019736 S31 M5 vr tm 10294

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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-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 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 RBD 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-2021-5683-1072656
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 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 RBD Coated Microspheres with Sera ................................ .12
9.5.Preparation of  Plate Washer ................................ ................................ ......... 13
9.6.  Secondary  Antibody  Preparation and Incubatio n ........................... 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 ................................ ................................ ................. 17
11.REFERENCES ................................ ................................ ................................ .................. 17
12.DOCUMENT VERSION MOD IFICAT IONS ................................ ................................ ..18
12.1. CRIF Number: VR -CRIF-20 -14620 ................................ ................................ .....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-2021-5683-1072657
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 31.HISTORICAL REVISION SUMMARY
Table 1.Chronological Summary of Pre vious Versions
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 thereceptor binding 
domain (RBD) of the spike (S) protein of severe acute re spirat ory disease coronavir us 2 
(SAR S-CoV-2).  The assay  method measures IgGantibody  levels for the RBD protein in 
human serum.
3.SCOPE
Personnel in Vacci ne Research & Developme nt (VRD) that perform the s ingle-plex 
SARS -CoV -2RBD IgGdLIAin regulated testing are required to follow t his 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 Definit ion
Ag Antigen
Assay Plate The 96 -well flat -bottom opaque plate us ed to per form the as say.
Batch
Beads Aterm interchangeable with microspheres .
COV19 Coronavirus Di sease 2 019
Dilution Plate,
dLIA Direct Lumi nex Immunoassay
Expiratio n Date Expiration 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 Immuno globulin 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.
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FDA-CBER-2021-5683-1072658
FDA-CBER-2021-5683-1072659
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 5Table 4.Roles and Responsibilities
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 talSOPs regarding safety , equipment use an d 
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 instr uments have been p roperly 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 Microbiolo gical and Biomedic al 
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, before perfo rming this 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 oratorysafet y guidelines con sult the Pearl Riv er 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/EQUIPMENT
All equipment 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 equivalent
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-2021-5683-1072660
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 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, vario us sizes
  Pipetting Device,  or equivalent
Vortex m ixer
Nutat ing m ixer
8.2.Materials


96-well flat bottom opaque white plates,  or equivalent
Polypropy lene 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 conta iners, various 
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-2021-5683-1072664
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 109.2.2. Manual Preparation of Dilution Plates
4.See Figure 1for the Dilution Pla te Dia gram.
Figure 1. Dilution Plate Diagram
9.2.2.1. Reference Standard 
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FDA-CBER-2021-5683-1072665
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 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-2021-5683-1072666
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 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 completion steps .
9.3.2. Manual P reparation of Assay Plat es
9.4.Primary Incubation of RBD Coat ed Microspheres with Sera
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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-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 139.5. Preparation of  PlateWasher
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.  Seconda ry Anti body Preparat ion a nd Incubation
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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-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 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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VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 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 plates in a Bi ohazard waste receptacle .
9.10. Electronic Documentation
VR-TM-10294-FM01 is the assay  completion worksheet used whensample 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-10294-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 batches wh ere met adata is recorded in L IMS.
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FDA-CBER-2021-5683-1072670
FDA-CBER-2021-5683-1072671
VACCINE RESEA RCH TEST METHOD PEA RL RIVER, NY 
TITLE: Single -plex Luminex Assay for Qu antitation of 
IgGAntibodi es toSARS -CoV-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1710.INTERPRETA TION OF RES ULTS
A qualified data reviewer anal yzes the data accordi ng to pre -specified acceptance criteria, as 
descri bed i n VR -SOP-LC-1112 0.
11.REFE RENCES
Table 5.General References
Document Title
VR-SOP-QU-10004 Documen tation 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 Report
VR-SOP-LC-10788 Labeling and Traceability of Reagents and Solutions
Table 6.Form References
Form Title
VR-TM-10294-FM01 Assay Complet ion Worksheet for SARS -CoV -2RBD IgG dLIA
VR-TM-10294-FM02 Manual Sample Preparation Wo rksheet for SARS -CoV -2 RBD IgGdLIA
VR-SOP-LC-10601 -FM14 Gene ric L uminex Assay Data Package Chain ofCustody
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  Assays
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 LIMS 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-PlexSystem Operation and Maintenance
VR-SOP-LC-10586 Liquid Handling Techniques for Regulated Laborato ryProcedures
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, Main tenance and Per formance Verificati on of  
Microplate Washers
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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-2RBD Protein in Human 
SerumDoc Number: VR-TM-10294 Version: 1.0
Doc. Alias: NA
PFIZER CO NFIDENTIAL
Page 1812.DOCUMENT VERSION MOD IFICATION S
12.1. CRIF Number: VR-CRIF-20-14620
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-2021-5683-1072673
Document Approval Record
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