shitar

David DiGiusto Ph.D.
Director-Laboratory for Cellular Medicine
y of Hope/Beckman
p /
Research Institute
City




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,
the identity,
identity
purity, potency and safety of the products.
When products are genetically modified, additional
assays should
h ld b
be d
developed
l
d to assess the
h extent off
genetic modification and justify the methods used
in manufacturing.
g
When viral vectors are used, assays should be
developed to test for the presence of replication
competent virus
virus.

Release Criteria (Safety and Purity)
◦ Viability ≥ 70% (later changed to 5 x 105 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
PCR ffor ttransgene copy number
b
Growth Kinetics and Phenotype
 Liquid Culture
 Stromal co-culture
RCL Testing of cell products
DiGiusto et al Sci Trans Med. June 16 2010
rHIV7-shI-TAR-CCR5RZ
CMV R U5
G
AU
U
5’-GCGGAGACAGCG
5
GACGAAGAGC
3’-UUCGCCUCUGUCGC
CUGCUUCUCG
G
UU
G
U
U6
shI
RRE flap WPRE
ψ
shI
hI
U6
TAR
VA1 CCR5RZ
U3 R U5
Standard Curves
pHIV-7
Serial Dilutions
p
HI
V7
p
HI
V7
WPRE
Ct Value
C
HIV7 Plasmid
Pl
id
pHIV7
(WPRE)
qPCR
p
HI
V7
Log Copies of DNA
Genomic DNA
p
HI
V7
Ct Value
p
HI
V7
Cellular Reference Gene
(ApoB)
ApoB
Log Cell Number
qPCR
Test Article DNA
Interpolate copies of WPRE and cell
number from standard curves
Copies of WPRE
Number of Cells
= Avg. Copies/cell
Apo B
40
40
2
r =0.998
N=5
30
25
20
15
0
r2 =0.999
N=10
35
Ctt values
35
Ctt values
WPRE
30
25
20
1
2
3
Log10 Cell Number
4
15
1
2
3
4
Log10 Copy Number
5
6
35
r²
0.998
Ct va
alue
Slope
-3.18 ± 0.0831
30
25
20
1
2
3
4
5
Log10 Copy Number
Number of values
G
Geometric
t i mean
Lower 95% CI of geo. mean
Upper 95% CI of geo. mean
100000
8
20 6
20.6
20.4
20.7
WPRE Copy Number
10000 1000 100
8
8
8
24
27 3
27.3
30 5
30.5
23.9
27.2
30.3
24.1
27.4
30.7
10
8
33 2
33.2
32.9
33.5
Standard Curve
Test Article
Green- untd
Red – TD Liquid
Blue – TD Stromal

Standard Curves:
◦
◦
◦
◦





R2 >0.99 (linearity)
1/Slope
/ p -0.28 → -0.33 ((efficiency)
y)
Melt Curve is ± 1°C (specificity)
Each point on curve is within expected 95% CI
Inter/Intra-assay
y and inter-operator
p
variability
y 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
(LOD
= 0.025%))
cell equivalents
q
(
We can reliably measure 10 copies of (WPRE) DNA
(LOQ = 0.05%)
Assay limits set using 8 runs according to SOP
Sample
CO
COH7-1
COH7-2
COH7-3
COH7-4
COH7-5
COH7-6
COH7-7
COH7-8
COH7-9
COH7-10
COH9 C2 Flask
COH10-C1 Wave
COH10-C1 Bag
COH10-C2 Flask
COH10- Non TD
MOI
0.1
0.1
0.3
0.3
1
1
3
3
0
0
1
1
1
1
0
Transductions Effective MOI WPRE/ApoB
1
2
1
2
1
2
1
2
0
0
2
2
2
2
0
0.1
0.2
0.3
0.6
1
2
3
6
0
0
2
2
2
2
2
0.15
0.18
0.29
0.41
0.33
0.68
0.58
1.03
0.00
0.00
0.11
0.58
0.62
0.73
0.00
D. DiGiusto/B. Levine
WPRE
UCPIN008/022510
UCPIN008/022510
UCPIN008/022510
UCPIN050/031110
UCPIN050/031110
UCPIN050/031110
UCPIN015/031810
UCPIN015/031810
UCPIN015/031810
UCPIN0091/041110
UCPIN0091/041110
UCPIN0091/041110
UCPIN106/040810
UCPIN106/040810
UCPIN106/040810
UCPIN108/042210
UCPIN108/042210
UCPIN108/042210
Avg
SD
23,400
23
400
22,980
20,650
142,800
134,100
,
117,400
76,530
76,910
64,100
54 660
54,660
49,150
30,570
77,080
80,070
76,310
80,700
77,860
71,450
ApoB
12,480
12
480
11,420
8,614
22,010
22,630
,
21,040
22,930
21,200
16,510
24 490
24,490
23,410
24,630
22,970
20,980
16,880
24,400
21,850
18,760
19 845
19,845
4,659
WPRE/ApoB
1.9
1
9
2.0
2.4
6.5
5.9
5.6
3.3
3.6
3.9
22
2.2
2.1
1.2
3.4
3.8
4.5
3.3
3.6
3.8
Avg
2.1
6.0
3.6
1.9
3.9
3.6




A rapid,
rapid sensitive RCL test was needed to
allow for product release.
RCL would likely use VSV-G
VSV G envelope spike
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.
Ct valu
ue
35
30
25
20
10 1
10 2
10 3
10 4
10 5
Copies VSV-G
Number of values
Mean
Lower 95% CI of mean
Upper 95% CI of mean
100000
9
19.3
19.0
19.6
10000
9
22.7
22.3
23.1
1000
9
25.9
25.4
26.4
100
9
29.2
28.6
29.8
10
9
32.5
31.5
33.5
Spike Recovery of VSV-G DNA
9
Mean
Std. Deviation
Std. Error
3.59
0.780
0.260
6.84
1.13
0.376
35.4
4.46
1.49
Lower 95% CI of mean
Upper 95% CI of mean
2.99
4.19
5.97
7.70
32.0
38.9
Sum
32.3
61.5
319
40
30
20
15
10
5
0
50
9
50
9
10
Number of values
10
5
5
Detectted Cop
pies
50
S ik d Copies
Spiked
C i
A. Cao
SampleAssay 1 Assay 2 Assay 3 Average
UCPIN008/022510
51
5.1
40
4.0
46
4.6
UCPIN050/031110
33.3
28.1
30.7
UCPIN015/031810
3.0
2.4
2.7
UCPIN091/040110
45
4.5
36
3.6
40
4.0
UCPIN106/040810
1.5
1.5
UCPIN108/042210
0.7
0.7
<5
N
N
Y
N
Y
Y
<10
Y
N
Y
Y
Y
Y


qPCR testing for VSV-G DNA is robust, sensitive
and reproducible.
Assay validation standards include
◦
◦
◦
◦

Melt Curve is ± 1°C (specificity)
R2 >0.99
>0 99 (linearity)
1/Slope -0.28 → -0.33 (efficiency)
Each point on curve is within expected 95% CI
Spike Recovery Assay
o
o
Not all spiked VSV-G DNA is recovered
l bl recovery is 5 copies off VSV-G DNA
Minimum reliable
 Requires
validation against culture assay.
Pre-Clinical development ongoing
as part of a Phase I/II Stem cell
gene therapy program for HIV.




Must replace discontinued cell washing device
(Cytomate).
(Cytomate)
HPC-A collections of 2-4 x1010 MNC need to
be washed free of platelets and RBC.
RBC
A high efficiency yield of CD34+ cells is
required to provide >2 x106 CD34+ cells/kg
pt. weight for transplantation.
The process must be robust and
accommodate the downstream production
requirements
Separation of cells by buoyant density
% of cells in
n fraction
n
100
N 3
N=3
75
Platelets
RBC
WBC
50
25
CD34CD34
selection
0
1
2
3
4
5
Elutriation Fraction
C. Tran/M. Coronado/A. Gardner
4
0.00% 10 0.62%
3
10
C D 34-P E
CD34-PE
1.68%
2
10
1
10
0
0.02%
3
10
2
10
1
10
89.24%
9.08%
10
0
1
2
3
4
10 10 10 10 10
CD3-APC-Alexa 750
Fraction 2
0
Exp_10_0047 015.LMD
Live
Exp_10_0047 011.LMD
Live
C D 34-P E
4
10
Exp_10_0047 007.LMD
Live
4
10
1.46%
0.05%
3
10
C D 34-P E
Exp_10_0047 003.LMD
Live
2
10
1
66.89
10
0
1
2
3
4
10 10 10 10 10
CD3-APC-Alexa 750
3
0
5.71%
0.03%
68.44%
25.81
3
10
2
10
1
10
10
32.47%
4
10
23.37%
75.12
10
0
1
2
3
4
10 10 10 10 10
CD3-APC-Alexa 750
4
0
10
0
1
2
3
4
10 10 10 10 10
CD3-APC-Alexa 750
5
% To
otal CD34
4+ Cells
in e
each Fra
action
60
50
M ean = 48.4%
N=7
40
30
M ean = 20.1%
20
10
0
F1
F2
F3
F4
F5
Elutriation Fraction
C. Tran/M. Coronado/A. Gardner
Experiment
Exp‐10‐0082
Exp 10 0084
Exp‐10‐0084
Exp‐10‐0085
Exp‐10‐0086
Exp‐10‐0089
Exp‐10‐0090
Exp‐10‐0101 E1
Exp‐10‐0101 E2
g
Average:
SD:
Total Number of CD34 Total
Number of CD34
Selected
Selected All Fractions ll
Fractions Yield Before After Yield (%)
(%)
Elutriation Elutriation
227
214
94.4
94.4
80 8
80.8
72 1
72.1
89 2
89.2
89 2
89.2
284
200
70.5
59.32
233
176
75.5
71.5
282
153
54.3
50.9
149
143
96
93.3
251
189
75.1
74.1
278
265
95.2
94
223.1
176.5
81.3
78.3
72.5
56.7
14.9
17.0
Selected Fractions
Fractions/Debulk
Fractions/Debulk
Fractions/Debulk
F2+F4+F5
F2+F4+F5
F4 + F5
F4 + F5
F4 + F5
E2 Product Fraction




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
l in h
high
h purity off target cells.
ll
PCR Assay Development
Lijing
Liji Li,
Li
Annie Cao
Anitha Rao
Supported by
NIAID grant number – AI61839
CIRM Disease Team – DR1-01490
Elutriation of HPC-A
Chy-Anh Tran
Agnes Gardner
M
i C
d
Monica
Coronado