Scalable manufacturing process to produce recombinant lentiviral vectors in serum-free suspension cell culture system

Abstract

Methods for preparing highly purified rLV vector formulations at the scale needed to meet anticipated demand for human gene therapy are provided.

What is claimed is: 
     
         1 . A method for large scale viral vector purification, comprising;
 a) harvesting recombinant viral vectors comprising a transgene from serum free suspension culture;   b) clarifying the harvest of step a) via filtration;   c) subjecting the clarified suspension of step b) to tangential flow filtration to reduce volume and exchange buffer;   d) harvesting the filtrate from step c) and exposing said filtrate to benzonase digestion to remove DNA/RNA impurities;   e) subjecting the the filtrate of step d) to PEG-modulated weak anion exchange column chromatography, thereby isolating said viral vectors;   f) further purifying the viral vectors obtained from step e) via tangential flow filtration to reduce the volume and buffer exchange;   g) subjecting the filtrate of step f) to size exclusion column chromatography to further purify said viral vectors;   h) subjecting the vectors of step g) to tangential flow filtration, and thereby obtaining final vector titer; and   i) filtering a vector solution obtained from step h) through a 0.45 um filter; and   j) storing said purified viral vectors.   
     
     
         2 . The method of  claim 1 , wherein said vector is a recombinant lentiviral vector (rLV) and said rLV is produced at approximately 3×10 8 /ml. 
     
     
         3 . A rLV vector formulation comprising rLV particles purified using the method of  claim 1  in a pharmaceutically acceptable carrier. 
     
     
         4 . The method of  claim 1 , wherein said transgene encodes a nucleic acid selected from the group consisting of a siRNA, an antisense molecule, and a miRNA a ribozyme and a shRNA. 
     
     
         5 . The method of  claim 1  wherein said transgene 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. 
     
     
         6 . The method of  claim 1 , wherein said transgene 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, 
     
     
         7 . The method of  claim 1 , wherein said transgene comprises a nucleic acid 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. 
     
     
         8 . The method of  claim 7 , wherein the gene product is Factor VIII or Factor IX.