Performance comparison of suspended bed and batch contactor chromatography

In some applications, the purification and recovery of biomolecules is performed via a cascade of batch adsorption and desorption stages using agitated contactors and related filtration devices. Suspended bed chromatography is a recent process-scale innovation that is applicable to these separations. This hybrid technique exploits the benefits of combining batch adsorption in an agitated contactor with elution in an enclosed column system. To some extent, the process is similar to batch contactor chromatography but can be fully contained and significantly quicker. The process has two steps; first the fluid containing the sample is mixed with the adsorbent in a stirred tank. Second, the slurry suspension is transferred directly into a specialized column, such as an IsoPak column. The media with the adsorbed product is formed as a packed bed, whilst the suspension liquid is passed out of the column. The product is then eluted from the packed bed utilizing standard column-chromatography techniques. The performance of the suspended bed and the agitated contactor operations are demonstrated both by full-scale experimental results and process simulations. The purification of ovalbumin from a hen-egg white feedstock by anion-exchange chromatography was used as a case study in order to prove the concept. With the availability of both pump-packed systems and shear-resistant media, suspended bed chromatography is a better alternative for a range of applications than the traditional batch separations using agitated contactors.     

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.     

Direct enzyme adsorption from an unclarified microbial feedstock using suspended bed chromatography

Suspended bed chromatography (SBC) is a new hybrid technique concomitantly benefiting from batch adsorption, the process advantages of an enclosed system, and its compatibility with established commercial chromatographic contactors and adsorbents. SBC was evaluated in the anion-exchange capture and chromatographic fractionation of native glyceraldehyde-3-phosphate dehydrogenase (G3PDH) from the complex mixture of molecular and particulate moieties that constitute wet-milled bakers' yeast. Method scouting established operating conditions exploiting Whatman Express-Ion Exchanger Q at pH 7.5 and a disrupted biomass concentration equivalent to 3.5% (wet mass/v original intact cells). Partially purified G3PDH was recovered directly from the yeast disruptate in a scaled-down process developed at 1/756 process scale. This was used to establish operating parameters to facilitate process scale-up to exploit a 44 cm I.D. Millipore IsoPak column, 18 kg (swollen mass) of Express-Ion Q anion-exchange cellulose and 275 1 of 3.5% (wet w/v) bakers' yeast disruptate. The generic utility of SBC was demonstrated for direct product adsorption from feedstocks characterised by a modest content of bioparticulates (equivalent to < 4% (wet w/v) disrupted cells). Analyses illustrated an enrichment of G3PDH in respect of enzyme concentration and significant reduction in product turbidity and Pico-Green reactivity (correlated with double stranded (ds) DNA content). The application niche for this new approach to primary protein recovery is discussed with particular reference to the downstream processing of coarsely clarified whole broths, cell disruptates and biological extracts.     

Evaluation of new high-density ion exchange adsorbents for expanded bed adsorption chromatography

New adsorbents Q HyperZ and CM HyperZ composed of hydrogel-filled porous zirconium oxide particles were evaluated for expanded bed adsorption applications in the present work. The HyperZ adsorbents have wet density of 3.16 g ml(-1), particle size of 44.5-100.8 microm and average sphere diameter of 67 microm. The bed expansion as the function of flow velocity and fluid viscosity was measured and correlated with Richardson-Zaki equation. The suitable expansion factor was considered less than 2.5, while the corresponding flow velocity was about 450 cmh(-1). Liquid mixing in the bed was determined to evaluate the stability of expanded bed. The Bodenstein numbers tested were higher than 40 and the axial mixing coefficients (D(ax)) were between 0.5 and 9.7x10(-6)m(2)s(-1), which demonstrated that a stable expanded bed could be formed under suitable operation conditions. Bovine serum albumin (BSA) and lysozyme were used as model proteins to estimate the adsorption capacities of Q and CM HyperZ, respectively. The maximum equilibrium adsorption of Q and CM HyperZ could reach 45.7 and 27.2 mg g(-1) drained adsorbents, respectively. It was found that yeast cells had little influence on the adsorption capacities of the two adsorbents tested. The dynamic adsorption capacity of BSA at 10% breakthrough with Q HyperZ was 35.9 mg g(-1) drained adsorbent at flow velocity of 100 cm h(-1) for packed bed adsorption. The values for expanded bed adsorption were 34.4 mg g(-1) drained adsorbent at flow velocity of 200 cm h(-1), 33.6 mg g(-1) drained adsorbent at 300 cm h(-1) and 31.7 mg g(-1) drained adsorbent 400 cm h(-1). The results demonstrated that Q HyperZ and CM HyperZ are suitable for expanded bed adsorption of biomolecules.     

Analysis of aminofluorescein-fatty acid derivatives by capillary electrophoresis with laser-induced fluorescence detection at the attomole level: application to mycobacterial fatty acids

In the present work, a new method of purification for antithrombin was developed using an expanded bed chromatography technique. Milk fat was removed by centrifugation and caseins were precipitated selectively by addition of zinc chloride. Crude skim milk was then directly fed to an expanded bed column containing the ion-exchange matrix. The use of a cation-exchanger (P-11) resulted in 100% adsorption and 13% recovery whereas the use of an anion-exchanger (DE-52) resulted in 100% adsorption and 84% recovery and up to five-fold purification of antithrombin. The buffer, 25 mM Tris-HCl pH 8.0; the eluting agent, 2 M (NH4)2SO4; and 100% expansion of settled bed were determined to be the optimum conditions for the purification of antithrombin by ion-exchange expanded bed chromatography. A comparison of column diameters revealed that the elution yields increase by two-fold while the column diameter increases from 1 to 2.5 cm. However, antithrombin III was concentrated to a higher degree by using the column with an internal diameter of 1 cm.     

Effect of moisture on the extraction efficiency of polycyclic aromatic hydrocarbons from soils under atmospheric pressure by focused microwave-assisted extraction

The performance of a large commercial chromatographic column was investigated using a short pulse of a tracer and an extension of the reverse-flow technique. This technique permits separate determination of the unavoidable irreversible microscopic processes and the reversible effects of flow maldistribution, and allows for the separation of flow maldistribution in the flow distributors from flow maldistribution inside the packed bed. This analysis was performed on a 0.44 m Millipore IsoPak column using Cellufine GC 700, cellulosic-based media with an average particle diameter of 75 microm, for the stationary phase. The column efficiency was quantified by analysis of the effluent curve from a short pulse of a 5% aqueous acetone tracer. The study examined behavior of beds of different lengths (10-24 cm) and beds packed from different slurry concentrations (10-75% v/v). The slurry-packed columns were very uniform, and no significant macroscopic flow maldistribution was observed inside the column. The observed bed plate heights conformed to the predictions of available one-dimensional continuum models. Dispersion in the flow distributors was significant, corresponding to 15-25% of the intracolumn dispersion when the full 24 cm available bed length was used and a proportionally larger increase for shorter bed lengths. Thus, the headers are shown to produce a significant increase in the observed plate height.     

Continuous counter-current extraction on an industrial sample using dual-flow counter-current chromatography

Both batch and continuous separations were performed on an industrial liquor using a specially built continuous counter-current extraction centrifuge. Changing the flow regime for different batch separations showed that the elution of components from the respective ends of the coil depends on the flow rates of both upper and lower phases. It was shown that, within the scope of the study, the elution of the components was not affected by the concentration of the injected reaction liquor and more importantly that continuous processing with a counter-current chromatography centrifuge was feasible. This research represents an important step forward in making continuous counter-current chromatography (or true moving bed chromatography) accessible for the pharmaceutical industry.     

Hydrophobic interaction chromatography of proteins. IV. Protein adsorption capacity and transport in preparative mode

The adsorption isotherms of four model proteins (lysozyme, α-lactalbumin, ovalbumin, and BSA) on eight commercial phenyl hydrophobic interaction chromatography media were measured. The isotherms were softer than those usually seen in ion-exchange chromatography of proteins, and the static capacities of the media were lower, ranging from 30 to 110 mg/mL, depending on the ammonium sulfate concentration and the protein and adsorbent types. The protein-accessible surface area appears to be the main factor determining the binding capacity, and little correlation was seen with the protein affinities of the adsorbents. Breakthrough experiments showed that the dynamic capacities of the adsorbents at 10% breakthrough were 20-80% of the static capacities, depending on adsorbent type. Protein diffusivities in the adsorbents were estimated from batch uptake experiments using the pore diffusion and homogeneous diffusion models. Protein transport was affected by the adsorbent pore structures. Apparent diffusivities were higher at lower salt concentrations and column loadings, suggesting that adsorbed proteins may retard intraparticle protein transport. The diffusivities estimated from the batch uptake experiments were used to predict column breakthrough behavior. Analytical solutions developed for ion-exchange systems were able to provide accurate predictions for lysozyme breakthrough but not for ovalbumin. Impurities in the ovalbumin solutions used for the breakthrough experiments may have affected the ovalbumin uptake and led to the discrepancies between the predictions and the experimental results.     

Purification of L-Lysine in Simulated Moving Bed and Fixed-Bed Chromatography

 L-Lysine was produced by a microbial process utilizing a Corynebacterium glutamicum (ATCC 21799) strain. L-Lysine was purified from the cultivated medium by fixed-bed and simulated moving bed （SMB） chromatography. The separation conditions including pH, eluent concentration and Lys+ and Lys2+ adsorption isotherms were studied in batch adsorption. The column capacity, eluent flow rate and eluent concentration have been studied in fixed-bed chromatography. Maximum purification rate of lysine was obtained as 0.066 g/(g·h) (per gram resin and per hour) at an eluent flow rate of 10 mL/min in fixed-bed chromatography. The results obtained from SMB were 0.11 g/(g·h) for L-lysine purification rate and 96% for L-lysine recovery.     

Recovery of actinorhodin from fermentation broth

In the present work, a new method of purification for actinorhodin was developed using an expanded bed chromatography technique in which antibiotic capture, feedstock clarification, centrifugation, dialysis and concentration are done in one step. The cation-exchanger (P-11) resulted in 26% adsorption and 2% recovery whereas the anion-exchanger (DE-52) resulted in 99% adsorption and 56% recovery of adsorbed antibiotic using methanol buffer and 2 M NH4Cl as eluting agent. Streamline DEAE anion-exchanger, which is especially designed for EBA applications, yields 82% adsorption and 50% elution of actinorhodin fed into the chromatography column directly from the fermentation broth. Isocratic elution resulted in extremely efficient yield compared to linear gradient elution, i.e. 13.5-fold more recovery in the column with an aspect ratio (L:D) of 4. Expansion by 150% of settled bed resulted in the best recovery of actinorhodin among 100 and 200% expansions. A comparison of breakthrough profiles in packed and expanded bed adsorption showed that the performance of the expanded bed is better (by 33%) at allowing more volume of the fermentation broth to pass through the chromatography column.     

Simulated moving bed technology with a simplified approach for protein purification. Separation of lactoperoxidase and lactoferrin from whey protein concentrate

Simulated moving bed (SMB) technology is a continuous chromatographic technique proven to have many advantages compared to conventional batch chromatography, such as: raised productivity and product concentration, reduced buffer consumption as well as more efficient use of raw material. In this study a 20 column SMB process for the separation of lactoperoxidase and lactoferrin from whey protein concentrate (WPC) was developed. A simplified approach with data from a single column experiment was used when designing the process. The SMB process data were compared to a theoretical scale-up of the breakthrough experiment reflecting the same 20 column set-up run in non-moving bed mode. The outcome of the comparison is a 48% raise in productivity, a 4.3 times decrease in buffer consumption, 6.5 times raise in target protein concentration with a raw material utilization which is slightly better for the SMB process.     

DNA binding during expanded bed adsorption and factors affecting adsorbent aggregation

DNA-induced aggregation and contraction of expanded bed adsorption chromatography beds have been examined using strong anion exchanger Q HyperZ and calf thymus DNA in buffers containing added NaCl. Two batches of adsorbent with different ionic capacities were used allowing the effects of different ligand densities to be examined. Very high dynamic binding capacities at 10% breakthrough were found in the absence of added salt. However, the highest binding capacities ( approximately 10 and approximately 19mgDNAml(-1) gel) were found in buffers containing added salt at concentrations of either 0.25 or 0.35M, for the low and high ligand density adsorbents, respectively. Bed contraction was observed, but did not correlate with dynamic binding capacity or with the amount of DNA loaded. No differences in bed contraction were seen by varying the concentration of DNA loaded in the range of 20-80mugml(-1) even though the dynamic binding capacity was reduced as DNA concentration was increased. The extent of bed contraction during DNA loading was found to be a function of added salt concentration and ligand density of the adsorbent. The results imply that ligand density significantly affects the salt tolerance of anion exchangers when binding DNA. However, more importantly, with the adsorbents examined here, attempts to reduce bed aggregation by feedstock conditioning with added salt may increase DNA binding leading to a reduction in expanded bed adsorption performance compromising protein capture in real feedstocks.     

Adsorbent usage for VOC removal in synthetic latices

In the synthetic latex industry, there is a trend towards lower VOC levels. This paper describes an alternative to the current steam stripping or chemicals injection processes, namely using adsorbents for VOC removal for a styrene‐butadiene latex. Both batch and continuous adsorption experiments were performed to screen adsorbents, experimental conditions and simulate plant scale‐up. The removal effect of the adsorbents was determined using gas chromatography (GC). It was found that activated carbon had the best VOC removal efficiency, and optimum operating conditions existed at a pH of 10.5, and ambient temperature. It was further found in the carbon bed experiments that the VOC level could be lowered down to 150 ppm, which was the minimum attainable. The regeneration of the spent carbon using solvent extraction was also investigated. Several solvents were tested, and toluene was found to be the most efficient.     

Macroporous copolymer matrix. IV. Expanded bed adsorption application

Macroporous crosslinked hydroxyethyl methacrylate-ethylene dimethacrylate copolymeric beads (HEG beads) were synthesized by suspension polymerization in the presence of a pore generating agent. These beads were coupled to alpha-cyclodextrin through a urethane spacer. These modified copolymer beads (affinity-HEG beads) so prepared were evaluated for their suitability in expanded bed chromatography. The optimum thickness of the distributor plate for stable expanded bed for use in expanded bed adsorption (EBA) was established. The affinity-HEG beads are comparable in density to Streamline diethyl amino ethane (DEAE) and exhibit better mechanical stability at higher superficial velocity under fluidization. The affinity-BEG beads were used as affinity chromatography matrices for the purification of cyclodextrin glycosyltransferase. Feeding of 5-fold diluted fermented broth to the column containing affinity-HEG beads of settled bed height 7.5 cm (I.D. 26 mm and length 42 cm) at double bed expansion resulted in a sharp breakthrough curve of alpha-cyclodextrin glycosyltransferase (CGTase). The adsorbed enzyme was eluted from the bed in 50 mM Tris-HCl buffer containing 10 mM CaCl2 at 25 degrees C in packed bed configuration.     

A practical approach to maximizing peak capacity by using long columns packed with pellicular stationary phases for proteomic research

Maximization of peak capacity is a very important step in developing one-dimensional separations of complex samples. In recent work, it was shown that the use of small particles in combination with the new technique of ultrahigh pressure liquid chromatography (UHPLC) was able to generate very high peak capacities. Here we show the ability of conventional HPLC instrumentation to give comparable peak capacities to those obtained in UHPLC for the important case of complex mixtures of peptides but at much lower pressures by using a 60 cm long set of columns packed with 5 microm pellicular (superficially porous) particles. We first show, in complete agreement with the well known results of the theory of isocratic separations that, when time is not limiting, the best peak capacities in gradient elution chromatography are obtained by using large particles and the longest column that can be operated at the pump's pressure limit. Two different types of 5 microm particles (superficially porous and totally porous) were compared for their efficiency in gradient chromatography of peptides. We find that the pellicular material gave about 50% higher peak capacity compared to the analogous porous material. A 60 cm column set packed with pellicular particles was made by connecting short columns in series; a peak capacity of about 460 was obtained in 4 h at room temperature. Increasing the column temperature to 70 degrees C reduced the analysis time to 2 h and further increased the peak capacity to more than 500. The number of peaks observed in the separation of bovine serum albumin tryptic peptides was greatly increased and the separation quality was significantly improved.     

Effect of phenyl sepharose ligand density on protein monomer/aggregate purification and separation using hydrophobic interaction chromatography

In the large-scale manufacturing and purification of protein therapeutics, multiple chromatography adsorbent lots are often required due to limited absorbent batch sizes or during replacement at the end of the useful column lifetime. Variability in the adsorbent performance from lot to lot should be minimal in order to ensure that consistent product purity and product quality attributes are achieved when a different lot or lot mixture is implemented in the process. Vendors of chromatographic adsorbents will often provide release specifications, which may possess a narrow range of acceptable values. Despite relatively narrow release specifications, the performance of the adsorbent in a given purification process could still vary from lot to lot. In this case, an alternative use test (one that properly captures the lot to lot variability) may be required to determine an acceptable range of variability for a specific process. In this work, we describe the separation of therapeutic protein monomer and aggregate species using hydrophobic interaction chromatography, which is potentially sensitive to adsorbent lot variability. An alternative use test is formulated, which can be used to rapidly screen different adsorbent lots prior to implementation in a large-scale manufacturing process. In addition, the underlying mechanism responsible for the adsorbent lot variability, which was based upon differences in protein adsorption characteristics, was also investigated using both experimental and modeling approaches.     

Direct recovery of recombinant nucleocapsid protein of Nipah virus from unclarified Escherichia coli homogenate using hydrophobic interaction expanded bed adsorption chromatography

A direct recovery of recombinant nucleocapsid protein of Nipah virus (NCp-NiV) from crude Escherichia coli (E. coli) homogenate was developed successfully using a hydrophobic interaction expanded bed adsorption chromatography (HI-EBAC). The nucleic acids co-released with the recombinant protein have increased the viscosity of the E. coli homogenate, thus affected the axial mixing in the EBAC column. Hence, DNase was added to reduce the viscosity of feedstock prior to its loading into the EBAC column packed with the hydrophobic interaction chromatography (HIC) adsorbent. The addition of glycerol to the washing buffer has reduced the volume of washing buffer applied, and thus reduced the loss of the NCp-NiV during the washing stage. The influences of flow velocity, degree of bed expansion and viscosity of mobile phase on the adsorption efficiency of HI-EBAC were studied. The dynamic binding capacity at 10% breakthrough of 3.2 mg/g adsorbent was achieved at a linear flow velocity of 178 cm/h, bed expansion of two and feedstock viscosity of 3.4 mPa s. The adsorbed NCp-NiV was eluted with the buffer containing a step gradient of salt concentration. The purification of hydrophobic NCp-NiV using the HI-EBAC column has recovered 80% of NCp-NiV from unclarified E. coli homogenate with a purification factor of 12.5.     

Concentration and purification of orotic acid directly from whey with an expanded bed adsorption system

The development of an expanded bed process for the concentration and purification of orotic acid directly from whey is described. Different commercially available adsorbents were tested in series of pilot batch adsorption experiments to determine the most suitable separation system. Best results were achieved using a weak anion-exchange matrix. An elution protocol was established using MgCl2 as eluting agent to recover the adsorbed orotic acid with approximately 85% yield and 10-fold concentration. Purified orotic acid was precipitated under acid conditions with a yield of 95%.     

Impact of adsorption isotherm parameters on the performance of enantioseparation using simulated moving bed chromatography

Often there are several chromatographic systems, i.e., combinations of mobile and stationary phases, available to solve a certain separation problem. Essential differences of these chromatographic systems are the separation factors and the efficiencies. For preparative applications in addition also the column saturation capacities and solubility limits are of importance. The impact of all these parameters appears to be rather well understood for conventional overloaded elution chromatography using a single column. In the last years the continuous simulated moving bed (SMB) process was increasingly used as a powerful alternative to batch elution since increased productivities and reduced solvent consumptions could be realised. However, the selection of suitable chromatographic systems is more sophisticated for this process. In this paper five different chromatographic systems capable of separating the enantiomers of mandelic acid are compared based on the achievable productivities using SMB chromatography. For these five systems the adsorption isotherms have been determined experimentally. Subsequently, an analysis of the SMB process was performed numerically using a well-established model.