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Custom Bispecific Antibody Production Tailored to Your Research Needs
Biointron2025-03-31Read time: 8 mins
Bispecific antibodies (bsAbs) have emerged as powerful therapeutic and research tools due to their ability to simultaneously bind two distinct antigens or epitopes. This dual specificity enables novel mechanisms of action, such as redirecting immune cells to tumor sites, crosslinking receptors for enhanced signaling modulation, or selectively engaging target cells based on multiple surface markers. As the applications of bsAbs expand, the need for highly customized and scalable production services becomes critical for biotech firms seeking to translate bsAb constructs into clinical and preclinical settings.
This article outlines the strategic considerations and technologies involved in custom bispecific antibody production and how specialized CRO services—such as Biointron’s bispecific antibody expression platform—can support your research and development pipeline.
Choosing the Right Bispecific Format
The design of a bispecific antibody must align with its intended biological function, expression system, and downstream application. Common bsAb formats include:
IgG-like formats: Incorporating full-length Fc regions, such as the dual-variable domain IgG, CrossMab, and knob-into-hole (KiH) technologies.
Fragment-based formats: Lacking the Fc region, such as BiTEs (bispecific T-cell engagers), DARTs (dual-affinity re-targeting molecules), and diabodies.
Each format presents specific advantages and manufacturing challenges. IgG-like bsAbs benefit from established Fc-mediated effector functions and extended half-life, but may suffer from chain mispairing during expression. Fragment-based formats avoid mispairing but generally have shorter serum half-lives and may require conjugation to half-life extension moieties or PEGylation.
Biointron's bispecific antibody development services support a wide range of bsAb formats, offering platform flexibility based on target biology and production goals.
A major technical hurdle in bsAb development is the correct pairing of heavy and light chains to ensure proper assembly and function. Mispaired chains can result in non-functional antibodies, reducing yield and complicating purification.
Several engineering strategies are employed to promote correct chain pairing:
Knob-into-hole (KiH) mutations: Introduce steric complementarity into the CH3 domains of the heavy chains, promoting heterodimerization.
CrossMab designs: Swap domains between the light and heavy chains to force correct pairing.
Biointron’s antibody engineering platform incorporates these strategies to enhance bsAb structural fidelity during the expression phase. Coupled with high-throughput screening, clients can assess multiple designs in parallel.
Heterodimerization of light and heavy chains. DOI: 10.1016/j.trecan.2024.07.002
Expression Systems and Yield Optimization
Production of bispecific antibodies requires expression platforms capable of high yield and product homogeneity. Mammalian expression systems, particularly CHO and HEK293 cells, are preferred for their ability to produce correctly folded, glycosylated antibodies with consistent quality.
Key variables optimized during bsAb expression include:
Transient vs. stable expression: Transient systems are suitable for rapid prototyping and screening, while stable clones are essential for scale-up and GMP manufacturing.
Codon optimization and gene synthesis: Improves transcription efficiency and protein translation.
Expression vector configuration: Dual-promoter systems, single-chain constructs, or 2A peptide-based polycistronic designs can be selected based on bsAb architecture.
Biointron’s CHO-based expression system for bispecific antibodies enables fast turnaround for transient expression, with scalable production up to gram quantities in serum-free suspension cultures.
Due to their structural complexity, bsAbs often require tailored purification protocols to achieve sufficient purity and activity. The choice of purification approach depends on the bsAb format and the presence or absence of Fc domains.
Standard purification steps include:
Protein A chromatography: Applicable to Fc-containing bsAbs for initial capture.
Ion exchange chromatography (IEX): Used to separate charge variants and remove aggregates or mispaired species.
Size exclusion chromatography (SEC): Assesses aggregation and final polishing.
Affinity chromatography with antigen-specific resins: Useful for confirming dual specificity or removing misassembled products.
Biointron’s downstream purification workflows for bispecific antibodies are optimized for high recovery rates and low impurity profiles, supporting both research-grade and preclinical-grade material production.
Analytical Characterization of Functional BsAbs
Robust characterization is essential for confirming the structural integrity, specificity, and functional potency of bispecific antibodies. A comprehensive analytical suite includes:
SDS-PAGE and Western blot: Confirm correct molecular weight and chain expression.
SEC-HPLC and DLS: Evaluate aggregation and homogeneity.
Mass spectrometry: Determines intact mass and verifies post-translational modifications.
SPR/BLI assays: Measure binding kinetics to each antigen independently.
Cell-based assays: Quantify biological activity, such as cytotoxicity, receptor blocking, or T-cell activation.
Supporting Therapeutic Modalities
Bispecific antibodies are central to a range of therapeutic strategies:
Immune cell redirection: T-cell engagers (e.g., anti-CD3 x tumor antigen) have shown clinical success in hematologic malignancies.
Dual receptor blockade: In oncology and immunology, simultaneous inhibition of two signaling pathways can overcome resistance mechanisms.
Tissue-specific targeting: One arm of the bsAb binds a tissue-restricted marker, enhancing specificity and reducing off-target effects.
Half-life modulation: Incorporation of FcRn-binding domains or fusion to albumin-binding moieties can optimize pharmacokinetics.
Biointron’s bsAb expression services are compatible with custom designs for targeted immune engagement, receptor antagonism, or half-life extension strategies.
DOI: 10.1016/j.apsb.2024.03.027
T-cell immunotherapy continues to advance with three main strategies: immune checkpoint inhibitors (ICIs), CAR-T cell therapy, and T-cell redirecting bispecific antibodies (BsAbs). Among them, BsAbs offer a promising off-the-shelf alternative by directly engaging T cells with tumor cells, inducing rapid cytotoxicity. Unlike ICIs, which broadly release immune inhibition and may cause off-target effects, BsAbs provide more targeted activity. Compared to CAR-T therapies, BsAbs are more scalable and cost-effective, avoiding complex patient-specific manufacturing. However, BsAb development in solid tumors faces challenges, including cytokine release syndrome (CRS), antigen escape, limited T-cell infiltration, and immunosuppressive tumor microenvironments (TMEs). CRS, often driven by TNF-α secretion, can be mitigated by using low-affinity anti-CD3 arms or co-administering cytokine inhibitors like IL-1β and IL-6 blockers.
Many BsAbs target differentiation antigens (e.g., CEA, EGFR, EpCAM, HER2), which are also expressed in normal tissues, narrowing the therapeutic window. To improve tumor specificity, strategies such as dual- or tri-specific targeting and conditional activation—via pH-sensitive designs or enzymatic masking—are being employed. For example, pH-dependent antibodies show selective binding in acidic TMEs, enhancing tumor targeting while reducing off-target toxicity. Additionally, T-cell exhaustion in the TME remains a major barrier, often requiring combination therapies. Current approaches integrate BsAbs with ICIs, immune agonists, or oncolytic viruses to restore T-cell function and enhance intratumoral killing. As clinical trials expand, deeper insights into BsAb structure–function relationships will guide the development of more effective, durable, and safe T-cell redirecting therapies.
Customization for Research and Screening Applications
Beyond therapeutics, bispecific antibodies serve as versatile tools for basic and translational research:
Bivalent labeling reagents: Enable co-localization studies in imaging applications.
Signal modulation: Bispecifics targeting receptor-ligand interactions are useful for dissecting signaling pathways.
Diagnostic agents: Dual-binding capability increases specificity in ELISA or flow cytometry.
Biointron offers rapid production of research-grade bispecific antibodies with options for custom tags, Fc modifications, and conjugation handles for flexible downstream use.
Intellectual Property Considerations
Given the proprietary nature of many bsAb platforms, companies must navigate IP constraints when selecting formats for custom development. A qualified CRO can assist in freedom-to-operate analyses, designing around patented formats, and identifying licensable scaffold technologies.
