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Separation of Plasmid DNA Topological Forms, Messenger RNA, and Lipid Nanoparticle Aggregates Using an Ultrawide Pore Size Exclusion Chromatography Column
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  • Alexandre Goyon*
    Alexandre Goyon
    Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
    *Email: [email protected]
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    Orcidhttps://orcid.org/0000-0002-7562-0074
  • Shijia Tang
    Shijia Tang
    Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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    Orcidhttps://orcid.org/0000-0002-5303-4293
  • Szabolcs Fekete
    Szabolcs Fekete
    Consumables and Lab Automation, Waters Corporation, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland
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    Orcidhttps://orcid.org/0000-0002-7357-0691
  • Daniel Nguyen
    Daniel Nguyen
    Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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  • Kate Hofmann
    Kate Hofmann
    Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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  • Shirley Wang
    Shirley Wang
    Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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    Orcidhttps://orcid.org/0000-0003-2898-3347
  • Whitney Shatz-Binder
    Whitney Shatz-Binder
    Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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    Orcidhttps://orcid.org/0000-0003-2996-501X
  • Kiel Izabelle Fernandez
    Kiel Izabelle Fernandez
    Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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  • Elizabeth S. Hecht
    Elizabeth S. Hecht
    Microchemistry, Proteomics, and Lipidomics, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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  • Matthew Lauber
    Matthew Lauber
    Consumables and Lab Automation, Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States
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  • Kelly Zhang
    Kelly Zhang
    Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
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Analytical Chemistry

Cite this: Anal. Chem. 2023, 95, 40, 15017–15024
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https://doi.org/10.1021/acs.analchem.3c02944
Published September 25, 2023

Copyright © 2023 The Authors. Published by American Chemical Society. This publication is licensed under

CC-BY 4.0 .

Abstract

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Health authorities have highlighted the need to determine oligonucleotide aggregates. However, existing technologies have limitations that have prevented the reliable analysis of size variants for large nucleic acids and lipid nanoparticles (LNPs). In this work, nucleic acid and LNP aggregation was examined using prototype, low adsorption ultrawide pore size exclusion chromatography (SEC) columns. A preliminary study was conducted to determine the column’s physicochemical properties. A large difference in aggregate content (17.8 vs 59.7 %) was found for a model messenger RNA (mRNA) produced by different manufacturers. We further investigated the nature of the aggregates via a heat treatment. Interestingly, thermal stress irreversibly decreased the amount of aggregates from 59.7 to 4.1% and increased the main peak area 3.3-fold. To the best of our knowledge, for the first time, plasmid DNA topological forms and multimers were separated by analytical SEC. The degradation trends were compared to the data obtained with an anion exchange chromatography method. Finally, unconjugated and fragment antigen-binding (Fab)-guided LNPs were analyzed and their elution times were plotted against their sizes as measured by DLS. Multi-angle light scattering (MALS) was coupled to SEC in order to gain further insights on large species eluting before the LNPs, which were later identified as self-associating LNPs. This study demonstrated the utility of ultrawide pore SEC columns in characterizing the size variants of large nucleic acid therapeutics and LNPs.

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Note Added after ASAP Publication

This paper was originally published ASAP on September 25, 2023. In the title, “Topology” was changed to “Topological”, and the corrected version reposted on September 25, 2023.

Introduction

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Plasmid DNA (pDNA) plays an essential role in cell and gene therapy and can be delivered via different methods, including electroporation for ex vivo therapy or lipid nanoparticles for in vivo delivery. (1,2) Topological forms such as the open-circular impurities or multimers need to be characterized as they may affect the transfection efficiency. (3) Their separation is typically performed by capillary gel electrophoresis (CGE) and anion exchange chromatography (AEC). (4) Limitations in the AEC analysis of pDNA include adsorption issues more pronounced for the open-circular forms and limited resolution between the various topological forms. (5) Conversely, CGE suffers from poor reproducibility and challenges associated with the identification of unknown impurity peaks.
Messenger RNAs (mRNAs) as pharmaceutical drugs have garnered significant interest since their successful use in lipid nanoparticle (LNP) vaccines to keep the coronavirus disease 2019 (COVID-19) under control and reduce the risks of life-threatening events. (6) The accelerated approval of these vaccines by health authorities (HAs) have curbed the pandemic emergency. However, questions remain about the nature of LNP and mRNA impurities and their potential effects on efficacy and safety. Specifically, mRNA aggregates are not listed in the COVID-19 vaccine specifications and the justification of specifications is not readily available to the public. (7,8) Both the mRNA drug substance (DS) and LNP drug product (DP) may self-associate to form aggregates. The large sizes of mRNA and LNP and extreme differences in their respective physicochemical properties are major obstacles to their analytical characterization.
The determination of LNP size and polydispersity is conventionally performed by dynamic light scattering (DLS). (7,8) However, limitations exist with DLS, including the inability to differentiate bimodal size populations. (9) LNP structure and morphology are commonly visualized by imaging or scattering techniques such as cryo-transmission electron microscopy (cryo-TEM), scanning electron microscopy (SEM), and small-angle X-ray scattering (SAXS). (10) However, these techniques are not quantitative by nature, have limited throughput, or suffer from measurement challenges. (11) There is an unmet analytical need for methods that look specifically at LNP-mRNA aggregation assemblies and potentially even the encapsulation of aggregated mRNA as cargo. It is impossible to know the impact of such alternative forms on the DP efficacy and safety without an analytical method capable of distinguishing ultrahigh molecular weight forms. It is also important that the analytical method preserves the alternative forms during analysis.
The public assessment reports by the European Medicines Agency (EMA) list the specifications of the Comirnaty (Pfizer-BioNTech) and Spikevax (Moderna) vaccines. (7,8) Metrics include the DP size determination by DLS and purity/integrity determination by reversed-phase liquid chromatography (RP-LC) or CGE. The purity determination method by RP-HPLC typically involves a high column temperature and the use of an ion-pairing agent in combination with an organic solvent. (12) The integrity determination of mRNA by CGE involves the use of high amounts of a denaturing agent, i.e., 4–8 M urea or formamide under nonaqueous conditions. (13,14) In both methods, the harsh denaturing conditions used prevent detection of noncovalent aggregates.
The sizes of mRNA and LNP are usually between 100 and 500 Å (15,16) and between 600 and 1000 Å, (17) respectively. The large size requires the use of SEC columns with ultrawide pores, which pose heightened challenges with regard to column particle stability. The stability of a packed bed strongly depends on the average pore diameter and has to be carefully considered when large pore particles are packed. (18) The Zenix SEC-300 column (300 Å pore size) and SRT SEC-1000 column (1000 Å pore size) are indicated for the determination of mRNA aggregates. (14) However, aggregates of mRNA and LNPs would be unlikely to be chromatographically resolved on these columns, given that they are larger than both of these pore sizes. Some providers recently commercialized SEC columns with 1000 and 2000 Å nominal pore diameters; however, inertness of the packing material and column hardware is also important to limit the loss of nucleic acids on metal surfaces in liquid chromatography. (19) Thus, there is an unmet need to develop new columns capable of separating these large species.
In this work, prototype ultrawide pore size SEC columns were investigated for their utility to separate ultralarge nucleic acids and their size variants. Studies were first performed in order to investigate the column’s physiochemical properties before assessing their metrics with DP applications. A heat treatment of enhanced green fluorescent protein (EGFP) mRNAs obtained from two commercial vendors was performed. The purity differences between the samples, characterized as the quantity and type of aggregates, was determined. Next, the thermal degradation of a 3.2 kilobase pairs (kbps) pDNA was evaluated using SEC and compared to an in-house AEC method. Finally, the SEC column was used to characterize a series of LNP formulations. The elution times of various LNPs were plotted against their sizes as determined by DLS. Coupling multi-angle light scattering (MALS) and differential refractometer (dRI) to SEC provided in-depth sizing and geometry information that enabled us to make insights into the order of the self-assembled species.

Experimental Section

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Chemicals

OmniTrace Ultra grade hydrochloric acid, BioUltra grade potassium chloride, and tris(hydroxymethyl)aminomethane (Tris) base were purchased from MilliporeSigma (St. Louis, Missouri, USA). Water was generated using a Milli-Q water purification system from MilliporeSigma, and the pH of the mobile phase was determined using a SevenExcellence pH meter from Mettler Toledo (Columbus, Ohio, USA).

Samples and Stress Conditions

mRNA and LNP Sample Preparation

Two EGFP mRNAs (980–996 nucleotides (nts)) were purchased from TriLink BioTechnologies (San Diego, Calfornia, USA) and GenScript (Piscataway, New Jersey, USA) at 1 mg/mL in 1 mM sodium citrate buffer (pH 6.4–6.5). The manufacturers are named “vendor A” and “vendor B” throughout the manuscript. The Cre mRNA (1350 nts) was provided by TriLink BioTechnologies at 1 mg/mL in 1 mM sodium citrate buffer (pH 6.4) and further diluted to 0.1 mg/mL with RNase-free water. The mRNAs were modified with 5-methoxyuridine (5moU) residues, 3′ poly(A) tail (100–120 nts) and 5′-cap-1. The pDNA sample was available at 1 mg/mL in water.
LNP samples were produced in-house using a NanoAssemblr Benchtop (Precision NanoSystems, BC, Canada). LNPs 1, 2, and 3 correspond to LNP samples prepared with various polyethylene glycol (PEG)–lipid compositions. Each LNP was either unconjugated (“–A”), or conjugated to Fab 1 (“–B”) or Fab 2 (“–C”).

mRNA and pDNA Stress Conditions

The same mRNA samples as described above were heated to study the nature of their aggregates. Samples were heated to 70 °C for 2 min.
Samples of pDNA in water were each prepared as 150 μL aliquots without dilution. Samples were heated at 50 °C and subsequently transferred to the HPLC sampler for analysis after 1, 3, 7, and 14 days.

Instrumentation

″LC system 1″ was used for the SEC-UV analysis of the mRNAs and mRNA-LNPs. The setup was composed of a biocompatible Vanquish Binary pump module, a Split Sampler HT module, a thermostatic rapid separation (RS) column compartment, and an UltiMate 3000 diode array detector (DAD) with a 2.5 μL cell volume (Thermo Scientific, Waltham, Massachusetts). UV absorption was monitored at 260 and 220 nm (10 Hz data collection rate and 0.5 s response time).
The AEC and SEC analyses of the pDNA samples were performed using a herein described “LC system 2″. A Vanquish quaternary pump F, Split Sampler FT, and column compartment H were coupled to a DAD-FG module equipped with a 2.5 μL flow cell volume and operated at 260 nm (Thermo Fisher Scientific, Waltham, Massachusetts).
″LC system 3″ was used for SEC-MALS-dRI analysis. For separations, an Agilent 1260 series instrument with a quaternary pump, thermostated column compartment, autosampler, and DAD was employed (Agilent Technologies, Santa Clara, California). For detection, the LC-DAD system was further coupled to a DAWN MALS Detector 8 (Wyatt Technology, Santa Barbara, California) and an Optilab dRI Detector (Wyatt Technology, Santa Barbara, California) for molecular weight (MW) and radius of gyration (Rg) analysis, respectively.

Methods

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SEC

Two prototype SEC columns were manufactured by Waters Corporation (Milford, Massachusetts, USA) using 100% silica particles and a two-step silanization procedure involving the use of a hydroxy PEG (OH-PEG), trifunctional silane reagent in the first reaction, and a methoxy PEG (MeO-PEG) monofunctional silane reagent in a second, subsequent reaction step. Pore size and pore volume were measured using an AutoPore V9600 porosimeter machine from MicroMeritics (Norcross, Georgia). The average particle diameter corresponding to a 50% volume distribution was considered. The experiments were conducted at mercury pressures ranging from vacuum pressure to 400 MPa. To avoid the inclusion of the void between particles, pore size and pore volume were calculated when the intrusion pressure was greater than 2 MPa, which corresponds to a pore size around 5000 Å. To ensure accuracy, a reference sample with a known average pore diameter of 1500 Å was also measured. Particle size was measured with a Beckman Coulter's Multisizer 4e instrument (Brea, California). Surface area was measured by nitrogen adsorption using a MicroActive instrument (MicroMeritics, Norcross, Georgia), while percent carbon was measured by thermogravimetric analysis. Coverages for the OH-PEG and MeO-PEG bondings were thereby calculated according to the elemental compositions of the applied silane reagents.
The pDNA was analyzed using the column packed with “particle A” (4.6 mm I.D. × 150, 3.0 μm, with 1360 Å average pore diameter and wide pore size distribution). mRNA and mRNA-LNP analyses were performed using the column packed with “particle B” (4.6 mm I.D. x 300, 3.0 μm, with a 1275 Å average pore diameter and narrow pore size distribution). The mobile phase contained 50 mM Tris and 200 mM potassium chloride (pH adjusted to 7.5 with hydrochloric acid). The column temperature and flow rate were set to 25 °C and 0.1 mL/min. The injection volumes were set to 1.0, 2.5, and 5.0 μL for the pDNA, mRNA, and mRNA-LNP samples, respectively. For MALS-dRI analyses, LC system 3 was used with a 4.6 mm I.D. × 300 mm column packed with “particle B”. Injection volumes were set to 20–30 μL for mRNA-LNP samples, and all other method conditions were identical with those in the SEC-UV experiment.

AEC

A BioPro QF column (4.6 mm I.D. × 100 mm, 5.0 μm, nonporous) was purchased from YMC America (Fort Devens, Massachusetts, USA). Mobile phase A contained 50 mM Tris in water (pH 7.5), while mobile phase B contained 50 mM Tris and 1.0 M potassium chloride in water (pH 7.5). Column temperature and flow rate were set to 20 °C and 0.7 mL/min. The autosampler was operated at 5 °C, and the injection volume was set to 5.0 μL. The mobile phase B composition was increased from 75 to 100% over 20 min and maintained at 100% for 2.5 min, followed by re-equilibration of the column for 7.5 min at 75% mobile phase B.

Software

LC systems 1, 2, and 3 were controlled by Chromeleon 7.2.10 (Thermo Fisher Scientific), Empower 3 Software build 7.50.00 (Waters Corporation), and ChemStation A.01.04 (Agilent Technologies), respectively. Astra 8 Software was used for data analysis in SEC-MALS-dRI. The dRI detector was used for MW determination with the refractive index increment (dn/dc) value of 0.165 mL/g.

Results and Discussion

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Physicochemical Characterization of the Prototype Ultrawide Pore SEC Columns

The average pore diameters were measured as 1360 and 1275 Å for ”particle A” and “particle B”, respectively. The ”particle A” material possesses a broad pore size distribution, while ”particle B” has a narrow pore distribution (Figure 1). Table 1 lists the physicochemical properties of the two types of particles. Both were treated with a two-step PEGylation silane reaction.
Table 1. Physicochemical Properties of the Two Prototype Ultrawide Pore SEC Particles
 ”particle A””particle B”
average pore size (Å)13601275
pore volume (mL/g)0.610.66
average particle diameter (μm)2.492.47
surface area (m2/g)12.416.0
carbon content after OH-PEG bonding (wt %)0.460.56
carbon content after MeO-PEG bonding (wt %)0.651.12
OH-PEG coverage (μmol/m2)1.361.28
MeO-PEG coverage (μmol/m2)0.681.68

Figure 1

Figure 1. Experimentally measured pore diameter distributions of the prototype ultrawide pore SEC materials.

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The two types of particles were then packed into low adsorption column hardware. The surface of the hardware is composed of an ethylene-bridged siloxane polymer that is formed on metal substrates using a vapor deposition process. (19) Besides a reduction in electrostatic interactions versus metal surfaces (i.e., stainless steel hardware), the ethylene-bridged hybrid surface is predicted to exhibit lower hydrophobicity than some alternative polymeric surfaces. Therefore, we expected that such column hardware would be well suited to the analysis of challenging nucleic acids.

Characterization of Multiple Nucleic Acid Products and Attributes

mRNA and pDNA are characterized by their long length, > 1000 nts for mRNA and >3 kbps for pDNA, and hydrophilic properties. However, they differ by the type of impurities commonly observed. mRNAs are single-stranded RNA molecules that can self-associate to form secondary structures such as hairpins and loops and interact with other mRNA molecules resulting in the formation of aggregates. The main impurities reported in the literature thus far are shortmer impurities missing the polyA tail for mRNA, (20) while topological forms and multimers are often observed with pDNA products. (5) Topological forms have the same MW with the desired supercoiled pDNA product but differ in the arrangement of their strands, such as open-circular impurities. The large size of both mRNA and pDNA and their extreme polarity and charge have been an obstacle to their characterization by SEC. Their large size requires ultrawide pore columns, while the presence of numerous phosphate groups requires column materials that limit nonspecific interactions. (19)

Determination of mRNA Aggregates

The presence of aggregates was compared for EGFP mRNA produced by different manufacturers (Figure 2).

Figure 2

Figure 2. SEC-UV profiles obtained for EGFP mRNA supplied by two vendors. The profiles were obtained before heating the samples (blue traces) and after heating (red traces). The mRNAs have the same nominal concentration of 1 mg/mL and similar length (980–996 nts).

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Significant differences were observed between the unstressed samples from two different vendors (blue traces in Figure 2). The material supplied by vendor B had a significantly reduced peak area compared to vendor A for the mRNA peak eluting at 31.26 min. In addition, vendor B’s sample showed a greater amount of aggregates (59.7 vs 17.8%), with species eluting between 17.25 and 30.20 min. A major peak eluted at 37.39 min, corresponding to smaller nucleotide fragments or excipients.
The nature of the aggregates was also investigated by performing a heat treatment (red traces in Figure 2). With the vendor A material, the intact mRNA absolute peak area remained similar upon heating (2% relative difference) while the mRNA peak area increased 3.3-fold for the mRNA supplied by vendor B. Heating vendor A’s mRNA resulted in the formation of an additional peak at 36.80 min (0.8% relative peak area), suggesting the formation of fragments. Identification of these species was not pursued, although these peak fractions might be amenable to analysis by denaturing IPRP or CGE. A significant reduction of aggregates from 59.7 to 4.1% was observed for the mRNA supplied by vendor B after the heat treatment, while a more modest reduction of aggregates was observed with the mRNA supplied by vendor A (17.8 to 15.1%, respectively). These results suggest that the aggregates detected in the vendor B’s mRNA were predominantly noncovalent in nature. Given that the heated samples were prepared 24 h before being analyzed by SEC-UV, we hypothesize that the dissociation of the mRNA aggregates may be irreversible or may reform at a very slow kinetic rate. However, further studies would be needed to confirm the hypothesis.
The differences in mRNA and aggregate content between the two mRNA samples highlight the value of SEC to compare the quality of materials at a batch and vendor-specific levels. SEC facilitated characterization of the nature of the aggregates, which differed in elution time and size. The heat treatment experiment showed the importance of using a nondenaturing method to quantify the amount of aggregates. It is unclear at this time how the heat treatment may affect the mRNA structures and whether it would affect the potency of the final product. The fast sample manipulation could also ensure mRNA content consistency between different batches as well as reduce potential safety risks that may be associated with the aggregates, i.e., immunogenicity and off-target toxicity.

Determination of pDNA Topological Forms and Multimers

The analytical characterization of plasmid impurities can be used to predict the transfection efficiency and ultimately guide the control strategy. Plasmid DNA topological forms and multimers are commonly separated by agarose gel electrophoresis (AGE), CGE, and AEC. The supercoiled form often referred to as covalently closed circular (ccc) form is preferred while other forms are considered unusable forms of pDNA. (4) Sousa et al. demonstrated that the transfection efficiency was improved by using supercoiled pDNA. (21) However, arguments exist against the necessity of having a high supercoiled content depending on plasmid use. (22) The USP 35 <1047> Gene Therapy Products guidance released in 2012 lists AEC for the quantification of the individual topological isoforms. (4) However, adsorption issues have been observed and are a disproportionate problem in particular for the open-circular forms, which were underestimated in several studies. (5,23) The development of ultrawide pore SEC columns would enable the characterization of these undesired nucleic acid forms. Figure 3 shows the SEC and AEC profiles of a pDNA and the changes in its profile after exposure to thermal stress.

Figure 3

Figure 3. SEC and AEC profiles of a pDNA sample stressed at 50 °C for up to 14 days.

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Two major impurity peaks were observed for the unstressed pDNA samples on both the SEC and AEC profiles (black traces in Figure 3). Broader peaks were observed on the SEC profiles due to the nonretentive nature of the size-based separation and the poor diffusion of the large pDNA. SEC and AEC separations achieved some degree of orthogonality. In the SEC profile, the two impurity peaks eluted before the main peak (tE = 21.71 min). In the AEC trace, the main peak eluted at 10.32 min, with one impurity peak eluting after and one eluting before. The relative peak areas of the two impurities were 2.3% (tE 19.44 min), 2.9% (tE 20.12 min) in SEC and 2.5% (tR 9.05 min), 2.7% (tR 10.65) in AEC. The SEC data indicated that both impurities had larger hydrodynamic radii than the supercoiled pDNA, and the AEC data indicated that one impurity had an increased local net charge distribution and one had a decreased local net charge distribution relative to the original structure. Multimers of DNA can bear a greater number of charges, suggesting that this corresponded to the AEC peak at 10.65 min. The degradation trends were evaluated in order to gain further insights into the formation and structures of these impurities.
After 1 day (yellow traces on Figure 3), a significant increase of the first eluting impurity by AEC (tR 9.05 min) and second eluting peak by SEC (tE 20.12 min) was observed. The open-circular impurity forms during thermal stress of pDNA. (23) The formation of the open-circular form at elevated temperature has been attributed to the presence of residual endogenous nucleases. (24) Thus, the second and growing AEC impurity peak at 9.05 min was assigned to the open-circular impurity. This correlated well with the SEC data. The open-circular form on the SEC trace grew to a 52% relative peak area and a 51% relative peak area, as determined by AEC. The open-circular impurity was the most abundant species observed on the SEC profiles after 3 days (orange traces on Figure 3).
A significant decrease in the amount of DNA species was observed after 7 days (red traces in Figure 3), which could be due to the formation and precipitation of insoluble species. A large amount of impurities were also observed to elute earlier than the open-circular species on the SEC profiles, which could suggest the formation of multimers. The formation of a secondary degradation of impurities was observed after 14 days (dark red traces on Figure 3) with the presence of a late eluting peak on the SEC profile (tE > 21 min) and early eluting peak on the AEC profile (tR < 10 min). The broad peak observed on both AEC and SEC profiles was associated with heterogeneous fragment size populations. The production of fragments after the appearance of denaturation products would be expected later in the thermal stress period. Despite the possible formation of heterogeneous impurities, the AEC and SEC profiles of the control sample (black traces in Figure 4) bracketing the stressed sample injections remained the same. This demonstrated the suitability of the methods and instrumentation.

Figure 4

Figure 4. SEC-UV profiles of mRNA-LNP samples and the corresponding Cre mRNA standard. The LNP sizes determined by DLS were plotted against the SEC elution time of the main peak. LNPs 1, 2, and 3 correspond to LNPs prepared with various PEG-lipid compositions. Each LNP was either unconjugated (“-A”), or conjugated to Fab 1 (“-B”) or Fab 2 (“-C”).

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Sample carryover was evaluated for the open-circular form, supercoiled form, and dimeric impurity. Using AEC, the relative peak areas of the open-circular form, supercoiled form, and dimeric impurity in a blank sample were 3.9, 0.2, and 4.3% to those integrated for the corresponding peaks of a pDNA standard injected before. With SEC, the relative peak areas were reduced to 1.2, 0.5, and <0.1% for the dimeric impurity, open-circular form, and supercoiled form, respectively. The low carryover achieved by SEC could help in improving the accuracy of open-circular impurity measurements.
Overall, the SEC and AEC traces provided orthogonal information that enabled more certain identification of the impurities present in the pDNA samples. The employed prototype ultrawide pore SEC columns showed powerful resolution of multimeric species that would likely not be achievable on columns packed with smaller pore size particles.

Determination of Multiple Attributes for mRNA-LNPs

Multiple attribute methods that facilitate simultaneous measurements can streamline analytical characterization. The determination of free mRNA DS present in the final mRNA-LNP DP informs the efficiency of the encapsulation process and stability of the DP. As discussed in the introduction, the large size of both the mRNA and LNP is an obstacle to their separation using SEC columns with a pore size lower than 300 Å. Figure 4 shows the SEC profiles obtained for a Cre mRNA standard (1350 nts) and various mRNA-LNP samples. The various LNPs represent real-world samples and are more heterogeneous and complex in nature than the monodisperse markers usually tested to characterize SEC materials.
The Cre mRNA standard eluted at 30.08 min (black traces on Figure 4) while the main peak of the various LNPs eluted between 24.39 and 25.43 min (blue, orange, and green traces on Figure 4), demonstrating the ability of the SEC columns to resolve the free mRNA from the intact LNPs. Interestingly, various elution times were observed for the LNP 1, 2, and 3 batch traces. The largest differences in elution times were observed between LNPs with or without conjugation to Fab ligands (LNPs B–C), which eluted earlier than the unconjugated LNPs (LNPs A).
We then further evaluated whether these elution time differences could be correlated to the LNP size, as determined by DLS (Figure 4B). First, the elution times of the unconjugated LNP1-A, LNP2-A, and LNP3-A correlated with their size determined by DLS (R2 > 0.99), demonstrating the effect of changing the PEG-lipid on the LNP size. In addition, for the LNP2 and LNP3 series, SEC elution times were found to be correlated with the size measured by DLS (R2 > 0.96). However, there was no correlation with LNP 1 series. This may be related to the difference in PEG-lipids used to synthesize LNPs 1, 2, and 3. PEGylation of lipids helps to prevent or limit the aggregation of LNPs by shielding their hydrophobic surface. (25) It was particularly interesting to see that the size determined by DLS for LNP 1-B and LNP 1-C was greater than any other LNP, and a multimodal size distribution was observed for the LNP 1-B in DLS. It might be that sample heterogeneity affected the DLS measurements, (9) thus causing the poor correlation for LNP 1 series in Figure 4.
We also observed a minor prepeak in the SEC chromatograms of all LNPs after surface Fab conjugation (LNPs B–C). Combining the MALS detector and a concentration detector, dRI, can measure the light scattered by an analyte in multiple angles and determine their MW and shape. Therefore, SEC-MALS-dRI investigations were performed in order to gain additional insights from the MW and the size analysis of the LNPs (Figure 5). The MW analysis suggested that the prepeak had a ∼1-fold increase in MW compared with the main peak (Figure 5A). In addition, the radius of gyration (Rg), the center of mass, also doubled after conjugation (Figure 5B). Given that the hydrodynamic radii of Fab ligands are normally smaller than 50 Å, (26) coating a layer of Fab ligands onto a ∼1000 Å LNP would be unlikely to cause the size to increase significantly. Therefore, the Rg increase was attributed to particle–particle aggregation. Although the unfunctionalized mRNA-LNPs (LNP As) remained as monomeric particles due to the incorporation of PEGylated lipids, the Fab functionalized mRNA-LNPs (LNP B–Cs) showed a higher tendency to aggregate. The presence of Fab ligands on the particle surface modified the LNP surface charges and increased surface hydrophobicity, thus causing more interparticle association. This study demonstrates the versatility of SEC analysis in combination with alternative detection techniques, providing online MW/sizing analysis and thus impurity (i.e., aggregates) identification for mRNA-LNPs. The SEC method can capture a small amount of particle aggregate that may not be resolved by alternative sizing techniques such as DLS. Detection of particle aggregates in various formulations can support process development to optimize conjugation chemistries and improve particle physical stability.

Figure 5

Figure 5. SEC-MALS-dRI results of mRNA-LNP samples (LNP As) and the corresponding Fab-conjugated mRNA-LNP samples (LNP Bs and Cs). (A) MW and (B) Rg analyses by SEC-MALS-dRI.

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Conclusions

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To our knowledge, this study is the first to characterize mRNA and LNP aggregates and plasmid topological forms and multimers by SEC. Prototype SEC columns packed with sub-5 μm particles and ultrawide pore morphologies were utilized to achieve these results. The physicochemical properties of the SEC packing materials were systematically evaluated. It was found that their average pore size and pore size distribution are well suited to the analysis of mRNA, LNP aggregates, and plasmid topological forms and multimers.
Significant differences in aggregate content (59.7 vs 17.8%) were observed for EGFP mRNA supplied by different manufacturers. Interestingly, most aggregates from vendor B were dissociated or may reform at a very slow kinetic rate upon heat treatment. On the same time scale, the main mRNA peak area increased 3.3-fold. The degradation trends of a pDNA (3.2 kbps) were compared using SEC and AEC. The information provided by orthogonal chromatographic modes enabled the identification of open-circular and multimer impurities that would have remained ambiguous when characterized by a singular method. The comparison of AEC and SEC profiles revealed a multistep degradation process involving the formation of (1) open-circular forms, (2) large multimers and insoluble species, and (3) fragments. Lower sample carryover was observed using SEC in comparison to AEC.
Multiple critical attributes of complex mRNA-LNP samples conjugated to Fab ligands were investigated using the ultrawide pore SEC columns. The mRNA was separated from the intact LNPs. Correlations between SEC elution time and size measured by DLS were established for two LNP series (R2 > 0.96). Further identification of a prepeak eluting before the main peak of LNPs conjugated to Fab ligands was performed using SEC-MALS-dRI, suggesting the presence of particle aggregates.

Author Information

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  • Corresponding Author
  • Authors
    • Shijia Tang - Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States;  Orcidhttps://orcid.org/0000-0002-5303-4293
    • Szabolcs Fekete - Consumables and Lab Automation, Waters Corporation, CMU-Rue Michel Servet 1, Geneva 4 1211, Switzerland;  Orcidhttps://orcid.org/0000-0002-7357-0691
    • Daniel Nguyen - Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
    • Kate Hofmann - Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
    • Shirley Wang - Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States;  Orcidhttps://orcid.org/0000-0003-2898-3347
    • Whitney Shatz-Binder - Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, California 94080, United States;  Orcidhttps://orcid.org/0000-0003-2996-501X
    • Kiel Izabelle Fernandez - Pharmaceutical Development, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
    • Elizabeth S. Hecht - Microchemistry, Proteomics, and Lipidomics, Genentech, 1 DNA Way, South San Francisco, California 94080, United States
    • Matthew Lauber - Consumables and Lab Automation, Waters Corporation, 34 Maple Street, Milford, Massachusetts 01757, United States
    • Kelly Zhang - Synthetic Molecule Analytical Chemistry, Genentech, 1 DNA Way, South San Francisco, California 94080, United States;  Orcidhttps://orcid.org/0000-0001-6515-544X
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors would like to thank Mingcheng Xu, Yuehong Xu, Justin McLaughlin, Steven Byrd, and Yeliz Sarisozen from Waters Corporation for their work to synthesize, characterize, and optimize the packing of prototype widepore SEC particles. We would also like to thank Peggy Ko, Matthew O’Brien Laramy, Chun-Wan Yen, Stefan G. Koenig, and Yareli Maciel Cebrero from Genentech, Inc., for providing the pDNA and mRNA samples.

References

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    It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufg. process. Several challenges encountered in the gene therapy field have emphasized the need for the development of novel platforms that allow the recovery of gene vectors and enable efficient transfection of cells. The use of plasmid DNA-based therapeutics relies on procedures that efficiently purify the supercoiled (s.c.) plasmid isoform. Plasmid DNA (pDNA) purifn. strategies that use amino acids as immobilized ligands have recently yielded interesting results. The present study describes a strategy that uses arginine-chromatog. to specifically purify s.c. pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate. Control anal. shows that the purity of the s.c. pDNA is 100% with a homogeneity higher than 97% of s.c. Furthermore, no RNA was detectable, the protein content was lower than 10 μg/mL and a 117-fold redn. on genomic DNA contamination and 95% endotoxin removal were accomplished. The chromatog. process demonstrated an impressive performance on s.c. isoform recovery (79% yield). Furthermore, the s.c. transfection efficiency of COS-7 cells (62%) was significantly higher compared to the efficiency (25%) achieved with a pDNA control. With the simplified s.c. pDNA purifn. process, a high yield was achieved, s.c. pDNA was purified under mild conditions and shown to be extremely efficient with respect to cell transfection. Arginine-chromatog. is thus an interesting option for use as a late stage plasmid purifn. step.
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    Biotechnology and Bioengineering (1999), 66 (3), 189-194CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
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  • Abstract

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    Figure 1

    Figure 1. Experimentally measured pore diameter distributions of the prototype ultrawide pore SEC materials.

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    Figure 2

    Figure 2. SEC-UV profiles obtained for EGFP mRNA supplied by two vendors. The profiles were obtained before heating the samples (blue traces) and after heating (red traces). The mRNAs have the same nominal concentration of 1 mg/mL and similar length (980–996 nts).

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    Figure 3

    Figure 3. SEC and AEC profiles of a pDNA sample stressed at 50 °C for up to 14 days.

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    Figure 4

    Figure 4. SEC-UV profiles of mRNA-LNP samples and the corresponding Cre mRNA standard. The LNP sizes determined by DLS were plotted against the SEC elution time of the main peak. LNPs 1, 2, and 3 correspond to LNPs prepared with various PEG-lipid compositions. Each LNP was either unconjugated (“-A”), or conjugated to Fab 1 (“-B”) or Fab 2 (“-C”).

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    Figure 5

    Figure 5. SEC-MALS-dRI results of mRNA-LNP samples (LNP As) and the corresponding Fab-conjugated mRNA-LNP samples (LNP Bs and Cs). (A) MW and (B) Rg analyses by SEC-MALS-dRI.

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      Newbury, Dale E.; Ritchie, Nicholas W. M.
      Scanning (2013), 35 (3), 141-168CODEN: SCNNDF; ISSN:0161-0457. (John Wiley & Sons, Inc.)
      A review. SEM/energy dispersive x-ray spectrometry (SEM/EDS) is a widely applied elemental microanal. method capable of identifying and quantifying all elements in the periodic table except H, He, and Li. By following the "k-ratio" (unknown/std.) measurement protocol development for electron-excited wavelength dispersive spectrometry (WDS), SEM/EDS can achieve accuracy and precision equiv. to WDS and at substantially lower electron dose, even when severe x-ray peak overlaps occur, provided sufficient counts are recorded. Achieving this level of performance is now much more practical with the advent of the high-throughput silicon drift detector energy dispersive x-ray spectrometer (SDD-EDS). However, three measurement issues continue to diminish the impact of SEM/EDS: (i) In the qual. anal. (i.e., element identification) that must precede quant. anal., at least some current and many legacy software systems are vulnerable to occasional misidentification of major constituent peaks, with the frequency of misidentifications rising significantly for minor and trace constituents; (ii) The use of standardless anal., which is subject to much broader systematic errors, leads to quant. results that, while useful, do not have sufficient accuracy to solve crit. problems, e.g. detg. the formula of a compd.; (iii) EDS spectrometers have such a large vol. of acceptance that apparently credible spectra can be obtained from specimens with complex topog. that introduce uncontrolled geometric factors that modify x-ray generation and propagation, resulting in very large systematic errors, often a factor of ten or more.
    12. 12
      Currie, J.; Dahlberg, J. R.; Eriksson, J.; Schweikart, F.; Nilsson, G. A.; Örnskov, E. Stability Indicating Ion-Pair Reversed-Phase Liquid Chromatography Method for Modified MRNA. ChemRxiv 2021, 1 10,  DOI: 10.26434/chemrxiv-2021-mgx9q .
      12
      Stability indicating ion-pair reversed-phase liquid chromatography method for modified mRNA
      Currie, Jonathan; Dahlberg, Jacob R.; Eriksson, Jonas; Schweikart, Fritz; Nilsson, Gunilla A.; Oernskov, Eivor
      ChemRxiv (2021), (), 1-10CODEN: CHEMWF; ISSN:2573-2293. (American Chemical Society)
      Modified mRNA (mRNA) represents a promising new class of therapeutic drug product. Development of robust stability indicating methods, for control of product quality, are therefore crit. to support successful pharmaceutical development. This paper presents the first ion pair reversed phase liq. chromatog. (IP-RPLC) method to characterize modified mRNA exposed to a wide set of stress-inducing conditions, relevant for pharmaceutical development of an mRNA drug product. The optimized method could be used for sepn. and anal. of large RNA, sized up to 1000 nucleotides. The impacts of column temp., mobile phase flow rate and ion-pair selection were studied and optimized. Baseline sepns. of the model RNA ladder sample were achieved using all examd. ion-pairing agents. We established that the optimized method, using 100mM Triethylamine, enabled the highest resoln. sepns. for the largest fragments in the RNA ladder (750/1000 nucleotides), in addn. to the highest overall resoln. for the selected modified mRNA compd. (eGFP mRNA, 996 nucleotides). The stability indicating power of the method was demonstrated by analyzing the modified eGFP mRNA, upon direct exposure to heat, hydrolytic conditions and treatment with RNases. Our results showed that the resulting degrdn. products, which appeared as shorter RNA fragments in front of the main peak, could be well visualized, using the optimized method, and the relative stability of the mRNA under the various stressed conditions could be assessed.
    13. 13
      Lu, T.; Klein, L. J.; Ha, S.; Rustandi, R. R. High-Resolution Capillary Electrophoresis Separation of Large RNA under Non-Aqueous Conditions. J. Chromatogr. A 2020, 1618, 460875  DOI: 10.1016/j.chroma.2020.460875
      13
      High-Resolution capillary electrophoresis separation of large RNA under non-aqueous conditions
      Lu, Tian; Klein, Lee J.; Ha, Sha; Rustandi, Richard R.
      Journal of Chromatography A (2020), 1618 (), 460875CODEN: JCRAEY; ISSN:0021-9673. (Elsevier B.V.)
      Large RNAs including mRNAs (mRNAs) are promising candidates for development of new drug products and vaccines. Development of high resoln. methods for direct anal. of large RNAs, esp. for purity in general and size or length in particular, is crit. to support new drug development and manuf. However, resoln. based on size or length for large RNAs is limited even by capillary electrophoresis (CE), which is one of the most efficient sepn. methods for nucleic acids in general. This paper presents a capillary gel electrophoresis (CGE) method for sepg. large RNA mols. by size or length under strongly denaturing, non-aq. conditions. We believe that our work constitutes the first time that a gel suitable for CGE prepd. with high mol. wt. polymers and using only formamide as solvent has been successfully employed to analyze large RNAs on the basis of their size or length with high resoln. With an eye toward application for mRNAs in particular, sepn. conditions in this work were optimized for RNAs approx. 2000 nucleotides (nt) in length. As compared to a std. CGE method using an aq. gel, resoln. for com.-available RNA ladder components at 1500 and 2000 nt is increased approx. 6-fold. The impacts of polymer type, mol. wt. of the polymer, and polymer concn. on the sepn. were studied and optimized. Anal. of the results presented here also provides guidance for optimization of sepn. conditions for RNAs with different sizes as needed for particular applications in the future.
    14. 14
      United States Pharmacopeia. Analytical Procedures for mRNA Vaccine Quality (Draft Guidelines) , 2023, . https://www.uspnf.com/notices/analytical-procedures-mrna-vaccines-20220210.
      There is no corresponding record for this reference.
    15. 15
      Buschmann, M. D.; Carrasco, M. J.; Alishetty, S.; Paige, M.; Alameh, M. G.; Weissman, D. Nanomaterial Delivery Systems for MRNA Vaccines. Vaccines 2021, 9 (1), 65,  DOI: 10.3390/vaccines9010065
      15
      Nanomaterial delivery systems for mRNA vaccines
      Buschmann, Michael D.; Carrasco, Manuel J.; Alishetty, Suman; Paige, Mikell; Alameh, Mohamad Gabriel; Weissman, Drew
      Vaccines (Basel, Switzerland) (2021), 9 (1), 65CODEN: VBSABP; ISSN:2076-393X. (MDPI AG)
      The recent success of mRNA vaccines in SARS-CoV-2 clin. trials is in part due to the development of lipid nanoparticle delivery systems that not only efficiently express the mRNA-encoded immunogen after i.m. injection, but also play roles as adjuvants and in vaccine reactogenicity. We present an overview of mRNA delivery systems and then focus on the lipid nanoparticles used in the current SARS-CoV-2 vaccine clin. trials. The review concludes with an anal. of the determinants of the performance of lipid nanoparticles in mRNA vaccines.
    16. 16
      Zhang, H.; Rombouts, K.; Raes, L.; Xiong, R.; De Smedt, S. C.; Braeckmans, K.; Remaut, K. Fluorescence-Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts. Adv. Biosyst. 2020, 4 (5), 2000057,  DOI: 10.1002/adbi.202000057
      16
      Fluorescence-Based Quantification of Messenger RNA and Plasmid DNA Decay Kinetics in Extracellular Biological Fluids and Cell Extracts
      Zhang, Heyang; Rombouts, Koen; Raes, Laurens; Xiong, Ranhua; De Smedt, Stefaan C.; Braeckmans, Kevin; Remaut, Katrien
      Advanced Biosystems (2020), 4 (5), 2000057CODEN: ABDIHL; ISSN:2366-7478. (Wiley-VCH Verlag GmbH & Co. KGaA)
      Extracellular and intracellular degrdn. of nucleic acids remains an issue in non-viral gene therapy. Understanding biodegrdn. is crit. for the rational design of gene therapeutics in order to maintain stability and functionality at the target site. However, there are only limited methods available that allow detg. the stability of genetic materials in biol. environments. In this context, the decay kinetics of fluorescently labeled plasmid DNA (pDNA) and mRNA (mRNA) in undiluted biol. samples (i.e., human serum, human ascites, bovine vitreous) and cell exts. is studied using fluorescence correlation spectroscopy (FCS) and single particle tracking (SPT). It is demonstrated that FCS is suitable to follow mRNA degrdn., while SPT is better suited to investigate pDNA integrity. The half-life of mRNA and pDNA is ≈1-2 min and 1-4 h in biol. samples, resp. The resistance against biodegrdn. drastically improves by complexation with lipid-based carriers. Taken together, FCS and SPT are able to quantify the integrity of mRNA and pDNA, resp., as a function of time, both in the extracellular biol. fluids and cell exts. This in turn allows to focus on the important but less understood issue of nucleic acids degrdn. in more detail and to rationally optimize gene delivery system as therapeutics.
    17. 17
      Schoenmaker, L.; Witzigmann, D.; Kulkarni, J. A.; Verbeke, R.; Kersten, G.; Jiskoot, W.; Crommelin, D. J. A. MRNA-Lipid Nanoparticle COVID-19 Vaccines: Structure and Stability. Int. J. Pharm. 2021, 601, 120586  DOI: 10.1016/j.ijpharm.2021.120586
      17
      mRNA-lipid nanoparticle COVID-19 vaccines: Structure and stability
      Schoenmaker, Linde; Witzigmann, Dominik; Kulkarni, Jayesh A.; Verbeke, Rein; Kersten, Gideon; Jiskoot, Wim; Crommelin, Daan J. A.
      International Journal of Pharmaceutics (Amsterdam, Netherlands) (2021), 601 (), 120586CODEN: IJPHDE; ISSN:0378-5173. (Elsevier B.V.)
      A review. A drawback of the current mRNA-lipid nanoparticle (LNP) COVID-19 vaccines is that they have to be stored at (ultra)low temps. Understanding the root cause of the instability of these vaccines may help to rationally improve mRNA-LNP product stability and thereby ease the temp. conditions for storage. We discuss proposed structures of mRNA-LNPs, factors that impact mRNA-LNP stability and strategies to optimize mRNA-LNP product stability. Anal. 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. The mRNA hydrolysis is the detg. factor for mRNA-LNP instability. It is currently unclear how water in the LNP core interacts with the mRNA and to what extent the degrdn. 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 compn. 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.
    18. 18
      Unger, K. K. Porous Silica, its properties and use as support in column liquid chromatography. 1st ed.; Elsevier Scientific Pub. Co., 16 1979.
      There is no corresponding record for this reference.
    19. 19
      DeLano, M.; Walter, T. H.; Lauber, M. A.; Gilar, M.; Jung, M. C.; Nguyen, J. M.; Boissel, C.; Patel, A. V.; Bates-Harrison, A.; Wyndham, K. D. Using Hybrid Organic–Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC. Anal. Chem. 2021, 93 (14), 5773– 5781,  DOI: 10.1021/acs.analchem.0c05203
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      Using Hybrid Organic-Inorganic Surface Technology to Mitigate Analyte Interactions with Metal Surfaces in UHPLC
      DeLano, Mathew; Walter, Thomas H.; Lauber, Matthew A.; Gilar, Martin; Jung, Moon Chul; Nguyen, Jennifer M.; Boissel, Cheryl; Patel, Amit V.; Bates-Harrison, Andrew; Wyndham, Kevin D.
      Analytical Chemistry (Washington, DC, United States) (2021), 93 (14), 5773-5781CODEN: ANCHAM; ISSN:0003-2700. (American Chemical Society)
      Interactions of analytes with metal surfaces in high-performance liq. chromatog. (HPLC) instruments and columns have been reported to cause deleterious effects ranging from peak tailing to a complete loss of the analyte signal. These effects are due to the adsorption of certain analytes on the metal oxide layer on the surface of the metal components. We have developed a novel surface modification technol. and applied it to the metal components in ultra-HPLC (UHPLC) instruments and columns to mitigate these interactions. A hybrid org.-inorg. surface, based on an ethylene-bridged siloxane chem., was developed for use with reversed-phase and hydrophilic interaction chromatog. We have characterized the performance of UHPLC instruments and columns that incorporate this surface technol. and compared the results with those obtained using their conventional counterparts. We demonstrate improved performance when using the hybrid surface technol. for sepns. of nucleotides, a phosphopeptide, and an oligonucleotide. The hybrid surface technol. was found to result in higher and more consistent analyte peak areas and improved peak shape, particularly when using low analyte mass loads and acidic mobile phases. Reduced abundances of iron adducts in the mass spectrum of a peptide were also obsd. when using UHPLC systems and columns that incorporate hybrid surface technol. These results suggest that this technol. will be particularly beneficial in UHPLC/mass spectrometry investigations of metal-sensitive analytes.
    20. 20
      Patel, H. K.; Zhang, K.; Utegg, R.; Stephens, E.; Salem, S.; Welch, H.; Grobe, S.; Schlereth, J.; Kuhn, A. N.; Ryczek, J.; Cirelli, D. J.; Lerch, T. F. Characterization of BNT162b2MRNA to Evaluate Risk of Off-Target Antigen Translation. J. Pharm. Sci. 2023, 112 (5), 1364– 1371,  DOI: 10.1016/j.xphs.2023.01.007
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      Characterization of BNT162b2 mRNA to Evaluate Risk of Off-Target Antigen Translation
      Patel, Himakshi K.; Zhang, Kun; Utegg, Rachael; Stephens, Elaine; Salem, Shauna; Welch, Heidi; Grobe, Svenja; Schlereth, Julia; Kuhn, Andreas N.; Ryczek, Jeff; Cirelli, David J.; Lerch, Thomas F.
      Journal of Pharmaceutical Sciences (Philadelphia, PA, United States) (2023), 112 (5), 1364-1371CODEN: JPMSAE; ISSN:0022-3549. (Elsevier Inc.)
      MRNA vaccines have been established as a safe and effective modality, thanks in large part to the expedited development and approval of COVID-19 vaccines. In addn. to the active, full-length mRNA transcript, mRNA fragment species can be present as a byproduct of the cell-free transcription manufg. process or due to mRNA hydrolysis. In the current study, mRNA fragment species from BNT162b2 mRNA were isolated and characterized. The translational viability of intact and fragmented mRNA species was further explored using orthogonal expression systems to understand the risk of truncated spike protein or off-target antigen translation. The study demonstrates that mRNA fragments are primarily derived from premature transcriptional termination during manufg., and only full-length mRNA transcripts are viable for expression of the SARS-CoV-2 spike protein antigen.
    21. 21
      Sousa, F.; Prazeres, D. M. F.; Queiroz, J. A. Improvement of Transfection Efficiency by Using supercoiled Plasmid DNA Purified with Arginine Affinity Chromatography. J. Gene Med. 2009, 11 (1), 79– 88,  DOI: 10.1002/jgm.1272
      21
      Improvement of transfection efficiency by using supercoiled plasmid DNA purified with arginine affinity chromatography
      Sousa, Fani; Prazeres, Duarte M. F.; Queiroz, Joao A.
      Journal of Gene Medicine (2009), 11 (1), 79-88CODEN: JGMEFG; ISSN:1099-498X. (John Wiley & Sons Ltd.)
      It is well known that the success of gene transfer to cells and subsequent expression is strictly affected by the vector manufg. process. Several challenges encountered in the gene therapy field have emphasized the need for the development of novel platforms that allow the recovery of gene vectors and enable efficient transfection of cells. The use of plasmid DNA-based therapeutics relies on procedures that efficiently purify the supercoiled (s.c.) plasmid isoform. Plasmid DNA (pDNA) purifn. strategies that use amino acids as immobilized ligands have recently yielded interesting results. The present study describes a strategy that uses arginine-chromatog. to specifically purify s.c. pDNA from other isoforms and Escherichia coli impurities present in a clarified lysate. Control anal. shows that the purity of the s.c. pDNA is 100% with a homogeneity higher than 97% of s.c. Furthermore, no RNA was detectable, the protein content was lower than 10 μg/mL and a 117-fold redn. on genomic DNA contamination and 95% endotoxin removal were accomplished. The chromatog. process demonstrated an impressive performance on s.c. isoform recovery (79% yield). Furthermore, the s.c. transfection efficiency of COS-7 cells (62%) was significantly higher compared to the efficiency (25%) achieved with a pDNA control. With the simplified s.c. pDNA purifn. process, a high yield was achieved, s.c. pDNA was purified under mild conditions and shown to be extremely efficient with respect to cell transfection. Arginine-chromatog. is thus an interesting option for use as a late stage plasmid purifn. step.
    22. 22
      Prazeres, D. M.; Ferreira, G. N.; Monteiro, G. A.; Cooney, C. L.; Cabral, J. M. Large-Scale Production of Pharmaceutical-Grade Plasmid DNA for Gene Therapy: Problems and Bottlenecks. Trends Biotechnol. 1999, 17 (4), 169– 174,  DOI: 10.1016/S0167-7799(98)01291-8
      22
      Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: problems and bottlenecks
      Prazeres, Duarte M. F.; Ferreira, Guilherme N. M.; Monteiro, Gabriel A.; Cooney, Charles L.; Cabral, Joaquim M. S.
      Trends in Biotechnology (1999), 17 (4), 169-174CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Science Ltd.)
      A review with 30 refs. Gene therapy is a promising process for the prevention, treatment and cure of diseases such as cancer, AIDS, and cystic fibrosis. One of the methods used to administer therapeutic genes is the direct injection of naked or lipid-coated plasmid DNA, but this requires considerable amts. of plasmid DNA. There are several problems and bottlenecks assocd. with the design and operation of large-scale processes for the prodn. of pharmaceutical-grade plasmid DNA for gene therapy.
    23. 23
      Cook, K. S.; Luo, J.; Guttman, A.; Thompson, L. Vaccine Plasmid Topology Monitoring by Capillary Gel Electrophoresis. Curr. Mol. Med. 2021, 20 (10), 798– 805,  DOI: 10.2174/1566524020666200427110452
      There is no corresponding record for this reference.
    24. 24
      Monteiro, G. A.; Ferreira, G. N. M.; Cabral, J. M. S.; Prazeres, D. M. F. Analysis and Use of Endogenous Nuclease Activities in Escherichia Coli Lysates during the Primary Isolation of Plasmids for Gene Therapy. Biotechnol. Bioeng. 1999, 66 (3), 189– 194,  DOI: 10.1002/(SICI)1097-0290(1999)66:3<189::AID-BIT7>3.0.CO;2-Z
      24
      Analysis and use of endogenous nuclease activities in Escherichia coli lysates during the primary isolation of plasmids for gene therapy
      Monteiro, G. A.; Ferreira, G. N. M.; Cabral, J. M. S.; Prazeres, D. M. F.
      Biotechnology and Bioengineering (1999), 66 (3), 189-194CODEN: BIBIAU; ISSN:0006-3592. (John Wiley & Sons, Inc.)
      Two important issues in the downstream processing of plasmids for gene therapy are the stability of plasmids in the process streams, and the presence of contaminating host RNA. Results with a 4.8-kb plasmid harbored in a non-nuclease-deficient strain of Escherichia coli show that, in spite of the harsh conditions during alk. lysis, a fraction of endogenous nucleases remains active, degrading both RNA and genomic and plasmid DNA. Although it is possible to minimize plasmid degrdn. by decreasing temp. and reducing processing times, the presence of endogenous nucleases can be used advantageously to purify the plasmid streams. The kinetics of nucleic acid degrdn. showed that, by controlling the incubation at 37°, it was possible to degrade RNA selectively, while maintaining plasmid integrity. A redn. of 40% in RNA content was obtained, corresponding to a 1.5-fold increase in plasmid purity using high-performance liq. chromatog. (HPLC). This strategy is simple and straightforward, and the increase in processing time and the assocd. plasmid loss (9%) are fully justified by the purity increase. Furthermore, the use of endogenous RNase activity is clearly advantageous over alternative procedures, such as the addn. of external RNase, in terms of cost, validation, and compliance with guidelines from regulatory agencies.
    25. 25
      Suk, J. S.; Xu, Q.; Kim, N.; Hanes, J.; Ensign, L. M. PEGylation as a Strategy for Improving Nanoparticle-Based Drug and Gene Delivery. Adv. Drug Delivery Rev. 2016, 99, 28– 51,  DOI: 10.1016/j.addr.2015.09.012
      25
      PEGylation as a strategy for improving nanoparticle-based drug and gene delivery
      Suk, Jung Soo; Xu, Qingguo; Kim, Namho; Hanes, Justin; Ensign, Laura M.
      Advanced Drug Delivery Reviews (2016), 99 (Part_A), 28-51CODEN: ADDREP; ISSN:0169-409X. (Elsevier B.V.)
      Coating the surface of nanoparticles with polyethylene glycol (PEG), or "PEGylation", is a commonly used approach for improving the efficiency of drug and gene delivery to target cells and tissues. Building from the success of PEGylating proteins to improve systemic circulation time and decrease immunogenicity, the impact of PEG coatings on the fate of systemically administered nanoparticle formulations has, and continues to be, widely studied. PEG coatings on nanoparticles shield the surface from aggregation, opsonization, and phagocytosis, prolonging systemic circulation time. Here, we briefly describe the history of the development of PEGylated nanoparticle formulations for systemic administration, including how factors such as PEG mol. wt., PEG surface d., nanoparticle core properties, and repeated administration impact circulation time. A less frequently discussed topic, we then describe how PEG coatings on nanoparticles have also been utilized for overcoming various biol. barriers to efficient drug and gene delivery assocd. with other modes of administration, ranging from gastrointestinal to ocular. Finally, we describe both methods for PEGylating nanoparticles and methods for characterizing PEG surface d., a key factor in the effectiveness of the PEG surface coating for improving drug and gene delivery.
    26. 26
      Armstrong, J. K.; Wenby, R. B.; Meiselman, H. J.; Fisher, T. C. The Hydrodynamic Radii of Macromolecules and Their Effect on Red Blood Cell Aggregation. Biophys. J. 2004, 87 (6), 4259– 4270,  DOI: 10.1529/biophysj.104.047746
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      The hydrodynamic radii of macromolecules and their effect on red blood cell aggregation
      Armstrong, J. K.; Wenby, R. B.; Meiselman, H. J.; Fisher, T. C.
      Biophysical Journal (2004), 87 (6), 4259-4270CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      The effects of nonionic polymers on human red blood cell (RBC) aggregation were investigated. The hydrodynamic radius (Rh) of individual samples of dextran, polyvinylpyrrolidone, and polyoxyethylene over a range of mol. wts. (1500-2,000,000) were calcd. from their intrinsic viscosities using the Einstein viscosity relation and directly measured by quasi-elastic light scattering, and the effect of each polymer sample on RBC aggregation was studied by nephelometry and low-shear viscometry. For all three polymers, despite their different structures, samples with Rh <4 nm were found to inhibit aggregation, whereas those with Rh >4 nm enhanced aggregation. Inhibition increased with Rh and was maximal at ∼3 nm; above 4 nm, the pro-aggregant effect increased with Rh. For comparison, the Rh of 12 plasma proteins were calcd. from literature values of intrinsic viscosity or diffusion coeff. Each protein known to promote RBC aggregation had Rh >4 nm, whereas those with Rh <4 nm either inhibited or had no effect on aggregation. These results suggest that the influence of a nonionic polymer or plasma protein on RBC aggregation is simply a consequence of its size in an aq. environment and that the specific type of macromol. is of minor importance.