Comparison of three AUC techniques for the determination of the loading status and capsid titer of AAVs.

Due to the rise of adeno-associated viruses (AAVs) as gene therapy delivery vectors, boundary sedimentation velocity analytical ultracentrifugation (boundary SV-AUC) has been developed into a widely used quality control assay even for release analytics. It can be considered as the "gold standard" for the determination of the loading status of empty, partially filled, and full capsids especially when conducted in multiwavelength (MWL) mode. It can be considered to provide the most accurate determination of the loading status, and it also provides information on the capsid titer, aggregates, and potential contaminants such as free DNA. MWL boundary SV-AUC can be regarded as a multi-attribute (MAM) method for the characterization of AAVs. One major drawback of the method is the high sample consumption both in terms of concentration and volume. Here, we compare two alternative AUC techniques, band SV-AUC and analytical CsCl density gradient sedimentation equilibrium AUC (CsCl SE-AUC) with the boundary SV-AUC and the MWL-SV-AUC experiment. Our data show a high consistency of the determined full/empty ratios between these techniques if the appropriate wavelengths and extinction coefficients are used.

A comparison between analytical approaches for molecular weight estimation of proteins with variable levels of glycosylation.

Biotherapeutics, such as mAbs and fusion proteins, are a major and rapidly growing class of pharmaceuticals. Majority of the biopharmaceuticals are glycoproteins, wherein about 1 to 30% of their molecular weight (MW) are contributed by the glycans. Determination of MW of heavily glycosylated proteins, such as Fc-fusion proteins, is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation influences the expected size of the glycoprotein, which leads to disproportionate MW estimation, in size-dependent methods. Hence, in this study, we have demonstrated the advantages and limitations of four widely used MW estimation techniques for three proteins having varying levels of glycosylation. It was proven that glycosylation had least impact on MW determination by SEC-MALS and SV-AUC. However, MW estimation by LC-MS and SDS-PAGE was extensively hampered by the degree of glycosylation. It is, thus, essential to consider the structural characteristics of proteins while selecting a technique for determining their MW.

Sedimentation Velocity Analytical Ultracentrifugation Analysis of Marketed Rituximab Drug Product Size Distribution.

PURPOSE: Analytical methods suitable for intact drug products are often necessary to evaluate the equivalence in physicochemical properties between two drug products (DP) containing the same drug substance (DS), e.g., an innovator biologic drug and its proposed biosimilar. Analytical Ultracentrifugation (AUC) is a biophysics technique applied to the analysis of size and shape of biomolecules. However, the application of AUC to formulated monoclonal antibody (mAb) DP at high concentration has not been reported. METHODS: A sedimentation velocity (SV) AUC procedure with a short-pathlength centerpiece was applied to two marketed rituximab DPs, Rituxan® (US) and Reditux® (India), without any buffer exchange or dilution. Detailed precision analysis was performed. RESULTS: Highly reproducible sedimentation coefficient values (S) and peak areas were obtained for the dominant (> 84%) monomeric rituximab peak. The minor mAb fragment peaks had large variation in both S values and peak areas (3-12%). The identification of oligomer peaks was only reproducible once the abundance was higher than 2%. CONCLUSIONS: SV-AUC provides an orthogonal characterization tool for protein size distribution, composition and assay, which could be informative for biosimilar drug developers who mostly only have access to formulated mAb. However, AUC needs thorough validation on its accuracy, precision and sensitivity.

Methodological Validation of Sedimentation Velocity Analytical Ultracentrifugation Method for Adeno-Associated Virus and Collaborative Calibration of System Suitability Substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and high sedimentation coefficient substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF (high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the relative standard deviation (RSD) could be within 5%. Even for empty or partially less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4 × 1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235 nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Because of the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control) laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Quantitation of Enterovirus A71 Empty and Full Particles by Sedimentation Velocity Analytical Ultracentrifugation.

The enterovirus A71 (EV71) inactivated vaccine is an effective intervention to control the spread of the virus and prevent EV71-associated hand, foot, and mouth disease (HFMD). It is widely administered to infants and children in China. The empty particles (EPs) and full particles (FPs) generated during production have different antigenic and immunogenic properties. However, the antigen detection methods currently used were established without considering the differences in antigenicity between EPs and FPs. There is also a lack of other effective analytical methods for detecting the different particle forms, which hinders the consistency between batches of products. In this study, we analyzed the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) in characterizing the EPs and FPs of EV71. Our results showed that the proportions of the two forms could be quantified simultaneously by SV-AUC. We also determined the repeatability and accuracy of this method and found that both parameters were satisfactory. We assessed SV-AUC for bulk vaccine quality control, and our findings indicated that SV-AUC can be used effectively to analyze the percentage of EPs and FPs and monitor the consistency of the process to ensure the quality of the vaccine.

Challenges in the analysis of pharmaceutical lentiviral vector products by orthogonal and complementary physical (nano)particle characterization techniques.

Lentiviral vectors (LVVs) are used as a starting material to generate chimeric antigen receptor (CAR) T cells. Therefore, LVVs need to be carefully analyzed to ensure safety, quality, and potency of the final product. We evaluated orthogonal and complementary analytical techniques for their suitability to characterize particulate matter (impurities and LVVs) in pharmaceutical LVV materials at development stage derived from suspension and adherent manufacturing processes. Microfluidic resistive pulse sensing (MRPS) with additional manual data fitting enabled the assessment of mode diameters for particles in the expected LVV size range in material from adherent production. LVV material from a suspension process, however, contained substantial amounts of particulate impurities which blocked MRPS cartridges. Sedimentation-velocity analytical ultracentrifugation (SV-AUC) resolved the LVV peak in material from adherent production well, whereas in more polydisperse samples from suspension production, presence of particulate impurities masked a potential signal assignable to LVVs. In interferometric light microscopy (ILM) and nanoparticle tracking analysis (NTA), lower size detection limits close to âˆ¼ 70 nm resulted in an apparent peak in particle size distributions at the expected size for LVVs emphasizing the need to interpret these data with care. Interpretation of data from dynamic light scattering (DLS) was limited by insufficient size resolution and sample polydispersity. In conclusion, the analysis of LVV products manufactured at pharmaceutical scale with current state-of-the-art physical (nano)particle characterization techniques was challenging due to the presence of particulate impurities of heterogeneous size. Among the evaluated techniques, MRPS and SV-AUC were most promising yielding acceptable results at least for material from adherent production.

Application of Size Exclusion Chromatography with Multiangle Light Scattering in the Analytical Development of a Preclinical Stage Gene Therapy Program.

To provide safe recombinant adeno-associated viruses (rAAV) to patients, scalable manufacturing processes are required. However, these processes may introduce impurities that impact the performance and quality of the final drug product. Empty rAAV capsids are product-related impurities. Regulatory guidance requires that accurate analytical methods be implemented early in product development to measure the level of empty capsids. A process confirmation vector, produced from 200 L production, was used to develop and optimize a size exclusion chromatography with UV and multiangle light scattering (SEC-MALS) method. Vector produced from a 500 L production was used to assess the full-to-empty ratio using the following analytical methods: sedimentation velocity analytical ultracentrifugation (SV-AUC), droplet digital PCR (ddPCR) with capsid enzyme-linked immunosorbent assay (ELISA), bulk absorbance at 260/280 nm, cryogenic electron microscopy, and SEC-MALS. This test article was used for a 30-day, non-Good Laboratory Practices animal study that assessed biodistribution of the product (STRX-330). SEC-MALS outperformed the other methods and correlated well with SV-AUC values of full-to-empty particles. In addition, SEC-MALS agreed with ddPCR and ELISA measurements for vector genomes/mL and capsid particles/mL, respectively. SEC-MALS was linear, accurate, and precise while achieving chromatography quality control (QC) recommendations. Compared to other stability-indicating assays, SEC-MALS performed similarly to ddPCR, capsid ELISA, and infectivity assays in accelerated stress studies. In response to alkaline, but not acidic stress, SEC-MALS indicated distinct changes in the DNA content of the monomer Adeno-associated viruses (AAV) peak for STRX-330, which was supported by ddPCR data. Conversely, acidic treatment resulted in more aggregated vector, but did not impact the DNA content. This work indicates that SEC-MALS is a valuable analytical tool in the analytical development and QC testing of AAV. In addition, this work suggests that SEC-MALS can provide fundamental understanding of AAV in response to environmental stress. This may impact steps of the manufacturing process to minimize conditions that reduce performance.

Deciphering the High Viscosity of a Therapeutic Monoclonal Antibody in High Concentration Formulations by Microdialysis-Hydrogen/Deuterium Exchange Mass Spectrometry.

High concentration formulations of therapeutic monoclonal antibodies (mAbs) are highly desired for subcutaneous injection. However, high concentration formulations can exhibit unusual molecular behaviors, such as high viscosity or aggregation, that present challenges for manufacturing and administration. To understand the molecular mechanism of the high viscosity exhibited by high concentration protein formulations, we analyzed a human IgG4 (mAb1) at high protein concentrations using sedimentation velocity analytical ultracentrifugation (SV-AUC), X-ray crystallography, hydrogen/deuterium exchange mass spectrometry (HDX-MS), and protein surface patches analysis. Particularly, we developed a microdialysis HDX-MS method to determine intermolecular interactions at different protein concentrations. SV-AUC revealed that mAb1 displayed a propensity for self-association of Fab-Fab, Fab-Fc, and Fc-Fc. mAb1 crystal structure and HDX-MS results demonstrated self-association between complementarity-determining regions (CDRs) and Fc through electrostatic interactions. HDX-MS also indicated Fab-Fab interactions through hydrophobic surface patches constructed by mAb1 CDRs. Our multi-method approach, including fast screening of SV-AUC as well as interface analysis by X-ray crystallography and HDX-MS, helped to elucidate the high viscosity of mAb1 at high concentrations as induced by self-associations of Fab-Fc and Fab-Fab.

Pre-Clinical In-Vitro Studies on Parameters Governing Immune Complex Formation.

The success of biotherapeutics is often challenged by the undesirable events of immunogenicity in patients, characterized by the formation of anti-drug antibodies (ADA). Under specific conditions, the ADAs recognizing the biotherapeutic can trigger the formation of immune complexes (ICs), followed by cascades of subsequent effects on various cell types. Hereby, the connection between the characteristics of ICs and their downstream impact is still not well understood. Factors governing the formation of ICs and the characteristics of these IC species were assessed systematically in vitro. Classic analytical methodologies such as SEC-MALS and SV-AUC, and the state-of-the-art technology mass photometry were applied for the characterization. The study demonstrates a clear interplay between (1) the absolute concentration of the involved components, (2) their molar ratios, (3) structural features of the biologic, (4) and of its endogenous target. This surrogate study design and the associated analytical tool-box is readily applicable to most biotherapeutics and provides valuable insights into mechanisms of IC formation prior to FIH studies. The applicability is versatile-from the detection of candidates with immunogenicity risks during developability assessment to evaluation of the impact of degraded or post-translationally modified biotherapeutics on the formation of ICs.