Data from original development of the method for electrophoretic mobility determination in red blood cells (1988) have been reprocessed according to QbD principles (2)(3)(4) in 4 sequential steps: 1) Method development: Analytical Target Profile (ATP), Critical Method Parameters (CMP) and Critical Method Attributes (CMA) and their acceptance criteria were established. 2) Risk assessment: factors affecting the analytical behavior were analyzed using a Priorization Matrix and Failure Mode and Effects Analysis (FMEA). 3) Method robustness: Design of Experiments (DoE) tools were used to determine Method Operable Design Reg ion (MODR) where the CMA acceptance criteria are met, and Control Space which defines the best conditions for analytical determinations. 4) Method ruggedness: Measurement System Analysis (MSA) techniques were used to quantify equipment, analysts and samples contribution to total method variability.

MODR

MODR

APPLICATIONOFQbDAPPROACHTOTHEDEVELOPMENTOFACYTOPHEROMETRICANALYSISOFHUMANREDBLOODCELLS:COMPARISONWITHTRADITIONALDEVELOPMENTMETHODOLOGY.

FernandoFERRÁNDIZ-VINDEL*andAdriánGARCÍADEMARINABAYO.GlaxoSmithKline,S.A.*/InkemiaIUCTGroup

fernando.ferrandiz@wanadoo.es

Cytopherometry has been used to measure the electrophoretic mobility of normal and pathological human red blood cells . A c c o r d i n g t o t h e o r i g i n a l m e t h o d development (1), the most convenient methodology consists of the application of a 10 mA electric field to fresh-not-washed erythrocytes (preserved at 4ºC if necessary), suspended in physiological saline solution (pH = 5.65, ionic strength = 0.1 M) and measured within a thermostatted chamber at 25-30ºC.

1)Ferrándiz,F.,Ródenas,S.,DelCastillo,B.(1988):“Análisiscitoferométricodeeritrocitoshumanos”.AnálisisClínicos,XIII,117-1202)http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Quality/(access25/07/2018)3)Borman,P.,Nethercote,P.,Chatfield,M.,Thompson,D.,Truman,K.(2007):“TheApplicationofQualitybyDesigntoAnalyticalMethods”.Pharm.Tech.31(10),142-152.4)Nethercote,P.,Ermer,J.(2012):“QualitybyDesignforAnalyticalMethods”.Pharm.Tech.Europe,24(10),52-56.5)DeepaM.,RavindraReddyK.,SatyanarayanaS.V.(2017):“Areviewonqualitybydesignapproachforanalyticalmethoddevelopment”.J.Pharm.Res.11(4),272-277.6)ArgentineM.,BarnettK.,ChatfieldM.,HewittE.,JacksonP.,KarmarkarS.,MarolewskiA.,PlessA.,RignallA.,SeminD.,TroneM.D.,WangQ.,WilliamsZ.,ZhaoY.(2017):“Evaluatingprogressinanalyticalqualitybydesign”.Pharm.Tech.Europe29(4),36-42.

XFactorsORIGINAL FOLLOWINGQbD

Workingconditions MODR(DesignSPace)

ControlSpace

SuspensionmediapH 5.65 5.00-5.75 5.65Mediaionicstrength 0.1M 0.1–0.2 0.1MElectrophoretic chambertemperature

25–30ºC 25–31ºC 25ºC

Electricalcurrentintensity 10mA 10–15mA 10mASamplestoragetemperature 4ºC N/A 4ºCRed blood cells (RBC)washing

No N/A No

UseoffreshRBC Yes N/A Yes

Results obtained following QbD show an enhancement in the knowledge and control of the cytopherometric method originally developed in 1988, giving more flexibility on CMP management and appropriate tools to control the principal sources of analytical variability. This behavior has been widely found when using analytical QbD (5)(6).

Pp/Ppk19.3653.3956.79056.79

% Proc5.3814.8315.78015.78

% Tol9.6826.728.4028.4

% Contrib11.6288.381000100Total

ANOVA AnalysisSourceEquipmentAppraiserGRRParts

DoE

MSA

VariableVariable

type (X/N/C)

Accuracy Precision Repeteability Reproducibility Selectivity Global Score

Ionic Strength X 9 5 5 5 5 29pH X 9 5 5 5 5 29Current intensity X 9 9 9 9 9 45Chamber temperature X 9 9 9 9 9 45Suspension media composition C 9 5 5 5 5 29

Process Possible error Consequences Probability (1 to 5)

Severity (1 to 5) Risk Validation

implications

Staff trainingStaff not enough trained in

sample preparation

High dispersion of results (high %CV)-

Reproducibility3 4 12 HIGH

Correct staff training

EquipmentInappropriate maintenance

and use records

High dispersion of results (high %CV)-

Repeatibility2 4 8 MEDIUM

Appropriate maintenance

Equipment Inappropriate placementHigh dispersion of

results (high %CV)-Reproducibility

5 4 20 HIGHUse appropriate

placement for the equipment

InletIncorrect sample inlet

(bubbles)

High dispersion of results (high %CV)-

Repeatibility1 2 2 LOW

Equipment11.62%

Appraiser88.38%

Parts,0,00%

Equipment

Appraiser

Parts

QbDterms Commments

AnalyticalTargetProfile(ATP)

Humanerythrocyteselectrophoreticmobilityaccuratedeterminationbycitopherometry,withabilitytodiscriminate between normal and pathological cells andwithout interference fromother particles insuspension.

CriticalMethodParameters(CMP)

• Ionicstrengthofsuspensionmedia• pHofsuspensionmedia• Electricalcurrentintensity• Temperature• Other(tobedetermined)

CriticalMethodAttributes(CMA)

Accuracy N/A(originaldevelopment)%Error ≤ 9,3% (QbD based onstatisticalanalysis)

Precision% CV≤ 10,0% (originaldevelopment, based onbibliography)

%CV≤ 7,16% (QbD based onstatisticalanalysis)

Selectivity: cells distribution(histogramplotter)

Erythrocytes subpopulations determination using histogramplotter. Normal erythrocytes should show only one population