ProteoGenix gives you access to the best expertise in bispecific antibody development: 3 therapeutic antibodies on the market, 300+ monoclonal antibodies successfully developed, scientists having more than 25 years experience in therapeutic antibody development. Choose the best knowledge in bispecific antibody format generation to build your blockbuster.

ProteoGenix’ Bispecific Antibody Production process

Project study

Bispecific antibody format definition

Bispecific antibody design

Gene synthesis

Bispecific antibody production

Bispecific antibody potency assessment

ProteoGenix’s Bispecific Antibody Production Process

ProteoGenix offers the best-in-class one-stop solution for your bispecific antibody (Bsab) development project. Get access to:

  • The expertise of renowned scientists with more than 25 years of experience in the development of therapeutic monoclonal antibodies.
  • Experts which have successfully developed more than 300 monoclonal antibodies in 15 years, including 3 therapeutic monoclonal antibodies currently on the market.

Send us your requirements, our team:

  • Defines the most accurate format for your application and for further pharmaceutical production (“developability”).
  • Adapts the available format to your antibody sequences.
  • Expresses and purifies complex bispecific antibody formats.
  • Optimizes and characterizes the best bispecific antibody formats.

Our complete bispecific antibody development service includes:

  • Your optimized and purified bispecific antibody.
  • A complete report with a description of the experimental steps, results, comments and conclusion.
  • Optional: an optimized bispecific antibody production process directly transferrable to your CMO (parameters optimized includes: HC/LC ratio, expression system, linkers and many others depending on the project).
  • All the property rights of the work done by ProteoGenix is transferred to the client.

Choosing ProteoGenix also means getting access to our best-in-class proprietary cell culture line XtenCHO™. Why best-in-class? Because it is the only cell line that has already overperformed the ExpiCHO cell line, the reference on the market, for the transient expression of therapeutic antibodies such as Pembrolizumab and many others (yields of up to 800 mg/L)..

Unleash the potential of your project by choosing ProteoGenix as your privileged partner!

How much does bispecific antibody production cost?

ProteoGenix offers a comprehensive bispecific antibody production service with a flexible pricing structure to accommodate a wide range of projects. Our prices start at a few thousand euros while the specific cost varies depending on several factors. The chosen bispecific antibody format plays a significant role in determining the price, as each format may require different production processes and resources.

Additionally, the overall product volumes required for the project influence the pricing, as larger-scale production may involve higher costs. To further tailor the service to meet individual customer needs, ProteoGenix offers various options that can be added to the production process. These additional options may include specific modifications or customizations, which contribute to the overall pricing.

Our goal is to provide customers with the best value for their investment while ensuring high-quality bispecific antibody production. By offering a range of pricing options and personalized solutions, ProteoGenix aims to support researchers and industry professionals in achieving their project objectives efficiently and cost-effectively.

How long does it take to produce a bispecific antibody?

The time required to produce a bispecific antibody can vary based on multiple factors, including the complexity of the desired format, antibody production, optimization, quality control, and specific project requirements of the customer.

At ProteoGenix, the entire process of bispecific antibody production typically spans from 3 to 4 weeks. This timeline encompasses crucial stages such as the project study, bispecific antibody format definition and design, engineering phase, actual production, and comprehensive quality control measures. Our efficient and streamlined workflow ensures the timely delivery of high-quality bispecific antibodies, meeting the diverse needs of our customers with precision and reliability.

How to assess the bispecific antibody potency

Measuring the potency of Bispecific antibodies (BsAbs) can be challenging due to their unique properties. However, several tests can aid customers in identifying the most effective BsAbs for their specific needs.
bispecific antibody (Bsab) development
To measure antibody affinity, ELISA (Enzyme-Linked Immunosorbent Assay) and KD (Dissociation Constant) determination are employed. ELISA allows the quantification of BsAb binding to specific antigens, providing valuable information about its binding strength. KD determination calculates the equilibrium dissociation constant, offering insights into the strength of the antibody-antigen interaction.

To assess solubility and aggregation, SEC-HPLC (Size-Exclusion Chromatography – High-Performance Liquid Chromatography) is used. This method separates BsAbs based on size, allowing the identification of any aggregated or insoluble forms.

DSL (Differential Scanning Calorimetry) is utilized to check BsAb thermostability. By measuring the heat-induced unfolding, this technique provides valuable data on the stability of the antibody structure.

Avidity, which refers to the cumulative strength of multiple interactions between BsAbs and antigens, is another crucial parameter. Specific assays, such as Surface Plasmon Resonance (SPR) or Biacore, are used to evaluate avidity.

By employing these tests and methodologies, customers can gain a comprehensive understanding of the potency and performance of Bispecific antibodies, enabling them to make informed decisions for their research or therapeutic development projects.

Overview of Bispecific Antibodies and their Significance in Target Therapy

Bispecific antibodies as powerful tools in cancer immunotherapies

Antibodies are one of the fastest growing classes of therapeutics mainly because they have demonstrated their ability to improve the treatments of several complex diseases such as cancer or inflammatory disorders.
Bispecific antibodies have revolutionized the field of medicine by offering a versatile and precise approach to targeting multiple disease pathways simultaneously. These innovative molecules are designed to bind to two distinct targets, such as two different proteins or cells, leading to enhanced therapeutic efficacy.

One of the most exciting applications of bispecific antibodies lies in their ability to treat patients with cancer. By simultaneously engaging both cancer cells and immune cells, these antibodies can orchestrate a powerful immune response against tumors, boosting the body’s natural defenses.

This targeted approach holds immense promise for treating various types of cancers, offering new hope to patients and potentially transforming the landscape of oncology.

To date, three bispecific antibodies were approved for clinical use:

    • Catumaxomab (REMOVAB®) is a T-cell recruiter developed by Trion Pharma which binds EpCAM and CD3. Catumaxomab is a nonhuman bispecific IgG-like rat/mouse antibody produced using the quadroma technology which was indicated as a treatment for malignant ascites (discontinued in 2014).
    • Blinatumomab (BLINCYTO®) is a BiTE developed by Amgen and Micromet. It is a T-cell recruiter which targets CD3 positive T-cell and CD19 positive B-cells. Blinatumomab is indicated for the treatment of acute lymphoblastic leukemia.
    • Emicizumab (HEMLIBRA®) is a therapeutic antibody developed by Roche and Chugai. This bispecific antibody induces the dimerization of its two targets (Factor IX and Factor X) in order to reactivate the latter. Emicizumab is indicated for the treatment of hemophilia A.

The most common application of bispecific antibodies relies on retargeting effector cells to tumor cells for cancer therapy. BiTE (bispecific T-cell Engager) is an example of format (developed by Micromet) based on this application. BiTE can be considered as a scFv tandem which targets a T-cell specific molecule, on the one hand, and a tumor specific antigen on the other hand, to improve patient’s immune response.

Bispecific antibodies can also overcome other limitations of monospecific antibodies. An example is their inability to cross the blood-brain barrier as the low permeability of the BBB hinders the development of antibodies which target an antigen in the CNS. A strategy currently discussed to cross the BBB consists of engineering bispecific antibodies that recognize a BBB receptor (to induce transcystosis) and the target within the CNS.

Due to their high diversity, bispecific antibodies can present many other therapeutic applications such as:

  • Inhibiting two pathways at the same time.
  • Enhancing antigen binding affinity.
  • Increasing target specifity.
  • And even more…

Bispecific Antibody Production; Beyond Cancer Immunotherapies

In addition to their promising role in cancer treatment, bispecific antibodies have shown immense potential in various clinical applications across a range of diseases. Here are a few notable examples:

● Autoimmune Disorders: Bispecific monoclonal antibodies can be developed to simultaneously bind to specific immune cells and target proteins involved in autoimmune disorders. By redirecting immune responses, these antibodies can help to treat patients with aberrant immune activity and reduce autoimmune-related inflammation.

● Infectious Diseases: Bispecific antibody development has been explored as a potential therapy for infectious diseases caused by viruses, bacteria, and other pathogens. They can be engineered to bind to viral or microbial antigens as well as immune cells, facilitating targeted neutralization of pathogens and enhancing the immune system’s ability to clear infections.

● Inflammatory Disorders: Bispecific monoclonal antibodies can target specific pro-inflammatory cytokines or receptors involved in chronic inflammatory conditions like rheumatoid arthritis, inflammatory bowel disease, and psoriasis. By blocking inflammatory signals and modulating immune responses, they have the potential to alleviate symptoms and improve patient outcomes.

● Hematological Disorders: Bispecific antibody development has been explored as therapeutic option for hematological malignancies, such as lymphomas and leukemias. They can be engineered to simultaneously target cancer cells and immune effector cells, activating the immune system’s anti-tumor response and improving the specificity of treatment.

These examples highlight the versatility of bispecific antibodies as a promising class of therapeutic agents, with the potential to transform the treatment landscape across a wide range of diseases.

How Bispecific antibodies can simultaneously bind to 2 different targets?

Bispecific antibodies possess a remarkable capability to simultaneously bind to two or more distinct targets, making them potent tools for diverse research and therapeutic applications. This unique functionality arises from their specialized design, where two different antigen-binding sites are incorporated within a single antibody molecule.

The most common approach to achieving bispecificity involves combining two different monoclonal antibodies into a single entity. By engineering hybrid antibodies, each equipped with its own binding specificity, researchers can effectively create Bispecific antibodies with the ability to target multiple antigens simultaneously.
Another strategy to confer bispecificity involves fusing together single variable domains from two separate antibodies. This fusion results in a single molecule with dual antigen-binding capabilities, allowing for simultaneous recognition of multiple targets.

Moreover, advances in antibody engineering technologies have led to the creation of innovative formats like CrossMAb and DuoBody, which enable controlled Fab arm exchange or separate expression of specific mutations to generate Bispecific antibodies efficiently.

Overall, the ability of Bispecific antibodies to target two or more different antigens makes them indispensable tools in a wide range of applications, including cancer immunotherapy, infectious disease research, and autoimmune disorder treatment, offering significant potential for advancements in biology and medicine.

Bispecific antibody engineering:
get access to the largest variety of structures

Bispecific antibodies (Bsabs) were initially produced using the quadroma method (hybrid hybridoma). This technique allows the production of antibodies in a single host cell but leads to a mixture of tailored, non-functional, and homodimer compounds. Therefore, producing Bsabs with sufficiently high yields to reach bioproduction was a real challenge due to heavy chain and light chain mispairing.

Recent engineering methods and recombinant antibody production have allowed the development of several strategies to address this problem, for example:

  • The heavy chain mispairing challenge was overcome in the 90s thanks to the engineering of the CH3 domains which controls heavy chain dimerization. Several strategies were employed to force antibody heavy chain heterodimerization over homodimerization such as the well-known “knob-into-hole” approach or electrostatic steering mutations.
  • To avoid light chain mispairing, several approaches have also been developed over the last decade. One of these consists circumventing the issue by using identical light chains. Other examples of the strategy include the separate expression of monoclonal antibodies before assembly in a single-host cell (Duobody, Genmab) or rearrangement of the heterodimerization interface by swapping between a heavy and a light chain domain (CrossMAb, Roche).

The capacity to assemble a large variety of building blocks opens the door to an infinite number of bispecific antibody formats. Controlling the various engineering technics means gaining access to the capacity to perfectly modulate the pharmacological properties of the desired compound and thus develop your blockbuster. For this reason, choosing a company with more than 15 years of experience in antibody engineering like ProteoGenix ensures the success of your project. Our scientific experts have already overcome the most difficult engineering challenges, that’s why it’s time to put yours in safe hands!

How to choose a bispecific antibody format?

Selecting the right Bispecific antibody (BsAb) format is a critical decision in research and therapeutic development.
At ProteoGenix, we offer two distinct options:
● IP-free and
● patented BsAbs,
These options provide our customers with the freedom to choose the most suitable intellectual property arrangement for their needs. Our extensive portfolio also encompasses a wide variety of BsAb formats, offering design flexibility.

When deciding on a Bispecific antibody format, several essential parameters come into play. These include
● The presence of the Fc region,
● BsAb symmetry
● BsAb specificity
● BsAb length
● number of BsAb binding sites
● BsAb immunogenicity
● BsAb half-life

It’s essential to recognize that a single format cannot be universally applicable to all research or therapeutic needs. Hence, different formats should be systematically evaluated within the specific application of interest to ensure optimal performance and efficacy.

At ProteoGenix, our expert team collaborates closely with customers to understand their unique requirements and guide them in selecting the most appropriate Bispecific antibody format for their projects. By offering diverse options and comprehensive support, we empower researchers and developers to make informed decisions that drive success and innovation in their pursuits.

Bispecific antibody formats

BiTE antibody format


Diabody bispecific antibody format


DART bsMAb format


Bispecific IgG and IgG-like

CrossMab bsAb format


Κλ-body bispecific antibody format


Duobody (Fab arm exchange)


Appended IgG

DVD-Ig antibody format


IgG(H)-scFv bsMAb format


scFv-(L)IgG bsAb format


Fusion protein

HSAbody bispecific antibody format


Dock and lock antibody format

Dock and Lock

F(ab’)2 antibody format


Bispecific antibody conjugates

SPRi KD determination


Cov-X-Body bispecific antibody format


F(ab’)2 antibody format


Crossmab: Bispecific IgG-like

CrossMAb is a bispecific IgG format developed by Roche to overcome the LC/HC mispairing. This technology is based on the exchanging of the light chain and heavy chain domain within a unique Fab arm to induce specific interaction while maintaining the original antigen binding affinity.

CrossMAb can be combined with the KiH approach to control both HC/HC and LC/HC pairing.

As a bispecific IgG, CrossMAb combines the advantages of IgG (long pharmacokinetic in vivo half-life, effector functions) and bispecific antibodies.

DuoBody: Bispecific IgG-like

The DuoBody platform is a technology invented by Genmab based on a controlled Fab arm exchange. This technique is based on the separate expression of two IgG1, each of them containing a single matched mutation in the CH3 domain. The antibodies obtained are further mixed and separated into half molecules under reducing conditions. Single point mutations introduced in each Fab arm, coupled with oxidizing conditions, control the reassembly of half molecules leading to yields of bispecific IgG of up to 95%.

Like CrossMAb, Duobody combines the advantages of IgG and bispecific antibodies.

DART: Bispecific Antibody Fragment

The DART format can be classified as a bispecific antibody fragment. DART is a diabody composed of the variable domains of two different antibodies. In this format, the variable heavy domain of the first antibody is connected via a short peptide linker to the variable light domain of the second antibody and vice versa leading to two scFv. Generation of DART is obtained by further stabilization via disulfide bond.

Like most small formats, DART are characterized by a high potency but a short serum half-life but can be further engineered to overcome this limitation, for example by fusion to a Fc region.

Nanobody: Bispecific Antibody Fragment

Nanobodies correspond to single domain antibody fragments which can be obtained from various species, such as llama or alpaca. Bispecificity can be obtained by connecting two nanobodies via short linkers.

The high-potential of nanobodies comes from their small size which permits antigen access that would not be sterically accessible to other antibodies.

Like DART, bispecific nanobodies are characterized by a high potency but a short half-life which can be overcome by fusional connection to a protein such as human serum albumin (HSA).

DVD-Ig: Appended IgG

DVD-Ig (dual variable domain-Ig) is part of the appended IgG family and was developed by Abbott. Appended IgG correspond to monoclonal IgG that have been further engineered to introduce bispecifity by addition of antigen binding sites at the N- or C- termini of either light chains or heavy chains. DVD-Ig are obtained by appending VL and VH domain of an antibody to an IgG and results in a bispecific and bivalent antibody for each antigen.

κλ-body: Bispecific IgG

κλ-body is a fully-human bispecific IgG recently developed by Novimmune to solve the LC/HC mispairing issue. This format is characterized by a common heavy chain coupled with two different light chains κ and λ governing the antigen-binding specificity. The process developed by Novimmune is based on the selection of light chains via phage display.

SEEDbody: Bispecific IgG-like

SEEDbody is a bispecific antibody format developed by Merck Serono which overcomes the HC/HC mispairing issue. The SEED method is based on the alternation of human IgG and IgA sequences in the CH3 domain in order to favor the heterodimerization.

SEEDbody is a bispecific IgG and thus combines the advantages of IgG and bispecific antibodies.

Triomab: Bispecific IgG-like

Triomab is a format developed by Trion Pharma and was the first bispecific antibody approved on the market for therapeutic application. Triomab is based on the quadroma production technique which is generally associated with HC/HC and LC/HC mispairing problems. Trion Pharma overcame both of these issues by generating a mouse/rat hybrid hybridoma.

  • LC/HC mispairing was solved by combining mouse IgG2a and rat IgG2b.
  • HC/HC mispairing was solved during the purification steps as mouse IgG2a have strong affinity for protein A on the contrary to rat IgG2b.

Bispecific antibody development services that will meet your expectations and surpass your expectations!

At ProteoGenix, we know that bispecific antibody engineering and design is only one step of the road to success. For this reason, we attach particular importance to guiding you from the development of your bispecific antibody through to its bioproduction. ProteoGenix has an extensive experience in the development of stable cell lines reaching high production yields and no unwanted side products. With ProteoGenix, you can rest assured that our scientists will be fully committed to converting your ideas into a blockbuster.