Recombinant Expression Strategies for Bispecific Antibodies: What You Need to Know？

In recent years, bispecific antibodies (BsAbs) have emerged as a major focus in the therapeutic antibody field. By simultaneously binding two distinct targets or two different epitopes on the same target, BsAbs can achieve biological functions unattainable by conventional monoclonal antibodies—such as bridging immune cells to tumor cells or blocking dual signaling pathways. This "bispecificity" endows BsAbs with significant therapeutic advantages, particularly in oncology. However, their manufacturing processes remain complex, primarily due to challenges in chain pairing. This article discusses key strategies for addressing chain-pairing issues, improving expression efficiency, and optimizing downstream purification.

1. The Core Challenge in BsAbs Recombinant Expression: Chain Pairing

The primary technical hurdle in BsAbs recombinant expression lies in the precise control of chain assembly. A classical IgG-like BsAbs comprises two distinct heavy chains (HCs) and two distinct light chains (LCs). When these genes are co-transfected into host cells (e.g., CHO or HEK293), up to ten different IgG combinations can theoretically be generated, of which only one is the desired bispecific construct. Random mispairing of light chains with heavy chains, alongside heavy-chain homodimerization, often results in an extremely low assembly yield of the target product—approximately 25% under conventional methods. This generates a plethora of difficult-to-separate byproducts, significantly increasing downstream purification complexity and cost.

2. Optimization Strategies for BsAbs Recombinant Expression

(1) Heavy-Chain Pairing: The "Knobs-into-Holes" Strategy

Knobs-into-Holes remains one of the most classic technologies for resolving heavy-chain mispairing. By introducing spatially complementary "knob" and "hole" mutations into the Fc regions of the two distinct heavy chains, steric hindrance effects compel preferential heterodimerization over homodimerization. This approach effectively enhances the assembly efficiency of the desired BsAbs.

图1 Knobs-into-Holes设计原理图.png

Figure 1. Schematic representation of the Knobs-into-Holes design principle.

(2) Light-Chain Pairing: The "Common Light Chain" Strategy

Light-chain mispairing poses a more intractable challenge due to the high specificity of LC–HC variable-region interactions. The Common Light Chain approach offers a robust solution: by genetically engineering antibody-transgenic mice or designing molecules that share an identical light chain across all candidates, one can screen for heavy chains specific to two different targets and pair them with the common LC. This strategy intrinsically eliminates light-chain mispairing from the outset. Platform data indicate that this approach can increase correct BsAbs assembly rates from approximately 25% (traditional methods) to over 95%, substantially shortening the R&D timeline.

(3) Single-Vector Bidirectional Promoter Systems: Optimizing Expression Efficiency

Beyond molecular design, expression vector architecture itself offers room for optimization. Recent studies have reported a single-vector system featuring a bidirectional CMV promoter (e.g., peaSKY). Compared to traditional dual-vector co-transfection, this system eliminates redundant backbone sequences, increasing the expression cassette proportion by 17% and enabling synchronized transcription of heavy and light chains—thereby facilitating proper protein folding and assembly. In transient expression assays, this system yielded a two-fold increase in BsAbs titers relative to the dual-vector system.

(4) Purification Innovations: Alkali-Resistant Protein L

Even with enhanced engineering to increase target product ratios, efficient purification remains critical for process feasibility. Wakabayashi and Kuramochi highlighted recent advances in affinity chromatography, particularly the development of alkali-resistant Protein L resins. Protein L binds to the κ variable region of antibody light chains, offering unique advantages for purifying fragment-type BsAbs that lack an Fc region, or as a complementary step to Protein A affinity chromatography. The alkali-resistant properties enable robust clean-in-place (CIP) protocols, significantly improving process economics and operational convenience.

3. Concluding Remarks

Strategies for recombinant expression of BsAbs—whether Knobs-into-Holes, Common Light Chain platforms, optimized expression vectors, or novel chromatographic media—all converge on a single goal: navigating the complexity of chain pairing to achieve high-purity, high-yield, and high-quality pharmaceutical products. Concurrently, continued advances in antibody genetic engineering offer diverse possibilities, enabling efficient production of complex molecules and the incorporation of novel functionalities into antibodies.

About the Author/Company

Tek Biotech (Tianjin) Co., Ltd. brings years of project experience and expertise in bispecific antibody development. Leveraging phage display and yeast display technologies, the company has established a comprehensive early-stage antibody discovery platform. Tailored to specific project requirements, Takeda Biotechnology designs customized BsAbs structures and expression/purification strategies, and utilizes a robust mammalian expression and fermentation platform to deliver high-quality bispecific antibody products that meet diverse client needs.

References

[1] Carter, P. J., & Rajpal, A. (2022). Designing antibodies as therapeutics. Cell, 185(15), 2789–2805.

[2] Brinkmann, U., & Kontermann, R. E. (2017). The making of bispecific antibodies. mAbs, 9(2), 182–212.

[3] Wakabayashi, T., & Kuramochi, T. (2025). Discovery and development of bispecific antibodies. Protein Expression and Purification, Volume 236, 2025,106787, ISSN 1046-5928.

[4] Merchant, A., Zhu, Z., Yuan, J. et al. An efficient route to human bispecific IgG. Nat Biotechnol 16, 677–681 (1998).

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