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Cited by 1,566RNA Interference and Gene DeliveryNanoparticle-Based Drug DeliverySARS-CoV-2 and COVID-19 Research

The DOI matches its Crossref record; 82 of the 85 references it cites were found in a registry or an index; none of the 84 sources its claims cite is checked for support yet.

mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability

Open access
Schoenmaker, Linde, Witzigmann, Dominik, Kulkarni, Jayesh A., Verbeke, Rein, Kersten, Gideon, Jiskoot, Wim, Crommelin, Daan J.A.·2021·International Journal of Pharmaceutics
DOI 10.1016/j.ijpharm.2021.120586PMC PMC8032477
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Abstract

A drawback of the current mRNA-lipid nanoparticle (LNP) COVID-19 vaccines is that they have to be stored at (ultra)low temperatures. Understanding the root cause of the instability of these vaccines may help to rationally improve mRNA-LNP product stability and thereby ease the temperature conditions for storage. In this review we discuss proposed structures of mRNA-LNPs, factors that impact mRNA-LNP stability and strategies to optimize mRNA-LNP product stability. Analysis of mRNA-LNP structures reveals that mRNA, the ionizable cationic lipid and water are present in the LNP core. The neutral helper lipids are mainly positioned in the outer, encapsulating, wall. mRNA hydrolysis is the determining factor for mRNA-LNP instability. It is currently unclear how water in the LNP core interacts with the mRNA and to what extent the degradation prone sites of mRNA are protected through a coat of ionizable cationic lipids. To improve the stability of mRNA-LNP vaccines, optimization of the mRNA nucleotide composition should be prioritized. Secondly, a better understanding of the milieu the mRNA is exposed to in the core of LNPs may help to rationalize adjustments to the LNP structure to preserve mRNA integrity. Moreover, drying techniques, such as lyophilization, are promising options still to be explored.

Overview

AI snapshot

Written from the paper’s full text.

TL;DR

This review synthesizes the structure and storage stability of mRNA-LNP COVID-19 vaccines, concluding that mRNA exposure to water is likely the main driver of instability and that frozen storage remains necessary. It outlines analytical methods and stabilization opportunities such as excipient optimization and lyophilization, while highlighting major gaps in public stability data.

Key findings

  • Current mRNA-LNP COVID-19 vaccines require frozen storage: Moderna’s vaccine is kept at −15 to −25 °C and Pfizer/BioNTech’s at −60 to −90 °C; at 2–8 °C, Pfizer/BioNTech and Moderna vaccines are stable for 5 and 30 days, respectively, while CureVac’s candidate is reported stable for 3 months at refrigerator temperatures and at −60 °C.
  • mRNA instability, rather than LNP instability, likely dictates storage conditions: siRNA-LNP Onpattro has a 3-year shelf life at 2–8 °C, and siRNA-LNPs remained stable at 2 °C for 156 days at pH 7 and at 4 °C for 1.5 years, whereas naked mRNA in aqueous solution can be stored at 4 °C for only a few days with RNase protection.
  • mRNA-LNPs are about 60–100 nm, with examples nCoVsaRNA at 75 nm and ARCoV at 89 nm; encapsulation efficiencies are typically >90%, and the isotropic LNP core may contain 24% water by volume fraction, potentially exposing mRNA to water and hydrolysis.
  • mRNA engineering includes 1-methyl-pseudouridine (m1Ψ) in Moderna and Pfizer/BioNTech vaccines and GC-enrichment in CureVac; conventional mRNA vaccines use 30 µg (Pfizer/BioNTech), 100 µg (Moderna), and 12 µg (CureVac) intramuscular doses, while self-amplifying mRNA constructs are about 9 kb versus 2–4 kb for non-replicating mRNA.

Methods

  • Narrative review; no primary experiments or new data are reported.
  • Draws on published literature, product and regulatory documents, and tables comparing BNT162b2/Comirnaty, mRNA-1273, CVnCoV, Onpattro, and self-amplifying mRNA candidates.
  • Synthesizes proposed mRNA-LNP structural models from cryo-TEM, SAXS/SANS, and NMR, mRNA degradation chemistry, analytical assays, and storage-stability evidence.

Limitations

  • Stated: little public data exist on long-term storage of mRNA-LNP formulations, the extent to which encapsulation affects storage stability is unclear, and very little is known about LNP structure/morphology, chemical stability of LNP components, and colloidal stability of the mRNA-LNP system.
  • Stated: no research report was available in the public domain on the integrity of both mRNA and LNPs in mRNA-LNP formulations, and questions remain about mRNA-LNP structure and mRNA–lipid interactions.
  • Evident: as a review, it relies on indirect evidence from siRNA-LNPs, naked mRNA, and product/regulatory documents rather than controlled head-to-head stability studies; some quality information comes from leaked EMA documents and public summaries rather than full public specifications.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1016/j.ijpharm.2021.120586

Cites

82 of 82 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Verification

Claims the paper makes

0 of 84 cited sources checked · 0 supported · 0 flagged

  • Findingcitation

    The 2′ OH group on the ribose is crucial in mRNA hydrolysis; the transesterification reaction leading to a strand break starts by nucleophilic attack by the 2′OH group on the phosphate ester bond, breaking the P-O5′ ester bond.

    “The 2′ OH group on the ribose plays a crucial role as the transesterification reaction leading to a mRNA strand break starts by a nucleophilic attack by the 2′OH group on the phosphate ester bond leading to a break at the P-O5′ ester bond (Fig. 5).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pogocki and Schöneich, 2000
  • Findingcitation

    The base sequence and secondary structure of mRNA influence the rate of hydrolysis.

    “In two publications on mRNA hydrolysis the authors state that the base sequence and secondary structure of mRNA influence the rate of hydrolysis (Kaukinen et al., 2002, Mikkola et al., 2001).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kaukinen et al., 2002 source
    • cites Mikkola et al., 2001 source
  • Findingcitation

    Hydrolysis of mRNA predominantly occurs via the phosphodiester bonds that form the backbone of the mRNA molecule.

    “Hydrolysis predominantly occurs via the phosphodiester bonds that form the backbone of the mRNA molecule (Fig. 5 ).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pogocki and Schöneich, 2000
  • Findingcitation

    Base-stacking may decrease the cleavage rate of phosphodiester bonds.

    “Specifically, base-stacking may decrease the cleavage rate of phosphodiester bonds.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Kaukinen et al., 2002 source
    • cites Mikkola et al., 2001 source
  • Findingcitation

    Chemical degradation of mRNA in vitro mainly occurs through hydrolysis and oxidation.

    “Chemical degradation of mRNA in vitro mainly occurs through hydrolysis and oxidation.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Houseley and Tollervey, 2009
    • cites Pogocki and Schöneich, 2000
  • It may state two separate claims as one. 27
  • Probably not a substantial claim. 10

  • It may state two separate claims as one.

    Findingcitation

    mRNA hydrolysis requires water and can be catalyzed by nucleases, by the mRNA molecule itself, and by exogenous factors such as Brønsted acids and bases.

    “This process requires water and can be catalyzed by nucleases, but also by the mRNA molecule itself and other exogenous factors like Brønsted acids and bases (Houseley and Tollervey, 2009, Pogocki and Schöneich, 2000).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Houseley and Tollervey, 2009
    • cites Pogocki and Schöneich, 2000

  • It may state two separate claims as one.

    Findingcitation

    Aggregation of LNPs during storage and fusion of LNPs has been reported.

    “Aggregation of LNPs during storage and fusion of LNPs has been reported (Ayat et al., 2019, Ball et al., 2016).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Ayat et al., 2019 source
    • cites Ball et al., 2016 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingcitation

    mRNA of 4000 nt is predicted to have a half-life of 941 days when stored at pH 7.4 and 5 °C.

    “Wayment-Steele et al. predict that mRNA (4000 nt) will have a half-life of 941 days when it is stored at pH 7.4 and 5 °C (Wayment-Steele et al., 2020).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wayment-Steele et al., 2020

  • It may state two separate claims as one.

    Findingcitation

    Polysarcosine-modified lipids stabilized lipid-based systems against aggregation while reducing the immunostimulatory response.

    “Introduction of polysarcosine-modified lipids stabilized lipid-based systems against aggregation while reducing the immunostimulatory response (Nogueira et al., 2020).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Nogueira et al., 2020 source

  • It may state two separate claims as one.

    Findingreasoning

    pH influences the hydrolysis rate of mRNA and also LNP stability.

    “pH optimization is also important for mRNA vaccine stability, as the pH influences the hydrolysis rate of mRNA and also LNP stability.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: mRNA is most stable in a weakly basic environment.

  • It may state two separate claims as one.

    Findingnone given

    Naked mRNA is hardly taken up by cells, degrades rapidly, and is therefore not available for translation.

    “mRNA that is not encapsulated (‘naked mRNA’) is hardly taken up by cells; besides, it degrades rapidly and is, therefore, not available for translation.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Findingcitation

    Oxidation can lead to cleavage of bases, strand break, and alteration of the secondary structure of mRNA.

    “Oxidation can lead to the cleavage of bases, strand break and the alteration of the secondary structure of the mRNA (Pogocki and Schöneich, 2000).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pogocki and Schöneich, 2000

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Findingcitation

    Longer mRNA sequences, such as SAMs, are more prone to hydrolysis.

    “They do note that longer mRNA sequences, such as SAMs, are more prone to hydrolysis.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Wayment-Steele et al., 2020

  • It may state two separate claims as one.

    Findingcitation

    Lipid oxidation can occur in unsaturated fatty acid moieties, which are not present in Comirnaty and mRNA-1273, and with cholesterol, potentially as a result of a hydroperoxide attack or an impurity present in the PEG-group of PEG2000-C-DMG.

    “Lipid oxidation can occur in unsaturated fatty acid moieties (not present in Comirnaty and mRNA-1273) and with cholesterol, potentially as a result of a hydroperoxide attack, an impurity present in the PEG-group of PEG2000-C-DMG (Jaeger et al., 1994, Wang et al., 2019).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Jaeger et al., 1994 source
    • cites Wang et al., 2019 source

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Findingnone given

    Denaturation comprises processes such as aggregation and precipitation, which negatively affect mRNA translation.

    “However, denaturation also comprises processes such as aggregation and precipitation, which negatively affect mRNA translation.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingnone given

    Encapsulation efficiencies of mRNA-LNPs are typically greater than 90%, and release of the RNA payload from LNPs during storage has not been reported as measured with the RiboGreen test.

    “Of note, encapsulation efficiencies are typically > 90% and release of the RNA payload from LNPs during storage has not been reported (as measured with the RiboGreen test).”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Findingcitation

    After lyophilization and reconstitution of mRNA lipid-like nanoparticles, the in vitro test showed no loss of activity, but the in vivo efficiency was lost.

    “Contrary to the data on siRNA-LNPs obtained by Shirane et al., they found that after lyophilization and reconstitution the in vitro test showed no loss of activity, but the in vivo efficiency was lost.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Zhao et al., 2020

  • It may state two separate claims as one.

    Premisecitation

    The ionizable cationic lipid contains positively charged ionizable amine groups (at low pH) to interact with the anionic mRNA during particle formation and facilitate membrane fusion during internalization.

    “The latter contains positively charged ionizable amine groups (at low pH) to interact with the anionic mRNA during particle formation and also facilitate membrane fusion during internalization (Evers et al., 2018, Reichmuth et al., 2016).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Evers et al., 2018 source
    • cites Reichmuth et al., 2016 source

  • It may state two separate claims as one.

    Premisecitation

    mRNA is hydrophilic; it can interact electrostatically and through hydrogen bonds with ionizable cationic lipids (apparent pKa < 6.5).

    “mRNA is hydrophilic; it can interact electrostatically and through hydrogen bonds with ionizable cationic lipids (apparent pKa < 6.5) (Buschmann et al., 2021).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Buschmann et al., 2021 source

  • It may state two separate claims as one.

    Premisecitation

    In mRNA-LNP formulations, the fraction of accessible mRNA is very low and encapsulation efficiencies are typically > 90%.

    “In mRNA-LNP formulations, such as those used in mRNA vaccines, the fraction of accessible mRNA is very low (Yanez Arteta et al., 2018, Patel et al., 2020) and thus, encapsulation efficiencies, derived from RiboGreen assays, are typically > 90%.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Yanez Arteta et al., 2018 source
    • cites Patel et al., 2020 source

  • It may state two separate claims as one.

    Premisecitation

    Inside the core, the main part of the ionizable cationic lipid, cholesterol (dependent on its concentration), water and mRNA are present.

    “Inside the core, the main part of the ionizable cationic lipid, cholesterol (dependent on its concentration), water and mRNA are present.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Yanez Arteta et al., 2018 source

  • It may state two separate claims as one.

    Interpretationreasoning

    The multilamellar vesicle model for mRNA-LNPs is unlikely to be correct because it does not correspond to the electron dense cores found by TEM.

    “The multilamellar vesicle model for mRNA-LNPs (Fig. 3A) is unlikely to be correct. It does not correspond to the electron dense cores found by TEM of the mRNA-LNPs.”
    Quote in the paperSubstantial claim

  • It may state two separate claims as one.

    Premisecitation

    There are indications that mRNA is located in the core of the LNPs and siRNA more towards the surface and mRNA can form 'blebs'.

    “Besides, there are indications that mRNA is located in the core of the LNPs and siRNA more towards the surface and mRNA can form ‘blebs’ (Fig. 2) (Viger-Gravel et al., 2018).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Viger-Gravel et al., 2018 source

  • It may state two separate claims as one.

    Interpretationcitation

    The mRNA is -at least partly- exposed to water inside the LNPs, which likely contributes to its instability upon storage under non-frozen conditions.

    “This would mean that the mRNA is –at least partly– exposed to water inside the LNPs, which likely contributes to its instability upon storage under non-frozen conditions (Arteta et al., 2018).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Arteta et al., 2018 source

  • It may state two separate claims as one.

    Premisereasoning

    mRNA is at least 100-fold larger than siRNA and this affects the structure of the LNPs.

    “mRNA is at least 100-fold larger than siRNA and this affects the structure of the LNPs.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Table 2

  • It may state two separate claims as one.

    Premisecitation

    A key aspect of LNPs is the presence of lipids in the core, although water is also present to some extent.

    “A key aspect of LNPs and the characteristic that makes them different from liposomes (spherical vesicles with at least one lipid bilayer and an aqueous core) is the presence of lipids in the core, although data from several studies indicate that water is also present to some extent (Yanez Arteta et al., 2018, Kulkarni et al., 2018, Brader et al., 2021).”
    Quote in the paperSubstantial claim0 of 3 sources checked
    • cites Yanez Arteta et al., 2018 source
    • cites Kulkarni et al., 2018 source
    • cites Brader et al., 2021 source

  • It may state two separate claims as one.

    Premisecitation

    Analysis of the components of the various mRNA-COVID-19 vaccines has shown that there are common features, but also significant differences.

    “Analysis of the components of the various mRNA-COVID-19 vaccines has shown that there are common features, but also significant differences (Table 1).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites Table 1

  • It may state two separate claims as one.

    Premisecitation

    For siRNA only weak electrostatic interactions were found with the ionizable cationic lipid, indicating that at least for siRNA-LNP formulations the pH inside approaches or equals the pH outside.

    “For siRNA only weak electrostatic interactions were found with the ionizable cationic lipid, indicating that at least for siRNA-LNP formulations the pH inside approaches or equals the pH outside (Viger-Gravel et al., 2018).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Viger-Gravel et al., 2018 source

  • Probably not a substantial claim. It may be too vague to check against evidence. The paper’s conclusions may not depend on it.

    Interpretationreasoning

    This could have implications for the stability of different formulations.

    “This, in turn, could have implications for the stability of different formulations.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    The shell of Arteta et al.'s mRNA-LNPs is a monolayer.

    “Another interesting finding by Arteta et al. is that the shell of their mRNA-LNPs is a monolayer (Yanez Arteta et al., 2018, Verbeke et al., 2019).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Yanez Arteta et al., 2018 source
    • cites Verbeke et al., 2019 source

  • It may state two separate claims as one.

    Interpretationreasoning

    The LNP formulation, the use of modified-nucleosides, the GC content and the differences in length between the conventional mRNA and SAM vaccines might influence the physical and chemical stability of these mRNA-COVID-19 vaccines during storage.

    “The LNP formulation, the use of modified-nucleosides, the GC content and the differences in length between the conventional mRNA and SAM vaccines might influence the physical and chemical stability of these mRNA-COVID-19 vaccines during storage.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The composition of mRNA-LNP vaccines is fundamental to their stability.

  • It may state two separate claims as one.

    Interpretationreasoning

    Assessment of the nature of the mRNA-LNP shell with these techniques is difficult and/or multiple types of mRNA-LNP structures may exist, dependent on the nature of the lipids and the preparation protocol.

    “These findings indicate that assessment of the nature of the mRNA-LNP shell with these techniques is difficult and/or that multiple types of mRNA-LNP structures may exist, dependent on, e.g., the nature of the lipids and the preparation protocol for the mRNA-LNPs.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. It may be too vague to check against evidence.

    Interpretationreasoning

    For mRNA vaccines, it is critical to monitor the integrity of the full molecule.

    “Consequently, for mRNA vaccines, it is critical to monitor the integrity of the full molecule.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim. The paper’s conclusions may not depend on it.

    Premisecitation

    Other researchers propose that the outer shell of mRNA-LNPs consists of one or multiple bilayers.

    “Other researchers propose that the outer shell of mRNA-LNPs consists of one or multiple bilayers, based on cryo-TEM analysis (Fig. 2) (Patel et al., 2020, Tanaka et al., 2020) or SANS (Sebastiani et al., 2021).”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites Patel et al., 2020 source
    • cites Tanaka et al., 2020 source
    • cites Sebastiani et al., 2021 source

  • It may state two separate claims as one.

    Interpretationcitation

    If the LNP shells are permeable to protons, the pH would be similar to the rest of the formulation, around 7 to 8, meaning that most of the ionizable cationic lipids would be uncharged.

    “If the LNP shells are permeable to protons –which is likely, as ionized dyes such as 2-(p-toluidino)-6-napthalene sulfonic acid (TNS) and thionine can enter the LNP core (Jayaraman et al., 2012, Brader et al., 2021) – the pH would be similar to the rest of the formulation, around 7 to 8, meaning that most of the ionizable cationic lipids would be uncharged.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites Jayaraman et al., 2012 source
    • cites Brader et al., 2021 source

  • It may state two separate claims as one.

    Interpretationreasoning

    mRNA inside LNPs is in an aqueous environment and therefore is susceptible to hydrolysis.

    “This would mean that the mRNA is in an aqueous environment and therefore is susceptible to hydrolysis.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim. It may be too vague to check against evidence.

    Interpretationreasoning

    Lyophilization would be a logical step to improve the long-term stability of mRNA-LNP formulations.

    “As the presence of water initiates degradation reactions in mRNA-LNPs, lyophilization would be a logical step to improve the long-term stability of mRNA-LNP formulations.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The presence of water initiates degradation reactions in mRNA-LNPs.

  • Probably not a substantial claim. The paper’s conclusions may not depend on it. It may state two separate claims as one.

    Premisecitation

    Chemical degradation plays a larger role in the degradation of small nucleic acids than physical instability, and this is probably even more true for large structures such as mRNA.

    “In a review on the stability of nucleic acids Pogocki and Schöneich state that chemical degradation plays a larger role in the degradation of small nucleic acids than physical instability and that is probably even more true for large structures such as mRNA (Pogocki and Schöneich, 2000).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites Pogocki and Schöneich, 2000

  • Probably not a substantial claim. It may be too vague to check against evidence.

    Interpretationreasoning

    Decreasing the exposure to water would be a promising approach for improving mRNA vaccine stability.

    “An implication of this is that decreasing the exposure to water would be a promising approach for improving mRNA vaccine stability.”
    Quote in the paperNot a substantial claim

  • It may state two separate claims as one.

    Interpretationreasoning

    Studies with either mRNA or LNPs suggest that lyophilization could increase the stability of the mRNA-LNP combination and allow storage at higher temperatures than currently required.

    “Thus, the studies with either mRNA or with LNPs suggest that lyophilization could be a possible way to increase the stability of the combination, mRNA-LNP, and could thereby allow for storage at higher temperatures than those currently required.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The presence of water initiates degradation reactions in mRNA-LNPs.
35 of 35

  • The paper relies on this without support.

    Currently, hardly any data is available in the public domain on what happens when mRNA-LNP formulations are stored for long periods of time.

    “Currently, hardly any data is available in the public domain on what happens when mRNA-LNP formulations are stored for long periods of time.”

  • The paper relies on this without support.

    It is unclear to what extent entrapping mRNA within LNPs influences the storage stability of the mRNA vaccine.

    “Moreover, it is unclear to what extent entrapping mRNA within LNPs influences the storage stability of the mRNA vaccine.”

  • The paper relies on this without support.

    Very little is known about the structure and morphology of LNPs formulated with mRNA, the chemical stability of the LNP components and the colloidal stability of the mRNA-LNP system.

    “Additionally, very little is known about the structure and morphology of LNPs formulated with mRNA, the chemical stability of the LNP components and the colloidal stability of the mRNA-LNP system.”

  • The paper relies on this without support.

    The degradation processes and the reasons why storage temperature requirements differ are not fully understood.

    “To date, the degradation processes and the reasons why storage temperature requirements differ, are not fully understood.”

  • The paper relies on this without support.

    The composition of mRNA-LNP vaccines is fundamental to their stability.

    “The composition of mRNA-LNP vaccines is fundamental to their stability.”

  • The paper relies on this without support.

    PEG-lipid is used to control the particle size and act as a steric barrier to prevent aggregation during storage.

    “In addition, PEG-lipid is used to control the particle size and act as a steric barrier to prevent aggregation during storage.”

  • The paper relies on this without support.

    One factor that strongly influences the required storage conditions is the stability of the mRNA.

    “One factor that strongly influences the required storage conditions is the stability of the mRNA.”

  • The paper relies on this without support.

    The integrity and purity of the mRNA are essential to safeguard efficacy and safety of mRNA vaccines.

    “As the integrity and purity of the mRNA are essential to safeguard efficacy and safety of mRNA vaccines, it is important to have tools to monitor these aspects.”

  • The paper relies on this without support.

    No information on LNP stability tests is available for the present mRNA COVID-19 vaccines.

    “No information on LNP stability tests could be found for the present mRNA COVID-19 vaccines, but the siRNA-LNP formulation of Onpattro (patisaran) has a three-year shelf life when kept between 2° and 8 °C (EMA, 2018).”

  • The paper relies on this without support.

    There is no research report available in the public domain on the integrity of both mRNA and LNPs in mRNA-LNP formulations.

    “To date, there is no research report available in the public domain on the integrity of both mRNA and LNPs in mRNA-LNP formulations.”

  • The paper relies on this without support.

    The presence of water initiates degradation reactions in mRNA-LNPs.

    “As the presence of water initiates degradation reactions in mRNA-LNPs, lyophilization would be a logical step to improve the long-term stability of mRNA-LNP formulations.”

  • The paper relies on this without support.

    mRNA is most stable in a weakly basic environment.

    “Generally, mRNA is most stable in a weakly basic environment.”

  • The paper relies on this without support.

    Quality specifications for the presently accepted mRNA-LNP COVID-19 products are still not available in the public domain.

    “Quality specifications for the presently accepted mRNA-LNP COVID-19 products are still not available in the public domain.”

  • The paper relies on this without support.

    Lyophilization of mRNA-LNP faces hurdles including high energy consumption and costs related to expanding lyophilization capacity.

    “Apart from challenges to preserve the integrity of mRNA-LNP through lyophilization, there are other hurdles such as the high energy consumption of the process and other costs, e.g., those related to the necessary, dramatic expansion of the lyophilization capacity worldwide in times of a pandemic.”
14 of 14

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Conflicting Pfizer/BioNTech storage-temperature ranges

    Minor

    One passage gives the required Pfizer/BioNTech storage range as −60 to −90 °C, while another gives it as −60 to −80 °C. The ranges disagree about whether temperatures below −80 °C are permitted.

1 of 1

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Metadata

Authors
Schoenmaker, L., Witzigmann, D., Kulkarni, J. A., Verbeke, R., Kersten, G., Jiskoot, W., & Crommelin, D. J. A.
Affiliations
  • Leiden University (NL)
  • University of British Columbia (CA)
  • Ghent University (BE)
  • Utrecht University (NL)
Corresponding author
  • Jiskoot, Wim
  • Crommelin, Daan J.A.
ORCID
Venue
International Journal of Pharmaceutics
Publisher
Elsevier BV
Published
2021-05
Volume
601
Pages
120586
ISSN
0378-5173 (ISSN-L)
DOI
10.1016/j.ijpharm.2021.120586
PMC
PMC8032477
OpenAlex
W3148255637
Cited by
1,566
References
85
Citations by year
2026: 267 · 2025: 351 · 2024: 317 · 2023: 291 · 2022: 259 · 2021: 81
FWCI
24.86
Type
journal article
Language
en
Open access
Licence
Locations
  • International Journal of Pharmaceutics (publishedVersion, cc-by)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
  • Leiden Repository (Leiden University) (submittedVersion, cc-by)
  • Ghent University Academic Bibliography (Ghent University) (submittedVersion, cc-by)
  • Utrecht University Repository (Utrecht University) (submittedVersion, other-oa)
  • Data Archiving and Networked Services (DANS) (submittedVersion, cc-by)
Funders
Universiteit Leiden
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
Keywords
COVID-19 vaccines, vaccine stability, ionizable cationic lipids, helper lipids, mRNA degradation, freeze-drying
MeSH terms
COVID-19, SARS-CoV-2, COVID-19 Vaccines, Humans, Lipids, RNA, Messenger, RNA, Small Interfering, Nanoparticles

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

A language model drew the claims from the paper on 2026-10-10, under version 3 of the claim definition (model deepseek-v4.1-flash, prompt extract-v3).

A second model labelled each claim on 2026-10-10 (model jev-1.13-20260917, definition revision gold-guideline-v0.2).

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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