Setting Biological Process Specifications

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?

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Areas of Concern

ToxicityCarcinogenicityImmunogenicityProduct quality

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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

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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.”

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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. “

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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

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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

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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?

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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!!!

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Tests for Protection

USP <671> Container – Performance Testing Light Transmission Water Vapor Permeation

Dye Ingression StudiesMicrobial Ingression Studies

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USP <660> Containers - Glass

Water AttackExtraction for release of alkali

Arsenic 1 ppm

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USP <661> Containers - Plastics

ExtractionNonvolatile residueResidue on IgnitionHeavy MetalsBuffering Capacity (for liquid products)Total Terephthaloyl Moieties (for PET &

PETG)Ethylene Glycol (for PET & PETG)

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USP <381> Elastomeric Closures for Injections

ExtractionTurbidityHeavy MetalsReducing AgentspH ChangeTotal Extractables

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Program Approach

ExtractIdentifyAssess the riskDevelop the MethodValidate the Method in Drug ProductPerform Leachable Study (Stability)

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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

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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

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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

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Size Range of Particulate Matter

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0.001 0.01 0.1 1 10 100 1000

Monomer

Soluble Aggregates

Subvisible Particles

Visible ParticlesAggregate Analysis

Particulate Analysis

Size (µm)

Protein Aggregates

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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

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Why the Interest

SafetyQualityGuidelines and Regulations

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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