Table of Contents
Response to CBER Comments Received on 20 November 2020
TABLE OF CONTENTS

1
2

Table 1. Emergency Supply Chain Manufacturing Nodes

3

Table 2. Lots Submitted to IND for Emergency Use

4

Table 3. Manufactured Lots Intended for Emergency Supplya

4

Table 4. Anticipated Distribution Dates

7

Table 5. Lot Genealogy with RNA Integrity and LMS Levels

10

Table 6. Characterization of LMS in DP Lots in addition to EG5411

12

Table 7. Accurate Mass Assignments of Nucleosides for Additional BNT162b2 DP
Lots via LC-UV/MS
15
Table 8. Accurate Mass Assignments of Nucleobases for Additional BNT162b2 DP
Lots via LC-UV-MS/MS
15
Table 9. Representative Stability Study Design

16

Table 10. RNA Integrity Test Results for the Study Performed with Lots# EJ1685aand EJ1688a (0.1 mg/mL in 0.9% Sodium Chloride)
18
Table 10. RNA Integrity Test Results for the Study Performed with Lots# EJ1685aand EJ1688a (0.1 mg/mL in 0.9% Sodium Chloride) Test Acceptance Criteria T0(5 days 2-8°C and 2 hours at 30 °C) 30 °C/75% RH
Vial 3 Hours 3 Hours 6
Hours 6 Hours 24 Hours
Vial Polycarbonate Syringe Vial Polycarbonate
Syringe Vial Lot # EJ1685 RNA Integrity Fragment Analyzer (CGE) T0 Vial
± 20% ( 50% Intact RNA) 64.8% 64.7% 64.9% 65.1% 64.0% 63.1%
ReportLMS result 9.7% 9.8% 9.8% 10.1% 10.0% 10.9% Lot # EJ1688 RNA
Integrity Fragment Analyzer (CGE) T0 Vial ± 20% ( 50% Intact RNA) 61.4%
59.5% 59.8% 58.4% 58.2% 55.8%
Report LMS result 11.0% 11.2% 11.9%
12.7% 13.0% 14.4% a. All vials used in this study were held for 5 days at 2-8 °C
plus 2 hours at 30 °C prior to dilution Abbreviations: CGE = capillary gelelectrophoresis; LNP = lipid nanoparticle
Table 11. Analytical Test Results
for the Study Performed with Lot# EH9899a (0.1 mg/mL in 0.9% Sodium Chloride)
19

Table 12. Analytical Test Results for the Study Performed with Lot# EJ0553a (0.1mg/mL in 0.9% Sodium Chloride)
20
Table 13. Lot Genealogy and Vials Rejected During Visual Inspection for Particles
23
Table 14. Lipids Used in BNT162b2 Drug Product Manufacturea

28

Table 15. Active Proteins Vendor Specification and Target Concentration During In
Vitro Transcription, DNase I Digestion and Proteinase K Steps
36
Table 16. Routine Process Controls Implemented During the Sanitization,
Equilibration, and Reuse procedure (Pfizer, Andover)38

Table 17. Routine Process Controls Implemented During the Sanitization,
Equilibration, and Reuse Procedure

39

Table 18. Performance Tests Implemented for Concurrent Validation for Membrane
Regeneration and Reuse
42
Table 19. Drug Substance Testing

44

Table 20. Drug Product Testing

45

Table 21. Validation Summary for the UV Spectroscopy Analytical Procedure(BioNTech)

46

Table 22. Validation Summary for the RT-PCR Analytical Procedure for Drug
Substance (BioNTech)

46

Table 23. Validation Summary for the Capillary Gel Electrophoresis Analytical
Procedure for Drug Substance (BioNTech)

46

Table 24. Validation Summary for the qPCR Analytical Procedure (BioNTech)

47

Table 25. Challenge Recovery Testing Results for Drug Substance (Rentschler)

48

Table 26. Inhibition/Enhancement Results for BNT162b2 Drug Substance(Rentschler)

49

Figure 1. CGE Electropherogram of DP Lot EJ1685

9

Figure 2. Ion Pairing RP-HPLC of RNA Extracted from Drug Product Lots

13

Figure 3. Analysis of Nucleosides in Additional BNT162b2 DP Lots by LC-UVMS/MS (A260 nm)

14

QUERY 13

QUERY 2

4

QUERY 3

6

QUERY 47

QUERY 59QUERY 69QUERY 712QUERY 821

QUERY 922

QUERY 1027

QUERY 1133

QUERY 1234

QUERY 1335

QUERY 1436

QUERY 1537

QUERY 1640

QUERY 1741

QUERY 1843

QUERY 1950

QUERY 2051

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736

Response to CBER Comments Received on 20 November 2020
Regarding Overall CMC Information

25 November 2020

PFIZER CONFIDENTIAL
Page 1COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

TABLE OF CONTENTSQUERY 1...................................................................................................................................3QUERY 2...................................................................................................................................4QUERY 3...................................................................................................................................6QUERY 4...................................................................................................................................7QUERY 5...................................................................................................................................9QUERY 6...................................................................................................................................9QUERY 7.................................................................................................................................12QUERY 8.................................................................................................................................21QUERY 9.................................................................................................................................22QUERY 10...............................................................................................................................27
QUERY 11...............................................................................................................................33
QUERY 12...............................................................................................................................34
QUERY 13...............................................................................................................................35
QUERY 14...............................................................................................................................36QUERY 15...............................................................................................................................37
QUERY 16...............................................................................................................................40QUERY 17...............................................................................................................................41QUERY 18...............................................................................................................................43QUERY 19...............................................................................................................................50QUERY 20...............................................................................................................................51

PFIZER CONFIDENTIAL
Page 2

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryREGARDING THE MANUFACTURE AND TESTING FACILITIES:QUERY 1
In amendment 119 (submitted on October 21, 2020) Table 1 - EUA and Commercial Supply
Chain Manufacturing Nodes, facilities are listed for use in “US and EU markets” (for initial EUA
and EUA supply-chain expansion) and “US and/or EU markets” (for commercial/EUA). Please
identify the facilities and manufacturing nodes for the production of emergency supply for the
US market.
RESPONSE 1
A simplified version of the table provided in amendment 119 (submitted on October 21, 2020) is
provided in Table 1 and includes facilities and manufacturing nodes for the production of
emergency supply for the US market.
Table 1.

Emergency Supply Chain Manufacturing Nodes
Emergency Supply

Drug
Substance

Pfizer Andover

DS Testing

Pfizer 5
Andover, 1,2 Chesterfield 2

LNP, DPPolymunPfizerPuurs

Fill/Finish

PfizerPuurs(Lines WSL5, FC2,
VC2)

PfizerKalamazoo
PfizerKalamazoo(Lines 8,18)

BNT Mainz,
andRentschler, Germany (Purification)
BNT Mainz,
BNT IMFS 4,
Rentschler, Germany
PolymunDermaPharm
Pfizer Puurs

DP Release andstability
Testing

PfizerPuurs(Line WSL5, FC2, VC2)

Pfizer
Pfizer
PfizerAndover,2Andover,2Andover,2Chesterfield,2Chesterfield,2Chesterfield,2
3
3
Puurs
Kalamazoo
Puurs 3
1 Microbial tests: endotoxin, bioburden.
2 Release and Stability testing for Identity, Composition, Strength, Product Purity and/or Process Related
Impurities.
3 Microbial tests: endotoxin, sterility. Back-up sterility test sites may be employed.
4 Poly(A) tail and 5’-Cap (Composition) tests may be performed for EUA supplies at Pfizer Andover or Pfizer
Chesterfield.
5 Double stranded RNA (Product Related Impurity) Test may be performed for EUA supplies at BNT IMFS.

Literature References
None
SUPPORTING DOCUMENTATIONNone
PFIZER CONFIDENTIAL
Page 3

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 2For each DP manufacturing node, please specify the number of GMP commercial scale DP lots
for which data will be available, and submitted for review, at the time of EUA. For DP
manufacturing nodes for which GMP commercial scale DP lot data will not be available at thetime of initial EUA, please provide an updated time table for submission of these data. Please
note that data from at least three GMP commercial-scale DP lots will be required from a DP
manufacturing node prior to initial authorization to distribute EUA supplies from that node.
RESPONSE 2
Six lots have been provided to the IND to date as listed in Table 2.
Table 2.

Lots Submitted to IND for Emergency Use

Lot
DS Manufacturing Site
LNP Production Site
Fill/Finish Site
EE8492
Pfizer AndoverEE8493
Pfizer Andover
Polymun ScientificPfizer, Puurs
EJ0553
Pfizer Andover
EJ1685a
BioNTech; Rentschler
EJ1686a
BioNTech; Rentschler
EK1768
Pfizer Andovera. IVE data for lots EJ1685 and EJ1686 are provided in 3.2.R BNT162b2 Comparability Report.

Lots manufactured but pending submission to the IND for Emergency Use are provided inTable 3
Table 3.
Lot

Manufactured Lots Intended for Emergency Supplya
DOM

EH98997-Oct-2020

DS
LNP
Manufacturing
Production Site
Site
Andover/Kalamazoo/KalamazooAndover
Kalamazoo

EK573022-Oct-2020

Andover

Kalamazoo

EK923104-Nov-2020

Andover

Kalamazoo

EL128311-Nov-2020

Andover

Kalamazoo

EL128417-Nov-2020

Andover

Kalamazoo

EL324619-Nov-2020

Andover

Kalamazoo

EL0141
EK4241

29-Oct-2020
12-Nov-2020

BioNTech;Rentschler/Polymun/Puurs
BNT;RNT
Polymunb
Puurs (WSL5)
BNT;RNT
Polymunb
Puurs (WSL5)

PFIZER CONFIDENTIAL
Page 4Fill/Finish Site

Kalamazoo(Line 8)Kalamazoo(Line 8)Kalamazoo(Line 18)
Kalamazoo(Line 18)
Kalamazoo(Line 18)
Kalamazoo(Line 8)

AnticipatedCoA
Availability
30-Nov-2020
30-Nov-2020
30-Nov-2020
14-Dec-2020
15-Dec-2020
29-Dec-2020
7-Dec-2020
15-Dec-2020

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 3.
Lot

Manufactured Lots Intended for Emergency Supplya
DOM

DS
LNP
Fill/Finish Site
Anticipated
Manufacturing
Production Site
CoA
Site
Availability
BioNTech;Rentschler/DermaPharm/Puurs
EL014029-Oct-2020
BNT;RNT
DermaPharm
Puurs (WSL5)
7-Dec-2020
EL0142
29-Oct-2020
BNT;RNT
DermaPharm
Puurs (WSL5)
7-Dec-2020
EK4237
5-Nov-2020
BNT;RNT
DermaPharm
Puurs (WSL5)
7-Dec-2020
EK4243
5–Nov-2020
BNT;RNT
DermaPharm
Puurs (WSL5)
7-Dec-2020
EK42445-Nov-2020
BNT;RNT
DermaPharm
Puurs (WSL5)
14-Dec-2020
EK424512-Nov-2020
BNT;RNT
DermaPharm
Puurs (WSL5)
16-Dec-2020
BioNTech;Rentschler/Puurs/Puurs
EL072530-Oct-2020
BNT;RNT
Puurs
Puurs (FC2)
7-Dec-2020
EL073903-Nov-2020
BNT;RNT
Puurs
Puurs (FC2)
7-Dec-2020
EL148404-Nov-2020
BNT;RNT
Puurs
Puurs (FC2)
7-Dec-2020
EJ679512-Nov-2020
Andover
Puurs
Puurs (FC2)14-Dec-2020
a: All lots are currently pending release.
b: Data from 6 GMP commercial-scale Polymun DP lots have already been submitted, thus complying with the 3
lot requirement to distribute EUA supplies from that node.
Abbreviations: BNT = BioNTech; RNT = Rentschler
Note: Final lot release by the Quality Unit occurs after CoA availability.

Literature References
None
SUPPORTING DOCUMENTATION
New or Replaced Supporting Documentation
3.2.R BNT162b2 Comparability Report, Replaced
Previously submitted supporting documentation
None

PFIZER CONFIDENTIAL
Page 5COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 3
Please note that data from all PPQ studies at all manufacturing nodes must be completed prior to
submission of a BLA.
RESPONSE 3
The sponsor acknowledges that data from all PPQ studies at all manufacturing nodes must becompleted prior to submission of a BLA.
Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
Page 6

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 4In the absence of adequate PPQ data for drug product manufactured at multiple sites, it will benecessary for you to submit final COAs for lots to be distributed under EUA at least 48 hours
prior to lot distribution. Please submit a plan and schedule for lot distribution under EUA.
RESPONSE 4
Pfizer plans to submit batches of available Certificates of Analyses (CoAs) twice a week (e.g. on
Mondays and Thursdays). This schedule may be increased prior to December 11 to facilitate
release of initial supplies and in accordance with the requirements laid out in Question 2. Lots
will continue to be manufactured in support of emergency supply and submitted to the IND atleast 48 hours prior to lot distribution.
Table 4.
Lot

Anticipated Distribution Dates
DOM

EH98997-Oct-2020

EK573022-Oct-2020

EK923104-Nov-2020

EL128311-Nov-2020

EL128417-Nov-2020

EL324619-Nov-2020

EE849205-Aug-2020

EE849305-Aug-2020

EJ055325-Sep-2020

EK1768

16-Oct 2020

EJ168505-Oct-2020

EJ168607-Oct-2020

EL0141

29-Oct-2020

EK4241

12-Nov-2020

DS
LNP
Fill/Finish
Manufacturing
Production
Site
Site
Site
Andover/Kalamazoo/KalamazooAndover
Kalamazoo
Kalamazoo(Line 8)
Andover
Kalamazoo
Kalamazoo(Line 8)
Andover
Kalamazoo
Kalamazoo(Line 18)
Andover
Kalamazoo
Kalamazoo(Line 18)
Andover
Kalamazoo
Kalamazoo(Line 18)
Andover
Kalamazoo
Kalamazoo(Line 8)
Andover/Polymun/Puurs
Andover
PolymunPuurs(WSL5)
Andover
PolymunPuurs(WSL5)
Andover
PolymunPuurs(WSL5)
Andover
PolymunPuurs(WSL5)
BioNTech;Rentschler/Polymun/Puurs
BNT;RNT
PolymunPuurs(WSL5)
BNT;RNT
PolymunPuurs(WSL5)
BNT;RNT
PolymunPuurs(WSL5)
BNT;RNT
PolymunPuurs(WSL5)PFIZER CONFIDENTIAL
Page 7

AnticipatedCoA
Availability

AnticipatedDistribution
Date

30-Nov-2020

14-Dec-2020

Authorization
Date
Authorization
Date
Authorization
Date
Dec 16-2020

15-Dec-2020

Dec 19-2020

29-Dec-2020

Dec 31-2020

Data provided

N/Aa

Data provided

N/Aa

Data provided

Authorization
Date
Authorization
Date

30-Nov-2020
30-Nov-2020

IVE Results
pending
Data provided
Data provided
7-Dec-2020
15-Dec-2020

Authorization
Date
Authorization
Date
Authorization
Date
Dec 19-2020

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 4.

Anticipated Distribution Dates

Lot

DOM

EL014029-Oct-2020

EL0142

29-Oct-2020

EK4237

5-Nov-2020

EK42445-Nov-2020

EK42435-Nov-2020

EK424512-Nov-2020

EL072530-Oct-2020

DS
LNP
Fill/Finish
Anticipated
Manufacturing
Production
Site
CoA
Site
Site
Availability
BioNTech;Rentschler/DermaPharm/Puurs
BNT;RNT
DermaPharm Puurs(WSL5)
7-Dec-2020
BNT;RNT
DermaPharm Puurs(WSL5)
7-Dec-2020
BNT;RNT
DermaPharm Puurs(WSL5)
7-Dec-2020
BNT;RNT
DermaPharm Puurs(WSL5)
7-Dec-2020
BNT;RNT
DermaPharm Puurs(WSL5)
14-Dec-2020
BNT;RNT
DermaPharm Puurs(WSL5)
16-Dec-2020
BioNTech;Rentschler/Puurs/Puurs
BNT;RNT
Puurs
Puurs (FC2) 7-Dec-2020

AnticipatedDistribution
Date
Authorization
Date
Authorization
Date
Authorization
Date
Authorization
Date
20-Dec-2020
30-Dec-2020

Authorization
Date
EL0739 03-Nov-2020
BNT;RNT
Puurs
Puurs (FC2) 7-Dec-2020
Authorization
Date
EL1484 04-Nov-2020
BNT;RNT
Puurs
Puurs (FC2) 7-Dec-2020
Authorization
Date
EJ679512-Nov-2020
Andover
Puurs
Puurs (FC2) 14-Dec-2020
30-Dec-2020
a. Not intended for distribution under Emergency Use in the United States due to differences in applied label
Abbreviations: BNT = BioNTech; RNT = Rentschler

Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
Page 8COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 5For lots with observable LMS, please describe the percent range of the LMS peak area detectedby CGE.
ANDQUERY 6
Please state the percentage of DP lots that have the LMS peak and provide a list of all impacted
DP lots, including information on the DP manufacturing site as well as the associated DS lots
and lipid lots/sources used for DP manufacture.
RESPONSE to 5 and 6
Table 5 is a list of drug product lots with results from capillary gel electrophoresis (CGE)
including the percent late migrating species. The RNA integrity assay reports the % timecorrected area of the main peak, with all other peaks (RNA fragments preceding main peak and
LMS RNA species trailing main peak) influencing the reported % RNA integrity value. Most
lots (14 out of 20) have some level of late migrating species reported.
The release specification for RNA integrity controls both RNA fragments and LMS since both ofthese species lead to lower RNA integrity. The release specification limit has been tightened to≥55% to ensure the integrity of RNA is maintained through the point of use. For lots that meet
release specification acceptance criteria, LMS ranged up to 16%. A representativeelectropherogram of a recent lot with 9% late migrating species is presented in Figure 1.
Figure 1. CGE Electropherogram of DP Lot EJ1685

PFIZER CONFIDENTIAL
Page 9

0

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 5.
LNP
Site

Lot Genealogy with RNA Integrity and LMS Levels
DP Lot
#

F/F
Site

LMS

RNA
Integrity
55%

ALC0315
Mfr
Avanti

ALC0315 Lot#
GALC0315-12

ALC0159
Mfr
Avanti

ALC0159 Lot#
GALC0159-12

PLY

EE8492

Puurs

< QLc

PLY

EE8493Puurs

KZO

EJ0701

DMP

< QLc

55%

Avanti

GALC0315-13

Avanti

GALC0159-12

Lipoid

KZO

17%

52%a

Croda

Avanti

Puurs

< QLc

71%

Avanti

PLY

EJ0553

Puurs

< QLc

68%

Avanti

ALC0159-105
ALC0159-105
GALC01
59-12

Avanti

EJ0724KZO

EH9899

KZO

< QLc

59%

Croda

EK1768

Puurs

< QLc

60%

Croda

ALC0159-105
ALC015
9-104

Avanti

PLY

DTP/465/1
GALC0315-14
GALC0315-12/
GALC03
15-13
DTP/465/3
1755889

PLY

EJ1685

Puurs

9%

66%

Croda

DTP/465/3

Avanti

GALC0159-12

Lipoid

PLY

EJ1686

Puurs

6%

69%

Croda

DTP/465/3

Avanti

LipoidDMP

EJ1688

Puurs

10%

63%

Croda

1755889

Avanti

PLY

EK4176

Puurs

10%

65%

Croda

1760275

Avanti

GALC0159-12/
ALC0159-104
ALC0159-105
ALC015
9-104

Avanti
Avanti

Avanti
AvantiPFIZER CONFIDENTIAL
Page 10

DSPC
Mfr

DSPC
Lot #

Cholesterol
Mfr

Cholesterol
Lot #Lipoid556500219039
5-01
556500219039
5-01
DSPCII
S-112
DSPCII
S-111
556500219039
5-01

Wilshireb

P60349Wilshireb

P90390AvantiSCHOLB105
SCHOLB105
P90390DSPCII
S-112
556500220042
1-01

AvantiWilshireb

SCHOLB105
P903905565002180372-01
5565002200421-01

Wilshireb

P90390Wilshireb

P90390AvantiSCHOLB105
P90390Avanti
LipoidLipoidAvanti
Lipoid5565002200421-01

DSPCII
S-111
556500220042

AvantiWilshireb

Wilshireb

1

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 5.

Lot Genealogy with RNA Integrity and LMS Levels

LNP
Site

DP Lot
#

F/F
Site

LMS

RNA
Integrity

ALC0315
Mfr

ALC0315 Lot#

ALC0159
Mfr

ALC0159 Lot
#

DSPC
Mfr

DMP

EK4175

Puurs

16%

58%

Croda

1760275

Avanti

AvantiDMP

EJ1691

Puurs

24%

51%a

Croda

1760275

AvantiKZO

EK5730

KZO

10%

62%

Croda

Avanti

DMP

EL0140

Puurs

6%

69%

Croda

DTP/465/3
1755889

PLY

EL0142

Puurs

6%

69%

Croda

1755889

Avanti

PLY

EL0141

Puurs

5%

67%

Croda

1755889

Avanti

ALC0159-106
ALC0159-106
ALC0159-105
ALC0159-106
ALC0159-106
ALC015
9-104

Puurs

EL0725

Puurs

9%

63%

Croda

Avanti

Avanti
Avanti
Avanti
Avanti
LipoidDTP/465 Avanti ALC015 Avanti
/3
9-106
DMP
EK4237 Puurs 9%64%
Croda 1755889
Avant ALC015 Avanti
and
i
9-107
1760275
Puurs
EL0739 Puurs 6%
67%
Croda
DTP/465 Avanti ALC015 Avanti/39-106
a. does not meet the tightened DP RNA Integrity release specification, not intended for emergency supply
b. Wilshire is now Evonik.
c. QL=quantitation limit (3%).

DSPC
Lot #
1-01
DSPCII
S-111
DSPCII
S-111
DSPCII
S-112
DSPCII
S-111
DSPCII
S-111
556500220042
1-01
DSPCII
S-112
DSPCII
S-111
DSPCII
S-112

Cholesterol
Mfr

Cholesterol
Lot #AvantiSCHOLB105
SCHOLB105
SCHOLB105
SCHOLS129SCHOLS129
P90390Avanti
Avanti
Avanti
AvantiWilshireb
Avanti
Avanti
AvantiSCHOLS129SCHOLS129SCHOLB105

QL= quantitation limit, DMP = Dermapharm, PLY = Polymun, KZO = Kalamazoo, LMS = late migrating species, LNP = lipid nanoparticle, F/F =fill/finish

PFIZER CONFIDENTIAL
Page 11

2

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 7
Your investigation regarding the identity of the LMS is based on the evaluation of one DPengineering lot (EG5411). Please provide similar data for other impacted DP lots. In addition,please provide data to support that the presence of LMS peak will have no impact on the stability
profile of DP.
RESPONSE 7
Characterization of additional DP lots are summarized below.
Table 6.

Characterization of LMS in DP Lots in addition to EG5411

Characterization Method
IP-RP-HPLC (Figure 2)

Lots (release LMS result)
EE8493 (< QL), EJ0724 (< QL),
00713968-0019-M01 (6%), EJ0701(17%)

Conclusion
Similar to EG5411, the same lateeluting peaks in IP-RP-HPLC areobserved at elevated levels in DP
lots with elevated LMS
Nucleoside and nucleotide analysis EE8493 (<QL), EJ0553 (<QL),
Similar to EG5411, for all tested
by LC/MS/MS (Figure 3, Table 3,
EJ0724 (<QL), 00713968-019-M01 lots with or without reportable
Table 4)(6%), EJ0701(17%), EH9978
levels of LMS, up to 35%, the(35%)
expected nucleosides andnucleobases are present, with nodetectable modifications greaterthan 0.01% (reportable limit),
besides the intended single 3′-Omethylated 7-methylguanosine and2′-O-methylated adenosine in the5′-Cap structure00713968-019-M01 is a small scale development lot. EH9978 is an early engineering lot. Other DP lots aredescribed in Table 1. QL= quantitation limit (3%)

In addition to the characterization data presented in P.2 on engineering lot EG5411, additional
data has been collected by the orthogonal ion-pairing RP-HPLC (IP-RP-HPLC) method
presented in P.2 on other representative lots (Figure 2). Similar to EG5411, the lots that havelate migrating species by CGE (EJ0701 and 00713968-0019-M01, a small scale development lot
with 6% late migrating species) also contain elevated levels of the same late-eluting peaks, ascompared to the lots without reported late migrating species (EE8493, EJ0724). As summarizedin P.2, the late eluting peaks from IP-RP-HPLC were characterized by UV multi-angle lightscattering (MALS) detection, denaturing agarose gel electrophoresis (AGE), and mass
spectrometry (MS). The late eluting peaks were characterized as RNA of the expected size that isconformationally folded or reversibly aggregated RNA and is not denatured in the sample
preparation of the CGE method.

PFIZER CONFIDENTIAL
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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Figure 2. Ion Pairing RP-HPLC of RNA Extracted from Drug Product Lots

DS 20Y513C201-RM is the DS reference material. EH9899 MOR is an in-process sample for the DP lot EH9899.
00713968-019-M01 is a small-scale development lot. Other DP lots are described in Table 1. Based on
characterization of the collected IP-RP-HPLC fractions, peak 1 contains fragments, peak 2 contains mostly intact
RNA, while peaks 3-5 are correlated with LMS.

In addition to analysis of additional lots by IP-RP-HPLC, nucleoside analysis by LC-UV with
online tandem mass spectrometry (MS/MS) was performed on additional lots that have elevatedlevels of late migrating species, EH9978 and EJ0701, for further confirmation that these lots
consist of RNA without detectable modification. Lot EH9978 is an early engineering lot (similar
to EG5411) with high levels of LMS (35%); EH9978 does not meet the RNA integrity
specification but was used for characterization purposes. Nucleoside analysis involved digestionof DS control and extracted RNA from DP lots with nuclease P1 and venom phosphodiesterase
at low and high pH, respectively, and then alkaline phosphatase to remove the phosphate group.
The resulting nucleosides were separated by reversed-phase ultrahigh performance liquid
chromatography with UV detection at A260 nm using an extended gradient in case of potential
modified residues (Figure 3). Each nucleoside displays an elution position and well resolved
peak that is consistent between all DP lots and the DS control. The observed accuratemonoisotopic masses for each respective peak were consistent with the theoretical masses of thefour expected nucleosides from BNT162b2 mRNA (Table 7). The corresponding observed
masses of the predominant fragment ions representing the four nucleobases also agreed with thetheoretical masses indicating no base modifications (Table 8). These data confirm that like
EG5411, a lot with higher levels of late migrating species, that the expected nucleosides andnucleobases are present, with no detectable modifications greater than 0.01% (reportable limit),
besides the intended single 3′-O-methylated 7-methylguanosine and 2′-O-methylated adenosinein the 5′-Cap structure (the relative abundances of 2′-O-methylated adenosine in 5′-Cap structure
range from 0.24 to 0.42% in the DP lots, similar to DS at 0.37%).
PFIZER CONFIDENTIAL
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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Figure 3. Analysis of Nucleosides in Additional BNT162b2 DP Lots by LC-UV-MS/MS(A260 nm)

Extraction Blank

AU

0.02
0.00

C

AU

0.20

AG

V

20Y513C201_DS RM

AU

0.00

EE8493

0.10

AU

0.00

EJ0553

0.10

AU

0.00

EJ0724

0.10

AU

0.00

EH9899 MOR

0.10

AU

0.00

00713968-0019_MO1

0.10

AU

0.00

EJ0701

0.10

AU

AU

0.00

EG54110.15

EH9978

0.10
0.00
0.00

2.00

4.00

6.00

8.00

10.00
Minutes

12.00

14.00

16.00

18.00

20.00

BNT162b2 nucleosides: cytidine (C); N1-methylpseudouridine (V); adenosine (A); guanosine (G)PFIZER CONFIDENTIAL
Page 14

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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 7.

Accurate Mass Assignments of Nucleosides for Additional BNT162b2 DP Lots
via LC-UV/MS

NucleosideDS
Reference
Theoreti Material
cal Mass

DP Lots

Engineering Run
DP Lots

00713968-0019_ EJ0701 EG5411 EH9978
MO1
244.0928 244.0925 244.0926 244.0926 244.0926 244.0927 244.0926 244.0925 244.0925 244.0925
20Y513C20
EE8493 EJ0553
1

EJ0724

EH9899MOR

Cytidine (C)N1methylpseudouridine 259.0925 259.0922 259.0922 259.0921 259.0922 259.0922 259.0923 259.0923 259.0922 259.0923(V)
Adenosine (A)268.1040 268.1036 268.1036 268.1036 268.1036 268.1037 268.1036 268.1037 268.1037 268.1036
Guanosine (G)284.0989 284.0988 284.0986 284.0988 284.0987 284.0988 284.0988 284.0988 284.0988 284.0988
Observed masses (monoisotopic) agree with theoretical masses to within 5 ppm, which is consistent with the accuracy andprecision of contemporary mass spectrometers

Table 8.

Accurate Mass Assignments of Nucleobases for Additional BNT162b2 DP Lots
via LC-UV-MS/MSDS
Reference(derived from Theoreti
Material
respective
cal Mass
diagnostic fragment
Nucleobase

ions)

DP Lots

Engineering Run
DP Lots

00713968-0019_ EJ0701 EG5411 EH9978
MO1
112.0511 112.0505 112.0502 112.0503 112.0502 112.0502 112.0503 112.0504 112.0503 112.0504
20Y513C20
EE8493 EJ0553
1

EJ0724

EH9899MOR

Cytosine (C)N1methylpseudouridine 139.0508 139.0500 139.0499 139.0501 139.0501 139.0500 139.0500 139.0500 139.0500 139.0501(V)
Adenine (A)136.0623 136.0617 136.0615 136.0615 136.0617 136.0618 136.0615 136.0616 136.0616 136.0616
Guanine (G)152.0572 152.0564 152.0564 152.0564 152.0564 152.0564 152.0565 152.0565 152.0565 152.0566
Observed masses (monoisotopic) agree with theoretical masses to within 10 ppm, which is consistent with the accuracy andprecision of MS/MS in contemporary mass spectrometers

Taken together, these data confirm that the late migrating species observed in additional lots isthe same as the EG5411 late migrating species used for detailed characterization, and the
additional MS characterization of enzymatically digested RNA confirm the presence of expectednucleosides in additional lots with LMS. The LMS has been characterized as conformationallyfolded or reversibly aggregated RNA that is not denatured in the CGE method. Lots that arewithin the RNA integrity specification have comparable in vitro expression as previously shownin P.2, Enhanced Characterization. The CGE method controls both LMS and fragments with a
specification limit on the intact RNA species (RNA integrity).

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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

The vaccine drug product is stored at a temperature of -70 °C. Emergency supply DP lots are
currently or will be enrolled in formal stability studies (Table 9) . Formal results of thesestudies will be provided as they become available.
Table 9.
Study
Condition

Representative Stability Study Design
Storage
Temperature

Time Points

Long Term-90 to -60 C

0, 1M, 3M, 6M, 9M, 12M, 18M, 24M

Accelerated-60 to -30 C

0, 1M, 3M, 6M, 9M, 12M, 18M, 24M

Accelerated-20  5 C

0, 1M, 3M, 6M, 9M, 12M, 18M, 24M

Accelerated

5  3 C

0, 1M, 3M, 6M

Thermal Stress25  2 C / 60 
5 % RH0, 1W, 2W, 1M

Analytical Procedures













Appearance
Potentiometry
Dynamic Light Scattering(LNP Size Polydispersity)
Fluorescence Assay (RNA
Encapsulation, Content)
HPLC-CAD (Lipid Content)
Cell-based Flow Cytometry(In vitro expression)
Capillary Gel Electrophoresis(RNA Integrity)
Subvisible Particles
Container Closure Integrity
Test
Endotoxin
Sterility

Current stability data that provide insight on impact of LMS on stability are available from
dilution and administration (DAI) simulation studies. Two BNT162b2 DP lots that contain9-10 % LMS at release (EJ1685 and EJ1688) were evaluated to demonstrate the product has
an acceptable RNA integrity at the time of dosing. The DP lots were removed from -70 °C
storage, thawed and held in refrigerated storage (2-8 °C) conditions for at least 5 days plus anadditional 2 hours at elevated ambient temperatures (30 °C/75% RH). The DP was thendiluted to 0.1 mg/mL with normal saline in the vial and held in contact with either the dosing
needles and syringes or in the vial for 6 hours at elevated ambient temperatures (30 °C/75%
RH). These conditions cover the maximum allowable storage and handling conditions at the
point of use. In addition, the diluted DP was held for 24 hours at elevated ambient
temperatures (30 °C/75% RH) in a vial to demonstrate the DP meets the specification beyond
the allowable conditions.
RNA integrity was acceptable through the allowed administration time period. The RNA
integrity data are presented in Table 10.
Additional data on complete DAI study results from two other lots are provided below(Table 11 and Table 12). Similar stability behavior of the RNA integrity level was observed,
all results met acceptance criteria and showed acceptable product quality.

PFIZER CONFIDENTIAL
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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

In conclusion, the stability profile of the drug product with LMS has been evaluated toconfirm acceptable storage and handling through the vaccine point of use, and will continue
to be monitored in stability studies. The RNA integrity attribute is a stability indicating assay
including for lots that contain LMS, and all lots showed similar stability behavior. The
specification for RNA integrity controls both fragment and LMS since both species would
lead to lower RNA integrity level. Vials that were thawed for 5 days followed by dilution insaline and a hold at room temperature for up to 6 hours were acceptable; the additional holdof a diluted vial in saline up to 24 hours also met specification including in vitro expression.

PFIZER CONFIDENTIAL
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8

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 10. RNA Integrity Test Results for the Study Performed with Lots# EJ1685a and EJ1688a (0.1 mg/mL in 0.9% Sodium Chloride)
Test

AcceptanceCriteria

T0 (5 days 2-8°C
and 2 hours at 30
°C)
Vial

30 °C/75% RH
3 HoursVial

3 Hours
Polycarbonate
Syringe

6 HoursVial

6 Hours
Polycarbonate
Syringe

24 Hours
Vial

Lot # EJ1685
RNA Integrity

Fragment
Analyzer(CGE)

T0 Vial ± 20%
64.8%(≥ 50% Intact
RNA)
Report LMS result 9.7%

64.7%

64.9%

65.1%

64.0%

63.1%

9.8%

9.8%

10.1%

10.0%

10.9%

T0 Vial ± 20%
61.4%(≥ 50% Intact
RNA)
Report LMS result 11.0%

59.5%

59.8%

58.4%

58.2%

55.8%

11.2%

11.9%

12.7%

13.0%

14.4%

Lot # EJ1688
RNA Integrity

Fragment
Analyzer(CGE)

a. All vials used in this study were held for ≥5 days at 2-8 °C plus 2 hours at 30 °C prior to dilution
Abbreviations: CGE = capillary gel electrophoresis; LNP = lipid nanoparticle

PFIZER CONFIDENTIAL
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9

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 11. Analytical Test Results for the Study Performed with Lot# EH9899a (0.1 mg/mL in 0.9% Sodium Chloride)
Test

AcceptanceCriteria

T0 (5 days 2-8°C
and 2 hours at 30
°C)
Vial

30 °C/75% RH
3 Hours

Off-whitesuspension
EFVP

RiboGreen
Fragment
Analyzer(CGE)

White to off-white Off-whitesuspension
suspension
Essentially Free of EFVP
Visible Particles(EFVP)
T0 Vial ± 20%
0.12 mg/mL
T0 Vial ± 20%
55.4%(≥50% Intact RNA)
Report LMS result 10.0%

3 Hours
6 Hours
Polycarbonate
Vial
Syringe
Off-white
Off-whitesuspension
suspension
EFVP
EFVP

0.12 mg/mL
54.2%

0.12 mg/mL
54.9%0.11 mg/mL
53.8%0.12 mg/mL
53.3%

0.12 mg/mL
50.2%

11.5%

10.9%

11.4%

11.7%

14.6%

RiboGreen

≥ 80%

94%

94%

94%

95%

95%

70 nm

69 nm

69 nm

0.19

0.18

0.17

79%

77%

69%

Vial
Appearance

VisualParticulate
Matter

RNA Content
RNA Integrity

RNA
Encapsulation
LNP Size95%

Dynamic Light 40 to 180 nm
67 nm
67 nm
70 nm
Scattering
LNP
Dynamic Light ≤ 0.3
0.16
0.16
0.19
Polydispersity Scattering
In-Vitro
Cell-based Flow ≥ 30% positive
86%
76%
69%
Expression
Cytometrycells
a. All vials used in this study were held for ≥5 days at 2-8 °C plus 2 hours at 30 °C prior to dilution
Abbreviations: CGE = capillary gel electrophoresis; LNP = lipid nanoparticle

PFIZER CONFIDENTIAL
Page 19

6 Hours
24 Hours
Polycarbonate
Vial
Syringe
Off-white
Off-whitesuspension
suspension
EFVP
EFVP

0

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 12. Analytical Test Results for the Study Performed with Lot# EJ0553a (0.1 mg/mL in 0.9% Sodium Chloride)
Test

AcceptanceCriteria

T0 (5 days 2-8°C
and 2 hours at 30
°C)
Vial

30 °C/75% RH
3 Hours

White to off-white Off-whitesuspension
suspension
Essentially Free of EFVP
Visible Particles(EFVP)
T0 Vial ± 20%
0.10 mg/mL

Off-whitesuspension
EFVP

3 Hours
6 Hours
Polycarbonate
Vial
Syringe
Off-white
Off-whitesuspension
suspension
EFVP
EFVP

0.11 mg/mL

0.10 mg/mL

0.10 mg/mL

0.11 mg/mL

0.11 mg/mL

T0 Vial ± 20%
67.2%(≥ 50% Intact
RNA)
Report LMS result 2.5%

67%

66.3%

66.1%

65.6%

64.2%

2.3%

2.7%

2.6%

2.4%

2.8%

≥ 80%

92%

92%

92%

94%

93%

70 nm

68 nm

76 nm

0.17

0.15

0.19

74%

71%

82%

Vial
Appearance

VisualParticulate
Matter

RNA Content

RiboGreen

RNA Integrity

Fragment
Analyzer(CGE)

RNA
Encapsulation
LNP Size

RiboGreen

93%

Dynamic Light 40 to 180 nm
69 nm
69 nm
74 nm
Scattering
LNP
Dynamic Light ≤ 0.3
0.18
0.17
0.18
Polydispersity Scattering
In-Vitro
Cell-based Flow ≥ 30% positive
79%
63%
75%
Expression
Cytometrycells
a. All vials used in this study were held for ≥5 days at 2-8 °C plus 2 hours at 30 °C prior to dilution
Abbreviations: CGE = capillary gel electrophoresis; LNP = lipid nanoparticle

PFIZER CONFIDENTIAL
Page 20

6 Hours
24 Hours
Polycarbonate
Vial
Syringe
Off-white
Off-whitesuspension
suspension
EFVP
EFVP

1

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryREGARDING THE INTRINSIC PARTICLES OBSERVED DURING VISUAL
INSPECTION, PLEASE PROVIDE THE FOLLOWING INFORMATION:QUERY 8
Please clarify the percentage of DP lots that contain the intrinsic visible particles and whether
particles occurred only in the most recent DP lots. Please comment on the possible
contributing factors that may lead to the formation of these particles.
RESPONSE 8
Table 13 (see Query 9) presents information for drug product lots and includes the inspection
method and the percentage of vials that were rejected due to particles during inspection. As
can be seen, in 29 out of 33 lots filled and inspected at the Pfizer Puurs and Pfizer
Kalamazoo sites including early lots produced, a small number of vials were detected duringvisual inspection and rejected from the lot due to particles (min 0.01% - max 3.68%). In
Pfizer’s experience with suspension vaccine products, these rejection rates are notuncommon. The particles specifically from EJ0553, EG5411 (bulk DP lot EG5477, Pfizer
Puurs) and EH9899 (bulk DP lot EH9783 Pfizer Kalamazoo) were characterized andidentified as product related and most likely comprised of lipids and cholesterol components,
as assessed by FTIR spectra. When a vial with particles was diluted with 1.8 mL of sterile
0.9% sodium chloride, Inj., mixed and the resulting solution drawn into a syringe, theparticles appeared to disperse.
No specific factors have been identified as definitively correlating with nor directly
contributing to the formation of these intrinsic particles. Particles have been observed to avarying degree across many lots spanning multiple manufacturing sites, including four sitesof LNP production and two fill finish sites, as listed in Table 13 and across different lipid
sources (vendors and batches) as listed in Table 14. Particles have been observed in thesterile holding vessel post-sterile filtration and before aseptic filling. Particles are light indensity and have a tendency to float. Because sterile bulk drug product flows from thebottom of the vessel to the filling line, floating particles tend to be observed more frequently
in vials filled towards the end of the filling process.
Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
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2

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 9For all lots that contain the visible intrinsic particles, please submit data on the percentage ofvials that contain the intrinsic particles (for example, through data obtained from the 100%
automated/manual inspection).
RESPONSE 9Previously submitted Section P.2 Pharmaceutical Development, Table P.2-17 Drug Product
Lot Genealogy and Usage is amended below as Table 13 with the inspection method (100%manual or 100% automated) and the percentage of vials that were rejected due to particles
during inspection. Information is presented for lots where fill finish was conducted at either
Pfizer Puurs or Pfizer Kalamazoo which are the fill finish sites intended for manufacture of
emergency supply and includes lots that are not intended for emergency supply (but are
manufactured at the same sites using the essentially the same process). Vials that are rejected
due to particles during 100% visual inspection are not reintroduced into the batch and are
discarded. The acceptance quality limit (AQL) sampling procedure assesses the robustness of
the inspection method, as a statistically determined number of vials that passed the inspection
method are reinspected manually. The appearance method and label reflect that after dilution,in the rare case particles are observed prior to administration, the product should not be
administered.

PFIZER CONFIDENTIAL
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3

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 13. Lot Genealogy and Vials Rejected During Visual Inspection for Particles
DP LotNumber(DP Name)

Fill
Volume(mL)

CTM10.4
BCV40720-P/
ED3938
CTM11
BCV40820-P/
EE3813
CTM12
BCV4/L05
EE8492
CTM13
BCV4/L06
EE8493
CTM14
BCV4/L07
EJ05530.45

BCV4/L08
EJ16850.45

BCV4/L09
EJ1686(BNT162b2)
BCV4/L10
EK1768(BNT162b2)

0.2
0.2

0.45
0.45

0.45
0.45

Drug Product (DP)LNP SiteFill/Finish(Process)
Site
PolymunScientific(Classical)
PolymunScientific(Classical)
PolymunScientific(Upscale)
PolymunScientific(Upscale)
PolymunScientific(Upscale)
PolymunScientific(Upscale)
PolymunScientific(Upscale)
PolymunScientific(Upscale)

Inspection
method% vials rejectedduring inspection
due to particles

Date of
Manufacture

Manufacturing
Scale (vials)

Drug Substance
Site of
Manufacture

Pfizer Puurs(S2F2)

16 Jul 2020

19,010

BioNTech

100% Manual
Visual Inspection

0.06%

Pfizer Puurs(S2F2)

29 Jul 2020

30,193

BioNTech

100% Manual
Visual Inspection

0.02%

Pfizer Puurs(WSL5)

05 Aug 2020

67,665

Pfizer Andover

100% automatedinspection at IL7

0.41%

Pfizer Puurs(WSL5)

05 Aug 2020

68,445

Pfizer Andover

100% automatedinspection at IL7

0.06%

Pfizer Puurs(WSL5)

25 Sep 2020

164,580

Pfizer Andover

100% automatedinspection at IL7

0.34%

Pfizer Puurs(WSL5)

05 Oct 2020

159,315

BioNTech;Rentschler

100% automatedinspection at IL7

0.16%

Pfizer Puurs(WSL5)

07 Oct 2020

147,615

BioNTech;Rentschler

100% automatedinspection at IL7

0.04%

Pfizer Puurs(WSL5)

16 Oct 2020

141,960

Pfizer Andover

100% automatedinspection at IL7

0.30%

EG5411

0.45

Pfizer Puurs

Pfizer Puurs(FC2)

3 Sep 2020

201,258

Pfizer Andover

100% automatedinspection at
Innoscan

0.29%

EJ0701

0.45

PfizerKalamazoo

PfizerKalamazoo(Line 18)

26 Sep 2020

200,265

Pfizer Andover

100% Manual
Visual Inspection

0%

EH99780.45

Pfizer Puurs

Pfizer Puurs(FC2)

23 Sept 2020

304,869

Pfizer Andover

100% automatedinspection at
Innoscan

1.30%

PFIZER CONFIDENTIAL
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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 13. Lot Genealogy and Vials Rejected During Visual Inspection for Particles
Drug Product (DP)LNP SiteFill/Finish(Process)
Site

DP LotNumber(DP Name)

Fill
Volume(mL)

EJ07240.45

Dermapharm

EH98990.45

PfizerKalamazoo

EJ16880.45

Dermapharm

EK4176

0.45

Polymun

EK41750.45

Dermapharm

EJ16910.45

Dermapharm

EK2808

0.45

Puurs

EK5730

0.45

PfizerKalamazoo

EL01400.45

Dermapharm

EL01420.45

Dermapharm

EL0141

0.45

Polymun

EL0725

0.45

Puurs

EK9231

0.45

PfizerKalamazoo

EK42370.45

Dermapharm

0.45

Puurs

EL0739Pfizer Puurs(WSL5)
PfizerKalamazoo(Line 8)
Pfizer Puurs(WSL5)
Pfizer Puurs(WSL5)
Pfizer Puurs(WSL5)
Pfizer Puurs(WSL5)
Pfizer Puurs(VC2)
PfizerKalamazoo(Line 8)
Pfizer Puurs(WSL5)
Pfizer Puurs(WSL5)
Pfizer Puurs(WSL5)
Pfizer Puurs(FC2)
PfizerKalamazoo(Line 18)
Pfizer Puurs(WSL5)
Pfizer Puurs(FC2)

Date of
Manufacture

Manufacturing
Scale (vials)

Drug Substance
Site of
Manufacture

29 Sep 2020

39,195

BioNTech;Rentschler

7 Oct 2020

179,400

Pfizer Andover

12 Oct 2020
16 Oct 2020
12 Oct 2020
16 Oct 2020

150.345131.625145.275
133.575

19 Oct 2020
22 Oct 2020
29 Oct 2020
29 Oct 2020

BioNTech;Rentschler
BioNTech;Rentschler
BioNTech;Rentschler
BioNTechRentschler

48,945

BioNTechRentschler

191,295

Pfizer Andover

155.610138.060

BioNTech;Rentschler
BioNTech;Rentschler
BioNTech;Rentschler

29 Oct 2020

156.195

30 Oct 2020

272,073

BioNTech;Rentschler

4 Nov 2020

230,685

Pfizer Andover

5 Nov 2020

140.985

BioNTech;Rentschler

3 Nov 2020

294,239

BioNTech;Rentschler

PFIZER CONFIDENTIAL
Page 24

Inspection
method% vials rejectedduring inspection
due to particles

100% automatedinspection at IL7
100% automatedinspection

0.01%

100% automatedinspection at IL7
100% automatedinspection at IL7
100% automatedinspection at IL7
100% automatedinspection at IL7
100% automatedinspection at
Innoscan
100% automatedinspection

0.06%

100% automatedinspection at IL7
100% automatedinspection at IL7
100% automatedinspection at IL7
100% automatedinspection at
Innoscan

0.52%

100% automatedinspection
100% automatedinspection at IL7
100% automatedinspection at
Innoscan

0.01%

0.03%
0.09%
1.17%
0.57%
0.09%

0.81%
0.15%
1.20%
0.02%
3.68%
1.34%

5

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 13. Lot Genealogy and Vials Rejected During Visual Inspection for Particles
Drug Product (DP)LNP SiteFill/Finish(Process)
Site

DP LotNumber(DP Name)

Fill
Volume(mL)

EL1484

0.45

Puurs

EL1283

0.45

PfizerKalamazoo

EL1284

0.45

PfizerKalamazoo

EL3246

0.45

PfizerKalamazoo

EJ6795

0.45

Puurs

PfizerKalamazoo(Line 18)
PfizerKalamazoo(Line 18)
PfizerKalamazoo(Line 8)
Pfizer Puurs(FC2)

EJ6796

0.45

Puurs

Pfizer Puurs(FC2)

EJ6797

0.45

Puurs

Pfizer Puurs(FC2)

Pfizer Puurs(FC2)

Inspection
method% vials rejectedduring inspection
due to particles

Date of
Manufacture

Manufacturing
Scale (vials)

Drug Substance
Site of
Manufacture

4 Nov 2020

277,608

BioNTech;Rentschler

100% automatedinspection at
Innoscan

0.58%

11 Nov 2020

245,895

Pfizer Andover

100% automatedinspection

0%

17 Nov 2020

214,305

Pfizer Andover

100% automatedinspection

0%

19 Nov 2020

204,360

Pfizer Andover

100% automatedinspection

0%

12 Nov 2020

282.645

Pfizer Andover

13 Nov 2020

293,828

Pfizer Andover

17 Nov 2020

293.526

Pfizer Andover

PFIZER CONFIDENTIAL
Page 25

100% automatedinspection at
Innoscan
100% automatedinspection at
Innoscan
100% automatedinspection at
Innoscan

0.89%
0.58%
0.48%

6

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Literature References
None
SUPPORTING DOCUMENTATION
None
Previously submitted supporting documentation
Section P.2 Pharmaceutical Development (modRNA), SN0137

PFIZER CONFIDENTIAL
Page 26

7

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 10
Please identify the source of the lipids used for the impacted DP lots.
RESPONSE 10
Previously submitted Section P.2 Pharmaceutical Development, Table P.2-18. Lipids Used in
BNT162b2 Drug Product Manufacture is amended below as Table 14 with the inspection
method and the percentage of vials that were rejected due to particles during inspection(same data as in Table 13). It now lists for each DP lot, the lipid lots used during
manufacturing, the related inspection method and percentage of vials rejected due toparticles. No correlation is observed between the frequency of vials with particles and thelipid lots used.

PFIZER CONFIDENTIAL
Page 27

8

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 14. Lipids Used in BNT162b2 Drug Product Manufacturea
ALC-0315
Drug
Product
Lot
BCV40720P /ED3938
BCV40820P /EE3813
BCV4/L05/EE8492
BCV4/L06/EE8493
BCV4/L07/EJ0553

Manufacturer

Lot
NumberAvanti

GALC031510
GALC031511

Avanti

GALC031512

Avanti

GALC031512

Avanti

GALC031513

Avanti

GALC031512/
GALC031513

ALC-0159
Manufacturer

DSPC

Lot
NumberAvanti

GALC015911

Avanti

GALC015911

Avanti

GALC015912

Avanti

GALC015912

Avanti

GALC015912

Manufacturer

Lot
NumberManufacturer

Lipoid556500219038801b

Lipoid556500219039501

Lipoid556500219039501

Lipoid556500219039501

Lipoid556500219039501

Wilshire

P90390Wilshire

P90390BCV4/L08/EJ1685

BCV4/L09/EJ1686

Cholesterol

Croda

DTP/465/3

Avanti

GALC015912

Lipoid556500218037201
556500220042101

Croda

DTP/465/3

Avanti

GALC015912/ALC0159104

Lipoid556500220042101

PFIZER CONFIDENTIAL
Page 28

Wilshire

Wilshire

Wilshire

Wilshire

Wilshire

Lot
Number

P60349P60349P60349P90390

P90390Inspection
method% vials
rejected
duringinspection
due toparticles100%
ManualVisualInspection
100%
ManualVisualInspection
100%
automatedinspection
at IL7100%
automatedinspection
at IL7100%
automatedinspection
at IL7100%
automatedinspection
at IL70.06%

100%
automatedinspection
at IL7

0.04%

0.02%

0.41%

0.06%

0.34%

0.16%

9

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 14. Lipids Used in BNT162b2 Drug Product Manufacturea
Drug
BCV4/L10/EK1768

ALC-0315

ALC-0159

DSPC

Cholesterol

Croda

1755889

Avanti

ALC0159104

Lipoid556500220042101

Wilshire

P90390EG5411

Croda

DTP/465/1

Avanti

GALC015912

AvantiDSPCIIS111

AvantiSCHOLB105

EJ0701

Croda

DTP/465/1

Avanti

ALC0159105

AvantiDSPCIIS112

AvantiSCHOLB105

EH9978Croda

DTP/465/1

Avanti

GALC015912

AvantiDSPCIIS111

AvantiSCHOLB105

EJ0724

Avanti

GALC031514

Avanti

ALC0159105

AvantiDSPCIIS111

AvantiSCHOLB105

EH9899Croda

DTP/465/3

Avanti

ALC0159105

AvantiDSPCIIS112

AvantiSCHOLB105

Avanti

ALC0159105

AvantiDSPCIIS111

AvantiSCHOLB105

Avanti

ALC0159104

Lipoid556500220042101

Avanti

ALC0159106

AvantiDSPCIIS111

Avanti

ALC0159106

AvantiDSPCIIS111

EJ1688

EK4176

EK4175

EJ1691

Croda

Croda

Croda

Croda

1755889

1760275

1760275

1760275

PFIZER CONFIDENTIAL
Page 29

Wilshire

P90390AvantiSCHOLB105

AvantiSCHOLB105

Inspection% vials

100%
automatedinspection
at IL7100%
automatedinspection
at Innoscan
100%
ManualVisualInspection
100%
automatedinspection
at Innoscan
100%
automatedinspection
at IL7100%
automatedinspection
100%
automatedinspection
at IL7100%
automatedinspection
at IL7100%
automatedinspection
at IL7100%
automatedinspection
at IL7

0.30%

0.29%

0%

1.30%
0.01%

0.01%
0.06%

0.03%

0.09%

1.17%

0

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 14. Lipids Used in BNT162b2 Drug Product Manufacturea
Drug

ALC-0315

ALC-0159

DSPC

Cholesterol

EK2808Croda

1760275

Avanti

ALC0159104

AvantiDSPCIIS111

AvantiSCHOLB105

EK5730

Croda

DTP/465/3

Avanti

ALC0159105

AvantiDSPCIIS112

AvantiSCHOLB105

AvantiDSPCIIS111

AvantiSCHOLS129

EL0140Croda

1755889

Avanti

ALC0159106

EL0142

Croda

1755889

Avanti

ALC0159106

AvantiDSPCIIS111

AvantiSCHOLS129

EL0141

Croda

1755889

Avanti

ALC0159104

Lipoid556500220042101

Wilshire

P90390EL0725

Croda

DTP/465/3

Avanti

ALC0159106

AvantiDSPCIIS112

AvantiSCHOLS129

EK9231

Croda

DTP/465/3

Avanti

ALC0159105

AvantiDSPCIIS112

AvantiSCHOLB105

EK4237

Croda

1755889,
1760275

Avanti

ALC0159107

AvantiDSPCIIS111

AvantiSCHOLS129

EL0739Croda

DTP/465/3

Avanti

ALC0159106

AvantiDSPCIIS112

AvantiSCHOLB105

EL1484

Croda

DTP/465/3

Avanti

ALC0159106

AvantiDSPCIIS112

AvantiSCHOLB105

PFIZER CONFIDENTIAL
Page 30

Inspection% vials

100%
automatedinspection
at Innoscan
100%
automatedinspection
100%
automatedinspection
at IL7100%
automatedinspection
at IL7100%
automatedinspection
at IL7100%
automatedinspection
at Innoscan
100%
automatedinspection
100%
automatedinspection
at IL7100%
automatedinspection
at Innoscan
100%
automatedinspection
at Innoscan

0.57%

0.09%
0.52%

0.81%

0.15%

1.20%

0.02%

3.68%

1.34%

0.58%

1

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 14. Lipids Used in BNT162b2 Drug Product Manufacturea
Drug

ALC-0315

ALC-0159

DSPC

Cholesterol

EL1283

Croda

DTP/465/3

Avanti

ALC0159105

AvantiDSPCIIS112

AvantiSCHOLB105

EL1284

Croda

1760275

Avanti

ALC0159105

AvantiDSPCIIS112

AvantiSCHOLB105

EL3246

Croda

1760275

Avanti

ALC0159105

AvantiDSPCIIS112

AvantiSCHOLS129

EJ6795

EJ6796

EJ6797

a.
b.

Croda

1760275

Croda

1760275,
DTP/465/3

Croda

0001781853

Avanti

ALC0159104

AvantiDSPCIIS112

AvantiSCHOLB105

Avanti

ALC0159104

AvantiDSPCIIS112

AvantiSCHOLB105

Avanti

ALC0159106

AvantiDSPCIIS111

AvantiSCHOLS129

Only lipids used for BNT162b2 manufacture are listed as BNT162b1 is no longer under development.
Wilshire is now Evonik.

PFIZER CONFIDENTIAL
Page 31

Inspection
100%
automatedinspection
100%
automatedinspection
100%
automatedinspection
100%
automatedinspection
at Innoscan
100%
automatedinspection
at Innoscan
100%
automatedinspection
at Innoscan% vials
0%
0%
0%
0.89%

0.58%

0.48%

2

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Literature References
None
SUPPORTING DOCUMENTATION
New or Replaced Supporting Documentation
None
Previously submitted supporting documentation
Section P.2 Pharmaceutical Development (modRNA), SN0137

PFIZER CONFIDENTIAL
Page 32

3

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 11
For DP lots with visible particles, please provide batch analysis data and stability data to support
that there is no impact on DP quality.
RESPONSE 11
As described in the responses to Query 9 and Query 10, visible particles have been observed
during 100% inspection in a small number of vials for nearly all DP lots. Therefore, the drug
product batch analysis data and stability data previously submitted to the IND are inclusive oflots with visible particles. Future emergency supply DP lots will also be enrolled in formal
stability studies and results of all studies will be provided as they become available.
Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
Page 33

4

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryREGARDING THE DS MANUFACTURING PROCESS:QUERY 12At BNT, there will be a hold/transportation step at 2-8°C (from Mainz to Rentschler). However,the duration of this step (“≥ 96”) is not accurate. Please specify the duration of this step.
RESPONSE 12
We acknowledge this inconsistency in the previous submission. The hold/transportation time of
the Proteinase K Pool is ≤ 96 h. The corresponding Table S.2.2.-4 in Section 3.2.S.2.2
Description of Manufacturing Process and Process Controls (modRNA) [BNT Mainz andRentschler] has been corrected.
Literature References
None
SUPPORTING DOCUMENTATION
New or Replaced Supporting Documentation
3.2.S.2.2 Description of Manufacturing Process and Process Controls (modRNA) [BNT Mainz
and Rentschler], Replaced
Previously submitted supporting documentation
None

PFIZER CONFIDENTIAL
Page 34

5

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 13
Regarding the DS manufacture at Pfizer, Andover, please specify the conditions and length ofhold time for the filtered DS before being dispensed into EVA containers, if applicable.
RESPONSE 13
The UFDF pool undergoes a bulk final 0.45/0.2 µm filtration into a flexible container. The
filtered DS is then mixed, sampled and dispensed into EVA containers. The whole process frombulk filtration to end of dispense into EVA containers takes less than 24 hours as described in
Section 3.2.S.2.5 Process Validation and/or Evaluation - Hold Times [Andover]. This isconsidered continuous processing and not evaluated as hold time.
Literature References
None
SUPPORTING DOCUMENTATION
New or Replaced Supporting Documentation
None
Previously submitted supporting documentation
Section S.2.5 Process Validation and/or Evaluation - Hold Times (modRNA) [Andover],SN0137PFIZER CONFIDENTIAL
Page 35

6

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 14
Please describe the target concentrations (both in terms of U/mL and mg/mL) for the active
proteins (DNase I, proteinase K, pyrophosphatase, RNase inhibitor, and T7 polymerase) used in
the In Vitro Transcription, DNase I Digestion, and Proteinase K Digestion steps.
RESPONSE 14
In Table 15 below, the vendor specifications are provided, as well as the target concentration in
the reaction for the active proteins used in the In Vitro Transcription, DNase I Digestion andProteinase K digestion steps. The vendor formulates the active proteins in units (U) per volume.
The concentration in mg/mL is provided by the vendor as report results in CoA for a subset ofthese enzymes and has not been used to set target concentration for the active proteins. Volumes
for the active proteins that are defined in the filing were determined using the target
concentration and the target value in the vendor specification.
Below, the vendor specifications are provided, as well as the target concentration in the reaction
for the active proteins used in the In Vitro Transcription, DNase I Digestion and Proteinase K
digestion steps. The vendor formulates the active proteins in units (U) per volume. The
concentration in mg/mL is provided by the vendor as report results in CoA for a subset of these
enzymes and has not been used to set target concentration for the active proteins. Target
concentrations for the active proteins are defined based on the vendor specifications for activity.
Table 15. Active Proteins Vendor Specification and Target Concentration During In Vitro
Transcription, DNase I Digestion and Proteinase K Steps
Raw material

Vendor Specification(U/µL solution)

Target Concentration(U/ L starting IVT volume)

RNase inhibitor

40 ± 650,000Pyrophosphatase0.10 ± 0.02

100

T7 polymerase

200 ± 21

16,000,000

DNase I

50 ± 9400,000

Proteinase K≥ 0.600

676

Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
Page 36

7

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 15
Please provide a description of the process controls implemented during the sanitization,equilibration, and reuse procedure for the ultrafiltration/diafiltration (UFDF) membrane.
RESPONSE 15
The Pfizer, Andover and Rentschler, Laupheim membrane sanitization, equilibration, and reuseprocedures are provided below. Process controls described in this section are included in amembrane lifetime protocol that will be provided for the BLA submission.
Pfizer, Andover
Sanitization
For sanitization, an initial WFI flush is performed followed by a 0.5 M NaOH solution flush andrecirculation for 60 min through the membrane. Following this sanitization, a subsequent WFIflush is performed prior to determining the normalized water permeability (NWP). The
membranes are then stored in 0.1 N NaOH.
Equilibration
Prior to UFDF execution, a WFI flush is performed to remove the 0.1 N NaOH storage solution.
Following the WFI flush, the membranes are equilibrated with equilibration buffer. As shown in
Table 16, bioburden and endotoxin samples are pulled from the retentate line after equilibrationis completed to demonstrate effective microbial control during membrane storage. Furthermore,
pH and conductivity are tested for retentate and permeate after equilibration.
Reuse
Membranes intended for re-use are flushed with DS formulation buffer after use and sanitized as
described above.
Table 16 below tabulates the process controls implemented for membrane reuse at commercial
scale.

PFIZER CONFIDENTIAL
Page 37

8

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 16. Routine Process Controls Implemented During the Sanitization, Equilibration,
and Reuse procedure (Pfizer, Andover)
Membrane Performance Test
Normalized membrane permeability post commissioning or cleaning (% recovery)
Equilibration retentate: bioburden (CFU/10mL)a
Equilibration retentate: endotoxin (EU/mL)a
Post diafiltration 2 permeate conductivity (mS/cm)
Post diafiltration 2 permeate pH
UFDF retentate pre-recovery product pool: bioburden (CFU/10mL)
UFDF retentate pre-recovery product pool: endotoxin (EU/mL)
Drug substance residual DNA template (ng DNA/mg RNA)
Step yield (%)
Abbreviations: CFU = colony forming units; EU = endotoxin units; UFDF = ultrafiltration/diafiltration; WFI =water for injection
a. Testing is performed as part of membrane lifetime validation and the testing frequency may be changed after
validation is complete

Rentschler, Laupheim
Sanitization
The TFF system and membrane are sanitized pre- and post-use. If the membrane was used
before, samples for bioburden and endotoxins are pulled between end of storage and pre-use
sanitization.
For sanitization, an initial WFI flush is performed followed by a 1.0 M NaOH solution flush andrecirculation for 60 – 65 min through the membrane. Subsequently, the normalized membrane
permeability (NMP) is measured. For NMP 0.1 M NaOH is used. The membranes are then stored
in 0.1 N NaOH.
Equilibration
Prior to UFDF batch execution, the membranes are equilibrated with equilibration buffer. As
shown in Table 17, bioburden and endotoxin samples are pulled from the retentate and permeateline after equilibration is completed to demonstrate effective microbial control during membranestorage. Furthermore, pH and conductivity are tested for retentate and permeate after
equilibration.
Reuse
Membranes intended for re-use are flushed with DS formulation buffer after use and sanitized as
described above.
Table 17 below tabulates the process controls implemented for membrane reuse at commercial
scale.

PFIZER CONFIDENTIAL
Page 38

9

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 17. Routine Process Controls Implemented During the Sanitization, Equilibration,
and Reuse Procedure
Step
Sanitization

Process controls
Post storage retentate and permeate: bioburden (CFU/10mL)
Post storage retentate and permeate: endotoxins (EU/mL)
Normalized membrane permeability post commissioning (%)
Pre-use: membrane integrity test
Equilibration
Post equilibration retentate and permeate: bioburden (CFU/10mL)
Post equilibration retentate and permeate: endotoxins (EU/mL)
Post equilibration retentate and permeate: pH
Post equilibration retentate and permeate: conductivity (mS/cm)
Reuse
Normalized membrane permeability post use (%)
Pre storage: bioburden (CFU/10mL)
Pre storage: endotoxins (EU/mL)
Pre storage retentate and permeate: conductivity (mS/cm)
Abbreviations: CFU = colony forming units; EU = endotoxin units;

Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
Page 39

0

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 16
Please note that all drug substance process steps must be performed under cGMP conditions.
Data to support complete process validation under cGMP conditions must be available at thetime of BLA submission.
RESPONSE 16
The sponsor confirms that all drug substance process steps are performed under cGMP
conditions. Complete data process validation details will be provided in the BLA.
Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
Page 40

1

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryREGARDING THE DP MANUFACTURING PROCESS:QUERY 17
Please provide a description of the process controls implemented during the sanitization,equilibration, and reuse procedure for the tangential flow filtration (TFF) membrane.
RESPONSE 17
Sanitization/Equilibration
Polymun, Austria and DermaPharm, Germany
Sanitization/Equilibration
The TFF membranes used in the drug product (DP) manufacturing process are currently used
once and then discarded. For Polymun, these filters are not supplied gamma irradiated/presterilized. However, for DermaPharm, these filters arrive gamma irradiated/pre-sterilized. In
both cases, the filters are sanitized by a flush and recirculation through the TFF membranes with
0.1 N sodium hydroxide (NaOH). The filters are then equilibrated for at least 24 hours with thesame NaOH buffer. If the TFF membranes are not used after 24 hours of the initial NaOH
sanitization step, the TFF membranes can stored in 0.1 N NaOH at 2-8 °C.
After this sanitization step, the filters are rinsed with WFI until conductivity of the permeate
stream reaches the conductivity of the WFI. For equilibration and pre-conditioning prior to use
in DP processing, a citrate buffer flush and recirculation are then performed until a permeate pHof 3.9-4.1 is achieved.
Reuse
A regeneration/reuse process for the TFF membranes in the DP manufacturing process is not
currently planned.
Pfizer Puurs, Belgium and Pfizer Kalamazoo, MI, US
Sanitization/Equilibration
The TFF membranes used in the DP manufacturing process are currently used once and thendiscarded. These filters arrive gamma irradiated/pre-sterilized. As such, a sanitization is not
required prior to use. However, a flush/recirculation through the TFF membrane is performed
with WFI or citrate buffer, after which a pre-use integrity test is performed with WFI or citratebuffer. If citrate buffer was not used in the prior step, an additional citrate buffer flush andrecirculation is performed for equilibration. The TFF membranes are then used for DP
processing.

PFIZER CONFIDENTIAL
Page 41

2

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryReuse
A regeneration/reuse process for the TFF membranes is targeted for implementation in GMP
manufacturing of drug product in December 2020. The implementation of a regeneration/reuseprocedure will include additional controls including TFF membrane cleaning with ≥20%
isopropyl alcohol (IPA), WFI and NaOH and a subsequent pre-use sanitization with NaOH as
well as additional sampling and testing through a concurrent validation protocol to ensure
appropriate membrane performance and microbial and residual carryover control. Performance
tests for the concurrent validation protocol for membrane reuse will include but not be limited tothose shown in Table 18.
Table 18. Performance Tests Implemented for Concurrent Validation for Membrane
Regeneration and Reuse
Membrane Performance Test
Normalized water permeability post commissioning or cleaning (% recovery)
Membrane integrity test
Post-CIP: bioburden
Post-CIP: endotoxin
Post TFF: bioburden (pre-bioburden reduction filtration)a
Post TFF: endotoxin (pre-bioburden reduction filtration)a
Yield (%)
a. Routine in-process test
Abbreviations: CFU = colony forming units; CIP = clean in place; EU = endotoxin units; TFF = tangential flow
filtration

Literature References
None
SUPPORTING DOCUMENTATIONNone

PFIZER CONFIDENTIAL
Page 42

3

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryREGARDING THE VALIDATION OF ANALYTICAL PROCEDURES:QUERY 18
In 2.3.1 Introduction p. 4 (Amendment 138; submitted on November 16, 2020), you stated that“the same method and validation information apply to each testing location”. However, thesummaries of qualification/validation study results provided thus far (3.2.S.4.3 and 3.2.P.5.3) do
not appear to contain site-specific information. Please explicitly specify that the same criticalreagents are used for each testing site. If so, please provide data to demonstrate comparable assay
performance between testing sites. If different SOPs/critical reagents are used, please provide
additional assay validation data.
RESPONSE 18
Additional method validation data are being provided for the BioNTech/Rentschler sites. The
test method standard operating procedure numbers for drug substance and drug product testing
are listed in Table 19 and Table 20 below. The Pfizer Andover and Chesterfield laboratories
perform all drug product testing and drug substance testing for DS manufactured at Pfizer,
Andover. The Pfizer laboratories use the same standard operating procedures. Both laboratories
were included in the co-validation of the test methods, and the intermediate precision determinedthrough the reproducibility studies were suitable to demonstrate consistent performance. The
compendial method verifications were performed at the Chesterfield or Andover site. The Pfizer
validation data were provided in Section S.4.3 Validation of Analytical Procedures (modRNA)of SN0137 (submitted 16 November 2020). The BioNTech and Rentschler laboratories perform
release and stability testing for drug substance manufactured at BioNTech/Rentschler sites. The
site and method of validation are provided in Table 19. The BioNTech/Rentschler validation data
are presented in Table 21 to Table 26 (excluding the BioNTech immunoblot validation fordsRNA, which was provided in a previous amendment). For methods which use critical reagents,
the same lot or an alternate qualified lot is used across testing sites. The intermediate precision
determined in the method validation studies are used to demonstrate consistent assay
performance between sites. As indicated in Table 19, all drug substance 5’-Cap and Poly(A) tailtesting are performed by Pfizer; all dsRNA testing is performed by BioNTech.

PFIZER CONFIDENTIAL
Page 43

4

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 19. Drug Substance Testing
Quality
Attribute

Analytical
Procedure

Test Method
Procedure
Number

PfizerVerification orValidation
Report #Clarity, Coloration

Appearance

TM100010539

VAL100131145

pH

Potentiometry

TM100010538

VAL100131145

Content (RNAConcentration)*

Platform UVSpectroscopy

TM100010308

VAL100188111
VAL100121467

Site Participatingin Validation

Test Method
Procedure
Number

BioNTech/Rentschler
Verification or
Site
Validation
Participating inReport #Validation

Verification ofcompendia
Verification ofcompendia

RL-SOP-00984/
RL-SOP-00994

Chesterfield, MO

RL-SOP-02645

RL-Report -09484

Rentschler

TM-072-038

MVR-20-0017

BioNTech, Mainz

TM-072-039

MVR-20-0018

BioNTech, Mainz

NA

NA

NA

NA

NA

NA

TM-072-028

MVR-20-0007

BioNTech, Mainz

Co-validation
performed at
Andover, MA and
Chesterfield, MO
Co-validation
performed at
Andover, MA and
Chesterfield, MO
Co-validation
performed at
Andover, MA and
Chesterfield, MO
Co-validation
performed at
Andover, MA and
Chesterfield, MO
Co-validation
performed at
Andover, MA and
Chesterfield, MO

RL-SOP-00995

RL-Statement00084
RL-Statement00084

Rentschler
Rentschler

Identity of Encoded
RNA Sequence

RT-PCR

TM100010407

VAL100121706

RNA Integrity

Capillary Gel
Electrophoresis

TM100010392

VAL100122020

5’ - Cap

RP-HPLC

TM100010578

VAL100123137

Poly(A) TailddPCR

TM100010379

VAL100123048

Residual Template
DNA

qPCR

TM100010388

VAL100121343

dsRNA

Immunoblot

NA

NA

NA

PAN-0720-K

VAL-2083-VB

BioNTech, IMFS

Bacterial
Endotoxin*

Endotoxin (LAL)

TM100010753

VAL100121189

Andover, MA

RL-SOP-00860

RL-Report-09494

Rentschler

Bioburden*

Bioburden

TM100010714

VAL100121185

Andover, MA

RL-SOP-00858

RL-Report-09493

Rentschler*Performed at Andover, MA and Rentschler as an IPC during DS manufacturing

PFIZER CONFIDENTIAL
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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 20. Drug Product Testing
Quality Attribute

Analytical
Procedure

Test Method
Procedure Number

Verification orValidation
Report #Site Participating in
Validation

Appearance(Visual, Particles)

Appearance

TM100010539

VAL100131145

Verification of compendia

Subvisible Particles

Subvisible
particulate matter

TM100010541

VAL100131145
VAL100126212

Verification of compendia

pH

Potentiometry

TM100010538

VAL100131145

Verification of compendia

Osmolality

Osmometry

TM100010540

VAL100131145

Verification of compendia

LNP Size and
Polydispersity

Dynamic Light
Scattering (DLS)

TM100010649

VAL100123376

RNA Encapsulation
and Content*

Fluorescence assay

TM100010402

VAL100123417

RNA Integrity

Capillary Gel
Electrophoresis

TM100010392

VAL100122020

Lipid Content and
Identity

HPLC-CAD

TM100010322

VAL100123165

Container Content
for injections

Volume ofInjections incontainers

TM100010614

VAL100131145

Identity of encoded
RNA sequence

RT-PCR

TM100010407

VAL100121706

In Vitro Expression

Cell-based flow
cytometry

TM100010380

VAL100122803

Bacterial Endotoxin

Endotoxin

Performed at DP
manufacturing site

NA***

Sterility

Sterility

Performed at DP
manufacturing siteNA***

Co-validation performed at
Andover, MA and
Chesterfield, MO
Co-validation performed at
Andover, MA and
Chesterfield, MO
Co-validation performed at
Andover, MA and
Chesterfield, MO
Co-validation performed at
Andover, MA and
Chesterfield, MO
Verification of compendia
Co-validation performed at
Andover, MA and
Chesterfield, MO
Co-validation performed at
Andover, MA and
Chesterfield, MO
Kalamazoo, MI
Puurs, Belgium
NA
Kalamazoo, MI
Puurs, BelgiumNA

RPT-49795,
Verification
INX100330679*Also run as an IPC during DP manufacturing (INX100426408). Supplemental verification performed(VAL100121467)**Run at T0 for stability batches only***Verification results pending(Note: Drug Product In-Process Controls, Sterility, and Endotoxin testing is performed at Pfizer Global Supply (PGS)
Drug Product manufacturing sites)
CCI**

Dye Incursion

TM100010635

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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 21. Validation Summary for the UV Spectroscopy Analytical Procedure (BioNTech)
Validation Parameter
Precision – Repeatability(System)
Precision – Repeatability(Method)

Results
RSD = ≤ 0.36%
RSD = ≤ 1.21%

Precision – Intermediate

RSD = ≤ 2.9%

AccuracySpecificity
Linearity

99.0 – 101.1%
Clear determination of the analyte in the presence of othersubstances.
Linearity plot is linear by visual inspection

Range
Robustness (prepared sample stability, other)

Coefficient of determination (R2) = 0.999
Range (A260) = 0.2 – 1.0
The method is robust against different lots of cuvettes

Table 22. Validation Summary for the RT-PCR Analytical Procedure for Drug Substance(BioNTech)
Validation Parameter
Specificity

Robustness: Amplification
extension time

Results
The RT-PCR assay was able to detect RNA from BNT162b2 DS only and there wasno cross reactivity with other non-specific RNA products. The positive signal wasobserved only in the positive control and the BNT162b2 DS samples. No positive
signal was found in the non-target samples and negative control.
The assay was able to detect RNA from DS using amplification extension times of31, 33, and 35 seconds in the thermocycle profile.

Table 23. Validation Summary for the Capillary Gel Electrophoresis Analytical Procedure
for Drug Substance (BioNTech)
Validation Parameter
Precision – Repeatability(System)
Precision – Repeatability(Method)
Precision – Intermediate
AccuracySpecificity
Linearity (Concentration)

Linearity (Integrity)

Results
RSD = 1.68 %RSD = 0.64 %RSD = 1.75 %Recovery: 100 -102 %Specificity shown. No interference in the area of the RNA peakLinearity plot is linear by visual inspection in the range of 30 – 250 %Coefficient of determination (R2) = 0.9832
Linearity plot is linear by visual inspection in the range of 23.1 – 71.6 %Coefficient of determination (R2) = 0.999

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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 24. Validation Summary for the qPCR Analytical Procedure (BioNTech)
Validation Parameter
Precision – Repeatability(System)Results
RSD (level: Ct values) = ≤ 0.7%
RSD (level: copies/mL) = ≤ 12.6%

Precision – Repeatability(Method)

RSD = 3.5%

Precision – Intermediate
AccuracySpecificity

RSD = ≤ 6.4%
98-101%
Specificity is proven.

Linearity
Detection Limit (DL)
Quantitation Limit (QL)

Specific signal using RNA drug substance in comparison to a negative control
sample.
Linearity plot is linear by visual inspection
Coefficient of determination (R2) = 0.9974
DL = 0.001 pg/mL
QL = 0.1 pg/mL

Validation for the Bioburden Analytical Procedure (Rentschler)
The validation of the bioburden analytical procedure was performed following the principles
described in USP <61>, Ph. Eur. 2.6.12, and JP 4.05 for BNT162b2 drug substance (DS). The
method validation (challenge recovery test) challenges the test method to ensure that the test
articles are non-inhibitory to the recovery of inoculated microorganisms.
The samples tested for the presence of bioburden are generally expected to be
non-bacteriostatic/non-fungistatic, or to be readily neutralized by membrane filtration. To
demonstrate this, the bioburden assay was performed with the addition of a microbial inoculum
composed of specified organisms (USP recommended). The challenge recovery testing was
performed for each test article with each microorganism along with the appropriate inoculum
control.
The acceptance criterion for this validation was as follows:
In the presence of the test article, the mean count of each test organism may not differ by a factor
greater than 2 from the value of the inoculum control.
Three batches of BNT162b2 DS (target concentration of 2.25 mg/mL), 10 mL in at least
duplicates per organism, were tested for the validation of the method.

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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 25. Challenge Recovery Testing Results for Drug Substance (Rentschler)
Batch
20E162001(Rentschler 1071539)

Organism
Recovery factor
A. brasiliensis
0.8
B. subtilis
1.0
C. albicans
0.8
P. aeruginosa
1.0
S. aureus
0.8
20E162002
A. brasiliensis
0.9(Rentschler 1071542)
B. subtilis
1.2
C. albicans
0.9
P. aeruginosa
1.3
S. aureus
1.0
20E162003
A. brasiliensis
1.3(Rentschler 1071544)
B. subtilis
0.9
C. albicans
1.6
P. aeruginosa
1.3
S. aureus
1.4
a. Challenge recovery testing was performed for each test article with each microorganism, as well as corresponding
inoculum control in triplicate.

Validation for the Bacterial Endotoxin Analytical Procedure (Rentschler)
The validation of the bacterial endotoxins test, using the kinetic turbidimetric limulus amebocyte
lysate (LAL) analytical procedure, for BNT162b2 drug substance (DS) was performed inalignment with the USP <85>, Ph. Eur. 2.6.14 and JP 4.01. The validation focused on thefollowing parameters:a. The criteria for the standard curve must be valid.
b. The sample solution must not interfere with the test (e.g. inhibition/enhancement)c. The sample must have a maximum valid dilution (MVD) established.
The MVD is the maximum allowable dilution of the test article at which the endotoxin limit canbe determined. The general equation to determine MVD is:d. MVD = (endotoxin limit)/ ()Where,
 = 0.01 EU/mL (label sensitivity of the lysate)
Three batches of BNT162b2 DS (target concentration, 2.25 mg/mL) were diluted and tested.
The inhibition/enhancement acceptance criterion for the spike recovery is 50-200%.

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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA Query

Table 26. Inhibition/Enhancement Results for BNT162b2 Drug Substance (Rentschler)
Batch Number
20E162001(Rentschler 1071539)

Sample Dilution
1:100

20E162002(Rentschler 1071542)

1:100

20E162003(Rentschler 1071544)

1:100

Spike Recovery (%)
101
100
110
121
113
130
115
108
100

Literature References
None
SUPPORTING DOCUMENTATION
New or Replaced Supporting Documentation
None
Previously submitted supporting documentation
Section S.4.3 Validation of Analytical Procedures (modRNA), SN0137

PFIZER CONFIDENTIAL
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Results (EU/mL)
0.50490.49850.54870.60790.56360.6506
0.57750.53870.4979

0

COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 19
Please provide the SOP and validation report for the in vitro expression assay for DP. With
continued manufacturing experience, please update the stringency of the specification according
to manufacturing process and assay capabilities.
RESPONSE 19
The SOP and validation report for the in vitro expression assay for the drug product are provided
with this response. The sponsor acknowledges CBER’s request to re-evaluate and update, if
necessary, the stringency of the specification according to manufacturing process and assay
capabilities.
Literature References
None
SUPPORTING DOCUMENTATION
New or Replaced Supporting Documentation
Test Method: TM1000010380, newValidation Report: VAL100122803, newPreviously submitted supporting documentation
None

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COVID-19 Vaccine (BNT162, PF-07302048)
BB-IND 19736
Response to 20-Nov-2020 FDA QueryQUERY 20
Please note, all analytical assays must be validated prior to BLA submission. Complete
validation reports should be submitted to the IND when they are available.
RESPONSE 20
Current validation data have been provided in the IND. The sponsor acknowledges that
additional validation data should be provided following transfers of the release and stability
methods to the additional commercial testing labs. After these transfers are completed, updated
validation reports will be provided.
Literature References
None
SUPPORTING DOCUMENTATIONNone

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