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International Journal of Vaccine Theory, Practice, and Research 3(1) January 26, 2023 | Page 787
https://doi.org/10.56098/ijvtpr.v3i1.68
Chemical-Physical Criticality and Toxicological Potential of
Lipid Nanomaterials Contained in a COVID-19 mRNA
Vaccine
Gabriele Segalla, PhD
Pure Chemistry (Organic Biological Chemistry), specialist in chemistry of micro-emulsions and colloidal systems, CEO
& Chief Scientist of Multichem R&D Italy, email: gabriele.segalla@gmail.com
ORCID: https://orcid.org/0000-0002-5969-3732
Abstract
The medicinal preparation called Comirnaty by Pfizer-BioNTech is an aqueous dispersion of lipid nanomaterials,
intended to constitute, after thawing and dilution, the finished product for intramuscular injection. In the
present study, we examine some evident chemical-physical criticalities of the preparation, regarding the
manifest instability of its qualitative-quantitative composition, as well as its consequent toxicological potential,
in this case related to the possible formation of ROS (reactive oxygen species), after intramuscular inoculation, in
different biological sites, such as, potentially, kidneys, liver, heart, brain, etc., causing dysfunctions and
alterations thereof. Of particular concern is the presence in the formulation of the two functional excipients,
ALC-0315 and ALC-0159, never used before in a medicinal product, nor registered in the European
Pharmacopoeia, nor in the European C&L inventory. The current Safety Data Sheets of the manufacturer are
omissive and non-compliant, especially with regard to the provisions of current European regulation on the
registration, evaluation, authorization and restriction of nanomaterials. The presence of electrolytes in the
preparation and the subsequent dilution phase after thawing and before inoculation raise well-founded
concerns about the precarious stability of the resulting suspension and the polydispersity index of the
nanomaterials contained in it, factors that can be hypothesized as the root causes of numerous post-vaccination
adverse effects recorded at statistical-epidemiological levels. Further immediate studies and verifications are
recommended, taking into consideration, if necessary and for purely precautionary purposes, the immediate
suspension of vaccinations with the Pfizer-BioNTech Comirnaty preparation.
Keywords: COVID-19 mRNA vaccine, LNP, lipid nanoparticles, nanomaterials, nanoforms, electrolytes, reactive
oxygen species, aggregate, agglomerate
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INTRODUCTION
The medicinal product called Comirnaty COVID-19 mRNA BNT162b2 is a concentrated semi-
finished product, based on a particular strand of mRNA encapsulated in lipid nanoparticles (LNPs),
and intended to constitute, after the phases of thawing and dilution with sodium chloride solution, a
dispersion of nanomaterials injectable intramuscularly. It was placed on the market in Europe, with
conditional marketing authorisation issued by EMA (European Medicines Agency) on 21 December 2020
and first Assessment Report on 19 February 2021 (EMA/707383/2020, 2021).
Nanomaterials (also called nanoparticles or nanoforms) are defined and described by ECHA (European
Chemicals Agency) as follows (with my emphasis in italics added here and throughout the remaining
quoted entries in this paper):
Nanomaterials are chemical substances or materials with particle sizes between 1 to 100 nanometers in at
least one dimension.
1
Due to an increased specific surface area by volume, nanomaterials may have different characteristics
compared to the same material without nanoscale features. As a result, the physicochemical properties of
nanomaterials may differ from those of bulk substances or particles of a larger size.
Many everyday products containing nanomaterials are already on the European market such as
batteries, coatings, anti-bacterial clothing and cosmetics. While nanomaterials may offer technical
and commercial opportunities, they may also pose risks to our health and the environment. Just like any
other substance on the EU market, it is important to ensure that their uses are properly assessed and that any
risks are adequately controlled.
Already in 2011, the European Commission published a “Recommendation” containing
the definition of nanomaterial, inviting member states, union agencies and economic
operators to use it in the adoption and implementation of legislation and strategic and
research programs related to nanotechnology products, in particular by making appropriate
amendments in several European regulations, including Regulation (EC) No 1907/2006
(REACH) and Regulation (EC) No 1272/2008 (CLP), in order to harmonize the way
nanomaterials were defined in the different legal frameworks.
This Recommendation was subsequently accepted and included in the Commission Regulation (EU)
2018/1881 entered into force on January 1, 2020, which, in addition to introducing some substantial
changes to the REACH Regulation, set out a much more articulated and complete definition of
nanomaterial, introducing specific indications for registration, evaluation, authorization and
restrictions concerning the so-called nanoforms.
On page 8 of this Regulation we read:
Definition of a nanoform and a set of similar nanoforms:
On the basis of the Commission Recommendation of 18 October 2011 on the definition of
nanomaterial, a nanoform is a form of a natural or manufactured substance containing particles, in an unbound
state or as an aggregate or as an agglomerate and where, for 50% or more of the particles in the number
size distribution, one or more external dimensions is in the size range 1 nm-100 nm, including also by
derogation fullerenes, graphene flakes and single wall carbon nanotubes with one or more external
dimensions below 1 nm. For this purpose, “particle” means a minute piece of matter with defined
physical boundaries; “agglomerate” means a collection of weakly bound particles or aggregates
1
It is important to realize that 1 nanometer = 1 billionth of a meter = 1 millionth of a millimeter, a size that is tens of
thousands of times smaller than the thickness of a human hair.
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where the resulting external surface area is similar to the sum of the surface areas of the individual
components and “aggregate” means a particle comprising of strongly bound or fused particles.
Why is it important to characterize and distinguish between the various types of nanoforms, such as
individual particles and their possible “aggregates” or “agglomerates”? It should be emphasized that
the criteria for assessing the hazard and toxicity of nanoforms are substantially those related to their
size. In fact, in the above legally binding definition, there is no reference to the chemical composition
(organic or inorganic) of the material under consideration, but only to the size of the particles that
constitute it, whether of natural, derived or synthetic origin. In particular, in order to assess their
toxicological profile, first of all the chemical-physical characteristics, and in particular the size of the
particles, their numerical size distribution, their shape and other morphological parameters (such as
crystallinity, information on the whole nanometric assembly, including for example shell structures
or hollow structures, etc.), their surface area (volume-specific, mass-specific area, or both) must be
taken into account, as well as their molecular structures (EU Commission Reg. 2018, p. 10).
COMPOSITION AND NANOMATERIALS OF THE COMIRNATY
COVID-19 mRNA VACCINE BNT162B2
As is now well known, the Pfizer-BioNTech COVID-19 vaccine, generally called “Comirnaty
BNT162b2”, contains a particular strand of mRNA encapsulated in lipid nanoparticles. These
nanoparticles have the primary function of protecting mRNA from enzymatic degradation and thus
allowing its penetration into the cells of the host organism, after intramuscular injection (Nance &
Meier, 2021).
Figure 1. Molecular structure of ALC-0315.
In the formulation, four specific lipid components are distinguished, capable of forming, in combination
with each other, nanoparticles dispersed in an aqueous medium:
a) ALC-0315 (ionizable, cationic functional lipid). Chemical name: ((4-hydroxybutyl (azanediyl)
bis (hexane-6,1-diyl) bis (2-hexyldecanoate)). CAS No. 2036272-55-4. Amphiphilic molecule
2
, of synthetic origin, consists of a tertiary amine structure with a hydroxy-butyl group and
two exilic groups esterified with 2-hexyldecanoic acid (Figure 1).
2
A molecule is called amphiphilic (also amphipathic) when it contains both a hydrophilic group (water-loving, polar) and a
lipophilic group (fat-loving, non-polar).
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b) ALC-0159 (functional lipid, non-ionic, polyethoxylated). Chemical name: 2
([polyethylene glycol]-2000)-N,N-ditetradecylacetamide. CAS No. 1849616-42-7.
Amphiphilic molecule, of synthetic origin, consisting of a di-myristil-amide of
hydroxyacetic acid, polyethoxylated with 45/50 moles of ethylene oxide (Figure 2).
c) DSPC (structural support phospholipid, “helper lipid”). Chemical name: 1,2-Distearoyl-sn-
glycero-3-phosphocholine. CAS No. 816-94-4. Molecule of semi-synthetic origin,
amphiphilic, consisting of a phosphoglyceride in which one group is phosphatidylcholine
and two groups are stearic acid chains (18:0) (Figure 3).
Figure 4. Schematic representation of the structure of a Pfizer-BioNTech
Comirnaty Vaccine nanoparticle (EMA/594686/2021 p. 15).
These four lipid components constitute the fundamental excipients of Comirnaty, instrumental to
the formation of lipid spheroidal nanoforms (Tenchov et al. 2021), i.e. lipid nanoparticles (LNPs )
Figure 2. Molecular structure of ALC-0159.
Figure 3. Cholesterol (lipid having functions, in this case, of structural support). Organic molecule
belonging to the class of sterols. CAS No. 57-88-5.
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of the type schematically represented in Figures 4 and 5, and intended to encapsulate, incorporate,
protect and convey the active substance, consisting of mRNA BNT162b2.
Figure 5. Suggested structures of lipid nanoparticle nucleic acid carriers: nucleic acids organized in
inverse lipid micelles inside the nanoparticle (A); nucleic acids intercalated between the lipid
bilayers (B) (Tenchov et al. 2021).
As stated by EMA in the aforementioned Comirnaty Assessment report of 19 February 2021
(EMA/707383, 2021), the nanoparticles, formed by the four lipids as described above, are solid
particles, held in suspension in an aqueous medium and in the presence of the so-called Phosphate
Buffered Saline (consisting of inorganic electrolytes), which maintains the pH at values between 6.9 and
7.9, and a sugar (sucrose), as a cryoprotective agent.
Figure 6. Assessment Report EMA/Pfizer-BioNTech Comirnaty, February 19, 2021,
page 140.
And, with regard to the two functional lipid ingredients, we read that, since the marketing
authorization is subject to conditions (EMA/707383, p. 140), the holder of this authorization (Pfizer-
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BioNTech) must complete, within the established timeframe, some specific tasks. Among these: “In
order to confirm the purity profile and ensure comprehensive quality control and batch-to-batch consistency throughout
the lifecycle of the finished product, the MAH
3
should provide additional information about the synthetic process and
control strategy” for both “new” lipid excipients ALC-0315 and ALC-0159. The expiry date of the
authorization for the delivery of this information is by July 2021, with interim reports scheduled for
January 2021 and April 2021. The final report on the clinical study “to confirm the efficacy and safety of
Comirnaty” is expected and required by December 2023 (Figure 6).
As is now known, at the date of this writing, the contents of the reports, presumably submitted by
the authorization holder within the scheduled dates (January 2021, April 2021, July 2021), have been
kept classified and undisclosed by EMA. For this reason, some inevitable and pressing questions
arise: does the additional information on the synthesis process and the control strategy, provided in the
interim reports, contain (or not) the evidences required by European legislation regarding the
registration and authorization of nanoforms? In other words, has all the information on the chemical-
physical and toxicological characteristics of the nanoforms of the medicinal product Comirnaty been provided?
And, if so, why keep it secret if it is obligatory by European law for every nanoform commercialized
in the European Community?
REGULATORY NON-COMPLIANCES AND ABSENCE OF
TOXICOLOGICAL STUDIES
LACK OF REGISTRATION IN PHARMACOPOEIA
All the ingredients of the medicinal product Comirnaty are known in the European Pharmacopoeia, except
ALC-0315 and ALC-0159. Both these nanomaterials are classified by EMA as “novel excipients” as
“never previously used in a medicinal product in Europe” and “not registered in the EU
Pharmacopoeia” (EMA/707383, p. 23).
It is disconcerting to see that a medicinal product that has been manufactured, authorized and
administered in billions of doses contains ingredients that have never been registered in the
Pharmacopoeia. The significance and gravity of such an omission is understood by reading the
description of the Purpose of the European Pharmacopoeia:
The European Pharmacopoeia is a single reference work for the quality control of medicines in
the signatory states of the Convention on its elaboration.
The official standards published within provide a legal and scientific basis for quality control during the
development, production and marketing processes.
They concern the qualitative and quantitative composition and the tests to be carried out on
medicines, on the raw materials used in production of medicines and on the intermediates of
synthesis. All producers of medicines and/or substances for pharmaceutical use must therefore apply these quality
standards in order to market their products in the signatory states of the Convention […]
The purpose of the European Pharmacopoeia is to promote public health by the provision of recognized common
standards for the quality of medicines and their components. Such standards are to be appropriate as a basis for
the safe use of medicines by patients. In addition, their existence facilitates the free movement of
medicinal products in Europe and beyond.
European Pharmacopoeia monographs and other texts are designed to be appropriate to the needs of:
3
Marketing Authorization Holder.
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- regulatory authorities;
- those engaged in the quality control of medicinal products and their constituents;
- manufacturers of medicinal products and their individual components.
The European Pharmacopoeia is widely used internationally. As globalization and expansion in
international trade present a growing need to develop global quality standards for medicines, the
Commission works closely with all users of the Pharmacopoeia worldwide. [my emphasis] (EU
Pharmacopoeia, 2023).
OMISSIVE AND NON-COMPLIANT SAFETY DATA SHEETS
In addition to being unknown to the European Pharmacopoeia, the two lipid components ALC-
0315 and ALC-0159 are not even reported in the C&L inventory.
4
Consequently, they do not have a
REACH registration number and their CLP classification is not known. In other words, their general
toxicological profile is not officially known — neither as substances, nor as nanoforms made up of
them. This is also confirmed by what is stated in section 3 (Composition/Ingredient Information) of the
Pfizer-BioNTech COVID-19 vaccine Product Safety Data Sheet, dated 7 December 2021 (Figure 7),
where, under the heading Classification according to Regulation (EC) No 1272/2008 (CLP) appears the
note “No data available”, and under the heading REACH Registration Number, no number appears.
Figure 7. Safety Data Sheet, section 3 dated 7 December 2021 of the Pfizer-BioNTech COVID-19 vaccine.
This contrasts with what we read on the official website of the European Union (Your Europe):
If you manufacture or import one ton or more per year of a chemical substance in the EEA, you
must record this in the REACH database. REACH stands for the Registration, Evaluation,
Authorisation and Restriction of Chemicals.
REACH applies to all chemical substances, both those needed for industrial processes and those
we use in our everyday lives, in paints, cleaning products, clothes, furniture and electrical
appliances, for example. It thus affects most businesses in the European Economic Area (EEA).
Non-registered substances must not be marketed or used. [my emphasis]
4
The C&L inventory is a database managed by ECHA that contains information on the classification and labelling of
substances placed on the European market. This database includes information on notified and registered substances,
but also the list of harmonised classifications and labelling according to Annex VI of the CLP Regulation
https://echa.europa.eu/information-on-chemicals/cl-inventory-database.
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The characteristics of the particles specifying the nanoform, as required and described in Annex VI
of the REACH Regulation, should also be indicated in the Safety Data Sheet of the manufacturer:
If the substance is registered and it covers a nanoform, the particle characteristics that specify the
nanoform, as described in Annex VI, shall be indicated.
If the substance is not registered, but the safety data sheet covers nanoforms, the particle characteristics of which
have impact on the safety of the substance, those characteristics shall be indicated. [my emphasis] (EU
Commission Reg. 2020/878, p.34).
On the contrary, although it is expressly indicated, in section 1 of the same Pfizer-BioNTech Safety
Data Sheet, that the product is a nanoform and belongs to the Chemical Family called lipid nanoparticles
Figure 8. In section 1.1 of the Pfizer-BioNTech Safety Data Sheet, dated 7 December 2021, version 3,
the nanoform configuration of the product is expressly indicated.
containing PF-07305885 (BNT162b2) and Lipids (Figure 8), the characteristics of the nanomaterial present in
the composition are not reported in any other section of the document, as opposed to the dictates of
Regulation (EU) 2020/878, which clearly prescribe, on page 45, how the characteristics of
nanoforms must be reported in subsection 9.1 of the Safety Data Sheet, as follows:
[…] (r) Particle characteristics:
Only apply to solids.
The particle size (median equivalent diameter, method of calculation of the diameter (number-,
surface- or volume-based) and the range in which this median value varies), shall be indicated.
Other properties may also be indicated, such as size distribution (e.g. as a range), shape and aspect
ratio, aggregation and agglomeration state, specific surface area and dustiness. If the substance is in
nanoform or if the mixture supplied contains a nanoform, those characteristics shall be indicated in this subsection,
or referred to if already specified elsewhere in the safety data sheet. [my emphasis]
Subsection 9.1 of the Pfizer-BioNTech Safety Data Sheet is shown in Figure 9. Under Particle
characteristics, it reads surprisingly:
No information available.
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Figure 9. Section 9.1 of the Safety Data Sheet, version 3, dated 7 December 2021, of the Pfizer-
BioNTech Comirnaty Vaccine.
NO CARCINOGENICITY, GENOTOXICITY AND MUTAGENICITY STUDIES
The analysis of the characteristics of nanoparticles (size, total surface area, state of aggregation or
agglomeration, polydispersity index, surface charge, etc.), as already described above and as expressly
reiterated in the aforementioned regulations, is essential in order to determine their possible
cytotoxic, genotoxic, mutagenic and carcinogenic potential. The state of agglomeration, in particular,
can in itself represent an important risk factor, as it can affect not only the translocation of
nanomaterials in or through various organs and tissues, but also the degree of accumulation within those
tissues and, consequently, the related catabolic elimination processes. (Bruinink et al., 2015). Despite this,
EMA, in its report dated 19 February 2021, regarding the assessment of the Comirnaty vaccine,
writes:
No genotoxicity nor carcinogenicity studies have been provided. The components of the vaccine
formulation are lipids and RNA that are not expected to have genotoxic potential. (EMA/707383, 2021,
p. 55)
As per guidance, no genotoxicity nor carcinogenicity studies were performed. The components of
the vaccine (lipids and mRNA) are not expected to have genotoxic potential. This is acceptable to the
CHMP.
5
[my emphasis] (EMA/707383, 2021, p. 56).
Note how, in expressing a hypothetical absence of genotoxicity and carcinogenicity of the lipid
components of the Comirnaty vaccine, EMA seems to ignore that the two novel excipients ALC-0315
(ionizable, cationic) and ALC-0159 (non-ionic, polyethoxylated) are not simple “lipids”, but, as
additionally and widely described in other sections of the same EMA report, they are “functional”
lipids, that is to say fundamental and determinant in order to carry out the formation, in situ, of lipid
nanoparticles, that is, substances subjected to all the aforementioned European provisions and
regulations on the registration, evaluation, authorization and restriction of Nanomaterials. Note that
these are provisions and regulations including, among other things, the obligation for manufacturers to
provide ALSO the appropriate genotoxicity and carcinogenicity tests specifically prescribed for nanoforms.
In particular, as stated on page 6 of the aforementioned Regulation (EU) 2018/1881 concerning
nanoforms:
The assessment should always include a statement as to whether the substance or, when
applicable, nanoforms thereof fulfils or does not fulfil the criteria given in Regulation (EC) No
5
CHMP: European Committee for Medicinal Products for Human Use.
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1272/2008 for classification in the hazard class carcinogenicity category 1A or 1B, in the hazard class
germ cell mutagenicity category 1A or 1B or in the hazard class reproductive toxicity category 1A or 1B.
[my emphasis]
It is now universally established that among the greatest risks to human health caused by
the exceptional penetrability and mobility of nanoforms within biological systems, those
related to genotoxicity and carcinogenicity must be taken into account. The related in vitro
assays are considered an extremely important, if not indispensable, tool for a thorough
understanding of the toxicity mechanisms and an adequate assessment of the health risks caused
by nanomaterials, especially in the medium to long term (Barone et al., 2017).
Equally non-compliant, and in conflict with the now consolidated regulatory-toxicological
practice relating to nanoforms, Section 11 (Toxicological information) of the Pfizer-
BioNTech Safety Data Sheet, with reference to the Comirnaty product says: Toxicological
properties have not been thoroughly investigated (Figure 10). The only toxicological information
reported in this section is that relating to the individual components, including, for
example, the toxicological profiles of sugar (sucrose) and common table salt (sodium
chloride), but excluding those of the aforementioned nano-functional lipids ALC-0315 and
ALC-0159. Also, there is no mention, in that section, of nanomaterials in the composition,
nor is there any reference to the toxicological assays required by law on nanoforms.
Figure 10. Section 11 (Toxicological Information) of the Pfizer-BioNTech Safety Data Sheet,
version 3, dated 7 Dec 2021.
REACTIVE OXYGEN SPECIES (ROS) FORMATION AND
NANOPARTICLE TOXICITY
It is also important to note that the main lipid component in the Pfizer-BioNTech
formulation, ALC-0315, being made up of a tertiary amine, tends to be protonated in a
moderately low pH environment, thus giving rise to the formation of cationic nanoforms, i.e.
having a positive surface charge. In fact, it is thanks to the attraction with the portions of the
mRNA having negative electric charge that the formation of spheroidal nanoforms takes
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place, such as those illustrated in Figure 5. What seems to be ignored in EMA's Assessment
Reports and Pfizer-BioNTech Safety Data Sheets is that experimental data show that
cytotoxic and genotoxic effects are enhanced if nanoparticles have a positive charge (Barone et al., 2017;
Fröhlich, 2012).
Nanoparticles consisting of monovalent cationic lipids, such as ALC-0315, have in fact been shown
to be significantly more efficient in inducing cell death through the production of reactive oxygen
species (ROS).
It is now confirmed by numerous studies that the toxic effects produced by nanoparticles in
biological systems are mainly and substantially due to the formation of ROS inside cells. ROS are
particles that contain oxygen, among which the most relevant are hydrogen peroxide (H2O2), superoxide
anion radical (O2-) and hydroxyl radicals (•OH).
They are predominantly
produced in cellular
organelles such as the
endoplasmic reticulum
(ER), peroxisomes, and
particularly in
mitochondria.
Nanoparticles
containing monovalent
cationic lipids have been
widely used in
anticancer therapies for
the administration of
nucleic acids such as
siRNA and
polypeptides, directly
into target cells.
However, several studies
have shown that cationic
liposomes induce ROS
formation and ROS-
mediated toxicity in
healthy cells and, at the
same time, reduce cell
viability. For example,
depending on lipid
concentration, surface
density of cationic lipids
and particle size,
nanoparticles containing
cationic lipids can lead
to ROS generation and
death of HepG2 liver cancer cells (Yun et al., 2016).
Figure 11. “Cellular events induced by Nanoparticles (NPs). ① NPs contribute to
the destruction of the cell membrane and to lipid peroxidation. ② The lysosomal
membrane is destroyed by NPs and results in the release of their contents. ③ The
mitochondrial membrane is damaged by NPs, leading to content release. NPs
reduce the generation of ATP and increase the production of ROS. ④ The ROS
induced by NPs results in the mistranslation of RNA. ⑤ NPs prevent the binding
of tRNA to the ribosome. ⑥ The ROS induced by NPs result in the
polymerization of proteins and DNA. ⑦ The ROS induced by NPs leads to DNA
mutations ⑧ The nuclear membrane is destroyed by NPs, resulting in the release
of its contents” (Yu et al., 2020).
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In almost all scientific studies on the subject, it is noted that, despite the undoubted benefits and
progress made in the use of nanomaterials in the biomedical field, concerns remain about the
potential toxicological effects of nanoparticles, especially in relation to their tendency to generate
reactive oxygen species. Due to their strong oxidation potential, excess ROS induced by
nanoparticles can cause damage to biomolecules and cell organelle structures. They can produce
oxidative carbonylation of proteins, lipid peroxidation, DNA/RNA breakdown, and destruction of
cell membranes, factors that can induce a complex of pathophysiological effects, such as
genotoxicity, necrosis, apoptosis, cytokine inflammation, fibrosis, metaplasia, hypertrophy,
carcinogenicity, or even mutagenesis impacting future generations (Yu et al., 2020; Figure 11).
Furthermore, Yu et al. point out that the extreme penetration and mobility of nanoparticles within the body
account for their easy entry into the systemic circulation and accumulation in organs such as kidneys, liver, heart,
brain, intestinal tract, and lungs, causing dysfunctions and alterations.
There is now overwhelming evidence that overproduction of ROS is the main cause of nanoparticle
biotoxicity. By concentrating mainly in lysosomes, mitochondria, and the nucleus of the cell, and
generating ROS at those sites, nanoparticles can cause devastating consequences. Numerous studies
irrefutably confirm that nucleotides components of cellular DNA and RNA constitute a
significantly vulnerable target to the aggression of ROS generated by nanomaterials. (Imlay et al.,
1988; Maki et al., 1992; Demple et al., 1994).
This can result in irreparable genetic damage, resulting in the development of genotoxicity, (Kang et
al., 2008; Singh et al., 2009; Chompoosor et al., 2010; Di Bucchianico et al., 2013; Proquin et al.,
2017), mutagenicity (Kirsch-Volders et al., 2002; Mateuca et al., 2006; Dufour et al., 2006; Levine et
al., 2017; Jena, 2012), carcinogenicity (Rusyn et al., 2004; Nel et al., 2006; Liou et al., 2010; Tretyakova et
al., 2015).
The accumulation of nanoparticles in the body can further induce inflammation and immune
responses, which in turn cause cell injury or apoptosis (cell death), dysfunction of vital organs and, finally,
stimulate the onset of numerous diseases, such as Alzheimer's, Parkinson's, inflammation of the liver, and
dysembryoplasia. (Yu et al., 2020, p. 9)
CHEMICAL-PHYSICAL CRITICALITIES OF NANOFORMS AND
CONSEQUENT TOXICOLOGICAL RISKS
The Polydispersity Index (PI)
As already mentioned, nanoparticles inserted in a dispersing medium, such as an aqueous solution as
in the Comirnaty preparation, tend to form aggregates or agglomerates of different shapes and sizes, thus
modifying their original dimensional characteristics, and, consequently, all those parameters crucial
for the evaluation of their toxicological profile (Figure 12). A fundamental parameter to which both
toxicologists and the European legislator assign great importance is definitely the degree of
agglomeration/aggregation (called Polydispersity index) of nanoparticles in an aqueous medium.
The Polydispersity index (PI) is a measure of the heterogeneity of a sample size of that nanomaterial
(Figure 13). Its value is included between 0 and 1: the closer it is to 0 the more the suspension is
monodisperse (uniform), while for indices close to 1 the suspensions are considered totally poly-dispersed
(non-uniform). International standardization organizations (ISOs) have established that PI values <
0.05 are specific to monodisperse samples, while values > 0.7 are related to distribution of large
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Figure 12. Formation of nanoparticle aggregates and agglomerates.
particles (polydisperse). In general, a suspension can be considered monodisperse for PI values ≤ 0.2,
on average polydisperse for 0.2 ≤ PI ≤ 0.5 and polydisperse for values greater than 0.6. PI can be
obtained from instruments using dynamic light scattering (DLS)
6
or electronic micrographs.
Figure 13. Monodisperse (uniform) and polydisperse (non-uniform)
nanomaterial suspensions.
The fact that the toxicological profile of a given nanomaterial is directly, though not exclusively,
linked to the Polydispersity index is easily understandable considering that, depending on how much
the primary nanoparticles aggregate or agglomerate with each other, larger secondary nanoparticles
are generated. These in turn could affect the exposure and bioavailability of the preparation in
different ways. For example, if primary particles aggregate or agglomerate with each other to form
larger, heterogeneous particulates with a higher PI, the material, or part of the material, may not
enter a cell and/or may deposit in tissues or organs not foreseen in its primary biological fate. The
heterogeneity of size distribution can, in other words, determine a considerable variability of the
potential impact both on the translocation of the different aggregates, and on the penetration of
biological barriers, such as crossing the blood-brain barrier, penetration into cells and subcellular
structures, and on the delivery into biological systems of any impurities or contaminants incorporated in the
6
ISO 22,412:2017 Particle size analysis — Dynamic light scattering (DLS)
https://www.iso.org/obp/ui/#iso:std:iso:22412:ed-2:v1:en
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particulate matter, especially where such impurities or contaminants are also of toxicological
significance.
7
At this point it is evident that, if a suspension of nanoforms, of the type of that of the medicinal
product Comirnaty, presented, at the time of inoculation, an index of excessive polydispersity (e.g. >
0.7), its efficacy (understood as the ability to penetrate through the cellular and subcellular
membranes and release the mRNA in the endosomal district, and from there in the cytosol of the
host cell) would be substantially inhibited, if not nullified. In this case we would therefore have a
totally ineffective medicinal product, as not able to perform the immunological task of releasing the
mRNA encoding the viral Spike protein inside the host cell. And, at the same time, the larger size
aggregates or agglomerates (often improperly called particulates), failing to penetrate into the cells, could
follow different and unexpected biological pathways or even settle in tissues from which they could
be metabolized or eliminated with difficulty, while triggering at the same time possible allergic or
anaphylactic reactions (Moghimi, 2021). An investigation published in the British Medical Journal in March
2021 shows that these problems have remained unresolved, raising serious concerns about the location of
such lipid nanoparticles in the body after inoculation. It is noteworthy that, in the entire EMA report of 19
February 2021, no reference is made to the actual value of the Polydispersity index of Comirnaty
lipid nanomaterials, although, on page 23, it is asserted that:
Visual particulate matter has occasionally [sic] been observed in finished product
batches [...] If particles are observed in the diluted vaccine the vial should be discarded.
[Figure 14]
At this point, however, it is
inevitable to ask: what does
“occasionally” mean in such
a pharmacological,
immunological,
toxicological, and regulatory
context? How frequently is
particulate matter observed?
In which and how many
batches? What were the PI
values for each specific
batch concerned? To which
specific phase of the
industrial process were these
“occasional” anomalies
related? Why did they
happen in certain batches
and not in others? What
hypotheses have been
formulated in order to
7
OECD - Guidance Manual for the Testing of Manufactured Nanomaterials: OECD’s Sponsorship Programme;
ENV/JM/MONO(2009)20/REV first revision – 02 Jun 2010, pp. 58
https://www.oecd.org/officialdocuments/publicdisplaydocumentpdf/?cote=env/jm/mono(2009)20/rev&doclanguage
=en
Figure 14. EMA/Pfizer-BioNTech Comirnaty Assessment Report, 19 February
2021, page 23.
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provide, in the shortest possible time, a plausible and logical technical explanation of the occurrence
of such a criticality? What were the remedies provided to avoid its recurrence?
These are valid questions, considering that, a few pages later, the author of the EMA report (2021,
p.37), acknowledges that:
Since mRNA integrity and polydispersity are CQAs
8
for the efficacy of the medicinal product, the finished
product acceptance criteria for these parameters should be revised as further data becomes
available from ongoing clinical trials and in line with manufacturing process capability. Due date:
July 2021, Interim reports: March 2021. [my emphasis]
It is therefore presumable, although not confirmed, that the anomalous variations relating to the
Polydispersity index of some batches were subsequently resolved and reported to EMA by July
2021.
What, then, were the stabilized PI mean values (that is not subject to “occasional” variability) of each
specific batch examined? To which specific phase of the industrial process were the previously
found “anomalies” in the polydispersity values related? Why did they happen in certain batches and
not in others? What were the root causes that, once identified, provided an unequivocal technical and
scientific explanation for the occurrence of such a criticality? What were the remedies adopted to
avoid its recurrence? What, ultimately, were the CA/PA (Corrective Actions/ Preventive Actions) adopted
in order to assure EMA (and, above all, the future patients subjected to inoculation) that such a
critical phenomenon could never occur again?
Unfortunately, these questions, at the date of this paper, still await detailed and exhaustive answers.
In the absence of sufficient information and official confirmations, we can, however, formulate
some hypotheses, which, once verified by the appropriate clinical or medico-legal authorities, could
provide further explanations and definitive confirmations both regarding the chemical-physical
instability of the Comirnaty preparation and the consequent immunological and toxicological risks
that such instability can cause and/or has already regrettably caused.
ZETA POTENTIAL AND INSTABILITY OF COLLOIDAL SYSTEMS
The Comirnaty medicinal preparation is, in essence, described, on a chemical-physical level, as:
A colloidal suspension, thermodynamically unstable, consisting of lipid nanoparticles and their aggregates or
agglomerates, characterized by a variable Polydispersity index
.
The term colloid derives from the Greek kòlla, glue, gluten, with the adjectival suffix -oid, which
indicates similarity, affinity, that is, similar to glue: it therefore appears as an amorphous mass that,
diluted in water, forms a more fluid colloidal dispersion (hence more suitable for parenteral
administrations).
A colloidal suspension is simply a mixture in which dispersed solid particles (in this case lipid
nanoparticles) remain suspended in an aqueous dispersing medium, for more or less long periods of
time. A suspension of very small particles (such as those formed by Comirnaty lipids) can
theoretically approach a real solution in appearance. In general, the system becomes more stable
(durable over time) if the dispersed particles are smaller, if the densities of the two phases
8
CQAs: Critical Quality Attributes
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(dispersed and dispersing) are made nearer the same, and if the density of the dispersing phase is
made greater (Stokes’ law).
The propensity of particles to
associate into aggregates or
agglomerates (and therefore
their polydispersity index)
depends on another important
parameter, which the
manufacturer of nanoforms is
required to measure, record, and
report to regulatory authorities:
the Zeta potential.
The Zeta potential, or
electrokinetic potential (referred
to the letter zeta “ζ” of the
Greek alphabet) is the potential
generated as a result of the
formation of an electric double
layer around the individual
particles. (Figure 15). It
represents the key factor for the
determination of electrokinetic
phenomena and stability of colloidal systems and, consequently, of the bioavailability of a
compound or drug carried by nanoforms and intended to cross cellular or subcellular membranes
(OECD, 2010, pp. 33, 63).
As described in an article (Barone et al., 2017) published by the Italian Agency ENEA (Energia
Nucleare Energie Alternative, Agency for New Technologies, Energy and Sustainable Economic
Development):
A nanoparticle placed in solution forms a colloidal system for a longer or shorter time. A greater
stability of the colloidal systems prevents the phenomenon of aggregation of particles as electrostatic
repulsions originate that favor their dispersion. The parameter used to calculate colloidal stability
is the Zeta potential which refers to the potential generated by a double layer of electric charges. When the
potential is low, attractive forces prevail over repulsive and therefore more aggregates will form.
The knowledge of the real concentration of the particles to which the biological system is
exposed is important to determine the estimation of the health risk and can be expressed both as
particle number and as total surface area and is strongly affected by the degree of aggregation of the
particles. In in-vitro experiments, the variation of these parameters can affect the greater or lesser
degree of endocytosis (internalization of particles by cells), which is important for defining the mode of action of
that nanomaterial. [my emphasis]
In summary, a high Zeta potential value (e.g. 40 to 60 mV) gives greater stability to colloidal systems,
as electrostatic repulsions arise that prevent the aggregation of dispersed particles. When the
potential is low (e.g. from 5 to 10 mV), attractive forces prevail over repulsive ones and therefore it is
easier for processes such as agglomeration, or even flocculation, to occur (OECD, 2010, p. 63). The latter
is nothing more than the formation of coarse particulate matter, sometimes, but not always, visible even
to the naked eye. This is the stage that could lead to
Figure 15. The Zeta potential basically defines the stability of the colloidal
suspension.
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coalescence, a phenomenon that
occurs when the film
surrounding the particles breaks
and the aggregates of various
sizes combine with each other
to form a larger agglomeration
(cluster), finally determining the
“breaking” of the dispersion
and the separation of the phases
(Fig. 16, 17).
CAUSES OF INSTABILITY
OF COLLOIDAL SYSTEMS
The causes that lead to the instability of a
colloidal biphasic system are many, and must
always be analyzed and identified case-by-case
with the appropriate laboratory instruments.
Among the most common, for example, are:
incorrect ratios between the dispersed phase and
the dispersing phase; wrong method of
processing; cooling or heating temperatures too
high or too low; excessive air absorption that
could change the ratios of the biphasic system;
and, above all, the presence of electrolytes
(Bushmanova et al., 1994).
An electrolyte is a substance that in solution or in
the molten state undergoes the division into ions
(electrically charged particles) of its molecules.
Substances that do not dissociate into electrically charged particles are called “non-electrolytes”. The
term “electrolyte” refers to the ability to conduct electric current thanks to the intervention of ions,
a peculiar characteristic of these chemical species. Inorganic mineral salts, such as sodium chloride,
are the most classic example (NaCl dissociates into Na+ and Cl- ions).
Electrolytes, depending on the concentrations involved, can considerably alter the Zeta potential of a
colloidal dispersion, causing the aggregation and agglomeration of nanoparticles, and their subsequent
flocculation by electrostatic attraction (Tadros, 2018). In other words: the addition of electrolytes is one of
the most common causes of the variation of the Zeta potential and the Polydispersity index and, consequently, of
the instability of the colloid, with all the easily predictable consequences that this entails, both with
regard to the ineffectiveness and to the toxicological risks that will characterize the preparation itself,
as already described above.
COMPOSITION OF THE MEDICINAL PRODUCT COMIRNATY
Comirnaty is originally supplied as a concentrated multi-dose liquid preparation (0.45 mL volume),
stored frozen between -90°C and -60°C in a 2 mL glass vial, and to be diluted shortly before
inoculation with a sodium chloride solution for injection.
Figure 16. Flocculation, coalescence and phase separation in an unstable
colloidal system.
Figure 17. “Breaking” of homogeneous dispersion
and separation of the two phases.
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The vial is thawed by keeping it in a refrigerator (2°C to 8°C) for 2 to 3 hours or at room
temperature (up to 25°C) for 30 minutes. Once returned to room temperature, the multi-dose vial is
diluted with 1.8 mL of the 9 mg/mL (0.9%) sodium chloride solution. After dilution, each vial of
Comirnaty contains 2.25 mL from which 6 doses of 0.3 mL of vaccine can be extracted. Each dose
contains 30 μg of the active ingredient (that is the mRNA BNT162b2, intended to code for the
SARS-CoV-2 spike glycoprotein) and the excipients listed in Table 1. After dilution, according to the
instructions of Pfizer-BioNTech, the vials are stored at a temperature between 2°C and 25°C and
should be used within 6 hours (FDA, 2021, p.4).
Table 1. Composition of one dose of Comirnaty vaccine after addition of a
physiological sodium chloride solution (*Electrolytes).
Ingredient
Function
Quantity per dose
BNT162b2
Active
30 μg
ALC-0315
Functional Lipid
0,43 mg
ALC-0159
Functional Lipid
0,05 mg
DSPC
Structural Lipid
0,09 mg
Cholesterol
Structural Lipid
0,2 mg
Sucrose
Cryoprotective
6 mg
Sodium chloride *
pH buffer & diluent
solution component
2,52 mg
Potassium chloride*
pH buffer component
0,01 mg
Sodium phosphate dibasic
dihydrate*
pH buffer component
0,07 mg
Potassium dihydrogen
phosphate*
pH buffer component
0,01 mg
Water for injection
Dispersing medium
q.s. to 0.3 ml
PRESENCE OF ELECTROLYTES IN THE COMPOSITION OF THE COMIRNATY MEDICINAL
PRODUCT
As shown in Table 1 and Figure 18, the formulation of the Pfizer-BioNTech COVID19 vaccine
contains 4 electrolytes (inorganic salts), components of the pH buffer, used to stabilize the pH of the
preparation at a value between 6.9 and 7.9: sodium chloride, potassium chloride, sodium dibasic
phosphate dihydrate, potassium dihydrogen phosphate.
Note that, in the final composition of Comirnaty, that is after dilution with a 0.9% sodium chloride
solution, the quantitative proportion (by weight) between the total amount of electrolytes present
and that of the two functional lipids is 5.44:1. In fact, for every dose of vaccine inoculated, we have
2.61 mg of electrolytes versus only 0.48 mg of ALC-0315 + ALC-0159. A quantity that turns out to be
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more than 5 times the amount of the two functional lipids responsible for the formation of nanoparticles in
suspension. The ratio by weight of lipid ALC-0315 (cationic) to mRNA (anionic) is 14:1.
Figure 18. Pfizer-BioNTech COVID19 Vaccine — FDA Document at
https://www.fda.gov/media/151733/download : Summary Basis for Regulatory Action -
11/08/2021.
The key question here is: can such a colloidal suspension be considered stable?
By diligently evaluating the above data regarding the Zeta potential and the Polydispersity Index, the
answer can only be negative: such a high relative concentration of electrolytes, in such a precarious
colloidal suspension, can only lead to a drastic reduction of the Zeta potential, with consequent
predictable phenomena of aggregation, agglomeration, and, finally, flocculation.
Moreover, examining the
EMA official document
Annex I Comirnaty Summary
of Product Characteristics, it is
clear that both the
manufacturer and the
authorizing bodies were well
aware of the risks relating to
its instability and the obvious
possibility of coarse
particulate formation in situ,
shortly before
administration. In fact, the
dilution instructions read:
• Gently invert the
diluted dispersion 10 times. Do not shake.
Figure 19. Dilution/mixing phase of Comirnaty lipid nanoparticles suspension.
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• The diluted vaccine should present as an off-white suspension, with no
particulates visible. Discard the diluted vaccine if particulates or discolouration are present
[Figure 19].
Leaving aside the improper use, in the text intended for vaccinating operators, of the term
particulates, rather than the more appropriate one of flocculates, we cannot avoid asking some
important questions concerning the assessment of the specific risks related to the inspection of the
vial, after the dilution phase:
- What is the value of the Zeta potential of nanoparticle suspension after dilution with
the sodium chloride electrolytic solution?
- What is the difference between the value of the Zeta potential after dilution and that
of the concentrated suspension (before dilution)?
- What is the value of the Zeta potential of the diluted suspension, under average
physiological conditions (such as pH 7.4; 2.2 mM Ca++) at 37°C, that is to say at the
conditions to which it is subjected a few moments after intramuscular inoculation?
- How accurate should the inspection of the vial be, after dilution, in order to minimize
errors (accidental and systematic) in assessing the presence or absence of aggregates or
agglomerates or flocculates?
- How numerous and “visible” must the “particulates” be in order to trigger in the
observer the decision-making act of discarding the non-compliant vial?
- How “gently” should the vial be turned upside down (that is to say inverted, but not
shaken)?
- What values of Zeta potential are obtained in case of minimum or excessive shaking
of the vial?
- What would be the possible risks related to an error (reasonably understandable and
most likely motivated by fatigue or nervous tension or absent-mindedness by the
vaccinating doctor) in the number of overturns of the vial? In other words, if the
reversals, instead of 10, were 8, or 12, or 5, what would be the risk, in these cases, of
obtaining an insufficient homogeneity (and therefore a greater instability) of the diluted
suspension?
- Who verifies and controls the evaluator of the dilution/ inverting/ visual inspection
procedures with regard to approval of compliant vials or rejection of non-compliant
vials?
- How many vials, statistically, have been detected as non-compliant? Are the statistical
findings significantly consistent among the various vaccination operators and the
different vaccination sites?
These are, of course, just some of the most relevant questions that emerge from elementary but
necessary evaluations and considerations related to fundamental parameters, such as the
Polydispersity index and the Zeta potential, and, consequently, to the degree of stability of the
resulting colloidal suspensions. However, these are questions that require accurate and prompt
answers, taking in consideration, above all, the serious consequences that any error, omission or
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negligence in the dilution phase could entail, from a statistical-epidemiological, clinical and medico-
legal point of view, for the safety of those who undergo intramuscular inoculation of a liquid
suspension of nanoparticles which could be excessively poly-dispersed, or even close to flocculation
or coalescence or phase separation.
ELIMINATION OF ELECTROLYTES IN THE COMPOSITION OF THE NEW MEDICINAL PRODUCT
COMIRNATY “TRIS”
On 2021, October 18, EMA announced, on its official website, that EMA’s human medicines committee
(CHMP) has approved two additional manufacturing sites for the production of Comirnaty, the COVID-19 vaccine
developed by BioNTech and Pfizer. One site, located in Monza, Italy, is operated by Patheon Italia S.P.A. The other
in Anagni, also in Italy, is operated by Catalent Anagni S.R.L. Both sites will manufacture finished product. These
sites will produce up to 85 million additional doses to supply the EU in 2021.
And, surprisingly, on the same page, it also announced that:
[…] The CHMP has approved a ready-to-use formulation of Comirnaty. This formulation does not require
dilution prior to administration, will be available in a 10-vial (60 dose) pack size and can be stored at 2-
8°C for up to 10 weeks. The current concentrated formulation requires dilution prior to
Figure 20. New composition (called Tris/Sucrose Finished Product) of the Pfizer-BioNTech Comirnaty vaccine,
electrolyte-free, ready to use, no longer requiring the dilution phase.
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administration, is available in a 195-vial (1,170 dose) pack size and can be stored at 2-8°C for up to
one month). These differences will provide improved storage, transport and logistic options for vaccine
distribution and administration. The new formulation will be available in a phased rollout starting
in early 2022. [my emphasis]
According to such peculiar announcement, the new Comirnaty formulation, ready for inoculation, no longer
requires dilution, with obvious advantages of storage, transport and logistics. It is assessed and authorized
under the same conditions as the previous one, but in a new EMA Assessment report, dated 2021,
October 14, entitled CHMP assessment report on group of an extension of marketing authorisation and
variations, in accordance with Reg. (EC) No 1234/2008, and with the premise that it is an Assessment
report as adopted by the CHMP with all information of a commercially confidential nature deleted.
And, on page 14 of such report, the new formulation is revealed (Figure 20), and, with it, some
details that tend to confirm, both on the chemical-physical and toxicological level, the above detailed
evaluation concerning the manifest instability and potential danger of the original Comirnaty flawed
formulation.
Oddly enough, in the new composition of Comirnaty, called Tris/Sucrose Finished Product, containing
the same active ingredient (mRNA chemically modified at nucleoside level), the same functional
lipids and the same supporting excipients (at the same concentrations), all the electrolytes that were
present in the previous electrolytic formulation (called, for the occasion, PBS/Sucrose Finished Product,
where PBS stands for Phosphate-Buffered Saline), have totally disappeared, of course without providing
the reader with any explanation.
Figure 21.Pfizer-BioNTech Comirnaty PBS/Sucrose vaccine (Electrolytic) Safety Data Sheet section
3.2., 2021, December 7.
This electrolyte purging operation may seem ordinary to non-experts, but in reality it is revealing to
experts in the field of colloidal systems, and even more explicit when comparing the relevant
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sections 3.2. of the Safety Data Sheets of Comirnaty PBS/Sucrose (the Electrolytic vaccine, Fig. 21)
and Comirnaty Tris/Sucrose (the Non-electrolytic vaccine) [Figure. 22].
Figure 22. Pfizer-BioNTech Comirnaty TRIS/Sucrose vaccine (NON-electrolytic)
Safety Data Sheet section 3.2., 2021, December 14.
From a technical point of view, in the new ready to use formulation, the previous pH buffer PBS
(electrolytic, inorganic) has been eliminated and replaced with another very common buffer called
Tris buffer, widely used in biology to prepare pH-controlled solutions (especially for nucleic acids), in
Figure 23. EMA Assessment Report, 2021, March 11, p. 26. Composition of Moderna's
COVID-19 Vaccine Spikevax.
pH ranges between 7 and 9. This is an organic buffer (meaning it does NOT contain inorganic
electrolytes), which stabilizes the pH of the Comirnaty Tris/Sucrose product at a physiological value
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of 7.4
9
, chemically consisting of tris(hydroxymethyl) aminomethane (also known as tromethamine or
tromethamol) and its hydrochloride. It is interesting to note that, eliminating the electrolyte buffer and
substituting the new organic buffer based on trometamol, the entire formulation of the new Pfizer-
BioNTech preparation called Tris Sucrose becomes, if not identical, at least very similar to that of
Moderna's Spikevax vaccine (the latter authorized by EMA on 2021, January 6, Assessment report 2021,
March 11). In fact, both of these vaccines include the following elements: a nucleoside-modified
mRNA + a cationic functional lipid + a polyethoxylated lipid + a neutral lipid (DSPC) + cholesterol
+ the non-electrolytic tromethamol-based pH buffer (Figure 23).
Figure 24. Comparison between lipids used in the Pfizer-BioNTech mRNA vaccine and
Moderna's mRNA vaccine (Tenchov et al., 2021).
As stated by the EMA itself, on page 22 of its Assessment report dated 14 October 2021
concerning the new Comirnaty Tris/Sucrose:
[...] the Tris/Sucrose finished product has been developed to provide a vaccine with an improved stability
profile and greater ease of use at administration sites. [my emphasis]
How much has the stability profile been improved, compared to that of the previous version, is
unfortunately not revealed. In other words, the key question here is: what is the variation between
the Polydispersity index of the non-electrolytic Tris/Sucrose vaccine and that of the electrolytic
PBS/Sucrose vaccine? What is the variation between the Zeta potential of the lipid nanoparticles of
the ready-to-use non-electrolytic Tris/Sucrose vaccine and that of the electrolytic PBS/Sucrose
vaccine diluted and gently inverted 10 times before inoculation? What is the Zeta potential of the
new Tris/Sucrose formulation, under physiological conditions (i.e., pH 7.4; 2.2 mM Ca++) and at the
9
In the absence of pathological states, the pH of the human body ranges between 7.35 to 7.45, with the average at 7.40
(Hopkins et al. 2022).
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temperature of 37°C, that is the temperature to which it is subjected a few instants after
intramuscular injection?
Unfortunately, the answers to these questions, may never arrive, since they are likely to be judged as
pertaining to information of a confidential commercial nature and therefore subject to suppression. But,
nevertheless, the logical observation that the Comirnaty PBS/Sucrose vaccine (injected in billions of
doses) had to be modified, not only for greater ease of use, but also, first and foremost, to improve its
stability, remains and will always remain insuppressible, as written in an official EMA report. And this
will remain, among other things, the first simple admission, albeit indirect and swamped by “logistic”
justifications, that the previous PBS/Sucrose electrolytic version, being NOT sufficiently stable,
consequently presented greater toxicological risks and subsequently had to be corrected by Pfizer-BioNTech
and grossly transmuted into a new version promptly authorized by EMA.
However, what seems very
odd is that the new non-
electrolytic version Tris/
Sucrose is presented on
the web only as a mere
pharmaco-technological
development (a simple
upgrade) as well as an
admirable solution to the
onerous problems of
storage, transport, and logistics,
without making any
mention of the
toxicological risks and
dangers to public health
that the previous
formulation implied. In
fact, the old electrolytic
version, though unstable
and to be diluted before
inoculation, remains
surprisingly on the market,
simply distinguished by a
purple cap (dilution necessary, for subjects aged 12 years and older), next to the new version with gray
cap (dilution NOT necessary, also for subjects aged 12 years or older). Both versions are in fact
authorized by EMA for placing on the market in Europe, always with the formula authorization subject
to conditions and considered equivalent and interchangeable with each other (Figure 25).
INSTABILITY AND TOXICOLOGICAL POTENTIAL OF THE
COMIRNATY PBS/SUCROSE
VERSION: CONFIRMATIONS AND CROSSCHECKS
In the light of the technical data set out above, it is now quite clear that the instability of the
colloidal system of lipid nanomaterials (and their consequent greater toxicological risk) of the first
version of Comirnaty is substantially due to the presence, in that formulation, of destabilizing factors,
such as, in fact, the excess electrolytic inorganic compounds, which make up the PBS pH-buffer
Figure 25. The two co-existing Pfizer/BioNTech Vaccines.
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therein used by Pfizer-BioNTech. In this regard, a clarifying definition of the concept of “stability”,
in relation to nanoparticles-based compositions, is reported in Moderna’s patent US 10,442,756 B2
Compounds and compositions for intracellular delivery of therapeutic agents:
Stability, stabilized and stable in the context of the present disclosure refers to the resistance of
nanoparticle compositions and/or pharmaceutical compositions disclosed herein to chemical or physical changes (e.g.,
degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing ,
preparation, transportation, storage and/or in-use conditions, e.g., when stress is applied such as shear
force, freeze/thaw stress, etc. [my emphasis]
Figure 26. BioNTech Patent US 10,485,884 B2 RNA Formulation for Immunotherapy - Nov
26, 2019; section Background of the invention, 1 (62-67), 2 (1-5).
In this same regard, however, what is reported in the patent of the same BioNTech (co-owner,
together with Pfizer, of the Comirnaty vaccine) US 10,485,884 B2 RNA Formulation for Immunotherapy
dated November 26, 2019 is even more explicit concerning “elevated toxicity” attributed to
“positively charged liposomes and lipoplexes”. The reference is to formulations of RNA
encapsulated in cationic lipid nanoparticles — of the same category as those used in Comirnaty —
and called, in this context, “lipoplexes”. In the description of the patent, it is explained, among other
things, how cationic nanoparticles containing RNA are formed mainly thanks to certain
mass/charge ratios between cationic (+) lipids and anionic (-) components of RNA, and how these
ratios play a fundamental role also with regard to the passage of RNA-containing nanoparticles
through the cell membrane and the consequent transfer of RNA inside the cell (transfection) to
modify its functional characteristics:
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Most natural membranes are negatively charged, and therefore the attractive electrostatic interaction between the
positively charged lipoplexes and the negatively charged biomembrane may play a role in cell binding and uptake of
the lipoplexes. Typical ranges of+/- ratios which are considered optimal for transfection are between
2 and 4. With lower excess positive charge, the transfection efficacy goes drastically down to
virtually zero. Unfortunately, for positively charged liposomes and lipoplexes elevated toxicity has been reported,
which can be a problem for the application of such preparations as pharmaceutical products. [my emphasis]
(Figure 26).
The reasons why pH-buffers of the PBS-type are absolutely not suitable in preparations based on
RNA-incorporating cationic nanoparticles are explained very clearly in the section Examples, Effects
of Buffers/Ions on Particle Sizes and Polydispersity Index of RNA Lipoplexes of the aforementioned
BioNTech patent; US 10,485,884 B2, 44 (47-50), 45 (4-6), 45 (31-33):
The use of buffer which is often necessary for pharmaceutical applications and ions can lead to
aggregation of lipoplexes which makes them unsuitable for parenteral application to patients […]
In PBS buffer, the same effect is more prominent. Lipoplexes with a positive or neutral charge
ratio form larger particles (partially stabilized by the positive charges […]
Under physiological conditions (i.e. pH 7.4; 2.2 mM Ca++), a negative charge ratio appears to be imperative due
to the instability of neutral or positively charged lipoplexes. [my emphasis] (Figure 27).
Figure 27. Figure 27. BioNTech Patent US 10,485,884 B2 RNA Formulation for Immunotherapy -
Nov. 26, 2019 (section Examples).
In other words, based on what is scientifically documented and reported in a patent of the same
BioNTech, additionally to what already described concerning the intrinsic dangerousness of positively
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https://doi.org/10.56098/ijvtpr.v3i1.68
charged lipid nanoparticles, we learn that a colloidal system of cationic lipid nanoparticles incorporating
mRNA
1. should NOT contain an ionic buffer such as PBS, in order to prevent aggregation, agglomeration,
flocculation of lipid nanoparticles, and all the toxicological consequences described above.
2. should NOT contain ionic compounds (such as sodium chloride), in order to prevent aggregation,
agglomeration, flocculation of lipid nanoparticles, and all the toxicological consequences
described above.
3. should NOT be injected intramuscularly, due to its instability when placed in the physiological
environment of the extracellular district (pH 7.4; 2.2 mM Ca++).
All three of these rigorous recommendations, reported in the aforementioned BioNTech patent of
2019, are shamelessly contradicted, or ignored, in 2020, both by Pfizer-BioNTech and by the
certifying bodies, both on the nature of the Comirnaty formulation (ionic/electrolytic) and on its
intended use (intramuscular injection).
In the final analysis, the medicinal preparation Comirnaty/PBS Sucrose from Pfizer-BioNTech,
authorized by EMA in 2020, presents serious and evident liabilities on the chemical-physical and
consequently toxicological level. Liabilities, which are, oddly and paradoxically, in open contrast even
with the specific and pertinent recommendations asserted by BioNTech itself in its aforementioned
patent US 10,485,884 B2. Liabilities, in open contrast with the specific and pertinent
recommendations asserted by BioNTech itself in its aforementioned patent.
On the basis of these confirmations and cross-checks, we can therefore hypothesize that the
addition of such an important share of electrolytic compounds to the already precarious equilibrium
of a colloidal system made of cationic nanoparticles, easily influenced by ionic charges, has
inevitably conditioned the stability, shelf life, functionality, and consequent toxicological potential of
the finished product Comirnaty PBS/Sucrose, causing in particular: unpredictable alterations of its
Polydispersity index and Zeta potential; possible consequent formation of aggregates, agglomerates,
flocculates, coalescences; different degrees of penetrability and mobility of nanolipid aggregates of
different sizes, after inoculation, in unexpected and unpredictable biological sites, with irregular ROS
formation at these sites; consequent heterogeneity of adverse effects (randomization), potentially
variable from batch to batch, from vial to vial, from vaccinator to vaccinator, from vaccinated to
vaccinated, in a sort of ineluctable, uncontrollable, and indecipherable Russian roulette (Santiago,
2022).
CONCLUSIONS
The Comirnaty COVID-19 mRNA BNT162b2 vaccine, in its original version and composition, called
PBS/Sucrose, presents numerous critical issues and drawbacks, examined in detail in this study and
summarized as follows:
- The two functional excipients responsible for the formation of lipid nanoparticles, ALC-
0315 and ALC-0159, are not registered in any Pharmacopoeia, nor are they among the
substances examined and classified in accordance with Regulation (EC) No 1272/2008 on
classification, labelling, and packaging of substances and mixtures in Europe (CLP).
- These excipients also do not appear in the inventory of substances registered in
accordance with Regulation (EC) No 1907/2006 concerning the Registration, Evaluation,
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Authorisation, and Restriction of Chemicals in Europe (REACH). Therefore, their
toxicological profile is not known in the first place.
- Not all the chemical-physical analysis procedures and toxicological tests required for
nanoforms of these substances have been carried out, contrary to Regulation (EU)
2018/1881 amending Regulation (EC) No 1907/2006 of the European Parliament and of
the Council concerning the Registration, Evaluation, Authorisation, and Restriction of
Chemicals (REACH), to include the nanoforms of substances.
- Carcinogenicity, genotoxicity and mutagenicity studies of the preparation have not been
carried out with the consent of the certifying body, although it has now been confirmed
by numerous scientific studies that Reactive Oxygen Species (ROS), generated by
nanoparticles, can have a high carcinogenic, genotoxic, and mutagenic potential.
- The Safety Data Sheets of the Comirnaty preparation do not report information on the
characteristics of the nanoforms present in the composition of the preparation itself,
contrary to the provisions of the aforementioned Regulation (EU) 2018/1881 and
Regulation (EU) 2020/878.
- The actual values of the Polydispersity index and the Zeta potential of the nanoparticles
present in the preparation are unknown. This leads to absolute uncertainty in the
determination of the chemical-physical stability of nanoparticles and their aggregates, with
consequent unpredictability inherent both to the potential efficacy of the vaccine and to
the degree of penetrability and mobility of its nanoparticles within the human body, as
well as their possible entry into the systemic circulation and accumulation in organs such
as kidneys, liver, heart, brain, lungs.
- The presence of electrolytes in the original preparation (meaningfully eliminated in the
subsequent Comirnaty Tris/Sucrose version) leads even more to the presumption that the
product called Comirnaty PBS/Sucrose may give rise to the formation of aggregates and
agglomerates before, during or after the inoculation procedure, and that it may therefore
be both ineffective (since not able to convey the mRNA encoding the viral Spike protein
of SARS-CoV-2 through the membranes of the host cell) and dangerous, as it would be
deposited in tissues or organs not foreseen in its primary biological fate.
In conclusion, it is considered urgent and indispensable that an accurate and long-term study be
carried out in the appropriate institutional, clinical or medico-legal seats, especially in relation to any
causal or con-causal links between what is presented here and the wide pathological heterogeneity of
serious or lethal adverse events that have occurred, or are occurring, after vaccinations, in order to
adopt and expedite all appropriate corrective and preventive actions to protect public health,
including discontinuing vaccinations with Pfizer-BioNTech Comirnaty PBS/Sucrose as soon as
possible, in accordance with the precautionary principle, and in the light of Article 10 of the
Nuremberg Code:
During the course of the experiment the scientist in charge must be prepared to terminate the
experiment at any stage, if he has probable cause to believe, in the exercise of the good faith,
superior skill and careful judgment required of him, that a continuation of the experiment is
likely to result in injury, disability, or death to the experimental subject.
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Funding and conflicts of interest
The author declares that he has not received any funding to influence what he says here and that the
research was conducted in the absence of any commercial or financial relationship that could be
construed as a potential conflict of interest.
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