Factors affecting the productivity of 4-Column Periodic Counter Current Chromatography (4C-PCC)

The advantages of continuous bioprocessing include steady state operation, reduced equipment footprint, high volumetric productivity, streamlined process flow, and reduced capital cost. These benefits have led to considerable interest in evaluating these technologies for the purposes of bioprocess intensification (Konstantinov & Cooney, 2014). It has been shown, through cost of goods modelling, that the cost of protein A is one of the most significant costs in a MAb process, and that decreasing the cost of protein A by maximising loading or increasing lifetime will have a significant effect on consumable costs (Broly, et al., 2010). Consequently, the application of continuous or semi continuous chromatography to primary capture of MAbs is of great interest for reducig overall product cost. A key factor affecting the productivity of periodic counter current chromatography is the loading time. This must be closely matched to the regeneration time in order to maximize productivity (Pollock, et al., 2013). At low feed stream product titres only modest productivity gains can be obtained when operating around the typical range of residence times for a protein A column. This is because the loading time is considerably longer than the regeneration time. However, by significantly reducing the residence time to less than a minute significant gains in productivity can be achieved. The productivity obtained using 8mL of silica based protein A media packed in four 2mL columns and run at a residence time of 12 seconds on a 4C-PCC system was compared to that obtained on a 40mL column packed with high capacity agarose based media run at a 4mins residence time in batch mode. The load concentrations were 0.3g/L and 0.44g/L respectively. The productivity gains were found to be about 90% better for the 4CPCC run with similar host cell protein clearance and recovery. The data from this comparison is simarised in table 1. In order to take full advantage of the benefits of continuous chromatography for primary capture of MAbs, a good understanding of the factors affecting productivity is required. This presentation will aim to highlight those factors and assess their relative importance in maximising the productivity of this downstream processing unit operatio

Development of Preparative Microfluidic Techniques for Lysis of Microbial Cells and Affinity Purification of Proteins

In order to fully realise the benefits of microscale mammalian cell culture and microbial fermentation systems, a device capable of online sample preparation to enable further investigation of product quality is a key requirement. The aim of this work is to move toward such a device by designing and characterising a microfluidic lysis device and microaffinity chromatography device that are compatible with each other. The resulting microfluidic lysis device is useful for preparatory lysis of microbial cells. It works by mixing a lysis reagent (BugBuster MastermixTM), with microbial culture, using a T-Piece connection. Lysis takes place in a 700µm internal diameter fused silica capillary. The device was able to successfully lyse microbial cells with similar active Glutathione S Transferase release to sonication. The operating flowrate range of the device was 3.207µL min-1 to 6.414 µL min-1 and the device volume was 30µL - 60µL. The microaffinity chromatography column performed well in studies with pure Glutathione S Transferase. It showed good loading and elution behaviour. The breakthrough and elution curves, and quantity of protein eluted per unit bed volume, were similar to lab scale. The difference being as a result of experimental error. The column also performed well with a 100% clarified Escherichia coli lysate containing recombinant Glutathione S Transferase from Schistosoma japonicum. The eluate had a purity of 55% and concentration of 2.24 mg/ml. The column was fabricated from inexpensive fused silica capillary. It had an internal diameter of 700µm, a length of 5cm (the same length as a typical lab scale Glutathione Affinity column), and a bed volume of approximately 19µL. The operating flowrate range for the column was the same as the microlysis device

The effect of feed quality due to clarification strategy on the design and performance of protein A periodic counter-current chromatography

The impact of two different quality feeds, derived using two different harvest clarification processes, on protein A periodic counter-current chromatography (PCC) design and performance is investigated. Data from batch experiments were input into a model to design optimal PCC operating parameters specific to each feed material. The two clarification methods were: depth filtration using a wetlaid matrix which has Q-functionality; and a combination of depth filtration and chromatographic clarification, using a Q-functional nonwoven with a high anion exchange capacity (Emphaze™ AEX Hybrid Purifier) in which key impurities such as host cell DNA (HCDNA) and host cell proteins (HCP) are removed. The model predicted 34% better productivity for the chromatographically clarified cell culture fluid (CCCF) using a 4 column system, and productivity gains of 28% using only 3 columns enabling the option to simplify the protein A PCC strategy. Experimental validation of the predicted optimized PCC operating parameters using industrially relevant monoclonal antibody (mAb) CCCF feedstock over 100 cycles showed productivity gains of 49% for the chromatographically clarified material. HCP concentration was 11-fold lower, and HCDNA concentration was reduced by 4.4 Log Reduction Value (LRV) in the protein A PCC eluates. This work, therefore, demonstrates that the removal of HCDNA and HCP during clarification is an effective strategy for improving protein A PCC performance. This was achieved using the Emphaze™ AEX Hybrid Purifier which can be easily incorporated into a batch or continuous process, in a scalable fashion, without adding additional separate unit operations. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018

The pressure dependence of electron transport in indium arsenide phosphide alloys.

Experimental and theoretical studies have been made of low and high field electron transport in n-type In AS1-X PX as a function of alloy composition and hydrostatic pressure. With the electron concentration and Hall mobility varying from 6 x 1015cm-3 and 3500 cm2/V/s respectively in InP to 2 x 1016cm-3 and 12500 cm2/V/s in In As, the total density of impurity was found to vary from about 1016cm-3 in InP to about 1017cm-3 in In As showing that impurity scattering became increasingly important with decreasing phosphorus content. A decrease in mobility with increasing pressure was observed. Taking the measured density of impurities, the variation in mobility with pressure could be accounted for by the theory when both polar optical and ionized impurity scatterings were taken into account. The drift mobility relevant to high field studies was determined by measuring the Hall mobility at a magnetic field of 9T. The results of high field measurements showed that at atmospheric pressure and x 0.3 by the Gunn effect. The threshold field, FT, was found to increase steadily with x from about 1 kV/cm in In As to close to 9 kV/cm in InP. In the avalanche region, (x < 0.3), increasing pressure and increasing x produced similar changes in both FT and VT if 1 k-bar was set equivalent to 1% increase in phosphorus. In the Gunn region, despite the increasing threshold field, vT measured remained almost constant with composition. By contrast, with increasing pressure FT remained almost constant and vT dropped about 16% in 15 k-bar. The results were simulated by Monte Carlo calculations of the velocity-field characteristics including an ionized impurity scattering corresponding to the low field mobility of the samples used. The parameters used and their variations with x and pressure were discussed. Reasonable agreement was obtained for the magnitudes of FT and VT and for their variations with pressure and composition. By reducing the impurity scattering to zero, estimates for the characteristics of the pure alloys were obtained. These indicated that vT increases from about 2.6 x 1017cm/s in InP to 3.05 cm/s in In As0.7 P0.3, suggesting this may be a useful material for microwave devices. Analysis of the transition from impact ionization to Gunn effect gave values of the sub-band gap, DeltaETL, of 0.71 and 0.79 eV for In AS0.7 P0.3 and In As0.79 P0.21 respectively. Electron loss to impurity levels normally above the Upsilon1c minimum was observed. The pressure coefficients of the levels are discussed and their energies are determined. A semiconductor-semimetal phase transition, which occurs at 60 k-bar in In As, is found to vary linearly to 100 k-bar in InP and is compared with the theory of Van Vechten

The pressure dependence of electron transport in indium arsenide phosphide alloys.

Experimental and theoretical studies have been made of low and high field electron transport in n-type In AS1-X PX as a function of alloy composition and hydrostatic pressure. With the electron concentration and Hall mobility varying from 6 x 1015cm-3 and 3500 cm2/V/s respectively in InP to 2 x 1016cm-3 and 12500 cm2/V/s in In As, the total density of impurity was found to vary from about 1016cm-3 in InP to about 1017cm-3 in In As showing that impurity scattering became increasingly important with decreasing phosphorus content. A decrease in mobility with increasing pressure was observed. Taking the measured density of impurities, the variation in mobility with pressure could be accounted for by the theory when both polar optical and ionized impurity scatterings were taken into account. The drift mobility relevant to high field studies was determined by measuring the Hall mobility at a magnetic field of 9T. The results of high field measurements showed that at atmospheric pressure and x 0.3 by the Gunn effect. The threshold field, FT, was found to increase steadily with x from about 1 kV/cm in In As to close to 9 kV/cm in InP. In the avalanche region, (x < 0.3), increasing pressure and increasing x produced similar changes in both FT and VT if 1 k-bar was set equivalent to 1% increase in phosphorus. In the Gunn region, despite the increasing threshold field, vT measured remained almost constant with composition. By contrast, with increasing pressure FT remained almost constant and vT dropped about 16% in 15 k-bar. The results were simulated by Monte Carlo calculations of the velocity-field characteristics including an ionized impurity scattering corresponding to the low field mobility of the samples used. The parameters used and their variations with x and pressure were discussed. Reasonable agreement was obtained for the magnitudes of FT and VT and for their variations with pressure and composition. By reducing the impurity scattering to zero, estimates for the characteristics of the pure alloys were obtained. These indicated that vT increases from about 2.6 x 1017cm/s in InP to 3.05 cm/s in In As0.7 P0.3, suggesting this may be a useful material for microwave devices. Analysis of the transition from impact ionization to Gunn effect gave values of the sub-band gap, DeltaETL, of 0.71 and 0.79 eV for In AS0.7 P0.3 and In As0.79 P0.21 respectively. Electron loss to impurity levels normally above the Upsilon1c minimum was observed. The pressure coefficients of the levels are discussed and their energies are determined. A semiconductor-semimetal phase transition, which occurs at 60 k-bar in In As, is found to vary linearly to 100 k-bar in InP and is compared with the theory of Van Vechten