WO2021247476A1 - Materials and methods for viral purification - Google Patents

Materials and methods for viral purification Download PDF

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Publication number
WO2021247476A1
WO2021247476A1 PCT/US2021/035113 US2021035113W WO2021247476A1 WO 2021247476 A1 WO2021247476 A1 WO 2021247476A1 US 2021035113 W US2021035113 W US 2021035113W WO 2021247476 A1 WO2021247476 A1 WO 2021247476A1
Authority
WO
WIPO (PCT)
Prior art keywords
mnase
aav particles
buffer
viral titer
fold
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2021/035113
Other languages
English (en)
French (fr)
Inventor
Brian E. Tomkowicz
Matthew P. ERCOLINO
Stephen T. SPAGNOL
Sakya Sing MOHAPATRA
William Lloyd PERRY, III
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Janssen Biotech Inc
Original Assignee
Janssen Biotech Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Janssen Biotech Inc filed Critical Janssen Biotech Inc
Priority to AU2021284250A priority Critical patent/AU2021284250A1/en
Priority to JP2022573726A priority patent/JP2023528406A/ja
Priority to CA3185729A priority patent/CA3185729A1/en
Priority to US18/000,525 priority patent/US20230212528A1/en
Priority to CN202180050117.9A priority patent/CN116096867A/zh
Priority to EP21818554.4A priority patent/EP4157301A4/en
Priority to KR1020237000108A priority patent/KR20230031281A/ko
Publication of WO2021247476A1 publication Critical patent/WO2021247476A1/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14123—Virus like particles [VLP]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14151—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/31—Endoribonucleases active with either ribo- or deoxyribonucleic acids and producing 3'-phosphomonoesters (3.1.31)
    • C12Y301/31001—Micrococcal nuclease (3.1.31.1)

Definitions

  • the method further comprises washing the solid support.
  • the washing comprises a high pH buffer.
  • the high pH buffer is greater than pH 9 0 In one embodiment, the high pH buffer is between pH 9.0 and pH 11 In one embodiment, the high pH buffer is about pH 9 5 In one embodiment, the high pH buffer is about pH 10 2 In one embodiment, the high pH buffer is about pH 10 3 In one embodiment, the high pH buffer is about pH 10 4
  • the low pH buffer is a citrate buffer, glycine buffer, or a phosphoric acid buffer. In one embodiment, the low pH buffer is a citrate buffer. In one embodiment, the low pH buffer is a phosphoric acid buffer.
  • the viral titer is increased about 2 fold to about 100 fold. In one embodiment, the viral titer is increased about 2 fold or greater. In one embodiment, the viral titer is increased about 3 fold or greater. In one embodiment, the viral titer is increased about 7 fold or greater. In one embodiment, the viral titer is increased about 80 fold or greater.
  • the purification method further comprises a high pH buffer prior to the low pH buffer.
  • the low pH buffer is less than about pH 3.0. In one embodiment, the low pH buffer is about pH 1.5 to about pH 2.5. In one embodiment, the low pH buffer is about pH 1.5. In one embodiment, the low pH buffer is about pH 2.5.
  • the elution further comprises ethanol.
  • the ethanol is about 5% to about 40%. In one embodiment, the ethanol is about 10% to about 30 %. In one embodiment, the ethanol is about 15% to about 25%. In one embodiment, the ethanol is about 20% ethanol.
  • the composition further comprises Benzonase®.
  • the viral titer is increased about 2 fold to about 100 fold. In one embodiment, the viral titer is increased about 2 fold or greater. In one embodiment, the viral titer is increased about 3 fold or greater. In one embodiment, the viral titer is increased about 7 fold or greater. In one embodiment, the viral titer is increased about 80 fold or greater.
  • the MNase is present in an amount sufficient to produce purified AAV particles comprising a melting temperature (Tm) within less than about 2°C of an aggregation temperature (Tagg), as measured by dynamic light scatter (DLS).
  • Tm melting temperature
  • Tg aggregation temperature
  • DLS dynamic light scatter
  • AAV adeno- associated viral
  • Various purification techniques for purifying AAV particles for small-scale production e.g ., density gradient centrifugation
  • large-scale production e.g., affinity chromatography, including ion exchange chromatography; size exclusion chromatography; and hydrophobic interaction chromatography; or a combination of such techniques
  • affinity chromatography including ion exchange chromatography; size exclusion chromatography; and hydrophobic interaction chromatography; or a combination of such techniques
  • the MNase is present in an amount sufficient to produce purified AAV particles comprising a melting temperature (Tm) within less than about 10°C of an aggregation temperature (Tagg), as measured by dynamic light scatter (DLS).
  • Tm melting temperature
  • Tg aggregation temperature
  • DLS dynamic light scatter
  • the MNase is present in an amount sufficient to produce purified AAV particles comprising a melting temperature (Tm) within less than about 5°C of an aggregation temperature (Tagg), as measured by dynamic light scatter (DLS).
  • Tm melting temperature
  • Tg aggregation temperature
  • DLS dynamic light scatter
  • the present disclosure also provides a composition for use in producing an AAV particle that is substantially free of chromatin-associated DNA, the composition comprising: (a) a supernatant comprising AAV particles; and (b) a chromatin-DNA nuclease.
  • the composition further includes Benzonase®.
  • the chromatin-DNA nuclease is MNase.
  • composition of embodiment C6, wherein the washing comprises a high pH buffer.
  • composition of embodiment C32, wherein the purification method comprises about 10% to about 30 % ethanol.
  • D16 The composition of embodiment Dll, wherein the MNase is present in a sufficient amount to reduce AAV particle impurities.
  • D26 The composition of any one of embodiments D20 to D23, wherein the viral titer is increased about 3 fold or greater.
  • D36 The composition of embodiment D34, wherein the full-to-empty capsid ratio is greater than about 60%.
  • D40 The composition of embodiment D11, wherein the MNase is present in an amount sufficient to produce purified AAV particles comprising a melting temperature (Tm) within less than about 10°C of an aggregation temperature (Tagg), as measured by dynamic light scatter (DLS).
  • Tm melting temperature
  • Tg aggregation temperature
  • DLS dynamic light scatter
  • E27 The kit of embodiment E26, wherein the full-to-empty capsid ratio is greater than about 50%.
  • E28 The kit of embodiment E26, wherein the full-to-empty capsid ratio is greater than about 60%.
  • G3 The system of embodiment G2, wherein the DNA binding protein comprises a histone.
  • G4 The system of embodiment Gl, wherein the impurity is a macroscopic impurity, a microscopic impurity, or both.
  • G8 The system of embodiments G5, wherein the viral titer comprises a functional viral titer.
  • G14 A system comprising a means for increasing a viral titer ratio of a product AAV particle fraction to a post-product AAV particle fraction.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Microbiology (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Virology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Plant Pathology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Solid-Sorbent Or Filter-Aiding Compositions (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
PCT/US2021/035113 2020-06-02 2021-06-01 Materials and methods for viral purification Ceased WO2021247476A1 (en)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU2021284250A AU2021284250A1 (en) 2020-06-02 2021-06-01 Materials and methods for viral purification
JP2022573726A JP2023528406A (ja) 2020-06-02 2021-06-01 ウイルス精製のための材料及び方法
CA3185729A CA3185729A1 (en) 2020-06-02 2021-06-01 Materials and methods for viral purification
US18/000,525 US20230212528A1 (en) 2020-06-02 2021-06-01 Materials and methods for viral purification
CN202180050117.9A CN116096867A (zh) 2020-06-02 2021-06-01 用于病毒纯化的材料和方法
EP21818554.4A EP4157301A4 (en) 2020-06-02 2021-06-01 Materials and methods for viral purification
KR1020237000108A KR20230031281A (ko) 2020-06-02 2021-06-01 바이러스 정제를 위한 재료 및 방법

Applications Claiming Priority (14)

Application Number Priority Date Filing Date Title
US202063033631P 2020-06-02 2020-06-02
US202063033549P 2020-06-02 2020-06-02
US202063033492P 2020-06-02 2020-06-02
US202063033731P 2020-06-02 2020-06-02
US202063033643P 2020-06-02 2020-06-02
US202063033531P 2020-06-02 2020-06-02
US202063033449P 2020-06-02 2020-06-02
US63/033,731 2020-06-02
US63/033,449 2020-06-02
US63/033,643 2020-06-02
US63/033,492 2020-06-02
US63/033,631 2020-06-02
US63/033,531 2020-06-02
US63/033,549 2020-06-02

Publications (1)

Publication Number Publication Date
WO2021247476A1 true WO2021247476A1 (en) 2021-12-09

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US (1) US20230212528A1 (https=)
EP (1) EP4157301A4 (https=)
JP (1) JP2023528406A (https=)
KR (1) KR20230031281A (https=)
CN (1) CN116096867A (https=)
AU (1) AU2021284250A1 (https=)
CA (1) CA3185729A1 (https=)
WO (1) WO2021247476A1 (https=)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024252024A1 (en) * 2023-06-09 2024-12-12 Sartorius Bia Separations D.O.O. A method of enhanced separation of full adeno-associated virus (aav) capsids
EP4578943A4 (en) * 2022-08-25 2026-02-25 Fujifilm Corp PROCESS FOR TARGETING VIRUS PRODUCTION AND CULTURE COMPOSITION

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6485976B1 (en) * 1999-04-30 2002-11-26 City Of Hope Use of adeno-associated virus (AAV) to deliver genes
US20120070890A1 (en) * 1997-04-24 2012-03-22 University Of Washington Targeted gene modification by parvoviral vectors
US20180243416A1 (en) * 2017-02-28 2018-08-30 The Trustees Of The University Of Pennsylvania Novel aav mediated influenza vaccines
US20190002841A1 (en) * 2015-12-11 2019-01-03 The Trustees Of The University Of Pennsylvania Scalable purification method for aavrh10

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6156303A (en) * 1997-06-11 2000-12-05 University Of Washington Adeno-associated virus (AAV) isolates and AAV vectors derived therefrom
BRPI0511764B8 (pt) * 2004-06-01 2021-05-25 Avigen Inc método de prevenção de agregação de vírions de vírus adeno-associado recombinante (raav) em uma preparação purificada de virions raav
ES2934848T3 (es) * 2015-12-11 2023-02-27 Univ Pennsylvania Método de purificación escalable para AAV8
CA3190596A1 (en) * 2020-08-07 2022-02-10 Janssen Biotech, Inc. Formulations for highly purified viral particles

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120070890A1 (en) * 1997-04-24 2012-03-22 University Of Washington Targeted gene modification by parvoviral vectors
US6485976B1 (en) * 1999-04-30 2002-11-26 City Of Hope Use of adeno-associated virus (AAV) to deliver genes
US20190002841A1 (en) * 2015-12-11 2019-01-03 The Trustees Of The University Of Pennsylvania Scalable purification method for aavrh10
US20180243416A1 (en) * 2017-02-28 2018-08-30 The Trustees Of The University Of Pennsylvania Novel aav mediated influenza vaccines

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP4157301A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP4578943A4 (en) * 2022-08-25 2026-02-25 Fujifilm Corp PROCESS FOR TARGETING VIRUS PRODUCTION AND CULTURE COMPOSITION
WO2024252024A1 (en) * 2023-06-09 2024-12-12 Sartorius Bia Separations D.O.O. A method of enhanced separation of full adeno-associated virus (aav) capsids

Also Published As

Publication number Publication date
KR20230031281A (ko) 2023-03-07
US20230212528A1 (en) 2023-07-06
AU2021284250A1 (en) 2023-02-09
EP4157301A1 (en) 2023-04-05
CN116096867A (zh) 2023-05-09
JP2023528406A (ja) 2023-07-04
EP4157301A4 (en) 2025-03-19
CA3185729A1 (en) 2021-12-09

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