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PF-07302048: Method Validation ofthe SARS -CoV-2 mNeonGreen Virus Microneutralization Assay
VR-MVR-10083, Ver. 1.0
PFIZER CONFIDENTIAL
Page 1Title:Method Validation 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 Vaccine Research and Development
401 N. Middletown Rd.
Pearl River, NY
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PF-07302048: Method Validation ofthe SARS -CoV-2 mNeonGreen Virus Microneutralization Assay
VR-MVR-10083, Ver. 1.0
PFIZER CONFIDENTIAL
Page 2Title:Method Validation of the SARS -CoV-2 mNeonGreen Virus Microneutralization
Assay
PRINCIPAL INVESTIGATORS: , Director
Ingrid L. Scully, Senior Director
, Director
CONTRIBUTING SCIENTIST S: , Senior Associate Scientist
Tyler Garretson, Principal Scientist
Kyle Jacobs, Associate Scientist
, Associate Scientist
, Senior Associate Scientist
George Phel an, Associate Scientist
, Scientist
, Senior Associate Scientist
, Associate Scientist
PREPARED BY:
APPROVED BY:
Tyler Garretson
Principal Scientist , Clinical and Diagnostic Assay Deve lopment
Ingrid L. Scully
Senior Director , Clinical and Diagnostic Assay Development
Charles Tan
Senior Director, Earl y Clinical Development Biostatistics
David Cooper
Executive Director, High
-throughput Clinical Immunoassay s & Diagnostics
Michael Pride
Executive DirectorBiology, Clinical and Diagnostic Assay Development
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PFIZER CONFIDENTIAL
Page 3Title:Method Validation of the SARS -CoV-2 mNeonGreen Virus Microneutralization
Assay
SYNOPSIS
This reportdescribesthe validation of the SARS-CoV-2 mNeonGreen Virus
Microneutralization Assay (SARS-CoV-2 mNG NT) used for the detection of serum
antibodies capable of neutralizing SARS- CoV-2. The assay readout of 50% virus
neutralization titer was validated per protocol VR-MVP-10074.
Based on dilutional linearity and assay precision, the lower limit of quantitation (LLOQ)
was determined to b e a titer equal to 41 and the upper limit of quantitation (ULOQ) was
determined to be a titer equal to 3,187.Samples with titers greater than the ULOQ may
be pre-diluted before testing to yield titers within thevalidated assay range.
Intermediate precis ion assessment demonstrated overall assay variability of 26.5% RSD.
Serum samples are run in replicate in the SARS -CoV-2 mNG NT. Based on the assay
performance, it was determined that the replicate titer ratio for a sample must be less than
or equal to 2.55 (sample extravariability rule).
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PFIZER CONFIDENTIAL
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. MATERIAL S AND MET HODS................................ ................................ ........................... 7
4.1. Critical Reagents ................................ ................................ ................................ .......7
4.2. Quality Control Samples ................................ ................................ ........................... 8
4.3. Negative Serum Diluent................................ ................................ ............................ 8
4.4. Validation Serum Sample Panels................................ ................................ ..............8
5. EXPERI MENTAL DESI GN................................ ................................ ............................... 10
6. STATI STICAL ANALY SIS................................ ................................ ............................... 10
6.1. Dilutional L inearity................................ ................................ ................................ .11
6.2. Precision................................ ................................ ................................ .................. 11
6.3. Limits of Quantitation ................................ ................................ ............................. 12
6.4. Assay Intermediate Precision ................................ ................................ .................. 12
6.5. Limit of Detection ................................ ................................ ................................ ...13
6.6. Extravariability of Replicates................................ ................................ .................. 13
7. RESUL TS AND DI SCUSSION................................ ................................ .......................... 13
7.1. Dilutional L inearity................................ ................................ ................................ .13
7.2. Precision ................................ ................................ ................................ .................. 14
7.3. Limits of Quantitation ................................ ................................ ............................. 15
7.4. Assay Intermediate Precision ................................ ................................ .................. 16
7.5. Limit of Detection ................................ ................................ ................................ ...16
7.6. Extravariability of Replicates................................ ................................ .................. 16
8. CONCLUSION ................................ ................................ ................................ .................... 17
9. DEVIATIONS ................................ ................................ ................................ ..................... 17
10. REFERENCES ................................ ................................ ................................ .................. 17
11. SUPPORTIVE TABLE S................................ ................................ ................................ ...19
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PFIZER CONFIDENTIAL
Page 511.1. Precision by Individual Sample ................................ ................................ .............19
12. SUPPORTIVE FIGUR ES................................ ................................ ................................ ..20
12.1. Assa y Design Schematic ................................ ................................ ....................... 20
12.2. Dilutional L inearity Plot by Individual Sample ................................ .................... 21
LIST OF TABLES
Table1. Terms and Definitions................................ ................................ ................ 7
Table2. Critical Reagents for the SARS- CoV-2 mNG NT Validation .................... 8
Table3. Quality Control Sample Specification Limits.............................................8
Table4. Validation Serum Sample Panels ................................ ................................ 9
Table5. Example Validation Run Schedule ................................ ........................... 10
Table6. Final Assay Range................................ ................................ .................... 15
Table7. of Samples Within the Quantitation Range................................ .....16
Table8. Assay Performance Near the L OD................................ ........................... 16
LIST OF FIGURES
Figure1. Dilutional L inearity Results Plot ................................ .............................. 14
Figure2. Precision Results Plot ................................ ................................ ...............15
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Page 6Title:Validation Report for the SARS -CoV-2 mNeonGreen Virus Microneutralization
Assay
Study Number: N/A
Functional Area: Vaccine Research and Development
Test Facility:Hackensack Meridi an Health
Nutley, NJ 07110
Study/Testing Initiation Date:01-Dec-2020
Study/Testing Completion Date: 20-Jan-2021
1.OBJECTIVES
This report describe sthe validation results for the SARS-CoV-2 mNeonGreen Virus
Microneutralization Assay (SARS-CoV-2 mNG NT) used for the detection of serum
antibodies capable of neutralizing SARS- CoV-2. The assay readout of 50% virus
neutralization titer was validated per protocol VR-MVP-10074.1 This validation provides
documented evidence that the SARS-CoV-2 mNG NT is suitable for its intended use when
performed in accordance with standard operating procedures b y qualified personnel .
2.INTRODUCTION
The SARS- CoV-2 mNG NT is a biofunctional assay that measures neutralizing antibodies
against SARS -CoV-2. The SARS- CoV-2 mNG virus is derived from the USA_WA1/2020
strain that had been rescued by reverse genetics and engineered to contain a mNeonGreen
(mNG) reporter gene in open reading frame 7 of the viral ge nome that produces green
fluorescence upon productive infection of cells.2This reporter virus generates similar plaque
morphologies and indistinguishable growth curves from wild- type virus.3
This assay is described in the test method VR-TM-10298.4 Briefly,serially diluted test
serum samples are mixed with SARS -CoV-2 mNG virus in a 96 -well plate to allow
virus-specific antibodies to bind to the virus. This serum-virus mixture is then transferred
onto aVero cell monolayer and incubated overnight to allow f or infection by non-neutralized
virus. Productive viral infection is detected b y enumerating green -fluorescent viral foci
using a cell -imaging reader. The total number of cells per well is calculated by enumerating
Vero cell nuclei stained blue with Hoechst 33342. An infection ratio is then calculated for
each well, whereb y the total number of virus infected (green) cells is divided by the total
number of cells present (blue nuclei). A sample titer is definedas the reciprocal serum
dilution at which a specific percentage of the virus is neutralized, eg, 50%, 80% or 90%
(termed “Titer Determining Value”, TDV) .
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Page 73.GLOSSARY
Table1.Terms and Definitions
Term Definition
Assay range Range of neutralization titers that can be measured in the assay with acceptable
dilutional linearity and precision.
COVID-19 Coronavirus Disease 2019
DL Dilutional linearity
GMT Geometric Mean Titer
LLOQ Lower Limit of Quantitation
LOD Limit of Detection
MDP Master Dilution Plate
NHS_dep Normal Human Serum -Antibody Depleted
NT Microneutralization Assay
QCS Quality Control Sample
Replicate An independent determination of an assay result ; the geometric mean of tw o replicate
titers is the reportable result for a sam ple tested in the SARS -CoV-2 mNG NT.
RSD Relative Standard Deviation
SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2; the etiologic 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 mNeonGreen reporter virus.
SAS Programming language and integrated software solution -set proprietary to SAS ®(the
company) that enables the coding of the various tasks associated w ith handling
datasets.
SOP Standard Operating Procedure
TDV Titer Determining Value; the threshold value in percent of measured viral green
particle counts that is used to report sample titers. Titers may be reported at 50%,
90% TDV.
Titer Inverse of serum dilution required to neutralize a specific percentage of the input
virus (eg 50% TDV).
TM Test Method
ULOQ Upper Limit of Quantitation
Vero African green monkey kidney epithelial cell line
4.MATERIALS AND METHOD S
General material supplies, reagents and equipment used are listed in SOPs VR-TM-10298,4
VR-SOP-LC-11299,5VR-SOP-LC-11294,6and VR-SOP-LC-11287.7
4.1.Critical Reagents
Critical reagents used for the validation assay s are listed in Table2.
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Page 8Table2.Critical Reagents for the SARS-CoV-2 mNG NT Validation
Reagent Name/Lot#
VirusStock SARS-CoV-2-mNG Lot 5
P2 Vero E6
Cell Line Vero cells; ATCC CCL81,
used from passage 134 to 155
4.2.Quality Control Samples
Quality control samples (QCS) were generated from pools of
QCS lower and upper titer limits
were established prior to the start of validation as docum ented in VR -RGR-RQ-107818and
arelisted in Table3.
Table3.Quality Control Sample Specification Limits
QCS Lower Specification Limit Upper Specification Limit
4.3.Negative Serum Diluent
was used as a negative
serum diluent for dilutional linearity testing during this validation .
4.4.Validation Serum Sample Panels
Panels comprised of serum samples from either
were selected to be used in the evaluation of assay dilutional linearity
and precision. Convalescent serum was obtained from
Negative samples
were collected from donors in 2013 (pre- COVID-19). All sera were heat- inactivated for
30min at 56°C prior to testing.
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Page 9Table4.Validation Serum Sample Panels
Intended Purpose Sample ID Serum Source
Dilutional Linearity Panel
Precision Panel
LOD Assessment
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Page 105. EXPERIMENTAL DESIGN
Validation of the SARS-CoV-2 mNG NT was performed as described in the validation
protocol, VR -MVP-10074.1 The assay readout of 50% virus neutralization titer(50% TDV)
was validated; 90% neutralization datasets were collected for exploratory purposes
only (VR-VTR-107429). Dilutional linearity and precision were evaluated in separate assay
runs according to the schedule shown in Tab le5. The factorial design includes assay runs,
completed on different day s, utilizing different sets of sample master dilution plate
(MDP) preparations, performed by different analy sts inorder to quantify the contribution
of these factors to the intermediate precision. Sample MDPs are prepared in deep -well
96-well plates and each are of sufficient volume to stamp out sample assay plates on each of
the assay days in the experimental des ign (refer to Supportive Figure12.1for the assay
design schematic).
For the dilutional linearity assessment, serum samples were tested
independentl y diluted
For the precision assessment, serum samples were tested (n= ) and
analyzed along with the dilutional linearity samples (n=8).
For theevaluation of the assay near the LOD, presumed negative serum
samples were tested .
The assay schedule for validation testing is shown in Table5.
Table5.Example Validation Run Schedule
Assay MDP SetaDay Analyst
6.STATISTICAL ANALYSIS
All analyses of titer data described in the statistical analy sis sections are based on the
reportable geometric mean titer (GMT). The reportable titer is the
Unless indicated otherwise, the word titer will be
understood to be reportable GMTat 50%TDV. All ranges a nd/or limits determined based
on the statistical analy ses will be applied for all non-clinical and clinical testing moving
forward.
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Page 11All statistical analy ses, including , were performed
using SAS ®version 9.4.
6.1.Dilutional Linearity
Each dilutional linearity sample wastested at dilutions and at dilutions of
in assay runs. For each assay run, a
dilution-adjusted titerwascalculated for each of the dilutions
.
10
6.2.Precision
Sample precision refers to the closeness of 2or more measurements of a sample to each other
and was reported as %RSD. The precision samples were tested in independent
runs(refer toTable5). The evaluation of precision consisted of using the titers of the
samples from the precision panel combined with the samples prepared for
dilutional linearity .
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Page 136.5.Limit of Detection
The limit of detection (LOD) was set to the low est possible dilution testedin the assay (titer
of 20). The LOD was used during assay development in lieu of an established LLOQ. Since
the LLOQ was determined through the validation experiments described here , the LLOQ will
be used in place of the LOD.However, t o demonstrate assay performance nearthe LOD, 8
presumed negative samples were tested in each of the assay runs, for a total of up
to reportable titer s. The data i s presented as a descriptive measure of assay performance .
6.6.Extravariability of Replicates
As defined in the data review SOP, VR -SOP-LC-11293,11reportable titers are the geometric
means of the
7. RESULTS AND DISCUSSI ON
Assay validation runs were performed by qualified anal ysts from01-Dec-2020through
20-Jan-2021,inclusive. The 9 assay runs were performed to assess dilutional linearity ,
precision, assay performance near the LOD,and to determine LLOQ and ULOQ . The raw
titer data for the dilutional linearity and precision experiments are listed in attachments
VR-MVR-10083-ATT0112and VR-MVR-10083-ATT02,13respectively . LOD titer data are
listedin VR-MVR-10083-ATT03.14 Documentation of anal yst training on the validation
protocol is provided in VR -MVR-10083-ATT04.15
7.1. Dilutional Linearity
A sample shows dilutional linearity when its titer changes in proportion to its dilution. Each
sample replicate was evaluated and at dilutions of
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Page 14For each of the dilutional linearity samples,
)
(Figure1). Supportive Figure 12.2shows
this data for all individual samples from the runs plotted together.
Figure1. Dilutional Linearity Results Plot
The dilutional linearity data displayed is based on samples, each assayed dilutions, tested in
independent runs. The dilutional linearity
7.2.Precision
The assay precision was calculated
. Precision results for individual
samples are display ed in Supportive Table 11.1.
Acceptable precision was demonstrated between titers of 41 and
3,187 (Figure2).
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Page 15Figure2.Precision Results Plot
Theprecision data displayed is based on precision samples, tested twi ce in independent runs ,and
, tested once in independent runs. The precision low er and upper titer
limitsare41 and 3,187 , respectively.
7.3.Limits of Quantitation
Table6displays the final
assay rangebounded b y the LLOQ and ULOQ.
Table6.Final Assay Range
Dilutional Linearity Range Precision
RangeFinal Assay Range
Lower Upper
41 3,187
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Page 167.4. Assay Intermediate Precision
The intermediate precision of the assay was evaluated using the
as described in Section 6.4and theresults are summarized in Table7.
The total %RSD is the estimated intermediate precision and the
Table7. S amples
%RSD
26.5
7.5.Limit of De tection
The limit of detection (LOD) was set to the lowest possible dilution tested inthe assay (titer
of 20). To demonstrate assay performance nearthe LOD, negative samples were tested
in each of the assay runs, for a total of up to titersor a maximum number of
titers per sample . The number of reportable titers for each sample as well as the percent
that the sample returned a negative titer is listed in Table8.
Table8. Assay Performance Near the LOD
Sample N of Resultsa% Negativeb
100
100
100
100
100
100
100
100
a.There were titers measured out of the titers possible . Sixteen titers were not resolved due to failed
assay suitability requirements on the plate.
b.A sample is considered negative if its titer is less than the LOD of 20 (ie, the low est measurable titer).
7.6.Extravariability of Replicat es
An upper limit for acceptable replicate titer ratios was computed from titer pairs by the
method defined in Section 6.6. This ratio limit was 2.55, indicating good agreement between
titers of replicate pairs .
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Page 178.CONCLUSION
The results documented in this validation report for the SARS-CoV-2 mNG NT provide
evidence that the assay is validated and suitable for its intended use in testing clinical,
epidemiological ,and non-clinical study samples. The assay demonstrated dilutional linearity
and precision that met predefined acceptance criteria. The LLOQ for the assay is a titer of
41 and the ULOQ a titer of 3,187 . Samples with titers greater than the ULOQ may be
pre-dilutedin assay bufferbefore testing to yield titers within thevalidated assay range.
Theperformance of the assay near the LOD is acceptable.
9.DEVIATIONS
During statistical anal ysis of the validation dataset, it was observed that SAS was
inappropriatel y assigninga final titer to samples with only one valid replicate titer. This
occurred 4 times in the validation and in each case the assay result was overridden to the
These assay results were then appropriatel y excluded from the final
data set and validation analysis. This deviation was recorded in Laboratory Deviation
Report VR -LDR-12674.The SAS data sy stem is being updated for future clinical
testing.
Section 5.4 of the validation protocol (VR -MVP-100741) incorrectl y stated that the
diluent would be obtained from , however it was
preparedat Pfizer.
Section 7.7 of the validation protocol (VR -MVP-100741) stated that a r eplicate titer ratio
limit of be applied to all replicate titer pairs during validation .However, a more
conservative limit of was applied after preliminary examination of the validation data.
10.REFERENCES
1.VR-MVP-10074: Validation Protocol for the SARS- CoV-2 mNeonGreen Virus
Microneutralization Assay .
2.Xie X, Muruato A , Lokugamage KG et al, An Infectious cDNA Clone of SARS -CoV-2.
Cell Host & Microbe 2020, 27 (5): 841-848.
3.Muruato AE, Fontes -Garfias CR, Ren P et al, A hi gh-throughput neutralizing antibody
assay for COVID- 19 diagnosis and vaccine evaluation. Nature Communications 2020,
11 (1):4059.
4.VR-TM-10298: Manual 96- well Microneutralization Assay for the Detection of
Functional Antibodies to SARS- CoV-2 in Test Serum.
5.VR-SOP-LC-11299: Reagent Preparation and Vero and Vero E6 (V- E6) Thawing,
Passaging, Harvesting and Plating.
6.VR-SOP-LC-11294: Preparation and Assessment of SARS -CoV-2-mNGStocks.
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PF-07302048: Method Validation ofthe SARS -CoV-2 mNeonGreen Virus Microneutralization Assay
VR-MVR-10083, Ver. 1.0
PFIZER CONFIDENTIAL
Page 187.VR-SOP-LC-11287: Preparation of Samples for SARS -CoV-2 Neutralization Assay
Using the Workstation.
8.VR-RGR-RQ-10781:Quality Control Sample Specification L imits for
in SARS -CoV-2-mNG Microneutralization Assay .
9.VR-VTR-10742: Exploratory Anal ysis of the SARS -CoV-2 mNG NT Validation Dataset
using 90% Virus Neutralization Titer Readouts.
10.
11.VR-SOP-LC-11293: Procedure for Data Review of the SARS -CoV-2-mNGManual
96-well Microneutralization Assay .
12.VR-MVR-10083-ATT01: Dilutional L inearity Data Listing for VR -MVR-10083.
13.VR-MVR-10083-ATT02: Precision Data Listing for VR -MVR-10083.
14.VR-MVR-10083-ATT03: LOD Data Listing for VR -MVR-10083.
15.VR-MVR-10083-ATT04: Training Documentation VR- MVP-10074 for
VR-MVR-10083.
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PF-07302048: Method Validation ofthe SARS -CoV-2 mNeonGreen Virus Microneutralization Assay
VR-MVR-10083, Ver. 1.0
PFIZER CONFIDENTIAL
Page 1911.SUPPORTIVE TABLES
11.1. Precision by Individual Sample
Data Source Sample Dilution N GMT %RSD
Precision
Dilutional
Linearity
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PF-07302048: Method Validation ofthe SARS -CoV-2 mNeonGreen Virus Microneutralization Assay
VR-MVR-10083, Ver. 1.0
PFIZER CONFIDENTIAL
Page 2012.SUPPORTIVE FIGURES
12.1. Assay Design Schematic
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PF-07302048: Method Validation ofthe SARS -CoV-2 mNeonGreen Virus Microneutralization Assay
VR-MVR-10083, Ver. 1.0
PFIZER CONFIDENTIAL
Page 2112.2. Dilutional Linearity Plotby Individual Sample
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Document Approval Record
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