We want to take stock of the situation together with you. Eight months have passed since July 2018 and in these length of time we have achieved extremely satisfying results. We have presented a ​research program ​and regarding the vaccines analysis we are able to make a point of reference, with the objectives achieved, those being finalised and those only planned for now.

To begin with, the analyses of 2 compounds for each vaccine have been verifyed by means of standards, 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, a total amount of those recognized as identities and those to be identified, which can be estimated within the order of 50 micrograms / mL, in contrast to the EMA / FDA guidelines.

These tests have given positive results, therefore they fully confirm the analysis method! The contaminations observed are probably due to different and variable manufacturing process’ phenomena and topics. What has been observed in the course of the studies is an “inter-batches” variation of the composition, which makes us assume that there are some steps along the whole product manufacturing process that are difficult to control.

Such analyses have allowed us to achieve the following steps:

• Conformity assessment of composition as outlined in the vaccine datasheet
• Screening for chemical and protein/peptide contaminations, as well as those deriving from genetic material
• Confirmatory study of chemical and protein target compounds through con standard certificates of inspection

The following vaccines have been submitted to an initial screening:

• Infanrix Hexa - GlaxoSmithKline Biologicals s.a.
• Priorix Tetra - GlaxoSmithKline S.p.A.
  
• Hexyon - Sanofi Pasteur Europe
• Gardasil 9 - MSD Vaccins

Sono stati analizzati questi altri vaccini come screening iniziale:

• Measles vaccine live B.P. - Poonawalla Group (Profarma AG, Baar)
• MMR vax Pro - MSD Vaccins, Francia
• PolioInfanrix - GlaxoSmithKline, Belgio
• Fluad -  Seqirus Srl, Siena
• Vivotif - PaxVax, Regno Unito

Used Technologies

The study is structured in:

Impurity and chemical and protein contamination analysis

• The LC-SACI / ESI-MS analysis system associated with the pioneering SANIST platform has been used  to perform an initial identification screening on the vaccines of interest, as well as to confirm it  with the control standards, with a minimum level between the nanograms and micrograms / dose
• MALDI-TOF-MS technology has been used to study the insoluble macromolecules found in the vaccines

Analysis of genetic material

• Test for the presence of nucleic acids (DNA / RNA) of human and animal origin and of microorganisms (viruses, bacteria) using the Next Generation Sequencing method, which made it possible to quantify the genetic material sequence contained in the vaccines in a highly specific and accurate way
• Verification of correspondence of bacterias and live attenuated and inactivated vaccine viruses genomic sequences (presence of genetic variants)

Quantitative analysis of metals

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

Analysis of chemical and protein contamination

After a first screening that had identified the presence of hundreds of chemical signals inside the vaccines, confirmation tests were carried out by means of standards of 2 compounds per vaccine, using certified control standards, chosen from those known for their critical hazard profile and for the non-residual quantity (such as to be considered components of the vaccines, therefore to be included in the technical data sheet and quantified).

These tests have given positive results, therefore they fully confirm the analysis method. So far only two compounds have been tested for economic reasons solely, in fact this study is not complete as it is limited by the considerable cost of the survey but we have chosen to identify the more relevant standards as for the regulatory restrictions and it is to be considered that this kind of in-depth investigation should not after all be up to us.

The observed contaminations are most likely due to different and variable manufacturing process’ phenomena and topics. What we have observed along the research steps is an inter-lot variation of the composition, which makes us assume  that there are some steps along the whole product manufacturing process that are difficult to control.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 unknown) that the contaminants are within the order of 50μg/mL . This is an important data because EMA/FDA guidelines is very clear about it. Contaminations should not be present (below the detection level) or if present they should be well identified and appropriate methods to reduce them should be proven to be apllied. In this case the contaminants are not below the capability level of the detection device (ng/mL) so we believe the producer is not applying any purification methods. As for the presence of adventitious viruses the problem does not exist, because they must not be present at all.

• IN PROGRESS - Interlaboratory confirmation analyses of these compounds are underway.
  The compounds identified by standard will be reconfirmed with the same technology by different laboratories. One of our goals is to coordinate this project also with the support and cooperation 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 vaccine batch.
  From the beginning of this project it was clear to us that the analyses, to meet the necessary requirements of the replicability and dat accuracy, had to be carried out with the most advanced instrumentation, and by monitoring any diversities within the same batch and between different batches. As we are proceeding with the investigations, other lots are being continuously analysed, obviously withing the budget and products availability.
  
  
• IN PROGRESS - Sequence analysis of  fetal DNA genome from the MRC-5 cell line.
  Since from the first screening we had the opportunity to verify by standard that the diploid lines used were MRC-5, as stated in the technical data sheet. We would like to deepen the study of this cell line also by virtue of the fact that its presence is remarkable in quantity and dimensions.
  
  
• IN PROGRESS - Study of minor variants (quasispecies) of measles, mumps and rubella viruses.
  Compared to the first report in July 2018 (https://bit.ly/2UCC2iS) we have commissioned an in-depth study of the minor variants since mutations in attenuated vaccine-viruses were not the only ones found. This issue too raises major doubts about the vaccine efficacy and safety. This result will take more time than the other reports because the mutants’ sequences’ verification has to be done manually.
  
  
• IN PROGRESS - Interlaboratory confirmation analysis
  We have identified foreign and national laboratories to which we have commissioned the analysis replication by standard. This result will be soon available.

Projects under evaluation

Study of the vaccine antigens potency: this study has to be conducted at a center accredited by the regulatory agencies for the preclinical study of vaccines. The aim is to verify if the vaccine antigens are able to produce or bind to specific antibodies (it is theoretically assumed that these antibodies are protective, but this analysis does not allow us to prove that the vaccine is able to protect against the disease). The cost of this step could be relevant, so the project is currently just an hypothesis.

Download: CORVELVA-Report-on-the-results-obtained.pdf

Translated by team CLiVa - www.clivatoscana.com