430 Engineering of Packaging Cell Lines for the Biomanufacturing of Retroviral Vectors hematopoietic stem cells (I ISC) and macrophage precursors The human ortholog of the FeLV C receptor, FLVCR, is a[.]
hematopoietic stem cells (I-ISC) and macrophage precursors The human ortholog of the FeLV-C receptor, FLVCR, is a ubiquitously expressed 12 transmembrane domain major facilitator superfamily member, recently demonstrated to be a heme exporter FLVCR is highly expressed on human CD34+ cells and maerophages Previous studies demonstrated FeLV-C pseudotyped vectors effectively targeted human HSC capable of repopulating multiple lineages in the in uterosheep model (Blood 106:51,2005) FLVCRis expressed at higher levels on immature cells than on more mature cells (Cell 118: 757, 2004), which is opposite from that of the GALV and ampho receptors Pit I and Pit 2, which increase expression during differentiation (Blood 106:51, 2005) Based upon these data, we decided to design the optimal FeLV-C-based packagingsystem, and compare it to existing oncorctroviral packaging systems Wetested whether an all FelY-based system was better than using murine gag and pol.Also, we testedwhetherendogenous FeLVLTRor the CMV promoter work best to drive envelope expression, and whether 3T3 or 293 cells work best Using the optimal components, we created a safe packaging line in two steps First, we transfected 293 cells with the LTR driven gag/pol expression vector and functionally screened for the two best clones Wethen transfeeted these with the envelope construct and screened clones for packaging function by supernatant titers We chose two independent clones (CatPac6 and CatPac7) with the highest titer supernatants for further analysis On all human cell linestested, CatPac supernatantsroutinely have to fold higherGFPtiters than either PhoenixGALVor Phoenix ampho, Transient transfeetion of CatPac cells with MSCV-basedrctroviral vectors results in high titer vector production (titcr->I x 106/ml) that retains nearly 100%transduction efficiency after storage at 4°C for 48 hours This long-term stability suggested it might be possible to concentrate CalPac supernatants, further increasing the titer Smallscale preps are easily concentrated over IO-fold (resulting titers -I 07/ml); largerscale preparationsare likelyto producemuch higher titer supernatants (35 to 100-fold concentration) Neither ampho or GALV vectors can readily be concentrated, which gives CatPac vectors an advantage in the ability to transduce difficult cells, and the ability to exchange the culture mediumcontainingthe vector,On human CD34+ cells, transduction rates reach a maximum of about 20%, likely including high numbers of HSC given the preferential expression ofFLVCR on less mature cells High levels ofFLVCR on macrophages and their ability to be infected with native FeLVC (Blood 81: 2585, 1993) suggest they might also be an excellent target for these pseudotyped vectors Preliminary studies confirm that CD34-derived monoeytes arc good targets This provides a novel approach for oncoretroviraltransduction of macrophage precursors for potential therapies such as lysosomal storage disorders, as well as HSC 429 Development of a cGMP Production Process for rAAV1 Vectors Produced Via Baculovirus Expression Kathyrn A Rizzo, I Robert A Ballinger; Karl Anderson; Joseph A Rininger,' I Process Development Protein Sciences Corporation, Meriden CT The Baculovirus Expression Vector System (BEVS) is now emerging as a new manufacturing platform for recombinant adenoassociated virus (rAAV)vectors The platform provides high yields as well as safety and likely regulatory advantages over mammalian production platforms Furthermore, given the scalability of the fermentation process, a limited number oflarger fermentations may be required to generate clinical grade rAAV As bioreactor working volumes increase to generate vector for therapeutic indications needing large quantities ofrAAV,strategies need to be developed to compensate for the increased harvest volumes and to immediately SI66 and efficientlyprocess the fermentation harvest for continued downstream processing This presentation will outline the strategy being undertaken to develop a cGMP production process with rAAVI produced via the baculovirus platform to be executed at 450L production scale The fermentation utilizes equal multiplicities of infection (MOl) of three recombinant baculoviruses, The purification process consists of a benzonase-free two-step hollow-fiberand diafiltrationstep for initialextraction ofrAAV I from the insectcells and removal of cellular protein contaminants The initial extraction step has resulted in rAAVI extraction solutions containing greater than I X 1015 total vector genomes per liter of fermentation volume by Q-PCR analysis The extracted rAAVis then applied to a series oftwo ion exchange chromatographysteps followed by diafiltration into the final formulation buffer.The applicability ofthis procedure to insect cell produced rAAV2 and rAAV8 will also be discussed 430 Engineering of Packaging Cell Lines for the Biomanufacturing of Retroviral Vectors Karim Ghani,I Amine Kamen,' Manuel Caruso.' 'Cancer Center: Laval University Quebec QC Canada; 2Animal Cell Technology Biotechnology Research Institute Montreal QC Canada The production of retroviral vectors for clinical gene therapy is cumbersome, costly, and lacks safety features because ofthe adherent nature of packaging cells and the necessity to supplement the culture media with bovine serum In this study, we report the constructionand the characterizationofthe first packagingcell linesthat produce rctroviral vectors in suspension without serum A clone of HEK293cells engineeredto producea high level of Moloneymurine leukemia virus (MLV)gag-pol proteins was firstconstructed.Then, the amphotropic (4070A), the gibbon ape leukemia virus (GLV) and the cat virus RDI14 envelopes (Env) genes were transfected separately in the gag-pol clone to generate packaging cell lines: 293GP-A2, 293GP-GLV9and 293GP-RD30.A GFPtransfer vector was next stably introduced in each packagingcell line to assess their potential as stable retrovirus producercell lines.Titers from 293GPA2,293GP-GLV9and293GP-RD30were4x J07, 1.9x 107 and 107 infectious viral particles (IVP/mL), respectively All the packaging cell line construction steps were performed with cells grown adherently and in presence of serum We next took on the challenge to produce retroviral vectors in suspension with serum-free media (SFM) For this purpose, packaging cells had to be adapted to these culture conditions in a low-calciumSFM Cells were transferredto a shake flask, and the serum concentration was slowly decreased over a I-month period until it was completely removed Once cells were fullyadapted,titers producedin suspensionand SFM were compared to those obtained adherently and in presence of serum Titers were not affected by the adaptation except with the 293GP-GLV9clone 293GP-A2, 293GP-GLV9 and 293GP-RDJO titers were x 107, 106 and x 1061VP/mL in suspension with SFM, respectively.The 293GP-A2 clone was stable since there was no drop in titer during a 3-month culture period The transduction with vector produced fromthis clone was assessedon adherentand lymphoidcell lines The adherent cell lines were highly transducible: mouse 3'1'3 cells were transducedat 95.7%, and human HTI080 andTE671 cells were infected at 88.3% and 72.2%, respectively DG75 (B lymphoid) and Jurkat (T lymphoid) cell lines were less infeetable with 16.3% and 12.6% transduction efficiency, respectively We are currently pursuing experimentsto test the transductionefficiencywith vectors produced by 293GP-GLV9and 293GP-RD30 cells In conclusion, this study shows for the first time the construction of packaging cell lines producing high-titer retroviral vectors in suspension and SFM The 293GP-A2, 293GP-GLV9and 293GP-RD30 packaging cell lines have the potential for the large-scale biomanufacturing of Molecular Therapy Volume 15, Supplemen t I ,\br 2007 Co pyright © Th e American Society o f G ene Thcr.Lp)· rctroviral vectors These cell lines wilI be ideal for the implementation of late phase gene therapy elinical trials that require a high number of patients 432 Process Characterization and GMP Manufacture of a Gene Transfer Vector for Leber Congenital Amaurosis 431 Large-Scale Production of Lentiviral Vectors Using Adherent and Suspension-Adapted Cells Bernd Hauck; Xingge Liu,' Sonali Joyce; Alex Tal,' Jeannette Bennicelli,? Olga Zelenaia,' Katherine A High, I J Fraser Wright I 'Center for Cellular and Molecular Therapeutics, Children s Hospital ofPhiladelphia Philadelphia I'll; 10p hthalmolog)\ Scheie Eye Institute, Philadelphia PA James P Brady,' Matthew Malehorn,' Joseph C Fratantoni,' Linda N Liu,' Madhusudan V Peshwa.' I Product Development MaxCyte Inc Gaithersburg MD Gene therapy vectors derived from lentiviruses offer significant advantages over other gene transfer vectors due to their ability to deliver therapeutic transgenes to both dividing and non-dividing celIs Lcntivirus is most commonly produced by transient, simultaneous transfection ofcells with multiple plasmids , which decreases the possibility of generating recombinant, replication competent lentivirus (RCL) Current transient transfection methods alIow production of small amounts of viral vectors , but the process is laborious, inconsistent and cannot be scaled-up beyond requirements of early-stage clinical trials Extensive effort has been focused on generating stable producing cell lines for lentivector production, but toxicity caused by Ientiviral gene products still remains unsolved Stable producing cell lines using inducible promoters generally yield unsatisfactory productivity compared to the transient method MaxCyte has developed a robust , consistent and scalable celI manipulation technology platform based on electroporation, which is being used in elinical trials in the US and Canada The MaxCyte platform was used to develop a scalable process for lentiviral vector production First, conditions were optimized for electro loading of adherent 293FT cells with components ofa four plasmid, HIV-based lentivector system encoding green fluorescent protein (OFP) Based on GFP prote in expression, cell transfection efficiency was >90% Viral supernatant was titrated on 293T cells, using FACS analysis to measure infectivity and transgene expression Leruivector titers > Ix 107 transducing units (TU)/mL were obtained in small-scale transfections (i.e Ix 107 cells/reaction) Scalability of lentivector production in adherent cells was demonstrated by electroporating 5xlO 293FT cells cultured in a 2-chamber Cell Factory (1264 cm-, Corning) Collections at 24 and 48 hrs post transfection revealed titers of both supernatants were> I x I0 TUlmL Total infectious lentivector part icles colIected from the 2-chamber Cell Factory exceeded 5x I09 • Efforts are under way to scale-up lentivector production to IO-chamber Cell Factories (6320 cm-) To demonstrate the feasibility of lcntivector production in suspension cells, 293FT cells were adapted to suspension growth in shake flasks Optimization of c1ectroloading of the suspension-adapted 293FT cells with GFP plasmid DNA resulted in >85% transfection efficiency, based on GFP protein expression, and >95% viabi lity, based on propidium iodide exclusion SmalI scale transfections of the 293FT suspension cells with the four lentivector component plasm ids yielded infectious lentiviral titers similar to those obtained with adherent cells (-lxI0 7TUlmL) Efforts are under way to scale-up lentivector production in suspension-adapted 293FT cells cultured in a WAVE bioreaetor In summary, the MaxCyte technology platform overcomes major hurdles associated with large-scale lcntivector production and offers the potential to manufacture lcntiviral vectors for clinical testing and commercial production in adherent and suspension-adapted cells 711is work was supported by SBJR Grant # I R43 HL088996-01 from NIH (NHLBI) Molecular Therapy Volume 15, Supplement I May2007 Coprright © The American Socie ty of Gf,.'f11; Th erap y We previously reported the manufacture and characterization of AAV2-hFIXI6, an investigation gene therapy vector for treatment of Hemophilia B (Hauck et al (2006) Mol Therapy 13:S196) Here we report detailed process characterization for our combined cation exchange chromatography / gradient ultracentrifugation purification process , and describe the manufacture of AAV2-hRPE65v2, an AAV2 based vector expressing retinal pigment epithelial 65kOa protein (hRPE65), an investigational gene transfer vector for Leber congenital amaurosis Vector biosynthesis / generation is achieved via transient transfection ofHEK293 cells in roller bottles Both cells and culture medium are harvested The harvest volume is reduced and low molecular weight cell culture impurities are removed by an initial tangential flow filtration process step Vector is release from the cells by microfluidization In this step AAV vectors as well as many soluble HEK293 proteins arc released from cells The mixture if subsequently filtered to remove insoluble debris prior to loading onto a cation exchange chromatography resin While the filtration step removes cellular debris, soluble non-vector proteins remain associated with the vector in the clarified filtrate Recovery of vector over this filtration step is 80-90% Purification of the vector using Poros 50HS cation exchange chromatography results in highly purified AAV2 particles as assessed by SOS-PAGE, exhibiting process step purification power similar to that achieved by affinity chromatography In the chromatography process step, the majority of non-vector proteins are separated from the vector, as shown by removal of non vector proteins present in the column feed; however, AAV2 VP proteins recovered in the product intermediate peak contain theAAV2 empty capsids Recovery of vector from the chromatography column process is approximately 50%, indicating an opportunity for further optimization The elution peak recovered from the Poros 50HS cation exchange resin is subjected to gradient ultracentrifugation in CsCI, a final purification step that removes trace impurities as well as AAV2 empty capsids Typically >lxlO'~ AAV genome-containing vector and >2xlO'~ AAV empty capsids are separated by a single gradient ultracentrifugation run At our current scale, we have achieved an average of9x1014 highly purified, empty-capsid free vector genomes per 100 roller bottles cell culture Our cGMP manufacturing campaign for AAV2-hRPE65v2 resulted in> Ix lOIS vg at the bulk drug substance stage The highly purified vector was formulated at a concentration of Ix I01) vg/mL in phosphate-buffered saline supplemented with 0.00 I% Pluronic F68, a formulation that was found to achieve high vector stability and prevent vector loss / inactivation during frozen storage and during dilution and administration SI67 ...rctroviral vectors These cell lines wilI be ideal for the implementation of late phase gene therapy elinical trials that require a high number of patients 432 Process Characterization... Canada The MaxCyte platform was used to develop a scalable process for lentiviral vector production First, conditions were optimized for electro loading of adherent 293FT cells with components ofa... Coprright © The American Socie ty of Gf,.''f11; Th erap y We previously reported the manufacture and characterization of AAV2-hFIXI6, an investigation gene therapy vector for treatment of Hemophilia