Biopharmaceutical Characterization According to ICHQ6B Harmonized Guidelines

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SGS Life Sciences’ range of services dedicated to biopharmaceutical characterization focus on the most recent technical and regulatory advances. A biologic characterization strategy designed to confirm structural and physicochemical properties will be presented in accordance with a uniform set of internationally accepted principals for characterization of new and biosimilar biopharmaceutical products (ICH Q6B).

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Biopharmaceutical Characterization According to ICHQ6B Harmonized Guidelines

  1. 1. PEGS, May 6, 2014 BIOPHARMACEUTICAL CHARACTERIZATION ACCORDING TO ICHQ6B HARMONIZED GUIDELINES Kenneth Warrington, PhD Director, Biosafety Business Development North America
  2. 2. 2 PEGS, May 6, 2014 DISCOVERY PRE SUBMISSION POST APPROVAL Discovery that you have a Bio-molecule that does something. This discovery phase is growing in the University, Small Company &Venture capital supported space Need to  Characterize molecule (chemical, biophysical)  Develop basic assays for potency, purity, identity  Cell line selection characterization and purification Proof of effectiveness, efficacy and acceptable safety profile  Certified reference material, and analytical standards (Stability)  Cell line optimization and characterization  Cell bank creation and management for future production  Validation of analytical methods for routine use and testing of submission batches, premarket stability.  Formulation development and packaging selection Regulatory approval and license to market obtained.  Routine testing with validated methods.  Management and re- characterization of reference materials & standards  Post-market stability studies The development life cycle may continue  Development of new strengths/more stable formulations, different dose formats, better analytical methods PRE-CLINICAL Extended proof of concept that the molecule is selective  Further characterization creation of reference materials  Development of fit for purpose assays  Tests in tissue culture and animal models for activity  Selection and characterization of producer cell lines  Cell toxicity, Biomarker analysis as indicators of areas and specificity of biological activity & effect  Forced degradation studies BIOPHARMACEUTICAL LIFE CYCLE
  3. 3. 3 PEGS, May 6, 2014 WHY CHARACTERIZE?  Product characterization is essential for product development and regulatory acceptance  Characterization is the basis of all knowledge and understanding of the product and it’s structure/function relationship  Understanding the product structure is key to all aspects of product and process development  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 pre- and post- approval
  4. 4. 4 PEGS, May 6, 2014  Co- and Post-Translational Modifications  Microheterogeneities  Immunogenicity WHY ARE BIOPRODUCTS A CHALLENGE? Acetylation Acylation Addition of lipid (palmitoylation) Amidation (deamidation) Carbamylation Carboxylation Formylation Gla (gamma carboxyglutamic acid) Glycosylation (N-linked, O-linked) Glycation Methylation Norleucine Phosphorylation Sulphation Proteolysis Methionine Oxidation Di-sulphide bond formation
  5. 5. 5 PEGS, May 6, 2014 WHAT REGULATIONS COVER PHYSICOCHEMICAL CHARACTERIZATION?  ICH Topic Q6B “Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products”  Structural characterization 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
  6. 6. 6 PEGS, May 6, 2014 HOW? ANTIBODY CHARACTERIZATION CASE STUDY  Typical analyses performed  Mass spectrometry of intact protein & released L &H chains  Amino Acid Composition Analysis  N-terminal sequencing  Peptide “MAPPING” Analysis (Sequence coverage: 100% LC and 100% HC)  Monosaccharide & sialic acid analysis  Oligosaccharide population analysis  SDS-PAGE analysis  Circular Dichroism  Analytical Ultracentrifugation
  7. 7. 7 PEGS, May 6, 2014 INTACT MASS MEASUREMENT
  8. 8. 8 PEGS, May 6, 2014 N-Linked biantennary core fucosylated with varying number of galactose residuesIgG Fuc Man – GlcNAc Asn - GlcNAc-GlcNAc- Man Man - GlcNAc - Gal - Gal Mab +2 x G0F Mab +1 x G0F + 1 x G1F Mab +2 x G1F Mab +1 x G1F + 1 x G2F G0F Mass shift = +1444 Da G1F Mass shift = +162 Da G2F Mass shift = +324 Da INTACT MASS: MONITORING GLYCOSYLATION
  9. 9. 9 PEGS, May 6, 2014 LC-MS: Heavy and light chain analysis REF STD BATCH 2 BATCH 1  Additional peak in development material  Mass 128 Da heavier than major HC component (GOF)  Basic from cIEF confirmed on C-terminal INTACT MASS: MONITORING MODIFICATIONS
  10. 10. 10 PEGS, May 6, 2014 +128Da + Lysine at C-terminus +162Da + Glycation ON-LINE LC/ES-MS MASS MEASUREMENT INTACT MASS: MONITORING MODIFICATIONS
  11. 11. 11 PEGS, May 6, 2014 ON-LINE LC/ES-MS TOTAL ION CURRENT INTACT MASS: MONITORING MODIFICATIONS
  12. 12. 12 PEGS, May 6, 2014 INTACT MASS: MONITORING MODIFICATIONS Light chain Light chain SS Bridged to Glutathione Light chain-Cysteinylated ON-LINE LC/ES-MS MASS MEASUREMENT
  13. 13. 13 PEGS, May 6, 2014 S S SH Disulphide bridged protein E E E Enzymic/Chemical digestion S S SH Mixture of peptides Identification by MS Followed by reduction And further MS CHARACTERIZATION OF S-S BRIDGES
  14. 14. 14 PEGS, May 6, 2014 2971.0 2989.6 3008.2 3026.8 3045.4 3064.0 Mass (m /z) 0 2567.0 0 10 20 30 40 50 60 70 80 90 100 %Intensity Voyager Spec #1 MC[BP = 3017.6, 2567] 3048.7 1154.0 1169.4 1184.8 1200.2 1215.6 1231.0 Mass (m /z) 1122.8 20 30 40 50 60 70 80 90 100 %Intensity Voyager Spec #1=>SM5[BP = 1662.4, 7089] 1955 1970 1985 2000 2015 2030 Mass (m /z) 754.3 10 20 30 40 50 60 70 80 90 100 %Intensity Voyager Spec #1=>SM5[BP = 1662.4, 7089]1062.6 1988.1 VTCVVVDISK 280 289 TCIVPEVSSVFIFPPKPK 252 269 KTCIVPEVSSVFIFPPKPK KVTCVVVDISK 252 269 280 289 Reduction CHARACTERIZATION OF S-S BRIDGES
  15. 15. 15 PEGS, May 6, 2014 MAPPING WORKFLOW
  16. 16. 16 PEGS, May 6, 2014 ANTIBODY ANALYSIS – GENERAL WORKFLOW
  17. 17. 17 PEGS, May 6, 2014 Q-TOF MS/MS of 785 [M+2H]2+ MS/MS AMINO ACID SEQUENCING
  18. 18. 18 PEGS, May 6, 2014  2 basic types of glycosylation normally observed.  N-linked to the amide of Asparagine (Asn) in the consensus sequence …Asn-X-Ser/Thr…where X is any AA except Pro.  O-linked to the hydroxyl functions of Serine (Ser) or Threonine (Thr).  The populations of sugars attached to an individual protein will depend on the cell type in which the protein is expressed and on the physiological status of the cell  Glycoproteins are mixtures of glycoforms i.e. the same polypeptide but different glycans PROTEIN GLYCOSYLATION
  19. 19. 19 PEGS, May 6, 2014 PROTEIN GLYCOSYLATION
  20. 20. 20 PEGS, May 6, 2014 ICH Topic Q 6 B Structural characterization and confirmation 6. Carbohydrate structure “For glycoproteins, the carbohydrate content (neutral sugars, amino sugars and sialic acids) is determined. In addition, the structure of the carbohydrate chains, the oligosaccharide pattern (antennary profile) and the glycosylation site(s) of the polypeptide chain is analysed, to the extent possible” WHAT REGULATIONS COVER GLYCOSYLATION CHARACTERIZATION?
  21. 21. 21 PEGS, May 6, 2014 COOH2HN S---S S---S N-Glycans O-Glycans Intact Mass by MALDI or ES MS Monosaccharide Composition Analysis (LC & MS) Reduction Carboxymethylation COOH2HN S-CM S-CMS-CMS-CM Reductive elimination Specific Protease Digest PNGase F Sep-pak 0% 20% 40% Permethylation MALDI, Nanospray-MS/MS & Linkage analysis LC & MS methods Monosaccharide Composition Glycan Population Screening Glycan Antennary Profile Glycosylation Site Linkage Analysis ANALYSIS OF GLYCOSYLATION
  22. 22. 22 PEGS, May 6, 2014 Column: CarboPac PA10 Eluent: 18 mM Sodium hydroxide Flow Rate: 1.5 mL/min Detection: Pulsed amperometry, gold electrode Sample: MAb 2 M TFA Hydrolysate Peaks: 1. Fucose 2. Rhamnose 3. Glucosamine 4. Galactose 5. Mannose 1 2 3 4 5 Minutes 0 10 20 30 nC MONOSACCHARIDE COMPOSITION ANALYSIS BY HPAEC-PAD
  23. 23. 23 PEGS, May 6, 2014 Column: CarboPac™ PA-100 Eluent: 0–250 mM Sodium acetate over 110 min in 100 mM Sodium hydroxide Flow Rate: 1 mL/min Detection: Pulsed amperometry, gold electrode Minutes 25 150 nA 25 190 nA 49% 35% 10% 19% 35% 14% 28% A B 1 2 3 4 1 2 3 5 0 10 20 30 40 50 25 300 nA 44% 37% 10% C 1 2 3  Possibility of semi-quantitative analysis  Isomers could, in very specific conditions, be separated  Possibility of batch to batch comparison N-glycan population profiling analysis of three different antibodies OLIGOSACCHARIDE POPULATION ANALYSIS BY HPAEC-PAD
  24. 24. 24 PEGS, May 6, 2014 OLIGOSACCHARIDE POPULATION ANALYSIS BY MALDI-TOF MS From CFG data (http://functionalglycomics.org)
  25. 25. 25 PEGS, May 6, 2014 FLD chromatogram TIC chromatogram  One molecule of N-glycan = one tag (response independent from N-glycan structural features)  Isomers could, in very specific conditions, be separated  Possibility of batch to batch comparison based on profile  Glycan structural identification could be obtained through coupling with MS 2-AB labelling and HPLC-FLD for profiling Oligosaccharide population Example of IgG N-glycans OLIGOSACCHARIDE PROFILING: LC- AND MS-BASED METHOD
  26. 26. 26 PEGS, May 6, 2014 TIC chromatogram Annotations based on MS data 2-AB labelling and HPLC-FLD for profiling Oligosaccharide population coupled with ESI-MS Example of IgG N-glycans OLIGOSACCHARIDE PROFILING: LC- AND MS-BASED METHOD
  27. 27. 27 PEGS, May 6, 2014 From Dell et al. Comprehensive Glycoscience, 2006 GLYCAN ANTENNAE PROFILING ANALYSIS BY MS/MS
  28. 28. 28 PEGS, May 6, 2014 Sample: Fetuin N-glycans linkage Acquired on: 17-Sep-2002 at 11:56:11 Job No: MS02 Sample No: MS02M-Scan Ltd. 10.000 11.000 12.000 13.000 14.000 15.000 16.000 17.000 18.000 19.000 20.000 rt0 100 % 16.752 14.001 13.171 13.471 14.941 Scan EI+ 117+118+129+159 3.33e6 RT FETNLIN t-Gal 2-Man 3-Gal 6-Gal 3,6-Man 4-GlcNAc 2,4-Man 4,6-GlcNAc LINKAGE ANALYSIS BY GC-MS
  29. 29. 29 PEGS, May 6, 2014 Imaging cIEF of Monoclonal Antibodies COMPARABILITY ON BASIS OF CHARGE
  30. 30. 30 PEGS, May 6, 2014  Spectroscopic method measuring the absorption of left and right handed circularly polarized light  Information on secondary structure such as α-helices and sheets HIGHER ORDER STRUCTURE: CD Far UVNear UV Monitor unfolding in presence of heat or denaturants 260-190nm320-250nm
  31. 31. 31 PEGS, May 6, 2014 Measures melting points (Tm’s) of IgG regions Good indicator of thermal stability HIGHER ORDER STRUCTURE: DSC
  32. 32. 32 PEGS, May 6, 2014  Common problem encountered during manufacture and storage of proteins  Undesirable due to potential immunogenicity (small aggregates) or problems with administration (large aggregates)  Regulatory authorities requesting Size Exclusion Chromatography (SEC) plus a column free technique such as Analytical Centrifugation (AUC) or Dynamic Light Scattering (DLS) to cross check data obtained from SEC as aggregates can potentially be lost by non-specific binding to an SEC column  Field Flow Fractionation (FFF) is also being used increasingly for analysis of protein aggregates PROTEIN AGGREGATION
  33. 33. 33 PEGS, May 6, 2014 Combination of UV, RI and MALS detection allow an overview of the aggregation state. Advantages Easy method to establish High throughput Straightforward data analysis Good resolution. Minimal sample preparation required Disadvantages Potential for loss of aggregates by non specific binding to the column Also potential for breaking aggregates during significant dilution effect following injection MONITORING AND QUANTIFYING AGGREGATION SEC-MALS: SIZE EXCLUSION CHROMATOGRAPHY WITH MULTI-ANGLE LASER LIGHT SCATTERING
  34. 34. 34 PEGS, May 6, 2014 Matrix free platform to qualify aggregation based in the interaction of scattered light with molecules of different size. Monitors the size of molecules and presence of hydrodynamic species other than the monomer (aggregates) with high molecular weight Advantages Non invasive/ Matrix free (avoidance of loss of aggregates by non specific binding to the column). Great sensitivity (trace concentrations) No sample prep required (only buffer filtration) Potential for moderate throughput (screening) Disadvantages Qualitative Lack of specificity Poor resolution MONITORING AND QUANTIFYING AGGREGATION 0 1 2 3 4 5 6 7 8 0.01 0.1 1 10 100 1000 10000 Intensity(%) Size(d.nm) Size Distribution by Intensity Record 13: 100734 1 Record 14: 100734 2 Record 15: 100734 3 Size (radius nm) Distribution by Volume Formulation C - 45 days Intensity(%) 0 2 4 6 8 10 12 14 16 18 0.01 0.1 1 10 100 1000 10000 Volume(%) Size(d.nm) Size Distribution by Volume Record 4: 100732 1 Record 5: 100732 2 Record 6: 100732 3 Size (radius nm) Distribution by Intensity Formulation A Formulation B Formulation C - 0 days Intensity(%) DLS: DYNAMIC LIGHT SCATTERING
  35. 35. 35 PEGS, May 6, 2014 SV-AUC provides a matrix free platform to quantify aggregation Monitors concentration distribution of solutes (migration pattern) in a centrifuge cell as a function of radius at different times Advantages Matrix free (avoidance of loss of aggregates by non specific binding to the column). Good resolution. No sample preparation required. Disadvantages Low throughput. Complicated data analysis. 5 10 15 20 0.0 0.5 1.0 1.5 2.0 85.05 ± 0.35% C(s)distribution Sedimentation coeficient (s) Cell 1 Cell 2 Cell 3 14.95 ± 0.35% Formulation A MONITORING AND QUANTIFYING AGGREGATION SV-AUC: ANALYTICAL ULTRACENTRIFUGATION SEDIMENTATION VELOCITY
  36. 36. 36 PEGS, May 6, 2014  Analytical characterisation is essential throughout all stages of biopharmaceutical development.  Advances in MS instrumentation and Proteomic/Glycomic strategies enable rapid identification of protein products and their PTMs, including glycosylation.  MS techniques alone are not enough and other orthogonal methods should also be included. SUMMARY
  37. 37. 37 PEGS, May 6, 2014 LABORATORY SERVICES - FROM BIOMARKERS TO BATCH ANALYSIS -
  38. 38. 38 PEGS, May 6, 2014 BIOPHARMACEUTICAL CHARACTERIZATION
  39. 39. 39 PEGS, May 6, 2014 LABORATORY SERVICES DETAIL OF BIOPHARMACEUTICAL ANALYSIS
  40. 40. 40 PEGS, May 6, 2014 Life Science Services Kenneth Warrington, Jr., PhD Director, Biosafety Business Development North America Phone: +1 (716) 796 4595 E-mail : kenneth.warrington@sgs.com Web : www.sgs.com/lifescience THANK YOU FOR YOUR ATTENTION

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