Cryo-EM reveals the molecular basis oflaminin polymerization and LN-lamininopathies.

Laminin polymerization is the major step in basement membranes assembly. Its failures cause laminin N-terminal domain lamininopathies including Pierson syndrome. We have employed cryo-electron microscopy to determine a 3.7 Å structure of the trimeric laminin polymer node containing α1, ß1 and γ1 subunits. The structure reveals the molecular basis of calcium-dependent formation of laminin lattice, and provides insights into polymerization defects manifesting in human disease.

Optimized Reversed-Phase Liquid Chromatography/Mass Spectrometry Methods for Intact Protein Analysis and Peptide Mapping of Adeno-Associated Virus Proteins.

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.

Routine Analysis of N-Glycans Using Liquid Chromatography Coupled to Routine Mass Detection.

Analysis of N-glycans are commonly conducted via enzymatic release, labeling, and liquid chromatography (LC) separation and fluorescent detection. Mass spectrometry (MS) has been increasingly used as an orthogonal detection method to provide additional structural information and increase the confidence of N-glycan analysis. In this chapter, we describe a method to perform routine analysis of N-glycans including the sample preparation with a signal-enhancement label, LC-MS data generation, and data analysis. Using this method, up to 24 N-glycan samples can be prepared at one time and analyzed by LC-MS. With the addition of automation platform, up to 96 N-glycan samples can be prepared and analyzed in a high-throughput manner.

Released N-Glycan Analysis for Biotherapeutic Development Using Liquid Chromatography and Mass Spectrometry.

In this chapter, we describe an LC-fluorescence (FLR)/MS-based method for released N-glycan analysis in the development of biotherapeutic proteins. The method includes enzymatic release and labeling of N-glycans with a signal-enhancing tag, LC-MS data collection, and data interpretation. Using the given protocol, up to 24 glycan samples can be prepared within 1 h, while the LC-FLR/MS data can be collected and analyzed using an established data processing method in a semi-automated manner.

High-Throughput Analysis of Fluorescently Labeled N-Glycans Derived from Biotherapeutics Using an Automated LC-MS-Based Solution.

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.

Chromatographic efficiency and selectivity in top-down proteomics of histones.

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-min gradient. Columns with C18 surfaces are compared for two different types of particle morphologies: 1) fully porous particles of 5µm in diameter, 2) superficially porous particles of 3µm in diameter with a shell of 0.2µm. 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.

Submicrometer particles and slip flow in liquid chromatography.

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.

Efficient separations of intact proteins using slip-flow with nano-liquid chromatography-mass spectrometry.

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.

One-pot colloidal chemistry route to homogeneous and doped colloidosomes.

Colloidosomes are usually produced from a series of building blocks with different sizes ranging from several nanometers to micrometers or various shapes, such as particles, microrods, and quantum dots. Colloidosomes can possess a variety of characteristics in terms of photics, electrology, mechanical strength, and selective permeability, derived from their building blocks. However, poor mechanical stability and complicated synthesis processes have limited the applications of colloidosomes. Here, we report a new one-pot colloidal chemistry route to synthesize phenol formaldehyde resin (PFR), Ag@PFR, and Au@PFR colloidosomes with high yields. The method can be modified to synthesize different kinds of doped colloidosomes with different components, which will provide a new approach to design colloidosomes with different functions.