Genome length determination in adeno-associated virus vectors with mass photometry.

Recombinant adeno-associated viruses (rAAVs) are attractive therapeutic viral vectors for gene delivery. To ensure the efficacy and safety of rAAV-based therapies, comprehensive characterization of the adeno-associated virus (AAV) capsids is essential. Mass photometry (MP) provides the advantage of short analysis times, low sample consumption, and high accuracy of molecular mass determination. Despite having just recently emerged, MP has already been used to characterize AAV genome content and quantify filled/empty capsid ratios. In this study, we explored three approaches for the application of MP to assess genome length in AAVs. In approach 1, genome length in intact AAVs was approximated with good precision (coefficient of variation [%CV] < 2.6%) and accuracy (±5%) by using a straightforward protein-based calibration. In approach 2, genome length was determined even more accurately (±1%, %CV < 2.9%) considering calibration with a set of additional AAVs of different genome length. In approach 3, genome length was assessed after genome release from the capsid by heating in 1% sodium dodecyl sulfate followed by surfactant removal with precision of %CV < 0.7% and accuracy of ±5%. In conclusion, the three developed MP-based approaches are fast, precise, and accurate methods for genome length determination in AAVs, differing in their calibration materials and efforts.

Purity and DNA content of AAV capsids assessed by analytical ultracentrifugation and orthogonal biophysical techniques.

Development and manufacturing adeno-associated virus (AAV)-based vectors for gene therapy requires suitable analytical methods to assess the quality of the formulations during development, as well as the quality of different batches and the consistency of the processes. Here, we compare biophysical methods to characterize purity and DNA content of viral capsids from five different serotypes (AAV2, AAV5, AAV6, AAV8, and AAV9). For this purpose, we apply multiwavelength sedimentation velocity analytical ultracentrifugation (SV-AUC) to obtain the species' contents and to derive the wavelength-specific correction factors for the respective insert-size. In an orthogonal manner we perform anion exchange chromatography (AEX) and UV-spectroscopy and the three methods yield comparable results on empty/filled capsid contents with these correction factors. Whereas AEX and UV-spectroscopy can quantify empty and filled AAVs, only SV-AUC could identify the low amounts of partially filled capsids present in the samples used in this study. Finally, we employ negative-staining transmission electron microscopy and mass photometry to support the empty/filled ratios with methods that classify individual capsids. The obtained ratios are consistent throughout the orthogonal approaches as long as no other impurities and aggregates are present. Our results show that the combination of selected orthogonal methods can deliver consistent empty/filled contents on non-standard genome sizes, as well as information on other relevant critical quality attributes, such as AAV capsid concentration, genome concentration, insert size length and sample purity to characterize and compare AAV preparations.

Characterization of Virus Particles and Submicron-Sized Particulate Impurities in Recombinant Adeno-Associated Virus Drug Product.

Characterization of particulate impurities such as aggregates is necessary to develop safe and efficacious adeno-associated virus (AAV) drug products. Although aggregation of AAVs can reduce the bioavailability of the virus, only a limited number of studies focus on the analysis of aggregates. We explored three technologies for their capability to characterize AAV monomers and aggregates in the submicron (<1 µm) size range: (i) mass photometry (MP), (ii) asymmetric flow field flow fractionation coupled to a UV-detector (AF4-UV/Vis) and (iii) microfluidic resistive pulse sensing (MRPS). Although low counts for aggregates impeded a quantitative analysis, MP was affirmed as an accurate and rapid method for quantifying the genome content of empty/filled/double-filled capsids, consistent with sedimentation velocity analytical ultracentrifugation results. MRPS and AF4-UV/Vis enabled the detection and quantification of aggregate content. The developed AF4-UV/Vis method separated AAV monomers from smaller aggregates, thereby enabling a quantification of aggregates <200 nm. MRPS was experienced as a straightforward method to determine the particle concentration and size distribution between 250-2000 nm, provided that the samples do not block the microfluidic cartridge. Overall, within this study we explored the benefits and limitations of the complementary technologies for assessing aggregate content in AAV samples.

Short-term study on in-use stability of opened bevacizumab biosimilar PF-06439535 vials.

OBJECTIVES: Aggregation is one of the key critical points limiting the stability of monoclonal antibodies in solution. The present study aimed to investigate the in-use stability of a residual monoclonal antibody solution after withdrawal of most of the filling volume of PF-06439535 (bevacizumab biosimilar), addressing the physical and chemical stability with respect to aggregation and fragmentation. METHODS: The stability of residual PF-06439535 solution (25 mg/mL) after withdrawal of 80% (12.8 mL) filling volume with a 20G needle was monitored over a light-protected storage period of 8 days at 2-8°C and 25°C with measurement time points at D0 (start of storage), D2, D4, and D8 (2, 4, and 8 days of storage after start, respectively). Unopened vials stored under the same conditions served as control. For this purpose, the analytical results from size exclusion chromatography, dynamic light scattering, and micro-flow imaging obtained after the individual measurement time points up to 8 days were compared with those obtained at D0 and with those obtained for unopened vials stored under the same conditions. RESULTS: No aggregation or ongoing fragmentation due to partial withdrawal of filling volume could be observed in the residual PF-06439535 solution. Moreover, no changes in the particle size distribution at D8 compared with the D0 values were identified upon storage at either 2-8°C or 25°C (both opened and unopened vials). The total concentration of particles ≥10 µm of all samples was <100 particles/mL. In addition, no variations in the pH values or in the visual appearance were detected over the whole study period in all samples at all storage conditions. CONCLUSIONS: Consequently, residual PF-06439535 solution (25 mg/mL) in opened vials may be regarded as stable when stored light-protected over a period of 8 days in the refrigerator (2-8°C) or at 25°C.

Towards quantification and differentiation of protein aggregates and silicone oil droplets in the low micrometer and submicrometer size range by using oil-immersion flow imaging microscopy and convolutional neural networks.

Biopharmaceutical product characterization benefits from the quantification and differentiation of unwanted protein aggregates and silicone oil droplets to support risk assessment and control strategies as part of the development. Flow imaging microscopy is successfully applied to differentiate the two impurities in the size range larger than about 5 µm based on their morphological appearance. In our study we applied the combination of oil-immersion flow imaging microscopy and convolutional neural networks to extend the size range below 5 µm. It allowed to differentiate and quantify heat stressed therapeutic monoclonal antibody aggregates from artificially generated silicone oil droplets with misclassification rates of about 10% in the size range between 0.3 and 5 µm. By comparing the misclassifications across the tested size range, particles in the low submicron size range were particularly difficult to differentiate as their morphological appearance becomes very similar.

Oil-Immersion Flow Imaging Microscopy for Quantification and Morphological Characterization of Submicron Particles in Biopharmaceuticals.

Flow imaging microscopy (FIM) is widely used to analyze subvisible particles starting from 2 µm in biopharmaceuticals. Recently, an oil-immersion FIM system emerged, the FlowCam Nano, designed to enable the characterization of particle sizes even below 2 µm. The aim of our study was to evaluate oil-immersion FIM (by using FlowCam Nano) in comparison to microfluidic resistive pulse sensing and resonant mass measurement for sizing and counting of particles in the submicron range. Polystyrene beads, a heat-stressed monoclonal antibody formulation and a silicone oil emulsion, were measured to assess the performance on biopharmaceutical relevant samples, as well as the ability to distinguish particle types based on instrument-derived morphological parameters. The determination of particle sizes and morphologies suffers from inaccuracies due to a low image contrast of small particles and light-scattering effects. The ill-defined measured volume impairs an accurate concentration determination. Nevertheless, FlowCam Nano in its current design complements the limited toolbox of submicron particle analysis of biopharmaceuticals by providing particle images in a size range that was previously not accessible with commercial FIM instruments.

Chemical and Biophysical Characteristics of Monoclonal Antibody Solutions Containing Aggregates Formed during Metal Catalyzed Oxidation.

PURPOSE: To physicochemically characterize and compare monoclonal antibody (mAb) solutions containing aggregates generated via metal catalyzed oxidation (MCO). METHODS: Two monoclonal IgG2s (mAb1 and mAb2) and one monoclonal IgG1 (rituximab) were exposed to MCO with the copper/ascorbic acid oxidative system, by using several different methods. The products obtained were characterized by complementary techniques for aggregate and particle analysis (from oligomers to micron sized species), and mass spectrometry methods to determine the residual copper content and chemical modifications of the proteins. RESULTS: The particle size distribution and the morphology of the protein aggregates generated were similar for all mAbs, independent of the MCO method used. There were differences in both residual copper content and in chemical modification of specific residues, which appear to be dependent on both the protein sequence and the protocol used. All products showed a significant increase in the levels of oxidized His, Trp, and Met residues, with differences in extent of modification and specific amino acid residues modified. CONCLUSION: The extent of total oxidation and the amino acid residues with the greatest oxidation rate depend on a combination of the MCO method used and the protein sequence.

Micro-flow imaging and resonant mass measurement (Archimedes)--complementary methods to quantitatively differentiate protein particles and silicone oil droplets.

Our study aimed to comparatively evaluate Micro-Flow Imaging (MFI) and the recently introduced technique of resonant mass measurement (Archimedes, RMM) as orthogonal methods for the quantitative differentiation of silicone oil droplets and protein particles. This distinction in the submicron and micron size range is highly relevant for the development of biopharmaceuticals, in particular for products in prefilled syringes. Samples of artificially generated silicone oil droplets and protein particles were quantified individually and in defined mixtures to assess the performance of the two techniques. The built-in MFI software solution proved to be suitable to discriminate between droplets and particles for sizes above 2 µm at moderate droplet/particle ratios (70:30-30:70). A customized filter developed specifically for this study greatly improved the results and enabled reliable discrimination also for more extreme mixing ratios (95:5-15:85). RMM showed highly accurate discrimination in the size range of about 0.5-2 µm independent of the ratio, provided that a sufficient number of particles (>50 counted particles) were counted. We recommend applying both techniques for a comprehensive analysis of biotherapeutics potentially containing silicone oil droplets and protein particles in the submicron and micron size range.

How subvisible particles become invisible-relevance of the refractive index for protein particle analysis.

The aim of the present study was to quantitatively assess the relevance of transparency and refractive index (RI) on protein particle analysis by the light-based techniques light obscuration (LO) and Micro-Flow Imaging (MFI). A novel method for determining the RI of protein particles was developed and provided an RI of 1.41 for protein particles from two different proteins. An increased RI of the formulation by high protein concentration and/or sugars at pharmaceutically relevant levels was shown to lead to a significant underestimation of the subvisible particle concentration determined by LO and MFI. An RI match even caused particles to become "invisible" for the system, that is, not detectable anymore by LO and MFI. To determine the influence of formulation RI on particle measurements, we suggest the use of polytetrafluoroethylene (PTFE) particles to test a specific formulation for RI effects. In case of RI influences, we recommend also using a light-independent technique such as resonant mass measurement (RMM) (Archimedes) for subvisible particle analysis in protein formulations.

Taylor dispersion analysis compared to dynamic light scattering for the size analysis of therapeutic peptides and proteins and their aggregates.

PURPOSE: To evaluate Taylor dispersion analysis (TDA) as a novel method for determination of hydrodynamic radius of therapeutic peptides and proteins in non-stressed and stressed formulations and to compare it with dynamic light scattering (DLS). METHODS: The hydrodynamic radius of oxytocin, bovine serum albumin, various monoclonal antibodies (type IgG) and etanercept at concentrations between 0.05 and 50 mg/ml was determined by TDA and DLS. IgGs and etanercept were stressed (elevated temperatures) and analyzed by TDA, DLS and HP-SEC. RESULTS: TDA and DLS were comparable in sizing non-stressed peptides and proteins in a concentration range of about 0.5 to 50 mg/ml. TDA performed well even at lower concentrations, where DLS tends to provide theoretically high values of the Z-average radius. However, because of differences in the detection physics, DLS was more weighted towards the detection of aggregates in stressed formulations than TDA. Advantageously, TDA was also able to size the small peptide oxytocin, which was not feasible by DLS. CONCLUSION: TDA allows the accurate determination of the hydrodynamic radius of peptides and proteins over a wide concentration range, with little interference from excipients present in the sample. It is marginally less sensitive than DLS in detecting size increase for stressed protein samples.

Comparison of analytical methods to detect instability of etanercept during thermal stress testing.

The aim was comparing the capability of a set of analytical methods to detect physical instability (focus on aggregation and structural changes) of etanercept during thermal stress testing as early as possible. Pre-filled syringes of Enbrel® 50mg from three batches were thermally stressed for one week at 50°C. Samples were taken at days 0, 1, 2, 3, 4 and 7, and analyzed with high-performance liquid size exclusion chromatography (HP-SEC), SDS-PAGE gel electrophoresis, dynamic light scattering (DLS), light obscuration, extrinsic fluorescence (Bis-ANS), far-UV circular dichroism (CD) spectroscopy, second derivative UV spectroscopy (UV), and enzyme-linked immunosorbent assay (ELISA). Thermal stress resulted in the formation of small soluble aggregates (HP-SEC, DLS) which were in part covalent (SDS-PAGE), and conformationally changed (Bis-ANS, CD, UV). No significant increase in subvisible particles was detected by light obscuration. An apparent increase in TNF-α binding to etancercept in the stressed formulations was found by ELISA. The three batches were comparable when unstressed, but showed slight differences in aggregation tendency. Bis-ANS fluorescence was most sensitive with respect to early-stage detection of heat-induced instability of etanercept (significant changes already at day 1), followed by HP-SEC (day 2) and DLS (day 3). This points towards a degradation mechanism involving exposure of hydrophobic patches due to partial unfolding followed by aggregation.