Director-Laboratory for Cellular Medicine City of Hope/Beckman Research Institute

Transcription

1 David DiGiusto Ph.D. Director-Laboratory for Cellular Medicine City of Hope/Beckman Research Institute

2 An Investigational New Drug Application (IND) is required for clinical use of all more than minimally manipulated (PHS 351) cell products. Assays must be performed to address the, identity, purity, potency and safety of the products. When products are genetically modified, additional assays should be developed d to assess the extent of genetic modification and justify the methods used in manufacturing. When viral vectors are used, assays should be developed to test for the presence of replication competent virus.

3 Release Criteria (Safety and Purity) Viability 70% (later changed to 5 x 10 5 CD34/kg) Sterility Gram/KOH (lack of bacterial and fungal elements) Retrospective USP bacterial and fungal Endotoxin (<5 EU/Kg/hr of infusion) Mycoplasma (retrospective PTC) Characterization - Identity/Potency (Strength) CD34% by flow cytometry CFU assay DNA qpcr for transgene copy number Growth Kinetics and Phenotype Liquid Culture Stromal co-culture RCL Testing of cell products DiGiusto et al Sci Trans Med. June

4 rhiv7-shi-tar-ccr5rz CMV RU5 ψ RRE flap WPRE U6 shi U6 TAR VA1 CCR5RZ U3 R U5 5 -GCGGAGACAGCG GACGAAGAGC 3 -UUCGCCUCUGUCGCUGCUUCUCG U G U A U U G G U shi

5 phiv-7 phiv7 Plasmid (WPRE) Serial Dilutions Standard Curves Ct Value WPRE p HI V- 7 p HI V- 7 p HI V- 7 Genomic DNA qpcr Log Copies of DNA p HI V- 7 p HI V- 7 Cellular Reference Gene (ApoB) Ct Value ApoB Log Cell Number qpcr Interpolate copies of WPRE and cell number from standard curves Test Article DNA Copies of WPRE Number of Cells = Avg. Copies/cell

6 Ct values 40 Apo B r 2 = N= Log 10 Cell Number Ct values 40 WPRE r 2 = N= Log 10 Copy Number

7 Ct va alue r² Slope ± Log 10 Copy Number WPRE Copy Number Number of values Geometric mean Lower 95% CI of geo. mean Upper 95% CI of geo. mean

8 Standard Curve Test Article Green- untd Red TD Liquid Blue TD Stromal

9 Standard Curves: R 2 >0.99 (linearity) 1/Slope (efficiency) Melt Curve is ± 1 C (specificity) Each point on curve is within expected 95% CI Inter/Intra-assay and inter-operator variability was insignificant Up to 20,000 cell equivalents of genomic DNA had no effect on the reaction We can detect as few as 5 copies of WPRE in 20,000 cell equivalents (LOD = 0.025%) We can reliably measure 10 copies of (WPRE) DNA (LOQ = 0.05%) Assay limits set using 8 runs according to SOP

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11 Sample MOI Transductions Effective MOI WPRE/ApoB COH COH COH COH COH COH COH COH COH COH COH9 C2 Flask COH10-C1 Wave COH10-C1 Bag COH10-C2 Flask COH10- Non TD D. DiGiusto/B. Levine

12 WPRE ApoB WPRE/ApoB Avg UCPIN008/ ,400 12, UCPIN008/ ,980 11, UCPIN008/ ,650 8, UCPIN050/ ,800 22, UCPIN050/ ,100 22, UCPIN050/ ,400 21, UCPIN015/ ,530 22, UCPIN015/ ,910 21, UCPIN015/ ,100 16, UCPIN0091/ ,660 24, UCPIN0091/ ,150 23, UCPIN0091/ ,570 24, UCPIN106/ ,080 22, UCPIN106/ ,070 20, UCPIN106/ ,310 16, UCPIN108/ ,700 24, UCPIN108/ ,860 21, UCPIN108/ ,450 18, Avg 19,845 SD 4,659

13 A rapid, sensitive RCL test was needed to allow for product release. RCL would likely use VSV-G envelopespike glycoprotein DNA sequences integrated during transduction. VSV-G DNA can be readily detected by qpcr The method would be predictive of RCL but p the sensitivity and reproducibility would have to be established.

14 35 Ct valu ue Copies VSV-G Number of values Mean Lower 95% CI of mean Upper 95% CI of mean

15 Spike Recovery of VSV-G DNA 50 Number of values Mean Std. Deviation Std. Error Lower 95% CI of mean Upper 95% CI of mean Sum Detect ted Cop pies Spiked Copies A. Cao

16 Sample- Assay 1 Assay 2 Assay 3 Average <5 <10 UCPIN008/ N Y UCPIN050/ N N UCPIN015/ Y Y UCPIN091/ N Y UCPIN106/ Y Y UCPIN108/ Y Y

17 qpcr testing for VSV-G DNA is robust, sensitive and reproducible. Assay validation standards include Melt Curve is ± 1 C (specificity) R 2 >0.99 (linearity) 1/Slope (efficiency) Each point on curve is within expected 95% CI Spike Recovery Assay o Not all spiked VSV-G DNA is recovered l bl f o Minimum reliable recovery is 5 copies of VSV-G DNA Requires validation against culture assay.

18 Pre-Clinical development ongoing as part of a Phase I/II Stem cell gene therapy program for HIV.

19 Must replace discontinued cell washing device (Cytomate). HPC-A collections of 2-4 x10 10 MNC need to be washed free of platelets and RBC. A high efficiency yield of CD34+ cells is required to provide >2 x10 6 CD34+ cells/kg pt. weight for transplantation. The process must be robust and The process must be robust and accommodate the downstream production requirements

20 Separation of cells by buoyant density

21 % of cells in fraction N= Elutriation Fraction Platelets RBC WBC CD34- selection C. Tran/M. Coronado/A. Gardner

22 Exp_10_ LMD Live Exp_10_ LMD Live Exp_10_ LMD Live Exp_10_ LMD Live CD34-PE % 0.00% CD34-PE % 9.08% CD3-APC-Alexa % 0.02% % CD3-APC-Alexa 750 CD34-PE % 0.05% % CD3-APC-Alexa 750 CD34-PE % 0.03% % CD3-APC-Alexa 750 Fraction

23 60 Mean = 48.4% 4+ Cells action otal CD34 each Fra % To in e 50 N= Mean = 20.1% F 1 F 2 F 3 F 4 F 5 Elutriation Fraction C. Tran/M. Coronado/A. Gardner

24 Total Number of CD34 Selected All Fractions Before After Fractions Yield Yield (%) Experiment Elutriation Elutriation (%) Selected Fractions Exp Fractions/Debulk Exp Fractions/Debulk Exp F2+F4+F5 Exp F2+F4+F5 Exp F4 + F5 Exp F4 + F5 Exp E F4 + F5 Exp E E2 Product Fraction Average: SD:

25 Elutriation is a useful (closed system) method for upfront processing of HPC-A products Platelets and RBC are efficiently separated from WBC ~70% of CD34+ cells are contained in fractions 4 and 5 CD34 selection of pooled fractions 4/5 results in high h purity of target cells.

26 PCR Assay Development Lijing Li, Annie Cao Anitha Rao Elutriation of HPC-A Chy-Anh Tran Agnes Gardner Monica Coronado Supported by NIAID grant number AI61839 CIRM Disease Team DR