A QUALITY DRIVEN BIOSIMILAR DEVELOPMENT - …

1 A QUALITY DRIVEN BIOSIMILAR . DEVELOPMENT . AN AVERSE RISK APPROACH. SEPTEMBER 17TH, 2015. Amman, Jordan AFTER LUNCH TEST: FIND THE DIFFERENCE. ? September 2015 / 2. CHALLENGES IN BIOSIMILAR DEVELOPMENT . Originator Similarity of BIOSIMILAR Structure Biological Activity Stability Safety Efficacy September 2015 / 3. OUTLINE. Understand the Complexity of Biologics Define the Originator QUALITY Target Product Profile Achieve Biosimilarity September 2015 / 4. N-GLYCOSYLATION BIOSYNTHESIS. September 2015 / 5. COMPLEXITY OF N-GLYCOSYLATION IN HUMANS. September 2015 / 6. TYPICAL BIOSIMILAR DEVELOPMENTS. Reference Produced in Produced in Active Ingredient Reference Product Active Ingredient Product Glycans per Chain Glycans per Chain CHO Nutropin Somatropin Avastin Bevacizumab 1xN (IgG1).

2 CHO. Octocog alpha Factor VIII. Avonex Interferon beta-1a 25xN+12xO. PEG-Interferon alpha- NS0 Pegasys Benlysta Belimumab 2a 1xN (IgG1). Proleukin Interleukin-2. Betaseron Interferon beta-1b CHO. Prolia Denosumab Copaxone chemical Glatiramer acetate 1xN (IgG2). SP2/0. CHO; Remicade Infliximab Enbrel 3xN, 10xO Etanercept 1xN (IgG1). (IgG1-Fus.) CHO. Rituxan Rituximab CHO; 1xN (IgG1). Epogen , Eprex 3xN, 1xO. Erythropoietin CHO. RoActemra Tocilizumab SP2/0; 1xN (IgG1). Erbitux 2xN (IgG1). Cetuximab Roferon Interferon alpha-2a CHO; Follicle-stimulating SP2/0. Gonal F Simponi Golimumab 4xN hormone 1xN (IgG1). CHO; SP2/0. Herceptin Trastuzumab Soliris Eculizumab 1xN (IgG1) 1xN (IgG2/4).

3 CHO, SP2/0. Humira Adalimumab Stelara Ustekinumab 1xN (IgG1) 1xN (IgG1). NS0. Lantus Insulin Tysabri Natalizumab 1xN (IgG4). CHO. Lucentis Ranibizumab Xolair Omalizumab 1xN (IgG1). CHO. Neupogen Filgrastim Yervoy Ipilimumab 1xN (IgG1). September 2015 / 7. INLFUENCE DURING MANUFACTURING. Glycation Deamidation Oxidation Glycosylation HCPs Clone DEVELOPMENT K-Clipping Degradation Aggregation Fragmentation Disulfide shuffling September 2015 / 8. QUALITY ATTRIBUTES OF MABS. September 2015 / 9. ANALYTICAL ASSAYS FOR BIOSIMILARITY ANALYSIS. Glyosylation Monosaccharide Analysis Oligosaccharide Analysis Structure/ Sequence Intact Mass MALDI-TOF. N- and C-terminus HILIC-HPLC/MS Surface Charge Amino acid analysis Peptide Mapping and Sequencing IEX.

4 Disulfide Linkage Analysis CZE. Intact mass (reduced and middle down) IEF/c-IEF. Activity Bioassays Aggregation Binding assays SEC. AUC. SDS-PAGE Higher Order AF4 CD. FT-IR. NMR. H/D Exchange X-Ray September 2015 / 10. OVERVIEW GLYCOSYLATION ANALYSIS. GlycosylationSite/. Chromatographic Saccharide Glycan Pool Glycan Profiles Site Occupancy/. Content Analysis Structure Site Specific Pool w/wo Deglycosylation w/wo Deglycosylation Exoglycosidase Degylcosylation w/wo Desialisation Site specific protein fragment Defucosylation Glycosylation Site and Monosaccharide HILIC-HPLC Site Occupancy by IEF gel/cIEF Content by HPAEC- HPAEC-PAD Linkage Analysis Peptide Mapping PAD GC-MS. LC-ESI-MS/MS, LC-UV.

5 Glycan distribution per LC-ESI/MALDI- Sialic Acid Content MALDI-MS of IEX-HPLC/RP-HPLC site by Peptide Mapping MS/MS. by HPAEC-PAD Released Glycans LC-ESI-MS/MS. Intact molecular 2D PAGE Intact molecular weight by weight by MALDI-MS NMR. 1D PAGE/cGE MALDI-MS or LC-ESI-MS. or LC-ESI-MS. September 2015 / 11. CHOICE TO SEE BIOSIMILARITY. Initially fast Upfront loaded, QUALITY DRIVEN High risk Lower risk Some QUALITY DRIVEN DEVELOPMENT Full QUALITY DRIVEN DEVELOPMENT Establishing Biosimilarity by Comparability to make a scientific principles September 2015 / 12. BIOSIMILAR DEVELOPEMENT - DEFINING QTPP. AND CQA. Detailed Characterization of Originator Batches Structural characterization QUALITY Target Poduct Profile Cell Line Dev Amino acid sequence and compos.

6 QTPP. N/C terminus May change during DEVELOPMENT Post translational modification ( Process Changes). Glycosylation Disulfide linkage Comparability Upstream Proc Dev Higher order structure Analytics Aggregates Bioassays Critical QUALITY Attributes Physicochemical properties Linking QUALITY to Safety and Effiicacy Downstrem Proc Dev Molecular weight or size Enabling QbD DEVELOPMENT Isoform pattern Extinction coefficient Electrophoretic pattern Liquid chromatographic pattern Formulation Spectroscopic profiles September 2015 / 13. MODULES ON REFERENCE MEDICINAL PRODUCT. Originator AA verification Originator Sourcing and Logistics QTPP by Originator Monitoring Program Originator Monitoring Program Method Qualification Upstream/ Eng.

7 Runs Cell Line Media Pre+Clinical Downstream and GMP Approval Develop Optimization Phase I+III. Develop Production September 2015 / 14. QUALITY TARGET PRODUCT PROFILE OF 6 RMP. QUALITY Attribute in %. Originator Batch ABC. BIOSIMILAR Candidate QUALITY Target by Originator Batch EDF. Considered not highly similar QUALITY Attribute in %. Originator Batch ABC. BIOSIMILAR Candidate Originator Batch EDF Assumed to be highly similar High number/high variability of characterized originator batches increases QUALITY target range for the BIOSIMILAR Candidate September 2015 / 15. CHANGE OF GLYCOSYLATION FOR MARKETED. PRODUCTS. Enbrel Rituxan/Mabthera September 2015 / 16. Schiestl et al.

8 , Nature Biotechnology, Vol 29, No 4 April 2011. ONGOING ORIGINATOR MONITORING. Detailed Monitoring program of 30 RMP Lots: 10 US, 10 EU, 10 ROW. Sourced internationally with highest variability in Lots and Exp. Dates; to include different Production sites and Process Changes of RMP. Sourced and analyzed in campaigns September 2015 / 17. METHOD QUALIFICATION FOR BIOSIMILARITY. The assays needs to be suitable to detect small differences Generation of Stress samples Testing of Stress samples Final method qualification with tested stress samples As an example to be able to qualify selected assays a variety of stress samples will be generated and tested with two different orthogonal method.

9 Subsequently the samples will be used as a reference Std. for Method Qualification September 2015 / 18. MODULES ON BIOSIMILAR CANDIDATE. Upstream/ Eng. Runs Cell Line Media Pre+Clinical Downstream and GMP Approval Develop Optimization Phase I+III. Develop Production Pool selection QTPP DRIVEN Clone selection Process DEVELOPMENT Media Selection Comparability studies Standardized and validated release testing Stability testing September 2015 / 19. QUALITY DRIVEN DEVELOPMENT . Pool selection QTPP DRIVEN Clone selection Process DEVELOPMENT Media Selection Initially fast Upfront loaded, QUALITY DRIVEN High risk Lower risk Not an overall cost argument ! September 2015 / 20. EXERPT OF QTPP.

10 September 2015 / 21. DEVELOPMENT BY QUALITY ATTRIBUTES. Maybe highly productive pool but not suitable Targeted attribute Limits for this attribute Clone Pool Single Clones Media Optimization QUALITY directed DEVELOPMENT planned in several iterations: Clone Pool Analysis Clone Selection Media Optimization Starting with >100 Samples and selected QUALITY Attributes Screening Assays optimized for parallelization, speed and sensitivity September 2015 / 22. EXAMPLE: ANALYSIS FOR CLONE SELECTION. Application: DEVELOPMENT of an IgG1 BIOSIMILAR MALDI-MS and subsequent comparison of glycosylation pattern 96 samples in less than 6h (including ProteinA, Glycan Release, MALDI, Data Evaluation).