Multiparametric Approach
for Defining Product
Quality
Dr Damian Marshall
Bioprocessing Summit
August 2016
The Cell and Gene Therapy Catapult
£70m Development Facility
•
1,200m2 Custom designed cell and gene
therapy development facility
•
Prime location in the heart of the London
clinical research cluster
•
120 permanent staff
£55m large scale manufacture center
•
7,200m2 manufacturing centre designed
specifically for cell and gene therapies
•
Located in the Stevenage biocatalyst
•
Opening 2017
The Cell and Gene Therapy Catapult
Therapy industrialisation
Process development
Analytical development
GMP process proving
Supply chain
Late clinical phase manufacturing
Business
Business development
Business models
Health economics
Clinical trial and regulatory
Regulatory
Clinical trial sponsor
Clinical operations
Pre-clinical safety
Product Characterisation
Product characterisation
Why characterise cell and gene therapy products ?
• Product characterisation demonstrates control of the manufacturing process
• Establishing specifications during manufacture helps ensure the quality and
lot-to-lot consistency of the final product
• Product parameters can be used to anticipate sub-optimal manufacture runs
• Characterisation can help assess product integrity and stability
Product characterisation challenges
What are the relevant parameters to measure?
Can cell quality be measured and monitored? What is cell quality?
Impact of the manufacturing process on cells – what is affected?
Analytical Inputs
Metabolomics
Protein expression
Cell count
Viability
Material
Characterisation
Morphology
Raw Materials
Process Outputs
Purity
Product knowledge
Purity
Gene expression
Surface markers
Identity
Sterility testing
Genomic stability
Flexible
manufacture
Sterility
Downstream
processing
Upstream processing
Manufacture
design space
Potency
Process
Control
Product
Consistency
Product characterisation challenges
What are the relevant parameters to measure?
Can cell quality be measured and monitored? What is cell quality?
Analytical Inputs
Impact of the manufacturing process on cells – what is affected?
Cell count
Viability
Raw Materials
Process Outputs
Cell count
Variable starting
material
Identity
Viability
Upstream processing
Sub-optimal
processing
Limited control
Sterility
Downstream
processing
High product
variability
The CGT Analytical Platform
iPS cell manufacture
Development of an automated iPS cell expansion process
• iPS cell expansion maintained in adherent culture for 40 days
• Cells undergo approximately 20-25 population doublings
Challenge – Development of an IPC framework which can monitor cell quality
and predict process success in manual and automated systems
Media
•
•
Metabolites
GF/Cytokine analysis
Cells
•
Morphology / Confluency
Passage
1
Passage
2
Passage
3
Cells
•
•
•
Cell enumeration / viability
Protein expression
Gene expression
Passage
4
Passage
5
……… Passage
10
Analytical Platform
Analytical Design
• QbD Analysis
• Analytical strategy
based on:
• Key unit operations
• Sample Availability
• Technology suitability
Quality by Design
• The cell and gene therapy catapult uses a quality by
design (QbD) approach to establish the appropriate
analytical strategy for a product
• QbD is a risk based framework for process design
which relates product and process attributes to
product quality
• The approach is based on knowledge of product biology and the
engineering used during its manufacture
• By establishing the critical quality attributes (biological, physical,
biochemical) for a product a design space can be established which
defines the expected range of each characteristic during manufacture
QbD is usually guided by the quality of the final product……..
QbD Toolkit
Analytical Design
Equipment
Rejected Product
• BSD
C Consumable
1
1
1
• P-Diagram
N
1
3
1
Cell
1 Product
1
1
1
1
1
1
1
1
1
1
Q
1
1
Risk
vsDonor
Assay or
data
COGS
1
required
Quality Attributes (and weightings)
Score
1
1
1
17
17
1
1
1
1
1
1
29
29
1
1
1
1
1
1
40
40
Quality Attribute 11
1
1
Quality Attribute 10
1
1
Quality Attribute 7
1
1
Quality Attribute 8
1
1
1
1
1
1
1
1
1
1
1
1
1
Process Operation 1
Input 1
C
1
1
1
1
1
1
1
1
1
1
1
1
Process Operation 1
Input 2
N
1
3
1
1
9
1
3
1
1
1
1
Process Operation 1
Input 3
X
1
1
9
9
9
1
1
1
Q
1
1
Process Operation Process Parameters C/N/X
Day 0
10 minutes
Sample
Assay or Donor
data
9
Weighted Score
9
Quality Attribute 9
9
Batch Completion
1
Quality Attribute 6
1
Quality Attribute 15
X
Quality Attribute 3
Input 3
1
1
Name the step in
bold
Put in detail
Decision
Yes
No
Rejected Product
Final Product
Weighted Score
Score
Quality Attribute 16
Quality Attribute 15
Quality Attribute 14
Quality Attribute 13
Quality Attribute 12
Quality Attribute 11
Quality Attribute 10
Quality Attribute 9
Quality Attribute 8
Batch Completion
Quality Attribute 6
Quality Attribute 7
1
• FMEA
Waste
1 • 1 COGS
1
1
1
1
Add
options
subprocesses
if
Sample
9
1
3
1
1
1
bold
Put in detail
Day 0
Input
1
120
minutes
Input 2
Template100x04
24/02/2015
24/02/2015
1Name1 the step
1 in1
Raw Material
Final Product
Quality Attribute 5
Quality Attribute 4
Process
Cell Product
Dissection
1
1
1
Quality Attribute 16
Yes
Analytical Lab
(Unclassified)
Quality Attribute 14
No
Analytical Lab
(Unclassified)
Quality Attribute 12
Decision
Sample
Assay or Donor
data
Equipment
Quality Attribute 4
Assay or Donor
data
Autoclave
(Unclassified)
Quality Attribute 5
• Gap
Analysis
Process Operation 1
• Analytical
Process Operation 1
Transfer
Process Operation 1
Process
Transfer
Tissue Culture
(Grade B)
Waste
Quality Attribute 13
Name the step in
bold
Put in detail
Cell Product
Quality Attribute 1
Step 1.2
Add
subprocesses if
required
Quality Attribute 2
Sample
Name the step in
bold
Put in detail
Step 1.1
Day 0
10 minutes
Step 1.1
Consumable
Step 1.2
Cell Product
Raw Material
Day 0
120 minutes
Template100x04
24/02/2015
24/02/2015
Project Code:
Last Modified:
Printed:
Quality Attribute 3
Project Code:
Last Modified:
Printed:
Quality Attribute 1
Process Operation Process Parameters C/N/X
Quality Attribute 2
Quality Attributes (and weightings)
Development
Equipment
1
1 Plan
1
Tissue Culture
1
1B)
(Grade
Autoclave
(Unclassified)
•1 Priority
1
1
1 Grid
1
17
17
Analytical Lab
•1 Implementation
(Unclassified)
1
1
1
1
29 29
Plan
1
1
Analytical Lab
(Unclassified)
1
1
1
40
Equipment
40
Analytical Platform
Analytical testing
• Screening strategy
• Integrated analysis
• Augment Information
• Stress testing
Image analysis
Analytical testing
Cell confluence tracking using quantitative imaging
Day 1
•
•
Day 4
confluence
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
Day 3
100%
100% 90%
90% 80%
80%
70% 70%
60% 60%
50%
50%
40%
30% 40%
20% 30%
10%
20%
0%
10%
0%
confluence
Day 2
3 independent biological replicates
Cells seeded at the same cell density
P1
1
P2
P3
P4
2
3
days
P5
4
P6
Texture analysis
Analytical testing
Texture of colonies
Day 1
PC2
day 4
Day 2
day 2
day 1
PC1
Day 3
Day 4
day 3
Flow cytometry analysis
Passage 2
FUNCTION
Stress
Stress
Stress
Stress
Stress
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Pluripotency
Ecotoderm
Ecotoderm
Ecotoderm
Ecotoderm
Ecotoderm
Mesoderm
Mesoderm
Mesoderm
Mesoderm
Mesoderm
Mesoderm
Mesoderm
Endoderm
Endoderm
Endoderm
Differentiation
Differentiation
Differentiation
Differentiation
Differentiation
Differentiation
Differentiation
Differentiation
Differentiation
Passage 4
Passage 6
Passage 8
Analytical testing
Passage 10
R1
R2
R3
R1
R2
R3
R1
R2
R3
R1
R2
R3
R1
R2
R3
0
0.11
0.04
0.1
0.17
95.6
99.2
99.9
5.29
7.6
96.9
70
37.8
9.87
66.2
79.8
63.3
1.08
20.1
20.5
1.69
96
99.9
1.26
4.11
0.28
0.13
1.47
1.01
0.83
0.22
75.3
3.9
2.04
2.7
15.4
0.05
0.68
0.58
0.16
2.28
7.29
0
0.03
0.06
0.06
0.09
96.4
99
99.9
5.25
5.75
97.5
77.7
47.2
14.3
65.2
83.9
63.2
1.19
76.6
98.3
85
87.1
99.2
0.42
1.91
0.37
0.11
1.29
2.53
0.8
0.13
83.1
10.7
2.89
0.65
14.5
0.06
0.78
0.52
0.24
3.64
1.71
0
0.18
0.03
0.07
0.08
95.5
98.4
99.4
5.55
4.79
97.9
72
42.3
12.2
62.4
83.5
55.6
0.93
85.8
98.8
91.2
84
99.4
1.49
2.56
0.23
0.26
1.16
0.69
0.69
0.36
78.6
6.32
3.17
0.76
17.1
0.04
0.43
0.54
0.31
3.92
0.47
0.11
0.11
0.17
0.15
0.19
86.4
99.8
100
3.8
45.3
90
85.9
50.8
20.2
60.3
97.8
95.8
0.97
93.6
95.3
62.7
12.3
75.2
0.47
14.9
3.94
0.22
2.57
0.66
0.93
0.24
73.1
2.16
7.02
1.18
2.28
0.13
1.17
0.21
0.11
3.22
0.12
0.09
0.22
0.13
0.36
0.19
86.2
99.6
99.9
2.87
31.4
88
86.7
35.6
19.4
57
97.2
96.1
0.92
85.7
91.2
35.6
10.3
70.2
0.62
12.4
3.27
0.25
1.87
0.55
0.97
0.37
67.2
1.43
7.36
0.76
2.27
0.14
1.05
0.23
0.13
3.71
0.17
0.12
0.18
0.13
0.33
0.13
85.4
99.8
100
2.92
33
87.6
85.6
39.8
20.8
61.1
96.7
97.6
0.93
11.7
81.2
14.5
7.6
74.4
0.54
11.6
2.84
0.11
1.5
0.84
0.72
0.27
70
0.5
7.43
1.09
1.14
0.14
0.81
0.19
0.08
2.85
0.18
0.13
0.21
0.37
0.23
0.35
72.4
99.5
100
1.03
74.8
96.1
92
35.6
61
76.3
95
96.7
1.07
75.5
85.5
71.7
7.13
99.8
1.9
11.1
2.91
0.17
1.61
0.92
0.53
0.65
28
0.28
6.18
0.71
18.9
0.01
0.72
0.19
0.1
7.91
1.81
0.08
0.13
0.58
0.28
0.28
75.8
99.5
99.9
1.29
80.8
96.5
91.8
43.5
59.7
79.1
95.4
95.8
2.36
74.4
81.8
64.8
21.9
98.1
9.39
13.3
4.9
0.25
1.95
1.14
0.47
0.75
42.4
0.47
8.99
0.72
27.9
0
0.77
0.23
0.11
8.27
2.15
0.05
0.06
0.37
0.17
0.1
75.9
99.7
99.9
0.94
86.8
96.8
93.6
47.4
61.4
78.4
96.5
96.9
1.4
75.3
82.9
42.3
17
98.4
1.81
11.3
6.29
0.19
1.31
1.3
0.46
0.36
26.1
0.44
9.97
0.74
26.9
0
0.94
0.07
0.11
6.88
0.99
0
0.73
0.01
2.96
0.93
86.2
98.9
99.5
5.66
43.3
98.8
67.3
35.7
57.1
36.1
92.4
53.4
0.88
84.7
89.2
90.3
51.8
97.8
43.2
10.4
0.75
0.18
0.46
4.2
0.3
0.53
1.43
5.37
4.97
3.67
92.7
0
0.31
8.27
2.43
15.7
89.1
0
2.2
0.08
3.64
2.34
89.3
98.4
99.6
5.87
40.5
97.9
66.6
36.9
63.1
44.4
89.3
58
0.83
80.1
83.9
83.3
51.5
96.8
67.4
9.47
0.95
0.21
0.47
4.63
0.28
0.57
1.13
5.94
4.86
4.5
92.3
0
0.26
7.79
5.23
24.3
89.3
0
1.38
0.03
2.09
1.79
83.4
98.4
99.6
2.74
46.7
99
68.5
27.2
63.2
33.5
86.1
43
0.54
82.6
83.1
82.9
47.4
96.6
65.1
19.7
0.82
0.19
0.49
2.79
0.31
0.41
1.52
2.15
4.65
5.62
91.9
0
0.44
13.9
1.98
29
91.7
0.01
0.48
0.02
10.2
1.06
79.6
97.9
99.8
2.43
31
88
69.5
19.9
48.9
43.6
81.5
10.3
1.17
92.5
95.1
67.1
42.1
99.5
33.9
10.7
0.58
0.16
3.82
2.87
0.64
1.94
1.29
6.38
7.03
7.87
90.7
0
0.3
15.1
12.9
12.6
56
0.06
0.33
0.02
11.4
0.94
76.9
98.6
99.8
2.38
20.7
86.1
67.4
25.6
46
44.7
79.6
9.74
1.28
92.6
94.7
61.2
34.9
99.9
23.3
5.1
1.87
0.22
2.31
1.72
0.59
15.4
0.55
4.19
7.9
16.6
82.8
0
0.26
9.7
8.59
10.8
55.2
0.04
0.33
0
13.9
1.22
80.1
98.3
99.7
2.24
26.1
84.1
69.3
22.8
42
44.8
74.4
12.2
2.28
93.2
94.8
57.1
21.2
99.9
37.6
7.49
1.07
0.19
3.78
3.53
0.73
7.09
2.19
4.93
7.44
4.89
67.7
0
0.24
11.7
13.8
6.15
58.5
endoderm
undifferentiated
mesoderm
ectoderm
P1
P10
P5
RT-qPCR analysis
Analytical testing
• Commercial screening technologies can be hard to interpret
• Reference samples may not be fit for purpose
• Established in house panels for ES and iPS cell manufacture
• Panel based on pluripotency, self renewal, differentiation and stress
markers
Undifferentiated
Endoderm
Ectoderm
Mesoderm
Mesoderm
iPS
Endoderm
Ectoderm
P1
P5
P10
P1
P5
P10
P1
P5
P10
P1
P5
P10
Extended metabolic screen
• Use of LC/MS to screen for
metabolic profile
• Wide range screen
• Automated sample
preparation and analysis
Analytical testing
Extended metabolic screen
Analytical testing
Analytical Platform
Biological Profiling
• Cluster Analysis
• Data Reduction
• Multivariate Analysis
Data integration
Biological Profiling
Multi-parametric data structure
183 parameters in total, 159 time points/conditions
live quantitative imaging
flow cytometry
gene expression
FLOW CYTOMETRY
10 passages (undifferentiated)
P0
MARKERS
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
day 1
day 2
day 3
day 4
R1
R2
R3
P1
R1
R2
R3
P2
R1
R2
R3
P3
R1
R2
R3
P4
R1
R2
R3
P5
10 Passages
R1
R2
R3
P6
R1
R2
R3
P7
R1
R2
R3
P8
R1
R2
R3
P9
R1
R2
R3
P 10
R1
R2
R3
Differentiation experiments
MESO
P0
R1
R2
R3
P5
R1
R2
R3
P 10
R1
R2
R3
differentiation
ENDO
P0
R1
R2
R3
P5
R1
R2
R3
P 10
R1
R2
R3
ECTO
P0
R1
R2
R3
P5
R1
R2
R3
P 10
R1
R2
R3
SCORECARDS
LIVE QUANTITATIVE IMAGING
CONTOURS
MASK
SLOPE
SLOPE
FRACTAL
FRACTAL
CONFLUENCE
MASK EROSION
TEXTURE PARAMETERS
cell counters
metabolites
VICELL, NUCLEOCOUNTER
METABOLITES (cubian)
Weight Network Expression Analysis
157 x 157 topological overlap matrix (TOM)
183 parameters in total,
159 time points/conditions
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
internetwork
connectivity
157 parameters filtered for network analysis
(Topological Overlap Matrix – TOM)
Modules = networks:
parameters with similar
expression patterns across
conditions, suggesting functional
relationships.
Connectivity:
• Intra-network = clusters along
diagonal
• Inter-network = red clusters
away from diagonal
Connectivity
high
modules
(networks)
low
Within a particular module, similar AND different types of markers
are mixed up and connected
Data reduction
Biological Profiling
The least connected markers are removed to leave a low resolution set of markers which
has the same overall connectivity as the full original dataset
original TOM
157 markers
Texture / morphology = 17
Metabolites = 8
Cell paramerters= 10
Genes = 80
Membrane markers = 42
reduced TOM
53 markers
30 markers
Data reduction
Conservation of information
Relationships between markers as well as
network connectivity is maintained
17 markers
Texture / morphology = 1
Metabolites = 3
Cell count= 1
Genes = 6
Membrane markers = 6
Inferential markers
Analytical Platform
Customised QC
• Inferential analysis
• Reference materials
• Automated analysis
Network Analysis –surrogate markers
• Potential inferential markers can be
identified based on their connectivity
with other markers.
Morphology
surface markers
• By looking for strong overlap
between modules it is possible to
identify coordinated expression
patterns – consistent throughout the
production process
• This allows the expression of one
marker to be used to indicate the
expression of others
surface markers,
genes, metabolites,
cell counts,
confluence
Inferential markers application
Soluble markers
RNA’s
CD markers
fail
marker expression
pass
time
end product
Surrogate markers application
Soluble markers
RNA’s
CD markers
marker expression
pass
time
end product
Summary
Analytical platform at the CGT
Build quality in the process by
Using a systematic approach to understanding product quality and
define what characteristics to measure in order to…..
• Allow control of the manufacturing process
• Support process optimisation to ensure the quality and lot-tolot consistency of the final product
• Have the analytical capacity to understand and control (or
terminate) the manufacture process
Summary – CGT analytical platform
Moving QC into the process
Providing the analytical platform for targeted characterisation to ensure that a
product is of intended quality, based on information collected during the
manufacturing process
Analytical Inputs
Metabolomics
Cell count
Cell
count
Viability
Material
Characterisation
Viability
Morphology
Process Outputs
Raw Materials
Product knowledge
Purity
Protein expression
Purity
Metabolites
Gene
expression
Surface markers
Identity
Sterility testing
Genomic stability
Flexible
manufacture
Sterility
Downstream
processing
Upstream processing
Manufacture
design space
Potency
Process
Control
Product
Consistency
“Helping cell and gene therapy
researchers and companies across the
world translate their research into
commercially viable and investable
therapies that are safe, effective,
scalable and affordable.”