Biologics Biopharmaceutical Testing Bioanalysis and Immunogenicity Biologics Characterization

Biologics Biopharmaceutical Testing Bioanalysis and Immunogenicity Biologics Characterization Quality Control and GMP Batch Release Typology of Analytical Projects §Biosimilars (proteins and monoclonal antibodies) §Innovative proteins, monoclonal antibodies and antibodies drug conjugates §Therapeutic peptides: innovative, generic and hybrid formulations §Polysaccharides: heparins and others §Non-living vaccines 01/04/2013 KYMOS 2

Biologics BIOANALYSIS & IMMUNOGENICITY 01/04/2013 KYMOS 3

Biologics bioanalysis Pharmacokinetic studies of peptides and proteins (EMA guideline on Clinical Investigation of the Pharmacokinetic of Therapeutic Proteins) Ø Development and validation of bioanalytical methods ü HPLC-MS/MS: triptic digestion of the protein and bioanalysis of characteristic peptides ü Immunoassay: § ECLA: electrochemiluminiscence by Meso. Scale Discovery § ELISA: direct, sandwich, bridge, competitive using different detection techniques (colorimetric, fluorescence, time resolved fluorescence, luminiscence). § RIA: radioimmunoassay testing using I 125 Ø Determination of drug levels in preclinical and clinical samples (phase I to III) 01/04/2013 KYMOS 4

Biologics immunogenicity Immunogenicity studies of proteins and peptides (EMA and FDA guidelines on Immunogenicity of Therapeutic Proteins) STUDIES Ø Determination of Binding Antibodies (Screening, confirmatory, titration) Ø Neutralizing Antibodies Ø Antibody Typology Ø Cellular Immune Response TECHNIQUES Ø Immunoassays (ECLA, RIA, ELISA) Ø Cell based assays (Fluorescence / Luminescence, Flow cytometry) Ø Surface plasmon resonance (SPR / Biacore) 01/04/2013 KYMOS 5

Biologics Added value services: • Studies phase I to III • Animal housing management in preclinical studies • Production of polyclonal antibodies as reagents for assays • Synthesis, purification and quality control of tracers and radiotracers • PK calculations, data analysis and reporting • e. CTD format if required 01/04/2013 KYMOS 6

Biologics BIOLOGICS CHARACTERIZATION QUALITY CONTROL & GMP BATCH RELEASE 01/04/2013 KYMOS 7

Biologics protein characterization OPTION A – TESTING SERVICE focused on analytical results In this option KYMOS performs individual analytical methods, under GLP or GMP if necessary, following four possible scenarios: a) Compendial Pharmacopoeia method b) CLIENT’s transferred method c) KYMOS’ standard method adapted to CLIENT’s product d) Specific method developed for CLIENT OPTION B – SERVICE PACK focused on the knowledge of the molecule In this option KYMOS provides a full knowledge of a particular aspect of the molecule in an orthogonal analytical approach, in different stages: 1 st stage General quotation 2 nd stage Proposal based on the information provided by the client, available bibliography and preliminary testing. Go / No go milestone 3 th stage Full testing, according to Proposal 01/04/2013 KYMOS 8

Biologics protein characterization PACK 1. - GENERAL IDENTIFICATION AND QUANTIFICATION Scope: General overview of the biomolecule Content: § Identification: Western Blot, PAGE, SDS-PAGE, Bioanalyzer, IEF § Purity & Impurities: PAGE, SDS-PAGE, Bioanalyzer, IEF § Quantification: Extinction Coeficient, Bradford, Lowry, BCA § Amino-acid Analysis: HPLC Acc. Q-Tag® (Waters) and OPA® (Agilent) § Antibody isotyping 01/04/2013 KYMOS 9

Biologics protein characterization PACK 2. - PRIMARY STRUCTURE AND POST TRANSLATIONAL MODIFICATIONS Scope: Confirmation of the sequence (and similarity) of a protein in all stages of product and process life cycle. Content: § Intact Mass: ESI-MS and MALDI-TOF § Peptide Mapping: digestion and peptide profile by HPLC-UV § Protein Sequencing: digestion and peptide sequencing by HPLC/MS-MS (QTOF, Q-TRAP) § N- and C- Terminal Sequence: MALDI-TOF ISD and EDMAN Degradation § Disulfide Bonds: HPLC-UV and HPLC-MS reductive and non-reductive § Aminoacid Modifications by HPLC-UV and HPLC-MS/MS § Glycosylation and Phosphorylation Sites by HPLC-UV, HPLC-MS/MS, ICP-MS § Glycosylation Profile by HPLC-FLD, HPLC-MS, HPLC-PAD (structural charactherization of glycans outsourced) 01/04/2013 KYMOS 10

Biologics protein characterization PACK 3. - HIGHER ORDER STRUCTURE Scope: Advanced characterization of secondary, tertiary and quaternary structure of a protein. Content: § Secondary and Tertiary structure: helix alpha, beta sheets, and others by Circular Dichroism, Fluorescence, UV, FTIR, § Quaternary: protein aggregates by AUC (outsourced) TO BE CONTRACTED AS TESTING SERVICES. NOT YET AVAILABLE 01/04/2013 KYMOS 11

Biologics protein characterization PACK 4. - PROCESS IMPURITIES Scope: Support to bioprocess development Content: § Host Cell Proteins: qualification of ELISA commercial kits (up to phase II) § Chemical contaminants by HPLC: kanamycin, ampicillin, PEG, polysorbates, cyanogen bromide, metotrexate, formamide, IPTG, tensioactives, … § Elemental Impurities: AAS, ICP/MS § DNA: colorimetric commercial kits § Virus and Mycoplasma Content: qualification of PCR commercial kits § Endotoxins: colorimetric test or gel-clot test Note: HCP specific assay for phase III requirements if antigen available. 01/04/2013 KYMOS 12

Biologics protein characterization PACK 5. - IDENTITY, CONTENT AND PROTEIN RELATED IMPURITIES Scope: Chromatographic characterization of target protein and related impurities Content: § Identification and Quantification of protein by chromatography: RP-HPLC, affinity cromatography, SEC-HPLC, … § Truncated and Aggregates: SEC/HPLC § Deamidated and Oxidated species: RP/HPLC, IEX/HPLC with UV and MS detectors § Glycosylation modifications by HPLC-FLD, HPLC-MS, HPLC-PAD 01/04/2013 KYMOS 13

Biologics protein characterization PACK 6. - BIOLOGICAL ACTIVITY Scope: Determination of protein functionality and potency Content: § Binding studies: Surface Plasmon Resonance by BIACORE § Cell Based Assay (proliferation, inhibition, etc. ): standard methods or transferred methods from the client (development of specific methods outsourced) 01/04/2013 KYMOS 14

Biologics protein characterization PACK 7. - BATCH TESTING FOR RELEASE Scope: Development and validation of quality control methods for batch release of API and FDF. Routine sample analysis for batch release. Content: § Appearance and description § Identity § Purity and Impurities § Potency § Quantification § General tests: p. H, osmolarity, visible and subvisible particles, elemental impurities, others (case by case) § Endotoxins and Sterility 01/04/2013 KYMOS 15

Biologicals vaccine characterization Characterization of vaccines (non living vaccines, proteins and allergens) ØAntigen characterization (FDA Guideline for Vaccines or related Products) ØAdjuvant characterization (EMA Guideline on Adjuvants in Vaccines for Human Use) ØDetermination of preservatives and other excipients Characterization of In-House Reference Preparations (IHRP) Potency assay of vaccines ØIn vivo immunization in different species ØRelevant antigen determination by immunoassays ØActivity determination by competitive immunoassays 01/04/2013 KYMOS 16

Biologicals Equipment ü ü ü ATKA Fast protein liquid chromatography system WIZARD gamma counter for RIA assays VICTOR 2 multilabel counter for ELISA assays SECTOR Imager 2400 Meso Scale Discovery for ECLA assays Guava PCA-96 flow cytometer for cell assays Electrophoresis: one and two dimensions, Western Blot Isogen Proxima Imaging System for electrophoresis data processing Pharmacia Phast. System High Speed Electrophoresis Symbiosis Pharma On line solid phase extraction system Agilent 2100 Bioanalyzer: microfluidics-based platform for cell, DNA, RNA and protein analysis Agilent 3100 Offgel fractionator for protein purification based in isoelectric focusing 01/04/2013 KYMOS ü LC-MS/MS AB SCIEX API 3. 000, 3. 200, 4. 000 Qtrap and 5. 500 Qtrap ü ICP/MS Agilent 7. 700 ü UHPLC: Agilent 1200 and Waters Acquity ü HPLC coupled to UV (diode array), fluorescence, refraction index, electrochemical and light scattering ü Tempo MDLC two dimensions nano HPLC and nano electrospray interface ü Zephyr Liquid Handler for automatic samples processing Other available in the PCB / UB: ü ü ü Agilent Capillary Electrophoresis ABSciex 4800 Maldi-TOF/TOF Micromass (Waters) QTOF Ultima Global 800 -300 Mhz NMR systems Biacore. T 100 SPR Jasco J-815 CD Spectropolarimeter 17