Mol Ther Methods Clin Dev
December 2024
Adeno-associated virus (AAV) vectors have attracted significant attention as the main platform for gene therapy. To ensure the safety and efficacy of AAV vectors when used as gene therapy drugs, it is essential to assess their critical quality attributes (CQAs). These CQAs include the genome packaging status, the size of the genome encapsidated within the AAV capsid, and the stoichiometry of viral proteins (VPs) that constitute the AAV capsids.
View Article and Find Full Text PDFThe adeno-associated virus (AAV) vector is one of the most advanced platforms for gene therapy because of its low immunogenicity and non-pathogenicity. The concentrations of both AAV vector empty particles, which do not contain DNA and do not show any efficacy, and AAV vector full particles (FPs), which contain DNA, are important quality attributes. In this study, a dual fluorescence-linked immunosorbent assay (dFLISA), which uses two fluorescent dyes to quantify capsid and genome titers in a single analysis, was established.
View Article and Find Full Text PDFThe higher-order structure (HOS) is a critical quality attribute of recombinant adeno-associated viruses (rAAVs). Evaluating the HOS of the entire rAAV capsid is challenging because of the flexibility and/or less folded nature of the VP1 unique (VP1u) and VP1/VP2 common regions, which are structural features essential for these regions to exert their functions following viral infection. In this study, hydrogen/deuterium exchange mass spectrometry (HDX-MS) was used for the structural analysis of full and empty rAAV8 capsids.
View Article and Find Full Text PDFProteins can be adsorbed on the air-water interface (AWI), and the structural changes in proteins at the AWI are closely related to the foaming properties of foods and beverages. However, how these structural changes in proteins at the AWI occur is not well understood. We developed a method for the structural assessment of proteins in the foam state using hydrogen/deuterium exchange mass spectrometry.
View Article and Find Full Text PDFMicrofiltration (MF) is an essential step during biopharmaceutical manufacturing. However, unexpected flux decay can occur. Although the flux decay profile and initial flux are important factors determining MF filterability, predicting them accurately is challenging, as the root cause of unexpected flux decay remains elusive.
View Article and Find Full Text PDFStudies of recombinant adeno-associated virus (rAAV) revealed the mixture of full particles with different densities in rAAV. There are no conclusive results because of the lack of quantitative stoichiometric viral proteins, encapsidated DNA, and particle level analyses. We report the first comprehensive characterization of low- and high-density rAAV serotype 2 particles.
View Article and Find Full Text PDFDuring biopharmaceutical development, particle monitoring and characterization are crucial. Notably, particles can be impurities considered as critical quality attribute, or active pharmaceutical ingredient (e.g.
View Article and Find Full Text PDFFlow imaging microscopy (FIM) is widely used to characterize biopharmaceutical subvisible particles (SVPs). The segmentation threshold, which defines the boundary between the particle and the background based on pixel intensity, should be properly set for accurate SVP quantification. However, segmentation thresholds are often subjectively and empirically set, potentially leading to variations in measurements across instruments and operators.
View Article and Find Full Text PDFAn increase in protein aggregates during transportation should be suppressed in therapeutic protein products because the aggregates have a potential risk of immunogenicity. In this study, three protein solutions in vials were exposed to tri-axial vibration with various combinations of frequency and acceleration using a transportation test system to investigate the relationship between low g-force stresses and protein aggregate generation. The number concentration of micron aggregates detected by flow imaging analysis increased markedly when the acceleration and frequency of agitation were within a specific range, in other words, above a threshold.
View Article and Find Full Text PDFSubvisible particles (SVPs) are a critical quality attribute of parenteral and ophthalmic products. United States Pharmacopeia recommends the characterizations of SVPs which are classified into intrinsic, extrinsic, and inherent particles. Flow imaging microscopy (FIM) is useful as an orthogonal method in both the quantification and classification of SVPs because FIM instruments provide particle images.
View Article and Find Full Text PDFThe interaction between IgG and Fc gamma receptor IIIa (FcγRIIIa) is essential for mediating immune responses. Recent studies have shown that the antigen binding fragment (Fab) and Fc are involved in IgG-FcγRIII interactions. Here, we conducted bio-layer interferometry (BLI) and isothermal titration calorimetry to measure the kinetic and thermodynamic parameters that define the role of Fab in forming the IgG-FcγRIII complex using several marketed therapeutic antibodies.
View Article and Find Full Text PDFRecombinant adeno-associated virus (rAAV) vectors have proven efficacy as gene therapy vehicles. However, non-specific adsorption of these vectors on solid surfaces is encountered during production, storage, and administration, as well as in quantification processes. Such adsorption has been reported to result in the loss of up to 90% of vector particles and can also result in high variability in vector genome quantification.
View Article and Find Full Text PDFProtein aggregate formation in prefilled syringes (PFSs) can be influenced by protein adsorption and desorption at the solid-liquid interface. Although inhibition of protein adsorption on the PFS surface can lead to a decrease in the amount of aggregation, the mechanism underlying protein adsorption-mediated aggregation in PFSs is unclear. This study investigated protein aggregation caused by protein adsorption on silicone oil-free PFS surfaces [borosilicate glass (GLS) and cycloolefin polymer (COP)] and the factors affecting the protein adsorption on the PFS surfaces.
View Article and Find Full Text PDFDuring the manufacturing of recombinant adeno-associated virus vectors, it is generally difficult to purify out vectors that lack nucleic acids (empty particles, EPs), contain incomplete nucleic acids (intermediate particles, IPs) or aggregates. These impurities may cause side effects and therefore it is essential to both quantify and reduce them; however, comprehensive identification of the size distribution and components of virus vectors have been lagging. We developed multiwavelength sedimentation velocity analytical ultracentrifugation to characterize EPs, full particles, IPs, and aggregates in adeno-associated virus vector samples.
View Article and Find Full Text PDFRecombinant adeno-associated virus is a leading platform in human gene therapy. The adeno-associated virus (AAV) capsid is composed of three viral proteins (VPs): VP1, VP2, and VP3. To ensure the safety of AAV-based gene therapy products, the stoichiometry of VPs of AAV vector should be carefully monitored.
View Article and Find Full Text PDFExpert Opin Drug Deliv
April 2021
Introduction: Several new biopharmaceutical dosage forms have developed over time, such as lyophilized vial, liquid vial, and liquid prefilled syringe formulations. This review summarizes major pharmaceutical dosage forms and their advantages, disadvantages, and countermeasures against the shortcomings of each formulation. The appropriate combination of active pharmaceutical ingredients, excipients, and containers should be selected for the safe and less burdensome administration to the patients.
View Article and Find Full Text PDFAs reported in the previous commentary (Ishii-Watabe et al., J Pharm Sci 2017), the Japanese biopharmaceutical research group is promoting collaborative multilaboratory studies to evaluate and standardize new methodologies for biopharmaceutical characterization and quality control. We have conducted the studies and held 2 annual meetings in 2018 and 2019.
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