(161f) Improved Settling in Continuous Aqueous Two-Phase System

Rapid and cost-effective vaccine production has life-saving potential, as demonstrated by the COVID-19 pandemic. Continuous processing promises increased flexibility and productivity compared to traditional batch processes. While biopharmaceutical manufacturers have successfully implemented continuous manufacturing for protein therapeutics, achieving up to a 75% reduction in footprint and 90% reduction in operator time [1], such innovations have not yet been explored sufficiently for viral vaccine production.

A continuous purification platform has been developed for purification of an influenza vaccine candidate using a two-step aqueous two-phase system (ATPS) [2]. ATPS is a liquid-liquid extraction method that facilitates efficient and scalable biomolecule purification while maintaining high product recovery. Our system is composed of a PEG 8 kDa and citrate salt as phase forming components. The bulk of the viral product is separated from the impurities in the first step, and the second step recovers the viral product from the viscous polymer phase to ease further processing.

The recombinant baculovirus expression system (BEVS) in Sf9 cells has been used to produce influenza virus-like particles (VLPs). We purified the influenza VLP produced by BEVS on our continuous ATPS platform. However, the surfactants in the Sf9 cell culture media delayed the settling of the phases in ATPS by lowering the surface tension difference between the phases. To overcome this challenge, sodium chloride and sodium sulphate salts at ionic strength ranging from 0.1M to 0.75M was added to decrease the settling time. Increased ionic strength decreases the critical micelle concentration of the surfactants [3]. Free surfactants reduce interfacial tension and micelle formation stops the addition of surfactant to the interface as surfactant concentration increases. Therefore, the settling and coalescence behavior of the ATPS with and without salt addition was monitored with a camera-assisted method to observe the phase separation over time. The results indicated that the ATPS settled 15% faster with 0.1M increased ionic strength and 30% faster with 0.75M increased ionic strength. This reduction in settling time is particularly advantageous for large-scale production, as minimizing settling time leads to a reduction in the settler size. However, it is possible that the increased ionic strength of the system could change the physicochemical properties of the ATPS that support purification. Introducing sodium chloride and sodium sulphate salts at ionic strength ranging from 0.1M to 0.75M did not significantly change the ATPS phase equilibrium concentrations. Partitioning of the virus and host-cell protein impurities did not change by addition of salts at these concentrations. These findings determined that increased ionic strength did not affect the ATPS purification characteristics.

In conclusion, increasing the ionic strength of the ATPS with salt addition significantly accelerated phase settling without negatively impacting purification characteristics. This presents a valuable strategy for improving the efficiency of continuous ATPS platforms specifically in large scale production. Our results demonstrate that a continuous two-step ATPS could be incorporated into a fully continuous purification process, potentially making influenza vaccine manufacturing more flexible and cost-effective as a proof-of-concept.

[1] Ramos, I., Sharda, N., Villafana, R., Hill‐Byrne, K., Cai, K., Pezzini, J., & Coffman, J. (2023). Fully integrated downstream process to enable next‐generation manufacturing. Biotechnology and Bioengineering, 120(7), 1869-1881.

[2] Nold, N. M., Kriz, S. A., Waldack, S., James, G., Colling, T., Sarvari, T., ... & Heldt, C. L. (2025). Purification of a Non-Enveloped Virus using Sequential Aqueous Two-Phase Extraction. Journal of Chromatography A, 465866.

[3] Khimani, M., Rao, U., Bahadur, P., & Bahadur, P. (2014). Calorimetric and scattering studies on micellization of pluronics in aqueous solutions: Effect of the size of hydrophilic PEO end blocks, temperature, and added salt. Journal of dispersion science and technology, 35(11), 1599-1610.