Document text
BioNTech SE
An der Goldgrube 12
55131 Mainz, Germany
Phone: +49 (0)6131 9084 -0
Telefax: +49 (0)6131 9084 -390
R&D DATA REPORT N o. R-20-0211
In Vitro Expression of BNT162b2 Drug Substance
and Drug Product
Version 0 2
Date: 17 SEP 2020
Reported by
Test item: BNT162b2
Key words: COVID19, modRNA, ATM material, Western blot, immunofluorescence ,
FACS
This R&D report consists of 22 pages.
Confidentiality Statement: The information contained in this document is the property and copyright of
BioNTech RNA Pharmaceuticals GmbH. Therefore, this document is provided in confidence to the recipient (e.g.
regulatory authorities, IECs/IRBs, investigators, auditors, inspectors). No information contained herein shall be
published, disclosed, or reproduced without prior written approval of the proprietors.
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TABLE OF CONTENTS
LIST OF FIGURES ................................ ................................ ............... 3
LIST OF TABLES ................................ ................................ ................. 3
LIST OF ABBREVIATION S ................................ ................................ .. 4
RESPONSIBILITIES ................................ ................................ ............. 5
1 SUMMARY ................................ ................................ ........................... 6
2 GENERAL INFORMATION ................................ ................................ .. 7
2.1 Sponsor and Test Facilities ................................ ................................ .. 7
2.2 Participating Personnel ................................ ................................ ......... 7
2.3 Study Dates ................................ ................................ .......................... 8
2.4 Guidelines and Regulations ................................ ................................ .. 8
2.5 Changes and Deviations ................................ ................................ ....... 8
2.6 Documentation and Archive ................................ ................................ .. 8
3 MATERIALS AND METHOD S ................................ .............................. 9
3.1 Test Item ................................ ................................ ............................... 9
3.2 Control Item ................................ ................................ .......................... 9
3.3 Test System ................................ ................................ .......................... 9
3.4 Materials ................................ ................................ ............................... 9
3.5 Methods ................................ ................................ .............................. 11
3.5.1 Study Design ................................ ................................ ...................... 12
3.5.2 Transfection of RNA Constructs in HEK293T Cells ............................ 12
3.5.3 Western Blot Analysis ................................ ................................ ......... 13
3.5.4 FACS Analysis ................................ ................................ .................... 13
3.5.5 Immunofluorescence ................................ ................................ .......... 14
4 TABLES AND FIGURES ................................ ................................ .... 15
5 CONCLUSION ................................ ................................ .................... 17
6 DOCUMENT HISTORY ................................ ................................ ...... 18
7 REFERENCES ................................ ................................ ................... 19
8 APPENDIX ................................ ................................ ......................... 20
Appendix 1: Certificates of Analysis ................................ .................... 20
BNT162b2 -RNA ................................ ................................ .................. 20
BNT162b2 ................................ ................................ .......................... 21
Appendix 2: FACS gating strategy ................................ ...................... 22
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LIST OF FIGURES
Figure 1: Western blot analysis for detection of BNT162b2 antigen expression .. 15
Figure 2: FACS analysis of transfection frequency and cell viability .................... 15
Figure 3: Immunofluorescence staining of transfected cells ................................ 16
LIST OF TABLES
Table 1: Equipment ................................ ................................ ............................... 9
Table 2: Consumables ................................ ................................ ......................... 10
Table 3: Reagents ................................ ................................ ............................... 10
Table 4: Antibodies and recombinant protein controls ................................ ......... 11
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LIST OF ABBREVIATION S
ATM Animal trial material
BNT162b2 Investigational vaccine in this study (DP)
BNT162b2 -RNA Investigational vaccine in this study (DS)
COVID -19 Coronavirus disease emerged in 2019
DNA Deoxyribonucleic acid
DP Drug product
DS Drug substance
ER Endoplasmic reticulum
FACS Fluorescence -activated cell sorting
GFP Green fluorescent protein
HEK Human embryonic kidney
modRNA modified RNA
PAGE Polyacrylamide gel electrophoresis
RBD Receptor binding domain
RNA Ribonucleic acid
S protein Spike protein
S1 Subdomain 1 of the S protein
SARS -CoV-2 Severe acute respiratory syndrome -Coronavirus -2
saRNA self-amplifying RNA
SDS Sodium dodecyl sulfate
SOP Standard operating procedure
uRNA unmodified RNA
V9 Antigen variant of the generated variants of the S protein
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2 GENERAL INFORMATION
2.1 Sponsor and Test Facilities
Sponsor
BioNTech RNA Pharmaceuticals GmbH
An der Goldgrube 12
55131 Mainz
Germany
Test Facilities
BioNTech RNA Pharmaceuticals GmbH
An der Goldgrube 12
55131 Mainz
Germany
2.2 Participating Personnel
Responsible person:
(as defined in SOP -100-024)
BioNTech RNA Pharmaceuticals GmbH
An der Goldgrube 12
55131 Mainz
Author:
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
Western blot , FACS
BioNTech RNA Pharmaceuticals GmbH
Experimenter:
Immunofluorescence
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2.3 Study Dates
Start of experiments: 22 JUN 2020
Completion of experiments: 14 AUG 2020
2.4 Guidelines and Regulations
All experiments are executed in accordance with the existing standard operating
procedures and described processes from BioNTech SE. Applicable documents are
listed below.
SOP -020-009 Ansetzen von Medien und Zusätzen für die Zellkultur
SOP -030-038 Standardisierte Kultivierung von Zellen
SOP -030-039 Zellzahlbestimmung mittels Neubauer Zählkammer
SOP -030-117 Du rchführung einer SDS Polyacrylamid Gelelektrophorese
(SDS -PAGE)
LA-50-255-000 Direkte / Indirekte Immunfluoreszenzfärbung
2.5 Changes and Deviations
Not applicable. There is no formal R&D plan available.
2.6 Documentation and Archive
Study plans and reports are st ored and archived according to SOP -100-003 Archiving
of Paper -Based Documents.
Raw data and evaluated data are saved at
P:\BioNTechRNA \RN9391R00_CoV -VAC\04_Preclinic \04_in
vitro\CorVac_IDV.invitro#050_b1_b2_b3c_DS_and_DP_IVE
P:\BioNTechRNA \RN9391R00_CoV -VAC\04_Preclinic \04_in
vitro\CorVac_IDV.invitro#059_HEK_IF_microscopy_at
P:\BioNTechRNA \RN9391R00_CoV -VAC\04_Preclinic \04_in
vitro\CorVac_IDV.invitro#064_mod9 -WB_IVE
Lab book MeGl, No. 2034
Lab book MeGl, No. 2035
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3 MATERIALS AND METHOD S
3.1 Test Item
BNT162b2 -RNA (ATM RNA ): For CoA see Appendix 1: Certificate s of Analysis .
BNT162b2 (ATM LNP): For CoA see Appendix 1: Certificate s of Analysis .
RNA batch: RNA -RF200321 -06, 97.5% integrity
Polymun batch RBP020.2 LNP with the lot: CoVVAC/270320
3.2 Control Item
Modified RNA GFP , p4.AGA_eGFP, RNA_RF200309_01c, 96% integrity
3.3 Test System
In vitro test system, cell culture, HEK293T cells
3.4 Materials
Table 1: Equipment
Product name Provider
Microscope IX53 Olympus Life Science
Microscope SP8 Leica
HeraCell 150i incubator Thermo Fisher Scientific
Vortexer Neolab
Biological Safety Cabinet HeraSafe2020 Thermo Fisher Scientific
Trans -Blot Turbo Transfer System Bio-Rad
Gel Dokumentation System ChemiDoc MP Imaging system ,
Detektor Supercooled CCD -30 °C, Pixel (Graustufen) 65535 Bio-Rad
Vacuum pump BVC -vacuu -control Vacuubrand
Pipette Eppendorf Research, 10 -100 μL Eppendorf
Pipette Eppendorf Research, 100 -1,000 μL Eppendorf
Pipette Eppendorf Research, 20 -200 μL Eppendorf
Centrifuge 5810R Eppendorf
FACSCanto II BD
Leica Application Suite LAS -X Version 3.1.5.16308 Leica
FlowJo software version 10.6.2 FlowJo LLC, BD Biosciences
Image Lab software version 5.0. Bio-Rad
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Table 2: Consumables
Product name Application/specification Article no. Provider
12-well plates Tissue culture 665180 Greiner -bio-one
96-well plates Tissue culture 650101 Greiner -bio-one
Safe-lock tubes 1.5 mL 0030120.086 Eppendorf
Tubes 15 mL 188271 Greiner -bio-one
Filter tips 20-200 μL 30077555 Eppendorf
Filter tips 100-1,000 μL 10212393 Eppendorf
Filter tips 0.1-10 μL 30077512 Eppendorf
Aspirating pipets 2 mL w/o plug 710183 Greiner -bio-one
Serological pipets 10 mL 607160 Greiner -bio-one
Table 3: Reagents
Product name Application/specification Article no. Provider
RiboJuice Transfection reagents TR-1013 Merck Millipore
FBS superior Fetal bovine serum 81D2925 Biochrom GmbH
DPBS No calcium, no magnesium 14190 -094 Thermo Fisher
Scientific
StemPro™ Accutase™ Cell Dissociation Reagent A11105 -01 Thermo Fisher
Scientific
Opti-MEM GlutaMAX Reduced serum medium 51985034 Thermo Fisher
Scientific
EDTA 0.5 M 03690 -
100ML Sigma -Aldrich
DMEM, high glucose,
GlutaMAX™ Supplement,
pyruvate Cell culture 31966047 Thermo Fisher
Scientific
BSA Bovine serum albumin GAUBSA01 -
64 Eurobio
Triton X -100 Immunofluorescence X100 -100ML Sigma Aldrich
Hoechst 1:5,000 H3570 Life Technologies
PFA, 32% Fixation 15714 -5 Electron Microscopy
ImmoMount media Mounting media 9990402 Life Technologies
4–15% Mini -PROTEAN®
TGX™ Precast Protein
Gels Polyacrylamide gel 4561083 Bio-Rad
cOmplete™ ULTRA
Tablets, Mini, EASYpack Protease Inhibitor Cocktail 5892970001 Roche
Transfermembran
Amersham™ Protran® NC Nitrocellulose membrane 4675.1 Carl Roth
Color Prestained Protein
Standard, Broad Range
(11–245 kDa) Molecular marker P7712 New England
BioLabs
10x Tris/Glycine/SDS Running Buffer 1610772 Bio-Rad
4 x Laemmli Sample Buffer Western blot 1610747 Bio-Rad
DTT Western blot A2948,0025 PanReac
AppliChem
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Product name Application/specification Article no. Provider
Fixable Viability Dye
eFluor™ 450 FACS 65-0863 -14 eBioscience
Fixation Buffer FACS 420801 Biolegend
Permeabilization Buffer
(10X) FACS 00-8333 -56 eBioscience
Ethanol Western blot 5054.4 Carl Roth
Nonfat dried milk powder Western blot A0830,1000 AppliChem
Tween -20 Western blot 9127.1 Carl Roth
Pierce™ ECL Western
Blotting Substrate Chemiluminescent substrate 32209 Thermo Fisher
Scientific
Tris Western blot 3170.2 Carl Roth
Glycin Western blot 3790.2 Carl Roth
SDS Western blot 0183.1 Carl Roth
NaCl Western blot 9265.2 Carl Roth
Sodium deoxycholate Western blot S1827 -100G Sigma Aldrich
EDTA Western blot 8040.3 Carl Roth
Table 4: Antibodies and recombinant protein controls
Product name Dilution Article no. Provider
SARS -CoV Spike S1 Subunit Protein
Antibody, Rabbit PAb 1:100 (IF);
1:1,000 (WB) 40150 -RP01 Sino Biological
SARS -CoV-2 (2019 -nCoV) Spike
Antibody, Rabbit Mab 1:400
(FACS) 40150 -R007 Sino Biological
Alexa Fluor™ 647 Antibody Labeling Kit N/A A20186 ThermoFisher
Scientific
Concanavalin A, Alexa Fluor™ 594
Conjugate 1:100 (IF) C11253 Invitrogen
Lectin GS -II From Griffonia simplicifolia,
Alexa Fluor™ 594 Conjugate 1:100 L21416 Invitrogen
Alexa Fluor® 488 AffiniPure Goat
Anti-Rabbit IgG (H+L) 1:400 (IF) 111-545-003 Jackson
ImmunoResearch
Anti-Rabbit IgG (whole molecule) –
Peroxidase antibody produced in goat 1:2,000 (WB) A0545 Sigma Aldrich
NCP -CoV(2019 -nCoV) Spike Protein (S1
Subunit, His Tag) N/A 40591 -V08H Sino Biological
3.5 Methods
Western blot and FACS assays were performed to analyze antigen expression;
immunofluorescence experiments were performed to assess correct localization in
cells.
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3.5.1 Study Design
The a im of this study was to analyze the express ion of BNT162b2 DS and DP in
HEK293T cells as a surrogate for a mammalian cell culture system . HEK293T cells
were transfected with DS BNT162b2 -RNA using a commercial transfection reagent or
with DP BNT162b2 to express the S protein of SARS -CoV-2 as the viral antigen.
Transfected HEK293T cells were allowed to express the S protein for 18 h before
analysis.
Expression of DS BNT162b2 -RNA was analyzed using Western blot to confirm
expression.
FACS analysis was performed in triplicates in permeabilized cells to assess
transfection frequencies and viability of cells transfected with either the DS BNT162b2 -
RNA, or with the DP BNT162b2 .
Finally, immunofluorescence microscopic analysis was used on cells transfected with
the DS BNT162b2 -RNA to assess processing of the expressed protein in the
endoplasmic reticulum ( ER). Since t he S protein contains a transmembrane domain it
is expressed on the cell surface, and therefore processing in the ER was p resumed .
3.5.2 Transfection of RNA Constructs in HEK 293T Cells
HEK293T cells were seeded in 12 -well plates with a cell number of 2 × 105 per well
one day before transfection or with a cell number of 4 × 105 per well 6 h before
transfection in DMEM with 10% FBS. For immunofluorescence experiments, HEK293T
cells were seeded in 12 -well plates with cover slips previously coated in collagen with
a cell number of 2 × 105 per well one day before transfection in DMEM with 10% FBS.
Cells were transfected with DS BNT162b2 -RNA using Ribojuice according to the
manufacturer’s protocol. As a transfection control, a modRNA construct encoding GFP
was used. Briefly, 1 µg of RNA was diluted in Opti -MEM and mixed with transfection
reagents. After an incubation of 4 min, 100 µL of the mixture was applied to the cells,
mixed gently , and incubated at 37°C/ 5% CO 2 for 18 h. For FACS analysis DP
BNT162b2 was transfected by diluting 1 µg of DP in 100 µl OptiMEM . The mixture was
applied to the cells, mixed gently and centrifuged by 500 ×g for 5 min at room
temperature before incubating at 37°C/5% CO 2 for 18 h.
Before proceeding with subsequent analyses, c ells transfected with GFP were
examined microscopically for successful transfection. Cells transfected with DS or DP
encoding for the antigen were either harvested for Western blot analysis or prepared
for subsequent immunofluorescence or FACS analysis
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3.5.3 Western Blot Analysis
Western blot analyses were used to evaluate whether the designed constructs were
expressed in HEK293T cells.
HEK293T cells were washed with PBS and detached from the well plate. Cells were
collected in a 1.5 ml Eppendorf tube, centrifuged for 5 min at 300 ×g/4°C. Supernatants
were discarded and the cell pellet was dissolved in 40 µL RIPA buffer (20 mM Tris,
0 15 M NaCl, 1% Triton X 100, 1% odium deo ycholate, 0 1% SDS, 10 mM EDTA)
with protease inhibitors and incubated for 30 min on ice. 13.3 µL 4x Laemmli sample
buffer with 10% DTT was added to the Eppendorf tubes and samples were heated for
5 min at 95°C. Recombinant protein controls were treated equally wit h 4x Laemmli
sample buffer/10% DTT diluted in PBS to achieve a 1x dilution. Afterwards, gels were
loaded with 25 µL of the samples and 3 µL of a marker. Gel electrophoresis was
performed with Tris/ glycine/SDS running buffer at 120 V. Proteins were transferred on
a nitrocellulose membrane for 30 min at 25 V (max. 1 A) using transfer buffer (43 mM
Tris, 35 mM glycine, 10% ethanol). The membranes were subsequently washed with
PBS/0.1% Tween -20, blocked for 1 h with blocking buffer (PBS, 0.1% Tween -20, 5%
nonfat dried milk powder) and incubated with the primary antibody in blocking buffer
overnight at 4°C.
After incubation, m embranes were washed with PBS/0.1% Tween -20 before
incubation with the secondary antibody for 1 h at room temperature, and washed again
before developing with chemiluminescent substrate and subsequent analysis on a
BioRad ChemiDoc system.
3.5.4 FACS Analysis
To assess transfection frequencies of DS BNT162b2 -RNA transfected cells using a
commercial transfection reagent or DP BNT162b2 transfected cells , FACS analysis
was performed. Cells were transferred to a 96 -well plate format and stained with 5 0 µl
fixable viability dye eFluor™ 450 diluted 1:500 for 15 min at room temperature. To
remove residual dye , cells were washed with FACS Buffer (1xDPBS, 1% BSA, 1%
0.5M EDTA) , centrifuged at 300xg for 5 min at 4°C and fixed with 100 µl Fixation Buffer
(Biolegend) for 12 minutes at room temperature. Cells were washed with 1x
Permeabilization Buffer (eBioscience) by centrifuging at 500xg for 5 min at 4°C and
stained with 50 µl anti -S1 antibody labelled with AF647 diluted 1:400 in 1x
permeabilization buffer fo r 30 min on ice. Afterwards, cells were washed twice with 1x
permeabilization buffer, centrifuging at 500xg for 5 min at 4°C. Cells were resuspended
in 100 µl FACS buffer before acquisition with a BD FACSCanto II.
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3.5.5 Immunofluorescence
Immunofluorescence staining of transfected cells was used to test whether the
construct was processed within the endoplasmic reticulum (ER) towards the cell
membrane leading to secretion or surface expression.
HEK293T cells were washed twice in PBS and fixed in 4% PFA for 1 0 min. Afterwards,
cells were washed three times for 5 min in PBS, permeabilized in PBS/0.2% Triton X -
100 for 5 min and blocked in PBS with 2% BSA and 5% goat sera for 30 min. Cells
were then incubated for 1.5 h with the primary antibod y in PBS/2% BSA (ant i-S1
antibody), washed three times in PBS for 5 min, and incubated with secondary
antibodies and conjugated Concanavalin A and Lectin GS -II in PBS/2%BSA for 2 h.
Afterwards, cells were stained with Hoechst for 3 min, washed three times in PBS for
5 min and were then mounted on slides and stored at 4°C until analysis. Cells were
analyzed with a Leica SP8 confocal microscope.
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4 TABLES AND FIGURES
Figure 1: Western blot analysis for detection of BNT162b2 antigen expression
Cells were transfected with DS BNT162b2 -RNA , and harvested after 18 h to allow antigen expression. Cells were
lysed and lysates were subjected to a sodium dodecyl sulfate (SDS) –polyacrylamide gel electrophoresis (PAGE)
system using a 4 –15% gradient polyacr ylamide gel followed by Western blot analysis . BNT162b2 has a predicted
size of 141.14 kDa. A recombinant SARS -CoV-2 S1 Subunit protein (76.5 kDa) was used as a positive control.
Figure 2: FACS analysis of transfect ion frequency and cell viability
Cells were transfected with BNT162b2 -RNA or BNT162b2 . After 18 h in culture, cells were stained with a viability
dye, fixed , permeabilized and stai ned with a monoclonal rabbit antibody recognizing the S1 protein subdomain
labelled with AF647. Non -transfected cells were used as a control.
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Figure 3: Immunofluorescence staining of transfected cells
Cells were transfected with BNT162b2 . After 18 h in culture, cells were fixed and stained for : the ER
(Concanavalin A, Alexa Fluor™ 594 conjugate and Lectin GS -II from Griffonia simplicifolia, Alexa Fluor™ 594
conjugate , red) , the S1 protein subdomain using a polyclonal antibody (anti-S1 antibody and Alexa Fluor® 488,
green) and deoxyr bonucleic acid (DNA) to define the nucleus (Hoechst, blue) . The merged colored picture shows
the co -localization of the two candidates within the ER (scale: 10 µm). A control , using non -transfected cells , is
shown in the bottom row.
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5 CONCLUSION
Western blot analysis confirmed the expression and size of the BNT162b2 antigen in
cell lysates of HEK 293T cells as a surrogate for correct expression in a eukaryotic
system .
FACS analysis was performed to assess transfection frequencies of HEK293T cells
transfected with either BNT162b2 drug substance (BNT162b2 -RNA) or drug product
(BNT162b2) . Both, BNT162b2 -RNA and BNT162b2 led to high frequencies of cells
being transfected, with BNT162b2 -transfected cells showing slightly higher
transfection frequencies compared to BNT162b2 -RNA transfect ed cells using a
commercial transfection reagent. There were no differences in cell viability after
transfection with BNT162b2 -RNA or BNT162b2 when comparing to non -transfected
cells.
Furthermore, co -localization of the S protein antigen with an ER marker was detected
by immunofluorescence experiments in HEK293T cells expressing BNT162b2 -RNA .
These result s show that the S protein is processed within the ER for s urface
expression .
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6 DOCUMENT HISTORY
Reasons for changes compared to previous version:
Section Version Version Reason for change
2.6 01 02 Specification of lab book numbers
3.4 01 02 Equipment was added
3.4 01 02 Correction of IF staining
3.5.5 01 02 Correction of IF staining
4 01 02 Correction of IF staining
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7 REFERENCES
Not applicable.
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8 APPENDIX
Appendix 1: Certificate s of Analysis
BNT162b2 -RNA
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BNT162b2
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Appendix 2: FACS gating strategy
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