The use of affinity adsorbents in expanded bed adsorption

The potential for the use of affinity ligands in expanded bed adsorption (EBA) procedures is reviewed. The use of affinity ligands in EBA may improve its use in direct recovery operations, as the enhanced selectivity of the adsorbent permits selective capture of the target from complex feedstocks and high degrees of purification. The properties of ligands suitable for use in EBA processes are identified and illustrated with examples. In addition to its use in the recovery of soluble products, such as proteins and nucleic acids, from particulate feedstocks, EBA can also be used to recover particulate entities, such as cells and packaged DNA (viruses and phages), from feedstocks. Affinity ligands coupled to appropriate chosen support materials will be required for such processes in order to achieve the necessary selectivity for the required particulate entity. The latter point is illustrated by the use of proteinaceous ligands immobilized to perfluorocarbon emulsions to achieve separations of microbial cells.     

Design and assembly of solid-phases for the effective recovery of nanoparticulate bioproducts in fluidised bed contactors

Practical recovery of nanoparticulate bioproducts from suspension feedstocks has been studied in batch, fixed bed and fluidised bed adsorptive contactors. The performance of five discrete configurations of adsorbent solid phase has been critically evaluated in the anion exchange recovery of mg quantities of BSA nanoparticles. These have served as surrogate size mimics of less easily sourced viral and plasmid gene therapy vectors, characterised by high value and a shortage of supply in quantities sufficient for research and development. Performance parameters of binding capacity, efficacy of washing, desorption efficiency and total cycle time were strongly influenced by the external and internal topographies of solid phases, together with the localised concentrations of interacting chemical ligands which modulate adsorption. In respect of a full operational recovery cycle, porous adsorbents developed for refined chromatographic fractionation of macromolecules, appear less suited overall than solid, non-porous particles, or solid particles coated with a shallow pellicle of active adsorbent material. Such findings have been confirmed in a detailed demonstration of the recovery of plasmid DNA (7.8 Kb) from chemical lysates of Escherichia coli.     

Galactosyl-mimodye ligands for Pseudomonas fluorescens beta-galactose dehydrogenase.

Protein molecular modelling and ligand docking were employed for the design of anthraquinone galactosyl-biomimetic dye ligands (galactosyl-mimodyes) for the target enzyme galactose dehydrogenase (GaDH). Using appropriate modelling methodology, a GaDH model was build based on a glucose-fructose oxidoreductase (GFO) protein template. Subsequent computational analysis predicted chimaeric mimodye-ligands comprising a NAD-pseudomimetic moiety (anthraquinone diaminobenzosulfonic acid) and a galactosyl-mimetic moiety (2-amino-2-deoxygalactose or shikimic acid) bearing an aliphatic 'linker' molecule. In addition, the designed mimodye ligands had an appropriate in length and chemical nature 'spacer' molecule via which they can be attached onto a chromatographic support without steric clashes upon interaction with GaDH. Following their synthesis, purification and analysis, the ligands were immobilized to agarose. The respective affinity adsorbents, compared to other conventional adsorbents, were shown to be superior affinity chromatography materials for the target enzyme, Pseudomonas fluorescensbeta-galactose dehydrogenase. In addition, these mimodye affinity adsorbents displayed good selectivity, binding low amounts of enzymes other than GaDH. Further immobilized dye-ligands, comprising different linker and/or spacer molecules, or not having a biomimetic moiety, had inferior chromatographic behavior. Therefore, these new mimodyes suggested by computational analysis, are candidates for application in affinity labeling and structural studies as well as for purification of galactose dehydrogenase.     

New developments in affinity chromatography

The design, synthesis and chromatographic operation of a new range of stable and selective immobilized dye affinity adsorbents for potential application in the purification of pharmaceutical proteins is described. Computer aided molecular design has been exploited to design novel dye ligands which show a predictable selectivity for the target protein and which, when coupled to stable perfluoropolymer supports, yield high capacity, low leakage adsorbents for affinity chromatography. It is anticipated that these new materials will withstand the rigorous conditions required for sanitization and cleaning in situ of industrial scale processes.     

Fabrication and characterisation of a novel pellicular adsorbent customised for the effective fluidised bed adsorption of protein products

A dense pellicular solid matrix has been fabricated by coating 4% agarose gel on to dense zirconia-silica (ZS) spheres by water-in-oil emulsification. The agarose evenly laminated the ZS bead to a depth of 30 μm, and the resulting pellicular assembly was characterised by densities up to 2.39 g/mL and a mean particle diameter of 136 μm. In comparative fluidisation tests, the pellicular solid phase exhibited a two-fold greater flow velocity than commercial benchmark adsorbents necessary to achieve common values of bed expansion. Furthermore, the pellicular particles were characterised by improved qualities of chromatographic behaviour, particularly with respect to a three-fold increase in the apparent effective diffusivity of lysozyme within a pellicular assembly modified with Cibacron Blue 3GA. The properties of rapid protein adsorption/desorption were attributed to the physical design and pellicular deployment of the reactive surfaces in the solid phase. When combined with enhanced feedstock throughput, such practical advantages recommend the pellicular assembly as a base matrix for the selective recovery of protein products from complex, particulate feedstocks (whole fermentation broths, cell disruptates and biological extracts).     

Studies on the binding sites of IgG2 monoclonal antibodies recognized by terpyridine‐based affinity ligands

This investigation has examined the origin of the molecular recognition associated with the interaction of monoclonal IgG2's with terpyridine‐based ligands immobilized onto agarose‐derived chromatographic adsorbents. Isothermal titration calorimetric (ITC) methods have been employed to acquire thermodynamic data associated with the IgG2‐ligand binding. These ITC investigations have documented that different enthalpic and entropic processes are involved depending on the nature of the chemical substituents in the core structure of the terpyridinyl moiety. In addition, molecular docking studies have been carried out with IgG2 structures with the objective to identify possible ligand binding sites and key interacting amino acid residues. These molecular docking experiments with the different terpyridine‐based ligands have shown that all of the examined ligands can potentially undergo favorable interactions with a site located within the Fab region of the IgG2. However, another favorable binding site was also identified from the docking poses to exist within the Fc region of the IgG2 for some, but not all, of the ligands studied. These investigations have provided a basis to elucidate the unique binding properties and chromatographic behaviors shown by several substituted terpyridine ligands in their interaction with IgGs of different isotype. Copyright © 2016 John Wiley & Sons, Ltd.     

Lumped parameter model for prediction of initial breakthrough profiles for the chromatographic capture of antibodies from a complex feedstock

A simple mathematical model to predict initial breakthrough profiles from preparative chromatographic separations of biological macromolecules has been developed. A lumped parameter approach was applied, employing Langmuirian adsorption kinetics to describe the rate of mass transfer (MT) from the bulk liquid in the column to the bound state. Equilibrium and kinetic adsorption data were determined for six different packed bed chromatographic adsorbents: two derivatised with rProtein A; and four functionalised with synthetic low molecular weight ligands. All adsorption isotherms were well described by the Langmuir model, whereas the data fitting to kinetic batch experiments showed that the model was inadequate after the first approximately 25 min of adsorption for four of the six adsorbents. The model underestimated the dynamic Ig breakthough on packed beds of rProtein A Sepharose FF, MabSelect, MBI HyperCel, and MabSorbent A1P, applying a feedstock of 20-100% (v/v) clarified rabbit antiserum. However, when employing a maximum adsorption capacity 25% greater than that determined in batch binding studies, excellent agreement was obtained at all antiserum strengths for most adsorbents. Useful insights into scale-up and process design can be obtained by applying the model, without determining tentative parameters specific for each adsorbent and target protein concentration. However, the model parameters are solvent dependent so a prerequisite for its true applicability is that binding is both Langmuirian and essentially independent of the ionic strength of the feedstock applied.     

Studies on endotoxin removal mechanism of adsorbents with amino acid ligands

In this paper, a series of adsorbents with different amino acid ligands for endotoxin removal were prepared and endotoxin adsorption capacities (EAC) in aqueous solution were studied using an affinity column. The results showed that the property and structure of amino acid ligands have great influence on EAC. As the increasing of isoelectric point and polarity of amino acids ligands, EACs of the adsorbents increased. In addition, computer simulation method was employed to a further investigation on the interaction between endotoxins and ligands. Based on the results, some adsorbents were applied to remove endotoxin from endotoxemia rabbit's serum. Similar adsorption results were observed and the removal efficiency of adsorbents with Arg, Ser ligands is up to 78%.     

Chromatographic removal of endotoxin from protein solutions by polymer particles

Endotoxins, constituents of cell walls of gram-negative bacteria, are potential contaminants of the protein solutions originating from biological products. Such contaminants have to be removed from solutions used for intravenous administration, because of their potent biological activities causing pyrogenic reactions. Separation methods used for decontamination of water, such as ultrafiltration, have little effect on endotoxin levels in protein solutions. To remove endotoxin from a solution of high-molecular-mass compounds, such as proteins, the adsorption method has proven to be most effective. In this review, we first introduce endotoxin-specific properties in an aqueous solution, and then provide various methods of chromatographic separation of endotoxins from cellular products using polymer adsorbents. We also provide the design of novel endotoxin-specific polymer adsorbents.     

Physical and biochemical characterization of a simple intermediate between fluidized and expanded bed contactors

Physical and biochemical comparison has been made of the performance of a simple fluidised bed contactor and a commercial expanded bed contactor, characterised by identical dimensions, and operated at various settled bed heights with two anion exchange adsorbents. The contactors were tested with various feedstocks comprising bovine albumin in the absence and presence of 20 g dry cell weight biomass litre^-1. Earlier classification of the simple contactor as a single-stage, well mixed fluidised bed is reviewed. The relative merits of STREAMLINE DEAE and DEAE Spherodex LS as fluidisable, anion exchange adsorbents are discussed.     

Rapid method for the isolation of hydroxylysylpyridinoline and lysylpyridinoline from concentrated bone hydrolysates by liquid chromatographic techniques

A rapid method for the isolation of hydroxylysylpyridinoline and lysylridinoline from bone by liquid chromatographic methods is described. Decalcified bone is hydrolysed in 7 M hydrochloric acid. After evaporation of the acid, the high molecular mass dark coloured degradation products are removed by adsorption on non-polar adsorbents. The pyridinolines are separated from the majority of the amino acids by adsorption on cellulose. Separation of HP and LP is performed either by cation-exchange chromatography or by reversed-phase ion-pari chromatography. The pyridinoline containing fractions are desalted by size-exclusion chromatography. The progress of the hydrolytic cleavage of collagen and the optimal parameters for purification and separation were examined. As a result the existing method allows the isolation of high amounts of pyridinolines with low amounts of adsorbents and chemical within a short time.     

Evaluation of gas chromatograph packings for the separation of butyric acid from serum-catalyzed hydrolysis of ethyl butyrate

Twelve synthetic and pilot adsorbents of different polarity and varying chemical composition were tested for the separation and quantitative determination of butyric acid from serum-catalyzed hydrolysis of ethyl butyrate. A gas chromatographic procedure with flame ionization detector (fld) using these adsorbents is satisfactory for the separation of butyric acid. The best results were obtained with Spheron SDA, Spheron BD, and Porapak R.     

Protein adsorbents intended for use in aqueous two-phase systems

Preparation of adsorbents with high partition coefficients in polyethylene glycol-dextran and polyethylene glycol-phosphate systems is described. These adsorbents may be used to carry to carry proteins away from the insoluble cell fragments generated during cell disruption. By chromatographic elution, proteins may be selectively desorbed in a reduced volume.     

Sequential membrane-based purification of proteins, applying the concept of multidimensional liquid chromatography (MDLC).

A purification method for formate dehydrogenase from Candida boidinii has been designed by using a sequence of membrane-based adsorbents. The membranes carrying ion-exchange and dye ligands (Sartobind) are general-purpose adsorbents for proteins. The membranes were used in an on/off mode. A sequence of cation exchange, dye-ligand and anion exchange allows to purify the enzyme as judged by SDS-PAGE and assay of specific activity. The sequence of ligands and the buffer conditions were chosen in such a way that the fraction eluted from one membrane could be directly loaded on the next membrane. Thereby an integrated version of MDLC was realized with membrane-based adsorbents. The excellent scaleability from micro- to laboratory scale (factor 40) indicates that the method could afford a general approach to develop purification trains since the micro-scale allows for a rapid screening of adsorbent types and sequences.     

Capture of human Fab fragments by expanded bed adsorption with a mixed mode adsorbent

A novel group of mixed mode adsorbents has been developed for purification of monoclonal and polyclonal antibodies from a broad range of raw materials such as hybridoma cell culture, ascites fluid, animal sera, milk, whey and egg yolk. The aim of this study was to determine whether such mixed mode adsorbents were also useful for the recovery of recombinant proteins from microbial feedstocks. This paper describes the performance of one of these adsorbents for expanded bed capture of a human Fab fragment from recombinant E. Coli cell extracts.It is concluded that the mixed mode adsorbent binds the Fab fragment efficiently from crude extracts without any requirement for preconditioning the extract by for example de-salting or dilution. The capacity of the mixed mode adsorbent is approx. 12 mg Fab/ml matrix.The novel mixed mode adsorbent can be useful during production of highly purified Fab fragments as the first step in a purification scheme. In this respect the mixed mode adsorbent is advantageous over alternative commercially available ion-exchange materials which require pre-conditioning of cell extract for Fab' capture. Together with the concentration and clarification effect a significant enrichment of the Fab fragment is obtained in one single high yield operation.     

A novel purification strategy for retrovirus gene therapy vectors using heparin affinity chromatography

Membrane separation and chromatographic technologies are regarded as an attractive alternative to conventional academic small-scale ultracentrifugation procedures used for retrovirus purification. However, despite the increasing demands for purified retroviral vector preparations, new chromatography adsorbents with high specificity for the virus have not been reported. Heparin affinity chromatography is presented here as a novel convenient tool for retrovirus purification. The ability of bioactive retroviral particles to specifically bind to heparin ligands immobilized on a chromatographic gel is shown. A purification factor of 63 with a recovery of 61% of functional retroparticles was achieved using this single step. Tentacle heparin affinity supports captured retroviral particles more efficiently than conventional heparin affinity chromatography supports with which a lower recovery was obtained (18%). Intact, infective retroviral particles were recovered by elution with low salt concentrations (350 mM NaCl). Mild conditions for retrovirus elution from chromatographic columns are required to preserve virus infectivity. VSV-G pseudotyped retroviruses have shown to be very sensitive to high ionic strength, losing 50% of their activity and showing membrane damage after a short exposure to 1M NaCl. We also report a complete scaleable downstream processing scheme for the purification of MoMLV-derived vectors that involves sequential microfiltration and ultra/diafiltration steps for virus clarification and concentration respectively, followed by fractionation by heparin affinity chromatography and final polishing by size-exclusion chromatography. Overall, by using this strategy, a 38% yield of infective particles can be achieved with a final purification factor of 2,000.     

Salt-independent binding of antibodies from human serum to thiophilic heterocyclic ligands

Several thiophilic adsorbents with mercaptoheterocyclic ligands have been analyzed for their ability to bind human serum proteins in a salt-independent way. In contrast to 2-mercaptopyrimidine, 2-mercaptopyridine derived ligands show a group-selective binding of immunoglobulins and α₂-macroglobulin, not only in the presence of high concentrations of sodium sulphate but in buffers with low ionic strength. The binding is restricted to thiophilic gels obtained by coupling 2-mercaptopyridine to a vinylsulphone-activated matrix and is not achieved on epichlorohydrin-activated gels. A novel thiophilic ligand based on mercaptonicotinic acid, containing a carboxylic group together with the thiophilic pattern of thioaromatic adsorbents, is demonstrated to be useful as an alternative purification scheme for antibodies.     

A mild hydrophobic interaction chromatography involving polyethylene glycol immobilized to agarose media refolding recombinant Staphylococcus aureus elongation factor G

Recombinant Staphylococcus aureus elongation factor G (EF-G) is difficult to refold by dilution due to the formation of large amounts of misfolded structures. However, refolding of EF-G by adsorption to a chromatographic column packed with immobilized polyethylene glycol 20,000 (PEG 20 K) followed by pulse elution with 8 M urea resulted in 88% mass recovery and 80% of correctly refolded structure. The PEG 20 K was coupled to brominated allyl group derivatized Sepharose High Performance to construct a mild hydrophobic adsorbent. Various other hydrophobic interaction adsorbents were also attempted to refold EF-G. However, ligands with high hydrophobicity tended to misfold EF-G, resulting in irreversible adsorption. Various solvents, detergents, and low temperature as well as 8 M urea were tried to release bound EF-G. Only pulse elution with 8 M urea was efficient. Urea concentrations favorable for efficiently refolding EF-G were investigated. Low urea concentration produced more misfolded structures.     

Separation of sterol acetates by column and thin-layer argentation chromatography

Column and thin-layer chromatographic systems employing silver nitrate-impregnated adsorbents are described for the separation of sterol acetates which differ in the number of double bonds in the steroid nucleus or side chain.     

Thioltrypsin. Chemical transformation of the active-site serine residue of Streptomyces griseus trypsin to a cysteine residue.

The active-site serine residue of Streptomyces griseus trypsin was converted to a cysteine residue, and the product, thioltrypsin, was purified through two chromatographic steps with organomercurial-Sepharose and soybean trypsin inhibitor-Sepharose as specific adsorbents. The purified preparation of thioltrypsin was found to contain a single residue of cysteine and to react with almost equimolar amounts of normality titrants. It exhibited only traces of catalytic activity toward typical trypsin substrates such as Nalpha-tosyl-L-arginine methyl ester, whereas it retained some activity toward "active ester" substrates such as Nalpha-carbobenzoxy-L-lysine p-nitrophenyl ester. The activity was inhibited by sulfhydryl-blocking reagents, but no inhibition was observed by reagents reactive with the active hydroxyl group of serine proteases. Leupeptin, a natural trypsin inhibitor of peptidyl nature, also inhibited thioltrypsin. Some difference in the mode of leupeptin inhibition, however, was detected between trypsin and thioltrypsin. The bindings of small synthetic ligands and soybean trypsin inhibitor to thioltrypsin were compared with those to trypsin.