Electrophoretic characterization of LNP/AAV-encapsulated nucleic acids: Strengths and weaknesses.

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.

Reversed Phase-Liquid Chromatography for Recombinant AAV Genome Integrity Assessment.

After decades of research, gene therapy products have reached market maturity in recent years. Recombinant adeno-associated viruses (rAAVs) are one of the most promising gene delivery vehicles and are currently under intense scientific investigation. These next-generation medicines remain very challenging when it comes to designing appropriate analytical techniques for quality control. One critical quality attribute is the integrity of ssDNA incorporated in these vectors. The genome is the active compound driving rAAV therapy and therefore requires proper assessment and quality control. Current techniques for rAAV genome characterization include next-generation sequencing, quantitative polymerase chain reaction, analytical ultracentrifugation (AUC), and capillary gel electrophoresis (CGE), yet each of them presents their limitations or lack of user-friendliness. In this work, we demonstrate for the first time the potential of ion pairing-reverse phase-liquid chromatography (IP-RP-LC) to characterize the integrity of rAAV genomes. The obtained results were supported by two orthogonal techniques, AUC and CGE. IP-RP-LC can be performed above DNA melting temperatures, avoiding the detection of secondary DNA isoforms, and does not require the use of dyes due to UV detection. We demonstrate that this technique is suitable for batch comparability, different rAAV serotypes (AAV2 and AAV8), internal vs external (inside vs outside the capsid) DNA analysis, and contaminated samples. Overall, it is exceptionally user-friendly, needs limited sample preparation, has high reproducibility, and permits fractionation for further peak characterization. All of these factors add significant value of IP-RP-LC to the analytical toolbox of rAAV genome assessment.

Using Peptide Nucleic Acid Hybridization Probes for Qualitative and Quantitative Analysis of Nucleic Acid Therapeutics by Capillary Electrophoresis.

The space of advanced therapeutic modalities is currently evolving in rapid pace necessitating continuous improvement of analytical quality control methods. In order to evaluate the identity of nucleic acid species in gene therapy products, we propose a capillary electrophoresis-based gel free hybridization assay in which fluorescently labeled peptide nucleic acids (PNAs) are applied as affinity probes. PNAs are engineered organic polymers that share the base pairing properties with DNA and RNA but have an uncharged peptide backbone. In the present study, we conduct various proof-of-concept studies to identify the potential of PNA probes for advanced analytical characterization of novel therapeutic modalities like oligonucleotides, plasmids, mRNA, and DNA released by recombinant adeno-associated virus. For single-stranded nucleic acids up to 1000 nucleotides, the method is an excellent choice that proved to be highly specific by detecting DNA traces in complex samples, while having a limit of quantification in the picomolar range when multiple probes are used. For double-stranded samples, only fragments that are similar in size to the probe could be quantified. This limitation can be circumvented when target DNA is digested and multiple probes are used opening an alternative to quantitative PCR.

Stronger together: Analytical techniques for recombinant adeno associated virus.

With recent FDA approval of two recombinant adeno-associated virus (rAAV)-based gene therapies, these vectors have proven that they are suitable to address monogenic diseases. However, rAAVs are relatively new modalities, and their production and therapy costs significantly exceed those of conventional biologics. Thus, significant efforts are made to improve the processes, methods, and techniques used in manufacturing and quality control (QC). Here, we evaluate transmission electron microscopy (TEM), analytical ultracentrifugation (AUC), and two modes of capillary electrophoresis (CE) for their ability to analyze the DNA encapsidated by rAAVs. While TEM and AUC are well-established methods for rAAV, capillary gel electrophoresis (CGE) has been just recently proposed for viral genome sizing. The data presented reflect that samples are very complex, with various DNA species incorporated in the virus, including small fragments as well as DNA that is larger than the targeted transgene. CGE provides a good insight in the filling of rAAVs, but the workflow is tedious and the method is not applicable for the determination of DNA titer, since a procedure for the absolute quantification (e.g., calibration) is not yet established. For estimating the genome titer, we propose a simplified capillary zone electrophoresis approach with minimal sample preparation and short separation times (<5 min/run). Our data show the benefits of using the four techniques combined, since each of them alone is prone to delivering ambiguous results. For this reason, a clear view of the rAAV interior can only be provided by using several analytical methods simultaneously.

Methionine oxidation of proteins analyzed by affinity capillary electrophoresis in presence of silver(I) and gold(III) ions.

Oxidative damage of biopharmaceuticals during manufacturing and storage is a key concern throughout pharmaceutical development. However, few simple and robust analytical methods are available for the determination of oxidation sites. Here, the potential of affinity capillary electrophoresis (ACE) in the separation of proteins with oxidized methionine (Met) residues is shown. Silver(I) and gold(I) ions have the attribute to selectively form complexes with thioethers over sulfoxides. The addition of these ions to the BGE leads to a selective complexation of Met residues and, thus, to a change of charge allowing separation of species according to the different oxidation states of Met. The mechanisms of these interactions are discussed and binding constants for peptides containing Met with silver(I) are calculated. Additionally, the proposed method can be used as an indicator of oxidative stress in large proteins. The presented technique is easily accessible, economical, and has rapid analysis times, adding new approaches to the analytical toolbox of Met sulfoxide detection.

Application of affinity capillary electrophoresis for charge heterogeneity profiling of biopharmaceuticals.

Charge heterogeneity profiling is important for the quality control (QC) of biopharmaceuticals. Because of the increasing complexity of these therapeutic entities [1], the development of alternative analytical techniques is needed. In this work, flow-through partial-filling affinity capillary electrophoresis (FTPFACE) has been established as a method for the analysis of a mixture of two similar monoclonal antibodies (mAbs). The addition of a specific ligand results in the complexation of one mAb in the co-formulation, thus changing its migration time in the electric field. This allows the characterization of the charged variants of the non-shifted mAb without interferences. Adsorption of proteins to the inner capillary wall has been circumvented by rinsing with guanidine hydrochloride before each injection. The presented FTPFACE approach requires only very small amounts of ligands and provides complete comparability with a standard CZE of a single mAb.