Characterization and Biological Relevanceof Protein aggregates and other particles100-200,000 nm in size (sub micron andsubvisible)Linda Narhi, PhD(and AAPS Protein aggregate and biologicalconsequences community)
Outline Introduction/historical perspectiveDescription of Techniques in USPResults of industry surveySummary and future plans
Biotherapeutics should be Efficacious– Achieve desired result at reasonable dose– With long enough half life (PK) to be effective Safe:––––No unexpected side effectsNo non-specific bindingNo ToxicityMinimized Immunogenicity (both neutralizing and non-neutralizingAbs) Manufacturable:– Stable shelf life for up to 2 years– Able to make consistently and efficiently– Fit existing facilities and process platforms much as possibleParticles are indicative or process control, sterililty, etcProtein aggregates could impact safety and efficacy 3
Proteins aggregate via different pathwaysHeterogeneous nucleation or interfacedependent aggregationAssemblyConformational or colloidal stabilitydependant aggregationStructural/chemicalChanges(e.g due to air-water/chemicalinterface)Silicone oil orNano-particlesStructural/ es can have different morphology based on the mechanism of formationSynthesis of work from multiple scientists including R. Thirumangalathu, J. Bee, SKrishnan, EY Ch, H-C Mahler, M. Joubert , Q Li, S. Shire, M Cromwell, L. Narhi, et al
Aggregates are a very heterogeneous population requiringmultiple descriptors* Size (Quaternary structure)–––– 100 nm (Nanometer)100-1000 nm (Sub-mm)1-100 µm (micron, SbVP) 100 µm (Visible particles havecompany-specific size range) ––––– *Narhi, Linda O., Schmit, J., Bechtold-Peters, K., Sharma, D.,Classification of Protein Aggregates (2012) J. Pharm. Sci. 101, 493-498. Chemical modificationCross-linkedReducible crosslinkNon-reducible crosslinkIntra-molecular modificationNo modificationMorphology–––––– nativepartially unfoldedunfoldedamyloidInherently disordedCovalent Modification––––––Reversibility– Reversible should be restricted toaggregates for which an equilibriumconstant can be measured. That is,the disassociation of proteins maybe observed on the experimentaltime scale simply by reverting tooriginal conditions.– Irreversible– Dissociable under physiologicalconditions– Dissociable with denaturant whenconditions that disrupt structure arerequired to dissociate the aggregateSecondary/Tertiary structureAspect ratioSurface roughnessInternal morphologyHomo and heteroaggregatesTranslucentHeterogeneousOptical properties: similar for allprotein particles
Aggregates are a Critical Quality attribute and shouldbe treated as suchTPP/CQA life cycle managementTarget Product Profile (TPP)Product QualityAttribute Assessments Indication & use Attribute definition Dosage & Product qualityadministration Tolerability Dosage forms &strength Efficacyattributeassessment Potential impactfor safety/efficacyQuality Target ProductProfile (QTPP) Critical qualityattribute selection Attribute rangedetermination Attribute focusedmolecule &process design Safety/side effects Value & accessCharacterization of particles and “what is normal” increase with progressionthrough development, enabling a phase appropriate, risked based approach
mAUAssay requirements change during the nscreening Qualified Compliant Highly Reliable Also used for stability testing More Detailed Structural Information Often Complex, Slow assays Used Less Frequently Data supports regulatory filings High Throughput Predictive Minimal Qualification Used for clone screening, supportof process developmentAggregate/Particles should be treated like all CQAs, acquiring knowledge early on toinform what is normal at lot release
History of Subvisible particles in USP/EP/JP Harmonized EP 2.9.19 Particulate Contamination: Subvisible Particles and USP 788 Particulate Matter in injectionsboth contained guidance on acceptability of 10 and 25 micronparticles (6000 and 600 per container) Essentially created to control levels of foreign particles in smallmolecule parenteral (extrinsic and intrinsic particles) Safety concerns were around capillary occlusion by these rigidSbVP, Also seen as indicating contamination, loss of process control,etc. No other regulatory guidance existed for subvisible particlesapart from the pharmacopoeias Lot release method, robust and reliable
For biologics, the focus on SbVP has changed to potentialimmunogenicityCOMMENTARY (by Authors from Academia and the FDA ) Overlooking Subvisible Particles in Therapeutic Protein Products: Gaps That May Compromise Product Quality, JohnF. Carpenter, Theodore W. Randolph, Wim Jiskoot, Daan J.A. Crommelin, C. Russell Middaugh, Gerhard Winter, Ying-xin Fan, Susan Kirshner, Daniela Verthelyi, StevenKozlowski, Kathleen A. Clouse, Patrick G. Swann, Amy Rosenberg, Barry Cherney J Pharm Sci. 2009 Apr;98(4):1201-5. doi: 10.1002/jps.21530. Original USP particulate testing was not designed to measure protein particle sizedistribution, or to address the potential risk of large protein aggregates to impactprotein immunogenicity. All formulated antibody drug products contain low levels of aggregates. The clinical immunogenic risk of aggregates is uncertain, resulting in a high riskfactor being assigned to the presence of protein aggregates in biologics. To reduce this uncertainty, the following should be defined: Aggregate attributes that cause a responseAmount of aggregate required to break the threshold of activationExtent and nature of the responseExtensive studies with different proteins, stresses, and model systems suggest the response dependson protein sequences, aggregate characteristics (including size, modification, and morphology),administration, and model systems or patient attributes. (Jiskoot et at, 2016, Ehab et al, 2016, etc) Analytical methods that can assess particulate characteristics (includingcomposition, amount and reversibility of the protein aggregate) are critical fordeveloping scientifically sound approaches for evaluating and mitigating riskto product quality caused by large protein aggregates and other particles
USP definitions: Visible and SbVP Particles can be assignedto one of three categories 1787 , 740 Extrinsic particles (from the outside) are materials that are not part of thedrug product, package, or process, but are present due to contamination.These are truly foreign particles that are unexpected in drug product (e.g.,insect parts, paint chips, clothing fragments, hair). Intrinsic particles (from the inside) are undesirable, non-protein materialfrom degradation of formulation components, or related to the manufacturingand packaging processes and the device itself (e.g., glass lamellae, particlesarising from packaging materials for drug product components, rubber fromstoppers, silicone oil).10
USP SbVP definitions Silicone oil droplets are important intrinsic particles resulting from the siliconeoil that is a necessary lubricant in glass pre-filled syringes. They can confoundthe analysis of the total subvisible particle population, and also have the potentialto interact with the protein depending on formulation conditions1-4 Inherent particles are particles which originate from the drug product, either theprotein therapeutic itself or formulation components. These particles can be anexpected characteristic of the drug product.11
The primary lot release method in the Pharmacopeia is LightObscuration Method 787 describe a method better suited forbiologics:– Test individual units (as much as possible)– Reduced sample volume 5mL– For many biologics individual units are less than 1 ml Release and stability testing: ( ) 5 mL/test– Characterization & investigation testing: 5 mL/test Qualification and validation: 100 mlFrom Beckman /lab-liquidparticle-counters/hiac-9703– Extend to multiple (e.g. 7) size channels: 2, 5, 10, 15,20, 25, 50 mm– Modify & improve sample handling procedure to reducefalse negatives and positives (micro bubbles, etc)– Improve performance compared to 788 Intended use for drug products:– Release and stability testing– Process and product characterization– InvestigationsCould be applied to all parenterals 1787 describes methods for particle/aggregate analysis beyond lot release 1788 includes best practices for Dynamic imaging and membrane microscopy12
The most commonly used orthogonal method isdynamic flow imaging, Camera Based TechnologiesMFI (micro-flowimaging)FlowcamTaken from instrument manufactures' information
Techniques for particle size and distributionanalysis from 1787 TechniquePrinciple of OperationTurbidimetry and Estimation of the particle sizeNephelometrydistribution is attained by measuringthe interaction of light with suspendedparticles, by the loss in intensity oftransmitted light (turbidimetry) or lightscattering (nephelometry).LightThe size of the particle in the productObscurationfluid is determined by the amount oflight that it blocks when passingbetween the source and the detector.Coulter:The size of the particle in product fluidelectricalor selected electrolyte is determined bysensing zonethe change in resistance as the particlepasses through a micro-channel(orifice).Mastersizer (laser Intensity and angle of scatteringdiffraction)generates a particle size distributioncurveRange0.035µm to50µm1 to 300µm1 to 1600µm0.01-3000micrometers
Techniques for size and morphology analysis from 1787 Principle of OperationRangeTechniquePhoton imaging of substances directly in productfluids or mounts or of isolated specimens onsubstrates.Digital image capture of the particles’ magnifiedimage in streaming product fluid, revealing size,shape, optical properties.0.3µm to mm’sElectron Microscopy(EM):Scanning EM,Scanningtransmission EM andtransmission EMElectron imaging of specimen isolates onsubstrates. High vacuum or near-ambientpressures required.Angstroms to mm’sFlow Cytometry:Forward scatteringchannelPassage of particle across light beam increaseslight scattering in forward direction. Lowrefractive index difference, irregular morphology1-100 micrometersLight MicroscopyDynamic ImagingAnalysis: FlowMicroscopy0.7 to 100µm for sizedistribution4 to 100µm formorphology
The absolute numbers and size of micronaggregate depends on the instrument usedHIAC, MFI and Coulter Counter gave different particle concentrations andparticle size distributions for protein particles in mAb Y drug substance at120 mg/mLSbVP mAb Y stressed DS at 120 Size (µm) HIAC has the lowest counts of particles through all sizes and dilutions Coulter has highest counts of smaller particles (2-5 µm) MFI has the highest counts of larger particles ( 5 µm). Dean Ripple has published on underlying causes for these types of discrepancies, and ways to addressthemRelative ranking of samples is usually consistent across techniques
Output from size and morphologyanalysisDynamic flow imageFlow cytometry output
Techniques for characterization from 1787 ospectroscopyElectronMicroscopy withEnergy-DispersiveX-ray Spectrometry[EDS]ElectronMicroscopy withElectron EnergyLoss Spectroscopy[EELS]Principle of OperationRangePhoton imaging of isolated specimens onsubstratesPhoton imaging of isolated specimens onsubstrates, or in product fluids or fluidmountsX-ray Photon emission from specimensenergized by a focused electron beam10µm to mm’sInelastic scattering from specimensenergized by a focused e-beam; e-loss ischaracteristic of the source element.Complementary to EDS.Angstroms tomm’s for imaging,0.5µm to mm’s forelementalcomposition0.5µm to mm’sAngstroms tomm’s for imaging,1µm to mm’s forelementalcomposition
What techniques are suitable for what phaseof development? AAPS PABC focus group did a survey withresponses obtained from 7 industrial and contractanalytical labs The participating labs rated the application of eachanalytical particle/aggregation method at eachphase of the product life cycle using a scale of 1-5(1 being not recommended, 5 being most oftenused or a requirement). The ratings were consolidated and discussed,including in a webinar, resulting in arecommendation for the phase-appropriateapplication of particle analytical methods
Analytical methods and phases of developmentincluded in surveyFormulation DevelopabilityAssessment andPre- FormulationVisual InspectionLight microscopyDark green most often used or a requirement(average scale rating 5)Light green typically used(average scale rating 4)Orange occasionally used(average scale rating 3)Yellow rarely used(average scale rating 2)Red not recommended(average scale rating 1)FluorescencemicroscopyLight obscurationdynamic imagingTurbidityNTARMMElectrical sensingzoneFlow cytometryDLSSLSSEM-EDXTEMFTIR microscopyRamanmicroscopyTOF-SIMSAF4 particlesAF4 HMWSEC/SEC-MALSAUCHydrophobic dyebindingPhase 1/FIHPhase 3QC GMPRelease andstabilityParticle rootcauseinvestigationduring GMPmanufacturing, (visible)
There were clear industry trends in what methods areused at what phase of development Initially predictive methods, that can be automated,are used to select the candidate and process tominimize aggregation, and provide relative rankings,require small volume, lot release methods areusually not appropriate (large volume) at this point During process and product development, from FIH tophase 3 multiple orthogonal methods are used tounderstand the aggregate, and to implement controlstrategy , including compendia methods For lot release LO is sufficient if backed up byprevious characterization, with other tools ready incase of NC or investigation.
Submicron Through IQ consortium determination of submicron particlespresent in marketed product using RMM and NTA has beencompleted, manuscript submitted, begin understanding clinical exposure and baseline of these species inmaterial administered to humansTo see if there are correlations between DP characteristics like volume,concentration, liquid or lyo and submicron populationEstablished best practices for sample handling and use of instrumentsAssessed variability of techniques
How do we test to see if particles do have risk ofimmunogenicity? In silico modeling based on sequences for Tcellrecognition In vitro cell based model systems (both Hu PBMC andcell lines) In vitro organelle model systems In vivo mouse models– Wild type, Xeno, and Xeno/Het MiceAll provide relative ranking for potential immunogenicity.
POINTS TO CONSIDER AND FUTUREPLANS
Key points to consider There is increased scrutiny on SbVP in protein products due topotential risk of immunogenicity Characterization with orthogonal methods is important (Coultercounter, MFI and other flow microscopy techniques, etc. in additionto light obscuration/HIAC) For all techniques it is important to verify results with expert analysts Characterization during development can both minimize particlespresent, and also result in understanding of “what is normal” andcontrol strategy This should enable use of LO as the lot release method based ondeep understanding of product gained during characterizationFor Internal Use Only. Amgen Confidential.25
Points to consider, cont’d The field is moving to a common nomenclature forprotein aggregates, Sample handling is critical, including effect of dilution onparticle size distribution, micro-particle removal, etc. Particle standards that are similar in optical propertiesand density to protein aggregates have been developedby NISTFor Internal Use Only. Amgen Confidential.26
Summary and future plans Protein aggregates occur due to multiple factors, inherent molecular properties,process conditions, and interactions with formulation and device. Our analytical ability and understanding of the biological consequences of micronprotein aggregates has improved significantly over the last few years.– Exploration of the applications of these techniques during product developmentcontinues All techniques have strengths and weaknesses. High concentration analysis isparticularly difficult for all of them. USP expert committee finalized 787 (Biologics specific chapter), 1787 ,informational chapter, and stimulus articles on submicron particles, and–is currently working on adding flow imaging (without specifications) to 1788 Bridging studies demonstrate that products that pass 787 will pass 788 as well,so companies do not have to file with both Discussion to add adjustments in 787 to harmonized chapters is ongoingFor Internal Use Only. Amgen Confidential.27
Future plans AAPS focus groups on Protein aggregation and BiologicalConsequences is planning cross lab experiment (16 labs fromindustry, academia, regulatory agencies and NIST), usingaggregate from same proteins (6), generated by same stresses,and characterize in the same assays examine variability of characterization assays, in vitro and invivo models, understand the variability of assays Identify CQA of aggregates that have some activity in invitro and in vivo assays The outcome will be 3 publications, one for each phase of thestudy.
Some related publications Rigidly organized protein arrays in the micron range may be highly immunogenic VSV-G and VLV and regularly spaced acrylamide polymers (5-10 nM) are immunogenicBachmann et al., Annu. Rev. Immunol, 15 (1997) 235-70.Denis et al., Virology, 363 (2007) 59-68.Dintzis et al., PNAS, 73 (1976) 3671-5.Chackerian et al, J Immunol, 169 (2002) 6120-6. Immune response of protein coated nanobeads and preferential internalization of protein coated aluminum adjuvantsby DCsFifis et al., J Immunol, 173 (2004) 3148-54.Morefield et al., Vaccine, 23 (2005) 1588-95. Reports of protein aggregate immunogenicity in vivo give conflicting results Aggregates of IFN-g: metal-catalyzed and pH/50 C induced aggregates (but not untreated, crosslinked, hydrogenperoxide or boiled) can break tolerance in transgenic mice.Hermeling et al., Pharm Res, 22 (2005) 1997-2006.Hermeling et al., J Pharm Sci, 95 (2006) 1084-96. Aggregates of FVIII: heat induced aggregates were less immunogenic than the monomeric protein.Purohit et al., J Pharm Sci, 95 (2006) 358-71. Aggregates of GH: freeze-thaw and agitation induced aggregates were not able to break the tolerance of transgenicmice (freeze-thaw and GH absorbed onto glass or alum particles showed an enhanced response in wild-type mice).Fradkin et al., J Pharm Sci, 98 (2009) 3247-64.Fradkin et al., J Pharm Sci, (2011) Only highly chemically modified aggregates (oligomers) broke tolerance in transgenic mouse modelBessa et al Pharm Res (2015) DOI 10.1007/s11095-015-1627-0 A weak transient response was obtained with aggregates in the 2-10 micron size range with some native structureand chemical oxidation in a Xeno-het modelBI et al J Pharm Sci 2013 102 (10): 3545-55For Internal Use Only. Amgen Confidential.29
Related Publications, cont’d John F. Carpenter, Theodore W. Randolph, Wim Jiskoot, Daan J.A. Crommelin, C. Russell Middaugh, Gerhard Winter, Ying-xin Fan, SusanKirshner, Daniela Verthelyi, Steven Kozlowski, Kathleen A. Clouse, Patrick G. Swann, Amy Rosenberg, Barry Cherney. Overlooking SubvisibleParticles in Therapeutic Protein Products: Gaps That May Compromise Product Quality J Pharm Sci. 2009 Apr;98(4):1201-5. doi:10.1002/jps.21530.Singh, Satish K. ; Afonina, Nataliya; Awwad, Michel; Bechtold-Peters, Karoline; Blue, Jeffrey T.; Chou, Danny; Cromwell, Mary ; Krause, HansJuergen ; Mahler, Hanns-Christian; Meyer, Brian K.; Narhi, Linda; Nesta, Doug P.; Spitznagel, Thomas . An Industry Perspective on theMonitoring of Subvisible Pa
SbVP_mAb Y stressed DS at 120 mg/mL HIAC MFI Coulter 1 10 100 1,000 10,000 100,000 1,000,000 0 20 40 60 HIAC, MFI and Coulter Counter gave different particle concentrations and particle size distributions for protein particles in mAb Y drug substance at 120 mg/mL Relative r
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