Establishment of a Comparability Strategy to Support a
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 www. diahome. org
Cell Line Changes During Clinical Development Driver Quality risk with initial cell line Examples • Genetic splicing or mutation identified • ASM exposure during cell line generation • Lack of assurance of clonality • Insufficient titer Initial cell line is • Insufficient cell line stability not commercially • Not consistent with manufacturing viable platform • Intellectual property issues www. diahome. org
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 www. diahome. org
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 www. diahome. org 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) www. diahome. org
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
Impact of Host Cell Line on Glycosylation www. diahome. org
Impact of Host Cell Line on Glycosylation CE-LIF Oligosaccharide Profiling www. diahome. org
Risks of Cell Line Changes • Different host cell line • Different clone from same host cell line • Additional round of cloning www. diahome. org 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 www. diahome. org
Clonal Variability in Glycosylation Fc Glycosylation of CHO-derived Ig. G 1 www. diahome. org
Clonal Variability in Glycosylation Fab Glycosylation of CHO-derived Ig. G 1 with 2 Glycosylation Sites www. diahome. org
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 m. Ab: 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 www. diahome. org
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) www. diahome. org
CQA Risk Assessments 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) www. diahome. org
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 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 www. diahome. org * Include co-mixture analysis of representative lots ** 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 MAb 1 Isotype Ig. G 4 Phase of Development Pre-Phase 2 Cell Line Change GS-NS 0 to GS-CHO-K 1 SV MOA Dependent upon Effector Function? Drivers for Cell Line Change No Elimination of non-human glycoforms Alignment with platform www. diahome. org
Case Study #1 Prior Knowledge: • Experience in GS-NS 0 to GS-CHO-K 1 SV 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 www. diahome. org
Case Study #1 Prior Knowledge: GS-NS 0 to GS-CHO-K 1 SV Cell Line Change Cation-Exchange Chromatography www. diahome. org
Case Study #1 Prior Knowledge: • Experience in GS-NS 0 to GS-CHO-K 1 SV 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 www. diahome. org
Case Study #1 Differences in glycosylation profiles were observed: Glycoforms GS-NS 0 -Derived MAb 1 Batch 2 a-Gal-containing 2. 0% 2. 4% Neu. Gc-containing 2. 8% 2. 4% 40. 8% 40. 9% GS-CHO-K 1 SVDerived MAb 1 Batch 2 Non-human glycoforms Not observed Human glycoforms b-Gal-containing www. diahome. org 26. 9% 27. 2%
Case Study #1 CHO-derived MAb 1 Differences in charge heterogeneity profiles were observed: Co-mixture NS 0 -derived MAb 1 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 des. Gly/amidation – Glycation www. diahome. org
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 MAb 1 www. diahome. org
Case Study #2 Property MAb 2 Isotype Ig. G 1 Phase of Development Pre-Phase 2 Cell Line Change DHFR-CHO-DG 44 to GS-CHO-K 1 SV MOA Dependent upon Effector Function? Drivers for Cell Line Change Yes Alignment with platform www. diahome. org
Case Study #2 Prior Knowledge: • No experience in DHFR-CHO-DG 44 to GS-CHO-K 1 SV 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 www. diahome. org
Case Study #2 Impact of Fucosylation on ADCC Activity of MAb 2 www. diahome. org
Case Study #2 Prior Knowledge: • No experience in DHFR-CHO-DG 44 to GS-CHO-K 1 SV 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 www. diahome. org
Case Study #2 Clonal Variability in Fucosylation of GS-CHO-K 1 SV-Derived MAb 2 www. diahome. org
Case Study #2 Similar fucosylation has been observed due to clone selection strategy & subsequent cell culture development: Glycoforms DG 44 -CHO-Derived MAb 2 GS-CHO-K 1 SVDerived MAb 2 Batch 1 Batch 2 Batch 3 Batch 1 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. www. diahome. org
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 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 MAb 2 (manufacture of clinical trial lots is ongoing) www. diahome. org
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) www. diahome. org
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