Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P50583 (asymmetrical)
 12,197 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The physicochemical properties of nanosized colloidal drug carrier systems are of great influence on drug efficacy. Consequently, a broad spectrum of analytical techniques is applied for comprehensive drug carrier characterization. It is the primary objective of this paper to present asymmetrical flow field-flow fractionation (AF4), coupled online with multiangle light scattering detection, for the characterization of gelatin nanoparticles. Size and size distribution of drug-loaded and unloaded nanoparticles were determined, and data were correlated with results of state-of-the-art methods, such as scanning electron microscopy and photon correlation spectroscopy. Moreover, the AF4 fractionation of gelatin nanoparticulate carriers from a protein model drug is demonstrated for the first time, proposing a feasible way to assess the amount of loaded drug in situ without sample preparation. This hypothesis was set into practice by monitoring the drug loading of nanoparticles with oligonucleotide payloads. In this realm, various fractions of gelatin bulk material were analyzed via AF4 and size-exclusion high-pressure liquid chromatography. Mass distributions and high-molecular-weight fraction ratios of the gelatin samples varied, depending on the separation method applied. In general, the AF4 method demonstrated the ability to comprehensively characterize polymeric gelatin bulk material as well as drug-loaded and unloaded nanoparticles in terms of size, size distribution, molecular weight, and loading efficiency.
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PMID:Asymmetrical flow field-flow fractionation and multiangle light scattering for analysis of gelatin nanoparticle drug carrier systems. 1505 51

Size-exclusion high-performance liquid chromatography (SE-HPLC, SEC) is the long-standing biopharmaceutical industry standard for quantitation of soluble protein aggregates. Recently, sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged as a possible orthogonal technique to SEC for soluble aggregate quantitation. Moreover, asymmetrical flow field flow fractionation (AF4) has shown early promise in quantifying protein aggregates, both soluble and insoluble. We report soluble aggreg ate quantities measured by SEC, AF4, and SV-AUC analyzed by SEDFIT/c(s) for acid stressed and unstressed samples of a recombinant humanized monoclonal antibody. In equivalent antibody samples, SV-AUC, and AF4 detect markedly higher total aggregate levels than SEC. Furthermore, SEC fails to detect higher molecular weight soluble aggregates apparent in SV-AUC and AF4 analyses. Pooled fractions containing soluble dimeric aggregates were purified and re-analyzed by both SV-AUC and SEC. Reinjection of purified dimer onto the SEC column induces formation of detectable quantities of monomer and trimer. All sample types show statistically significant (p-values<0.01) antibody losses through the SEC column. This incomplete mass recovery from SEC indicates probable antibody physical adsorption to gel filtration media. Analysis of the sedimentation behavior of high molecular weight components suggests increased molecular asphericity with increasing molecular weight. We present an aggregation model based on nearly linear end-to-end assembly of monomeric subunits which is shown to be consistent with SV-AUC, SEC, AF4, and dynamic light scattering (DLS) results.
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PMID:Quantitation of aggregate levels in a recombinant humanized monoclonal antibody formulation by size-exclusion chromatography, asymmetrical flow field flow fractionation, and sedimentation velocity. 1708 Apr 24

The PEGylation of colloidal drug carrier systems protects them from a rapid clearance from the blood stream and therefore prolongs their plasma half-lives. This fundamental concept is nowadays widely applied whereas the analytical description, i.e., the quantification of the PEGylation process, is still challenging due to the poor spectrophotometrical properties of PEG. The aim of this work is to quantify the PEGylation process of gelatin nanoparticles by utilizing the combination of asymmetrical flow field-flow fractionation (AF4) and refractive index (RI) detection and to demonstrate the potential of AF4 in the work with colloidal drug carrier systems. An AF4 separation mechanism of gelatin nanoparticles and PEG was developed without further sample preparation. After separation, the PEGylation could be directly quantified from the respective RI data and a threshold of a maximum amount of PEG that can be bound onto the surface of the nanoparticles could be determined. The PEGylation could be further visualized by atomic force microscopy (AFM). In sum, the presented results show the successful application of AF4 in the field of colloidal drug carrier systems, and in combination with AFM, both techniques can be stated as promising tools for the future analysis of colloidal drug carrier systems.
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PMID:Method for quantifying the PEGylation of gelatin nanoparticle drug carrier systems using asymmetrical flow field-flow fractionation and refractive index detection. 1750 21

Chitosan and its derivatives are an important group of polymers used extensively in pharmaceutics. Thus, their physicochemical properties are of considerable interest and need to be characterized carefully. The most important feature to determine is their molecular weight and molecular weight distribution while the molecular weight of polymers plays an important role as pharmaceutical excipients. In this study, the feasibility of using asymmetrical flow field-flow-fractionation (AF4) connected online to a multi-angle light scattering (MALS) detector to measure the molecular weight of chitosans, trimethyl chitosans were studied and compared with the results from some traditional measurement methods. It was found that the influence of trimethyl chitosan synthesis process on the resulting molecular weight decrease depends on the initial molecular weight of chitosan. Significant molecular weight decrease was observed when chitosan molecular weight was larger than 100 kDa. In contrast, the influence was marginal when the molecular weight of chitosan was less than 50 kDa. The AF4-MALS was found to be a suitable method for the characterization of pure chitosans and trimethyl chitosans.
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PMID:Characterization of chitosan and its derivatives using asymmetrical flow field-flow-fractionation: a comparison with traditional methods. 1789 38

This study illustrates the application of asymmetrical flow field-flow fractionation (AF4) and light scattering analysis during the development of a gene delivery vehicle based on virus-like particles (VLPs) derived from the human polyoma JC virus. The analytical system was created by connecting an AF4 apparatus to the following detectors: diode array, fluorescence, multiangle light scattering, dynamic light scattering, and refractometer. From a single analysis, the molar mass, root mean square and hydrodynamic radii, composition, and purity of the sample could be determined. The VLPs were purified from baculovirus-infected Sf158 insect cells overexpressing the recombinant VP1 protein using weak anion exchange chromatography. The VLPs were dissociated to VP1 pentamers, and the contaminating DNA and proteins were removed using strong anion exchange chromatography. The gene delivery vehicle was created by reassembling the VP1 pentamers in the presence of the desired DNA. The newly formed VLPs encapsulated the DNA and were shown to be capable of delivering the gene of interest to target cells where it was translated into protein. This paper describes the scalable process that was derived to produce the VLPs and demonstrates how the AF4-based analytical characterization was indispensable during the development process.
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PMID:Characterization of virus-like particle assembly for DNA delivery using asymmetrical flow field-flow fractionation and light scattering. 1834 13

The aim of this study was to develop an online fluorescent dye detection method suitable for high-pressure size exclusion chromatography (HP-SEC) and asymmetrical flow field flow fractionation (AF4). The noncovalent extrinsic fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (Bis-ANS) was added to the mobile phase or the sample, and the fluorescence emission at 488nm was recorded on excitation at 385nm. By combining HP-SEC and AF4 with online dye detection, it was possible to simultaneously detect heat-induced aggregation and structural changes of monomeric and aggregated immunoglobulin G (IgG); an increase in Bis-ANS fluorescence was observed in both the aggregate and monomer fractions. These structural changes of individual fractions, which were not detectable by online UV and multiangle laser light scattering (MALLS) or by stand-alone dynamic light scattering (DLS), intrinsic IgG fluorescence, and far-UV circular dichroism (CD), resulted in progressive aggregation on storage. The developed online fluorescent dye detection for HP-SEC or AF4 with Bis-ANS is a powerful method to detect both aggregation and structural changes of both monomeric and aggregated IgG in heat-stressed formulations.
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PMID:Online fluorescent dye detection method for the characterization of immunoglobulin G aggregation by size exclusion chromatography and asymmetrical flow field flow fractionation. 1845 94

Virus-like particles (VLPs) have been extensively explored as vaccine candidates since the mid-1980s. Numerous VLPs have been designed as vaccines for prevention of virus-induced infectious diseases and for the therapeutical treatment of chronic diseases and drug addiction. Recently, a vaccine against nicotine addiction, which is based on VLPs of the RNA phage Qb to which nicotine haptens are covalently coupled via succinimate linkers (NicQb), has attracted a great deal of interest. Phase II clinical trials with this vaccine have shown that it is efficacious for smoking cessation in humans when antinicotine antibody levels are sufficiently high. For commercialization, the development of stable formulations enabling storage for prolonged periods is required. Hereby, lyophilization, a well-established method leading to stable and dry formulations, is often applied. In this study, we investigated the influence of different pH values and various excipients such as surfactants, polyols, sugars, and salts on the stability of NicQb in liquid formulations, during freeze thawing, freeze drying, and finally upon storage of the dried product. Lyophilized NicQb formulations were developed which were stable over 6 months at ambient temperature with fully retained biological activity. Hereby, it was found that a combination of the surfactant polysorbate 20 and the disaccharide trehalose was capable to prevent NicQb aggregation and to preserve its integrity (nicotine binding and integrity of VLP shell). Furthermore, asymmetrical flow field-flow fractionation (AF4), a new, promising analytical tool, was established for the investigation of VLP stability.
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PMID:Rational design of a stable, freeze-dried virus-like particle-based vaccine formulation. 1901 59

A dual purpose multilane channel system to carry out isoelectric focusing (IEF) and asymmetrical flow field-flow fractionation (IEF-AFlFFF or IEF-AF4) was developed for the high-speed fractionation of a proteome in two dimensions (2D): isoelectric point (pI) and hydrodynamic diameter (d(s)). Separation of proteins is initially achieved by differences in pI using IEF in an open thin segment, which is formed by interconnecting the beginning part of six parallel flow FFF channels in the lateral direction. After IEF, each protein pool of a different pI interval is simultaneously separated in an orthogonal direction by d(s) in six individual AF4 channels. The developed IEF-AF4 multilane channel system provides ultimate nongel, elution based, and 2D protein separation at an improved separation speed; the entire separation can be processed within 30 min, compared to approximately 3 h with the previously developed capillary isoelectric focusing-hollow fiber FlFFF (CIEF-HFFlFFF or CIEF-HF5) (Kang, D.; Moon, M. H. Anal. Chem. 2006, 78, 5789-5798) or approximately 36 h with 2D-polyacryamide gel electrophoresis (2D-PAGE). An initial evaluation of IEF-AF4 was performed to investigate the influence of ampholyte concentration and IEF voltage on the separation of standard protein mixtures.
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PMID:Development of a multilane channel system for nongel-based two-dimensional protein separations using isoelectric focusing and asymmetrical flow field-flow fractionation. 1916 32

An online multilane channel system for isoelectric focusing and asymmetrical flow field-flow fractionation (IEF-AF4) is utilized for the two-dimensional separation (2D: isoelectric point, pI, and hydrodynamic diameter, d(s)) of a human proteome sample followed by the shotgun proteomic analysis using nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (nLC-ESI-MS-MS). IEF-AF4 was recently developed to carry out nongel-based high speed two-dimensional protein separation [ Kim , K. , et al. Anal. Chem. 2009, 81 , 1715 ]. In IEF-AF4, proteins are separated according to pI along an IEF channel located at the head of six AF4 channels, and then the fractionated protein bands are directed to multilane AF4 channels for size-based separation. In this report, the original IEF-AF4 system has been modified to avoid the possible adsorption of proteins onto the membrane wall of IEF segments during isoelectric focusing by isolating the IEF channel segments from the multilane AF4 channels. The performance of the modified IEF-AF4 system was tested with protein standards and was further applied for the 2D fractionation of the human urinary proteome sample under two ampholyte solutions with different pH ranges (pH 3-10 and 3-6). The entire 2D separation was achieved in less than 30 min. The collected protein fractions were digested for peptide analysis using nLC-ESI-MS-MS, resulting in the identification of 245 total urinary proteins, including 110 unique proteins that are not yet reported in literature. Our experiments also showed a higher efficiency in the identification of urine proteins using ampholyte solution in the narrower pH range.
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PMID:High speed two-dimensional protein separation without gel by isoelectric focusing-asymmetrical flow field flow fractionation: application to urinary proteome. 1965 98

Alterations of lipoproteins (LPs) and related lipid levels in blood serum are correlated to the risk of coronary artery disease (CAD). Fast, possibly automated methods to obtain complete, multi-parametric LP profiles are therefore welcome to be developed for routine, clinical analysis practice. In this work, asymmetrical flow field-flow fractionation (AF4) with on-line, dual post-fractionation reaction detection (PFRD) is applied to develop a method for single-run, simultaneous quantification of cholesterol (CHOL) and triglycerides (TGs) in each fractionated LP class. The enzymatic reagents used for the post-fractionation reaction are available as commercial kits for certified, standard clinical protocols for the analysis of CHOL and TGs in serum. Using CHOL and glycerol as reference standards, a new procedure is applied to optimize the experimental conditions for PFRD-based, quantitative analysis. Upon optimized PFRD and AF4 conditions, results obtained for the determination of total CHOL (TC), TGs, HDL-cholesterol (HDL-C), and LDL-cholesterol (LDL-C) in a set of serum samples from healthy donors are found in agreement with the values provided by a clinical laboratory. The intra-day and inter-day precisions of the method were found always lower than 10% (CV). When the method was applied to serum samples from patients affected by sepsis, differences in CHOL and TG profiles between patients and healthy donors were observed.
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PMID:Enzymatic determination of cholesterol and triglycerides in serum lipoprotein profiles by asymmetrical flow field-flow fractionation with on-line, dual detection. 1985 Jan 70


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