# US 2025/0207149 A1 · A pre-screening method and a methods for separating adeno-associated virus capsids > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 2025/0207149 A1 - Document ID: US20250207149A1 - Status: Published application - Kind: A1 - Application number: 18/847,585 - Filed: 2023-04-26 - Published: 2025-06-26 - Earliest claimed: 2022-04-27 - Assignees: Cytiva BioProcess R&D AB - Applicants: Cytiva Bioprocess R&D AB - Inventors: Åsa Hagner McWhirter; Jean-Luc Maloisel; Brigitta Németh - Web page: https://patentgrep.com/patent/US20250207149A1 ## CPC - C07K 14/005: from viruses - C12N 15/86: Viral vectors - B01D 15/166: {Fluid composition conditioning, e.g. gradient} - B01D 15/363: {Anion-exchange} - B01D 15/426: {Specific type of solvent} - B01J 20/285: based on polymers - B01J 20/289: bonded via a spacer - B01J 39/05: in the strongly acidic form - B01J 39/19: obtained otherwise than by reactions only involving unsaturated carbon-to-carbon bonds - B01J 39/26: Cation exchangers for chromatographic processes - B01J 41/12: Macromolecular compounds - B01J 47/014: in which the adsorbent properties of the ion-exchanger are involved, e.g. recovery of proteins or other high-molecular compounds - G01N 30/34: of fluid composition, e.g. gradient (G01N30/36 takes precedence) - G01N 30/88: Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 - C12N 2750/14143: viral genome or elements thereof as genetic vector - C12N 2750/14151: Methods of production or purification of viral material - G01N 2030/8813: {biological materials} - C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00) - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) - B01D: SEPARATION (separating solids from solids by wet methods B03B, B03D; by pneumatic jigs or tables B03B; by other dry methods B07; magnetic or electrostatic separation of solid materials from solid materials or fluids, separation by high-voltage electric fields B03C; centrifuges, vortex apparatus B04; presses per se for squeezing-out liquid from liquid-containing material B30B9/02; treatment of water C02F, e.g. softening by ion-exchange C02F1/42; {arrangements of air intake cleaners in gas turbine plants F02C7/05} ; arrangements or mounting of filters in air-conditioning, air-humidification or ventilation F24F13/28) - B01J: CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract The present disclosure is directed to a method for determining elution conditions suitable for separating adeno-associated virus (AAV) capsids fully packaged with genetic material from AAV capsids not fully packaged with genetic material, the method comprising: (a) adding a liquid sample comprising AAV capsids to a strong, or partially strong, anion exchange chromatography material comprising a surface extender, (b) eluting the AAV virus capsids from the chromatography material by applying an elution buffer comprising a step gradient of increasing conductivity, which increases by from about 0.5 to about 3 mS/cm per step, (c) based on an elution profile obtained in step (b), determining a first value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids not fully packaged with genetic material, and (d) based on the elution profile obtained in step (b), determining a second value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids fully packaged with genetic material. Further disclosed are methods for separating fully packaged AAV capsids from not fully packaged AAV capsids based on pre-determined first and second value of conductivity or conductivity-related parameter, as well as use of an anion exchange chromatography material for separating fully packaged AAV capsids from not fully packaged AAV capsids. ## Claims ### Claim 1 A method for determining elution conditions suitable for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising the following steps: a. adding a liquid sample comprising adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 1012 adeno-associated virus capsids/ml, of which at least 5% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material, to a strong, or partially strong, anion exchange chromatography material comprising a support and a ligand for binding to the adeno-associated virus capsids, wherein the chromatography material comprises a surface extender connecting the ligand to the support, wherein the surface extender is a polymer, wherein the polymer is selected from: (i) a polymer having a naturally occurring skeleton, such as a polysaccharide, such as starch, cellulose, dextran, or agarose, and (ii) a polymer having a synthetic skeleton, such as a polyvinyl alcohol, a polyacrylamide, a polymethacrylamide, or a polyvinyl ether; b. eluting the adeno-associated virus capsids not fully packaged with genetic material and the adeno-associated virus capsids fully packaged with genetic material from the chromatography material by applying an elution buffer comprising a step gradient of increasing conductivity, which starts at from about 0 to about 5 mS/cm, and which increases by from about 0.5 to about 3 mS/cm per step, at least up to and including a conductivity at which the adeno-associated virus capsids not fully packaged with genetic material and the adeno-associated virus capsids fully packaged with genetic material have been eluted from the chromatography material; c. based on an elution profile obtained in step (b), determining a first value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids not fully packaged with genetic material, and d. based on the elution profile obtained in step (b), determining a second value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids fully packaged with genetic material. ### Claim 2 (depends on 1) The method of claim 1, wherein the step gradient of increasing conductivity in step (b) is a step gradient of increasing salt concentration, optionally wherein the conductivity-related parameter in steps (c) and (d) is the salt concentration. ### Claim 3 (depends on 2) The method of claim 2, wherein the salt is a kosmotropic salt. ### Claim 4 (depends on 2, 3) The method of claim 2 or 3, wherein the salt comprises (i) an anion selected from a group consisting of CO32−, SO42−, S2O32−, H2PO4−, HPO42−, acetate−, citrate−, and Cl−, and (ii) a cation selected from a group consisting of NH4+, K+, Na+, and Li+; optionally wherein the salt is sodium acetate. ### Claim 5 The method of any preceding claim, wherein step (b) comprises adding a volume of the elution buffer corresponding to from about 1 to about 10 volumes of the chromatography material, per step of the step gradient. ### Claim 6 (depends on 1) A method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising steps (a)-(d) of claim 1, and further comprising the steps: e. adding a liquid sample comprising adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 1012 adeno-associated virus capsids/ml, of which at least 5% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material, the liquid sample originating from a cell culture harvest from which the liquid sample of step (a) originated, to the chromatography material as defined in step (a); f. eluting the adeno-associated virus capsids not fully packaged with genetic material by applying an elution buffer having the first value of conductivity or conductivity-related parameter as determined in step (c); and g. eluting the adeno-associated virus capsids fully packaged with genetic material by applying an elution buffer having the second value of conductivity or conductivity-related parameter as determined in step (d); wherein (i) the duration of step (f) is at least 3 times the duration of step (g), and/or (ii) the method comprises a step (f′) between step (f) and step (g), wherein step (f′) comprises applying an additional step elution and/or a gradient of increasing conductivity between the first value and the second value of conductivity or conductivity-related parameter, and the duration of steps (f) and (f′) is at least 3 times the duration of step (g). ### Claim 7 (depends on 6) The method of claim 6, wherein the elution buffer comprises a salt and the step gradient of increasing conductivity in step (b) is a step gradient of increasing salt concentration, optionally wherein the conductivity-related parameter in steps (c), (f) and (g) is the salt concentration. ### Claim 8 (depends on 7) The method of claim 7, wherein the salt is a kosmotropic salt. ### Claim 9 (depends on 7, 8) The method of claim 7 or 8, wherein the salt comprises (i) an anion selected from a group consisting of CO32−, SO42−, S2O32−, H2PO4−, HPO42−, acetate−, citrate−, and Cl−, and (ii) a cation selected from a group consisting of NH4+, K+, Na+, and Li+; optionally wherein the salt is sodium acetate. ### Claim 10 (depends on 6, 7, 8, 9) The method of any one of claims 6-9, wherein step (b) comprises adding a volume of the elution buffer corresponding to from about 1 to about 10 volumes of the chromatography material, per step of the step gradient. ### Claim 11 (depends on 1) A method for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, the method comprising: (I) adding a liquid sample comprising adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 1012 adeno-associated virus capsids/ml, of which at least 5% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material, to a strong, or partially strong, anion exchange chromatography material comprising a support and a ligand for binding to the adeno-associated virus capsids, wherein the chromatography material comprises a surface extender connecting the ligand to the support, wherein the surface extender is a polymer, wherein the polymer is selected from: (i) a polymer having a naturally occurring skeleton, such as a polysaccharide, such as starch, cellulose, dextran, or agarose, and (ii) a polymer having a synthetic skeleton, such as a polyvinyl alcohol, a polyacrylamide, a polymethacrylamide, or a polyvinyl ether; (II) eluting the adeno-associated virus capsids not fully packaged with genetic material by applying an elution buffer having a pre-determined first value of conductivity or conductivity-related parameter; (III) eluting the adeno-associated virus capsids fully packaged with genetic material by applying an elution buffer having a pre-determined second value of conductivity or conductivity-related parameter; the pre-determined first and second value of conductivity or conductivity-related parameter having been determined during separation of a liquid sample comprising adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 1012 adeno-associated virus capsids/ml, of which at least 5% of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material, the liquid sample originating from a cell culture harvest from which the liquid sample of step (I) originates, optionally having been determined by performing the method of steps (a)-(d) of claim 1; wherein (i) the duration of step (II) is at least 3 times the duration of step (III), and/or (ii) the method comprises a step (II′) between step (II) and step (III), wherein step (II′) comprises applying a step elution and/or a gradient of increasing conductivity between the first value and the second value of conductivity or conductivity-related parameter, and the duration of steps (II) and (II′) is at least 3 times the duration of step (III). ### Claim 12 The method of any preceding claim, wherein the chromatography material is defined by: i) Formula I: wherein R1 is selected from C1-C3 alkyl, and R2 and R3 are independently selected from C1-C3 alkyl, CH2OH, and CH2CHOHCH3; optionally wherein each of R1, R2, and R3 is CH3; or ii) Formula II: wherein: m is an integer of from 1 to 3; R1 and R2 are independently selected from a C1-C3 alkyl; R3, and R4 are independently selected from C1-C3 alkyl and CH2CHOHCH3; and R5 is selected from hydrogen, a C1-C3 alkyl and CH2CHOHCH3; provided that if m is 1, the chromatography material is defined by Formula III: wherein n is an integer of from 0 to 3; provided that if n is 0, R3 and R4 are independently selected from C1-C3 alkyl, and R5 is hydrogen or CH2CHOHCH3; optionally wherein the chromatography material is defined by Formula III and comprises a combination of two or more of the following structures (i)-(iv): (i) n is 0; R3 and R4 are ethyl; and R5 is hydrogen or CH2CHOHCH3; (ii) n is 1; R1, R2, R3, R4 are ethyl; and R5 is hydrogen or CH2CHOHCH3; (iii) n is 2; each R1 and R2 is ethyl; R3 and R4 is ethyl; and R5 is hydrogen or CH2CHOHCH3; (iv) n is 3; each R1 and R2 is ethyl; R3 and R4 is ethyl; and R5 is hydrogen or CH2CHOHCH3. ### Claim 13 The method of any preceding claim, wherein the surface extender is dextran; optionally wherein the dextran has a molecular weight of from about 10 to about 2000 kDa, such as about 40 kDa, and/or optionally wherein the density of dextran is from about 5 to about 30 mg dextran per ml of the strong anion exchange chromatography material. ### Claim 14 The method of any preceding claim, wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; optionally wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 9 (AAV9) or a variant thereof. ### Claim 15 (depends on 1, 6, 7, 8, 9, 10) The method of any one of claims 6-10, wherein the chromatography material is defined by Formula IV: wherein the elution buffer of step (b) of claim 1 and the elution buffer of steps (f) and (g) of claim 6 comprise sodium acetate, wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; optionally wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 9 (AAV9) or a variant thereof. ### Claim 16 (depends on 11) The method of claim 11, wherein the chromatography material is defined by Formula IV: wherein the elution buffer of steps (II) and (III) of claim 11 comprises sodium acetate; wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; optionally wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 9 (AAV9) or a variant thereof. ### Claim 17 Use of an anion exchange chromatography material comprising a support, a ligand, and a surface extender connecting the ligand to the support, and being defined by Formula IV: for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, comprising determining elution conditions suitable for separating adeno-associated virus capsids fully packaged with genetic material from adeno-associated virus capsids not fully packaged with genetic material, wherein said elution conditions are determined by performing the steps: a. adding a liquid sample comprising adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 1012 adeno-associated virus capsids/ml, of which at least 5%, such as 10%, of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material, to the chromatography material; b. eluting the adeno-associated virus capsids not fully packaged with genetic material and the adeno-associated virus capsids fully packaged with genetic material from the chromatography material by applying an elution buffer comprising a step gradient of increasing conductivity, which starts at from about 0 to about 5 mS/cm, and which increases by from about 0.5 to about 3 mS/cm per step, such as from about 1 to about 2 mS/cm per step, such as from about 1.2 to about 1.5 mS/cm per step, at least up to and including a conductivity at which the adeno-associated virus capsids not fully packaged with genetic material and the adeno-associated virus capsids fully packaged with genetic material have been eluted from the chromatography material; c. based on an elution profile obtained in step (b), determining a first value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids not fully packaged with genetic material, and d. based on the elution profile obtained in step (b), determining a second value of conductivity or conductivity-related parameter, which is suitable for eluting the adeno-associated virus capsids fully packaged with genetic material. ### Claim 18 (depends on 17) The use of claim 17, wherein the adeno-associated virus capsids fully packaged with genetic material are separated from adeno-associated virus capsids not fully packaged with genetic material by performing steps (a)-(d) of claim 17 and further by performing the steps: e. adding a liquid sample comprising adeno-associated virus capsids of a purity of at least 90% and of a concentration of at least 1012 adeno-associated virus capsids/ml, of which at least 5%, such as 10%, of the adeno-associated virus capsids are adeno-associated virus capsids fully packaged with genetic material, the liquid sample originating from a cell culture harvest from which the liquid sample of step (a) originated, to the chromatography material; f. eluting the adeno-associated virus capsids not fully packaged with genetic material by applying an elution buffer having the first value of conductivity or conductivity-related parameter as determined in step (c); and g. eluting the adeno-associated virus capsids fully packaged with genetic material by applying an elution buffer having the second value of conductivity or conductivity-related parameter as determined in step (d); wherein (i) the duration of step (f) is at least 3 times, such as 4 times, the duration of step (g), and/or (ii) the method comprises a step (f′) between step (f) and step (g), wherein step (f′) comprises applying an additional step elution and/or a gradient of increasing conductivity between the first value and the second value of conductivity or conductivity-related parameter, and the duration of steps (f) and (f′) is at least 3 times, such as 4 times, the duration of step (g). ### Claim 19 (depends on 17, 18) The use of claim 17 or 18, wherein the elution buffer of step (b) of claim 17, and when referring to claim 18 also the elution buffer of steps (f) and (g) of claim 18, comprises sodium acetate; wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 1 (AAV1), adeno-associated virus serotype 2 (AAV2), adeno-associated virus serotype 3 (AAV3), adeno-associated virus serotype 4 (AAV4), adeno-associated virus serotype 5 (AAV5), adeno-associated virus serotype 6 (AAV6), adeno-associated virus serotype 7 (AAV7), adeno-associated virus serotype 8 (AAV8), adeno-associated virus serotype 9 (AAV9), or adeno-associated virus serotype 10 (AAV10), or a variant thereof; optionally wherein the adeno-associated virus capsids are capsids of adeno-associated virus serotype 9 (AAV9) or a variant thereof.