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Bryan J. Harmon
Establishment of a Comparability Strategy to Support a Cell Line Change During Clinical Development of a Monoclonal Antibody
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The views and opinions expressed in the following PowerPoint slides are those of the individual presenter and should not be attributed to Drug Information Association, Inc. (“DIA”), its directors, officers, employees, volunteers, members, chapters, councils, Special Interest Area Communities or affiliates, or any organization with which the presenter is employed or affiliated.
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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
Need for & extent of each element driven
by risk assessments
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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
G0F
Man-5
G0F-1GlcNAc
G0
G1F
G2F
G2F+NeuGc
G1F+NeuGc
G2F+(1-3)Gal +NeuGc G1F+(1-3)Gal
G2F+(1-3)Gal
G2F+2(1-3)Gal
GlcNAc Man Fuc (1-4)Gal (1-3)Gal NeuGcGlcNAc Man Fuc (1-4)Gal (1-3)Gal NeuGc
“Non-human” structures: (1-3)Gal & NeuGc“Human-like” structures
CHO & NS0 NS0 Only
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Impact of Host Cell Line on Glycosylation
RF
U
G0F
G1FG2F
20 25 30 35 40Migration Time (min)
RF
UCHO-Derived IgG4
NS0-Derived IgG4
G0Man-5
CE-LIF Oligosaccharide Profiling
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Risks of Cell Line Changes
• Different host cell line
• Different clone from same host cell line
• Additional round of cloning
Increasing risk to
CQAs of molecule
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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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Clonal Variability in Glycosylation
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.84 0.86 0.88 0.90 0.92 0.94 0.96
Fuc/Glycan
Gal
/Gly
can
Fc Glycosylation of CHO-derived IgG1
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Clonal Variability in GlycosylationFab Glycosylation of
CHO-derived IgG1 with 2 Glycosylation Sites
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
0.00 0.20 0.40 0.60 0.80 1.00 1.20
NeuAc/Glycan
Gal
/Gly
can
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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
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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
Documented impact on toxicity, safety, efficacy,
PK/PD or immunogenicity?
Yes
No
Platform modification?
No
Located in region likely to impact activity?
YesYes
Impact on in vivo toxicity, safety, efficacy, PK/PD or
immunogenicity?
No
Yes
No
No
CQANon-CQA
Quality Attribute
Impact on in vitrobioactivity?
Yes or unknown
Pyroglutamation of HC Gln1
Loss of HC Lys449
Oxidation of HC Met252
Glycation of Fab & Fc lysines & N-termini
Deamidation of Fc Asn sites
Free thiol (incomplete disulfide)
427
369
427
369
323
263
323
263
193
13387
23
202
146
96
22
231231
23
87133
193
22
96
146
202
213
222
228 228 213
222
105 105
Cleavage at LC Asn93/Pro94
Glycosylation of HC Asn299
Oxidation of LC Met32
HC His226/Thr227 (hinge) cleavage
Oxidation of Fc Met sites
Aggregation
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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 TestsRelease 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**
Non-reduced CE-SDS*
Reduced CE-SDS*
Process-Related Impurities
Host cell protein Triton X-100
DNA Insulin
Protein A MSX
* Include co-mixture analysis of
representative lots
** Added based upon regulatory
feedback
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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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Case Study #1
Property MAb1
Isotype IgG4
Phase of Development Pre-Phase 2
Cell Line Change GS-NS0 to GS-CHO-K1SV
MOA Dependent upon Effector Function?
No
Drivers for Cell Line ChangeElimination of non-human glycoforms
Alignment with platform
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Case Study #1Prior 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
Cation-Exchange Chromatography
GS-NS0-Derived IgG4GS-CHO-K1SV-Derived IgG4
Incomplete pyroglutamate
Both HCOne HC
Prior Knowledge: GS-NS0 to GS-CHO-K1SV Cell Line Change
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Case Study #1Prior 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
GS-CHO-K1SV-Derived MAb1
Batch 1 Batch 2 Batch 1 Batch 2
Non-human glycoforms
-Gal-containing 2.0% 2.4%Not observed
NeuGc-containing 2.8% 2.4%
Human glycoforms
-Gal-containing 40.8% 40.9% 26.9% 27.2%
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Case Study #1
mV
10.00
90.00
mV
10.00
90.00
mV
10.00
90.00
mV
10.00
90.00
mV
10.00
90.00
Minutes10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
21.6
min
19.2
min
20.4
min
22.7 min
24.0
min
25.0 min
26.1
min
(a)
(b)
(c)
(d)
(e)
27.4
min
28.5
min
mV
10.00
90.00
mV
10.00
90.00
mV
10.00
90.00
mV
10.00
90.00
mV
10.00
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Minutes10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00 32.00 34.00
21.6
min
19.2
min
20.4
min
22.7 min
24.0
min
25.0 min
26.1
min
(a)
(b)
(c)
(d)
(e)
27.4
min
28.5
min
CHO-derived MAb1
NS0-derived MAb1
Co-mixture
Differences in charge heterogeneity profiles were observed:
LC-MS characterization of isolated CEX fractions identified small differences in proportions of typical sources of MAb charge variants:
– Heavy chain N-terminal pyroglutamate
– Heavy chain C-terminal lysine
– Heavy chain C-terminal desGly/amidation
– Glycation
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Case Study #1 - SummaryPhysico-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 Pre-Phase 2
Cell Line Change DHFR-CHO-DG44 to GS-CHO-K1SV
MOA Dependent upon Effector Function?
Yes
Drivers for Cell Line Change Alignment with platform
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Case Study #2Prior 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
0
50
100
150
200
250
300
0.86 0.88 0.90 0.92 0.94 0.96 0.98
Fucosylation (Fuc/oligo)
Rel
ativ
e A
DC
C A
ctiv
ity
Impact of Fucosylation on ADCC Activity of MAb2
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Case Study #2Prior 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
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.84 0.86 0.88 0.90 0.92 0.94 0.96
Fuc/Glycan
Gal
/Gly
can
Incr
easi
ng C
DC
Act
ivity
Increasing ADCC Activity
GS-CHO-K1SV Clones
DHFR-CHO-DG44-derived MAb2
Clonal Variability in Fucosylation of GS-CHO-K1SV-Derived MAb2
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Case Study #2
GlycoformsDG44-CHO-Derived MAb2
GS-CHO-K1SV-Derived 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.
Similar fucosylation has been observed due to clone selection strategy & subsequent cell culture development:
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Case Study #2 - SummaryPhysico-Chemical Testing:• No apparent adverse impact on structural integrity, product-related impurities or process-
related 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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ConclusionsCharacterization 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)
