1. First revision January 17, 2019
Study on the chemical composition profile of Gardasil 9
Introduction and description of the need
The quali-quantitative analysis of organic compounds is of great importance in the pharmacological field , as potential safety problems arise from
1
the new production processes of biological drugs and from the complex structural and biological characteristics of these products .
2
The review of the registration dossiers for military vaccines that we find in the final report of the Parliamentary Commission of Inquiry “Depleted
3
Uranium” revealed the presence of protein-chemical contaminants and impurities, which required further analytical study. Our association has
4
decided to take charge of it, as far as possible.
This project is part of the above-mentioned insights. It has been therefore necessary to develop a technology capable of analyzing a wide spectrum
of molecules of chemical, metabolic and protein origin in order to evaluate the quality of the obtained results. A method has been therefore
developed, based on SANIST -6
technology to test vaccines for purity and safety (further information below).
5
Results and Discussion
1. Analysis of the composition declared on the vaccine’s leaflet
Compound Precence Ionic species
Sodium chloride Not detected -
L-histidine yes [M+H]+
Polysorbate 80 Not detected -
Sodium borate Not detected -
L1 protein [Human papillomavirus type 6] yes [M+nH]n+
L1 protein [Human papillomavirus type 11] Not detected -
L1 protein [Human papillomavirus type 16] yes [M+nH]n+
L1 protein [Human papillomavirus type 18] yes [M+nH]n+
L1 protein [Human papillomavirus type 31] yes [M+nH]n+
L1 protein [Human papillomavirus type 33] yes [M+nH]n+
L1 protein [Human papillomavirus type 45] yes [M+nH]n+
L1 protein [Human papillomavirus type 52] yes [M+nH]n+
L1 protein [Human papillomavirus type 58] Not detected -
1
Lett Appl Microbiol. 2015 Feb;60(2):174-80. doi: 10.1111/lam.12355 (https://www.ncbi.nlm.nih.gov/pubmed/25376111)
2
Fuchs F., Biochimie. 2002 Nov;84(11):1173-9 (https://www.ncbi.nlm.nih.gov/pubmed/12595146)
3
http://www.camera.it/leg17/491?idLegislatura=17&categoria=022bis&tipologiaDoc=documento&numero=023&doc=pdfel
4
http://www.camera.it/leg17/436?shadow_organo_parlamentare=2588
5
Albini A. et al., Front Endocrinol (Lausanne). 2018 Apr 5;9:110.doi:10.3389/fendo.2018.00110. (https://www.ncbi.nlm.nih.gov/m/pubmed/29674995/)
2. First revision January 17, 2019
2. Protein fraction analysis
According to the manufacturer’s specifications, the Gardasil 9 vaccine contains some proteins. The sample has been analyzed for the identification
of these proteins.
2.1 - 1° analysis: Bradford’s assay
To ascertain whether proteins were presents, the Gardasil 9 vaccine was submitted to Bradford’s assay. 300 μL of osmotic H2O were added to
200 μL of vaccine to reach the appropriate volume: 500 μL of Bradford’s reagent were then added to the solution. Following visual analysis,
presence of proteins or peptides’ sequences was confirmed by the blue coloring of the sample.
Based on the previously determined calibration curve, the proteins’ concentration has been estimated at 0.062 mg/mL.
2.2 - 2a
analysis: Digestion as it is
After Bradford's assay, the vaccine’s sample was submitted to enzymatic digestion. 10 μL of the raw sample has been treated with 50 μL of
Trypsin and left at in thermoblock at 37°C overnight. 1 mg / mL hemoglobin control has been prepared and treated as the sample.
Presence of proteins in the sample has been detected with this analysis.
2.3 - 3a
Analysis: Digestion of the Precipitate
The vaccine sample was then submitted to a second analysis, whereby the solid and the liquid fractions were separated through centrifugation.
After centrifugation, all the supernatant was taken away. The remaining precipitated solid fraction was then treated with 30 μL of Trypsin and left
at in thermoblock at 37°C overnight. 1 mg / mL hemoglobin control was prepared and treated following the same procedure of the vaccine sample.
Following the digestion, both vaccine sample and control were submitted to centrifugation. The supernatant was extracted and placed in vials for
analysis. 20 μL of osmotic H2O were added to reach sufficient volume for the injection.
Presence of proteins in the sample has been detected with this analysis.
Name Molecular weight (KDa) Score Database
Chain A, High resolution cryo-EM maps of Human
Papillomavirus 16 reveal L2 location and heparin-induced
conformational changes
54 415/50 5KEP_A
major capsid protein L1 [Human papillomavirus type 33] 56 226/50 ACV84008.1
Chain A, Crystal Structure Of Hpv58 Pentamer In Complex
With The Fab Fragment Of Antibody A12a3
55 219/50 5Y9C_A
major capsid protein L1 [Human papillomavirus type 52] 59 175/50 ABU55765.1
L1 capsid protein [Human papillomavirus type 31] 56 139/50 AAA92894.1
major capsid protein L1, partial [Human papillomavirus type
16]
6 123/50 ADB97224.1
L1 protein [Human papillomavirus type 18] 64 94/50 AAP20601.1
L1 [Human papillomavirus type 45] 57 69/50 AAY86494.1
L1 [Human papillomavirus type 6] 56 72/50 AAF00066.1
3. First revision January 17, 2019
Toxicity evaluation for the sequences identified
It was not possible to evaluate the molecular toxicity index, based on the peptides’ sequences identified.
Presence of free peptides
No free peptides have been detected.
3. Metabolic fraction analysis
It should be emphasized that this screening study provides a semi-quantitative data, which correspond to a range from nanograms to micrograms
as an indicative order of magnitude. To obtain accurate quantitative data, it will be necessary to proceed using certified analytical standards of
known strength.
Below, we report the identification screening results obtained in the two batches under examination:
Lotto #1 (R013092)
Unknown compounds
Known compounds
In Batch #1 we identified 338 signals, of which only 22% known
NOTES FOR UNDERSTANDING: this is a first level analysis, which means an identification based on molecular weight. If the result
is univocal, (i.e. at a measured molecular weight only one compound matches) it is more likely to be accurate, but absolute certainty
is not possible in this phase. As you will notice, as far as certain compounds are concerned, different substances can correspond to
the same molecular weight. Further targeted analysis, of the appropriate standard are thus necessary to confirm their identity.
3.1. Gardasil vaccine
338 signals were detected, among which only 22% gave a potential classification (Table 1). It should be highlighted that the proportion of signals
is not exactly correlated to the proportion of compounds, since in mass-spectrometry a single compound can generate several mass/charge ratios
(m/z).
4. First revision January 17, 2019
It must be specified that compound identity is not certain and it should be confirmed by a second level screening carried out with certified
analytical standards.
In fact, during screening level, the device measures a particular data by its accurate molecular weight (measurement error <10 ppm). The empirical
formula is calculated on the basis of these measures. Some formulas might correspond to several compounds having the same molecular weight
but different chemical identity
NOTES FOR UNDERSTANDING: in essence, what has been ascertained is that we have several unknown signals, which could
not be identified through international databases.
Molecules potentially belonging to toxin category have been researched. They have been suggested on the basis of accurate m / z mode research
(error <10 ppm) using the toxic compounds database in the Metlin search engine. Table 2 shows the candidates obtained.
4. Fina Consideration
From the analyst performed on the two lots there is a significant variability in the content of contaminant and impurities, of these, most were not
characterized using the metabolic and protein reference databases (KEGG, NCBI-Prot and SwissProt). 8-9
There is a critical issue in the contamination of various compounds potentially or definitely harmful to human health.
In short, the first questions we asked ourselves, and the relative answers obtained, are the following:
1. Are the chemical substances in the data sheet present? Partly
2. Are there any chemical contaminations? Yes
3. How many contaminating compounds are there? Over 70
4. What are they potentially? Chemical toxins, chemical compounds
Next analysis
1. to identify with certainty the most interesting probable compounds
2. determine the exact amount of each contaminant
5. Future research developments
Confirmation and identity analyzes will be performed using the "Tandem Mass Spectrometry (MS / MS)" technique associated with the aid of
certified analytical standards. The analyzes will be performed in compliance with the European directives (EU directive 2002/657 / EC) useful for
the identification of compounds. 29
6. Description of SANIST technology
The internationally renowned SANIST platform, through publications in pre-referenced scientific journals - was used to perform a first identification
screening on the interest vaccinee.
7. Details related to the analytical method
5. First revision January 17, 2019
SANIST technology consists of:
a. kit for the extraction of the analytes (the unknown substances to be determined);
b. the analysis system LC-SACI / ESI-MS 6 which reduce the chemical noise of mass spectrometers and obtain a better detection
instrumental signals;
c. the SANIST -
data processing system consisting of a local and network bioinformatics platform capable of processing data through the 6 7
use of dedicated databases and customized algorithms. It is specified that, in the screening phase, the identification is carried out in the
scientific research context and through research in official banks (KEGG, NCBI-Prot and SwissProt) 8-9
without the use of certified 89
analytical standards. A second level analysis must be performed with certified analytical standards to confirm the identity.
8. Areas of application of SANIST technology
Until today the SANIST platform is applicable in the following fields:
a. In clinical research of disease markers and their direct application in the diagnostic field.
b. Food services, food traceability. Comparative studies to determine the quality of products based on their complex molecular composition.
Control of food counterfeiting.
c. Nutraceutical sector, development of the nutritional value of a food supplement based on its molecular composition. Forged search (for
example: addition drugs).
d. Pharmaceutical sector, drug control and research of active biomolecules.
e. Cosmetic industry: the molecular composition of cosmetic products can be carefully monitored and correlated with the quality of the
product.
9. How to read the tables
This is a screening phase, the instrument measures an accurate molecular weight data (measurement error <10 ppm). On the basis of these
measures a brute formula is calculated. Some formulas can correspond to several compounds having the same molecular weight but different
chemical identity (see table 1)
Example of a single associated component:
￭ (-)-Jasmonic acid C12H18O3 211.1313019
In this example, the instrument detected a signal with a molecular weight. By inserting the brute formula in the databases, it was possible to
associate a probable component.
10. Complete contamination tables
6
Albini A. et al., Rapid Commun Mass Spectrum. 2015 Oct 15;29(19):1703-10. doi: 20.1002/rcm.7270. (https://onlinelibrary.wiley.com/doi/full/10.1002/rcm.7270)
7
Cristoni S. et al., J Mass Spectrom. 2017 Jan;52(1):16-21. doi:10.1002/jms.3895. (https://www.ncbi.nlm.nih.gov/pubmed/27776380)
8
Kanehisa M. et al., Nucleic Acids Res. 2017 Jan 4;45(D1):D353-D361. doi:10.1093/nar/gkw1092. (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5210567/)
9
Cristoni S. et al., Expert Rev Proteomics. 2004 Dec; 1(4):469-83. (https://www.ncbi.nlm.nih.gov/pubmed/15966842
![First revision January 17, 2019
Study on the chemical composition profile of Gardasil 9
Introduction and description of the need
The quali-quantitative analysis of organic compounds is of great importance in the pharmacological field , as potential safety problems arise from
1
the new production processes of biological drugs and from the complex structural and biological characteristics of these products .
2
The review of the registration dossiers for military vaccines that we find in the final report of the Parliamentary Commission of Inquiry “Depleted
3
Uranium” revealed the presence of protein-chemical contaminants and impurities, which required further analytical study. Our association has
4
decided to take charge of it, as far as possible.
This project is part of the above-mentioned insights. It has been therefore necessary to develop a technology capable of analyzing a wide spectrum
of molecules of chemical, metabolic and protein origin in order to evaluate the quality of the obtained results. A method has been therefore
developed, based on SANIST -6
technology to test vaccines for purity and safety (further information below).
5
Results and Discussion
1. Analysis of the composition declared on the vaccine’s leaflet
Compound Precence Ionic species
Sodium chloride Not detected -
L-histidine yes [M+H]+
Polysorbate 80 Not detected -
Sodium borate Not detected -
L1 protein [Human papillomavirus type 6] yes [M+nH]n+
L1 protein [Human papillomavirus type 11] Not detected -
L1 protein [Human papillomavirus type 16] yes [M+nH]n+
L1 protein [Human papillomavirus type 18] yes [M+nH]n+
L1 protein [Human papillomavirus type 31] yes [M+nH]n+
L1 protein [Human papillomavirus type 33] yes [M+nH]n+
L1 protein [Human papillomavirus type 45] yes [M+nH]n+
L1 protein [Human papillomavirus type 52] yes [M+nH]n+
L1 protein [Human papillomavirus type 58] Not detected -
1
Lett Appl Microbiol. 2015 Feb;60(2):174-80. doi: 10.1111/lam.12355 (https://www.ncbi.nlm.nih.gov/pubmed/25376111)
2
Fuchs F., Biochimie. 2002 Nov;84(11):1173-9 (https://www.ncbi.nlm.nih.gov/pubmed/12595146)
3
http://www.camera.it/leg17/491?idLegislatura=17&categoria=022bis&tipologiaDoc=documento&numero=023&doc=pdfel
4
http://www.camera.it/leg17/436?shadow_organo_parlamentare=2588
5
Albini A. et al., Front Endocrinol (Lausanne). 2018 Apr 5;9:110.doi:10.3389/fendo.2018.00110. (https://www.ncbi.nlm.nih.gov/m/pubmed/29674995/)](https://image.slidesharecdn.com/corvelva-study-on-the-chemical-composition-profile-of-gardasil9-190130114915/85/Corvelva-study-on-the-chemical-composition-profile-of-gardasil9-1-320.jpg)
![First revision January 17, 2019
2. Protein fraction analysis
According to the manufacturer’s specifications, the Gardasil 9 vaccine contains some proteins. The sample has been analyzed for the identification
of these proteins.
2.1 - 1° analysis: Bradford’s assay
To ascertain whether proteins were presents, the Gardasil 9 vaccine was submitted to Bradford’s assay. 300 μL of osmotic H2O were added to
200 μL of vaccine to reach the appropriate volume: 500 μL of Bradford’s reagent were then added to the solution. Following visual analysis,
presence of proteins or peptides’ sequences was confirmed by the blue coloring of the sample.
Based on the previously determined calibration curve, the proteins’ concentration has been estimated at 0.062 mg/mL.
2.2 - 2a
analysis: Digestion as it is
After Bradford's assay, the vaccine’s sample was submitted to enzymatic digestion. 10 μL of the raw sample has been treated with 50 μL of
Trypsin and left at in thermoblock at 37°C overnight. 1 mg / mL hemoglobin control has been prepared and treated as the sample.
Presence of proteins in the sample has been detected with this analysis.
2.3 - 3a
Analysis: Digestion of the Precipitate
The vaccine sample was then submitted to a second analysis, whereby the solid and the liquid fractions were separated through centrifugation.
After centrifugation, all the supernatant was taken away. The remaining precipitated solid fraction was then treated with 30 μL of Trypsin and left
at in thermoblock at 37°C overnight. 1 mg / mL hemoglobin control was prepared and treated following the same procedure of the vaccine sample.
Following the digestion, both vaccine sample and control were submitted to centrifugation. The supernatant was extracted and placed in vials for
analysis. 20 μL of osmotic H2O were added to reach sufficient volume for the injection.
Presence of proteins in the sample has been detected with this analysis.
Name Molecular weight (KDa) Score Database
Chain A, High resolution cryo-EM maps of Human
Papillomavirus 16 reveal L2 location and heparin-induced
conformational changes
54 415/50 5KEP_A
major capsid protein L1 [Human papillomavirus type 33] 56 226/50 ACV84008.1
Chain A, Crystal Structure Of Hpv58 Pentamer In Complex
With The Fab Fragment Of Antibody A12a3
55 219/50 5Y9C_A
major capsid protein L1 [Human papillomavirus type 52] 59 175/50 ABU55765.1
L1 capsid protein [Human papillomavirus type 31] 56 139/50 AAA92894.1
major capsid protein L1, partial [Human papillomavirus type
16]
6 123/50 ADB97224.1
L1 protein [Human papillomavirus type 18] 64 94/50 AAP20601.1
L1 [Human papillomavirus type 45] 57 69/50 AAY86494.1
L1 [Human papillomavirus type 6] 56 72/50 AAF00066.1](data:image/gif;base64,R0lGODlhAQABAIAAAAAAAP///yH5BAEAAAAALAAAAAABAAEAAAIBRAA7)