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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 Product Specifications
David Lin, Ph.D.Senior Consultant
Biologics Consulting Group
IVT Method ValidationOctober 15, 2008
2010 by Biologics Consulting Group. All rights reserved. Reproduction in part or in whole without written permission is strictly prohibited.
Biological vs. Chemical Pharmaceutical Products
ProteinProducts
ChemicalProducts
Biological vs. Chemical Pharmaceutical Products
Raw MaterialsProduction ProcessesHandling ConditionsFormulationsMethods of Analysis
Physiochemical Characteristics
Stability Profile
Storage ConditionsExpiration Dating
Significant Differences In:
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
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
FDA Guidances Withdrawnin 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
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
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
Specifications
Which guidance is most relevant, Q6A or Q6B?
If synthetic, follow Q6A Low MW
If biologically source (e.g. recombinant), follow Q6B Higher MW
Specifications
Q6A allows for skip testingQ6A allow for parametric release Monitor sterilization cycle instead of
performing sterility testing Sterilization process validation and
demonstration of control critical
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
Product attributes
Lead to the specificationsLead to the method development
requirements
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
Establishing Relevant Specifications
Characterization Physicochemical properties Biological activity Immunochemical properties Purity Impurities
Data from development and after process changes
Product Attributes
Chemical PhysicalMicrobiologicalBiological Performance
Relate to: Efficacy Safety Quality
Method Measures DetectsCell-based potencyLigand binding
Biological Activity Overall integrity of the molecule
SDS-PAGEReduced and non-
reducedMALDI-TOF
Molecular Weight Subunit molecular massFragmentationCovalent crosslinking
RP-HPLC, HIC-HPLC Surface Hydrophobicity Chemical and conformational variantsIEF Net Surface Charge Charge variantsELISARIAWestern BlotSurface Plasmon Resonance
Antibody Recognition Epitope binding site integritySpecific contaminantsImmunogenicityAntigenicity
Peptide MapAmino Acid CompositionN and C terminal sequence
CompositionPost-translational modifications
Primary Structure variantsPrimary structure integrityIntegrity of PTMs
CD (Far UV) Optical Activity Secondary StructureUV AbsorbanceFluorescenceCD (Near UV)
Aromatic Amino AcidsSide Chains
Tertiary Structure
SEC HPLCAnalytical Ultracentrifugation
Hydrodynamic Radius Molecular sizeConformational changesAggregation/dissociation
Light Scattering Radius of Gyration Molecular sizeAggregation/dissociation
NMRX-ray Cystallography
Nuclear magnetic energyTransitionsX-ray diffraction
Complete molecular structure
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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
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
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
What is a Bioassay?
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?
Ana T. Menendez, Ph.D.Director of Bioassays and Biosafety Testing
Cardinal Health
Types of Bioassays
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
Ana T. Menendez, Ph.D.Director of Bioassays and Biosafety Testing
Cardinal Health
Why are Cell-Based Bioassays Different than other Analytical CMC Assays?
Involve recurring consistent source of live organisms
Need Master and Working Cell BanksCell lines require characterizationBiological reagents require qualificationResults are mostly calculated on 4-parameter
curveCVs and %Accuracy are less stringent than in
analytical techniques
Ana T. Menendez, Ph.D.Director of Bioassays and Biosafety Testing
Cardinal Health
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”
FDA Guidance 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
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
Raw Materials and Excipients
Meet quality standards for intended useRaw materials for production require
determination of adventitious agents Extractables/leachables from purification
resin materialsMinimum standards conform to
pharmacopoeia
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
Why Worry About Extractables and Leachables?
27
Areas of Concern
ToxicityCarcinogenicityImmunogenicityProduct quality
28
Required by Regulation
21 CFR 211.65(a) – Equipment21 CFR 600.11(b) - Equipment21 CFR 211.94(a) - Drug product
container closures21 CFR 600.11(h) – Containers and
closures
29
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
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
Public Health Notifications
PVC devices 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
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
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)
What Tests to Perform?
35
Primary Considerations
Information from supplierHas supplier intended use been modifiedConsider existing databasesUnderstand chemistry of materials Start with compendial tests
Need to justify if relevant to specific use
Don’t focus on just organics Equipment reuse (i.e., column resins, filters, etc.)
Be realistic!!!
36
Tests for Protection
USP <671> Container – Performance Testing Light Transmission Water Vapor Permeation
Dye Ingression StudiesMicrobial Ingression Studies
37
USP <660> Containers - Glass
Water AttackExtraction for release of alkali
Arsenic 1 ppm
38
USP <661> Containers - Plastics
ExtractionNonvolatile residueResidue on IgnitionHeavy MetalsBuffering Capacity (for liquid products)Total Terephthaloyl Moieties (for PET &
PETG)Ethylene Glycol (for PET & PETG)
39
USP <381> Elastomeric Closures for Injections
ExtractionTurbidityHeavy MetalsReducing AgentspH ChangeTotal Extractables
40
Program Approach
ExtractIdentifyAssess the riskDevelop the MethodValidate the Method in Drug ProductPerform Leachable Study (Stability)
41
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
Risk Assessment
Literature searchBiological testing
In-vitro - USP <87> In-vivo - USP <88>
Effect on release/stability methods
43
44From BioPharm International, Dec. 2002, Miller et.al.
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
“OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES”
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
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
“OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES”
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
FDA - CASE STUDY #2Container 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
“OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES”
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
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
FDA - CASE STUDY #3
“OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES”
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
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
“OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES”
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
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)
“OVERVIEW OF EXTRACTABLES AND LEACHABLES IN PROTEIN THERAPEUTICS: SOURCES, METHODS, AND CASE STUDIES”
Kathy Lee, FDA CDER, OBP (WCBP2006 Presentation)
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
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
Size Range of Particulate Matter
53
0.001 0.01 0.1 1 10 100 1000
Monomer
Soluble Aggregates
Subvisible Particles
Visible ParticlesAggregate Analysis
Particulate Analysis
Size (µm)
Protein Aggregates
54
0.001 0.01 0.1 1 10 100 1000
Monomer
Soluble Aggregates
Subvisible Particles
Visible Particles
Particulate Analysis
Oligomers: 10 nm- 0.1 µm
Size (µm)
Submicron: 0.1- 1 µmMicron: 1-125 µm
Visible: > 125 µm
What is Known
Many biologics form particulatesParticulates are generally undesirableParticulate formation is not well
understoodConsequences of particulates are not
well understoodVisible particulates are difficult to
measure objectively
55
Why the Interest
SafetyQualityGuidelines and Regulations
56
57
EMA Guideline
Guideline on 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”
Dosage Form Specifications
Compendial requirements Microbiological Content uniformity Volume in container Particulate matter
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?
Preservatives
Minimize the content in the productNeed to justify use Proof of effectiveness
Antioxidants
Justify amount by demonstrating lack of or less degradation
Antimicrobial Effectiveness Testing
Indicator organismsE. coliP. aeruginosaS. aureusC. albicansA. niger
Environmental isolates
Antimicrobial Preservatives
Evaluate antimicrobial properties during storage
Determine preservative content and degradation during storage
Confirm antimicrobial effectiveness at lower limit of preservative specification
Antimicrobial Effectiveness Testing-USP
USP <51> Category
Description Inoculum Acceptance CriteriaLog reduction
1 Injectables, other parenterals Sterile nasal products Aqueous based ophthalmic
products
105-106
CFU/mLBacteria: 7 d NLT 1.0
14 d NLT 3.028 d NI from 14
Yeast & molds: NI2 Aqueous based topical
products Nonsterile nasal products
105-106
CFU/mLBacteria: 14 d NLT 2.0
28 d NI from 14Yeast & molds: NI
3 Aqueous based oral products
105-106
CFU/mLBacteria: 14 d NLT 1.0
28 d NI from 14Yeast & molds: NI
4 Aqueous based antacids 103-104
CFU/mLBacteria, yeasts & molds: NI
Antimicrobial Effectiveness Testing-EP
EP Description Inoculum Acceptance CriteriaLog reduction
ParenteralsAqueous based
ophthalmic products
105-106 CFU/mL Bacteria: 6 h NLT 224 h NLT 328 d No recovery
Fungi: 7 d NLT 228 d NI
Topical products 105-106 CFU/mL Bacteria: 2 d NLT 27 d NLT 228 d NI
Fungi: 14 d NLT 228 d NI
Oral products 105-106 CFU/mL Bacteria: 14 d NLT 328 d NI from 14
Fungi: 14 d NLT 128 d NI
Osmotic Agents (salts)Chelators (EDTA, citrate)CationsSugars (mannose, maltose, dextrose) Amino Acids (arginine, glycine, glutamic acid)Redox Agents (ascorbate, reducing sugars)Solubilizers (Tween, Deoxycholate)Stabilizers (albumin, lipids)Solvents (aqueous, nonaqueous)
Common Formulation Excipients for Biotechnology-Based Products
Several of these compounds interfere with analytical technologies used for biotech products
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 AnalyticalTesting, BioPharm 14:5, pp 12-20.
Potentially Critical Assay ComponentsHPLC - 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.
Quality by Design Initiative
ICH Guidances Q8, Q9 & Q10Does 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
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
Thank You!!
Questions or Advice
David LinSenior Consultant
Biologics Consulting Group, Inc.
www.biologicsconsulting.com