Setting Biological Process Specifications
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The document discusses the setting of biological product specifications and highlights key differences between biological and chemical pharmaceuticals, including raw materials, production processes, and methods of analysis. It outlines various ICH guidelines relevant to the characterization, stability, and testing of biological products, emphasizing the significance of specifications in ensuring quality and consistency. Additionally, it addresses issues related to extractables and leachables from materials used in biologics, their impact on product quality, and the regulatory requirements for approval.
![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](https://image.slidesharecdn.com/settingbiologicalprocessspecifications-130228160815-phpapp02/85/Setting-Biological-Process-Specifications-30-320.jpg)
Setting Biological Process Specifications
- 1. Setting Biological Product Specifications David Lin, Ph.D. Senior Consultant Biologics Consulting Group IVT Method Validation October 15, 2008 2010 by Biologics Consulting Group. All rights reserved. Reproduction in part or in whole without written permission is strictly prohibited.
- 2. Biological vs. Chemical Pharmaceutical Products Protein Products Chemical Products
- 3. Biological vs. Chemical Pharmaceutical Products Significant Differences In: Raw Materials Production Processes Handling Conditions Formulations Methods of Analysis Physiochemical Characteristics Stability Profile Storage Conditions Expiration Dating
- 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. 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. 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. Definition of Specification “A list of tests, references to analytical procedures, and appropriate acceptance criteria which are numerical limits, ranges, or other criteria for the tests described. It establishes the set of criteria to which a drug substance, drug product, or materials at other stages of its manufacture should conform to be considered acceptable for its intended use. Conformance to specification means that the drug substance and drug product, when tested according to the listed analytical procedures, will meet the acceptance criteria. Specifications are critical quality standards that are proposed and justified by the manufacturer and approved by regulatory authorities as conditions of approval.” ICH Q6B Guidance, Aug. 1999
- 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. 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. 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. 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. Product attributes Lead to the specifications Lead to the method development requirements
- 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. Establishing Relevant Specifications Characterization Physicochemical properties Biological activity Immunochemical properties Purity Impurities Datafrom development and after process changes
- 15. Product Attributes Chemical Physical Microbiological Biological Performance Relate to: Efficacy Safety Quality
- 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-TOF Used 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. Protein Structural Elements Primary = Covalent or chemical structure. Completely defined by the amino acid sequence plus any disulfide bonds, and any post-translational modifications (e.g. glycosylation, phosphorylation) Secondary = Periodic structures within the overall conformation (e.g. alpha helix, various beta- structures) Tertiary = Total folding pattern of the polypeptide chain(s) Quaternary = Association of subunits of polypeptide chains
- 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. 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. 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. 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. Why are Cell-Based Bioassays Different than 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. 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. 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. 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. 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. Why Worry About Extractables and Leachables? 27
- 28. Areas of Concern Toxicity Carcinogenicity Immunogenicity Product quality 28
- 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 closures 29
- 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. 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. Public Health Notifications PVCdevices containing plasticizer DEHP (2002) http://www.fda.gov/MedicalDevices/Safety/ AlertsandNotices/PublicHealthNotifications /UCM062182 IV bags, blood bags, infusion tubing, etc. BPA in food (2010) http://www.fda.gov/NewsEvents/PublicHeal thFocus/ucm064437.htm 32
- 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 manufacturing 33
- 34. FDA Container-Closure Data Requirements for Biological Products Information to support the container and closure packaging used with bulk biological products is required to be included in the FDA submission (rather than simply referenced, as with traditional drugs) because there is a greater potential for adverse effects on the identity, strength, quality, purity and potency of biologics and protein products during storage or shipping. FDA Guidance: Container-Closures for Packaging Human Drugs and Biologics, Questions/Answers (2002)
- 35. What Tests to Perform? 35
- 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. Tests for Protection USP <671> Container – Performance Testing Light Transmission Water Vapor Permeation Dye Ingression Studies Microbial Ingression Studies 37
- 38. USP <660> Containers - Glass Water Attack Extraction for release of alkali Arsenic 1 ppm 38
- 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. USP <381> Elastomeric Closures for Injections Extraction Turbidity Heavy Metals Reducing Agents pH Change Total Extractables 40
- 41. Program Approach Extract Identify Assess the risk Develop the Method Validate the Method in Drug Product Perform Leachable Study (Stability) 41
- 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 vendor 42
- 43. Risk Assessment Literaturesearch Biological testing In-vitro - USP <87> In-vivo - USP <88> Effect on release/stability methods 43
- 44. 44 From BioPharm International, Dec. 2002, Miller et.al.
- 45. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES” 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. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES” 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. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE 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. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE 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. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE 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. “OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES” 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. 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 JP 51
- 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 µm 52
- 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. 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. 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 objectively 55
- 56. Why the Interest Safety Quality Guidelines and Regulations 56
- 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. Dosage Form Specifications Compendial requirements Microbiological Content uniformity Volume in container Particulate matter
- 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. Preservatives Minimize the content in the product Need to justify use Proof of effectiveness
- 61. Antioxidants Justifyamount by demonstrating lack of or less degradation
- 62. Antimicrobial Effectiveness Testing Indicator organisms E. coli P. aeruginosa S. aureus C. albicans A. niger Environmental isolates
- 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. Antimicrobial Effectiveness Testing-USP USP <51> Description Inoculum Acceptance Criteria Category Log reduction 1 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: NI 2 Aqueous based topical 105-106 Bacteria: 14 d NLT 2.0 products CFU/mL 28 d NI from 14 Nonsterile nasal products Yeast & molds: NI 3 Aqueous based oral 105-106 Bacteria: 14 d NLT 1.0 products CFU/mL 28 d NI from 14 Yeast & molds: NI 4 Aqueous based antacids 103-104 Bacteria, yeasts & molds: NI CFU/mL
- 65. Antimicrobial Effectiveness Testing-EP EP 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. Common Formulation Excipients for 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. 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 manufacturer Ritter, N and Wiebe, M (2001) Validating Critical Reagents Used in cGMP Analytical Testing, BioPharm 14:5, pp 12-20.
- 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 stoppers Ritter, N and Wiebe, M (2001) Validating Critical Reagents Used in cGMP Analytical Testing, BioPharm 14:5, pp 12-20.
- 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. 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. Thank You!!
- 72. Questions or Advice David Lin Senior Consultant Biologics Consulting Group, Inc. www.biologicsconsulting.com