A capillary with a pulled tip, densely packed with silica particles of 0.47 μm in diameter, is shown to provide higher peak capacity and sensitivity in the separation of intact proteins by reversed-phase liquid chromatography–mass spectrometry (LC–MS). For a C18 bonded phase, slip flow gave a 10-fold flow enhancement to allow for stable nanospray with a 4-cm column length. Model proteins were studied: ribonuclease A, trypsin inhibitor, and carbonic anhydrase, where the latter had impurities of superoxide dismutase and ubiquitin. The proteins were well separated at room temperature with negligible peak tailing. The peak capacity for ubiquitin was 195 for a 10-min gradient and 315 for a 40-min gradient based on Gaussian fitting of the entire peak, rather than extrapolating the full-width at half-maximum. Separation of a cell lysate with a 60 min gradient showed extremely high peak capacities of 750 and above for a peptide and relatively homogeneous proteins. Clean, low noise mass spectra for each model protein were obtained. The physical widths of the peaks were an order of magnitude narrower than those of conventional columns, giving increased sensitivity. All proteins except ubiquitin exhibited significant heterogeneity apparently due to multiple proteoforms, as indicated by both peak shapes and mass spectra. The chromatograms exhibited excellent reproducibility in retention time, with relative standard deviations of 0.09 to 0.34%. The results indicate that submicrometer particles are promising for improving the separation dimension of LC in top-down proteomics.

Recombinant adeno-associated viruses (AAVs) have emerged as the leading gene delivery platform owing to their nonpathogenic nature and long-term gene expression capability. The AAV capsid, in addition to protecting the viral genome, plays an important role in viral infectivity and gene transduction, indicating the value of the constituent viral proteins (VPs) being well-characterized as part of gene therapy development. However, the limited sample availability and sequence homology shared by the VPs pose challenges to adapt existing analytical methods developed for conventional biologics. In this study, we report the development of reversed-phase liquid chromatography/mass spectrometry-based methods for characterization of AAV capsid proteins at intact protein and peptide level with reduced sample consumptions. The developed methods allowed the measurement of VP expression with fluorescence detection and intact mass/post-translational modifications (PTMs) analysis through a benchtop time-of-flight mass spectrometer. The general applicability and validity of the methods for gene therapy product development were demonstrated by applying the optimized methods to multiple common AAV serotypes. A 1-h enzymatic digestion method was also developed using 1.25 μg of AAV VPs, providing >98% protein sequence coverage and reproducible relative quantification of various PTMs of the VPs. The efficient and sensitive analyses of AAV capsid proteins enabled by the reported methods provide further understanding and offer guidance in the development and manufacturing of AAV-related therapeutics.

Smaller particles have progressively led to higher efficiency in liquid chromatography, particularly for proteins, due to smaller diffusion distances. Particle diameter has recently entered the submicrometer region, with the back-pressure requirements alleviated by slip flow.

Protein glycosylation can impact the efficacy and safety of biotherapeutics and therefore needs to be well characterized and monitored throughout the drug product life cycle. Glycosylation is commonly assessed by fluorescent labeling of released glycans, which provides comprehensive information of the glycoprofile but can be resource-intensive regarding sample preparation, data acquisition, and data analysis. In this work, we evaluate a comprehensive solution from sample preparation to data reporting using a liquid chromatography–mass spectrometry (LC-MS)-based analytical platform for increased productivity in released glycan analysis. To minimize user intervention and improve assay robustness, a robotic liquid handling platform was used to automate the release and labeling of N-glycans within 2 h. To further increase the throughput, a 5 min method was developed on a liquid chromatography–fluorescence–mass spectrometry (LC-FLR-MS) system using an integrated glycan library based on retention time and accurate mass. The optimized method was then applied to 48 released glycan samples derived from six batches of infliximab to mimic comparability testing encountered in the development of biopharmaceuticals. Consistent relative abundance of critical species such as high mannose and sialylated glycans was obtained for samples within the same batch (mean percent relative standard deviation [RSD] = 5.3%) with data being acquired, processed, and reported in an automated manner. The data acquisition and analysis of the 48 samples were completed within 6 h, which represents a 90% improvement in throughput compared with conventional LC-FLR-based methods. Together, this workflow facilitates the rapid screening of glycans, which can be deployed at various stages of drug development such as process optimization, bioreactor monitoring, and clone selections, where high-throughput and improved productivity are particularly desired.

Histones are involved in epigenetic control of a wide variety of cellular processes through their multiple post-translational modifications. Their strongly cationic nature makes them challenging to separate with reversed-phase liquid chromatography coupled to mass spectrometry (RPLC-MS), where trifluoroacetic acid is avoided due to adduct formation. Columns with higher resolution are needed. In this work, RPLC-MS is performed on a histone sample using difluoroacetic acid and a 20-minute gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5 μm in diameter, 2) superficially porous particles of 3 μm in diameter with a shell of 0.2 μm. The resolution for the histone separation is better for the latter column, but only when the modifier is trifluoroacetic acid, which is used with UV absorbance detection. When difluoroacetic acid is used for LCMS, the peaks broaden enough to erase the advantage in efficiency for the superficially porous particles. The fully porous and superficially porous cases show similar performance in RPLC-MS, with slightly higher resolution for the fully porous particles. The expected advantage of the shorter diffusion distances for the superficially porous particles is shown to be outweighed by the lower selectivity of its bonded phase.