We want to take stock of the situation together with you. From July 2018 to today 8 months have passed and in these months we have achieved extremely satisfactory results. We have presented a research program and as regards the analysis of vaccines, we are able to make a first point of the situation, with the objectives achieved, those in the completion phase and those still in the planning stage.

We begin this update by saying that the analyzes by standard of 2 compounds for each vaccine have been confirmed, using certified control standards with a concentration in the order of micrograms / mL. The compounds we have chosen are among those known for their critical hazard profile. We are talking about a cumulative quantity, i.e. in total between those confirmed as identity and those to be identified, which can be estimated in the order of 50 micrograms / mL, in contrast with the EMA / FDA guidelines.

These confirmations gave positive results, therefore they fully confirm the analysis method! The contaminations observed are probably due to phenomena and variables associated with the manufacturing process. What has been observed during the studies is a long-term variation of this composition which leads to the assumption that there are processes that are difficult to control within the overall process of processing the product.

The analyzes carried out made it possible to conclude the following steps:

• Assessment of the conformity of the composition as indicated in the vaccine data sheet
• Screening of chemical, protein / peptide and genetic material contaminations
• Confirmation study of target compounds (chemical and protein) with certified control standards

The following vaccines have been analyzed "completely":

• Infanrix Hexa - GlaxoSmithKline Biologicals sa
• Priorix Tetra - GlaxoSmithKline SpA
  
• Hexyon - Sanofi Pasteur Europe
• Gardasil 9 - MSD Vaccines

These other vaccines were analyzed as initial screening:

• Measles vaccine live BP - Poonawalla Group (Profarma AG, Baar)
• MMR vax Pro - MSD Vaccins, France
• PolioInfanrix - GlaxoSmithKline, Belgium
• Fluad - Seqirus Srl, Siena
• Vivotif - PaxVax, United Kingdom

Used technologies

The study was articulated on:

Analysis of impurities and chemical and protein contaminations

• The LC-SACI / ESI-MS analysis system associated with the innovative SANIST platform was used both to perform an initial identification screening on the vaccines of interest, and for confirmation with the control standards, with a minimum limit between the nanograms and micrograms / dose
• MALDI-TOF-MS technology has been used to study the insoluble macromolecules present in vaccines

Genetic material analysis

• Test for the presence of nucleic acids (DNA / RNA) of human and animal origin and from microorganisms (viruses, bacteria) using the Next Generation Sequencing method, which allowed to quantify in a highly specific and accurate way the sequence of the genetic material contained in the vaccines examined

Quantitative analysis of metals

• ICP-MS technology has made it possible to quantify the metals present in vaccines with a minimum limit of 5ng / dose.

Analysis of chemical and protein contaminations

After an initial screening that identified hundreds of chemical signals in the vaccines, confirmations were performed using standard 2 compounds per vaccine, using certified control standards, chosen from those known for their critical hazard profile and non-residual quantity (such that they can be considered components of the vaccines, therefore to be included in the technical data sheet and quantified).

These confirmations have given positive results, therefore they fully confirm the analysis method. For now they have been carried out on only two compounds for purely economic reasons, in fact this study is not exhaustive because it is limited by the considerable cost of the investigation but we have chosen to identify the standards that have the greatest impact in terms of regulatory limitations and it is also true that this kind of thorough investigation would be up to us.

The contaminations observed are probably due to phenomena and variables associated with the manufacturing process. What has been observed during the studies is a long-term variation of this composition which leads to the assumption that there are processes that are difficult to control within the overall process of processing the product. Considering the usual analytical factors, it can be assumed that the concentration is in the order of micrograms / mL. As for the cumulative quantity, it is possible to estimate on the basis of a semi-quantitative evaluation (since most of the compounds are not known) that the contaminants are in the order of 50 μg / mL. This is important because the EMA / FDA guideline is clear in this regard. Contaminations must not be present (below the detection limit) or if they are, they must be well characterized and proven to apply appropriate methods to reduce them as much as possible. In our case the contaminants that we have found are not below the detection limit of the instrument (ng / mL) and therefore in our opinion the manufacturer is not applying any purification methods. As for the presence of adventitious viruses, the problem does not exist, because they must be completely absent.

• IN PROGRESS - The interlaboratory confirmation analyzes of these compounds are in progress.
  The compounds identified by standards will be reconfirmed with the same technology in other laboratories. One of the targets we have set ourselves is to coordinate this action also through the support of international associations.
• IN PROGRESS - Study of the precipitation kinetics of aluminum-bound antigens.

Metagenomic analysis

• IN PROGRESS - Analysis with additional DNA and RNA control standards of another batch per vaccine.
  Since the beginning of this project, it has been clear to us that the analyzes, in order to meet the necessary requirements for the repeatability and accuracy of the data, had to be carried out with cutting-edge instrumentation, and monitoring for any differences between the lots and between different lots. As we proceed with the investigations, other lots are continuously analyzed, obviously limited to the costs and availability of the products.
• IN PROGRESS - Sequencing analysis of the fetal DNA genome from the MRC-5 cell line.
  Already in the first screening we had the opportunity to confirm by standard that the diploid lines used were MRC-5, as stated in the data sheet. We want to deepen the study of this cell line also by virtue of the fact that it is present in considerable quantities and of absolutely considerable size.
• IN PROGRESS - Study of minor variants (quasispecies) of measles, mumps and rubella viruses.
  Compared to the first report of July 2018 (https://goo.gl/cSspVH) we commissioned the in-depth study of the minor variants since mutations of attenuated vaccine viruses were not the only ones found. This element also poses enormous doubts about the efficacy and safety of the vaccine. The result will take longer than the other reports because the verification of the sequences of the various mutants must be done manually.
• IN PROGRESS - Interlaboratory confirmation analysis
  We have identified foreign and national laboratories to which we have commissioned the replication of the analyzes by standard. This result will be available shortly.

Projects under evaluation

Study of the potency of vaccine antigens: this study must be carried out at a center accredited by regulatory agencies for the preclinical study of vaccines. The aim is to verify whether the vaccine antigens are capable of producing or binding to specific antibodies (it is theoretically assumed that these antibodies are protective, but this analysis does not allow to demonstrate that the vaccine is able to protect from the disease). The cost of this step could be considerable, therefore the project is hypothetical for now.

Download: CORVELVA-report-of-results-ottenuti.pdf