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PF-07302048 Validation Protocol fo r the SARS- CoV-2 mNeonG reen Virus Microneutralization Assay
VR-MVP -10074, Ver. 2.0
PFIZER CONF IDENTIAL
Page 1
Title: Validation Protocol for the SARS -CoV -2 mNeonGreen Virus Microneutralization
Assay
Study Number: N/A
Parent Compound Number(s): PF-07302048
Alternative Compound Identifiers: N/A
Pfizer Vaccine Research and Development
401 N. Middletown Rd.
Pearl River, NY
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PF-07302048 Validation Protocol fo r the SARS- CoV-2 mNeonG reen Virus Microneutralization Assay
VR-MVP -10074, Ver. 2.0
PFIZER CONF IDENTIAL
Page 3Title: Validation Protocol for the SARS -CoV -2 mNeonGreen Virus Microneut ralization
Assay
SYNOPSIS
This protocol descri bes the pr ocesses and requirements for validation of the SARS -CoV-2
mNeonGreen Virus Microneutralization Assay (SARS -CoV -2 mNG NT) used forthe
detection of serum antibodies capable of neutralizing SARS -CoV -2. This assay may be used
to ev aluateclinical t rial sera and support epidemiological and non- clinical studies. The
following paramet erswill be investigated :
1.Dilutional L inearity
2.Precision
3.Intermediate Precision (inter -assay variability )
4.Lower and Upper Limits of Quantitation (LLOQ and ULOQ)
5.Limit of Detection (LOD)
6.Extravariability Ratiofor Replicate Titers
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VR-MVP -10074, Ver. 2.0
PFIZER CONF IDENTIAL
Page 4TABLE OF CONTENTS
SYNOPSI S................................ ................................ ................................ ................................ .3
LIST OF TABLES ................................ ................................ ................................ ..................... 5
LIST OF FIGURES ................................ ................................ ................................ ................... 5
1.OBJECTI VES ................................ ................................ ................................ ........................ 6
2.INTRODUCTION ................................ ................................ ................................ ................. 6
3.GLOSSARY ................................ ................................ ................................ .......................... 7
4.ROL ES AND RESPONSIB ILIT IES................................ ................................ ..................... 7
5.MATERIAL S AND METHO DS................................ ................................ ........................... 8
5.1.Viral Stock ................................ ................................ ................................ ................. 8
5.2.Cell Culture ................................ ................................ ................................ ............... 8
5.3.Quality Control Samples ................................ ................................ ........................... 9
5.4.Negative Serum Diluent ................................ ................................ ............................ 9
5.5.Validation Sample Panels ................................ ................................ .......................... 9
5.6.Assay Methods ................................ ................................ ................................ .......... 9
6.PROTOCOL DESIGN ................................ ................................ ................................ ......... 10
6.1.Validation Parameters ................................ ................................ ............................. 10
6.2.Design Summary ................................ ................................ ................................ .....11
6.2.1. Design of Dilutional L inearit y and Precision Experiments ........................ 11
6.2.2. Dilutional L inearity Samples ................................ ................................ ......12
6.2.3. Precision Samples ................................ ................................ ....................... 12
6.2.4. Negative Samples ................................ ................................ ....................... 12
7.STATI STICAL ANALYSI S ................................ ................................ ............................... 13
7.1.Log Transformation................................ ................................ ................................ .13
7.2.Dilutional L inearity ................................ ................................ ................................ .13
7.3.Precision ................................ ................................ ................................ .................. 13
7.4.Limits of Quantitation ................................ ................................ ............................. 14
7.5.Assay Intermediate Precision ................................ ................................ .................. 14
7.6.Limit of Detection ................................ ................................ ................................ ...15
7.7.Extravariability of Replicate s................................ ................................ .................. 15
7.8.Titer Reporting Range ................................ ................................ ............................. 15
8.DEVIATIONS MANAGEMENT ................................ ................................ ....................... 15
9.VALIDAT ION REPORT ................................ ................................ ................................ ....16
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PF-07302048 Validation Protocol fo r the SARS- CoV-2 mNeonG reen Virus Microneutralization Assay
VR-MVP -10074, Ver. 2.0
PFIZER CONF IDENTIAL
Page 510.REFERENCES ................................ ................................ ................................ .................. 16
11.SUPPORTIVE TABLES ................................ ................................ ................................ ...17
11.1.Validation Run Plate Map for Dilutional L inearit y Runs ................................ .....17
11.2. Validation Run Plate M ap for Precision Runs ................................ ...................... 17
12.SUPPORTIVE FIGURES ................................ ................................ ................................ ..19
12.1. Assay Design Schematic ................................ ................................ ....................... 19
LIST OF TABLES
Table 1. Terms and Definitions ................................ ................................ ................ 7
Table 2. Roles and Responsibilities................................ ................................ .......... 7
Table 3. Validation Run Schedule ................................ ................................ .......... 12
LIST OF FIGURES
Figure 1. Assay Plate Lay out ................................ ................................ ................... 10
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VR-MVP -10074, Ver. 2.0
PFIZER CONF IDENTIAL
Page 6Title: Validatio n Protoc ol for the SARS- CoV -2 mN eonGreen Virus Microneut ralization
Assay
Study Number: N/A
Function al Area: Vaccine Resear ch and Develo pment
Test Facility :Hackensack Meridien He alth
Nutley , NJ 07110
Study/Testing Initiation Date: N/A
Study/Testing Comp letion Date: N/A
1.OBJECTIV ES
This protocol describe sthe processes and requirement s for vali dation of the SARS -CoV -2
mNeonGreen VirusMicroneutralization Assay (SARS-CoV -2 mNG NT) used forthe
detection of serum antibodies capable of neutralizing SARS -CoV -2.This validation will
provide documented evidence that the SARS -CoV -2 mNG NT is suitab le for its intended use
when performed in a ccordance w ith standard ope rating procedures by qualified perso nnel.
2.INTRODUCTION
The SARS- CoV -2 mNG NT is a biofunctional assay that measures neutr alizing antibodies
against SARS -CoV -2. The SARS- CoV -2 mNG virus i s derived fro m the USA_WA1/2020
strain t hat had bee nrescued by reverse genetics and engineered to cont ain a mNeonGreen
(mNG) reporter gene in open reading frame 7 ofthe v iralgenome that p roduces green
fluorescence upon productive infection of cells .1This reporter virus generat es similar plaqu e
morphologi es andindistinguis hable growth curves from wild -type virus.2
This assay is described in the test method VR-TM-10298.3 Briefl y,seriall y diluted test
serum samples are mi xed with SARS -CoV -2 mNG virus in a 96- well plate to allow
virus-specif ic antibodies to bind t o the virus . Thisserum -virus mixture is then transferred
ontoaVero cell monolayer and incubated overnight toallow for infection by non-neutralized
virus . Product iveviral infection is detected b y enumerating green fluore scent viral foc i using
acell-i magin g reader .The total number of cells per well is calc ulated b y visualizing Hoechst
33342 stained Vero cell nuclei, which are blue. An infection ratio is then calculated for each
well, whereb y the total number of virus i nfected (green) cells is divided by the total n umber
of cells present (blue nuclei). A sample tite r is defined as the reciprocal serum dilutio n at
which a specif icpercentage of the virus is neutralized ,eg, 50%, 80% or 90% (termed “Titer
Determining Valu e”, TDV ).
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VR-MVP -10074, Ver. 2.0
PFIZER CONF IDENTIAL
Page 73.GLOSSARY
Table 1.Terms an dDefinitions
Term Definition
Assayrange Range of antibody titers that can be measured in the ass aywith acceptable
dilutional linearity andprecision .
BSL-3 Biosafety Level 3
COVID -19 Corona virus D isease 2019
DL Dilutional linearity
GDMS Global Doc ument Manageme nt System
GMT Geometric Mean Titer
GxP Good Pra ctice Quality Guidelines
IR Infectionratio; the number of vir us infected cells (green) divided by the total
number of cells present (blue)
LLOQ Lower Li mit of Quantitation
LOD Limit of Dete ction
MDP Master Dilut ion Pl ate
NHS_dep Normal Human Serum -Antibody Depleted
NT Micro neutr alizationAssay
QCS Quality Control S ample
Replicate An independent de termination of an assay result
RSD Relative Standard Deviation
SARS -CoV -2 Severe acute re spiratory synd romecoronavirus 2;the etiologi c agent of COVID -19
SARS -CoV-2 mNG NT 96-well manual microneutralization assay for the detection of functional antibodies
to SARS -CoV -2 using the m NeonGreen reporter virus .
SAS Programming language and integrate d software s olution -set proprieta ry to SAS ®
(the company) that enables the coding of t he va rious tasks associated w ith handling
datasets .
SOP Standard Operating Procedure
TDV Titer Determinin g Value; the threshold value in percent of measured viral g reen
particl e counts that is used to repo rt sample titers. Titers may be reported at 50% ,
or 90% TD V.
TM Test Method
ULOQ Upper Limit of Quantitation
Vero Africangreen monkey kidney epithelial cell line
4.ROLES AND R ESPONSIBILIT IES
Table 2. Roles and Responsibilities
Role Responsibilities
AssayAnalyst Read, understand, and complete training on the applicable Standard Operating
Procedures(SOP) and Test Me thods (TM) used to perform the SARS -CoV- 2
mNG NT .
Execute this validation protocol.
Analyst Read, understand, an d com plete tra ining on the applicable Standard Operating
Procedures (SOP )and T est Methods (TM) u sed to prepa re the serum dilution
assay plates used in the SARS -CoV -2 mNG N T.
Prepare serum di lution assay pla tes for use in executing thi s protocol .
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PFIZER CONF IDENTIAL
Page 8Table 2. Roles and Responsibilities
Role Responsibilities
Reviewer Read , underst and, and complete training on the applicable Standard Operating
Procedures (SOP) and Test Metho ds (TM) used to perfo rm the SARS -CoV-2
mNG NT .
Read and underst and this validation proto col.
Revie w the SARS -CoV- 2 mNG NT documentation to ensure c ompleteness of
data entry and compliance with relevant poli cies. Di rect the As sayAnalyst to
correct documen tation errors.
Consul t with a Supe rvisor or Senior Manager if thereare any questi ons
related to t he procedure itself , the serum samples, aty pical event sor prot ocol
deviatio ns that may occur.
Supervisor Ensure tha t the Ass ay Analyst and Analyst executingthis protoc ol
are traine d on the appropriate SOPs a ndTMs.
Revie w and ev aluate data gene rated during execution of this validation
protoco l.
Author vali dation protocol and/or reports.
Ensure that validation protocol andreport are entered into GDM S.
Senior Manager Review and appro ve this validation protoco l.
Ensure the accurat e execution of t his validation protocol.
Revie w and evalu ate data gene rated dur ingexecution of this validation
protocol .
Statistician Works with the Supervisor to create a fin al experimental design for this
protocol prior to the init ialreview workflow .
Anal yzes the final data supplied by the Supervisor .
Collaborates to co author th e validat ionreport .
All personnel and posi tions referred to i n this procedure are considered tohave an altern ate. An alt ernate must
ensure that they are trained and knowledg eable with the p rocess .
5.MATERIALS AND METHOD S
General m aterial supplies, rea gents and equipment used in this protocol are listed in SOPs
VR-TM-102983(Assay Test Method) ,VR-SOP-LC-112994(Vero cell culture) , and
VR-SOP-LC-112875(Sample Prepar ation on Wor kstation) .
A tab le listing of critica l reagent lot numb ers will be provide d inthe Validat ion Repor t.
5.1. V iral Stock
The SARS -CoV -2 mNG vir alstock wasproduc edas describ ed in VR -SOP-LC-11294 ,6
aliquoted intosingle -use vials, and f rozen at -80°C.
5.2.Cell Culture
The Vero cellline (CCL81) waspurchased from ATCC and used to g enerat e anin-housecell
bank for long -term use. Cells are passage d acc ording t oVR-SOP-LC-112994for u p to
cellpassages .
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VR-MVP -10074, Ver. 2.0
PFIZER CONF IDENTIAL
Page 12precision runs as show nintheValidation Run Plate Maps inSupportive Table 11.1 and
11.2,respect ively.
Table 3.Validation Run Schedule
6.2.2. Di lutional L inearitySamples
(refe r to Supportive Table 11.1, for Dilutional L inearit y
Validati on Run Plate Map).
6.2.3. P recision Sam ples
(refer t o
Supportive Table 11.2, for Precision V alidation Run Plate Map ).
6.2.4. Negative Samples
A panel of negative serawill be tested in a total of assays generating a
maximum of repor table titers per sample . The negative serumsamples are distribu ted
across the Dilutional Linear ityand Pre cision runs according the Validation Run Plate Map s
inSupportive Table 11.1 and11.2, respective ly.The samples will be used todescriptivel y
evalu atethe assay performance near the LOD.
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VR-MVP -10074, Ver. 2.0
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Page 157.6.Limit of Detect ion
The limit of detec tion (LOD) is set to the lowest possible d ilution in the assay (titer o f 20).
TheLOD i s used during assay development in lieu of an establ ished LLOQ. Once the LLOQ
is determined thr oughthe validation experime nts,the LLOQ is used in place of t he LOD. To
demonstrate assay performance near the LOD, up to presumed negative samp les will
each betested in each of the assay runs, for a tota l of up to reportable t iters.
Thedata will be presented as a descriptive measur e of assa y performance .
7.7.Extravariability of Replicates
As defined in the d ata review SOP, VR-SOP-LC-11293 ,8reporta ble titers a re the geom etric
means of t he
7.8.Titer Reporting Range
The validated assay range of the SARS -CoV -2 mNG NT will be the range of sample titers
that are shown to have suita ble assay performance. This assay perfo rmance will define the
lowerand upper limits o f quantit ation (L LOQ and ULOQ, respectivel y) for SARS -CoV -2
neutralizi ng an tibodie s, as d escribed i nSection 7.4. The maximum limits for the titer
reporting r ange without pre -dilution of test ser a are neutralizat iontiters between 20and
, since the plate des ign can de termine titers in this range only . The LLOQ a nd ULO Q
determined upon execution of this validat ionprotocol m ay further limit the titer reporting
range for c linical serum sam ples tested in the SARS -CoV -2 mNG NT without pre -dilution .
8.DEVIATIONS MA NAGEMENT
All deviations encount ered during assay validation wi ll be docume nted in the assa y
validationsummary report.
Any devi ation that re quires testing to be stoppe d tempor arily will include an impact
statement in the summary re port.
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Page 16Typographical error s are not deviations and will be corrected b y the author , as required.
9.VAL IDATION REPORT
AValidation Reportwillbe writtento describe thestatistical analy ses, the interpr etatio n of
the data ,and conclusion s regarding the fitness to ca rry out the SARS -CoV-2 mNG N Tfor
routine serological testing .
10.REFERENC ES
1. Xi e X, Muruato M, Lokugama ge KG et al, A n Infect ious cDNA Clone of SARS-CoV -2.
Cell Host & Microbe 2020 , 27(5): 841 -848.
2.Murua to AE, Fontes -Garfias CR, Ren P et al, A high -throughput neut ralizing ant ibody
assay for COVID- 19 diagnosis and vaccine evaluation .Nature Communications 2020 , in
press .
3.VR-TM-10298 : Manual 96 -well Neutraliz ation Assay for the Detection of Fu nctiona l
Antib odies to SARS -CoV -2 inTest Se rum.
4.VR-SOP-LC-11299 : Reagent Preparation a nd Vero and Vero E6 (V -E6) Thawing,
Passaging, Harvesting and Plating .
5.VR-SOP-LC-11287 : Preparat ion of Sa mples fo r SARS -CoV -2 Neutralization Assay
Using the Workstation .
6.VR-SOP-LC-11294 : Preparation of S ARS -CoV -2 Stocks.
7.VR-MQR-10214 : Qualification of the SARS -CoV -2 mNeonGr een Virus
Microneutralization Assay .
8.VR-SOP-LC-11293 : Procedure forData Review of the SARS -CoV -2 Manual 96 -well
Neutralizatio n Assay .
9.
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Page 1711.SUPPORTIVE TABLES
11.1. Validation Run Plate Map for Dilutional Li nearity Runs
Sample MDP # SampleaSample MDP # Sample
11.2. Validation Run Pla te Map for Precision Runs
Sample MDP # SampleaSample MDP # Sample
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Page 1811.2. Validation Run Pla te Map for Precision Runs
Sample MDP # SampleaSample MDP # Sample
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