Biosimilar Product Structural Characterization - Strategies for Comparability Analysis.

Transcription

1 Biosimilar Product Structural Characterization - Strategies for Comparability Analysis. Dr Fiona M Greer Global Director, BioPharma Services Development, SGS M-Scan CASSS 8th Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology Industry

2 SUMMARY: WHY WHAT HOW? Why is analytical characterisation essential throughout all stages of development? What are the regulatory issues associated with characterisation of Biosimilars? - Characterisation of the Innovator - Side by Side Comparison of Innovator and Biosimilar How can we characterise complex protein/glycoprotein products? Which techniques, old & new, are suitable for Biosimilar comparability - Analytical Package for Biosimilars - Battery of Methods 2

3 WHY CHARACTERISE? Process and Analytical Development (GLP and/or GMP) Understand the chemical structure, physical properties, impurity profile and degradation pathways Determine the effect of process change on drug substance Formulation GMP Manufacture Guide to select specification, QC & stability assays Comparability studies - e.g. changes post-approval Biosimilars/ Follow-on Biologicals Comparability approach - at 3 levels: quality, safety and efficacy Comparable to Reference Determine if proteins have the same biochemical, biophysical and physiological attributes 3

4 BIOSIMILARS WHY TERMINOLOGY MATTERS* Copy biologicals True biosimilars Similar biotherapeutic products Follow-on protein products Subsequent entry protein products Biobetters A biosimilar is a copy version of an already authorized biological medicinal product (reference product) with demonstrated similarity in physicochemical characteristics, efficacy and safety, based on a comprehensive comparability exercise *Weise, M et al, Nature Biotechnology 29, (2011) 4

5 BIOSIMILARS IN EUROPE In 2006, EMEA issued guidelines on biosimilars Overarching Guideline on Similar Biological Medicinal Products Guidelines on Quality, Non-clinical and Clinical. 6 product classes: HGH, repo ( under review), rg-csf, rinsulin, (Low m.wt Heparins), Alpha - Interferon and Mab (in draft) Comparison against reference medicinal product 5

6 EU REFERENCE PRODUCT Must be authorised in the EEA ( Complete Dossier, Article 8 Directive 2001/83) Data exclusivity period expired. Active substance must be similar in molecular and biological terms Same pharmaceutical form, strength, route of administration Same comparator used throughout 6

7 EMA WORKSHOP ON BIOSIMILAR MABS Held at EMA 2 nd July 2009 Draft biosimilar mab guideline published Nov Consultation period ended 31 May 2011 Final guideline end of year? 7

8 BIOSIMILAR GUIDELINES EU -approved its first biosimilar in 2006 and now has 14 Brazil, Australia, Turkey, Taiwan, India, Malaysia, Argentina, Mexico, Japan, Canada, South Africa... WHO Guideline on Evaluation of Similar Biotherepeutic Products. Adopted Oct 2009 USA now have a legal pathway. 8

9 USA BIOSIMILARS Biologics Price Competition and Innovation Act (BPCIA), March 23rd Part of Affordable Care Act New pathway-351(k) Single reference product approved under a 351(a) Two steps: Biosimilar or Interchangeable Biosimilar Complex Patent disclosure provisions 9

10 FDA will produce guidances etc but cannot say when Steve Kozlowski, MD Director, Office of Biotechnology Products, CDER, FDA 4 May

11 Developing the Nation s Biosimilar Program Steve Kozlowski, MD, Janet Woodcock, MD, Karen Midthun, MD, and Rachel Behrman Sherman, MD, MPH. The New England Journal of Medicine 365;5 Aug

12 AUTHORISED BIOSIMILARS IN THE EU Trade Name Active substance Reference Product Therapeutic area Date of authorisation Owner of Trade Name Status Nivestim filgrastim Neupogen Hematopoietic Stem Cell TransplantationCancerNeutropenia Filgrastim Hexal filgrastim Neupogen NeutropeniaCancerHematopoietic Stem Cell Transplantation Zarzio filgrastim Neupogen CancerHematopoietic Stem Cell TransplantationNeutropenia 08/06/2010 Hospira Authorised 06/02/2009 Hexal Authorised 06/02/2009 Sandoz Authorised Biograstim filgrastim Neupogen Hematopoietic Stem Cell TransplantationNeutropeniaCancer 15/09/2008 CT Arzneimittel GmbH Authorised Filgrastim Ratiopharm filgrastim Neupogen NeutropeniaHematopoietic Stem Cell TransplantationCancer 15/09/2008 Ratiopharm Authorised Ratiograstim filgrastim Neupogen NeutropeniaCancerHematopoietic Stem Cell Transplantation 15/09/2008 Ratiopharm Authorised Tevagrastim filgrastim Neupogen NeutropeniaCancerHematopoietic Stem Cell Transplantation 15/09/2008 Teva Generics GmbH Authorised Retacrit epoetin zeta Eprex CancerAnemiaKidney Failure, ChronicBlood Transfusion, Autologous 18/12/2007 Hospira Authorised Silapo epoetin zeta Eprex AnemiaBlood Transfusion, AutologousCancerKidney Failure, Chronic Abseamed epoetin alfa Eprex Kidney Failure, ChronicAnemiaCancer 18/12/2007 Stada Arzneimittel 28/08/2007 Pütter Medice Arzneimittel GmbH & Co Authorised Authorised Binocrit epoetin alfa Eprex Kidney Failure, ChronicAnemia 28/08/2007 Sandoz Authorised Epoetin alfa Hexal epoetin alfa Eprex Kidney Failure, ChronicAnemiaCancer 28/08/2007 Hexal Authorised Valtropin somatropin Humatrope Dwarfism, PituitaryTurner Syndrome 24/04/2006 Biopartners Authorised Omnitrope somatropin Genotropin Turner SyndromeDwarfism, 12/04/2006 Sandoz Authorised PituitaryPrader-Willi Syndrome 12

13 NOT APPROVED Trade Name Active substance Reference Product Therapeutic area Status Date of withdrawal Reason Applicant Alpheon Interferon alfa-2a Referon-A Rejected 28/06/2006 Various BioPartners Insulin Human Rapid Marvel human insulin (soluble insulin) Humulin Insulin S Diabetes mellitus Withdrawn 20/12/2007 Comparability concerns Marvel LifeSciences Ltd Insulin Human Long Marvel human insulin (isophane insulin) Humulin Insulin I Diabetes mellitus Withdrawn 20/12/2007 Comparability concerns Marvel LifeSciences Ltd Insulin Human 30/70 Mix Marvel human insulin (30% soluble insulin and 70% isophane insulin) Humulin Insulins M3 Diabetes mellitus Withdrawn 20/12/2007 Comparability concerns Marvel LifeSciences Ltd Epostim Eoitin Withdrawn /4/2011 Reliance Genemedix 13

14 WHAT REGULATIONS COVER PHYSICOCHEMICAL CHARACTERISATION? ICH Topic Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products Structural characterisation and confirmation 1. Amino acid sequence 2. Amino acid composition 3. Terminal amino acid sequence 4. Peptide map 5. Sulfhydryl group(s) and disulfide bridges 6. Carbohydrate structure Physicochemical properties 1. Molecular weight or size 2. Isoform pattern 3. Extinction coefficient 4. Electrophoretic pattern 5. Liquid Chromatographic pattern 6. Spectroscopic profiles 14

15 Revision of EMEA Guideline on Production and Quality Control of Monoclonal Antibodies 15

16 Section 4.3 CHARACTERISATION OF MONOCLONAL ANTIBODIES 16

17 STAGE 1 Determination of the exact sequence of the Originator molecule. Usually involves MS/MS de novo sequencing Target molecule CQAs assessed 17

18 CASE STUDY 1 TROUBLE-SHOOTING PEPTIDE MASS MAP - UNEXPLAINED SIGNAL IN TRYPTIC MASS MAP Peptide of interest missing from Mass MAP ESQXXXXXXXR Searched for missed Tryptic cleavage ESQXXXXXXXRXXXXK Search for expected y ions within one of the MS/MS channels Found to be successful for the missed cleavage peptide However. 18

19 UNEXPLAINED SIGNAL IN TRYPTIC MASS MAP 19

20 UNEXPLAINED SIGNAL IN TRYPTIC MASS MAP Peptide of interest missing from Mass MAP ESQXXXXXXXR Searched for missed Tryptic cleavage ESQXXXXXXXRXXXXK Search for expected y ions within one of the MS/MS channels Peptide +10amu higher From MS/MS interpretation, this mass difference was attributed to site of second residue, possibly Serine to Proline change 20

21 STAGE 2 Comparison of the Biosimilar side-by-side with the Originator. Choice of reference material Batches of Biosimilar vs batches of Originator Changes in Originator product Strategies for Primary and Higher Order Structure-battery of techniques 21

22 NON TREATED ERYTHROPOIETIN ON cief IN 8M UREA Focused for 1 minute at 1500V and 5 minutes at 3000V. In 3% and 1% 5-8 Ampholytes. 22

23 CHARACTERISATION OF BIOPHARMACEUTICALS ICH Topic Q 6 B Physicochemical properties 5. Spectroscopic profiles The ultraviolet and visible absorption spectra are determined as appropriate. The higher-order structure of the product is examined using procedures such as circular dichroism, nuclear magnetic resonance (NMR), or other suitable techniques, as appropriate 23

24 de mol-1 dm3 cm-1 SECONDARY STRUCTURE ANALYSIS (Far UV CD Analysis nm) Circular dichroism of different batches of the same product Sample A Sample B Sample C Sample D Sample E -10 Wavelength (nm) Sample % α- Helix % other helix % β- sheet % Turns % Other A B C D E

25 CD\ mdeg CD\ mdeg TERTIARY STRUCTURE ANALYSIS (Near UV CD nm) Sample A Sample B Sample C Sample D Sample E Sample Sample Sample Wavelength (nm) Wavelength (nm) CD signals in the nm region arise from the aromatic amino acids and also disulphide bonds. The spectra have significant signals and this indicates well defined protein structure, as well as the presence of aromatic residues. Phenlyalanine signals arise between nm. Tyrosine signals are from nm and Tryptophans show intensity from nm. Disulphide bonds may give rise to broad weak signals throughout the spectra. 25

26 CASE STUDY 2 : ANTIBODY CHARACTERISATION Performed analyses: Mass spectrometry of intact protein and released L &H chains Amino Acid Composition Analysis N-terminal sequencing Peptide MAPPING Analysis (Sequence coverage: 100% LC and 100% HC) Monosaccharide and sialic acid analysis Oligosaccharide population analysis SDS-PAGE analysis Circular Dichroism Analytical Ultracentrifugation 26

27 CONCLUSION Advances in Mass Spectrometry instrumentation and Proteomic/Glycomic strategies enable rapid identification of protein products and their Post Translational Modifications, including glycosylation. Techniques are applicable to characterisation at all stages of product development, but particularly for comparison of Biosimilar with Reference products for regulatory purposes. MS techniques alone are not enough for full physicochemical characterisation and other orthogonal methods should also be included. 27

28 ACKNOWLEDGEMENTS SGS M-Scan GmbH SGS M-Scan Ltd M-Scan HQ Group-MSRTC Ltd Dr Matthias Berg Christian Wandschneider Dr Viv Lindo (now at Medimmune) Dr Mark Taylor Dr Mark Millichip Dr Andrew Reason Prof HR Morris 28

29 THANK YOU FOR YOUR ATTENTION Fiona Greer Life Science Services Global Director, BioPharma Services Development, SGS M-Scan SGS M-Scan Ltd 3 Millars Business Centre, Fishponds Close, Wokingham Berkshire, RG41 2TZ, UK Phone: +44 (0) Fax: +44 (0) services.ltd@m-scan.com f.greer@m-scan.com Web :