Methodological Considerations Regarding the Quantification of DNA Impurities in the COVID-19 mRNA Vaccine Comirnaty

Abstract

DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a specific limit value must be set for them during marketing authorisation. This particularly applies to mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore, when quantifying the total DNA content in the final product, we must observe that, in addition to the mRNA active ingredient, DNA impurities are also encased in lipid nanoparticles and are therefore difficult to quantify. In fact, the manufacturer of the mRNA vaccine Comirnaty (BioNTech/Pfizer) only measures DNA impurities in the active substance by means of a quantitative polymerase chain reaction (qPCR), whose DNA target sequence is less than just 1% of the originally added DNA template. This means that no direct DNA quantification takes place, and compliance with the limit value for DNA contamination is only estimated from the qPCR data using mathematical extrapolation methods. However, it is also possible to dissolve the lipid nanoparticles with a detergent to directly measure DNA contamination in the final product by using fluorescence spectroscopic methods. Experimental testing of this approach confirms that reliable values can be obtained in this way.

Full text

Citation: König, B.; Kirchner, J.O.
Methodological Considerations
Regarding the Quantification of DNA
Impurities in the COVID-19 mRNA
Vaccine Comirnaty®.Methods Protoc.
2024,7, 41. https://doi.org/
10.3390/mps7030041
Academic Editor: Philip Hublitz
Received: 12 March 2024
Revised: 6 May 2024
Accepted: 7 May 2024
Published: 8 May 2024
Copyright: © 2024 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Commentary
Methodological Considerations Regarding the Quantification of
DNA Impurities in the COVID-19 mRNA Vaccine Comirnaty®
Brigitte König 1,2 and Jürgen O. Kirchner 3, *
1Magdeburg Molecular Detections GmbH & Co. KG, 39104 Magdeburg, Germany;
brigitte.koenig@medizin.uni-leipzig.de
2Institute of Medical Microbiology and Virology, Faculty of Medicine, University of Leipzig,
04103 Leipzig, Germany
3Independent Researcher, 22307 Hamburg, Germany
*Correspondence: j.o.kirchner@email.de
Abstract: DNA impurities can impact the safety of genetically engineered pharmaceuticals; thus, a
specific limit value must be set for them during marketing authorisation. This particularly applies to
mRNA vaccines, as large quantities of DNA templates are used for their production. Furthermore,
when quantifying the total DNA content in the final product, we must observe that, in addition to the
mRNA active ingredient, DNA impurities are also encased in lipid nanoparticles and are therefore
difficult to quantify. In fact, the manufacturer of the mRNA vaccine Comirnaty (BioNTech/Pfizer)
only measures DNA impurities in the active substance by means of a quantitative polymerase chain
reaction (qPCR), whose DNA target sequence is less than just 1% of the originally added DNA
template. This means that no direct DNA quantification takes place, and compliance with the limit
value for DNA contamination is only estimated from the qPCR data using mathematical extrapolation
methods. However, it is also possible to dissolve the lipid nanoparticles with a detergent to directly
measure DNA contamination in the final product by using fluorescence spectroscopic methods.
Experimental testing of this approach confirms that reliable values can be obtained in this way.
Keywords: mRNA vaccines; Comirnaty; DNA impurities; fluorescence spectroscopy; Qubit fluorometry
1. Considerations
Among genetically engineered drugs, those with mRNA active ingredients are a spe-
cial case, as their cell-free biosynthesis requires high concentrations of DNA templates,
which must be removed before the products can be used as drugs. In the case of the
COVID-19 mRNA vaccine Comirnaty
®
produced by BioNTech/Pfizer (BNT162b2) (Mainz,
Germany), these templates are produced by plasmids obtained from bacterial cultures [1].
Thus, Comirnaty
®
has a special quality: DNA impurities are possible due to the manufac-
turing process; this may be relevant for all genetically engineered drugs, but it is otherwise
rarely a problem [
2
]. This is due to the fact that genetically engineered active substances
are predominantly proteins, which can be easily separated from DNA due to their chemical
differences. Accordingly, DNA impurities in genetically engineered medicinal products
have so far only been a marginal issue. However, the situation is quite different with mRNA
vaccines: contaminating DNA and active ingredient mRNA are both nucleic acids and
therefore chemically so similar that separation is far more difficult than separating DNA
during the purification of protein active ingredients [3].
The addition of highly concentrated DNA templates, which are, in fact, linearized
plasmids, to the reaction mixture that is used to produce an mRNA vaccine therefore poses
a particular challenge for COVID-19 mRNA vaccines in terms of quality assurance with
regard to contaminating DNA. In addition, the active substance in the form of mRNA only
has low stability compared to contaminating DNA. Even exposure to room temperature
Methods Protoc. 2024,7, 41. https://doi.org/10.3390/mps7030041 https://www.mdpi.com/journal/mps

Methods Protoc. 2024,7, 41 2 of 8
can lead to the decay of RNA, whereas DNA remains stable for decades under the same
conditions in the absence of degrading enzymes [
4
]. The lipid nanoparticles used for drug
formulation, whose function is to transport the mRNA into the cells of a vaccinated person,
appear to be even more sensitive. Their disintegration, which already occurs at room
temperature, makes it necessary to store Comirnaty
®
at very low temperatures. In order to
achieve a shelf life that meets practical requirements, storage at
−
60 to
−
90
◦
C is therefore
prescribed. Storage at 2 to 8
◦
C is also permissible but considerably shortens the product’s
shelf life [5].
In order to remove the DNA templates that were added during the production process
and the accompanying residues of genomic DNA of the host bacteria after the production of
the mRNA active ingredient, DNA digestion with the enzyme DNase I is first carried out in
the reaction mixture after the completion of cell-free mRNA synthesis. Subsequent filtration
is intended to remove the resulting DNA fragments, while the mRNA is retained [6].
This may sound simple, but it must be borne in mind that the considerable chemical
instability of the mRNA used can pose a problem. This is particularly because DNase
digestion takes place at temperatures above 35
◦
C and under stirring—i.e., under conditions
that could lead to significant losses of the mRNA active ingredient if the exposure lasts
long enough. This means that DNA digestion must be limited in every respect, so that the
mRNA yield remains economical while, at the same time, the DNA content is kept below a
limit to be set in each case. This limit was set as part of the authorisation of Comirnaty
®
,
with a limit value of 10 ng DNA per dose [
6
,
7
], corresponding exactly to the relevant WHO
recommendations for genetically engineered medicinal products [2].
The fact that this limit value was successfully met in the production of Comirnaty
®
was generally accepted as a given after its authorisation. However, this dogma had to
be reconsidered after the US scientist Kevin McKernan and his team made it public that
they had found large quantities of DNA impurities in Comirnaty
®
[
8
], most of which were
present in quantities that were several hundred times higher than the applicable limit
of
10 ng
DNA per dose. Other scientists followed with their own results, including the
Canadian group led by David Speicher [
9
] and the US cancer researcher Phillip Buckhaults,
who presented his findings to the South Carolina Senate [10].
Is it therefore possible that the DNA quantifications carried out for Comirnaty
®
as part
of batch testing were incorrect? In order to verify this, it is first necessary to examine the
methodical procedure employed. This question primarily stems from a European Medicines
Agency (EMA) document that was created as part of the approval procedure and dates
from 19 November 2020 [
6
]. This source states that DNA quantification takes place in the
active substance after DNase digestion and filtration have been carried out. This document
also states that the method of choice for this DNA analysis is a quantitative polymerase
chain reaction, abbreviated as qPCR, wherein the target sequence is only
69 base
pairs of
the total 7824-base-pair-long DNA template, whereby the sequence of the T7 promoter is
integrated, an important step for the transcription process for the production of the mRNA
active substance. Therefore, only the presence of this sequence is checked; the remaining
7755 base pairs, and thus 99% of the template, and any remaining genomic DNA of the
host bacterium remain undetermined.
According to further official information from the German government [
11
], a the-
oretical DNA content is extrapolated from the measured value, obtained via this qPCR
measurement, and compared with the limit value of 10 ng DNA per dose. What this
means in detail is explained in the EMA document from 19 November 2020, which has
already been cited above [
6
]. According to this document, a dilution series is produced
with the linearized plasmid, which serves as a DNA template for
in vitro
transcription and
which, in turn, is to be measured using qPCR. A standard curve is generated from the
data obtained when measuring the dilution series. Finally, the measurement results of the
active substance samples are mathematically compared with this standard curve through
extrapolation. However, it is not clear from the description of the above-mentioned EMA
document that the processes to which the DNA templates, i.e., the linearized plasmids,

Methods Protoc. 2024,7, 41 3 of 8
are subjected during the manufacturing process are taken into account in any way. This
applies in particular to
in vitro
transcription, in addition to DNase and proteinase digestion
and filtration, processes that remove small DNA and protein fragments into which the
DNA templates and the added enzymes have been degraded. Anything that affects the
linearized plasmids during the manufacturing process does not appear to be taken into
account when creating the standard curve. However, this would be necessary to allow the
standard curve to actually reflect what the qPCR measures. This applies in particular to
what actually happens during DNase digestion. With this in mind, the following questions
are of the utmost importance:
1.
Which DNA fragments are specifically formed during DNase digestion, and is the
qPCR target sequence actually degraded proportionally to the entirety of the remain-
ing fragments of the linearized plasmids? Are distinct sequences of the linearized plas-
mids degraded more frequently or significantly less frequently by DNase
than others
?
2.
What influence do the
in vitro
transcription conditions have on the sequence of the T7
promoter, which is part of the qPCR target sequence? It should be considered that
the T7 promoter has a special affinity for the polymerase used, so the target sequence
may be at least partially masked by the polymerase or its fragments resulting from
proteinase digestion and therefore be potentially unmeasurable using qPCR.
3.
Does the target sequence, which is only 69 base pairs long, actually remain in a quan-
tity that is proportional to the other sequences remaining after DNase digestion and
the subsequent filtration steps? If the proportionality is not given, any extrapolation
is bound to be wrong.
These questions show that when using DNA quantification via qPCR, it is difficult
to obtain reproducible values that correspond to the actual ratios for the given question,
i.e., whether the limit value of 10 ng DNA per dose of the end product is adhered to.
Against this background, it is no surprise that the European Pharmacopoeia 2.6.35.
Quantification And Characterization of Residual Host-Cell DNA [
12
] states that qPCR is the
method of choice for the quantification of specific DNA sequences, while the measurement
of total DNA is not assigned to qPCR but to other methods.
A further communication from the German Federal Government [
7
] also states that
batch testing in Europe is carried out according to a protocol [
13
] published by the European
Directorate for Quality in Medicine, EDQM. This document confirms the following: apart
from the singular measurement at the active substance level conducted by the manufacturer,
no further experimental DNA quantification is carried out for the vaccine, especially not
for the final product, not even in the context of official batch testing.
This approach raises the question of how this can be justified. The answer can also
be found in an official statement made by the German government [
11
]. According to
this statement, the quantification of DNA impurities should be carried out in the active
substance, as a measurement in the ready-to-use vaccine could be disturbed by the lipid
nanoparticles that it contains, which could lead to incorrect values. At first glance, this
sounds acceptable. However, further examination of the EDQM protocol shows that the
mRNA active ingredient—a nucleic acid like DNA—is quantified despite the lipid nanopar-
ticles contained in the final product. But if the quantification of mRNA is not disturbed by
lipid nanoparticles, this should, in principle, also apply to the quantification of DNA due
to the common properties of nucleic acids. Hence, how is it that the quantification of RNA
in the end product is accepted as feasible by state institutions, while the quantification of
DNA at the same level of production, i.e., in the ready-to-use vaccine, is not?
Documents published by the Australian Therapeutic Goods Administration (Aus-
tralian Government, Department of Health) provide important facts for answering this
question. Firstly, there is a batch release document for Comirnaty
®
that has been issued by
Sciensano, the National Laboratory of Belgium [
14
]. This document reveals that RNA is
determined in the final vaccine using a fluorescence spectrometric method. Another doc-
ument published by the Australian Therapeutic Goods Administration [
15
] reveals what
this method is, as it provides validation data concerning this method. According to this

Methods Protoc. 2024,7, 41 4 of 8
document, the RNA-specific fluorescent dye RiboGreen
®
is used for mRNA quantification
in Comirnaty
®
at the level of the finished product. This dye binds highly specifically to
RNA, resulting in fluorescence that is proportionally dependent on the amount of RNA
that is present and can be measured. RiboGreen
®
, in turn, is one of the fluorescent dyes
that are part of the fluorometric Quant-iT
®
system produced by ThermoFisher Scientific
(Dreieich, Germany) [16].
Since Quant-iT
®
measurements are carried out using the analysers that are regularly
available in quality control laboratories, it is necessary to validate a method specifically
on the device used. Such a validation was recorded in the aforementioned documentation
published by the Australian Government [
15
]. These validation data also reveal that
for RNA quantification, it is necessary to disintegrate the lipid nanoparticles in order to
release the mRNA that is bound in them and make it accessible for measurement, wherein
this disintegration of the lipid nanoparticles is in turn carried out using the detergent
Triton-X-100 (final concentration 1%).
In addition to the RNA-specific RiboGreen
®
, the analogue but DNA-specific fluores-
cent dye PicoGreen
®
is also available as an alternative, so that DNA quantification in the
final vaccine can be carried out just as reliably as RNA quantification after the disintegration
of the lipid nanoparticles using Triton-X-100 [
15
]. In addition to the Quant-iT
®
system,
ThermoFisher Scientific also offers the Qubit
®
system for specific quantification using
fluorescent dyes. While Quant-iT
®
enables a higher sample throughput with standard
laboratory equipment (microtitre plate readers), Qubit
®
is the method of choice in laborato-
ries where no equipment for extensive routine tests is available and a comparatively low
sample throughput is expected [
17
]. Qubit
®
uses an automated fluorescence spectrometer
that can be combined with standardised Qubit
®
test kits. These Qubit
®
kits are optimised
for either RNA or DNA quantification, and standardised kits for protein quantification are
also available [
18
]. Due to this versatility, Qubit
®
is standard equipment in many molecular
biology laboratories. The excellent selectivity of Qubit
®
has been extensively validated and
documented by the manufacturer, as has the low influence of impurities contained in the
samples. In particular, it was proven that high quantities of RNA do not alter Qubit DNA
quantification, while the Nanodrop
®
spectrophotometer failed in this regard [
19
]. Qubit
®
therefore has an advantage over Quant-iT
®
: certain test validations that are required when
using Quant-iT
®
on the standard device used can be omitted due to the manufacturer’s
calibration [
18
]. The two systems, Quant-iT
®
and Qubit
®
, therefore correspond to each
other in terms of functionality. This means that both Quant-iT
®
and Qubit
®
can distinguish
DNA and RNA with the highest reliability using highly specific binding fluorescent dyes.
It was therefore necessary to investigate the practical suitability of Qubit for the
quantification of total DNA in Comirnaty. To this end, a series of experiments were carried
out, involving both RNA and DNA quantification (details are given in the Supplementary
Materials, including data on possible confounding factors).
Figure 1shows the results of measuring mRNA with Qubit
®
in seven batches of
Comirnaty
®
without and after treatment with Triton-X-100. Four batches were already
expired, while three batches had a remaining shelf life of 11 to 13 months. The results clearly
show that the treatment of Comirnaty
®
with Triton-X-100 leads to a significant increase in
RNA values. In the specific series of tests carried out, this effect appears to depend partly
on whether the batch had already expired at the time of measurement or whether it still had
a long shelf life. This means that in two of four expired batches, also without Triton-X-100,
over 50% of the total RNA was measurable. This suggests that in expired batches, the lipid
nanoparticles are disintegrated, even without Triton-X-100, whereas in vaccines with a long
shelf life, 97 to 99% of the total RNA was only measurable after the lipid nanoparticles were
dissolved with Triton-X-100 (further details are included in the Supplementary Materials).

Methods Protoc. 2024,7, 41 5 of 8
Methods Protoc. 2024, 7, 41 5 of 8
on whether the batch had already expired at the time of measurement or whether it still
had a long shelf life. This means that in two of four expired batches, also without Triton-
X-100, over 50% of the total RNA was measurable. This suggests that in expired batches,
the lipid nanoparticles are disintegrated, even without Triton-X-100, whereas in vaccines
with a long shelf life, 97 to 99% of the total RNA was only measurable after the lipid na-
noparticles were dissolved with Triton-X-100 (further details are included in the Supple-
mentary Materials).
Figure 1. Quantification of total RNA in batches of Comirnaty® using Qubit® fluorometry without
and with the addition of Triton-X-100 as a detergent to disintegrate the lipid nanoparticles contained
in the vaccine formulation. The measured values shown as bars in the figure refer to the total RNA
content in ng per dose of ready-to-use diluted Comirnaty®. In all batches, it was found that the
measured RNA value increased considerably after treatment with Triton-X-100. As expected, this
could only be a consequence of the dissolution of the lipid nanoparticles and the resulting release
of the RNA that was bound in them. In batches 1 to 4, which had all expired, it was found that after
treatment with Triton-X-100, between 36 and 97% of the maximum measured RNA value had be-
come accessible for measurement due to the dissolution of the lipid nanoparticles, while in batches
5 to 7, which still had a shelf life of 11 to 13 months, this value was between 97 and 99% of the total
RNA. Two of four expired batches may have largely disintegrated even without treatment with a
detergent, whereas this was only caused by Triton-X-100 in the batches with a longer shelf life. Irre-
spective of this, however, very high RNA values were measurable in all batches after Triton-X-100
treatment, significantly exceeding the target value for one dose of 30 µg (30,000 ng). * Target value
for one dose: 30,000 ng (30 µg) of RNA (300 µL of ready-to-use Comirnaty). ** Total RNA ng/dose
after treatment with 1% Triton-X-100.
However, if the mRNA active substance was quantifiable using fluorescence spec-
trometry in the final mRNA vaccine after it was treated with Triton-X-100, this should also
be possible for the DNA impurities as an integrated part of the batch testing of Co-
mirnaty®.
In order to verify this assumption, corresponding DNA quantifications in ready-to-
use diluted Comirnaty® batches with and without Triton-X-100 were conducted. Figure 2
shows the results (further details are included in the Supplementary Materials): if Co-
mirnaty® is treated with Triton-X-100, the result is a significant increase in DNA values
for some of the batches but not for others. In the specific series of tests carried out, this
effect appears to depend on whether the batch had already expired at the time of meas-
urement or whether there was still a long shelf life of 11 or more months at the time of
measurement. This suggests, as already found via mRNA testing, that in expired batches,
the lipid nanoparticles are at least partly disintegrated even without Triton-X-100,
whereas in vaccines with a long shelf life, they are still largely intact and include the DNA
impurities, so they are not fully accessible for measurement due to this compartmentali-
sation.
Figure 1. Quantification of total RNA in batches of Comirnaty
®
using Qubit
®
fluorometry without
and with the addition of Triton-X-100 as a detergent to disintegrate the lipid nanoparticles contained
in the vaccine formulation. The measured values shown as bars in the figure refer to the total RNA
content in ng per dose of ready-to-use diluted Comirnaty
®
. In all batches, it was found that the
measured RNA value increased considerably after treatment with Triton-X-100. As expected, this
could only be a consequence of the dissolution of the lipid nanoparticles and the resulting release of
the RNA that was bound in them. In batches 1 to 4, which had all expired, it was found that after
treatment with Triton-X-100, between 36 and 97% of the maximum measured RNA value had become
accessible for measurement due to the dissolution of the lipid nanoparticles, while in batches 5 to 7,
which still had a shelf life of 11 to 13 months, this value was between 97 and 99% of the total RNA.
Two of four expired batches may have largely disintegrated even without treatment with a detergent,
whereas this was only caused by Triton-X-100 in the batches with a longer shelf life. Irrespective of
this, however, very high RNA values were measurable in all batches after Triton-X-100 treatment,
significantly exceeding the target value for one dose of 30
µ
g (30,000 ng). * Target value for one dose:
30,000 ng (30
µ
g) of RNA (300
µ
L of ready-to-use Comirnaty). ** Total RNA ng/dose after treatment
with 1% Triton-X-100.
However, if the mRNA active substance was quantifiable using fluorescence spectrom-
etry in the final mRNA vaccine after it was treated with Triton-X-100, this should also be
possible for the DNA impurities as an integrated part of the batch testing of Comirnaty®.
In order to verify this assumption, corresponding DNA quantifications in ready-to-use
diluted Comirnaty
®
batches with and without Triton-X-100 were conducted. Figure 2shows
the results (further details are included in the Supplementary Materials): if Comirnaty
®
is
treated with Triton-X-100, the result is a significant increase in DNA values for some of the
batches but not for others. In the specific series of tests carried out, this effect appears to
depend on whether the batch had already expired at the time of measurement or whether
there was still a long shelf life of 11 or more months at the time of measurement. This
suggests, as already found via mRNA testing, that in expired batches, the lipid nanoparticles
are at least partly disintegrated even without Triton-X-100, whereas in vaccines with a long
shelf life, they are still largely intact and include the DNA impurities, so they are not fully
accessible for measurement due to this compartmentalisation.
Methods Protoc. 2024, 7, 41 6 of 8
Figure 2. Quantification of total DNA in batches of Comirnaty® using Qubit® fluorometry without
and with the addition of Triton-X-100 as a detergent to disintegrate the lipid nanoparticles contained
in the vaccine formulation. The measured values shown as bars in the figure refer to the total DNA
content in ng per dose of ready-to-use diluted Comirnaty®. These measurement results must be
compared with the limit value for the total DNA content of 10 ng DNA per dose for Comirnaty®.
One dose consists of 300 µL of ready-to-use vaccine. In all batches, it was found that the measured
DNA value increased considerably after treatment with Triton-X-100. As expected, this could only
be a consequence of the dissolution of the lipid nanoparticles and the resulting release of the DNA
that was bound in them. In batches 1 to 4, which had all expired, it was found that after treatment
with Triton-X-100, between 16 and 81% of the maximum measured DNA value had become acces-
sible for measurement due to the dissolution of the lipid nanoparticles, while in batches 5 to 7, which
still had a shelf life of 11 to 13 months, this was even as high as between 93 and 97% of the total
DNA. This indicates that the lipid nanoparticles from expired batches may have largely disinte-
grated even without treatment with a detergent, whereas this was only caused by Triton-X-100 in
the batches with a longer shelf life. Irrespective of this, however, very high DNA values were meas-
urable in all batches after Triton-X-100 treatment, with these values ranging from 360 to 534 times
the permissible DNA limit or 3600 to 5340 ng DNA per dose. * Threshold of 10 ng of DNA/dose (300
µL of ready-to-use Comirnaty). ** Total DNA ng/dose after treatment with 1% Triton-X-100.
2. Conclusions
The available information and data indicate that the ready-to-use mRNA vaccine Co-
mirnaty contains DNA impurities that exceed the permied limit value by several hun-
dred times and, in some cases, even more than 500 times, and that this went unnoticed
because the DNA quantification carried out as part of batch testing only at the active sub-
stance level appears to be methodologically inadequate when using qPCR, as explained
above. Because of the conditions during the production of the mRNA active substance of
Comirnaty, the applied qPCR is designed so that a massive under-detection of DNA im-
purities appears to be the result. Here, we have to remember that qPCR is matchless if
specific DNA sequences are being quantified, but this is not the case if the aim is the quan-
tification of the total DNA content. However, DNA contamination in Comirnaty is about
total DNA, regardless of the sequences that it contains. Accordingly, it can be assumed
that a fluorescence spectrometric measurement of the total DNA in the end product, anal-
ogous to the quantification of the mRNA active ingredient, a process that is, in fact, carried
out in the end product, is not associated with a risk of under-detecting DNA contamina-
tions but rather provides reliable values and thus satisfies the required level of drug safety.
Against this background, experimental testing of the total DNA contained in the
ready-to-use diluted vaccine Comirnaty® via fluorescence spectrometric measurement,
which is to be carried out by the authorities as part of the legal mandate for official batch
testing, appears to be essential. Why this was systematically omied by the European con-
trol laboratories according to the statements by the German Federal Government cited
above should therefore be the subject of extensive expert discussions and reconsidera-
tions.
Figure 2. Quantification of total DNA in batches of Comirnaty
®
using Qubit
®
fluorometry without
and with the addition of Triton-X-100 as a detergent to disintegrate the lipid nanoparticles contained

Methods Protoc. 2024,7, 41 6 of 8
in the vaccine formulation. The measured values shown as bars in the figure refer to the total DNA
content in ng per dose of ready-to-use diluted Comirnaty
®
. These measurement results must be
compared with the limit value for the total DNA content of 10 ng DNA per dose for Comirnaty
®
.
One dose consists of 300
µ
L of ready-to-use vaccine. In all batches, it was found that the measured
DNA value increased considerably after treatment with Triton-X-100. As expected, this could only be
a consequence of the dissolution of the lipid nanoparticles and the resulting release of the DNA that
was bound in them. In batches 1 to 4, which had all expired, it was found that after treatment with
Triton-X-100, between 16 and 81% of the maximum measured DNA value had become accessible for
measurement due to the dissolution of the lipid nanoparticles, while in batches 5 to 7, which still
had a shelf life of 11 to 13 months, this was even as high as between 93 and 97% of the total DNA.
This indicates that the lipid nanoparticles from expired batches may have largely disintegrated even
without treatment with a detergent, whereas this was only caused by Triton-X-100 in the batches with
a longer shelf life. Irrespective of this, however, very high DNA values were measurable in all batches
after Triton-X-100 treatment, with these values ranging from 360 to 534 times the permissible DNA
limit or 3600 to 5340 ng DNA per dose. * Threshold of 10 ng of DNA/dose (300
µ
L of ready-to-use
Comirnaty). ** Total DNA ng/dose after treatment with 1% Triton-X-100.
2. Conclusions
The available information and data indicate that the ready-to-use mRNA vaccine
Comirnaty contains DNA impurities that exceed the permitted limit value by several
hundred times and, in some cases, even more than 500 times, and that this went unnoticed
because the DNA quantification carried out as part of batch testing only at the active
substance level appears to be methodologically inadequate when using qPCR, as explained
above. Because of the conditions during the production of the mRNA active substance
of Comirnaty, the applied qPCR is designed so that a massive under-detection of DNA
impurities appears to be the result. Here, we have to remember that qPCR is matchless
if specific DNA sequences are being quantified, but this is not the case if the aim is the
quantification of the total DNA content. However, DNA contamination in Comirnaty
is about total DNA, regardless of the sequences that it contains. Accordingly, it can be
assumed that a fluorescence spectrometric measurement of the total DNA in the end
product, analogous to the quantification of the mRNA active ingredient, a process that is,
in fact, carried out in the end product, is not associated with a risk of under-detecting DNA
contaminations but rather provides reliable values and thus satisfies the required level of
drug safety.
Against this background, experimental testing of the total DNA contained in the
ready-to-use diluted vaccine Comirnaty
®
via fluorescence spectrometric measurement,
which is to be carried out by the authorities as part of the legal mandate for official batch
testing, appears to be essential. Why this was systematically omitted by the European
control laboratories according to the statements by the German Federal Government cited
above should therefore be the subject of extensive expert discussions and reconsiderations.
Further, it should also be taken into account that DNA impurities in Comirnaty
®
are
apparently integrated into the lipid nanoparticles and are thus transported directly into the
cells of a vaccinated person, just like the mRNA active ingredient. What this means for the
safety risks, particularly the possible integration of this DNA into the human genome, i.e.,
the risk of insertional mutagenesis, should be a secondary focus of the discussion required,
which must go far beyond what could have been considered years before the so unexpected
introduction of mRNA pharmaceuticals into the global market.
Supplementary Materials: The following supporting information can be downloaded at
https://www.mdpi.com/article/10.3390/mps7030041/s1: Table S1. Qubit
®
fluorometry: Quan-
tification of total RNA in batches of Comirnaty
®
without and with Triton-X-100. Table S2. Qubit
®
fluorometry: Quantification of total DNA in batches of Comirnaty
®
without and with Triton-
X-100.
Table S3. Qubit®
fluorometry: Influence of DNA concentration on DNA quantification.

Methods Protoc. 2024,7, 41 7 of 8
Table S4. Qubit®
fluorometry: Influence of Triton-X-100 on DNA quantification. Table S5. Qubit
®
fluorometry: DNA quantification in the mRNA vaccine in the presence of 1% Triton-X-100.
Author Contributions: Conceptualization, B.K. and J.O.K.; methodology, B.K.; Writing—original
draft, J.O.K.; writing—review & editing, B.K. and J.O.K.; project administration, B.K. and J.O.K. All
authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The original contributions presented in the study are included in the
article/Supplementary Material, further inquiries can be directed to the corresponding author.
Acknowledgments: The authors wish to thank Irina Kouznetsova for technical and scientific support.
Conflicts of Interest: B.K. is CEO of Magdeburg Molecular Detection GmbH & Co. KG. The company
played no role in the design of the study, the collection, analysis, or interpretation of data, the writing
of the manuscript, or the decision to publish the article.
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