Factors affecting dispersion in expanded bed chromatography

Factors affecting the dispersion of solutes in expanded bed chromatography were experimentally investigated to characterize the behavior in small columns. Pulse response curves were measured with a vitamin B¹² tracer, and HETP (height equivalent to a theoretical plate) values were calculated from peak variance and retention time. Approximately 15 min were required to attain a stable steady state expanded bed height with constant HETP values. HETP values ranged from 0.8 to 1.6 cm and did not change appreciably with the degree of expansion (1.5–3.5 fold), column diameter (1.6 and 2.6 cm), column temperature (293–308 K) or settled bed height (ca. 4–11 cm). A very small column (1.6 cm diam. and 4.2 cm-settled bed height) was successfully expanded and axial mixing measured could be useful for conducting scale down experiments.     

Expanded bed adsorption on supermacroporous cross-linked cellulose matrix

Rigid spherical macroporous adsorbent beads (CELBEADS) prepared by cross-linking of cellulose were characterised and found eminently suitable for use as expanded bed affinity chromatography matrix. Chromatographic runs were performed on a 10 mm diameter column with three solutes tyrosine, papain and bovine serum albumin under non-retaining conditions on CELBEADS and Streamline^TM DEAE, a commercial agarose based expanded bed matrix. Performance of the runs was measured in terms of height equivalent to theoretical plate, HETP. Variation in HETP with velocity on Streamline^TM DEAE gave flat profiles in packed bed and increasing trend in expanded bed. On CELBEADS, the HETP curves in both packed and expanded bed modes followed profiles typical of macroporous adsorbents i.e. increasing and levelling with velocity. HETP values obtained for papain and bovine serum albumin on CELBEADS were lower than those obtained on Streamline^TM DEAE at all velocities. Lactate dehydrogenase was purified from porcine muscle homogenate using Cibacron blue conjugated to CELBEADS using a protocol reported for supports with surface hydroxyl groups. Elution of the enzyme was investigated both in packed mode as well as in expanded mode at a flow rate of 1 ml min^-1. The purification procedure took about 60 minutes and a purification fold of about 14 was achieved in both cases. The adsorbent could be cleaned in place with 5 M urea and used repeatedly without loss of performance.     

High gradient magnetic separation versus expanded bed adsorption: a first principle comparison

A robust new adsorptive separation technique specifically designed for direct product capture from crude bioprocess feedstreams is introduced and compared with the current bench mark technique, expanded bed adsorption. The method employs product adsorption onto sub-micron sized non-porous superparamagnetic supports followed by rapid separation of the loaded adsorbents from the feedstock using high gradient magnetic separation technology. For the recovery of Savinase® from a cell-free Bacillus clausii fermentation liquor using bacitracin-linked adsorbents, the integrated magnetic separation system exhibited substantially enhanced productivity over expanded bed adsorption when operated at processing velocities greater than 48 m h^–1. Use of the bacitracin-linked magnetic supports for a single cycle of batch adsorption and subsequent capture by high gradient magnetic separation at a processing rate of 12 m h^–1 resulted in a 2.2-fold higher productivity relative to expanded bed adsorption, while an increase in adsorbent collection rate to 72 m h^–1 raised the productivity to 10.7 times that of expanded bed adsorption. When the number of batch adsorption cycles was then increased to three, significant drops in both magnetic adsorbent consumption (3.6 fold) and filter volume required (1.3 fold) could be achieved at the expense of a reduction in productivity from 10.7 to 4.4 times that of expanded bed adsorption.     

Design of expanded bed supports for the recovery of plasmid DNA by anion exchange adsorption

In this study we detail the rational design of new chromatographic adsorbents tailored for the capture of plasmid DNA. Features present on current chromatographic supports that can significantly enhance plasmid binding capacity have been identified in packed bed chromatography experiments and blueprints for improved expanded bed adsorbents have been put forward. The characterisation and testing of small (20-40 m) high density (>3.7 g cm^–3) pellicular expanded bed materials functionalised with various anion exchange structures is presented. In studies with calf thymus DNA, dynamic binding capacities of 1.2 and 3.4 mg ml^–1 were recorded for prototype diethylaminoethyl-and polyethylene imine-linked adsorbents which were respectively 25 and 70 fold higher than those of equivalently derivatised commercial expanded bed materials. The prototype polyethylene imine-coupled material exhibited severe sensitivity to inter-particle bridging by nucleic acid polymers, gave low DNA recoveries (<37%) and proved difficult to regenerate. In contrast, few operational difficulties were experienced with the diethylaminoethyl-linked prototype adsorbent and successful high capacity (>0.8 mg ml^–1) capture of plasmid DNA from crude neutralised E. coli lysate was demonstrated.     

Plate height determination for gradient elution chromatography of proteins

A method for determining the plate height HETP from the elution curve obtained by the linear gradient elution (LGE) ion-exchange chromatography (IEC) of proteins is presented. The method was developed on the basis of the numerical solutions of a chromatography model which considers the zone sharpening and the distribution coefficient as a function of the salt concentration. The plate height HETP is determined from the peak width and the salt concentration at which the peak is eluted in LGE. The method was applied to the experimental results with various ion-exchange chromatography media. A calculation example based onthe present method is presented to show how the chromatographic and operating parameters should be tuned to obtain a desired resolution. A simplified calculation procedure for the peak profile is also described. (c) 1995 John Wiley & Sons, Inc.     

Expanded and packed bed albumin adsorption on fluoride modified zirconia

The expanded bed characteristics of 75-103microm fluoride-modified zirconia (FmZr) particles synthesized by a fed batch oil emulsion process were investigated. These particles are distinguished from commercially available expanded-bed adsorbents by virtue of their high density (2.8 g/cc) and the mixed mode protein retention mechanism which allows for the retention of both cationic and anionic proteins. The linear velocity versus bed porosity data agree with the Richardson-Zaki relationship with the terminal velocity in infinite medium of 2858.4 cm/h and a bed expansion index of 5.1. Residence time distribution (RTD) studies and bovine serum albumin (BSA) adsorption studies were performed as a function of the height of the settled bed to the column diameter (H:D) ratio and degree of bed expansion with superficial velocities of 440 to 870 cm/h. The settled bed, a 2x expanded bed, and a 3x expanded bed were studied for the H:D ratios of 1:1, 2:1, and 3:1. The dynamic binding capacity (DBC) at 5% breakthrough was low (2-8 mg BSA/mL settled bed) and was independent of the H:D ratio or the degree of bed expansion. The saturation DBC was 32.3 +/- 7.0 mg BSA/mL settled bed. The adsorption-desorption kinetics and intraparticle diffusion for protein adsorption on FmZr (38-75 micrometer) were investigated by studying the packed bed RTD and BSA adsorption as a function of temperature and flow rate. The data show that the adsorption-desorption kinetics along with intraparticle diffusion significantly influence protein adsorption on FmZr. Low residence times ( approximately 0.8 min) of BSA result in a DBC at 5% breakthrough which is 3.5-fold lower compared to that at 6-fold higher protein residence time. At low linear velocity (45 cm/h) the breakthrough curve is nearly symmetrical and becomes asymmetrical and more dispersed at higher linear velocity (270 cm/h) due to the influence of slow adsorption-desorption kinetics and intraparticle diffusion.Copyright 1998 John Wiley & Sons, Inc. Bioeng 60: 333-340, 1998.     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1     

Routine manufacture of recombinant proteins using expanded bed adsorption chromatography

Two different recombinant human proteins were purified directly from Pichia pastoris whole cell fermentation broth, containing 30–44% biomass (wet weight percent), by strong cation exchange expanded bed adsorption chromatography. Expanded bed adsorption chromatography provided clarification, product purification and product concentration in a single unit operation at large scale (2000-l nominal fermentation volume). The efficiency of expanded bed adsorption chromatography resulted in a short process time, high process yield, and limited proteolytic degradation of the target proteins. The separations were operated using a 60-cm (d) column run at 14 l/min. For one protein, expanded bed adsorption chromatography resulted in an average product recovery of 113% (relative to fermentation supernatant) and a purity of 89% (n=10). For the other protein, the average product recovery was 99% (relative to fermentation supernatant) and the purity was 62.1 (n=10). Laboratory experiments showed that biomass reduced product dynamic binding capacity for protein 2.     

Effect of adsorbent porosity on performance of expanded bed chromatography of proteins

Expanded bed or fluidized bed adsorption has emerged as an important unit operation in downstream processing of proteins. A number of specifically designed commercial adsorbents are available today for expanded bed purification of proteins. Protein purification essentially requires adsorbent matrices that have large pore size. Very large pore size or macroporous adsorbents can provide high efficiency in packed beds even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the macropores. This is reflected in leveling off of HETP (height equivalent to theoretical plate) versus flow curve after a threshold velocity. Expanded bed operation, on the other hand, can also show plateauing of the HETP curve, but not necessarily on account of macroporosity of adsorbent. It is shown in this article how any adsorbent intended for protein adsorption in expanded bed mode can give plateauing HETP curve, regardless of pore size. As a result, RTD measurements on an expanded bed can give equal, and at times better, performance than a corresponding packed bed. Large pore size, on the other hand, can result in lesser retention of biomass and easy flushing of the adsorbent to obtain an entirely particulate-free adsorbent prior to the product elution step. Adsorbent with larger pores is also shown to provide faster and more efficient elution both in packed and expanded bed modes.     

Studies on protein adsorption in a vortex flow reactor

The performance of a vortex flow reactor (VFR) with suspended particles for protein adsorption was studied under varying operating conditions, and resin volume fractions. The VFR behaved as an expanded bed in the regimen of laminar vortices flow. Streamline DEAE was used for bovine serum albumin (BSA) adsorption. Expanded bed VFR experiments were performed with varying geometric aspect ratios (14.6, 28.6 and 40.0) and axial superficial velocity (100–300 cm h⁻¹) to investigate their influence on productivity and dynamic capacity. The results are compared with literature data on an expanded bed column (EBC). Adsorption breakthrough curves were fitting to a simple two-parameter model.     

Characterisation of STREAMLINETM Phenyl

STREAMLINE Phenyl is a new hydrophobic interaction chromatography support designed for use in expanded bed adsorption. The phenyl groups are linked to STREAMLINE matrix via highly stable ether linkages. Within this development project the chemical and chromatographic stability as well as the breakthrough capacity for human IgG has been studied. The chemical stability was monitored as the carbon leakage from the matrix to the storage solution, pH 1–14 at 20 and 40 °C. The carbon content in the supernatant was determined with Total Organic Carbon (TOC) technique. In the chromatographic stability study STREAMLINE Phenyl was stored in eight different storage solutions under ambient conditions for 12 weeks and then tested in a chromatographic function test. The results show that the adsorbent is chemically stable and that the chromatographic properties are retained under the tested conditions. The breakthrough capacity study demonstrates the importance of the bed height for obtaining maximal dynamic capacity. Further, there is a good correlation between breakthrough data generated from packed bed and expanded bed runs.     

Surface thermodynamics of pellet formation in Aspergillus niger

The fungal pellets were formed from the surface thermodynamic balance between fungal cell and liquid media. The Gibbs free energy of pellet formation of the initial culture media (–73 –81 ergs/cm2) were increased to –13 –46 ergs/cm2 at 48 h. FTIR analysis showed that factors inducing pellet formation simultaneously increased the cell wall hydrophobicity of Aspergillus niger.     

Evaluation of radial chromatography versus axial chromatography, practical approach

Developments in packing and packing port design of radial columns in recent years have resulted in a claimed significant increase in performance of this process chromatography technology. In this first study, the main chromatographic parameters as efficiency, capacity factor, asymmetry and resolution were evaluated in a unique one-to-one comparison between a 120 ml bed-volume and 6 cm bed length radial chromatography mini-process column against a 50 mm diameter, 6 cm bed height and 120 ml bed-volume axial chromatography column. Radial chromatography showed an increase in efficiency by 31% in the number of plates per meter while the equilibration could be reduced by 0.4-0.5 column volumes. The asymmetry factor for bovine serum albumin in radial chromatography showed a reduction of 20% while the reduction of the asymmetry factor of the smaller protein ovotransferrin decreased even by 46% in comparison to the performance of the comparative axial chromatography column. Therefore in radial chromatography resolution improved up to 20%. The retention volume was similar in both cases. For radial chromatography, the decrease in "width at half height" at Height Equivalent of Theoretical Plates (HETP) measurements was 40% while the decrease of the over-all width of the peak was 27%. For adsorbed/desorbed proteins, the elution peak showed similar results: "width at half height" decreased to 45% while the over-all width of the peak decreased by 28%. The concentration of the non-retained protein in the flow-through (lysozyme), increased by 35% while the concentration of the eluted fraction (serum albumin bovine), increased with 40% in the radial chromatography columns. The better results obtained with the radial column were probably the consequence of the geometrical design of this device (larger inlet surface area and small outlet surface area which concentrate the eluted fraction).     

Submerged Macrophyte-bed Effects on Water-Column Phosphorus,Chlorophyll <Emphasis Type="Italic">a</Emphasis>, and Bacterial Production

Submerged macrophytes are a major component of freshwater ecosystems, yet their net effect on water column phosphorus (P), algae, and bacterioplankton is not well understood. A 4-month mass-balance study during the summer quantified the net effect of a large (5.5 ha) undisturbed macrophyte bed on these water-column properties. The bed is located in a slow-flowing (0.05–0.1 cm s^–1) channel between two lakes, allowing for the quantification of inputs and outputs. The P budget for the study period showed that, despite considerable short-term variation, the macrophyte bed was a negligible net sink for P (0.06 mg m^–2 day^–1, range from –0.76 to +0.79 mg m^–2 day^–1), demonstrating that loading and uptake processes in the weedbed roughly balance over the summer. Chlorophyll a was disproportionately retained relative to particulate organic carbon (POC), indicating that the algal component of the POC was preferentially trapped. However, the principal contribution of the weedbed to the open water was a consistent positive influence on bacterioplankton production over the summer. Conservative extrapolations based on measured August specific exports (m^–2 day^–1) of P and bacterial production exiting the weedbed applied to five regional lakes varying in lake morphometry and macrophyte cover suggest that even in the most macrophyte dominated of lakes (66% cover), P loading from submerged weedbeds never exceeds 1% day^–1 of standing epilimnetic P levels, whereas subsidization of bacterioplankton production can reach upward of 20% day^–1. The presence of submerged macrophytes therefore differentially modifies algae and bacteria in the water column, while modestly altering P dynamics over the summer.     

A method for the analysis of acetate turnover in a coastal marine sediment

The concentrations of volatile fatty acids were measured in the pore water of sediment from the Limfjorden, Denmark. The pore water was freeze-dried and the acids, which were redissolved in formic acid, were analyzed by gas chromatography on a Carbopack column. The limit of detection was 0.1 mol l^–1 pore water. The concentration ranges (mol l^–1 pore water) were as follows: 0.1 to 6.0 for acetate; <0.1 to 0.6 for propionate, and <0.1 to 0.5 for butyrate. The rate constants for the disappearance of injected tracer concentrations of U-¹⁴C-acetate were measured at 2 cm depth intervals in sediment strata (0 to 10 cm). The rate constant for acetate turnover at 4 to 6 cm depth did not vary greatly with season, 2.1 h^–1, SD 0.6 for 7 values. In spring, the rate constants were highest in the 0 to 2 cm stratum and decreased with sediment depth. The calculated rates for acetate turnover of 7.2 mmol m^–2 day^–1 for early spring (2°C) and of 19.6 mmol m^–2 day^–1 for late autumn (7°C) were higher than would be expected from published values for carbon oxidation by sulfate in these sediments.     

Dialysis cultures with immobilized hybridoma cells for effective production of monoclonal antibodies

An industrial scale reactor concept for continuous cultivation of immobilized animal cells (e.g. hybridoma cells) in a radial-flow fixed bed is presented, where low molecular weight metabolites are removed via dialysis membrane and high molecular products (e.g. monoclonal antibodies) are enriched. In a new nutrient-split feeding strategy concentrated medium is fed directly to the fixed bed unit, whereas a buffer solution is used as dialysis fluid. This feeding strategy was investigated in a laboratory scale reactor with hybridoma cells for production of monoclonal antibodies. A steady state monoclonal antibody concentration of 478 mg l^-1 was reached, appr. 15 times more compared to the concentration reached in chemostat cultures with suspended cells. Glucose and glutamine were used up to 98%. The experiments were described successfully with a kinetic model for immobilized growing cells. Conclusions were drawn for scale-up and design of the large scale system.Abbreviations: c_Glc – glucose concentration, mmol l^-1; c_Gln – glutamine concentration, mmol l^-1; c_Amm – ammonia concentration, mmol l^-1; c_Lac – lactate concentration, mmol l^-1; c_MAb – MAb concentration, mg l^-1; D – dilution rate, d^-1; D_i – dilution rate in the inner chamber of the membrane dialysis reactor, d^-1; D₀ – dilution rate in the outer chamber of the membrane dialysis reactor, d^-1; q*_FB,Glc – volume specific glucose uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1; q*_FB,Gln – volume specific glutamine uptake rate related to the fixed bed volume, mmol l_FB ^-1 h^-1.     

Comparison of exhaled breath condensate pH using two commercially available devices in healthy controls,asthma and COPD patients

Background

Analysis of exhaled breath condensate (EBC) is a non-invasive method for studying the acidity (pH) of airway secretions in patients with inflammatory lung diseases.

Aim

To assess the reproducibility of EBC pH for two commercially available devices (portable RTube and non-portable ECoScreen) in healthy controls, patients with asthma or COPD, and subjects suffering from an acute cold with lower-airway symptoms. In addition, we assessed the repeatability in healthy controls.

Methods

EBC was collected from 40 subjects (n = 10 in each of the above groups) using RTube and ECoScreen. EBC was collected from controls on two separate occasions within 5 days. pH in EBC was assessed after degasification with argon for 20 min.

Results

In controls, pH-measurements in EBC collected by RTube or ECoScreen showed no significant difference between devices (p = 0.754) or between days (repeatability coefficient RTube: 0.47; ECoScreen: 0.42) of collection. A comparison between EBC pH collected by the two devices in asthma, COPD and cold patients also showed good reproducibility. No differences in pH values were observed between controls (mean pH 8.27; RTube) and patients with COPD (pH 7.97) or asthma (pH 8.20), but lower values were found using both devices in patients with a cold (pH 7.56; RTube, p < 0.01; ECoScreen, p < 0.05).

Conclusion

We conclude that pH measurements in EBC collected by RTube and ECoScreen are repeatable and reproducible in healthy controls, and are reproducible and comparable in healthy controls, COPD and asthma patients, and subjects with a common cold.     

The effect of tubificid oligochaetes on the uptake of zinc by Lake Erie sediments

A laboratory experiment was conducted to determine the effect of tubificid worms on the flux of zinc into lake sediments. Forty-six cores of Lake Erie sediment, with and without (control) tubificid worm populations, were exposed to aquarium water with a zinc concentration of about 5 mg 1^–1 for 139 days. Pore water and exchangeable particulate zinc concentrations in the top 12 cm of sediment were periodically determined in pairs of cores — one with worms and one without worms — at 1 cm depth increments. After 139 days, pore water zinc concentrations in sediments with and without worms were nearly identical in the 0–1 cm interval (4.1 and 4.3 mg 1^–1 respectively), but were significantly greater in the sediments with worms in the 1–2 cm (4.4 vs. 0.3 mg^1–1) and the 2–3 cm (1.3 vs. 0.3 mg 1^–1) intervals. Exchangeable particulate zinc concentrations in the 0–1, 1–2, and 2–3 cm intervals in sediments with worms were 612.3, 750.7, and 191.5 µg g^–1 dry sediment respectively, whereas in sediments without worms, concentrations were 375.4, 5.9, and 3.2 µg g^–1 dry sediment. The increased flux of zinc into tubificid-inhabited sediments was caused by the conveyor belt feeding activity of the worms, which continuously exposed sedimentary particles to the overlying water. Movement of zinc into sediments with worms was dominated by adsorption and by particle movement, whereas movement of zinc into control sediments was by adsorption at the sediment-water interface and diffusion. The increased concentration of zinc in tubificid-inhabited sediments has important implications with respect to the trophic transfer of zinc through the aquatic food chain.     

Independence of water and solute pathways in human RBCs

We have investigated the permeability of the human red blood cell to four di-hydroxy alcohols, 1,2PD (1,2 propanediol), 1,3PD (1.3 propanediol), 1,4BD (1,4 butanediol), and 2,3BD (2,3 butanediol), and to water by using a recently developed ESR stopped-flow method which is free from artifacts found in light scattering methods. Numerical solutions of the Kedem-Katchalsky equations fit to experimental data yielded the following permeability coefficients: P_1,2PD = 3.17 × 10^–5 cm sec^–1, p_1,3pd = 1.75 × 10^–5 cm sec^–1, P_1,4BD = 2.05 × 10⁵ cm sec^–1, P_2,3BD = 7.32 × 10^–5 cm sec^–1. Reflection coefficients () were evaluated by comparing data fit with assumed values of = 0.6,0.8 and 1.0. In all four cases the best fit was obtained with = 1.0. Treatment of cells with PCMBS (para-chloro mercuri-benzenesulfonate) was followed by a large (> 10-fold) decrease in water permeability with virtually no change in alcohol permeability. We conclude that these alcohols do not permeate the water channels to any significant extent, and discuss some of the problems in light scattering measurements of reflection coefficients that could lead to erroneous values for .We would like to thank Professor Lenore W. Yousef (Dept. of Biology, California State Univ., Fresno) for valuable discussions and critical comments. We thank Lidia Mannuzzu for measurements of ESR spectra in the presence and absence of alcohol. We are also indebted to Kate Van Fossen for her dedicated technical support. This work was supported by NIH grant No. HL-20985.     

Anion exchange purification of plasmid DNA using expanded bed adsorption

Recent developments in gene therapy with non-viral vectors and DNA vaccination have increased the demand for large amounts of pharmaceutical-grade plasmid DNA. The high viscosity of process streams is of major concern in the purification of plasmids, since it can cause high back pressures in column operations, thus limiting the throughput. In order to avoid these high back pressures, expanded bed anion exchange chromatography was evaluated as an alternative to fixed bed chromatography. A Streamline 25 column filled with 100 ml of Streamline QXL media, was equilibrated with 0.5 M NaCl in TE (10 mM Tris, 1 mM EDTA, pH=8.0) buffer at an upward flow of 300 cmh^-1, E. coli lysates (obtained from up to 3 liters of fermentation broth) were injected in the column. After washing out the unbound material, the media was allowed to sediment and the plasmid was eluted with 1 M NaCl in TE buffer at a downward flow of 120 cmh^-1. Purification factors of 36±1 fold, 26±0.4 plasmid purity, and close to 100% yields were obtained when less than one settled column volume of plasmid feed was injected. However, both recovery yield and purity abruptly decreased when larger amounts were processed–values of 35±2 and 5±0.7 were obtained for the recovery yield and purity, respectively, when 250 ml of feedstock were processed. In these cases, gel clogging and expansion collapse were observed. The processing of larger volumes, thus larger plasmid quantities, was only possible by performing an isopropanol precipitation step prior to the chromatographic step. This step led to an enhancement of the purification step.