Setting Biological Process Specifications


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This presentation from IVT Network's Method Validation Conference covers required and suggested regulations and guidances for biological process specifications. It also covers dosage form considerations and specifications for other components.

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Setting Biological Process Specifications

  1. 1. Setting Biological Product Specifications David Lin, Ph.D. Senior Consultant Biologics Consulting Group IVT Method Validation October 15, 20082010 by Biologics Consulting Group. All rights reserved. Reproduction in part or in whole without written permission is strictly prohibited.
  2. 2. Biological vs. ChemicalPharmaceutical Products Protein Products Chemical Products
  3. 3. Biological vs. Chemical Pharmaceutical ProductsSignificant Differences In: Raw Materials Production Processes Handling Conditions Formulations Methods of Analysis Physiochemical Characteristics Stability Profile Storage Conditions Expiration Dating
  4. 4. Guidances/Guidelines ICH Q5A Viral Safety Evaluation of Biotechnology Products Derived From Cell Lines of Human or Animal Origin, Sep 1998 ICH Q5B Quality of Biotechnological Products: Analysis of the Expression Construct in Cells Used for Production of r- DNA Derived Protein Products, Feb 1996 ICH Q5C Quality of Biotechnological Products: Stability Testing of Biotechnological/Biological Products, Jul 1996 ICH Q5D Quality of Biotechnological/Biological Products: Derivation and Characterization of Cell Substrates Used for Production of Biotechnological/Biological Products, Sep 1998 ICH Q5E Comparability of Biotechnological/ Biological Products Subject to Changes in Their Manufacturing Process, Jun 2005
  5. 5. Guidances/Guidelines ICH Q6A Guidance on Q6A Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances, Dec 2000 ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/ Biological Products, Aug 1999 ICH Q8 Pharmaceutical Development, May 2006 ICH Q8(R) Pharmaceutical Development Revision, Jun 2009 ICH Q9 Quality Risk Management, Jun 2006 Q10 Pharmaceutical Quality System, Apr 2009
  6. 6. FDA Guidances Withdrawn in May-June 2006 Submission of CMC Information for Synthetic Peptides, Nov 1994 Format and Content of the CMC Section of an Application, Feb 1987 Submitting Documentation for the Stability of Human Drugs and Biologics, Feb 1987 Stability Testing of Drug Substances and Drug Products, Jun 1998 Analytical Procedures and Method Validation - CMC Documentation, Aug 2000 BACPAC I: CMC Documentation, Feb 2001 Drug Product: CMC Information, Jan 2003 Drug Substance: CMC Information, Jan 2004
  7. 7. Definition of Specification“A list of tests, references to analytical procedures,and appropriate acceptance criteria which arenumerical limits, ranges, or other criteria for thetests described. It establishes the set of criteria towhich a drug substance, drug product, or materialsat other stages of its manufacture should conformto be considered acceptable for its intended use.Conformance to specification means that the drugsubstance and drug product, when testedaccording to the listed analytical procedures, willmeet the acceptance criteria. Specifications arecritical quality standards that are proposed andjustified by the manufacturer and approved byregulatory authorities as conditions of approval.”ICH Q6B Guidance, Aug. 1999
  8. 8. Definition of Acceptance Criteria “Numerical limits, ranges, or other suitable measures for acceptance of the results of analytical procedures which the drug substance or drug product or materials at other stages of manufacture should meet.”ICH Q6B Guidance, Aug. 1999
  9. 9. Specifications  Which guidance is most relevant, Q6A or Q6B?  If synthetic, follow Q6A  Low MW  Ifbiologically source (e.g. recombinant), follow Q6B  Higher MW
  10. 10. Specifications  Q6A allows for skip testing  Q6A allow for parametric release  Monitor sterilization cycle instead of performing sterility testing  Sterilization process validation and demonstration of control critical
  11. 11. Specifications  Q6A references Q3A for establishing impurity levels in peptide drug substance, Q3C for residual solvents and Q3B for the drug product  Q6A does not discuss bioassay but Q6B discusses potency as a measure of biological activity
  12. 12. Product attributes Lead to the specifications Lead to the method development requirements
  13. 13. Purpose of Specifications  One part of control strategy to ensure quality and consistency  Subset of product characterization and serves as surrogate of full product characteristics  Complements product understanding derived from process control, in-process testing, raw material control, stability testing
  14. 14. Establishing RelevantSpecifications  Characterization  Physicochemical properties  Biological activity  Immunochemical properties  Purity  Impurities  Datafrom development and after process changes
  15. 15. Product Attributes  Chemical  Physical  Microbiological  Biological  Performance  Relate to:  Efficacy  Safety  Quality
  16. 16. Method Measures Detects Cell-based potency Biological Activity Overall integrity of the molecule Ligand binding SDS-PAGE Molecular Weight Subunit molecular mass Reduced and non- Fragmentation reduced Covalent crosslinking MALDI-TOFUsed with Permission from Jay Schrier, 2004 RP-HPLC, HIC-HPLC Surface Hydrophobicity Chemical and conformational variants IEF Net Surface Charge Charge variants ELISA Antibody Recognition Epitope binding site integrity RIA Specific contaminants Western Blot Immunogenicity Surface Plasmon Antigenicity Resonance Peptide Map Composition Primary Structure variants Amino Acid Composition Post-translational Primary structure integrity N and C terminal modifications Integrity of PTMs sequence CD (Far UV) Optical Activity Secondary Structure UV Absorbance Aromatic Amino Acids Tertiary Structure Fluorescence Side Chains CD (Near UV) SEC HPLC Hydrodynamic Radius Molecular size Analytical Conformational changes Ultracentrifugation Aggregation/dissociation Light Scattering Radius of Gyration Molecular size Aggregation/dissociation NMR Nuclear magnetic energy Complete molecular structure X-ray Cystallography Transitions X-ray diffraction
  17. 17. Protein Structural ElementsPrimary = Covalent or chemical structure.Completely defined by the amino acid sequenceplus any disulfide bonds, and any post-translationalmodifications (e.g. glycosylation, phosphorylation)Secondary = Periodic structures within the overallconformation (e.g. alpha helix, various beta-structures)Tertiary = Total folding pattern of the polypeptidechain(s)Quaternary = Association of subunits of polypeptidechains
  18. 18. Functional Characterization  Potency provides a different level of analytical information on the structural integrity of the molecular entity  Assessment of product potency typically requires in vitro and/or in vivo bioassays  A bioassay may directly reflect the mechanism of action of the product, or it may be a surrogate functional assessment (e.g. if MOA is not known, or is not able to be replicated in a potency assay)  R&D likely used a bioassay to facilitate discovery of the molecular entity; it may serve as a starting point for development of a potency assay
  19. 19. Bioassay To assess the activity/potency of the protein molecule To serve as measurement of the biological activity and structural integrity (e.g., correct conformation) of the protein molecule
  20. 20. What is a Bioassay? Ana T. Menendez, Ph.D. Director of Bioassays and Biosafety Testing Cardinal Health Any biological activity that can demonstrate that the product that was expressed by the host cell or organism is going to perform as it should. The activity can be cellular, for example:  can the product kill cancer cells?  can the product make cells grow?  can the product stimulate cells to produce cytokines? The activity can be microbiological, for example:  can the product cause an immune response?  can the product lyse bacteria?  can the product prevent virus from replicating in cells?
  21. 21. Types of Bioassays Ana T. Menendez, Ph.D. Director of Bioassays and Biosafety Testing Cardinal Health  Cellular:  Cell growth or inhibition  Cell lysis  Angiogenesis induction or inhibition  Induction of cytokines  Differentiation  Bacterial  Immunogenic response  Cell lysis or inhibition  Viral  Enzymatic  Immunological
  22. 22. Why are Cell-Based Bioassays Differentthan other Analytical CMC Assays? Ana T. Menendez, Ph.D. Director of Bioassays and Biosafety Testing Cardinal Health Involve recurring consistent source of live organisms Need Master and Working Cell Banks Cell lines require characterization Biological reagents require qualification Results are mostly calculated on 4-parameter curve CVs and %Accuracy are less stringent than in analytical techniques
  23. 23. IND Development FDA Guidance for Phase I Studies (11/1995)  “Validation data and established specifications ordinarily need not be submitted at the initial stage of drug development”  “However, for well characterized biologicals preliminary specifications and additional validation data may be needed….. to ensure safety in Phase I” FDAGuidance for Phase 2 and Phase 3 Studies, CMC Information (5/2003)  Acceptance criteria refined based on batch analyses  Limits on new impurities based on manufacturing experience, stability data and safety  Data from stress studies crucial
  24. 24. In-Process Specifications  Confirm consistency by measuring at critical process steps  Action limits or acceptance criteria?  Safety attributes such as adventitous agents should be based on acceptance criteria  Testing results could serve to eliminate testing of drug substance or drug product  Internal action limits serves as indicator of potential consistency issue or control issue
  25. 25. Raw Materials and Excipients  Meet quality standards for intended use  Raw materials for production require determination of adventitious agents  Extractables/leachables from purification resin materials  Minimum standards conform to pharmacopoeia
  26. 26. Containers and Closures  Extractables/leachables not just from the primary container and closure, but also any delivery system required for administration  Product formulation specific evaluation is needed  Equivalent materials based on pharmacopoeia standards might not be adequate for specific formulation
  27. 27. Why Worry About Extractables and Leachables?27
  28. 28. Areas of Concern Toxicity Carcinogenicity Immunogenicity Product quality28
  29. 29. Required by Regulation 21 CFR 211.65(a) – Equipment 21 CFR 600.11(b) - Equipment 21 CFR 211.94(a) - Drug product container closures 21 CFR 600.11(h) – Containers and closures29
  30. 30. Regulatory Citations  Evans Vaccine (2003)  The inspection noted the lack of filter extractable validation studies on filtered […..] monovalent and trivalent bulks  Similasan AG (August 2005)  “Further it is unclear to us whether you have conducted filter extractable and leachable testing with product. If you have this data, provide it to us. If you do not, let us know when you will be able to provide it to us.”30
  31. 31. Regulatory Citations  Wyeth (2006)  “Your previous investigation into various unknown peaks occurring in your drug products had identified phenol as a packaging extractable originating from ink used to print package inserts. However your firm later identified the unknown peak as Caprolactarn, an extractable that potentially originated from Nylon components used to pack the drug”  GTC Biotherapeutics (2009)  “There were no leachable and extractable testing performed for --b(4)--- materials used in buffer preparation. “31
  32. 32. Public Health Notifications  PVCdevices containing plasticizer DEHP (2002)  AlertsandNotices/PublicHealthNotifications /UCM062182  IV bags, blood bags, infusion tubing, etc.  BPA in food (2010)  thFocus/ucm064437.htm32
  33. 33. Differences in Safety Consideration for Biologics  Proteins are large molecules with complex configurations that are affected by E&Ls  Larger surface for interactions with E&Ls  Product administered in high dose so total E&L exposure is higher  Lower molar concentration of protein in product  Exposure to different materials during manufacturing33
  34. 34. FDA Container-Closure DataRequirements for Biological ProductsInformation to support the container and closurepackaging used with bulk biological products isrequired to be included in the FDA submission(rather than simply referenced, as with traditionaldrugs) because there is a greater potential foradverse effects on the identity, strength, quality,purity and potency of biologics and proteinproducts during storage or shipping.FDA Guidance: Container-Closures for Packaging Human Drugs andBiologics, Questions/Answers (2002)
  35. 35. What Tests to Perform?35
  36. 36. Primary Considerations  Information from supplier  Has supplier intended use been modified  Consider existing databases  Understand chemistry of materials  Start with compendial tests  Need to justify if relevant to specific use  Don’tfocus on just organics  Equipment reuse (i.e., column resins, filters, etc.) Be realistic!!!36
  37. 37. Tests for Protection  USP <671> Container – Performance Testing  Light Transmission  Water Vapor Permeation  Dye Ingression Studies  Microbial Ingression Studies37
  38. 38. USP <660> Containers - Glass  Water Attack  Extraction for release of alkali  Arsenic  1 ppm38
  39. 39. USP <661> Containers - Plastics  Extraction  Nonvolatileresidue  Residue on Ignition  Heavy Metals  Buffering Capacity (for liquid products)  Total Terephthaloyl Moieties (for PET & PETG)  Ethylene Glycol (for PET & PETG)39
  40. 40. USP <381> Elastomeric Closures for Injections  Extraction  Turbidity  Heavy Metals  Reducing Agents  pH Change  Total Extractables40
  41. 41. Program Approach  Extract  Identify  Assess the risk  Develop the Method  Validate the Method in Drug Product  Perform Leachable Study (Stability)41
  42. 42. Identify  Analytical testing  TOC  UV  GC/FID, GC/MS, GC/IR  LC/UV, LC/MS, LC/NMR  FTIR  pH  Conductivity  ICP/MS  Work with vendor42
  43. 43. Risk Assessment  Literaturesearch  Biological testing  In-vitro - USP <87>  In-vivo - USP <88>  Effect on release/stability methods43
  44. 44. 44 From BioPharm International, Dec. 2002, Miller
  45. 45. “OVERVIEW OF EXTRACTABLES AND LEACHABLES INPROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASESTUDIES” PROBLEMS ASSOCIATED WITH LEACHABLES  Increase in drug product impurities  Interaction with active ingredient, vehicle or excipients  May cause toxicity of a drug product  Interference with drug product assays  Interference with medical diagnostic tests Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
  46. 46. “OVERVIEW OF EXTRACTABLES AND LEACHABLES INPROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASESTUDIES” FDA - CASE STUDY #1 Process Change: Lyophilized to liquid formulation Source: release of divalent metal cation from rubber stopper Mechanism: activation of a contaminating metalloproteinase in the product caused product degradation Impact: Increase in protein degradation Resolution: chelator added to formulation buffer Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
  47. 47. “OVERVIEW OF EXTRACTABLES AND LEACHABLES INPROTEIN THERAPEUTICS: SOURCES, METHODS, ANDCASE STUDIES” FDA - CASE STUDY #2  Container closure: prefilled syringe  Tungsten wires are used perforate the syringe barrel during syringe manufacturing  Source: release of tungsten oxide from the syringe into the product  Impact: increase in protein oxidation followed by aggregation  Resolution:switch to tungsten-free wires to perforate syringe barrels Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
  48. 48. “OVERVIEW OF EXTRACTABLES AND LEACHABLES INPROTEIN THERAPEUTICS: SOURCES, METHODS, ANDCASE STUDIES” FDA - CASE STUDY #3 Process Change: lyophilized product changed to a lower dosage form Impact:decrease in protein stability at room temperature after reconstitution Hypothesis: leachables from rubber stopper at increased ratio of leachables to the protein cause for instability Resolution: product storage temperature changed from controlled room temperature to 2-8 C Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
  49. 49. “OVERVIEW OF EXTRACTABLES AND LEACHABLES INPROTEIN THERAPEUTICS: SOURCES, METHODS, ANDCASE STUDIES” FDA - CASE STUDY #4  Process Change: from vials to prefilled syringes  Source: solvent from partially dried epoxy glue used for needle attachment to syringe barrel leached into the product  Outcome: increase in protein oxidation followed by aggregation  Resolution:syringe barrels allowed to dry for 6 months prior to use Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
  50. 50. “OVERVIEW OF EXTRACTABLES AND LEACHABLES INPROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASESTUDIES” FDA - LESSONS LEARNED  Leachables can have a great impact on the quality and safety of protein therapeutics  Compendial tests often do not provide adequate sensitivity and specificity (e.g., did not detect tungsten oxide)  Important to monitor leachables over time (e.g., extended time points reflective of product dating period should be included) Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
  51. 51. Particulate Matter Definition  USP <788> for Injections states that in “particulate matter in injections and parenteral infusions consists of extraneous mobile undissolved particles, other than gas bubbles, unintentionally present in the solutions”  Harmonized with Ph.Eur. and JP51
  52. 52. USP <788> Criteria  Volume < 100 mL  NMT 3000 > 10 µm  NMT 300 > 25 µm  Volume > 100 mL  NMT 12/mL > 10 µm  NMT 2/mL > 25 µm52
  53. 53. Size Range of Particulate Matter Particulate Analysis Aggregate Analysis Visible Particles Subvisible Particles Soluble Aggregates Monomer 0.001 0.01 0.1 1 10 100 1000 Size (µm)53
  54. 54. Protein Aggregates Particulate Analysis Visible Particles Oligomers: 10 nm- 0.1 µm Subvisible Particles Soluble Aggregates Visible: > 125 µm Micron: 1-125 µm Monomer Submicron: 0.1- 1 µm 0.001 0.01 0.1 1 10 100 1000 Size (µm)54
  55. 55. What is Known  Many biologics form particulates  Particulates are generally undesirable  Particulate formation is not well understood  Consequences of particulates are not well understood  Visible particulates are difficult to measure objectively55
  56. 56. Why the Interest  Safety  Quality  Guidelines and Regulations56
  57. 57. EMA Guideline  Guidelineon Development, Production, Characterisation and Specifications for Monoclonal Antibodies and Related Products  Effective July 1, 2009  “The formation of aggregates, subvisible and visible particulates in the drug product is important and should be investigated and closely monitored on batch release and during stability studies. In addition to the pharmacopoeial tests for particulate matter, other orthogonal analytical methods…”  “Visible and sub-visible particulate matter in drug product should comply with the requirements set forth in the European Pharmacopoeia”57
  58. 58. Dosage Form Specifications  Compendial requirements  Microbiological  Content uniformity  Volume in container  Particulate matter
  59. 59. Dosage Form Specifications  Product specific  Water content for lyophilized dosage form  Preservative content for multi-use dosage form  Antioxidant content  Osmolality  pH  Container closure integrity during stability testing » Replaces sterility? » Dye ingress, microbial ingress?
  60. 60. Preservatives  Minimize the content in the product  Need to justify use  Proof of effectiveness
  61. 61. Antioxidants  Justifyamount by demonstrating lack of or less degradation
  62. 62. Antimicrobial EffectivenessTesting Indicator organisms  E. coli  P. aeruginosa  S. aureus  C. albicans  A. niger Environmental isolates
  63. 63. Antimicrobial Preservatives  Evaluate antimicrobial properties during storage  Determine preservative content and degradation during storage  Confirm antimicrobial effectiveness at lower limit of preservative specification
  64. 64. Antimicrobial Effectiveness Testing-USPUSP <51> Description Inoculum Acceptance CriteriaCategory Log reduction1  Injectables, other parenterals 105-106 Bacteria: 7 d NLT 1.0  Sterile nasal products CFU/mL 14 d NLT 3.0  Aqueous based ophthalmic 28 d NI from 14 products Yeast & molds: NI2  Aqueous based topical 105-106 Bacteria: 14 d NLT 2.0 products CFU/mL 28 d NI from 14  Nonsterile nasal products Yeast & molds: NI3  Aqueous based oral 105-106 Bacteria: 14 d NLT 1.0 products CFU/mL 28 d NI from 14 Yeast & molds: NI4 Aqueous based antacids 103-104 Bacteria, yeasts & molds: NI CFU/mL
  65. 65. Antimicrobial Effectiveness Testing-EPEP Description Inoculum Acceptance Criteria Log reduction Parenterals 105-106 CFU/mL Bacteria: 6 h NLT 2 24 h NLT 3 Aqueous based 28 d No recovery ophthalmic Fungi: 7 d NLT 2 products 28 d NITopical products 105-106 CFU/mL Bacteria: 2 d NLT 2 7 d NLT 2 28 d NI Fungi: 14 d NLT 2 28 d NIOral products 105-106 CFU/mL Bacteria: 14 d NLT 3 28 d NI from 14 Fungi: 14 d NLT 1 28 d NI
  66. 66. Common Formulation Excipientsfor Biotechnology-Based ProductsOsmotic Agents (salts)Chelators (EDTA, citrate)CationsSugars (mannose, maltose, dextrose)Amino Acids (arginine, glycine, glutamic acid)Redox Agents (ascorbate, reducing sugars)Solubilizers (Tween, Deoxycholate)Stabilizers (albumin, lipids)Solvents (aqueous, nonaqueous) Several of these compounds interfere with analytical technologies used for biotech products
  67. 67. Hidden Sources of Variability:Assay Materials and Reagents Potentially “Critical” Assay Reagents for Biotech Methods:  Complex molecules, often biologically derived  Demonstrated to be a key assay component  Sensitive to operational or assay conditions  Selected characteristics may vary from lot to lot  Limited concurrent availability of multiple lots  Single-source product manufacturerRitter, N and Wiebe, M (2001) Validating Critical Reagents Used in cGMP AnalyticalTesting, BioPharm 14:5, pp 12-20.
  68. 68. Potentially Critical Assay Components HPLC - columns (resins and packing procedures), unique mobile phase components Capillary electrophoresis - capillaries, electrode buffers, prepared kit components Gel electrophoresis - gel matrix components, unique buffers, precast gels, stains, dyes Immunoassays - immunoreagents, detection agents, unique blocking materials Peptide maps - reduction/alkylation reagents, digestion enzymes, HPLC columns Colormetric methods - commercial standards, chromogenic agents, prepared assay kits Amino acid analysis - hydrolysis reagents, derivatization reagents Protein sequencing - coupling, cleavage and conversion reagents; de-blocking enzymes Bioactivity assays - substrates, cofactors, ligands, cell cultures, media components Sample preparation - unique buffer components, filters, membranes, culture plates, vials and stoppersRitter, N and Wiebe, M (2001) Validating Critical Reagents Used in cGMP Analytical Testing,BioPharm 14:5, pp 12-20.
  69. 69. Quality by Design Initiative  ICH Guidances Q8, Q9 & Q10  Does this affect how specifications are established or used?  Specifications are linked to manufacturing process  Specifications should account for stability  Specifications are linked to preclinical and clinical studies
  70. 70. Conclusions  Specifications contains two components, the test method and the acceptance criterion  Specifications are established as surrogates of characterization tests  Specifications selected to ensure quality of material for safety and efficacy  Specifications based on manufacturing process, stability and preclinical/clinical data  Limited batch data can be compensated for by more thorough understanding of manufacturing process and link between quality attributes and clinical outcome
  71. 71. Thank You!!
  72. 72. Questions or Advice David Lin Senior Consultant Biologics Consulting Group, Inc.