The influence of mixing on lysozyme renaturation during refolding in an oscillatory flow and a stirred-tank reactor

Protein refolding is a key unit operation in many processes that produce recombinant biopharmaceuticals using Escherichia coli. Yield in this step generally controls overall process yield, and at industrially relevant protein concentrations is limited by aggregation. While most refolding operations are optimised with respect to chemical environment, the physical processes affecting yield have been neglected. In this study, we demonstrate that refolding yield for the model protein lysozyme is dependent on mixing intensity during dilution refolding. This is shown for two different reactor configurations: a standard stirred-tank reactor and a novel oscillatory flow reactor. We further show that the effect of mixing is dependent on the type of chaotrope employed for denaturation. Yield falls significantly when mixing intensity is decreased following urea denaturation, while the effect of mixing is not apparent when guanidine hydrochloride is employed as the denaturant. In batch tests we further confirm that, for urea, the “path” of dilution affects yield, and hence the observed sensitivity to mixing is not unexpected. We conclude that mixing is a critical parameter that must be optimised in industrial reactors, along with the usual chemical and protein-specific parameters.     

Comparison of axial and radial flow chromatography on protein separation speed and resolution

Fluid dynamic behavior, particularly the relationship between pressure drop and liquid flowrate, inside an axial and a radial flow Chromatographic column packed with compressible porous media was theoretically analyzed using the modified Kozeny-Carman equation. The results were compared with experimental observations obtained using compressible DEAE-agarose as a model medium. At the 2–9 psi pressure drop range studied, theoretical derivation accounting for the gel compression’ effect predicted simple Langmuirian-type response of volumetric flowrate to changes in pressure drop. On the other hand, the experimental result was more or less sigmoidal. At the same pressure drop, radial column yielded 2–3 times higher flowrates than those of axial column both theoretically and experimentally. Using r-HBsAg crude extract, protein resolution effects between the two types of columns at various flowrates were compared side-by-side. Though general Chromatographic behaviors were very similar, the axial column was somewhat superior in terms of rHBsAg recovery yield and specificity. However, the number of theoretical plates analysis indicated the protein resolution effects were comparable.     

The effect of temperature on the flow field in a continuous rotating annular electrophoresis column

The effect of a non-uniform temperature profile on the flow field in a continuous rotating annular electrophoresis column is investigated. The momentum balance equation is solved for packed as well as unpacked columns using a centered-difference finite difference technique with a known temperature profile. The packed column analysis utilizes a parallel annular channel model similar to the capillary channel model used in packed bed columns. Results indicate that the changes in density and viscosity as result of the temperature variation have a significant effect on the flow field. Effects of voltage gradient and column geometry are also discussed.     

Effects of separation space diameter on the performance of a novel reverse flow cyclone

ABSTRACT

A numerical study was carried out to investigate the effect of separation space diameter on the performance of a novel reverse flow tangential inlet cyclone design by using the Eulerian-Lagrangian approach. The design of this cyclone is based on the idea of increasing vortex length and decreasing pressure drop compared with traditional cyclones. This novel cyclone differs from the traditional cyclones with separation space and vortex limiter instead of the conical part. A qualitative numerical study was performed to analyze the effect of separation space diameter on the cyclone performance at different flow rates by evaluating velocity profile, pressure drop, fractional and overall efficiencies. The results show that the collection efficiency of smaller particles increases while pressure drop decreases significantly with the increase in separation space diameter for D₁/D < 0.5.     

Effects of double-layer polarization and electroosmotic flow on the electrophoresis of a finite cylinder along the axis of a cylindrical pore

The electrophoresis of a rigid, finite cylindrical particle along the axis of a long cylindrical pore is analyzed theoretically under the conditions of arbitrary surface potential and double-layer thickness. The effects of double-layer polarization and electroosmotic flow on the electrophoretic behavior of the particle are discussed. We show that if both the particle and the pore are positively charged, the mobility of the particle has a local minimum as the thickness of double layer varies. Also, if the level of the surface potential of the particle is sufficiently high and its aspect ratio is sufficiently large, then its mobility may change its sign twice as the thickness of double layer (or the concentration of electrolytes) varies. These findings are of practical significance in designing an electrophoresis operation because it will influence the prediction of the charged conditions on a particle if electrophoresis is used as an analytical tool, and the separation efficiency, if it is adopted as a separation technique.     

Numerical simulation of the effects of the design feature of a cyclone and the inlet flow velocity on the separation of CO2 particles from a CO2-COF2 mixture

In synthesizing COF₂ from CO, a considerable amount of CO₂ is produced. A method of solidifying CO₂ at low temperature and separating CO₂ particles from the COF₂ gas using a cyclone was designed and the separation efficiency according to the cyclone feature was studied. Optimal sizing and operation conditions of the cyclone were investigated by reviewing the flow velocity profile and the particle trajectory using a numerical analysis with computational fluid dynamics (CFD). The effects of the inlet flow velocity and the ratio of the cyclone diameter to the cone length (D/L) on the recovery efficiency were estimated. Results revealed that the separation efficiency increases with an increase in the ratio of D/L and a decrease in the cyclone size. The recovery efficiency of CO₂ increases with the increase in the inlet flow velocity. Based on these results, we could propose a concept and methodology to design the optimal features and sizing of a cyclone suitable for separating solid CO₂ from gaseous COF₂ at low temperature.     

Effect of different operating parameters on the recovery of proteins from casein whey using a rotating disc membrane ultrafiltration cell

In this study, effects of different operating parameters in ultrafiltration (UF) of casein whey in rotating disk ultrafiltration module, fitted with a 30 kD followed by a 5 kD molecular weight cut-off (MWCO) membrane have been analyzed and the benefits of membrane rotation with respect to stationary membrane module are highlighted. Prior to UF, centrifugation and microfiltration (MF) were carried out with the raw casein whey with an aim to remove the major membrane foulants like colloidal matters, suspended casein particles and lipid. The effects of pH, membrane rotation and transmembrane pressure (TMP) on UF flux and rejection were studied thoroughly, giving an emphasis on the effect of operating conditions in pressure and mass transfer controlled region as well. It was observed that solution pH was having a strong effect on UF flux and rejection for treating casein whey, which was explained in terms of prevailing monomer-dimer equilibrium of β-lactoglobulin, a major constituent of casein whey, as well as due to conformational changes of protein molecules with respect to isoelectric point. It was observed that for the 30 kD membrane, after 20 min of operation, 28.5% higher flux was obtained at pH 2.8 compared to pH 5.5 at a pressure of 490 kPa for stationary membrane, whereas the same figure for rotating membrane was 49.5%. Further, 38.7% higher UF flux at 300 rpm membrane speed compared to stationary membrane in 20 min of operation at pH 2.8 and TMP 490 kPa suggests the importance of shear across the membrane in minimizing the effects of concentration polarization. In the 5 kD membrane α-lactalbumin, which was mainly in the permeate stream from the 30 kD membrane, was separated from the other low molecular weight component of whey, such as lactose.