Clinical trial (study C4591001)

Both Rapporteurs raise major objections, and several other concerns are identified. Both rapporteurs raise a major objection related to comparability between batches used in clinical trials (Process 1) and representative commercial batches (Process 2).

The linear DNA template is not part of the final product but defines the sequence of the mRNA product and therefore it is fundamental to ensure its adequate control. 

Therefore, the level of details included in the dossier with respect to the manufacturing process and the control strategy for this starting material, although shortly described, is not yet considered adequate to allow for a proper assessment.
The circular plasmid DNA is filtered via 0.2 μm filtration and stored frozen at -60 to -90 °C; the hold time for this intermediate is not defined. 
No additional information nor data are provided to support stability. 
The reference material for plasmid identity testing is not described.
No descriptions of the analytical methods used for the control of the linear DNA template nor evidence regarding their qualification/validation have been yet provided. This information is, however, considered critical for quality of the final product. 

To be noted, the assessment (manufacturing process) is based on the currently submitted data and therefore the final evaluation of the control strategy cannot be made at this point.
A severe deficiency of the characterisation section is that no biological characterisation is presented and that the mode of action is not described. 
Even though two methods, namely agarose gel electrophoresis and capillary gel electrophoresis, have been applied to determine RNA integrity of BNT162b2 DS, no characterisation data on truncated forms is presented and the potential safety risks associated with truncated RNA isoforms are not addressed.
Some of the analytical methods are not presented in sufficient detail and often method descriptions are based on “examples” of procedures, controls and standards as well as on “typical” system operating parameters. 
Several concerns are raised for specific assays requesting additional information on critical procedures, reagents, standards and equipment. 
The results for the comparability of the commercial PPQ-batches versus the clinical supply batches of DP is pending and will be provided for assessment during the procedure. 
The proposed acceptance criteria (LNP size) of 40 to 180 nm seem wide compared to clinical batch data that is found in the range of 59-74 nm for the small scale clinical batches (“classical LNP process) and 68-71 nm for the emergency supply (“upscale” LNP process). 
The proposed acceptance criteria (encapsulation) of ≥80% seem wide compared to clinical batch data that is found in the range of 92-94%.
The proposed acceptance criteria of ≥50% intact RNA for RNA integrity as measured by capillary gel electrophoresis seem wide compared to clinical batch data that is found in the range of 69-81%.
The applicant should therefore discuss, and present comparative results for DS and DP, on RNA integrity. 
Therefore, method description and validation summary of the rapid sterility test should be provided during the procedure.
The assay (potency) was implemented recently and the proposed acceptance criteria of ≥30% cells positive seem wide compared to the limited batch release data available to date, i.e. emergency supply lots that is in the range of 63-65%. 
no information is provided on the lipid-related impurities originating from the degradation of the lipid nanoparticles and such data needs to be provided.
The characterisation of BNT162b2 DS is currently not found acceptable in relation to the CQA mRNA integrity. 
The identities and abundances of truncated forms should be sufficiently characterised, and consistency between batches should be addressed. 
Furthermore, the potential safety risks associated with truncated RNA isoforms should be thoroughly discussed 
The applicant needs to provide an estimation of the non-neutralizing antibodies in the whole antibody response. 
The applicant is asked to comment on the differences in the kinetics of the two novel excipients as well as on the relatively long liver clearance of ALC-0315 
Given that the acetamines have been reported to be carcinogenic in animals, including liver tumors, potentially based on genotoxic mechanism, the applicant is asked to provide a discussion on the distribution and metabolism of the ALC-0159 focusing on the acetamide moiety 
Since almost no unchanged ALC-3015 was detected in urine or faeces, metabolism may play a bigger role in the elimination of ALC-0315 than ALC-0159.
The Applicant is invited to further discuss the risk that the mRNA vaccine can trigger potential autoimmune responses and how they plan to possibly evaluate their occurrence 
In view of potential acute immunotoxicity mediated by LNPs, does the Applicant possess data on other timepoints (earlier than 6h or beyond) regarding the cytokines measurements? 
Representative CoAs or full specifications should be provided for starting and non-compendial raw materials used in the manufacturing Puurs, Belgium LNP Production, Bulk Drug Formulation.