Binary protein mixture separation and purification by a cassette having an internally staged ultrafiltration membrane stack

Streptolysin O (SLO) is a cholesterol-dependent cytolysin produced by the important human pathogen, group A Streptococcus (Streptococcus pyogenes or GAS). In addition to its cytolytic activity, SLO mediates the translocation of GAS NAD-glycohydrolase (NADase) into human epithelial cells in vitro. Production of both NADase and SLO is associated with augmented host cell injury beyond that produced by SLO alone, but the mechanism of enhanced cytotoxicity is not known. We have now shown that expression of NADase together with SLO dramatically enhanced the lytic activity of GAS culture supernatants for erythrocytes but had no effect on SLO-mediated poration of synthetic cholesterol-rich liposomes. This result revealed a previously unknown contribution of NADase to the cytolytic activity associated with GAS production of SLO. Purified recombinant SLO bound NADase in vitro, supporting a specific, physical interaction of the two proteins. Exposure of human keratinocytes to wild-type GAS, but not to a NADase-deficient mutant strain, resulted in profound depletion of cellular NAD+ and ATP. Furthermore, expression of recombinant GAS NADase in yeast, in the absence of SLO, induced growth arrest, depletion of NAD+ and ATP, and cell death. These findings have provided evidence that the augmentation of SLO-mediated cytotoxicity by NADase is a consequence of depletion of host cell energy stores through the enzymatic action of NADase. Together, the results have provided mechanistic insight into the cytotoxic effects of a unique bipartite bacterial toxin.

A new preparative electrophoresis method, multichannel flow electrophoresis (MFE), is proposed in this paper. Mechanisms for different operation modes of MFE are described and a simulation of continuous MFE is proposed. A membrane spaced 5-compartment electrolyzer is developed in which the central compartment and its neighboring compartments are respectively used for sample loading and product discharging. The mass transfer behavior of MFE is investigated by measuring the equivalent migration speed and mobility of bovine serum albumin (BSA) and hemoglobin bovine blood (HBB) at different buffering pH, electric field strength, and sample loading flow rate. The continuous separation of a mixture composed of BSA and HBB, buffered with 0.01 M Tns-HAc at pH 5.9, yields 13.2 mg BSA and 20.0 mg HBB per hour. The application of MFE is exemplified through purification of anti-urokinase mouse IgG. The preliminary results confirm the workability of MFE and point out its promising application potentials in th...

With the recent development of biomaterials and biodevices with reduced dimensionality, it is critical to comprehend protein adhesion processes to nanoscale solid surfaces, especially those occurring in a competitive adsorption environment. Complex sequences of adhesion events in competitive adsorption involving multicomponent protein systems have been extensively investigated, but our understanding is still limited primarily to macroscopic adhesion onto chemically simple surfaces. We examine the competitive adsorption behavior from a binary protein mixture containing bovine serum albumin and fibrinogen at the single protein level. We subsequently evaluate a series of adsorption and displacement processes occurring on both the macroscopic homopolymer and nanoscopic diblock copolymer surfaces, while systematically varying the protein concentration and incubation time. We identify the similarities and dissimilarities in competitive protein adsorption behavior between the two polymeric surfaces, the former presenting chemical uniformity at macroscale versus the latter exhibiting periodic nanointerfaces of chemically alternating polymeric segments. We then present our novel experimental finding of a large increase in the nanointerface-engaged residence time of the initially bound proteins and further explain the origin of this phenomenon manifested on nanoscale diblock copolymer surfaces. The outcomes of this study may provide timely insight into nanoscale competitive protein adsorption that is much needed in designing bioimplant and tissue engineering materials. In addition, the fundamental understanding gained from this study can be beneficial for the development of highly miniaturized biodevices and biomaterials fabricated by using nanoscale polymeric materials and interfaces.

Chemical modification of protein a chromatography ligands with polyethylene glycol. II: Effects on resin robustness and process selectivity

FYWHCLDE-based affinity chromatography of IgG: Effect of ligand density and purifications of human IgG and monoclonal antibody

Efficient downstream processing represents a significant challenge in the rapidly developing field of therapeutic viruses. While it is known that the terminal sterile filtration step can be a major cause of product loss, there is little known about the effect of host cell impurities (DNA and protein) on filtration performance. In this study, fractions of relatively pure Vero host cell protein and DNA were spiked into a highly pure preparation of vesicular stomatitis virus (VSV). Then, the resulting solutions were sterile filtered using two commercially available 0.22 µm rated microfiltration membranes. A combination of transmembrane pressure measurements, virus recovery measurements, and post-filtration microscopy images of the microfiltration membranes was used to evaluate the sterile filtration performance. It was found that increasing the amount of host cell protein from approximately 1 µg/mL (in the un-spiked VSV preparation) to 25 µg/mL resulted in a greater extent of membrane fouling, causing the VSV recovery to decrease from 89% to 65% in experiments conducted with the highly asymmetric Express PLUS PES membrane and to go as low as 48% in experiments conducted with the symmetric Durapore PVDF membrane. Similar effects were not seen when bovine serum albumin, a common model protein used in filtration studies, was spiked into the VSV preparation, which indicates that the sterile filtration performance is critically dependent on the complex composition of the mixture of host cell proteins rather than the presence of any protein. The results presented in this work provide important insights into the role of host cell impurities on the performance of sterile filtration processes for therapeutic viruses.

Glutathione functionalized magnetic 3D covalent organic frameworks combined with molecularly imprinted polymer (magnetic 3D COF–GSH MIPs) were developed for the selective recognition and separation of bovine serum albumin (BSA). Ultrasonication was used to prepare magnetic 3D COFs with high porosity (~1 nm) and a large surface area (373 m2 g−1). The magnetic 3D COF–GSH MIP nanoparticles had an imprinting factor of 4.79, absorption capacity of 429 mg g−1, magnetic susceptibility of 32 emu g−1, and five adsorption–desorption cycles of stability. The proposed method has the advantages of a shorter equilibrium absorption time (1.5 h), higher magnetic susceptibility (32 emu g−1), and larger imprinting factor (4.79) compared with those reported from other studies. The magnetic 3D COF–GSH MIPs used with BSA had selectivity factors of 3.68, 2.76, and 3.30 for lysozyme, ovalbumin, and cytochrome C, respectively. The successful recognition and separation of BSA in a real sample analysis verified the capability of the magnetic 3D COF–GSH MIP nanoparticles.

Improved separation of bovine serum albumin and lactoferrin mixtures using charged ultrafiltration membranes

In this work, a novel chip-based enantioselective open-tubular capillary electrochromatography (OT-CEC) was developed employing bovine serum albumin (BSA)-conjugated graphene oxide–magnetic nanocomposites (GO/Fe3O4) as stationary phase. GO/Fe3O4 nanocomposites with high magnetic responsivity, excellent solubility, and high dispersibility in water were prepared through a facile and controllable in situ chemical deposition strategy. BSA was then adsorbed onto the GO/Fe3O4 surface to form GO/Fe3O4/BSA conjugates, which were then locally packed into PDMS microchannels with the help of magnets. The resultant GO/Fe3O4/BSA conjugates not only have the magnetism of Fe3O4 NPs that make them easily manipulated by an external magnetic field, but also have the larger surface and excellent biocompatibility of graphene, which can incorporate much more biomolecules and well maintain their biological activity. In addition, the successful BSA decorations endowed GO/Fe3O4/BSA conjugates with pH-tunable water solubility related to the isoelectric point of BSA (pI 4.7) and led to enhanced stability against high ionic strength. Compared with the native PDMS microchannel, the modified surfaces exhibited more stable and suppressed electroosmotic mobility and less nonspecific adsorption toward analytes. Successful separation of tryptophan, threonine, and propranolol enantiomers were achieved in less than 80 s with resolution factors of 1.22, 1.9, and 2.1, respectively, utilizing a separation length of 37 mm coupled with in column amperometric detection. The presented on-chip enantioselective OT-CEC protocol simplifies the protein immobilization methodology and has the potential to provide a platform for high-throughput screening of enantiomer candidates.

Low fouling negatively charged hybrid ultrafiltration membranes for protein separation from sulfonated poly(arylene ether sulfone) block copolymer and functionalized multiwalled carbon nanotubes

Novel ultrafiltration membranes with adjustable charge density based on sulfonated poly(arylene ether sulfone) block copolymers and their tunable protein separation performance

Two types of ionic modification approaches (i.e., sulfonation and triethylamination) were applied with the aid of dual-layer hollow fiber technology in this work to fine tune the pore size and pore size distribution, introduce the electrostatic interaction, and reduce membrane fouling for long-term high-performance protein separation. A binary protein mixture comprising bovine serum albumin (BSA) and hemoglobin (Hb) was separated in this work. The sulfonated fiber exhibits an improved BSA/Hb separation factor at pH = 6.8 compared with as-spun fibers but at the expense of BSA sieving coefficient. On the other hand, the triethylaminated fiber reveals the best and most durable separation performance at pH = 4.8. Its BSA/Hb separation factor is maintained above 80 for 4 days and maximum BSA sieving coefficient reaches 33%. Therefore, this study documents that an intelligent combination of both size-exclusion and electrostatic interaction can synergistically enhance protein separation performance in both purity and concentration. © 2008 American Institute of Chemical Engineers AIChE J, 2009

Lipid-based nanoparticles (LNPs) are transforming the field of drug delivery, with one gene therapy product and four mRNA vaccines approved at the time of this report. As with other novel nanomedicines, the development of reliable and standardized methods for evaluating their quality attributes-factors that are essential for quality control and regulatory compliance-is essential to support their bench-to-bedside transition. Frit-Inlet Asymmetric Flow Field-Flow Fractionation (FI-AF4) combined with ultraviolet (UV), multi-angle light scattering (MALS) and dynamic light scattering (DLS) online detectors is widely recognized as a robust and versatile technique for the physicochemical analysis of LNPs. A robust protocol for FI-AF4 method development was established for a mixture of MC3-LNPs and bovine serum albumin (BSA), guided by preliminary particle size and polydispersity assessments using lower-resolution techniques such as DLS and nanoparticle tracking analysis (NTA). The FI-AF4 technique was subsequently applied to characterize particle size and morphology, and to evaluate the most suitable MALS fitmodels across different analytical approaches. The coupling of FI-AF4 with online MALS and DLS enabled the simultaneous separation and online analysis of large sub-populations contained in the sample (methods 2-4), which could not be detected using method 1. The change in particle morphology was found to be significant amongst different methods for each subpopulation (shape factor 0.709-0.793 for LNP peak 1, 0.765-0.853 for LNP peak 2, and 1.069-1.263 for LNP peak 3). MALS fit models indicate that the coated sphere and random coil models were the most appropriate fits for the three LNP sub-populations (R2 > 0.95 and RMSE < 0.009). In summary, implementing the FI-AF4-UV-MALS-DLS protocol provided successful separation of LNPs from protein-containing media with additional analytical approaches not previously described by ISO guidelines. Collecting robust and reproducible information on LNP attributes is key to the several phases of drug development that ideally transform a drug in the pre-clinical phase, from bench-to-bedside.

Porous metal membranes have broad applications in separation processes, membrane reactors, electronic catalysis, and membrane supports. These applications require a high membrane surface area and packing density to achieve high efficiency. Hollow fiber membrane configurations offer such high packing densities and thus significantly enhance the membrane module performance. However, regulating the pore size of metal hollow fiber membranes remains challenging. In this study, the pore size of fabricated nickel (Ni) hollow fiber membranes (NHFMs) was tuned under redox conditions. The influence of the oxidation–reduction temperature on pore morphology and separation performance was systematically investigated. Optimized redox conditions yielded NHFMs with a narrow pore size distribution (mean pore size ∼60 nm, maximum ≤70 nm) and high porosity (∼47%), achieving a N2 permeance of 2.7 × 104 GPU. Additionally, the pore size of NHFMs was tuned to a mean pore size of 162–177 nm, which demonstrated excellent bovine serum albumin (BSA) separation, exhibiting a 96.6% rejection rate and a water flux of 71 L/(m2·h). This work presents a simple strategy for tailoring the pore size of Ni hollow fiber membranes for diverse applications.

Passive scalar transport in turbulent channel flow subject to spanwise system rotation is studied by direct numerical simulations. The Reynolds number $Re=U_{b}h/\unicode[STIX]{x1D708}$ is fixed at 20 000 and the rotation number $Ro=2\unicode[STIX]{x1D6FA}h/U_{b}$ is varied from 0 to 1.2, where $U_{b}$ is the bulk mean velocity, $h$ the half channel gap width and $\unicode[STIX]{x1D6FA}$ the rotation rate. The scalar is constant but different at the two walls, leading to steady scalar transport across the channel. The rotation causes an unstable channel side with relatively strong turbulence and turbulent scalar transport, and a stable channel side with relatively weak turbulence or laminar-like flow, weak turbulent scalar transport but large scalar fluctuations and steep mean scalar gradients. The distinct turbulent–laminar patterns observed at certain $Ro$ on the stable channel side induce similar patterns in the scalar field. The main conclusions of the study are that rotation reduces the similarity between the scalar and velocity field and that the Reynolds analogy for scalar-momentum transport does not hold for rotating turbulent channel flow. This is shown by a reduced correlation between velocity and scalar fluctuations, and a strongly reduced turbulent Prandtl number of less than 0.2 on the unstable channel side away from the wall at higher $Ro$. On the unstable channel side, scalar scales become larger than turbulence scales according to spectra and the turbulent scalar flux vector becomes more aligned with the mean scalar gradient owing to rotation. Budgets in the governing equations of the scalar energy and scalar fluxes are presented and discussed as well as other statistics relevant for turbulence modelling.