Osmolyte enhanced aqueous two‐phase system for virus purification

Abstract

Due to the high variation in viral surface properties, a platform method for virus purification is still lacking. A potential alternative to the high-cost conventional methods is aqueous two-phase systems (ATPSs). However, optimizing virus purification in ATPS requires a large experimental design space, and the optimized systems are generally found to operate at high ATPS component concentrations.The high concentrations capitalize on hydrophobic and electrostatic interactions to obtain high viral particle yields. This study investigated using osmolytes as driving force enhancers to reduce the high concentration of ATPS components while maintaining high yields. The partitioning behavior of porcine parvovirus (PPV), a nonenveloped mammalian virus, and human immunodeficiency virus-like particle (HIV-VLP), a yeast-expressed enveloped VLP, were studied in a polyethylene glycol (PEG) 12 kDa-citrate system. The partitioning of the virus modalities was enhanced by osmoprotectants glycine and betaine, while trimethylamine N-oxide was ineffective for PPV. The increased partitioning to the PEG-rich phase pertained only to viruses, resulting in high virus purification. Recoveries were 100% for infectious PPV and 92% for the HIV-VLP, with high removal of the contaminant proteins and more than 60% DNA removal when glycine was added. The osmolyte-induced ATPS demonstrated a versatile method for virus purification, irrespective of the expression system.

Design Rules for the Sequestration of Viruses into Polypeptide Complex Coacervates

“…64 HRV-14 strain 1059 (ATCC VR-284) was propagated in H1HeLa cells (ATCC CRL-1958) and cultured in EMEM, as described previously. 65 Virus stocks were clarified to remove cell debris by spinning at 4500 rpm (3850 × g) in a Sorvall ST16R centrifuge (Thermofisher). The clarified stocks were stored at −80 °C and thawed before use, unless otherwise noted.…”

Downstream processing of virus‐like particles with aqueous two‐phase systems: Applications and challenges

“…ATPS represents a promising downstream processing tool for various biomolecules including VLPs. Its high yields, robust, continuous bioprocessing capability, scalability, cost viable, and more advan-tages have attracted researchers to perform further investigations on different types of biomolecules [10,11,13,31,32].…”

“…A thorough discussion regarding the applications of ATPSs in extracting VLPs is summarized in Table 2. Several studies had employed polymer/saltbased ATPSs to separate and purify VLPs, yielding remarkable recovery rates and separation efficiency of product [32,[59][60][61][62][63][64]. For instance, Benavides et al [59] utilized PEG/salt ATPSs to primary recover nonenveloped double-layered rotavirus-like particles (dlRLP) expressed with insect cell baculovirus vector.…”

“…A thorough discussion regarding the applications of ATPSs in extracting VLPs is summarized in Table 2 . Several studies had employed polymer/salt‐based ATPSs to separate and purify VLPs, yielding remarkable recovery rates and separation efficiency of product [32, 59–64]. For instance, Benavides et al.…”

“…The osmolytes utilized in the mentioned study (particularly glycine or betaine) served as the driving force enhancers to diminish the high concentration of phase‐forming agents by altering the TLL and partitioning behavior of viral particles in ATPS, while maintaining the high recovery yield. As a result, the production cost will be greatly reduced due to the low usage of phase‐forming components in ATPS besides attaining a promising VLPs yield [32]. Collectively, this demonstrated that the modified ATPSs can increase productivity and flexibility besides reducing the capital and manufacturing costs.…”