This study was designed to evaluate the effect of shear on the supercoiled circular (SC) form of plasmid DNA. The conditions chosen are representative of those occurring during the processing of plasmid-based genes for gene therapy and DNA vaccination. Controlled shear was generated using a capillary rheometer and a rotating disk shear device. Plasmid DNA was tested in a clari®ed alkaline lysate solution. This chemical environment is characteristic of the early stages of plasmid puri®cation. Quantitative data is reported on shear degradation of three homologous recombinant plasmids of 13, 20 and 29 kb in size. Shear sensitivity increased dramatically with plasmid molecular weight. Ultrapure plasmid DNA redissolved in 10 mM Tris/ HCl, 1 mM EDTA pH 8 (TE buffer) was subjected to shear using the capillary rheometer. The shear sensitivity of the three plasmids was similar to that observed for the same plasmids in the clari®ed alkaline lysate. Further experiments were carried out using the 20 kb plasmid and the rotating disk shear device. In contrast with the capillary rheometer data, ultrapure DNA redissolved in TE buffer was up to eight times more sensitive to shear compared to plasmid DNA in the clari®ed alkaline lysate. However, this enhanced sensitivity decreased when the ionic strength of the solution was raised by the addition of NaCl to 150 mM. In addition, shear damage was found to be independent of plasmid DNA concentration in the range from 0.2 lg/ml to 20 lg/ml. The combination of shear and air-liquid interfaces caused extensive degradation of the plasmid DNA. The damage was more evident at low ionic strength and low DNA concentration. These ®ndings show that the tertiary structure of plasmid DNA can be severely affected by shear forces. The extent of damage was found to be critically dependent on plasmid size and the ionic strength of the environment. The interaction of shear with air-liquid interfaces shows the highest potential for damaging SC plasmid DNA during bioprocesses.

No abstract

Computational fluid dynamics was used to model the high flow forces found in the feed zone of a multichamber-bowl centrifuge and reproduce these in a small, high-speed rotating disc device. Linking the device to scale-down centrifugation, permitted good estimation of the performance of various continuous-flow centrifuges (disc stack, multichamber bowl, CARR Powerfuge) for shear-sensitive protein precipitates. Critically, the ultra scale-down centrifugation process proved to be a much more accurate predictor of production multichamber-bowl performance than was the pilot centrifuge.

No abstract

No abstract

Extracellularly expressed anti-hen egg lysozyme single-chain antibody fragments (scFv) produced by Aspergillus awamori were recovered using filtering centrifugation. Two filtering centrifuges with 0.5- and 30-L capacities were used to represent laboratory- and pilot-scale equipment, respectively. Critical regime analysis using the computational fluid dynamics (CFD) technique provided information about the local energy dissipation rates in both units. Experimental data indicated loss of scFv activity for energy dissipation rates above about 2.0 x 10(4) W kg(-1). This loss of activity increased in the presence of gas-liquid interfaces during filtering centrifugation. An ultra scaledown filtering centrifuge with a maximum working volume of 35 mL was designed to mimic the operating conditions identified by the critical regime analysis for the laboratory- and pilot-plant-scale units. The recovered scFv activity levels and the separation performance of the three units were comparable when operated at equal maximum energy dissipation rates.

No abstract

Numerical simulations are presented showing the effects of operating and geometrical parameters on the transition of laminar to Taylor vortex¯ow for induced rotational-axial¯ow in the gap of a pair of rotating cylinders. These simulations indicate that annular rotational ow becomes more stable in the presence of a small degree of axial¯ow and as gap width increases. The effect of rotational speed on the breakdown of laminar¯ow is more complex and for given radius ratio and axial¯ow rate depends on both the speed ratio and the direction of the rotation of the cylinders, counter-rotating¯ow generally producing a more stable¯ow than co-rotating. Limited experimental data are provided on the residence time distribution for¯ow of Newtonian liquids through the gap of two rotating cylinders. The data include results from experiments in which¯ow transition occurred from laminar to Taylor vortex¯ow. The ®ndings from these experiments are successfully analyzed and assessed using the simulations studies. List of symbolsDimensionless differential operator D m 2 s À1 Axial dispersion coef®cient L m Distance between two measuring points M ± Dimensionless angular velocity, À Xr X 0 Á N rps Rotational speed of cylinder P Pa Pressure P ± Parameter de®ned by Eq. (17) Pe ± Peclet number, À WL D Á Re ± Axial Reynolds number, À WR 2 ÀR 1 m Á R 2 Y R 1 m Radii of outer and inner cylinders R m m Mean radius, À R 1 R 2 2 Á r m Radial coordinate s ± Growth rate of disturbances T c ± Critical Taylor number given by Eq. (15) Ta ± Taylor number, À À 4AX 0 R 2 ÀR 1 4 m 2 Á Ta c ± Critical Taylor number t s Time Ur m s À1 Radial velocity component Vr m s À1 Tangential velocity component W m s À1 Axial¯ow velocity Wr m s À1 Axial velocity component x ± Transformed dimensionless radial coordinate z m Axial coordinateGreek symbols a ± Radius ratio,Wave number l kg m À1 s À1 Viscosity of the working liquid m kg m À1 s À1 Kinematic viscosity of the working liquid r ± Dimensionless growth rate Dr 2 h ± Dimensionless variance difference X 2 , X 1 rad À1