Downstream processing of XXXX -like particles: Single-stage and multi-stage aqueous two-phase extraction.

@entity1 @entity778 @entity691 @entity66 @entity7257 @entity375 @entity970
UNASSIGNED: The demand for vaccines against @entity66 has enforced the research and development of @entity778 -like particle (VLP) based vaccine candidates in recent years. Significant progress has been made in increasing VLP titres during upstream processing in bacteria, @entity375 and insect cells. Considering downstream processing, the separation of host cell impurities is predominantly achieved by time-intensive ultracentrifugation processes or numerous chromatography and filtration steps. In this work, we evaluate the potential of an alternative separation technology for VLPs: aqueous two-phase extraction ( @entity7257 ). The benefits of @entity7257 have been demonstrated for various biomolecules, but capacity and separation efficiency were observed to be low for large biomolecules such as VLPs or viruses. Both performance parameters were examined in detail in a case study on @entity1 B19 parvovirus-like particles derived from Spodoptera frugiperda Sf9 insect cells. A solubility-guided approach enabled the design of @entity691 ( @entity970 ) salt aqueous two-phase systems with a high capacity of up to 4.1mg/mL VLPs. Unique separation efficiencies were obtained by varying the molecular weight of @entity970 , the pH value and by using neutral salt additives. Further improvement of the separation of host cell impurities was achieved by multi-stage @entity7257 on a centrifugal partition chromatography (CPC) device in 500mL scale. While single-stage @entity7257 enabled a DNA clearance of 99.6%, multi-stage @entity7257 improved the separation of host cell proteins (HCPs). The HPLC purity ranged from 16.8% (100% VLP recovery) for the single-stage @entity7257 to 69.1% (40.1% VLP recovery) for the multi-stage @entity7257 . An alternative two-step downstream process is presented removing the ATPS forming polymer, cell debris and 99.77% DNA with a HPLC purity of 90.6% and a VLP recovery of 63.9%.
