Mechanistic model-based characterization of size-exclusion-mixed-mode resins for removal of monoclonal antibody fragments.

Although antibody fragments are a critical impurity to remove from process streams, few platformable purification techniques have been developed to this end. In this work, a novel size-exclusion-mixed-mode (SEMM) resin was characterized with respect to its efficacy in mAb fragment removal. Inverse size-exclusion chromatography showed that the silica-based resin had a narrow pore size distribution and a median pore radius of roughly 6.2 nm. Model-based characterization was carried out with Chromatography Analysis and Design Toolkit (CADET), using the general rate model and the multicomponent Langmuir isotherm. Model parameters were obtained from fitting breakthrough curves, performed at multiple residence times, for a mixture of mAb, aggregates, and an array of fragments (varying in size). Accurate fits were obtained to the frontal chromatographic data across a range of residence times. Model validation was then performed with a scaled-up column, altering residence time and feed composition from the calibration run. Accurate predictions were obtained, thereby illustrating the model's interpolative and extrapolative capabilities. Additionally, the SEMM resin achieved 90% mAb yield, 37% aggregate removal, 29% [Formula: see text] removal, 54% Fab/Fc removal, 100% Fc fragments removal, and a productivity of 72.3 g mAbL×h. Model predictions for these statistics were all within 5%. Simulated batch uptake experiments showed that resin penetration depth was directly related to protein size, with the exception of the aggregate species, and that separation was governed by differential pore diffusion rates. Additional simulations were performed to characterize the dependence of fragment removal on column dimension, load density, and feed composition. Fragment removal was found to be highly dependent on column load density, where optimal purification was achieved below 100 mg protein/mL column. Furthermore, fragment removal was dependent on column volume (constant load mass), but agnostic to whether column length or diameter was changed. Lastly, the dependence on feed composition was shown to be complex. While fragment removal was inversely related to fragment mass fraction in the feed, the extent depended on fragment size. Overall, the results from this study illustrated the efficacy of the SEMM resin in fragment and aggregate removal and elucidated relationships with key operational parameters through model-based characterization.

A contoured gap coaxial plasma gun with injected plasma armature.

A new coaxial plasma gun is described. The long term objective is to accelerate 100-200 microg of plasma with density above 10(17) cm(-3) to greater than 200 km/s with a Mach number above 10. Such high velocity dense plasma jets have a number of potential fusion applications, including plasma refueling, magnetized target fusion, injection of angular momentum into centrifugally confined mirrors, high energy density plasmas, and others. The approach uses symmetric injection of high density plasma into a coaxial electromagnetic accelerator having an annular gap geometry tailored to prevent formation of the blow-by instability. The injected plasma is generated by numerous (currently 32) radially oriented capillary discharges arranged uniformly around the circumference of the angled annular injection region of the accelerator. Magnetohydrodynamic modeling identified electrode profiles that can achieve the desired plasma jet parameters. The experimental hardware is described along with initial experimental results in which approximately 200 microg has been accelerated to 100 km/s in a half-scale prototype gun. Initial observations of 64 merging injector jets in a planar cylindrical testing array are presented. Density and velocity are presently limited by available peak current and injection sources. Steps to increase both the drive current and the injected plasma mass are described for next generation experiments.

Production of beta-glucosidase using immobilised Piromyces sp. KSX1 and Orpinomyces sp. 478P1 in repeat-batch culture.

Two anaerobic fungi, one a monocentric strain ( Piromyces sp. KSX1) and the other a polycentric strain ( Orpinomyces sp. 478P1), were immobilised in calcium alginate beads and cultured in sequential batches where spent medium (containing 0.25% cellobiose) was repeatedly drained and replaced. beta-Glucosidase production with KSX1 was maintained for 45 days over six repeated batch cultures yielding a maximum level of 107 mIU/ml. For 478P1, beta-glucosidase production was maintained for 30 days over four repeated batches yielding a maximum level of 34 mIU/ml. Although repeat-batch cultures of KSX1 produced more beta-glucosidase than strain 478P1, the maximum specific beta-glucosidase produced from these immobilised cultures was similar. The immobilised polycentric strain proved to be operationally superior to strain KSX1, as strain 478P1 did not produce any growth in the culture liquor.