019736 S31 M5 vr tm 10293

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VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1
TABLE OF CONTENTS
LIST OF TABLES ................................ ................................ ................................ ..................... 2
LIST OF FIGURES ................................ ................................ ................................ ................... 2
1.HISTORICAL REVI SIONSUMMARY ................................ ................................ .............. 3
2.PURPOSE ................................ ................................ ................................ .............................. 3
3.SCOPE ................................ ................................ ................................ ................................ ...3
4.GLOSSARY ................................ ................................ ................................ .......................... 3
5.GENERAL ................................ ................................ ................................ ............................. 4
6.RESPONSIBILIT IES................................ ................................ ................................ ............ 4
7.SAFETY ................................ ................................ ................................ ................................ 5
8.MATERIAL S/EQUIPMENT ................................ ................................ ................................ 5
8.1. Equipment ................................ ................................ ................................ ................. 5
8.2.Materials ................................ ................................ ................................ .................... 6
8.3.Reag ents................................ ................................ ................................ .................... 7
8.3.1. Standard and QCS, Prepared as Described in VR -RGR -RS-10747............. 7
8.3.2. Microspheres ................................ ................................ ................................ .7
8.3.3. Secondary  Antibody ................................ ................................ ..................... 7
8.3.4. ................................ ................................ ................................ ............... 7
8.3.5. Buffers ................................ ................................ ................................ .......... 7
9.PROCEDURE ................................ ................................ ................................ ........................ 8
9.1.Preparation of Single -plex S1 Microspheres ................................ ............................. 9
9.2.Preparation of Sample Dilution Plates ................................ ................................ ......9
9.2.1. Robotic Preparation of Dilution Plates ................................ ......................... 9
9.2.2. Manual Preparation of Dilution Plates ................................ ....................... 10
9.2.2.1. Reference Standard ................................ ................................ ...10
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FDA-CBER-2021-5683-1072637
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 29.2.2.2. Unknown Specimen and QCS................................ ................... 11
9.2.2.3. Samples Requiring Pre -dilutions ................................ ............... 11
9.2.2.4. Dilution Plate Completion ................................ ......................... 12
9.3.Preparation of Assay Plates ................................ ................................ ..................... 12
9.3.1. Robotic Preparation of Assay  Plates................................ ........................... 12
9.3.2. Manual Preparation of Assay  Plates ................................ ........................... 12
9.4.Primary  Incubation of S1 Coated M icrospheres with Sera ................................ .....12
9.5.Preparation of  Plate Washer ................................ ................................ ......... 13
9.6.PE-conjugated Secondary  Antibody  Preparation and Incubation ........................... 13
9.7.Assay  Completion ................................ ................................ ................................ ...14
9.8.Reading Assay  Plates on the Bio -Plex  I nstrument ................................ ........... 14
9.9.Rereading of Assay  Plates Due to Errors ................................ ................................ 15
9.10. Electronic Documentation ................................ ................................ ..................... 15
9.11. Data Export and Primary  Data Review ................................ ................................ .16
10.INTERPRETATION OF R ESULTS ................................ ................................ ................. 16
11.REFERENCES ................................ ................................ ................................ .................. 17
12.DOCUMENT V ERSION MODIFICATIONS ................................ ................................ ..18
12.1. CRIF Number: VR -CRIF-20 -14611 ................................ ................................ .....18
LIST OF TABLES
Table 1. Chronological Summary  of Previous Versions ................................ .......... 3
Table 2. Vaccine Research Functional Units ................................ ............................ 3
Table 3. Terms and Definitions ................................ ................................ ................ 3
Table 4. Roles and Responsibilities................................ ................................ .......... 5
Table 5. General References ................................ ................................ ................... 17
Table 6. Form References ................................ ................................ ....................... 17
Table 7. Specific References ................................ ................................ .................. 17
Table 8. Detailed Changes ................................ ................................ ...................... 18
LIST OF FI GURES
Figure 1. Dilution Plate Diagram ................................ ................................ ............. 10
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FDA-CBER-2021-5683-1072638
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 31.HISTORICAL REVISION SUMMARY
Table 1.Chronological Summary of Previous Ve rsions
Versio n 
#Effective Date Originator Summary
1.0 Current Guy Singh New  Document
2. PURPOSE
This p rocedure descri bes the method to quantitate Ig Gantibody  levels to subunit 1 ( S1) of 
the spike (S) protein of severe acute respirator y disease coro navir us 2 ( SAR S-CoV-2).  The 
assay  method measures IgGantibody  levels for the S1protein in human serum.
3.SCOPE
Personnel in Vacci ne Research & Developme nt (VRD) that perform the s ingle-plex 
SARS -CoV -2 S1 IgGdLIAin regulated testing are required to follow this test method .
Table 2.Vacci ne Research Functio nal Units
Functional Units Location
Vaccine Re search & Development Pearl River
4.GLOSSARY
Table 3.Terms and Definitions
Term Definition
Ag Antigen
Assay Plate The 96 -well flat -bottom opaque plate us ed to per form the as say.
Batch
Beads A term interchangeable with microspheres .
COV19 Coronavirus Di sease 2 019
Dilution Plate,
dLIA Direct Luminex Immunoassay
Expira tion Date Expirat ion dates of r eagents will be assign ed based on the av ailable s tability data.   
Specific expi ration dates may be recorded on reagen t labels, or within lab oratory 
notebooks or documentation workboo ks refere nced by the reagent label.
IgG Imm unoglobulin G
Labware LIMS TheLabw are brand of a Lab oratory Information Management System whic h is a 
database used to track specimens and their associated tests.
LNB Laboratory Notebook
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FDA-CBER-2021-5683-1072639
FDA-CBER-2021-5683-1072640
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 5Table 4.Roles and Responsi bilities
Role Responsibilities
Analyst Performs the test method foll owing the p rocedures described in this SOP.
Com pletesdocumentation of the w ork perform ed and any deviations that occurred .
Follows applicable depar tmen tal SOPs regarding safety, equipme nt use and 
documentation practices .
Initiates LDRs .
Notifies superviso r of any at ypical ev ents.
Supervisor or 
designeeReviews and signs documentation. 
Ensures that analysts have been trained on appropriate SOPs.
Ensures that equ ipment and instruments ha ve been prop erly calibrated and maintained.
Ensures LDRs are initiated and proper ly invest igated .
7.SAFETY
Biosafet y lev el 2 precautions should be taken when handling human sera. For current 
guidelines refer to the CD C/NI H Biosafe ty in Microbiological and Biomedical 
Laboratories Manual (BMBL), 5th Ed., 2009: 
http://www.cdc.gov/biosafe ty/public ations/bmb l5/index.htm.
Personnel must be trained on current labora torysafet y procedures and made aware of the 
Safety  Data Shee ts (SDS) for chemicals being used, bef ore performi ngthis procedure. 
For SDS information, refer to link at 
All laboratory  work is to be carried ou t incompliance with Pfizer safet y policies.  For 
general lab oratory safet y guidelines consult the Pearl River Environmental Health and 
Safety  Laboratory  Manual located at: 
Lab coats, safet yglasses and disposable gloves must be worn at all times when handling 
human specimens .
8.MATERIALS/E QUIPMENT
Allequipment and materials used in regulated testing must co nform to cu rrent dep artmental 
guidelines for calib ration, maintenance and qualification.
8.1.Equipment
Bio-Plex Reader, Bio -Rad Cat. #  orequivalent
Platform Shaker, or equivale nt
Scientific freezers, -80°C ±15°C and -20°C -15/+5 °C
Scientific ref rigerator ,2 to 8°C
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FDA-CBER-2021-5683-1072641
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 6 (magnetic bead) plate washer or equiva lent
Alarm timer
Single channel pipettes, various sizes
Multi -channel pipe ttes, manual and electronic, various si zes
  Pipetting Device,  or equivalent
Vortex m ixer
Nutat ing m ixer
8.2.Materials


96-well flat bottom opaque white plates,  or equ ivalent
Polypropylene pipette tips, various sizes 
Sterile disposable serological plastic p ipettes, various sizes
Dispos able polypropy lene tubes or container s, various sizes
Disposable light protective amber pol yprop ylene centrifug e tubes or containers, va rious 
sizes
Aluminum sealing tape for 96 well plates
Reagent Reservoir s, various sizes
Polypropy lene micro snap cap tube s
Tube racks
Magnetic separators
Bioh azardous waste receptacle
Biohazard sharps waste containers
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FDA-CBER-2021-5683-1072643
FDA-CBER-2021-5683-1072644
FDA-CBER-2021-5683-1072645
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 109.2.2. Manual Preparation of Dilution Plates
4.See Figure 1for the Dilution Plat e Diagram.
Figure 1. Dilution Plate Diagram
9.2.2.1. Reference Standard 
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FDA-CBER-2021-5683-1072646
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 119.2.2.2. Unknown S pecim enand QCS
9.2.2.3. Samples Requ iring Pre- dilutions
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FDA-CBER-2021-5683-1072647
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 129.2.2.4. Dilution Plate Completion
9.3.Preparation of Assa y Plates
9.3.1. Robotic Preparat ion of Assay Plates
Refer to VR-SOP-LC-11186for assay plate p reparation using the  
Robot.  Continue to Section 9.4to begin a ssay comp letion steps.
9.3.2. Manual P reparation of Assay Plat es
9.4.Primary Incubation of S1Coat ed Microspheres with Sera
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FDA-CBER-2021-5683-1072648
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 139.5. Preparation of PlateWashe r
1.Prepare  plate washer for use as described in VR -SOP-FE-10887.  Record the 
activit ies, as required, in the appropriate equipment logbook .
2.Ensure the Magnetic Bead Plate Adapto r is inserted for mag netic bead plate wash.
9.6.PE-conjugated Se conda ry Antibo dy Preparatio n and Incubation
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FDA-CBER-2021-5683-1072649
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 149.7.Assay Completion
9.8.Reading Assay Plat es on the Bio- Plex  Instrument
1.Prepare the Bio-P lex reader for use as described in VR -SOP-FE-10111.  Rec ord the 
activities, as required in the appropri ate equipment logbook.
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FDA-CBER-2021-5683-1072650
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 159.9. Rereading of Assay Plate s Due to Errors
6.Prepare assay  plates for disposal by  emptying the liquid off the plates.  Discard empty
assay plat es in a Bi ohazard waste receptacle .
9.10. Electronic Documentation
VR-TM-10293-FM01 is the assay  completion worksheet used w hensample pr eparatio n 
is performed either manually  or by automated methods.  A n assay  completion worksheet 
is not required for fully electronic /paperless batches where metadata is recorded in LIMS.
VR-TM-10293-FM02 is the manual sample preparation workshe et used when a utomate d 
sample preparation is not performed .  An assay  completio n worksheet is not required for 
fully  electronic /pape rless batche s where meta data is recorded in L IMS.
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FDA-CBER-2021-5683-1072651
FDA-CBER-2021-5683-1072652
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1711.REFERENCES
Table 5.General References
Document Title
VR-SOP-QU-10004 Document ation Pract ices in Vacci ne Research
VR-GWP -10041 Metadata Entry Pr ocedure in LabWare LIMS for Luminex Immunoassays
VR-SOP-QU-10726 Laboratory Devi ation Rep ort
VR-SOP-LC-10788 Labeling and Traceability of Reagents and Solutions
Table 6.Form References
Form Title
VR-TM-10293-FM01 Assay Complet ion Worksheet for SARS -CoV -2S1 IgG dLIA
VR-TM-10293-FM02 Manual Sample Preparation Work sheet for SARS -CoV -2 S1 IgG dLIA
VR-SOP-LC-10601 -FM14 Gene ric L uminex Assay Data Package Chain of Custody
Table 7. Speci fic References
Document Title
VR-SOP-LC-10222 Procedure for M aking Sample Predilutions with  
Robot
VR-SOP-LC-10627 Preparation of Assay Buffers and Solutions for  Assa ys
VR-SOP-LC-11114 Data Review Procedures for Dir ect Luminex I mmunoass ays Using Input Files or 
LabWare v5 LIMS
VR-SOP-LC-1112 0 Data Review Procedures f or Direct Luminex Immunoassays in LIM S v6.
VR-SOP-LC-11186 Runni ng the Method Using  Robot
VR-SOP-LC-11190 Standard Operating Procedure f or Ru nning Predilute Sample Method using 
 Robot
VR-SOP-LC-11295 Preparation of and Evaluation of  Microsp heres for use in Direct 
Luminex Assays
VR-RGR -RS-10747 Preparat ion of Reference Standard Seru m and Quality Control Samples for 
SARS -CoV -2 S1 and RBD Direc t Luminex Immunoass ays
VR-SOP-FE-10111 Bio-Plex Sys tem Operation and Maintenance
VR-SOP-LC-10586 Liquid Handling Techniques for Regulated Laboratory P rocedures
VR-SOP-LC-10601 Data P ackage Chain of Custody
VR-SOP-LC- 10774 Specimen Ma nagement Record Using LabWare LIMS
VR-SOP-FE-1088 7 Operation, Maintenance and Per formance Verificati on of  
Microplate Washers
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FDA-CBER-2021-5683-1072653
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2 S 1 Protein in Human 
SerumDoc Number: VR-TM-10293 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1812.DOCUMENT VERSION MOD IFICAT ION S
12.1. CRIF Number: VR-CRIF -20-14611
Table 8. Detailed Changes
List detailed changes for document( s) 
Include section number(s) for eachList rati onale for each chan ge
New NA
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FDA-CBER-2021-5683-1072654
Document Approval Record
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