Sedimentation velocity of sodium hyaluronate-lysozyme mixtures

A study of the sedimentation behaviour of lysozyme in sodium hyaluronate (Na-HA) solution and of the Na-HA medium itself, has been carried out to determine whether the strongly basic enzyme lysozyme forms complexes with Na-HA at physiological ionic strength. At typical physiological salt concentration, 0.146 m NaCl, and also in 0.100 M NaCl, lysozyme sedimentation in an Na-HA solution can be adequately described as independent sedimentation of a slightly associated protein through a three-dimensional network acting partially as a macromolecular sieve. The s_20,w of lysozyme when determined in 0.146 M NaCl, indicated partial aggregation of the enzyme at this salt concentration. Decreases in sedimentation coefficients of lysozyme with increase in Na-HA concentration show a pronounced sieving effect by the equality of observed sedimentation coefficient of lysozyme and Na-HA at higher Na-HA concentrations, but typically individual sedimentation coefficients when the macromolecular mixture was diluted approximately ten-fold.     

Sedimentation velocity analytical ultracentrifugation for characterization of therapeutic antibodies

Sedimentation velocity analytical ultracentrifugation (SV-AUC) coupled with direct computational fitting of the observed concentration profiles (sedimentating boundary) have been developed and widely used for the characterization of macromolecules and nanoparticles in solution. In particular, size distribution analysis by SV-AUC has become a reliable and essential approach for the characterization of biopharmaceuticals including therapeutic antibodies. In this review, we describe the importance and advantages of SV-AUC for studying biopharmaceuticals, with an emphasis on strategies for sample preparation, data acquisition, and data analysis. Recent discoveries enabled by AUC with a fluorescence detection system and potential future applications are also discussed.     

X-Ray Characterization of Melanins—I

The intrinsic local structure characterization of natural sepia melanin and L-dopa and tyrosine synthetic melanin powder has been carried out by X-ray diffraction using synchrotron radiation. The derived structure factor, S(q), shows six significant diffuse peaks within the q-range from 0.3 Å^-1 to 16 Å^-1 in the reciprocal space (q= (4π sin θ)/λ, 2θ is the scattering angle). The Fourier transform of S(q), which yields the radial distribution function (RDF), gives us information in real space of a 1.42 Å distance averaged over the C-C, C-O and C-N bond lengths as well as peaks at 2.40-2.41 Å, 3.67-3.71 Å and 4.67-4.70 Å discrete neighbor distances. There is a great similarity in the scattering intensity profiles of the natural and synthetic melanins indicating that the synthetically prepared material may be essentially similar to “real” melanin in its local atomic arrangements. An evidence of a prepeak at q = 0.45 Å^-1 has been confirmed which indicates a preferred length scale of ～ 13-20 Å that corresponds to the initial particle size in colloidal melanin solutions.     

Sedimentation velocity of myxomycete spores

Sedimentation velocities were measured for seven myxomycete species and one fungus. Values for these first measurements for Myxomycetes were fitted with the formula of Stoke’s law for the terminal velocity of small spherical bodies in air. The obtained correlation coefficient of R=0.939 indicates that sedimentation velocities of myxomycete spores follow Stoke’s law well. With spore density as a parameter, the fit estimated a mean density of 0.74 g/cm³ for air-dried spores. The importance of the stalked spore case as well as the spore diameter for dispersal abilities of Myxomycetes is discussed.     

A composite sensor array impedentiometric electronic tongue Part I. Characterization

A study aimed at the characterization of five compounds with different chemical characteristics and gustative perceptions by measuring the variations of the electrical impedance of a composite sensor array is presented. The array was composed of five sensors of three different types based on carbon nanotubes or carbon black dispersed in polymeric matrices and doped polythiophenes. Measurements were carried out by evaluating the electrical impedance of the sensor array at a frequency of 150 Hz, and the data acquisition process was automated; a mechanical arm and a rotating platform controlled by a data acquisition card and a dedicated software allowed the sequential dipping of sensors in the test solutions. Fifty different solutions eliciting the 5 basic tastes (sodium chloride, citric acid, glucose, glutamic acid and sodium dehydrocholate for salty, sour, sweet, umami and bitter, respectively) at 10 concentration levels comprising the human perceptive range were analysed. More than 100 measurements were carried for each sample in a 4-month period to evaluate the system repeatability and robustness. The impedentiometric composite sensor array is shown to be sensitive, selective and stable for use in an electronic tongue.     

Sedimentation velocity separation: a preparation method for cervical samples.

A preparation procedure, aiming at monolayer deposition of cervical exfoliative material on glass slides for high resolution prescreening has been developed. The main features of this procedure are centrifugal deposition after suspension and sedimentation of samples over isopycnic medium of 1.026 density. Fractioning of the separation column after centrifugation at 50 X g yields two preparations with leukocytes, bacteria and cellular debris predominantly located on the first slide and epithelial cells on the second one. The degree of spatial cellular isolation as well as the amount of diagnostically relevant cells per slide seem to fit the requirements of automated high resolution analysis.     

Physical Characterization and Quantification of Bacteria by Sedimentation Field-Flow Fractionation

Studies in microbial ecology require accurate measures of cell number and biomass. Although epifluorescence microscopy is an accepted and dependable method for determining cell numbers, current methods of converting biovolume to biomass are error prone, tedious, and labor-intensive. This paper describes a technique with sedimentation field-flow fractionation to enumerate bacteria and determine their density, size, and mass. Using cultured cells of different shapes and sizes, we determined optimum values for separation run parameters and sample-handling procedures. The technique described can separate and detect 4′, 6-diamidino-2-phenylindole-stained cells and generate a fractogram from which cell numbers and their size or mass distribution can be calculated. A direct method for estimating bacterial biomass (dry organic matter content) which offers distinct advantages over present methods for calculating biomass has been developed.     

Fraction I protein concentration in plants by analytical ultracentrifuge.

The fraction I protein content of plants was determined by centrifuging extracts in the analytical ultracentrifuge until the protein boundary was well separated. The refractive index increase across the boundary was determined with the interference optical system and converted to fraction I protein concentration with the factor 0.245 mg/ml/fringe.     

Sedimentation velocity analytical ultracentrifugation and SEDFIT/c(s): limits of quantitation for a monoclonal antibody system

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has emerged in the biopharmaceutical industry as a technique to detect small quantities of protein aggregates. However, the limits of detection and quantitation of these aggregates are not yet well understood. Although diverse factors (molecule, instrument, technique, and software dependent) preclude an all-encompassing measurement of these limits for the complete system, it is possible to use simulated data to determine the quantitation limits of the data analysis software aspect. The current study examines the performance of the SEDFIT/c(s) data analysis tool with simulated antibody monomer/dimer and monomer/aggregate systems. Under completely ideal conditions (zero noise, known meniscus, and shape factor homogeneity), the software limit of quantitation was 0.01% for the monomer/aggregate system and 0.03% for the less well-resolved monomer/dimer system. Under more realistic conditions (0.005 OD root mean square [RMS] noise, shape factor variability, and long solution column), the software limits of quantitation were 0.2 and 0.6% (0.002 and 0.006 OD) for the monomer/aggregate and monomer/dimer systems, respectively. Interestingly, diminished quantitation accuracy at very low levels of oligomer was not accompanied by deterioration of fit quality (as measured by root mean square deviation [RMSD] and residuals bitmap images).     

Facial recognition — Part 1

Out of all the biometric technologies, the facial recognition sector has received the most attention in recent months. Although the technology is not as accurate as some others, such as fingerprint or iris recognition, it does have unique features that elude other biometric modalities, and that make it an attractive biometric to use in a multitude of scenarios. The first part of this two-part survey looks at the issues surrounding this technology, predicted market sizes and the types of technology on offer. Part two will look in depth at the companies and individual products in this exciting market, as well as listing full contact details for the main players.     

Sedimentation velocity studies on microgram quantities of rat intestinal goblet cell mucin.

A procedure is outlined by which sedimentation analyses of small quantities of mucin glycoproteins can be performed. Rat intestinal goblet cell mucin was stained with periodic acid-Schiff reagent to permit detection by light absorption at 555 nm. PAS treatment resulted in chemical modification of sialic acid and 55% of fucose residues in the mucin. No other chemical or physical alterations were detected. The stained mucus was subjected to band ultracentrifugation using D₂O-containing solvents. Sedimentation was monitored by scanning at 555 nm. Results compared favorably with those reported earlier for conventional boundary ultracentrifugation of intact goblet cell mucin. Because of the low concentrations of mucin used in band ultracentrifugation (0.2–1.5 μg protein/ml), S_20,w values are comparable to sedimentation coefficients at zero concentration (S^o values), determined by conventional means.     

Process analytical technology (PAT) for biopharmaceutical products: Part I. concepts and applications

Process analytical technology (PAT) has been gaining momentum in the biotech community due to the potential for continuous real‐time quality assurance resulting in improved operational control and compliance. In this two part series, we address PAT as it applies to processes that produce biotech therapeutic products. In the first part, we address evolution of the underlying concepts and applications in biopharmaceutical manufacturing. We also present a literature review of applications in the areas of upstream and downstream processing to illustrate how implementation of PAT can help realize advanced approaches to ensuring product quality in real time. In the second part, we will explore similar applications in the areas of drug product manufacturing, rapid microbiology, and chemometrics as well as evolution of PAT in biotech processing. Biotechnol. Bioeng. 2010; 105: 276–284. Published 2009 Wiley Periodicals, Inc.     

X-Ray Characterization of Melanins—II

Structural modeling of amorphous eumelanin has been carried out by comparing calculated data, S(q) and RDF(r), in reciprocal and real space, respectively, for limited random network models with the experimental X-ray scattering data of tyrosine melanin (Cheng et al., 1994). A basic picture of the atomic arrangements in amorphous eumelanin, which accounts for the short and intermediate range order, has been formulated. This reveals domains of a fundamental “particle” dimension of R～ 15 Å, consisting of a paracrystalline array of disordered planar networks polymerized by 4-8 DHI monomers with a graphite-like stacking spacing of ～ 3.45 Å, 4-5 layers thick.