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Analytical Development of Biosimilar Mabs: From Vision to Reality

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The approval of the first biosimilar mAbs in Europe last June (Celltrion’s Remsima™ and Hospira’s Inflectra™ versions of infliximab) paves the way for the advent of more widespread access to biological treatments in indications such as rheumatology and oncology. One year on, lessons learned from these approvals will guide the global regulatory framework for such complex biomolecules. Indeed, several more biosimilar mAbs are currently in late-stage clinical trials and can be expected to be submitted to Regulatory Authorities shortly. Read more or listen to live streaming http://bit.ly/SGSBiosimilarWebinar

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Analytical Development of Biosimilar Mabs: From Vision to Reality

  1. 1. SGS LIFE SCIENCE SERVICES WEBINAR ANALYTICAL DEVELOPMENT OF BIOSIMILAR MABS: FROM VISION TO REALITY June 25, 2014 Dr. Fiona M. Greer SGS Life Science Services
  2. 2. LIFE SCIENCE SERVICES OVERVIEWLIFE SCIENCE SERVICES OVERVIEW 1© SGS SA 2014 ALL RIGHTS RESERVED
  3. 3. SPEAKERSPEAKER  Dr. Fiona M. Greer Global Director for Biopharma Services D l tDevelopment SGS Life Science Services 2© SGS SA 2014 ALL RIGHTS RESERVED
  4. 4. POLL QUESTIONPOLL QUESTION  What stage of development are you currently in with your Biosimilar? (select all that apply) 3© SGS SA 2014 ALL RIGHTS RESERVED
  5. 5. ANALYTICAL AND CLINICAL DEVELOPMENT OF BIOSIMILAR mABs FROM VISION TO REALITY MEETING REGULATORYMEETING REGULATORY CHARACTERIZATION EXPECTATIONSEXPECTATIONS Dr Fiona M GreerDr Fiona M Greer Global Director, BioPharma Services Development SGS M-Scan
  6. 6. AGENDA: MEETING REGULATORY CHARACTERIZATION EXPECTATIONS  Introduction  What are the regulatory guidelines What are the regulatory guidelines associated with structural characterization and comparability/biosimilarity testing ofp y y g Biosimilars?  Wh i l ti l h t i ti i d? When is analytical characterization required?  Which techniques old & new are suitable Which techniques, old & new, are suitable  Package of analytical tools/ battery of methods  Strategies for primary and higher order structure 5© SGS SA 2014 ALL RIGHTS RESERVED
  7. 7. INTRODUCTION: REGULATORY PERSPECTIVE ON BIOSIMILAR MABS  June 2013, EMA approval of the first biosimilar mAbs in Europe (Celltrion’s Remsima™ and Hospira’s Inflectra™ versions of infliximab)  Many more biosimilar mAbs are currently in late- stage trials and can be expected to be submittedstage trials and can be expected to be submitted to Regulatory Authorities shortly  Jan 2014 Celltrion received S Korean MFDS Jan 2014, Celltrion received S. Korean MFDS approval for Herzuma, a version of Roche’s Herceptin trastuzumab  April 2014, Biocad received first approval of a biosimilar mAb in Russia for it’s rituximab, A llBi 6© SGS SA 2014 ALL RIGHTS RESERVED AcellBia
  8. 8. GLOBAL REGULATORY BACKGROUNDGLOBAL REGULATORY BACKGROUND EUROPE  2005, EMEA issued guidelines on “Similar UNITED STATES  Biologics Price Competition and Innovation Act (BPCIA) REST OF WORLD  Brazil, Australia, Turkey, Taiwan Malaysiaguidelines on Similar biological medicinal products”. Many updated since then. ( ) March 23rd 2010. New pathway-351(k) in PHS Act.  Feb 2012, FDA issued draft Guidances (2 plus Q&A). Taiwan, Malaysia, Argentina, Mexico, Japan, Canada, S.A. and others have some form of pathway.  Approved 1st Biosimilar in 2006 and now has 16 including 2 mAbs. E i d l t ( p )  April 2013, draft Guidance on Formal Meetings Between the FDA and Biosimilar Biological product p y  Some adopted EMA guides, others wrote their own.  Experienced regulatory authority.  Many non-European companies targeting this g p Sponsors or Applicants. • May 2014, 5th draft Guidance.  Oct 2009, WHO “Guideline on Evaluation of Similar Biotherapeutic Products” 7© SGS SA 2014 ALL RIGHTS RESERVED companies targeting this market. Products
  9. 9. EU REGULATIONS: ARTICLE 10(4) DIRECTIVE 2001/83/EC Overarching Guideline (CHMP/437/04). “G id li Si il Bi l i l M di i l P d t ”Defines principles “Guideline on Similar Biological Medicinal Products”Defines principles Quality General Non- clinical Clinical General guidelines Quality / Safety Efficacy IFN-Epoetin LMWHGCSFSomatropinInsulin mAbs follitropin-a IFN-bpp Product class specific data requirements p Non- clinical Cli i l Non- clinical Cli i l Non- clinical Cli i l Non- clinical Cli i l Non- clinical Cli i l Non- clinical Cli i l Non- clinical Cli i l Non- clinical Cli i l Non- clinical Cli i l 8© SGS SA 2014 ALL RIGHTS RESERVED requirements Clinical Clinical Clinical ClinicalClinicalClinical Clinical ClinicalClinical
  10. 10. US REGULATORY PATHWAY (1/2)US REGULATORY PATHWAY (1/2)  New pathway-351(k) requires comparison with a single reference product approved under the normal 351(a). The application must include information demonstrating biosimilarity based on data derived from:based on data derived from:  Analytical studies demonstrating that the biological product is “highly similar” to the reference product notwithstanding minor differences in clinicallynotwithstanding minor differences in clinically inactive components  Animal studies and  A clinical study or studies A clinical study or studies  Two basic types of Biosimilar, from two steps:  Biosimilar requires analytics (“highly similar”) Biosimilar - requires analytics ( highly similar ), preclinical & clinical (including immunogenicity) studies, any of which can be waived  Interchangeable Biosimilar switching studies 9© SGS SA 2014 ALL RIGHTS RESERVED  Interchangeable Biosimilar - switching studies
  11. 11. US REGULATORY PATHWAY (2/2)US REGULATORY PATHWAY (2/2)  FDA will consider the “totality of the data and information submitted”  Suggest the use of a “meaningful fingerprint-like analysis algorithm”  May 13 2014, draft Guidance on “Clinical Pharmacology Data to Support a demonstration of Biosimilarity to a Reference Product”. Introduces 4 categories: » Not similar » Similar » Highly similar» Highly similar » Highly similar with fingerprint-like similarity Steven Kozlowski, M.D. Director, Office of Biotechnology Products OPS/CDER/US FDA 10© SGS SA 2014 ALL RIGHTS RESERVED
  12. 12. WHEN IS ANALYTICAL CHARACTERIZATION REQUIRED 11© SGS SA 2014 ALL RIGHTS RESERVED
  13. 13. REVISED EMA QUALITY GUIDELINE: COMPARABILITY EXERCISE U t t f t l ti l th l th d Use state-of-art analytical, orthogonal methods  Comparative characterization studies should include assessment of composition physicalinclude assessment of composition, physical properties, primary and higher order structures, purity, product-related substances (e.g. isoforms) and impurities, and biological activity with “ ffi i tl iti l ti l t l ” p g y “sufficiently sensitive analytical tools”  Quantitative ranges for quality attributes establishedestablished  Use material from final process for clinical trials (i.e. avoid additional comparability exercises)(i.e. avoid additional comparability exercises)  The suitability of the formulation should be demonstrated (need not be identical) 12© SGS SA 2014 ALL RIGHTS RESERVED
  14. 14. WHAT REGULATIONS COVER PHYSICOCHEMICAL CHARACTERIZATION? ICH T i Q6B “S ifi ti T t ICH Topic Q6B “Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products” S l h i i d fi i Structural characterization and confirmation 1. Amino acid sequence 2. Amino acid composition 3. Terminal amino acid sequence3. 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 coefficient3. Extinction coefficient 4. Electrophoretic pattern 5. Liquid Chromatographic pattern 6. Spectroscopic profiles 13© SGS SA 2014 ALL RIGHTS RESERVED
  15. 15. POTENTIAL ANALYTICAL TOOLSPOTENTIAL ANALYTICAL TOOLS  Amino acid sequence and modifications: MS, peptide mapping, chromatography  Glycosylation: Anion exchange, enzymatic digestion, peptide mapping, CE, MSmapping, CE, MS  Folding: MS S-S bridge determination, calorimetry, HDX and ion mobility MS, NMR, circular dichroism, Fourier transform spectroscopy, fluorescence  PEGylation & isomers: chromatography, peptide mapping  Aggregation: Analytical ultracentrifugation, size-exclusion chromatography, field flow fractionation, light scattering, microscopymicroscopy  Proteolysis: electrophoresis, chromatography, MS  Impurities: proteomics, immunoassays, metal & solvents analysis  S b it i t ti h t h i bilit MS Subunit interactions: chromatography, ion mobility MS  Heterogeneity of size, charge, hydrophobicity: Chromatography; gel & capillary electrophoresis, light scattering, IM-MS 14© SGS SA 2014 ALL RIGHTS RESERVED
  16. 16. CASE STUDY: ANTIBODY CHARACTERIZATION  Mass spectrometry of intact t i d l d L &H h iprotein and released L &H chains  Amino Acid Composition Analysis  N- and C-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 15© SGS SA 2014 ALL RIGHTS RESERVED
  17. 17. INTACT MASS MEASUREMENT N-Linked biantennary core fucosylated with varying number of galactose residues (MONITORING GLYCOSYLATION) Fuc Man – GlcNAc Asn - GlcNAc-GlcNAc- Man Man - GlcNAc - Gal - Gal mAb +1 x G0F + 1 x G1F mAb +2 x G1F G0F Mass shift = +1444 G1F Mass shift = +162 G2F Mass shift = +324 + 1 x G1F mAb +2 x G0F mAb +1 x G1F + 1 x G2F 16© SGS SA 2014 ALL RIGHTS RESERVED
  18. 18. INTACT MASS COMPARISON OF THREE BIOSIMILAR MABS 17© SGS SA 2014 ALL RIGHTS RESERVED
  19. 19. PEPTIDE MAPPING WORKFLOWPEPTIDE MAPPING WORKFLOW 18© SGS SA 2014 ALL RIGHTS RESERVED
  20. 20. ANTIBODY ANALYSIS – GENERAL WORKFLOW 19© SGS SA 2014 ALL RIGHTS RESERVED
  21. 21. SULFHYDRYL GROUP(S) AND DISULFIDE BRIDGES 20© SGS SA 2014 ALL RIGHTS RESERVED
  22. 22. CHARACTERIZATION OF S S BRIDGESCHARACTERIZATION OF S-S BRIDGES Disulphide bridged 2567.0100 Voyager Spec #1 MC[BP = 3017.6, 2567] Disulphide bridged protein Enzymic/Chemical di ti S S SH Mixture of peptides 40 50 60 70 80 90 %Intensity KTCIVPEVSSVFIFPPKPK 252 269 S S SH E E digestion 2971.0 2989.6 3008.2 3026.8 3045.4 3064.0 Mass (m/z) 00 10 20 30 40 C SS KVTCVVVDISK 280 289 SH E E Identification by MS Followed by reduction A d f th MS Reduction And further MS 1122.8 80 90 100 Voyager Spec #1=>SM5[BP = 1662.4, 7089] 754.3 80 90 100 Voyager Spec #1=>SM5[BP = 1662.4, 7089] 1062.6 1988.1 VTCVVVDISK 280 289 TCIVPEVSSVFIFPPKPK 252 269 30 40 50 60 70 %Intensity 30 40 50 60 70 %Intensity 21© SGS SA 2014 ALL RIGHTS RESERVED 1154.0 1169.4 1184.8 1200.2 1215.6 1231.0 Mass (m/z) 20 30 1955 1970 1985 2000 2015 2030 Mass (m/z) 10 20
  23. 23. GLYCOSYLATION CHARACTERIZATIONGLYCOSYLATION CHARACTERIZATION Structural characterisation and confirmation of carbohydrate.y For glycoproteins, the following should be determined:  Carbohydrate content (neutral sugars, amino sugars and sialic acids))  Structure of the carbohydrate chains, the oligosaccharide pattern, the antennary profile, linkage  Glycosylation site(s) on the protein chain 22© SGS SA 2014 ALL RIGHTS RESERVED
  24. 24. ANALYSIS OF GLYCOSYLATIONANALYSIS OF GLYCOSYLATION N-Glycans Intact Mass by MALDI or COOH2HN S---S S---S N-Glycans O-Glycans Intact Mass by MALDI or ES MS Monosaccharide Composition Analysis (LC &Reduction Carboxymethylation MS) COOH2HN S-CM S-CMS-CMS-CM Specific Protease DigestSpecific Protease Digest PNG FPNGase F Sep-pak Monosaccharide Composition Reductive elimination 0% 20% 40% Permethylation MALDI, Nanospray MS/MS & Linkage analysis Composition Glycan Population Screening Glycan Antennary Profile Glycosylation Site 23© SGS SA 2014 ALL RIGHTS RESERVED Nanospray-MS/MS & Linkage analysis LC & MS methods Linkage Analysis
  25. 25. GLYCOMIC / GLYCOPROTEOMIC WORKFLOW Anomeric specific Intermonosaccharide Glycan C iti MALDI-MS Linkages Glycan Profile HPAEC-PAD Heterogeneity & Extent of Composition Glycan MALDI-MS Native Glycans Intact mass vs. Deglycosylated ES MS / MALDI MS Glycosylation y Sequence MALDI-MS/MS A t SampleGlycoprotein ES-MS / MALDI-MS Quantitative Monosaccharide Composition Derivatised Glycans Antennary Profile ESI-MS Glycopeptides Composition GC-MS Quantitative PMAA GC MS Inter- monosaccharide Linkages Peptide mapping Qualitative Site-specific Glycosylation HPAEC-PAD Quantitative Sialic Acid Content PMAA GC-MS Quantitative Glycan profile 24© SGS SA 2014 ALL RIGHTS RESERVED LC-ES-MS 2AB-LC-MS y p
  26. 26. OLIGOSACCHARIDE PROFILING LC- AND MS-BASED METHODS 2-AB labelling and HPLC-FLD for profiling Oligosaccharide population coupled with ESI-MS TIC chromatogram Annotations based on MS dataAnnotations based on MS data 25© SGS SA 2014 ALL RIGHTS RESERVED Example of IgG N-glycans N-acetylglucosamine Galactose Mannose Fucose N-acetylneuraminic acid N-glycolylneuraminic acid
  27. 27. CHARGE COMPARABILITYCHARGE COMPARABILITY Imaging cIEF ofImaging cIEF of Monoclonal Antibodies 26© SGS SA 2014 ALL RIGHTS RESERVED
  28. 28. BIOPHYSICAL TECHNIQUES FOR HIGHER ORDER STRUCTURE CONFORMATION ANDORDER STRUCTURE, CONFORMATION AND AGGREGATION Technique Reports on Advantages DisadvantagesTechnique Reports on Advantages Disadvantages Circular Dichroism Secondary/ Tertiary Structure Quantitative Sensitive to helix content Formulation buffers can interfere Quantitative FTIR Secondary Structure Sensitive to sheet content Less prone to buffer interfence Intrinsic Fluorescence Local Tertiary Structure Sensitive Potential for moderate HTP Qualitative Extrinsic Fluorescence Surface hydrophobicity Sensitive Ensemble tertiary structure-no local Potential for moderate HTP Qualitative UV-VIS (2ndderivative) Local Tertiary Structure Simultaneous to concentration determination QualitativeUV VIS (2 derivative) Local Tertiary Structure determination Potential for moderate HTP Qualitative Differential Scanning Calorimetry Thermal Stability Screening method for formulation (HTP) Qualitative SV-AUC Oligomers/ aggregates Matrix free, quantitative, resolution Slow, DLS HMW aggregates Sensitivity, moderate for HTP Poor resolution, qualitative 27© SGS SA 2014 ALL RIGHTS RESERVED SEC-MALS Oligomers/ aggregates Direct MW determination, rapid analysis Matrix present High shear forces
  29. 29. SUMMARYSUMMARY  The development of a biosimilar requires comprehensive physicochemical structural characterization at many stages.  Initially, batches of originator are studied to determine the exact protein sequence PTMs and variability of quality attributesprotein sequence, PTMs and variability of quality attributes. These data form the Quality Target Product Profile (QTPP).  Advances in MS instrumentation and Proteomic/Glycomic strategies enable rapid identification of QTPP including PTMsstrategies enable rapid identification of QTPP including PTMs.  At early stage, characterization surveys may help to guide choice of an appropriate cell line. Build similarity concept from start.  Various regulatory guidelines then require side-by-side comparative data to demonstrate “Biosimilarity”.  M ltiple orthogonal anal tical methods are sed to define Multiple orthogonal analytical methods are used to define “fingerprint” comparison. Strategies for primary and higher order structure determination are required.  Increasing importance on HOS to link with biological activity 28© SGS SA 2014 ALL RIGHTS RESERVED  Increasing importance on HOS to link with biological activity.
  30. 30. THANK YOU FOR YOUR ATTENTIONTHANK YOU FOR YOUR ATTENTION Life Science Services Fiona Greer Global Director, BioPharma Services Development SGS M Scan Ltd Phone: +44 (0) 118 989 6940 + 41 22 739 9548SGS M-Scan Ltd Phone: +44 (0) 118 989 6940 2-3 Millars Business Centre, Fax: +44 (0) 118 989 6941 Fishponds Close, Wokingham E-mail : fiona.greer@sgs.com Berkshire, RG41 2TZ, UK Web : www.sgs.com/biosimilars + 1 866 SGS 5003 + 65 637 90 111 + 33 1 53 78 18 79 + 1 877 677 2667 + 33 1 41 24 87 87 29© SGS SA 2014 ALL RIGHTS RESERVED + 1 877 677 2667

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