MANUFACTURING ISSUES OF TWO « BIOLOGICALS » AND REGULATION

1. MANUFACTURING ISSUES OF TWO « BIOLOGICALS » AND REGULATION Youssou NDAO Anne-Félice PELLET, Laure TIQUET, Florent ZOONEKYND, Khadra BUBAKER

2. ACTUAL SITUATION 3 7 MammalianCells Summary of productcharacteristics (www.ema.europa.eu/ & EvaluatePharma, June 2012

3. PROTEIN PRODUCTION PIPELINE SALVAGE APPROACHES Target Selection Target Optimization Gene Cloning Selection of Expression Vector Selection of Expression Host Expression Analysis Scaling up Fermentation Purification Purification Optimization Characterization Concentration & Storage http://www.sciencedirect.com/science/article/pii/S1046202311001605

4. GENE CLONING Donor DNA Recombinant Vectorwith insert 1 or 2 Restriction frangments Transformation Replication, Amplification & cell division Clone of donor fragment 1 4

5. BUILDING THE MASTER CELL BANK Collection of cells of uniform composition derivedfromselectedcell clonecontaining the expression construct MCB is cryopreserved in aliquots stored in the liquidnitrogen • Working Cell Bank derived from one or more vials of cells from the MCB • Cells from the MCB expanded by serial subculture up to a passage number selected by the manufacturer and approved by regulator ICH Q5D: Derivation and characterization of cell substrates used for production of Biotechnological/Biological

6. CHARACTERIZATION OF MCB • Manufacturers should perform Quality control testsfor all cell banks include: Identity: expression construct Sterility: Test for the presence of contaminating cell lines, viruses, mycoplasma, and bacteria Stability: coding region (for recombinant cell banks) must be determined during cultivation and storage 6 ICH Q5D: Derivation and characterization of cell substrates used for production of Biotechnological/Biological

7. CELL PREPARATION FOR FREEZING Refeed cells to ensure log phase of growth Label cryotubes with cell line, cells/vial, date, MCB Use trypan blue for viability Count cells Select a freezing media Select a cryoprotective agent (DMSO or glycerol) => Place cryovials gradualy infreezer -40°C/-80°C, and in liquid nitrogen (-196°C) Cell preparation for freezing: Dana M. Hopkins Wm. Davies, Jr. Career & Technical HS, Rochester, NY

8. THAWING CELL BANK Cells are damaged to a certain degree DMSO may be toxic to cells after thawing Cells should be thawed rapidly (37°C) and then diluted slowly into warm growth medium Removal of DMSO by changing media quickly (dilution) www.corning.com/lifesciences

9. CELL CULTURE MEDIA http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958569/#R53

10. PROCESS CHARACTERIZATION AND VALIDATION http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958569/#R53

11. PROCESS TECHNOLOGY TRANSFER • A “gap analysis” should be conducted • Limitations and potential risks Facility and equipment modifications and qualification activities should be completed prior to full-scale production runs • Pre- and post-change product not identical, but: • . Biological activity highly comparable, • . Changes have no impact upon the safety or efficacy of the product http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958569/#R53

12. 12 http://www.sciencedirect.com

13. CHARACTERIZATION OF PRODUCT ICH Q6B: Test procedures and acceptance criteria for Biotechnological/Biological products

14. STORAGE FACILITIES Auditing process to ensure that maintenance and documentation are kept up to date • Process changes and degradation products during storage • Heterogeneity patterns in the material used during preclinical and clinical development Stability profile to proof that changes will be detected http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2958569/#R53

15. PROBLEMATIC OF POST-TRANSCRIPTIONAL MODIFICATION Proteome-widepost-translational modification statistics: frequencyanalysis and curation of the swiss-protDatabase. George A. Khoury, Richard C. Baliban & Christodoulos A. Floudas.

16. PROBLEMATIC OF POST-TRANSCRIPTIONAL MODIFICATION N-glycosylation Proteome-widepost-translational modification statistics: frequencyanalysis and curation of the swiss-protDatabase. George A. Khoury, Richard C. Baliban & Christodoulos A. Floudas.

17. N-GLYCOSYLATION IN HUMAN Sialyltransferase Galactosyltransferase Fucosyltransferase Mannosidases N-acetylglucosaminyltransferase SUBSTRATES KINETICS Glucosidases Oligosaccharyl transferase NIH Public Access, Metabolism, Cell Surface Organization, and Disease James W. Dennis, Ivan R. Nabi, and Michael Demetriou

18. N-GLYCOSYLATION IN HUMAN Glycosylation = Dynamic Tri-antennarycomplex Leu Ser Asn Gly … Arg Ile Cys COO- NH3+ Thr Asn Ser Glu NIH Public Access, Metabolism, Cell Surface Organization, and Disease James W. Dennis, Ivan R. Nabi, and Michael Demetriou

19. N-GLYCOSYLATION IN HUMAN Glycosylation = Dynamic SEVERAL GLYCOFORMS Tri-antennarycomplex Leu Ser Asn Gly … Arg Ile Cys COO- NH3+ Thr Asn Ser High-Mannose type Glu No glycan NIH Public Access, Metabolism, Cell Surface Organization, and Disease James W. Dennis, Ivan R. Nabi, and Michael Demetriou

20. N-GLYCOSYLATION IN HUMAN Example of humanIgG High Throughput Isolation and Glycosylation Analysis of IgG–Variability and Heritability of the IgGGlycome in Three Isolated Human Populations, The American Society for Biochemistry and Molecular Biology, Inc. 2011

21. EXAMPLE OF N-GLYCOSYLATION PROBLEMATIC IN CHO • Ex : IgG • Increase serum clearance • Reduced ADCC Knockout Lowglucose / glutamine concentration Optimal and consistent protein glycosylation in mammalian cell culture, P Hossler, SF Khattak, ZJ Li - Glycobiology, 2009 - SocGlycobiology

22. EXAMPLE OF N-GLYCOSYLATION PROBLEMATIC IN CHO T1/2 Lowtemperature Overexpression of CMP-SAT Glycerol Ammonia Optimal and consistent protein glycosylation in mammalian cell culture, P Hossler, SF Khattak, ZJ Li - Glycobiology, 2009 - SocGlycobiology

23. EXAMPLE OF N-GLYCOSYLATION PROBLEMATIC IN CHO Example of recombinant EPO CLINICAL TRIALS Activity 4 Activity 2 Activity 1 Activity 3 = … Product efficacy Half-life 4 Half-life 3 Half-life 2 Half-life 1 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3449730/pdf/10616_2004_Article_267657.pdf

24. DEGRADATION PRODUCTS • Desiredproduct: • Proteinwhich has the expected structure • Proteinexpectedfrom the DNA sequence • Anticipated post-translational modification (glycoforms),

25. II – ETUDES DE CAS

26. PERJETA®- Pertuzumab

27. PERJETA®- Pertuzumab Recombinant humanized IgG1 Indication : Her2-positive metastatic breast cancer CombinationwithTrastuzumab and Docetaxel Aggressive tumor and a poor prognosis Clinical pharmacology and biopharmaceutics review of perjeta http://www.ncbi.nlm.nih.gov.doc-distant.univ-lille2.fr/pmc/articles/PMC3462608/

28. MECHANISM OF ACTION Clinical pharmacology and biopharmaceutics review of perjeta

29. MECHANISM OF ACTION Pertuzumab • Subdomain II of HER2 Trastuzumab Pertuzumab • Subdomain IV of HER2 HER2 Induce ADCC Trastuzumab http://clincancerres.aacrjournals.org/content/17/15.cover-expansion

30. ADCC : Antibody-dependentcell-mediatedcytotoxicity FcγR Macrophages NK Cells Neutrophils Tumorcell Lysis of the targetcell Comment améliorer les propriétés effectrices des anticorps monoclonaux thérapeutiques ? Christophe Carnoy

31. CLINICAL TRIALS / « CLEOPATRA » 12.4 monthswithPlacebo VS 18.4 monthswithPertuzumab => 6.1 months of improvement (p < 0.001) Baselga J, Cortés J, Kim SB, et al. Pertuzumab plus trastuzumab plus docetaxel for metastaticbreast cancer. N Engl J Med. 2012;366:109-119.

32. COST OF TREATMENT • Perjeta® • Herceptin® • $5,900 • per month • $4,500 • per month • $71,000 • per year • $54,000 • per year $125,000 for 1 year Pharmaceutical Approval Update Marvin M. Goldenberg, PhD, RPh, MS / Vol. 37 No. 9 • September 2012 • P&T

33. MANUFACTURING Recombinant DNA technology in CHO FDA pre-approval inspection • Failure rate for the WorkingCell Bank thawduring the 2012 manufacturingcampaign

34. CENTER FOR DRUG EVALUATION AND RESEARCH APPLICATION NUMBER:125409Orig1s000 CHEMISTRY REVIEW(S) « Wediscoveredthat the Sponsor wasexperiencingserious issues with the thaw and subsequent propagation of cellsfrom WCB »

35. http://www.bionique.com/mycoplasma-resources/technical-articles/certified-working-cell-bank.htmlhttp://www.bionique.com/mycoplasma-resources/technical-articles/certified-working-cell-bank.html

36. HISTORY • December 2011 • Genentech filed applications for approval in US and EU • Dec 13, 2012 • Positive opinion from the CHMP • June 8, 2012 • FDA’s Approval • (Product launch about 2 weeks post-approval) • February 2012 • FDA grants pertuzumab “Priority Review” 2011 2012 • May 2012 • Submission in Japan • December 7, 2011 • Improvement PFS by 6.1 months (NEJM) • March 2012 • FDA Pre-license drug inspection • August 2012 • Approval in Switzerland • Failure rate for WCB growth PDL BiopharmaCorporateOverview July 2012

37. TO RECAP Issues with : Thaw & Propagation cell Approval for campaign 2010 FDA What about campaign 2012 ?? Consequences ??

38. RESTRICTIONS ON APPROVAL Restriction from FDA : Only campaign 2010 approved Potentialshortage MANUFACTURING ISSUES Could have ramifications for the development and approval of biosimilars Genentech’s Perjeta Clears FDA despite Unresolved Manufacturing IssuesElsevier Business Intelligence: 'The Pink Sheet' - June 18, 2012

39. RECOMMENDATIONS ON APPROVABILITY Three concurrent plans to resolve the cell growth issues • Manufacturingfrom the Master Cell Bank • Manufacturingusing a modifiedprocessfromWorkingCell Bank • Developing a new WCB and manufacturingfromthis new WCB Center for drugevaluation an d research – 125409Orig1s000 Chemistry Review0

40. APPROVAL’S CONDITIONS Letterfrom Division of Monoclonal Antibodies (DMA) 13 PerjetaCommitments 40

41. 1- Stabilitystudy of the drug substance manufacturedfromspecificthaws 2012 pertuzumabcampaign Real time Confirm the use-by-date Stressedstabilitytesting Evaluate the intrinsicstability of the active molecule Identifydegradationproducts Chooseanalyticalmethods Predict the stability of a drug formulation Shortenedshelf life  Risk of lack of biologicaleffects http://www.hc-sc.gc.ca/dhp-mps/prodpharma/applic-demande/guide-ld/chem/stabt_stabe-fra.php#1.3

43. Stabilitystudies : EMA’s guideline “…proteins and/or polypeptides, maintenance of molecular conformation and, hence of biological activity, is dependent on noncovalent as well as covalent forces. The products are particularly sensitive to environmental factors such as temperature changes, oxidation, light, ionic content, and shear. In order to ensure maintenance of biological activity and to avoid degradation, stringent conditions for their storage are usually necessary.” Guideline Quality of Biotechnological Products: Stability Testing of Biotechnological/BiologicalProducts

44. 2- Stabilitystudies of the Master Cell Bank at more frequentintervalsthan the currentlyproposed 10 years Check the stability of the MCB every 4 years

45. 3- Reassess release and stabilityspecifications for pertuzumabdrug substance and drugproductthroughJune 30, 2014

46. Process validation study to support manufacture of pertuzumabfrom 4- Master Cell Bank 5- WorkingCell Banks by a modifiedprocess 6- A new WorkingCell Bank

47. Process validation study Objective : check that all manufacturing steps lead to a product in compliance with standard of stability and reproducibility. Drug substance manufacturing campaigns are able to deliver consistent product with comparable strength, quality, purity and potency to that used in the phase 3 clinical trial • Reproducibility : glycosylation profile, ADCC activity, and purity

49. Process validation studies : EMA’s guideline “The evaluation/validation data provide essential information on the reproducibility and robustness of the process steps and are an important element to guarantee consistency in the quality of the product.”

50. “Effective process validation contributes significantly to assuring drug quality • … • Quality, safety, and efficacy are designed or built into the product. • Quality cannot be adequately assured merely by in-process and finished-product inspection or testing. • Each step of a manufacturing process is controlled to assure that the finished product meets all quality attributes including specifications.”