Bispecific antibodies (BsAbs) have emerged as one of the most dynamic and promising segments in biotherapeutic development, marked by a striking upward trend in regulatory approvals and industry investment. In 2024, the FDA’s CDER approved 16 novel biologics, and BsAbs accounted for ~17% of these—marking a huge leap from a decade ago when they were niche¹. Today, BsAbs rank as the FDA’s second-largest antibody-based therapeutic category, trailing only monoclonal antibodies (mAb).

Their commercial allure is strong. Grand View Research projects the global BsAb market to grow at a CAGR over 44% through 2030², thanks to their unique ability to target two molecules and drive synergistic effects—opening new options for refractory cancers and autoimmune diseases. A recent article in Nature highlighted them as accounting for a quarter of the top 20 highest-grossing deals in 2025, potentially marking the start of a new default modality³.

With hundreds of BsAb candidates in clinical trials, first-mover advantage is pivotal, while delayed therapies deprive patients with unmet needs. Thus, accelerating BsAb development—without sacrificing quality and safety—has become an urgent priority for the biopharma industry. To meet this challenge, it is essential to understand the unique hurdles that BsAbs inherently present.

The Intricate Challenges of BsAb Development

The surge in BsAb development is accompanied by inherent complexities that distinguish them sharply from mAbs and pose substantial barriers to efficient translation. Unlike mAbs, which rely on a single, homogeneous heavy-light chain pair, BsAbs need two distinct heavy and light chains, which randomly form mispaired byproducts. Even engineered solutions leave impurities, cutting target yields and complicating purification due to structural similarities between impurities and the desired molecules.

Yield and stability issues compound this, with BsAb titers sometimes lower than those of mAbs. Yield is reduced by structural constraints that cause protein degradation, aggregation or fragmentation, while product heterogeneity is common due to co-expressing multiple chains leading to mispairing challenges. This makes traditional mAb processes less effective; extra specialized steps are required. For example, as mispaired impurities may share biochemical traits with BsAbs, diverse BsAb formats need customized, more advanced techniques during downstream purification. Structural heterogeneity also makes them sensitive to manufacturing/storage conditions—pH, shear stress, or temperature shifts can induce aggregation.

This complexity translates to longer development timelines, higher manufacturing costs, and greater technical uncertainty—challenges that have limited the number of approved BsAbs relative to conventional modalities. Effective development therefore mandates an integrative approach blending molecular engineering, cell line development (CLD), manufacturing process optimization, comprehensive analytical controls, formulation and drug product development, as well as project management to ensure fast track development.

Optimizing Molecular Assembly and Stable Expression

Early-stage Research Pool Study

Producing high-quality BsAbs requires overcoming common issues that can reduce yields and increase immunogenicity risk, such as chain mispairing and homodimer formation. Early-stage “research pool” studies enable systematic tuning of key parameters, including signal peptides, codon usage, vector design, chain expression ratios, and host cell platforms, establishing a foundation for successful cell line generation.

Research pool studies usually take about 6 weeks. The optimal stable transfection condition can then be selected using a multi-parametric evaluation framework encompassing productivity, purity, signal peptide residue risk and mRNA splicing risk. Figure 1 illustrates a case study evaluating the effects of vector design, codon, signal peptide, and chain ratio on productivity and product quality attributes. This systematic analysis enables the identification of optimal conditions for stable transfection.

Meanwhile, concurrently developed analytical methods help ensure the early detection of critical quality attributes and key byproducts. This proactive approach enables early risk identification in BsAb programs through implementation of “gatekeeper” criteria at the stable transfection milestone, ultimately delivering cell lines with optimized performance while establishing essential quality control measures. Taken together, these concurrent steps can greatly accelerate early BsAb development.

Building on the foundation established by research pool studies, the choice of host cell platform plays a pivotal role in further boosting the efficiency and stability of BsAb production.

Advanced Host Cell Platforms

The WuXia cell line is our extensively adopted platform for BsAbs, utilized in the development of over 1,000 cell lines for clinical and commercial manufacturing applications. During the COVID-19 pandemic, we successfully accelerated CMC timelines for therapeutic antibodies to 6 months or less, demonstrating the feasibility of rapid biologics manufacturing under pandemic conditions. Building on this accelerated platform, we have refined the methodology for non-pandemic scenarios. Our current approach integrates stability-driven CLD with top clone bioreactor evaluation, enabling differentiated development pathways: 2.5-month CLD supports an accelerated 6-month CMC timeline, while the 4.5-month CLD supports a 10–12-month CMC timeline.

WuXi Biologics recently established a novel, robust Chinese hamster ovary (CHO) cell line, WuXia TrueSite, which leverages site-specific integration technology to rapidly develop stable cell lines with remarkable productivity. It has demonstrated considerable titer improvement and >99% stability, with less than 20% titer reduction after 60 population doubling levels (PDLs) (Figure 2). This means that cell line stability is no longer a rate-limiting step for final clone selection, and it may even be possible to eliminate the need for cell line stability studies from the critical path, directly accelerating development timelines. Specifically, WuXia TrueSite enables acceleration of Master Cell Bank (MCB) establishment to 9-10 weeks, cutting the overall conventional development timeline in half (Figure 3). To date, WuXia TrueSite has been applied to eight BsAb development programs, with an average pool titer of 6.5 g/L (range: 5.8–7.9g/L) and a monomeric purity of at least 90%.

With a robust and high-performing host cell platform secured, the next key focus is on optimizing cell culture conditions that maximize yield while preserving BsAb stability.

Tailoring Cell Culture to Boost Yield While Ensuring Stability

BsAbs present unique challenges in cell culture compared to traditional mAbs due to their potentially lower expression levels and the presence of heterogeneous molecular variants such as aggregates, fragments and chain mispairing. These complexities necessitate specially tailored cell culture strategies to maximize the yield of the desired product while ensuring robust process scale-up.

Early control of difficult-to-remove impurities in upstream processes lays a crucial foundation for downstream purification. Detailed characterization of molecular variants in the early stages provides in-depth insights into impurity profiles, thereby informing the rational design of cell culture processes and purification strategies. Employing diverse clone pools during pre-development allows efficient screening of culture parameters, which can be implemented in clone evaluation and cell culture process optimization to accelerate process development timelines, especially when integrated with high-throughput (HTP) bioreactor platforms.

Beyond traditional fed-batch (TFB) culture, intensified fed-batch and advanced continuous culture modes, such as WuXi Biologics’ proprietary ultra-intensified fed-batch platform WuXiUI and perfusion platform WuXiUP, further enable the clinical and commercial-scale manufacturing of BsAbs. WuXiUP achieves expression levels up to 30-80g/L for challenging BsAb molecules, representing a 5 to 20-fold increase in productivity compared to conventional fed batch. Benefiting from continuous culture and harvest mode, challenging molecules that are prone to aggregation or cleavage are released from stress in the culture environment, resulting in high monomer purity or integrity levels up to 98% (Figure 4).

Meanwhile, WuXiUI enables ultra-high cell density production in a fed-batch mode combining with 3-4 cycles of intermittent perfusion for refreshed cell culture environment and sustained cellular machinery for active product synthesis while maintaining a desirable product quality profile. Figure 5 shows a case exhibiting a threefold increase in BsAb upstream titers and improvements in product purity of 12% and 18%, as determined by SEC and CE-NR, respectively. WuXiUI has demonstrated broad applicability across diverse complex recombinant proteins, delivering up to a six-fold increase in upstream titers while enhancing key product quality attributes.

Collectively, these integrated cell culture strategies address the intrinsic complexities of BsAbs, facilitating efficient transition from clone selection through to commercial manufacturing.

Overcoming Downstream and Analytical Complexities

Tailored Downstream Processing

Despite various engineering strategies being employed to improve the correct pairing of heavy and light chains, BsAb purification is challenging and time-consuming because mispaired byproducts, unassembled chains/half molecules and high levels of aggregates cannot be completely avoided. In addition, different BsAb formats necessitate different downstream processes.

Our experience with over 150 BsAb projects reveals that some commonalities can be leveraged for greater efficiency. Although BsAbs differ in format, many share some of the same engineering strategies or key features, such as T cell receptor (TCR) constant domains, single-chain variable fragment (scFv), single-variable domain on heavy chain (VHH), common light chain, knobs-into-holes (KIH), or charge pairing. Experience gained from these common elements has enabled us to tailor downstream strategies that can be applied to new BsAb with similar designs. For example, the low isoelectric point of TCR in WuXibody design makes ion-exchange chromatography an effective strategy for removing the half antibody and homodimer⁴. ScFv typically causes 10-20% aggregates that can be efficiently removed by mixed-mode chromatography. VHH sometimes triggers truncated variants that can be removed through polishing steps. Charge pairing usually produces varying degrees of mispaired byproducts.

In addition, the capability of chromatographic techniques to remove various byproducts has been intensively investigated, helping to accelerate the development of the BsAb purification process, maximizing yield and purity (Table 1). In summary, by relying on our expertise in chromatography technology, and our database of over 150 BsAb projects, we can develop successful downstream processes in a short timeline.

Table 1. Optimal BsAb quality and maximum yield can be achieved through the application of chromatography techniques.

Sophisticated Analytical Control with Tiered Toolbox Approaches

The diverse and complex structures of BsAb pose significant analytical challenges. FDA guidance for BsAb development emphasizes specific considerations for different formats, including aggregates, fragments, homodimers, and other mispaired species, antigen specificity, affinity, avidity, potency, and on/off rates.

Among these, developing bioassays for BsAbs is highly challenging due to the unique designs that reflect their complicated and innovative mechanism of actions (MoA), as well as dual targets/epitopes. Therefore, phase-appropriate approaches, per regulatory guidance, are widely adopted. A combination of potency assays is highly preferable to address scientific, medical and regulatory aspects of biological activities. For example, driven by the MoA, dual binding Enzyme-Linked Immunosorbent Assays (ELISA) assays that reflect simultaneous binding can be well-developed as robust and QC-friendly release assays in early phases. Characterization bioassays such as single binding ELISA and binding kinetics by surface plasmon resonance (SPR) can provide a comprehensive understanding of biological activities. More complex and MoA-reflective cell-based assays can be established as characterization assays in early phases or developed into robust and QC-friendly release assays in later phases.

However, the greatest analytical challenge lies in monitoring mismatched species like heavy chain-heavy chain homodimers and heavy chain-light chain mismatches. For instance, a 4-chain BsAb, composed of two distinct heavy chains, and two distinct light chains, could theoretically form nine different 4-chain byproducts. Among these, the light chain-swapped species could display the exact same molecular weight and similar physiochemical features as the target molecule. This challenges the limits of conventional analytical capabilities.

To implement the concept of QbD in BsAb CMC development, it is crucial to establish a Quality Target Product Profile (QTPP) at the very beginning, based on the MoA and structural characteristics of the BsAb molecule. In particular, the existence of mismatched byproduct species needs to be experimentally confirmed at an early stage, and they need to be thoroughly evaluated. Each existent mismatch species needs to be monitored by appropriate analytical methods, to guide clone selection and process development. A powerful analytical toolbox is essential. In addition to conventional separation methods such as liquid chromatography with different separation mechanisms, it is necessary to apply methods that differentiate between target molecules and mismatched byproducts. Mass spectrometry (MS) methods are more suited to mismatched byproduct monitoring, due to superior sensitivity.

Analytical capabilities for BsAb process development require state-of-the-art methods and are deployed in a phase appropriate manner. For example, in a case study of an asymmetric 4-chain BsAb, intact MS was utilized in the chain ratio study, and more focused on heavy chain mispairing. When the project entered CLD, a subunit MS method was added to monitor the light chain-heavy chain mispairing. When the project progressed to process development, a hydrophobicity interaction chromatography (HIC) method was developed to enable fast testing turnaround. The HIC method was continuously optimized to establish the QL as 2% and became QC ready when the process was locked and ready to move into GMP production (Figure 6).

This case demonstrated how a smart analytical strategy guided by QTPP, based on advanced tiered toolboxes, is key to successful acceleration of BsAb development. A tiered analytical toolbox empowers a thorough understanding of BsAbs, provides guidance to accelerate process development, and realizes QTPP-based quality risk management (Figure 7). Together with intact molecule level methods (Tier 1) and subunit level methods (Tier 2), RP-MS, MS coupled with HIC, SEC, IEX, CIEF, and even more complex coupling such as MS with IEX-RP or SEC-RP and biophysical methods like MALS, AUC and DLS have been more and more commonly used to identify and quantify byproducts and/or variants of BsAbs (Tier 3).

Ensuring Stability, Manufacturability and Accurate Dosing

Robust Formulation and Process Development

BsAbs face unique stability challenges that emerge during both manufacturing and storage. Long-term storage often leads to physical instability, such as aggregation or fragmentation, as well as chemical modifications like oxidation or deamidation. These issues are exacerbated by sensitivity to environmental stressors, including temperature fluctuations, light exposure, and mechanical agitation. During manufacturing processes, shear stress from pumping, mixing, filtration, or filling steps can further destabilize BsAbs, particularly those with fragile structural architectures.

To address these challenges, an elaborate development workflow is essential. Early-stage developability assessments play a pivotal role in identifying molecules with stability liabilities, lead molecule selection, and subsequent formulation design by profiling key molecular characteristics such as solubility, aggregation propensity and degradation pathways. In addition, HTP methods (e.g. kD, B22 indicating protein-protein interactions) can be applied to predict protein stability. In the CMC phase, formulations are optimized by testing combinations of pH buffers, excipients and surfactants to stabilize the BsAbs and meet requirements for long-term storage. For BsAbs that exhibit physical and/or chemical instability during long-term storage, or have a limited development timeline prior to IND filing, freezing or lyophilizing is an option. Current capabilities enable lyophilization of products in vials as well as dual-chamber cartridges. These delivery systems can enhance stability as well as patient compliance (Figure 8). Biologic-device combination products, e.g. pre-filled syringes and auto-injectors could also be further developed in the post-IND stage.

Customized process parameters are developed, and related consumables are evaluated based on the molecule’s sensitivity as well as the manufacturing facility requirements. Lab-scale models simulating shear forces during pumping, mixing, filtration or filling help define tolerable process limits. Compatibility testing with storage bags, mixers, filters, tubing and primary packaging materials ensures that in-process interactions do not compromise product quality. Moreover, light sensitivity and oxidation sensitivity, particularly for facilities utilizing vaporized hydrogen peroxide (VHP) sterilization, are assessed to evaluate the impacts of light or VHP exposure on molecule stability, guiding the selection of dosage forms, drug product storage conditions and secondary packaging.

Accurate Dosing Delivery with Proper Approaches

Clinical in use (CIU) studies help to evaluate the stability of diluted drug product in the diluents (e.g. saline or 5% dextrose) as well as the compatibility of contacting components. For BsAbs, especially T-cell engager (TCE) BsAbs, the required dose for clinical use is quite low due to high potency. This induces further challenges, such as qualified analytical method development, the stability of the diluted solution and accurate dose delivery. Currently, WuXi Biologics has developed several methods including Meso Scale Discovery (MSD), Liquid Chromatography-Mass Spectrometry (LC-MS), Homogeneous Time-Resolved Fluorescence (HTRF), Spectro Fluorimetry (i.e., Duetta), ELISA, Size Exclusion Chromatography-Fluorescence Detector (SEC-FLD) to support the protein concentration as low as the level of microgram or nanogram per milliliter. Protein adsorption to contacting material, especially the in-line filter, results in protein loss. The detailed adsorption mechanisms with different proteins, diluents or in-line filters have been investigated and the corresponding proper mitigation plans are suggested, including the development of Intravenous Solution Stabilizer (IVSS) and/or the selection of appropriate diluents or in-line filters (Figure 9)⁵.

Conclusion

From CLD and cell culture process development, through downstream processes to manufacture, storage and dosing, BsAbs present particular challenges in protein expression, purity and stability. Added to this is the constant demand for accelerated development timelines, as companies race to bring their products to market more quickly. Addressing these issues throughout the BsAb lifecycle requires in-depth knowledge, expertise and modern tools such as our proprietary advanced technology platforms (e.g. WuXiUP, WuXia TrueSite). Only then is it possible to optimize the molecular assembly and expression of these products, as well as ensuring stability, purity and manufacturability.

The impact of integrating multidisciplinary solutions is profound: developers can now shorten timelines significantly—from a traditional 12-month CMC process down to 6 months, while simultaneously achieving titers without compromising product quality. Moreover, by embedding advanced analytical frameworks and stability strategies early on, risks are minimized, and regulatory pathways become more predictable, paving the way for smoother approvals and successful commercialization.

References

1. FDA. Novel Drug Approvals for 2024, https://www.fda.gov/drugs/novel-drug-approvals-fda/novel-drug-approvals-2024.
2. Grand View Research. Bispecific Antibodies Market (2023 – 2030), www.grandviewresearch.com/industry-analysis/bispecific-antibodies-market-report.
3. Marshall A. The year of the bispecific in oncology and beyond. Nature, December 1, 2025.
4. Chen T et al. Monitoring removal of hole-hole homodimer by analytical hydrophobic interaction chromatography in purifying a bispecific antibody. Protein Expr Purif. 2019;164:105457.
5. Mu X et al. Protein adsorption of in-line intravenous infusion filter and the corresponding mitigation plans. J Pharmaceut Sci 2025;114:103846.

Dr. Sherry Gu serves as Executive Vice President and Chief Technology Officer at WuXi Biologics, where she leads Biologics Development — a world’s leading technology and capability platform for biologics CMC development and clinical manufacturing with proven 6-month DNA-to-IND capability. With 29+ years of biopharmaceutical expertise, she has directly led numerous early- and late-stage CMC projects, including DNA to EUA approval of Sotrovimab through the VIR-GSK collaboration in the combat of the worldwide COVID-19 pandemic. Before joining WuXi Biologics, she held leadership roles at Eli Lilly and Company and Bristol Myers Squibb. She holds a Ph.D. in Biochemical Engineering from the Massachusetts Institute of Technology (MIT).