Manufacturing of recombinant adeno-associated viral vectors for clinical trials

The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings. The ability to elicit robust and long-term transgene expression in vivo together with minimal immunogenicity and little to no toxicity are only a few features that make recombinant adeno-associated virus (rAAV) vectors ideally suited for many gene therapy applications. Successful preclinical studies have encouraged the use of rAAV for therapeutic gene transfer to patients in the clinical setting. Nevertheless, the use of rAAV in clinical trials has underscored the need for production and purification systems capable of generating large amounts of highly pure rAAV particles. To date, generating vector quantities sufficient to meet the expanding clinical demand is still a hurdle when using current production systems. In this chapter, we will provide a description of the current methods to produce clinical grade of rAAV under current good manufacturing practice (cGMP) settings. Significant advancements in the efficacy of gene delivery to treat human diseases using adeno-associated viral vectors (AAV), together with recent breakthroughs in manufacturing methods, has stimulated an unprecedented interest toward drug development for gene therapies and commercialization, often as a concerted and collaborative effort between academic and industry institutions. The growing interest in AAV-based therapies lies on a series of features that make rAAV vectors ideally suited for gene transfer approaches: its ability to elicit robust and long-term transgene expression in animals and humans and its safety, demonstrated in several phase 1/2 trials, with lack of toxicity, absence of adverse events following administration and manageable immune responses.1Carter BJ Adeno-associated virus vectors in clinical trials.Hum Gene Ther. 2005; 16: 541-550Crossref PubMed Scopus (165) Google Scholar,2Carnes A Williams J. High-yield plasmid DNA production.Gen Eng News. 2012; 32Google Scholar The first AAV clinical trial was conducted in subjects with cystic fibrosis in the mid-nineties,3Flotte T Carter B Conrad C Guggino W Reynolds T Rosenstein B et al.A phase I study of an adeno-associated virus-CFTR gene vector in adult CF patients with mild lung disease.Hum Gene Ther. 1996; 7: 1145-1159Crossref PubMed Scopus (219) Google Scholar with today more than 70 approved clinical trials worldwide for a wide variety of diseases.4Gemcris.www.gemcris.od.nih.govGoogle Scholar In 2012, Glybera, an AAV1-based drug for the treatment of Lipoprotein lipase deficiency in adults, was the first rAAV to be market-approved in Europe.5UniQure.www.uniqure.comGoogle Scholar To date, no AAV drugs have been approved nor submitted for approval in the United States. The use of rAAV in the clinical setting has underscored the urgent need for production and purification systems capable of generating very large amounts of highly pure rAAV particles. Typical FDA-approved investigational new drug include extensive preclinical studies for toxicology, safety, dose, and bio-distribution assessments, with vector requirements often reaching the 1E15 to 1E16 vector genome range. Manufacturing such amounts, although technically feasible, still represents an incredible effort when using the current production systems. In this review, we will provide a description of the methods that have been used to produce clinical grade rAAV under current the good manufacturing practice (cGMP) settings. It is noteworthy that methods used in clinical manufacturing are often not published and therefore this review will provide general insights into the methods used to date from information gathered in detailed references or from general websites.4Gemcris.www.gemcris.od.nih.govGoogle Scholar,6Trials C.www.clinicaltrials.govGoogle Scholar Transfection of plasmid DNA into eukaryotic cells was the first and remains the most commonly used protocol for clinical grade manufacturing of AAV. The most traditional approach is a calcium phosphate plasmid precipitation on human embryonic kidney 293 cells (HEK293)7Zolotukhin S Byrne BJ Mason E Zolotukhin I Potter M Chesnut K et al.Recombinant adeno-associated virus purification using novel methods improves infectious titer and yield.Gene Ther. 1999; 6: 973-985Crossref PubMed Scopus (1023) Google Scholar or HEK293-T8Allay JA Sleep S Long S Tillman DM Clark R Carney G et al.Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.Hum Gene Ther. 2011; 22: 595-604Crossref PubMed Scopus (106) Google Scholar with equimolar amount of the vector construct plasmid (prAAV-promoter-transgene) and the helper plasmid that provide the AAV Rep and Cap functions as well as the Ad5 genes (VA RNAs, E2A, and E4OEF6).9Grimm D Kern A Rittner K Kleinschmidt JA Novel tools for production and purification of recombinant adenoassociated virus vectors.Hum Gene Ther. 1998; 9: 2745-2760Crossref PubMed Scopus (569) Google Scholar,10Xiao X Li J Samulski RJ Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J Virol. 1998; 72: 2224-2232Crossref PubMed Google Scholar HEK293 cells constitutively express E1a/b, the fourth Ad function required for AAV replication. Cells are amplified in Corning cellstacks or roller bottles (see Table 1). A variation to this protocol is a two-helper method (or triple transfection) with AAV and Ad5 functions provided from separate plasmids.10Xiao X Li J Samulski RJ Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J Virol. 1998; 72: 2224-2232Crossref PubMed Google Scholar, 11Collaco RF Cao X Trempe JP A helper virus-free packaging system for recombinant adeno-associated virus vectors.Gene. 1999; 238: 397-405Crossref PubMed Scopus (68) Google Scholar, 12Flotte TR Conlon TJ Poirier A Campbell-Thompson M Byrne BJ Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites.Hum Gene Ther. 2007; 18: 245-256Crossref PubMed Scopus (45) Google Scholar, 13Wright JF Manufacturing and characterizing AAV-based vectors for use in clinical studies.Gene Ther. 2008; 15: 840-848Crossref PubMed Scopus (123) Google Scholar Typically up to 80% of cells are transfected with virus production peaking between 48–72 hours in the cell harvest. The versatility of this method has been tested by the production of a variety of AAV serotypes and capsid variants including 1, 2, 2.5, 2i8, 2 triple-tyrosine mutant, 7.4, 8, 9, and rh10 for clinical trials (referenced in Table 1), as well as double-stranded AAV.8Allay JA Sleep S Long S Tillman DM Clark R Carney G et al.Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.Hum Gene Ther. 2011; 22: 595-604Crossref PubMed Scopus (106) Google Scholar,14Koilkonda RD Yu H Chou TH Feuer WJ Ruggeri M Porciatti V et al.Safety and effects of the vector for the Leber hereditary optic neuropathy gene therapy clinical trial.JAMA Ophthalmol. 2014; 132: 409-420Crossref PubMed Scopus (68) Google ScholarTable 1Institutions and rAAV GMP manufacturing technologiesCenter, locationProductionCells and platformPurificationSerotypesRemoval emptiesReferencePowell Gene Therapy Center, Human Applications Laboratory, University of Florida, Gainesville, FL2-plasmid Transfection (CaPO4)HEK293 CellSTACKSCell harvest microfluidization or acidic flocculation and lysis, Benzonase, Heparin AC, IEC: SP, POROS, PS, HA, Hollow fiber tangential flow filtration (TFF)1, 2, 2(Y444, 500, 730F), 9No14Koilkonda RD Yu H Chou TH Feuer WJ Ruggeri M Porciatti V et al.Safety and effects of the vector for the Leber hereditary optic neuropathy gene therapy clinical trial.JAMA Ophthalmol. 2014; 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9: 1164-1172Abstract Full Text Full Text PDF PubMed Scopus (502) Google ScholarTargeted Genetics Corporation, Seattle, WAwtAd5 InfectionHEK293 Producer HeLa Cell line WAVE and stir Tank bioreactorsDepth filtration, Benzonase, Ion exchange, UF/TFF, Chrom step, Heat inactivation, Chrom step, NanofiltrationPolishingFormulation1, 294Thorne BA Takeya RK Peluso RW Manufacturing recombinant adeno-associated viral vectors from producer cell clones.Hum Gene Ther. 2009; 20: 707-714Crossref PubMed Scopus (57) Google ScholarAGTC, FL at SAFC, CAHSV InfectionsBHK WAVE bioreactorsDetergent lysis, Benzonase, Depth filtration, TFF, Ion Exchange chromatography, Affinity chromatography, TFF1No38Clément N Knop DR Byrne BJ Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies.Hum Gene Ther. 2009; 20: 796-806Crossref PubMed Scopus (79) Google Scholar,41Knop DR Harrell H Bioreactor production of recombinant herpes simplex virus vectors.Biotechnol Prog. 2007; 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Open table in a new tab The information in the table was assembled utilizing information gathered in detailed references or from general websites. The main limitation to the transfection method of adherent HEK293 cells is its inherent lack of scalability. Scaling-up a method relying on adherent cells would require the linear multiplication of the number of flasks/surface area needed. A typical GMP manufacturing campaign requires more than a hundred cellstacks for yields often lower than 1E15 vg of clinical product (Clement N, unpublished data).8Allay JA Sleep S Long S Tillman DM Clark R Carney G et al.Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.Hum Gene Ther. 2011; 22: 595-604Crossref PubMed Scopus (106) Google Scholar15Grieger JC Soltys SM Samulski RJ Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector.Mol Ther. 2015; (epub ahead of print)PubMed Google Scholar Based on these numbers, generating >1E16 vg would require greater than 500 cell factories. Although technically possible, it is likely not a viable option for most if not all manufacturing facilities with regard to the man power, space, and cost limitations. An attractive option to further expand the capacity of vector production using transfection is the cell suspension format. Grieger et al. at the University of North Carolina Gene Therapy Center Vector Core developed a scalable, transfection-based manufacturing process utilizing suspension HEK293 cells. HEK293 cells from a qualified clinical master cell bank were adapted to grow in suspension in an animal component and antibiotic-free media, in shaker flasks, WAVE bioreactors and stir tank bioreactors. Yields generated upon triple transfection with PEImax, are typically greater than 105 vector genome containing particles vg/cell in crude lysates or greater than 1 × 1014 vg/l of cell culture (1 × 106 cells/ml) after 48 hours incubation.15Grieger JC Soltys SM Samulski RJ Production of recombinant adeno-associated virus vectors using suspension HEK293 cells and continuous harvest of vector from the culture media for GMP FIX and FLT1 clinical vector.Mol Ther. 2015; (epub ahead of print)PubMed Google Scholar Parameters including selection of a compatible serum-free, chemically defined suspension media optimal for both cell growth and transfection, selection of a transfection reagent, transfection conditions and cell density, were fully optimized. Early process development data supports a linear increase in rAAV yields with respect to cell density, achieving prepurification yields of ∼1 × 1015 vg/l of high cell density cell culture in both shaker flasks and small scale stir tank bioreactors to be scaled to 250L single-use bioreactors (Grieger JC, unpublished data). Increased yield recovery using improved purification methods will achieve >4 × 1014 purified AAV vector per L of transfected high cell density culture (Grieger JC, unpublished data). This platform has all the prerequisites to enable rapid and scalable rAAV production for larger-scale manufacturing campaigns. The overall advantages of the transfection method are: simple protocols, ease and relatively low cost to generate and test the raw materials (plasmid preparations) due to current technologies available to manufacture high plasmid yields (0.5–1 g/l),2Carnes A Williams J. High-yield plasmid DNA production.Gen Eng News. 2012; 32Google Scholar versatility to generate different rAAV constructs and serotypes in a relatively short turn-around time, which is ideal as no need to generate and characterize several cells clones (MCBs and WCBs) and viral banks (MVBs and WVBs for Ad, Bac or herpes simplex virus (HSV)), high particle yield per cell in crude lysates (>105 vg/cell) with optimized protocols, standard physical-to-infectious particle ratios and the largest portfolio of historical data pertaining to clinical safety. Most of the current rAAV GMP manufacturing campaigns for use in phase 1/2 human gene therapy clinical trials have been generated using transfection and are summarized in Table 1. Stable cell lines, usually derived from HeLa cells, are engineered by introducing either the AAV rep and cap genes (packaging cell lines) and/or the rAAV genome to be produced (producer cells).16Clark KR Recent advances in recombinant adeno-associated virus vector production.Kidney Int. 2002; 61: S9-15Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 17Gao GP Qu G Faust LZ Engdahl RK Xiao W Hughes JV et al.High-titer adeno-associated viral vectors from a Rep/Cap cell line and hybrid shuttle virus.Hum Gene Ther. 1998; 9: 2353-2362Crossref PubMed Scopus (161) Google Scholar, 18Liu X Voulgaropoulou F Chen R Johnson PR Clark KR Selective Rep-Cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines.Mol Ther. 2000; 2: 394-403Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar One interesting feature of packaging and producer cell-based production relates to their ability to generate relatively high rAAV particle-per-cell yield. Yields as high as 105 vg/cell have been reported with vg/infectious unit (i.u) ratio comparable to transfection-produced rAAV. rAAV is produced from packaging cell lines upon transfection of the AAV construct and coinfection with an Ad helper virus, or alternatively, upon infection with a recombinant Ad/AAV hybrid helper. For producer cells containing both AAV rep/cap and rAAV genome, production is a single-step infection with an Ad helper virus.19Martin J Frederick A Luo Y Jackson R Joubert M Sol B et al.Generation and characterization of adeno-associated virus producer cell lines for research and preclinical vector production.Hum Gene Ther Methods. 2013; 24: 253-269Crossref PubMed Scopus (45) Google Scholar,20Martin JN Wolken N Brown T Dauer WT Ehrlich ME Gonzalez-Alegre P Lethal toxicity caused by expression of shRNA in the mouse striatum: implications for therapeutic design.Gene Ther. 2011; 18: 666-673Crossref PubMed Scopus (80) Google Scholar Several AAV clinical lots were produced using HeLa producer cell lines with Ad or Ad/AAV infection (Table 1), including tgAAVCF for cystic fibrosis (HeLa JL L-14), tgAAV94 for arthritis (HeLa 51-H5), and an AAV2 (α, β, γ, or Δ) for limb girdle muscular dystrophy (HeLa B50).21Stedman H Wilson JM Finke R Kleckner AL Mendell J Phase I clinical trial utilizing gene therapy for limb girdle muscular dystrophy: alpha-, beta-, gamma-, or delta-sarcoglycan gene delivered with intramuscular instillations of adeno-associated vectors.Hum Gene Ther. 2000; 11: 777-790Crossref PubMed Scopus (76) Google Scholar Clinical data reported no adverse event related to the AAV product.22Frank KM Hogarth DK Miller JL Mandal S Mease PJ Samulski RJ et al.Investigation of the cause of death in a gene-therapy trial.N Engl J Med. 2009; 361: 161-169Crossref PubMed Scopus (72) Google Scholar A more scalable approach takes advantage of the HeLaS3 cell line, a HeLa derivative capable of growing in both adherent and suspension formats. The cell line was engineered using traditional stable transformation procedures in flasks, and the large-scale manufacturing can occur in suspension format in spinner or bioreactors. Mydicar (AAV1), a product for h