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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 1Title:Qualification Report for a Single-plex Direct L uminex Ass ay (dLIA) for Quantitation
of IgG Antibodies to SARS- CoV-2 S1 Protein in Human Sera
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
090177e194a89aaf\Approved\Approved On: 18-Aug-2020 23:11 (GMT)
FDA-CBER-2021-5683-1072675
FDA-CBER-2021-5683-1072676
PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 3Title:Qualification Report for a Single-plex Direct L uminex Ass ay (dLIA) for Quantitation
of IgG Antibodies to SARS- CoV-2 S1 in ProteinHuman Sera
SYNOPSIS
This reportdocuments the results of the statistical anal yses from assay qualification
experiments of a single-plex direct Luminex immunoassay (dLIA) for q uantitation of human
IgG antibodies to subunit 1 (S1) of the spike (S) protein of severe acute respiratory disease
coronavirus 2 (SARS -CoV-2)in human sera . The performance parameters examined in the
assay qualification were linearity, precision and standard curve bias . From these
data, the assay range, lower limit of quantitation, and descriptive statistics of assay
parameters were determined. Key qualification outcomes are highlighted in the table below.
Key Qualification Outcomes
Antigen Assay Range
Lower Bound Plate
Well IgG
Concentrat ion
(U/mL)aAssay Range
Upper Bound Plate
Well IgG
Concentration
(U/mL)aLLOQbSample
Dilution Adjusted
IgGConcentration
(U/mL)aAssay Precision
c
COVS1d0.002533 0.128000 1.2665
a.Units/mL .
b.Lower Limit of Quantitation .
c. .
d.Subunit 1 of the Spike protein of severe acute respiratory disease coronavirus 2 .
The data provided in this report support the qualification of the single-plex dLIA for
quantitation of human IgG antibodies that bind to the S1 protein of SARS- CoV-2 and
confirm tha t the assay is suitable for its intended use when performed in accordance w ith
standard operating procedures by qualified personnel.
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 4TABLE OF CONTENTS
SYNOPSI S................................ ................................ ................................ ................................ .3
LIST OF TABLES ................................ ................................ ................................ ..................... 5
LIST OF FIGURES ................................ ................................ ................................ ................... 6
1. OBJECTI VES................................ ................................ ................................ ........................ 7
2. INTRODUCTION ................................ ................................ ................................ ................. 7
3. GLOSSARY ................................ ................................ ................................ .......................... 8
4. EXPERI MENTAL OUTL INE................................ ................................ ............................... 9
4.1. Mater ials and Methods ................................ ................................ .............................. 9
4.1.1. Methods................................ ................................ ................................ ........9
4.1.2. Critical Reagents ................................ ................................ ........................... 9
4.2. Experimental Design ................................ ................................ ............................... 10
4.2.1. Linearity ................................ ................................ .................... 10
4.2.2. Precision ................................ ................................ ................................ .....11
4.2.3. Standard Curve Bias ................................ ................................ ................... 11
5. STATI STICAL METHO DS................................ ................................ ................................ 11
5.1. Sampl e Results................................ ................................ ................................ ........11
5.2. Statistical Analy ses................................ ................................ ................................ .11
5.2.1. Linearity ................................ ................................ .................... 12
5.2.2. Precision ................................ ................................ ................................ .....12
5.2.3. Standard Curve Bias ................................ ................................ ................... 13
5.2.4. Assay Range................................ ................................ ............................... 13
5.2.5. Assay Precision (Intermediate Precision) ................................ ................... 14
5.2.6. Sample Quantitation Range ................................ ................................ ........14
5.2.6.1. L ower Limit of Quantitation ................................ ..................... 14
5.2.6.2. Upper Limit of Quantitation ................................ ...................... 15
5.2.7. Assay Run Performance ................................ ................................ .............15
5.2.7.1. ................................ ................................ .15
5.2.7.2. Standard Curve Parameters ................................ ....................... 15
5.2.7.3. Quality Control Samples ................................ ........................... 15
6. RESUL TS AND DI SCUSSION................................ ................................ .......................... 16
6.1. L inearity Evaluation ................................ ................................ ...............16
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 56.2. Precision Evaluation ................................ ................................ ................................ 17
6.3. Standard Curve Bias Evaluation................................ ................................ .............18
6.4. Assay Range Based on L inearity, Precision, and Standard Curve
Bias................................ ................................ ................................ ............................ 19
6.5. Assay Precision (Intermediate Precision) ................................ ............................... 19
6.6. Sample Quantitation Range ................................ ................................ ..................... 20
6.6.1. Lower Limit of Quantitation ................................ ................................ .......20
6.6.2. Upper Limit of Quantitation ................................ ................................ .......20
6.7. Assay Run Performance ................................ ................................ .......................... 20
6.7.1. ................................ ................................ .................. 20
6.7.2.Standard Curves ................................ ................................ .......................... 21
6.7.3. Quality Control Samples.............................................................................21
7. CONCLUSION ................................ ................................ ................................ .................... 22
8. DEVIATIONS ................................ ................................ ................................ ..................... 22
9. SUPPORTING DOCUME NTATION ................................ ................................ ................. 22
10. SUPPL EMENTAL INF ORMATION ................................ ................................ ...............22
11. SUPPORTIVE FIGUR ES................................ ................................ ................................ ..24
12. SUPPORTIVE TABLE S................................ ................................ ................................ ...25
LIST OF TABLES
Table1. Terms and Definitions................................ ................................ ................ 8
Table 2. SOPs and Robotic Methods within Scope of this Protocol ........................ 9
Table 3. List of Critical Reagents ................................ ................................ ...........10
Table4. Well Concentration Range Based on L inearity Data..............17
Table5. Well Concentration Range Based on Precision Data ............................... 18
Table6. Well Concentration Range Based on Reference Standard Curve
Bias................................ ................................ ................................ ...........19
Table7. Final IgG Assay Range – Well Concentration (Units/mL) ...................... 19
Table 8. ................................ 19
Table 9. Lower Limit of Quantitation (LLOQ) ................................ ...................... 20
Table10. ................................ ................................ ................ 21
Table11. Descriptive Statistics for Standard Curve Parameters.............................. 21
Table12. Descriptive Statistics for QCS ................................ ................................ ..22
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 6LIST OF FIGURES
Figure1. Linearity Plot for S1 IgG dLIA ................................ ...............16
Figure 2. Precision Plot for S1 IgG dLIA ................................ ................................ 17
Figure3. Standard Curve Bias Plot for S1 IgG dLIA ................................ ..............18
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FDA-CBER-2021-5683-1072684
FDA-CBER-2021-5683-1072685
PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 125.2.1. Linearity
5.2.2.Precision
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 135.2.3.Standard Curve Bias
5.2.4.Assay Range
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FDA-CBER-2021-5683-1072689
PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 166. RESULTS AND DISCUSSI ON
Qualification data used for the results of the anal yses described herein are listed in
VR-MQR-10211-ATT01(see Section 9).
6.1. Linearity Evaluation
Figure1. Linearity Plot for S1IgG dLIA
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 18Table5.Well Concentration Range Based on Precision Data
6.3.Standard Curve Bias Evaluation
Figure3. Standard Curve Bias Plot for S1IgG dLIA
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 206.6.Sample Quantitation Range
6.6.1.Lower Limit of Quantitation
The LLOQ is the lowest sample IgG concentration in the assay that is precise
and falls into the linear assay range. Unlike the lower limit of the assay
range that is defined as a well concentration, the LLOQ is a dilution-adjusted sample
concentra tion. As described in Section 5.2.6.1
, LLOQ is defined as the dilution -adjusted
lower limit of the assay range.Table 9lists the LLOQ value for the SARS CoV -2 S1 IgG
dLIA.
Table 9.Lower Limit of Quantitation (LLOQ)
Antigen LLOQ (U nits/mL)
COVS1a1.2665
a.Subunit 1 of the Spike protein of severe acute respiratory disease coronavirus 2 .
6.6.2.UpperLimit of Quantitation
6.7.Assay Run Performance
6.7.1.
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 21Table10.
6.7.2.Standard Curve s
Descriptive statistics for the reference standard curves for S1IgGare listed in Table11.
Table11presents suitability limits on standard curves which were calculated as described in
Section 5.2.7.2.
Table11. Descriptive Statistics for Standard Curve Pa rameters
6.7.3.Quality Control Samples
Descriptive statistics for the QCS samples from the qualification data for the SARS-CoV-2
S1 IgG dLIA are listed in Table12.
. Table12also shows QCS limits on the plates (refer to
Section 5.2.7.3).
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 22Table12. Descriptive Statistics for QCS
7.CONCLUSION
The data provided in this report support the qualification of the single-plex SARS-CoV-2 S1
IgGdLIAfor quantitating S1 specific IgGinhuman sera . The assay is suitable for the
intended use when performed in accordance with standard operating procedures by qualified
personnel. The assay limits established from the data generated in this qualification will be
used to support assay suitability during clinical testing until such limits are refined in a future
validation .
8.DEVIATIONS
NA
9. SUPPORTING DOCUMENTA TION
1.VR-TM-10293, Single -plex Luminex Assay for Quantitation of IgG Antibodies to
SARS-CoV-2 S1 protein in Human Serum.
2.VR-MQR-10211-ATT01, Supportive Data for VR- MQR-10211.
3.VR-SOP-LC-11120, Data Review Procedures for Direct Luminex I mmunoassay s in
LIMS v6.
10. SUPPLEMENTAL INFORMA TION
1.VR-SOP-LC-11295, Preparation and Evaluation of Coated Microspheres
for use in Direct Luminex Assays.
2.VR-SOP-LC-10627, Preparation of Assay Buffers and Solution.
3.VR-SOP-LC-11186, Standard Operating Procedure for Running Method
Using Microlab STAR Robot .
4.
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 235.VR-DTN-11380, Planned Deviation from VR- SOP-FE-10111 Bio- Plex Operation
and Maintenance .
6.VR-SOP-FE-10111, BioPlex System Operation and Maintenance .
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 2411.SUPPORTIVE FIGURES
11.1. Plate Lay out for L inearity Experiments ................................ ..........24
11.2. SARS -CoV-2 S1 IgG dLIA Assay Plate Layout for Routine Testing ............................ 24
11.1. Plate Layout for Linearity Experiments
11.2.SARS-CoV-2 S1 IgG dLIA Assay Plate Layout for Routine Testing
The assay plate layout in Supportive Figure 11.2is used for routine testing.
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 2512.SUPPORTIVE TABLES
12.1. Linearity Samples ................................ ................................ .......................... 25
12.2. Sample Panel for Precision Evaluation ................................ ................................ ...........26
12.3. Precision of COVS1 Well Titers Sample................................ ....26
12.4. ................................ .................. 28
12.1. Linearity Samples
Sample Number Sample ID
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG Antibodies to SARS -CoV-2 S1 Protein in Human
Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 2612.2. Sample Panel for Precision Evaluation
Sample Number ID Sample Number ID Sample Number ID Sample Number ID
12.3.Precision of COVS1 Well Titers Sample
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG Antibodies to SARS -CoV-2 S1 Protein in Human
Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 2712.3.Precision of COVS1 Well Titers Sample
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PF-07302048: Qualification Report for a Single-plex Direct Luminex Ass ay (dLIA) for Quantitation of IgG
Antibodies to SARS -CoV-2 S1 Protein in Human Sera
VR-MQR-10211, Ver. 2.0
PFIZER CONFIDENTIAL
Page 28
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