estimation of protein aggregate density using ......richard cavicchi, dean ripple, jason king, cayla...

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Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7 # Biomolecular Measurement Division, NIST # West Virginia Wesleyan College ESTIMATION OF PROTEIN AGGREGATE DENSITY USING SEDIMENTATION COMBINED WITH MICRO-PARTICLE TRACKING

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Page 1: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

RichardCavicchi,DeanRipple,JasonKing,CaylaColle7#

BiomolecularMeasurementDivision,NIST#WestVirginiaWesleyanCollege

ESTIMATIONOFPROTEINAGGREGATEDENSITYUSINGSEDIMENTATIONCOMBINEDWITHMICRO-PARTICLE

TRACKING

Page 2: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

1.  PossibleimprovementstoFlowMicroscopyAnalysis

a.  Shapeeffect

b.  Newimageprocessingalgorithm

2.  AggregateDensitymeasurementsbasedonsedimentaMon

3.  NewReferenceMaterials

a. Fluoropolymeraggregatesimulants

b. Othermaterialsunderdevelopment

Outline

Page 3: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

LightobscuraMon FlowImaging

ElectricalSensingZone(Coulter)

SubvisibleParCcleSizingMethods

ΔR~volume

(ESZ)

(FI)(LO) areaΔI~area

ResonanceMass ΔM

ParMcletrackingD(relatedtodhydrodynamic)

Page 4: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Measureddiameter,concentraConforpolystyrenebeads.

Goodagreement

Page 5: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

ForProteinaggregates,methodsdon’tagree…

Ripple,D.C.andZ.S.Hu(2016)PharmaceuMcalResearch33(3):653-672.

Page 6: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Proteinaggregatesvaryinsize,shape,intensity…

Page 7: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

VariableThresholdMethodforimprovedParCcleBoundaryDetecCon

CanwegetimprovedboundarydetecMonwithoutlosingnearlytransparentparMcles?

Newalgorithm,breakanalysisintotwoparts:a)  FindparMcleswithasinglelowthresholdb)  EvaluateparMcleboundariesusinga

thresholdappropriatetotheintensityofeachindividualparMcle

Page 8: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

eba

d

c

VariableThresholdMethodforimprovedParCcleBoundaryDetecCon

Result:

Page 9: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

(e)

(b)(a)

(d)

(c)

Strategyforfragments

a. Connectfragments

b.  Drawtheconvexhull

c.andd.Perform“AND”operaMonwithoriginalthesholdedarea

e. ResulMngboundarysuperimposedonimage

Page 10: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Collagewithoverlays

ImplementedinImageJ/FIJI,opensourcesoeware

ResultstableofanalyzedparCcles

Page 11: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

CollagestripsofsamebeadsanalyzedwithsinglethresholdandVariableThreshold(redbackground)

10µm

2µm

10µm

5µm

20µm

(ImagesobtainedwithFlowcam10x)

TestofVariableThresholdMethod

Page 12: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

10µm

ECD13.4µm

SEMimage

VariableThreshold

Page 13: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

10µm

VariableThreshold

TestofVariableThresholdMethodproteinaggregates

Page 14: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

PossibleimprovementstoFlowMicroscopyAnalysis

a.  Shapeeffect,canuseperimetertoimproveequivalentcirculardiametercalculaMonforelongatedparMcles.

b.  ImageprocessingalgorithmyieldsMghterboundariesforintenseparMcles,withoutlosingcountsonnearlytransparentparMcles.

i)be7eranalysisforsampleswithvaryingparMcleproperMes

ii)sameinstrumentseingsforcalibraMonandoperaMon

iii)maybeusefulforimprovedclassificaMonofparMcles

Page 15: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Whatisthedensityofaproteinaggregateandwhyisitimportant?

Differentmethodscharacterizedifferentaspectsofsize:Flowimaging:spaMalextent(A~V)ElectricalSensingZone:liquidexcludedvolume(V’)ResonanceMass:MParMcleTracking:VhTocompareparMclecountsaboveacertainsize,orsizedistribuMonfromthesemethods,adensityvalueforproteinaggregatesisneeded.

ρ =VM

Page 16: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Forasphere

SedimentaCon/ParCcletracking

2gR29µv

Δρ= µviscoscityvvelocityg9.8m/s2

Rhydrodynamicradiusofsphere

ToreduceconvecMon,useRectangularglasscapillary50µmor100µminsidethicknessx1mm,sealedatbothends

MeasuringRfromimage,andmeasuringvbyfollowingimages,candeduceΔρ

Page 17: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

AggregatesfromNISTMaB

100µm

Page 18: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Forasphere

BrownianMoCon-MicroparCcleTrackingAnalysis

2Dt<x2>= Ddiffusionconstant

Rhydrodynamicradiusofsphere6πµR

kBTD =

2gR29µv

Δρ=

SedimentaCon:StokesLaw

Whereµ isviscoscity,gisgravitaMonconstant,visthevelocity(iniMal–finalverMcalposiMon/Mme),andRisparMcleradiusfromimageorBrowniantrajectoryanalysis(below)

<x2>ismeansquaredisplacement,calculatedfromparMcletrajectory(seeD.Ernst&J.Kohler,Phys.Chem.Chem.Phys.,2013,15,845)

Page 19: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Whatisthedensityofaproteinaggregate?

Page 20: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

100µm

3mmPolystyrenebeadsinwater

Page 21: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

100µm

3mmPolystyrenebeadsinwater,backgroundsubtracted

Page 22: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

100µm

3mmPolystyrenebeadsinwater,withtracks

ParMclesanalyzedbycustomImageJpluginTracksdeterminedusingTrackmate

Page 23: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Beadtrajectories-MicroparCcletrackinganalysis

1µm 2µm

5µm 10µm

Polystyrenebeadsinwater

3µm

Page 24: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Results1µmbead

Page 25: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Results2µmbead

Page 26: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Results5µmbead

Page 27: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Results10µmbead

Page 28: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Beads:ImageDiametervsTrackingDiameter

MSDImage1 54.2431 66.78522 51.9681 69.55425 43.1026 41.079310 53.313 43.664

Beads:DensitybyMSD&Image(aaerdiamcorrecCon)

Page 29: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Beads:ImageDiametervsTrackingDiameter

MSDImage1 54.2431 66.78522 51.9681 69.55425 43.1026 41.079310 53.313 43.664

Beads:DensitybyMSD&Image

Page 30: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

100µm

AggregatesfromNISTMaBwithtracks

ParMclesanalyzedbycustomImageJpluginTracksdeterminedusingTrackmate

Page 31: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

DensityvsTrackingDiameter

Average

Avgdensity~1040kg/m3Aggregatedensityincreaseswithdecreasingsize

Page 32: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

Conclusions•  Improvementsinanalysiscanprovidebe7eraccuracyinparMclesizingforFlowimaging:variablethresholdpluginforImageJ/FIJI

•  ParMcledensitycriMcalforrelaMngcountsfromdifferentinstruments

•  MicroparMcletrackinggivesusefuldimensionsupto~5µm•  ResultsbasedonsedimentaMonof1µm-7µmparMclesindicatesadensityof1040kg/m3,withdensityincreasingwithdecreasingsize.Thisdensityislowerthangenerallyassumedintheliterature.

Page 33: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

20µm

Silicabeadsn=1.33fluid

Silicabeadsn=1.43fluid

VariableThreshold

6µm

4µm

4µm

6µm

TestofVariableThresholdMethod

Page 34: ESTIMATION OF PROTEIN AGGREGATE DENSITY USING ......Richard Cavicchi, Dean Ripple, Jason King, Cayla Colle7# Biomolecular Measurement Division, NIST #West Virginia Wesleyan College

5µm

Proteinaggregateatdifferentfocaldistances