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PF-073020 48: Qualificati on of the SARS -CoV -2 mNeon Green Virus Micron eutralization Assay
VR-MQR -1021 4, Ver. 2.0
PFIZER CONFIDENTIAL
Page 1
Title: Qualification of the SARS- CoV -2 mNeonGreen Virus Microneutralization Assay
Study Number: N/A
Parent Compound Number(s): PF-07302048
Alternative Compound Identifiers: N/A
Pfizer Vacc ine Research and Development
401 N. Mi ddletow n Rd.
Pearl River, NY
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FDA-CBER-2021-5683-0779353
FDA-CBER-2021-5683-0779354
PF-073020 48: Qualificati on of the SARS -CoV -2 mNeon Green Virus Micron eutralization Assay
VR-MQR -1021 4, Ver. 2.0
PFIZER CONFIDENTIAL
Page 3Title: Qualification of the SARS- CoV -2 mNeonGreen Virus Microneutralization Assay
SYNOPSIS
This report documents the qualification of the SA RS-CoV-2 mNeon Green virus
microneutra lizatio n assay(SARS -CoV -2 mN G NT) . The SARS -CoV-2 mNG NTmeasures
funct ional antibodies present in serum capable of neutralizing SARS -CoV -2. Twoassay
endpoints were examined : 50% and 90% viral neutralization . The 9 0% neutralization titer
analyses were prepar edfor int ernal purposes only; in routine clinic al testing onl y the 50%
neutralization titers will be reported. The data provided in this repor tindicate the
SAR S-CoV -2 mN G NT has the precision expecte d of a cell-based biofunctional manual
assay , acceptab le performance near the assa y LOD (l ow false negative and false positive
rates), and quality control sera are perform ing as exp ected. Together, the data confirm that
the assay issuitabl e for itsintended us e wh en performed in accordanc ewith stand ard
operating procedures b y qualifie d personnel.
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PF-073020 48: Qualificati on of the SARS -CoV -2 mNeon Green Virus Micron eutralization Assay
VR-MQR -1021 4, Ver. 2.0
PFIZER CONFIDENTIAL
Page 4TABLE OF CO NTENTS
SYNOPSI S................................ ................................ ................................ ................................ .3
LIST OF TABLES ................................ ................................ ................................ ..................... 5
LIST OF FIGURES ................................ ................................ ................................ ................... 5
1.OBJECTI VE................................ ................................ ................................ .......................... 6
2.INTRODUCTION ................................ ................................ ................................ ................. 6
3.GLOSSARY ................................ ................................ ................................ .......................... 7
4.MATERIAL S AND METHO DS................................ ................................ ........................... 7
4.1.Materials ................................ ................................ ................................ .................... 7
4.1.1. Precision Sample Pa nel................................ ................................ ................. 8
4.1.2. Quality Control Samples...............................................................................9
4.2.Methods ................................ ................................ ................................ ..................... 9
4.2.1. Assay Methods..............................................................................................9
4.2.2. Precision Assessment Experimental Design ................................ ................. 9
4.3.Statistical Methods ................................ ................................ ................................ ..10
4.3.1. Precision ................................ ................................ ................................ .....10
4.3.2. Intermediate Precision ................................ ................................ ................ 10
4.3.3. Assessment of Assay Performance Near the LOD ................................ .....11
4.3.4. Assessment of Performance ................................ ........... 11
5.RESUL TS AND DI SCUSSION ................................ ................................ .......................... 12
5.1.Precision Assessment ................................ ................................ .............................. 12
5.1.1. Precision ................................ ................................ ................................ .....12
5.1.2. Intermediate Precision ................................ ................................ ................ 12
5.2.Assessment of Assay Performance Near the LOD ................................ .................. 13
5.3.Assessment of Performance ................................ ........................ 13
5.4.Infection Rati o Consistency ................................ ................................ .................... 16
6.CONCL USION ................................ ................................ ................................ .................... 17
7.SUPPORTING DOCUMENTATION ................................ ................................ ................. 17
8.SUPPL EMENTAL INFORMATI ON................................ ................................ ................. 18
9.SUPPORTIVE TABLES ................................ ................................ ................................ .....19
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PF-073020 48: Qualificati on of the SARS -CoV -2 mNeon Green Virus Micron eutralization Assay
VR-MQR -1021 4, Ver. 2.0
PFIZER CONFIDENTIAL
Page 5LIST OF TABLES
Table 1. Terms and Definit ions................................ ................................ ................ 7
Table 2. SARS -CoV -2 mNG NT Assay Critical Reagents ................................ ......7
Table 3. Precision Sample Panel ................................ ................................ ............... 8
Table 4. Precision Assessm ent Run Schedule ................................ .......................... 9
Table 5. ................................ ................................ 12
Table 6. Database Prof ile for the SARS -CoV -2 Control Data Baseline b y
Control Serum Descriptive Information ................................ ................... 13
Table 7. Control Data Profile for SARS -CoV -2 mNG NT Assay b y QC
Serum Process ............................... 14
Table 8. Specification Limits for SARS- CoV -2 mNG NT by Control Serum
................................ ................................ ................................ .......14
LIST OF FIGURES
Figure 1. ................................ ........................... 15
Figure 2. ................................ ........................... 16
Figure 3. Infection Rati o Chart ................................ ................................ ................ 17
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PF-073020 48: Qualificati on of the SARS -CoV -2 mNeon Green Virus Micron eutralization Assay
VR-MQR -1021 4, Ver. 2.0
PFIZER CONFIDENTIAL
Page 6Title: Qualification of theSARS-CoV- 2 mNeonGre en Virus Micro neutraliza tionAssay
Study N umber: N/A
Functional Are a:Vaccine Research an dDevelopme nt
Test Facility:Univer sity of Texas Medica l Branc h, 224 11th St reet, Galveston, TX 77555
Stud y Initiation Date: 30-May-2020
Study Completion Date: 03-Jun-2020
1.OBJEC TIVE
This reportsummarize sthemethodology , statistical analysis and results of the qualification
of theSARS -CoV -2 mNeonGr eenMicron eutralization Assay (SARS -CoV-2 mNG NT),
including assay precision, assay performance near the L OD, and assessment of qua lity
control sample (QCS) performance.
2. INTRODU CTION
The SARS -CoV-2 mNG NT is abiofunctional assay thatmeasures neutraliz ing antibodies
againstSARS -CoV -2.The SARS -CoV -2 mNG virus is derived from the USA -WA1/ 2020
strain that had bee n rescued by reverse ge netics and e nginee red to containa mNe onGreen
(mNG) reporter gene into op en reading frame 7 of the vira l genome thatproduces green
fluores cence upon productive infection of cells (Xieet al,2020 ).1This report er virus
generates similar plaque morphologies and indistinguishable gr owth cur ves fro m wild -type
virus (Muruato et al, 2020).2
This assay is descri bed in the test method SHI-SOP-10011 (see Section 7).Briefly, the
SARS -CoV-2 mNG NT is a t hreeday manual 96-w ellassay . On Day 0, Vero CCL -81 cells
areseeded into 96-well tissue -culture treated read -out pl ates (R OPs). On Day 1, serial
dilutions of heat-inactivated testsera are inc ubated with theSARS -CoV-2 mNG virus to
allow an y virus -specific antibodies to bind to the virus. The serum -virus mixture is then
transferred onto the Verocell monolay erand incubated for 16-24hours to allo wforinfectio n
by non-neutralized virus. On Day 2, productiv e viral i nfection is enumerated by visualiz ing
fluorescent viral foci on a C ytation-7 Cell Imag ingMulti-Mode Reade r. Total number of
cells per well is cal culated by visualizing Hoechst 33342 stained Vero cel l nuclei , which ar e
blue. An infe ction ratio i s then c alculated fo r each wel l, whereb y the totalnumb er of virus
infected ( green )cells is divid edby the t otal n umber of cells present (blue nuclei ). A sample
titeris defined as the recipro cal se rum dilution at which a specific pe rcentage of the virus is
neutralized : 50% o r90% of t he virus (termed “TiterDetermi ning Value”, T DV)
(seeSection 4.2.1 ).
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PF-073020 48: Qualificati on of the SARS -CoV -2 mNeon Green Virus Micron eutralization Assay
VR-MQR -1021 4, Ver. 2.0
PFIZER CONFIDENTIAL
Page 7Thequalification is being conducted to demonstr ate that theSARS -CoV -2 mNG NT yields
consi stent a ndacceptable results for the test ingof clinica l samples . This rep ort documen ts
the pr ecision assessment exper imental design ,as well as the statistical a nalysis of the
precisionassessment data, perf ormance near the assay LOD and QCS performance.
3.GLOSSA RY
Table 1.Terms and De finitions
Term Definition
IR Infection Ratio
GMT Geom etric Mean Titer
LOD Limit of Detection
QCS Quality Control Sample
ROP Read O ut Plate
RSD Relative Standard D eviation
SARS -CoV- 2 mNG NT 96-wellmanual microneutralizat ion assay for the detection of functional antibo dies to
SARS -CoV-2using themNe onGreen reporter virus
SOP Standard Operatin g Procedure
TDV Titer Determining Val ue; the threshol d value in percen tof measured viral green
particl e counts that is used to report sample ti ters. Titers may be report ed at 5 0%or
90% TDV.
Vero African green monk ey kid ney epithelial cell line
4.MATERIALS AND METHODS
4.1.Mate rials
Reagents, s uppliesand equipment used in the precisi on assessment are listed in the t est
method , SHI-SOP-10 011 ( see Section 7 ). Thecritical reagents used in these precision
assessment runs are listed in Table2.The viral bank is aliquote dinsingle-use vials and is at
a concentration o f The Vero ce lls w ere purchased f rom ATCC .A Vero
cellbank was prepared and each cell bankvialis used f or up to 35 passages.
Table2.SARS -CoV -2 mNG NT Assay Critic al Reage nts
Reagent Nam e/Lot #
SARS -CoV-2 mNGvirus SAR S-CoV-2- mNEON
P1Vero E6
04.21.20 AM
Cell Line Vero cells;ATCC CC L81, used from pas sage 1to35
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VR-MQR -1021 4, Ver. 2.0
PFIZER CONFIDENTIAL
Page 135.2.Assessm ent of Assay Performance Near the LOD
5.3.Assessment of Performance
To monitor for as say variabili tyover time
, abaseline database w as establish ed by inclu ding
data collec tedfrom 23- Jun-2020 through 07-July-2020, inclusive . QCS
data f or the SARS- CoV -2-mNG NT is listed in VR -MQR -10214 -ATT02 (see Section 7).
Descriptive infor mation such as the number of data points ( N) and the dates of the first and
last runs included in the database a re sho wn in Table 6.
Table 6. Database Profile for the SARS -CoV-2Contr ol Data Baseline by Cont rol
Serum Descript ive Informa tion
Assay Control DaysaNbMin
TitercMax
TiterdFirst
DateeLast
Datef
SARS -CoV-2 mNG
NT23JUN2020 07JUL2020
23JUN2020 07JUL2020
a.Days, Num ber of D
b.N, N umber of observations .
c.Min Ti ter, Low estobserved t itervalue .
d.Max Titer, Highest observed t iter value .
e.First Date, Earliest run d ate..
f.Last Date, Las t run d ate
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PFIZER CONFIDENTIAL
Page 165.4.Infection Ratio Consistency
The number of Vero cells p lated per well and the amount of v irusadded aretargeted to yield
an infection ratio (IR) between 10- 30%. IRdata f or the SA RS-CoV -2-mNG NT is listed in
VR-MQR -10214 -ATT0 3(Section 7).Scatter pl ots of IR are sh own in Figure 3. Thisplot
indicate sthattheIR consistently falls between
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