Demonstration of the use of windows of operation to visualize the effects of fouling on the performance of a chromatographic step.

The high resolution afforded by packed bed chromatography makes it an indispensable operation in the downstream processing of therapeutic molecules. Packed bed performance is however inherently susceptible to changes in feed stream characteristics and fouling processes. The impact of fouling is seldom considered during the early stages of bioprocess development which is concerned with the selection of purification conditions. Instead these are performed with rigorously clarified feeds. Under such conditions, chromatography is effectively treated as an isolated step, independent from its preceding unit operations. In this study, we demonstrate how windows of operation could be used to visualize the impact of changes in the preceding clarification step on the fouling response of a subsequent cation exchange capture step. Laboratory columns (2,5 and 12 cm height) were subjected to varying fouling challenges of Escherichia coli lysate containing different amounts of solids carried over from the previous step. Changes in trans-column pressure drop and breakthrough of the target protein (Fab') were monitored. The limits of operability of the resin were determined with respect to the process material's properties. This information was used to extract the parameters for the adsorption kinetics used in the general rate (GR) model to create windows of operation for manufacturing scale operation.

Inactivation of epoxide hydrolase by catalysis-induced formation of isoaspartate.

Epoxide hydrolases catalyze hydrolytic epoxide ring-opening, most often via formation of a covalent hydroxyalkyl-enzyme intermediate. A mutant of Agrobacterium radiobacter epoxide hydrolase, in which the phenylalanine residue that flanks the invariant catalytic aspartate nucleophile is replaced by a threonine, exhibited inactivation during conversion when the (R)-enantiomer of para-nitrostyrene epoxide was used as substrate. HPLC analysis of tryptic fragments of the epoxide hydrolase, followed by MALDI-TOF and TOF/TOF analysis, indicated that inactivation was due to conversion of the nucleophilic aspartate into isoaspartate, which represents a novel mechanism of catalysis-induced autoinactivation. Inactivation occurred at a lower rate with the (S)-enantiomer of para-nitrostyrene epoxide, indicating that it is related to the structure of the covalent hydroxyalkyl-enzyme intermediate.

Studies on aerosol delivery of plasmid DNA using a mesh nebulizer.

Aerosol delivery of plasmid DNA therapeutic solutions is promising for the treatment of respiratory diseases. However, it poses challenges, most significantly the need to protect the delicate supercoiled (sc) structure of plasmid during aerosolization. Nebulizers for liquid aerosolization using meshes appear a better method for delivery than conventional jet and ultrasonic nebulizers. This paper explores their application to the delivery of plasmid DNA. A computational fluid dynamics model of the dynamics of fluid flow through the nozzle of the MicroAIR mesh nebulizer indicated high strain rates (>10(5) s(-1)) near the nozzle exit capable of causing damage to the shear-sensitive plasmid DNA. Knowledge of the strain rates predicted using CFD and molecule size determined using atomic force microscopy (AFM) enabled estimation of the hydrodynamic force and whether damage of shear-sensitive therapeutics was likely. Plasmids of size 5.7 and 20 kb were aerosolized in the mesh nebulizer. The sc structure of the 5.7-kb plasmid was successfully delivered without damage, while aerosolization of the 20-kb plasmid led to disintegration of the pDNA sc structure as observed in AFM. Subsequent formulation of the sc 20-kb plasmid with PEI resulted in successful aerosol delivery. The maximum hydrodynamic forces computed for the aerosolization of structures of the size of 5.7-kb and PEI formulated 20-kb plasmids were less than the forces reported to damage the structure of double-stranded DNA. A combination of CFD analysis and structure analysis may be used to predict successful aerosol delivery in such a mesh nebulizer.

Effect of fouling on the capacity and breakthrough characteristics of a packed bed ion exchange chromatography column.

This study examined the impact of fouling with yeast homogenate on capacity and breakthrough performance of an ion exchange packed bed column. Column performance was assessed by analysis of breakthrough curves obtained with BSA as a test protein. The overall impact of fouling on breakthrough performance depended heavily on the level of clarification of the feed stream. Challenging the column with particulate-free homogenate caused no change in column performance. Loading successive small volumes of poorly clarified homogenate, interspersed with frequent column salt washes, did not alter significantly the column capacity. By contrast, when the column was challenged with an equivalent cumulative volume of poorly clarified homogenate, dynamic binding capacity decreased significantly and changes in breakthrough curves suggested increased intraparticle and external mass transfer limitations. These changes were ascribed to deposition of solid particulates in void spaces in the bed and colloidal contaminants in the bead pores.