In situ real time monitoring of emulsification and homogenization processes for vaccine adjuvants.

Adjuvants are commonly employed to enhance the efficacy of a vaccine and thereby increase the resulting immune response in a patient. The activity and effectiveness of emulsion-based adjuvants has been heavily studied throughout pharmaceuticals; however, there exists a lack in research which monitors the formation of a stable emulsion in real time. Process analytical technology (PAT) provides a solution to meet this need. PAT involves the collection of in situ data, thereby providing real time information about the monitored process as well as increasing understanding of that process. Here, three separate PAT tools - optical particle imaging, in situ particle analysis, and Raman spectroscopy - were used to monitor two key steps involved in the formation of a stable emulsion product, emulsification and homogenization, as well as perform a stability assessment. The obtained results provided new insights-particle size decreases during emulsification and homogenization, and molecular changes do not occur during either the emulsification or homogenization steps. Further, the stability assessment indicated that the coarse emulsion product obtained from the emulsification step is stable over the course of 24 hours when mixed. To the best of our knowledge, this is the first report of an analytical methodology for in situ, real time analysis of emulsification and homogenization processes for vaccine adjuvants. Using our proposed analytical methodology, an improved understanding of emulsion-based vaccine adjuvants can now be achieved, ultimately impacting the ability to develop and deliver successful pharmaceuticals.

Single Cell Proteomics by Data-Independent Acquisition To Study Embryonic Asymmetry in Xenopus laevis.

Techniques that allow single cell analysis are gaining widespread attention, and most of these studies utilize genomics-based approaches. While nanofluidic technologies have enabled mass spectrometric analysis of single cells, these measurements have been limited to metabolomics and lipidomic studies. Single cell proteomics has the potential to improve our understanding of intercellular heterogeneity. However, this approach has faced challenges including limited sample availability, as well as a requirement of highly sensitive methods for sample collection, cleanup, and detection. We present a technique to overcome these limitations by combining a micropipette (pulled glass capillary) based sample collection strategy with offline sample preparation and nanoLC-MS/MS to analyze proteins through a bottom-up proteomic strategy. This study explores two types of proteomics data acquisition strategies namely data-dependent (DDA) and data-independent acquisition (DIA). Results from the study indicate DIA to be more sensitive enabling analysis of >1600 proteins from ∼130 µm Xenopus laevis embryonic cells containing <6 nL of cytoplasm. The method was found to be robust in obtaining reproducible protein quantifications from single cells spanning the 1-128-cell stages of development. Furthermore, we used micropipette sampling to study intercellular heterogeneity within cells in a single embryo and investigated embryonic asymmetry along both animal-vegetal and dorsal-ventral axes during early stages of development. Investigation of the animal-vegetal axis led to discovery of various asymmetrically distributed proteins along the animal-vegetal axis. We have further compared the hits found from our proteomic data sets with other studies and validated a few hits using an orthogonal imaging technique. This study forms the first report of vegetal enrichment of the germ plasm associated protein DDX4/VASA in Xenopus embyos. Overall, the method and data presented here holds promise to enable important leads in developmental biology.

Local collection, reaction and analysis with theta pipette emitters.

A mobile nanofluidic device based on theta pipettes was developed for "collect-react-analyze" measurements of small volumes of a sample collected locally from biological samples. Specifically, we demonstrate execution of local reactions inside single cells and on Pseudomonas aeruginosa biofilms for targeted analysis of metabolites. Nanoliter volumes of the sample, post-reaction, were delivered to a mass spectrometer via electrospray ionization (ESI) for chemical analysis. A new strategy was developed where the additional barrel of a theta pipette was utilized both to enable chemical manipulations after sample collection and to electrospray the nanoliter sample volumes collected directly from the pipette tip. This strategy proved a robust method for ESI from nanometer sized tips without clogging or degradation of the emitter and obviated the need to coat glass pipettes with a conductive metal coating. Chemical reactions investigated include acid catalyzed degradation of oligosaccharides inside the pipette tip to increase the detection sensitivity of minor metabolites found in Allium cepa cells. Additionally, phenylboronic acid complexation of carbohydrates from single cells and liposaccharides from biofilms was also performed inside the pipette tip for selective detection of carbohydrates and liposaccharides with cis-diols.

Segmented flow sampling with push-pull theta pipettes.

We report development of a mobile and easy-to-fabricate theta pipette microfluidic device for segmented flow sampling. The theta pipettes were also used as electrospray emitters for analysis of sub-nanoliter segments, which resulted in delivery of analyte to the vacuum inlet of the mass spectrometer without multiple transfer steps. Theta pipette probes enable sample collection with high spatial resolution due to micron or smaller sized probe inlets and can be used to manipulate aqueous segments in the range of 200 pL to tens of nanoliters. Optimized conditions can enable sampling with high spatial and temporal resolution, suitable for chemical monitoring in biological samples and studies of sample heterogeneity. Intercellular heterogeneity among Allium cepa cells was studied by collecting cytoplasm from multiple cells using a single probe. Extracted cytoplasm was analyzed in a fast and high throughput manner by direct electrospray mass spectrometry of segmented sample from the probe tip.

Real-time in situ monitoring of CRM-197 and polysaccharide conjugation reaction by fluorescence spectroscopy.

Aims: Process analytical technology (PAT) is increasingly being adopted within the pharmaceutical industry to build quality into a process. Development of PAT that provides real-time in situ analysis of critical quality attributes are highly desirable for rapid, improved process development. Conjugation of CRM-197 with pneumococcal polysaccharides to produce a desired pneumococcal conjugate vaccine is a significantly intricate process that can tremendously benefit from real-time process monitoring. Methods: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time. Results & conclusion: In this work, a fluorescence-based PAT methodology is described to elucidate CRM-197-polysacharide conjugation kinetics in real time.

Process monitoring of polysaccharide deketalization for vaccine bioconjugation development using in situ analytical methodology.

Pneumococcal conjugate vaccines (PCVs) are formed by bioconjugation of a carrier protein to the purified capsular polysaccharide (Ps) from multiple serological strains of Streptococcus pneumoniae. The associated bioconjugation chemistry relies on initial selective modifications to the Ps backbone structure. Among these modifications, removal of a ketal functional group, termed deketalization, is one that is important for pharmaceutical PCV production. Herein, we report a process monitoring investigation into the deketalization of a polysaccharide relevant to PCV process development. We have applied process analytical technology (PAT) for in situ process monitoring to study the deketalization reaction in real time. We find that in situ FTIR spectroscopy elucidates multiple classes of reaction kinetics, one of which correlates strongly with the deketalization reaction of interest. This PAT approach to real time reaction monitoring offers the possibility of improved process monitoring in the pharmaceutical production of PCVs. To our knowledge, this report represents the first PAT investigation into Ps deketalization. Our findings suggest that broader application of PAT to the chemical modifications associated with PCV bioconjugation, as well as other pharmaceutically relevant bioconjugation processes, carries the power to enhance process understanding, control, and efficiency through real time process monitoring.

Design and Study of PEG Linkers That Enable Robust Characterization of PEGylated Proteins.

Several PEGylated therapeutic proteins are approved drugs, and more are under development. However, the synthesis and characterization of these bioconjugates, especially heterogeneous mixtures of PEGylated proteins, are challenging. The present study focuses on the development of PEG linkers that can be installed through biocatalytic route and render much simpler and insightful analytical characterization of PEG-protein conjugates. This linker enables traditional peptide mapping assay to determine protein sequence coverage, natural PTMs, and PEG attachment sites. Novel PEG linkers are cleavable during traditional sample preparation, leaving behind reporter amino acids to allow the determination of PEG attachment sites by peptide mapping. Products of transglutaminase-catalyzed bioconjugation of 5K PEG to Interferon α-2b were analyzed, and K31, K134, and K164 were identified as the PEGylation sites; the former two being newly determined sites demonstrates the sensitivity of the approach. In another instance, conjugation sites on Interleukin-2-PEG conjugation were found to be K31, K47, K48, and K75.

Bacterial colonization reprograms the neonatal gut metabolome.

Initial microbial colonization and later succession in the gut of human infants are linked to health and disease later in life. The timing of the appearance of the first gut microbiome, and the consequences for the early life metabolome, are just starting to be defined. Here, we evaluated the gut microbiome, proteome and metabolome in 88 African-American newborns using faecal samples collected in the first few days of life. Gut bacteria became detectable using molecular methods by 16 h after birth. Detailed analysis of the three most common species, Escherichia coli, Enterococcus faecalis and Bacteroides vulgatus, did not suggest a genomic signature for neonatal gut colonization. The appearance of bacteria was associated with reduced abundance of approximately 50 human proteins, decreased levels of free amino acids and an increase in products of bacterial fermentation, including acetate and succinate. Using flux balance modelling and in vitro experiments, we provide evidence that fermentation of amino acids provides a mechanism for the initial growth of E. coli, the most common early colonizer, under anaerobic conditions. These results provide a deep characterization of the first microbes in the human gut and show how the biochemical environment is altered by their appearance.

Role of Chloride for a Simple, Non-Grignard Mg Electrolyte in Ether-Based Solvents.

Mg battery operates with Chevrel phase (Mo6S8, ∼1.1 V vs Mg) cathodes that apply Grignard-based or derived electrolytes, which allow etching of the passivating oxide coating forms at the magnesium metal anode. Majority of Mg electrolytes studied to date are focused on developing new synthetic strategies to achieve a better reversible Mg deposition. While most of these electrolytes contain chloride as a component, and there is a lack of literature which investigates the fundamental role of chloride in Mg electrolytes. Further, ease of preparation and potential safety benefits have made simple design of magnesium electrolytes an attractive alternative to traditional air sensitive Grignard reagents-based electrolytes. Work presented here describes simple, non-Grignard magnesium electrolytes composed of magnesium bis(trifluoromethane sulfonyl)imide mixed with magnesium chloride (Mg(TFSI)2-MgCl2) in tetrahydrofuran (THF) and diglyme (G2) that can reversibly plate and strip magnesium. Based on this discovery, the effect of chloride in the electrolyte complex was investigated. Electrochemical properties at different initial mixing ratios of Mg(TFSI)2 and MgCl2 showed an increase of both current density and columbic efficiency for reversible Mg deposition as the fraction content of MgCl2 increased. A decrease in overpotential was observed for rechargeable Mg batteries with electrolytes with increasing MgCl2 concentration, evidenced by the coin cell performance. In this work, the fundamental understanding of the operation mechanisms of rechargeable Mg batteries with the role of chloride content from electrolyte could potentially bring rational design of simple Mg electrolytes for practical Mg battery.

Nanopipettes: probes for local sample analysis.

Nanopipettes (pipettes with diameters <1 µm) were explored as pressure-driven fluid manipulation tools for sampling nanoliter volumes of fluids. The fundamental behavior of fluids confined in the narrow channels of the nanopipette shank was studied to optimize sampling volume and probe geometry. This method was utilized to collect nanoliter volumes (<10 nL) of sample from single Allium cepa cells and live Drosophila melanogaster first instar larvae. Matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) was utilized to characterize the collected sample. The use of nanopipettes for surface sampling of mouse brain tissue sections was also explored. Lipid analyses were performed on mouse brain tissues with spatial resolution of sampling as small as 50 µm. Nanopipettes were shown to be a versatile tool that will find further application in studies of sample heterogeneity and population analysis for a wide range of samples.