Towards Better Characterization of Recombinant AAV Gene Transfer Vectors: Case Studies Illustrating Challenges with Dose Determining Vector Concentration methods J. Fraser Wright, Ph.D. Well Characterized Biologic Products 2011 15 th Symposium on the Interface of Regulatory and Analytical Sciences for Biotechnology Health Products Washington DC, January 10-12, 12, 2011
Adeno associated virus (AAV) AAV member of Parvoviridae family 25 nm diameter (small), non-enveloped (stable) ssdna genome of 4.7 kb dependent upon helper virus for replication - adenovirus, herpes simplex virus, others >12 serotypes described, other capsid variants no known disease association attractive vector for therapeutic gene transfer because: - lack of pathogenicity / low immunogenicity - long-term transgene expression in several animal models - serotypes demonstrate varying tissue tropism
MAADGYLPDW LEDTLSEGIR QWWKLKPGPP PPKPAERHKD DSRGLVLPGY KYLGPFNGLD KGEPVNEADA AALEHKKAYD RQLDSGKNPY LKYNHADAEF QERLKEDTSF GGNLGRAVFQ AKKRVLEPLG LVEEPVKT AP GKKRPVEHSP VEPDSSSGTG KAGQQPARKR LNFGQTGDAD SVPDPQPLGQ PPAAPSGLGT NT MATGSGAP MADNNEGADG VGNSSGNWHC DSTWMGDRVI TTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI NNNWGFRPKR LNFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQG CLPPFPADVG MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFT FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNT PSGTTTQSRL QFSQAGASDI RDQSRNWLPG PCYRQQRVSK TSADNNNSEY SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKFFPQSGVL IFGKQGSEKT NVDIEKVMIT DEEEIRTTNP VATEQYSGSVS TNLQRGNRQA ATADVNTQGV LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP SPLMGGFGLK HPPPQILIKN TPVPANPSTT FSAAKFASFI TQYSTGQVSV EIEWELQKEN SKRWNPEIQY TSNYSKSVNV DFTVDTNGVY SEPRPIGTRY LTRNL 1 51 101 151 201 251 301 351 401 451 501 551 601 651 701 AP GKKRPVEHSP VEPDSSSGTG KAGQQPARKR LNFGQTGDAD SVPDPQPLGQ PPAAPSGLGT NT MATGSGAP MADNNEGADG VGNSSGNWHC DSTWMGDRVI TTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI NNNWGFRPKR LNFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQG CLPPFPADVG MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFT FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNT PSGTTTQSRL QFSQAGASDI RDQSRNWLPG PCYRQQRVSK TSADNNNSEY SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKFFPQSGVL IFGKQGSEKT NVDIEKVMIT DEEEIRTTNP VATEQYSGSVS TNLQRGNRQA ATADVNTQGV LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP SPLMGGFGLK HPPPQILIKN TPVPANPSTT FSAAKFASFI TQYSTGQVSV EIEWELQKEN SKRWNPEIQY TSNYSKSVNV DFTVDTNGVY SEPRPIGTRY LTRNL 139 151 201 251 301 351 401 451 501 551 601 651 701 MATGSGAP MADNNEGADG VGNSSGNWHC DSTWMGDRVI TTSTRTWALP TYNNHLYKQI SSQSGASNDN HYFGYSTPWG YFDFNRFHCH FSPRDWQRLI NNNWGFRPKR LNFKLFNIQV KEVTQNDGTT TIANNLTSTV QVFTDSEYQL PYVLGSAHQG CLPPFPADVG MVPQYGYLTL NNGSQAVGRS SFYCLEYFPS QMLRTGNNFT FSYTFEDVPF HSSYAHSQSL DRLMNPLIDQ YLYYLSRTNT PSGTTTQSRL QFSQAGASDI RDQSRNWLPG PCYRQQRVSK TSADNNNSEY SWTGATKYHL NGRDSLVNPG PAMASHKDDE EKFFPQSGVL IFGKQGSEKT NVDIEKVMIT DEEEIRTTNP VATEQYSGSVS TNLQRGNRQA ATADVNTQGV LPGMVWQDRD VYLQGPIWAK IPHTDGHFHP SPLMGGFGLK HPPPQILIKN TPVPANPSTT FSAAKFASFI TQYSTGQVSV EIEWELQKEN SKRWNPEIQY TSNYSKSVNV DFTVDTNGVY SEPRPIGTRY LTRNL 203 251 301 351 401 451 501 551 601 651 701 AAV2: More detailed structure and composition A1 A69 B1 protein 6 VP1 735 aa 6 VP2 597 aa 48 VP3 533 aa Particle radius: 25nm Molecular weight: protein (74%) M r ~ 3750 kda DNA (26%) M r ~ 1350 kda total virus M r ~ 5100 kda relief Xie, Qing et al. (2002) Proc. Natl. Acad. Sci. USA 99, 10405-10410 charge
Certified raw materials / components: HEK293 MCB Plasmid DNA DMEM FBS Trypsin Process buffers (PBS) Benzonase TM Cesium chloride Excipient buffer (PBS) Poloxamer 188 Manufacturing process: HEK293 MCB T75 flask T225 flasks RB (2, 10, 100) Transfection Serum free medium Crude Cell Harvest Concentration (TFF) Cell lysis (HPM) Filtration (0.45 / 0.2 μm) Chromatography (IEX) Filtration (0.2 μm) Gradient centrifugation * Buffer exchange (TFF) Filtration (0.2 μm) Bulk Drug Substance Dilution Filtration (0.2 μm) Vial Fill / Finish Final Product cgmp Manufacturing overview Rigorous QC Testing Safety - microbial and viral contaminants - physicochemical Purity - product- and processrelated impurities Potency - vector concentration and functional activity Stability - safety, purity and potency monitoring for 5 years
AAV vectors: Evolution of the clinical manufacturing process PROCESS 1 (E14 vg) cell growth in T-flasks Transfection medium exchange Harvest cell lysis (freeze / thaw) Centrifugation DNase digestion / CaCl 2 Centrifugation PEG precipitation Centrifugation CsCl ultracentrifugation (2X) UF / DF (TFF) formulation / F&F PROCESS 2 (E15 vg) * cell growth in roller bottles transfection medium exchange harvest volume reduction (TFF) cell lysis (microfluidization) filtration ion exchange chromatography gradient ultracentrigation * buffer exchange (TFF) formulation / F&F CLINICAL PROCESS OBJECTIVES FOR Ph III / COMMERCIAL (E16+ vg) Vector generation: - suspension culture - serum free media - helper virus free - productivity: > 10 6 vg/cell - low vector-related subpopulations Vector purification: - < 0.1% DNA impurities * - < 1% non capsid protein impurities - < 10% empty capsids - no ultracentrifugation - yield: > 80%
AAV vector characterization / certification for clinical use - Characterize AAV vectors prepared to support clinical studies -Assess consistency in clinical lots - Ensure successful process transfer from R&D to clinical manufacturing - Basis for product comparability studies MW 2x10 10 vg VP1 VP2 VP3 PARAMETER Identity: AAV capsid protein vector genome Purity: Protein impurities Residual plasmid DNA Residual mammalian DNA Residual cesium chloride Residual Benzonase Potency: vector genomes in vitro transduction infectivity titer Safety: Adventitious viruses Mycoplasma WT AAV USP sterility Endotoxin ph Osmolality Aggregation Appearance METHOD SDS-PAGE SS / WB restriction digest / SB SDS-PAGE SS / CB optical Density 260 / 280nm Q-PCR Q-PCR ICP-MS ELISA Q-PCR transduction / transgene ELISA limiting dilution / Q-PCR in vitro assay for viral contaminant agar cultivable and non-cultivable ICA with Ad four media, direct inoculation LAL potentiometry osmometry dynamic light scattering visual inspection
from: Wright (2008) Gene Therapy * * *
Model for qpcr efficiencies on DNA templates PCR amplification efficiency: <<100% <100% 100%
Titer by qpcr 3.82 x 10 10 vg/ml Inter-laboratory variability RSD = 78% range: 0.13 12.7x10 10 vg/ml
Effect of qpcr assay variability on other parameters typically calculated for vector characterization: titer P:I ratio * residual DNA (vg/ml) (vg/iu) (pg / 10 9 vg) Mean 1SD 0.85 x10 10 4.5 90 Mean 3.82 x10 10 20 20 Mean +1SD 6.79 x10 10 35 11 * Particle to Infectious unit ratio
Quantification of vector genomes and capsid particle concentration by Optical Density (OD) Assays used to assess vector genome capsid particle concentration are complex and derivative (Q-PCR, capsid protein ELISA) Development of a novel assay to assess these parameters by optical density measurement of purified AAV vector useful as a supplemental assay to confirm titers and assess purity Based on: A 260 260 = DNA 260 + Protein 260, A 280 = DNA 280 + Protein 280
Determination of extinction coefficients for AAV capsid proteins Theoretical: W Y F VP / capsid W / capsid Y / capsid F / capsid VP1 15 32 32 5 75 160 160 VP2 12 26 28 5 60 130 140 VP3 12 26 27 50 600 1300 1350 Tot 60 735 1590 1650 260 nm 280 nm A 260 /A 280 ratio ε of W 3,600 5,700 0.63 ε of Y 530 1,300 0.41 ε of F 144 2 72 ε of capsid (per mole) 3.73x10 6 6.26x10 6 0.595 predicted: 10 12 capsids / ml 0.0062 0.0104 0.595 Experimental: - Prepared highly purified empty capsids (LN431) - Performed quantitative amino acid analysis on LN431, giving 310 µg/ml A 260 / A 280 = 0.523 ε 260 = 3.72x10 6 mol -1 ε 280 = 6.61x10 6 mol -1 Sommer et al, 2003
Titer determination by qpcr for single stranded (ss) and self complementary (sc) AAV vectors Vector lot genome config n Titer by qpcr (x 10 13 ) Titer by OD (x 10 13 ) OD ratio 260/280 Titer ratio qpcr / OD Res pdna (pg / 10 9 vg) single-stranded vectors: 003A ss 1.35 1.49 1.5 0.91 - A2P1-001 ss 1.10 1.28 1.42 0.86 - A2P1-002 ss 1.01 1.13 1.45 0.89 39.1 0907C ss 0.78 1.09 1.53 0.72 46.6 self-complementary vectors: 0804 sc 0.50 1.53 1.49 0.33-1001 sc 0.18 0.71 1.52 0.25 131 493 sc 0.36 1.4 1.55 0.26 -
AAV vector genome concentration by OD method - comparison with Q-PCRQ vg/ml (Absorbance) 1.2e+13 1.0e+13 8.0e+12 6.0e+12 4.0e+12 2.0e+12 - linearized plasmid standard - self complementary AAV genome (m > 1, ~ 2-5) - linearized plasmid standard - single stranded AAV genome (m = 1.1, R 2 = 0.98) - supercoiled plasmid standard - single stranded AAV genome (m < 1, ~ 0.4) 0.0 0.0 2.0e+12 4.0e+12 6.0e+12 8.0e+12 1.0e+13 1.2e+13 vg/ml (Q-PCR)
Acknowledgements Clinical Vector Core: Process Development Regulatory Affairs Research Core Bernd Hauck Olga Zelenaia Liza Africa Xingge Liu Guang Qu Jennifer McDonnell Greg Podsakoff Shangzhen Zhou Sonali Joyce Alex Tai Research Collaborations Valder Arruda Linda Couto Katherine High Center for Cellular and Molecular Therapeutics The Children s Hospital of Philadelphia Jean Bennett, Al Maguire RPE65 Team University of Pennsylvania School of Medicine