Accelerating attribute-focused process and product development through the development and deployment of autonomous process analytical technology platform system.

Enabling real-time monitoring and control of the biomanufacturing processes through product quality insights continues to be an area of focus in the biopharmaceutical industry. The goal is to manufacture products with the desired quality attributes. To realize this rigorous attribute-focused Quality by Design approach, it is critical to support the development of processes that consistently deliver high-quality products and facilitate product commercialization. Time delays associated with offline analytical testing can limit the speed of process development. Thus, developing and deploying analytical technology is necessary to accelerate process development. In this study, we have developed the micro sequential injection process analyzer and the automatic assay preparation platform system. These innovations address the unmet need for an automatic, online, real-time sample acquisition and preparation platform system for in-process monitoring, control, and release of biopharmaceuticals. These systems can also be deployed in laboratory areas as an offline analytical system and on the manufacturing floor to enable rapid testing and release of products manufactured in a good manufacturing practice environment.

A DNA aptamer for binding and inhibition of DNA methyltransferase 1.

DNA methyltransferases (DNMTs) are enzymes responsible for establishing and maintaining DNA methylation in cells. DNMT inhibition is actively pursued in cancer treatment, dominantly through the formation of irreversible covalent complexes between small molecular compounds and DNMTs that suffers from low efficacy and high cytotoxicity, as well as no selectivity towards different DNMTs. Herein, we discover aptamers against the maintenance DNA methyltransferase, DNMT1, by coupling Asymmetrical Flow Field-Flow Fractionation (AF4) with Systematic Evolution of Ligands by EXponential enrichment (SELEX). One of the identified aptamers, Apt. #9, contains a stem-loop structure, and can displace the hemi-methylated DNA duplex, the native substrate of DNMT1, off the protein on sub-micromolar scale, leading for effective enzymatic inhibition. Apt. #9 shows no inhibition nor binding activity towards two de novo DNMTs, DNMT3A and DNMT3B. Intriguingly, it can enter cancer cells with over-expression of DNMT1, colocalize with DNMT1 inside the nuclei, and inhibit the activity of DNMT1 in cells. This study opens the possibility of exploring the aptameric DNMT inhibitors being a new cancer therapeutic approach, by modulating DNMT activity selectively through reversible interaction. The aptamers could also be valuable tools for study of the functions of DNMTs and the related epigenetic mechanisms.

Mapping Molecular Structure of Protein Locating on Nanoparticles with Limited Proteolysis.

The molecular structure of a protein could be altered when it is attached to nanoparticles (NPs), affecting the performance of NPs present in biological systems. Limited proteolysis coupled with LC-MS/MS could reveal the changes in protein structure when it binds to a variety of entities, including macro-molecules and small drugs, but it has not yet been applied to study protein-NP interaction. Herein, adsorption of proteins, transferrin, and catalase on the polystyrene (PS) or iron oxide (IO) NPs was analyzed with this method. Both increased and decreased proteolytic efficiency in certain regions on the proteins were observed. Identification of the peptides affected by protein-NP interaction led to proper prediction of alterations to protein function as well as to colloidal stability of NPs. Overall, the present work has demonstrated the utility of limited proteolysis in helping to elucidate the potential biological outcomes of the protein-NP conjugate, obtaining knowledge to guide improvement of the rational design of the protein-conjugated NPs for biomedical applications and to understand the biological behaviors of the engineered NPs.

Highly Efficient Exosome Isolation and Protein Analysis by an Integrated Nanomaterial-Based Platform.

Exosomes play important roles in mediating intercellular communication and regulating a variety of biological processes, but clear understanding of their functions and biogenesis has not been achieved, due to the high technical difficulties involved in analysis of small vesicular structures that contain a high proportion of membrane structures. Herein, we designed a novel approach to integrate two nanomaterials carrying varied surface properties, the hydrophilic, macroporous graphene foam (GF) and the amphiphilic periodic mesoporous organosilica (PMO), for efficient exosome isolation from human serum and effective protein profiling. The high specific surface area of GF, after modification with the antibody against the exosomal protein marker, CD63, allowed highly specific isolation of exosomes from complex biological samples with high recovery. Since the organic solvent, methanol, turned out to be the most effective lysis solution for releasing the exosomal proteins, the amphiphilic PMO was employed to rapidly recover the exosomal proteins, including the highly hydrophobic membrane proteins. The fine pores of PMO also acted as the nanoreactors to accelerate protein digestion that produced peptides subject to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. A total of 334 proteins with 111 membrane proteins [31% of these contained >2 transmembrane domains (TMD)] were identified using the integrated GF/PMO platform. In contrast, with the commercial exosome isolation kit and the in-solution protein digestion method, only 151 proteins were found, with 28 being membrane proteins (only one contained three TMDs). Our results support that the integrated GF/PMO platform is of great value to facilitate the comprehensive characterization of exosomal proteins for better understanding of their functions and for identification of more exosome-based disease markers.

A Single Extracellular Vesicle (EV) Flow Cytometry Approach to Reveal EV Heterogeneity.

Extracellular vesicles (EVs) actively participate in intercellular communication and pathological processes. Studying the molecular signatures of EVs is key to reveal their biological functions and clinical values, which, however, is greatly hindered by their sub-100 nm dimensions, the low quantities of biomolecules each EV carries, and the large population heterogeneity. Now, single-EV flow cytometry analysis is introduced to realize single EV counting and phenotyping in a conventional flow cytometer for the first time, enabled by target-initiated engineering (TIE) of DNA nanostructures on each EV. By illuminating multiple markers on single EVs, statistically significant differences are revealed among the molecular signatures of EVs originating from several breast cancer cell lines, and the cancer cell-derived EVs among the heterogeneous EV populations are successfully recognized. Thus, our approach holds great potential for various biological and biomedical applications.

Fluorescamine Labeling for Assessment of Protein Conformational Change and Binding Affinity in Protein-Nanoparticle Interaction.

Protein adsorption alters the "biological identity" of nanoparticles (NPs) and could affect how biosystems respond to invading NPs. Study of protein-NP interaction can help understand how the physicochemical properties of NPs impact the interaction and thus potentially guide the design of safer and more effective NPs for biomedical or other applications. Binding affinity between proteins and NPs and the occurrence of protein conformational change upon binding to NPs are two important aspects to be learned, but few methods are currently available to assess both simultaneously in a simple way. Herein, we demonstrated that the fluorescamine labeling method developed by our group not only could reveal protein conformational change upon adsorption to NPs, owing to its capability to label the primary amines exposed on protein surface, but also could be applied to measure the binding affinity. By screening the interaction between a large number of proteins and four types of NPs, the present study also revealed that protein adsorption onto NPs could be strongly affected by structure flexibility. The proteins with high structure flexibility experienced high degrees of conformation change when binding to the polystyrene NPs, which could potentially influence protein function. Overall, we demonstrate that our assay is a quick, simple, and high-throughput tool to reveal potential impacts on protein activity and evaluate the strength of protein-NP binding.

Rapid Enrichment and Sensitive Detection of Multiple Metal Ions Enabled by Macroporous Graphene Foam.

Nanomaterials have shown great promise in advancing biomedical and environmental analysis because of the unique properties originated from their ultrafine dimensions. In general, nanomaterials are separately applied to either enhance detection by producing strong signals upon target recognition or to specifically extract analytes taking advantage of their high specific surface area. Herein, we report a dual-functional nanomaterial-based platform that can simultaneously enrich and enable sensitive detection of multiple metal ions. The macroporous graphene foam (GF) we prepared displays abundant phosphate groups on the surface and can extract divalent metal ions via metal-phosphate coordination. The enriched metal ions then activate the metal-responsive DNAzymes and produce the fluorescently labeled single-stranded DNAs that are adsorbed and quenched by the GF. The resultant fluorescence reduction can be used for metal quantitation. The present work demonstrated duplexed detection of Pb2+ and Cu2+ using the Pb- and Cu-responsive DNAzymes, achieving a low detection limit of 50 pM and 0.6 nM, respectively. Successful quantification of Pb2+ and Cu2+ in human serum and river water were achieved with high metal recovery. Since the phosphate-decorated GF can enrich diverse types of divalent metal cations, this dual-functional GF-DNAzyme platform can serve as a simple and cost-effective tool for rapid and accurate metal quantification in determination of human metal exposure and inspection of environmental contamination.

Photochemical Bionanoreactor for Efficient Visible-Light-Driven in Vitro Drug Metabolism.

In light of the significance of cytochrome P450 (CYP) catalyzed drug metabolism for drug development and toxicity screening, it is very important to imitate natural metabolic pathways accurately and efficiently in vitro. Herein, a novel and simple photochemical bionanoreactor has been constructed for efficient visible-light-driven in vitro drug metabolism based on eosin-Y-functionalized macroporous ordered silica foams (MOSF-EY). Because of the unique transfer of photoinduced electrons from photosensitizers to CYP heme domain, CYP catalyzed drug metabolism can be in vitro driven by the MOSF-EY nanoreactor under the irradiation of visible light. In such a case, the utilization of expensive electron donors, such as NADPH, can be avoided. Meanwhile, the in vitro drug metabolism approach exhibits high efficiency because of the fast adsorption of both CYP and drug molecules from the bulk solution into the nanopores of MOSF-EY, where the enzyme and substrate are highly concentrated and confined in nanospace to achieve a high reaction rate. Taking advantage of these attractive merits, the first example of photochemical bionanoreactor has been successfully applied in in vitro metabolism of both purified drug molecules and real tablets. Not only excellent CYP-catalyzed drug metabolism but also enzyme inhibition assay has been performed with the MOSF-EY photochemical bionanoreactor.

High-throughput profiling of nanoparticle-protein interactions by fluorescamine labeling.

A rapid, high throughput fluorescence assay was designed to screen interactions between proteins and nanoparticles. The assay employs fluorescamine, a primary-amine specific fluorogenic dye, to label proteins. Because fluorescamine could specifically target the surface amines on proteins, a conformational change of the protein upon interaction with nanoparticles will result in a change in fluorescence. In the present study, the assay was applied to test the interactions between a selection of proteins and nanoparticles made of polystyrene, silica, or iron oxide. The particles were also different in their hydrodynamic diameter, synthesis procedure, or surface modification. Significant labeling differences were detected when the same protein incubated with different particles. Principal component analysis (PCA) on the collected fluorescence profiles revealed clear grouping effects of the particles based on their properties. The results prove that fluorescamine labeling is capable of detecting protein-nanoparticle interactions, and the resulting fluorescence profile is sensitive to differences in nanoparticle's physical properties. The assay can be carried out in a high-throughput manner, and is rapid with low operation cost. Thus, it is well suited for evaluating interactions between a larger number of proteins and nanoparticles. Such assessment can help to improve our understanding on the molecular basis that governs the biological behaviors of nanomaterials. It will also be useful for initial examination of the bioactivity and reproducibility of nanomaterials employed in biomedical fields.

Distribution profiling of circulating microRNAs in serum.

Circulating microRNAs (miRNAs) are potential biomarkers useful in cancer diagnosis. They have been found to be bound to various carriers like proteins, lipoprotein particles, and exosomes. It is likely that only miRNAs in particular carriers, but not the overall quantity, are directly related to cancer development. Herein, we developed a method for rapid separation of different miRNA carriers in serum using asymmetrical flow field flow fractionation (AF4). Sera from two healthy individuals (control) or from two cancer patients (case) were fractionated. Six fractions enriching different types of miRNA carriers, such as the lipoprotein particles and exosomes, were collected. The quantities of eight selected miRNAs in each fraction were obtained by RT-qPCR to yield their distribution profiles among the carriers. Larger changes in miRNA quantity between the control and the case were detected in the fractionated results compared to the sum values. Statistical analysis on the distribution profiles also proved that, the quantities of 4 miRNAs within particular fractions showed significant difference between the controls and the cases. On the contrary, if the overall quantity of the miRNA was subject to the same statistical analysis, only 2 miRNAs exhibited significant difference. Moreover, principle component analysis revealed good separation between the controls and the cases with the fractionated miRNA amounts. All in all, we have demonstrated that, our method enables comprehensive screening of the distribution of circulating miRNAs in the carriers. The obtained distribution profile enlarges the miRNA expression difference between healthy individuals and cancer patients, facilitating the discovery of specific miRNA biomarkers for cancer diagnosis.

Prediction of protein corona on nanomaterials by machine learning using novel descriptors.

Effective in silico methods to predict protein corona compositions on engineered nanomaterials (ENMs) could help elucidate the biological outcomes of ENMs in biosystems without the need for conducting lengthy experiments for corona characterization. However, the physicochemical properties of ENMs, used as the descriptors in current modeling methods, are insufficient to represent the complex interactions between ENMs and proteins. Herein, we utilized the fluorescence change (FC) from fluorescamine labeling on a protein, with or without the presence of the ENM, as a novel descriptor of the ENM to build machine learning models for corona formation. FCs were significantly correlated with the abundance of the corresponding proteins in the corona on diverse classes of ENMs, including metal and metal oxides, nanocellulose, and 2D ENMs. Prediction models established by the random forest algorithm using FCs as the ENM descriptors showed better performance than the conventional descriptors, such as ENM size and surface charge, in the prediction of corona formation. Moreover, they were able to predict protein corona formation on ENMs with very heterogeneous properties. We believe this novel descriptor can improve in silico studies of corona formation, leading to a better understanding on the protein adsorption behaviors of diverse ENMs in different biological matrices. Such information is essential for gaining a comprehensive view of how ENMs interact with biological systems in ENM safety and sustainability assessments.

A supramolecular sensor array for selective immunoglobulin deficiency analysis.

A host-guest based fluorescence sensor array can sense small differences in protein structure. The combination of three cavitand hosts and two fluorophores to form a 4-component array is sufficient to fully discriminate five structurally similar Ig protein isotypes. The array can be applied to recognize Ig deficiencies in serum, when combined with a Protein L-based extraction process, allowing analysis of immunodeficiency in a simpler, lower cost manner than tests that require multiple specific antibodies.

Metal-assisted selective recognition of biothiols by a synthetic receptor array.

A synergistic combination of a deep cavitand host, fluorophore guests and transition metal ions can be used to sense small molecule thiols of biological interest with good efficiency and selectivity in complex aqueous media.

Encapsulation of ionic nanoparticles produces reactive oxygen species (ROS)-responsive microgel useful for molecular detection.

Encapsulation of ionic nanoparticles in a hydrogel microparticle, i.e. microgel, produces a target-stimulated probe for molecular detection. Selective reactive oxygen species (ROS) release the enclosed cations from the microgel which subsequently turn on the fluorogenic dyes to emit intense fluorescence, permitting rapid detection of ROS or ROS-producing molecules. The ROS-responsive microgel provides the advantages of simple fabrication, bright and stable signals, easy handling, and rapid response, carrying great promise in biomedical applications.

ZrO2 Nanofiber as a Versatile Tool for Protein Analysis.

Phosphorylation is one of the most important post-translational modifications in proteins. Their essential roles in the regulation of cellular processes and alteration of protein-protein interaction networks have been actively studied. However, phosphorylated proteins are present at low abundance in cells, and ionization of the modified peptides is often suppressed by the more abundant species in mass spectrometry. Effective enrichment techniques are needed to remove the unmodified peptides and concentrate the phosphorylated ones before their identification and quantification. Herein, we prepared ZrO2 nanofibers by electrospinning, a straightforward and easy fabrication technique, and applied them to enrich phosphorylated peptides and proteins. The fibers showed good size homogeneity and porosity and could specifically bind to the phosphorylated peptides and proteins, allowing their separation from the unmodified analogues when present in either simple protein digests or highly complex cell lysates. The enrichment performance was superior to that of the commercially available nanoparticles. Moreover, modifying the solution pH could lead to selective adsorption of proteins with different pI values, suggesting the fibers' potential applicability in charge-based protein fractionation. Our results support that the electrospun ZrO2 nanofibers can serve as a versatile tool for protein analysis with great ease in preparation and handling.

Probing and quantifying DNA-protein interactions with asymmetrical flow field-flow fractionation.

Tools capable of measuring binding affinities as well as amenable to downstream sequencing analysis are needed for study of DNA-protein interaction, particularly in discovery of new DNA sequences with affinity to diverse targets. Asymmetrical flow field-flow fractionation (AF4) is an open-channel separation technique that eliminates interference from column packing to the non-covalently bound complex and could potentially be applied for study of macromolecular interaction. The recovery and elution behaviors of the poly(dA)n strand and aptamers in AF4 were investigated. Good recovery of ssDNAs was achieved by judicious selection of the channel membrane with consideration of the membrane pore diameter and the radius of gyration (Rg) of the ssDNA, which was obtained with the aid of a Molecular Dynamics tool. The Rg values were also used to assess the folding situation of aptamers based on their migration times in AF4. The interactions between two ssDNA aptamers and their respective protein components were investigated. Using AF4, near-baseline resolution between the free and protein-bound aptamer fractions could be obtained. With this information, dissociation constants of ∼16nM and ∼57nM were obtained for an IgE aptamer and a streptavidin aptamer, respectively. In addition, free and protein-bound IgE aptamer was extracted from the AF4 eluate and amplified, illustrating the potential of AF4 in screening ssDNAs with high affinity to targets. Our results demonstrate that AF4 is an effective tool holding several advantages over the existing techniques and should be useful for study of diverse macromolecular interaction systems.

Identification of key licorice constituents which interact with cytochrome P450: evaluation by LC/MS/MS cocktail assay and metabolic profiling.

Licorice has been shown to affect the activities of several cytochrome P450 enzymes. This study aims to identify the key constituents in licorice which may affect these activities. Bioactivity assay was combined with metabolic profiling to identify these compounds in several complex licorice extracts. Firstly, the inhibition potencies of 40 pure licorice compounds were tested using an liquid chromatography/tandem mass spectrometry cocktail method. Significant inhibitors of human P450 isozymes 1A2, 2C9, 2C19, 2D6, and 3A4 were then selected for examination of their structural features by molecular docking to determine their molecular interaction with several P450 isozymes. Based on the present in vitro inhibition findings, along with our previous in vivo metabolic studies and the prevalence of individual compounds in licorice extract, we identified several licorice constituents, viz., liquiritigenin, isoliquiritigenin, together with seven isoprenylated flavonoids and arylcoumarins, which could be key components responsible for the herb-drug interaction between cytochrome P450 and licorice. In addition, hydrophilic flavonoid glycosides and saponins may be converted into these P450 inhibitors in vivo. These studies represent a comprehensive examination of the potential effects of licorice components on the metabolic activities of P450 enzymes.

Investigation of the binding sites and orientation of caffeine on human serum albumin by surface-enhanced Raman scattering and molecular docking.

Fluorescence, normal Raman and surface-enhanced Raman scattering (SERS) were introduced to explore the absorptive geometry of caffeine on Human Serum Albumin (HSA) at physiological condition. The molecular docking was also employed to make a better understanding of the interaction between caffeine and HSA as well as to elucidate the detailed information of the major binding site. The results showed that caffeine could bind to HSA via the hydrophobic force of aromatic stacking and the main binding group on caffeine could be the pyrimidine ring. In addition, a consecutive set of changes in the orientation of caffeine molecule had been demonstrated during the process of caffeine binding to HSA, and the primary binding site was considered to be a hydrophobic cavity formed by Leu198, Lys199, Ser202, Phe211, Trp214, Val344, Ser454 and Leu481 in domain II.