mature B cells (already challenged by a specific antigen and containing affinity matured antibodies) with a myeloma partner to produce a new hybrid and immortalized cell line.

These new cell lines can easily produce antibodies in vitro indefinitely, making the selection of clones with the highest affinity towards a specific antigen very straightforward.

Unlike in vitro methods of antibody generation, hybridomas capitalize on the extensive natural antibody repertoires and the elegant and efficient process of affinity maturation via in vivo recombination. In this way, these cell lines produce antibodies with:

  • The highest possible affinity towards a specific antigen
  • Maximal stability with naturally long shelve lives, making them easier to store
  • Optimal developability, making them naturally easier to produce in recombinant systems

Because the hybridoma technology capitalizes on this natural process, the early stages of antigen design, production, and immunization strategy development remain the most important of the entire process.

Immunization strategies and hybridoma
production for therapeutic applications

Choosing an immunization strategy often dictates the success of a hybridoma production project.This choice depends on several factors, including:

  • The antigen’s origin
  • The antigen’s native conformation and nature
  • The ease of production of a given antigen
  • The purpose of the antibody produced by the hybridoma technology

Most of the relevant clinical targets for cancer and auto-immune conditions are membrane-bound human cell surface receptors. Likewise, for viral diseases, most of the biologically important targets are structural proteins bound to the viral envelope. In these cases, antibodies able to block the interaction between the structural protein and its receptor (i.e.neutralizing antibodies) are considered valuable biotherapeutics.

These proteins are challenging to produce due to their glycosylation profiles and complex native conformations. These molecules are part hydrophobic (membrane-bound) and part hydrophilic (soluble), rendering them unstable under typical laboratory conditions.

We can employ one of two approaches to overcome this constraint:

  • Producing only the soluble part of the protein in linear form (peptide) or conserving part of its secondary structure (protein)
  • Producing the integral membrane-bound protein by DNA immunization

The latter is a great solution when working with complex membrane-bound antigens. These proteins are hard to produce even when using high-productivity recombinant expression systems. Moreover, they are hard to purify and cannot be efficiently used as antigens due to their unstable nature.

In these cases, the genetic immunization approach remains cheaper since it combines the ease of gene synthesis processes with the flexibility of building self-adjuvant vectors (i.e. by including bacterially methylated DNA or toxin-encoding genes) that forgo the need to use chemical adjuvants.

However, genetic immunization is not a one-size-fits-all solution. For instance, when developing therapeutic antibodies for bacterial diseases or when targeting soluble antigens, DNA immunization may not always be the best approach.

In these cases, choosing protein-based or peptide-based immunization strategies is advisable. These targets tend to be more stable than membrane-bound proteins and easier to produce and purify. Moreover, using protein or peptide antigens guarantees greater control over the region or epitopes that will be recognized by the hybridoma-generated antibody.

Adapting hybridoma-generated antibodies
for therapeutic applications

Hybridomas offer unique advantages for therapeutic antibody development. Nevertheless, since most hybridomas are generated in mice or other rodents, the resulting molecules (murine antibodies) can prompt the development of the human anti-murine antibody (HAMA) response.

This response can be described as an allergic reaction to the xenogeneic antibodies and it can lead to their quick elimination from the human organism reducing their clinical efficiency. Several efficient strategies have been developed to overcome this constraint, the most important of which is the antibody humanization strategy.

Humanizing an antibody implies selecting antigen-binding domains from a murine antibody and grafting these domains into a fully human framework. This technique has been so successful that most therapeutic antibodies currently licensed for clinical use (almost 50%) are humanized IgG1 or IgG4 molecules obtained through the mice hybridoma technology.

This trend has been consistently observed year after year, reaffirming the hybridoma technology as an efficient and relevant approach for therapeutic antibody production.

Clinical development of therapeutic antibodies with the hybridoma technology

The preclinical and clinical evaluation of an antibody drug is a vital step in the development process. Even if hybridoma-generated antibody drugs display the best stability and affinity in vitro, it remains vital to validate their therapeutic efficiency in preclinical models.

With this purpose in mind, the development of anti-idiotypic antibodies arose as a robust way to measure clinical efficiency. These antibodies are generally used in a Fab format and can be classified according to their function:

  • Antigen blocking: antibodies that bind to the paratope (epitope-binding region) of a specific antibody drug and are used for measuring the amount of free drug (not bound to the target)
  • Non-blocking: antibodies that bind the antibody drug outside the paratope region allowing total drug quantification (bound and not bound to the target)
  • Complex specific: antibodies that bind the antibody-antigen complex allowing the quantification of antibodies that have efficiently bound the target antigen

These antibodies are vital for establishing the pharmacokinetics (PK), pharmacodynamics (PD), and immunogenicity of therapeutic antibodies. For this reason, they are also considered vital tools for optimal therapeutic antibody development.

The unique advantages of using the hybridoma
technology for therapeutic antibody development

Antibody production in hybridomas is one of the most successful approaches for the discovery of therapeutic antibodies. These hybridoma-derived
drugs have inherently higher stabilities, better developability profiles, and improved affinity in comparison to their in vitro generated counterparts.

Part of what makes these antibodies so successful is the cost-effective process of in vivo affinity maturation. This process ensures that the selection of the best binders from a naïve repertoire and optimization of their affinity through extensive recombination is performed in a naturally effective way, forgoing the need for costly in vitro affinity maturation processes.

These antibodies have been successfully used for the treatment of cancer, autoimmune diseases, rare diseases, and infectious diseases, among others. Moreover, they can serve as support tools for measuring the clinical efficiency of therapeutic antibodies (anti-idiotypic antibodies).