WHITE PAPER - m RNA vaccine production Quality control- regulatory and toxicological aspects

WHITE PAPER
GRAPHENEand DERIVATES : PHYSICO- CHEMICAL and TOXICOLOGY properties in
the m- Rna VACCINE MANIFACTURING STRATEGY
Needed specific proof of absence for the regulatory aspects
AUTHORS
1)Luisetto M IMA academy Marijnskaya , professorship toxicology , pharmacology
natural science branch italy
2) Tarro G ,President of the T & L de Beaumont Bonelli Foundation for Cancer
Research, Naples Italy
3) Edbey K, Professor, Departmentof Chemistry, Libya PhysicalChemistry,
University of Benghazi, Libya
4) Hamid Gamal Abdul Professor heamtology –oncology Aden university YEMEN
5)Nili B. Ahmadabadi ,Nano Drug Delivery, (a ProductDevelopment Firm), United
States
6)Oleg YurevichLatyshev IMA academy President
Keywords : Graphene, covid -19 , m RNA, vaccine, side effects ,
toxicology,chemistry manifacturing tecnhnology and materials, contaminations,
impurity
Corresponding author :luisetto M maurolu65@gmail.com +393402479620 italy PC
29121

Abstract
Observing thefact that every year various registerd and authorized drugs are
recalled by the public regulatory agency ( in example last Ranitidin cases due by the
possibility of impurity presence of a cancerogenic sustantia ) it is interesting to
observethe innovative production strategy of somem RNA vaccine .
Also for the biotechnological drugs product the regulatory agency ask quality
stantard for impurity for the obiously safety implications.
The biotechnological drugs , as the classic chemical drugs , due by the complex
manifacturing process useraw material and industrialprocedurethat require
Great quality monitoring according the GMP normativerules.
So it is no to strange to discuss of impurities in this new kind of products
And also for the covid-19 vaccine.
Introduction
This work startby recent and really interesting new evicence ( DISINFECTIONad
Natura Docet Darkfield microscopeanalysis of the blood of 1006 symptomatic
subjects affer vaccination whit two types of mRNA vaccine n 1/ 2022 GIOVANNINI
et al ) and other research works published also by university professorsand other
professionals ( CAMPRA P univerisy of ALMEIRA , YOUNG R O ,YOUNG Mi LEE et al) .
Of interest also a recent publication ( aug 25th 2022) by SCIENCEWORLD JOURNAL
OF PHARMACEUTICAL SCIENCE of the research article “Grapheneand Derivates :
Physico- Chemical and Toxicology Properties in the M Rna Vaccine Manifacturing
Strategy”
Before of all it was done a review of interesting article related the chemico- physical
and toxicological property of graphene derivates ( also woks published before the
Covid-19 Pandemia) as well as distribution profile related the way of
subministration.

Itwas also analyzed ADR charactheristics that somerecent Covid-19 vaccinehave
reported in the pharmacovigilance center ( national and international ) .
Great attention was done towards the verify of the manifacturing technology used
in field Of m RNA vaccinesince form the research in oncology.
Fromliterature Grapheneand Grafene Oxide GO and other derivates are subjectof
many use in biotechnological research : in diagnostic field but also in production
and expecially in last decades.
Itis possibleto report Carrier or adiuvant or extractive property ( there are study
that show increase in immune responceafter their use) , and objectof research for
INTRA NASAL covid-19 vaccine.
Properties to improve RNA extraction- purification in manifacturing field.
( absrobtion property, increaseRNA stability )
In someliterature are described use of pegylated nanolipids whit graphene oxide
In m RNA Vaccineresearch development. ( some producers can providethis raw
material and the technical and security sheet are easy available on internet ) .
On literature as well as by bio-technological producers there are variuos products
made of MAGNETIC MICROBEAS used for extraction of RNA derivates.
This are all use Knowed at today and easily verifible in biomedical database since
not suspicious times .
Interesting also to note as there is also a PREPRINT written by the CEO of a great
pharmaceutical industry that produces innovative covid-19 vaccineand other
researcher of the same indutry reportamong the reagent for production of a covid-
vaccine per non human primates magnetic micro beads . ( see reference on article
under publication as reported ).
(And on literature are reported article works about grapheneGO - magnetic
microbeads.).

Materials andmethods
With an observationalpoint of view somerelevant literature is analyzed in order to
producea global conclusion related the topics of this work .
After this reviw part it si submitted an experimental projecthypotesys usefulfor to
Producea global conclusion.
Results
We have performed as a review part an searching activity on biomedical database
like Pub med and other in order to find relevant literature for this work.
All using keywords usefullto limit the references to only the crucial ones in order to
cover the various argument: independent researcher results in findings graphene
like particle in some vials of covid-19 vaccine, or related the chemico -physical
properties of this class of compounds , ore the biotechnological usein bio-
pharmaceutical production .
As we have see : In literature also before COVID-19 pandevia various researcher
published works Related the role of graphenederivates in example to better purify
m RNA vaccine production ( also for oncologic research) , as carrier, or as adiuvant
and also recently studied for intranasalflu vaccine new products.
In last decades this innovative material was introduced in various settings and also
io biomedical or in biotechnology ( in esample for testing – biosensor, tissue
regeneration , antibacterial properties and other ).
Beacuse this productcan increaseextraction property of mRna in manifacturing
bio-pharmaceuticals as showed by researcher ( about 170% vs calssic methods
accordingXuan-Hung Pham et al using magnetic beads ) it is relevant to verify if
some producers of raw material Use this technology.
So Itis Necessary to verify the impurity level for raw material and also in the final
biopharmaceutical products
( using a right analytical chemical methods: with pretreatement of the sample with
solvent in order to extract - before to test the analite from nanolipids particles
avoiding interferences in the cases of m RNA VACCINENLP ).

This becausethe toxicity profile of this class of products requiredeeply
investigation.( Rare severe ADR like thrombosys, miocarditys , pericarditys )
The fact that some regulatory agency after GMP visit in production site asked
officialy to the final producer of some m RNA VACCINEto complete the quality
Profile of someeccipents tell us to more clarify this aspects .
To confimr this proposalthere are the evidence collected in some university as in
some research study reported ( TURIN UNIVERSITY- CHEMISTRYfaculty ,
CAMPRA P. - UNIVERSITYOF ALMERIA professor CHEMICAL SCIENCE).
Regarding the contaminants it is of interest to report thay in 26 August2021 BBC
news Coronavirus pandemic Japan suspends 1.6 million Moderna doses over
contamination fears A staff of Japan"s supermarketgroup Aeon receives a doseof
the Moderna vaccine“Japan has suspended the use of about 1.63 million doses of
the Moderna vaccine due to contamination.
A metallic substantia the react with magnete finded in some lots .
Of interest finally to remember two recent italian judgesententia : PISA EFIRENZE
2022 it was recognized in one cause- effect relationship between a covid-19
vaccination and a Thromobocitopenia reaction in an 16 years old ,and in the other
case the judge written that this vaccine are experimental , Dna invasive, potentially
of irreversibleeffect and not prevedile at today .
Even if this drugs areofficially registered by regulatory agency during the last years
In example somecountries restricted uses of some covid-19 vaccinein determinate
subpopulation even initially not done .
The same some technical sheet was recomded to be updated by EMA PRAC
introducing some Rare ADR not presentin the first approved VACCINE oneor also
changed the name of the vaccinein order To update registrativedocuments
reporting new controindications.
For this reason it is necessary thatregulatory agency official asks to the producers to
verify in official way the presenceod absence of graphenederivates in the final
products using qualitative but also quantitative methods (with control ).
And the results must to be certified for all lots released.

Last version of European pharmacopea allow the use of Raman spettroscopy
For production quality controlscope and also as non destructive methods – direct.
But independe researcher used a classic destructive methods using solvent to
pretreat nanolipids in their analitycal procedureto find graphenepresence in vials
of vaccine m -Rna.
The different results obtained by regulatory agency ( ABSENCE ) and by some
independent researcher ( PRESENCE of graphenelike particle ) need a more better
Verify .
According a Personalopinion of a university full Professsor Phd chemicalscience
received ( 24-08-2022)
“In my opinion GO and other non-declared substances mustbe located by
microscopicaltechniques coupled with spectroscopy (Raman, XPS, e
diffraction).Otherwisetheanalyses will yield negative identification, as their amount
is low and they show as dispersed particles”
Fromliterature :
Results
Are reported reference ( 1-50)
Experimental project hypotesys
In order to verify the absence/presence of grapheederivates in vials of some bio-
pharmaceutical compounds it is needed to test 100 sampleof a new technological
products (In example m RNA vaccine in nanolipids) .
This using analitycal procedureofficially CGMP approved ( RAMAN spettroscopy )
and with the accettable sensibility. ( one procedurewith a classic destructive
method and using also a non destructivemethod).

1) Method as approved EUROPENA PHARACOPOEIA likedirect non desctructive
method
2) Method as reported by some rearcher ( with extraction in a classic chemical
methods befor test, destructivemethod )
This sample mustdivided in group of 20 and sended blinded to various and
different accreditated chemical laboratory and independent.
Itis needed a controlgroup, all sample blinded .
The sample must to be tretated for the pre-analitycal need ( extraction) before to be
analyzed.
This in order to verify in the same condition the inside nanolipids included and
outiside of this.
Results: verify is there is or not significative presenceof grapheneor its derivated
in the final approved vials. ( p < 0,005)
The results m ustto be divided using a destructive method and a non destructive
one.
Discussion
So related to all of this , matching the evidence of sometest on vials of vaccine as
well as reseacth on dark filed miscrosocpy and related the fact that the toxicological
profile of this particles seem to correlate with the ADRreported ( rare thrombosys
, pericarditis and other ).
Also the chemico-phyicalproperties of this particles are so peculiar and make
possibleto give explanation of somephenomena.
According the authors it is clear that it is needed to deeply investigate this evidence
verifying the manifacturing technological process in order to clear if are presence
or not possible pollutants residues of production even if this are not declared and
reported on official techincal sheet and registered by public health authorities.
(In the raw materials , reagents of final product).

The pharmaceutical producers useoften to buy raw materials form other producers
The same author submitto the researcher to analyzea more wider and significative
number of vials of the various vaccinefor covid-19 , troughtvarious and certified
independet laboratory and using the blind technique.
To confirm this proposalthere are the evidence collected in some university as in
some research study reported ( TURIN UNIVERSITY- CHEMISTRYfaculty ,
CAMPRA P. - UNIVERSITYOF ALMERIA professor CHEMICAL SCIENCE).
Regarding the contaminants it is of interest to report thay in 26 August2021 BBC
news Coronavirus pandemic Japan suspends 1.6 million Moderna doses over
contamination fears A staff of Japan"s supermarketgroup Aeon receives a doseof
the Moderna vaccine“Japan has suspended the use of about 1.63 million doses of
the Moderna vaccine due to contamination.
A metallic substantia the react with magnete finded in some lots .
Of interest finally to remember two recent italian judgesententia : PISA EFIRENZE
2022 it was recognized in one cause- effect relationship between a covid-19
vaccination and a Thromobocitopenia reaction in an 16 years old ,and in the other
case the judge written that this vaccine are experimental , Dna invasive, potentially
of irreversibleeffect and not prevedile at today .
Even if this drugs areofficially registered by regulatory agency during the last years
In example somecountries restricted uses of some covid-19 vaccinein determinate
subpopulation even initially not done .
The same some technical sheet was recomded to be updated by EMA PRAC
introducing some Rare ADR not presentin the first approved VACCINE oneor also
changed the name of the vaccinein order To update registrativedocuments
reporting new controindications.

Conclusion
As global concusion it is possibleto ask to the innovative vaccine producers to
provideofficial written proof of chemico-analytical absence of graphene and
derivates in the final vaccine product.( the same by the international regulatory
agency ).
The same to providefull clarification of the bio-technological manifacturing process
Even if there are patent of industrialsecret
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Biointerfacing 2D Nanomaterials and Engineered Heterostructures
GrapheneOxide Nano-Concentrators Selectively Modulate RNA Trapping According
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Marco De Spirito, Wanda Lattanzi and Massimiliano Papi
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48)Luisetto M , Almukthar N, Tarro G , B. NILI A, Edbey K, F.H. Khan , G.A.Hamid,
Fiazza C, Cabianca L.,Ilman I, Rafa YA , MashoriG.R., Gadama P.G, Latyshev O.Y
(2022). Grapheneand Derivates: Physico-Chemicaland Toxicology Properties in the
mRNA Vaccine Manifacturing Strategy . Sci World J Pharm Sci, 1(2);1-23

48)Bio-pharmaceutical manifacturing large scale production process: The graphene
- derivates role and m RNA vaccine Luisetto M , Tarro G et al aug 2022 article
accepted for publication by editor
49)RAMAN ( RS) SPETTROSCOPYfor BIOPHARMACEUTICAL QUALITYCONTROL and
PAT.RAW MATERIAL - FINAL PRODUCTS : THE NANOLIPIDS EFFECTONSIGNAL
INTENSITY.REGULATORY AND TOXICOLOGICAL ASPECTS luisetto M, Khaled edbey
Et al aug 2022 article accepted by editor ( reviewed)
50) Book SELF-ASSEMBLING PROPERTYOF GRAPHENEDERIVATES
CHEMICO -PHYSICAL and TOXICOLOGICAL IMPLICATIONS luisetto M
Edbey khaled, Gamal Abdul HAMID et al LAMBERT academic publishing 2022
Other additional references
A)https://www.reuters.com/business/healthcare-pharmaceuticals/japan-finds-
stainless-steel-particles-suspended-doses-moderna-vaccine-2021-09-01/
B)https://www.aifa.gov.it/-/aifa-dispone-divieto-di-utilizzo-di-un-lotto-astrazeneca-
accertamenti-in-corso-in-coordinamento-con-ema
C)https://www.pharmaceutical-technology.com/news/moderna-rovi-recall-
vaccines/
D)https://www.fdanews.com/articles/207362-moderna-recalls-nearly-800000-
doses-of-its-covid-19-vaccine
E)https://www.pfpdocs.com/jj-vaccine-recall
F) Dodicesimo rapporto AIFA farmacovigilanza - ADRGRAVI vederedocumento (
rare-molto rare)

G)EMA covid-19-vaccine-safety-update/vederetutti gli aggiornamenti dei vari
vaccini a m RNA e non
H) https://www.ema.europa.eu/en/news/astrazenecas-covid-19-vaccine-ema-finds-
possible-link-very-rare-
cases-unusual-blood-clots-low-blood
I) https://www.reuters.com/business/healthcare-pharmaceuticals/japan-finds-
stainless-steel-particles-
suspended-doses-moderna-vaccine-2021-09-01/
L) https://www.cdc.gov/coronavirus/2019-ncov/vaccines/safety/adverse-
events.html
M) OPEN LETTER for TRASPERENCY to ITALIAN MINISTRY OFHEALTH : Lettera aperta per la
trasparenza per MINISTERO DELLA SALUTEitaly, AIFA, ASSOBIOTEC Tarro, Luisetto,
Monsellato , agosto 2022

RESEARCH ARTICLE
TITLE
RAMAN ( RS) SPETTROSCOPY for BIOPHARMACEUTICAL QUALITY CONTROL and
PAT.
RAWMATERIAL - FINAL PRODUCTS : THE NANOLIPIDS EFFECTON SIGNAL
INTENSITY.
REGULATORY AND TOXICOLOGICAL ASPECTS
Authors:
1) Luisetto M IMA academy Marijnskaya, professorship in toxicology and
pharmacology member of the Chemical technology and Chemical industry IMA
branch , curriculum studiorum in Turin , Pavia and Parma university italy 29121
2)Nili B. Ahmadabadi ,Medicinal chemist ,Nano Drug Delivery, (a Product
Development Firm), United States, Curriculum studiorum in Turin university
3) Edbey K, Professor, Departmentof Chemistry, Libya PhysicalChemistry,
4) Gamal A. Hamid Professor Hematology Oncology, University of Aden, Yemen
5) Tarro G , Professor of oncological virology,Chairman of the Committee on
Biotechnologies of VirusSphere, World Academy of Biomedical Technologies
(WABT), Paris
6) Cabianca L. bio-medical laboratory turin italy Citta’ della Salute
7) Oleg Yurevich Llatyshev IMA presidentRU

Abstract:
The Biopharmaceuticals production is based on a GMP system of quality control
used for the patients health and for the regulatory scope.
Itis relevant for this role the chemico- analytical procedures applied , the specificity
and sensibility of the methods ( to test raw materials but also the final products
before commercialization).
Aim of this work is to verify the role played by nanolipids on Raman ( RS)
Spettroscopy that encapsulate active principle API or other substantia unsing the
different procedurein use today :
1) destructivemethods or
2) non destructive technique.
This is relevant becauseregualtory agency authorized ( EMA) for GMP rules the use
also of the non destructivemethods like direct- RAMAN ( RS) spettroscopy in
various stageof the manifacturing
process ( for raw materials used and to test the final product- drug).
Corresponding author : mauro luisetto maurolu65@gmail.com+393402479620
Keywords: biopharmaceuticals , m RNA vaccine , GMP, European pharmacopeia ,
EMA procedures , PAT, quality control , raw material ,Final products , destructive ,
non destructive direct methods , intensity of signal, sample pre-treatement of the
sample , extraction, toxicology.

INTRODUCTION
In last decades RAMAN ( RS) spettroscopy was deeply introduced in various settings
and also in pharmaceutical Drugs production becauseinnovative, non invasive and
easy to usetechnology.
Fig. n 1 fromhttps://www.edinst.com/us/blog/Raman ( RS)-scattering-blog/
This work start due by the interesting facts that single independent researchers
finded GRAPHENEdeivates in some Vials of m RNA covid-19 vaccine .
This whas notconfirmed by EUROPEAN regulatory agency that written
In an official document ( EMA ) : Lastupdated: 27 January 2022
Parliamentary question - P-000303/2022(ASW)
European Parliament
Answer given by Ms Kyriakides on behalf of the European Commission

8.3.2022Written question
“In the EU a marketing authorisation is granted to a medicinal- productonly after its
quality, safety and efficacy have been evaluated and a positive benefit-risk balance
related to its use has been concluded. For EU authorisations of COVID-19 ( sars cov-
2 ) vaccines this assessmentis carried out by the EMA.
EMA has analysed reports describing the analysis of severalvials of COVID-19 ( sars
cov-2) vaccines suggesting the presenceof grapheneand they concluded that the
currently available data do not show presenceof graphene in the vaccines
concerned. The analysis by EMA’s working party for biological- medicines included
an input on the Raman ( RS)- spectroscopy from theEuropean Directorate for
Quality of Medicines and the independent national testing labs. responsiblefor the
batch release (OMCLs).
Grapheneoxide GO is not used in the manufactureor formulation of any of the
COVID-19 ( sars-cov-2) vaccines or other medicines, so it would not be present at
manufacturing -facilities and there is no obvious way that it could get into the
vaccines. Quality controltesting CQ and quality assurancereview, by the vaccine
manufacturers and OMCLs responsiblefor batch/lots release, confirm that each
batch met all quality standards prior to the release. No product complaints have
been received for the batches mentioned in the paper. The presence of graphene or
graphenederivatives in the vaccines thereforeare not plausible.
The Commission and EMA do not consider that any further- actions are necessary at
this stage.”
But if we read the work of one of this researcher: Campra, P. (2021, June28).
Grapheneoxide detection in aqueous suspension:

ANALYTICAL METHODOLOGY
“Fundamentals of the micro -Raman ( RS) technique Dueto the characteristics of the
sample and to the dispersion of objects with a grapheneappearance of micro-metric
sizein a complex matrix of an indeterminate composition, the direct application of
spectro-scopic methods does not allow characterization of the nano-particles
studied here without a previous microscopic- localization or fractionation from the
original sample.
Therefore, microscopy coupled to RAMAN ( RS) spectroscopy (micro-RAMAN( RS))
was selected as an effective technique for an exhaustive screening of micro metric
objects visible under the optical micro-scope.” (1)
So it is possibleto verify that in example Young RO reported a pretreatment of the
sample beforetest with
Other technique:
Young, R. O. (2021, February 5). Scanning & Transmission Electron Microscopy
Reveals GrapheneOxide in CoV-19 Vaccines. Dr. RobertYoung.
https://www.drrobertyoung.com/post/transmission-electron-microscopy-reveals-
graphene-oxide-in-cov-19-vaccines
“Steps of Analysis of Vaccine Aqueous Fractions
Refrigerated samples were processed under sterile conditions, using laminar -flow
chamber and sterilized lab ware.
Steps for analyses were:
1. Dilution in 0.9% sterile physiologicalsaline (0.45 ml+ 1.2 ml)
2. Polarity fractionation: 1.2 ml hexane + 120 ul of RD1 sample
3. Extraction of hydrophilic- aqueous pHase
4. UV- absorbanceand fluorescencespectroscopy scanning” (2)
Itcan be considered a distructivemethod.

And according the Eurpean pharmacopeia EP : Among the methods established for
quality control of classicalmedicines the so called “non-invasive”, non-destructive,
techniques, such as near-infrared and Raman ( RS)- spectroscopy havebeen applied
for molecular imaging and analytics in process -analyticaltechnology (PAT) and are
implemented in quality by design (QbD) concepts
But What is Raman ( RS) Spectroscopy?
Raman ( RS) spectroscopy is an chemico - analytical technique wherescattered light
is used to measurethe vibrational - energy modes of a sample. Itis named after the
Indian physicistresearcher C. V. Raman ( RS) who, together with his research
partner K. S. Krishnan, was thefirstto observeRaman ( RS) scattering in the year
1928.
Raman ( RS) spectro scopy can provideboth chemical- structuralinformation, as
well as the identification of substances through their characteristic Raman ( RS)
‘finge-rprint’. Raman ( RS) spectro-scopy extracts this information through the
detection of Raman ( RS) scattering from the sample.
This method is based on the phenomena of diffusion of an electro-magnetic mono-
cromatic radiation laser
By the sample tested.
Itis obviousand clear that it is relevant to separatethe sample from its chemical
contest in order to avoid other shade or interference form other molecule inside
the samesample.
This last molecule makepossible not to obtain reviability results.
For this kind of reason often it is used to extract the analite to be detected with
solvent– or diluent beforetest .

And What is the Raman ( RS) Scattering?
When light is scattered by molecule, the oscillating electro magnetic -field of a
photon induces a polarisation of the molecular -electron cloud which leaves the
molecule in a higher energy- state with the energy of the photon -transferred to the
molecule.
This can be considered as the formation of a very short-lived complex between the
photon and molecule which is commonly called the virtual -state of the molecule.
The virtual- state is not stable and the photon is re-emitted almost immediately, as
scattered light.
In the vastmajority of scattering- events, the energy of the molecule is un-changed
after its interaction with the photon; and the energy, the wavelength, of the
scattered photon is equal to that of the incident photon. This is called elastic
(energy of scattering particle is conserved) or Rayleigh scattering and is the
dominant process.
In a much rarer event ( 1 in 10 million photons)Raman ( RS) scattering occurs, which
is an inelastic scattering process with a transfer of energy between the molecule and
the scattered photon.
If the molecule gains energy fromthe photon during the scattering (excited to a
higher vibrationallevel) then scattered- photon loses energy and its wave-length
increases which is called Stokes -Raman ( RS) scattering . In inverseway , if the
molecule loses energy by relaxing to a lower vibrational level the scattered photon
gains the corresponding energy and its wavelength decreases; which is called Anti-
Stokes Raman ( RS)- scattering. Quantum mechanically Stokes and Anti-Stokes are
equally likely processes.With an ensemble of molecules, the majority of molecules
will be in the ground vibrationallevel ( as Boltzmann distribution) and Stokes scatter
is the statistically more probable process. As a result, the Stokes- Raman ( RS)
scatter is always more intense than the anti-Stokes and for this reason, it is nearly
always the Stokes Raman ( RS) scatter that is measured in the Raman- ( RS)-
spectroscopy.

Figure2 JablonskiDiagramshowing the origin of Rayleigh, Stokes and Anti-Stokes
Raman ( RS) Scatter
Raman ( RS) Shift
Itis clear fromthe abovefigure , that the wavelength of the Raman ( RS) scattered
light will depend on the wavelength of the excitation -light. This makes the Raman- (
RS) scatter wavelength an impractical number for comparison between the spectra
measured using different lasers. The Raman ( RS)- scatter position is therefore
converted to a Raman ( RS) shift away from excitation wave -length:

Raman ( RS) shift equation
The first term is the wave number Raman- ( RS) shift in cm-1, λ(0) is the wavelength
of excitation -laser in nm, and λ(1) is the wavelength of the Raman ( RS) scatter in
nm.
Vibrational Modes
Figurereported shows thatRaman ( RS)- spectroscopy measures theenergy gap
between the vibrational levels of the molecule. The ladder of vibrational- levels
shown in Figurereported is for a single vibrational mode of the molecule. Poly-
atomic molecules will contain many vibrational modes, each with their own ladder
of vibrational- levels.
Fig. n 3
Fig n 4 fromRaman ( RS) Spectroscopy and Related Techniques in Biomedicine
by Andrew Downes ,Alistair Elfick
Schoolof Engineering, The University of Edinburgh, Edinburgh EH9 3JL, UK
Sensors 2010, 10 https://doi.org/10.3390/s100301871

Fig. n 5 f orm REVIEW article Front. Microbiol., 12 June 2018
Sec. Food Microbiology
https://doi.org/10.3389/fmicb.2018.01236
Detection of FoodbornePathogens by SurfaceEnhanced Raman ( RS) Spectroscopy
Xihong Zhao, Mei Li and Zhenbo Xu
FromEuropean pharmacopea : 32.3 july 2020 APPLICATIONS
“Raman ( RS) spectro-scopy is commonly used for qualitative and quantitative
applications and can be applied to solid, liquid and gaseous samples. Raman ( RS)
spectro-scopy is a rapid and non-invasiveanalyticalmethod and can be performed
off-line, at-line, on-line or in-line, e.g. for process analytical technology (PAT)-
Process analyticaltechnology. Raman ( RS) spectrometers can be situated far from
the point of measurementusing long-distance optical fibres to collect the Raman (
RS) signal. Raman ( RS) spectro-scopy has a wide variety of applications, for
example:
– identification of materials, active substances or excipients;
– determination of solid-stateproperties, polymorphism and solvated state;
– quality control, assay, uniformity of dosageunits;
– process analysis, monitoring of biological and chemical reactions, synthesis,
crystallisation, granulation, mixing, drying, lyophilisation, extrusion, encapsulation
and coating;

– detection of falsified products;
– mapping, imaging and depth profiling of pharmaceutical forms, distribution of
chemical compounds, detection of un-known substances.
EQUIPMENT
2 types of Raman ( RS) spectrometers can be distinguished depending on the
detection principle,
dispersiveand Fourier transform(FT) instruments. Thesemay be benchtop
instruments
(including microscope-coupled devices, portable-instruments) or hand-held
instruments.
RESPONSE-INTENSITYSCALE
The absolute and relative intensities of Raman ( RS) signals are affected by variations
of severalfactors
including:
– polarisation of the irradiating -light
– polarisation of the Raman ( RS) scattered -light
– intensity of the irradiating- light
– instrumentresponse
– focus and geometry at sample
– packing density of particles in solid samples
– refractiveindex n or change of n (Δn) between analyte and the environment
– the particle- size and particle-sizedistribution
– the scattering cross-section
– the absorption cross-section
The verification of the response-intensity scaleis principally performed for
quantitativemethods.

PROCEDURE
PREPARATIONOF THESAMPLE
Raman ( RS) spectra can be obtained from solids, liquids or gases directly, in
suitable glass or plastic
containers or through films (provided that un-wanted signalcontributions are under
control),
generally with-outprior the sample preparation or dilution.
QUALITATIVEMETHODS
Since frequency shiftpositions are employed for identification, identical laser
intensity for both the reference standard and the material to be examined may not
be necessary. The material to be examined is measured in the same physical -state (
liquid, solid) as the referenceor library material. Raman ( RS) techniques offer the
advantageof non-invasivemeasurements of the material to be examined with-out
removalfrom the packaging. Some packaging materials may lead to additional
signals in the Raman ( RS)- spectrum. This is especially the case when the packaging
material absorbs atthe laser’s excitation wave-length.
QUANTITATIVEMETHODS
Quantitative determination requires that the reference – standard RS and the
material to be examined mustbe measured at the same laser -intensity and
frequency. Ensurethat the material to be examined is measured in the same
physicalstate ( liquid, solid) and concentration range as the reference standard or
library used for calibration. While the Beer-Lambertlaw is not valid for
Raman ( RS) spectroscopy, Raman ( RS) -intensity is directly proportionalto the
concentration of the Raman ( RS) scattering analytes; For solid samples and
suspensions theRaman ( RS) intensity may be affected by the matrix ( owing to
fluorescenceand self-absorption).

The Raman ( RS) signal is influenced by the refractiveindex of the material, the
particle sizeand the particle-size distribution (wheresmall -particles give a relatively
more- intense Raman ( RS) scattering than the large particles), the packing density,
the scattering cross-section, theabsorption cross-section.”
Material and methods
With an observationalmethod some relevant scientific literature and figure (1-18)
are reported and then analizyed.
After this review part an experimental projecthypotesys is submitted to the
reseacher in order to providea complexive glogal conclusion related the topic of this
article.
All literature comes fromscientific bio medical database .
Are also reported some Documents form EU regulatory agency .
Results
Fromliterature :
04 August2016
Raman ( RS) spectroscopy as a process analyticaltechnology for pharmaceutical
manufacturing and bioprocessing
Karen A. Esmonde-White, Maryann Cuellar, Carsten Uerpmann, Bruno Lenain & Ian
R. Lewis
Analytical and Bioanalytical Chemistry
“Adoption of Quality by Design principles, regulatory supportof QbD, process
analytical technology ( named PAT), and continuous manufacturing aremajor
factors effecting new approaches to pharmaceutical manufacturing and bio
processing.

In this review work , we highlight new technology developments, data analysis
models, and applications of Raman ( RS) spectro-scopy, which haveexpanded the
scopeof Raman ( RS) spectro-scopy as a process analyticaltechnology. Emerging
technologies such as transmission and enhanced reflection Raman- ( RS), and new-
approaches to using available technologies, expand the scopeof Raman ( RS)
spectroscopy in pharmaceutical manufacturing process , and now Raman ( RS)
spectro scopy is successfully integrated into real-time release testing, continuous
manufacturing, and statistical process control. Since the last major review of Raman
( RS) as a pharmaceutical PAT in 2010, many new Raman ( RS) applications in bio
processing haveemerged. Exciting work reports of in situ Raman ( RS) spectro scopy
in bi-oprocesses complementa growing scientific field of biological and bio-medical
Raman ( RS) spectroscopy. Raman ( RS) spectro-scopy has madea positive impact as
a process analytical and control tool for pharmaceutical manufacturing and bio
processing, with demonstrated scientific and financial benefits throughouta
product’s lifecycle. Raman ( RS) spectro-scopy is an optical spectro-scopy technique
that provides a “molecular finger-print” of a sample.
As optical- method, Raman ( RS) enables non-destructiveanalysis of chemical
composition and molecular structure. Applications of Raman ( RS) spectro scopy in
polymer, pharmaceutical, bio processing, and bio medical analysis havesurged in
the past3 decades as laser sampling and detector technology has improved.
Because of these technological advances, Raman ( RS) spectro-scopy is a practical
analysis technique inside and outside the laboratory. The Raman ( RS) spectro
scopy is an established PAT tool. Since 1980s, Raman ( RS) spectro scopy has been
used to study active pharmaceutical ingredients (API). Raman ( RS) spectro scopy as
a tool for API analysis has been described for various applications, as polymorph
identification, quantitative analysis QA , in situ crystallization monitoring, real-time
release testing, pharmaceutical -unit operations PUO, and process-induced
transformations . In addition to identifying isolated poly-morphic forms, mixtures of
forms can be analyzed and quantified . The diversestructures that have been
measured by Raman ( RS), fromthe discovery lab. to the manufacturing
environment, show that Raman ( RS) can reliably providequantitative data. In-line
Raman ( RS) spectro scopy can control critical process parameters, enables real-time
process corrections, and ensures consistentproduction of the correctAPI form.

We highlight the new applications in API synthesis and crystallization, real-time
release testing, flow or continuous- manufacturing, and new developments in
Raman ( RS) spectroscopy for understanding and controlling bio processes
Regulatory- perspectives and also guidance.
A philosophicalchange in pharmaceuticalmanufacturing quality, which is strongly
encouraged by regulatory agencies, has created opportunities to integrate real-time
process analytics into manufacturing processes. In 2002, theU.S. FDA launched an
initiative to encourageinnovation in manufacturing technology and quality system
approaches. The FDA 2004 PAT- framework stronglyemphasized a shiftfrom tested-
in quality after the drug productwas produced to building in quality throughout
production with “continuous real time quality assurance” . The EMA established a
PAT team in 2003, which released guidance documents on process PAT, quality by
design (QbD), and real-time release testing. InternationalConference on
Harmonization (ICH) Q8, Q9, Q10, and Q11 documents reinforced FDA and EMA
guidance, which has been implemented in the USA, EU, and Japan since2009.
The FDA and ICH documents provided a strategic- guidance, rather than
prescriptive- guidance, on developing an approach to understand and manage the
risks that might affect critical quality attributes. PAT has an crucial role in this new
framework to understand and manage risk throughouta pharmaceutical product’s
lifecycle. Recently, these principles were extended to bio processing. As a PATin
pharmaceutical manufacturing and bio processing, Raman ( RS) spectro scopy has
demonstrated value fromscientific understanding to process control. Over the past
25 years, Raman -( RS) spectroscopy instrumentation has evolved from home-built
academic lab. instruments to robustcommercially available solutions-based
systems. Theadvent of stable laser sources, high-speed optical fibers, volume holo
graphic gratings, and low-noisechargecoupled device detectors enabled robust
commercial Raman ( RS)- spectroscopy instruments. Newer commercialinstruments
are straight-forward to usebecausethey do not require constantrealignment or
sophisticated knowledge of optics, are equipped with instrumentcontrol -software
CS , and are integrated with Raman ( RS) spectral -libraries. Thus, Raman ( RS)
spectroscopy is accessibleto scientists and environments beyond the academic
research environmentworld . Modern instrumentation has been reviewed in detail
elsewhere .

There are three basic components of a Raman ( RS) spectro-graph, including a laser,
sampling optics, and detector. Modern Raman- ( RS) instruments optimizes the
amount of inelastically scattered -photons and their detection. Modern Raman ( RS)
instruments usea laser as the illumination sourcebecause it is a high-intensity
mono-chromatic sourceof light.
While the laser wavelength can vary from the UV to the near-infrared (λ = 200–1064
nm), most pharmaceutical or bio-processing applications usenear-infrared
wavelengths (λ = 785 or 830 nm), primarily to minimize fluorescenceinterferences.
Articles, bubbles, or droplets with sizes approaching the excitation wave-length
exhibit Lorenz-Miescattering, which causes aqueous systems to become turbid.
Photons can be scattered multiple- times, resulting in photons being diffusely
distributed in a turbid- media. API or excipient particles and cellular organelles, like
mitochondria and nuclei, also strongly scatte-r light . Understanding photon -
transportin turbid media is an important consideration for the quantitative Raman
( RS) spectroscopy applications in content uniformity, real-time release RTR testing,
and in situ bio process control. Much research has been devoted in developing
Raman ( RS) spectroscopy for pharmaceuticalsolids analysis, taking into
consideration process compatibility, validation, and ease of use. Figure reported
shows thevariants of Raman ( RS) spectro scopy that utilize fiber optic probes.
Within the process environment, the sampling flexibility of Raman -( RS) spectro
scopy means that Raman ( RS) can be employed as an off-line, at-line, on-line, or in-
line (or in situ) PAT. Pharmaceutical excipient chemical and physical -properties are
typically a critical process parameter becausethey affect manufacturability, bio-
availability, and risk of process-induced API transformations. Raman ( RS)-
spectroscopy measures excipientmaterial attributes non-destructively and rapidly,
with handheld systems typically used for this application. A comprehensivedatabase
of commonly used pharmaceutical- excipients contains both the Raman ( RS)
spectrum and band assignments . The excipient spectrum can be affected by
different crystalforms, amorphous -content, or other process variations. In-house
preparation of excipients or bio pharmaceutical formulations may require its own
risk-based manufacturing approach . (3)”

Journalof Pharmaceutical and Biomedical Analysis
Volume 76, March 2013
In situ monitoring of powder blending by non-invasiveRaman ( RS) spectrometry
with wide area illumination
Pamela Allana ,LukeJ.Bellamya, Alison Nordona ,David Little johna ,John Andrews
,Pau lDallin
https://doi.org/10.1016/j.jpba.2012.12.003
“A 785 nm diode -laser ( and probewith a 6 mm spotsize) wereused to obtain
spectra of stationary- powders and powders mixing at 50 rpm in a high shear
convective blender. 2 methods of assessing theeffect of particle characteristics on
the Raman ( RS) sampling depth for micro-crystallinecellulose (Avicel), aspirin or
sodium- nitrate werecompared: (A) the information depth, based on the
diminishing Raman ( RS) signal of TiO2 in a reference plate as the depth of powder
prior to the plate was increased, and (B) the depth at which a sample became
infinitely thick, based on the depth of powder at which the Raman ( RS)- signalof
the compound became constant.
The particle size, shape, density and/or light absorption capability of the
compounds wereshown to affect the “information” and “infinitely- thick” depths of
individual compounds.
When different sized -fractions of aspirin were added to Avicel as the main
component, the depth values of aspirin ASA were the same and matched that of the
Avicel: 1.7 mm for the “information” depth and 3.5 mm for the “infinitely- thick”
depth. This latter value was considered to be the minimum Raman ( RS) sampling
depth when monitoring the addition of aspirin to Avicel in the blender. Mixing
profiles for aspirin ASA were obtained non-invasively through the glass- wall of the
vesseland could be used to assess how theaspirin blended into the main
component, identify the end- point of the mixing process (which varied with the
particle sizeof the aspirin ASA ), and determine the concentration of aspirin in real
time.

The Raman ( RS) procedurewas compared to 2 other non-invasivemonitoring
techniques, near infrared (NIR) spectro-metry and broadband acoustic emission
spectro-metry. Thefeatures of the mixing profiles generated by the 3 techniques
were similar for addition of aspirin to Avicel. Even if Raman ( RS) was less sensitive
than NIRspectrometry, Raman ( RS) allowed compound specific mixing profiles to be
generated by studying the mixing behaviour of an aspirin a High lights
Powder blending monitored non-invasively by widearea Raman ( RS) spectro
metry. Effect of the particle sizeon sampling depth and Raman ( RS) -signal
investigated for wide area illumination. Raman ( RS) measurements used to monitor
mixing dynamics, determine end-point and perform quantitative analysis. Higher
chemical specificity of Raman -( RS) compared to near infrared- spectrometry offers
advantages for multi-component mixtures spartame/Avicelmixture.” (4)
Fig. n 6 fromUse of In-lineRaman ( RS) Spectroscopy as a Non-destructiveand
Rapid Analytical Technique to Monitor Aggregation of a Therapeutic Protein
Monday, November 1, 2010 Amol Mungikar, Ph.D
Madhav Kamat, Ph.D Bristol-Myers Squi

Fig. n 6-1 fromRaman ( RS) Spectroscopy: a non-destructive, non-contactand
simple technique to characterizecarbon materials - part 1: Carbon nanotubes
VENERDÌ, 02 OTTOBRE2020 07:53
From ABCS website
https://www.abcs.it/it/blog/caratterizzazione-materiali/Raman ( RS)-spectroscopy-
a-non-destructive-non-contact-and-simple-technique-to-characterize-carbon-
materials-part-1-carbon-nanotubes
Why Raman ( RS) spectroscopy has been used?
Advantages of Raman ( RS) spectroscopy
Very small samples
No special preparation of samples
Ease of use
Non-destructiveand non-contactanalysis
Measurement of various types of samples (liquids, solids, powders, etc.)
Raman ( RS) Spectro scopy needs relative shorttime. So we can do Raman ( RS)
Spectro scopy detection very quickly.

Raman ( RS) spectro scopy is one of the most informative probes for studies of
material properties under extreme conditions of high pressureand low-
temperature
Depth analysis
https://www.contractpharma.com/issues/2021-09-01/view_features/Raman ( RS)-
spectroscopy-for-pharmaceutical-analysis-quality-control/
Raman ( RS) Spectroscopy for PharmaceuticalAnalysis & Quality Control
Raman ( RS) spectroscopy helps ensurequality along the pharma supply chain of
materials—fromincoming raw materials through to finished product.
Jacques Ledru, Head of Characterization, Catalent, Nottingham 2021
“Raman ( RS) spectro scopy has many applications within the pharmaceutical-
industry. Itcan be used to identify polymorphs, to analyze active pharmaceutical
ingredient (API) forms and their distribution within formulated -products. Butwhat
is it, and how can it be applied in practice filed ?
In contrastto the standard infrared (IR)- spectro-scopy, which identifies the specific
frequencies of radiation that are absorbed by a sample, Raman ( RS) spectro-scopy
studies the way light is scattered by the molecules. As a laser beam passes through
the sample, much of the light passes through and scatters with its energy un-
changed; this is known as Rayleigh- scattering.
Some of its photons collide with the molecules and lose -energy, in a phenomenon
known as a Stokes -shift. Others may pick up energy from the excited molecules and
emerge with a higher- energy level, or an anti-Stokes shift. In Raman- ( RS) spectro-
scopy, the light that emerges is collected, and that which is scattered without
changing energy is filtered out. What remains provides a unique spectral- pattern
for that individual molecule. This finger print can be used to identify the molecule by
comparing the pattern to an knowed reference substantia.

Transmission Raman ( RS) spectrometry, mean while, often gives better results
when sampling solids than a conventional backscatter Raman ( RS) technique as the
radiation passes through the sample analyzing a much larger volume. As the
technique is a non-evasiveand non-destructive, it can be used for the direct
analysis of batches of hundreds of whole -tablets, or capsules, that can be scanned
in minutes, and can quantify both the API (down to less than 1% drug loading) and
the excipient in a single measurement using appropriately developed partial least-
squares calibration- models.
In this kind of technique, the incident light is passed through an objective - lens, and
focused onto a very small spot. This allows resolution down to fractions of a micron
to be achieved. The distribution of components within a sample can be determined
in this way, the laser can be focus on the sed on specific areas of concern. This may
be to determine the presence/ identification of a suspected contaminant, particle
or other un expected feature, and as such, Raman- ( RS)- microscopy is much more
sensitivethan techniques used for the analysis of a material’s bulk -properties.”
Talanta
Volume 250, 1 December 2022, 123719
Talanta
Raman ( RS)-based detection of ciprofloxacin and its degradation in pharmaceutical
formulations
Chen Liu Lisa Müller-Bötticher ChangLiudeJürgen Poppa Dagmar Fischerg Dana
Cialla-Mayab
“A Raman ( RS)-based label-free analytical method was developed to detect
antibiotic ciprofloxacin (CIP) in various pharmaceuticalformulations in the presence
of different matrices ( ear drops, eye drops and infusion- solutions)”

Fig. n 7
Fig n . 8 Figure1. Illustration of the portable Raman ( RS) device used: (A) sample
holder, (B) the device, (C) mean Raman ( RS) spectrum of the de-ionised water
collected froma glass vial, and (D) a Raman ( RS) spectrum of glass. From Molecules
n Situ Water Quantification in Natural Deep Eutectic Solvents Using Portable Raman
( RS) Spectroscopy by S. Elderderi ,Laura Wils ,Charlotte Leman-Loubière ,Hugh J.
Byrne,I. Chourpa ,Cécile Enguehard-Gueiffier ,E. Munnier,Abdalla A. Elbashir ,Leslie
Boudesocque-Delaye,FranckBonnier

from
Silge, A., Bocklitz, T., Becker, B. et al.
Raman ( RS) spectroscopy-based identification of toxoid vaccine products. npj
Vaccines (2018).
https://doi.org/10.1038/s41541-018-0088-y
“European Pharmacopoieia (Ph. E.), provides the legislative framework for product
testing and regulatory- bodies such as the European Directorate for Quality of the
Medicines (EDQM) prequalify methods for these purposes, including the biological-
standards to be used to obtain comparability. Between the methods established for
quality control of classicalmedicines the so called “non-invasive”, e.g., non-
destructive, techniques, such as near-infrared and the Raman -( RS)- spectroscopy
have been applied for molecular- imaging and analytics in process analytical-
technology and are implemented in quality by design (QbD) concepts.
Recent technical developments and works in the field of the Raman ( RS) -
technology now enable manufacturers to usethis technique for analysis of more-
complex biological products including protein mixtures in bio reactors and cell-based
and tissue-engineered products. Raman ( RS) -micro spectroscopy is an inelastic light
scattering-based method usefulfor the non-destructiveanalysis of bio chemical
samples. Itprovides a wealth of molecular information on a specimen by the
sample’s own inherent vibrational -signatures.
As the bio-chemical composition of a sample is mirrored in the Raman ( RS)
spectrum, mathematical methods including analytical modelling translate the
physically recorded Raman -( RS) data into higher level information, which can
further be exploited for comparativeanalyses. The fingerprint-likespecificity of
spectral -signatures can be utilized to setup a reference data baseof tested
biological -products for identification purposes” (5)
Analytical and Bioanalytical Chemistry Anal Bioanal Chem. 2022
doi: 10.1007/s00216-021-03727-4

The role of Raman ( RS) spectroscopy in biopharmaceuticals from development to
manufacturing
Karen A. Esmonde-White, Maryann Cuellar, and Ian R. Lewis
Raman ( RS) spectroscopy as a process analyticaltechnology (PAT) in bioprocessing
“Advances in cell -engineering, process control, and media composition are credited
with improving the volumetric yield of cell- culture bio processes, making bio
pharmaceutical manufacturing morecost-effective and practical . Adoption of the
PAT and Quality by Design (QbD) principles is an important contributor to
improvements in bio- process control. PATprovides real-time understanding which
helps to manage risk throughouta bio pharmaceutical product’s lifecycle. The PAT-
framework is an integrated approach using historical process knowledge, modeling,
and analyses. Many types of physicaland chemical analyses areused for bio
processing. Traditionalparameters like pH, temperature, dissolved oxygen, feed
composition, and feed timing are measured in situ.
Bio-chemical -parameters such as nutrients, metabolites, amino acids, proteins, cell
viability, and biomass can be measured by spectro-scopy, electro-chemicalsensors,
bio-chemical assay, or chromatog-raphy. ThesebiochemicalPATs can be used in
situ, integrated with an automated sampler for at-line measurements, or off-line.
Spectro-scopy- PATtechniques are based on light’s interactions with materials. They
providea fast, label-free, non-invasive, and non-destructive chemical analysis of a
material” (6)

Fig. n 9 Raman ( RS) spectra of AuNP100nm@lipid (blue) and liposome(orange),
obtained wit h total lipid concentrations of 1 and 100 mM, respectively. Lipid-
compositions wereDOPC/Chol(60/40). Allthe samples weremeasured at 25 °C. At
least three reproduciblespectra were obtained for each system. Raw- spectraldata
are shown in the Supporting Info.
And in Article 2017
AdenosineTriphosphate-Encapsulated Liposomes with Plasmonic Nanoparticles for
SurfaceEnhanced Raman ( RS) Scattering-Based Immunoassays
Xuan-Hung Pham,Eunil Hahm ,Tae Han Kim ,Hyung-Mo Kim ,Sang Hun Lee ,Yoon-Sik
Lee ,DaeHong Jeong Bong-Hyun Jun
Sensors 2017
“Preparation of ATP-Encapsulated Liposomes and SiO2@Au@Ag NPs
We designed and fabricated ATP-en-capsulated liposomes that could release ATP
only when the liposome structurewas ruptured for SERS-based immuno-assays as
shown in the Scheme reported. So For this, ATP en-capsulated lipo-somes and gold-
silver alloy (Au@Ag)-assembled silica NPs (SiO2@Au@Ag) wereprepared,
separately. Both liposomes and SiO2@Au@Ag NPs alonewere inactive for SERS -
measurement. When the liposome’s structureis broken, and the ATP is released, a
strong SERS signal could be obtained, because the released ATPs are immobilized on
SiO2@Au@Ag NPs.” (7)

Fig. n 10 Surfaceenhanced Raman ( RS) scattering (SERS) spectra of (i) SiO2@Au@Ag
NPs, (ii) 10 mM ATP, and (iii) SiO2@Au@Ag NPs in the presenceof 10 mM ATP. The
concentrations of SiO2@Au@Ag NPs were 1 mg/mL in ethanol solution,
respectively.
Fromhttps://patents.google.com/patent/US20130273561
(19) United States(12) Patent Application Publication (10) Pub. No.: US
2013/0273561 A1 Walker etal. US 20130273561A1 (43) Pub. Date: Oct. 17, 2013

FIG. 11 shows thestability of MGITC-lipid-coated particles and Rho-lipid-coated-
particles in which;
A) shows theSERS spectrum of MGITC-lipid-coated particles collected on day of
synthesis, 12 days, and since 25 days after synthesis; and
B) shows theSERS spectrum of Rho-lipid-coated-particles collected on day of
synthesis, and 7 days after synthesis, in which for both cases, ( particles werestored
in water at 4 deg C. between the measurements)

Nanomaterials (Basel). 2019 Mar
2019 Mar 3. doi: 10.3390/nano9030341
Raman ( RS) Imaging of Nanocarriers for Drug Delivery
Sally Vanden-Hehir, William J. Tipping, Martin Lee, Valerie G. Brunton, Anna
Williams, and Alison N. Hulme1
“A major advantageof the Raman ( RS) is that it allows direct imaging of the
nanocarriers, and notthe payload en-capsulated within them” (8)
Fig. n 12 fromDaria Petrenko et al

JournalList Nanomaterials (Basel) v.9(3); 2019 Mar PMC6474004
Logo of nanomat
Nanomaterials (Basel). 2019 Mar doi: 10.3390/nano9030341
Williams, and Alison N. Hulme
Fig n.13 Raman imaging of nano carriers. (a) Representation of different materials
which can be fabricated into nano-carriers, such as biopolymers ( alginate), synthetic
polymers (PLGA), and lipids ( liposomes and micelles). (b) Energy- level diagrams
showing the processes of spontaneous Raman ( RS), stimulated -Raman ( RS)
scattering (SRS), and coherent anti-Stokes Raman- ( RS)- scattering (CARS). (c)
Spontaneous Raman ( RS) spectra showing the characteristic peaks in microglia (top,
green spectrum) and PLGA, a common polymer for drug delivery (bottom, black
spectrum). The Spectra are normalized and offsetfor clarity. (d) SRS -images of
microglia when Ω = 2939 cm−1 (CH3, proteins, grey), 2856 cm−1 (CH2, lipids, cyan),
and 1663 cm−1 (amide I, magenta). Scale bars = 5 µm.

Nanomaterials (Basel). 2019 Mar 2019 Mar 3. doi: 10.3390/nano9030341
Williams, and Alison N. Hulme
“There are various ways of fabricating materials into nano carriers, depending on
the desired properties of the final formulation and the drug to be en-capsulated.
The polymer is dissolved in an organic solventprior to emulsification with an
aqueous -phaseto form nano-sized droplets, which become the nano carriers upon
evaporation of the organic solvent. Hydrophobic drugs can be added into the
organic phase with the polymer, whilst the process can be modified to a double
water-in-oil-in-water emulsion to en-capsulatehydrophilic drugs. Liposomes are
generally formed by a lipid -film hydration method , and micelles will self-assemble
in an aqueous -solution abovethe critical micelle- concentration ” (8)
fig n . 14 Measured Raman ( RS) spectra of graphite, grapheneoxide, and reduced
grapheneoxide
FormAppl. Phys. Lett. (2015); https://doi.org/10.1063/1.4928124
Sreekanth Perumbilavila, P. Sankara, T. Priya Rose, and Reji Philipb

Fig n 15 from Royal Society of Chemistry Issue43, 2015 From thejournal:
PhysicalChemistry Chemical Physics Effects of the molecular level dispersion of
grapheneoxide GO on the free volume characteristics of poly(vinylalcohol) and its
impact on the thermal and mechanical properties of their nanocomposites.
S. K. Sharma, J. Prakashb ,P. K. Pujari

Fig n 16 FTIRspectra of pure ATO ( atorvastatin), free NLC (Nano structured lipid
carrie )and optimized ATO-NLC formulation. From settings
Hypolipidemic Activity of OliveOil-Based Nano structured Lipid Carrier Containing
Atorvastatin
by Heba S. Elsewedy ,Tamer M. Shehata ,Mervt M. Almostafa and Wafaa E. Soliman
Academic Editors: Rosalia Bertorelli, Gemma Gutierrez and Maria Matos
Nanomaterials , https://doi.org/10.3390/nano12132160
23 June 2022

Fig. n 17 Raman ( RS) spectra of heparin and those of AuHep-NPs and AgHep-NPs.
FromAntifungal and Cyto-toxic Evaluation of Photo chemically Synthesized Heparin-
Coated Gold and Silver Nanoparticles June 2020 Molecules 25(12) DOI:
10.3390/molecules25122849
Project: Micro and nano-structured novelbiomaterials for the inhibition of micro
organisms thatcause oral infections.
Lab: Nano-estructuras y Biomateriales, Laboratorio de Investigación Interd. (LII)
María Del Pilar Rodrígueet al

Fromhttps://www.pei.de/EN/newsroom/press-releases/year/2018/20-raman-
spectroscopy-allows-fast-analysis-of-vaccines.html
20/2018
“Vaccines are complex kin of bio medicines ( drugs) composed of a number of
different molecules. In the manufacturing- process and beforemarketing
authorisation, extensive research is required to verify the identity, quality, efficacy
and safety of the products. Fast, cost-effectiveyet reliable chemico analytical -
methods are necessary and can contribute to fighting against counterfeit medicines.
Researchers fromPaul-Ehrlich-Institut haveshown with scientists from Jena that
the Raman spectroscopy (RS) is a suitable tool for this. The results are reported by
NPJ Vaccines in its edition of 04.10.2018.
In Raman -spectroscopy (RS), molecules or solids are exposed with laser light. The
inelastic scattering of the light and the associated differences in frequency with the
incident light allow conclusions to be drawn about the examined substance. The so-
called molecular finger-printallows the quick and easy identification of any
molecule. The method is used, for example, to study the material properties of semi
conductors or for infection diagnostics. The process is also used in the quality
control of chemical- medicines (tablet form), in drug manufacturing (fermenter) and
for the identification of counterfeit -medicines.
Researchers applied the Raman maps to analyze specific Raman signatures fromair-
dried samples of combination vaccines containing antigens (AG) from tetanus,
diphtheria, and pertussis (DTaP vaccines). In fact, the vaccines could be identified
and distinguished using these specific signatures”.

Silge A, Bocklitz T, Becker B, Matheis W, Popp J, Bekeredjian-Ding I (2018). Raman
spectroscopy-1 based identification of toxoid vaccine products.
NPJ Vaccines NPJ Vaccines 3, Article number: 50 (2018)
https://doi.org/10.1038/s41541-018-0088-y
“Vaccines are complex bio medicines. Manufacturing is time consuming and
requires a high level of quality control(QC) to guarantee consistentsafety - potency.
An increasing global demand has led to the need to reducetime and cost of
manufacturing. The evolving concepts for QC and the up coming threat of
falsification of bio medicines define a new need for methods that allow the fast and
reliable identification of vaccines. Raman spectroscopy- (RS) is a kind of non-
destructivetechnology already established in QC of classicalmedicines. We
hypothesized that Raman- spectro scopy RS could be used for identification and
differentiation of vaccine products. Raman- maps obtained from air-dried samples
of combination vaccines containing antigens from tetanus, diphtheria , pertussis
(DTaP -vaccines) weresummarized to compile product-specific Raman signatures.
Sources of technical variancewere emphasized to evaluate the robustness and
sensitivity in down -streamdata analysis. The data management approach corrects
for spatial in-homogeneities in the dried sample while offering a proper
representation of the original samples inherent chemical -signature. Reproducibility
of the identification was validated by a leave-one-replicate-out cross-validation. The
results high lighted the high specificity and sensitivity of Raman RS measurements
in identifying DTaP vaccine -products. Theresults pave the way for further
exploitation of the Raman technology for identification of vaccines in batch release
and cases of the suspected falsification.
Preparation of the sample
For Raman (RS) measurements vaccine suspensions wereextracted from the
containers into THE Eppendorf tubes, homogenized by vortexing and 1 μl was
applied onto a CaF2 slide and dried at the roomtemperature Tta . The sample size
was predicted using the learning -curveresulting in 5 replicates to be necessary.

We planned with 10 in every group to ensurea valid statistical outcome. 20 minutes
after the preparation- samples werevisibly dry and the measures started with the
firstreplicate. The last replicate was measured after ~2 hour later.
Technical reasons for varianceof Raman spectra RS
While examining air-dried vaccine samples by means of Raman micro-spectroscopy
2 sources of technical variance wereidentified. First, the drying -procedureof the
colloidal vaccine droplets caused a spatial in-homogeneity of the vaccine material
during evaporation, which is known as coffee-ring effect.
Concentration gradients within the air-dried droplet resulted in spatial in-
homogeneities and so thereforein variation of Raman signal intensity at different
grid points .
Second, vaccine products containing phenoxy-ethanol showed variations within
their Raman- signaturethat roughly correlated with the drying time.
In Fig. reported the mean spectra of dTaP-IPV2,IPV, and dT2 are depicted. Each
spectrum summarizes the Raman- RS signatures of one replicate mapped after the
indicated drying interval. The mean spectra of the first replicates were measured
after a drying interval of about 20 min.
These spectra were dominated by Raman RS signals that were previously assigned
to phenoxy-ethanol by
Badawi et al. These signals diminished in intensity the longer the vaccine
suspensions weredried at roomtemperature, albeit to varying extents: For the
vaccine products dTaP-IPV2 and IPV the
signals disappear almost completely after a drying period of 2 h (Fig. reported )
while in dT2 the phenoxy-ethanolsignals remain prominent in related Raman -
signaturewithin the observed time span.
The dried spots of a vaccine suspension arenot identical preparations, they rather
representtechnical replicates.Slight differences in the drying behaviour were
obtained in the microscopeimages of distinct vaccinespots ( as in Fig. reported).
Also the evaporation behaviour of the phenoxy-ethanolis subjected to such
fluctuations, Fig. reported.

We suggest, each replicate has its individual drying -kinetic.The phenoxyethanol
Raman signals can still be presentafter 100 min drying time in one spot while they
were disappeared after 90 min drying in another spot(Fig reported ). Aside from
such fluctuations, the trend of the decreasing Raman -signalintensities of phenoxy-
ethanol withongoing drying time due to evaporation is evident in the mean spectra
of Fig. reported . For the statistical modelling, it is important to be aware of such
variation.
The resulting spectrum is formed by the super-position and the reciprocal -influence
of the underl-ying spectral values simultaneously recorded from all chemical
constituents,like, vaccine antigens, adjuvants, buffer components and other kind of
excipients. These signals are further influenced by the presenceof
solvents, the pH and the physical - properties, such as formation of crystals or
amorphous particles within the vaccine suspension.
Thus, the mixture itself and other interfering effects complicatethe analysis of the
spectraldata. Well-established computational -methods were applied to correct for
the influence of the instrumental setup or background noise. A common method for
dimension reduction is the principal
component analysis (PCA). PCA transforms a setof possibly correlated response
variables into a new set of non-correlated variables, referred to as principal
components (PC). The output of the PCA is the components in the order of
significance.
Components with less significance (assigned to noise) can be ignored. So the
dimension of the data is reduced with-outloss of information.” (12)

Analyst. 2006 Oct; doi: 10.1039/b605299a. Epub 2006 Aug 25.
Raman spectroscopy as a process analytical technology tool for the understanding
and the quantitative in-line monitoring of the homogenization process of a
pharmaceutical suspension
T R M De Beer 1, W R G Baeyens, J Ouyang, C Vervaet, J P Remon
DOI: 10.1039/b605299a
“The aim of this study work was to proposea Process AnalyticalTechnology
strategy for the quantitative in-line monitoring of an aqueous pharmaceutical
suspension using RS Raman -spectroscopy. A screening design was used to study
the significanceof process variables (mixing speed and height of the stirrer in the
reactor) and of formulation variables (concentration of the active pharmaceutical
ingredient (API) ibuprofen and the viscosity enhancer ( the xanthan- gum)on the
time required to homogenizean aqueous pharmaceutical model suspension as
responsevariable.
Ibuprofen conc. (10% and 15% w/v ) and the height of stirrer (position 1 and 2) were
discrete variables, whereas the viscosity enhancer (conc. range: 1-2 g L-1) and the
mixing -speed (700-1000 rpm) werecontinuous variables. Next, a multilevel full-
factorial design was applied to study the effect of the remaining significant variables
upon the homogenization- process and to establish the optimum conditions for the
process. Interactions between these kind of variables were investigated as well.
During each design experiment, the conformity index (CI) method was used to
monitor homogeneity of the suspension mixing system in real-time using the Raman
spectroscopy RS in combination with a fibre optical immersion probe. A principal
component regression- (PCR) modelwas developed and evaluated to perform
quantitative real-time and in-line measurements of the API during the mixing -
process. Theexperimental design results showed that the suspension
homogenization- process is an irregular- process, for which it is impossibleto model
the studied variables upon the measured responsevariable. Applying PCR model it
is possibleto predict in-line and real-time the concentration of the API in a
suspension during a mixing process.

In this research work , it is shown that Raman spectroscopy RS is a suitable PATtool
for the control of the homogenization -process of an aqueous -suspension. Raman
spectroscopy notonly allowed real-time monitoring of the homogeneity of the
suspension, butalso helped (in combination with experimental design) to
understand the global process. The technique allowed real-time and in-line
quantification of the API during mixing -process.” (13)
Pharmaceutics Article
Analytical Techniques for the Assessmentof Drug-Lipid Interactions and theActive
SubstanceDistribution in Liquid Dispersions of Solid Lipid Microparticles (SLM)
Produced de novo and Reconstituted from Spray-Dried Powders
Eliza Wolska et al
15 July 2020
“ Raman spectroscopy (RS) detects the vibrations of molecules after excitation by an
intensive laser
beam . This technique has already been used as a tool to identify and localize
specific components
in various liquid - solid dosageforms . The use of Raman spectroscopy RS to
characterizethe colloidal and micro-particulate lipid systems is rare.
Raman spectro-scopy is a useful technique, as it involves no sample- preparation
and, most importantly, allows measurements in the presence of water. At the
currentstage of our research, it has not broughtthe expected results, and the
attempt to confirmwith this technique the localization of the API on the surfaceof
the SLMwas un-successful.
Eevn if both CsA and SPIRwereidentified on the SLMsurface, the dominant
components on the Raman maps werelipids and polysorbate(or PVP).
Discrimination on the spectra of the bands derived from the API and lipids (or other
excipients) was impossible, mainly due to the spectral- properties of the tested API
and their low concentrations in the formulations.“.

Fig. n 18 Raman maps (20×resolution) of F3L prepared “de novo” microparticles
formulations. Red and green correspond to the API ( active substantia) and lipids,
respectively.
Experimental projecthypotesys
In order to verify the absence/presence of graphenederivates in vials of some bio-
pharmaceutical compounds it is needed to test 100 sampleIn example m RNA
vaccine- nanolipids .
This tests mustto be performed using : A) classic chemico analitycal procedureand
B) officially GMP approved ( RAMAN ( RS) direct spettroscopy ) , all with the
accettable sensibility.
1) Method as reported by somerearcher ( with solvent extraction in a classic
chemical methods befor test, destructive method )
2) Method as approved EUROPENA PHARACOPOEIA likedirect RAMAN
spettroscopy non desctructivemethod
This sample mustdivided in group of 20 and sended blinded to various and
different accreditated- certified chemical laboratory and independent.
Itis needed a controlgroup, it is needed to be used use standard solution .

To be Performed qualitative and quantitative analisys.
The sample must to be treated for the pre-analitycalneed ( extraction) before to be
analyzed.
This in order to verify in the same condition the nanolipids inclusion and outiside of
this nanoparticles.
Results: it is necessary to verify is there is or not significative presenceof
grapheneor its derivated in the final approved vials. ( p < 0,005)
The results must to be divided using a destructivemethod and a non destructive
one.
For a quantitative test it is necessary to use standard ( due by charactheristics of the
sample: lipids nanoparticles)
Discussion
it is interesting to observethe analitical behavior of nanoparticles- liposome with
encapsulated molecule like eccipients or impurity in a RAMAN ( RS) spectra vs the
non encapsulated ones.
Observing fig 9 it is possibleto say that encapsulated particle producea reduced
intensity in Raman ( RS) Spettroscopy.
The heparin molecule show greater intensity signalvs the heparin AU -hep - NPS
(17)
Also of interest to observethe kinetics during time of some nanoparticles as
reported (11) and the fact that
After 1 -12 -25 days the signalgradually increase.
Of great interest also that some independent researcher ( as published by Young
R.O) using other method
Pre- treated the samplein order to have extraction before test.
And as reported Anja Silge “For Raman measurements vaccinesuspensions were
extracted fromthe containers into Eppendorf tubes, homogenized by vortexing
and 1 μl was applied onto a CaF2 slide and dried at room temperature”

P. CAMPRA associate Professor ALMEIRA university Phd in Chemical sciences
written :
“Fundamentals of the micro -Raman ( RS) technique Dueto the characteristics of the
sample and to the dispersion of objects with a grapheneappearance of micro-metric
sizein a complex matrix of indeterminate composition, the direct application of
spectroscopic methods does not allow characterization of the nano-particles studied
here without a previous microscopic- localization or fractionation from the original
sample.”
According Sally Vanden-Hehir et al
“A major advantageof Raman ( RS) is that it allows direct imaging of the
nanocarriers, and notthe payload
en-capsulated within them”
for quality control of final drugs and raw material :
EUROPEN PHARMACOPEIA reportthatit can be used for classical drugs CQ -RAMAN
( RS) SPETTROSCOPY aslo in non destructive direct method. ( GMP)
The “Assesmentreport“of a famous m RNA covid-19 VACCINEEMA in febr. 2021
Provided specific obbligation to the producer in order to complete post-
authorization measure for the conditionate marketing authorization:
Additional information are needed for 1 eccipient ALC-0315 and the syntetic
process .
Also as reported in the technical sheet of a m RNA covid-19 vaccinedec 2021 : “
11.1. Information on hazard classes as defined in Regulation (EC) No 1272/2008”
General Information: Toxicological properties havenot been thoroughly
investigated. The following information is available for the individual ingredients.
11.1. Information on hazard classes as defined in Regulation (EC) No 1272/2008
General Information: Toxicological properties havenot been thoroughly
investigated. The following information is available for the individual ingredients.

Related the works of some independent reseacher ( P CAMPRA, Young RO, Ki-Yeob
Jeon, Young MI Lee , Giovanniniet al ), the methods used before to test ( pre –
treat the sample with solvent) and their evidences :
it is of great interest to match this results with the EMA statement that in a written
responceconfirm that graphenederivates was not presentin the sample tested (
observig the RAMAN spectra in the laboratory of proof related).
So becauseGraphene derivates are used in many bio technological process dueto
their properties in absorbtion, extracion , purification , carrier, adiuvant and many
other : it is needed to verify the productiveprocess in manifacturing of new
biopharmaceuticals to verify if impurity are present , what kind of this and in what
concentration. ( also in m RNA covid-19 vaccine)
All this for toxicological and patients safety needs obviously .
Of interest it is the fact that Scientific literature show different entity in RAMAN (
RS) -INTENSITYfor theencapusalted molecule andfor the non encapulates ones (
nanoparticles- nanolipids- liposome).
Direct RAMAN ( RS) - technique is moreefficacy in testing the nanoparticles ( and
not their payload) (8)
The charcteristic kinetic destiny of this nanoparticle during the times it is also of
interest : after Various days the signal increase( disruption of the nanoparticle
contribute to makenacked the encapsuled molecule ? )seeas reported in
references.
So considering all this facts : it is recomended to whom it concern to test as
reported an experimental projecthypotesys thepresence / absence of graphene
GO in :
100 vials of the m RNA covid-19 vaccine - nanolipids using the method of classic
analytical chemistry
Like RAMAN ( RS) destructivemethod with pretreatment – extraction of the
sample by solvent
and 100 vials samplewith the method as reported In EP like RAMAN ( RS)
spettroscopy non destructive direct method.

Itis needed to send the sample to various certified labs using also control ( blinded)
The results must to be collected and the analized in statistical way in order to verify
if there is similar results between the two groups or there are significative deviation.
Conclusion
After this review part , but related :
- the recent new evidences about graphene derivates finded in somevials of covid-
19 vaccine by independed researcher , that seem not coerent whit the Regulatory
agency (analitic reportand statement )
-the fact that the status of encapsulated molecule show differentprofile of intensity
signal in RAMAN ( RS) spettroscopy
it is stretcly recomend to Performthe experimental hypotesys project submitted
using this two methods
( classic chemical pre-treatement of the sample before Raman ( RS) and compared
with a NON destructivedirect Method as permitted by EP- EMA GMP).
Itis crucial to verify the entity of the nanolipids particles EFFECT in the RAMAN ( RS)
SIGNAL of an encapsuled molecule to be searched: it can be relevant for the CQ?
What happen to the signal when dissolved nanolpidis? And nanolipids can
influence/reduce intensity of RAMAN ( RS) spectra of an analite to be detected ?
According the authors only after seeing this results it will be possibleto solvethis
apparent contraddiction. ( Between what showed by some independent researcher
and the regulatory agency related a sameanalita).
The only way it is to pre-treat the sample in the same way beforeregister Raman
( RS) for the two groups even if not requestby the direct non destructive methods .

Finally about the entity of this phenomena :the reduction of intensity of the signal
of the payolad in a nanoparticle :What kind of implication can haveon GMP - CQ ,
PAT , regulatory process and for the toxicological Profileof a new innovative
biopharmaceutical product ?
Impurity in classic drugs was observed in somecases even in registered and
authorized drugs so why non deeply investigate the impurity profile also of m RNA
covid-19 vaccine?
Expecially when some manifacturing procedureare not fully knowed also by
regulatory agency and when for some eccipent used the controlauthority asked to
the producer to providecomplete information related quality testof raw materials
used.
Itis opinion of the authors that the responceprovided by EMA related written
question on graphenederivate presence or not in viaslof covid-19 vaccinemustto
be integrated with written information about the Intereanalitical process used in
the controllab ( also related pre-treatement ) .
Conflict of interest : no

References
1) Campra, P. (2021, June28). Grapheneoxide detection in aqueous suspension:
2)Young, R. O. (2021, February5). Scanning & Transmission Electron Microscopy
Reveals GrapheneOxide in CoV-19 Vaccines. Dr. RobertYoung.
3)Published: 04 August2016
Raman ( RS) spectroscopy as a process analyticaltechnology for pharmaceutical
manufacturing and bioprocessing
Karen A. Esmonde-White, Maryann Cuellar, Carsten Uerpmann, Bruno Lenain & Ian
R. Lewis
Analytical and Bioanalytical Chemistry volume409, pages637–649 (2017)
4)Journalof Pharmaceutical and Biomedical Analysis Volume76, 25 March 2013,
Pages 28-35
Journalof Pharmaceutical and Biomedical Analysis
In situ monitoring of powder blending by non-invasiveRaman ( RS) spectrometry
with wide area illumination
Pamela Allana Luke
J.Bellamya Alison Nordona David Little johna John Andrews PaulDallin
https://doi.org/10.1016/j.jpba.2012.12.003

5)FromMolecules settings
In Situ Water Quantification in Natural Deep Eutectic Solvents Using PortableRaman
( RS) Spectroscopy
Suha Elderderi ,Laura Wils ,Charlotte Leman-Loubière ,Hugh J. Byrne,Igor Chourpa
Cécile Enguehard-Gueiffier ,Emilie Munnier ,Abdalla A. Elbashir ,Leslie Boudesocque-
Delaye and Franck Bonnier
Silge, A., Bocklitz, T., Becker, B. et al. Raman ( RS) spectroscopy-based identification
of toxoid vaccine products. npj Vaccines (2018). https://doi.org/10.1038/s41541-
018-0088-y
6)Analyticaland Bioanalytical Chemistry
Anal Bioanal Chem. 2022
doi: 10.1007/s00216-021-03727-4
The role of Raman ( RS) spectroscopy in biopharmaceuticals from development to
manufacturing
Karen A. Esmonde-White, Maryann Cuellar, and Ian R. Lewis
7) And in Article 2017
AdenosineTriphosphate-Encapsulated Liposomes with Plasmonic Nanoparticles for
SurfaceEnhanced Raman ( RS) Scattering-Based Immunoassays
Xuan-Hung Pham,Eunil Hahm ,Tae Han Kim ,Hyung-Mo Kim ,Sang Hun Lee ,Yoon-Sik
Lee ,DaeHong Jeong Bong-Hyun Jun
Sensors 2017
8) Nanomaterials (Basel). 2019 Mar 2019 Mar 3. doi: 10.3390/nano9030341
Raman ( RS) Imaging of Nanocarriers for Drug Delivery Sally Vanden-Hehir, William J.
Tipping, Martin Lee, Valerie G. Brunton, Anna Williams, and Alison N. Hulme

9) EMA - 19 February 2021 - EMA/707383/2020 Corr.1
- Committee for Medicinal Products for Human Use
(CHMP) AssessmentreportComirnaty - Common
name: COVID-19 mRNA vaccine(nucleoside-modified
- ProcedureNo. EMEA/H/C/005735/0000, https://
www.ema.europa.eu/en/documents/assessment-report/
comirnaty-epar-public-assessment-report_en.pdf pp. 8
10) Safety sheet Cominarty dec 2021
11) Darkfield microscopeanalysis of the blood of 1006 symptomatic subjects affer
vaccination whit two types of mRNA vaccine” (F. Giovannini, R. Benzi Capelli, G.
Pisano) -disinfection 1/ 2022 organo di A.T.T.A. (AssociazioneTossicologieTecnici
Ambientali) Analisi al microsopio in campo scuro sulsanguedi 1006 soggetti
sintomatici dopo vaccinazionecon due tipi di vaccino a m RNA
12)SilgeA, Bocklitz T, Becker B, Matheis W, Popp J, Bekeredjian-Ding I (2018).
Raman spectroscopy-1 based identification of toxoid vaccine products.
NPJ Vaccines NPJ Vaccines 3, Article number: 50 (2018)
https://doi.org/10.1038/s41541-018-0088-y
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T R M De Beer 1, W R G Baeyens, J Ouyang, C Vervaet, J P Remon
DOI: 10.1039/b605299a

14) Pharmaceutics Article
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American J Epidemiol Public Health. 2022 January 12;6(1): 001-006.doi:
10.37871/ajeph.id50

APPRECIATION LETTERS :
Related articled SELF-ASSEMBLING PROPERTYOF GRAPHENEDERIVATES
CHEMICO -PHYSICAL and TOXICOLOGICAL IMPLICATIONS- in publication
Professor gamalA. H
mer 24 ago, 16:10
mail To luisetto mauro :
Dear Professor
Greetings
Itis great work Congratulations
Gamal
Related BOOK-RAMANSPETTROSCOPYfor BIOP-PHARMACEUTICAL QUALITY
CONTROL and PAT- RAW MATERIAL - FINAL PRODUCTS -THENANOLIPIDS EFFECTON
SIGNAL INTENSITY -REGULATORYAND TOXICOLOGICAL ASPECTS
FromProfessor khaled edbey
10:22
To mauro Luisetto
Thank you very much, I really appreciate it.
Regards

For article RAMANSPETTROSCOPY in CQ Biopharmaceuticals
Comment fromProfessor G. H. HAMID
Dear Professor Mauro
Greetings.
Great work; you explain a lot about Raman Spectroscpy.
The aim of the study was clear and definitely explain what you need;
Aim of this work is to verify the role played by nanolipids on Raman Spectroscopy
encapsulating active principle or other substances using differentprocedure:1)
destructive2) non destructive technique.This is relevant because regulatory agency
authorized ( EMA) for cGMP rules the usealso of nondestructivemethods like
RAMAN spectroscopy in various stageof manufacturing drugs ( for raw material and
final product).
In chapters 6,7,8 thelanguage and punctuation can be corrected by grammarly
program.
Congratulations Gamal
Personal opinion of a university full Professsor Phd chemicalscience received ( 24-
08-2022)
“In my opinion GO and other non-declared substances mustbe located by
microscopicaltechniques coupled with spectroscopy (Raman, XPS, e
diffraction).otherwisetheanalyses will yield negative identification, as their amount
is low and they show as dispersed particles”

RESEARCH ARTICLE
TITLE : SELF-ASSEMBLING PROPERTY OF GRAPHENEDERIVATES
CHEMICO -PHYSICAL and TOXICOLOGICAL IMPLICATIONS
AUTHORS
1)Luisetto M IMA academy Marijnskaya, professorship in toxicology and
pharmacology,Chemicaltechnology and Chemical industry branch science Branch
italy 29121
2) Khaled E , Professor, Departmentof Chemistry, Libya PhysicalChemistry,
3)GamalA. Hamid Professor Hematology Oncology, University of Aden, Yemen
4)Tarro G , Professor of oncologicalvirology,Chairman of the Committee on
Biotechnologies of VirusSphere, World Academy of Biomedical Technologies
(WABT), Paris
5)Nili B. Ahmadabadi ,Nano Drug Delivery, (a ProductDevelopment Firm), United
States
6)Cabianca L. bio-medical laboratory turin italy Citta’ della Salute
7)Oleg Yurevich Latyshev IMA Academy President

WHITE PAPER - m RNA vaccine production Quality control- regulatory and toxicological aspects

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