Manufacturers are being pushed beyond mAb‑era platforms into a new design model built around molecular engineering, complex analytics and amplified upstream and downstream processing.
The advent of multispecific antibody therapeutics has upended CDMO development architecture. To keep pace with their robust progress, manufacturers are adapting to a new design model built around molecular engineering, deep analytics and intensified upstream and downstream process configurations.
Multispecifics are engineered biologics that can bind to more than one target at the same time.
Bispecifics established the first generation of multitarget biologics. They have dominated trade news headlines as Chinese biotechs launched an unprecedented wave of bispecific programs between 2020 and 2025. During those years, CRDMOs and CDMOs such as WuXi Biologics, Lonza Biologics and Samsung Biologics announced technology platforms for developing bispecifics and multispecifics.
The next wave of multispecifics include trispecifics, T-cell engagers with co‑stimulation, NK engagers and dual agonists. Their development poses new challenges beyond bispecifics’ inherent complexity— mispairing, instability and heterogeneity— and force CDMOs to revamp their development design and build.
“The greatest impact with multispecific molecules is typically in cell line development (CLD), analytical characterization and downstream purification which will define a robust control strategy for manufacturing,” said Laura Daley, senior director, Cell Biology & Product Development at Catalent.
Molecular engineering changes
Multispecifics often struggle with chain mispairing and product heterogeneity. To address these concerns, engineering strategies such as knobs‑into‑holes are still frequently used as well as common light chains are often helpful, said Randal Bass, executive vice president, Process Design and Innovation at Just-Evotec Biologics.
Knobs-into-holes is a bioengineering technique that modifies the shapes of two different protein halves so they fit together like matching puzzle pieces, ensuring they pair up correctly during manufacturing. While knobs-into-holes forces the two inner heavy chains to pair correctly, a common light chain uses the exact same piece for both outer light chains so they can never mispair.
Multispecifics often require controlled expression of multiple chains at defined ratios, creating challenges that do not exist with traditional monoclonal antibodies, Daley said. Catalent has invested in its GPEx® Lightning platform to support complex biologics, including multispecifics, through precise control of gene integration, expression balancing and stable cell line generation.
Of course, engineering the rest of the construct requires expertise, Bass said. “The domains you are trying to pull together, often Fab fragments, VHHs, a receptor or ligand fragment, also need to be stable,” he said. Fab fragments and VHHs are small pieces of proteins derived from antibodies that bind to specific targets.
“If you can get the fundamental pieces stable that makes putting them together easier,” Bass said. Just-Evotec Biologics has a suite of in silico tools (Abacus) to help with these engineering tasks.
The bioprocessing platforms themselves are becoming more amenable to multispecifics, Bass noted. CLD workflows employing high throughput transfections and automation makes screening multiple DNA vector chain ratios faster and easier. This assists in having the correctly assembled molecule.
In terms of expression levels for trispecific formats compared to bispecifics or monoclonals, “few things are going to express as well as a fully human monoclonal antibody,” Bass said. However, multispecifics are still going to express well in modern cell lines and vector systems. Additionally, with so much advancement with intensified bioreactor processes employing ATF or TFF perfusion, high volumetric productivity can be obtained.
ATF and TFF are techniques used to retain living cells inside a bioreactor while continuously adding fresh nutrients and harvesting the liquid product (perfusion).
Analytical capabilities crucial
New analytical methods have become essential for multispecific characterization.
By far, mass spectrometry tools have become foundational, Bass said, adding that whole mass analysis and reduced whole mass analysis “can go a long way” to identify correctly paired molecules. Traditional size exclusion chromatography, coupled with nonreduced and reduced capillary electrophoresis, can mostly take care of quantifying impurities and the final assembled molecule, once it is characterized. Finally, the multiattribute method is indispensable as it detects bad modifications, as well as clipping sites that are more prevalent in complex molecules, Bass said.
Multispecifics require significantly deeper analytical characterization than traditional monoclonal antibodies, Daley said. Key focus areas include chain pairing confirmation, structural characterization, variant identification, aggregate analysis and product heterogeneity assessment. Catalent has expanded integrated analytical development capabilities that support characterization and release testing throughout development and manufacturing, enabling rapid feedback loops between cell line development, process development and manufacturing.
The rise of multispecifics has also changed CDMOs’ quality control (QC) burdens, especially around binding and potency assays, Bass said. This is because some physical modifications to the multispecifics can affect binding to targets and subsequent potency. Thus, for every new target an assay can potentially be added.
“Developers increasingly want faster decision-making, greater process understanding and reduced release timelines,” Daley said. “While industry-wide adoption remains gradual, technologies such as process analytical technologies, automated data review, real-time process monitoring and AI-assisted analytics are becoming increasingly important.”
Upstream & downstream process development
Although intensified upstream technologies are gaining attention to increase productivity and shorten timelines, they are not always required to achieve commercial success, Daley said. Catalent’s GPEx® Lightning technology routinely generates high-expressing cell lines across complex multispecific modalities in short timelines. It combines high-titer cell lines with an integrated development and manufacturing model.
Many sponsors are requesting intensified upstream processes, such as perfusion and continuous feeding, Bass said.
A challenge is that low titers, stability issues and proteolytic clipping can be common upstream issues with multispecifics, he said. By intensifying perfusion in the upstream process, low titers can be overcome. Additionally, a continuous capture step out of that reactor can mitigate proteolytic clipping and other stability concerns, like deleterious post-translation modification.
Removing the molecule from the milieu of cell fragments, enzymes and metabolites in the bioreactor continuously is key, Bass said. For complex engineered multispecifics, they may contain molecular liabilities that could preclude them from being produced in a fed-batch process.
Downstream is often the greater challenge than upstream, Daley noted. “Multispecifics frequently require molecule-specific purification strategies to address chain mispairing, aggregate control or product-related variants,” she said. “Rather than redesigning facilities, successful CDMOs invest in flexible downstream infrastructure and high-throughput development capabilities that allow chromatography workflows to be rapidly optimized and scaled.”
Generally, the downstream process employs multiple chromatography steps (and even some depth filter removal of partial molecules) that can remove molecules that are not the final product, Bass said. While Just-Evotec Biologics has been able to use standard resins for this purification, it does employ scouting out more resins and more operating space to ensure complex multi-specifics can achieve an appropriate level of purity.
“Fortunately, automation and plate-based or robo-column screening along with machine learned optimization analysis greatly aids finding the optimal resin and condition,” Bass said.
