430 engineering of packaging cell lines for the biomanufacturing of retroviral vectors

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

Previ-ous 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 testedwhetherendogenousFeLVLTRor 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 expressionvector 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 furtheranalysis On

all human cell linestested, CatPac supernatantsroutinely have2 to 5

fold higherGFPtiters than either PhoenixGALVor Phoenixampho,

Transient transfeetion of CatPac cells with MSCV-basedrctroviral

vectors results in high titer vector production(titcr->Ix 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

Small-scale preps are easily concentrated over IO-fold (resulting titers

-I07/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 exchangethe culture mediumcontainingthe vector,On

human CD34+ cells, transduction rates reach a maximum ofabout

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

FeLV-C (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

pre-cursors for potential therapies such as lysosomal storage disorders,

as well as HSC

Process for rAAV1 Vectors Produced Via

Baculovirus Expression

KathyrnA Rizzo,IRobert 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

adeno-associated 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

work-ing 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

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and efficientlyprocessthe fermentation harvestforcontinued down-stream 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 purifica-tion process consists ofa 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 1015total vector genomes per liter offermentation 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

the Biomanufacturing of Retroviral Vectors

Karim Ghani,IAmine Kamen,'Manuel Caruso.'

'Cancer Center: Laval University Quebec QC Canada; 2 Animal Cell Technology Biotechnology Research Institute Montreal.

QC. Canada

The production of retroviral vectors for clinical gene therapy is cumbersome, costly,and lackssafety features because ofthe adher-ent nature of packaging cells and the necessity to supplemadher-ent the culture media with bovine serum In this study, we report the con-structionand the characterizationofthe first packagingcell linesthat produce rctroviral vectors in suspension without serum A clone of HEK293cells engineeredto producea high levelof Moloneymurine leukemiavirus (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 3 packaging cell lines: 293GP-A2,293GP-GLV9and 293GP-RD30.A GFPtransfer vector was nextstably introducedin each packagingcell line to assess their potential as stable retrovirus producercell lines.Titers from 293GP-A2,293GP-GLV9and293GP-RD30were4xJ07,1.9x 107and 107

infectious viral particles (IVP/mL), respectively.All the packaging cell line construction steps were performed with cells grown ad-herently 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 conditionsin a low-calciumSFM.Cells were transferredto a shake flask,and the serum concentrationwas slowly decreased over

a I-month period until it was completely removed Once cells were fullyadapted,titers producedin suspensionand SFM werecompared

to those obtained adherently and in presence ofserum Titers were not affected by the adaptation except with the 293GP-GLV9clone 293GP-A2,293GP-GLV9 and 293GP-RDJO titers were 4 x 107,

106and 5 x 1061VP/mLin suspension with SFM,respectively.The 293GP-A2 clone wasstable since there was no drop in titer during

a 3-month culture period The transduction with vector produced fromthis clonewas assessedon 3 adherentand 2 lymphoidcell lines The 3 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 pursuingexperimentsto 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 biomanufacturingof

Molecular Therapy Volume 15 , Supplemen t I ,\b r 2007

Co pyright © Th e Ame rican Societ y o f G ene Thcr.Lp) ·

rctroviral vectors These cell lines wilI be ideal for the

implemen-tation of late phase gene therapy elinical trials that require a high

number of patients

Vectors Using Adherent and Suspension-Adapted

Cells

James P Brady,' Matthew Malehorn,' Joseph C Fratantoni,'

Linda N Liu,' Madhusudan V Peshwa.'

I Product Development MaxCyte Inc Gaithersburg MD.

Gene therapy vectors derivedfrom 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,

simulta-neous 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

ma-nipulation 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 loadingof

adherent 293FT cells with components ofa four plasmid, HIV-based

lentivector system encoding green fluorescent protein (OFP) Based

on GFP proteinexpression,cell transfection efficiency was >90%

Viral supernatant was titrated on 293T cells,using FACS analysis

to measure infectivity and transgene expression Leruivector titers

> Ix 107transducing units (TU)/mL were obtained in small-scale

transfections (i.e Ix 107cells/reaction) Scalability of lentivector

production in adherent cells was demonstrated by electroporating

5xlO8293FT 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 I07TUlmL Total infectious

lentivector particles colIected from the 2-chamber Cell Factory

exceeded 5x I09•Efforts are under way to scale-up lentivector

pro-duction 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

Optimiza-tion of c1ectroloading of the suspension-adapted 293FT cells with

GFP plasmid DNA resulted in >85% transfection efficiency, based

on GFP protein expression, and >95% viability,based on propidium

iodide exclusion SmalI scale transfections of the 293FT

suspen-sion cells with the four lentivector component plasm ids yielded

infectious lentiviral titers similar to those obtained with adherent

cells (-lxI07TUlmL).Efforts are under way to scale-up

lentivec-tor 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

vec-tors 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).

MolecularTherapy V olume 15 ,Sup plementI May2 007

C oprright © Th eAmerican Socie tyo f G f,.'f11;Th erapy

Manufacture of a Gene Transfer Vector for Leber Congenital Amaurosis

Bernd Hauck; Xingge Liu,' Sonali Joyce; Alex Tal,'Jeannette Bennicelli,?Olga Zelenaia,' Katherine A High,IJ Fraser Wright I

'Center for Cellular and Molecular Therapeutics, Childrens Hos-pital ofPhiladelphia Philadelphia I'll; 10 phthalmolog)\ Scheie Eye Institute, Philadelphia PA.

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 purifica -tion 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 inter-mediate 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 of9x1014highly 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

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