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NZ747126B2 - Column-based fully scalable raav manufacturing process - Google Patents

Column-based fully scalable raav manufacturing process

Info

Publication number
NZ747126B2
NZ747126B2 NZ747126A NZ74712617A NZ747126B2 NZ 747126 B2 NZ747126 B2 NZ 747126B2 NZ 747126 A NZ747126 A NZ 747126A NZ 74712617 A NZ74712617 A NZ 74712617A NZ 747126 B2 NZ747126 B2 NZ 747126B2
Authority
NZ
New Zealand
Prior art keywords
column
raav vector
vector particles
produce
lysate
Prior art date
Application number
NZ747126A
Other versions
NZ747126A (en
Inventor
Lin Lu
Younghoon Oh
Guang Qu
John Fraser Wright
Original Assignee
Spark Therapeutics Inc
Filing date
Publication date
Application filed by Spark Therapeutics Inc filed Critical Spark Therapeutics Inc
Priority claimed from PCT/US2017/025396 external-priority patent/WO2017173283A1/en
Publication of NZ747126A publication Critical patent/NZ747126A/en
Publication of NZ747126B2 publication Critical patent/NZ747126B2/en

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/34—Size-selective separation, e.g. size-exclusion chromatography; Gel filtration; Permeation
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/362—Cation-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/36—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving ionic interaction, e.g. ion-exchange, ion-pair, ion-suppression or ion-exclusion
    • B01D15/361—Ion-exchange
    • B01D15/363—Anion-exchange
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D15/00—Separating processes involving the treatment of liquids with solid sorbents; Apparatus therefor
    • B01D15/08—Selective adsorption, e.g. chromatography
    • B01D15/26—Selective adsorption, e.g. chromatography characterised by the separation mechanism
    • B01D15/38—Selective adsorption, e.g. chromatography characterised by the separation mechanism involving specific interaction not covered by one or more of groups B01D15/265 and B01D15/30 - B01D15/36, e.g. affinity, ligand exchange or chiral chromatography
    • B01D15/3804—Affinity chromatography
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2315/00—Details relating to the membrane module operation
    • B01D2315/16—Diafiltration
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
    • B01D61/14—Ultrafiltration; Microfiltration
    • B01D61/145—Ultrafiltration
    • 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
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/06—Lysis of microorganisms
    • 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
    • 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
    • C12N2750/14142—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
    • 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
    • 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
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles
    • 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

Abstract

accordance with the invention, provided herein are methods for purifying recombinant adeno-associated (rAAV) vector particles.

Claims (29)

WHAT IS CLAIMED IS:
1. A method for purifying recombinant adeno-associated (rAAV) vector particles said method comprising the steps of: (a) harvesting cells and cell e supernatant comprising rAAV vector particles to produce a harvest; (b) optionally concentrating said harvest produced in step (a) to e a concentrated harvest; (c) lysing said harvest produced in step (a) or said concentrated harvest produced in step (b) to produce a lysate; (d) treating the lysate produced in step (c) to reduce contaminating nucleic acid in the lysate thereby producing a nucleic acid reduced lysate; (e) filtering said nucleic acid reduced lysate produced in step (d) to produce a clarified lysate, and optionally diluting said clarified lysate to produce a diluted clarified lysate; (f) subjecting said ied lysate or d clarified lysate produced in step (e) to anion exchange column chromatography to produce a column eluate comprised of rAAV vector particles, and optionally concentrating said column eluate to produce a concentrated column eluate; (g) ting said column eluate or said concentrated column eluate produced in step (f) to size exclusion column chromatography to produce a second column eluate comprised of rAAV vector les thereby separating rAAV vector particles from protein ties, and optionally diluting said second column eluate to produce a diluted second column eluate; (h) ting said second column eluate or said diluted second column eluate produced in step (g) to cation exchange column chromatography to produce a third column eluate comprised of rAAV vector particles thereby separating rAAV vector particles from protein or other production impurities, and ally concentrating said third column eluate to produce a concentrated third column eluate; and (i) ing said third column eluate or said concentrated third column eluate produced in step (h) thereby producing purified rAAV vector particles.
2. A method according to claim 1, wherein said concentrating of step (b) and/or step (f) and/or step (h) is via is by ultrafiltration/diafiltration, optionally by tangential flow filtration.
3. A method according to claim 1, wherein said concentrating of step (b) reduces the volume of said harvested cells and cell culture supernatant by about 2-10 fold.
4. A method according to claim 1, n said trating of step (f) reduces the volume of said column eluate by about 5-20 fold.
5. A method according to claim 1, wherein said lysing of said harvest ed in step (a) or said concentrated harvest produced in step (b) is by microfluidization.
6. A method according to claim 1, n step (d) comprises treating with a nuclease thereby reducing contaminating nucleic acid.
7. A method according to claim 6, wherein the nuclease comprises benzonase.
8. A method according to claim 1, wherein said filtering said clarified lysate or said diluted clarified lysate of step (e) is via a filter having a pore er of between about 0.1 and 0.8 microns, inclusive.
9. A method according to claim 1, wherein said diluting said clarified lysate or said diluted clarified lysate of step (e) is with an aqueous buffered acetate solution.
10. A method according to claim 1, wherein said diluting of said second column eluate of step (g) is with an s buffered acetate solution.
11. A method according to claim 10, n said aqueous buffered acetate solution has a pH of between about 4.0 and 7.0, inclusive.
12. A method ing to claim 1, wherein said rAAV vector particles resulting from step (i) are formulated with a tant to produce an AAV vector formulation.
13. A method according to claim 1, wherein said anion exchange column chromatography of step (f) comprises polyethylene glycol (PEG) modulated column chromatography.
14. A method according to claim 13, wherein said anion exchange column chromatography of step (f) comprises washing said column with a PEG solution prior to n of said rAAV vector les from the column.
15. A method according to claim 14, n the PEG in said PEG solution has an e molecular weight in a range of about 1,000 to 50,000, inclusive.
16. A method according to claim 1, wherein said anion exchange column of step (f) comprises washing said column with an aqueous surfactant solution prior to elution of said rAAV vector particles from the column.
17. A method according to claim 1, wherein said cation exchange column of step (h) comprises washing said column with a surfactant solution prior to elution of said rAAV vector particles from the column.
18. A method according to any of claims 16-17, wherein said surfactant solution comprises an aqueous Tris-Cl/NaCl buffer or an aqueous ate/NaCl buffer.
19. A method according to claim 18, wherein said NaCl buffer comprises between about 20-250 mM NaCl, inclusive, or between about 50-200 mM NaCl, inclusive.
20. A method according to claim 1, wherein said rAAV vector particles are eluted from said anion ge column of step (f) in an s Tris-Cl/NaCl buffer.
21. A method according to claim 20, n said Tris-Cl/NaCl buffer comprises 50-200 mM NaCl.
22. A method according to claim 1, wherein said rAAV vector particles are eluted from said cation exchange column of step (h) in an aqueous phosphate/NaCl buffer.
23. A method according to claim 22, wherein said phosphate/NaCl buffer comprises between about 100-500 mM NaCl, inclusive.
24. A method according to claim 1, wherein said anion exchange column of step (f) comprises a nary ammonium functional group such as quaternized yleneimine.
25. A method according to claim 1, wherein said size exclusion column of step (g) has a tion rage (Molecular weight) between about 10,000 and 600,000, inclusive.
26. A method according to claim 1, wherein said cation exchange column of step (h) comprises a sulfonic acid or functional group such as sulphopropyl.
27. A method according to any of claims 1-26, n the method excludes a step of cesium chloride gradient ultracentrifugation.
28. A method according to any of claims 1-27, wherein said rAAV vector particles comprise a transgene that encodes a nucleic acid selected from the group consisting of a siRNA, an antisense molecule, miRNA, a ribozyme, and a shRNA.
29. A method according to any of claims 1-28, wherein said rAAV vector les comprise a transgene that encodes a gene product selected from the group ting of insulin, on, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle ating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), oietins, angiostatin, granulocyte colony stimulating factor (GCSF), erythropoietin (EPO), connective tissue growth factor (CTGF), basic fibroblast growth factor (bFGF), acidic fibroblast growth factor (aFGF), epidermal growth factor (EGF), transforming growth factor a , platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFß, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT
NZ747126A 2017-03-31 Column-based fully scalable raav manufacturing process NZ747126B2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201662316252P 2016-03-31 2016-03-31
PCT/US2017/025396 WO2017173283A1 (en) 2016-03-31 2017-03-31 Column-based fully scalable raav manufacturing process

Publications (2)

Publication Number Publication Date
NZ747126A NZ747126A (en) 2025-05-02
NZ747126B2 true NZ747126B2 (en) 2025-08-05

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