Multi-step filtration and clarification methods for recombinant adeno-associated virus purification

Abstract

Provided are systems and methods for separating adeno-associated viral (rAAV) particles from host cell derived impurities and other byproducts in a preparation produced from a host cell culture.

Description

MULTI-STEP FILTRATION AND CLARIFICATION METHODS FOR RECOMBINANT ADENO-ASSOCIATED VIRUS PURIFICATIONCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application Nos. 63 / 637,054, filed on April 22, 2024. and 63 / 644,663, filed on May 9. 2024, the disclosures of each which are incorporated by reference herein in its entirety for all purposes.BACKGROUND

[0002] Recombinant adeno-associated viruses (rAAVs) engineered to carry' a heterologous nucleic acid of interest (e g., a gene encoding a therapeutic protein, an antisense nucleic acid molecule, a ribozyme, a miRNA, an siRNA, a nucleic acid encoding a CRISPR / Cas system, or the like) are increasingly being explored as therapeutic agents for various diseases. These rAAVs are engineered by deleting, in whole or in part, the internal portion of the AAV genome and inserting the heterologous nucleic acid of interest between the inverted terminal repeats (ITRs). The ITRs remain functional in such rAAVs, allowing replication and packaging of the rAAV particle containing the nucleic acid cargo enclosed within the AAV capsid. Typically, the heterologous nucleic acid is operably linked to regulatory sequences (e g., promoter and / or enhancer sequences) capable of driving expression of the cargo in a patient’s target cells.

[0003] There is a drive towards rAAV cell culture production methods using increasing cell densities for large scale manufacturing to reduce cost-of-goods and increase patient access to rAAV -based therapies. However, large scale manufacturing of these rAAVs may suffer from presence of impurities in rAAV preparations that may include particulate impurities, host cell debris, host cell proteins and helper virus particles. Conventional filtration schemes utilize depth filters designed for purification of traditional biologies (e.g., monoclonal antibodies and recombinant proteins). The filters and filtration schemes known in the art are not optimized for impurity profiles of a crude harvest derived from a rAAV producer cell line. At cell densities that yield high volumetric productivity of rAAV, the increased impurity burden prohibits the practical use of conventional filtration schemes.

[0004] This application addresses the challenge of effectively removing impurities from a rAAV preparation obtained from a producer cell line by implementing a filtration scheme tailored to remove specific impurities that predominantly contribute to fouling of clarification filters. The present disclosure provides addition of a pre-filter to reduce cells, cell aggregates, and cellfragments to enhance the capacity of the primary depth filter, and substitution of a traditional depth filter with an anion-adsorber medium to remove negatively charged colloids and increase the throughput across the bioburden reduction filter.SUMMARY

[0005] Clarification of rAAV producer cell lysate for removing contaminants including but not limited to host cell derived impurities and helper viral particles, is an important step before purifying the rAAV product. Most clarification methods known in the art rely on combinations of one or more depth filters with a bioburden reduction filter. There is a need in the art for new clarification methods and systems for effectively removing impurities from rAAV producer cell lysates to produce high purity preparations for further downstream purification of rAAV based drug product. The present disclosure provides methods and systems using a combination of prefilter, depth filter, anion exchange chromatography (AEX) filter and a bioburden reduction filter, for clarifying rAAV producer cell lysates. Such combinations depart from known rAAV clarification methods and systems and overcome prior operational issues including potential loss of product rAAV material and back logging of filter media.

[0006] The present disclosure provides methods of clarifying a cell lysate produced from a cell culture expressing recombinant adeno-associated virus (rAAV) particles, comprising: (a) passing the cell lysate through a prefilter medium to generate a first filtrate; (b) passing the first filtrate through a depth filter medium to generate a second filtrate; (c) passing the second filtrate through an anion adsorber medium to generate a third filtrate; and (d) passing the third filtrate through a bioburden reduction filter to generate a clarified cell lysate.

[0007] In some embodiments of the methods of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about IxlO6to about 2xl07cells / mL. In some embodiments of the methods of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density of at least about 106cells / mL. In some embodiments of the methods of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density' of at least about 8x106to about 2x107cells / mL.

[0008] In some embodiments of the methods of the present disclosure, the cell lysate has a pH of about 7.5 to about 8.0. In some embodiments of the methods of the present disclosure, the cell lysate has a pH of about 7.7 to about 7.9.

[0009] In some embodiments of the methods of the present disclosure, the cell lysate comprises rAAV particles and one or more of cellular complexes, cellular aggregates, colloidal aggregates, nucleases, nucleic acids, host cell proteins, lysed cell fragments, and nucleotide fragments.

[0010] In some embodiments of the methods of the present disclosure, the clarified cell lysate comprises the rAAV particles. In some embodiments of the methods of the present disclosure, the clarified cell lysate retains at least 80% of the total rAAV particles present in the cell lysate. In some embodiments of the methods of the present disclosure, the clarified cell lysate retains >85% of the total rAAV particles present in the cell lysate.

[0011] In some embodiments of the methods of the present disclosure, the cell lysate comprises helper virus particles. In some embodiments of the methods of the present disclosure, the helper virus particles are adenovirus particles. In some embodiments of the methods of the present disclosure, the adenovirus particles are adenovirus serotype 5 (Ad5) particles.

[0012] In some embodiments of the methods of the present disclosure, the cell lysate comprises the Ad5 particles at a density of about IxlO12to about 3xl012GC / ml. In some embodiments of the methods of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the anion adsorber filter is about 3xl016to about 5xl016GC / L of membrane volume (GC / L-MV). In some embodiments of the methods of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the anion adsorber filter is about 3xl016to about 7.25x 1016GC / L-MV.

[0013] In some embodiments of the methods of the present disclosure, the volume of the cell lysate is at least 50L. In some embodiments of the methods of the present disclosure, the volume of the cell lysate is at least 250L. In some embodiments of the methods of the present disclosure, the volume of the cell lysate is between about 50L and about 250L.

[0014] In some embodiments of the methods of the present disclosure, the prefilter medium has a pore size of about 20 pm to about 75 pm. In some embodiments of the methods of the present disclosure, the prefilter medium has a pore size of about 50 pm.

[0015] In some embodiments of the methods of the present disclosure, passing the cell lysate through the prefilter medium increases the volumetric capacity7of the depth filter by at least 25%. In some embodiments of the methods of the present disclosure, passing the cell lysate through the prefilter medium increases the filter capacity7of the depth filter by about 30 to about 50 L / m2

[0016] In some embodiments of the methods of the present disclosure, the flow rate of the cell lysate in the prefilter medium is about 225 to about 925 L / m2h. In some embodiments of the methods of the present disclosure, the prefilter medium has a volumetric capacity of about 300 to about 700 L / m2. In some embodiments of the methods of the present disclosure, the differential pressure in the prefilter medium is about 1 to about 5 psi.

[0017] In some embodiments of the methods of the present disclosure, the depth filter medium comprises any one of a cellulose-based, glass-based and acrylic-based filter medium. In some embodiments of the methods of the present disclosure, the depth filter medium comprises 2 to 8 layers of the filter medium. In some embodiments of the methods of the present disclosure, the depth filter further comprises any one of a diatomaceous earth (DE) filter aid and a silica filter aid. In some embodiments of the methods of the present disclosure, the depth filter medium comprises at least one polypropylene fiber pulp, a non-woven fiber layer or a combination thereof. In some embodiments of the methods of the present disclosure, the depth filter medium further comprises a cationic binder.

[0018] In some embodiments of the methods of the present disclosure, the flow rate of the first filtrate in the depth filter medium is about 50 to about 150 L / m2h. In some embodiments of the methods of the present disclosure, the volumetric capacity of the depth filter medium is about 70 to about 200 L / m2. In some embodiments of the methods of the present disclosure, the differential pressure in the depth filter medium is about 1 to about 18 psi.

[0019] In some embodiments of the methods of the present disclosure, the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder. In some embodiments of the methods of the present disclosure, the depth filter medium comprises a first layer of non-woven fiber, a second layer of polypropylene fiber pulp, and a third and fourth layer, the third and fourth layers comprising polyacrylic fiber pulp, silica filter aid and cationic binder. In some embodiments of the methods of the present disclosure, the depth filter medium comprises six layers of polypropylene fiber and non-woven fiber, a second layer of cellulose fibers, and a third layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder. In some embodiments of the methods of the present disclosure, the anion adsorber medium is any one of a strong anion exchange chromatography (AEX) medium or an AEX hybrid medium.

[0020] In some embodiments of the methods of the present disclosure, the strong AEX medium is any one of Sartobind® Q medium, Mustang® Q medium or Natrix® Q medium. In someembodiments of the methods of the present disclosure, the AEX hybrid medium comprises AEX chromatography medium and a polyamide membrane. In some embodiments of the methods of the present disclosure, the AEX hybrid medium comprises AEX chromatography medium and polyether sulfone (PES) membrane. In some embodiments of the methods of the present disclosure, the AEX chromatography medium is a quaternary ammonium salt functional AEX medium. In some embodiments of the methods of the present disclosure, the quaternary ammonium salt functional AEX medium is non-woven medium. In some embodiments of the methods of the present disclosure, the AEX chromatography medium is any one of a Harvest® RC medium or Emphaze® AEX medium.

[0021] In some embodiments of the methods of the present disclosure, the pore size of the polyamide membrane or the PES membrane is about 0. 1 to about 0.5 pm. In some embodiments of the methods of the present disclosure, the pore size of the polyamide membrane or the PES membrane is 0.2 pm. In some embodiments of the methods of the present disclosure, the flow rate of the first filtrate in the anion adsorber medium is about 50 to about 200 L / m2h. In some embodiments of the methods of the present disclosure, the volumetric capacity of the anion adsorber medium is about 70 to about 200 L / m2In some embodiments of the methods of the present disclosure, the differential pressure of the anion adsorber medium is about 1 to about 10 psi. In some embodiments of the methods of the present disclosure, the conductivity of the filter load of the adsorber medium is about 16 to about 40 mS / cm. In some embodiments of the methods of the present disclosure, the conductivity of the filter load is about 18 to about 37 mS / cm.

[0022] In some embodiments of the methods of the present disclosure, the bioburden reduction filter comprises any one of a poly vinylidene fluoride (PVDF)-based filter, a PES-based filter, and a polyamide-based filter. In some embodiments of the methods of the present disclosure, the flow rate of the third filtrate through the bioburden reduction filter is about 300 to about 700 L / m2h. In some embodiments of the methods of the present disclosure, the volumetric capacity of the bioburden reduction filter is about 500 to about 750 L / m2. In some embodiments of the methods of the present disclosure, the differential pressure limit of the bioburden reduction filter is about 20 to about 25 psi.

[0023] The present disclosure also provides a system for clarifying a cell lysate produced from a cell culture expressing recombinant adeno-associated virus (rAAV) particles, comprising: (a) a first module comprising a prefilter medium that filters the cell lysate to remove particulate matter of size > 50 pm from the cell lysate to generate a first filtrate; (b) a second module comprising adepth filter medium that filters the first filtrate to remove particulate matter of size of >lpm to <50 pm from the first filtrate to generate a second filtrate; (c) a third module comprising an anion adsorber medium that filters the second filtrate to remove adenovirus particles from the second filtrate to generate a third filtrate; and (d) a fourth module comprising a bioburden reduction filter that filters the third filtrate to remove microbial contaminants from the third filtrate to generate a clarified cell lysate.

[0024] In some embodiments of the systems of the present disclosure, the cell lysate is produced from a rAAV expressing cell culture with a cell density of about IxlO6to about 2xl07cells / mL. In some embodiments of the systems of the present disclosure, the cell lysate is produced from a rAAV expressing cell culture with a cell density of about 106cells / mL. In some embodiments of the systems of the present disclosure, the cell lysate is produced from a rAAV expressing cell culture with a cell density' of about 6x106to about 2x107cells / mL.

[0025] In some embodiments of the systems of the present disclosure, the cell lysate has a pH of about 7.5 to about 8.0. In some embodiments of the systems of the present disclosure, the cell lysate has a pH of about 7.7 to about 7.9.

[0026] In some embodiments of the systems of the present disclosure, the helper virus adenoviral particles are adenoviral particles. In some embodiments of the systems of the present disclosure, the adenoviral particles are Ad5 particles. In some embodiments of the systems of the present disclosure, the cell lysate comprises the Ad5 particles at a density' of about IxlO12to about 3x1012GC / ml. In some embodiments of the methods of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the anion adsorber filter is about 3xl016to about 5x1016GC / L-MV. In some embodiments of the methods of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the anion adsorber filter is about 3x1016to about 7.25x1016GC / L-MV.

[0027] In some embodiments of the systems of the present disclosure, the volume of the cell lysate is at least 50L. In some embodiments of the systems of the present disclosure, the volume of the cell lysate is at least 250L.

[0028] In some embodiments of the systems of the present disclosure, the prefilter medium is any one of polypropylene fleece based pre-filter, adsorptive glass fleece material based pre-filter, mixed cellulose esters, nylon membrane pre-filter, glass and quartz fiber pre-filter, polyvinyl chloride and polytetrafluoroethylene. In some embodiments of the systems of the presentdisclosure, the prefilter medium has a pore size of about 20 micrometers (pm) to about 75 pm. In some embodiments of the systems of the present disclosure, the prefilter medium has a pore size of about 50 pm.

[0029] In some embodiments of the systems of the present disclosure, the depth filter medium is any one of a cellulose-based, glass-based and acrylic-based filter medium. In some embodiments of the systems of the present disclosure, the depth filter medium comprises between 2-8 layers of the filter medium. In some embodiments of the systems of the present disclosure, the depth filter further comprises any one of diatomaceous earth (DE) filter aid and a silica filter aid. In some embodiments of the systems of the present disclosure, the depth filter medium comprises at least one polypropylene fiber pulp, a non-woven fiber layer or a combination thereof. In some embodiments of the systems of the present disclosure, the depth filter medium further comprises a cationic binder.

[0030] In some embodiments of the systems of the present disclosure, the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder. In some embodiments of the systems of the present disclosure, the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder, the depth filter medium comprises a first layer of non-woven fiber layer, a second layer of polypropylene fiber pulp, and a thirst and fourth layer of polyacrylic fiber pulp, silica filter aid and cationic binder. In some embodiments of the systems of the present disclosure, the depth filter medium comprises six layers of polypropylene fiber and non-woven fiber, a second layer of cellulose fibers, and a third layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder.

[0031] In some embodiments of the systems of the present disclosure, the anion adsorber medium is any one of a strong AEX medium or an AEX hybrid medium. In some embodiments of the systems of the present disclosure, the strong AEX medium is any one of a Sartobind® Q medium, Mustang® Q medium or Natrix® Q medium. In some embodiments of the systems of the present disclosure, the AEX hybrid medium comprises AEX chromatography medium and a polyamide membrane. In some embodiments of the systems of the present disclosure, the AEX hybrid medium comprises AEX chromatography medium and poly ether sulfone (PES) membrane. In some embodiments of the systems of the present disclosure, the AEX chromatography medium is a quaternary ammonium salt functional AEX medium. In some embodiments of the systems of the present disclosure, the quaternary ammonium salt functional AEX medium is non-wovenmedium. In some embodiments of the systems of the present disclosure, the AEX chromatography medium is any one of a Harvest® RC medium or Emphaze® AEX medium. In some embodiments of the systems of the present disclosure, the pore size of the polyamide membrane or the PES membrane is between about 0.1 to about 0.5 pm. In some embodiments of the systems of the present disclosure, the pore size of the polyamide membrane or the PES membrane is 0.2 pm. In some embodiments of the systems of the present disclosure, the bioburden reduction filter can be any one of a PVDF- based filter, a PES-based filter, and a polyamide-based filter.BRIEF DESCRIPTION OF THE DRAWINGS

[0032] FIGs. 1A-1B depict a schematic of a conventional (FIG. 1A) and the multi-stage filtration train of the present disclosure (FIG. IB), for clarification of high cell-density rAAV productions.

[0033] FIGs. 2A-2B depict a comparison of depth filter pressure profiles with a pre-filter (FIG. 2A), and without a pre-filter (FIG. 2B). The x-axis depicts throughput velocity (L / m2), and y-axis depicts differential pressure (psid). The differential pressure profile curve with respect to throughput v elocity in presence of depth filter and pre-filter are as indicated.

[0034] FIGs. 3A-3C depict a comparison of the effect of conventional depth filter and adsorptive filters on the throughput capacity of the bioburden reduction filter. The following filters connected in series, as indicated: i) conventional depth filter and bioburden reduction filter (FIG. 3A); ii) adsorptive filter A and bioburden reduction filter (FIG. 3B); and iii) adsorptive filter B and bioburden reduction filter (FIG. 3C).

[0035] FIG. 4 depicts a comparison of pressure profiles of various adsorptive media. The x- axis indicates the throughput of rAAV preparation passed through the indicated filter media and y-axis indicates the differential pressure.

[0036] FIG. 5 depicts harvest clarification filter pressure profiles of the multi-stage filtration train for a 50-L scale production. The y-axis indicates the inlet pressure for each filter of the multistage filter train, corresponding to the throughput velocity indicated by the x-axis.

[0037] FIG. 6 depicts a comparison of turbidity measurement between the crude harvest and the clarified filtrate generated from two pilot-scale batches (production lot 1 and production lot 2, as indicated) employing the clarification and filtration systems disclosed herein. The y-axis indicates the turbidity measurement (Nephelometric Turbidity Units, NTU) of samples from feed material (empty bars) and clarified filtrates (black bars) of production lots 1 and 2, as indicated.

[0038] FIG. 7 depicts an elution profile of rAAV particles and conductivity change (dashed lines) corresponding to elution volume (x-axis). The level of rAAV particles eluted is indicated by absorption at UV280 and UV254 represented by thick black and thin black lines, respectively, as indicated in the left y-axis. FIG. 7 also depicts the change in relative conductivity (corresponding to the right y-axis), with increasing elution volume.

[0039] FIG. 8 depicts the dynamic binding capacity of the anion adsorber filter and Ad5 breakthrough concentration in the loading material. The level of retention of Ad5 corresponding to the amount of Ad5 in the loading material (GC / L-MV) is indicated on the x-axis. The sampling frequency (number of samples obtained per second) is indicated on the y-axis. The percentage Ad5 retention from the loading material on the anion adsorber filter is as indicated corresponding to the amount of Ad5 that is within the filter’s dynamic binding capacity (DBC), amount that results in acceptable Ad5 breakthrough, and amount that results in undesirable breakthrough ranges, as indicated.DETAILED DESCRIPTION OF CERTAIN EMBODIMENTSDefinitions

[0040] Where the use of the term “about” is before a quantitative value, the disclosure encompasses the specific quantitative value itself, (e.g., “about 10% to about 20%” should be understood to include “10% to 20%”, as well as “about 10% to 20%” and “10% to about 20%”), unless specifically stated otherwise. As used herein, the term “about” refers to a ± 10% variation from the nominal value unless otherwise indicated or inferred.

[0041] Throughout the present specification, numerical ranges are provided for certain quantities or parameters. It is to be understood that these ranges comprise endpoints and all subranges therein, including each integer in and between a disclosed range. Thus, the range “from 50 to 80” includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.) as well as each individual integer from 50 to 80 (e.g., 50, 51, 52, 53, 54, etc.). Where ranges are provided in the form of fractions, percentages, decimals, and the like, such ranges likewise include all possible subranges therein and each individual fraction, percentage, decimal, etc. in and between the disclosed range. For example, the range “from 0. 1 to 1.0” includes all possible ranges therein (e.g., 0.2 to 0.9, etc.) and each individual l / 10th decimal from 0.1 to 1.0 (e.g., 0.1, 0.2, 0.3, 0.4, etc.). Furthermore, all values within a given range may be an endpoint for the rangeencompassed thereby (e g., the range 50-80 includes the ranges with endpoints such as 55-80, SOS, etc.).

[0042] The term “a” or “an” refers to one or more of that entity; for example, “a medium” refers to one or more media or at least one medium. As such, the terms “a” (or “an”), “one or more” and “at least one” are used interchangeably herein. In addition, reference to “a” component by the indefinite article “a” or “an” does not exclude the possibility’ that more than one of the components is present, unless the context clearly requires that there is one and only one of the components.

[0043] The verb “comprise” as is used in this description and in the claims and its forms / conjugations are used in the non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The present invention may suitably “comprise”, “consist of; or “consist essentially of; the steps, elements, and / or reagents described in the claims.

[0044] As used herein, the term “adeno-associated virus” (AAV) refers to a small, replicationdefective, non-enveloped virus that infects humans and some other primate species. AAV are made up of a proteinaceous shell (referred to generally as a “capsid") surrounding a core which comprises the viral genome. AAV is not known to cause disease and elicits a very mild immune response. Gene therapy vectors that utilize AAV can infect both dividing and quiescent cells and can persist in an extrachromosomal state without integrating into the genome of the host cell. These features make AAV an attractive viral vector for gene therapy. There are currently 13 recognized naturally occurring serotypes of AAV (AAV1 - 13) and numerous variants, including, e.g., AAVhu.37 and AAVrh. 10. The term “recombinant adeno-associated virus” (rAAV) refers to AAV engineered to carry a heterologous nucleic acid of interest by deleting, in whole or in part, the internal portion of the AAV genome and inserting the heterologous nucleic acid of interest between the inverted terminal repeats (ITRs). The full ITR-to-ITR DNA sequence of a rAAV is referred to as a “vector genome”.

[0045] Unless otherwise noted, where the term “between” or “from” is used to refer to a numerical range, the range includes the specified endpoints. For example, the range “between 1 pM and 10 pM” includes 1 pM, 10 pM, and values greater than 1 pM but less than 10 pM.

[0046] As used herein, the term “a recombinant adeno-associated virus particle” or “rAAV particle” refers to a particle that comprises at least one viral capsid protein which (i) encapsidatesa nucleic acid, e g., a vector genome or a portion thereof, and / or (ii) forms a structure surrounding a core.

[0047] The term “full” or “full capsid” in reference to a rAAV particle or its encapsidated vector genome refers to a capsid particle that comprises a complete vector genome, that is, a vector genome that comprises a heterologous nucleic acid of interest flanked on both sides by AAV ITRs.

[0048] The term “intermediate” in reference to a rAAV particle or its encapsidated vector genome refers to a capsid particle that comprises a portion of a vector genome, that is, less in size or length (as measured in total base pairs) than the full vector genome that comprises a heterologous nucleic acid of interest flanked on both sides by AAV ITRs.

[0049] The term “empty” in reference to a rAAV particle refers to a capsid particle that does not contain an encapsidated vector genome.

[0050] As used herein, the term “inverted terminal repeat” (abbreviated “ITR”) refers to a symmetrical nucleic acid sequence in the genome of adeno-associated viruses required for efficient replication. ITR sequences are located at each end of the AAV DNA genome. The ITRs serve as the origins of replication for viral DNA synthesis and are essential cis components for packaging the AAV DNA genome into the vector capsid generating rAAV vectors.

[0051] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0052] As used herein, the terms “filtration system”, “filtration scheme”, “filtration train”, “filtration chain” “filtration series”, “system”, “scheme”, “train”, “chain” and “series” are used interchangeably and refer to a series of filtration modules or filter types wherein a rAAV feed material is passed through each of the filter types of the system to generate final clarified rAAV preparation.

[0053] As used herein, the term “filtrate”, “fraction”, “eluent” or "flowthrough" refers to the whole or part of a “feed material”, “cell lysate”, “cell harvest” or “rAAV preparation” that is obtained or collected after passing or flowing the feed material or cell lysate through “a filter”, “a medium” or “a resin”. As used herein the term “filtrate”, “fraction”, “eluent” or “flowthrough” also refers to whole or part of an already obtained filtrate after passing it through “a filter”, “a medium” or “a resin”.

[0054] As used herein, the term '‘feed material”, “cell lysate”, “cell harvest” or “rAAV preparation” refers to a suspension, mixture, cell culture milieu or supernatant obtained from a rAAV producing host cell culture producing rAAV, wherein the solution is expected to contain rAAV particles and impurities.

[0055] As used herein, the term “impurities” refers to non-rAAV particle, including but not limited to cell debris, cellular compartments, proteins, peptides, metabolites and nucleic acids and fragments thereof, that are released from a rAAV producer host cell culture when producing rAAV. The impurities can also include helper virus particles.

[0056] As used herein, “obtaining” or “to obtain” — with respect to a “filtrate”, “fraction”, “eluent” or “flowthrough”, e.g., a filtrate or flowthrough collected after a step of passing through or applying a cell lysate or a previously collected filtrate to a filter medium, that can be a prefilter medium, a depth filter, an anion adsorber medium or a bioburden reduction filter — means that the fraction or eluent is generated during and until the end of that step. A fraction that is “obtained” or “generated” may or may not be collected.

[0057] As used herein, the term “clarified lysate”, “clarified filtrate” or “clarified rAAV preparation” refers to a last or final filtrate containing the rAAV particles obtained after passing a cell lysate or a feed material from a rAAV producing host cell culture through one or more filter media, wherein the final filtrate contains about 0-50% less impurities as compared to the cell lysate or feed material.

[0058] As used herein, the term “recombinant,” may be used to describe, e.g., a nucleic acid molecule that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or by the artificial manipulation of isolated segments of nucleic acid molecules, such as by genetic engineering techniques.

[0059] A “recombinant adeno-associated virus preparation” or “rAAV preparation.” refers to a product that results from a method of manufacturing recombinant AAV in a host cell (e.g., in a mammalian cell or an insect cell). In some embodiments, a recombinant AAV preparation includes a mixture of full rAAV particles and empty rAAV particles. In some embodiments, a recombinant AAV preparation has been subjected to one or more downstream operations after initial upstream operations, e.g., nuclease treatment, filtration to remove host-cell impurities, and / or affinity purification using ligands that bind AAV capsids. In some embodiments, a recombinant AAV preparation has been subjected to treatment with one or more nuclease(s) including but not limitedto endonuclease, deoxyribonuclease and ribonuclease. In some embodiments, a recombinant AAV preparation has been subjected to treatment with one or more endonuclease(s). In some embodiments, a recombinant AAV preparation has been subjected to treatment with one or more endonuclease(s) including but not limited to Benzoase®, EndoCleave®, DENARASE®, OmniCleave* and ArcticZymes®.

[0060] As used herein, the term “vector’7may refer to a nucleic acid molecule allowing insertion of, or having inserted, foreign nucleic acid without disrupting the ability of the vector to replicate and / or integrate in a host cell. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to allow transcription and translation of inserted gene or genes. The term “vector” is also used in a more general sense to refer to a carrier or vehicle, such as in the case of a viral vector intended to deliver a heterologous DNA to a target cell or tissue, e.g., for purposes of gene therapy, such as an AAV vector.

[0061] As used herein, the term “module” refers to a set up or a system that can comprise a filter. The module as described herein can further comprise suitable mechanisms for passing and / or detecting passage of a cell lysate or a previously obtained filtrate through the filter. Such mechanisms can be pumps, syringes, tubing, feeding containers, filtrate collection containers, sensors (including but not limited to pressure sensor, conductivity sensor, flux sensor, or a combination thereof).

[0062] As used herein, the terms “filter”, “filter medium.” “filtration medium”, “medium,”, and the like, refer to a physical structure, such as column packed with resins or a monolith or a membrane, a fiber, or a woven fibrous structure, and the like, to which a rAAV preparation is applied in order to achieve separation of certain fractions and impurities of the rAAV preparation. For example, a rAAV preparation may be applied to a filter, which filter may then be washed with one or more solutions to pass the rAAV in the preparation (and collect rAAV containing fractions) while holding back the impurities and fractions not containing rAAV (or vice versa; i.e., to pass the impurities and fractions not containing rAAV while holding back the rAAV in the preparation). In some embodiments, a separation medium is an anion-exchange medium. In some embodiments, a separation medium is a mixed-modal medium that can serve as an anion-exchange medium. In some embodiments, a separation medium is a column (e.g.. a monolithic column or particles in a packed column). In some embodiments, a filter medium is a membrane. The term “medium”typically refers to one substance or substrate, and the term “media” is typically used as the plural for “medium”, meaning more than one medium. However, it is common in the art to use medium and media interchangeably. Consequently, reference to “media” throughout this description does not necessarily imply or require more than one “medium” unless context clearly indicates otherwise.

[0063] As used herein, the term “GC / L of Bed Volume” or “GC / L-BV” or “GC / L of Membrane Volume” or “GC / L-MV” refers to the total concentration of rAAV or adenoviral particles in the volume of filtrate loaded onto a filter (e.g., an anion adsorber filter or membrane).

[0064] As used herein, the term “conductivity” refers to the ability of a solution, a cell lysate, a cell harvest, a previously obtained filtrate or a feed material to conduct electricity measured by determining the resistance of the solution between two flat or cylindrical electrodes separated by a fixed distance. The conductivity of a solution as used herein is due to the presence of one or more cations or electrolytes in the solution including but not limited to H~, Li+, Na+, K+, NHZ, Ag+, Ca2+, CO(NHS)63+, OH", Cl’, Br’, T, NOs’, CHsCOO’, ClOty F", SO42’, C2O42; HC2OF, HCOO' , CO32-, HSCh2' and SCh2'. The conductivity of a solution passed through a filter determines if the rAAV will be retained in the filter medium or pass through with the filtrate. The conductivity / ionic strength of the filter load (i.e., preparation to be filtered) is critical, such that adjusting the filter load conductivity to a specific range (optimal conductivity range) allows the rAAV to pass through the filter, while retaining impurities, including the helper virus (or vice versa). As an example, if the filter load conductivity is higher than the optimal conductivity range, the rAAV can bind to the filter along with the impurities and / or helper virus. If the filter load conductivity is lower than the optimal conductivity range, on the other hand, the impurities and / or helper virus can also pass through the filter along with the rAAV. The optimal conductivity range can differ depending on the specific filter medium and the solute. For example, for high ligand density AEX filter (e.g., a containing positively charged quaternary amine functional groups)), the helper virus can elute at ~40 mS / cm while the rAAV can elute at -9-12 mS / cm, thus allowing the operation of the AEX filter at 36 mS / cm. As described herein, the conductivity of the feed material or previously collected filtrate containing the rAAV and impurities can be adjusted (increased or decreased) to prevent from binding of the rAAV particles in the feed material but allowing binding of the impurities to the filter (or vice versa). As described herein, the conductivity of the feed material or previously collected filtrate can be adjusted by increasing or decreasing the electrolyte or cation or anion content of the feed material or previously collected filtrate. The cation or anion content of the feed material or previously collected filtrate can be increased by adding into the feedmaterial or previously collected filtrate an amount of a suitable ionic solution. The cation or anion content of the feed material or previously collected filtrate can be decreased by diluting the feed material or previously collected filtrate with a suitable diluent.

[0065] As used herein, the term dynamic binding capacity (DBC) of a chromatography filter or medium (e.g., an anion adsorber filter, like Q-filter) describes the maximum amount of target protein or viral particles (e.g., rAAV particles, or adenovirus 5 particles) that can be loaded onto the filter without causing undesirable breakthrough, measured under realistic experimental conditions (operating flow-rate, representative impurity matrix).Methods of producing AAV

[0066] Methods of producing AAV, including rAAV, are described in the art (see, for example: WO 2023 / 172491 Al (incorporated herein by reference in its entirety); WO 2020 / 154607 Al (incorporated herein by reference in its entirety); and WO 2022 / 112218 Al (incorporated herein by reference in its entirety)). Such methods may utilize host cells for production of AAV vectors including rAAV. In some embodiments, the host cells may comprise a recombinant nucleic acid molecule, viral vector, e.g., an AAV vector, or a rAAV disclosed herein. The host cells may be suitable for propagation and / or production of AAV.

[0067] Any know n AAV production host cell can be used. Examples of know n host cell types include bacteria cells, yeast cells, insect cells (such as Sf9 cells), and mammalian cells, etc. In some embodiments, the host cell can be a cell (or a cell line) appropriate for production of AAV (e.g., rAAV), for example, a HeLa cell, Cos-7 cell, HEK293 cell (and HEK293 derivative cell lines), AGEI.hn cells, A549 cell, BHK cell, Vero cell, RD cell, ARPE-19 cell, CAP cells, HEK293, PER.C6, NS01, CHO, CV1, VERO, MDCK, BRL3A, W138, or HepG2 cells. In some embodiments, stable inducible AAV producer cell lines, such as those according to the doxycycline-inducible CAP cells and HEK293 cells described in WO 2022 / 112218 Al and HeLa PCLs described in W02020154607 and WO2023172491 (incorporated herein by reference in their entirety) are utilized in the systems and methods described herein.

[0068] Recombinant nucleic acid molecules or vectors can be delivered into the host cell culture using any suitable method known in the art (e.g., transfection-based methods). In some embodiments, a stable host cell line that has a recombinant nucleic acid molecule or vector inserted into its genome by transfection is utilized. Furthermore, a stable cell line can be established and utilized according to protocols known in the art, such as those described in Clark, KidneyInternational Vol 61 (2002):S9-S15, and Yuan et al, Human Gene Therapy 201 1 May;22(5):613- 24.

[0069] The host cells utilized for AAV production may be engineered to be AAV producer cells. An AAV producer cell line (PCL) thus may be utilized to generate AAV producer cell harvest for use in the systems and methods described herein.

[0070] Various cellular / viral components are needed for AAV production. For example, components for AAV replication, vector genome packaging, and structural components of the capsid all must be produced to generate an AAV particle. The AAV Rep gene encodes four proteins that are involved in packaging and replication, and the cap gene encodes three structural capsid proteins (called VP1, VP2, and VP3). Wild-type AAV is replication deficient and requires co-infection of cells by a helper virus, e.g., a herpes virus or adenovirus, e.g.. Ad5 virus, in order to replicate. For example, Ad5 virus supplies Ad5 helper virus function factors / genes, such as Ela, Elb, E40rf6, E2a and / or virus-associated (VA) RNA, that mediate AAV replication. See, e.g., Nayak et al. J Virol. 81.5(2007):2205-12. Recombinant AAV vectors permit integration of a gene of interest, or transgene, into a viral vector such that the transgene is transmitted, encoded, and / or expressed by the viral machinery’. In some cases, a recombinant AAV vector comprises inverted terminal repeats (ITRs) that serve as origins of replication and / or packaging. In some embodiments, the recombinant AAV vector comprises a transgene flanked by ITRs (one ITR on either side of the transgene). See, e.g., Carter B. Adeno- Associated Virus and AA V Vectors for Gene Delivery, in Gene and Cell Therapy, 4th Edition, N.S. Templeton. Editor. 2015. CRC Press.

[0071] AAV PCLs can be generated that contain one or more of these components required for AAV production. An exemplary list of AAV components includes (a) a nucleic acid sequence comprising a transgene, e.g., a therapeutic payload: (b) a nucleic acid sequence comprising an inverted terminal repeat (ITR), e.g., one or two ITRs, e.g., where the two ITRs flank one or more additional AAV components (e.g., a trans gene); (c) a nucleic acid sequence encoding one or more AAV replication and / or packaging proteins (e.g., encoded by the AAV rep gene); (d) a nucleic acid sequence encoding one or more AAV structural capsid proteins (e.g.. encoded by the AAV cap gene, e.g., VP1, VP2, or VP3 protein); (e) one or more AAV replication and / or packaging proteins; (f) one or more AAV structural capsid proteins; and / or (g) one or more helper virus components, e.g., Ad5 helper virus components (e.g., Ela, Elb. E40rf6, E2a and / or VA RNA; or Ad5 helper virus). As used herein, an “AAV PCL” or “AAV producer cell line” refers to a cell, e.g., a cell described herein, that comprises one or more of (a)-(g) above and, when supplied withany necessary helper virus component(s), is capable of producing an AAV under suitable production conditions that are known in the art.

[0072] Any cell line suitable for use in AAV production can be used as an AAV production host cell to generate AAV product for clarification and purification according to the systems and methods described herein, including suitable mammalian and insect cell lines (including Sf9 cells). For example, a mammalian cell line comprising one or more components needed for AAV production, e.g., a PCL derived from any mammalian cell line, can be used in any of the systems and methods described herein. In some embodiments, the systems and methods described herein comprise culturing mammalian cells, e.g., human cells or non-human mammalian cells, e.g., mammalian PCLs, e.g., human PCL or non-human mammalian PCLs. Exemplary ty pes of cells include but are not limited to: BALB / c mouse myeloma line (NSO / 1, ECACC No: 85110503); human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et ah, J. Gen Virol., 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells + / -DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23 :243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2 or HeLa S3, ECACC Catalog No. 87110901); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et ah, Annals N.Y. Acad. Sci., 383:44-68 (1982)); FS4 cells; a human hepatoma line (Hep G2); and / or a PCL version of any of the cell types described herein. In some embodiments, a PCL version of a cell type described herein comprises the cell t pe having been engineered to possess one or more components needed for AAV production.

[0073] In some embodiments, the systems and methods described herein comprise culturing HeLa cells (e.g., HeLa producer cell lines (PCLs), CHO cells (e.g., CHO PCLs), Sf9 cells, Sp2 / 0 cells, ELEVECTA™ producer cell line (Cytiva), HEK cells (e.g., HEK PCLs, HEK293), CAP cells, AGElhn cells. PER.C6. NS01, COS cells, BHK. CV1, VERO, MDCK, MDBK cells, BRL3A, W138, and HepG2 cells. In some embodiments, the systems and methods described herein comprise culturing HeLa cells, e.g., HeLa PCLs. In some embodiments, stable inducible AAV producer cell lines, such as those according to the doxycycline-inducible CAP cells and HEK293 producer cell lines described in WO 2022 / 112218, and HeLa PCLs described inW02020154607 and WO2023172491 (incorporated herein by reference in their entirety), utilized in the systems and methods described herein.

[0074] As noted above, in some instances the cells will be selected or engineered to include one or more components necessary7for AAV production (e.g., engineered into PCLs). Alternatively or additionally, engineered PCLs may comprise genetic modifications to reduce expression and / or activity of one or more genes and / or proteins, which modifications increase AAV titers. Exemplary engineered PCLs useful in the systems and methods of the disclosure include those described in US Patent Application Publication Number US 2020 / 0325455 AL

[0075] AAV may be produced for downstream purification according to the methods described herein using either conventional batch mode production methods or enhanced methods implementing fed-batch and / or perfusion strategies to increase AAV titer. Generally, in batch mode AAV production, AAV PCLs are cultured in a N-l culture vessel to achieve a high viable cell density. N-l culture cells are then used to seed a N culture vessel at a particular seeding density7. The seeded cells in the N culture vessel may be cultured under conditions that permit production of AAV. Generally, modified batch processes involve preparing a vessel with grow th medium for AAV producing cell growth stage culture. In some embodiments, the vessel may be a single vessel intended for culturing both growth and production stage cultures, i.e., a ‘"combined growth / production vessel.” Such vessel is subsequently inoculated with AAV producing cells. Perfusion may be initiated in the inoculated growth or combined growth / production vessel to initiate AAV producing cell growth. Once a final cell density target is achieved, production stage culture can be initiated. In some embodiments, production stage initiation involves infecting the growth stage culture with a helper virus to initiate AAV production. AAV production can be initiated by alternative means not dependent on AAV helper virus infection. Such alternative means include, for example, use of triple transfection approaches or use of producer cells with stable integration of components necessary to produce AAV, such as adenovirus helper vims functions, AAV replicase, and capsid genes (e.g., the ELEVECTA® (CEVEC) doxycycline- inducible AAV producing cells, and similar systems). AAV production stage can be maintained, e.g., by providing feed, exchanging medium for production medium, and / or initiating perfusion to sustain production. AAV produced accordingly can be harvested using any suitable separation technology. The AAV harvest can then be clarified and purified using methods described herein.

[0076] In some embodiments, the AAV are produced using a final perfusion production stage carried out in a production vessel, at, e.g., 2000L scale. The production vessel may be a vesselsuited to any culture volume for AAV production at any scale. For example, the production vessel may be a sub-liter, IL, 2L, 5L, 10L, 20L, 50L, 100L, 150L, 200L, 250L, 500L, WOOL, WOOL, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, 6000L, 6500L, 7000L, 7500L, 8000L, 8500L, 9000L, 9500L, 10000L, 10500L, 1 WOOL, 1 WOOL, 12000L vessel, or greater.

[0077] In some embodiments, the N-stage growth culture for producing AAV may target a final producer cell density of at least 5x W6viable cells (vc) / mL. The N- stage growth may target any final density suitable for seeding a production stage AAV bioreactor, including, for example, 1x106, 2xW6, 3xW6, 4xW6, 5xW6, 6xW6, 7xW6, 8xW6, 9xW6, 10xW6, 20xW6, 30xW6, 40xW6, or up to 50xW6vc / mL. or greater.

[0078] In some embodiments, any pre-production stage culture is carried out any appropriate scale for AAV producing cell growth phase. For example, any pre-production stage culture maybe carried out at sub-liter to 6000L scale. Accordingly, the present systems and methods can utilize a sub-liter, I L, 2L, 5L, WL, 20L, 50L, W0L, 150L, 200L, 250L, 500L, WOOL, WOOL, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L vessel in the bioreactor for pre- production or growth stage culture.

[0079] In some embodiments, the production stage culture is carried out any appropriate scale for AAV production. For example, the production stage culture may be carried out at sub-liter to 6000L scale. Accordingly, the present systems and methods can utilize a sub-liter, IL. 2L. 5L, WL, 20L, 50L, W0L, 150L, 200L, 250L, 500L, WOOL, WOOL, 2000L, 2500L, 3000L, 3500L, 4000L, 4500L, 5000L, 5500L, or 6000L vessel in the bioreactor for production stage culture.

[0080] In some embodiments, the methods described herein further comprise collecting or harvesting AAV product from the production stage culture. Standard methods of collecting or separating viral particles can be used, e.g., including but not limited to filtration or centrifugation. Further downstream purification processes can be utilized to purify collected AAV product.

[0081] In accordance with the methods described herein, the AAV titer, e.g., in the N culture, can be determined, e.g., on any one or more days of the N culture. An exemplary AAV titer measurement is vector genome copies per cell (GC / cell). GC / cell can be determined by standard methods in the art, e.g., including but not limited to dot blot, quantitative PCR or ddPCR, spectroscopy, or fluorimetry. See, e.g., Dorange et al. Cell Gene Therapy Insights 4.2(2018): 119- 129.

[0082] In some embodiments, the methods described herein are capable of producing an AAV titer, e.g., in the production stage culture, of at least about IxlO9vector genomes (vg) / mL, at least about 2xl09vg / mL, at least about 3xl09vg / mL. at least about 4xl09vg / mL, at least about 5xl09vg / mL, at least about 6xl09vg / mL, at least about 7xl09vg / mL, at least about 8xl09vg / mL. at least about 9xl09vg / mL, at least about IxlO10vg / mL, at least about 2xlO10vg / mL, at least about 3xlO10vg / mL, at least about 4xlO10vg / mL, at least about 5xlO10vg / mL, at least about 6xlO10vg / mL, at least about 7x1010vg / mL, at least about 8x1010vg / mL, at least about 9x1010vg / mL, at least about IxlO11vg / mL, at least about 2xlOnvg / mL, at least about 3xl0nvg / mL, at least about 4xlOnvg / mL, at least about 5xl0nvg / mL, at least about 6xlOnvg / mL. at least about 7xlOnvg / mL, at least about 8xl0nvg / mL, at least about 9xlOnvg / mL, at least about IxlO12vg / mL, at least about 2xl012vg / mL, at least about 3xl012vg / mL, at least about 4xl012vg / mL, at least about 5xl012vg / mL, or greater.Clarification methods

[0083] Clarification of producer cell harvest containing rAAV is an important step for removal of impurities before subjecting the cell harvest to methods of purifying the rAAV. Clarification processes fall in two broad categories: (a) primary clarification step for removal of the bulk of large particles, whole cells, and cell debris and (b) secondary clarification step for removal smaller particles present in the resulting filtrate from primary clarification. Common primary clarification methods include centrifugation, transient flow filtration (TFF) and depth filtration, while common secondary’ clarification methods include depth filtration and bioburden-reduction filters. Depth filters are particularly useful as a single-use clarification solution in biopharmaceutical development. The overall performance of commercially available depth filters is known, in that they are effective and comparable at small and pilot scale. However, effective clarification and recovery of rAAV during scale up of production from high cell density cultures of producer cell line is often severely limited by clogging of filters and hold-up volume due to an increase in differential pressure beyond the capacity of traditional depth filters, (i.e., a recommended value of 20-25 psi).

[0084] The present disclosure provides improved clarification systems comprising a series of different filter types and methods of using same for removing impurities from scale-up level volume of rAAV containing high producer cell density harvests while maintaining the industry operational standards of filtration, that can reduce downstream purification costs and time.

[0085] The present disclosure provides methods of clarifying a cell lysate produced from a cell culture expressing recombinant adeno-associated virus (rAAV) particles, comprising: (a) passing the cell lysate through a prefilter medium to generate a first filtrate; (b) passing the first filtrate through a depth filter medium to generate a second filtrate; (c) passing the second filtrate through an anion adsorber medium to generate a third filtrate; and (d) passing the third filtrate through a bioburden reduction filter to generate a clarified cell lysate.

[0086] In some embodiments of the methods of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about IxlO6to about 2xl07cells / mL (e.g., about IxlO6to about 2xl06cells / mL, about 2xl06to about 3xl06cells / mL. about 3xl06to about 4xl06cells / mL, about 4xl06to about 5xl06cells / mL, about 5xl06to about 6xl06cells / mL, about 6xl06to about 7xl06cells / mL, about 7xl06to about 8xl06cells / mL, about 8xl06to about 9xl06cells / mL, about 9xI06to about IxlO7cells / mL, about IxlO7to about 2xl07cells / mL, about 2xl06to about IxlO7cells / mL, about 3xl06to about IxlO7cells / mL, about 3xl06to about 9xl06cells / mL, about 4xl06to about 8xl06cells / mL, about 5xl06to about 7xl06cells / mL. and all integers including and in between about IxlO6to about 2xl07cells / mL). In some embodiments of the methods of the present disclosure, the cell lysate is produced from a rAAV- expressing cell culture with a cell density of at least about 106cells / mL. In some embodiments of the methods of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density of at least about 8xl06to about 2xl07cells / mL (e.g., about 8xl06to about 8.5xl06cells / mL, about 8.5xl06to about 9xl06cells / mL, about 9xl06to about 9.5xl06cells / mL, about 9.5xl06to about IxlO7cells / mL, about IxlO7to about 1.5xl07cells / mL, about 1.5xl07to about 2xl07cells / mL, about 8.5xl06to about 1.5xl07cells / mL, about 9xl06to about IxlO7cells / mL, and all integers including and in between about 8xl06to about 2xl07cells / mL). In some embodiments of the methods of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density' of equal to or greater than 2xl07cells / mL.

[0087] In some embodiments of the methods of the present disclosure, the cell lysate has a pH of about 7.5 to about 8.0 (e.g., about 7.5 to about 7.6, about 7.6 to about 7.7, about 7.7 to about 7.8, about 7.8 to about 7.9, about 7.9 to about 8.0, about 7.6 to about 7.9, about 7.5 to about 7.7, about 7.6 to about 7.9, and all integers including and in between about 7.5 to about 8.0). In some embodiments of the methods of the present disclosure, the cell lysate has a pH of about 7.7 to about 7.9 (e.g., about 7.7 to about 7.8, about 7.8 to about 7.9, and all integers including and in between about 7.7 to about 7.9).

[0088] In some embodiments of the methods of the present disclosure, the cell lysate comprises rAAV particles and one or more of cellular complexes, cellular aggregates, colloidal aggregates, nucleases, nucleic acids, host cell proteins, lysed cell fragments, and nucleotide fragments. In some embodiments of the methods of the present disclosure, the cell lysate comprises one or more nuclease(s) including but not limited to endonuclease, deoxyribonuclease and ribonuclease. In some embodiments of the methods of the present disclosure, the cell lysate comprises one or more endonuclease(s). In some embodiments of the methods of the present disclosure, the cell lysate comprises one or more endonuclease(s) including but not limited to Benzoase®, EndoCleave®, DENARASE®. OmniCleave® and ArcticZymes®. For instance, the cell lysate may have been subjected to one or more nuclease(s) including but not limited to endonuclease, deoxyribonuclease and ribonuclease. Likewise, the cell lysate may have been subjected to one or more endonuclease(s) including but not limited to Benzoase®, EndoCleave®, DENARASE®, OmniCleave® and ArcticZymes®.

[0089] In some embodiments of the methods of the present disclosure, the clarified cell lysate comprises the rAAV particles. In some embodiments of the methods of the present disclosure, the clarified cell lysate retains at least 80% of the total rAAV particles present in the cell lysate. In some embodiments of the methods of the present disclosure, the clarified cell lysate retains >85% (e.g., 85%, 86%, 87%, 88%. 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) of the total rAAV particles present in the cell lysate.

[0090] In some embodiments of the methods of the present disclosure, the cell lysate comprises helper virus particles. In some embodiments of the methods of the present disclosure, the helper virus particles can be any one of adenovirus particles, herpesvirus particles and papillomavirus particles. In some embodiments of the methods of the present disclosure, the helper herpesvirus particles are herpes simplex virus (HSV) particles, human cytomegalovirus particles (HCMV) particles or a variant or mutant version thereof. In some embodiments of the methods of the present disclosure, the helper virus particles are adenovirus particles. In some embodiments of the methods of the present disclosure, the helper adenovirus particles are adenovirus serotype 5 (Ad5) particles, adenovirus serotype 12 (Adl2) particles, or a variant or mutant version thereof. In some embodiments of the methods of the present disclosure, the adenovirus particles are Ad5 particles or a variant or mutant version thereof. In some embodiments of the methods of the present disclosure, the papillomavirus particles are human papillomavirus type 16 particles (HPV-16) particles or a variant or mutant version thereof.

[0091] In some embodiments of the methods of the present disclosure, the cell lysate comprises the Ad5 particles at a density of about IxlO12to about 3xl012GC / ml (e.g., about IxlO12to about 1.2xl012GC / ml, about 1.2xl012to about 1.4xl012GC / ml, about 1.4xl012to about 1.6xl012GC / ml. about 1.6xl012to about 1.8xl012GC / ml. about 1.8xl012to about 2xl012GC / ml. about 2xl012to about 2.2xl012GC / ml, about 2.2xl012to about 2.4xl012GC / ml, about 2.4xl012to about 2.6xl012GC / ml, about 2.6xl012to about 2.8xl012GC / ml, about 2.8xl012to about 3xl012GC / ml, about 1.2xl012to about 2.8xl012GC / ml, about 1.4xl012to about 2.6xl012GC / ml, about 1.6xl012to about 2.4xl012GC / ml, about 1.8xl012to about 2.2xl012GC / ml, and all integers including and in between about IxlO12to about 3xl012GC / ml).

[0092] In some embodiments of the methods of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the volume of anion adsorber filter is about 3xl016to about 5xl016GC / L of membrane volume (GC / L-MV) (e.g., about 3xl016to about 3.25xl016GC / L-MV, about 3.25xl016to about 3.5xl016GC / L-MV, about 3.5xl016to about 3.75xl016GC / L-MV, about 3.75xl016to about 4xl016GC / L-MV, about 4xl016to about 4.25xl016GC / L- MV, about 4.25xl016to about 4.5xl016GC / L-MV, about 4.5xl016to about 4.75xl016GC / L-MV, about 4.75xl016to about 5xl016GC / L-MV, about 3.25xl016to about 4.75xl016GC / L-MV, about 3.5xl016to about 4.25xl016GC / L-MV, and all integers including and in between about 3xl016to about 5xl016GC / L-MV).

[0093] In some embodiments of the methods of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the anion adsorber filter is about 3xl016to about 7.25xl016GC / L-MV (e.g.. about 3xl016to about 3.25xl016GC / L-MV. about 3.25xl016to about 3.5xl016GC / L-MV, about 3.5xl016to about 3.75xl016GC / L-MV, about 3.75xl016to about 4xl016GC / L-MV, about 4xl016to about 4.25xl016GC / L-MV, about 4.25xl016to about 4.5xl016GC / L-MV, about 4.5xl016to about 4.75xl016GC / L-MV, about 4.75xl016to about 5xl016GC / L- MV, about 5xl016to about 5.25xl016GC / L-MV. about 5.25xl016to about 5.5xl016GC / L-MV, about 5.5xl016to about 5.75xl016GC / L-MV, about 5.75xl016to about 6xl016GC / L-MV, about 6xl016to about 6.25xl016GC / L-MV, about 6.25xl016to about 6.5xl016GC / L-MV, about 6.5xl016to about 6.75xl016GC / L-MV, about 6.75xl016to about 7xl016GC / L-MV, about 7xl016to about 7.25xl016GC / L-MV, about 3.25x1016to about 7xl016GC / L-MV. about 3.5x1016to about 6.75xl016GC / L-MV, about 3.75xl016to about 6.5xl016GC / L-MV, about 4xl016to about 6xl016GC / L-MV, about 4.25xl016to about 5.75xl016GC / L-MV, about 4.5xl016to about 5.5xl016GC / L-MV and all integers including and in between about 3x1016to about 7x1016GC / L-MV).

[0094] In some embodiments of the methods of the present disclosure, the volume of the cell lysate is at least 50L. In some embodiments of the methods of the present disclosure, the volume of the cell lysate is at least 250L. In some embodiments of the methods of the present disclosure, the volume of the cell lysate is between about 50L and about 250L (e.g., about 50L and about 250L and all integers including and in between about 50L and about 60L, about 60L and about 70L, about 70L and about 80L, about 80L and about 90L, about 90L and about 100L, about 100L and about 120L, about 120L and about 150L, about 150L and about 170L, about 170L and about 200L, about 200L and about 220L. about 220L and about 250L, about 60L and about 220L. about 70L and about 200L, about 80L and about 170L, about 90L and about 150L, and all integers including and in between about 50L and about 250L).

[0095] In some embodiments of the methods of the present disclosure, the pre-filter is a particulate filter. In some embodiments of the methods of the present disclosure, the prefilter medium comprises any one of a polypropylene fleece based pre-filter, an adsorptive glass fleece material based pre-filter, a mixed cellulose ester based pre-filter, a nylon membrane based prefilter. a glass and quartz fiber based pre-filter, polyvinyl chloride based filter and polytetrafluoroethylene based filter.

[0096] In some embodiments of the methods of the present disclosure, the prefilter medium comprises a polypropylene fleece based pre-filter. In some embodiments of the methods of the present disclosure, the polypropylene fleece based pre-filter is any one of a Sartopure® PP3, BECO PROTECT PG® pre-filter and EMD Millipore® polypropylene pre-filter. In some embodiments, the prefilter medium comprises an adsorptive glass fleece material based pre-filter. In some embodiments of the methods of the present disclosure, the adsorptive glass fleece material based pre-filter is any one of Sartopure® PP3 Maxicaps® filter and Sartorius Stedim Biotech™ Sartopure™ filter. In some embodiments of the methods of the present disclosure, the prefilter medium comprises a mixed cellulose ester based pre-filter. The mixed cellulose ester based prefilter can comprise nitrocellulose, and an appropriate amount of cellulose acetate, acetone, n- butanol or ethanol. In some embodiments of the methods of the present disclosure, the mixed cellulose ester based pre-filter is any one of Fisherbrand™ Mixed Cellulose Ester Membrane, Whatman™ Mixed Cellulose Ester Membrane, Advantec® Mixed Cellulose Ester Membrane, Sterlitech® Nitrocellulose Mixed Ester Membrane Filters and Immobilon® -NC Membrane Triton-free Mixed Cellulose Ester filter.

[0097] In some embodiments of the methods of the present disclosure, the pre-filter comprises a nylon membrane based pre-filter. In some embodiments of the methods of the present disclosure, the nylon membrane based pre-filter is any one of Sterlitech® Nylon Membrane Filter, Hydrofil™ Nylon 6.6 Membrane Filter, PALL 66606 Nylaflo™ Nylon Membrane Filter, Nalgene® Rapid- Flow™ Tissue Culture Filter and Tisch Scientific® Nylon Membrane Filter. In some embodiments of the methods of the present disclosure, the pre-filter comprises a glass and quartz fiber based pre-filter. In some embodiments of the methods of the present disclosure, the glass and quartz fiber based pre-filter is any one of Advantec® Glass and Quartz Fiber Filter and EMD Millipore® Glass and Quartz Fiber Filter. In some embodiments of the methods of the present disclosure, the pre-filter comprises a polyvinyl chloride based filter. In some embodiments, the polyvinyl chloride based filter is any one of EMD Millipore® PVC Membrane Filter, CytivaGLA- 5000TM PVC membrane disc filter and Durapore® Membrane Filter. In some embodiments of the methods of the present disclosure, the pre-filter comprises a polytetrafluoroethylene based filter. In some embodiments of the methods of the present disclosure, the polytetrafluoroethylene based filter is any one of Sterlitech® PTFE Laminated Membrane Filter, Fluoropore™ PTFE Membrane Filter and Omnipore™ PTFE Membrane Filter, Omnipore™ Membrane Filter and Mitex™ Membrane Filter.

[0098] In some embodiments of the methods of the present disclosure, the prefilter medium has a pore size of about 20 pm to about 75 pm (e.g.. about 20 pm to about 25 pm, about 25 pm to about 30 pm, about 30 pm to about 35 pm, about 35 pm to about 40 pm, about 40 pm to about 45 pm, about 45 pm to about 50 pm, about 50 pm to about 55 pm, about 55 pm to about 60 pm, about 60 pm to about 65 pm, about 65 pm to about 70 pm, about 70 pm to about 75 pm, about 25 pm to about 70 pm, about 30 pm to about 65 pm, about 35 pm to about 60 pm, about 40 pm to about 55 pm, and all integers including and in between about 20 pm to about 75 pm). In some embodiments of the methods of the present disclosure, the prefilter medium has a pore size of about 50 mm.

[0099] In some embodiments of the methods of the present disclosure, passing the cell lysate through the prefilter medium increases the volumetric capacity of the depth filter by at least 25%. In some embodiments of the methods of the present disclosure, passing the cell lysate through the prefilter medium increases the volumetric capacity of the depth filter by about 25% to about 50% (e.g., about 25% to about 30%, about 30% to about 35%, about 35% to about 40%, about 40% to about 45% , about 45% to about 50% , about 30% to about 50%, about 35% to about 45%, about35% to about 50%, about 25% to about 45%, and all percentages including and in between about 25% to about 50%). In some embodiments of the methods of the present disclosure, passing the cell lysate through the prefilter medium increases the filter capacity of the depth filter by about 30 to about 50 L / m2(e.g.. about 30 to about 32 L / m2, about 32 to about 34 L / m2, about 34 to about 36 L / m2, about 36 to about 38 L / m2, about 38 to about 40 L / m2, about 40 to about 42 L / m2, about 42 to about 44 L / m2, about 44 to about 46 L / m2, about 46 to about 48 L / m2, about 48 to about 50 L / m2, about 32 to about 48 L / m2, about 32 to about 46 L / m2, about 34 to about 44 L / m2, about 36 to about 42 L / m2, and all integers including and in between about 30 to about 50 L / m2).

[0100] In some embodiments of the methods of the present disclosure, the flow rate of the cell lysate in the prefilter medium is about 225 to about to about 925 L / m2h (about 225 to about to about 275 L / m2h, about 275 to about 325 L / m2h, about 325 to about 375 L / m2h, about 375 to about 425 L / m2h, about 425 to about to about 475 L / m2h, about 475 to about 525 L / m2h, about 525 to about 575 L / m2h, about 575 to about 625 L / m2h, about 625 to about 675 L / m2h, about 675 to about 725 L / m2h, about 725 to about 775 L / m2h, about 775 to about 825 L / m2h, about 825 to about 875, about 875 to about 925 L / m2h, about 275 to about 875L / m2h, about 325 to about 825 L / m2h, about 375 to about 775 L / m2h, about 425 to about 725 L / m2h, about 475 to about 675 L / m2h, about 525 to about 625 L / m2h, and all integers including and in between about 250 to about 925 L / m2h).

[0101] In some embodiments of the methods of the present disclosure, the prefilter medium has a volumetric capacity of about 300 to about 700 L / m2(e.g., about 300 to about 350 L / m2, about 350 to about 400 L / m2, about 400 to about 450 L / m2, about 450 to about 500 L / m2, about 500 to about 550 L / m2, about 550 to about 600 L / m2, about 600 to about 650 L / m2. about 650 to about 700 L / m2, about 350 to about 650 L / m2, about 400 to about 600 L / m2, about 450 to about 650 L / m2, about 350 to about 550 L / m2, and all integers including and in between about 300 to about 700 L / m2). In some embodiments of the methods of the present disclosure, the differential pressure in the prefilter medium is about 1 to about 5 psi (e.g., about 1 to about 1.5 psi, about 1.5 to about 2 psi, about 2 to about 2.5 psi, about 2.5 to about 3 psi, about 3 to about 3.5 psi, about 3.5 to about 4 psi, about 4 to about 4.5 psi, about 4.5 to about 5 psi, about 1.5 to about 4.5 psi, about 2 to about 4 psi, about 2.5 to about 3.5 psi, about 3 to about 5 psi, about 2 to about 4 psi, about 1 to about 3 psi, and all integers including and in between about 1 to about 5 psi).

[0102] In some embodiments of the methods of the present disclosure, the depth filter medium comprises any one of a cellulose-based, glass-based and acrylic-based filter medium. In some embodiments of the methods of the present disclosure, the depth filter medium comprises 2 to 8layers of the filter medium. In some embodiments of the methods of the present disclosure, the depth filter further comprises any one of a diatomaceous earth (DE) filter aid and a silica filter aid. In some embodiments of the methods of the present disclosure, the depth filter medium comprises at least one polypropylene fiber pulp, a non-woven fiber layer or a combination thereof. In some embodiments of the methods of the present disclosure, the depth filter medium further comprises a cationic binder.

[0103] In some embodiments of the methods of the present disclosure, the depth filter comprises a cellulose-based filter medium. In some embodiments of the methods of the present disclosure, the cellulose-based filter medium is any one of a Sartoclear® depth filter, Seitz® ZD pure cellulose depth filter, EMD Millipore® mixed cellulose ester filter, Millistak+® depth filter, Stax™ mAx depth filter, Allegro™ Connect depth filter, Stax™ single use depth filter and Stax™ mAx CF cake filter.

[0104] In some embodiments of the methods of the present disclosure, the depth filter comprises a glass-based filter medium. In some embodiments of the methods of the present disclosure, the glass-based filter medium is any one of Cytiva® 934- AH glass microfiber filter, Cytiva® EX / THICK glass fiber filter. Sartorius® glass microfiber filter, Sterlitech® glass fiber filter, Merck® glass fiber filter and Borosilicate glass microfiber filter.

[0105] In some embodiments of the methods of the present disclosure, the depth filter comprises an acrylic-based filter medium. In some embodiments of the methods of the present disclosure, the acry lic-based filter medium is any one of Millistak+ ® HC Pro Pod depth filter.

[0106] In some embodiments of the methods of the present disclosure, the flow rate of the first filtrate in the depth filter medium is about 50 to about 150 L / m2h (e.g., about 50 to about 75 L / m2h, about 75 to about 100 L / m2h, about 100 to about 125 L / m2h, about 125 to about 150 L / m2h, about 75 to about 125 L / m2h, about 100 to about 150 L / m2h, about 75 to about 150 L / m2h. about 50 to about 100 L / m2h and all integers including and in between about 50 to about 150 L / m2h). In some embodiments of the methods of the present disclosure, the volumetric capacity7of the depth filter medium is about 70 to about 200 L / m2(e.g.. about 70 to about 100 L / m2, about 100 to about 125 L / m2. about 125 to about 150 L / m2, about 150 to about 175 L / m2, about 175 to about 200 L / m2, about 100 to about 175 L / m2, about 125 to about 175 L / m2, about 150 to about 200 L / m2, about 125 to about 175 L / m2, and all integers including and in between about 70 to about 200 L / m2). In some embodiments of the methods of the present disclosure, the differential pressure in the depth filter medium is between about 1 to about 18 psi (e.g., about 1 to about 3 psi, about 3 to about 6psi, about 6 to about 9 psi, about 9 to about 12 psi, about 12 to about 15 psi, about 15 to about 18 psi, about 3 to about 15 psi, about 3 to about 9 psi, about 6 to about 12 psi, about 9 to about 15 psi, about 12 to about 18 psi, and all integers including and in between about 1 to about 18 psi).

[0107] In some embodiments of the methods of the present disclosure, the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder. In some embodiments of the methods of the present disclosure, the depth filter medium comprises a first layer of non-woven fiber, a second layer of polypropylene fiber pulp, and a third and fourth layer, the third and fourth layers comprising polyacrylic fiber pulp, silica filter aid and cationic binder. In some embodiments of the methods of the present disclosure, the depth filter medium comprises six layers of polypropylene fiber and non-woven fiber, a second layer of cellulose fibers, and a third layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder.

[0108] In some embodiments of the methods of the present disclosure, the anion adsorber medium is any one of a strong AEX medium or an AEX hybrid medium. In some embodiments of the methods of the present disclosure, the strong AEX medium is any one of Sartobind™ Q medium, Mustang™ Q medium or Natrix™ Q medium. As used herein, the term “AEX hybrid medium” refers to a combination of an AEX filter and a non- AEX filter. In some embodiments of the methods of the present disclosure, the non-AEX filter is a fine particle, bioburden reduction membrane. As used herein, the term “strong AEX medium” refers to an AEX filter medium alone without any additional non-AEX filter medium. In some embodiments of the methods of the present disclosure, the AEX hybrid medium comprises AEX chromatography medium and a polyamide membrane. In some embodiments of the methods of the present disclosure, the AEX hybrid medium comprises AEX chromatography medium and poly ether sulfone (PES) membrane. In some embodiments of the methods of the present disclosure, the AEX chromatography medium is a quaternary ammonium salt functional AEX medium. In some embodiments of the methods of the present disclosure, the quaternary ammonium salt functional AEX medium is non-woven medium. In some embodiments of the methods of the present disclosure, the AEX chromatography medium is any one of a 3M™ Polisher ST, Harvest™ RC medium or Emphaze™ AEX medium.

[0109] In some embodiments of the methods of the present disclosure, the pore size of the polyamide membrane or the PES membrane is about 0. 1 to about 0.5 pm (e.g., about 0.1 to about 0.2 pm, about 0.2 to about 0.3 pm, about 0.3 to about 0.4 pm, about 0.4 to about 0.5 pm, about 0.2 to about 0.4 pm, about 0.1 to about 0.3 pm, about 0.3 to about 0.5 pm, and all integersincluding and in between about 0.1 to about 0.5 pm. In some embodiments of the methods of the present disclosure, the pore size of the polyamide membrane or the PES membrane is 0.2 pm. In some embodiments of the methods of the present disclosure, the flow rate of the second filtrate in the anion adsorber medium is between about 50 to about 200 L / m2h (e.g., about 50 to about 75 L / m2h, about 75 to about 100 L / m2h, about 100 to about 125 L / m2h, about 125 to about 150 L / m2h, about 150 to about 175 L / m2h, about 175 to about 200 L / m2h, about 75 to about 175 L / m2h. about 100 to about 150 L / m2h, about 125 to about 175 L / m2h, about 150 to about 200 L / m2h, about 50 to about 100 L / m2h and all integers including and in between about 50 to about 200 L / m2h). In some embodiments of the methods of the present disclosure, the volumetric capacity of the anion adsorber medium is between about 70 to about 200 L / m2(e.g., about 100 to about 120 L / m2, about 120 to about 130 L / m2, about 130 to about 140 L / m2, about 140 to about 150 L / m2, about 150 to about 160 L / m2, about 160 to about 170 L / m2, about 170 to about 180 L / m2. about 180 to about 190 L / m2, about 190 to about 200 L / m2, about 120 to about 190 L / m2, about 130 to about 180 L / m2, about 140 to about 170 L / m2, and all integers including and in between about 100 to about 200 L / m2). In some embodiments of the methods of the present disclosure, the differential pressure of the anion adsorber medium is between about 1 to about 10 psi (e.g., about 1 to about 2 psi, about 2 to about 3 psi, about 3 to about 4 psi, about 4 to about 5 psi, about 5 to about 6 psi, about 6 to about 7 psi, about 7 to about 8 psi, about 8 to about 9 psi, about 9 to about 10 psi, about 2 to about 9 psi, about 3 to about 8 psi, about 4 to about 7 psi, about 5 to about 8 psi, about 6 to about 9 psi, and all integers including and in between about 1 to about 10 psi). In some embodiments of the methods of the present disclosure, the conductivity of the filter load of the anion adsorber medium is between about 16 to about 40 mS / cm (e g., about 16 to about 20 mS / cm, about 20 to about 25 mS / cm, about 25 to about 30 mS / cm, about 30 to about 35 mS / cm, about 35 to about 40 mS / cm. about 20 to about 35 mS / cm, about 25 to about 40 mS / cm, about 16 to about 30 mS / cm, and all integers including and in between about 16 to about 40 mS / cm). In some embodiments of the methods of the present disclosure, the conductivity of the filter load is between about 18 to about 37 mS / cm (e.g., about 18 to about 20 mS / cm, about 20 to about 22 mS / cm, about 22 to about 24 mS / cm, about 24 to about 26 mS / cm, about 26 to about 28 mS / cm, about 28 to about 30 mS / cm, about 30 to about 32 mS / cm. about 32 to about 34 mS / cm, about 34 to about 37 mS / cm, about 20 to about 34 mS / cm, about 22 to about 32 mS / cm, about 24 to about 30 mS / cm, and all integers including and in between about 18 to about 37 mS / cm).

[0110] In some embodiments of the methods of the present disclosure, the bioburden reduction filter comprises any one of a PVDF-based filter, a polyethersulfone (PES)-based filter, and apolyamide-based filter. In some embodiments of the methods of the present disclosure, the flow rate of the third filtrate through the bioburden reduction filter is about 300 to about 700 L / m2h (e.g., about 300 to about 350 L / m2h, about 350 to about 400 L / m2h, about 400 to about 450 L / m2h, about 450 to about 500 L / m2h, about 500 to about 550 L / m2h, about 550 to about 600 L / m2h, about 600 to about 650 L / m2h, about 650 to about 700 L / m2h, about 300 to about 500 L / m2h, 350 to about 650 L / m2h, about 400 to about 600 L / m2h, about 450 to about 550 L / m2h about, about 500 to about 700 L / m2h, about 550 to about 650 L / m2h, and all integers including and in between about 300 to about 700 L / m2h). In some embodiments of the methods of the present disclosure, the volumetric capacity of the bioburden reduction filter is about 200 to about 750 L / m2(e.g.. about 200 to about 250 L / m2, about 250 to about 300 L / m2, about 300 to about 350 L / m2, about 350 to about 400 L / m2, about 400 to about 450 L / m2, about 450 to about 500 L / m2, about 500 to about 550 L / m2, about 550 to about 600 L / m2, about 600 to about 650 L / m2, about 650 to about 700 L / m2. about 700 to about 750 L / m2, about 250 to about 700 L / m2, about 300 to about 650 L / m2, about 350 to about 600 L / m2, about 400 to about 550 L / m2, and all integers including and in between about 200 to about 750 L / m2). In some embodiments of the methods of the present disclosure, the differential pressure limit of the bioburden reduction filter is between about 20 to about 25 psi (e.g., about 20 to about 21 psi, about 21 to about 22 psi, about 22 to about 23 psi, about 23 to about 24 psi. about 24 to about 25 psi. about 21 to about 24 psi, about 20 to about 23 psi, about 22 to about 25 psi, and all integers including and in between about 20 to about 25 psi).

[0111] In some embodiments of the methods of the present disclosure, the bioburden reduction filter comprises a PVDF-based filter. In some embodiments of the methods of the present disclosure, the bioburden reduction filter is any one of Durapore® membrane filter, Sterlitech® hydrophilic PVDF Membrane, SteriLUX® PVDF membrane and Sartorius Microsart™ PVDF membrane. In some embodiments of the methods of the present disclosure, the bioburden reduction filter comprises a PES-based filter. In some embodiments of the methods of the present disclosure, the PES-based filter is any one of PVDF-based filter Cytiva® PES filter, Sterlitech® PES filter, EZFlow® PES membrane disc filter, Pharmsteri™ PES filter and Millipore Express® filter. In some embodiments of the methods of the present disclosure, the bioburden reduction filter comprises a polyamide-based filter. In some embodiments of the methods of the present disclosure, the polyamide-based filter is any one of Sartorius® polyamide membrane filter, Sterlitech® polyamide filter, POREX® porous polyamide media and Millipak® Express filter.

[0112] In some embodiments of the methods of the present disclosure, the passing of one or more of the cell lysate, the first filtrate, second filtrate or the third filtrate through a filter ormedium is done manually or using a mechanical device. In some embodiments of the methods of the present disclosure, the mechanical device is a peristaltic pump.

[0113] In some embodiments of the methods of the present disclosure, the method of the present disclosure comprises passing a wash solution through a filter or a medium before and / or after passing one or more of the cell lysate, the first filtrate, second filtrate or the third filtrate through the filters or medium. In some embodiments of the methods of the present disclosure, the wash solution can comprise any one of water, monovalent salt, divalent salt, quaternary' ammonium salt, nonionic surfactant or a combination thereof.

[0114] In some embodiments of the methods of the present disclosure, the method of the present disclosure comprises passing an equilibration buffer through a pre-filter medium before passing the cell lysate through the pre-filter medium. In some embodiments of the methods of the present disclosure, the equilibration buffer is a non-ionic buffer. In some embodiments, the equilibration buffer comprises NaCl, (NHfhSC , Na2SO4, KC1, CH3COONH4 or a combination thereof. In some embodiments, the equilibration buffer comprises NaCl. Any of a variety' of equilibrating solutions may be suitable for use in accordance with methods of the present disclosure. In some embodiments of the methods of the present disclosure, the pH and ionic strength of the buffer equilibrating solution is chosen based on characteristics of the viral capsid preparation and / or the type of anion exchange medium or media being used.

[0115] In some embodiments of the methods of the present disclosure, the equilibration buffer comprises NaCl at a concentration of about 100 millimolar (mM) to about 400 mM (e.g., about 100 mM to about 150 mM, about 150 mM to about 200 mM, about 200 mM to about 250mM, about 250 mM to about 300 mM, about 300 mM to about 350 mM, about 350 mM to about 400 mM, about 150 mM to about 350 mM, about 200 mM to about 300 mM, about 250 mM to about 350 mM, and all integers including and in between about 100 mM to about 400 mM). In some embodiments, the equilibrating solution comprises NaCl at a concentration of about 350 mM.

[0116] In some embodiments of the methods of the present disclosure, the equilibrating solution further comprises Tris. In some embodiments of the methods of the present disclosure, the equilibrating solution comprises Tris at a concentration of about 10 mM to about 50 mM (e.g., about 10 mM to about 15 mM, about 15 mM to about 20 mM, about 20 mM to about 25 mM, about 25 mM to about 30 mM, about 30 mM to about 35 mM, about 35 mM to about 40 mM, about 40 mM to about 45 mM, about 45 mM to about 50 mM, about 15 mM to about 45 mM, about 20 mM to about 40 mM, about 25 mM to about 35. and all integers including and in betweenabout 10 mM to about 50 M). In some embodiments of the methods of the present disclosure, the equilibrating solution comprises Tris at a concentration of about 20 mM.

[0117] In some embodiments of the methods of the present disclosure, the equilibrating solution further comprises nonionic surfactant. In some embodiments, the nonionic surfactant can be any one of a fatty alcohol, ether, ester or block polymer. In some embodiments of the methods of the present disclosure, the nonionic surfactant can be a poloxamer. In some embodiments, the poloxamer can be any one of Poloxamer 188™, Poloxamer 237™, Poloxamer 338™, and Poloxamer 407™. In some embodiments of the methods of the present disclosure, the equilibrating solution comprises Poloxamer 188™ at a concentration of about 0.0005% (w / v) to about 0.01% (w / v) (e.g., about 0.0005% (w / v) to about 0.0006% (w / v), about 0.0006% (w / v) to about 0.0007% (w / v), about 0.0007% (w / v) to about 0.0008% (w / v), about 0.0008% (w / v) to about 0.0009% (w / v), about 0.0009% (w / v) to about 0.001% (w / v), about 0.001% (w / v) to about 0.005% (w / v), about 0.005% (w / v) to about 0.01% (w / v), about 0.0006% (w / v) to about 0.005% (w / v), about 0.0007% (w / v) to about 0.0009% (w / v), about 0.0007% (w / v) to about 0.005% (w / v), and all percentages including and in between about bout 0.0005% (w / v) to about 0.01% (w / v)). In some embodiments of the methods of the present disclosure, the equilibrating solution comprises poloxamer 188 at a concentration of about 0.001% (w / v)

[0118] In some embodiments of the methods of the present disclosure, the equilibrating solution has a pH of about 7.0 to about 9.0 (e.g., about 7.0 to about 7.5, about 7.5 to about 8.0, about 8.0 to about 8.5, about 8.5 to about 9.0, about 7.5 to about 8.5, about 7.0 to about 8.0, about 8.0 to about 9.0. and all integers including and in between about 7.0 to about 9.0). In some embodiments of the methods of the present disclosure, the equilibrating solution has a pH of about 8.0. As a non-limiting example, in some embodiments, the equilibrating solution is 20 mM Tris, 350 mM NaCl, 0.001% (w / v) Poloxamer 188.

[0119] In some embodiments, the methods of the present disclosure further comprise collecting the clarified lysate generated from the bioburden reduction filter. In some embodiments, the methods of the present disclosure further comprise detecting the presence of one or more of cellular complexes, cellular aggregates, colloidal aggregates, nucleases, nucleic acids, host cell proteins, lysed cell fragments, and nucleotide fragments in the collected clarified lysate.

[0120] In some embodiments of the methods of the present disclosure, the amount of impurities of size >1 mM to <50 mM in the clarified cell lysate is <50% as compared to the cell lysate before passing through the pre-filter medium. In some embodiments of the methods of the presentdisclosure, the amount of impurities of size > 50 mM in the clarified cell lysate is <50% as compared to the cell lysate before passing through the pre-filter medium.Clarification systems

[0121] The present disclosure also provides systems for clarifying a cell lysate produced from a cell culture expressing recombinant adeno-associated virus (rAAV) particles, comprising: (a) a first module comprising a prefilter medium that filters the cell lysate to remove particulate matter of size > 50pm from the cell lysate to generate a first filtrate; (b) a second module comprising a depth filter medium that filters the first filtrate to remove particulate matter of size of >lpm to <50pm (e.g., 1pm to 5pm, 5pm to 10pm, 10pm to 20pm, 30pm to 40pm, 40pm to 49.9pm, 5pm to 49.9pm. 10pm to 30pm, 20pm to 40pm, and all integers including and in between 1pm to 49.9pm) from the first filtrate to generate a second filtrate; (c) a third module comprising an anion adsorber medium that filters the second filtrate to remove adenovirus particles from the second filtrate to generate a third filtrate; and (d) a fourth module comprising a bioburden reduction filter that filters the third filtrate to remove microbial contaminants from the third filtrate to generate a clarified cell lysate.

[0122] In some embodiments of the systems of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about IxlO6to about 2xl07cells / mL (e.g., about IxlO6to about 2xl06cells / mL, about 2xl06to about 3xl06cells / mL. about 3xl06to about 4xl06cells / mL, about 4xl06to about 5xl06cells / mL, about 5x106 to about 6xl06cells / mL, about 6xl06to about 7xl06cells / mL, about 7xl06to about 8xl06cells / mL, about 8xl06to about 9xl06cells / mL, about 9xl06to about IxlO7cells / mL, about IxlO7to about 2xl07cells / mL, about 2xl06to about IxlO7cells / mL, about 3xl06to about IxlO7cells / mL, about 3xl06to about 9xl06cells / mL, about 4xl06to about 8xl06cells / mL, about 5xl06to about 7xl06cells / mL, and all integers including and in between about IxlO6to about 2xl07cells / mL). In some embodiments of the systems of the present disclosure, the cell lysate is produced from a rAAV- expressing cell culture with a cell density of at least about 106cells / mL. In some embodiments of the systems of the present disclosure, the cell lysate is produced from a rAAV-expressing cell culture with a cell density of at least about 8xl06to about 2xl07cells / mL (e.g., about 8xl06to about 8.5xl06cells / mL, about 8.5xl06to about 9xl06cells / mL, about 9xl06to about 9.5xl06cells / mL, about 9.5xl06to about IxlO7cells / mL, about IxlO7to about 1.5xl07cells / mL, about 1.5xl07to about 2xl07cells / mL, about 8.5xl06to about 1.5xl07cells / mL, about 9xl06to about IxlO7cells / mL, and all integers including and in between about 8xl06to about 2xl07cells / mL).

[0123] In some embodiments of the systems of the present disclosure, the cell lysate has a pH of about 7.5 to about 8.0 (e.g., about 7.5 to about 7.6, about 7.6 to about 7.7, about 7.7 to about 7.8, about 7.8 to about 7.9. about 7.9 to about 8.0, about 7.6 to about 7.9, about 7.5 to about 7.7, about 7.6 to about 7.9, and all integers including and in between about 7.5 to about 8.0). In some embodiments of the systems of the present disclosure, the cell lysate has a pH of about 7.7 to about 7.9 (e.g., about 7.8 to about 7.8, about 7.8 to about 7.9, and all integers including and in between about 7.7 to about 7.9).

[0124] In some embodiments of the systems of the present disclosure, the helper virus particles are adenoviral particles. In some embodiments of the systems of the present disclosure, the adenoviral particles are Ad5 particles. In some embodiments of the systems of the present disclosure, the cell lysate comprises the Ad5 particles at a density of about IxlO12to about 3xl012GC / ml (e.g., about IxlO12to about 1.2xl012GC / ml, about 1.2xl012to about 1.4xl012GC / ml, about 1.4xl012to about 1.6xl012GC / ml, about 1.6xl012to about 1.8xl012GC / ml, about 1.8xl012to about 2xl012GC / ml, about 2xl012to about 2.2xl012GC / ml, about 2.2xl012to about 2.4xl012GC / ml. about 2.4xl012to about 2.6xl012GC / ml, about 2.6xl012to about 2.8xl012GC / ml. about 2.8x1012to about 3xl012GC / ml, about 1.2xl012to about 2.8xl012GC / ml, about 1.4xl012to about 2.6xl012GC / ml, about 1.6xl012to about 2.4xl012GC / ml, about 1.8xl012to about 2.2xl012GC / ml, and all integers including and in between about IxlO12to about 3x1012GC / ml).

[0125] In some embodiments of the systems of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the anion adsorber filter is about 3xl016to about 5xl016GC / L-MV (e.g., about 3xl016to about 3.25xl016GC / L-MV, about 3.25xl016to about 3.5xl016GC / L-MV, about 3.5xl016to about 3.75xl016GC / L-MV, about 3.75xl016to about 4xl016GC / L-MV, about 4xl016to about 4.25xl016GC / L-MV, about 4.25xl016to about 4.5xl016GC / L-MV, about 4.5xl016to about 4.75xl016GC / L-MV, about 4.75xl016to about 5xl016GC / L- MV, about 3.25xl016to about 4.75xl016GC / L-MV, about 3.5xl016to about 4.25xl016GC / L-MV, and all integers including and in between about 3x1016to about 5x1016GC / L-MV).

[0126] In some embodiments of the systems of the present disclosure, the total amount of Ad5 particles in the volume of filtrate loaded onto the anion adsorber filter is about 3x1016to about 7.25xl016GC / L-MV (e.g., about 3xl016to about 3.25xl016GC / L-MV, about 3.25xl016to about 3.5xl016GC / L-MV, about 3.5xl016to about 3.75xl016GC / L-MV, about 3.75xl016to about 4xl016GC / L-MV, about 4xl016to about 4.25xl016GC / L-MV, about 4.25xl016to about 4.5xl016GC / L-MV, about 4.5xl016to about 4.75xl016GC / L-MV, about 4.75xl016to about 5xl016GC / L-MV, about 5xl016to about 5.25xl016GC / L-MV, about 5.25xl016to about 5.5xl016GC / L-MV, about 5.5xl016to about 5.75xl016GC / L-MV, about 5.75xl016to about 6xl016GC / L-MV, about 6xl016to about 6.25xl016GC / L-MV, about 6.25xl016to about 6.5xl016GC / L-MV, about 6.5xl016to about 6.75xl016GC / L-MV, about 6.75xl016to about 7xl016GC / L-MV, about 7xl016to about 7.25xl016GC / L-MV, about 3.25x1016to about 7xl016GC / L-MV, about 3.5x1016to about 6.75xl016GC / L-MV, about 3.75xl016to about 6.5xl016GC / L-MV, about 4xl016to about 6xl016GC / L-MV, about 4.25xl016to about 5.75xl016GC / L-MV, about 4.5xl016to about 5.5xl016GC / L-MV and all integers including and in between about 3xl016to about 7.25xl016GC / L-MV).

[0127] In some embodiments of the systems of the present disclosure, the volume of the cell lysate is at least 50L. In some embodiments of the systems of the present disclosure, the volume of the cell lysate is at least 250L. In some embodiments of the systems of the present disclosure, the volume of the cell lysate is about 50L to about 250L (e.g., about 50L and about 250L and all integers including and in between about 50L and about 60L, about 60L and about 70L, about 70L and about 80L, about 80L and about 90L, about 90L and about 100L, about 100L and about 120L, about 120L and about 150L, about 150L and about 170L, about 170L and about 200L. about 200L and about 220L, about 220L and about 250L, about 60L and about 220L, about 70L and about 200L, about 80L and about 170L, about 90L and about 150L, and all integers including and in between about 50L and about 250L).

[0128] In some embodiments of the systems of the present disclosure, the prefilter medium is any one of polypropylene fleece based pre-filter, adsorptive glass fleece material based pre-filter, mixed cellulose esters, nylon membrane pre-filter, glass and quartz fiber pre-filter, polyvinyl chloride and polytetrafluoroethylene.

[0129] In some embodiments of the systems of the present disclosure, the prefilter medium comprises a polypropylene fleece based pre-filter. In some embodiments of the systems of the present disclosure, the polypropylene fleece based pre-filter is any one of a Sartopure® PP3, BECO PROTECT PG® pre-filter and EMD Millipore® polypropylene pre-filter. In some embodiments of the systems of the present disclosure, the prefilter medium comprises an adsorptive glass fleece material based pre-filter. In some embodiments of the systems of the present disclosure, the adsorptive glass fleece material based pre-filter is any one of Sartopure® PP3 Maxicaps® filter and Sartorius Stedim Biotech™ Sartopure™ filter. In some embodiments of the systems of the present disclosure, the prefilter medium comprises a mixed cellulose ester based pre-filter. The mixed cellulose ester based pre-filter can comprise nitrocellulose, and anappropriate amount of cellulose acetate, acetone, n-butanol or ethanol. In some embodiments of the systems of the present disclosure, the mixed cellulose ester based pre-filter is any one of Fisherbrand™ Mixed Cellulose Ester Membrane, Whatman™ Mixed Cellulose Ester Membrane, Advantec® Mixed Cellulose Ester Membrane. Sterlitech® Nitrocellulose Mixed Ester Membrane Filters and Immobilon® -NC Membrane Triton-free Mixed Cellulose Ester filter.

[0130] In some embodiments of the systems of the present disclosure, the pre-filter comprises a nylon membrane based pre-filter. In some embodiments of the systems of present disclosure, the nylon membrane based pre-filter is any one of Sterlitech® Nylon Membrane Filter, Hydrofil™ Nylon 6.6 Membrane Filter, PALL 66606 Nylaflo™ Nylon Membrane Filter, Nalgene® Rapid- Flow™ Tissue Culture Filter and Tisch Scientific® Nylon Membrane Filter. In some embodiments of the systems of the present disclosure, the pre-filter comprises a glass and quartz fiber based pre-filter. In some embodiments of the systems of the present disclosure, the glass and quartz fiber based pre-filter is any one of Advantec® Glass and Quartz Fiber Filter and EMD Millipore® Glass and Quartz Fiber Filter. In some embodiments of the systems of present disclosure, the pre-filter comprises a polyvinyl chloride based filter. In some embodiments of the systems of present disclosure, the polyvinyl chloride based filter is any one of EMD Millipore® PVC Membrane Filter, Cytiva GLA-5000TM PVC membrane disc filter and Durapore® membrane filter. In some embodiments of the systems of present disclosure, the pre-filter comprises a polytetrafluoroethylene based filter. In some embodiments of the systems of present disclosure, the polytetrafluoroethylene based filter is any one of Sterlitech® PTFE Laminated membrane filter, Fluoropore™ PTFE membrane filter, Omnipore™ PTFE membrane filter. Omni pore™ membrane filter and Mi tex™ membrane filter.

[0131] In some embodiments of the systems of the present disclosure, the prefilter medium has a pore size of about 20 pm to about 75 pm (e.g., about 20 pm to about 25 pm, about 25 pm to about 30 pm, about 30 pm to about 35 pm, about 35 pm to about 40 pm, about 40 pm to about 45 pm, about 45 pm to about 50 pm, about 50 pm to about 55 pm, about 55 pm to about 60 pm, about 60 pm to about 65 pm. about 65 pm to about 70 pm, about 70 pm to about 75 pm, about 25 pm to about 70 pm, about 30 pm to about 65 pm, about 35 pm to about 60 pm, about 40 pm to about 55 pm, and all integers including and in between about 20 pm to about 75 pm). In some embodiments of the systems of present disclosure, the prefilter medium has a pore size of 50 pm.

[0132] In some embodiments of the systems of the present disclosure, the depth filter medium is any one of a cellulose-based, glass-based and acrylic-based filter medium. In someembodiments of the systems of present disclosure, the depth filter medium comprises between 2- 8 layers of the filter medium. In some embodiments of the systems of the present disclosure, the depth filter further comprises any one of diatomaceous earth (DE) filter aid and a silica filter aid. In some embodiments of the systems of present disclosure, the depth filter medium comprises at least one polypropylene fiber pulp, a non-woven fiber layer or a combination thereof. In some embodiments of the systems of present disclosure, the depth filter medium further comprises a cationic binder.

[0133] In some embodiments of the systems of present disclosure, the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder. In some embodiments of the systems of present disclosure, the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder, the depth filter medium comprises a first layer of non-woven fiber layer, a second layer of polypropylene fiber pulp, and a thirst and fourth layer of polyacrylic fiber pulp, silica filter aid and cationic binder. In some embodiments of the systems of present disclosure, the depth filter medium comprises six layers of polypropylene fiber and non-woven fiber, a second layer of cellulose fibers, and a third layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder.

[0134] In some embodiments of the systems of present disclosure, the depth filter comprises a cellulose-based filter medium. In some embodiments of the systems of present disclosure, the cellulose-based filter medium is any one of a Sartoclear® depth filter, Seitz® ZD pure cellulose depth filter, EMD Millipore® mixed cellulose ester filter, Millistak+® depth filter, Stax™ mAx depth filter. Allegro™ Connect depth filter, Stax™ single use depth filter and Stax™ mAx CF cake filter.

[0135] In some embodiments of the systems of present disclosure, the depth filter comprises a glass-based filter medium. In some embodiments of the systems of present disclosure, the glassbased filter medium is any one of Cytiva® 934- AH glass microfiber filter. Cytiva® EX / THICK glass fiber filter. Sartorius® glass microfiber filter. Sterlitech® glass fiber filter, Merck® glass fiber filter and Borosilicate glass microfiber filter.

[0136] In some embodiments of the systems of present disclosure, the depth filter comprises an acrylic-based filter medium. In some embodiments of the systems of present disclosure, the acrylic-based filter medium is any one of Millistak+® HC Pro Pod depth filter, Versapor® acrylic copolymer membrane disc filter and Minisart® with Acrylic-based MBS Housing filter.

[0137] In some embodiments of the systems of present disclosure, the anion adsorber medium is any one of a strong AEX medium or an AEX hybrid medium. In some embodiments of the systems of present disclosure, the strong AEX medium is any one of a Sartobind Q medium, Mustang Q medium or Natrix Q medium. In some embodiments of the systems of present disclosure, the AEX hybrid medium comprises AEX chromatography medium and a polyamide membrane. In some embodiments of the systems of present disclosure, the AEX hybrid medium comprises AEX chromatography medium and polyether sulfone (PES) membrane. In some embodiments of the systems of present disclosure, the AEX chromatography medium is a quaternary ammonium salt functional AEX medium. In some embodiments of the systems of present disclosure, the quaternary ammonium salt functional AEX medium is non-woven medium. In some embodiments of the systems of present disclosure, the AEX chromatography medium is any one of a Harvest RC medium or Emphaze AEX medium. In some embodiments of the systems of present disclosure, the pore size of the polyamide membrane or the PES membrane is between about 0.1 to about 0.5 pm (e.g., about 0.1 to about 0.2 pm, about 0.2 to about 0.3 pm, about 0.3 to about 0.4 pm, about 0.4 to about 0.5 pm, about 0.2 to about 0.4 pm, about 0. 1 to about 0.3 pm, about 0.3 to about 0.5 pm, and all integers including and in between about 0.1 to about 0.5 pm). In some embodiments of the systems of present disclosure, the pore size of the polyamide membrane or the PES membrane is 0.2 pm. In some embodiments of the systems of present disclosure, the bioburden reduction filter can be any one of a PVDF-based filter, a PES-based filter, and a polyamide-based filter.

[0138] In some embodiments of the systems of present disclosure, the bioburden reduction filter comprises a PVDF-based filter. In some embodiments of the systems of present disclosure, the bioburden reduction filter is any one of Durapore® membrane filter, Sterlitech® hydrophilic PVDF Membrane, SteriLUX® PVDF membrane and Sartorius Microsart™ PVDF membrane. In some embodiments of the systems of present disclosure, the bioburden reduction filter comprises a PES-based filter. In some embodiments of the systems of present disclosure, the PES-based filter is any one of PVDF-based filter Cytiva® PES filter, Sterlitech® PES filter, EZFlow® PES membrane disc filter, Pharmsteri™ PES filter and Millipore Express® filter. In some embodiments of the systems of present disclosure, the bioburden reduction filter comprises a polyamide-based filter. In some embodiments of the systems of present disclosure, the polyamide- based filter is any one of Sartorius® polyamide membrane filter, Sterlitech® polyamide filter, POREX® porous polyamide media and Millipak® Express filter.Filter combinations for clarification methods and systems of the present disclosure

[0139] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based filter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF -based filter.

[0140] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of acrylic-based filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a PVDF -based filter.

[0141] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0142] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0143] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of an acry lic-based filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0144] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0145] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0146] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of acellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0147] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0148] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0149] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a cellulose- based depth filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0150] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0151] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0152] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0153] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0154] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF -based filter.

[0155] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF -based filter.

[0156] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a glassbased depth filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0157] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0158] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a glassbased filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PVDF-based filter.

[0159] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0160] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0161] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8layers of acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF -based filter.

[0162] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF -based filter.

[0163] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF -based filter.

[0164] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of an acry lic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0165] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0166] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0167] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0168] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0169] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0170] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a cellulose- based depth filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0171] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0172] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0173] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0174] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0175] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0176] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of aglass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF -based filter.

[0177] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a glassbased depth filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF -based filter.

[0178] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF -based filter.

[0179] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a glassbased filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF -based filter.

[0180] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PVDF-based filter.

[0181] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based filter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0182] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of acry lic-based filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0183] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0184] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0185] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0186] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0187] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0188] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0189] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0190] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0191] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based depth filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0192] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0193] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0194] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0195] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0196] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0197] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0198] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a glassbased depth filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0199] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0200] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a glassbased filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a PES-based filter.

[0201] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0202] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0203] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0204] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0205] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0206] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of anacrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0207] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0208] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0209] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0210] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0211] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0212] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a cellulose- based depth filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0213] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0214] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0215] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0216] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0217] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0218] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0219] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a glassbased depth filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0220] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0221] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0222] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a PES-based filter.

[0223] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based filter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide -based filter.

[0224] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of acrylic-based filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0225] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0226] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0227] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0228] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0229] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0230] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0231] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0232] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0233] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a cellulose- based depth filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0234] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0235] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0236] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of acellulose-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0237] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0238] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0239] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0240] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a glassbased depth filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0241] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0242] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a glassbased filter medium, the anion adsorber medium is an AEX hybrid medium and the bioburden reduction filter is a polyamide-based filter.

[0243] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0244] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0245] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0246] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0247] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of an acrylicbased filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0248] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0249] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0250] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of an acrylic-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0251] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of acellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0252] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0253] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0254] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a cellulose- based depth filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0255] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0256] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0257] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0258] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polypropylene fleece based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0259] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is an adsorptive glass fleece material based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0260] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a mixed cellulose ester based pre-filter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0261] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a nylon membrane based prefilter, the depth filter comprises 2 to 8 layers of a glassbased depth filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0262] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a glass and quartz fiber based prefilter, the depth filter comprises 2 to 8 layers of a glass-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0263] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polyvinyl chloride based prefilter, the depth filter comprises 2 to 8 layers of a glassbased filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.

[0264] In some embodiments of the methods and systems of the present disclosure, the prefilter medium is a polytetrafluoroethylene based prefilter, the depth filter comprises 2 to 8 layers of a cellulose-based filter medium, the anion adsorber medium is a strong AEX medium and the bioburden reduction filter is a polyamide-based filter.Viral capsid preparationsMethods and systems of the present disclosure are useful for separating rAAV capsid particles from host producer cell derived impurities like nucleic acids, proteins, peptides, lipopolysaccharides, cell membranes, and helper viral particles in a viral capsid preparation, e.g., a viral capsid preparation that results from a process intended to generate a recombinant virus particle comprising a heterologous nucleic acid. Typically, the rAAV capsid particles in a given viral capsid preparation are capsid particles of the same virus and same seroty pe. In someembodiments, the capsid is from an AAV capsid of serotype 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, rhlO, or hu37 or a variant thereof. In some embodiments, the capsid is from an AAV capsid of serotype 8 or 9 or a variant thereof. In some embodiments, the capsid is from an AAV capsid of serotype 8 or a variant thereof. In some embodiments, the capsid is from an AAV capsid of serotype 9 or a variant thereof. In some embodiments, the capsid is from an AAV capsid of serotype rhlO or hu37 or a variant thereof. In some embodiments, the capsid is from an AAV capsid of serotype rhlO or a variant thereof. In some embodiments, the capsid is from an AAV capsid of serotype hu37 or a variant thereof.

[0265] Throughout the description, where systems or methods are described as having, including, or comprising specific components or specific steps, it is contemplated that, additionally, there are systems or methods of the present disclosure that consist essentially of, or consist of, the recited components or the recited steps.

[0266] In the application, where an element or component is said to be included in and / or selected from a list of recited elements or components, it will be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0267] Further, it will be understood that elements and / or features of a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and disclosure(s). For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of the disclosure(s) described and depicted herein. All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose.

[0268] It will be understood that the expression "at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use. The expression “and / or” in connection with three or more recited objects will be understood to have the same meaning unless otherwise understood from the context.

[0269] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, will be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0270] The use of any and all examples, or exemplary language herein, for example, “for instance”, “such as”, “for example”, “e.g..”. or “including” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of the disclosure unless claimed. No language in the specification will be construed as indicating any non-claimed element as essential to the practice of the subject matter of the present disclosure.

[0271] It is understood that this disclosure is not limited to the particular methodology7, protocols, materials, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present disclosure, which will be encompassed by the appended claims.

[0272] All publications, patents and patent applications, including any drawings, sequences, and appendices therein referred to throughout the present description are incorporated by reference in their entirety7for all purposes to the same extent as if each individual publication, patent or patent application, drawing, sequence, or appendix was specifically and individually indicated to be incorporated by reference in its entirety7for all purposes.EXAMPLES

[0273] The inventive systems and methods now being generally described, will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.

[0274] Clarification of harvest collected from high cell density producer cell lines for producing rAAV particles with the aid of helper virus to separate is an essential step of bioprocessing and manufacturing rAAV-based drug products. The following studies disclose the testing and identification of specific filters for use in development of the clarification and filtration systems of the present disclosure to remove contaminants, impurities including cell aggregates, and helper viruses from harvested rAAV producer cell lysate.Example 1: Effect of adding pre-filter on depth filter differential pressure of filtration train

[0275] Objective and study design: In this study, two different filtration trains were used to process identical feed materials to demonstrate the influence of the addition of a pre-filter for primary filtration (FIG. 1A-1B). The identical feed materials containing same levels of rAAV particles, impurities and Ad5 particles were obtained from the same lot of lysate derived from AAV PCL grown at a peak cell density of 1.5xl07cells / ml in 250L scale bioreactor using enhanced methods implementing fed-batch and / or perfusion strategies described herein. For the first train (FIG. 2A), the feed (crude har est) w as processed through a 23 cm2Millistak+® HC Pro DOSP (MilliporeSigma; MD0SP23CL3) filter. To evaluate the effect of the addition of a pre-filter, for the second train (FIG. 2B), the feed was processed through a 0.018 m250-pm Sartopure® PP3 (Sartorius; 5051350P4) pre-filter followed by a 23 cm2Millistak+® HC Pro DOSP filter. The filtration was conducted under constant flow wherein the flowrates were controlled through peristaltic pumps (Watson-Marlow 120). Inlet pressures of each filter were monitored by singleuse pressure transducers (PendoTECH; PRESS-S-000) and a PendoTECH data acquisition system. The operating flux was fixed at 75 L / m2h relative to the DOSP filter. Prior to processing the feed material, water was flushed through the PP3 and DOSP filters. A system equilibration is performed with a solution containing 20 mM Tris, 350 mM NaCl, 0.001% (w / v) Poloxamer 188 (pH 8.0). After priming and equilibration, the crude harvest was processed through the filtration trains until the feed was consumed or if the DOSP differential pressure exceeded ~16 psid.

[0276] Results: The results described herein show that in absence of the pre-filter, the flow of the crude harvest through the filtration trains was arrested beyond a flow rate of 150 L / m2with a maximum differential pressure saturation of 16 psid (FIG. 2 A). However, with addition of the prefilter the flow- rate of the filtration train was extended to 200 L / m2, with a differential pressure of about 20 psid (FIG. 2B).

[0277] Conclusion: Based on the results of the study described herein, addition of a pre-filter with the appropriate pore size, extends the throughput (L / m2) of the DOSP depth filter. This is likely due to a staging effect wherein larger particulates are retained by the pre-filter, decreasing the likelihood of fouling on the DOSP depth filter.Example 2: Comparison of conventional depth filter and adsorptive filters on the throughput capacity of the bioburden reduction filter

[0278] Obj ective and study design: In the study described herein, three different filtration trains were used to process comparable feed material to demonstrate the effect of using an adsorptive filter for secondary filtration (FIG. IB). For all three trains, filtration was conducted under constant flow wherein the flowrates were controlled through peristaltic pumps (Avantor® MasterFlex* L / S®; MFLX07522-20). For the first train (depth filter plus bioburden reduction filter) (FIG. 3A), the feed (crude harvest) was processed through a 0.018 m250-pm Sartopure PP3 (Sartorius; 5051350P4) pre-filter, the outlet was split into two and processed through two 0.027 m2(total filter area of 540 cm2) Millistak+® HC Pro D0SP (MilliporeSigma; MD0SP027H1) filters, the filtrate of the two D0SP filters were consolidated and processed through a 0.027 m2Millistak+* F0HC (MilliporeSigma; MF0HC027H1) filter, and finally through a 0.014 m2Millipore Express SHC (MilliporeSigma; KHGES015FB3) 0.5 / 0.2-pm filter. The operating flux was fixed at 75 L / m2h relative to the D0SP filter. For the second (FIG. 3B) and third train (FIG. 3C), an intermediate pool was generated to serve as the feed. A crude harvest feed, comparable to that of the first train, was processed through a 0.018 m250-pm Sartopure PP3 filter, the outlet was split into two and processed through two 0.027 m2(total filter area of 540 cm2) Millistak+® HC Pro D0SP filters. The process fluid from the filtration train was collected and this intermediate served as the feed to the second and third train. In the second train, the D0SP filtrate intermediate was processed through a 0.0340 m2Harvest RC (3M Purification Inc.; EMP513HRC2FA) AEX-based filter (adsorptive filter A), followed by a 0.014 m2Millipore Express SHC 0.5 / 0.2-pm filter. In the third train, the D0SP filtrate intermediate was processed through a 0.0170 m2Emphaze (3M Purification Inc.; EMP513AEX020R) AEX-based filter (adsorptive filter B), followed by a 0.014 m2Milhpore Express SHC 0.5 / 0.2-pm filter. For the primary filtration step (PP3 and D0SP), the operating flux was fixed at 75 L / m2h relative to the D0SP filter. For the secondary filtration step (adsorptive filter and SHC), the operating flux was fixed at 50 and 100 L / m2-h relative to the adsorptive filter A (second train) and adsorptive filter B (third train), respectively. Inlet pressures of each filter were monitored by single-use pressure transducers (PendoTECH; PRESS-S-000) and a PendoTECH data acquisition system. Prior to processing the feed material, pre-use flushes were performed for each filter. A system equilibration is performed with a solution containing 20 mM Tris, 350 mM NaCl, 0.001% (w / v) Poloxamer 188 (pH 8.0). After priming and equilibration, the feed material was processed through the filtration trains until the feed was consumed or if the inlet pressure exceeded 20 psi.

[0279] Results: The results described herein show that the flow of the crude harvest through the first filtration train comprising a conventional depth filter and a bioburden reduction filter, was arrested beyond a flow rate of about 300 L / m2with a maximum differential pressure saturation of 20 psid. The second filtration train with adsorptive filter A extended the flow rate up to 650 L / m2corresponding to a differential pressure of 12 psid. The second filtration train with adsorptive filter A extended the flow rate up to 800 L / m2corresponding to a differential pressure of 12 psid.

[0280] Conclusion: The results described herein show that the use of an AEX-based clarification filter in secondary filtration extends the throughput (L / m2) of the bioburden reduction filter (0.5 / 0.2-pm filter). The observed extension in flow rate with reduced differential pressure can be because the fouling / pore-plugging species are better retained in these filters as opposed to a conventional depth filter, such as an F0HC filter.Example 3: Comparison of pressure profiles of various commercially available AEX-based adsorptive media.

[0281] Objective and study design: In the study described herein, three different adsorptive separation media were evaluated without being limited by the pressure build-up on a downstream bioburden reduction filter. An AKTATM Avant 25 unit (Cytiva) w as used to perform the filtration experiments. Inlet pressures were monitored by the in-line pressure sensors, a sample-loop valve, an outlet valve, several versatile valves, with which different flow paths can be applied, a conductivity sensor, a pH sensor, a UV detector (0.2 cm path length), and a built-in fraction collector. For all three devices testing the three different adsorptive separation media, filtration was conducted under constant flow wherein the flowrates were controlled through the Avant 25 dual-head piston pump. For the second (FIG. 3B) and third train (FIG. 3C), an intermediate pool was generated to serve as the feed. A crude harvest feed, comparable to that of train one, w as processed through a 0.018 m250-pm Sartopure PP3 (Sartorius; 5051350P4) filter, the outlet is split into tw o and processed through two 0.027 m2(total filter area of 540 cm2) Millistak+® HC Pro D0SP (MilliporeSigma; MD0SP027H1) filters. The process fluid from the filtration train was collected and this intermediate served as the feed for the comparison between the three adsorptive separation media. Three different AEX-based devices were compared: 1) 0.2-mL Natrix® Q (MilliporeSigma; NXF-00) (Adsorptive hydrogel); 2) 1.4mL Harvest RC (3M Purification Inc.; BC4, EMP201HRC2FA) (Adsorptive filter A); and 3) 1.4mL Emphaze (3M Purification Inc.; BV1. EMP201AEX020R) (Adsorptive filter B). Prior to processing the feed material, the vendor recommended pre-use flushes were performed for each filter. A system equilibration wasperformed with a solution containing 20 mM Tris, 350 mM NaCl, 0.001% (w / v) Poloxamer 188 (pH 8.0). After priming and equilibration, the feed material was processed through the AEX-based devices until the feed was consumed or if the delta pressure exceeded 30 psid. Flow rates were held at 0.5 media volumes per min.

[0282] Result: The study described herein shows that the adsorptive hydrogen demonstrated quick clogging indicated by maximum differential pressure of more than 15 psid reached with increase in loading volume from 25 to 125 throughput units. In contrast, the two AEX based adsorptive filters, Adsorptive Filter A and Adsorptive Filter B, depicted extension of the loading volume capacity up to about 150 throughput units, respectively, to reach the maximum differential pressure of less than about 2 psid (FIG. 4), thus showing reduced clogging.

[0283] Conclusion: Multiple factors determine the adsorption capacity and throughput of the adsorptive filter. The adsorptive filter devices used in the clarification studies were specifically designed for clarification. In contrast, other AEX media on the market that have comparable or higher AEX capacity' are designed for chromatography applications. Natrix® Q is considered to have the highest AEX capacity among AEX-based adsorbers available on the market. Presumably, due to the smaller pore size and device design, Natrix® Q (Adsorptive Hydrogel) exhibited immediate pore-plugging. There are also design differences between the adsorptive filters Harvest® RC and Emphaze®. The former has a lower AEX capacity7, and a tighter built-in bioburden reduction filter compared to the latter. The results disclosed herein show that Emphaze (Adsorptive Filter B) displayed a greater throughput than Harvest RC (Adsorptive Filter A) before building up pressure. One of the two factors, AEX capacity or pore-size distribution of the built- in bioburden reduction filter may pre-dominantly contribute to the difference in throughput.Example 4: Harvest clarification filter pressure profiles of the multi-stage filtration train for a 50-L scale production

[0284] Objective and study7design: The study described herein is a 50-L scale-up demonstration to evaluate the full filtration assembly using exemplary7, process-scale devices of the cell lysate clarification and filtration system disclosed herein. The feed (crude harvest) was processed through a 0. 12 m250-pm Sartopure® PP3 (Sartorius; 5055350P8) pre-filter, challenged to 351 L / m2at a flux of 345 L / m2h. Post pre-filtration the process feed was fed into two 0.33 m2(total filter area of 66 m2) Millistak+® HC Pro D0SP (MilliporeSigma; MD0SP03FS 1) depth filters, challenged to 64 L / m2at a flux of 63 L / m2h. Depth-filtration was followed by processing through two 0.23 m2(total filter area of 46 m2) Emphaze® (3M Purification Inc.;EMP710AEX020R) AEX-based adsorptive filters, challenged to 92 L / m2at a flux of 90 L / m2h. The filtration train ended with a 0.13 m2Opticap™ XLT3 (MilliporeSigma; KHGES03EIH3) 0.5 / 0.2-pm bioburden reduction filter, challenged to 324 L / m2at a flux of 318 L / m2h. Prior to processing the feed material, pre-use flushes were performed for each filter. A system equilibration was performed with a solution containing 20 mM Tris, 350 mM NaCl, 0.001% (w / v) Poloxamer 188 (pH 8.0). After priming and equilibration, the feed material was processed.

[0285] Results: The study described herein shows that the differential pressure of each of the filtration steps remained below about 10 psid, with the correspondingly increasing throughput volumes (L / m2), which was below the recommended bench apparatus differential pressure limit of 20 psid. The results described herein helped determine the operation parameters defined by the optimal limits of flux, volumetric challenge and differential pressure limits of the different filtration components of the clarification and filtration system disclosed herein.Table 1 : Operating parameters of the disclosed clarification and filtration system

[0286] Conclusion: This scale-up evaluation at the 50-L scale suggested comparable performance (throughput) using process-scale devices as with using bench-scale devices.Example 5: Turbidity comparison of the crude harvest against the clarified filtrate for two pilot-scale batches employing an adsorptive filter for secondary filtration

[0287] Objective and study design: The study described herein was done to evaluate the turbidity of aliquots of the crude (pre-filtration) and clarified harvest (post-filtration) using theclarification and filtration systems of present disclosure, from two different production lots with comparable feed material, under filtration conditions described below. Change in turbidity of the filtrate with respect to the feed material was used as an indication of the effectiveness of the disclosed clarification and filtration system in removing impurities from the feed material.

[0288] Production Lot 1 was a scale-up demonstration to evaluate the full filtration assembly using representative, process-scale devices. The feed (crude harvest) was processed through a 0.65 m250-pm Sartopure® PP3 (Sartorius; 5057350P1 ) filter, challenged to 389 L / m2at a flux of 248 L / m2h. The process feed was then fed into three 0.77 m2(total filter area of 2.3 m2) Millistak+® HC Pro DOSP (MilliporeSigma; MD0SP07FS1) filters, challenged to 109 L / m2at a flux of 70 L / m2h. This was followed by processing through two 1.61 m2(total filter area of 3.22 m2) Harvest™ RC (3M Purification Inc.; EMP770HRC2FA) filters, challenged to 78 L / m2at a flux of 50 L / m2h. The filtration train ended with two 0.49 m2(total filter area of 0.98 m2) Opticap® XLT10 (MilliporeSigma; KHGES03HH3) 0.5 / 0.2-pm filters, challenged to 258 L / m2at a flux of 329 L / m2h. Production Lot 2 comprised aliquots taken from the same filtration experiment and conditions, as described in Example 4.

[0289] Results: The study described herein shows that the clarification and filtration train used in both production lots 1 and 2, significantly reduced the turbidity of the final filtrate generated as compared to the respective feed material that was fed into the system (FIG. 6).

[0290] Conclusion: Based on the results described herein, the clarified harvest pool turbidity indicates that the filtrate generated by filtering a feed material through the clarification and filtration systems of present disclosure is of sufficient purity to process forw ard with minimal risk of impacting subsequent unit operations for purifying drug product.Example 6: Anion adsorber filter load conductivity evaluation

[0291] Objective and study design: The study described herein determined the effect of anion adsorber filter (Q-filter) loading conductivity on rAAV recovery and impurity level reduction (determined by turbidity) from incoming filtrate applied to the Q-filter. To demonstrate that rAAV bound to the membrane, a linear conductivity gradient phase followed the sample applicate / flow- through phase. Specifically, the effect of low, medium and high conductivity of loading material on rAAV recovery and purified filtrate turbidity was determined.

[0292] Result: The elution profile of the rAAV capsids (elution profile at UV254 and UV280) as a function of linearly increasing loading material conductivity, and loading volume wasdetermined. There was no apparent change in the UV absorbance traces for either 280 or 254 nm wavelengths, indicative of stripping of the rAAV from the Q-filter (FIG. 7). The percentage recovery of rAAV and turbidity- from the incoming filtrate applied to the Q-filter filtrate is depicted in Table 2 below. As shown in Table 2, at low load conductivity, several impurity populations bind to the Q-filter along with the product rAAV particles, resulting in low percentage recovery' of the product rAAV and low turbidity- of the filtrate. At high load conductivity-, the impurity' populations and the rAAV do not stick to the adsorptive filter, resulting in high percentage recovery of product rAAV but with high turbidity- filtrate. Adjustment to a medium load conductivity balanced high rAAV recovery and high impurity- removal as depicted by low turbidity of the filtrate.Table 2: Load conductivity optimization

[0293] Conclusion: The results of the study described herein show that the recovery of rAAV and reduction of impurity from the incoming filtrate applied to an anion adsorber filtrate is optimum at medium conductivity of the loading material.Example 7: Adenovirus 5 (Ad5) loading density simulation on anion adsorber filter

[0294] Objective and study design: The study described herein was a loading densitysimulation conducted to account for typical process variability in filtration of a feed material. Process variability in terms of the expected volumetric loading, Ad5 loading in terms of genome copy per loading volume (Ad5, GC per L-MV), and breakthrough for AEX Emphaze capsules was evaluated assuming a conservative dynamic binding capacity (DBC) for a fixed filter bed volume of 78.4 L. The probability distribution values for expected upstream processing output (bioreactor working volume and Ad5 titer) were based on historical therapeutic transgene carrying rAAV, pilot batches, and small-scale Ambr250 bioreactor studies. Input, fixed, and output variables as used are provided in Table 3, Table 4, and Table 5, respectively. 1000 iterations were simulatedto generate the output data. A histogram of the loading density events is show n in FIG. 8. 1000 iterations were simulated to generate the output data.

[0295] Table 3. Loading Density Simulations - Input VariablesTable 4. Loading Density Simulations - Fixed VariablesTable 5. Loading Density Simulations - Output Variables

[0296] Result: The results from the loading density simulations of the study described herein are summarized in FIG. 8. Based on the results described herein, at an Ad5 loading challenge of 3xl06to 5xl06GC per loading volume (L-MV), 91.8% of the Ad5 is retained by the anion adsorber filter, denoting the Ad5 concentration in feed material below the DBC.

[0297] Conclusion: Based on the results described herein the delineation between loading regimes for utilizing the dynamic binding capacity of an anion adsorber filter for use in the methods and systems of the present disclosure are as follows: (a) incoming Ad5 concentration infiltrate entering the anion adsorber filter is less than 0.1% of the DBC of the filter; (b) incoming Ad5 concentration in filtrate entering the anion adsorber filter is greater than 0. 1% of the DBC of the filter and less than 1% of the DBC of the filter. The expected Ad5 concentration is projected to be ~2xlO10GC / mL, which is in the range typical for a rAAV producing HeLa cell based platform described herein; and (c) incoming Ad5 concentration in filtrate entering the anion adsorber filter is greater than 1 % of the DBC of the filter. A Mustang Q step filter can be expected to remove this level of Ad5 concentration, but there may be a threshold Ad5 concentration that increases the rate of fouling on the bioburden reduction filter.

Claims

CLAIMSWe claim:

1. A method of clarifying a cell lysate produced from a cell culture expressing recombinant adeno-associated virus (rAAV) particles, comprising:(a) passing the cell lysate through a prefilter medium to generate a first filtrate;(b) passing the first filtrate through a depth filter medium to generate a second filtrate;(c) passing the second filtrate through an anion adsorber medium to generate a third filtrate; and(d) passing the third filtrate through a bioburden reduction filter to generate a clarified cell lysate.

2. The method of claim 1, wherein the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about 1x106to about 2xl07cells / mL.

3. The method of claim 2, wherein the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about 106cells / mL.

4. The method of claim 1 or 2, wherein the cell lysate is produced from a rAAV-expressing cell culture with a cell densify of about 8xl06to about 2xl07cells / mL.

5. The method of any one of claims 1-4, wherein the cell lysate has a pH of about 7.5 to about 8.0.

6. The method of claim 5, wherein the cell lysate has a pH of about 7.7 to about 7.9.

7. The method of any one of claims 1-6, wherein the cell lysate comprises rAAV particles and one or more of cellular complexes, cellular aggregates, colloidal aggregates, nucleases, nucleic acids, host cell proteins, lysed cell fragments, and nucleotide fragments.

8. The method of any one of claims 1-7, wherein the clarified cell lysate retains at least 80% of the total rAAV particles present in the cell lysate.

9. The method of claim 7 or 8, wherein the cell lysate comprises helper virus particles.

10. The method of claim 9, wherein the helper virus particles are adenoviral particles.

11. The method of claim 10, wherein the adenoviral particles are adenovirus type 5 (Ad5) particles or a variant or mutant version thereof.

12. The method of claim 11, wherein the cell lysate comprises the Ad5 particles at a densify of about IxlO12to about 3xl012GC / ml.

13. The method of any one of claims 1-12, wherein the volume of the cell lysate is at least 50L.

14. The method of any one of claims 1-13, wherein the volume of the cell lysate is at least15. The method of any one of claims 1-14, wherein the prefilter medium comprises any one of a polypropylene fleece based pre-filter, an adsorptive glass fleece material based pre-filter, a mixed cellulose ester, a nylon membrane pre-filter, a glass and quartz fiber pre-filter, polyvinyl chloride based filter and polytetrafluoroethylene based filter.

16. The method of any one of claims 1-15, wherein the prefilter medium has a pore size of about 20 pm to about 75 pm.

17. The method of claim 16, wherein the prefilter medium has a pore size of about 50 pm.

18. The method of any one of claims 1-17, wherein passing the cell lysate through the prefilter medium increases the volumetric capacity of the depth filter by at least 25%.

19. The method of any one of claims 1-18, wherein passing the cell lysate through the prefilter medium increases the filter capacity of the depth filter by about 30 to about 50 L / m220. The method of any one of claims 1-19, wherein the flow rate of the cell lysate in the prefilter medium is about 225 to about 925 L / m2h.

21. The method of any one of claims 1-17, wherein the prefilter medium has a volumetric capacity of about 300 to about 700 L / m2.

22. The method of any one of claims 1-21, wherein the differential pressure in the prefilter medium is about 1 to about 5 psi.

23. The method of any one of claims 1-22, wherein the depth filter medium comprises any one of a cellulose-based, glass-based and acrylic-based filter medium.

24. The method of claim 23, wherein the depth filter medium comprises 2 to 8 layers of the filter medium.

25. The method of claim 24, wherein the depth filter further comprises any one of a diatomaceous earth (DE) filter aid and a silica filter aid.

26. The method of any one of claims 23-25, wherein the depth filter medium comprises at least one polypropylene fiber pulp, a non-woven fiber layer or a combination thereof.

27. The method of any one of claims 23-26, wherein the depth filter medium further comprises a cationic binder.

28. The method of any one of claims 23-27. wherein the flow rate of the first filtrate in the depth filter medium is about 50 to about 150 L / m2h.

29. The method of any one of claims 23-28, wherein the volumetric capacity of the depth filter medium is about 70 to about 200 L / m2.

30. The method of any one of claims 23-29, wherein the differential pressure in the depth filter medium is about 1 to about 18 psi.

31. The method of any one of claims 23-30. wherein the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder.

32. The method of any one of claims 23-30, wherein the depth filter medium comprises a first layer of non-woven fiber, a second layer of polypropylene fiber pulp, and a third and fourth layer, the third and fourth layers comprising poly acrylic fiber pulp, silica filter aid and cationic binder.

33. The method of any one of claims 23-30, wherein the depth filter medium comprises six layers of polypropylene fiber and non-woven fiber, a second layer of cellulose fibers, and a third layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder.

34. The method of any one of claims 1-33, wherein the anion adsorber medium is any one of a strong anion exchange chromatography (AEX) medium or an AEX hybrid medium.

35. The method of claim 34, wherein the strong AEX medium is any one of Sartobind® Q medium, Mustang® Q medium or Natrix® Q medium.

36. The method of claim 34, wherein the AEX hybrid medium comprises AEX chromatography medium and a polyamide membrane.

37. The method of claim 34, wherein the AEX hybrid medium comprises AEX chromatography medium and polyether sulfone (PES) membrane.

38. The method of any one of claims 36-37, wherein the AEX chromatography medium is a quatemaiy ammonium salt functional AEX medium.

39. The method of claim 38, wherein the quaternary ammonium salt functional AEX medium is non-woven medium.

40. The method of claim 38, wherein the AEX chromatography medium is any one of a 3M Polisher® ST medium, Elarvest® RC medium or Emphaze® AEX medium.

41. The method of any one of claims 36-40, wherein the pore size of the polyamide membrane or the PES membrane is about 0. 1 to about 0.5 mm.

42. The method of claim 41, wherein the pore size of the polyamide membrane or the PES membrane is 0.2 mm.

43. The method of any one of claims 34-42, wherein the flow rate of the second filtrate in the anion adsorber medium is about 50 to about 200 L / m2h.

44. The method of any one of claims 34-43. wherein the volumetric capacity of the anion adsorber medium is about 70 to about 200 L / m2.

45. The method of any one of claims 34-44. wherein the differential pressure of the anion adsorber medium is about 1 to about 10 psi.

46. The method of any one of claims 34-45. wherein the conductivity of the filter load of the anion adsorber medium is about 16 to about 40 mS / cm.

47. The method of claim 46, wherein the conductivity of the filter load is about 18 to about 37 mS / cm.

48. The method of any one of claims 1-47, wherein the bioburden reduction filter comprises anyone of a poly vinylidene fluoride (PVDF) based filter, a PES-based filter, and a polyamide- based filter.

49. The method of any one of claims 1-48, wherein the flow rate of the third filtrate through the bioburden reduction filter is about 300 to about 700 L / m2h.

50. The method of any one of claims 1-49, wherein the volumetric capacity- of the bioburden reduction filter is about 200 to about 750 L / m251. The method of any one of claims 1-50, wherein the differential pressure limit of the bioburden reduction filter is about 20 to about 25 psi.

52. A system for clarifying a cell lysate produced from a cell culture expressing recombinant adeno-associated virus (rAAV) particles, comprising:(a) a first module comprising a prefilter medium that filters the cell lysate to remove particulate matter of size > 50 pm from the cell lysate to generate a first filtrate;(b) a second module comprising a depth filter medium that filters the first filtrate to remove particulate matter of size of > 1 pm to <50 pm from the first filtrate to generate a second filtrate;(c) a third module comprising an anion adsorber medium that filters the second filtrate to remove helper virus particles from the second filtrate to generate a third filtrate; and(d) a fourth module comprising a bioburden reduction filter that filters the third filtrate to remove microbial contaminants from the third filtrate to generate a clarified cell lysate.

53. The system of claim 52, wherein the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about IxlO6to about 2xl07cells / mL.

54. The system of claim 53, wherein the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about 106cells / mL.

55. The system of claim 53, wherein the cell lysate is produced from a rAAV-expressing cell culture with a cell density of about 8xl06to about 2xl07cells / mL.1056. The system of any one of claims 52-55, wherein the cell lysate has a pH of about 7.5 to about 8.0.

57. The system of claim 56, wherein the cell lysate has a pH of about 7.7 to about 7.9.

58. The system of any one of claims 52-57, wherein the helper virus particles are adenoviral particles.

59. The system of claim 58, wherein the adenoviral particles are Ad5 particles or a variant or mutant version thereof.

60. The system of claim 59, wherein the cell lysate comprises the Ad5 particles at a density of about IxlO12to about 3xl012GC / ml.

61. The system of any one of claims 52-60, wherein the volume of the cell lysate is at least 50L.

62. The system of any one of claims 52-60, wherein the volume of the cell lysate is at least 250L.

63. The system of any one of claims 52-62, wherein the prefilter medium is any one of polypropylene fleece based pre-filter, adsorptive glass fleece material based pre-filter, mixed cellulose esters, nylon membrane pre-filter, glass and quartz fiber pre-filter, polyvinyl chloride and polytetrafluoroethylene.

64. The system of claim 63, wherein the prefilter medium has a pore size of about 20 pm to about 75 pm.

65. The system of claim 64, wherein the prefilter medium has a pore size of about 50 pm.

66. The system of claim 63, wherein the depth filter medium is any one of a cellulose-based, glass-based and acrylic-based filter medium.

67. The system of claim 66, wherein the depth filter medium comprises between 2-8 layers of the filter medium.

68. The system of claim 67, wherein the depth filter further comprises any one of diatomaceous earth (DE) filter aid and a silica filter aid.

69. The system of any one of claims 63-68, wherein the depth filter medium comprises at least one polypropylene fiber pulp, a non-w oven fiber layer or a combination thereof.

70. The system of any one of claims 63-69, wherein the depth filter medium further comprises a cationic binder.

71. The system of any one of claims 63-70, wherein the depth filter medium comprises a first layer of cellulose fibers and a second layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder.

72. The system of any one of claims 63-70, wherein the depth filter medium comprises a first layer of non-woven fiber layer, a second layer of polypropylene fiber pulp, and a thirst and fourth layer of polyaciylic fiber pulp, silica filter aid and cationic binder.

73. The system of any one of claims 63-70, wherein the depth filter medium comprises six layers of polypropylene fiber and non-woven fiber, a second layer of cellulose fibers, and a third layer of cellulose, diatomaceous earth (DE) filter aid and cationic binder.

74. The system of claim 52, wherein the anion adsorber medium is any one of a strong AEX medium or an AEX hybrid medium.

75. The system of claim 74. wherein the strong AEX medium is any one of a Sartobind Q medium, Mustang Q medium or Natrix Q medium.

76. The method of claim 75, wherein the AEX hybrid medium comprises AEX chromatography medium and a polyamide membrane.

77. The method of claim 75, wherein the AEX hybrid medium comprises AEX chromatography medium and polyether sulfone (PES) membrane.

78. The method of any one of claims 74-77, wherein the AEX chromatography medium is a quaternary ammonium salt functional AEX medium.

79. The method of claim 78, wherein the quaternary ammonium salt functional AEX medium is non-woven medium.

80. The method of claim 79, wherein the AEX chromatography medium is any one of a 3M Polisher® ST medium, Harvest® RC medium or Emphaze® AEX medium.

81. The method of any one of claims 76-80, wherein the pore size of the polyamide membrane or the PES membrane is between about 0. 1 to about 0.5 pm.

82. The method of claim 81, wherein the pore size of the polyamide membrane or the PES membrane is 0.2 pm.

83. The method of claim 82. wherein the bioburden reduction filter can be any one of a PVDF- based filter, a PES-based filter, and a polyamide-based filter.

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