Purification of viruses, virus-like particles, and globular biomolecules by filtration-supported polyalkylene glycol precipitation

A method for the purification of viruses, virus-like particles, extracellular vesicles, proteins having a diameter of 5 nm or more, protein complexes having a diameter of 5 nm or more, nucleic acids having a diameter of 5 nm or more, protein-nucleic acid complexes having a diameter of 5 nm or more, and/or molecules having a diameter of 5 nm or more, by filtration-supported polyalkylene glycol precipitation, comprising the steps of:

• (a) providing a filtration module, said filtration module comprising:
• (i-α) one stack or two separate stacks of 2 to 9 layers of a stabilized, non-ionic, hydrophilic porous structure, said layers being deposited on top of each other, wherein each of said stack(s) provide(s) a homogenous flow resistance and has a total thickness of 300 μm or more and 2000 μm or less, or
• (i-β) one monolithic stabilized, non-ionic, hydrophilic porous structure, or two separate monolithic stabilized, non-ionic, hydrophilic porous structures, wherein each of said porous structure(s) provide(s) a homogenous flow resistance and has a total thickness of 300 μm or more and 2000 μm or less, and
• (ii) a housing encasing said stack(s) of porous structure layers or said monolithic porous structure(s), said housing comprising a fluid inlet and a fluid outlet, wherein fluid entering the filtration module through the fluid inlet has to pass through said stack(s) of porous structure layers or said monolithic porous structure(s) in order to exit the filtration module through the fluid outlet, and wherein the housing is adapted to distribute the feed solution entering the housing via the fluid inlet evenly over the entire accessible surface area of the first layer of said stack(s) of porous structure layers or over the entire accessible surface area of said monolithic porous structure(s),
• (b) providing a feed solution, containing said viruses, virus-like particles, extracellular vesicles, proteins having a diameter of 5 nm or more, protein complexes having a diameter of 5 nm or more, nucleic acids having a diameter of 5 nm or more, protein-nucleic acid complexes having a diameter of 5 nm or more, and/or molecules having a diameter of 5 nm or more, to said filtration module via said fluid inlet at a surface area-dependent flow rate of at least 1.0 mL per minute per cm2 of accessible surface area of the first layer of said stack(s) of layers of a porous structure or of the monolithic porous structure(s) (1.0 mL min−1·cm−2), whereby the viruses, virus-like particles, extracellular vesicles, proteins having a diameter of 5 nm or more, protein complexes having a diameter of 5 nm or more, nucleic acids having a diameter of 5 nm or more, protein-nucleic acid complexes having a diameter of 5 nm or more, and/or molecules having a diameter of 5 nm or more, adhere to the porous structure, and
• (c) eluting the viruses, virus-like particles, extracellular vesicles, proteins having a diameter of 5 nm or more, protein complexes having a diameter of 5 nm or more, nucleic acids having a diameter of 5 nm or more, protein-nucleic acid complexes having a diameter of 5 nm or more, and/or molecules having a diameter of 5 nm or more, from said porous structure and thereby harvesting purified viruses, virus-like particles, extracellular vesicles, proteins having a diameter of 5 nm or more, protein complexes having a diameter of 5 nm or more, nucleic acids having a diameter of 5 nm or more, protein-nucleic acid complexes having a diameter of 5 nm or more, and/or molecules having a diameter of 5 nm or more.