Rapid isolation of monosialogangliosides from bovine brain gangliosides by selective-overload chromatography.

A procedure for rapid isolation of monosialogangliosides from purified bovine brain gangliosides has been developed. It utilizes the selective difference in association between monosialogangliosides and polysialogangliosides for the ion-exchange resin Q-Sepharose. When the ion-exchange column is overloaded with a bovine brain ganglioside mixture in the proper ganglioside to column bed-volume ratio, the polysialogangliosides are selectively retained by the column while the monosialogangliosides emerge with the void volume without the use of salt for elution. With the critical ganglioside to bed-volume ratio (1 g:8.32 ml), and an appropriate column bed-height to column radius ratio of 6.9, monosialogangliosides are reproducibly obtained in high purity with greater than 90% yield. The method has been used at both the analytical and preparative scale. We call this separation technique selective-overload chromatography.     

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.     

HETP values of large-scale columns for liquid chromatography

Two columns of 100 mm and 200 mm in diameter for bench-scale liquid chromatography were designed to obtain a small dead volume and an appropriately uniform distribution of liquid, and the values of the height equivalent to a theoretical plate were measured for three kinds of packing beads, Sepharose 4B, Cellulofine GC-700 m and Silica. A suitable pressure drop through a distribution plate and/or a packed bed helps to distribute liquid flow uniformly over the column diameter, and reasonable HETP values were obtained.     

Packing of large‐scale chromatography columns with irregularly shaped glass based resins using a stop‐flow method

Rigid chromatography resins, such as controlled pore glass based adsorbents, offer the advantage of high permeability and a linear pressure‐flow relationship irrespective of column diameter which improves process time and maximizes productivity. However, the rigidity and irregularly shaped nature of these resins often present challenges in achieving consistent and uniform packed beds as formation of bridges between resin particles can hinder bed consolidation. The standard flow‐pack method when applied to irregularly shaped particles does not yield well‐consolidated packed beds, resulting in formation of a head space and increased band broadening during operation. Vibration packing methods requiring the use of pneumatically driven vibrators are recommended to achieve full packed bed consolidation but limitations in manufacturing facilities and equipment may prevent the implementation of such devices. The stop‐flow packing method was developed as an improvement over the flow‐pack method to overcome these limitations and to improve bed consolidation without the use of vibrating devices. Transition analysis of large‐scale columns packed using the stop‐flow method over multiple cycles has shown a two‐ to three‐fold reduction of change in bed integrity values as compared to a flow‐packed bed demonstrating an improvement in packed bed stability in terms of the height equivalent to a theoretical plate (HETP) and peak asymmetry (A_s). © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1319–1325, 2014     

Monolithic chromatography on conjoint liquid chromatography columns for speciation of platinum-based chemotherapeutics in serum of cancer patients

BackgroundMonolithic chromatography using convective interaction media (CIM) disks or columns can be used in the separation step of speciation analysis. When different monolithic disks are placed in one housing, forming conjoint liquid chromatography (CLC) monolithic column, two-dimensional separation is achieved in a single chromatographic run.MethodsHere, we assembled low-pressure (maximum 50 bar) CLC monolithic column, which consists of two 0.34 mL shallow CIM monolithic disks and high-pressure CLC column (maximum 150 bar) from 0.1 mL analytical high performance short bed CIMac monolithic disks. Both the CLC columns constructed from affinity Protein G and weak anion exchange diethylamine (DEAE) disks, were applied for the speciation of cisplatin, oxaliplatin and carboplatin in spiked standard serum proteins, spiked human serum and serum of cancer patients. The analytical performances of the CLC columns used were evaluated by comparing their robustness, selectivity, repeatability and reproducibility. The separated serum proteins were detected on-line by ultraviolet (UV) and eluted Pt species by inductively coupled plasma mass spectrometry (ICP-MS). For accurate quantification of the separated Pt species (unbound Pt-based chemotherapeutic from species associated to transferrin (Tf), human serum albumin (HSA) and Immunoglobulin G (IgG)), post column isotope dilution (ID)-ICP-MS was used.ResultsThe data from analyses showed that both tested CLC monolithic columns gave statistically comparable results, with the low-pressure CLC column exhibiting better resolving power and robustness. It also enables more effective cleaning of monolithic disks and to analyse larger series of serum samples than the high-pressure CLC column. Analyses of serum samples of cancer patients treated with cisplatin or carboplatin showed that Pt-chemotherapeutics were bound preferentially to HSA (around 80%). The portion of unbound Pt in general did not exceed 2%, up to 5% of Pt was associated with Tf and approximately 20% with IgG. Column recoveries, calculated as a ratio between the sum of concentrations of Pt species eluted and concentration of total Pt in serum samples, were close to 100%.ConclusionsLow-pressure CLC column exhibited greater potential than high-pressure CLC column, and can be thus recommended for its intended use in speciation analysis of metal-based biomolecules.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%-60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Effects of adsorbent properties on zone spreading in expanded bed chromatography

The mixing performance as well as the adsorption performance in expanded bed chromatography (EBC) was investigated by using various types of adsorption media (average particle size = 100–700 m, density = 1100–1700 kg/m³, base matrix = hydroxyapatite, styrene-divinylbenzene, cross-linked agarose). The scale down study with 0.8 cm diameter columns was also attempted. Pulse response curves were measured with vitamin B₁₂ as a tracer [Residence time distribution RTD experiments], and the HETP (height equivalent to a theoretical plate or plate height) values were calculated from the peak variance and the peak retention time. The HETP values for different types of packing media tested showed very similar values (0.5–1.0 cm), which did not depend on the flow-rate or the column diameter (0.8–2.6 cm). Dynamic binding capacity (DBC) values of lactic acid on a Dowex anion-exchange resin were determined from breakthrough curve (BTC) measurements for both EBC and fixed bed chromatography (FBC). The DBC values for EBC were similar to those for FBC. When the liquid feed contained insoluble particles (yeast cells) the degree of mixing increased. However, the contribution of the mixing to the total spreading of BTCs for EBC was usually small so that this increase in the mixing did not affect the adsorption performance or the DBC values significantly.     

Induced binding of proteins by ammonium sulfate in affinity and ion-exchange column chromatography

In general, proteins bind to affinity or ion-exchange columns at low salt concentrations, and the bound proteins are eluted by raising the salt concentration, changing the solvent pH, or adding competing ligands. Blue-Sepharose is often used to remove bovine serum albumin (BSA) from samples, but when we applied BSA to Blue-Sepharose in 20 mM phosphate, pH 7.0, 50%–60% of the protein flowed through the column; however, complete binding of BSA was achieved by the addition of 2 M ammonium sulfate (AS) to the column equilibration buffer and the sample. The bound protein was eluted by decreasing the AS concentration or by adding 1 M NaCl or arginine. AS at high concentrations resulted in binding of BSA even to an ion-exchange column, Q-Sepharose, at pH 7.0. Thus, although moderate salt concentrations elute proteins from Blue-Sepharose or ion-exchange columns, proteins can be bound to these columns under extreme salting-out conditions. Similar enhanced binding of proteins by AS was observed with an ATP-affinity column.     

Technical advance: identification of plant actin-binding proteins by F-actin affinity chromatography

Proteins that interact with the actin cytoskeleton often modulate the dynamics or organization of the cytoskeleton or use the cytoskeleton to control their localization. In plants, very few actin-binding proteins have been identified and most are thought to modulate cytoskeleton function. To identify actin-binding proteins that are unique to plants, the development of new biochemical procedures will be critical. Affinity columns using actin monomers (globular actin, G-actin) or actin filaments (filamentous actin, F-actin) have been used to identify actin-binding proteins from a wide variety of organisms. Monomeric actin from zucchini (Cucurbita pepo L.) hypocotyl tissue was purified to electrophoretic homogeneity and shown to be native and competent for polymerization to actin filaments. G-actin, F-actin and bovine serum albumin affinity columns were prepared and used to separate samples enriched in either soluble or membrane-associated actin-binding proteins. Extracts of soluble actin-binding proteins yield distinct patterns when eluted from the G-actin and F-actin columns, respectively, leading to the identification of a putative F-actin-binding protein of approximately 40 kDa. When plasma membrane-associated proteins were applied to these columns, two abundant polypeptides eluted selectively from the F-actin column and cross-reacted with antiserum against pea annexins. Additionally, a protein that binds auxin transport inhibitors, the naphthylphthalamic acid binding protein, which has been previously suggested to associate with the actin cytoskeleton, was eluted in a single peak from the F-actin column. These experiments provide a new approach that may help to identify novel actin-binding proteins from plants.     

Performance of affinity chromatography columns

Performance of affinity chromatography columns was studied by measuring the rates of adsorption and elution of trypsin in a Sepharose 4B-soybean trypsin inhibitor column and a Sepharose 4B-arginine peptides column. The volumetric coefficient for trypsin transfer was evaluated from the break-through curves of trypsin, and elution profiles bed height of Sepharose 4B-STI column was estimated based on these results.     

Improved packing of preparative biochromatography columns by mechanical vibration

The bioprocessing industry relies on packed-bed column chromatography as its primary separation process to attain the required high product purities and fulfill the strict requirements from regulatory bodies. Conventional column packing methods rely on flow packing and/or mechanical compression. In this work, the application of ultrasound and mechanical vibration during packing was studied with respect to packing density and homogeneity. We investigated two widely used biochromatography media, incompressible ceramic hydroxyapatite, and compressible polymethacrylate-based particles, packed in a laboratory-scale column with an inner diameter of 50 mm. It was shown that ultrasonic irradiation led to reduced particle segregation during sedimentation of a homogenized slurry of polymethacrylate particles. However, the application of ultrasound did not lead to an improved microstructure of already packed columns due to the low volumetric energy input (~152 W/L) caused by high acoustic reflection losses. In contrast, the application of pneumatic mechanical vibration led to considerable improvements. Flow-decoupled axial linear vibration was most suitable at a volumetric force output of ~1,190 N/L. In the case of the ceramic hydroxyapatite particles, a 13% further decrease of the packing height was achieved and the reduced height equivalent to a theoretical plate (rHETP) was decreased by 44%. For the polymethacrylate particles, a 18% further packing consolidation was achieved and the rHETP was reduced by 25%. Hence, it was shown that applying mechanical vibration resulted in more efficiently packed columns. The application of vibration furthermore is potentially suitable for in situ elimination of flow channels near the column wall.     

The displacement effect of phenylalanine on lysozyme elution in hydrophobic interaction chromatography

The volume, retention time, and shape of the lysozyme peak eluted from a hydrophobic interaction chromatography column (TosoHaas 650 M Phenyl) was influenced by the presence and concentration of phenylalanine in the elution buffer. Lysozyme peak retention time decreased by a factor of 2.5 with the addition of 86 mM phenylalanine to the elution buffer.     

Characterization of supermacroporous monolithic polyacrylamide based matrices designed for chromatography of bioparticles

Supermacroporous monolithic acrylamide (AAm)-based cryogels were prepared by radical cryo-polymerizaton (polymerization in the moderately frozen system) of AAm with functional monomers and cross-linker N,N'-methylene-bis-acrylamide (MBAAm). Electron microscopy studies revealed supermacroporous structure of the developed cryogels with pore size of 5-100 microm. Cryogel porosity depended on cryo-polymerization conditions. More than 90% of the monolithic bed volume is the interconnected supermacropores filled with water and less than 10% of the monolithic volume is pore walls. The total protein binding capacity (lysozyme in the case of immobilized metal affinity chromatography (IMAC) column and bovine serum albumin (BSA) in the case of anion-exchange (AE) column) was independent of the flow rates till 600 cm/h. Chromatographic behavior of E. coli cells when a cell suspension was applied to ion-exchange cryogel columns depended on both the density of functional ligand and the porosity of the cryogel.     

High-performance hydrophobic interaction chromatography of proteins

A new, weakly hydrophobic, high-performance liquid chromatography column has been developed for the separation of native proteins based on their relative hydrophobicities. Starting with a covalently bound, hydrophilic polyamine matrix, packing materials were synthesized through acylation with anhydrides and acid chlorides of increasing chain length to obtain increasingly hydrophobic surfaces. Proteins in aqueous buffers were induced to bind hydrophobically to the columns by the use of high salt concentrations in the mobile phase. Elution was achieved by decreasing the ionic strength of the solvent in a linear gradient. A mixture of cytochrome c, conalbumin, and beta-glucosidase was used as a standard to test the resolving power of newly synthesized columns. On a 4-cm butyrate column, baseline resolution was achieved in 20 min with a gradient of 3.0 mu sodium sulfate in 0.1 M potassium phosphate buffer, pH 7.0, to water. The static loading capacity for each column was determined using a hemoglobin binding assay. Capacities normally ranged between 150 and 180 mg of hemoglobin per gram of support. Since proteins are not denatured in hydrophobic interaction chromatography, enzymes eluted from the column retained enzymatic activity. Samples of alpha-amylase and beta-glucosidase ranging in size from 10 to 200 micrograms were recovered from the butyrate column with greater than 92% enzymatic activity in all cases. In a single trial, the enzyme citrate synthase was recovered from the benzoate column with 92% retention of enzymatic activity.     

Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.     

Modeling of transient flow through a viscoelastic preparative chromatography packing

The common method for purification of macromolecular bioproducts is preparative packed‐bed chromatography using polymer‐based, compressible, viscoelastic resins. Because of a downstream processing bottleneck, the chromatography equipment is often operated at its hydrodynamic limit. In this case, the resins may exhibit a complex behavior which results in compression–relaxation hystereses. Up to now, no modeling approach of transient flow through a chromatography packing has been made considering the viscoelasticity of the resins. The aim of the present work was to develop a novel model and compare model calculations with experimental data of two agarose‐based resins. Fluid flow and bed permeability were modeled by Darcy's law and the Kozeny–Carman equation, respectively. Fluid flow was coupled to solid matrix stress via an axial force balance and a continuity equation of a deformable packing. Viscoelasticity was considered according to a Kelvin–Voigt material. The coupled equations were solved with a finite difference scheme using a deformable mesh. The model boundary conditions were preset transient pressure drop functions which resemble simulated load/elution/equilibration cycles. Calculations using a homogeneous model (assuming constant variables along the column height) gave a fair agreement with experimental data with regard to predicted flow rate, bed height, and compression–relaxation hysteresis for symmetric as well as asymmetric pressure drop functions. Calculations using an inhomogeneous model gave profiles of the bed porosity as a function of the bed height. In addition, the influence of medium wall support and intraparticle porosity was illustrated. The inhomogeneous model provides insights that so far are not easily experimentally accessible. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:958–967, 2013     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Large-scale ion-exchange column chromatography of proteins. Comparison of different formats

This review describes the performance of various column designs available to process-scale users of low-pressure chromatography for protein purification. By carrying out a range of ion-exchange separations using Whatman microgranular ion-exchange celluloses we are able to compare and contrast the practical performance issues associated with several designs of axial and radial flow columns.     

Tresyl activation of a hydroxyalkyl ligand for coupling to a hydrazide gel: stable immobilization of T-2 toxin for affinity purification of T-2 antibody

A stable T-2 hydrazide gel is prepared by activating T-2 toxin with tresyl chloride followed by coupling to agarose-adipic acid hydrazide. Utilized as an affinity chromatography column, this T-2 hydrazide gel purifies a monoclonal antibody for T-2 in high yield directly from ascites fluid. Specific antibody trapped on the column is eluted either with excess T-2 or at pH 11.6. Much less successful are two other T-2 affinity columns that were prepared and evaluated: T-2 bovine serum albumin Affi-Gel 15 and T-2 hexylamine Sepharose.     

Expanded bed adsorption at production scale: Scale-up verification, process example and sanitization of column and adsorbent

Expanded bed adsorption is a technique for recovery of biomolecules directly from unclarified feedstocks. The work described here demonstrates that expanded bed adsorption is a scaleable technique. The methods used to test scaleability were “determination of degree of bed expansion”, “determination of axial dispersion” and “determination of protein breakthrough capacity”. The performance of a production scale expanded bed column with 600?mm diameter was tested using these methods and the results were found to be consistent with the results obtained from lab scale and pilot scale expanded bed columns. The scaleability and function of the expanded bed technique was also tested by performing a “process example”: a purification mimicking a real process using a yeast culture spiked with bovine serum albumin as feedstock. The results show that the 600?mm diameter production scale column was as efficient as a 25?mm diameter lab scale column in recovering bovine serum albumin from the unclarified yeast culture. The production scale runs were fully automated using a software controlled system containing an adaptor position sensor and an adsorbent sensor. A cleaning study was performed which showed that after use of a proper cleaning protocol, no surviving microorganisms could be detected in the column or in the adsorbent.