Bispecific antibodies (bsAbs) are engineered immunoglobulins capable of simultaneously binding two distinct antigens or epitopes, offering unique mechanistic advantages over conventional monoclonal antibodies. This dual specificity enables bsAbs to mediate functions such as redirected cytotoxicity, dual-pathway inhibition, or targeted delivery, depending on their format and therapeutic context.
The bispecific market size is estimated at about $12 billion in 2024 and is expected to balloon further to $50 billion by 2030 based on current projections. This rapid growth is fueled by clinical uptake for bispecific antibodies, first-in-class drugs in development, and expanding opportunities in important territories such as the US and China.1
The development of bsAbs involves technical challenges that are different from traditional monoclonal antibody (mAb) production. These include selecting the right format, preventing heavy and light chain mispairing, reducing aggregation, improving expression yields, and managing complex downstream processing—all of which stem from the structure of BsAbs, especially their heterodimeric configurations.
At Biointron, we turn these challenges into strengths. Our experienced team applies proven design strategies, optimized expression systems, and robust purification workflows to deliver high-quality bsAbs efficiently and reliably.
Format Selection and Its Implications for Expression
BsAbs can be engineered in numerous formats, each with different structural, functional, and manufacturing profiles. Selecting the right format is necessary to balancing pharmacokinetics, manufacturability, and therapeutic efficacy. Below are some of the most widely adopted bispecific formats in drug development today:
IgG-like formats (e.g., CrossMab, DuoBody):
These resemble conventional IgG antibodies and retain the Fc region, which imparts several key advantages, including prolonged serum half-life via neonatal Fc receptor (FcRn) recycling and engagement of immune effector functions such as ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). They offer advantageous properties in stability, immunogenicity, ease of purification, production yield, and compatibility with standard antibody manufacturing platforms.
For example, teclistamab by Johnson & Johnson, is a BCMAxCD3 DuoBody that has been marketed for relapsed and/or refractory (R/R) multiple myeloma (MM) treatment. It was generated by using the Genmab DuoBody technology and contains the S228P/L234A/L235A mutations in the Fc region to stabilize and minimize its immunological effector functions.2
Fragment-based formats (e.g., BiTEs – Bispecific T-cell Engagers, DARTs – Dual-Affinity Re-Targeting molecules):
These formats are constructed from antibody fragments (e.g., single-chain variable fragments or scFvs) and typically lack an Fc region, resulting in smaller molecular size and enhanced tissue penetration. Their smaller size also enables faster clearance, which can be beneficial or limiting depending on the clinical application. BiTEs, for example, are designed to bring cytotoxic T cells into close proximity with tumor cells, facilitating direct cell killing. Due to their unique mechanism of action and structural simplicity, they are often expressed in bacterial or mammalian systems and require continuous infusion to maintain efficacy in vivo.
For example, blinatumomab by Amgen, is a CD19/CD3 BiTE of two linked, independent scFvs, approved for treatment of B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) and more later on.3
Appended IgG Formats (e.g., DVD-Ig, Triomab, HC/LC Fusion Constructs):
Appended IgGs are a category of bispecific antibodies designed to improve valency, binding diversity, and biological function. These formats can be subclassified based on fusion site:
- HC Fusions: Appending variable domains or whole Fab fragments to the heavy chain allows for tri- or tetravalent binding while maintaining Fc-mediated functions (e.g., Triomab, Fab-scFv fusions).
- LC Fusions: Appending domains to the light chain can provide alternative orientation and valency configurations.
- HC & LC Fusions Combined: Formats like DVD-Igs have dual variable domains on each Fab arm, allowing for simultaneous binding to two targets for each arm, thus improving functional avidity and target engagement.
Mispairing and Aggregation in BsAb Production
Mispairing refers to the incorrect assembly of heavy and light chains, leading to product heterogeneity and reduced functional activity. Strategies to address this include:
- Heavy chain engineering, such as Knobs-into-holes (KiH) mutations, to promote heterodimer formation.
- Common light chain technology that uses a single light chain compatible with both heavy chains, while maintaining the natural molecular shape of the antibody.
- CrossMab technology, which involves Fab domain swapping to enforce correct pairing.
- Orthogonal Fab interfaces that introduce mutation between Fab interface to enhance correct heavy-light chain pairing.
Biointron’s Technical Solutions: High-Yield, High-Purity Bispecific Antibodies—Fast
With over 50,000 multispecific antibodies delivered annually across 400 unique formats, Biointron stands at the forefront of bispecific antibody (BsAb) production. Our proven process, from gene synthesis to delivery, ensures speed, precision, and reliability, even for structurally complex constructs.
Key strengths of our service include:
- All Formats Covered: Any bispecific format—IgG-scFv, BiTE, DVD-Ig, and more—can be expressed
- Customization Solution: Personalized approach tailored to your research goals.
- Efficient Turnaround: From sequence submission to purified bsAbs in as fast as 2–3 weeks.
- Scale with Speed: Transient transfection for scaled-up production (up to 200 L), stable line construction is not a necessity.
- Optimized Expression: Codon optimization and high-expression vector systems support exceptional yields (e.g., >250 mg/L), even for challenging constructs.
- Streamlined Purification & QC: One-step affinity purification combined with stringent QC (SDS-PAGE >95%, SEC-HPLC purity >96%) ensures bioactivity and batch consistency.
- High-Throughput & Automation: Our automated platforms and scalable production capabilities allow rapid handling of both small and large projects without compromising quality.
From initial construct to purified product, Biointron's bispecific antibody production service is designed to eliminate common challenges—such as mispairing and aggregation—so you can focus on what matters most: advancing your therapeutic pipeline with confidence.
Learn more about antibody production:
Visit Biointron’s Learning Center or contact our expert team at info@biointron.com or +1(732)790-8340.
References:
- KuicK Research. (2025, March 17). Bispecific Antibodies Market Size Approved Bispecific Antibody Sales Price Patent Market Forecast 2030. GlobeNewswire News Room; KuicK Research. https://www.globenewswire.com/news-release/2025/03/17/3043524/0/en/Bispecific-Antibodies-Market-Size-Approved-Bispecific-Antibody-Sales-Price-Patent-Market-Forecast-2030.html
- Pillarisetti, K., Powers, G., Luistro, L., Babich, A., Baldwin, E., Li, Y., Zhang, X., Mendonça, M., Majewski, N., Nanjunda, R., Chin, D., Packman, K., Elsayed, Y., Attar, R., & Gaudet, F. (2020). Teclistamab is an active T cell–redirecting bispecific antibody against B-cell maturation antigen for multiple myeloma. Blood Advances, 4(18), 4538. https://doi.org/10.1182/bloodadvances.2020002393
- Burt, R., Warcel, D., & Fielding, A. K. (2018). Blinatumomab, a bispecific B-cell and T-cell engaging antibody, in the treatment of B-cell malignancies. Human Vaccines & Immunotherapeutics, 15(3), 594. https://doi.org/10.1080/21645515.2018.1540828