Document text
BioNTec h SE
An der Goldgrube 12
55131 Mainz, Germany
Phone: +49 (0)6131 9084 -0
Telefax: +49 (0)6131 9084 -390
R&D STUDY REPORT No. R-20-0072
EXPRESSION OF LUCIFERASE-ENCODING
MODRNA AFTER I. M. APPLICATION O F GMP-
READ Y ACUITAS LIPID NANOPARTICLE
FORMULATION
Version 03
Date: 27 Nov 2020
Reported by
Test item : modRNA encoding luciferase
Key words: COVID-19 , modRNA, biodistribution , mouse , bioluminescence assay
This R&D report consi sts of 36 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
TABLE OF CONTENTS ................................ ................................ ........................ 2
LIST OF FIGURES ................................ ................................ ................................ 3
LIST OF TABLES ................................ ................................ ................................ .. 3
LIST OF ABBREVIATION S ................................ ................................ ................... 4
RESPONSIBILITIES ................................ ................................ .............................. 5
1 SUMMARY ................................ ................................ ........................... 6
2 GENERAL INFORMATION ................................ ................................ .. 8
2.1 Sponsor and Test Facilities ................................ ................................ .. 8
2.2 Participating Personnel ................................ ................................ ......... 8
2.3 Study Dates ................................ ................................ .......................... 8
2.4 Guidelines and Regulations ................................ ................................ .. 9
2.5 Changes and Deviations ................................ ................................ ....... 9
2.6 Documentation and Archive ................................ ................................ .. 9
3 INTRODUCTION ................................ ................................ ................ 10
3.1 Background ................................ ................................ ........................ 10
3.2 Objectives ................................ ................................ ........................... 10
3.3 Study Design ................................ ................................ ...................... 11
4 MATERIALS AND METHOD S ................................ ............................ 12
4.1 Test Item ................................ ................................ ............................. 12
4.2 Control Item ................................ ................................ ........................ 12
4.3 Test System ................................ ................................ ........................ 12
4.4 Materials ................................ ................................ ............................. 13
4.5 Methods ................................ ................................ .............................. 14
4.5.1 Animal Care ................................ ................................ ........................ 14
4.5.2 Animal Monitoring ................................ ................................ ............... 15
4.5.3 Endpoint of Experiment / Ter mination Criteria ................................ .... 15
4.5.4 Injection of Test and Control Items ................................ ..................... 15
4.5.5 Bioluminescence Measurements ................................ ........................ 16
4.5.6 Blood Sampling v ia the Vena Facialis ................................ ................. 16
4.5.7 Luminex -based Multiplex Assay (ProcartaPlex Multiplex Immunoassay)
................................ ................................ ................................ ............ 16
4.5.8 Luciferase -specific ELISA ................................ ................................ ... 17
4.5.9 ELISpot Analysis ................................ ................................ ................. 17
4.5.10 Statistical Analysis ................................ ................................ .............. 18
5 RESULTS ................................ ................................ ........................... 19
5.1 Bioluminescence Measurements ................................ ........................ 19
5.2 Liver Expression vs LNP8 ................................ ......................... 21
5.3 Luminex -based Multip lex Assay ................................ ......................... 21
5.4 Luciferase -specific ELISA ................................ ................................ ... 23
5.5 IFN- ELISpot Assay ................................ ................................ ........... 23
6 CONCLUSION ................................ ................................ .................... 25
7 DOCUMENT HISTORY ................................ ................................ ...... 26
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8 REFERENCES ................................ ................................ ................... 27
9 APPENDIX ................................ ................................ ......................... 28
Appendix 1: Animal Observations ................................ ................................ ........ 28
Appendix 2: Certificates of Analysis ................................ ................................ .... 29
Appendix 3: Raw Data IFN - ELISpot ................................ ................................ .. 32
Appendix 4: Statistical Analysis ................................ ................................ ........... 33
LIST OF FIGURES
Figure 1: Bioluminescence measurement using the LNP -formulated modRNA
encoding for luciferase ................................ ................................ ........................ 20
Figure 2: Bioluminescence measurement in the liver using the LNP -formulated
modRNA encoding for luciferase ................................ ................................ ......... 21
Figure 3: Activation of the innate immune system by LNP -formulated modRNA
encoding for luciferase ................................ ................................ ........................ 22
Figure 4: Luciferase -specific IgG ELISA on days -1 and 9 ................................ .. 23
Figure 5: ELISpot analysis using splenocytes on day 9 ................................ ....... 24
LIST OF TABLES
Table 1: Study design ................................ ................................ .......................... 11
Table 2: Lipid component formulations ................................ ................................ 12
Table 3: Materials ................................ ................................ ................................ 13
Table 4: Software ................................ ................................ ................................ 14
Table 5: Peptide pool for stimulation of splenocytes ................................ ............ 14
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LIST OF ABBREVIATIONS
ALC-0159 Proprietary PEG -lipid included as an excipient in the LNP formulation from
Acuitas
ALC-0315 Proprietary amino -lipid included as an excipient in the LNP formulation from
Acuitas
ALP Alkaline phosphatase
ANOVA Analysis of variance
AUC Area under the curve
BALB/c Mouse strain used in this study
BCIP 5-Bromo -4-chloro -3-indolyl -phosphate
BNT162 BioNTech’s vaccine program against COVID -19
ConA Concanavalin A
COVID -19 Coronavirus disease 2019
DPBS Dulbecco’s phosphate -buffered saline
ELISA Enzyme -linked immunosorbent assay
ELISpot Enzyme -linked immuno sorbent spot
FELASA Federation of European Laboratory Animal Science Associations
GMP Good manufacturing practice
HRP Horseradish peroxidase
IFN Interferon
IgG Immunoglobulin G
IL Interleukin
i.m. Intramuscularly
IP-10 Interferon -gamma induced protein 10
IVIS In vivo imaging system
LNP Lipid nanoparticle
Luc Luciferase (from firefly Pyractomena lucifera )
MCP -1 Monocyte chemotactic protein 1
MHC Major histocompatibility complex
MIP-1 Macrophage inflammatory protein 1
modRNA Nucleoside -modified mRNA
NBT Nitro blue tetrazolium
OD Optical density
p/s Photons per second
SD Standard deviation
SOP Standard operating procedure
S protein Spike protein
saRNA Self-amplifying mRNA
SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
TNF Tumor necrosis factor
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1 SUMMARY
BioNTech is developing RNA -based vaccines designed to protect against the novel
coronavirus disease that emerged in 2019 (COVID -19). The BNT162 project involves
testing three RNA platforms, which are under development at BioNTech with the
surface or spike protein (S protein) of the novel coronavirus (SARS -CoV-2) as the viral
antigen. These RNAs will be formulated with a GMP -compatible LNP formulation
provided by Acuitas.
In this study, the GMP -ready formulation containing the amino -lipid ALC -0315 and the
PEG -lipid ALC -0159 (in this report r eferred to as LNP 8 which is the identical
composition as used in BNT162 ) was tested in comparison with a
by Acuitas , and an in-house formulation , to characterize the
biodistribution of luciferase expressed by LNP -formulated nucleoside -modified mRNA
(modRNA ). Activation of the innate immune system, formation of antibodies against
luciferase , and T -cell activation were also assessed .
Four groups of three BALB/c mice were injected intramuscularly (i.m.) with a total dose
of 2 µg/animal of LNP8 - or -formulated modRNA encoding luciferase
or with buffer (DPBS) as control. At 6 h, 24 h, 48 h, 72 h, 6 d, and 9 d after injection,
the in vivo luciferase expression was measured by luciferin application. Serum
samples were taken 1 day before and 6 h after immunization as well as on day 9 for
quantification of the acti vation of the innate immune system in a Luminex -based
multiplex assay and antigen -binding antibody analysis in an IgG -specific ELISA.
Splenocytes were isolated on 9 d to assess the T -cell response by IFN- ELISpot
Assay .
An approximately 20 -fold higher luc iferase expression at the injections site was
observed for modRNA -Luciferase and the GMP -ready modRNA -Luciferase
LNP8 when compared to modRNA formulated with The difference between
the area under the curve for and LNP8 -formulated modRNA compared to buffer
control as well as to formulated modRNA was statistically significant. In
addition, luciferase expressed from the -formulated modRNA showed limited
drainage to the liver compared to LNP8 -formulated modRNA .
A multiplex assay showed that the innate immune system was temporally activated by
and LNP8 -formulated modRNA. The activation was more pronounced for
formulated modRNA than for modRNA formulated with LNP8, indicating a formulation -
related effect rather than a payload or expression level effect.
Treatment with modRNA with all tested LNP formulations did not induce the format ion
of luciferase -specific IgGs on day 9. However, a strong antigen -specific IFN - T-cell
response was measured by ELISpot assay on day 9 for and LNP8 -formulated
modRNAs, with statistically significant differences between these test groups , the
buffe r control , and the group.
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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 Facility
BioNTech SE
An der Goldgrube 12
55131 Mainz
Germany
2.2 Participating Personnel
Responsible p erson:
(as defined in SOP -100-024)
BioNTech
Author:
BioNTech SE
Experimenter:
Immunization, blood sampling,
ELISA, ELISpot BioNTech
Experimenter:
Luminex -based multiplex assay
BioNTech
2.3 Study Dates
Start of experiments: 14 JAN 2020
Completion of experiments: 23 JAN 2020
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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.
Animal test application approval number: G18-12-007
SOP -030-071 Abtöten von Mäusen
SOP -030-072 Fixiergriff und Ohrmarkierung bei Mäusen
SOP -030-073 Betäubung bei Mäusen
SOP -030-074 Blutentnahme bei Mäusen
SOP -030-078 Isolierung muriner Splenozyten
SOP -030-079 Intramuskuläre Applikation bei Mäusen
SOP -030-110 IFN - ELISpot murin
SOP -090-013 Biological safety in laboratories
2.5 Changes and Deviations
Not applicable. There is no formal R&D plan available .
2.6 Documentation and Archive
Study reports are stored and archived according to SOP -100-003 Archiving of Paper -
Based Documents.
Raw data and ev aluated data are saved at :
P:\BioNTechRNA \RN_R0030_AIRVAC \24_Preclinic \01_Vakzine Testing in
vivo Luc \IM#88 GMP ready LNP Acuitas modRNA
Lab book s:
o No. 1455 (complete study p lan including results)
o No. 1835 (IVIS images and quantification, Luciferase ELISA ,
ELISpot )
o No. 1593 page 71-84 (Luminex -based multiplex assay)
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3 INTRODUCTION
3.1 Background
In December 2019, an outbreak of pneumonia of unknown cause in Wuhan, Hubei
province in China started. The disease spread rapidly and in January 2020, the agent
was identified. By 1 April 2020, infection with the novel coronavirus ( SARS-CoV-2) was
confirme d in approximately 820,000 people with more than 40,000 casualties1. A
vaccine is urgently needed and BioNTech decided to develop a rapid vaccine project
(BNT162) with the surface or spike protein (S protein) of the virus as the viral antigen.
The developm ent of in vitro transcribed RNA as an active platform for the use in
infectious disease vaccines is based on the extensive knowledge of the company in
RNA technology, which has been gained over the last decade. The core innovation is
based on in vivo delivery of a pharmacologically optimized, antigen -coding RNA
vaccine to induce robust neutralizing Abs and accompanying/concomitant T -cell
response s to achieve protective immunization with minimal vaccine doses
(Vogel et al. 2017 , Moyo et al. 2018 , Pardi et al. 2017 ).
At BioNTech, three different RNA platforms formulated with lipid nanoparticles (LNPs)
are under development, namely non-modified uridine -containing mRNA ( uRNA),
nucleoside -modified mRNA (modRNA) and self -amplifying RNA (saRNA). In the
present study, an LNP -formulated modRNA encoding luciferase was used
representatively to investigate the in vivo biodistribution and the immune response of
the vaccine candida tes.
LNP formulation s from a third party provider (Acuitas) were tested in comparison to the
in-house formulation Acuitas
Acuitas also provided a n LNP
formulation that is cGMP -ready , namely LNP8 , which contains two p roprietary lipid s
(ALC -0159 and ALC -0315) and has the identical composition as the LNP formulatio n
used in the BNT162 program .
3.2 Objectives
The objective of this study was to investigate the biodistribution of luciferase expressed
by the LNP-formulated modRNA using bioluminescence measurements in BALB/c
mice , as well as innate immune system activation, formation of antibodies against
luciferase and T -cell activation .
1 Coronavirus diseas e (COVID -2019) situation report 72, World Health Organization;
/www.who.int/emergencies/diseases/novel -coronavirus -2019/situation -reports
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3.3 Study Design
Four groups of three BALB/c mice were injected intramuscularly (i.m.) in the right and
left hind leg with each 1 µg of LNP -formulated modRNA encoding luciferase or with
buffer a s control on day 0. At 6 h, 24 h, 48 h, 72 h, 6 d, and 9 d after injection, the
in vivo luciferase expression was measured by luciferin application.
In addition , serum samples were collected on day -1 and 6 h and 9 d post immunization
and cytokine /chemokine level determination (multiple ) and on day 1 and 9 for
luciferase -specific ELISA . On day 9, spleens were resected for immunological analysis
using IFN - ELISpot assays .
Table 1: Study design
Group Treatment Dose [µg/
mouse] Formulation Treatment
schedule End of
experiment Sample collection
1 Buffer control N/A N/A Day 0 Day 9
Serum ELISA on
days -1 and 9 ,
serum for Multiplex
assay on day -1, 6 h,
and 9 d;
splenocytes for
ELISpot on day 9 2 modRNA -
Luciferase 2 µg
(1 µg/leg) Day 0 Day 9
3 modRNA -
Luciferase 2 µg
(1 µg/leg) Acuitas proprietary Day 0 Day 9
4 modRNA -
Luciferase
LNP8 (GMP -
ready) 2 µg
(1 µg/leg) ALC-0315:ALC -
0159:DSPC:Chol Day 0 Day 9
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4 MATERIALS AND METHODS
4.1 Test Item
LNP-formulated modRNA encoding luciferase diluted to 0.05 mg/m L to obtain
a dose of 1 µg in 20 µL application volume . For CoA s see Appendix 2:
Certi ficate s of Analysis of RNA and LNPs.
Acuitas LNPs:
o
o LNP8 m odRNA Luc, RNA -EH190611 -01c, batch FM-1074 -D, 90%
encapsulation, 1.0 mg/m L encapsulated RNA, diameter 71 nm,
polydispersity 0.053, s torage temperature -80°C.
BioNTech LNP:
o
Table 2: Lipid component formulation s
Formulation Lipids
Functional l ipid 1 Functional l ipid 2 Structural l ipid 1 Structural l ipid 2
Acuitas
proprietary Acuitas
proprietary Acuitas
proprietary Acuitas
proprietary
LNP8 ALC-0315 ALC-0159 DSPC Cholesterol
4.2 Control Ite m
DPBS
4.3 Test System
Mus musculus : 12 female BALB/c mice at an age of 9 weeks at study start with
a body weight of approximately 25 g
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4.4 Materials
Table 3: Materials
Product name Application/specification Article no. Working
dilution Provider
Dulbecco’s
phosphate -buffered
saline ( DPBS ) Buffer control 14190 -094 1× Thermo Fisher
Scientific
Syringes 0.3 mL 30 G Insulin syringes for i.m.
application 4144150 N/A BD
Syringes 0.5 mL 29 G Insulin syringes for i.p.
application 324824 N/A BD
Luciferin Substrate for in vivo
luciferase imaging 122799 -10 150 mg/kg Perkin -Elmer
Microvette 500 Z -gel
tubes Blood sampling 2021 -01-
31 N/A Sarstedt
Xenogen IVIS®
Spectrum In vivo BLI imager - N/A Caliper Life
Sciences
Maxi Sorp plate ELISA 439454 N/A Thermo Fisher
Scientific
QuantiLum
recombinant
luciferase Positive control E1701 100 ng/µL Promega
Casein blocking buffer 10× B6429 1× Sigma -Aldrich
TMB ONE ECO -TEK Chromogenic substrate for
horseradish peroxidase 4380H N/A BIOTREND
Sodium bicarbonate 7.5% NaHCO 3 S8761 -
100 ml N/A Sigma -Aldrich
HCl Hydrochloric acid solution
volumetric, 0.1 M HCl 2104 -50 ml N/A Sigma -Aldrich
RPMI1640 medium Cell culture medium 61870 N/A Gibco
Biotek Epoch ELISA plate reader - N/A Biotek
Mouse IFN -
ELISpotPLUS kit Kit for enumeration of cells
secreting mouse IFN - 3321 -
4APT -2 N/A Mabtech
ImmunoSpot® S5
Versa Analyzer ELISpot plate reader - N/A Cellular
Technology Ltd .
RPMI1640 medium Cell culture medium 61870 N/A Gibco
Multiplex PROCARTAPLEX 10
PLEX PPX-10 -
MXU63C9 N/A Life
Technologies
GmbH
Bio-Plex 200 Multiplex reader - N/A Bio-Rad
Anti-firefly luciferase
antibody (mAb21 ), Assay control ELISA ab64564 1:1,000 Abcam
Mouse IgG isotype Assay control ELISA 0107 -08 1:100 as
starting
dilution Southern
Biotech
Goat anti-mouse IgG
HRP Secondary antibody ELISA 115-035-
071 1:15,000 ( if
stored in
50%
glycerol
1:7,500) Jackson
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Table 4: Software
Product name Application Provider
Living image In vivo BLI quantification Perkin -Elmer
Prism Analysis Graph Pad Software Inc.
Excel Animal monitoring, raw data ELISA Microsoft Corp.
Bio-Plex Manager (6.1) Bio-Plex reader Bio-Rad
Gen5 Absorbance reader Biotek
ImmunoCapture V6.3 ELISpot analysis Cellular Technology Ltd .
Table 5: Peptide pool for stimulation of splenocytes
Peptide pool MHC -I
Name Sequence
Firefly luciferase -1 GFQSMYTFV
Firefly luciferase -2 VPFHHGFGM
Firefly luciferase -3 VALPHRTAC
Consensus sequences from Limberis et al. 2009
4.5 Methods
4.5.1 Animal Care
4.5.1.1 General Information
BALB/c mice were delivered at the age of at least six weeks. Delivered mice were used
for experiments after approximately one week of acclimatization. All experiments and
protocols were approved by the local authorities (local welfare committee), conducted
according to the FELASA recommendations and in compliance with the German
animal welfare act and Directive 2010/63/EU. Only animals with an unobjectionable
health status were selected for testing procedures.
All animals were registered upon arrival in the lab animal colony management system
PyRAT (Scionics Computer Innovation GmbH, Dresden, Germany) and tracked until
death. Each cage was label ed with a cage card indicating the mouse strain, gender,
date of bi rth, and number of animals per cage. At the start of an experiment additional
information was added such as the project and license number, the start of the
experiment and details on interventions. Where necessary for identification, animals
were arbitrari ly numbered with earmarks.
4.5.1.2 Housing Condition and Husbandry
Mice were housed at BioNTech SE ’s animal facility under barrier and SPF conditions
(An der Goldgrube 12, 55131 Mainz) in individually ventilated cages (Sealsafe GM500
IVC Green Line, TECNIPLAST, Hohenpeißenberg, Germany; 500 cm²) with a
maximum of five animals per cage. The temperature and relative humidity in the cages
and animal unit was kept at 20 -24°C and 45 -55%, respectively, and the air change
(AC) rate in the cages at 75 AC/hour. The cages with dust -free bedding made of
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debarked chopped aspen wood (Abedd LAB & VET Service GmbH, Vienna, Austria,
product code: LTE E -001) and additional nesting material were changed we ekly.
Autoclaved sniff M -Z food (sniff Spezialdiäten GmbH, Soest, Germany; pr oduct code:
V1124) and autoclaved water (tap water) were provided ad libitum and changed at
least once weekly. All materials were autoclaved prior to use.
4.5.2 Animal Monitoring
Routine animal monitoring wa carried out daily and included in pection for dead
animals and control of food and water supplies. Each animal ’s health was closely
assessed at least once weekly. The general physical condition was assessed with
regard to the following parameters:
Body weight change
Macroscopic assessment of activity level/ behavior
Macroscopic assessment of general discomfort: drop in body temperature
determined by touch and by visual inspection of ears and paws. Ears and paws
appear pink in a healthy mouse, white in a mouse with discomfort indicated by
reduced blood circula tion
Macroscopic assessment of fur condition and appearance of eyes, inspection
of body cavities/ fluids
Macroscopic assessment of irregularities in breathing ability
Indication of pain
Macroscopic assessment for signs of automutilation and or fighting
4.5.3 Endpoint of Experiment / Termination Criteria
Animals were euthanized in accordance with §4 of the German animal welfare act and
the recommendation of GV -SOLAS by cervical dislocation or by exposure to carbon
dioxide. Additionally, termination criteria applie d according to the specification within
the respective animal test approval as listed below. Body weight losses exceeding
20%, or a high severity level in any of the parameters found in Section 4.5.2 were on
their own sufficient reason for immediate euthanasia.
4.5.4 Injection of Test and Control Items
Animals were anesthetized by inhalation of 2.5% isoflurane in oxygen and the injection
site (hind leg) was s haved. Buffer or dissolved test item was applied i.m. into the
musculus gastrocnemius at a volume of 20 µL. All mice received 1 µg in each leg. After
injection and a short recovery phase from anesthesia, the animals were observed for
any signs of discomfor t due to the injection procedure .
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4.5.5 Bioluminescence Measurements
The mice were monitored over a period of 9 days using in vivo imaging system (IVIS)
measurements. Briefly, the Xenogen IVIS® Spectrum device was used for in vivo
imaging according to the manufa cturer’s instruction. Approximately 6 h after LNP
administration and 5 min prior to imaging, animals were injected for the first time
intraperitoneally (i.p.) with luciferin (150 mg/kg, dosing volume: 300 µL, 29 G needle).
Mice were anesthetized (2.5% isof lurane/O 2) and placed in the imager, first with the
dorsal side exposed and then with the ventral side exposed, and luciferase activity was
measured. Images were taken with exposure time and sensitivity set to 1 s, 10 s, and
1 min and bin 2, bin 4, or bin 8, respectively. The dorsal and ventral images were
analyzed by visual inspection after aligning of sensitivities of each picture and used for
illustration of findings. The images were analyzed using Living Image in vivo imaging
3.0 software, where the regions to be quantified (radiance) were drawn manually and
calculated automatically (region of interest, ROI), to follow kinetics of the total fluxes
(p/s) over time in a GraphPad file.
4.5.6 Blood Sampling via the Vena Facialis
Blood was sampled via the vena facialis according to SOP -030-074. In short, without
prior anesthesia, mice were held tightly and using a lancet, the v. facialis was
punctured in a precise and short movement. Blood was collected into Microvette 500
Z-gel tubes, subsequently the restr aining grip was loosened. Blood samples were
centrifuged at 10 ,000 ×g (room temperature) for 5 min and serum transferred to a pre -
labeled 1.5 mL reagent tube before storage at -20°C.
4.5.7 Luminex -based Multiplex Assay (Procarta Plex Multiplex
Immunoassay)
The assay was performed according to manufacturer’s protocol. Briefly, magnetic
beads were added to the provided 96 -well flat bottom plate and the beads were washed
(wash buffer 1 ×) with the help of a hand -held magnetic plate washer. The antigen
standard was reconstituted in universal assay buffer (1 ×), pooled in one tube, the
volume adjusted to a final volume of 250 µL, serial diluted (4 -fold serial dilution steps) ,
and 50 µL was added to the designated wells. Serum samples were diluted 1:1 with
the unive rsal assay buffer and 50 µL added to the wells. The standard was measured
in duplicates and the serum samples in triplicates. The plate was incubated on a plate
shaker at 500 rpm) for 2 h covered with a black lid. After three wash steps, 25 µL of
the ready -to-use detection antibody was added, incubated for 30 min on the shaker
and washed three times. Streptavidin -PE (50 µL) were added and the plate incubated
for 30 min on the shaker and washed three times. The beads were resuspended in
120 µL reading buffer , the plate was sealed , and data were acquired in the Bio -Plex
200 Luminex system.
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4.5.8 Luciferase -specific ELISA
Luciferase -specific IgGs in serum samples obtained on study days 1 and 9 were
detected using ELISA. Recombinant luciferase (100 ng/100 µL) protein was utilized to
coat MaxiSorp plates at 4°C overnight. Upon washing and blocking using casein -based
blocking buffer, serum samples were screened for luciferase -specific antibodies by
incubation on plates for 1 h at 37°C. An anti-firefly luciferase antibody (mAb21 ) as well
as a mouse IgG isotype were included as assay controls. Subsequently, plates were
incubated with horseradish peroxidase (HRP) -labeled secondary anti -mouse IgG
antibody for another 45 min at 37°C before 3, 3’, 5, 5’ -tetramethylbenzi dine (TMB) ONE
substrate was applied. Colorimetric detection was monitored and optical density read
at 450 nm calculated to a wavelength reference of 620 nm (ΔOD 450–620 nm).
4.5.9 ELISpot Analysis
4.5.9.1 T-cell epitope prediction
The respective peptides for stimulatio n of splenocytes (Table 5) were used as
published by Limberis et al. 2009 , where the authors mapped the dominant and minor
T-cell epitopes in BALB/c mice (GFQSMYTFV and VPFHHGFGM, VALPHRTAC,
respectively) for monitoring cellular responses in vivo. No modifications have been
added to the published peptides before peptide synthesis by JPT technologies Gmb H.
4.5.9.2 Sample Collection and Processing
Spleens were removed on day 9 after euthanizing the mice , and single -cell
suspensions were prepared (SOP -030-078). In brief, the removed organs were
pressed through a 70 µm cell mesh using the plunger of a syringe to rel ease the cells
from the organ into a tube. After washing with PBS the cell pellet was incubated with
erythrocyte lysis buffer, washed in PBS , and passed again through a 70 µm cell mesh.
Resulting cells were resuspended in medium and counted.
4.5.9.3 IFN-γ ELISpot Assay
The IFN - ELISpot assay was used to measure IFN - release after in vitro stimulation
of T cells as an indicator for the induction of antigen -specific T cells. ELISpot analysis
was performed using the Mabtech Mouse IFN - ELISpotPLUS kit. Isolated splenocytes
were seeded to pre -coated ELISpot plates at 5 × 105 cells/well in 200 µL medium and
stimulated with antigen -specific peptide pools composed of single peptides and a final
concentration of 2 µg/mL per peptide, predicted as describ ed in Section 4.5.9.1
overnight in a humidified incubator at 37°C. As peptide controls, splenocytes were
incubated with 6 µg/mL of an irrelevant AH1 peptide derived f rom the endogenous
retroviral gene product envelope glycoprotein 70 (gp70; AH1: amino acids 6 to 14).
Splenocytes were incubated with medium alone as a negative control or with 2 µg/mL
Concanavalin A (ConA) as an internal positive control, confirming the f unctionality of
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the assay. Spots were visualized with a biotin -conjugated anti -IFN- antibody followed
by incubation with streptavidin -alkaline phosphatase (ALP) and 5 -bromo -4-chloro -3-
indolyl -phosphate/nitro blue tetrazolium (BCIP/NBT) substrate. Spot num bers were
counted and analyzed using the ImmunoSpot® S5 Versa ELISpot Analyzer, the
ImmunoCaptureTM image acquisition software, and the ImmunoSpot® Analysis
software version 5. The quality control ( QC) function of ImmunoSpot analysis software
was used to l imit false positive spot counts. All tests were performed in triplicate and
spot counts were summarized as median values for each triplicate.
4.5.10 Statistical Analysis
GraphPad Prism 8 software (La Jolla, USA) was used for statistical analysis and figure
genera tion. All groups were compared by a one -way ANOVA with Tukey’s multiple
comparison post -test on each measurement day ( area under the curve for
bioluminescence assay, ELISA , and ELISpot analysis ).
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5 RESULTS
5.1 Bioluminescence Measurements
The biodistribution of luciferase expressed by the LNP -formulated modRNA after i.m.
injection was assessed by bioluminescence measurements. Mice received a total dose
of 2 µg LNP8 , or -formulated modRNA, the control group received
20 µL DPBS o nly.
Mice were monitored over nine days and Luciferase signal was recorded and
quantified (Figure 1).
Time
24 h 48 h 72 h 9 d 6 d 6 h
Buffer control
modRNA -Luciferase
LNP8A
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Figure 1: Bioluminescence measurement using the LNP -formulated modRNA encoding for luciferase
BALB/c mice were injected i.m. in the right and left hind leg with each 1 µg of LNP -formulated modRNA encoding
luciferase or DPBS only . A) At different time points after in jection, the luciferase expression in vivo was measured
by luciferin application. After 9 d, the reporter expression dropped to background levels . B) Quantification of
luciferase signal over time and C) as total area under the curve ( AUC, ±SD). p/s: photon s per second
All formulations resulted in a modRNA -typical expression over time (Figure 1). Highest
signal was detected at the first time points after immunization at the injection site and
the signal declined slowly over time unt il day 9 (Figure 1B). Luciferase expressed by
the modRNA formulate d with Acuitas LNP8 drained to the liver as visualized by
luciferase expression at 6 h in the liver region
Acuitas formulations (Figure 1A and Figure 2).
Group mean l uciferase expression from RNA formulated with Acuitas LNP8
in the muscle at 6 h was approximately 1 × 109 p/s,
Hence, Acuitas -formulated
modRNA started at about 20-fold higher signal levels , stayed more than 20 -fold higher
until 72 h (~7 × 107 p/s for LNP8 vs ) and
declined then to a low level
on day 9 (~3–5 × 105 p/s).
Area under the curve calculation allow ed comparing overall expression level s over the
course of the experiment ( Figure 1C). Statistical significance was assessed by one -
way ANOVA with Tukey’s multiple comparison post-test comparing all groups with
each other. Total luciferase expression from modRNA formulated with
while total luciferase expression from modRNA formulated
with LNP8 was approximately 1.5 × 109 p/s, respectively.
The difference between the area under the curve for LNP8 -formulated
modRNA compared to buffer control was
1051061071081091010Total AUCTotal Flux [p/s]
6 h24 h 48 h 72 h6 d 9 d1041051061071081091010Luciferace signal over time
Time after injectionTotal Flux [p/s]
Buffer control modRNA-Luciferase LNP8B C
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statistically significant (p < 0.0001).
5.2 Liver Expression LNP8
As mentioned in Section 5.1, luciferase expressed by the modRNA formulated with
Acuitas LNP8 drained to the liver as visualized by luciferase expression at 6 h in the
liver region Acuita
(Figure 2A). Here the luciferase signal of modRNA formulated with LNP8
was quantified for better comparison . Group mean luciferase expression from RNA
formulated with Acuitas in the liver at 6 h was while
luciferase expression of RNA formulated with LNP8 was at about 4.94 × 107 p/s.
Hence, luciferase expression from Acuitas
compared to LNP8 (Figure 2B). The liver luciferase expression f rom RNA
formulated with Acuitas LNP 8 dropped to 2.4 × 106 p/s at 24 h, while the luciferase
signal from RNA formulated with Acuitas No liver signal
was detected at 48 h post immunization. Statistical analysis was not performed.
Figure 2: Bioluminescence measurement in the liver using the LNP -formulated modRNA encoding for
luciferase
BALB/c mice were injected i.m. in the right and left hind leg with each 1 µg of LNP -formulated modRNA encoding
luciferase. A) At 6 h, 24 h, and 48 h after injection, the luciferase expression in vivo was measured by luciferin
application. B) Qua ntification of luciferase signal in the liver over time ( mean ±SD). p/s: photons per second .
5.3 Luminex -based Multiplex Assay
Activation of the innate i mmune system was assessed in a Luminex -based multiplex
assay (Procarta immunoassays) . Serum samples (day -1 (pre) , 6 h, and day 9) were
tested for levels of the following chemokines and cytokines: MCP -1, MIP -1, TNF -,
IFN-, IFN -, IL-2, IL-6, IL-10, IL -1, IP-10 (Figure 3). No cytokines/chemokines were
detected in the pre -serum.
Immunization with LNP 8 induced slightly increased levels of MCP -1, IL-
6, and IP-10 at 6 h post immunization
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All chemokine/cytokine levels dropped to
background levels at day 9. Statistical analysis was not performed for this assay.
Figure 3: Activation of the innate immune system by LNP-formulated modRNA encoding for luciferase
BALB/c mice were injected i.m. in the right and left hind leg with each 1 µg of LNP -formulated mo dRNA encoding
luciferase or DPBS only . Serum samples ( day -1 (pre), 6 h, and 9 d) were assessed for presence of indicated
chemokines/cytokines in a L uminex -based multiplex immunoassay.
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd9050100150MCP-1Serum MCP-1 (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd902468
MIP-1Serum MIP-1 (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd902468
TNF-Serum TNF- (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd90.00.20.40.60.81.0
IFN-Serum IFN- (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd9051015
IFN-Serum IFN- (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd90.00.20.40.60.81.0
IL-2Serum IL-2 (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd9050100150200
IL-6Serum IL-6 (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd90.00.20.40.60.81.0
IL-10Serum IL-10 (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd90.00.20.40.60.81.0
IL1-Serum IL1- (pg/ml)
pre 6hd9 pre 6hd9 pre 6hd9 pre 6hd9020406080100
IP-10Serum IP-10 (pg/ml)
Buffer control modRNA-Luciferase LNP8
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5.4 Luciferase -specific ELISA
Luciferase -specific IgGs in serum samples obtained on study days -1 and 9 were
investigated by ELISA.
Before immunization, no luciferase -specific IgGs were detected ( day -1, Figure 4).
Treatment with modRNA with all tested LNP formulations did not induce the formation
of luciferase -specific IgGs on day 9 post immunization.
Figure 4: Luciferase -specific IgG ELISA on days -1 and 9
BALB/c mice were immunized with 1 μg/leg luciferase -encoding modRNA on day 0. Serum sa mples were collected
on days -1 and 9 and the total amount of antigen -specific immunoglobulin G (IgG) was measured via ELIS A. The
serum 1:10 diluted . Individual ΔOD values are shown by dots; group mean values ar e indicated by horizontal bars
(±SD ).
5.5 IFN- ELISpot A ssay
Mice were euthanized on day 9 and splenocytes were isolated to assess T -cell
responses by ELISpot analysis. Splenocytes were stimulated with luciferase -specific
peptide pools (Table 5) and IFN - secretion was detected. Statistical significance was
assessed by one -way ANOVA with Tukey’s multiple comparison post -test comparing
all groups with each ot her. Control measurements were performed using an irrelevant
peptide pool, medium only , or Concanavalin A.
Stimulation of splenocytes with MHC I-specific peptide pools induced IFN - responses
in T cells of animals immunized with all modRNA LNP candidates (Figure 5). Group
mean values of 53 spots per 5 × 105 cells were counted for animals injected with buffer
control after stimulation with MHC I-specific luciferase peptide pools. The high spot
count in can be attributed to reactivity of T cells of one mouse in group 1 . Splenocytes
of this mouse react also to the stimulation with AH1, the negative control. The group
mean values are also 44 spots be 5 × 105 cells for the AH1 control , thus the response
0123
Day -1 (1:10)OD (450-620nm)
0123
Day 9 (1:10)
Buffer control
modRNA-Luciferase LNP8
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of the control group to the luciferase -specific peptide pool can be considered
unspecific. Group mean spot counts after stimulation with MHC I-specific peptide pools
were
519 spots per 5 × 105 cells for the group treated with LNP8 -
formulated modRNA. The reactivity of splenocytes of the treatment groups to the
negative control was very low (6 -8 spots per 5 × 105 cells) , thus activation of T -cells
with luciferase peptides is hig hly specific in the treatment groups.
The differences between the g roups
treate d LNP8 -formulated modRNAs compared to buffer control were
statistically significant ( p = 0.0051 for
modRNA -luciferase LNP8) . The and LNP8 groups displayed significantly higher
MHC I-specific IFN - secretion
p = 0.0163 for modRNA -
luciferase LNP8 ).
Figure 5: ELISpot analysis using splenocytes on day 9
ELISpot assay was performed using splenocytes is olated on day 9 after prime immunization. Splenoc ytes were
stimulated with MHC I-specific luciferase peptide pools and IFN - secretion was measured to assess T -cell
responses. Individual spot counts are shown by dots; group mean values are indicated by horizontal bars (± SD).
05001000LuciferaseSpots per 5*105cells
05001000AH1
Buffer control modRNA-Luciferase LNP8
Medium ConA0500100015002000
Medium and ConA controls
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6 CONCLUSION
After injection of LNP -formulated modRNAs , an approximately 20 -fold higher luciferase
expression at the injections site was observed in a bioluminescence assay for
the GMP -ready modRNA -Luciferase LNP 8
The difference between the area
under the curve for LNP8 -formulated modRNA compared to buffer control
was statistically significant.
A multiplex assay showed that the innate immune system was temporally activate d by
LNP8 -formulated modRNA .
Treatment with modRNA with all tested LNP formulations did not ind uce the formation
of luciferase -specific IgGs on day 9 as measured by ELISA.
However, a strong antigen -specific IFN - T-cell response measured by ELISpot assay
on day 9 for LNP8 -formulated modRNAs , with statistically significant
differences betwe en these test groups and the buffer control
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7 DOCUMENT HISTORY
Reason s for change s compared to previous version:
Minor editorial changes, such as the correction of typing errors, are not listed.
Section s Version 01 Version 02 Reason for change
1
- More detailed
information
about LNP8
added Clarification that LNP8 contains ALC -0159 and
ALC-0315 3.1
6
4.1 Table added Details of LNP lipid component formulations
Section s Version 0 2 Version 03 Reason for change
4.1 - Test item
information
updated Diameter of LNP8 modRNA was corrected to
71 nm
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8 REFERENCES
Limberis MP , Bell CL, Wilson JM. Identification of the murine firefly luciferase -specific
CD8 T -cell epitopes. Gene Ther. 2009;16(3):441 -7.
Moyo N, Vogel AB, Buus S, Erbar S, Wee EG, Sahin U et al. Efficient Induction of
T Cells against Conserved HIV -1 Regions by Mosaic Vaccines Delivered as Self -
Amplifying mRNA. Molecular therapy. Methods & clinical development. 2018; 12,
32-46.
Pardi N, Hogan MJ, Pelc RS, Muramasu H, Andersen H, DeMaso CR et al. Zika virus
protection by a single low -dose nucleoside -modified mRNA vaccination. Nature. 2017;
543 (7644), 248 -251.
Vogel AB, Lambert L, Kinnear E, Busse D, Erbar S, Reuter KC et al. Self-ampl ifying
RNA vaccines give equivalent protection against influenza to mRNA vaccines but at
much lower doses. Molecular therapy: the journal of the American Society of Gene
Therapy. 2017; 26 (2), 446 -455.
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9 APPENDIX
Appendix 1: Animal O bservations
treatment Lab IDweight
in ghealthweight
in g% change
weighthealthweight
in g% change
weighthealthweight
in g% change
weighthealth
1-1 21.4 No observations 21.7 101.0 No observations 21.5 100.4 No observations 21.7 101.3 No observations
1-2 22.5 No observations 22.8 101.2 No observations 22.2 98.6 No observations 22.7 100.8 No observations
1-3 23.0 No observations 23.3 101.4 No observations 23.5 102.3 No observations 23.4 101.5 No observations
2-1 20.7 No observations 21.0 101.4 No observations 20.9 100.7 No observations 20.8 100.3 No observations
2-2 21.5 No observations 22.5 104.6 No observations 22.3 103.8 No observations 23.2 107.9 No observations
2-3 21.2 No observations 21.4 100.8 No observations 21.3 100.4 No observations 21.3 100.5 No observations
3-1 21.3 No observations 21.5 100.8 No observations 21.4 100.5 No observations 21.0 98.4 No observations
3-2 21.2 No observations 21.2 99.9 No observations 21.0 99.1 No observations 21.0 99.2 No observations
3-3 20.9 No observations 21.1 101.0 No observations 22.3 107.0 No observations 21.7 104.1 No observations
4-1 22.2 No observations 23.2 104.7 No observations 22.9 103.2 No observations 23.5 106.1 No observations
4-2 21.6 No observations 22.4 103.8 No observations 22.5 104.3 No observations 21.9 101.6 No observations
4-3 20.5 No observations 20.8 101.3 No observations 20.9 101.7 No observations 20.9 101.9 No observations 09.01.2020-5 days p.a.
Buffer control
modRNA-
Luciferase LNP8
(GMP-ready)modRNA-
Luciferase
Acuitas LNPs
modRNA
luciferase1 days p.a.
15.01.20206 days p.a.
20.01.20203 days p.a.
17.01.2020
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Appendix 2: Certi ficate s of Analysis
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Appendix 3: Raw Data IFN - ELISpot
Plate 1: Left, luciferase peptides ; right , AH1 ELISpot (irrelevant peptide)
Group 1: Buffer control; Group 2: (2x1 µg); Group 3:
(2x1 µg); Group 4: modRNA -luciferase LNP8 (GMP -ready) (2x1 µg)
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Appendix 4: Statistical Analysis
Bioluminescence assay
Group mean values , bioluminescence assay, luciferase signal over time
Time point Buffer control modRNA -
Luciferase LNP8
N = 3 N = 3
6 h 128046,667 1,2589e+009
24 h 227766,667 7,310667e+008
48 h 139995 2,1038333e+008
72 h 132585 7,8667e+007
6 d 162383,333 2920333,333
9 d 76573,333 509000
Please note that commas are used as decimal separators.
Descriptive statistics, bioluminescence assay, area under the curve
Buffer control modRNA -
Luciferase LNP8
Number of values 3 3
Minimum 657790 1352000000
Maximum 889908 1848000000
Range 232118 496000000
Mean 765040 1652666667
SD 117058 264244836
SEM 67583 152561827
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Tukey’s multiple comparisons post -test, bioluminescence
assay, area under the curve
ANOVA summary
F 80,68
P value <0,0001
P valu e summary ****
Significant diff. among means (P < 0.05)? Yes
R square 0,9680
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Tukey’s multiple
comparisons test Mean diff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer control vs.
modRNA -Luciferase
LNP8 -1651901626 -2072407467 to -
1231395786 Yes **** <0,0001
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
Luciferase -Specific ELISA
Descriptive statistics, luciferase -specific ELISA , day 9
Buffer control modRNA -
Luciferase LNP8
Number of values 3 3
Minimum 0,0110 0,0100
Maximum 0,0190 0,0220
Range 0,00800 0,0120
Mean 0,0153 0,0177
SD 0,00404 0,00666
SEM 0,00233 0,00384
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
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One-way ANOVA with Tukey’s multiple comparisons post -test, bioluminescence
assay, area under the curve
ANOVA summary
F 0,4597
P value 0,6520
P valu e summary ns
Significant diff. among means (P < 0.05)? No
R square 0,1209
No post -test for non-significant main test.
ELISpot analysis
Descriptive statistics, ELISpot analysis, day 9
Buffer control modRNA -
Luciferase LNP8
Number of values 3 3
Minimum 4,00 381
Maximum 148 749
Range 144 368
Mean 53,0 519
SD 82,3 201
SEM 47,5 116
Please note that commas are used as decimal separators. SD: Standard deviation. SEM: Standard error of the
mean.
One-way ANOVA with Tukey’s multiple comparisons post -test, bioluminescence
assay, area under the curve
ANOVA summary
F 19,90
P value 0,0005
P valu e summary ***
Significant diff. among means (P < 0.05)? Yes
R square 0,8819
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FDA-CBER-2021-5683-0013959
R&D Report R-20-0072 Version 03 Page 36 of 36
Strictly Confidential
Tukey’s multiple
comparisons test Mean diff. 95,00% CI of diff. Significant? Summary Adjusted P
value
Buffer control vs.
modRNA -Luciferase
LNP8 -465,7 -769,0 to -162,3 Yes ** 0,0051
Please note that commas are used as decimal separators. F: F -statistic . P values ≤ 0.05 indicate statistically
significant difference. R square: Coefficient of determination. CI: Confidence interval. n.s.: Not significant.
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FDA-CBER-2021-5683-0013960
1Von:
Gesendet: Freitag, 27. November 2020 07:24
An:
Cc:
Betreff: signatures: R-20-0072v3.0 (BNT162)
Anlagen: R-20-0072 Report V3.0 Biolumines cence in vivo_final_sig.pdf
Kennzeichnung: Zur Nachverfolgung
Kennzeichnungsstatus: Gekennzeichnet
Hello
With this email I’m giving my approval for the R&D report R-20-0072 version 3 update.
Best,
BioNTech SE
090177e195a2e6f3\Approved\Approved On: 27-Nov-2020 16:30 (GMT)
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FDA-CBER-2021-5683-0013961