Quality by Design for enabling RNA platform production processes

Pfizer-BioNTech's (BNT162b2) and Moderna's (mRNA-1713) vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are the first RNA-based biologics to be approved for human use and mass produced.RNA technology holds great promise beyond infectious disease prophylaxis, from cancer and gene therapy to treatments against cardiovascular and autoimmune diseases.Current production processes have been developed and scaled up at an unprecedented speed, mostly under a Quality by Testing paradigm.Recent research has highlighted a strong link between product and process development.The application of advanced analytical and modeling technologies could rapidly reshape and digitize manufacturing processes. RNA-based products have emerged as one of the most promising and strategic technologies for global vaccination, infectious disease control, and future therapy development. The assessment of critical quality attributes (CQAs), product–process interactions, relevant process analytical technologies, and process modeling capabilities can feed into a robust Quality by Design (QbD) framework for future development, design, and control of manufacturing processes. QbD implementation will help the RNA technology reach its full potential and will be central to the development, pre-qualification, and regulatory approval of rapid response, disease-agnostic RNA platform production processes. RNA-based products have emerged as one of the most promising and strategic technologies for global vaccination, infectious disease control, and future therapy development. The assessment of critical quality attributes (CQAs), product–process interactions, relevant process analytical technologies, and process modeling capabilities can feed into a robust Quality by Design (QbD) framework for future development, design, and control of manufacturing processes. QbD implementation will help the RNA technology reach its full potential and will be central to the development, pre-qualification, and regulatory approval of rapid response, disease-agnostic RNA platform production processes. The coronavirus disease 2019 (COVID-19) pandemic and emergence of safe and efficient RNA vaccines have brought RNA technology to the forefront of medical innovations [1.Corbett K.S. et al.SARS-CoV-2 mRNA vaccine design enabled by prototype pathogen preparedness.Nature. 2020; 586: 567-571Crossref PubMed Scopus (740) Google Scholar,2.Vogel A.B. et al.BNT162b vaccines protect rhesus macaques from SARS-CoV-2.Nature. 2021; 592: 283-289Crossref PubMed Scopus (318) Google Scholar]. The rapid development and production timelines, in combination with recent genotyping methods, make RNA technology suitable to respond to emerging infectious threats and variants [3.Kis Z. et al.Emerging technologies for low-cost, rapid vaccine manufacture.Biotechnol. J. 2019; 14e1800376Crossref Scopus (19) Google Scholar]. While most conventional vaccines and biopharmaceuticals require the use of inherently variable cell cultures, RNA manufacturing is based on a relatively simple, scalable, and affordable cell-free production system [4.Kis Z. et al.Resources, production scales and time required for producing RNA vaccines for the global pandemic demand.Vaccines. 2020; 9: 3Crossref PubMed Scopus (61) Google Scholar]. Given its mechanism of action, the therapeutic scope of RNA technology is wide, and production processes are versatile (Box 1) [5.Batra K. et al.An insight on RNA based therapeutics and vaccines: challenges and opportunities.Curr. Top. Med. Chem. 2021; 21: 2851-2855Crossref PubMed Scopus (0) Google Scholar]. The potential clinical applications encompass infectious disease prophylaxis; rare disease treatment; and gene, cancer, and protein replacement therapy [6.Halloy F. et al.Innovative developments and emerging technologies in RNA therapeutics.RNA Biol. 2022; 19: 313-332Crossref PubMed Scopus (0) Google Scholar, 7.Sahin U. Türeci Ö. Personalized vaccines for cancer immunotherapy.Science. 2018; 359: 1355-1360Crossref PubMed Scopus (551) Google Scholar, 8.Pardi N. et al.mRNA vaccines – a new era in vaccinology.Nat. Rev. Drug Discov. 2018; 17: 261-279Crossref PubMed Scopus (2081) Google Scholar]. Different products could be manufactured using the same raw materials (excluding DNA template), consumables, equipment, unit operations, and analytical methods. However, a multiproduct platform technology still requires proof of scientific and industrial mastery to be approved and truly disruptive.Box 1Current RNA-based drug productRNA-based vaccines and therapeutics are composed of two key elements: the RNA active substance encoding a protein of interest and the LNP structure as the delivery vehicle. Typically, the protein can be a viral antigen, such as the SARS-CoV-2 spike protein, a cancer marker, or a missing protein. Regardless of the route of administration, the RNA enters the cytosol through a receptor-mediated mechanism. Then, the mRNA active substance uses the host cell translation machinery, while saRNA also encodes its own replication machinery [9.Kim J. et al.Self-assembled mRNA vaccines.Adv. Drug Deliv. Rev. 2021; 170: 83-112Crossref PubMed Scopus (170) Google Scholar]. This is one of the major differences between these two classes of RNA-based products. Currently, approved vaccines and most of the clinically advanced candidates are based on nonreplicating mRNA systems. However, saRNA could be advantageous because lower doses of RNA are potentially sufficient for enhanced and prolonged protein expression, thereby also reducing production costs and the occurrence of some adverse reactions [4.Kis Z. et al.Resources, production scales and time required for producing RNA vaccines for the global pandemic demand.Vaccines. 2020; 9: 3Crossref PubMed Scopus (61) Google Scholar]. The saRNA nucleotide sequence is also longer (e.g., approximately 10 kb compared with 4.5 kb for mRNA COVID-19 vaccines), implying potential manufacturing differences [10.Spencer A.J. et al.Heterologous vaccination regimens with self-amplifying RNA and adenoviral COVID vaccines induce robust immune responses in mice.Nat. Commun. 2021; 12: 2893Crossref PubMed Scopus (74) Google Scholar]. In this review, the mRNA and saRNA systems are collectively referred to as 'RNA technology'. RNA-based vaccines and therapeutics are composed of two key elements: the RNA active substance encoding a protein of interest and the LNP structure as the delivery vehicle. Typically, the protein can be a viral antigen, such as the SARS-CoV-2 spike protein, a cancer marker, or a missing protein. Regardless of the route of administration, the RNA enters the cytosol through a receptor-mediated mechanism. Then, the mRNA active substance uses the host cell translation machinery, while saRNA also encodes its own replication machinery [9.Kim J. et al.Self-assembled mRNA vaccines.Adv. Drug Deliv. Rev. 2021; 170: 83-112Crossref PubMed Scopus (170) Google Scholar]. This is one of the major differences between these two classes of RNA-based products. Currently, approved vaccines and most of the clinically advanced candidates are based on nonreplicating mRNA systems. However, saRNA could be advantageous because lower doses of RNA are potentially sufficient for enhanced and prolonged protein expression, thereby also reducing production costs and the occurrence of some adverse reactions [4.Kis Z. et al.Resources, production scales and time required for producing RNA vaccines for the global pandemic demand.Vaccines. 2020; 9: 3Crossref PubMed Scopus (61) Google Scholar]. The saRNA nucleotide sequence is also longer (e.g., approximately 10 kb compared with 4.5 kb for mRNA COVID-19 vaccines), implying potential manufacturing differences [10.Spencer A.J. et al.Heterologous vaccination regimens with self-amplifying RNA and adenoviral COVID vaccines induce robust immune responses in mice.Nat. Commun. 2021; 12: 2893Crossref PubMed Scopus (74) Google Scholar]. In this review, the mRNA and saRNA systems are collectively referred to as 'RNA technology'. In addition, the rapid production of safe and efficient vaccines was only possible thanks to a high-risk financing strategy, government support, and strong incentives for industrial adaptation [20.Funk C.D. et al.A snapshot of the global race for vaccines targeting SARS-CoV-2 and the COVID-19 pandemic.Front. Pharmacol. 2020; 11: 937Crossref PubMed Scopus (135) Google Scholar]. In the long run, this approach is neither sustainable nor desirable. International technology transfer and distributed manufacturing are urgently needed. Additionally, despite the high level of safety and efficacy observed in these approved vaccines, product quality remains a critical issue [21.Walsh E.E. et al.Safety and immunogenicity of two RNA-based COVID-19 vaccine candidates.N. Engl. J. Med. 2020; 383: 2439-2450Crossref PubMed Scopus (1570) Google Scholar]. An example is the occurrence of rare severe adverse events, such as myocarditis and anaphylactic reactions, which could hamper vaccination campaigns and strengthen public mistrust in this new technology [22.Milano G. et al.Myocarditis and COVID-19 mRNA vaccines: a mechanistic hypothesis involving dsRNA.Futur. Virol. 2021; 17: 191-196Crossref Google Scholar]. Their clinical success should not overshadow the current need for booster doses and the failures of other mRNA vaccine candidates [23.Dolgin E. CureVac COVID vaccine let-down spotlights mRNA design challenges.Nature. 2021; 594: 483Crossref PubMed Scopus (0) Google Scholar]. The two components, the RNA active substance and the lipid nanoparticle (LNP) (see Glossary) (Box 1), are both unstable and prone to degradation [13.Schoenmaker L. et al.mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability.Int. J. Pharm. 2021; 601120586Crossref PubMed Scopus (385) Google Scholar]. For instance, RNA integrity in the BNT162b2 vaccine is estimated to be approximately 70% at the end of production, with further degradation expected during distribution [24.Tinari S. The EMA covid-19 data leak, and what it tells us about mRNA instability.BMJ. 2021; 372n627PubMed Google Scholar]. This is partially reflected in the low temperature requirements for product storage, which further complicates the vaccine supply chain [25.Crommelin D.J.A. et al.Addressing the cold reality of mRNA vaccine stability.J. Pharm. Sci. 2021; 110: 997-1001Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar]. Finally, RNA technology is still under intense development and should prove its therapeutic versatility in clinical trials while new manufacturing requirements are anticipated [26.Heine A. et al.Clinical and immunological effects of mRNA vaccines in malignant diseases.Mol. Cancer. 2021; 20: 52Crossref PubMed Scopus (19) Google Scholar]. Therefore, a deeper understanding of both product and process appears necessary to face these multiple challenges. The absence of rigid RNA-specific regulatory guidelines leaves further room for continuous innovation [27.Knezevic I. et al.Development of mRNA vaccines: scientific and regulatory issues.Vaccines. 2021; 9: 81Crossref Scopus (15) Google Scholar,28.Liu M.A. et al.WHO informal consultation on regulatory considerations for evaluation of the quality, safety and efficacy of RNA-based prophylactic vaccines for infectious diseases, 20–22 April 2021.Emerg. Microbes Infect. 2022; 11: 384-391Crossref PubMed Scopus (0) Google Scholar]. In particular, the application of a QbD approach to this new class of drugs could be a paradigm shift and could unlock the potential of RNA manufacturing technology (Figure 1). This review discusses how these QbD principles can be applied and tuned for RNA-based products and how this new technology can specifically benefit from them. The emerging literature in this rapidly evolving field is analyzed herein and is interpreted through the prism of a multiproduct and patient-centric manufacturing approach. This knowledge assessment offers the first risk-based review of product quality attributes, process parameters, and their potential interactions. From this, the potential avenues for the development of characterization and modeling tools to underpin an enhanced QbD approach can be identified. Eventually, a theoretical and holistic manufacturing framework encompassing and integrating CQAs, critical process parameters (CPPs), and the requirements of current and future RNA-based products can be drawn. At this early stage of development, this analysis further brings new perspectives and a roadmap for the rapid deployment of a versatile, distributed, and affordable RNA platform technology. In QbD, CQAs are at the basis of production process development, design, monitoring, control, and life-cycle management. The first comprehensive identification of potential CQAs for RNA-based biologicals is presented in Table 1. It is established on the basis of prior knowledge, current structure–function understanding, strategic nonclinical studies, and relevant real-world experience. Furthermore, this list is in accordance with nascent and existing regulatory guidelines and encompasses the specification of mRNA-1713 and BNT162b2 productsi,ii [27.Knezevic I. et al.Development of mRNA vaccines: scientific and regulatory issues.Vaccines. 2021; 9: 81Crossref Scopus (15) Google Scholar,28.Liu M.A. et al.WHO informal consultation on regulatory considerations for evaluation of the quality, safety and efficacy of RNA-based prophylactic vaccines for infectious diseases, 20–22 April 2021.Emerg. Microbes Infect. 2022; 11: 384-391Crossref PubMed Scopus (0) Google Scholar]. The sources, risk assessment methodology, and detailed rationale for each attribute are displayed in Tables S1–S3 in the supplemental information online.Table 1Identification of quality attributes of RNA-based productaAbbreviations: AS, active substance; CQA, critical quality attribute; DP, drug product; dsRNA, double-stranded RNA; LNP, lipid nanoparticle; poly(A), polyadenylation; pCQA, potential critical quality attribute; QA, quality attribute.,bRNA yield and RNA recovery can be identified as key performance indicators and not CQAs, but they remain central in the process control strategy and are also indicators of process consistency and hazardous deviations from normal operating conditions.Criticality levelAS attributesAS-related impuritiesDP attributesDP-related impuritiesAdditional attributes and compendial testingCQAsRNA contentRNA purityRNA contentLipid–RNA species impurityImmunogenicityRNA sequence identitydsRNA speciesLipid contentPotency/in vitro expressionRNA sequence integrityShorter RNA speciesLNP sizeEndotoxins5′ capping efficiencyLNP polydispersityBioburdensPoly(A) tail lengthLNP surface chargeSterilityPoly(A) tail levelLipid identitypHRNA encapsulationOsmolalityParticulate matterpCQAsRNA structural integrityRNA precipitatesIndividual lipid impuritiesLNP morphologyResidual enzymesTotal lipid impuritiesResidual host cell proteinsResidual solventAdditional residual impuritiesQAsResidual DNAAppearanceViscositya Abbreviations: AS, active substance; CQA, critical quality attribute; DP, drug product; dsRNA, double-stranded RNA; LNP, lipid nanoparticle; poly(A), polyadenylation; pCQA, potential critical quality attribute; QA, quality attribute.b RNA yield and RNA recovery can be identified as key performance indicators and not CQAs, but they remain central in the process control strategy and are also indicators of process consistency and hazardous deviations from normal operating conditions. Open table in a new tab More specifically, activation of the innate immune system is a central factor in assessing RNA-based biologics' safety and efficacy. Inflammatory reactions are indeed related to most frequent and rare adverse events [21.Walsh E.E. et al.Safety and immunogenicity of two RNA-based COVID-19 vaccine candidates.N. Engl. J. Med. 2020; 383: 2439-2450Crossref PubMed Scopus (1570) Google Scholar,32.Baden L.R. et al.Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine.N. Engl. J. Med. 2021; 384: 403-416Crossref PubMed Scopus (5663) Google Scholar]. Although immunogenicity can be considered a priori as an advantageous feature for product efficacy, providing adjuvant-like properties, it could simultaneously reduce RNA translation as a result of the activation of stress genes and a cellular trade-off between innate immune and translation machinery mechanisms [33.Nelson J. et al.Impact of mRNA chemistry and manufacturing process on innate immune activation.Sci. Adv. 2020; 6eaaz6893Crossref Scopus (127) Google Scholar]. Although minimizing inflammation seems to emerge as the best approach for prophylactic vaccines and a requirement in gene therapies, this stimulation remains promising in cancer treatment because type I interferon activation is correlated with favorable disease outcomes [34.Zitvogel L. et al.Type I interferons in anticancer immunity.Nat. Rev. Immunol. 2015; 15: 405-414Crossref PubMed Scopus (764) Google Scholar]. More indirectly, inflammation can affect potency by limiting the dose regime, as occurred in the second phase of CureVac's COVID-19 vaccine trials [16.Blakney A.K. et al.Polymeric and lipid nanoparticles for delivery of self-amplifying RNA vaccines.J. Control. Release. 2021; 338: 201-210Crossref PubMed Scopus (6) Google Scholar]. Regarding the drug product, in vivo administration of naked RNA and empty LNP triggers immune stimulation, confirming the immunogenic nature of both structures [35.Ndeupen S. et al.The mRNA-LNP platform's lipid nanoparticle component used in preclinical vaccine studies is highly inflammatory.iScience. 2021; 24103479Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. Particular emphasis should be placed on polyethylene glycol (PEG) lipids suspected to be related to the observed anaphylactic reactions [36.Kozma G.T. et al.Anti-PEG antibodies: properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals.Adv. Drug Deliv. Rev. 2020; 154–155: 163-175Crossref PubMed Scopus (236) Google Scholar,37.Packer M. et al.A novel mechanism for the loss of mRNA activity in lipid nanoparticle delivery systems.Nat. Commun. 2021; 12: 6777Crossref PubMed Scopus (3) Google Scholar]. On the one hand, potential lipid–RNA reactions and hybridizations also require careful consideration because they have been observed during the mRNA-1713 manufacturing process and could lead to inactivation of the active substance potency and enhanced degradation [20.Funk C.D. et al.A snapshot of the global race for vaccines targeting SARS-CoV-2 and the COVID-19 pandemic.Front. Pharmacol. 2020; 11: 937Crossref PubMed Scopus (135) Google Scholar,34.Zitvogel L. et al.Type I interferons in anticancer immunity.Nat. Rev. Immunol. 2015; 15: 405-414Crossref PubMed Scopus (764) Google Scholar]. On the other hand, numerous RNA-related impurities are also potentially immunogenic (Table 1), and longer double-stranded RNA (dsRNA) species represent one of the major risks [38.Wang Y. et al.Length dependent activation of OAS proteins by dsRNA.Cytokine. 2020; 126154867Crossref Scopus (11) Google Scholar]. These heterogeneous byproducts are potent pathogen-associated molecular patterns and can completely deplete RNA translation. Structural elements, such as 5′-cap and polyadenylation [poly(A)] tail, and their integrity play another key role in product reactogenicity and translational efficiency [39.Kim S.C. et al.Modifications of mRNA vaccine structural elements for improving mRNA stability and translation efficiency.Mol. Cell. Toxicol. 2022; 18: 1-8Crossref PubMed Scopus (4) Google Scholar]. While the criticality of RNA primary sequence is obvious, the importance of the RNA secondary and tertiary structure is still being assessed [40.Zhang H. et al.A new method of RNA secondary structure prediction based on convolutional neural network and dynamic programming.Front. Genet. 2019; 10: 467Crossref PubMed Scopus (41) Google Scholar]. The higher-order structures of BNT162b2 active substance have been evaluated during characterization studies and are expected to influence the RNA thermostability and half-life [23.Dolgin E. CureVac COVID vaccine let-down spotlights mRNA design challenges.Nature. 2021; 594: 483Crossref PubMed Scopus (0) Google Scholar,41.Wayment-Steele H.K. et al.Theoretical basis for stabilizing messenger RNA through secondary structure design.Nucleic Acids Res. 2021; 49: 10604-10617Crossref PubMed Scopus (5) Google Scholar]. Ultimately, LNP structural characteristics, such as the lipid content, size distribution, and surface charge, are of critical importance in determining immunogenicity, biodistribution, cellular uptake, endosomal escape, and circulation time [42.Delehedde C. et al.Intracellular routing and recognition of lipid-based mRNA nanoparticles.Pharmaceutics. 2021; 13: 945Crossref PubMed Scopus (4) Google Scholar]. Although risks are now thoroughly identified, there are still major knowledge gaps in our understanding of product structure, inflammatory pathways, and their links with clinical performance. First, as described in Box 1, a diversity of LNP structural and morphological features, potentially affecting product activity, have been observed, but very few of them have been tested or screened during development. Once better understood, the consistency of the LNP morphology could also be checked during initial process development, scale-up, or technology transfer. Second, elucidating the activation of certain inflammatory pathways, including on- and off-target effects, would be of great help to optimize safety and efficacy profiles [43.Leonardelli L. et al.Literature mining and mechanistic graphical modelling to improve mRNA vaccine platforms.Front. Immunol. 2021; 12738388Crossref PubMed Scopus (1) Google Scholar]. For instance, clinical studies of RNA vaccines inform us that a Th1-type bias response and specific cytokine signatures could be predictors of a potent antibody response [44.Bettini E. Locci M. SARS-CoV-2 mRNA vaccines: immunological mechanism and beyond.Vaccines. 2021; 9: 147Crossref PubMed Scopus (138) Google Scholar,45.Bergamaschi C. et al.Systemic IL-15, IFN-γ, and IP-10/CXCL10 signature associated with effective immune response to SARS-CoV-2 in BNT162b2 mRNA vaccine recipients.Cell Rep. 2021; 36109504Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar]. This will also help us to define appropriate endpoints and design relevant models and assays for activity testing, which remain fundamental in current process development and quality control strategy [28.Liu M.A. et al.WHO informal consultation on regulatory considerations for evaluation of the quality, safety and efficacy of RNA-based prophylactic vaccines for infectious diseases, 20–22 April 2021.Emerg. Microbes Infect. 2022; 11: 384-391Crossref PubMed Scopus (0) Google Scholar]. Finally, in line with the scope expansion of the RNA technology, identifying optimal product characteristics for a given route of administration, therapeutic field, or targeted organ will be crucial. While the optimal immunogenic profile and LNP morphology are both likely to be highly variable, targets for other CQAs could also vary [46.Zhang H. et al.Rational design of anti-inflammatory lipid nanoparticles for mRNA delivery.J. Biomed. Mater. Res. A. 2022; 110: 1101-1108Crossref PubMed Scopus (0) Google Scholar]. Among others, while neutral particle surface is desired in current RNA vaccine, surface charge appears to be a crucial parameter in organ targeting and in determining lymph node- and mucus-penetrating ability [47.Nakamura T. et al.The effect of size and charge of lipid nanoparticles prepared by microfluidic mixing on their lymph node transitivity and distribution.Mol. 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This is especially important in the case of repeated RNA administration or high-dose regimens, such as in chronic administration or protein replacement therapy [54.Sedic M. et al.Safety evaluation of lipid nanoparticle–formulated modified mRNA in the Sprague-Dawley rat and cynomolgus monkey.Vet. Pathol. 2018; 55: 341-354Crossref PubMed Scopus (107) Google Scholar,55.Trepotec Z. et al.Delivery of mRNA therapeutics for the treatment of hepatic diseases.Mol. Ther. 2019; 27: 794-802Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar]. Taken together, addressing these issues could reshape future product and process development. The next step in the QbD approach is to assess the relationships between identified CQAs and CPPs within all critical unit operations. The large-scale Current Good Manufacturing Practice (cGMP) manufacturing processes and corresponding CPPs are mapped in Figure 2, while relevant methodology and details behind individual CQA–CPP relationships are displayed in Tables S4–S11 in the supplemental information online. Despite the scarcity of large-scale and RNA-specific data, numerous in-process risks can still be identified due to the repurposing of multiple unit operations and our increased mechanistic understanding of RNA and LNPs as biophysical systems [56.Kulkarni J.A. et al.On the formation and morphology of lipid nanoparticles containing ionizable cationic lipids and siRNA.ACS Nano. 2018; 12: 4787-4795Crossref PubMed Scopus (232) Google Scholar, 57.Has C. Pan S. Vesicle formation mechanisms: an overview.J. 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On the nanoparticle side, LNP degradation is limited not just to LNP-related impurities but also to particle aggregation, fusion, RNA leakage, or other structural modifications such as lipid phase transitions [13.Schoenmaker L. et al.mRNA-lipid nanoparticle COVID-19 vaccines: structure and stability.Int. J. Pharm. 2021; 601120586Crossref PubMed Scopus (385) Google Scholar,14.Larson N.R. et al.pH-dependent phase behavior and stability of cationic lipid-mRNA nanoparticles.J. Pharm. Sci. 2022; 111: 690-698Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. First, numerous manufacturing options appear in upstream processing, which can significantly affect the quality of the active substance. The most striking one is the RNA capping strategy: although Pfizer-BioNTech has opted for a cotranscriptional capping using the recently developed CleanCap system, Moderna has adopted an enzymatic capping approach. This last option requires extensive intermediate purification steps, impacting overall recovery and RNA integrity, but yields almost 100% capped RNA, even for hard-to-cap structures [62.Henderson J.M. et al.Cap 1 messenger RNA synthesis with co-transcriptional CleanCap analog by in vitro transcription.Curr. Protoc. 2021; 1e39Crossref Scopus (1) Google Scholar]. Second, in vitro transcription (IVT) can be performed in either batch or fed-batch mode. While nucleotide feeding increases the amount of RNA produced by a DNA template by two- to threefold, many RNA-related attributes are deteriorated by prolonged reaction time [63.Elich, J. et al. ModernaTx, Inc. Fed-batch in vitro transcription process, WO2020185811.Google Scholar]. Besides these options, IVT process condition ranges are currently wide, as protocols have not been optimized for long RNA molecules with therapeutic application. Potential optimization paths focus on increasing RNA quality and, crucially, avoiding the formation of RNA-relate