Making COVID-19 mRNA vaccines accessible: challenges resolved

ABSTRACT

Introduction

The rapid spread of SARS-CoV2 infection allowed testing of mRNA vaccines that translate the target antigen, unlike introducing antigens in traditional vaccines. It proved safer and more effective and, as a chemical vaccine, much easier to develop and manufacture.

Areas covered

The science and technology behind the mRNA vaccines are pertinent to establishing low-cost manufacturing of reverse-engineered mRNA vaccines, as suggested by the WHO. A stepwise approach to establishing a compliant manufacturing facility, testing, supply chain, regulatory submissions, and intellectual property handling is presented.

Expert opinion

mRNA technology is more straightforward, and the cost of establishing a manufacturing facility is affordable, even in developing countries. The technology and supplies are widely available; however, based on experience, several misconceptions and misunderstandings about mRNA vaccines need to be removed, such as the regulatory and intellectual property issues that are resolved in this paper.

KEYWORDS:

• COVID-19
• vaccines
• mRNA vaccine
• LNP
• SARS-CoV2
• WTO
• TRIPS
• LDCs
• developing countries
• FDA
• EMA

1. Introduction

The COVID-19 pandemic, arriving a century after the Spanish Flu, has created global havoc infecting half a billion and killing 6.5 million [1] compared to 12–50,000 deaths per year from flu every year [2]. The cost of managing this pandemic has been astronomical; the US alone spent $4.6 trillion, more than the combined yearly spending of the 203 out of 228 countries [3,4], yet still lost more than one million citizens. A good part of the US expenditure went into assisting the development and supply of new vaccines against COVID-19. The developers of the mRNA vaccines against COVID-19 sold over $54B in 2021 and are expected to sell $40B in 2022, mainly in the West, while the rest of the world is still awaiting the vaccine. Such costs were unaffordable to more than 80% of the world, creating a major gap in the supply chain of the vaccine, more particularly the newest type and the most effective messenger RNA (mRNA) against COVID-19.

There is a dire need to make mRNA vaccine accessible globally to convert the pandemic into endemic and then continue protecting against COVID-19, as this is not going away soon or ever. One of the positive sides of the fast-spreading of COVID-19 allowed quicker approval of the mRNA vaccine that demonstrated an efficacy of 95% and higher than the FDA acceptance range of 50% or more.

The mRNA vaccines went further in demonstrating effectiveness against the viral mutants of SARS-CoV-2, an unexpected outcome adding to the value of mRNA vaccines [5] (Figure 1). Interestingly, while the spreading of the Delta and Omicron variants of SARS-CoV-2 was much higher, the mortality rates were the same or lower than the Alpha strain; this helped contain the death rates despite increased infections.

Figure 1. Infectiveness and mortality from infections, including SARS-CoV-2 strains [5,6] .

2. Scientific understanding

Traditional vaccines prevent infections by introducing attenuated infecting organism or its antigens (such as surface protein) into the body, while the mRNA vaccines, a single strand of nucleosides that enter the cell and translate the surface protein encoded in it to produce the antigen, to induce humoral or antibody-mediated immunity [7,8]. Humoral immunity is achieved as the helper T cells, and B cells differentiate into plasma B cells to produce antibodies; the cellular immunity occurs inside infected cells and is mediated by T lymphocytes. Helper T cells release cytokines that help activated T cells bind to the infected cells’ MHC-antigen complex and differentiate the T cell into a cytotoxic T cell to lyse the infected cells (Figure 2).

Figure 2. mRNA in vitro transcription and innate immunity activation. (A) mRNA in vitro transcription. Using DNA with the antigen-encoding sequence as a template, mRNA in vitro transcription products contain single-stranded RNA (ssRNA), double-stranded RNA (dsRNA), etc. The ssRNA structure typically includes a five-prime cap (5’ cap), a five-prime untranslated region (5’ UTR), an open reading frame (ORF) region, a three-prime untranslated region (3’ UTR) and a poly (A) tail structure. (B) RNA translation and antigen presentation. Through endocytosis, mRNAs enter the cytoplasm. Some mRNAs combine with ribosomes of the host cell and translate successfully. Antigen proteins can be degraded to antigenic peptides by the proteasome in the cytoplasm and presented to cytotoxic T lymphocytes (CTLs) via the major histocompatibility complex (MHC) I pathway. Or, they can be released out of the host cell and taken up by DCs. Then, they are degraded and presented to helper T cells and B cells via the MHC-II pathway. B cells can also recognize released antigen proteins. (C) Self-adjuvant effect. Various pattern recognition receptors (PRRs) can recognize mRNA in vitro transcription products. Endosomal innate immune receptors can recognize ssRNA (e.g. Toll-like receptor 7 (TLR7), TLR8). Endosomal innate immune receptors can recognize dsRNA (e.g. TLR3) and cytoplasmic innate immune receptors (e.g. protein kinase RNA-activated (PKR), a retinoic acid-inducible gene I protein (RIG-I), and melanoma differentiation-associated protein 5 (MDA5), and 2’-5’-oligoadenylate synthase (OAS). Based on those, mRNA products can stimulate the secretion of pro-inflammatory cytokines and type I interferon (IFN), leading to activation of antigen-presenting cells (APCs) and inflammatory reaction. However, they can also activate antiviral enzymes that cause stalled mRNA translation and mRNA degradation [8]. [Under the Creative Commons Attribution 4.0 International license] .

While the focus of most mRNA vaccines is to induce antibody responses, T-cell responses are also generated by mRNA vaccines, including CD4 + T-helper cells, which are needed for optimal antibody responses [9,10]. While the antibody responses are easier to measure, it is difficult to assess the exact role of T cells in correlating with immunity, as no vaccines based solely on T-cell responses have been developed. Nevertheless, both types of T cells’ role in generating antibody responses (T-cell help) and their ability to kill virally infected cells (cytolytic T lymphocytes) add this other arm of immunity to mRNA vaccines. For example, anti-SARS-CoV-2 T cells may be necessary since neutralizing antibodies wane over time [11,12].

3. Historical perspective

The mRNA was discovered more than 60 years ago [7]. Still, its application in gene delivery did not arrive until 1990s, when the direct injection of ‘naked’ mRNA was shown to be capable of resulting in in vivo expression of the encoded protein. Even then, further development and its use were halted because of many hurdles: instability of mRNA in vivo due to the near-ubiquitous presence of RNAse enzyme; high immunogenicity of the mRNA, stimulating innate responses with a concomitant decrease in the translation of the mRNA [13].

The delivery and protection of the mRNA vaccine began resolution in the late 1990s when the US government embarked on a multibillion-dollar quest to find a vaccine to prevent HIV infections. The HIV vaccine had failed due to the destruction of the mRNA as a natural response of the body to protect against foreign RNA such as the viral RNAs. The destruction apparatus in the cells comprises toll-like receptors (TLRs) in the cytoplasm; the TLRs are a class of proteins that are single-pass membrane-spanning receptors usually expressed on sentinel cells, such as macrophages and dendritic cells that recognize the structurally conserved molecules derived from microbes, the antigens or in this case the RNA. To prevent destruction by TLRs, the mRNA sequences had to be modified to make them look more like endogenous mRNA. One such modification is that using pseudouridine in place of uridine in the mRNA improves stability and increases translational capability [14]. Incidentally, the name of the famous mRNA company Moderna comes from ‘modified RNA.’

Another challenge in the use of mRNA was protecting it from the RNAse enzyme, found in abundance in the body and that disintegrates mRNA almost instantly upon contact. The solution came as a lipid nanoencapsulation to protect the mRNA and expedite delivery into the cytoplasm [15].

A significant reason for the enthusiastic embrace of mRNA for a COVID-19 vaccine was the speed with which mRNA vaccines can be developed once the genetic sequence of a target antigen is established. From the knowledge of sequence to the final product ready for testing may take only days, instead of months and years for traditional vaccines. For example, Moderna developed the mRNA vaccine against COVID-19 within two days after receiving the genome sequence of the coronavirus surface protein from the NIH [16].

While biological vaccines require enormous investment, the chemical vaccine, the only of its kind, is a lower-cost venture. The manufacturing size is also small as the dosing is generally in micrograms. Further dose reduction can come if a self-amplifying type of mRNA that replicates itself in the cell is used–making more copies of the protein antigen. However, the currently approved vaccines are non-amplifying.

The only missing thing in validating mRNA vaccines was real-time testing to demonstrate their safety and efficacy. While vaccines often take decades of testing to collect sufficient data to establish safety and efficacy, the COVID-19 pandemic allowed testing into 30–40,000 to secure at least 140–150 positive responses [17,18] that were enough to establish secure regulatory approval. Surprisingly, the safety and efficacy of the COVID-19 mRNA vaccines came out far superior to other vaccines approved in the past.

The first FDA approval of an mRNA vaccine came in 2021 to prevent the spread of COVID-19 [19], a historical moment that matches the paradigm shift of Edward Jenner, the founder of vaccinology, who injected a vaccine against smallpox about 225 years ago [7].

The regulatory agencies must be commended for their fast response in approving these new vaccines. The FDA guideline for approval of the COVID-19 vaccines still suggests a minimum efficacy of 50% [20]; the mRNA vaccines far exceeded the minimum expectations. The success of the Pfizer and Moderna mRNA vaccines garnering sales of over $60B in 2021 and anticipated to achieve the same in 2022 [21] made other traditional vaccines look inferior such as the Chinese Sinopharm [22] and the Russian Sputnik vaccines [23]. The Russian vaccine Sputnik continues to be rejected by the WHO [24]. However, the sales volume does not correctly represent the number of doses given.

4. Near perspective

The mRNA COVID-19 vaccine needs of poorer countries were supposed to be met through Covax, an international body meant to facilitate global vaccine distribution, but donations have been slow and limited [25]. To overcome the dire accessibility of the mRNA vaccines, the WHO suggested reverse-engineering the mRNA vaccines [26] and assisting developing countries in producing their vaccine supply. This is the main topic of this paper.

Most estimates put the cost of setting up the production of mRNA vaccines at $100 million to $200 million [27,28], a cost that is not prohibitive even in developing countries, especially if they can reach out to several international funding agencies [29]. The awareness to assist developing countries in producing their vaccines is gaining attention; the EU recently committed $45 million to help six countries to develop their mRNA vaccines [30]. However, all except one country in the list will be subject to intellectual property issues. Therefore, this promised aid from the EU is insufficient and improbable to transfer technology quickly. Consequently, I recommend that developing countries not wait for any funding offers that come with many constraints, as in this paper, based on my hands-on experience, how to reduce the cost substantially based on novel approaches in technology development and regulatory planning [31,32].

A supply line of mRNA vaccines can be highly profitable, despite reducing the cost by more than 90% of the current market price [28,33,34]. A one dollar per dose cost is realistic, and these could be sold to the WHO Covax program and other agencies at $3–4 per dose readily. Furthermore, the developers should sell the vaccines in bulk to other countries to reduce their capital expenditure in establishing or expanding their fill and finish facility. I suggest the developers plan a capacity of one billion doses per year; when operational, this could bring billions of dollars of cash flow; this is still much less than what Pfizer and Moderna have earned, but a significant amount for the developing countries. There can be no better business that takes care of humanitarian and humanity’s needs.

5. Manufacturing solutions

Commercial manufacturing of mRNA vaccines is a relatively simple and small-scale process [28,33,34] since the dose administered is small (e.g. 30 μg for the Pfizer vaccine), and the manufacturing involves a chemical purification process that is well defined. There are two stages in vaccine production, an upstream process to produce DNA in a recombinant E. coli and a downstream process of in vitro translation (IVT), followed by lipid nanoparticle (LNP) formulation and packaging (Figure 3).

Figure 3. Process flow of mRNA vaccine manufacturing. With permission [28] .

5.1. Establish facility

A commercial mRNA vaccine facility is small; to produce one billion doses per year, a facility of about 10,000 square feet will be sufficient. The mRNA production is a four-stage process: creating linearized DNA (upstream), in vitro transcription into mRNA (downstream), LNP (formulation), and fill and finish. Unlike the production of recombinant therapeutic proteins, the upstream area will be non-GMP, where the plasmid is made and the DNA linearized, while the downstream area should be class 10,000. Since the downstream process is smaller, this can be achieved by using laminar downdraft or standard hoods to reduce the cost. MRNA production can be established in any space available without spending millions on building a qualified facility. The manufacturing can begin in less than six months; the bulk of the cost goes into chemical materials for production. The equipment requirements are standard, including a smaller 30 L bacterial fermentation reactor to produce pDNA, single-use mixing chambers, chromatography systems, and filtration systems. Other methods of producing DNA, like the PCR techniques, are under development for commercial scaling; when available, they will eliminate the need for the biological production of pDNA, further reducing the cost of mRNA production [35].

Special equipment is required to formulate LNP, and these mixers are in high demand requiring early acquisition. The fill and finish part is a standard injectable facility as a clean room, and using dedicated filling heads prevents cross-contamination.

5.2. cGMP compliance

A GMP certificate is not required for manufacturing and testing starting materials for ATMPs [36,37] (EU designation of mRNA products). For mRNA products, this includes creating plasmid and DNA linearization. However, the FDA has not yet guided GMP compliance of plasmid and linearization, but I do not see any different requirements by the FDA [38]. This means that the high cost of GMP compliance can be avoided, at least in the upstream manufacturing stage of plasmid production and DNA linearization.

5.3. Supply chain

The following chemicals are required for the downstream IVT process: 1-methyl-pseudouridine-5’-triphosphate (mod-UTP); Acetic acid; Adenosine-5’-triphosphate (ATP); Calcium chloride (CaCl2); Cholesterol; Citric acid; CleanCap AG; cytidine-5’-triphosphate (CTP); Deoxyribonuclease I (DNase I); Disodium phosphate (Na2HPO4); Dithiothreitol (DTT); Ethyl alcohol (ethanol); Guanosine-5’-triphosphate (GTP); Ionizable lipid; Linear template DNA; Magnesium chloride (MfgCl2); Monopotassium phosphate (KH2PO4); Phospholipid; Polyethylene glycol (PEG) lipid; Potassium chloride (KCl); Pyrophosphatase; RNase enzyme inhibitor; Sodium acetate; Sodium chloride (NaCl); Sodium citrate; Sodium hydroxide (NaOH); Spermidine; Sucrose; T7 RNA polymerase; Tris hydrochloride (Tris HCl); Water for injection (WFI), RNAse free. Developers should start early to procure these chemicals, which are often in short supply. Fortunately, given the surge in demand for these chemicals, many new suppliers are now coming to the market, so hoarding them is not a good idea because of their high cost.

5.4. Vaccine selection

This must be an mRNA vaccine that has received full regulatory approval, not only the Emergency Use Authorization. This choice comes from the regulatory plan where you would present comparison data, and you can do this only with an approved (or licensed in the US) product. Only two mRNA vaccines have received full approval or licensing: the Pfizer BioNTech and Moderna vaccines. Current effectiveness data shows similar efficacy and safety, so the choice should depend on cost and intellectual property constraints. The dose of the Pfizer vaccine is 30 μg vs. 100 μg for the Moderna vaccine; thus, the latter is almost three times more expensive to produce since the bulk of the cost of manufacturing goes into chemicals. Therefore, I would recommend using the Pfizer vaccine as the target product to copy. There were issues relating to the storage conditions 0 f − 80°C required for the Pfizer vaccine that was difficult to maintain, but that has changed to the same condition needed for the Moderna vaccine. This change came from Pfizer’s attempt to outrun Moderna by selecting a storage condition that may not require stability testing; once approved, Pfizer changed the storage requirement using the three-month time advantage to conduct stability testing.

5.5. Target protein sequence

The selected vaccine translates its targeted protein, which is the same in both approved mRNA vaccines. However, a developer may choose another target protein with time if the mutations lead to vaccines becoming ineffective.

The complete sequencing of SARS-CoV-2 isolate Wuhan-Hu-1 has been completed [39], including the surface spike protein sequence (Table 1). All three mRNA COVID-19 vaccines translate the same sequence comprising 1273 amino acids. The engineered sequence substitutes two amino acids – both prolines – for lysine (K) and valine (V), in the viral spike to stabilize the spike protein, keeping it from folding up and thus preventing it from inducing antibodies that may not be specific to virus spike protein if it has a different shape (see the bold print in Table 1). The purpose of these mutations is to prevent the cleavage of the spike protein at the site of the two proline mutations. This cleavage occurs typically in the early stages of the viral infection process after the virus has bound to its cellular receptor. The cleavage allows the viral fusion process with the cell membrane to occur, thus the invasion of the virus into the interior of the cell, where it begins to replicate. It is known that this cleavage alters the way the protein is presented to the immune system. This altered conformation of the spike protein results in the generation of less effective antibodies against the intact virus before cell-binding occurs.

Table 1. The SARS-Cov-2 virus surface protein sequence and the modified sequence (shown in BOLD) translated into the three mRNA vaccines

P0DTC2 (SPIKE_SARS2) Protein Sequence

BNT162b2, mRNA-1273, CVnCov Protein Sequence

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

5.6. mRNA sequence

The size of an mRNA vaccine is approximately [(3x#amino acids) × (~1.10)]. For example, the Pfizer BioNTech (Code name: BNT162b2; brand name: Comirnaty®) has 4284 nucleosides to translate into a surface protein having a chain length of 1273 amino acids [174; Moderna (Code: mRNA-1273, brand Spikevax®) solves the same surface protein. The complete sequence of the Pfizer vaccine is known; the sequence of the Moderna and CureVac vaccine are not reported fully, but it can be easily analyzed [40].

The mRNA sequence of the target vaccine is analyzed by first extracting it in a phenol-chloroform mixture using TRIzol Reagent (a complete, ready-to-use reagent for the isolation of total RNA. This monophasic solution of phenol and guanidine isothiocyanate is designed to isolate separate fractions of RNA, DNA, and proteins from cell and tissue samples of human, animal, plant, yeast, or bacterial origin, within one hour) with intactness assessed by a bioanalyzer before and after extraction. The RNA is then fragmented by heating to 94°C, primed with a random hexamer-tailed adaptor, amplified through a template-switch protocol, and finally sequenced using a sequencing instrument with paired-end 778-per-end sequencing for the COVID-19 vaccines. An RNA of known concentration and sequence (such as from bacteriophage MS2) is used as the reference for the COVID-19 vaccine. Table 2 lists the structure, and Table 3, the BioNTech-Pfizer vaccine.

Table 2. BioNTech-Pfizer Vaccine Structure and Modifications (mRNA Sequence (4284) (Uridine is replaced with Ψ = 1-methyl-3’-pseudouridylyl) [26]

Element	Description	Position
cap	A modified 5’-cap1 structure (m7G+m3’-5’-ppp-5’-Am)	1–2
5’-UTR	5´-untranslated region derived from human alpha-globin RNA with an optimized Kozak sequence.	3–54
sig	S glycoprotein signal peptide (extended leader sequence), which guides translocation of the nascent polypeptide chain into the endoplasmic reticulum.	55–102
S protein_mut	Codon-optimized sequence encoding full-length SARS-CoV-2 spike (S) glycoprotein containing mutations K986P and V987P to ensure the S glycoprotein remains in an antigenically optimal pre-fusion conformation; stop codons: 3874–3879.	103–3879
3’-UTR	The 3´ untranslated region comprises two sequence elements derived from the amino-terminal enhancer of split (AES) mRNA and the mitochondrial encoded 12S ribosomal RNA to confer RNA stability and high total protein expression.	3880–4174
poly(A)	A 110-nucleotide poly(A)-tail consisting of a stretch of 30 adenosine residues, followed by a 10-nucleotide linker sequence and another 70 adenosine residues.	4175–4284

Table 3. Sequence of BioNTech-Pfizer mRNA Vaccine (Uridine is replaced with Ψ = 1-methyl-3’-pseudouridylyl) [26]

GAGAAΨAAAC	ΨAGΨAΨΨCΨΨ	CΨGGΨCCCCA	CAGACΨCAGA	GAGAACCCGC	50
CACCAΨGΨΨC	GΨGΨΨCCΨGG	ΨGCΨGCΨGCC	ΨCΨGGΨGΨCC	AGCCAGΨGΨG	100
ΨGAACCΨGAC	CACCAGAACA	CAGCΨGCCΨC	CAGCCΨACAC	CAACAGCΨΨΨ	150
ACCAGAGGCG	ΨGΨACΨACCC	CGACAAGGΨG	ΨΨCAGAΨCCA	GCGΨGCΨGCA	200
CΨCΨACCCAG	GACCΨGΨΨCC	ΨGCCΨΨΨCΨΨ	CAGCAACGΨG	ACCΨGGΨΨCC	250
ACGCCAΨCCA	CGΨGΨCCGGC	ACCAAΨGGCA	CCAAGAGAΨΨ	CGACAACCCC	300
GΨGCΨGCCCΨ	ΨCAACGACGG	GGΨGΨACΨΨΨ	GCCAGCACCG	AGAAGΨCCAA	350
CAΨCAΨCAGA	GGCΨGGAΨCΨ	ΨCGGCACCAC	ACΨGGACAGC	AAGACCCAGA	400
GCCΨGCΨGAΨ	CGΨGAACAAC	GCCACCAACG	ΨGGΨCAΨCAA	AGΨGΨGCGAG	450
ΨΨCCAGΨΨCΨ	GCAACGACCC	CΨΨCCΨGGGC	GΨCΨACΨACC	ACAAGAACAA	500
CAAGAGCΨGG	AΨGGAAAGCG	AGΨΨCCGGGΨ	GΨACAGCAGC	GCCAACAACΨ	550
GCACCΨΨCGA	GΨACGΨGΨCC	CAGCCΨΨΨCC	ΨGAΨGGACCΨ	GGAAGGCAAG	600
CAGGGCAACΨ	ΨCAAGAACCΨ	GCGCGAGΨΨC	GΨGΨΨΨAAGA	ACAΨCGACGG	650
CΨACΨΨCAAG	AΨCΨACAGCA	AGCACACCCC	ΨAΨCAACCΨC	GΨGCGGGAΨC	700
ΨGCCΨCAGGG	CΨΨCΨCΨGCΨ	CΨGGAACCCC	ΨGGΨGGAΨCΨ	GCCCAΨCGGC	750
AΨCAACAΨCA	CCCGGΨΨΨCA	GACACΨGCΨG	GCCCΨGCACA	GAAGCΨACCΨ	800
GACACCΨGGC	GAΨAGCAGCA	GCGGAΨGGAC	AGCΨGGΨGCC	GCCGCΨΨACΨ	850
AΨGΨGGGCΨA	CCΨGCAGCCΨ	AGAACCΨΨCC	ΨGCΨGAAGΨA	CAACGAGAAC	900
GGCACCAΨCA	CCGACGCCGΨ	GGAΨΨGΨGCΨ	CΨGGAΨCCΨC	ΨGAGCGAGAC	950
AAAGΨGCACC	CΨGAAGΨCCΨ	ΨCACCGΨGGA	AAAGGGCAΨC	ΨACCAGACCA	1000
GCAACΨΨCCG	GGΨGCAGCCC	ACCGAAΨCCA	ΨCGΨGCGGΨΨ	CCCCAAΨAΨC	1050
ACCAAΨCΨGΨ	GCCCCΨΨCGG	CGAGGΨGΨΨC	AAΨGCCACCA	GAΨΨCGCCΨC	1100
ΨGΨGΨACGCC	ΨGGAACCGGA	AGCGGAΨCAG	CAAΨΨGCGΨG	GCCGACΨACΨ	1150
CCGΨGCΨGΨA	CAACΨCCGCC	AGCΨΨCAGCA	CCΨΨCAAGΨG	CΨACGGCGΨG	1200
ΨCCCCΨACCA	AGCΨGAACGA	CCΨGΨGCΨΨC	ACAAACGΨGΨ	ACGCCGACAG	1250
CΨΨCGΨGAΨC	CGGGGAGAΨG	AAGΨGCGGCA	GAΨΨGCCCCΨ	GGACAGACAG	1300
GCAAGAΨCGC	CGACΨACAAC	ΨACAAGCΨGC	CCGACGACΨΨ	CACCGGCΨGΨ	1350
GΨGAΨΨGCCΨ	GGAACAGCAA	CAACCΨGGAC	ΨCCAAAGΨCG	GCGGCAACΨA	1400
CAAΨΨACCΨG	ΨACCGGCΨGΨ	ΨCCGGAAGΨC	CAAΨCΨGAAG	CCCΨΨCGAGC	1450
GGGACAΨCΨC	CACCGAGAΨC	ΨAΨCAGGCCG	GCAGCACCCC	ΨΨGΨAACGGC	1500
GΨGGAAGGCΨ	ΨCAACΨGCΨA	CΨΨCCCACΨG	CAGΨCCΨACG	GCΨΨΨCAGCC	1550
CACAAAΨGGC	GΨGGGCΨAΨC	AGCCCΨACAG	AGΨGGΨGGΨG	CΨGAGCΨΨCG	1600
AACΨGCΨGCA	ΨGCCCCΨGCC	ACAGΨGΨGCG	GCCCΨAAGAA	AAGCACCAAΨ	1650
CΨCGΨGAAGA	ACAAAΨGCGΨ	GAACΨΨCAAC	ΨΨCAACGGCC	ΨGACCGGCAC	1700
CGGCGΨGCΨG	ACAGAGAGCA	ACAAGAAGΨΨ	CCΨGCCAΨΨC	CAGCAGΨΨΨG	1750
GCCGGGAΨAΨ	CGCCGAΨACC	ACAGACGCCG	ΨΨAGAGAΨCC	CCAGACACΨG	1800
GAAAΨCCΨGG	ACAΨCACCCC	ΨΨGCAGCΨΨC	GGCGGAGΨGΨ	CΨGΨGAΨCAC	1850
CCCΨGGCACC	AACACCAGCA	AΨCAGGΨGGC	AGΨGCΨGΨAC	CAGGACGΨGA	1900
ACΨGΨACCGA	AGΨGCCCGΨG	GCCAΨΨCACG	CCGAΨCAGCΨ	GACACCΨACA	1950
ΨGGCGGGΨGΨ	ACΨCCACCGG	CAGCAAΨGΨG	ΨΨΨCAGACCA	GAGCCGGCΨG	2000
ΨCΨGAΨCGGA	GCCGAGCACG	ΨGAACAAΨAG	CΨACGAGΨGC	GACAΨCCCCA	2050
ΨCGGCGCΨGG	AAΨCΨGCGCC	AGCΨACCAGA	CACAGACAAA	CAGCCCΨCGG	2100
AGAGCCAGAA	GCGΨGGCCAG	CCAGAGCAΨC	AΨΨGCCΨACA	CAAΨGΨCΨCΨ	2150
GGGCGCCGAG	AACAGCGΨGG	CCΨACΨCCAA	CAACΨCΨAΨC	GCΨAΨCCCCA	2200
CCAACΨΨCAC	CAΨCAGCGΨG	ACCACAGAGA	ΨCCΨGCCΨGΨ	GΨCCAΨGACC	2250
AAGACCAGCG	ΨGGACΨGCAC	CAΨGΨACAΨC	ΨGCGGCGAΨΨ	CCACCGAGΨG	2300
CΨCCAACCΨG	CΨGCΨGCAGΨ	ACGGCAGCΨΨ	CΨGCACCCAG	CΨGAAΨAGAG	2350
CCCΨGACAGG	GAΨCGCCGΨG	GAACAGGACA	AGAACACCCA	AGAGGΨGΨΨC	2400
GCCCAAGΨGA	AGCAGAΨCΨA	CAAGACCCCΨ	CCΨAΨCAAGG	ACΨΨCGGCGG	2450
CΨΨCAAΨΨΨC	AGCCAGAΨΨC	ΨGCCCGAΨCC	ΨAGCAAGCCC	AGCAAGCGGA	2500
GCΨΨCAΨCGA	GGACCΨGCΨG	ΨΨCAACAAAG	ΨGACACΨGGC	CGACGCCGGC	2550
ΨΨCAΨCAAGC	AGΨAΨGGCGA	ΨΨGΨCΨGGGC	GACAΨΨGCCG	CCAGGGAΨCΨ	2600
GAΨΨΨGCGCC	CAGAAGΨΨΨA	ACGGACΨGAC	AGΨGCΨGCCΨ	CCΨCΨGCΨGA	2650
CCGAΨGAGAΨ	GAΨCGCCCAG	ΨACACAΨCΨG	CCCΨGCΨGGC	CGGCACAAΨC	2700
ACAAGCGGCΨ	GGACAΨΨΨGG	AGCAGGCGCC	GCΨCΨGCAGA	ΨCCCCΨΨΨGC	2750
ΨAΨGCAGAΨG	GCCΨACCGGΨ	ΨCAACGGCAΨ	CGGAGΨGACC	CAGAAΨGΨGC	2800
ΨGΨACGAGAA	CCAGAAGCΨG	AΨCGCCAACC	AGΨΨCAACAG	CGCCAΨCGGC	2850
AAGAΨCCAGG	ACAGCCΨGAG	CAGCACAGCA	AGCGCCCΨGG	GAAAGCΨGCA	2900
GGACGΨGGΨC	AACCAGAAΨG	CCCAGGCACΨ	GAACACCCΨG	GΨCAAGCAGC	2950
ΨGΨCCΨCCAA	CΨΨCGGCGCC	AΨCAGCΨCΨG	ΨGCΨGAACGA	ΨAΨCCΨGAGC	3000
AGACΨGGACC	CΨCCΨGAGGC	CGAGGΨGCAG	AΨCGACAGAC	ΨGAΨCACAGG	3050
CAGACΨGCAG	AGCCΨCCAGA	CAΨACGΨGAC	CCAGCAGCΨG	AΨCAGAGCCG	3100
CCGAGAΨΨAG	AGCCΨCΨGCC	AAΨCΨGGCCG	CCACCAAGAΨ	GΨCΨGAGΨGΨ	3150
GΨGCΨGGGCC	AGAGCAAGAG	AGΨGGACΨΨΨ	ΨGCGGCAAGG	GCΨACCACCΨ	3200
GAΨGAGCΨΨC	CCΨCAGΨCΨG	CCCCΨCACGG	CGΨGGΨGΨΨΨ	CΨGCACGΨGA	3250
CAΨAΨGΨGCC	CGCΨCAAGAG	AAGAAΨΨΨCA	CCACCGCΨCC	AGCCAΨCΨGC	3300
CACGACGGCA	AAGCCCACΨΨ	ΨCCΨAGAGAA	GGCGΨGΨΨCG	ΨGΨCCAACGG	3350
CACCCAΨΨGG	ΨΨCGΨGACAC	AGCGGAACΨΨ	CΨACGAGCCC	CAGAΨCAΨCA	3400
CCACCGACAA	CACCΨΨCGΨG	ΨCΨGGCAACΨ	GCGACGΨCGΨ	GAΨCGGCAΨΨ	3450
GΨGAACAAΨA	CCGΨGΨACGA	CCCΨCΨGCAG	CCCGAGCΨGG	ACAGCΨΨCAA	3500
AGAGGAACΨG	GACAAGΨACΨ	ΨΨAAGAACCA	CACAAGCCCC	GACGΨGGACC	3550
ΨGGGCGAΨAΨ	CAGCGGAAΨC	AAΨGCCAGCG	ΨCGΨGAACAΨ	CCAGAAAGAG	3600
AΨCGACCGGC	ΨGAACGAGGΨ	GGCCAAGAAΨ	CΨGAACGAGA	GCCΨGAΨCGA	3650
CCΨGCAAGAA	CΨGGGGAAGΨ	ACGAGCAGΨA	CAΨCAAGΨGG	CCCΨGGΨACA	3700
ΨCΨGGCΨGGG	CΨΨΨAΨCGCC	GGACΨGAΨΨG	CCAΨCGΨGAΨ	GGΨCACAAΨC	3750
AΨGCΨGΨGΨΨ	GCAΨGACCAG	CΨGCΨGΨAGC	ΨGCCΨGAAGG	GCΨGΨΨGΨAG	3800
CΨGΨGGCAGC	ΨGCΨGCAAGΨ	ΨCGACGAGGA	CGAΨΨCΨGAG	CCCGΨGCΨGA	3850
AGGGCGΨGAA	ACΨGCACΨAC	ACAΨGAΨGAC	ΨCGAGCΨGGΨ	ACΨGCAΨGCA	3900
CGCAAΨGCΨA	GCΨGCCCCΨΨ	ΨCCCGΨCCΨG	GGΨACCCCGA	GΨCΨCCCCCG	3950
ACCΨCGGGΨC	CCAGGΨAΨGC	ΨCCCACCΨCC	ACCΨGCCCCA	CΨCACCACCΨ	4000
CΨGCΨAGΨΨC	CAGACACCΨC	CCAAGCACGC	AGCAAΨGCAG	CΨCAAAACGC	4050
ΨΨAGCCΨAGC	CACACCCCCA	CGGGAAACAG	CAGΨGAΨΨAA	CCΨΨΨAGCAA	4100
ΨAAACGAAAG	ΨΨΨAACΨAAG	CΨAΨACΨAAC	CCCAGGGΨΨG	GΨCAAΨΨΨCG	4150
ΨGCCAGCCAC	ACCCΨGGAGC	ΨAGCAAAAAA	AAAAAAAAAA	AAAAAAAAAA	4200
AAAAGCAΨAΨ	GACΨAAAAAA	AAAAAAAAAA	AAAAAAAAAA	AAAAAAAAAA	4250
AAAAAAAAAA	AAAAAAAAAA	AAAAAAAAAA	AAAA		4284

5.7. Design a plasmid DNA template

The next step is to design a plasmid DNA that contains the gene, restriction site, 3’ primer site, origin of replication, antibiotic resistance gene, a selectable marker, promoter, 5’ primer site, restriction site. This design is readily created using online resources such as www.en.vectorbuilder.com.

5.8. Create a recombinant cell line

The gene of interest (above) is inserted into the pDNA, which is then introduced into host bacteria (typically E. coli) in a process called transformation, usually by electroporation.

5.9. Grow host cells to produce DNA

E. coli growth in a small bioreactor, not more than 30 L in size, to produce about 30 g of plasmid DNA. PCR technology to produce DNA is fast approaching the stage where it can replace pDNA production using E. coli. The PCR-based DNA is purer and thus reduces the amount needed, reducing the cost; it is also produced fast, though at a higher cost that should come down soon with technology improvement.

5.10. Extract pDNA and linearize

When the projected cell density is achieved, cells are harvested, and pDNA extracted, typically by alkaline lysis. Next, the lysate is clarified and DNA linearized by restriction digestion using enzymes that generate blunt ends or 5’-overhangs.

5.11. Perform In Vitro Transcription (IVT)

The IVT reaction mixture in a rocking bioreactor contains ribonucleotides, a viral RNA polymerase (e.g. T7 RNA polymerase), and the linearized DNA. The cap analog is also added to the reaction mixture that additionally includes a source of magnesium ions (e.g. MgCl2) and a polyamine, such as spermidine; magnesium is required for RNA polymerases to work, and polyamines help enhance transcription. After transcription, a deoxyribonuclease (DNase) is added to break down the DNA template and facilitate its subsequent removal. By the end of IVT, the reaction mixture contains a variety of impurities: buffer components, enzymes, unused nucleotides, cap analogs, truncated RNA/RNA fragments, dsRNA, DNA template, Mg2+, spermidine, etc. CleanCap AG; Deoxyribonuclease I (DNase I); RNase enzyme inhibitor; T7 RNA polymerase; linear template DNA; 1-methylpseudouridine-5’-triphosphate (mod-UTP).

5.12. Purification

Purification starts with a crossflow filter to remove small impurities and condition the mRNA in an appropriate buffer for the subsequent affinity (oligo-dT) chromatography step that is operated in a capture mode, removing most of the remaining impurities. Another step of hydrophobic chromatography does the final polishing. A second crossflow filtration allows the exchange of the buffer and the adjustment of the mRNA concentration, followed by formulation in a lipid nanoparticle.

5.13. Formulation

The lipid nanoparticle formulation of mRNA vaccines is the most critical step. Table 4 lists the lipids in the two mRNA vaccines.

Table 4. Lipid nanoparticle formulation of mRNA vaccines

mRNA Vaccine	Lipid Name	Lipid amount per dose [mg]
NIH-Moderna; mRNA-1273	Ionizable lipid a	1.09
	Phospholipid b	0.24
	Cholesterol	0.47
	PEG lipid c	0.13
	Total lipids	1.93
BioNTech-Pfizer; BNT162b2	Ionizable lipid d	0.43
	Phospholipid e	0.09
	Cholesterol	0.2
	PEG lipid f	0.05
	Total lipids	0.77
CureVac; CVnCoV	Ionizable lipid g	0.17
	Phospholipid h	0.04
	Cholesterol	0.08
	PEG lipid i	0.02
	Total lipids	0.31

5.14. Testing

The vaccine is tested for nanoparticle characteristics, encapsulation, transfection efficiencies, in vitro cytotoxicity, and stability and storability. The vaccine’s safety is assessed in Balb/c mice injected with the vaccine containing 10 µg of spike-encoding mRNA. The vaccine efficacy is tested by inducing an immune response against SARS-CoV-2 in Balb/c and C57BL/6 mice (receiving 1 or 10 µg of mRNA) and rhesus macaque monkeys (infused with the vaccine containing 30–100 µg of mRNA). The ELISA and virus-neutralizing test (VNT) results will show a significant augmentation in the level of neutralizing antibodies against SARS-CoV-2. Moreover, the ELISA assay will show virus-specific IFN-γ secretion in immunized mice as a TH1 cell-based immune response marker. In contrast, favorably, no change in the production of IL-4 is detected. GLP.

5.15. Fill and finish

The vaccine is filled in vials after passing through a sterilizing filter.

6. Regulatory solutions

The mRNA products are listed by EMA under its advanced therapy medicinal product (ATMP) program [36,37]. ATMPs can be classified into three main types: gene therapy medicines, somatic cell therapy, and tissue-engineered medicines. An equivalent program at the FDA is the regenerative medicine advanced therapy (RMAT) classification [38] that includes cell therapy, therapeutic tissue engineering product, human cell and tissue product, or any combination product using such therapies or products, except human cells, tissues, cellular and tissue-based products. Additionally, this designation also includes if the drug is intended to treat, modify, reverse, or cure a serious or life-threatening disease or condition; and preliminary clinical evidence indicates that the drug has the potential to address unmet medical needs for such disease or condition.

Before the first regulatory application for an mRNA vaccine was filed with the FDA, developers assumed that this would be treated as a gene therapy product [39]. However, the FDA licensed the two products as biological products under CBER 351(a), where all vaccines fall. However, the FDA classifies the siRNA, a double-strand RNA, as a chemical drug, not a biological product.

In managing the COVID-19 pandemic, the FDA has provided final guidance to suggest that vaccines that are highly similar to an approved mRNA vaccine, ‘generic mRNA vaccine’ as defined in this paper, can be approved in a fast-track requiring fewer clinical studies. In addition, this FDA guidance has opened the doors to developing generic mRNA vaccines because their chemical structure can be copied entirely. The US FDA guideline staters:

“COVID-19 vaccine development may be accelerated based on knowledge gained from similar products manufactured with the same well-characterized platform technology, to the extent legally and scientifically permissible. Similarly, with appropriate justification, some aspects of manufacture and control may be based on the vaccine platform, and in some instances, reduce the need for product-specific data.” [39].

While this FDA advice pertains to the COVID-19 vaccine, it should equally apply to other mRNA vaccines arriving in the future. However, there is no ‘generic’ provision in any current regulatory guideline.

The mRNA vaccine copies will also be filed under the 351(a) clause of the PHS Act with the FDA, where it will be licensed as a new or standalone biological product within the CBER. This will also be a new product if filed with the EMA. What will make this filing different is the inclusion of public domain data to support safety and efficacy – in this case, the registration dossier of the originator product and any published studies. Since the chemical structure of the generic mRNA product is the same (preferably identical) as the originator product, several concessions will be allowed if proper arguments are presented in the regulatory filing. Since the translated antigen will be the same, the cellular response is expected to be the same. In addition, whether differences in the LNP formulation are relevant can be readily tested. These tests will likely include, among others, mRNA-LNP stability and storability, in vitro cytotoxicity of mRNA-LNPs, humoral response in animal models, investigation of the humoral immune response, virus neutralization test (VNT), and investigation of the cell-mediated immune response.

Based on what the FDA has stated [39], reduced testing of the copies of mRNA vaccines will require an intuitive plan as follows:

• Establish structural similarity of the nucleotides and demonstrate no differences, including in the poly(A) tails. If more than one mRNA vaccine licensed (not under EUA) has different nucleotides, you may not switch back and forth; the generic vaccine must be the same as your targeted vaccine. Think of it as a reference product, and you are manufacturing a biosimilar product, and this attribute is the primary sequence.

• Create a formulation of LNP using precisely the same chemicals and process as used by the licensed vaccine. This information is widely available, and a particle size comparison can be easily made in side-by-side testing with the licensed vaccine. However, here you have some leeway if you can show in side-by-side testing in an animal model a similar efficacy profile that will validate the delivery system that serves as the vector [40].

• Keep the dosing, primary, secondary, and tertiary packaging the same as the licensed vaccine; the storage conditions can be different if you have sufficient data to prove stability. For example, Pfizer vaccine for COVID-19 chose stringent storage conditions to get a head start for the approval since, at those conditions, there is little possibility of any degradation; Pfizer has since secured approval of modified conditions for the vials to be stored in the refrigerator at 2°C to 8°C (35°F to 46°F) for up to 1 month. Previously, thawed, undiluted vaccine vials could be stored in the refrigerator for up to 5 days. If these conditions change over time, a similar change can be brought to the generic version. One reason to follow this advice is to reduce special handling requirements at the point of use, to make the generic version more acceptable.

• Identify critical quality attributes. In addition to the usual drug quality attributes, mRNA drug substances have a unique set of attributes to consider, including mRNA integrity and concentration, residual DNA template, and residual double-stranded RNA; all impact the registration dossier.

• Test the generic vaccine side-by-side with the licensed vaccine to include nucleotide sequence, mRNA-LNP stability, storability, in vitro cytotoxicity, rodent testing in Balb/c mouse, non-human primate test, humoral immune response test, and cellular immune response study.

The most crucial element for regulatory approval is demonstrating safety and efficacy in humans. However, developers should argue with the regulatory agencies that a phase 3 placebo-control study will be unethical since there is already an approved vaccine, a view accepted by the regulatory agencies [41]. Instead, present testing of safety and efficacy in a comparative model in a parallel design wherein one group gets a licensed vaccine and the other its proposed generic version in a double-blind protocol with the licensed vaccine. The side effects within 48 hours of administration are compared, as most side effects of mRNA vaccines appear within a few days [42]. Additionally, the antibody profiles over two weeks are compared to ascertain efficacy.

The 351(a) filing pathway allows the developers to conduct meetings with the FDA without paying a registration fee for 351(k). Therefore, it is advisable to meet with the FDA or EMA as early as possible to launch a generic version of an mRNA vaccine.

While the discussion above used the terminology more familiar to the US FDA guidelines, these suggestions apply equally to the EMA guidelines. The ICH guidelines like ICH Q6B will be most relevant.

The above understanding of regulatory filing may not apply to other agencies, particularly those where scientific evaluation is impossible, as with most developing nation agencies. Since the purpose of the ‘generic’ mRNA vaccine is to make it available in the developing countries, not the US or EU, where there is already an abundance of these vaccines, filing a registration application with the FDA or EMA is not advised. However, both agencies allow initial meetings and suggest establishing safety and efficacy. Therefore, I recommend all developers, regardless of their jurisdiction, to engage in a Type B meeting with the FDA and a preliminary meeting with the EMA to secure, in writing, what they would consider being an acceptable proof of safety and efficacy. These documents will clarify the agencies and assure faster approval when presented globally to any other regulatory agency.

7. Intellectual property solutions

The mRNA vaccine technology is heavily protected under intellectual property by the leading developers, manufacturers, and suppliers of formulation components. A recent (May 2021) analysis provides details of this landscape [43]. Moderna owns several patents, allowing their use without claiming infringement [44]. So, if one were to copy the Moderna vaccine, it can be marketed without infringing any patents of Moderna. In addition, Moderna has secured patent rights to chemicals used; the developers can secure the same licensing. However, recently this path has been muddied by the claims of the chemical suppliers to Moderna refusing to allow the use of their chemicals without licensing.

Pfizer has taken an opposite position disputing the US government’s suggestion [45] to remove patents related to Covid-19 products claiming [46] that it will bring a shortage of raw materials they are using. This argument does not hold. Pfizer also states that such a move will kill innovation listing the billions they have invested. And there is indeed a shortage of chemical supplies; however, many suppliers have recently emerged to fill in the supply chain gap (www.hzymes-global.com). So, despite much news about the shortage of chemicals, it is not a hurdle in manufacturing anymore.

The types of patents involved in the design of mRNA vaccines include nucleoside modifications, the LNP formulation, the cap structure, and the poly(A) tail sequences. In addition, the coding region of an mRNA vaccine is generally not patentable since the sequence of the translated protein is in the public domain. While these legal challenges appear to challenge the development of generic mRNA vaccines, the intellectual property landscape, and its enforcements are subject to humanitarian use solutions. No patents are allowed to claim the sequence of the surface proteins (being a natural product). The patents may include the nucleotide modification, the cap structure, the poly(A) tails, the formulation of LNP, and managing the process controls.

However, not all the IP developed and allowed in US or EU is relevant globally since a patent must be registered and permitted in the jurisdiction of the use; regulatory approval does not constitute an affirmation of the intellectual property rights.

The patents that protect the current COVID-19 vaccine can be challenged, even though this view is not popular among the developers. First, we know that the US and EU governments provided billions of dollars to vaccine developers to complete the testing of their products. The pre-purchase further supported the developers and provided them with other benefits of Operation Warp-Speed [45], where several agencies collaborated. OWS has funded JNJ-78436735 (Janssen), mRNA-1273 (Moderna), NVX CoV2373 (Novavax), V590 (Merck/IAVI), V591 (Merck/Themis), AZD1222 (AstraZeneca/University of Oxford), and the candidate developed by Sanofi and GlaxoSmithKline. Within OWS, the US National Institutes of Health (NIH) has partnered with more than 18 biopharmaceutical companies in ACTIV to fast-track the development of drug and vaccine candidates for COVID-19. The COVID-19 Prevention Trials Network (COVPN) combines clinical trial networks funded by the National Institute of Allergy and Infectious Diseases (NIAID): the HIV Vaccine Trials Network (HVTN), HIV Prevention Trials Network (HPTN), Infectious Diseases Clinical Research Consortium (IDCRC), and the AIDS Clinical Trials Group.

Patents can also be challenged on the legal ground of government support that allows the citizens to claim the rights since their tax money benefitted these companies. The WTO has undertaken the responsibility of deciding whether patents related to COVID-19 can be enforced; a decision is expected soon. The US government has already expressed its position; they will nullify the US patents. So, while the IP remains a significant issue, it seems like the humanitarian aspect of COVID-19 treatment and prevention can be exploited. The WTO’s agreement on intellectual property – the TRIPS (Trade-Related Aspects of Intellectual Property Rights) Agreement allow several options.

To invoke compulsory licensing, a prospective manufacturer should first ask for licensing of the patent if the patent is registered in the jurisdiction [47]. Should the patent owners deny such a request, a compulsory license is issued by the government; however, the patent owner should be given a royalty; the TRIPS Agreement says ‘the right holder shall be paid adequate remuneration in the circumstances of each case, taking into account the economic value of the authorization.’ Still, it does not define ‘adequate remuneration’ or ‘economic value.’ Generally, the country invoking the licensing decides it. The fee is generally no more than 5%. The first use of compulsory licensing to gain patent rights came when HIV drugs were produced and supplied in Africa. Conclusion: There is no reason developing countries cannot adopt the same pathway for the COVID-19 vaccines and drugs for its treatment. It is not breaking any laws; it is making the best use of the allowances already made in global treaties. An example of compulsory is the historic WIPO actions regarding HIV treatment drugs [48], where manufacturers were allowed to supply despite the patents being in place.

The resolution of intellectual property in the case of the 46 Least Developed Countries (LDCs) is even simpler [49]. In November 2015, the World Trade Organization announced its approval of a limited 17-year extension of a 2001 waiver of obligations in the TRIPS Agreement, set to expire at the end of 2015, the terms of which exempt Least Developed Countries (LDCs) from requirements to grant patents or related intellectual property rights on pharmaceutical products [50,51]. As a result, the LDCs can use any technology without infringement issues. Therefore, they remained listed as an LDC and distributed only to other LDCs, or where a country may exercise the compulsory licensing. This situation should serve as an incentive to LDCs and other developing countries to expand their technology base.

More recently, in March 2022, an agreement was reached for the waiver of all COVID-19 patents[53] ; however, this waiver can still be muddled with several cross-patent issues where the material suppliers may have their patents that were sublicensed.

8. Discussion

More lives have been saved throughout the history of humankind by the use of vaccines than any other measure. Vaccination began about 200 years ago when the death rate was about 3000 per million people dying of infections in England [52]. In the first 50 years of vaccination, this number dropped to 400, and when vaccines were enforced in 1889, it went down to less than 10.

Today, most of the world population in underdeveloped countries remains unvaccinated against COVID-19 because of supply and affordability issues that can be resolved if the mRNA vaccine manufacturing facilities vaccines are installed in developing countries. It is projected that the cost per dose of the COVID-19 vaccine will not exceed one dollar per dose compared to the current cost of $25-$28 per dose. Developers can also supply the vaccine to other countries in bulk at a much lower cost and seek financial and marketing support from the Covax program. If there are still financial constraints, developing countries can form consortiums to pool their resources.

Self-sufficiency in producing mRNA vaccines will bring many rewards in the future, including managing future pandemics, converting traditional vaccines into lower cost and more effective mRNA vaccines, and benefitting from the vaccines against autoimmune disorders that will arrive soon.

9. Conclusions

The COVID-19 pandemic has taken a significant toll on everyone living and interrupted billions of lives – all caused by a non-living strip of chemicals; the solution came as another non-living chemical strip – the mRNA vaccine. Though the COVID-19 vaccines provided a ready answer, most of the world still awaits them, unable to afford them. The COVID-19 pandemic also brought us a gift of mRNA vaccine that can be produced at an affordable cost, but we still do not know how to get the vaccine into the hands (or arms) of most humans on earth?

The regulatory pathway is evident because a generic mRNA vaccine is an exact copy of an approved vaccine – it is a chemical, not a biological drug. In addition, the FDA and EMA have already made it clear how a ‘generic’ form of vaccine will be approved without labeling it as such for various legal and scientific reasons.

The most debated issue about the mRNA vaccine is the intellectual property rights owned by the vaccine developers and chemical suppliers. However, for least developed countries, this is not an issue, and it will be an incentive for the countries to form a consortium to manufacture and distribute these vaccines in all 46 LDCs and other countries that may choose to allow compulsory licensing, making it a remarkable economic opportunity as well.

10. Expert opinion

Universal vaccinations are required to control pandemics, but when a crisis strikes, the more affluent countries get first and complete access to these protections, as seen in the COVID-19 pandemic. Holding back the accessibility to most of the world population is the availability and affordability of vaccines. This gap can be filled by encouraging developing countries to manufacture these vaccines by copying approved vaccines as suggested by the WHO; however, this applies only to the mRNA vaccines since these are chemical vaccines and happen to be most effective and safer than any other vaccine type. It is not possible to copy other types of vaccines and claim them as equivalent to an approved vaccine

New vaccines will be based on the mRNA technology, and over time, so will the existing vaccines. The possibility of combining vaccines in lipid nanoparticles offers the opportunity for producing vaccines like HIV and HPV combined. The mRNA vaccines will also bring the first prevention modality for autoimmune disorders for which no treatment exists, turning the healthcare focus quickly. There are over 200 mRNA products under development. However, the cost of developing new products is high. Still, with the proven safety of mRNA vaccines, the regulatory approval bar will likely be reduced, as seen in the approval of mRNA vaccines against COVID-19.

The technology for mRNA vaccines is readily available, and the cost of setting up manufacturing is minimal compared to any other type of vaccine. There is also financial support available through international agencies. This is also a good investment in healthcare and the financial well-being that is equally in need of help.

Developers can begin technology transfer and proof of concept studies in their existing laboratories under laminar flow hoods since the process is of small scale; once the technical dossier has been developed, additional facilities can be added if these cannot be obtained due to cost constraints. In addition, there are no biosafety concerns in the design of manufacturing facilities. The upstream process is non-GMP since it makes a ‘pre-API.’ Major efforts will come in the testing that may have to be outsourced since it involves animal testing and requires other advanced technologies.

Regulatory approval of mRNA vaccines that are copies of approved vaccines is relatively short since a placebo-control study is not allowed. It will only involve testing side effects over 48 hours and matching the antibody profile over 2–4 weeks from a parallel blinded study. However, the developers may run into difficulties securing approval of the copies of their mRNA vaccines because of a lack of knowledge and expertise within the regional regulatory agencies. I anticipate WHO coming forward with rational approval guidelines soon. However, one suggestion to developing countries is to have their product wetted by the FDA or EMA (there is no fee) and then present this evaluation to their Agencies.

Intellectual property hindrances associated with mRNA vaccines can be overcome by 1) securing rights to these patents; 2) seeking a compulsory licensing pathway; 3) manufacturing in one of the LDCs that will also allow distribution to 45 other LDCs that are primarily unvaccinated. Recentlly, the US and EU have entered an agreement with Covid-19 patents and allowing it for open use [53].

The new era of mRNA vaccines has forever changed the future of medicine; however, this technology should be acquired by developing countries, where the needs have historically remained unmet. Therefore, this is the time for global equalization of the benefits of innovative medicines. It is time for a paradigm shift for the developed countries to consider healthcare an essential element that cannot be left to others to provide. It is time for the developing countries to remove the dogma that modern technologies belong to the West only [53].

Article highlights

• The COVID-19 pandemic introduced the chemical mRNA vaccines decades in the making as the most effective and the safest vaccines.

• Unlike all other vaccines, mRNA is also the cheapest vaccine and requires a minor investment to set up manufacturing, as the plan submitted shows.

• Developing countries should become self-sufficient in their vaccine supplies for the COVID-19 vaccine to protect against future pandemics.

• Many new mRNA vaccines are under development to prevent autoimmune disorders, making it the most critical technology.

• Besides protecting the health, the mRNA vaccines offer remarkable revenue generation opportunities, now and in the future.

Declaration of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

Reviewer disclosures

Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.

Disclaimer

The author is not advising any legal violations when suggesting how to secure the rights to intellectual property.

Additional information

Funding

This paper was not funded.