Peter R. Levison

Recent developments of magnetic beads for use in nucleic acid purification

The performance of Magarose, an agarose-based bead containing a paramagnetic component has been evaluated. The anion exchanger DEAE-Magarose is effective at binding DNA from a crude cell lysate. The plasmid pBluescript was isolated from 1.5 ml Escherichia coli JM109 cell culture, following alkaline lysis yielding 8.2 micrograms high-quality DNA. Under similar binding conditions 21 micrograms of salmon sperm DNA bound to the ion exchangers. The affinity medium oligo-dT Magarose was demonstrated to bind 75 mumol of an oligo-dA probe/g of medium by hybridization. Under similar conditions mRNA could be isolated from a preparation of baby hamster cell total RNA. The magnetic susceptibility of Magarose is very high, facilitating the use of this separation technique for rapid batch chromatographic processes.

Performance comparison of low-pressure ion-exchange chromatography media for protein separation

The physical and functional performance of 70 different anion- and cation-exchange media based on cellulose, agarose, dextran and polymeric/composite materials has been evaluated. Physical tests such as swelling, flow performance and column packing densities and functional tests including small-ion capacity, protein capacity and chromatographic performance were carried out on each grade. The data, descriptive rather than prescriptive, demonstrates significant performance differences from medium to medium and suggest that a rigorous media screening exercise be carried out prior to developing an ion-exchange separation process, in order to optimise the efficiency of the process.

Economic considerations important in the scale-up of an ovalbumin separation from hen egg-white on the anion-exchange cellulose DE92

The scale-up of the separation of hen egg-white proteins has been investigated using Whatman DE92 anion-exchange cellulose. Having developed suitable chromatographic conditions, a maximum binding capacity of 100 mg protein/ml packed DE92 was determined in a 25-ml column. The process was scaled up 1000-fold and the influence of batch and column techniques on the chromatographic step assessed. Data indicate column processes to be more efficient than batch in the adsorptive stage.

New approaches to the isolation of DNA by ion-exchange chromatography

The performance of different anion-exchange media have been compared for the isolation of plasmid DNA and genomic DNA from bacterial cells and human whole blood. Whatman DEAE-Magarose, based on an agarose bead containing a paramagnetic component, has been compared with prepacked gravity-flow columns containing a derivatised silica matrix. In each case the DNA isolation at various scales of operation was similar both in terms of yield and quality. The magnetic susceptibility of DEAE-Magarose is very high, facilitating the use of this separation technique for rapid flexible batch chromatographic processes, a limitation of the prepacked column techniques.

Influence of flow-rate on the chromatographic performance of agarose- and cellulose-based anion-exchange media

Abstract The influence of flow-rate on the separation of hen egg-white proteins was investigated using Whatman Express-Ion Exchanger Q and Pharmacia Q-Sepharose Fast Flow. Using 25-ml columns, breakthrough studies demonstrated that Express-Ion Q had an ovalbumin capacity of ca. 70 mg/ml, ca. 20% (w/w) greater than Q-Sepharose Fast Flow. Breakthrough for each exchanger was rapid. Using 25-l columns ca. 3.9 kg of protein was loaded on to each exchanger with Express-Ion Q having a capacity of ca. 2.1 kg compared with ca. 1.8 kg for Q-Sepharose Fast Flow. The desorption kinetics of the agarose were slower than those of the cellulose, as indicated by reduced chromatographic resolution with increasing flow-rate. Express-Ion Q was shown to give consistent chromatography over 100 chromatographic cycles with periodic clean-in-place.

Direct isolation of monoclonal antibodies from tissue culture supernatant using the cation-exchange cellulose Express-Ion S

The chromatography of the murine hybridoma cell C595/102 culture supernatant expressing the therapeutic monoclonal antibody C595, on the cation-exchange cellulose Whatman Express-Ion Exchanger S has been investigated. Initial method scouting studies using purified C595 in 1-ml mini columns demonstrated that binding capacity and binding efficiency were dependent not only on decreasing pH but also on the buffer salts used to prepare the mobile phase. Under optimised conditions of 0.1 M sodium acetate buffer, pH 5.0, we were able to separate purified C595 from BSA, the major contaminant in tissue culture fluid. Under these conditions immunoreactive C595 could be isolated directly from tissue culture supernatant. A scale-down study was carried out using a 25-ml column operated at a flow-rate of 150 cm/h which also yielded purified immunoreactive antibody. This procedure should now be suitable for scale-up.

Influence of column design on process-scale ion-exchange chromatography

The performance of two new designs of pump-packed axial flow process chromatography columns have been evaluated for the preparative anion-exchange chromatography of hen egg-white proteins using Whatman Express-Ion Exchanger Q. A 16 1 Side-Pack column and a 24 1 IsoPak column containing Express-Ion Q were used in this study. In each case ca. 20 1 feedstock containing 5-7 g protein/l, was applied per litre packed bed at flow-rates of ca. 150 and 300 cm/h. In each case the ovalbumin binding capacity was ca. 70 g/l packed bed with ca. 100% (w/w) recovery of applied protein. A clean-in-place procedure involving storage in 0.5 M NaOH was effective in maintaining chromatographic performance in all cases. These data were consistent with our previous work using the more traditionally configured slurry-packed axial flow columns. Each of these column designs were easy to use facilitating rapid packing with this adsorbent and in the case of IsoPak rapid pump unpacking. The introduction of these column designs significantly improves the task of column packing, hitherto a labour intensive, physically demanding and potentially unreproducible process.

Process-scale evaluation of a fast-flowing anion-exchange cellulose

Abstract The scale-up of the separation of hen egg-white proteins was investigated using Whatman DEAE-cellulose/D856, a fast-flowing microgranular anion-exchange cellulose. Under suitable conditions the maximum binding capacity for ovalbumin was determined to be 84 mg protein/ml packed DEAE-cellulose/D856 in a 25-ml column. The process was scaled-up 1000-fold and using a sub-maximum loading a working capacity of 56 mg protein/ml bed was obtained at a linear flow-rate of 150 cm/h. This reflects 100% recovery of applied ovalbumin with utilization of 66% of the theoretical maximum capacity of the medium. It is estimated that the productivity of this system would be up to 20 kg ovalbumin/m 3 · h based on these data.

Validation studies in the regeneration of ion-exchange celluloses

The effectiveness of a clean-in-place procedure involving treatment with 0.5 M NaOH for 16 h at room temperature has been examined following process-scale chromatography of 1.85 kg hen egg-white proteins on the DEAE-cellulose, Whatman Express-Ion Exchanger D and the QA-cellulose, Whatman Express-Ion Exchanger Q in 251 columns operating at flow-rates of 150 cm/h. Treatment of the media with 0.5 M NaOH did not affect the performance of the media after re-equilibration. The NaOH treatment was effective for sanitization and depyrogenation of columns of Express-Ion D and Express-Ion Q following gross microbial contamination. Furthermore, hydrolysis of the DEAE functional groups from Express-Ion D during treatment with 0.5 M NaOH was beyond the limits of detection, i.e. <0.01% of the total DEAE content of the exchanger.

Suspended bed chromatography, a new approach in downstream processing

A new technique in downstream processing, suspended bed chromatography has been developed. This hybrid technique exploiting the benefits of batch adsorption and the process advantages of an enclosed column system can be carried out using established contactors and adsorbents. A 44 cm I.D. IsoPak column and the anion-exchange cellulose Express-Ion Exchanger Q were used in the purification of ovalbumin from hen-egg white. After suspension of 16.25 kg Express-Ion Q in 500 l of feedstock containing 5 g protein/l, adsorption was effected by recirculation of the suspension using the IsoPak slurry preparation station. Protein-loaded adsorbent was collected in the IsoPak column unit, where it was washed and protein desorbed using gradient elution at a flow-rate of 300 cm/h. The entire process was complete in under 3 h. With the introduction of pump-packed column systems and the availability of mechanically strong adsorbents suitable for column separations, suspended bed chromatography offers a new approach to downstream processing and provides a less challenging alternative to batch separations.