Versatile recovery of interferon alpha-2b via polymer molecular weight tailoring: From liquid-liquid extraction to
Leonor S Castro1, Mara G Freire1, Augusto Q Pedro1
1CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193, Aveiro, Portugal.
Abstract:
In biomanufacturing, downstream processing encompasses the recovery, purification, and polishing of protein therapeutics, representing the major contributor to overall production costs. The development of efficient recovery strategies is therefore critical to enhance product yield and stability, while reducing impurity load and extending the operational lifetime of chromatographic resins and membranes. In this context, this work demonstrates that polymer-polymer aqueous biphasic systems (ABS) and related three-phase partitioning (TPP) strategies constitute effective and versatile platforms for the recovery of interferon alpha-2b (IFNα-2b) from Komagataella phaffii culture broths. IFNα-2b, fused to the alpha mating factor to enable extracellular secretion, was successfully produced in K. phaffii, reaching maximum titers of 78.1 mg·L-1 upon supplementation of the culture medium with Tween 80. Following upstream production, downstream recovery was investigated using ternary systems composed of water, polypropylene glycol (PPG 400), and polyethylene glycol (PEG) of varying molecular weights (400-6000 g·mol-1). In ABS formed with low-molecular-weight PEGs, IFNα-2b preferentially partitions into the PEG-rich phase, with recovery yields increasing as the PEG molecular weight decreases. Conversely, the use of high-molecular-weight PEGs promotes the formation of TPP systems, in which IFNα-2b is recovered as a precipitate at the interphase, achieving recovery yields as high as 99% with PEG 6000 g·mol-1. These results highlight the pivotal role of polymer molecular weight in dictating phase behavior, protein partitioning, and transition in the separation platform, from liquid-liquid extraction to three-phase partitioning. Overall, this work demonstrates that polymer-based ABS/TPP can be strategically tailored to integrate multiple separation stages, offering a versatile approach for protein recovery in downstream bioprocessing.
