Capillary-Based Physicochemical Characterization of Lipid Nanoparticles.

Characterization of lipid nanoparticles using macro mass photometry: Insights into size and mass.

Theory of the correlation between capillary and free-flow zone electrophoresis and its use for the conversion of analytical capillary separations to continuous free-flow preparative processes: Application to analysis and preparation of fragments of insulin

The number of nucleic acid therapeutics is set to grow within the pharmaceutical industry sector, deploying nanocarrier-based delivery systems as drug products. Poly(A) is a widely used model sequence used in lipid nanoparticle (LNP) formulations for which there are no reported critical quality attributes (CQAs) such as molecular weight, chain length, and impurity profile. In this study, we analyze Poly(A) from three different vendors to measure any existing differences in their CQAs. Poly(A) from these brands was encapsulated in SM102 LNPs using microfluidics to produce three branded Poly(A)-based LNPs. We utilized an orthogonal analytical pipeline approach for both Poly(A) drug substance and LNP drug product CQA evaluation, which included a combination of dynamic light scattering and flow field flow fractionation multiplexed with inline UV, dynamic, and multiangle light-scattering detectors. Similar purity (260/280) values of >3 were obtained across all three brands of Poly(A); however, distinct differences in molecular weight and chain length distributions were identified across Poly(A) brands, with this study representing the first to apply EAF4 methodology for in-depth characterization of model RNA drug substances. Brand A produced a smaller and broader molecular weight distribution, followed by Brand B, and then Brand C produced the largest molecular weight species and the most uniform molecular weight distribution. On encapsulation in LNPs, differences seen in Poly(A) CQAs did not translate to CQA differences in resultant LNPs. We show that a deeper understanding of drug substance CQAs and their subsequent impact on resultant overall drug product characteristics is needed on a case-by-case basis. We show correlations between analytical pipelines, with future work investigating the impact of RNA molecular weight in LNP formulations with different lipid compositions and using these correlations in AI or machine learning to further enhance our knowledge of the correlation between drug substance and resultant drug product CQAs.

Lipid nanoparticles (LNPs), most commonly recognised for their role in COVID-19 mRNA vaccines, are important delivery vehicles for nucleic acid (mRNA, siRNA) therapies. The physicochemical attributes, such as size, nucleic acid encapsulation and electric charge, may have a significant impact on the efficacy of these medicines. In this study, adjustments to aqueous to lipid phase ratios were assessed for their impact on LNP size and other critical quality attributes (CQAs). It was observed that minor adjustments of aqueous-to-organic lipid phase ratios can be used to precisely control the size of ALC-0315-formulated LNPs. This was then used to evaluate the impact of phase ratio and corresponding size ranges on the in vitro and in vivo expression of these LNPs. In HEK293 cells, larger LNPs led to higher expression of the mRNA cargo within the LNPs, with a linear correlation between size and expression. In THP-1 cells this preference for larger LNPs was observed up to 120 d.nm after which there was a fall in expression. In BALB/c mice, however, LNPs at the lowest phase ratio tested, >120 d.nm, showed reduced expression compared to those of range 60–120 d.nm, within which there was no significant difference between sizes. These results suggest a robustness of LNP expression up to 120 d.nm, larger than those <100 d.nm conventionally used in medicine.

Lipid nanoparticles (LNPs) are currently the most advanced non-viral clinically approved messenger ribonucleic acid (mRNA) delivery systems. The ability of a mRNA vaccine to have a therapeutic effect is related to the capacity of LNPs to deliver the nucleic acid intact into cells. The role of LNPs is to protect mRNA, especially from degradation by ribonucleases (RNases) and to allow it to access the cytoplasm of cells where it can be translated into the protein of interest. LNPs enter cells by endocytosis and their size is a critical parameter impacting their cellular internalization. In this work, we studied different formulation process parameters impacting LNPs size. Taylor dispersion analysis (TDA) was used to determine the LNPs size and size distribution and the results were compared with those obtained by Dynamic Light Scattering (DLS). TDA was also used to study both the degradation of mRNA in the presence of RNases and the percentage of mRNA encapsulation within LNPs.

We offer a highly sensitive and reproducible dielectric-spectroscopy assay of deoxyribonucleic acid (DNA) sequence on a platform of quantum graphene-like structures arranged on nanoporous alumina to correctly identifying an infectious agent in a native double-stranded (ds) DNA. The hybridization of complementary target DNA with probe DNA in the sensor sensitive layer leads to penetration of the formed single-stranded (ss) target DNA into the underlayer nanoporous anodic alumina through the nanocavities of LB-film from organometallic complexes. This results in linking of MWCNT ends, shielding of Helmholtz double layer and following decrease of electrical capacitance of the sensor. The novel electrochemical impedimetric DNA sensor with self-organized multi-walled carbon nanotube (MWCNT) bundles decorated by organometallic complexes as transducer has been utilized to detect the viral DNA in the biological samples of patients with virus infection at DNA concentration as low as 1.0–1.3 ng/μL.

Identification of a monoclonal antibody clipping variant by cross-validation using capillary electrophoresis – sodium dodecyl sulfate, capillary zone electrophoresis – mass spectrometry and capillary isoelectric focusing – mass spectrometry

Messenger RNA vaccines have come into the spotlight as a promising and adaptive alternative to conventional vaccine approaches. The efficacy of mRNA vaccines relies on the ability of mRNA to reach the cytoplasm of cells, where it can be translated into proteins of interest, allowing it to trigger the immune response. However, unprotected mRNA is unstable and susceptible to degradation by exo- and endonucleases, and its negative charges are electrostatically repulsed by the anionic cell membranes. Therefore, mRNA needs a delivery system that protects the nucleic acid from degradation and allows it to enter into the cells. Lipid nanoparticles (LNPs) represent a nonviral leading vector for mRNA delivery. Physicochemical parameters of LNPs, including their size and their charge, directly impact their in vivo behavior and, therefore, their cellular internalization. In this work, Taylor dispersion analysis (TDA) was used as a new methodology for the characterization of the size and polydispersity of LNPs, and capillary electrophoresis (CE) was used for the determination of LNP global charge. The results obtained were compared with those obtained by dynamic light scattering (DLS) and laser Doppler electrophoresis (LDE).

The in vitro product of mouse leukemia virus deoxyribonucleic acid (DNA) polymerase can be separated into two fractions by sedimentation in sucrose gradients. These two fractions were analyzed for their content of single-stranded DNA, double-stranded DNA, and DNA-ribonucleic acid (RNA) hybrid by (i) digestion with enzymes of known specificity and (ii) equilibrium centrifugation in Cs(2)SO(4) gradients. The major fraction early in the reaction contained equal amounts of single-stranded DNA and DNA-RNA hybrid and little double-stranded DNA. The major fraction after extensive synthesis contained equal amounts of single-and double-stranded DNA and little hybrid. In the presence of actinomycin D, the predominant product was single-stranded DNA. To account for these various forms of DNA, we postulate the following model: the first DNA synthesis occurs in a replicative complex containing growing DNA molecules attached to an RNA molecule. Each DNA molecule is displaced as single-stranded DNA by the synthesis of the following DNA strand, and the single-stranded DNA is copied to form double-stranded DNA either before or after release of the single strand from the RNA. Actinomycin blocks this conversion of single-to double-stranded DNA.

A new approach to selectively enhance the ultraviolet (UV) detection sensitivity of titania (TiO2), albeit in the presence of silica (SiO2), alumina (Al2O3), and zinc oxide (ZnO), nanoparticles in capillary electrophoresis (CE) analysis was developed. Interactions of Triton X-100 (TX-100), polyethylene glycol (PEG),and deoxyribonucleic acid (DNA) with TiO2 nanoparticles produced larger CE-UV peaks at various enhancement factors. Single-stranded DNA (ssDNA) was a more effective adsorbate than double-stranded DNA (dsDNA) due to its flexible molecular structure that participated in a stronger interaction with TiO2 nanoparticles via its sugar-phosphate backbone. Disaggregation of TiO2 nanoparticles upon DNA binding due to electrosteric stabilization was validated using dynamic light scattering. PEG coating of TiO2-DNA nanoparticles further enhanced the UV detection sensitivity in CE analysis by providing extra electrosteric stabilization. This analytical technique, which involves binding of TiO2 nanoparticles with DNA followed by coating with PEG, has allowed us to achieve progressively an enhancement factor up to 13.0 ± 3.0 - fold in analytical sensitivity for the accurate determination of disaggregated TiO2 nanoparticles. Graphical Abstract Selective enhancement of UV detection sensitivity for TiO2 nanoparticles via electrosteric stabilization using ssDNA and PEG.

Physicochemical profiling of drug candidates using capillary-based techniques

Two capillary electrophoresis (CE) methods have been developed for the separation of charge and mass variants of human insulin and its recombinant analogue lispro. Since the capillary zone (CZE) and Capillary gel electrophoresis (CGE) are based on different principles of separation, they can be used to detect different impurities of insulin and its analogues. Application of CZE enabled a separation of compounds with different m/z ratio, therefore CZE is a suitable method for the separation of deamidation products of insulin. After the optimization, this method is validated according ICH requirements. CGE method was used for the separation of higher molecular weight transformation products. Experimental data have shown that CZE and CGE are simple, fast and robust methods which could be used as a routine analysis for quality control of insulin formulations.

Lipid nanoparticles (LNPs) are one of the most clinically advanced candidates for delivering nucleic acids to target cell populations, such as hepatocytes. Once LNPs are endocytosed, they must release their nucleic acid cargo into the cell cytoplasm. For delivering messenger RNA (mRNA), delivery into the cytosol is sufficient; however, for delivering DNA, there is an added diffusional barrier needed to facilitate nuclear uptake for transcription and therapeutic effect. Here, we use fluorescence microscopy to investigate the intracellular fate of different LNP formulations to determine the kinetics of localization to endosomes and lysosomes. LNPs used in the studies were prepared via self-assembly using a NanoAssemblr for microfluidic mixing. As the content of polyethylene glycol (PEG) within the LNP formulation influences cellular uptake by hepatocyte cells, the content and hydrocarbon chain length within the formulation were assessed for their impact on intracellular trafficking. Standard LNPs were then formed using three commercially available ionizable lipids, Dlin-MC3-DMA (MC3), Dlin-KC2-DMA (KC2), and SS-OP. Plasmid DNA (pDNA) and mRNA were used, more specifically with a mixture of Cyanine 3 (Cy3)-labeled and green fluorescence protein (GFP) producing plasmid DNA (pDNA) as well as Cy5-labeled GFP producing mRNA. After formulation, LNPs were characterized for the encapsulation efficiency of the nucleic acid, hydrodynamic diameter, polydispersity, and zeta potential. All standard LNPs were ~100 nm in diameter and had neutral surface charge. All LNPs resulted in encapsulation efficiency greater than 70%. Confocal fluorescence microscopy was used for the intracellular trafficking studies, where LNPs were incubated with HuH-7 hepatocyte cells at times ranging from 0-48 h. The cells were antibody-stained for subcellular components, including nuclei, endosomes, and lysosomes. Analysis was performed to quantify localization of pDNA to the endosomes and lysosomes. LNPs with 1.5 mol% PEG and a hydrocarbon chain C14 resulted in optimal endosomal escape and GFP production. Results from this study demonstrate that a higher percentage of C14 PEG leads to smaller LNPs with limited available phospholipid binding area for ApoE, resulting in decreased cellular uptake. We observed differences in the localization kinetics depending on the LNP formulation type for SS-OP, KC2, and MC3 ionizable lipids. The results also demonstrate the technique across different nucleic acid types, where mRNA resulted in more rapid and uniform GFP production compared to pDNA delivery. Here, we demonstrated the ability to track uptake and the sub-cellular fate of LNPs containing pDNA and mRNA, enabling improved screening prior to in vivo studies which would aid in formulation optimization.

The biopharmaceutical market is one of the fastest growing biotechnology markets. In order to ensure affordable and reliable therapeutics, the biopharmaceutical process has to be closely monitored. Capillary electrophoresis (CE) has proven to be a valuable technique for the analysis of product concentration, critical quality attributes, product and process-related impurities, and nutrients and metabolites in the cell culture medium. Capillary zone electrophoresis, capillary gel electrophoresis, and capillary isoelectric focusing are extensively used for product concentration, and size or charge heterogeneity determination. CE has a number of benefits for the analysis of upstream and downstream process-intermediates, including the ability to handle highly complex matrices found in process-intermediates, high resolving power, little sample preparation requirements, rapid analysis, and low solvent and sample consumption. The small sample volumes (nL range) are especially beneficial for microbioreactor analysis or clone selection experiments. The simple setup and the possibility for miniaturisation and automation using microchip CE provides great opportunities for on-site, real-time monitoring of the process. This review discusses CE applications in upstream and downstream processing of the last decade.

The use of nucleic acids (NAs) has revolutionized medical approaches and ushered in a new era of combating various diseases. Accordingly, there is an increasing demand for accurate identification, localization, quantification, and characterization of NAs encapsulated in nonviral or viral vectors. The vast spectrum of molecular dimensions and intra- and intermolecular interactions presents a formidable obstacle for NA analytical development. Typically, the comprehensive analysis of encapsulated NAs, free NAs, and their spatial distribution poses a challenge that is seldom tackled in its complete complexity. The identification of appropriate physicochemical methodologies for large nonencapsulated or encapsulated NAs is particularly intricate and necessitates an evaluation of the analytical outcomes and their appropriateness in addressing critical quality attributes. In this work, we examine the analytics of non-encapsulated or encapsulated large NAs(>500 nucleotides) utilizing capillary electrophoresis (CE) and liquid chromatography (LC) methodologies such as free zone CE, gel CE, affinity CE, and ion pair high-performance liquid chromatography (HPLC). These methodologies create a complete picture of the NA's critical quality attributes, including quantity, identity, purity, and content ratio.