Lineage of “For Research Use Only” Vectors
The Rapporteur Rolling Review Critical Assessment Report, which assessed the application materials submitted by BioNTech to the EMA, contains a plasmid map of pST4-1525 (7,824 bp), the plasmid used for the manufacture of BNT162b2. However, this plasmid map does not display the SV40-derived sequence present in the plasmid backbone.[1]
An important fact is that what is omitted from this map is not simply a generic SV40 sequence, but an SV40-derived sequence originating from pCMV-Script, a vector designated “For Research Use Only.” In other words, what is omitted from the Report is an SV40-derived sequence originating from the pCMV-Script, which Stratagene (now Agilent) commercialized as a research-use product rather than as a product for pharmaceutical or clinical use.
Tracing the backbone containing the SV40-derived sequence found in BioNTech's mRNA vaccine manufacturing plasmids leads back to the Stratagene's pCMV-Script. The pCMV-Script is a mammalian expression vector commercialized by Stratagene in 1997. The designation of this vector for research use can be traced not merely to Agilent's current labeling, but to Stratagene's own trademark record dating from the time the product was first commercialized in 1997.
On July 29, 1997, Stratagene filed a U.S. trademark application for “PCMV-SCRIPT” (Serial No. 75332337; Registration No. 2178551).[2] The trademark record identifies Stratagene as both the Original Applicant and Original Registrant and describes the goods as: “kits for gene cloning containing laboratory reagents for research use; laboratory reagents for use in molecular biology”
The record further identifies August 1997 as both the First Use Anywhere and the First Use in Commerce. Thus, the “research use” designation can be traced back to the original commercialization of PCMV-SCRIPT by Stratagene in 1997, when it was registered as a research-use product for gene cloning and molecular biology.
This designation was maintained in subsequent manufacturer documentation. Agilent's official Instruction Manual for the pCMV-Script Vector itself (Catalog #212220) states on its cover:
“For Research Use Only. Not for use in diagnostic procedures.”[3]
An even more important fact is that the relationship between the pCMV-Script and the Mainz/BioNTech pST plasmid lineage was expressly documented by the Mainz research group, the group from which BioNTech emerged.
In a 2006 paper, Holtkamp, Kreiter, Türeci, Şahin and colleagues at Johannes Gutenberg University Mainz reported that pST1-2β-globin UTR-A(120) was constructed by introducing a T7 promoter, two tandem human 3′ β-globin UTRs, a poly(A) tail, and a neomycin-resistance gene into the pCMV-Script Vector (Stratagene, La Jolla, CA), and described this construct as the “ancestor” of a subsequent series of vectors.[4]
Another paper from the same Mainz group describes this relationship even more directly:
“All vectors used were variants of the pST1-A120 plasmid, which we engineered from the pCMV-Script vector…”[5]
Accordingly, the vector ancestry from the Stratagene pCMV-Script (research use) to the Mainz pST1 (research use) is not an inference based solely on sequence similarity; it is directly documented in publications by the developers themselves. The pST vector platform subsequently developed in Mainz evolved into a DNA-template platform for the production of mRNA by in vitro transcription (IVT).
The Rolling Review Critical Assessment Report, which assessed the BNT162b2 quality documentation submitted by BioNTech to the EMA, states that the drug substance is manufactured using a linear DNA template and that this template is produced from plasmid DNA amplified in DH10B Escherichia coli.
That plasmid is pST4-1525, which is described as follows:
“The plasmid, pST4-1525, is a 7,824 base pair plasmid designed for the production of BNT162b2.”[1]
Figure S.2.3-1, “pST4-1525 Plasmid Map,” in the same document displays the T7 promoter, 5′ UTR, Kozak sequence, S protein, FI element, poly(A), KanR, ori, and other features, whereas the SV40-derived sequence present in the backbone is not identified as a feature on the map.[1]
Independently of this documentary genealogy, publicly available nucleotide sequences are also consistent with this lineage. The complete sequence of pCMV-Script is deposited in NCBI GenBank as AF028239.1, a 4,278-bp “Mammalian expression vector pCMV-Script.”[6] The Pfizer bivalent BNT162b2 expression vector is deposited in GenBank as OR134577.1, with a length of 7,810 bp.[7]
Direct comparison of the two sequences identifies a continuous 3,469-bp corresponding region between: AF028239.1 nt 810–4278, and OR134577.1 nt 118–3586
Of these 3,469 nucleotides, 3,468/3,469 (99.97%) are identical. There is only a single nucleotide difference, which is explicable by the inheritance of a known pUC-lineage backbone mutation, and there is no insertion or deletion in the alignment. The conserved region also contains the SV40-derived sequence at issue.[6,7] Thus, the SV40-derived sequence found in OR134577.1 does not exist as an isolated SV40 fragment; rather, it occurs within an approximately 3.47-kb conserved pCMV-Script backbone region.
Thus, on the basis of the developmental continuity can be traced as follows:
Stratagene pCMV-Script (Research Use Only) → Mainz pST1 → pST family → pST4-1525 → BNT162b2 manufacturing, with the pCMV-Script-derived research-use backbone retained through the lineage.
Taken together, the evidence establishes the following facts concerning pCMV-Script:
(1) The pCMV-Script is a mammalian expression vector commercialized by Stratagene in 1997 as a research-use product. This designation can be traced back to Stratagene's PCMV-SCRIPT trademark record from the time of the product's original commercialization.[2]
(2) The official Instruction Manual for the pCMV-Script itself states “For In Vitro Use Only,” and Agilent currently designates the product “For Research Use Only.”[3]
(3) Şahin, Türeci, Holtkamp, Kreiter and colleagues in Mainz themselves reported that pST1 was engineered from this pCMV-Script vector.[4,5]
(4) The publicly available Pfizer bivalent expression vector OR134577.1 contains an approximately 3.47-kb continuous region that is 99.97% identical to the pCMV-Script, and this conserved region includes the SV40-derived sequence.[6,7]
(5) The Rolling Review Critical Assessment Report assessing the BNT162b2 quality documentation submitted by BioNTech to the EMA identifies pST4-1525, a member of the pST lineage, as the plasmid used for BNT162b2 production and reproduces its plasmid map.[1]
(6) However, the Rolling Review Critical Assessment Report does not identify on the pST4-1525 plasmid map the SV40-derived sequence carried forward through the vector lineage from pCMV-Script, a vector designated “For Research Use Only” and “For In Vitro Use Only.”[1,3,6,7]
