Transcript In vitro - Drug Information Association

Establishment of a Comparability
Strategy to Support a Cell Line
Change During Clinical Development
of a Monoclonal Antibody
Bryan J. Harmon
www.diahome.org
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Outline
• Drivers for Cell Line Changes
• Elements of Comparability Strategy
• Case Studies
• Conclusions
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Cell Line Changes During Clinical Development
Driver
Examples
Quality risk with
initial cell line
• Genetic splicing or mutation identified
• ASM exposure during cell line
generation
• Lack of assurance of clonality
Initial cell line is
not commercially
viable
• Insufficient titer
• Insufficient cell line stability
• Not consistent with manufacturing
platform
• Intellectual property issues
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Types of Cell Line Changes
• Additional round of cloning
• Different clone from same host cell line
• Different host cell line
Cell line changes:
• Are considered the biggest risk among process changes
• Have been practiced very conservatively in the industry
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Elements of Integrated Comparability Strategy
1. Host cell line & clone selection
criteria
2. Analytical comparability
testing strategy
3. In vitro biological testing
4. Nonclinical PK, PD &
immunogenicity assessments
5. Clinical assessments
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Need for &
extent of each
element driven
by risk
assessments
Risk Assessments – FMEA Analysis
1. Severity – impact on toxicity, safety, efficacy or PK/PD
2. Occurrence – likelihood of being outside preclinical &
clinical experience (process capability, control &
robustness)
3. Detection – capability of analytical methods to detect
occurrence
Risk Rating = Severity x Occurrence x Detection
Risk assessments must be cross-functional
(toxicology, medical, analytical, process scientists)
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Host Cell Line & Clone Selection Criteria
In evaluating risk of cell line change, must consider:
• Post-translational modification capabilities of
potential new host cell line
• Clonal variability of chosen host cell line in
product quality attributes
• Capability to mitigate comparability risks through
process development/optimization
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Impact of Host Cell Line on Glycosylation
CHO & NS0
G0F
NS0 Only
G0
G1F
G0F1GlcNAc
G2F
Man-5
“Human-like” structures
GlcNAc
Man
G2F+2(1-3)Gal
G2F+NeuGc
Fuc
G1F+NeuGc
G2F+(1-3)Gal
G2F+(1-3)Gal
+NeuGc
G1F+(1-3)Gal
“Non-human” structures: (1-3)Gal & NeuGc
(1-4)Gal
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(1-3)Gal
NeuGc
Impact of Host Cell Line on Glycosylation
G0F
CHO-Derived IgG4
RFU
G1F
G2F
Man-5
G0
NS0-Derived IgG4
RFU
CE-LIF
Oligosaccharide
Profiling
20
25
30
Migration Time (min)
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35
40
Risks of Cell Line Changes
• Different host cell line
• Different clone from same
host cell line
• Additional round of cloning
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Increasing
risk to
CQAs of
molecule
Host Cell Line & Clone Selection Criteria
In evaluating risk of cell line change, must consider:
• Post-translational modification capabilities of
potential new host cell line
• Clonal variability of chosen host cell line in
product quality attributes
• Capability to mitigate comparability risks through
process development/optimization
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Clonal Variability in Glycosylation
Fc Glycosylation of
CHO-derived IgG1
0.6
Gal/Glycan
0.5
0.4
0.3
0.2
0.1
0.0
0.84
0.86
0.88
0.90
Fuc/Glycan
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0.92
0.94
0.96
Clonal Variability in Glycosylation
Fab Glycosylation of
CHO-derived IgG1 with
2 Glycosylation Sites
1.6
1.5
Gal/Glycan
1.4
1.3
1.2
1.1
1.0
0.9
0.8
0.7
0.6
0.00
0.20
0.40
0.60
0.80
NeuAc/Glycan
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1.00
1.20
Comparability Risk Mitigation During Clone Selection
Greater emphasis on product quality parameters that are:
•
•
•
Enzymatic processes that are likely to be clone specific: e.g., glycosylation,
proteolytic clipping
Genetic issues: e.g., mutations, frame shifts, splices
Critical to the biological activity of the mAb: e.g.,
– ADCC → Fucosylation
– CDC → Galactosylation
Lesser emphasis on product quality parameters that are:
•
Optimized through purification process development; e.g., host cell protein,
aggregation
– Caveat: aggregation could be an indicator of other issues (e.g., splicing, disulfide
reduction)
•
Chemical mechanisms that are less likely to be clone specific; e.g., oxidation,
deamidation, glycation
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Host Cell Line & Clone Selection Criteria
In evaluating risk of cell line change, must consider:
• Post-translational modification capabilities of
potential new host cell line
• Clonal variability of chosen host cell line in
product quality attributes
• Capability to mitigate comparability risks through
process development/optimization
www.diahome.org
Physico-Chemical Comparability Testing
Assess impact of cell line change on CQAs of MAb based upon risk assessment of
quality attributes
•
Additional testing to satisfy regulatory concerns; e.g.,
–
•
Glycosylation analysis for MAb whose MOA is not dependent upon effector function
Co-mixture analysis of representative lots where appropriate (e.g., LC-MS
peptide mapping, SEC, CEX, CE-SDS)
Assessment of impact on degradation mechanisms (e.g., stressed or
accelerated stability study)
Pre-defined acceptance criteria:
•
•
–
At early stages of development:
•
•
–
Insufficient data to establish statistical limits tighter than specifications at early stages of
development
Qualitative criteria for characterization assays
Allowance for investigative testing (e.g., source of differences in charge
heterogeneity)
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CQA Risk Assessments
Pyroglutamation of HC Gln1
22
96
105
96
146
105
146
202
22
Oxidation of LC Met32
202
23
23
87
133
222
193
213
228 228
231
213
231
263
263
323
323
369
369
427
427
Cleavage at LC Asn93/Pro94
87
133
222
193
HC His226/Thr227 (hinge) cleavage
Oxidation of HC Met252
Glycosylation of HC Asn299
Quality Attribute
Oxidation of Fc Met sites
Deamidation of Fc Asn sites
Free thiol (incomplete disulfide)
Loss of HC Lys449
Glycation of Fab & Fc lysines & N-termini
Aggregation
Documented impact on
toxicity, safety, efficacy,
PK/PD or immunogenicity?
No
Yes
Yes
Impact on in vitro
bioactivity?
Located in region likely to
impact activity?
No
No
No
Platform modification?
Yes
Impact on in vivo toxicity,
safety, efficacy, PK/PD or
immunogenicity?
Yes or unknown
No
Yes
Non-CQA
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CQA
Physico-Chemical Comparability Testing
Assess impact of cell line change on CQAs of MAb based upon risk assessment of
quality attributes
•
Additional testing to satisfy regulatory concerns; e.g.,
–
•
Glycosylation analysis for MAb whose MOA is not dependent upon effector function
Co-mixture analysis of representative lots where appropriate (e.g., LC-MS
peptide mapping, SEC, CEX, CE-SDS)
Assessment of impact on degradation mechanisms (e.g., stressed or
accelerated stability study)
Pre-defined acceptance criteria:
•
•
–
At early stages of development:
•
•
–
Insufficient data to establish statistical limits tighter than specifications at early stages of
development
Qualitative criteria for characterization assays
Allowance for investigative testing (e.g., source of differences in charge
heterogeneity)
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Typical Physico-Chemical Comparability Tests
Release Tests
Characterization Tests
Potency/Biological Activity
Bioassay
Surface plasmon resonance
Structural Integrity (Primary, Secondary & Tertiary)
Intact LC-MS
Partial reduction LC-MS
LC-MS peptide mapping*
Far & near UV circular dichroism
Free thiol analysis
Calorimetry**
Molecular Heterogeneity
Cation-exchange chromatography*
Oligosaccharide profiling
Product-Related Impurities
Size-exclusion chromatography*
Analytical ultracentrifugation**
* Include co-mixture
analysis of
representative lots
Non-reduced CE-SDS*
Reduced CE-SDS*
Process-Related Impurities
Host cell protein
Triton X-100
DNA
Insulin
Protein A
MSX
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** Added based upon
regulatory feedback
Physico-Chemical Comparability Testing
Assess impact of cell line change on CQAs of MAb based upon risk assessment of
quality attributes
•
Additional testing to satisfy regulatory concerns; e.g.,
–
•
Glycosylation analysis for MAb whose MOA is not dependent upon effector function
Co-mixture analysis of representative lots where appropriate (e.g., LC-MS
peptide mapping, SEC, CEX, CE-SDS)
Assessment of impact on degradation mechanisms (e.g., stressed or
accelerated stability study)
Pre-defined acceptance criteria:
•
•
–
At early stages of development:
•
•
–
Insufficient data to establish statistical limits tighter than specifications at early stages of
development
Qualitative criteria for characterization assays
Allowance for investigative testing (e.g., source of differences in charge
heterogeneity)
www.diahome.org
Case Study #1
Property
MAb1
Isotype
IgG4
Phase of Development
Cell Line Change
Pre-Phase 2
GS-NS0 to GS-CHO-K1SV
MOA Dependent upon Effector
Function?
Drivers for Cell Line Change
No
Elimination of non-human glycoforms
Alignment with platform
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Case Study #1
Prior Knowledge:
•
Experience in GS-NS0 to GS-CHO-K1SV cell line changes suggested risk of:
–
Changes in glycosylation profile
–
Changes in charge heterogeneity resulting from differences in proportions of charge variants
Risk Assessment:
•
Expected differences presented low risk to the safety and efficacy of molecule
Comparability Strategy:
•
Extraordinary efforts would not be made in clone selection and process development to
eliminate these differences
•
Demonstrate comparability through:
–
Physico-chemical testing
–
In vitro biological assays
–
Non-clinical in vivo PK, PD and immunogenicity studies
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Case Study #1
Prior Knowledge: GS-NS0 to GS-CHO-K1SV Cell Line Change
Cation-Exchange Chromatography
Incomplete pyroglutamate
One HC
Both HC
GS-CHO-K1SVDerived IgG4
GS-NS0-Derived IgG4
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Case Study #1
Prior Knowledge:
•
Experience in GS-NS0 to GS-CHO-K1SV cell line changes suggested risk of:
–
Changes in glycosylation profile
–
Changes in charge heterogeneity resulting from differences in proportions of charge variants
Risk Assessment:
•
Expected differences presented low risk to the safety and efficacy of molecule
Comparability Strategy:
•
Extraordinary efforts would not be made in clone selection and process development to
eliminate these differences
•
Demonstrate comparability through:
–
Physico-chemical testing
–
In vitro biological assays
–
Non-clinical in vivo PK, PD and immunogenicity studies
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Case Study #1
Differences in glycosylation profiles were observed:
Glycoforms
GS-NS0-Derived
MAb1
Batch 1
Batch 2
2.0%
2.4%
GS-CHO-K1SVDerived MAb1
Batch 1
Batch 2
Non-human glycoforms
-Gal-containing
Not observed
NeuGc-containing
2.8%
2.4%
40.8%
40.9%
Human glycoforms
-Gal-containing
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26.9%
27.2%
Project Name:
21.6 min
mV
Case Study #1
KY743B_LA294_2008
25.0 min
28.5 min
19.2 min
20.4 min
22.7 min
(a)
27.4 min
90.00
26.1 min
Ronald L. Kow le (c051710)
24.0 min
Reported by User:
10.00
90.00
(b)
mV
CHO-derived MAb1
10.00
90.00
(c)
Co-mixture
mV
Differences in charge
heterogeneity profiles
were observed:
10.00
90.00
(d)
mV
NS0-derived MAb1
10.00
90.00
mV
(e)
LC-MS characterization of isolated
CEX fractions identified small
differences in proportions of typical sources of MAb charge variants:
10.00
12.00
14.00
16.00
18.00
– Heavy chain N-terminal10.00pyroglutamate
– Heavy chain C-terminal lysine
20.00
22.00
24.00
Minutes
26.00
28.00
SampleName CHO GMP 1month 2-8; Lot PP0113-08
SampleName CHO TOX 1month 2-8; Lot 16673-47
SampleName C1 (Co-mix); Lot 15648-139-C1
SampleName NS0 TOX 1month 2-8; Lot 14278-117
SampleName NS0 GMP 1month 2-8; Lot 04-0466
– Heavy chain C-terminal desGly/amidation
– Glycation
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30.00
32.00
34.00
Case Study #1 - Summary
Physico-Chemical Testing:
•
No apparent adverse impact observed in structural integrity, product-related impurities or
process-related impurities
•
Minor differences observed in molecular heterogeneity
–
Glycosylation
–
Charge heterogeneity
In vitro Biological Assays
•
No apparent differences observed in potency
Nonclinical PK, PD & Immunogenicity Assessment
•
No apparent differences observed
The cell line change presents low risk
to the safety or efficacy of MAb1
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Case Study #2
Property
MAb2
Isotype
IgG1
Phase of Development
Cell Line Change
Pre-Phase 2
DHFR-CHO-DG44 to GS-CHO-K1SV
MOA Dependent upon Effector
Function?
Drivers for Cell Line Change
Yes
Alignment with platform
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Case Study #2
Prior Knowledge:
•
No experience in DHFR-CHO-DG44 to GS-CHO-K1SV cell line changes
•
Knowledge of clonal variability suggested risk of changes in glycosylation profile:
– Core fucosylation → impact ADCC activity
– Terminal -galactose → impact CDC activity
Risk Assessment:
•
Changes in glycosylation could present significant risk to the safety and efficacy of
molecule
Comparability Strategy:
•
Glycosylation as criterion for clone selection to mitigate comparability risk
–
•
Fucosylation prioritized based upon proposed MOA
Demonstrate comparability through:
–
Physico-chemical testing
–
In vitro biological assays (including ADCC & CDC)
–
Non-clinical in vivo PK, PD & immunogenicity studies
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Case Study #2
Impact of Fucosylation on ADCC Activity of MAb2
Relative ADCC Activity
300
250
200
150
100
50
0
0.86
0.88
0.90
0.92
0.94
0.96
Fucosylation (Fuc/oligo)
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0.98
Case Study #2
Prior Knowledge:
•
No experience in DHFR-CHO-DG44 to GS-CHO-K1SV cell line changes
•
Knowledge of clonal variability suggested risk of changes in glycosylation profile:
– Core fucosylation → impact ADCC activity
– Terminal -galactose → impact CDC activity
Risk Assessment:
•
Changes in glycosylation could present significant risk to the safety and efficacy of
molecule
Comparability Strategy:
•
Glycosylation as criterion for clone selection to mitigate comparability risk
–
•
Fucosylation prioritized based upon proposed MOA
Demonstrate comparability through:
–
Physico-chemical testing
–
In vitro biological assays (including ADCC & CDC)
–
Non-clinical in vivo PK, PD & immunogenicity studies
www.diahome.org
Case Study #2
Clonal Variability in Fucosylation of GS-CHO-K1SV-Derived MAb2
DHFR-CHO-DG44derived MAb2
0.5
Gal/Glycan
Increasing CDC Activity
0.6
0.4
GS-CHO-K1SV Clones
0.3
0.2
0.1
0.0
0.84
0.86
0.88
0.90
0.92
0.94
Fuc/Glycan
Increasing ADCC Activity
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0.96
Case Study #2
Similar fucosylation has been observed due to clone selection strategy
& subsequent cell culture development:
DG44-CHO-Derived MAb2
Glycoforms
GS-CHO-K1SVDerived MAb2
Batch 1
Batch 2
Batch 3
Batch 1
Fucose/oligosaccharide
0.93
0.94
0.95
0.96
-Galactose/oligosaccharide
0.55
0.51
0.54
0.40
In vitro biological assays indicate comparable ADCC activity.
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Case Study #2 - Summary
Physico-Chemical Testing:
•
No apparent adverse impact on structural integrity, product-related impurities or processrelated impurities
•
Minor differences observed in molecular heterogeneity
–
Lower -galactosylation levels
In vitro Biological Assays
•
No apparent differences observed in ADCC activity
Nonclinical PK, PD & Immunogenicity Assessment
•
No apparent differences observed
Thus far, cell line change presents low risk to the
safety or efficacy of MAb2
(manufacture of clinical trial lots is ongoing)
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Conclusions
Characterization during clone selection can mitigate risks associated with a
cell line change
•
Integrated comparability strategy for a cell line change should start prior to clone
selection
•
MAb’s MOA & clonal variability in CQAs should drive clone selection strategy
Cross-functional risk assessments play a critical role throughout; e.g.,
•
Defining CQAs for MAb
•
Defining clone selection strategy
•
Defining physico-chemical testing protocol & acceptance criteria
•
Defining need for & extent of nonclinical PK, PD & immunogenicity assessments
•
Assessing potential impact of observed differences
When possible, comparability plan/protocol should be shared with FDA
prior to execution (e.g., briefing document, IND amendment)
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