Scalable manufacturing platform for viral vector purification and viral vectors so purified for use in gene therapy

• US 9,408,904 B2
• Filed: 07/30/2012
• Issued: 08/09/2016
• Est. Priority Date: 01/28/2010
• Status: Active Grant

• Current Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Original Assignee: Children's Hospital of Philadelphia (Children's Hospital of Philadelphia Foundation)
• Inventors: Wright, John Fraser, Qu, Guang, Hauck, Bernd, High, Katherine A.
• Primary Examiner(s): Gill, Rachel B
• Application Number:
  US13/561,753
• Publication Number:
  US 20130072548A1
• Time in Patent Office: 1,471 Days
• US Class Current: 1/1
• CPC Class Codes: A61K 38/465 acting on ester bonds (3.1), e.g. lipases, ribonucleases
• CPC Class Codes: A61K 39/23 Parvoviridae, e.g. feline panleukopenia virus
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61K 48/0091 Purification or manufacturing processes for gene therapy compositions
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/04 Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
• CPC Class Codes: C07K 2/00 Peptides of undefined number of amino acids; Derivatives thereof
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 15/864 Parvoviral vectors , e.g. parvovirus, densovirus
• CPC Class Codes: C12N 15/8645 Adeno-associated virus
• CPC Class Codes: C12N 2750/14011 Parvoviridae
• CPC Class Codes: C12N 2750/14051 Methods of production or purification of viral material
• CPC Class Codes: C12N 2750/14111 Dependovirus, e.g. adenoassociated viruses
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2750/14151 Methods of production or purification of viral material
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00
• CPC Class Codes: C12Y 301/01064 Retinoid isomerohydrolase (3.1.1.64)

Abstract

Methods for preparing highly purified AAV vector formulations are provided. The highly pure AAV formulations described herein are superior for clinical use.

Claims

1. A method for producing a highly purified adeno-associated (AAV) vector formulation, said method comprising the steps of:

(a) harvesting cells and cell culture supernatant comprising recombinant AAV vector particles;

(b) concentrating said cells and said cell culture supernatant harvested in step (a) via tangential flow filtration to produce a concentrated harvest;

(c) lysing said concentrated harvest produced in step (b) by microfluidization to produce a lysate;

(d) filtering said lysate produced in step (c) to produce a clarified lysate;

(e) subjecting said clarified lysate produced in step (d) to ion exchange column chromatography to produce a column eluate comprised of purified AAV vector particles, and optionally concentrating said column eluate by tangential flow filtration to produce a concentrated column eluate;

(f) mixing said column eluate or said concentrated column eluate produced in step (e) with cesium chloride to produce a mixture, and subjecting said mixture to gradient ultracentrifugation to substantially separate said bona fide AAV vector particles from empty capsid AAV vector particles and other AAV vector related impurities;

(g) collecting said bona fide AAV vector particles separated in step (f) and subjecting said collected bona fide AAV vector particles to a buffer exchange by tangential flow filtration;

(h) formulating said bona fide AAV vector particles resulting from step (g) with surfactant to produce an AAV vector formulation; and

(i) filtering said AAV vector formulation produced in step (h) to produce a highly purified AAV vector formulation in which at least 90% of the AAV vector particles in said highly purified AVV vector formulation are bona fide AAV particles.

2. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles of the eluate of step (e) are present at a concentration of about 100 mg/mL.

3. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles are present in said highly purified AAV vector formulation produced in step (i) at a concentration of 10¹⁵ particles per mL.

4. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles are present in said highly purified AAV vector formulation produced in step (i) at a concentration of 10¹⁶ particles per mL.

5. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles are present in said highly purified AAV vector formulation produced in step (i) at a concentration of 10¹⁷ particles per mL.

6. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles are derived from an AAV selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9.

7. An AAV vector particle formulation comprising bona fide AAV vector particles purified using the method of claim 1 in a pharmaceutically acceptable carrier, wherein at least 90% of the AAV particles in said formulation are bona fide AAV vector particles, and wherein the AAV particles in the formulation are present at a concentration of at least 10¹⁵ particles per mL.

8. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles comprise a transgene that encodes a nucleic acid selected from the group consisting of sRNA, an antisense nucleic acid molecule, miRNA, ribozyme, and shRNA.

9. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of insulin, glucagon, growth hormone (GH), parathyroid hormone (PTH), growth hormone releasing factor (GRF), follicle stimulating hormone (FSH), luteinizing hormone (LH), human chorionic gonadotropin (hCG), vascular endothelial growth factor (VEGF), angiopoietins, 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 α

(TGFα

), platelet-derived growth factor (PDGF), insulin growth factors I and II (IGF-I and IGF-II), TGFβ

, activins, inhibins, bone morphogenic protein (BMP), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophins NT-3 and NT4/5, ciliary neurotrophic factor (CNTF), glial cell line derived neurotrophic factor (GDNF), neurturin, agrin, netrin-1 and netrin-2, hepatocyte growth factor (HGF), ephrins, noggin, sonic hedgehog and tyrosine hydroxylase.

10. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles comprise a transgene that encodes a gene product selected from the group consisting of thrombopoietin (TPO), interleukins (IL1 through IL-17), monocyte chemoattractant protein, leukemia inhibitory factor, granulocyte-macrophage colony stimulating factor, Fas ligand, tumor necrosis factors α

and β

, interferons α

, β

, and γ

, stem cell factor, flk-2/flt3 ligand, IgG, IgM, IgA, IgD and IgE, chimeric immunoglobulins, humanized antibodies, single chain antibodies, T cell receptors, chimeric T cell receptors, single chain T cell receptors, class I and class II MHC molecules.

11. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles comprise a transgene encoding a protein useful for correction of in born errors of metabolism selected from the group consisting of carbamoyl synthetase I, ornithine transcarbamylase, arginosuccinate synthetase, arginosuccinate lyase, arginase, fumarylacetacetate hydrolase, phenylalanine hydroxylase, alpha-1 antitrypsin, glucose-6-phosphatase, porphobilinogen deaminase, factor V, factor VIII, factor IX, cystathione beta-synthase, branched chain ketoacid decarboxylase, albumin, isovaleryl-coA dehydrogenase, propionyl CoA carboxylase, methyl malonyl CoA mutase, glutaryl CoA dehydrogenase, insulin, beta-glucosidase, pyruvate carboxylate, hepatic phosphorylase, phosphorylase kinase, glycine decarboxylase, RPE65, H-protein, T-protein, a cystic fibrosis transmembrane regulator (CFTR) sequence, and a dystrophin cDNA sequence.

12. A method for producing a highly purified AAV vector formulation according to claim 11, wherein said gene product is Factor VIII or Factor IX.

13. A method for producing a highly purified AAV vector formulation according to claim 1, comprising collecting the empty capsid fraction separately in step (f).

14. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said empty capsids are present in said filtrate of step (i) in an amount of 10% or less.

15. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said bona fide AAV vector particles are present in said filtrate of step (i) in an amount of at least 95%.

16. A method for producing a highly purified AAV vector formulation according to claim 15, wherein said empty capsids are present in said filtrate of step (i) in an amount of 5% or less.

17. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said centrifugation in step (f) is conducted in a single step.

18. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said centrifugation in step (f) is density gradient ultracentrifugation.

19. A method for producing a highly purified AAV vector formulation according to claim 1, wherein said column eluate in step (e) is concentrated by tangential flow filtration to produce a concentrated column eluate.

20. A method for producing a highly purified AAV vector formulation according to claim 1, further comprising adding a nuclease to the lysate produced in step (c).

21. A method for producing a highly purified adeno-associated (AAV) vector formulation, said method comprising the steps of:

(a) harvesting cells and cell culture supernatant comprising recombinant AAV vector particles;

(b) concentrating said cells and said cell culture supernatant harvested in step (a) via tangential flow filtration to produce a concentrated harvest;

(c) lysing said concentrated harvest produced in step (b) by microfluidization to produce a lysate;

(d) filtering said lysate produced in step (c) to produce a clarified lysate;

(e) subjecting said clarified lysate produced in step (d) to ion exchange column chromatography to produce a column eluate comprised of purified AAV vector particles, and optionally concentrating said column eluate by tangential flow filtration to produce a concentrated column eluate;

(f) mixing said column eluate or said concentrated column eluate produced in step (e) with cesium chloride to produce a mixture;

(g) subjecting said mixture in step (f) to gradient ultracentrifugation conducted in a single step to substantially separate said bona fide AAV vector particles from empty capsid AAV vector particles and other AAV vector related impurities;

(h) collecting said bona fide AAV vector particles separated in step (g) and subjecting said collected bona fide AAV vector particles to a buffer exchange by tangential flow filtration;

(i) formulating said bona fide AAV vector particles resulting from step (h) with surfactant to produce an AAV vector formulation; and

(j) filtering said AAV vector formulation produced in step (i) to produce a highly purified AAV vector formulation in which at least 90% of the AAV vector particles in said highly purified AVV vector formulation are bona fide AAV particles, and wherein said bona fide AAV vector particles are present in said highly purified AAV vector formulation at a concentration of 10¹⁵ particles per mL.