Modular High-Throughput Platform for scFv and Antibody Therapeutic Purification

Numab Therapeutics (Horgen, Switzerland) and Cytiva (Uppsala, Sweden) researchers investigated the purification of single-chain fragment variables and multispecific antibodies using new automated workflows. Their findings were published in the Journal of Chromatography A (1).

Multispecific antibodies (msAbs) are novel drug modalities that can simultaneously bind to two or more target proteins or bridge two different cells, eliciting therapeutic effects (2). These modalities have led to various advancements in therapeutics, showing promise for enhancing therapeutic efficacy and specificity in the treatment of complex diseases, such as cancer and autoimmune diseases. MsAbs are typically assembled from fragment antigen binding (Fab), single-chain fragment variable (scFv), fragment crystallizable (Fc) and single domain antibodies (VHH), which are interconnected by genetic fusion or heterodimerization.

Efficient and fast manufacturing of scFvs and msAbs is essential for screening hundreds of early-stage drug discovery candidates, which can allow for pharmacodynamic and biophysical profiling with the aim to identify functional and biophysical superior molecules before entering the lead optimization phase. Various high-throughput systems for antibody purification have been described in literature; however, using large, expensive, and customized robotic systems make their use in drug discovery unattractive for biotechnology and academic institutions. Further, conventional high-throughput methods typically use plate-based or tip-column formats, which often do not provide sufficient amounts of antibodies with acceptable monomeric purity for extended biophysical characterization and stability studies to assess developability and manufacturability.

One alternative to traditional protein A resins that has grown popular in recent years is highly efficient protein A membrane adsorbers, which are said to offer quicker purification cycles without compromising binding capacity. In this research, the scientists developed new routines based on automated ÄKTA system-based purification protocols in to streamline and accelerate the production of scFvs and msAbs. This type of liquid chromatography system, created by Cytiva, is claimed to allow for quick customization with its modular design (3). The procedures involve autosampler systems to automate sample injections during overnight and idling times, all while incorporating one-, two- and three-column schemes to facilitate purification workflows. Further, a novel membrane affinity chromatography technology was used to decrease residence times by increasing flowrates, shortening purification method run time per cycle.

With these procedures and further efforts to implement versatile and automated multi-column purification protocols, the scientists enabled the cost-effective and rapid capture of up to 51 single-chain variable fragments per day with high monomeric purities over 90%. Automated protocols facilitated the daily purification of up to 22 msAbs. Further, to increase the monomeric target purity of selected scFvs and msAbs, size-exclusion was automated using an autosampler system, which enabled with the polishing of up to 15 scFvs or msAbs per day.

The original purification processes developed and implemented in this study proved effective for scFvs and msAbs-based therapeutics. Using automation systems and multi-column schemes, workflows were simplified in multiple ways, especially during overnight and idling operation, enhancing throughput, accuracy, and reproducibility. The membrane affinity chromatography columns also bettered the throughput while shortening purification run times. Overall, this automated platform was shown to be a modular, reliable, and cost-effective scaffold for designing complex purification methods.

References

(1) Franke, B.; Québatte, J.; Wolniak, S.; et al. Modular High-Throughput Platform for the Purification of scFvs and Multispecific Antibody-Based Therapeutics. J. Chromatogr. A 2025, 1757, 466096. DOI: 10.1016/j.chroma.2025.466096

(2) Multispecific Antibodies: Next Generation Therapeutics Targeting Complex Disease Biology. AstraZeneca 2025. https://www.astrazeneca.com/r-d/next-generation-therapeutics/multispecifics.html (accessed 2025-7-16)

(3) ÄKTA Pure™ Chromatography System. Cytiva 2025. https://www.cytivalifesciences.com/en/us/shop/chromatography/chromatography-systems/akta-pure-p-05844?srsltid=AfmBOoqexlkqzgUJFurND1fkYkLPpthBQgZzAFlbf3p8w1RMtliM-gFQ (accessed 2025-7-16)