The Use Of Quality-by-Design And DOE Tools For BioAssay .
The Use of Quality-by-Designand DOE Tools for BioAssayDevelopment:Part 1: Component OptimizationDr. Laureen E. LittleCopyright by Quality Services and Laureen Little1
Contact InformationLaureen Little, Ph.D.Quality ServicesPrincipal Consultantphone:951-659-1957email: Biotech@ix.netcom.comCopyright by Quality Services and Laureen Little2
Legal Stuff U.S. Copyright Law protects the course notes from unauthorizedduplication. COPYRIGHTDISCLAIMER:The information contained in these course notes has been compiled from varioussources and is believed to be reliable and to represent the best current opinionrelative to this course. FasTrain offers no warranty, guarantee or representation asto its absolute correctness or sufficiency. FasTrain assumes no responsibility inconnection therewith; nor should it be assumed that all acceptable safety andregulatory measures are contained herein, or that other or additional informationmay be required under particular or exceptional conditions or circumstances.Copyright by Quality Services and Laureen Little3
Before we start: You have a transceiver. These are to allow us to dosome interactive things. When the clock appears in the bottom right hand sidepush a number for your answer. A green light will appear. If it remains green and thengoes out your answer was accepted. If the lightbecomes red your answer was not received. Try again. If you hit the wrong answer – just answer again. Thefirst answer will be removed and replaced with themost recent answer. (only 1 answer allowed pertransceiver.)Copyright by Quality Services and Laureen Little4
About YouWhat Kind of Company do you work for?1.Contract Organization2.Small Biopharmaceutical ( 50employees)3.Mid-Size Biopharmaceutical (50– 300)4.Global Pharmaceutical5.Consultant6.Research Institute7.OtherCopyright by Quality Services and Laureen Little56%60%50%40%30%20% 15%12%7%10%2% 6%1%0%510
EU RESULTS6
Where are the bioassays developed?1.In–house for own products (I’m product developer/manufacturer)2.Contracted out (I’m product developer/manufacturer)3.1 24.In-house (I’m a contract organization)5.By product developer/manufacturer client (I’m a contract organization)6.4 5Copyright by Quality Services and Laureen Little710
50%45%40%35%30%25%20%15%10%5%0%In-house (I’m acontractorganization)84 51%7%By productdeveloper/manufacturer client (I’m Copyright by Quality Services and Laureen Little1 2Contracted out(I’m productdeveloper/manuf In–house for ownproducts (I’mproduct Where?43%31%15%2%
EU Responses9
Stages at which assay(s) used1.Preclinical development2.Phase 1 / Phase 2 / Phase 33.Post-marketing4.Preclinical development / Phase 1 / Phase 2 / Phase 35.Phase 1 / Phase 2 / Phase 3 / Post-marketing6.Preclinical development / Phase 1 / Phase 2 / Phase 3 / Postmarketing7.We have biosimilar products – therefore the above doesn’t make sense8.OtherCopyright by Quality Services and Laureen Little1010
Copyright by Quality Services and Laureen Little0%8%7%Other7%We have biosimilarproducts – Preclinicaldevelopment / 17%Phase 1 / Phase 2 /Phase 3 / Post- Preclinicaldevelopment / 3%Post-marketing45%40%35%30%25%20%15%10%5%0%Phase 1 / Phase 2 /Phase 3PreclinicaldevelopmentWhen?42%15%11
EU Responses
Functional or Ligand Binding?1.Cell –Based functional primarily2.Animal tests primarily3.Binding Primarily4.1 35.1 26.1-3Copyright by Quality Services and Laureen Little1310
Types of Assays65%Copyright by Quality Services and Laureen Little141-34%1 21%1 34%BindingPrimarily14%Animal testsprimarily12%Cell –Basedfunctionalprimarily70%60%50%40%30%20%10%0%
EU Response
Functional Assay types used1.cell-based2.cell-free3.cell-based and functional cell-free4.Binding (ligand, receptor, cofactor, .)5.cell-based binding6.cell-free binding7.cell-based functional cell-free bindingCopyright by Quality Services and Laureen Little1610
Functional Assay types ht by Quality Services and Laureen Little3%1%0%17
EU Response
Binding Assay type (primarily)1.Immunoassay2.functional &/or binding3.SPR4.qPCR5.FTIR6.OtherCopyright by Quality Services and Laureen Little1910
Ligand Binding Assays70%60%50%40%30%20%10%0%60%27%Copyright by Quality Services and Laureen Little8%2%2%202%
EU Responses
How many bioassay systems do you run?1.None2.13.2-54.5 - 105.10 - 206.More than 20Copyright by Quality Services and Laureen Little50%40%30%20%10%0%44%32%12%2% 3%227%10
EU ResponseCopyright by Quality Services and Laureen Little23
DOE in your labWhat is your current use of DOE?Never2.Just starting3.Use for robustness only4.Use for trouble shooting5.Use for componentoptimization6.3 and 47.3 and 58.3, 4 and 59.OtherCopyright by Quality Services and Laureen Little32%35%30%25%16%20%14%12%15%10%10%10%1% 4%1%5%0%NeverJust startingUse for Use for Use for 3 and 43 and 53, 4 and 5Other1.2410
EU ResponseCopyright by Quality Services and Laureen Little25
Design of Experiments (DOE)in Bioassays DOE is a tool which can be used throughoutthe entire development cycle. It is best used sequentially (i.e. don’t try todesign one experiment to ask all yourdevelopment questions). Current bioassay field uses DOE to determinerobustness. While this is a fabulous tool – if itis your only use, then you are starting toolate!Copyright by Quality Services and Laureen Little2626
Your DesignsHow do you design you DOEs1.We have a statistician (eitheremployed or consultant)2.Use a software and design myown.3.Design my own withoutsoftwareCopyright by Quality Services and Laureen Little50%40%30%20%10%0%47%37%16%2710
EU ResponseCopyright by Quality Services and Laureen Little28
Types of DoE Screening methodologies : which are designed todetermine what factors are important. Fractional Factorials Specialized designs such as Taguchi (PlackettBurman)Full Factorials: which are designed to determinethe best conditions of the factors you know to beimportant Most common one we see: 23 factorialCopyright by Quality Services and Laureen Little29
Your Design (Continued)What type of designs do you use?1.Full Factorial only2.Specific screening design only(such as a placket-burman)3.Fractional Factorial4.A mixture of the above5.Too early in our use of DOE tobe able to answer this questionCopyright by Quality Services and Laureen Little40%35%30%25%20%15%10%5%0%35% 33%16%8%8%3010
EU ResponseCopyright by Quality Services and Laureen Little31
Cell Culture Example This was a screening design – we were trying tooptimize a component – the cell culture – we didn’tknow what was important. Choose 5-6 factors and design a FractionalFactorial. This can be done twice. We may find that most of the factors we think weshould study – don’t actually impact the method.Therefore it is smart to figure out which factorsare critical and then study them.Copyright by Quality Services and Laureen Little3232
This is a Sequential DOEApproach Following is a sequential methoddevelopment – using sequential DOE. The following example is a design todetermine the best growing conditions fora cell-based potency assay. Why? It is themost crucial component for achievinglow imprecision.Copyright by Quality Services and Laureen Little3333
Choosing the Right Response Most of the DOEs that I have seen have notcarefully thought through what should be themeasured response. This is especially important if you are tryingto optimize the assay by improving a specificcharacteristic or component of the assay. IN THIS EXAMPLE WE DID NOT HAVE DRUGPRESENT!! Since we were only trying tooptimize the cells we only looked at aviability dye.Copyright by Quality Services and Laureen Little3434
In Other Assay Systems Iwould have: Perhaps looked at specific receptor expression on thecell Looked at zero and high drug concentrations Perhaps other viability marker? But when optimizing components – you normally do notwant to look at the entire assay.Copyright by Quality Services and Laureen Little35
Additional Examples ELISA assays We were having a non-specific binding problem. We usedsequential DOE to identify blocking reagents andprocedures to essentially eliminate this. Our read out wasaverage of 5 blank samples and 5 high samples. Wecalculated results for both Blank average and Signal/Noise(Z’ factor)Cell based assay (biomarker) We were having dilutional linearity problems. We usedsequential DOE. Here we used 4 single point dilutions ofseveral patient samples and looked at “average” relativebias numbers. We found a sample diluent whichcompletely solved the problem. (This took 5 placketburman runs, followed by 2 full factorials)Copyright by Quality Services and Laureen Little36
Back to example at Hand:Some Interesting Things about this Design It was chosen to do this in 16 plates (basedupon the tissue culture analyst that couldhandle 8 plates) Note the analysts actually informed me theycould easily handle 16 plates per day. I assumedthat in any type of DOE throughput drops by 50%because of the complexity of the individual assayruns.We chose 6 variables.Copyright by Quality Services and Laureen Little3737
First Select the Factors and LevelsNamePassage #Seeding DensityTryp concentrationTryp IncubFBS lotsDay FeedingR1R2UnitsTypeLowHighpassagecells percm2mlMinutesLottimes perweek%CV*BowlRatio**Factor P10 Response12Copyright by Quality Services and Laureen LittleResponse3838
Initial Screen from StatEase Number of runsThis gave us a 26-2 Fractional Factorial.This is a level 4 resolution.What does this mean?Copyright by Quality Services and Laureen Little3939
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What is the Response? This is component optimization– not assay optimization. Therefore, what are the characteristics wewould like to see? Well-to-wellconsistency of growth (This can bemeasured by an Alamar Blue dye, cell-titer glo,whatever viability assay you have – then reportedout as an average and %CV.) Lackof systematic bias: Experience tells us that thebowl ratio is the most common growth pattern:Therefore, let’s take Avg OD outer/Avg OD innerCopyright by Quality Services and Laureen Little4141
Run DesignStd Run 516P# P6 P6 P6 P6 P6 P6 P6 P6 P12 P12 P12 P12 P12 P12 P12 P12Seedingcells per 0500010000100001000010000[Tryp] Try Incubml2424424242422424Copyright by Quality Services and Laureen B2B1B2B1B1B2B1B2B2B1B2B1B1B2B1B2X /week %CV Bowl Ratio21212121121212124242R1R2
Results All of the results had a serious positionalproblem. None of the factors studied had an impact. Did a second round of experimentation lookingat more of the technique issues.Copyright by Quality Services and Laureen Little4343
NameUnitsTypeLowHighFactorSimple10 xInversion inversionY/NFactorup/down2 x inpipette on rotarypipette typeFactor12 nutes)Factor515R1%CV*ResponseR21st row vslast rowResponseInitial mixing of cellsMixing prior todispensingpipette type usedTemperature (media)pipette tipsTrypsinzationY/NCopyright by Quality Services and Laureen Little4444
Copyright by Quality Services and Laureen Little4545
Original Run Lay Out Selectedby SoftwareProblem is that having mixing procedures intertwined is procedurally difficult.Copyright by Quality Services and Laureen Little4646
Sorted by Factor 1Still had a practical problem: Next sorted by Factor TwoCopyright by Quality Services and Laureen Little4747
Final Run Lay OutCopyright by Quality Services and Laureen Little4848
Insert Data for AnalysisCopyright by Quality Services and Laureen Little4949
R1 – Diagnostic PlotCopyright by Quality Services and Laureen Little5050
R1 – Diagnostic PlotCopyright by Quality Services and Laureen Little5151
This Demonstrates theImportance of the InteractionCopyright by Quality Services and Laureen Little5252
Based on R2 (Ratio of 1st to Last Row)Copyright by Quality Services and Laureen Little5353
Now Went Back to the FirstDesignNamePassage #Seeding DensityTryp concentrationTryp IncubFBS lotsDay FeedingR1R2UnitsTypeLowHighpassagecells percm2mlMinutesLottimes perweek%CV*BowlRatio**Factor P10 Response12Copyright by Quality Services and Laureen LittleResponse5454
Results Found pre-mixing (mixing prior todispensing) important. Repeated the first screening and foundpassage number and trypsinzationconditions were also important Did a 23 full factorial and limited thepassage number to less than 20 (based onother available data – FACs studies to lookat stability of the receptor expression).Copyright by Quality Services and Laureen Little5555
Design SelectedCopyright by Quality Services and Laureen Little5656
Sorted by Passage NumberCopyright by Quality Services and Laureen Little5757
Diagnostic PlotIndicates that the PassageNumber and the Length of theTrypsin Incubation have anEffectCopyright by Quality Services and Laureen Little5858
Choose Model vs. Error TermsCopyright by Quality Services and Laureen Little5959
D Trypsin incubation A Passage numberCopyright by Quality Services and Laureen Little6060
Take Home Messages This example was to indicate that the DOE can beused during optimization – not just finalcharacterization or verification of assayperformance. Sequential DOE studies is an excellent approach. Don’t panic if the first design doesn’t yield “results”– perhaps you didn’t select the appropriate factors tostudy. Although a statistician is a real asset with modernsoftware – you can still use DOE and have it reallyaccelerate your development time.Copyright by Quality Services and Laureen Little6161
What to Do? This specific result should not be taken as a universaldecision. Specifically, not every cell line should be pre-mixed asdescribed here. Not every cell line will be sensitive to Tryspinization orhave the same passage number restrictions. I would suggest instead, that you might be able tocome up with a universal screening design – offactors into which specific levels for a given cell linecould be inserted. Then, require that as part of development, each newproposed cell-line would be tested in this universaldesign.Copyright by Quality Services and Laureen Little6262
But .DOE isn’t the Panaceafor all Component Assay WoesIt is a great tool to quickly differentiate criticalparameters from those which have little impact.It doesn’t eliminate the need for focused scientificproblem solving.Always start with potential scientific root causes toperformance problems63
Copyright by Quality Services and Laureen LittleBut .What about 69One Low 11120.3780.3990.3450.3450.3890.3990.3870.358
Potential Causes Poorly calibrated pipette Insufficient Number of Cells Too concentrated of some biologicalcomponent which is killing cells orinhibiting growth Reader Problem (off-set detector)Copyright by Quality Services and Laureen Little65
Copyright by Quality Services and Laureen LittleYet another case 9Gradual Increase Across the 430.6670.4470.6310.5550.549
Possible Causes Settling of cells during initial pipetting Fragile cells breaking during mixing during plating Increased or Decreased concentration of a criticalcomponent because of dilution scheme Time differences due to manipulation of the cellsCopyright by Quality Services and Laureen Little67
Your ComponentsWe have talked a lot about cells. Do you optimize well-to-wellcharacteristics of your plates?1.Sometimes2.We don’t usually need tobecause we get our cells from apotency group which hasoptimized our cells3.AlwaysCopyright by Quality Services and Laureen Little60%50%40%30%20%10%0%50%38%13%6810
EU ResponseCopyright by Quality Services and Laureen Little69
Ready-to-UseDo you use Ready to Use cells?1.Never2.Always3.Whenever possible4.Are just implementing5.Not applicable to our productsCopyright by Quality Services and Laureen Little50%40%30%20%10%0%45%26%21%4%4%7010
EU ResponseCopyright by Quality Services and Laureen Little71
Have you had any discussion onDOE with regulators?1.No relevant discussion with regulator2.Subject raised but no comment from regulator3.Subject raised and regulator suggested use4.Regulator spontaneously suggested use5.Data based on DOE submitted – no comment6.Data based on DOE submitted – favourable response7.Data based on DOE submitted – modifications suggested8.Data without DOE submitted – regulator required use9.Different responses in different casesCopyright by Quality Services and Laureen Little7210
Discussion with ht by Quality Services and Laureen Little0%16%4%0%734%
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Appendix: DOE BasicsThese are for your convenience and were not coveredin the talk.Copyright by Quality Services and Laureen Little75
DOE Designs If you only have a few conditions (such as with stability) –typically do full-factorial designs If you have many conditions and you are interested in findingwhich (if any) are important, you will do partial factorial(screening) designs E.g. Plackett-Burman, Fractional FactorialsCopyright by Quality Services and Laureen Little7676
DOE - Basics Factors The assay conditions or reagents thatyou vary (e.g.: incubation temperature,dilution, incubation time, etc. Usually assignthese letters Level The condition of the factor which youtest (e.g.: 25 minutes vs. 35 minutes, 1:1000vs. 1:2000 dilution, etc.) In the followingexample:High level Plus ( ) (or can be capital letters)Low Level Minus (-) (or can be small letters)Copyright by Quality Services and Laureen Little7777
Example # 1 Few Conditions: Therefore factorial designat two levels: 23 look at three factors This is three factorsThis tells you there are two levels.An example would be :FactorTimeTemperaturepHCopyright by Quality Services and Laureen Little 2h4h20 C 37 C6.587878
What is Factorial Design? Set of experiments so that more than onevariable can be tested at the same time. This is done by running all the possiblecombinations-of each factor at each level. Therefore 22 2*2 experiments: 4experiments And 23 2*2*2 experiments: 8Copyright by Quality Services and Laureen Little7979
Note that allof thesedesigns arebalanced.This is a keyaspect ofDOECopyright by Quality Services and Laureen Little8080
This type of design allows us todetermine the effect of changing eachvariable (aka: the main effect):NB: Y is themeasuredresponse81Copyright by Quality Services and Laureen Little81
NB: E is ameasureof theeffectCopyright by Quality Services and Laureen Little8282
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Design of Experiments (DOE) in Bioassays DOE is a tool which can be used throughout the entire development cycle. It is best used sequentially (i.e. don’t try to design one experiment to ask all your development questions). Current bioassay field uses DOE to d
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