Stability Testing for Biotechnology/Biologic Products

Stability Testing for Biotechnology/Biologic Products

David Lin, Ph.D.Senior ConsultantBiologics Consulting Group

IVT Forum on StabilityDecember 8, 2010

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Biological vs. Chemical Pharmaceutical Products

ProteinProducts

ChemicalProducts

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Biological vs. Chemical Pharmaceutical Products

Raw MaterialsProduction ProcessesHandling ConditionsFormulationsMethods of Analysis

Physiochemical Characteristics

Stability ProfileStorage ConditionsExpiration Dating

Significant Differences In:

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Areas Affected by Stability

Drug Substance

Excipients

Drug Product Finished ProductManufacturing

Packaging

API Stability

Interactions with DS

Impurities from Manufacturing

Container Closure

Stability during Storage

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Stability Concerns

Raw materials- bulk drug substance and excipients

R&D formulationsClinical trial materialsMarketed productPost-approval changesDistribution channelsPatient use In vivo stability

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Critical to Effective Stability Evaluation

Degradation pathways

Analytical method development

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Degradation Modes

ChemicalPhysicalMicrobiological

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Elements of Stability Program

Commitment to qualityScientific understandingKnowledge of regulatory policies &

guidelines, and pharmacopoeial standardsCommunication between R&D, production,

QC/QA, regulatory affairsUnderstanding of analytical methodsStability budgetSkills to manage stability program

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Guidances/Guidelines

ICH Q1A (R2) Stability Testing of New Drug Substances & Products, Feb 2003

ICH Q1B Photostability, Nov 1996ICH Q1C Stability for New Dosage

Forms, Nov 1996ICH Q1D Bracketing & Matrixing, Feb

2002

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Guidances/Guidelines (cont)

ICH Q1E Evaluation of Stability Data, Feb 2003

ICH Q1F Data Package for Climatic Zones III & IV, Feb 2003

ICH Q5C Stability Testing of Biotechnological/Biological Products, Jul 1996

FDA Stability Guidance (draft), Jun 1998

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ASEAN Guideline on Stability Study of Drug Product, Feb 2005

WHO Guidelines on Stability Testing of Active Pharmaceutical Ingredients and Finished Pharmaceutical Products, WHO Technical Report, No. 593, Annex 2, 2009.

Panama Stability Study Standards, Nov 2005WHO Eastern Mediterranean Region, Stability

Testing of Active Substances and Pharmaceutical Products, Apr 2006 (draft)

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Content and Format of Investigational New Drug Applications (INDs) for Phase 1 Studies of Drugs, Including Well-Characterized, Therapeutic Biotechnology-Derived Products (November 1995)

INDs for Phase 2 and 3 Studies: Chemistry, Manufacturing, and Controls Information (May 2003)

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FDA Guidance for Industry for the Submission of Chemistry, Manufacturing and Controls Information for Synthetic Peptide Substances, Nov 1994

FDA Guidance for Industry: Drug Substance Chemistry, Manufacturing and Controls Information (draft), May 2004

FDA Guidance for Industry: Drug Product Chemistry, Manufacturing and Controls Information (draft), Jan 2003

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Legal Consequences

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Warning Letter Example #1

“There is no data to show that the method used for XX stability testing has been validated as stability-indicating with respect to acid and base hydrolysis and photolysis; there was inadequate data for oxidation and thermal degradation.”

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“The written stability testing program is inadequate to assess the stability characteristics of drug products and for determining appropriate storage conditions and expiration dates [21 CFR 211.166(a)]. In addition, expiration dates on drug product labeling have not been determined by appropriate stability testing [21 CFR 211.137(a)]. “

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FDA Response

“Your response to the Form FDA-483 asserted that stability failure of a few batches of a drug product is a minor deficiency and that the expiration date for the product is still valid. The frequency of stability failures outlined above is significant, and there is no evidence that your drug products meet the standards of strength, quality and purity at the time of their use within the expiration period. Your response did not specify the corrective measures that you will take in the event of these and any future stability failures. In addition you have provided no rationale for these failures and no corrective actions.”

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“Stability samples were not tested at the scheduled intervals.”

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FDA Response

“Three batches of [ ] API were not tested at the [ ] month interval. The previous FDA-483 issued to your firm on 7 November 2003 cited the same observation where 12 batches of API were not tested at 15 scheduled intervals……..”

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“Adequate written procedures for the storage of drug products under appropriate conditions of temperature, humidity, and light so that the identity, strength, quality, and purity of the drug products are not affected have not been established and followed. [21 CFR 211.142(b)]”

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FDA Response

“We acknowledge your revision of the SOP and your commitment to improve your temperature monitoring system by conducting temperature mapping studies and installing appropriate recorders (data loggers). However, your response does not provide information on the corrective and preventive actions taken to ensure that the warehouse temperature can be maintained and controlled within the acceptable USP storage requirement for (b)(4), (e.g., preserved in tight containers, protected from light, at controlled room temperature). Please revise your SOP and address this issue with supportive documents.”

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“Failure to follow stability testing programs established for [ ], as well as other different ophthalmic ointments, in accordance with 21 CFR 211. 166(a)(1), resulting in a significant. number of omitted test intervals in product stability studies, ……..”

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“Failure of the quality assurance unit to fully and adequately investigate stability out-of-specification incidents for several lots of [ ] products in accordance with 21 CFR 211 .20(a) (3) and 211.160 (we refer you to FDA-483 Observations 3(c), 8(a) & 8(b))……..”

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Main Sources of Impurities

Process-relatedElement of manufacturing process

control strategy

Product-related ImpuritiesVariants

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Process Related Impurities

Cell substrate derivedHost cell proteins

»Risk of immunogenic potentialHost cell nucleic acids (DNA)

»Risk of genotoxicity

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Cell culture derivedAntibioticsMedia components

» Soy proteins, tropolone, albumin, transferrin, etc.

» Risk of Immunogenic potential, TSE transmission

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Downstream processingChemical additives

»Methotrexate, DTT, guanidine HCl, etc.

»Risk of toxicity Leachables

»Protein A, resin, heavy metals, filter preservatives, etc.

»Risk of toxicity

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Host Cell Proteins (HCP)

Purified from recombinant protein of interest

Highly complex mixture of polypeptidesDeveloping assay is critical Product purity Process consistency

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HCP Assay

Typically ELISA basedSensitivity is a strengthKey limitation is nonimmunoreactive or

weakly immunoreactive proteinsOrthogonal method needed SDS-PAGE 2D-PAGE

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Multi-Product or Product-Specific ELISA?

Consider cell type and expression system

Consider analysis pointConsider potential for process

changesAssess applicability of commercial

“kits”

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Biologics Product-Related Impurities/Degradants

AdductsAggregatesCarbamylation (isocyanate addition)Fragmentation

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Biologics Product-Related Impurities/Degradants

DeamidationDisulfide formation or scramblingGlycosylationN-terminal acetate and pyroglutamateOxidation

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Degradation Pathways of Biologics

Adducts: from cysteine or glutathione used as reducing agent

Covalent Aggregation: re-arranged disulfide bonds alter intramolecular bridges to form different linkages; can result from reaction with trace metals (copper or iron), or incomplete reduction of protein

Kats, M. “Forced Degradation Studies: Regulatory Considerations and Implementation”, BioPharm Intl, Jul 1, 2005.

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Degradation Pathways of Proteins

Non- Covalent Aggregation: multimeric association of monomers due to denaturation or interaction of protein with air: fluid interface; can result from heating, freezing, agitation, or desiccation of solution

Carbamylation: isocyanate addition to free amino groups; due to high urea concentrations at high pH and temp. that promote cyanate production

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Fragmentation: C or N-terminal truncations, or internal cleavage; caused by acidic or basic pH internal cleavages might not be observed if

disulfide linkages present

Deamidation: conversion of asparagine or glutamine to a free caroboxylic acid; can be induced by changes in pH, ionic strength, temperature and humidity (in lyophilized protein

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Disulfide exchange: misfolded and thermodynamically unstable protein

Glycosylation: correct N and O-glycosylation sites; asparigine, serine and threonine

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N-Terminal Modifications: acetylation on serine and alanine. Pyroglutamate formation in proteins with N-terminal glutamate or glutamine.

Oxidation: addition of an oxygen to methionine, cysteine, histidine, tryptophan or tyrosine residues; can result from exposure to atmospheric oxygen under conditions of light, heat, moisture or agitation

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ICH Q5C: Characterization of Biotechnological Product Degradants

“Whenever significant qualitative or quantitative changes indicative of product degradation is detected during long-term, accelerated, or stress studies, consideration should be given to the potential hazards and to the need for characterization and quantitation of degradants.”

Potential Safety Concern:Immunogenicity of Degradants

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Safety Concerns

Sequences of amino acids can constitute epitope sites

Epitopes presented to the immune system can trigger an antibody response

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Safety Concerns (cont.)

Patient antibodies may neutralize the intended product

Patient antibodies may also neutralize any endogenous protein, impacting patient safety

Immunogenicity of protein products has recently been the cause of a major adverse event (EPO)

Immunogenicity ELISAs are now being requested by FDA starting at Phase 1 with continuing use through post-market surveillance

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Stability-Indicating Method“The evaluation of stability may necessitate complex analytical methodologies.”

“Appropriate physicochemical, biochemical and immunochemical methods for the analysis of the molecular entity and the quantitative detection of degradation products should also be a part of the stability program whenever purity and molecular characteristics of the product permit use of these methodologies.”

ICH Q5C: Stability Testing of Biotechnological/Biological Products (1995)

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Stability Indicating Methods –FDA Expectations

No single method or parameter will profile all biotech product stability characteristics.

The nature of the biotech product will determine the types of test methods that should be used.

For purposes of stability, biotech products should be tested for purity with more than one method, and the focus should be on the ability to determineproduct degradants.

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Stability-Indicating Methods: Using Forced Degradation

AggregationPrecipitationFragmentationDeamidation HydrolysisDisulfide bond exchangePhotosensitivityOxidation

Chemical/Physical Treatments that Promote:

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Acid-Base Hydrolysis

0.1 M HCl 0.1 M H3PO4

0.5% TFA

Kats, M. “Forced Degradation Studies: Regulatory Considerations and Implementation”, BioPharm Intl, Jul 1, 2005.

0.01 N NaOH 0.01 M Na2HPO4 at pH

8.0

Acid Conditions Base Conditions

Degradation Mechanism:N and/or C terminus; adjacent to Asp bonds

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Disulfide Bond Exchange

Cu (II) or Fe (II) ions 1-2 M GnHCl 1-2 M urea 0.01 M DTT disulfide isomerase enzyme

Yu, J. “Intentionally Degrading Protein Pharmaceuticals to Validate Stability-Indicating Analytical Methods”, BioPharm 13:11 (2000), p.46.

Conditions:

Degradation Mechanism:Re-arranged Cys-Cys bonds

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Aggregation

Agitation Thermal treatment (e.g. 500C to 600C; 5 – 120

min) Freezing/thawing (e.g. 3, 5, and 10 cycles)

Conditions:

Degradation Mechanisms:Dissociable (noncovalent) or Non-dissociable (covalent)


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Deamidation

Increased pH Increased temperature Increased ionic strength Presence of borate or phosphate ions 0.1 M Tris pH 9.0 1-2 M urea or GnHCl L-asparaginase or L-glutaminase

Conditions:

Degradation Mechanisms:Asparagine to aspartic acid (esp at Asn-Gly); Glutamine to glutamic acid


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Oxidation

Atmospheric O2

Bubbling O2 gas plus 0.1% TFA

0.05% H2O2 in 0.1% TFA

Conditions:

Degradation Mechanisms:Side chains of Cys, His, Trp, Tyr and esp. MET


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Photo Irradiation

Follow ICH Q1B for UV/Vis conditions

Utilize 1, 5, and 10 times the confirmatory conditions

Conditions:

Degradation Mechanisms:Oxidation, aggregation, fragmentation


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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)

Common Formulation Excipients for Biotechnology-Based Products

Several of these compounds interfere with analytical technologies used for biotech products

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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http://www.fda.gov/MedicalDevices/Safety/AlertsandNotices/PublicHealthNotifications/UCM062182�



http://www.fda.gov/NewsEvents/PublicHealthFocus/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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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



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



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



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



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)



Particulate Matter Definition

USP <788> for Injections states that “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 can be difficult to

measure objectively

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Why the Scrutiny?

SafetyQualityGuidelines and Regulations

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EU Guidelines and Standards

EMEA Guideline: Guideline on Development, Production, Characterization and Specifications For Monoclonal Antibodies and Related Products, Jul 2009

EP Mab Monograph (2031) EP Parenteral Monograph (0502) EP Particulate Contamination: Visible Particles

(2.9.20) EP Particulate Contamination: Sub-visible

Particles (2.9.19)

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EMEA 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”

EP Parenterals (0520) and Sub-Visible Particulates (2.9.19)

Parenterals and solutions for injection, examined under suitable conditions are clear and practically free of particles Definition of “practically free of particles”? Visual inspection is subjective

Sub-Visible Particulates Microscopy and light obscuration methods Limits same as for USP Can smaller particulates be quantitated with current

methods?

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EP Monoclonal Antibodies (2031)

2004 Monograph “Liquid preparations are clear or slightly

opalescent, colourless or slightly yellow liquids, without visible particles”

2009 Monograph “Monoclonal antibodies are without visible

particles, unless otherwise justified and authorised”

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Challenges Around Guidelines and Standards

Particulates in Mabs is relatively common so compliance to the EP needs to be considered early in formulation development

Inconsistency between EP Mab monograph and EP Parenteral monograph

“Without visible particles” is an absolute statement, without room for flexibility and justification

There is no distinction between intrinsic and extraneous particulates

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Protein Aggregates

Usually formed from partially unfolded protein that form dimer species to multimers Processing, storage, shipping, handling, etc.

Foreign material from packaging or process equipment can serve as sites for nucleation

Dimers and other smaller size aggregates are usually soluble

Particulates generally refer to larger size aggregates But can encompass visible, subvisible and sub-

subvisible size ranges

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What is the Concern?

Large protein assemblies induce immune responses

Vaccine development show that adsorbing antigenic proteins to particles increase immunogenicity

Is this also the case for therapeutic proteins?Maybe!But still a regulatory safety concernCannot predict in-vivo effects of different sizes,

types and quantities of aggregates Develop relationship during clinical trials between

aggregates in clinical material and clinical performance

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Shipping: FDA Expectations

GMP Inspection Shipping conditions validated

» Containers and methods Shipping for intermediates validated and

process validated Shipper to ensure proper shipping

conditions

Compliance Program Guidance Manual, Inspection of Biological Drug Products (CBER) 7345.848, Dec 2004

Compliance Program Guidance Manual, Drug Quality Assurance (CDER), 7356.002M, Oct 2003

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Considerations During Shipping

Direct shipment within a country or region (EU) usually less than 24 hours Need to ensure that shipping container can

maintain required temperature for expected time period However, consider receipt of shipment and

instructions for temporary storage

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Considerations During Shipping (cont)

Shipment between countries and regions may be considerably longer Even if shipping time is known Need to consider potential delay in

customs Storage of shipment in customs

ICH Q5E: Comparability of Biotechnological/ Biological Products Subject to Changes in Their Manufacturing Processes (2005)

During product development, it is expected that multiple changes in the manufacturing process will occur that could impact drug product quality, safety, and efficacy.

Comparability exercises are generally performed to bridge nonclinical and clinical data generated with pre-change to post-change product in order to facilitate further development and, ultimately, to support the marketing authorization.

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ICH Q5E: Comparability of Biotechnological/ Biological Products Subject to Changes in Their Manufacturing Processes (2005)

Any change with the potential to alter protein structure or purity and impurity profiles should be evaluated for its impact on stability, since proteins are frequently sensitive to changes, such as those to buffer composition, processing and holding conditions, and use of organic solvents.

Furthermore, stability studies might be able to detect subtle differences that are not readily detectable by the characterization studies.

Accelerated and stress stability studies are often useful tools to establish degradation profiles and provide a further direct comparison of pre-change and post-change products.

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ICH Q5E: Comparability of Biotechnological/Biological Products Subject to Changes in Their Manufacturing Processes (2005)

Comparability studies conducted for products in development are influenced by factors such as…

the stage of product development the availability of validated analytical

procedures the extent of product and process knowledge

…which are limited at times due to the available experience that the manufacturer has with the process.

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Methods for Comparability Assessment

Peptide Sequencing Amino acid analysis RP-HPLCHPLC (Size-

exclusion) Peptide mapping Isoelectric Focusing

(IEF) Protein-related

impurities

Mass Spectroscopy (MS)

Circular DichroismUV ELISANMRBioassay Process-related

impuritiesHCP

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Managing Changes after Approval

Implementation of changes will depend on the “potential to have an adverse effect on identity, strength, quality, purity, or potency of the product

Substantial potential - Prior approval supplement

Moderate potential - Changes being effected supplement

Minimal potential - Annual report

FDA Guidance: Changes to an Approved Application for Specified Biotechnology and Specified Synthetic Biological Products, July 1997

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Stability Data Package?

No clear current guidance 1987 FDA Stability guidance is the only one

to referenceCurrent expectation is 6 months of

data for 3 batches and compared to pre-change stability data

Drug product stability data need to be considered

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#1:Complex Recombinant Protein

Need for media change results in cell line change

Potential change in fermentation conditions Potential change in modified protein

Scale-up failures during consistency batches Should the data be usedWill FDA conclude that there was no

demonstration of consistency

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#2:Recombinant Protein

Unmodified proteinNumerous small scale batches (>15)Batches used in clinical studies

However, upon scale-upColumn purification resulted in different

impurity profileUsed theoretical column load

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Conclusions

Understand degradation pathwayDevelop appropriate analytical method to

evaluate stability profile

Critical component of comparability assessment for linking of manufacturing process or formulation changes

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Thank You!!

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Questions???

David LinSenior Consultant

Biologics Consulting Group, Inc.

www.biologicsconsulting.com

http://www.biologicsconsulting.com/�

Documents

Stability Testing for Biotechnology/Biologic Products