Document text
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 1TABLE OF CONTENTS
LIST OF TABLES ................................ ................................ ................................ ..................... 2
LIST OF FIGURES ................................ ................................ ................................ ................... 2
1.HISTORICAL REVI SIONSUMMARY ................................ ................................ .............. 3
2.PURPOSE ................................ ................................ ................................ .............................. 3
3.SCOPE ................................ ................................ ................................ ................................ ...3
4.GLOSSARY ................................ ................................ ................................ .......................... 4
5.GENERAL ................................ ................................ ................................ ............................. 5
6.RESPONSIBILIT IES................................ ................................ ................................ ............ 6
7.SAFETY ................................ ................................ ................................ ................................ 8
8.MATERIAL S/EQUIPMENT ................................ ................................ ................................ 8
8.1. Equipment ................................ ................................ ................................ ................. 8
8.2.Reagents ................................ ................................ ................................ .................... 9
8.3.Supplies ................................ ................................ ................................ ..................... 9
9.PROCEDURE ................................ ................................ ................................ ...................... 10
9.1.General Points ................................ ................................ ................................ ......... 10
9.2.Assay Set Up ................................ ................................ ................................ ........... 11
9.3. Recording of Equipment and Reagent Information and Use of Barcode
Scanner ................................ ................................ ................................ ...................... 11
9.4.Preparation of ROPs ................................ ................................ ................................ 12
9.5.Preparation of SAPs ................................ ................................ ................................ 12
9.6. Processing of SAPs ................................ ................................ ................................ .13
9.7.Add Assay Media to Cell Control We lls in SAPs ................................ ................... 14
9.8.Preparation of Viral Stock Solution ................................ ................................ ........ 14
9.9. Addition of Viral Stock Solution to SAPs and Neutralization of Virus.................. 15
9.10. Transfer of Neutralization Reaction onto ROPs and Decontamination of
SAPs ................................ ................................ ................................ .......................... 17
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FDA-CBER-2021-5683-0779588
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 29.11. Assay –Day 2 ................................ ................................ ................................ .......19
9.12. Preparation and Addition of Hoechst 33342 Solution to ROPs ............................ 19
9.13. Image Acquisition and Counting on the C ytation 7................................ .............. 21
9.13.1. Scanning Quality Check and Correction of Barcode Reader Errors ........ 25
9.14. Raw Data and Image Export ................................ ................................ ................. 25
9.15. Data Acquisition ................................ ................................ ................................ ....27
9.16. Analy sis of Data and Calculation of SARS -CoV -2-mNG_NT Titers .................. 27
9.17. SARS -CoV -2-mNG_NT D ata and Document Review ................................ ......... 28
10.TABLES, FIGURES, DI AGRAMS ................................ ................................ .................. 28
11.REFERENCES ................................ ................................ ................................ .................. 29
12.DOCUMENT VERSION MO DIFICATIONS ................................ ................................ ..30
12.1. CRIF Number: VR -CRIF-21 -14875 ................................ ................................ .....30
LIST OF TABLES
Table 1. Chronological Summary of Previous Versions ................................ .......... 3
Table 2. Vaccine Research Functional Units ................................ ............................ 3
Table 3. Method Developm ent, Qualification and Validation Status ....................... 3
Table 4. Terms and Definitions ................................ ................................ ................ 4
Table 5. Roles and Responsibilities................................ ................................ .......... 6
Table 6. Vero Cell Concentration in ROP for SARS -CoV -2-mNG_NT ................ 11
Table 7. Sample Assay Plate Map ................................ ................................ .......... 13
Table 8. Assay Job Aid 1 - Comparison of Assay Runs Using Oracle -LIMS
Based SAS and File -based SAS ................................ ............................... 28
Table 9. General Ref erences ................................ ................................ ................... 29
Table 10. Form References ................................ ................................ ....................... 29
Table 11. Specific References ................................ ................................ .................. 30
Table 12. Detailed Changes ................................ ................................ ...................... 30
LIST OF FIGURES
Figure 1. Posit ions................................ ................................ .......... 15
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(b) (4)
(b) (4)
FDA-CBER-2021-5683-0779589
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 31.HISTORICAL REVISION SUMMARY
Table 1.Chronological Summary of Previou sVersions
Version # Effective Date Originator Summary
3.0 Current Updated to improve quality assurance of data in the event
LIMSis unavaila bleby com pletion ,upload ,andreview
of VR -TM-10298 -FM0 2.
2.0 06-Jan-2021 Updated setti ngs of Cytation 7 ,manual steps on Day 2
involving the Cytation 7 , and raw data and image export
section
1.0 29-Jul-2020 Tyler Garretson New
2. PURPOSE
This doc ument describe s the proc edure for a manual 96- well SARS -CoV -2-mNG reporter
virus microneutral ization assay (SARS -CoV -2-mNG_NT) designed to measure functional,
neutralizing antibodies specific for SARS -CoV -2 in test serum. The assay can be performed
utiliz ing LIMS or el ectronical ly filling in VR -TM-10298 -FM02 (in case LIMS is down)
while following th e assay paperless guide VR -TM-10298 -FM01. In the case of file -based
(FB) -SAS assay runs, VR -TM-10298 -FM03 should be completed electronically .
3.SCOPE
This procedur e applies to all personnel responsible for executing VR- TM-10298 at
Pfizer, Pearl River or Hac kensack Meridian Health Center for Discovery and I nnovation ,
Nutley , NJ.
Table 2.Vaccine Research Functional Units
Functional Units Location
Pfizer , Vaccine Research & Developmen t Pearl River, NY and Nutley, NJ
Table 3. Method Development, Qualification and Validation Status
Project Developed per Qualified per Validated per
General Use N/A VR-MQR -10214 N/A
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FDA-CBER-2021-5683-0779590
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 44.GLOSSARY
Table 4.Terms and Definitions
Term Definition
Ab Antibody
Batch
BSC Biological Safety Cabinet
BSL Biosafety Level
CDTAS Control Data Tracking and Analysis System
CMDP Control Master Dilution Plate
Cytation 7 Cell imaging m ulti-mode reader manufactured by Biotek
DAP Data Analysis Portal
FBS Fetal Bovine S erum
Gen5 Software asso ciated w ith the Cytat ion 7
GMT Geom etric Mean Titer
GPC Green Posit iveCells; SARS -CoV -2-mNG infected cells
HBSS Hanks' Balanced Salt Solution
Hoechst 33342 Cell-perm eant nuclear counterstain that emits blue fluorescence when bo und
todsDNA
LIMS Labor atory Information Man agem ent System
PPE Personal Protective Equipmen t
PBS Phosphate Buffered Saline
QCS Quality Control Sample
QNS Quantity Not Sufficient
ROP Read Out Plate
SAP Sample Assay Plate
SAP O Sample Assay Plate ou tputfile prod uced by workstation
SARS -CoV -2 Severe acute respiratory syndrome corona virus 2
SARS -CoV -2-mNG_NT Manual 96 -well micro neutralization assay for the detection of functional
antibodies to SARS -CoV -2
SARS -CoV -2-mNG SARS -CoV-2- mNeonGree n reporter vir us
SAS Programming language an d integrated software solution -set proprietary to
SAS® (thecompany) that enables the coding of the various tasks associated
with handling and analyzing data -sets.
SMDP Sample Master Dilution Plate
SMDP O Sampl e Mast er Dilut ion Plate output file produced by w orkstation
SOP Standard Operating Pr ocedure
TDV Titer Determining Value. The threshold value in recovered vira l focus
forming units that is used to determine test serum titers in the
microneutral ization assay.
Titer The reciprocal of the d ilution of a test serum required to neutralize vir usat a
specified percentage of the viral input in a neutralization assay .
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FDA-CBER-2021-5683-0779591
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 5Table 4.Terms and Definitions
Term Definition
VI Viral input is the number of recovered viral focus forming units (FFUs) or
green po sitivecells ( GPC) measu red in neutralization assay reactions
incubated without the presence o f immune serum. The VI value is equal to
100% recovered virus for the purpos e of calculating assay titers.
VSS Viral Stock Solution
Vero African green monkey kidney epitheli al cell s
Handheld Electronic 96 -Channel Pipette
5.GENE RAL
The SARS- CoV -2-mNG- NT is a biofunctional assay designed to measure total serum
antibody levels capable of neutralizing SARS -CoV -2 infection of cells in cultur e.
TheSARS -CoV-2-mNG- NTis a three -dayassay , performed manuall y. On Day 0,
Vero cells are seeded into 96 -well tissue -culture treated plates. On Day 1, serial
dilutions ofheat-inactivated immun e sera in sample assay plates (SAPs) are incubated
with SAR S-CoV-2 -mNG re porter vir us to allow antigen -specific antibodies to bind to the
virus. The rea ction is then transferred onto the Vero cell monolay erread-out plates (ROPs)
and allowed to incubate overnight; onl y non -neutralized virus is able to infect the ce lls. On
Day 2, produc tive viral infection is detected in live cells using the mNeonGreen exp ressed b y
the reporter virus. Live cells are stained with the nuclear d ye Hoechst 33342. Both viral foci
and live cells are enumerated from the ROPs on a Cy tation 7 reader.
A sample t iter is calculated asthe reciprocal serum dilution at which a specifi c percentage of
the virus is neutralized ( eg, 50% or 90% of the virus). The process for analy sis and review of
sample titer data generated dur ing the execution o f VR-TM-10298 is detaile d in
VR-SOP-LC-11293.
A qualified technician (ie, SARS -CoV -2-mNG- NT an alyst) ty picall y anal yzes up to SAPs
per run (batch). SAPs are produced u sing the workstation per VR -SOP-LC-11287 .
The SAP lay out accommodates serum titrations of samples tested in replicate. A maximum
of samples are tested in a - plate batch.
Plates that contain the Vero monolay er (ROPs)
are produc edas per VR-SOP-LC-1129 9. Plating of Vero o nto the ROPs occurs at least
hours prior to th e infection of the monolay er. This incubation time allows the cells to
adher e to the plate surface and grow to a confluent monolay er to allow for consistent
meas urement of inf ection.
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(b)(4)
FDA-CBER-2021-5683-0779592
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 66.RESPONSIBILITIES
All pe rsonnel and positions referred to in this procedu re are considered to have an
alternate.
An alternate must ensure that they are trained and knowledgeable with the process.
Table 5.Roles and Responsibi lities
Role Responsibilities
SARS -CoV -2-
mNG_NT AnalystRead s, understand s, and completestraining and qualification on this test method.
Read s, understand s, and com pletes training on applicable equipment operating
manuals, procedures and test methods tha t are requ ired to execute the p rocedure
described in this document.
If LIMS is u sed, logs in as Analyst role.
Execute sthis test method and complete steps in LIMS by follow ing the paperless
guide VR -TM-10298 -FM01. If LIMS is unavailable, com pletes
VR-TM-10298-FM02 a nd fills into LIMS whe n functional ; attaches completed VR -
TM-10298 -FM02as a batch comment . In case of FB -SAS or non -paperless runs,
complete sVR-TM-10298 -FM03.
Consult swith supervisor if there are any q uestions related to the procedure itself or
serum samples, if an atypical event is observed or if a protoc ol deviation o ccurs.
SARS -CoV -2-
mNG_NT
CoordinatorProvides detailed scheduling of all SARS -CoV-2- mNG_NT runs.
If LIMS is used, uses Lab Manager, Rev iewer, and/or Analyst role .
Creates assay run batches in LIMS and corrects run inf ormation w ith QNS
inform ation from the annotated VR -SOP-LC-11287 -FM01 printout or LIMS
comments (Paperless) received from the Operator.
Coordinates with Vero analysts to ensure the correct numbe rs of ROPs are available
for SARS -CoV-2- mNG_NT runs.
Coordinate s with analysts to schedule pr oduction of
SARS -CoV -2-mNG NT assay plates .
Analyst Read s, understand sand com plete straining on applicable equipmen t operating
manuals, procedures and test methods that are required to operate
work stations and p roduce serum d ilution pla tes for SARS -CoV -2-mNG_NT .
Serially dilutes test sera and control sera in master dilution plates using the
workstatio n according to VR-SOP-LC-11287.
Prepare sSARS -CoV-2- mNG_NT assay plates from master dilutio n plates using the
workstation according to VR-SOP-LC-11287.
Provide sSARS -CoV -2-mNG_NT analysts with sample assay plates (SAPs) on the
day of their assay r uns.
Coordinates with SARS -CoV -2-mNG_NT Coordinator to schedule production of
SAR S-CoV-2-mNG_NT assay pla tes.
For non -pape rless as says, provides the SARS -CoV -2-mNG_NT Analysts and
SARS -CoV-2- mNG_NT Coordinator with appropriate run documentation
(ie,SMDPo, QNS -Annotated LIMS run file, and VR -SOP-LC-11287 printouts)
describing the proce ssing of a ssay pla tes an dQNS sam ples.
For Paperl ess assays, enters SMDP, SAP, and QNS data into LIMS.
If LIMS is used, uses Robotics role.
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(b)(4)
(b)(4)
FDA-CBER-2021-5683-0779593
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 7Table 5.Roles and Responsibi lities
Role Responsibilities
Vero Analyst Maintain sand grow s Vero cells .
Plates the appropriate concentration of Vero cells onto ROPs according to
VR-SOP-LC-1129 9.
Providesan appro priate numb er of ROPs with a Vero monolayer to each
SARS -CoV-2- mNG_NT analyst on the day of the assay.
Provide sworksheet VR-SOP-LC-11299 -FM07 to each analyst.
SAS Operator,
Data AnalystRead s, understand sandcompletestrainin g on VR-SOP-LC-10336, VR -TM-10298,
and VR-SOP-LC-11293.
For Paperless assays, executes the steps of VR -TM-10298 -FM01, Table 13.
For FB -SAS and non -paperless assays, executes the steps of VR -TM-10298 -FM03,
Table 20.
This person may be the same per sonas the SARS -CoV-2- mNG_NT analyst.
This person may not be the Reviewer for the same batch.
Reviewer Read s,understand s and completes training on this test method.
Reads, u nderstands and c omplete s training on VR -SOP-LC-11293.
Revie w SARS -CoV -2-mNG_NT documentatio n to ens ure co mpleteness of data
entry and com pliance with Pfizer policies. If VR-TM- 10298 -FM02 is used during a
paperless assay, review VR -TM-10298 -FM02 in LIMS. Direct the SARS -CoV -2-
mNG_NT Analyst to correct documentation errors.
Applies data revie w rules according to VR -SOP-LC-11293.
Consult swith SARS -CoV -2-mNG_NT supervisor ormanagem ent if t here a re any
questions related to the procedure itself or serum samples, if an atypical event is
observed or a protocol deviation occurs.
Upload s SARS -CoV -2-mNG_NT data into LIMS , if appli cable.
If LIMS is used, uses Lab Review er role.
Thisperson must notbe th eSARS-CoV -2-mNG_NT analy st,SAS operator , or Data
analyst for the same batch.
Lab Manager
(Arole specific to
LIMS)Verifies that sample data is corr ectly entered and amended in LI MS.
Transfers quality control serum data into CDTA S.
Verifie s that q uality control data is correctl y entered and amended in CDTAS.
This person may not be the SARS -CoV -2-mNG_NT analyst, SAS Operator, or
Revie wer for the same b atch.
Lab authorizes batches.
Releases data from LIMS to Data Management after QA authoriza tion is obtain ed,
where required.
SARS -CoV -2-
mNG_NT
Supervisors and
Senior ManagersRead sand understand sthis testmethod.
Revie wsand approve sthis testmethod.
Ensure sthat SARS -CoV -2-mNG_NT analysts executing this testmethod are trained
in theapprop riate pr ocedur es and test methods.
Prov idesguidance to SARS -CoV -2-mNG_NT analysts and reviewers and answer
any questions that arise during the execution of this pro cedure .
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FDA-CBER-2021-5683-0779594
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 87.SAFETY
Biosafet y leve l 2(BSL -2) precautions should be taken when handl inghuman and
non-human prim ate sera. Biosafet y level 3 (BSL -3) precautions should be taken when
handling live SARS -CoV -2-mNG. For current guidelines refer to the CDC/NIH
Biosafet y in Microbiological and Biomedical Laboratories Manual (BMBL), 5th Ed.,
2009:http://www.cdc .gov/b iosafety/publications/bmb l5/index.htm.
Personnel must be trained on current laboratory safet y procedures and made aware of the
Safety Data Sheets for c hemicals being used before performing this procedure.
Prior to starting work at B SL3:
Regis ter as B SL3 wo rkerand complete the training and access requirements for BSL3
facilities:
oBSL3 training course
oBSL3 facilit y specific training and access checklist f orm
oBSL3 check off
oBSL3 mentorship
All laboratory work is to be carried out in co mpliance w ith Pfiz er saf ety policies.
Forgenera l laboratory safety guidelines consult the Pearl River Environmental Health
and Safet y Laboratory Manual located at:
http://
8.MATERIALS/EQUIPMENT
8.1.Equip ment
channel h andh eld pipette , , or equivalent
Standard Plate Holder, , or equivalent
-Channel Pipetting Head 5 - 125 µL, , or
equival ent
Light Microsco pe, with fluorescence source, or equivalent
Temperature and CO 2Incubator (equilibrated to 37 ± 2°C, 4-6% CO 2),
CO 2Incubator or equivalent.
Biosafe tyCabinet, , or equivale nt
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FDA-CBER-2021-5683-0779595
FDA-CBER-2021-5683-0779596
FDA-CBER-2021-5683-0779597
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 11completed form must be attached to the batch as a LIM S comment . The reason why the
form was complet edshould also be incl uded in the LIMS comment section.
The assay workshe et VR -TM-10298 -FM03 isused during assay runs where FB -SAS
processes are necessary .
Assay plates containing test samples and QCS are provided b y the group and
are prepared as described in VR -SOP-LC-11287.
The Vero cells are prepared by the Vero analyst as described i n VR-SOP-LC-11299 ,
“Thawing, Passaging and Harvesting of Vero Cells”. Generall y, the
SARS -CoV -2-mNG _NT coordinat or communicates plate requirements to the Vero
coordinator at least 1 week in advance. Required plating densities are sh own Table 6.
Table 6. Vero Cell Concentration in ROP for SARS- CoV -2-mNG_NT
Virus ROP T ype Cell concentration Volume per well
SARS -CoV-2- mNG Black µCLEAR flat -bottom 96-well
plates (Greiner Bio -one 655090), or
equivalent
The SARS -CoV -2-mNG_ NT scheduler, in cooperation with analysts,
coordinates the ass essment of sample sera b y SARS -CoV -2-mNG_ NT, including
determining the number of SARS -CoV -2-mNG_ NT runs on each day and the number of
assay plate s in each SARS -CoV -2-mNG_ NTrun.
9.2.Assay Set Up
1.If using paperless LIMS processes, move to Section 9.3. If using form
VR-TM-10298 -FM03, electronicall y place documents in batch folder received fro m the
SARS -CoV-2-mNG _NT Coordinator along with VR-TM-10298 -FM03. This may
include:
a.SAP to ROP barcode linkage file ( heading is
).
b. outputfile (heading is ).
9.3.Recordi ng of Equipment and R eagent Infor mation and U se of Barcode Scanner
1.Equipment and reagent information is recorded for each assay . This information includes
the equipm ent ba rcode and calibration due date, reagent expiration dates and lot numbers.
2.Equipment and reagent information is record ed in one of two way s:
a.It can be t yped or scanned into VR -TM-10298 -FM02 or VR- TM-10298 -FM03.
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FDA-CBER-2021-5683-0779598
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 12b.It can be s canned with the use of a hand -held scanner or entered directl y into fields in
the batch electronicall y into LIMS. Comp lete all information requested b yLIMS.
Note: Equipment conditions (temperature, CO 2, etc.), Equipment Calibrations and Reagent
Expiration s should be visually inspecte dto be within tolerances prior to Performing the
Assay .
9.4.Preparation of ROPs
Note: Refer to Table 1 of VR -TM-10298-FM01 or Table 4 of VR-TM-10298 -FM03.
1.Obtain ROPs from Vero anal yst. Ensure ROPs have incubated at least hour s prior to
execution of Section 9.10, Transfer of Neutralizat ionReaction onto ROPs.
2. If using Paperless LIMS:
a.Enter information from the cell plate distribution worksheet,
VR-SOP-LC-11299 -FM07, into LIMS.
3.Ifusing form VR -TM-10298 -FM03:
a.Fill in CWS # of Cell Plate Distribution Worksheet VR-SOP-LC-11299 -FM07.
4. Inspect 6wells at random of 2 ROPs per run under the light microscope to ensure the
monolay er is healthy with no microbial contamination and has ≥80% c onflue ncy.
Ahealth y Vero monolayer is evenl y dispersed squamous cells adhered to the bo ttom of
the ROP well . Record abn ormalities in the Aty pical Event(s)box in
VR-TM-10298 -FM03 or the Comment Field in Paperless LIMS.
5.In a BSC, l abel and number e ach pl ate ap propriately (typically 1 -20) with a marker and
store in 37°C/5% CO 2until transfer step(Section 9.10).
9.5.Preparation of SAPs
1.SAPs are prepared b y a workstation as des cribed in VR- SOP-LC-11287 .
2. SAPs are prepared according to a standard plate map as shown in Table 7. The initia l
serum dil ution for the SARS- CoV -2-mNG_NT is 1:20, which, along with a 9- point
2-fold serum dilution series, ena bles a potential plate titer range of 20 to 5,120. Samples
that receive titers greater than 5,120 (or the upp er limit of quantita tionas deter mined
during qualification, whichever is the lower value) are retested after pre -dilution in assay
buffer. Sampl es tha tfailto neutralize at the TDV when tested at the lowest serum
dilution of 1: 20 are reported to have a n eutralizing titer o f .
3.Test samples are assay ed in replicate and a t ypical SAP contains sample titrations in rows
B throug hG. Each SAP ty picall y contains 3 test sera. For example, a -plate assay
contains slots for test samples, which translates t o sample s tested in replicate.
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FDA-CBER-2021-5683-0779599
FDA-CBER-2021-5683-0779600
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 144.Inspect plates to confirm that each well of eac h SAP contain approximately µLliquid
and that volu mes are consistent from well to we ll. Use the At ypical Event(s) box in
VR-TM-10298 -FM03 or the Comment Field in Paperless LIMS to record th e loca tion o f
any wells that have no liquid or appear to have a volume other than µL . Do not use a
pipette to me asure well volumes; thisstep is a visual i nspection only .
5.Number each SAP with a marker. The hand- written number dictates the order in which
theSAPs will receive a reagent and corresponds to its respective ROP.
6.Store SAP s at room temperature until a ddition of virus (Section 9.9). Do not let SAPs sit
at room temperature long er tha n 8 ho urs.
9.7. Add Assay Media to Cell Control Wells in SAPs
1.Warm Assay media in 37 °C water bath. Keep warm for activities in Section 9.8.
2.Perform all work below in BSC with a 8 -channel pipette.
3.Fill reservoir wit h assa ymedi a.
4. Place SAPs in the BSC and one at a time remove the SAP lid and sealer .
5. A ttach 8 pipette tips tothe 8- channel pipe ttor and add µL of ass ay media to all wells
in of SAP –as seen in T able 7.
6. R eplace lid and repeat steps 4-5 unt il all SAPs have this assay media in .
9.8. Preparation of Viral S tock Solution
Note: If using Paperless LIMS, the bank information and volumes required to prepare the
viral stock solution will be display ed in LIMS. Follo wthe inst ructions on Table 4 of
VR-TM-10298 -FM01. If using VR -TM-10298 -FM03, refer to Table 9 of
VR-TM-10298 -FM03.
Calculations for Viral Stock Solution (VSS):
Total Volume of VSS = (4.0 mL * total number of SAPs in batch) + 10mL
Volume of Virus = Tota l Volu me of VSS ÷ Viral Dilution
Volume of Assay Media = Total Volume of VSS –Volume of Virus
1.Perfo rm all manipulations of reagents and active virus in a BSC within the BSL -3
laboratory using sterile techniques.
2.Label a sterile bottle “ VSS” .
3.If using VR -TM-1029 8-FM03, circle appropriate p arameters needed to prepare viral
stock so lution in Table 9 . If u sing VR -TM-10298 -FM02, indicate parameters needed in
Table 9.
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FDA-CBER-2021-5683-0779601
FDA-CBER-2021-5683-0779602
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 163.Remove tips from column 12 (should not a dd virus to column 12 wells).
4.Select custo mprotocol “ .” (custom protocols outlined in
SOP, VR -SOP-FE-11315 ).
5.Press action button to initialize preset .
6.Place r eservoir for VSS in on Carefully add VSS to reserv oir.
7.Prepare reservoir with disinfectant a ndplace to side in BSC.
8.Lower onto tips , press blue flashing button to secure tips, and raise
9. Remove lid of SAP an d remove tip box in
10.Place SAP in on
11.Move over VSS in
12.Lowe r into VS S solution and aspirate µL of VSS.
13. Switch position of over SAP in , lower into SAP volume,
and dispense µLof VSS into eachwell ofSAP.
14.Press action butt on to mix contents.
15.Raise
16.Rem ove SAP from Retu rn lid to SAP.
17.Move disinfectant reservoir to
18. Lower into disinfectant and decontaminate tips by pressing action button.
19.Raise and remove disinfectant rese rvoir from
20.Add empty tip box (for tip waste) to
21. Lower to and eject tips into the waste tip box.
22.Remove the tip box waste from and replace with new clean tip s (be carefu l to
avoid cross contaminatin g waste and clean tips).
23.Repeat steps 5 – 22 for all remaining SAP s in batch.
24. Place stacks of 4 SAPs in secondary container in the BSC and place the SAPs in the
secondary contain er. Latch the secondary container sh ut and disin fect the exterio r.
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FDA-CBER-2021-5683-0779603
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 1725.Place the secondary container in the incubator and open thesecondary contai nerlid
and remove from incubator .
26.Incubate SAPs for 60 minutes at 37°C/5% CO 2instacks of no more than 4 SAPs.
27.Using a serological pipette, add equ al volume of disinfectant in VSS reservoi r. Wait
15minutes, then pour it down sink in BSL -3.
28.For Paperles s LIMS, record the times in LIMS. For VR -TM-10298 -FM03 assay s, record
the start and end times for this incubati on in Table 10in VR-TM-10298 -FM03.
9.10. Transfe rof Ne utralization Re action onto R OPs and Decontamination of SAPs
Note: If using Paperless LIMS, there will be prompts for scanning SAP and ROP barcodes.
Follow the instructions on Table 6 of VR -TM-10298 -FM01. If using VR -TM-10298 -FM03,
refer to Ta ble 11 of VR -TM-10298 -FM03.
1.Identify theappropriate size and quantity of tip box es. Obtain one additional tip box for
tip waste. Place tip boxes inside the BSC and appropriatel y label the tip boxes as
“Clean” and “W aste” to avoid cross contamination .
2.Remove seconda ry container wi th SAPs fro m the incubator, tr ansfer the container to the
BSC, open the lid, and place SAPs inside the BSC.
3.Place appropriate clean tip box in on .
4.Select custom prot ocol (custom protocols out lined in SOP,
VR-SOP-FE-11315 ).
5. R emove lid from RO P and place ROP in on .
6. Prepare reservoir with disinfectant and place to side in BSC.
7.Lower onto tips in pres s blue button to secure tips, and rais e
8.Press ac tion button to initializ e
9.Remove lid of SAP and remove tip box at
10.Place SAP in on
11.Scan barcode on SAP and ROP.
12.Move over SAP in
13. L ower into SAP and aspir ate µL of solution. Rai se the
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(b)(4)
FDA-CBER-2021-5683-0779604
FDA-CBER-2021-5683-0779605
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 199.11. A ssay – Day 2
Note: Refe r toTable 7 -8of VR -TM-10298 -FM01 or Table 13-16 of VR -TM-10298 -FM03.
1.Day 2 activities may be performed b y the same analy st who performed day 1 activities or
by another SARS -CoV -2-mNG NT analyst. For P aperless LIM S, the day 2 anal yst will
login to LIMS a nd select the correct batch and follow the instructions on
VR-TM-10298 -FM01. For VR -TM-10298 -FM03, the individual who performed day 2
signs the “ SARS -CoV -2-mNG_NT anal yst, day 2” line of Table 1and ent ers the date
these steps were completed.
2.Record the date and time that ROPs were removed from the incubator in LIMS, for
Paperless assay s, or in Table 15 of VR -TM-10298-FM03.
3. C arefull yexamine theinstrument Cytation ™ 7 andtheworkplace. Ensu re no used plates
or box esareblocking theplate carrier. Power ontheinstrument, wait atleast 5minutes
towarm uptheinstrument. Green light willdisplay ontheinstrument when it isready.
9.12. Preparation a nd Addition of Hoechst 33342 Solutionto ROP s
Calculation s for Hoechs t 33342 Solution (HS):
Total Volume of HS = (2.5 mL * total number of ROPs in batch) + 2 0 mL
Volume of Hoechst 33342 = Total Volume of HS ÷ Hoechst 3334 2 Dilution
Volume of HBSS = Tota lVolume of HS – Volume of Hoec hst33342
1.Prepare Hoechst 33342 solution by performing a 1:500 dilution of Hoe chst 33342 to
HBSS ( 1-partHoechst 33342 stock to 499 parts HBSS).
2.Secure a lid to another secondary container and remove 5 ROPs from incubator .
3.Place ROPs inside the secondar y container, close the lid, a nd transfer to the BSC.
Open the lid and place the ROPs in the BSC.
4.Place reservoir forHoeschst 33342 Solution in on
5.Carefull y add Hoechs t 33342 Solution to reservoir .
6.Select custom protocol (custom prot ocols outline d in SOP ,
VR-SOP-FE-11315 ).
7.Press action butt on to set .
8.Place appropriate tip box in below lower and attach tips
to by press ing the blue flashing button .
9.Remove tip box f rom
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FDA-CBER-2021-5683-0779606
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 2010.Remo velid of ROP and place R OP in
11.Lower i nto Hoeschst 33342 Solution in and aspirate 125 µL of
solution.
12.Switch position of over ROP position, lower above ROP liquid, and
dispense ofHoeschst 33342 Solution into each wel lof ROP.
13.Raise , r emove RO P from and r eturn lid to ROP.
14.Repeat steps 11-13forup to 4 more plates.
15.Repeat ste ps 6-14for the rest of batch.
16.Raise
17.Prepare reservoir with dis infecta nt andplace in
18.Lower and tips into disinfectant an d mix 25 µL of disinfectant five tim es.
19.Remove the disinfectant reservoir.
20.Place waste tip bo x on in
21.Transfer over tip box waste. Eject tips.
22.Place theROPs in a secondary cont ainer in stacks of no more than 4 ROPs. L atch the
container and disinfect the exte rior. Transport the container to the incubato r.
23. Incubate ROPs at 37°C/5% CO2 for .
24.Record start and end time for Hoechst inc ubation.
25. A t the end of the incubation, secure a lid to another secondary container and add one
ROP from the incubator andplace inside the BSC .
26.Remove lid from ROP a ndseal plate with Sealing Tape
.
27.Repeat steps 25 - 26 until nomore platesremain .
28.Using a ser ological pipette, add equal volume of disinfec tant in Hoe chst reservoir.
Wait 15 minutes, then pour it down sink in BSL-3.
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FDA-CBER-2021-5683-0779607
FDA-CBER-2021-5683-0779608
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 226.Counting and Data Reduction p aramete rs tha t are specific to the SAR S-CoV -2-mNG_NT
are listed below:
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(b) (4)
FDA-CBER-2021-5683-0779609
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 237.Press “Run Proto colin sof tware.
8.Selec toption: and hit OK.
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FDA-CBER-2021-5683-0779610
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 24
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(b) (4)
FDA-CBER-2021-5683-0779611
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 2513.After reading the ROPs on the Cytation 7, place the ROPs in the BSC and remove the
adherent film. Disinfect the ROP sby spray ing disinfectant over the entire plate.
9.13.1. Scanning Quality Check and Correction of Barcode Reader Er rors
9.14. Raw Data and Image Export
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(b) (4)
(b) (4)
FDA-CBER-2021-5683-0779612
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 26
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(b) (4)
FDA-CBER-2021-5683-0779613
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 279.15. Da ta Acquisition
9.16. Analysis of Data and Calculation of SARS -CoV -2-mNG_NT Tit ers
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(b) (4)
(b) (4)
FDA-CBER-2021-5683-0779614
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 289.17. SARS -CoV -2-mNG_NT Data and Document Review
3. R eview and processing of data are described in VR-SOP-LC-11293 .
10.TABLES, FIGURES, DIA GRAMS
Table 8.Assay Job Aid 1 - Compar ison of Assay R uns Using Oracle -LIMS Based
SAS and File -based SAS
Runs Processed Through
Oracle SASRuns Processed Through File-based SAS or
VR-SOP -LC-11293 -FM02
Documentation N/A VR-SOP-LC-10601 -FM30,
VR-TM-10298 -FM03,
QNS Annotated File (if appli cable),
VR-SOP-LC-11287 -FM01 (ifapplicable),
VR-SOP-LC-11287 -FM02 (if appli cable),
SAP Oprintout (if applicable),
VR-SOP-LC-11299 -FM0 7 (if applicable),
Metadata and plate -count printouts,
supplem ental instructions from
SARS -CoV-2- mNG_NT coordinator (if applica ble)
SAS printouts
Reagent and Equipment
InformationEntered into LIMS and shown
in the SAS prin toutEntered into .Run file and shown in the SAS printout
Entered by hand onto VR -TM-10216-FM01
Plate Identification Barcoded plates Barcoded plates
Non-barcoded plates
Assay plate preparation Using a Workstation Using a Workstation
Manual Preparation
Sample Tes ting Replicate Replicate
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(b) (4)
(b) (4)
(b)(4)
(b)(4)
FDA-CBER-2021-5683-0779615
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 29Table 8.Assay Job Aid 1 - Compar ison of Assay R uns Using Oracle -LIMS Based
SAS and File -based SAS
Runs Processed Through
Oracle SASRuns Processed Through File-based SAS or
VR-SOP -LC-11293 -FM02
Data Analysis Automated using the web -
based SAS application found
in Data Analysis Portal (DAP)Auto matedusing the web -based SAS application
found in Data Analysis Portal (DAP ).
Semi-autom ated using f orm
VR-SOP-LC-11293 -FM02, if applicable
Quality control serum
titertrackingAutomated using the web -
based SAS application and
LIMS to upload titers to the
control database tracking and
analysis systemEntered into CDTAS manually
11.REFERENCES
Table 9.General References
Document Title
N/A N/A
Table 10.Form References
Form Title
VR-TM-10298 -FM01 Manual 96 -wellSARS -CoV-2 Micron eutralization Assay Using
(paperless LIMS) Worksheet
VR-TM-10298 -FM02 Manual 96 -well SAR S-CoV- 2 Micron eutralization Ass ay Using
(LIMS metadata ) Worksheet
VR-TM-10298 -FM03 Manual 96 -well SARS -CoV- 2 Micron eutralizatio n Assay Using
(FB-SAS) Works heet
VR-SOP-LC-11287 -FM01 Preparation of SMDPs for Sample Test ing of SARS -CoV -2in a
Neutrali zation Assay Usin g the Head
Workstation
VR-SOP-LC-11287 -FM02 Preparation of Assay Plates from SMDPs for S ample Testing
ofSARS -CoV -2 in a Neutr alization Assay Using the
Workstation
VR-SOP-LC-11287 -FM03 Preparation of 96 w ell CMDPs for Sample Testing of
SARS -CoV- 2 in a Neutralization Assay Usi ng the
Workstation
VR-SOP-LC-11299 -FM07 Worksheet for Dist ribution of Vero and Vero E6 (V -E6) Cells
VR-SOP-LC-11293 -FM02 Titer Calculatio n Worksheet fo r VR -SOP-LC-11293
VR-SOP-LC-10601-FM30 NT COV2 Data Package Chain of Custody
VR-SOP-FE-11315 Operation of Pipetting System
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(b)(4)
(b)(4)
(b)(4)
(b)(4)
FDA-CBER-2021-5683-0779616
VACCINE RESEARCH TEST METHOD PEARL RIVER, NY
TITLE: Manual 96 -well Micron eutralization Assay for the
Detection of Functional Antibodies to SAR S-CoV -2 in
Test SerumDoc Numbe r: VR-TM-10298 Version: 3.0
Doc. Alias: NA
PFIZER CONFIDENTIAL
Page 30Table 11.Specif ic References
Document Title
VR-SOP-LC- 11287 Preparation of Samples for SARS -COV-2 Neutralization Assay
Using the Head Workstation
VR-SOP-LC-11293 Proced ure for Data Review of the SARS -CoV -2-mNG Manual
96-well Micron eutralization Assay
VR-SOP-LC-10336 Analyzing Bio -functio nal Assay Data Using the Web SAS
VR-SOP-LC-11299 Thaw ing, Passaging and Harvesting of Vero Cells
VR-RI-10072 -RS01 Requirements Specif ication for COV2 -mNG Neut
VR-SOP-FE-11315 Operation of Pipettin g System
12.DOCUMENT VERSION MOD IFICATIONS
12.1. CRIF Number: VR -CRIF -21-14875
Table 12.Detailed Changes
List detail ed changes for document(s)
Includ e section numb er(s) for ea chList rationale for each change
Updated roles and responsibilities on Table 5. Up dated to reflect proc edure of using VR -TM-10298-FM02 in
the eve nt LIMS is unavailable.
Updated p rocedure for the event LIMS is
unavailable in Section 9.1.Updated to imp rove quality assurance of data in the event LIMS
is unavaila bleby com pletion and upload of
VR-TM-10298 -FM0 2.
Updated review er steps for the event LIM S is
unavailable in Section 9.17.Updated pro cedure for data reviewers to include revie wing
VR-TM-10298 -FM02 i n the eventLIMS was unavailab le for
analyst .
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(b)(4)
FDA-CBER-2021-5683-0779617
Document Approval Record
Document Name:
Document Title:
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FDA-CBER-2021-5683-0779618