1. Establishment of a Comparability Strategy to Support a Cell Line Change During Clinical Development of a Monoclonal Antibody
Bryan J. Harmon

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3. Outline Drivers for Cell Line Changes
Elements of Comparability Strategy
Case Studies
Conclusions

4. 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

5. 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

6. Elements of Integrated Comparability Strategy
Host cell line & clone selection criteria
Analytical comparability testing strategy
In vitro biological testing
Nonclinical PK, PD & immunogenicity assessments
Clinical assessments
Need for & extent of each element driven by risk assessments

7. Risk Rating = Severity x Occurrence x Detection
Risk Assessments – FMEA Analysis
Severity – impact on toxicity, safety, efficacy or PK/PD
Occurrence – likelihood of being outside preclinical & clinical experience (process capability, control & robustness)
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)

8. 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

9. Impact of Host Cell Line on Glycosylation

10. Impact of Host Cell Line on Glycosylation
CE-LIF Oligosaccharide Profiling

11. 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

12. 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

13. Clonal Variability in Glycosylation
Fc Glycosylation of CHO-derived IgG1

14. Clonal Variability in Glycosylation
Fab Glycosylation of CHO-derived IgG1 with 2 Glycosylation Sites

15. 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

16. 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

17. 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)

18. CQA Risk Assessments

19. 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)

20. Characterization Tests
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**
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

21. 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)

22. 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 Change
Elimination of non-human glycoforms
Alignment with platform

23. Case Study #1 Prior Knowledge: Risk Assessment:
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

24. Case Study #1 Prior Knowledge: GS-NS0 to GS-CHO-K1SV Cell Line Change
Cation-Exchange Chromatography

25. Case Study #1 Prior Knowledge: Risk Assessment:
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

26. GS-CHO-K1SV-Derived MAb1
Case Study #1
Differences in glycosylation profiles were observed:
Glycoforms
GS-NS0-Derived MAb1
GS-CHO-K1SV-Derived MAb1
Batch 1
Batch 2
Non-human glycoforms
a-Gal-containing
2.0%
2.4%
Not observed
NeuGc-containing
2.8%
Human glycoforms
b-Gal-containing
40.8%
40.9%
26.9%
27.2%

27. Case Study #1
CHO-derived MAb1
Differences in charge heterogeneity profiles were observed:
Co-mixture
NS0-derived MAb1
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

28. 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

29. 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

30. Case Study #2 Prior Knowledge: Risk Assessment:
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 b-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

31. Case Study #2
Impact of Fucosylation on ADCC Activity of MAb2

32. Case Study #2 Prior Knowledge: Risk Assessment:
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 b-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

33. Clonal Variability in Fucosylation of GS-CHO-K1SV-Derived MAb2
Case Study #2
Clonal Variability in Fucosylation of GS-CHO-K1SV-Derived MAb2

34. GS-CHO-K1SV-Derived MAb2
Case Study #2
Similar fucosylation has been observed due to clone selection strategy & subsequent cell culture development:
Glycoforms
DG44-CHO-Derived MAb2
GS-CHO-K1SV-Derived MAb2
Batch 1
Batch 2
Batch 3
Fucose/oligosaccharide
0.93
0.94
0.95
0.96
b-Galactose/oligosaccharide
0.55
0.51
0.54
0.40
In vitro biological assays indicate comparable ADCC activity.

35. (manufacture of clinical trial lots is ongoing)
Case Study #2 - Summary
Physico-Chemical Testing:
No apparent adverse impact on structural integrity, product-related impurities or process-related impurities
Minor differences observed in molecular heterogeneity
Lower b-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)

36. 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)