Anion exchange membrane adsorbers for flow‐through polishing steps: Part I. clearance of minute virus of mice

Membrane adsorbers may be a viable alternative to the packed‐bed chromatography for clearance of virus, host cell proteins, DNA, and other trace impurities. However, incorporation of membrane adsorbers into manufacturing processes has been slow due to the significant cost associated with obtaining regulatory approval for changes to a manufacturing process. This study has investigated clearance of minute virus of mice (MVM), an 18–22 nm parvovirus recognized by the FDA as a model viral impurity. Virus clearance was obtained using three commercially available anion exchange membrane adsorbers: Sartobind Q®, Mustang Q®, and ChromaSorb®. Unlike earlier studies that have focused on a single or few operating conditions, the aim here was to determine the level of virus clearance under a range of operating conditions that could be encountered in industry. The effects of varying pH, NaCl concentration, flow rate, and other competing anionic species present in the feed were determined. The removal capacity of the Sartobind Q and Mustang Q products, which contain quaternary ammonium based ligands, is sensitive to feed conductivity and pH. At conductivities above about 20 mS/cm, a significant decrease in capacity is observed. The capacity of the ChromaSorb product, which contains primary amine based ligands, is much less affected by ionic strength. However the capacity for binding MVM is significantly reduced in the presence of phosphate ions. These differences may be explained in terms of secondary hydrogen bonding interactions that could occur with primary amine based ligands. Biotechnol. Bioeng. 2013; 110: 491–499. © 2012 Wiley Periodicals, Inc.     

The effects of buffer condition on the fouling behavior of MVM virus filtration of an Fc-fusion protein

A combined pore blockage and cake filtration model was applied to the virus filtration of an Fc-fusion protein using the three commercially available filters, F-1, F-2, and F-3 in a range of buffer conditions including sodium-phosphate and tris-acetate buffers with and without 200 mM NaCl at pH 7.5. The fouling behaviors of the three filters for the feed solutions spiked with minute virus of mice were described well by this combined model for all the solution conditions. This suggests that fouling of the virus filters is dominated by the pore blockage mechanism during the initial stage of the filtration and transformed to the cake filtration mechanism during the later stage of the filtration. Both flux and transmembrane resistance can be described well by this model. The pore blockage rate and the rate of increase of protein layer resistance over blocked pores are found to be affected by membrane properties as well as the solution conditions resulting from the modulation of interactions between virus, protein, and membrane by the solution conditions.     

The Effect of Maturity Status on Biochemical Composition,Antioxidant Activity,and Anthocyanin Biosynthesis Gene Expression in a Pomegranate (Punica granatum L.) Cultivar with Red Flowers,Yellow Peel,and Pinkish Arils

Pomegranate (Punica granatum L.) has widely been used as a fruit and in folk medicine since ancient civilizations in the world. It is now known that bioactive compounds present in pomegranates attribute to its therapeutic potential. Harvesting at the correct maturity stage is one of the key factors deciding the quality of harvest for consumption in fresh or value-added forms. Identification of the correct maturity stage (harvesting index) is particularly difficult for cultivars having yellowish peel and pinkish arils (sarcotesta). We studied the changes in total phenolic content (TPC), antioxidant activity (AOX), punicalagin α and β contents, color indices, total soluble solids (TSS), and expression of anthocyanin biosynthetic pathway genes from flowering to harvesting in the pomegranate cultivar Nimali, having red flowers, yellow peels, and pinkish arils at maturity over two growing seasons. Interestingly, there was no seasonal variation observed in any of the parameters over two cultivation seasons. Although the β punicalagin content did not change, the TPC, AOX, punicalagin α contents in both peel and arils gradually decreased from flowering to maturity. Though the TPC of both peels and arils, AOX and total punicalagin content of peel did not change significantly 140 days after flower initiation, the TPC and the total punicalagin of arils reached a stable level at 160 days. The TSS in both peels and arils increased significantly with the maturity having the highest values at 180 days. The peel color changed from green to yellow with maturity with a significant increase in l* and b* values and significant decrease in a* value. Nevertheless, the aril color changed from pale white to pink with the maturity with significant reduction of l* and b* values and significant increase of a* value. Changes in pomegranate dihydroflavonol 4-reductase (DFR), flavanone 3-hydroxylase (F3H), and leucoanthocyanidin dioxygenase (anthocyanidin synthase-ANS) gene expression in Nimali arils correlated with its color changes during maturity. These findings support to identify the harvesting index of Nimali ensuring the maximum nutritional and health benefits of pomegranate flower, arils, and peels for different downstream uses.

     

Obtaining the shear stress versus shear rate relationship and yield stress of blood from capillary viscometry data by Tikhonov regularization

This paper describes a procedure, based on Tikhonov regularization, for extracting the shear stress versus shear rate relationship and yield stress of blood from capillary viscometry data. The relevant equations and the mathematical nature of the problem are briefly described. The procedure is then applied to three sets of capillary viscometry data of blood taken from the literature. From each data set the procedure computes the complete shear stress versus shear rate relationship and the yield stress. Since the procedure does not rely on any assumed constitutive equation, the computed rheological properties are therefore model-independent. These properties are compared against one another and against independent measurements. They are found to be in good agreement for shear stress greater than 0.1 Pa but show significant deviations for shear stress below this level. A possible way of improving this situation is discussed.     

Types of the cerebral arterial circle (circle of Willis) in a Sri Lankan Population

Background  

The variations of the circle of Willis (CW) are clinically important as patients with effective collateral circulations have a lower risk of transient ischemic attack and stroke than those with ineffective collaterals. The aim of the present cadaveric study was to investigate the anatomical variations of the CW and to compare the frequency of prevalence of the different variations with previous autopsy studies as variations in the anatomy of the CW as a whole have not been studied in the Indian subcontinent.     

Clearance of minute virus of mice by flocculation and microfiltration

Clearance of minute virus of mice (MVM) from CHO cell suspensions by flocculation and microfiltration has been investigated. MVM is a parvovirus that is recommended by the U.S. Food and Drug Administration for validating clearance of parvoviruses. The feed streams were flocculated using a cationic polyelectrolyte. Virus clearance in excess of 10,000-fold was obtained in the bulk permeate for flocculated feeds streams. However, the level of clearance was only about 10- to 100-fold for unflocculated feed streams. The results suggest that virus clearance involves interactions between the MVM particles, the cationic polyelectrolyte, and the CHO cells present. Validating virus clearance is a major concern in the biotechnology industry. New unit operations are frequently added to the purification train simply to validate virus clearance. However, many of these unit operations are less effective at validating clearance of nonenveloped viruses. Validating clearance of parvoviruses is often particularly problematic as they are nonenveloped and the virus particles are small (18 to 24 nm), making physical removal difficult. The results obtained herein indicate that addition of the cationic polyelectrolyte not only results in significant clearance of MVM but also leads to an increase in permeate flux.     

Mass transfer in blood oxygenators using blood analogue fluids

Mass transfer correlations for hollow fiber blood oxygenators have been determined experimentally using Newtonian and non-Newtonian blood analogue fluids. The Newtonian fluids consisted of deionized water and glycerol/water mixtures. The non-Newtonian fluids were prepared by adding small amounts of xanthan gum to the Newtonian blood analogue fluids. The rheological behavior of the non-Newtonian blood analogue fluids was modeled using the power law. The diffusion of oxygen into and out of the Newtonian and non-Newtonian blood analogue fluids has been studied. The liquid stream flowed outside and across bundles of woven hollow fibers, while the gas stream flowed inside the fibers.     

Resource partitioning among the fishes of rainforest streams in Sri Lanka

The fish assemblage typical of small rainforest streams of Sri Lanka was investigated to see if the fishes were characterized by a high degree of specialization, expected of equilibrium communities, or if they were relatively unspecialized, expected of more stochastic (non-equilibrium) communities. Morphological features, habitats, microhabitats and diets of the 20 most abundant fish species were measured. The fishes included 11 Cyprinidae (including seven Barbus spp. and two Rasbara spp.), two Gobiidae, two Channidae, and one species each in the Cobitidae, Cyprinodontidae, Belontidae, Belonidae, and Mastacembelidae. Microhabitats were determined by measuring (for 6760 fish) water column depth, distance of fish from bottom, mean water column velocity, water velocity at fish, and substrate. Morphologically, the species showed a high degree of specialization, especially in structures related to feeding. Although several species were habitat generalists, most species occurred in distinct habitats. Within habitats, microhabitat overlap among co-occuring species was low, particularly in relation to position in the water column. Principal component analysis of the microhabitat measurements produced three new variables. Most species not clearly segregated by the overlap analysis showed segregation on the new variables. Fishes not segregated by habitat or microhabitat tended to show low dietary overlaps. Specialization in feeding habits was more pronounced among the Sri Lankan fishes than noted for fishes in small rainforest streams elsewhere, in part because of the greater reliance of the Sri Lanka fishes on autochthonous foods. Overall, the fish assemblage had the characteristics expected of an equilibrium (deterministic) assemblage.     

Enhanced virus removal by flocculation and microfiltration

In this work we have investigated the feasibility of virus clearance by flocculation and tangential flow microfiltration. Chinese hamster ovary cell feed streams were spiked with minute virus of mice and then flocculated using cationic polyelectrolytes prior to tangential flow microfiltration. Our results indicate that flocculation prior to microfiltration leads to more than 100 fold clearance of minute virus of mice particles in the permeate. Today, validation of virus clearance is a major concern in the manufacture of biopharmaceutical products. Frequently new unit operations are added simply to validate virus clearance thus increasing the manufacturing cost. The results obtained here suggest that virus clearance can be obtained during tangential flow microfiltration. Since tangential flow microfiltration is frequently used for bioreactor harvesting this could be a low cost method to validate virus clearance.     

Use of scanning electron microscopy and energy dispersive X-ray spectroscopy to identify key fouling species during alternating tangential filtration

Alternating tangential flow filtration (ATF) has become one of the primary methods for cell retention and clarification in perfusion bioreactors. However, membrane fouling can cause product sieving losses that limit the performance of these systems. This study used scanning electron microscopy and energy dispersive X-ray spectroscopy to identify the nature and location of foulants on 0.2 μm polyethersulfone hollow fiber membranes after use in industrial Chinese hamster ovary cell perfusion bioreactors for monoclonal antibody production. Membrane fouling was dominated by proteinaceous material, primarily host cell proteins along with some monoclonal antibody. Fouling occurred primarily on the lumen surface with much less protein trapped within the depth of the fiber. Protein deposition was also most pronounced near the inlet/exit of the hollow fibers, which are the regions with the greatest flux (and transmembrane pressure) during the cyclical operation of the ATF. These results provide important insights into the underlying phenomena governing the fouling behavior of ATF systems for continuous bioprocessing.