WO2025193698A1 - Purification of viral particles with two stage aqueous two-phase extraction - Google Patents

Abstract

Disclosed herein are methods of isolating and concentrating viruses from a sample using two aqueous two-phase systems (ATPSs). Also disclosed herein is a system comprising a first ATPS and a second ATPS.

Description

Docket No.066040-0021-WO01 PURIFICATION OF VIRAL PARTICLES WITH TWO STAGE AQUEOUS TWO-PHASE EXTRACTION CROSS-REFERENCE TO RELATED APPLICATION(S) [0001] This application claims priority to U.S. Provisional Patent Application No.63/563,789, filed on March 11, 2024, which is incorporated herein by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH [0002] This invention was made with government support under grant CBET-1818906 awarded by the National Science Foundation (NSF) and grant number 1R01FD007461 awarded by the Food and Drug Administration (FDA). The government has certain rights in the invention. FIELD [0003] This disclosure relates to methods of isolating and concentrating viruses from a sample using two aqueous two-phase systems (ATPSs). The disclosure also relates to a system comprising a first ATPS and a second ATPS. INTRODUCTION [0004] Virus-based pharmaceuticals hold incredible ability to prevent disease as vaccines and potential to cure disease as gene therapies. However, they need to be manufactured safely and cost-effectively. Viruses are more difficult to produce than protein-based therapeutics due to their complex structure, difficult detection, poor overall process recoveries, and sensitivity to degradation. Density gradient ultracentrifugation is a traditional method of virus purification, but it is a highly manual process and cannot be easily scaled for manufacturing. Virus purification trains increasingly incorporate liquid chromatography. Chromatography is an easily scalable purification method commonly used for protein therapeutics. However, the relatively large size of viruses prevents them from accessing all of the binding sites in the resin, resulting in low binding capacity. Chromatography-based purification processes generally yield only 25- 50% of the virus produced upstream, which is poor compared to protein therapeutics. Furthermore, the affinity resins compatible with viruses are quite expensive; for example, an affinity resin specific to adeno-associated virus costs $25K per liter and is only used once in some manufacturing processes due to caustic instability. High chromatography resin costs and low product yields drive many of the costs in viral product purification. Docket No.066040-0021-WO01 [0005] Aqueous two-phase systems (ATPS) offer a cost-effective alternative for viral vector purification. ATPS generally consists of a polymer/polymer or polymer/salt solution that spontaneously separates above a critical concentration into two component-rich phases. For polymer-salt ATPS, the non-electrolyte polymer is salted out of the electrolyte salt above the critical concentration. Purification can occur when biological species partition into separate phases. ATPS has been used to purify various biologics, including viruses. A PEG-sulfate ATPS was able to recover 97% of adenovirus into the PEG-rich phase with comparable in vivo activity to ultracentrifuge-purified adenovirus. The foot-and-mouth disease virus was purified by multi-step 6 kDa polyethylene glycol (PEG) and sodium citrate ATPS with 72% total recovery and 90% host cell protein (HCP) removal. A PEG-citrate ATPS using 12 kDa PEG yielded 79% recovery of infectious porcine parvovirus (PPV) in the polymer-rich phase of a PEG-citrate salt ATPS. A 6 kDa PEG-citrate ATPS recovered 95% of influenza virus and 96% of adenovirus to the interface between the phases with 43% and 71% protein removal, respectively. While the recoveries sound promising, purified viral products collected in the PEG-rich phase or at the interface between phases are problematic for further processing. PEG is viscous, resulting in difficulties processing downstream of the ATPS. Partitioning to the interface is likewise undesired because it requires extremely skilled, precise, and labor-intensive recovery processes that would be difficult to perform at manufacturing scale. An automated process without painstaking interfacial recovery or high viscosity transfers would make ATPS more desirable to industry. [0006] Some work has been done to remove virus from the PEG-rich phase after purification. PEG precipitation is commonly used at research scale to concentrate viruses, but the long incubation times required make it a difficult method to use in manufacturing. Quick, continuous precipitation is now possible for protein therapeutics due to high bioreactor titers, but the inherently lower titers of viral products make this continuous precipitation unlikely to succeed. Osmolytes have successfully sped virus precipitation compared to PEG precipitation with over 80% virus recovery, but multi-hour incubation times are still required. To simplify ATPS integration into the purification train, there is a need for another method that reduces product viscosities and forgoes long incubation times. [0007] A second ATPS step could recover the purified viral product from the PEG-rich phase into the citrate-rich phase. Attempts at extracting biological products from the PEG-rich phase to the salt-rich phase have varied in success, ranging from 32-99% recovery. Only one of these studies focused on a non-enveloped viral product rather than a protein product and Docket No.066040-0021-WO01 reported 72% overall viral recovery after a three-step ATPS with 86% protein removal. Thus, there is a need for a multi-step ATPS purification strategy that consistently delivers high overall viral product recoveries with more complete DNA and protein impurity removal. SUMMARY [0008] In an aspect, the disclosure relates to a method of isolating and concentrating viruses from a sample comprising: (i) mixing a first phase component and a second phase component to form a first aqueous two-phase system (ATPS); (ii) mixing the ATPS with a sample comprising a virus, wherein the first phase component comprises from about 20 wt.% to about 30 wt.% of a polymer dissolved in an aqueous solution, wherein the second phase component comprises from about 6.4 wt.% to about 10.8 wt.% of a salt dissolved in an aqueous solution, wherein the pH of the first ATPS is from about 7 to about 9, and wherein the viruses concentrate in the first phase component to form a first virus concentrated phase; (iii) mixing a third phase component with the first virus concentrated phase to form a second ATPS, wherein the third phase component comprises from about 8 wt.% to about 13 wt.% of a salt dissolved in an aqueous solution, wherein the first virus concentrated phase comprises from about 7.9 wt.% to about 12 wt.% of the polymer dissolved in an aqueous solution, wherein the pH of the second ATPS is from about 4.5 to about 7, and wherein the viruses concentrate in the third phase component to form a second virus concentrated phase; and (iv) recovering the viruses from the second virus concentrated phase. In an embodiment, recovering the viruses from the second virus concentrated phase comprises pipetting or pumping the second virus concentrated phase away from the first virus concentrated phase. In another embodiment, the method further comprises recovering the first virus concentrated phase before mixing with the third phase component. In another embodiment, recovering the viruses from the first virus concentrated phase comprises pipetting or pumping the first virus concentrated phase away from the second phase component. In another embodiment, the first virus concentrated phase comprises from about 0 wt.% to about 3 wt.% of the salt. In another embodiment, the pH of the second ATPS is from about 5 to about 6. In another embodiment, the polymer is polyethylene glycol (PEG). In another embodiment, the molecular weight of the PEG is from about 4 kDa to about 12 kDa. In another embodiment, the molecular weight of the PEG is from about 6 kDa to about 12 kDa. In another embodiment, the molecular weight of the PEG is from about 8 kDa to about 12 kDa. In another embodiment, the salt is sodium citrate. In another embodiment, the aqueous solution is water. In another embodiment, the viruses are enveloped viruses, non-enveloped viruses, virus-like particles, or combinations thereof. In another embodiment, the viruses are Porcine Docket No.066040-0021-WO01 Parvovirus, Herpes Simplex Virus, Adeno-Associated Virus, Influenza B, Lentivirus, influenza A, influenza A virus-like particles, or combinations thereof. In another embodiment, the first ATPS is at a high tie line length (TLL). In another embodiment, the TLL is from about 24 to about 42. In another embodiment, the first ATPS is at a high tie line ratio (TLR). In another embodiment, the TLR is from about 0.3 to about 0.5. In another embodiment, the second ATPS is at a low tie line length (TLL). In another embodiment, the TLL is from about 12 to about 24. In another embodiment, the second ATPS is at a low tie line ratio (TLR). In another embodiment, the TLR is from about 0.3 to about 3.7. [0009] In a further aspect, the disclosure relates to a system comprising the first ATPS and the second ATPS described herein. [00010] The disclosure provides for other aspects and embodiments that will be apparent in light of the following detailed description and accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS [00011] FIGS.1A-B show the two-stage ATPS process for viral purification that includes a primary step to partition the virus from cell culture impurities, followed by a second step to extract the virus from the viscous PEG phase. FIG.1A is a schematic of the two-step ATPS. PPV is purified from host cell impurities in the first ATPS step. Virus-laden PEG-rich phase is then mixed with fresh citrate at different chemical conditions to recover PPV into the salt-rich phase in the second ATPS step. FIG.1B is an ATPS binodal curve showing system partitioning concentrations. Above the binodal curve, the polymer and salt separate into two phases. Lower TLLs (gray line) are closer to the axis and have lower phase component concentrations. Lower TLR (gray cross) are closer to the y axis have a higher proportion of PEG-rich phase volume than citrate-rich phase volume. The reverse is true for the high TLR. [00012] FIGS.2A-H show tie lines for 12 kDa and 8 kDa PEG ATPS for a range of pH. FIG. 2A is a graph showing tie lines for 8 kDa PEG ATPS with pH 8 citrate. FIG.2B is a graph showing tie lines for 8 kDa PEG ATPS with pH 7 citrate. FIG.2C is a graph showing tie lines for 8 kDa PEG ATPS with pH 6 citrate. FIG.2D is a graph showing tie lines for 12 kDa PEG ATPS with pH 8 citrate. FIG.2E is a graph showing tie lines for 12 kDa PEG ATPS with pH 7 citrate. FIG.2F is a graph showing tie lines for 12 kDa PEG ATPS with pH 6 citrate. FIG.2G is a graph showing tie lines for 8 kDa PEG ATPS with pH 7 glycine and a 0.5 M glycine addition. FIG.2H is a graph showing tie lines for 8 kDa PEG ATPS with pH 6 phosphate. Docket No.066040-0021-WO01 [00013] FIGS.3A-C show binodal curves for 12 kDa and 8 kDa PEG ATPS for a range of pH. FIG.3A is a graph showing binodal curves for 12 kDa and 8 kDa PEG ATPS at pH 8. FIG.3B is a graph showing binodal curves for 12 kDa and 8 kDa PEG ATPS at pH 7. FIG.3C is a graph showing binodal curves for 12 kDa and 8 kDa PEG ATPS at pH 6. [00014] FIGS.4A-C show comparisons of ATPS 2-step conditions. ATPS compositions (first row), where “1” indicates step 1 composition and “2” indicates step 2 composition, PPV recoveries (second row), and corresponding partition coefficients (third row). FIG.4A is graphs showing comparisons between pH-shifted 8 kDa PEG systems including pH 8 to 6 (8A), pH 7 to 6 (8B), and, as a control, pH 7 to 7 (8C). Similar TLL and TLR conditions were used. FIG.4B is graphs showing comparisons between high and low back-extraction TLRs (8A versus 8E) using 8 kDa PEG with similar pH and TLL conditions. FIG.4C is graphs showing comparisons between 12 kDa and 8 kDa PEG-containing systems (12A versus 8D) with no pH shift, and similar TLL and TLR conditions. The box-and-whisker plots are based on the recovery results from three to ten replicates. * = p < 0.1. ** = p <0.05. [00015] FIGS.5A-D show microspecies distributions according to pH. FIG.5A is a graph showing microspecies distribution of sodium citrate. FIG.5B is a structure of neutral citrate molecule. FIG.5C is a graph showing microspecies distribution of sodium phosphate. FIG.5D is a structure of neutral citrate molecule. Data from chemicalize.com. [00016] FIGS.6A-D show impurity removal by ATPS for the system with the highest recovery (8B, TABLE 1). FIG.6A is a graph showing log reduction of host cell DNA and protein impurities after the complete two-step ATPS process. FIG.6B is a graph showing protein concentrations in PPV starting material and in final citrate product for three ATPS experiments. FIG.6C is a graph showing DNA concentrations in PPV starting material and in final citrate product for three ATPS experiments. FIG.6D is an image showing SDS-PAGE of crude PPV starting material, step 1 PEG-rich and citrate-rich phases, and step 2 PEG-rich and citrate-rich phases. The SDS-PAGE is only sensitive enough to detect contaminating proteins and not PPV capsids. [00017] FIGS.7A-B show isolated step 2 ATPS performance. FIG.7A is a graph showing a comparison between recovery using PPV-laden PEG from step 1 versus isolated step 2 conditions directly using crude PPV. Similar TLL, TLR, and pH conditions with 8 kDa PEG were used. The overall recovery for the isolated step 2 conditions is projected using the step 1 Docket No.066040-0021-WO01 recovery shown. FIG.7B is a graph showing partition coefficients for the two-step versus the isolated step 2 conditions. Box-and-whisker plots are based on the recovery results from three to ten experiment replicates. * = p < 0.1. ** = p <0.05. [00018] FIGS.8A-C show the effect of 0.5 M glycine addition on ATPS performance. FIG. 8A is a graph showing step 1 and step 2 conditions. FIG.8B is a graph showing PPV recovery comparison for 8 kDa PEG, pH 7-to-6 shifted citrate ATPS with (8GA/8GB) and without (8B) a 0.5 M glycine addition during the first step. Two glycine-utilizing systems are included at a medium (8GB) and higher (8GA) TLL. FIG.8C is a graph showing natural logs of the viral particle partition coefficients for systems 8B, 8GA, and 8GB. Box and whisker plots are based on three or four experiment replicates. * = p < 0.1. ** = p <0.05. [00019] FIGS.9A-F show liquid chromatography – mass spectrometry results for glycine- containing samples. FIGS.9A, C, and E show relative abundance of sample components. This should not be confused with recovery of inputted glycine. FIGS.9B, D, and F show mass spectrometry results confirming glycine’s identity. FIG.9A is a graph showing LC profile of glycine-added Step 1 citrate phase. FIG.9B is a graph showing MS profile of glycine-added Step 1 citrate phase. FIG.9C is a graph showing LC profile of glycine-added Step 2 citrate phase. FIG.9D is a graph showing MS profile of glycine-added Step 2 citrate phase. FIG.9E is a graph showing LC profile of 0.5 mM glycine control. FIG.9F is a graph showing MS profile of 0.5 mM glycine control. [00020] FIGS.10A-C show the effect of phosphate salt on step 2 ATPS performance. FIG. 10A is a graph showing step 1 and step 2 ATPS compositions identified on the binodal curve and tie line graphs. FIG.10B is a graph showing PPV recovery comparison for an 8 kDa PEG, pH 7-to-6 shifted ATPS where both systems use citrate as the salt phase during step 1, but one system (solid line) uses citrate during step 2 (8B) while the other (dashed line) uses phosphate during step 2 (8PA). FIG.10C is a graph showing natural logs of the viral particle partition coefficients for systems 8B and 8PA. Box and whisker plots are based on three or four experiment replicates. * = p < 0.1. ** = p <0.05. [00021] FIGS.11A-D show additional optimization studies. FIG.11A is a graph showing a 12 kDa PEG ATPS with a pH shift from 7 to 6 and a high back-extraction tie line ratio was compared to the previously mentioned system with a pH hold at 7. Only two replicates were performed for the pH-shifted system due to the poor results. FIG.11B is a graph showing Docket No.066040-0021-WO01 natural logs of the partition coefficients for systems 12A and 12B. FIG.11C is a graph showing the order of phase component addition was tested to determine their effect on back-extraction recovery. There was no difference between adding PEG or citrate to the system first. FIG.11D is a graph showing the addition of citrate as a dry powder was compared to its usual addition as a stock solution. No significant difference was seen between these two methods. All results are based on three or more replicates unless otherwise noted. [00022] FIG.12 is a diagram showing a continuously-processing purification system which uses multi-stage aqueous two-phase extraction to purify porcine parvovirus from cell culture impurities. Tangential flow filtration is used to concentrate the virus solution before purification and to formulate the solution after purification. [00023] FIGS.13A-B show continuously-processing ATPS for viral vectors. FIG.13A is a diagram showing a continuously-processing system. FIG.13B is an image of a continuously- processing system. [00024] FIG.14 shows total virus recovery for a range of viruses. PPV-porcine parvovirus; AAV2 and AAV9-adenoassociated virus; HSV-herpes simplex virus; LV-lentivirus; IBV-influenza B virus. FIG.14 is a graph showing total virus recovery at various pH values. The first value is the pH for step 1 and the second value is the pH for step 2. [00025] FIG.15 is a graph showing partition coefficients for three viruses at various pH values. When one pH value is shown, it is step 1 and when two pH values are shown, the first value is the pH for step 1 and the second value is the pH for step 2. DETAILED DESCRIPTION [00026] Described herein are methods of isolating and concentrating viruses from a sample. The methods may comprise: (i) mixing a first phase polymer component and a second phase salt component to form a first aqueous two-phase system (ATPS); (ii) mixing the ATPS with a sample comprising a virus, wherein the viruses concentrate in the first phase component to form a first virus concentrated phase; (iii) mixing a third salt phase component with the first virus concentrated phase to form a second ATPS, wherein the viruses concentrate in the third phase component to form a second virus concentrated phase; and (iv) recovering the viruses from the second virus concentrated phase. Docket No.066040-0021-WO01 [00027] Virus-based vaccines and therapies require a purification method that is both cost- effective and easily scalable. An aqueous two-phase system (ATPS) comprising polyethylene glycol (PEG) and citrate salt has been proven to deliver high virus recoveries along with high impurity removal. However, these systems often place the virus into a viscous PEG-rich phase or at the two-phase interface, leading to difficulties in subsequent downstream processes. Herein, a second ATPS to extract the virus product back into the citrate-rich phase by changing the chemical conditions was used. ATPS performance was tested as a function of phase component concentration, phase component volume ratios, PEG molecular weight, salt type, pH, glycine addition, and phase component addition order to identify the most impactful parameters for the extraction of non-enveloped or enveloped viruses. By shifting the pH, lowering phase component concentrations, and increasing the volume ratio of the citrate-rich phase between the first and second ATPS steps, significantly more virus was recovered with 2.0 logs of host cell protein removal and 1.0 logs of host cell DNA removal. Using a PEG molecular weight of from about 4 kDa to about 12 kDa enabled a pH shift between the first and second ATPS steps without precipitation. Glycine addition during the first step of ATPS, phosphate salt use during the second step of ATPS, and component addition order did not significantly increase the overall recovery. This process may be used in a continuously-processing format for continuous and low-cost viral vector manufacturing. 1. Definitions [00028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The meaning and scope of the terms should be clear. In case of conflict, the present document, including definitions, take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. [00029] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms Docket No.066040-0021-WO01 “a,” “and,” and “the” include plural references unless the context clearly dictates otherwise. The present disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. [00030] For the recitation of numeric ranges herein, each intervening number there between with the same degree of precision is explicitly contemplated. For example, for the range of 6-9, the numbers 7 and 8 are contemplated in addition to 6 and 9, and for the range 6.0-7.0, the number 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. [00031] The term “about” or “approximately” as used herein as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In certain aspects, the term “about” refers to a range of values that fall within 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Alternatively, “about” can mean within 3 or more than 3 standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value. [00032] “Adeno-associated virus” or “AAV” as used interchangeably herein refers to a small virus belonging to the genus Dependovirus of the Parvoviridae family that infects humans and some other primate species. AAV is not currently known to cause disease and consequently the virus causes a very mild immune response. [00033] The terms “control,” “reference level,” and “reference” are used herein interchangeably. The reference level may be a predetermined value or range, which is employed as a benchmark against which to assess the measured result. “Control group” as used herein refers to a group of control subjects or samples. The predetermined level may be a cutoff value from a control group. The predetermined level may be an average from a control group. Cutoff values (or predetermined cutoff values) may be determined as known in the art. Docket No.066040-0021-WO01 [00034] “Herpes Simplex Virus” or “HSV” as used interchangeably herein refers to members of the Herpesviridae family. They are viruses that produce viral infections in humans and some other primate species and can cause painful blisters or ulcers. [00035] “Influenza A” or “IAV” as used interchangeably herein refers to a species of the genus Alphainfluenzavirus of the virus family Orthomyxoviridae. It is a pathogen with strains that infect birds and some mammals, as well as causing seasonal flu in humans. [00036] “Influenza B” or “IBV” as used interchangeably herein refers to a species in the genus Betainfluenzavirus in the virus family Orthomyxoviridae. Influenza B virus is a negative- sense single-strand RNA virus known only to infect certain mammal species, including humans, ferrets, pigs, and seals. It can cause the seasonal flu in humans. [00037] “Lentivirus” as used herein refers to a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. [00038] “Porcine Parvovirus” or “PPV” as used interchangeably herein refers to a virus in the species Ungulate protoparvovirus 1 of genus Protoparvovirus in the virus family Parvoviridae. It causes reproductive failure of swine characterized by embryonic and fetal infection and death, usually in the absence of outward maternal clinical signs. [00039] “Sample” or “test sample” as used herein can mean any sample in which the presence and/or level of a virus is to be detected or determined or any sample comprising a virus or component thereof as detailed herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid. In other embodiments, the sample comprises a cell culture preparation. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering Docket No.066040-0021-WO01 components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art. [00040] “Virus-like particles” or “VLPs” as used interchangeably herein refer to non-infectious particles that resemble viruses in structure and appearance. They are composed of viral structural proteins, such as capsid proteins, but lack the viral genome and therefore cannot replicate. 2. Aqueous Two-Phase System (ATPS) [00041] Provided herein are aqueous two-phase systems (ATPSs). An ATPS consists of two distinct liquid phases, which are semi-immiscible with each other and used to form the ATPS, the ratios of which can be adjusted. For example, phase component concentration, phase component volume ratios, polymer molecular weight, salt type, pH, and phase component addition order may be adjusted. Biomolecules suspended in the ATPS partition into one of the two aqueous phases based on their physicochemical properties (e.g., hydrophilicity and interfacial tension of the two phases), thereby concentrating biomolecules of interest. For example, the ATPS may be used to isolate and/or concentrate one or more viruses from a sample. Different molecules in a mixture are distributed differentially between the two phases due to their different properties, and it is possible to separate and concentrate target molecules using the ATPS with minimal set up and human intervention. For example, no power or equipment is necessary to bring about the phase separation. a. Two ATPS System [00042] Provided herein is a system comprising a first ATPS and a second ATPS. The system may be used to isolate and/or concentrate viruses from a sample. [00043] The first ATPS may include a first phase component and a second phase component. The first ATPS may have a pH of from about 7.0 to about 9.0, about 7.5 to about 9.0, about 8.0 to about 9.0, about 8.5 to about 9.0, about 7.0 to about 8.5, about 7.0 to about 8.0, or about 7.0 to about 7.5. [00044] The first ATPS may be at a high tie line length (TLL). The TLL may be from about 24 to about 42, about 28 to about 42, about 32 to about 42, about 36 to about 42, about 40 to about 42, about 24 to about 38, about 24 to about 34, about 24 to about 30, or about 24 to about 26. Docket No.066040-0021-WO01 The first ATPS may be at a high tie line ratio (TLR). The TLR may be from about 0.3 to about 0.5, about 0.4 to about 0.5, or about 0.3 to about 0.4. [00045] The first phase component may comprise from about 20 wt.% to about 30 wt.%, about 22 wt.% to about 30 wt.%, about 24 wt.% to about 30 wt.%, about 26 wt.% to about 30 wt.%, about 28 wt.% to about 30 wt.%, about 20 wt.% to about 28 wt.%, about 20 wt.% to about 26 wt.%, about 20 wt.% to about 24 wt.%, or about 20 wt.% to about 22 wt.% of a polymer dissolved in an aqueous solution. The polymer may be polyethylene glycol (PEG), dextran (Iqbal et al., Biological Procedures Online, 2016; 18, 1-18), polyethylene glycol dimethyl ether (PEGDME), polypropylene glycol (PPG), polyethylene oxide (PEO; Reschke et al., Fluid Phase Equilibria, 2015; 387, 178-189), poly vinyl alcohol (PVA; Elversson and Millqvist-Fureby, International journal of pharmaceutics, 2005; 294: 1-2, 73-87), or a combination thereof. In some embodiments, the polymer may be PEG. The molecular weight of the PEG may be from about 4 kDa to about 12 kDa, about 6 kDa to about 12 kDa, about 8 kDa to about 12 kDa, about 10 kDa to about 12 kDa, about 4 kDa to about 10 kDa, about 4 kDa to about 8 kDa, or about 4 kDa to about 6 kDa. The aqueous solution may be water. [00046] The second phase component may comprise from about 6.4 wt.% to about 10.8 wt.%, about 7.4 wt.% to about 10.8 wt.%, about 8.4 wt.% to about 10.8 wt.%, about 9.4 wt.% to about 10.8 wt.%, about 10.4 wt.% to about 10.8 wt.%, about 6.4 wt.% to about 9.8 wt.%, about 6.4 wt.% to about 8.8 wt.%, about 6.4 wt.% to about 7.8 wt.%, or about 6.4 wt.% to about 6.8 wt.% of a salt dissolved in an aqueous solution. The salt may be kosmotropic salts, chaotropic salts, inorganic salts containing cations such as straight or branched trimethyl ammonium, triethyl ammonium, tripropyl ammonium, tributyl ammonium, tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium and tetrabutyl ammonium, and anions such as phosphates, sulphate, nitrate, chloride and hydrogen carbonate. The salt may be any citrate salt. The salt may be NaCl, Na₃PO₄, NaH₂PO₄, Na₂HPO₄, KPO₄, KH₂PO₄, K₂HPO₄, Na₂SO₄, NaHSO₄, potassium citrate, dipotassium citrate, tripotassium citrate, (NHA)₂SO₄, sodium citrate, disodium citrate, trisodium citrate, sodium phosphate, sodium sulfate, sodium chloride, sodium acetate, or combinations thereof. Other salts, e.g., ammonium acetate, may also be used. Two or more salts may be used for adjusting the pH value or altering the interfacial tension between the phases. In an embodiment, the salt may be sodium citrate. The aqueous solution may be water. Docket No.066040-0021-WO01 [00047] If the concentrations of the polymer and salt are less than the above lower limits, an ATPS is not formed. On the other hand, if the concentrations thereof are higher than the above upper limits, a long period of time is required to dissolve the polymers and surface tension is high between the two phases, making it difficult to dissolve a third solute such as a sample comprising viruses. It will also increase the time to separate the phases. Also, at some point in time, the solubility limit of the polymer and salt will be reached. [00048] Viruses from a sample mixed with the first ATPS concentrate in the first phase component of the first ATPS to form a first virus concentrated phase. A sample may include, but is not limited to, cell culture fluid, tissues, blood, plasma, serum, cerebrospinal fluid (CSF), urine, saliva, fecal matters, and discharges such as tears, sputum, nasopharyngeal mucus, vaginal discharge, penile discharge. [00049] The first virus concentrated phase may comprise from about 0 wt.% to about 3 wt.%, about 1 wt.% to about 3 wt.%, about 2 wt.% to about 3 wt.%, about 0 wt.% to about 1 wt.%, or about 0 wt.% to about 2 wt.% of the salt from the second phase component. Any virus may be isolated with a system described herein. The viruses may be enveloped viruses, non-enveloped viruses, virus-like particles, or combinations thereof. The viruses may be Porcine Parvovirus (PPV) and other parvoviruses, Herpes Simplex Virus (HSV) and other herpes viruses, Adeno- Associated Virus (AAV), Influenza B, Lentivirus, influenza A, influenza A virus-like particles, Vesicular stomatitis Indiana virus (VSIV or VSV), Bacteriophage MS2, Adenovirus (Ad), Human Immunodeficiency virus (HIV) and other retroviruses, Venezuelan Equine Encephalitis (VEE) virus and other alpha viruses, vaccinia virus (VACV) and myxoma virus (MYXV) and other poxviruses, Newcastle disease virus, Measles virus, or combinations thereof. [00050] The second ATPS may include a third phase component and the first virus concentrated phase. The second ATPS may have a pH of from about 4.5 to about 7.0, about 5.0 to about 7.0, about 5.0 to about 6.0, about 5.5 to about 7.0, about 6.0 to about 7.0, about 6.5 to about 7.0, about 4.5 to about 6.5, about 4.5 to about 6.0, about 4.5 to about 5.5, or about 4.5 to about 5.0. The viruses concentrate in the third phase component to form a second virus concentrated phase. The viruses may be recovered from the second virus concentrated phase. [00051] The second ATPS may be at a low tie line length (TLL). The TLL may be from about 12 to about 24, about 15 to about 24, about 18 to about 24, about 21 to about 24, about 12 to about 21, about 12 to about 18, or about 12 to about 15. The second ATPS may be at a low Docket No.066040-0021-WO01 TLR. The TLR may be from about 0.3 to about 3.7, about 0.9 to about 3.7, about 1.5 to about 3.7, about 2.1 to about 3.7, about 2.7 to about 3.7, about 3.3 to about 3.7, about 0.3 to about 3.1, about 0.3 to about 2.5, about 0.3 to about 1.9, about 0.3 to about 1.3, or about 0.3 to about 0.7. [00052] The third phase component may comprise from about 8 wt.% to about 13 wt.%, about 9 wt.% to about 13 wt.%, about 10 wt.% to about 13 wt.%, about 11 wt.% to about 13 wt.%, about 12 wt.% to about 13 wt.%, about 8 wt.% to about 12 wt.%, about 8 wt.% to about 11 wt.%, about 8 wt.% to about 10 wt.%, or about 8 wt.% to about 9 wt.% of a salt dissolved in an aqueous solution. The salt may be kosmotropic salts, chaotropic salts, inorganic salts containing cations such as straight or branched trimethyl ammonium, triethyl ammonium, tripropyl ammonium, tributyl ammonium, tetramethyl ammonium, tetraethyl ammonium, tetrapropyl ammonium and tetrabutyl ammonium, and anions such as phosphates, sulphate, nitrate, chloride and hydrogen carbonate. The salt may be any citrate salt. The salt may be NaCl, Na₃PO₄, NaH₂PO₄, Na₂HPO₄, KPO₄, KH₂PO₄, K₂HPO₄, Na₂SO₄, NaHSO₄, potassium citrate, dipotassium citrate, tripotassium citrate, (NHA)₂SO₄, sodium citrate, disodium citrate, trisodium citrate, sodium phosphate, sodium sulfate, sodium chloride, sodium acetate, or combinations thereof. Other salts, e.g., ammonium acetate, may also be used. Two or more salts may be used for adjusting the pH value or altering the interfacial tension between the phases. In an embodiment, the salt may be sodium citrate. The aqueous solution may be water. [00053] The first virus concentrated phase comprises from about 7.9 wt.% to about 12.0 wt.%, about 8.9 wt.% to about 12.0 wt.%, about 9.9 wt.% to about 12.0 wt.%, about 10.9 wt.% to about 12.0 wt.%, about 11.9 wt.% to about 12.0 wt.%, about 7.9 wt.% to about 11.0 wt.%, about 7.9 wt.% to about 10.0 wt.%, about 7.9 wt.% to about 9.0 wt.%, or about 7.9 wt.% to about 8.0 wt.% of the polymer dissolved in an aqueous solution. [00054] The use of a high-molecular weight PEG for the upper phase (e.g., ≥4 kDa PEG as opposed to <4 kDa PEG) and the use of a pH shift between the first and second stages improve product recovery. The systems described herein result in purified viruses in the salt-rich phase, where they can easily be further processed by filtration. Typically in the art, virus recovery is into the polymer-rich phase or interface between the phases; these processes are not easily adaptable to large-scale manufacturing. Docket No.066040-0021-WO01 3. Methods of Isolating and Concentrating Viruses [00055] Provided herein are methods of isolating and concentrating viruses from a sample. The methods may include: (i) mixing a first phase component described herein and a second phase component described herein to form a first ATPS described herein; (ii) mixing the ATPS with a sample comprising a virus, wherein the viruses concentrate in the first phase component to form a first virus concentrated phase as described herein; (iii) mixing a third phase component described herein with the first virus concentrated phase to form a second ATPS described herein, wherein the viruses concentrate in the third phase component to form a second virus concentrated phase as described herein; and (iv) recovering the viruses from the second virus concentrated phase. [00056] Recovering the viruses from the second virus concentrated phase may comprise pipetting or pumping the second virus concentrated phase away from the first virus concentrated phase. [00057] The method may further comprise recovering the first virus concentrated phase before mixing with the third phase component. For example, the first virus concentrated phase (e.g., the polymer-rich phase comprising the viruses) may be pipetted or pumped away from the second phase component (e.g., salt-rich phase comprising contaminants such as host cell residual DNA (HC-DNA) and proteins) and then mixing the recovered first virus concentrated phase with the third phase component. [00058] The purity of the viruses contaminated with proteins and HC-DNA may be increased, thus enabling various downstream applications for use of the viruses such as therapeutic use of the virus or viral vector like gene therapy, disease diagnosis, vaccine research and therapy, a drug delivery vehicle, and the like. 4. Kits [00059] Provided herein is a kit, which may be used to isolate and/or concentrate viruses. The kit comprises a first ATPS as described herein or components for making the first ATPS, for concentrating viruses in a first phase component, as described above, and instructions for using and making the first ATPS. The kit also comprises a second ATPS as described herein or components for making the second ATPS, for concentrating viruses in a third phase component, as described above, and instructions for using and making the second ATPS. In some Docket No.066040-0021-WO01 embodiments, the kit comprises a polymer dissolved in an aqueous solution as described herein and two solutions where a salt is dissolved in an aqueous solution as described herein, and instructions for making the first ATPS and second ATPS as well as using the first and second ATPSs. [00060] Instructions included in kits may be affixed to packaging material or may be included as a package insert. While the instructions are typically written on printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this disclosure. Such media include, but are not limited to, electronic storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like. As used herein, the term “instructions” may include the address of an internet site that provides the instructions. 5. Examples [00061] The foregoing may be better understood by reference to the following examples, which are presented for purposes of illustration and are not intended to limit the scope of the invention. The present disclosure has multiple aspects and embodiments, illustrated by the appended non-limiting examples. Example 1 Summary of Two-Step Extraction Process using Aqueous Two-Phase Systems [00062] A two-step extraction process using aqueous two-phase systems (ATPS) for the purpose of purifying virus-based vaccines and viral gene therapy vectors was developed. This process is illustrated in FIG.1A. Joshi et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2019; 1126-1127: 121744 details the system composition selection for the first step in this process. A crude virus stock is added to a 12 kDa or 8 kDa polyethylene glycol (PEG) and citrate ATPS which separates into two phases at sufficiently high concentrations. Components of the crude virus stock added to the ATPS including virus, host cell proteins, and host cell DNA, are separated based on their differing affinities for the PEG or citrate. Joshi et al. reported that up to 79% of porcine parvovirus can be recovered in the PEG phase in a single stage, while the host cell protein and DNA impurities largely partition to the citrate phase. [00063] A second step developed herein extracts the virus-based vaccine from the viscous PEG phase product of the first stage into a salt-rich phase for further processing. By decreasing Docket No.066040-0021-WO01 the concentration of phase forming components and the pH of the systems, the virus-based vaccines can be coaxed from the PEG-rich phase to the citrate-rich phase. This project found that efficiency of back-extraction from PEG to citrate is comparable using either 8 kDa and 12 kDa PEG. However, the use of 8 kDa enables a pH shift between the first and second ATPS steps. When 12 kDa PEG is used in a pH-shifted system, there appears to be inactivation or precipitation of the viral product. So far, the technology has been optimized for porcine parvovirus (PPV; vaccine), adeno-associated virus (AAV; gene therapy vector), herpes simplex virus (HSV; gene therapy vector), influenza B virus (IBV; vaccine), influenza A virus like particle (IAV VLP; vaccine) and lentivirus (gene therapy vector). This work shows system’s versatility with different virus structures. [00064] The process parameters this two-step ATPS process purification platform for multiple viral products include: a system pH of pH 7 or 8 during the first step with a shift to pH 5 or 6 during the second step; a polyethylene glycol molecular weight of 8 kDa PEG; a salt type of sodium citrate; step 1 component concentrations (pH 7 and 8) of 20.5 w/w% PEG and 10.8% sodium citrate; step 2 component concentrations (pH 6 only) of 7.9 w/w% PEG and 10.2% sodium citrate; and step 2 component concentrations (pH 5 only) of 7.0 w/w% PEG and 13.0% sodium citrate. [00065] The process performance for multiple viral products is shown in TABLE 1. TABLE 1. Performance overview. Size Total Protein DNA ) [00066] The detection methods used for the detection of the viruses and impurities are shown in TABLE 2. TABLE 2. Analytical methods. Docket No.066040-0021-WO01 Virus Detection Method Impurity detection methods PPV Infectivity assay (MTT) B df d t i ti t in [00067] Infectivity assay (MTT assay). Virus concentration is determined by how completely the viruses kill cultured cells. This assay is used whenever possible because it proves the virus is still active after purification. It cannot be used for AAV or lentivirus since they are non- replicative. The MTT assay is described in Heldt et al., J Virol Methods.2006; 135(1): 56-65. [00068] Droplet digital polymerase chain reaction (ddPCR). Highly precise method for measuring virus genome. BioRad^® ddPCR kit was used (Vericheck ddPCR Empty-Full Capsid Kits). The Bio-Rad (Hercules, CA) application manual was followed to conduct the assay. [00069] Quantitative PCR (qPCR). Similar technology to ddPCR, but less precise. Used before ddPCR was available. [00070] Transduction assay. Lentivirus vector activity was confirmed by looking for change in cells after infection. I.e. if the lentivirus codes for green fluorescent protein, cells should become fluorescent. [00071] Bradford assay. Protein concentration was measured by colorimetric method. The assay color indicator gets darker when more protein is available to bind to the reporter protein. The protocol from Thermo Fisher (Waltham, MA) was used for this assay. [00072] Picogreen assay. DNA concentration was measured by fluorescent detection. Fluorescence increases when more DNA is available to bind to the reporter molecule. The protocol from Thermo Fisher (Waltham, MA) was used for this assay. Example 2 Materials and Methods [00073] Materials. Cell culture supplies including Eagle’s minimum essential media, sodium bicarbonate, fetal bovine serum (USDA-approved), penicillin-streptomycin (10,000 U/mL), Docket No.066040-0021-WO01 0.25% trypsin-EDTA solution, and phosphate-buffered saline (pH 7.2) were purchased from Life Technologies (Grand River, NY). For cell viability assays, 2-(3,5-diphenyltetrazol-2-ium-2-yl)- 4,5-dimethyl-1,3thiazole bromide (MTT, 98%) was purchased from Alfa Aesar (Ward Hill, MA). Sodium dodecyl sulfate (Bioreagent, ≥98.5%) was purchased from Sigma Aldrich (St. Louis, MO). [00074] ATPS supplies including polyethylene glycol 8000 (PEG 8 kDa), polyethylene glycol 12000 (PEG 12 kDa), polyethylene glycol 6000 (PEG 6 kDa), polyethylene glycol 4000 (PEG 4 kDa), sodium citrate tribasic dihydrate (ACS reagent, ≥99%), and sodium phosphate dibasic heptahydrate (ACS reagent, ≥98%) were purchased from Sigma Aldrich (St. Louis, MO). Citric acid monohydrate was purchased from Fisher Scientific (Hampton, NH). Sodium phosphate monobasic monohydrate was purchased from Merck & Co. (Kenilworth, NJ). For pH titration, hydrochloric acid was purchased from Fisher Scientific (Hampton, NH) and sodium hydroxide was purchased from J.T. Baker (Radnor, PA). For solution filtration, 0.2 μm Nalgene^® bottle top filters were purchased from Thermo Fisher Scientific (Waltham, MA) and 0.22 μm cellulose acetate syringe filters were purchase from VWR (Radnor, PA). [00075] For citrate removal, 3 kDa molecular weight cut-off Amicon spin filters were purchased from Merck Millipore (Cork, Ireland). For gel electrophoresis prep, Laemmli SDS 4X Sample Reducing Buffer was purchased from Thermo Fisher Scientific (Waltham, MA), β- mercaptoethanol (55 mM in PBS) was purchased from Life Technologies (Grand River, NY), and NuPAGE 4-12% Bis-Tris gels were purchased from Life Technologies (Grand River, NY). To run and stain the gels, Precision Plus Protein Dual Color Standard was purchased from Bio- Rad (Des Plaines, IL), NuPAGE MOPS SDS Running Buffer was purchased from Invitrogen (Waltham, MA), and SimplyBlue™ SafeStain was purchased from Invitrogen (Waltham, MA). [00076] Virus Propagation and Analysis. Porcine parvovirus (PPV), a non-enveloped viral vaccine model, was gifted by Dr. Ruben Carbonell at North Carolina State University and propagated using porcine kidney (PK-13) cells (CRL-6489TM, ATCC, Manassas, VA). Cell culture and virus propagation were carried out as previously described (Heldt et al., J Virol Methods.2006; 135(1): 56-65). PPV titer was determined using the 3-(4,5-dimethylthiazol-2-yl)- 2,5-diphenyltetrazolium bromide (MTT) colorimetric cell viability assay as previously described (Tafur et al., Antiviral Res.2013; 99(1): 27-33), and titer units are expressed as log(MTT₅₀/mL). HSV-1 strain F (VR-733 ^TM, ATCC) was propagated using Vero cells (CCL-81^TM, ATCC) grown in DMEM as described previously (Meingast et al., Biotechnol J.2021; 16 (7) e2000342). Virus Docket No.066040-0021-WO01 stocks clarified of cell debris were aliquoted and stored at -80°C. PPV and HSV were titrated using the MTT assay as described previously (Meingast et al., Biotechnol J.2021; 16 (7) e2000342). [00077] ATPS Characterization. In preparation for ATPS, 40 w/w% stocks of PEG, 25 w/w% stocks of sodium citrate, and 30 w/w% stocks of sodium phosphate were made in Nanopure™ water. Salt solutions were pH titrated using hydrochloric acid or sodium hydroxide and 0.2 µm- filtered before use. Binodal curves were mapped using the turbidity method, as described previously (Vijayaragavan et al., J Chromatogr B Analyt Technol Biomed Life Sci.2014; 967: 118-26). Tie lines were identified by first relating the citrate conductivity of the citrate-rich ATPS phase to a citrate weight percentage. A tie line extrapolated from this composition through the global composition of the tested ATPS connected to the PEG-rich phase composition on the opposite edge of the binodal curve. The calculations for tie line length (TLL) and tie line ratios (TLR) are described previously (Joshi et al., J Chromatogr B Analyt Technol Biomed Life Sci. 2019; 1126-1127: 121744). For glycine-containing systems, all solutions used contained 0.5 M glycine so that the concentration was consistent for all compositions and dilutions. [00078] ATPS Experiments. To form ATPS, 40 w/w% PEG stocks, 25 w/w% citrate stocks, 30 w/w% phosphate stocks, and Nanopure™ water were used to create systems on a two-gram scale. 10 v/v% of crude cell lysate was added to each system before vortexing for 30 seconds. The polymer stock was added to the system first, followed by the salt stock, the water, and the virus stock. Samples were centrifuged at about 250 × g for three minutes using a Thermo Scientific™ ST16R centrifuge and 30 x 2 mL fixed angle rotor. PEG- and citrate-rich phase volumes were visually estimated using microcentrifuge tube markers before harvesting for infectivity, DNA, and protein assays. Step recovery of PPV for step 1 ATPS was determined using the following equation: 1_{0்ುಶಸ/^^^ ∗ ^^ ^^^^^^^^ 1 ^^^^^^ ^^^^^^^^^^^^^^^^ ^%^ ^ாீ/^^௧ ∗ 100} [00079] where T per , volume of the phase (mL), PEG is PEG-rich phase, Cit is the citrate-rich phase, and S is the PPV stock added into the ATPS. Step recovery of PPV for step 2 ATPS was determined using the following equation: 1_{0்ುಶಸ/^^^ ∗ ^^ ^^^^^^^^ ^^^^^^ ^^^^^^^^^^^^^^^^ ^ாீ/^^௧ ∗ 100} Docket No.066040-0021-WO01 [00080] where P1 is the titer and volume harvested from the step 1 PEG-rich phase. The partition coefficients (K) for steps 1 and 2 were calculated using the following equation: ^_{^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ^^^^ ^^^^^^ െ ^^^^^^ℎ ^^ℎ^^^^^^ ^^^^^^^^ ൌ ^^^^ ൬ ^^^^^^^^^^ ^^^^^^^^^^^^^^^^^^ ^^^^ ^^^^^^^^^^^^^^ െ ^^^^^^ℎ ^^ℎ^^^^^^} ^ [00081] Impurity was quantified using the Quant-iT™ PicoGreen™ dsDNA Assay Kit from Life Technologies (Grand River, NY). Host cell protein in the citrate-rich phase was quantified using Pierce™ Coomassie (Bradford) Protein Assay Kit from Thermo Fisher Scientific (Waltham, MA). DNA and protein concentrations were quantified according to the manufacturer’s instructions. Samples were spin-filtered to remove interfering citrate content. PEG-rich samples were not tested because the PEG could not be removed by spin filtration. Recovery for both impurities was calculated for batch experiments using the equation below: ^_{^ ^^^^^^^^^^^^^^^^ ^^^^^^^^^^^^^^^^ ^%^ ൌ ^^௧ ∗ ^^^^௧ ∗} [00082] where ^^ is the phase (^^^^^^) or the stock (^^). ^^ is the volume of the citrate-rich phase or the stock. [00083] Host cell protein was qualitatively analyzed using sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS-PAGE). Before electrophoresis, all experimental samples were treated with 2x Laemmli SDS Sample Reducing Buffer and 8 mM β- mercaptoethanol before incubation at 90°C for 10 minutes. NuPAGE™ 4-12% Bis-Tris gels were loaded with 20 μL of treated experimental samples and 10 μL of Precision Plus Protein™ Dual Color Standard. Gels were run for 50 minutes at 200 V in NuPAGE™ MOPS SDS Running Buffer. After electrophoresis, the gels were stained using SimplyBlue™ SafeStain according to manufacturer instructions. [00084] Glycine Detection. Glycine concentrations in the salt phase were quantified using LC-MS. An electrospray ionization method on a Thermo Scientific™ Ultimate 3000™ HPLC paired with a Thermo Fisher Orbitrap Elite™ mass spectrometer was used to detect glycine in ATPS samples by comparing each sample to a stable glycine isotope and standard curve of a known concentration. The LC-MS method and analysis were described previously (Joshi et al., Biotechnol Bioeng.2021; 118(8): 3251-3262). Docket No.066040-0021-WO01 [00085] Conductivity and Tie line length determination. The tie lines were determined by conductivity measurements of the citrate-rich (bottom) phase using a VWR^® sympHony™ conductivity meter (Radnor, PA), and intrapolating the citrate compositions from a standard curve. The endpoints of the tie lines were determined by calculating the remaining amount of citrate in the PEG-rich (top) phase by completing a mass balance of the system. The tie lines were characterized by their tie line length (TLL), calculated as, T_{LL ൌ ^∆x ଶ ^^ୋ ^ ∆x^} ^ଶ ୟ_୪^ [00086] where Δx² _PEG is the PEG in the top phase and bottom phase and Δx² _salt is the concentration difference of citrate in the top phase and bottom phase. Tie line ratios were calculated as l_{ength ^} ^ത _T ^ത _S ^ത _{^ TL ratio ൌ length^} ^ത _S ^തത _B ^ത _^ [00087] where S is the system composition point, T is the top node, and B is the bottom node. The volume ratios were calculated as ^Volume^ V_{R ൌ ^୭୮୮୦ୟ^^} ^Volume^_{ୠ୭^^୭୫୮୦ୟ^^} [00088] Viscosity Measurements. A Malvern Panalytical Kinexus^® Lab+ shear rheometer (Malvern Panalytical, Malvern, United Kingdom) was used to collect viscosity data across a range of shear rates. Preequilibrated 12 kDa PEG-rich phases at TLL 12 and 32 and 8 kDa PEG phases at tie line lengths 20 and 34 were homogenized on a shaker table for 30 minutes at 100 rpm. Once homogenized, each sample was transferred to a Couette cup and thermally equilibrated to 25.0 ± 0.1°C for 5 minutes using a Peltier cylinder cartridge (Malvern Panalytical). Each sample underwent a logarithmic shear rate ramp from 0.01 to 3000 s^-1 over seven minutes, with ten data points stored per shear rate decade. PEG phases from each tie line length were measured in triplicate. [00089] Statistical Analysis. Minitab^® 20.4 (62-bit) (State College, PA) was used to determine the statistical significance of the datasets. The Grubbs test with a confidence level of 0.05 was used to identify and remove outliers. The 2-sample t-tests without assumed equal variances were used to confirm whether datasets were significantly different. Partition Docket No.066040-0021-WO01 coefficients (K) were converted out of log form before performing the 2-sample t-test. PPV titration using the MTT assay included three repeats and three to ten replicates for each step and condition. Example 3 Two-step ATPS [00090] The chemical conditions of a two-step ATPS were optimized for the non-enveloped virus PPV. PPV, a non-enveloped, single-stranded DNA virus with a relatively small diameter of 18-26 nm and an isoelectric point of about 5.0. PPV is infectious to swine and is in the same family as canine parvovirus and adeno-associated virus. This makes PPV a relatively safe virus model for both parvovirus vaccine and gene therapy purification. [00091] The two-step ATPS was designed for purification and recovery of PPV. During step 1, as shown in FIG.1A, PPV is partitioned into the PEG-rich phase and purified from the cell culture impurities that partition to the citrate-rich phase or the interface. However, to process the PPV further downstream, the PPV cannot remain in the viscous PEG-rich phase. In step 2, the virus-laden PEG is extracted, and fresh citrate is added to change the chemical conditions and encourage PPV to partition into the citrate-rich phase. PEG molecular weight, salt type, phase component concentration, phase component ratios, pH, and glycine addition were all investigated to find optimized conditions for PPV recovery after two-step ATPS. [00092] ATPS are liquid-liquid extractions defined by binodal curves unique to their chemical properties. Below the binodal curve (FIG.1B) is the one-phase region where PEG and citrate are miscible, while above the binodal curve are compositions at which PEG is salted-out into a second phase. ATPS whose equilibria represent the same Gibb’s free energy lie on the same tie line, which are described by the tie line length (TLL). As the concentrations of PEG and citrate increase, the Gibb’s free energy increases, leading to stronger driving forces, higher phase component concentrations, and a higher TLL. All points on the same tie line have the same PEG-rich and citrate-rich phase concentrations, and are where the tie line intersects the binodal curve. Different compositions on the same tie line have different volume ratios of each phase. ATPS volume ratio is expressed as the tie line ratio (TLR), where a TLR less than one indicates there is a higher proportion of PEG-rich phase and a TLR greater than one indicates a higher proportion of citrate-rich phase. Binodal curves must be characterized when chemical properties such as pH, PEG MW, and PEG manufacturing lots change; therefore, distinct Docket No.066040-0021-WO01 binodal curves were determined for each PEG molecular weight, salt type, and system pH (FIGS.2A-H). The slight binodal curve shifts between 12 kDa and 8 kDa PEG for three distinct pHs are shown in FIGS.3A-C. In two chemically different systems, the same TLL may relate to different PEG-citrate concentrations, and therefore, to two different Gibb’s free energy states. Characterization of binodal curves is essential to understanding system thermodynamics and the resulting purification outcomes. [00093] Binodal curves and tie lines were used to plan and explore step 1 and step 2 ATPS conditions. It has been shown that both TLL and TLR highly impact the partitioning of viruses. A variety of ATPS were tested to optimize PPV purification, as shown in TABLE 1 with detailed compositions in TABLE 2. Step 1 was conducted at a high TLL and TLR to increase PPV recovery in the PEG-rich phase. Step 2 was conducted at a low TLL and TLR to encourage the PPV to partition back to the salt-rich phase. Conditions were optimized using the parameters of pH (FIG.4A), TLR (FIG.4B), and PEG MW (FIG.4C). A summary of PPV recoveries for all conditions can be found in TABLE 3 with experimental titers in TABLE 4. The high variability in PPV recovery results is largely due to the 0.5 log error associated with the cell-based MTT infectivity assay. PPV can also be recovered by ddPCR (TABLE 5). TABLE 1. ATPS Compositions Tested. Step 1 Step 2 Tie Line Tie Line e osp ae sa use o sep . TABLE 2. ATPS Compositions. Docket No.066040-0021-WO01 PEG Step Tie Line Tie PEG Salt Volume MW (1 or pH Len th Line Concentration Concentration Ratio t) TABLE 3. PPV Recoveries and Partition Coefficients from Tested Systems. ATPS Step 1 Step 2 Overall Step 1 Step 2 (Table 1) Recovery Recovery Process Partition Partition experiments using the same step 1 condition. † Same as 8B Same as 8A TABLE 4. Experimental PPV Titers. Label (Table Starting Titer Step 1 PEG Titer Step 2 Citrate Titer Docket No.066040-0021-WO01 8.7 7.5 6.9 8.7 7.9 N/A 87 75 72 TABLE 5. Experimental PPV Titers and ATPS recovery by ddPCR. Step 1 Step 2 Step 1 Step 2 Overall Starting i i i y Docket No.066040-0021-WO01

Docket No.066040-0021-WO01 Example 4 pH Shift Effect, TLR, and PEG MW Effect on ATPS Performance [00094] Among the factors studied, the greatest improvement in PPV recovery was found by implementing a pH shift between steps. Initial pH shift experiments were done using 8 kDa PEG. Shifting from pH 8 or 7 in the first step to pH 6 in the second step resulted in 71-73% higher PPV step 2 ATPS recovery compared to keeping a constant pH 7 for both steps, as shown in FIG.4A. This translated to 52-66% recovery overall after the two ATPS steps. Since the isoelectric point of PPV is 5.5, the viral net charge is negative at pH 7 and 8 but is close to neutral at pH 6. The citrate salt carries three negative charges at pH 7 and 8, and mainly carries two negative charges at pH 6 (TABLE 6, FIGS.5A-D). For these two reasons, the repulsive electrostatic force that initially drove the PPV into the PEG-rich phase is greatly reduced when step 2 ATPS is at pH 6, thus allowing the PPV to partition back to the citrate-rich phase. The pH 7 binodal curve is shifted slightly away from the axes compared to the pH 8 and pH 6 curves; this may be due to slight differences in buffer conductivity after pH adjustment. TABLE 6. Ionic Species Percentage. Charge -1 -2 -3 Density Polarizability Polarizability [00095] Adjustment of the TLR at a constant TLL can significantly adjust the step 2 recovery of PPV in PEG-citrate ATPS. Increasing the TLR of step 2 ATPS, thereby increasing the proportion of the citrate-rich phase, successfully increased PPV recovery in 8 kDa PEG ATPS. A higher TLR of 3.7 improved the average step 2 ATPS recovery by 47% compared to the low TLR of 0.2 (FIG.4B). Increasing the ratio of the citrate-rich phase may drive the PPV particles towards that phase to maintain PPV concentrations, with the effect of increasing the overall recovery in that phase. Docket No.066040-0021-WO01 [00096] High molecular weights of PEG have been shown to drive higher recoveries into the PEG-rich phase due to increased amphiphilic forces between the viral product and the PEG. 12 kDa PEG and 8 kDa PEG ATPS were compared for their ability to recover PPV first into PEG- rich phase at a high phase component concentration, and then into citrate-rich phase at a lower phase component concentration, without a pH shift for comparison to past results (FIG.4C). The 8 kDa binodal curve is slightly shifted away from the axes compared to the 12 kDa curve (FIGS.3A-C), which is expected due to its lower molecular weight and likelihood of being salted out. The 12 kDa PEG showed better performance when the pH condition was kept at pH 7 for both steps, but showed a drastic reduction in recovery once a pH shift was implemented. There was no significant difference between PPV recovery into 12 kDa or 8 kDa PEG during step 1. At a constant pH, 12 kDa condition yielded 36% higher PPV recovery into the citrate-rich phase during step 2 (12A versus 8D). Both conditions used pH 7 for both steps and a tie line ratio of 0.5 or less for step 2 ATPS. [00097] A 12 kDa PEG system shifting from pH 7 to pH 6 was also explored. Surprisingly, poor step 2 ATPS recovery resulted from the 12 kDa PEG pH-shifted ATPS with an overall PPV recovery of 10%, as shown in FIG.4C (ATPS 12B). The combined step recovery for the PEG- rich and citrate-rich step 2 ATPS phases was below 20%, indicating that PPV precipitated or was lost at the interface between phases. This unexpected result could be due to precipitation of PPV by PEG; 8 kDa PEG has been used to precipitate PPV at concentrations as low as 7.5 w/w% when supplemented with 1.5 M NaCl, and PEG-induced precipitation of proteins increases with molecular weight. 8 kDa PEG was used in subsequent ATPS experiments utilizing a pH shift to avoid potential precipitation and PPV loss. Moreover, 8 kDa PEG ATPS is significantly less viscous than 12 kDa PEG ATPS (TABLE 7) and is thus easier to work with at manufacturing scale. The partition coefficients of PPV for systems 12A and 12B are shown in FIG.4C. TABLE 7. PEG Viscosities in ATPS. PEG MW ATPS Label PEG Conc. Step 1 PEG Step 2 PEG s) Example 5 Docket No.066040-0021-WO01 ATPS Impurity Removal [00098] ATPS must deliver high impurity removal along with product recovery to be a feasible purification method. Batch ATPS studies for the 8 kDa PEG, pH 7-to-6 shifted system which had the highest PPV recovery (8B in TABLE 1) showed 2.0 ± 1.1 logs of removal for host cell protein and 1.0 ± 0.3 logs of removal for host cell DNA after the two-step ATPS process (FIG. 6A). The final titers of host cell protein and DNA appear consistent with final titers being 13 ± 10 µg/mL and 8 ± 4 ng/mL, respectively, despite widely varying impurity levels present in the crude virus loaded at the beginning of the process (FIGS.6B-C). There is variation in starting protein and DNA titers since these are byproducts of the virus propagation process and their final titers highly depend on the extent of virus infection before harvest. Multi-stage ATPS has previously delivered close to a log of protein removal, and this study furthermore shows that higher log removals are possible for contaminating DNA and protein depending on the starting impurity levels. These results indicate that ATPS can deliver predictable impurity output levels even with variable input levels, making ATPS a robust purification method. Protein removal was visualized using SDS-PAGE to show a pure end product (FIG.6D). Protein and DNA impurity concentrations are listed in TABLE 8. TABLE 8. Experimental DNA and Protein Amounts. Starting Amount Step 1 Citrate Step 2 Citrate l Example 6 Isolated Step 2 ATPS [00099] To better understand the second ATPS step, it was performed in isolation with crude PPV, and not using step 1 purified PPV. The PPV stock was mixed directly with 8 kDa PEG and pH 6 citrate at TLL 24 and TLR 3.7 as used for 8A and 8B step 2 ATPS (TABLE 1). This was done to see if coating the PPV in PEG, as likely occurred during the step 1 ATPS where the Docket No.066040-0021-WO01 virus was recovered in the PEG-rich phase, would affect the step 2 recovery of the virus into the salt-rich phase. [000100] The recovery of PPV into the citrate-rich phase drastically increased when step 2 ATPS conditions were performed in isolation versus when they are used as a secondary ATPS step (FIGS.7A-B). However, the isolated recovery values may be artificially high, as demonstrated by an average recovery of 200%. Since the step 2 ATPS recovery for a two-step process is already near 100%, it does not appear that the existing amphiphilic attraction between the virus and PEG-rich phase restricts the virus transport back into the citrate-rich phase. The artificially high recoveries are likely an artifact of the MTT infectivity assay, which has inherently high variability due to its reliance on living cells and can be difficult to get consistent results when the virus is in the highly viscous PEG-rich phase. Example 7 Other Explored ATPS: Glycine Addition, Phosphate Salt Phase, and ATPS Order of Addition [000101] Glycine was added to the first step of the most successful 8 kDa PEG, pH 7-to-6 shifted citrate ATPS. Glycine has additionally been shown to increase PPV partition to the PEG-rich phase by osmotic displacement. Glycine addition shifts the binodal curve towards the axis, which indicates that it drives salting-out of PEG by the salt phase (FIG.8A). Improved recovery with glycine addition appears to be dependent on TLL. Two TLLs were tested: TLL 33 which was close in TLL to the glycine-free control’s TLL 34, and TLL 42, which contained the same global compositions of PEG and citrate as the glycine-free control. The difference between TLL and global compositions occurs because the glycine shifts the binodal curve towards the axis, as shown in FIG.8A. Both glycine-added systems and the control had the same glycine-free pH 6 citrate ATPS for step 2. There was no significant difference in the step 1 PPV recovery or partitioning between the control and the TLL 42 glycine conditions (FIGS. 8B-C). The step 1 recovery of the TLL 33 condition appears very poor, but an inflated step 2 recovery indicates that the step 1 recovery is more similar to the other conditions than it appears. Glycine addition did not significantly improve overall PPV recovery in the 8 kDa PEG ATPS. [000102] Both steps 1 and 2 of the glycine-containing systems were analyzed using LC-MS to determine whether glycine was carried over to the second step. Near 100% of the added Docket No.066040-0021-WO01 glycine was recovered into the citrate-rich phase during step 1 ATPS (FIGS.9A-F). Only 0.5% of the added glycine was detected in the step 2 ATPS citrate-rich phase. Therefore, glycine likely does not affect the partitioning during step 2 ATPS. Past successes with glycine were achieved with 12 kDa PEG ATPS, so larger PEG MW or hydrophobicity may be required to achieve gains in PPV recovery using glycine addition. [000103] Previously, PEG-phosphate ATPS showed PPV partitioning towards the interface compared to PEG-citrate ATPS. It was hypothesized that PPV would more readily partition into the salt phase if phosphate were used as the step 2 salt because phosphate typically carries one negative charge at pH 6 while citrate carries two or three negative charges (TABLE 6, FIGS.5A-D). However, at similar pH and concentration PEG-phosphate step 2 ATPS provided 75% lower step 2 recovery (FIG.10B). The near-zero partition coefficients seen for PEG- phosphate ATPS indicate that PPV is being lost at the interface between the polymer and salt- rich phases (FIG.10C). The PEG-phosphate binodal curve is shifted towards the axes compared to PEG-citrate curves, indicating greater salting-out ability of PEG. The binodal curve shift agrees with the phosphate ATPS reduced step 2 recovery of PPV as compared to citrate. The strong salting-out of both PEG and PPV by phosphate indicates that there is more to the salting-out and salting-in behavior of viruses than charge alone. [000104] One explanation for the poor PPV recovery in phosphate stems from how the phosphate ion interacts with the virus surface. Molecular simulations of ion-protein interactions have shown that specific protein backbone and side chain interactions influence salting-out behavior beyond what is expected simply based on ion hydration. Non-enveloped viruses like PPV have a protein capsid for their outer surface, so ion-protein interactions should similarly affect salting-out. When net negatively charged PPV capsids are repelled from highly negative salt ions, their hydrophobic residues are more likely to interact. This dual electrostatic and hydrophobic action drives the salting-out of the virus. [000105] Phosphate ions have a similar charge density to citrate ions at pH 6, so charge density is not responsible for its stronger salting-out behavior. However, the phosphate ions have a relatively low proportion of polarizability, which describes the tendency of a molecule to form a dipole (TABLE 6, FIGS.5A-D). Citrate may show weaker salting-out behavior due to its ability to polarize and have a more dispersed charge across the ion. PPV may be able to return to the citrate-rich phase at pH 6 since citrate’s negative charge is less concentrated. In future Docket No.066040-0021-WO01 work with different viruses, it will be interesting to see how virus net charge and charge patterns interact with citrate polarizability to drive recovery into the citrate phase. [000106] The order of addition of PEG or citrate was studied to see if this could change the recovery of virus in ATPS. Pre-exposure of protein to PEG or salt before incorporation into the ATPS has been shown to affect step 2 recovery. Bovine serum albumin recovery was reduced by 40% during two-step ATPS when the protein was pre-exposed to PEG before the addition of phosphate. In this work, step 2 PPV recovery did not change significantly whether PEG or citrate was mixed with PPV before adding the other ATPS components (FIG.11A). Interactions between PEG and PPV appear more reversible than interactions between PEG and bovine serum albumin. For this study, PPV was typically added with the citrate stock. [000107] The extent and direction of water transport between step 1 and step 2 ATPS was tested to understand if water movement can facilitate viral transport. When one ATPS component is mixed with water before adding the second component, water transport flows towards the phase that is dominated by the second component to create equilibrium. It was tested whether water transport in the direction of the desired viral transport significantly improved recovery. Dry citrate salts were used during step 2 ATPS instead of the pre-dissolved citrate stocks, as used for all other experiments. As water was drawn out of the PEG-rich phase to dissolve the citrate salt and form the salt-rich phase, it was expected that PPV would be caught in the convective transport and driven into the citrate-rich phase, therefore increasing recovery. However, no significant increase in PPV recovery was seen using citrate salts or citrate stocks (FIG.11B). Example 8 Guidance for ATPS Development [000108] One of the main drawbacks of ATPS for industrial applications is that the design space is too large for the time often given for downstream process development. The findings of this study can guide the development of ATPS for new viruses to reduce the design space. Based on the results, development should begin by considering the isoelectric point of the virus. A pH that will result in a net negative viral charge should be chosen for step 1 ATPS to drive the salting-out of the virus into the PEG-rich phase. The pH must be shifted in step 2 ATPS to neutralize the virus’s isoelectric point. Based on these studies, it appears that the diffuse citrate ion makes it particularly well-suited to recover the virus from PEG into the salt-rich phase. Docket No.066040-0021-WO01 [000109] Once appropriate pHs have been chosen for both ATPS steps, it is critical to map ATPS binodal curves and tie lines; high TLL with low TLR should be chosen for step 1 ATPS and low TLL with high TLR should be chosen for step 2 ATPS. It is essential to map the ATPS system since variabilities in the polydisperse high molecular weight PEG can result in shifting of the binodal curve. The step 2 ATPS is especially sensitive to binodal curve shifts since it lies close to the binodal curve. Were ATPS implemented into an industrial process, a vendor that can provide a less polydisperse high molecular weight PEG could reduce variability in the process. Future studies should confirm the consistency of ATPS impurity outputs despite variabilities in starting impurity profiles. ATPS is notorious for experiment-intensive development. By using this study as a starting point, the screening of ATPS conditions for new viruses can be simplified. Example 9 ATPS Purification Process is an Effective and Efficient Two-Step Extraction Process [000110] ATPS is a low-cost, generic materials-based purification method for viral products that can be adapted to operate continuously. However, most viral ATPS studies report partitioning into the PEG-rich phase or interface, which complicates downstream process steps. This study reports an optimized two-step ATPS that eases downstream processing and continuous processing by back-extracting the viral product from the viscous PEG-rich phase into the salt-rich phase. ATPS using 8 kDa PEG and a pH 7-to-6 shifted citrate yields an average of 66% infectious PPV overall recovery with 2.0 logs of protein removal and 1.0 logs of DNA removal. During the first ATPS step, high TLL, low TLR, and pH above the isoelectric point of PPV work together to drive the virus into the PEG-rich phase. During the second ATPS step, TLL close to the binodal curve, high TLR, and pH near the isoelectric point of PPV work together to draw PPV back into the salt-rich phase. Glycine addition during step 1 and phosphate use during step 2 were not found to be influential. The failure of phosphate salt to improve recoveries indicates that the high polarizability of the citrate ion may encourage its interaction with PPV when the virus is near its isoelectric point. 8 kDa PEG was chosen over 12 kDa PEG because of its lower viscosity and potential viral precipitation encountered with the 12 kDa PEG. Product recovery was not significantly improved by influencing water transport during step 2 ATPS. Docket No.066040-0021-WO01 [000111] In summary, an ATPS purification process was developed for PPV that is reliant on inexpensive generic materials but with competitive product recovery compared to traditional purification methods. The design of an effective and efficient two-step extraction paves the way for ATPS to become a powerful unit operation in the field of viral particle manufacturing. Such a two-step system could be adapted to continuous processing for rapid industrial-scale production of vaccine or gene therapy candidates. Example 10 Shifted pH and Concentration Improve Multi-Stage Aqueous Two-Phase Extraction Recoveries for a Non-Enveloped Virus [000112] The COVID-19 pandemic accentuated the value of time- and cost-effective vaccine manufacturing in an effort to quickly distribute doses to patients. Continuous manufacturing could reduce the manufacturing time and footprint necessary to produce vaccines. Aqueous two-phase extraction (ATPE) provides a purification method more cost-effective and more compatible with continuous processing than traditional liquid chromatography purification. ATPE is here composed of semi-miscible polyethylene glycol (PEG) polymer and citrate salt solutions. By leveraging hydrophobic and electrostatic interactions between the viral product and the ATPE components, the partitioning behavior of the viral product can be adjusted. By changing pH, component concentrations, and volume ratios, a non-enveloped vaccine model porcine parvovirus (PPV) was partitioned initially to the polymer phase in stage one and secondly to the salt phase in stage two to facilitate purification, recovery, and processability. [000113] During the first stage of ATPE, PPV must be partitioned to the PEG phase in order to separate it from cell culture impurities. At a neutral pH, PPV is negatively charged due to its low isoelectric point. The citrate salt solution also contains a net negative charge at this pH and exerts a repulsive force on the PPV. Meanwhile, PPV’s amphiphilic nature interacts favorably with the amphiphilic PEG molecules, coaxing an average of 60% of the PPV particles into the PEG phase with removal of 95% of host cell DNA and 89% of host cell proteins. However, the PEG solution is highly viscous. PPV must be back-extracted into the salt solution to allow for further processing. This can be achieved by reversing the aforementioned conditions. By harvesting the PPV-laden PEG and introducing a more dilute citrate, the repulsive and amphiphilic forces fade to permit an average of 99% of PPV particles into the citrate phase. Docket No.066040-0021-WO01 This process yields an average 49% recovery of PPV particles. PPV recovery may be improved by supplementing the first stage with osmolytes to further displace PPV into the PEG phase. [000114] When adapted to process continuously, the PPV recoveries have corresponded to batch experiments. This continuous purification framework presents a strategy for more efficient vaccine manufacturing at a lower cost compared to traditional methods, which could reduce cost and increase access to patients in need of therapies. [000115] The ATPS can also be used for continuous processing of viral vectors (FIG.12 and FIGS.13A-B). Example 11 Virus Recovery Data [000116] TABLE 9 shows that the two step ATPS described can also be used for AAV9. TABLE 9. AAV9 Virus Recovery. n n S^o _i ^t %y^r o n n e ⁱ _t D o S ⁱ _t ⁿ _l o p n ) ⁱ _t ^k o n_l ⁱ _t D S 7 62 29 0⁵ .2 Docket No.066040-0021-WO01 pH 8.5 shift from 8.5 in the → 6 first step to 6 in the second step [000117] TABLE 10 shows that the two step ATPS described can also be used for HSV. TABLE 10. Human simplex virus (HSV) Recovery. ) n ₎ n ₎ n _y ^K ₍ ^K ₍ n o_{i l} ^{r %} oⁱ _t D n oⁱ _t n oⁱ _t D S .0 .8 .6 .8 Docket No.066040-0021-WO01 [000118] TABLE 11 shows that the two step ATPS described can also be used for AAV2. TABLE 11. Adeno Associated virus 2 (AAV2) Recovery. ) n ₎ n ₎ n o _{y i} ^t _{i l} ^{r %} o^{i K a} _e ^, _{t t (}n o^{i K L} _{t (} n n o^{i L} _{t t} v ^p _i D ^p _i D ^p _i D S .0 .0 0 .0 [000119] TABLE 12 shows that the two step ATPS described can also be used for lentivirus. TABLE 12. Lentivirus Recovery. ) ₎ n _{y )} n ^K ₍ n ^K ₍ n o _l ^{r %} oⁱ oⁱ oⁱ D S NA NA NA NA Docket No.066040-0021-WO01 [000120] TABLE 13 shows that the two step ATPS described can also be used for IBV. All the data in TABLE 13 are from HA assay except for pH7-6 TLR which is from ddPCR. ^{TABLE 13. Influenza B virus (IBV) Recovery.} y^r ₎ n ₎ n ₎ n ^% o^{i K} ₍ o^{i K} ₍ noⁱ D S 3.4 2.1 2.1 0.5 2.4 [000121] Summary of the recovery data is also shown in FIG.14. [000122] FIG.15 shows the partition coefficients found for different two step ATPS for HSV, AAV2, and LV. [000123] TABLE 14 shows how much protein impurity is removed for different viral vectors after purification with ATPS. TABLE 14. Protein impurity removal. Docket No.066040-0021-WO01 r ^l n ₎ o gu no o _{a )} ⁱ t S v% _t ^{( p} g_l ^{i a} _t P o _i D o_v ^p _i D S 0 .0 0 0 0 0 [000124] TABLE 15 shows the DNA removal for PPV. ^{TABLE 15. DNA Removal.} )L _{) )} L _{) )} Docket No.066040-0021-WO01 9^{284 1857 104 72 20 18 466 97.8} _{% of host cell DNA 1.7} ^{log removal of} 74 98 118 83 83.5 impurity remo host cell DNA 8200 1640 98 val of 0.8 crude HSV by impurity of crude S a, om ep p o^f A de S a, om ep p o^f A de S a, om ep p o^f A de S a, om ep p o^f A de S a, om ep p o^f A de with nd 8 in Docket No.066040-0021-WO01 the first step to 6 in the first step to 6 the second step in the second step o^f A de S a, om ep p o^f A de PS a, om st he p *** [000125] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. [000126] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents. [000127] All publications, patents, patent applications, and/or other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as Docket No.066040-0021-WO01 if each individual publication, patent, patent application, and/or other document were individually indicated to be incorporated by reference for all purposes. [000128] For reasons of completeness, various aspects of the invention are set out in the following numbered clauses: [000129] Clause 1. A method of isolating and concentrating viruses from a sample comprising: (i) mixing a first phase component and a second phase component to form a first aqueous two-phase system (ATPS); (ii) mixing the ATPS with a sample comprising a virus, wherein the first phase component comprises from about 20 wt.% to about 30 wt.% of a polymer dissolved in an aqueous solution, wherein the second phase component comprises from about 6.4 wt.% to about 10.8 wt.% of a salt dissolved in an aqueous solution, wherein the pH of the first ATPS is from about 7 to about 9, and wherein the viruses concentrate in the first phase component to form a first virus concentrated phase; (iii) mixing a third phase component with the first virus concentrated phase to form a second ATPS, wherein the third phase component comprises from about 8 wt.% to about 13 wt.% of a salt dissolved in an aqueous solution, wherein the first virus concentrated phase comprises from about 7.9 wt.% to about 12 wt.% of the polymer dissolved in an aqueous solution, wherein the pH of the second ATPS is from about 4.5 to about 7, and wherein the viruses concentrate in the third phase component to form a second virus concentrated phase; and (iv) recovering the viruses from the second virus concentrated phase. [000130] Clause 2. The method of clause 1, wherein recovering the viruses from the second virus concentrated phase comprises pipetting or pumping the second virus concentrated phase away from the first virus concentrated phase. [000131] Clause 3. The method of clause 1 or clause 2, wherein the method further comprises recovering the first virus concentrated phase before mixing with the third phase component. [000132] Clause 4. The method of clause 3, wherein recovering the viruses from the first virus concentrated phase comprises pipetting or pumping the first virus concentrated phase away from the second phase component. [000133] Clause 5. The method of any one of clauses 1-4, wherein the first virus concentrated phase comprises from about 0 wt.% to about 3 wt.% of the salt. Docket No.066040-0021-WO01 [000134] Clause 6. The method of any one of clauses 1-5, wherein the pH of the second ATPS is from about 5 to about 6. [000135] Clause 7. The method of clause 1, wherein the polymer is polyethylene glycol (PEG). [000136] Clause 8. The method of clause 7, wherein the molecular weight of the PEG is from about 4 kDa to about 12 kDa. [000137] Clause 9. The method of clause 7, wherein the molecular weight of the PEG is from about 6 kDa to about 12 kDa. [000138] Clause 10. The method of clause 7, wherein the molecular weight of the PEG is from about 8 kDa to about 12 kDa. [000139] Clause 11. The method of any one of clauses 1-10, wherein the salt is sodium citrate. [000140] Clause 12. The method of any one of clauses 1-11, wherein the aqueous solution is water. [000141] Clause 13. The method of any one of clauses 1-12, wherein the viruses are enveloped viruses, non-enveloped viruses, virus-like particles, or combinations thereof. [000142] Clause 14. The method of clause 13, wherein the viruses are Porcine Parvovirus, Herpes Simplex Virus, Adeno-Associated Virus, Influenza B, Lentivirus, influenza A, influenza A virus-like particles, or combinations thereof. [000143] Clause 15. The method of any one of clauses 1-14, wherein the first ATPS is at a high tie line length (TLL). [000144] Clause 16. The method of clause 15, wherein the TLL is from about 24 to about 42. [000145] Clause 17. The method of any one of clauses 1-16, wherein the first ATPS is at a high tie line ratio (TLR). [000146] Clause 18. The method of clause 17, wherein the TLR is from about 0.3 to about 0.5. Docket No.066040-0021-WO01 [000147] Clause 19. The method of any one of clauses 1-18, wherein the second ATPS is at a low tie line length (TLL). [000148] Clause 20. The method of clause 19, wherein the TLL is from about 12 to about 24. [000149] Clause 21. The method of any one of clauses 1-20, wherein the second ATPS is at a low tie line ratio (TLR). [000150] Clause 22. The method of clause 21, wherein the TLR is from about 0.3 to about 3.7. [000151] Clause 23. A system comprising the first ATPS and the second ATPS of any one of clauses 1-22.

Claims

Docket No.066040-0021-WO01 CLAIMS What is claimed is: 1. A method of isolating and concentrating viruses from a sample comprising: (i) mixing a first phase component and a second phase component to form a first aqueous two-phase system (ATPS); (ii) mixing the ATPS with a sample comprising a virus, wherein the first phase component comprises from about 20 wt.% to about 30 wt.% of a polymer dissolved in an aqueous solution, wherein the second phase component comprises from about 6.4 wt.% to about 10.8 wt.% of a salt dissolved in an aqueous solution, wherein the pH of the first ATPS is from about 7 to about 9, and wherein the viruses concentrate in the first phase component to form a first virus concentrated phase; (iii) mixing a third phase component with the first virus concentrated phase to form a second ATPS, wherein the third phase component comprises from about 8 wt.% to about 13 wt.% of a salt dissolved in an aqueous solution, wherein the first virus concentrated phase comprises from about 7.9 wt.% to about 12 wt.% of the polymer dissolved in an aqueous solution, wherein the pH of the second ATPS is from about 4.5 to about 7, and wherein the viruses concentrate in the third phase component to form a second virus concentrated phase; and (iv) recovering the viruses from the second virus concentrated phase. 2. The method of claim 1, wherein recovering the viruses from the second virus concentrated phase comprises pipetting or pumping the second virus concentrated phase away from the first virus concentrated phase. 3. The method of claim 1 or claim 2, wherein the method further comprises recovering the first virus concentrated phase before mixing with the third phase component. Docket No.066040-0021-WO01 4. The method of claim 3, wherein recovering the viruses from the first virus concentrated phase comprises pipetting or pumping the first virus concentrated phase away from the second phase component. 5. The method of claim 1, wherein the first virus concentrated phase comprises from about 0 wt.% to about 3 wt.% of the salt. 6. The method of claim 1, wherein the pH of the second ATPS is from about 5 to about 6. 7. The method of claim 1, wherein the polymer is polyethylene glycol (PEG). 8. The method of claim 7, wherein the molecular weight of the PEG is from about 4 kDa to about 12 kDa. 9. The method of claim 7, wherein the molecular weight of the PEG is from about 6 kDa to about 12 kDa. 10. The method of claim 7, wherein the molecular weight of the PEG is from about 8 kDa to about 12 kDa. 11. The method of claim 1, wherein the salt is sodium citrate. 12. The method of claim 1, wherein the aqueous solution is water. 13. The method of claim 1, wherein the viruses are enveloped viruses, non-enveloped viruses, virus-like particles, or combinations thereof. 14. The method of claim 13, wherein the viruses are Porcine Parvovirus, Herpes Simplex Virus, Adeno-Associated Virus, Influenza B, Lentivirus, influenza A, influenza A virus-like particles, or combinations thereof. 15. The method of claim 1, wherein the first ATPS is at a high tie line length (TLL). 16. The method of claim 15, wherein the TLL is from about 24 to about 42. Docket No.066040-0021-WO01 17. The method of claim 1, wherein the first ATPS is at a high tie line ratio (TLR). 18. The method of claim 17, wherein the TLR is from about 0.3 to about 0.5. 19. The method of claim 1, wherein the second ATPS is at a low tie line length (TLL). 20. The method of claim 19, wherein the TLL is from about 12 to about 24. 21. The method of claim 1, wherein the second ATPS is at a low tie line ratio (TLR). 22. The method of claim 21, wherein the TLR is from about 0.3 to about 3.7. 23. A system comprising the first ATPS and the second ATPS of claim 1.