With the continuous advancement of biomedical technologies, multispecific antibodies (MSAs) have demonstrated enormous potential in the therapeutic field. Capable of targeting multiple epitopes simultaneously owing to their unique molecular structure, these antibodies have become a research hotspot for the treatment of cancer, autoimmune diseases and other complex disorders. Despite the resolution of many early developmental bottlenecks via molecular design and genetic engineering, several challenges persist—particularly concerning product stability and redox reaction processes—which compromise the final product quality and production efficiency. This article focuses on the challenges related to stability and redox reactions in the bioprocess development of multispecific antibodies, and explores strategies to address these technical bottlenecks.
1. Stability Issues: Low-pH Instability and Homodimer Mutations
While sophisticated molecular design and genetic engineering have resolved most early stability problems in multispecific antibody development, structural inherent stability challenges still remain. For instance, low-pH instability is a common drawback when charge-based design strategies are adopted.
In the molecular construction of multispecific antibodies, mutations are frequently introduced into homodimers to facilitate the correct assembly of heterodimers. These mutations typically involve charged amino acids, which are designed to destabilize homodimers and drive the formation of target heterodimers. However, such structural lability may cause antibodies to dissociate into half-antibody fragments under conventional conditions even without the addition of reducing agents. This phenomenon impairs overall antibody stability, and homodimer dissociation is prone to occur especially under low-pH environments.
To mitigate this issue, researchers have optimized molecular design by selectively modulating targeted mutations to minimize or eliminate low-pH-induced stability impairment. Practical process strategies include elevating the pH of elution buffers during Protein A affinity chromatography capture, or rapidly neutralizing the pH of product pools immediately after elution to avoid prolonged exposure of antibodies to low-pH conditions.
2. Challenges in Redox Reactions
The production of multispecific antibodies generally relies on redox reactions to reconstruct the native disulfide bond configuration. Two mainstream process strategies are applied based on different manufacturing workflows: one starts with producing homodimers as isolated half-antibodies, and the other adopts production of intact full-length antibodies.
In the half-antibody workflow, each half-antibody carries a distinct antigen-binding epitope. The knobs-into-holes technology is commonly deployed to drive accurate assembly of two half-antibodies into the desired multispecific antibody format.
For the full-antibody workflow, two types of intact parent antibodies with different epitope specificities are manufactured. Mutations are introduced at the hinge and CH3 domains of both parent antibodies to enable controlled reduction and subsequent oxidative rearrangement, ultimately yielding assembled multispecific antibodies.
The core of redox reactions lies in the reduction and reformation of disulfide bonds, especially inter-heavy-chain disulfide bonds. Conventional strong reducing agents such as dithiothreitol (DTT), β-mercaptoethanol (BME) and tris(2-carboxyethyl)phosphine (TCEP) are widely used for this purpose. However, these potent reductants non-specifically break not only target inter-chain disulfide bonds but also unintended intra-chain and heavy-light chain disulfide bonds, leading to irreversible structural disruption of antibodies.
To address this limitation, mild reducing agents including reduced glutathione (GSH), cysteamine and cysteine have been increasingly adopted. These mild reductants selectively act on inter-heavy-chain disulfide bonds without over-reducing heavy-light chain or intra-domain disulfide bonds, thereby minimizing unnecessary structural damage to antibody molecules.
3. Critical Process Parameters for Redox Reactions
Redox reaction is an indispensable unit operation in multispecific antibody production, governed by multiple key factors: pH value, buffer capacity, temperature, reductant-to-protein ratio, protein molar ratio, mixing mode and oxygen level. Optimization of these parameters is critical to maximizing the yield and purity of correctly assembled multispecific antibodies.
pH Value and Buffer Capacity: Redox reactions are typically performed under alkaline conditions with pH maintained above neutrality. A moderately elevated pH accelerates reaction kinetics, while excessively high pH may trigger protein instability and chemical modifications such as asparagine deamidation. Rational pH optimization is therefore essential to balance reaction efficiency and structural stability.
Temperature Effect: Temperature significantly influences the rate of redox reactions. Elevated temperatures (30–37°C) can speed up reaction progress, yet excessive heat may induce protein denaturation and aggregation. Ambient temperature is commonly preferred for redox processing to ensure stable and consistent reaction performance.
Reductant-to-Protein Ratio: Reductant dosage directly determines reaction rate, which increases with higher reductant concentration until saturation is reached. Over-dosage of reducing agents, nevertheless, may cause antibody fragmentation and degradation, necessitating precise dosage control.
Oxygen Level: Sufficient oxygen supply is a prerequisite for oxidative refolding and disulfide bond reformation. Aeration or headspace air overlay can be applied to maintain adequate oxygen availability. In oxygen-deficient scenarios, chemical oxidation or buffer exchange can be implemented to remove residual reductants and drive complete oxidation.
4. Potential Side Effects During Reduction Processes
Although controlled reduction is an essential step in multispecific antibody manufacturing, improper process design may induce adverse side reactions. For example, depth filtration during harvest and clarification may inadvertently trigger disulfide bond reduction, generating undesired impurities such as antibody fragments. Accordingly, process development must fully anticipate such risks, with targeted mitigation strategies implemented to avoid adventitious reduction and impurity accumulation.
5. Summary and Prospect of Multispecific Antibody Bioprocess Development
The bioprocess development of multispecific antibodies is inherently challenging, with stability maintenance and controlled redox reactions standing as major technical hurdles. These bottlenecks can be largely overcome through optimized molecular design, precise regulation of redox reaction conditions, and rational selection of reducing agents and process temperatures, ensuring consistent product stability and quality. Given the structural uniqueness of each multispecific antibody candidate, continuous innovation and customized process optimization remain imperative to manufacture high-quality drug products.
With the relentless advancement of biotechnology, multispecific antibodies are poised to play an increasingly pivotal role in immunotherapy, cancer treatment and other clinical applications in the future.
