Affinity chromatography of nucleases

The review concerns isolation and purification of nucleases by affinity chromatography. Different stationary ligands and the methods for their immobilization on supports are described, along with diverse eluents and various procedures for a nuclease detachment from the affinity sorbents. The data on the affinity chromatography application for measuring the dissociation constants of the enzyme complexes with either immobilized or soluble ligands are compiled.     

蛋白质层析用离子交换和疏水作用层析介质的发展概况

层析是蛋白质纯化的关键技术之一 ,作为层析技术的核心———层析介质一直以来是层析技术研究的一个热点。近年来 ,越来越多的新型层析介质被开发出来 ,如粒度均匀的交联多糖、人工合成的大孔聚合物、触角型吸附剂、软胶包裹在硬胶表面等介质。主要介绍应用较为广泛的IEC和HIC介质的组成、特性及其在蛋白质纯化中的应用 ,还研究了与HIC技术相关的两种新技术 :亲硫层析和疏水电荷诱导层析 (HCIC) ,重点介绍了HCIC的介质及其应用 ,同时也讨论了在蛋白质纯化中应用的三相纯化策略 (富集、中间纯化和精制 )。结合我国的实际情况 ,就当前蛋白质纯化的离子交换和疏水层析介质面临的挑战和未来的发展进行讨论并提出了建议     

Controlled porous glass (CPG) with reactive epoxy groups as support for affinity chromatography I. Optimization of CPG modification and the binding of glucose with modified surface

The selective and complementary interaction between the ligand bound to support surface and isolated substance is the key of the affinity chromatography. This is the reason of the research in the field of certain sorbents. They are obtained by chemical reaction of matrix with proper ligands. It is carried out mainly by using bifunctional substances which make the reaction with the ligand in question possible. This paper deals with the preparation of controlled porosity glass with reactive epoxy groups. In order to obtain such support and avoid application of a proper epoxy silane compound, the double-step reaction using simple agents was employed. The sorbent syntheses were optimized. The prepared sorbents were applied for coupling some carbohydrates and amino acids.     

Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA)

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.     

A new approach to the depletion of albumin and immunoglobulin G from human serum

The use of proteomic analysis to find potential diagnostic biomarkers is limited by the presence of serum albumin (HSA) and immunoglobulin (IgG) at high concentrations in patients’ blood; these substances impede the detection of serum proteins with similar molecular weights. Recombinant HSA- and IgG-binding polypeptides are used as ligands in creating sorbents for complete removal of the proteins by affinity chromatography. The binding specificity of the sorbents for HSA and IgG is higher than that of the conventionally used antibodies. A composite sorbent enabling the depletion of HSA and IgG from serum by single-step affinity chromatography was obtained. The developed sorbents were used to prepare serum for proteomic analysis.     

Affinity chromatography of proteolytic enzymes on silica-based biospecific sorbents

New biospecific sorbents for affinity chromatography of proteolytic enzymes were prepared by the attachment of the cyclopeptide antibiotics bacitracin, bacilliquin or gramicidin S to aminosilochrom via a reaction with p-benzoquinone. The content of the cyclopeptide ligands within the sorbents varied from 2 to 46 mumol/g. The sorbents prepared by this reaction were successfully applied in the purification of the carboxylic proteinases produced by fungi, Russula decolorans (a basidiomycete) and Trichoderma lignorum, as well as crude pepsin. Serine proteinases from Thermoactinomyces vulgaris, Trichoderma koningii, Trichoderma lignorum and bacilli (subtilisins) were also submitted to chromatography on these materials. The yields of purified enzymes approached quantitative levels, sometimes being higher as a result of elimination of inhibitors. An important advantage of these sorbents is their stability against the enzymes degrading the carbohydrate matrixes of affinity sorbents synthesized on the basis of agarose, dextran or cellulose derivatives.     

Chromatography of carboxylic proteinases on sorbents containing the 2,4-dinitrophenyl group. Role of ionic interactions]

The chromatography of porcine pepsin on biospecific sorbents (Sepharose-4B-epsilon-DNP-aminocapronylhydrazide and Sepharose-4B-N-DNP-N'-acetylhexamethylenediamine) was studied. The sorbents in question differ from the previously used hydrophobic sorbent Sepharose-4B-DNP-hexamethylenediamine by the lack of strongly basic groups in the site of the ligand binding to the polymeric matrix. No qualitative differences in the pepsin chromatography on the three sorbents were observed. Presumably the decrease of the pepsin binding to the sorbents, containing the dinitrophenyl group, at pH values above the isoelectric point may be due to the effects of the salt on the binding site in the enzyme molecule rather than to the screening of the positive charges of the sorbent by chlorine ions. A commercial preparation of pepsin was purified 2-fold on the sorbent Sepharose-4B-epsilon-DNP-animocapronylhydrazide. The synthesis of sorbents is described.     

Hydrophobic interaction chromatography of proteins. II. Binding capacity,recovery and mass transfer properties

Hydrophobic interaction chromatography media suited for large scale separations were compared regarding dynamic binding capacity, recovery and mass transfer properties. In all cases, pore diffusion was the rate limiting step. Reduced heights equivalent to a theoretical plate for bovine serum albumin derived from breakthrough curves at reduced velocities between 60 and 1500 ranged from 10 to 700. Pore diffusion coefficients were derived from pulse response experiments for the model proteins alpha-lactalbumin, lysozyme, beta-lactoglobulin, bovine serum albumin and immunoglobulin G. Diffusivity of lysozyme did not follow the trend of decreasing diffusivity with increasing molecular mass, as observed for the rest of the proteins. In general, mass transfer coefficients were smaller compared to ion-exchange chromatography. Dynamic binding capacities for the model protein bovine serum albumin varied within a broad range. However, sorbents based on polymethacrylate showed a lower dynamic capacity than media based on Sepharose. Some sorbents could be clustered regarding binding capacity affected by salt. These sorbents exhibited a disproportional increase of binding capacity with increasing ammonium sulfate concentration. Recovery of proteins above 75% could be observed for all sorbents. Several sorbents showed a recovery close to 100%.     

Preparation of affinity sorbents with immobilized synthetic ligands for therapeutic apheresis

The practical aspects of preparation and stability of medical sorbents are considered. A simple and convenient technique has been developed for synthesis of highly effective biospecific autoclavable sorbents based on the polysassharide matrix; synthetic ligands (amino acid, oligopeptide, or oligosaccharide) containing primary amino group were immobilized to the matrix via a spacer. The developed approach may be used for preparation of various affinity sorbents suitable for application in medicine and biotechnology.     

Study of conformational effects of recombinant interferon γ adsorbed on a non-porous reversed-phase silica support

Reversed-phase chromatography is a powerful method for separating recombinant interferon γ and one of its analogues differing only by a single amino acid residue. Structural differences of the proteins explain this separation ability as demonstrated from adsorption studies on a non-porous reversed-phase support. To reveal the structural differences occurring in the adsorbed state, two different and independent methods were employed. The variation of the retention with the slope of the linear gradient gave information about the molecular contact area of the protein with the support. For different experimental conditions, these data were correlated with the adsorbent capacities measured on an n-octadecyl-modified non-porous silica support. These supports are useful for these types of experiments because the protein is adsorbed exclusively at the external surface of the beads. Moreover, a small amount of protein is necessary to saturate the column, owing to its low capacity.     

Isolation and purification of proteolytic enzymes on organo-silica sorbents with immobilized gramicidin S

Biospecific sorbents for affinity chromatography of proteolytic enzymes have been synthesized by attaching cyclopeptide antibiotic gramicidin S to organo-silica supports. It is shown possible to attach gramicidin S to the organo-silica supports using glutaric aldehyde, p-benzoquinone, soluble and insoluble carbodiimides. The sorbents prepared by these methods were successfully applied for the purification of the crude pepsin from horse gastric juice and proteolytic complex produced by Acremonium chrysogenum.     

A new chromatographic method for the preparative isolation of phosphoinositides and other anionic phospholipids

A new chromatographic procedure for preparative isolation of mono-, di- and triphosphoinositides and other anionic phospholipids with the use of adsorbents containing primary amino groups is described. Sorbents with immobilized neomycin, L-lysine and aminoalkyl groups were tested. Conditions for isolation of chromatographically pure phospholipids of separate classes on the above sorbents were developed. Isolation of polyphosphoinositides on the amino sorbents represents a new type of chromatography involving bioaffinity and ion-exchange interaction.     

Surface-enhanced laser desorption-ionization retentate chromatography mass spectrometry (SELDI-RC-MS): a new method for rapid development of process chromatography conditions

Protein biochip arrays carrying functional groups typical of those employed for chromatographic sorbents have been developed. When components of a protein mixture are deposited upon an array's functionalized surface, an interaction occurs between the array's surface and solubilized proteins, resulting in adsorption of certain species. The application of gradient wash conditions to the surface of these arrays produces a step-wise elution of retained compounds akin to that accomplished while utilizing columns for liquid chromatography (LC) separations. In retentate chromatography-mass spectrometry (RC-MS), the "retentate" components that remain following a wash are desorbed and ionized when a nitrogen laser is fired at discrete spots on the array after treatment with a laser energy-absorbing matrix solution. Ionized components are analyzed using a time-of-flight mass spectrometer (TOF MS). The present study demonstrates that protein biochips can be used to identify conditions of pH and ionic strength that support selective retention-elution of target proteins and impurity components from ion-exchange surfaces. Such conditions give corresponding behavior when using process-compatible chromatographic sorbents under elution chromatography conditions. The RC-MS principle was applied to the separation of an Fab antibody fragment expressed in Escherichia coli as well as to the separation of recombinant endostatin as expressed in supernatant of Pichia pastoris cultures. Determined optimal array binding and elution conditions in terms of ionic strength and pH were directly applied to regular chromatographic columns in step-wise elution mode. Analysis of collected LC fractions showed favorable correlation to results predicted by the RC-MS method.     

Designing new monoclonal antibody purification processes using mixed-mode chromatography sorbents

Current platforms for purification of monoclonal antibodies, mostly relying on Protein A as a first capture step, are robust and efficient but significantly increase downstream purification costs, mainly due to Protein A resins. To decrease manufacturing costs, industry is increasingly considering the use of purification schemes without affinity Protein A resins. Mixed-mode chromatography can be used as a powerful alternative to standard purification platforms as it offers new selectivity and separation mechanisms exploiting a combination of both ionic and hydrophobic characteristics of antibodies and contaminating proteins. By using a design of experiments (DoE) approach and high throughput screening in 96-well plates, we developed four different two-steps MAb purification processes, based on the use of mixed-mode sorbents. Finally, three of the tested processes resulted in final purified Mab fractions containing less than 100 ppm of residual CHO proteins (CHOP), with overall process yields above 70%. These data show that mixed-mode chromatography sorbents, used at capture or intermediate purification steps, really expand the options of MAb purification process development with or without Protein A affinity chromatography.     

Progress in packed column supercritical fluid chromatography: materials and methods

This article summarizes recent developments in packed column supercritical fluid chromatography. Silica-based chemically bonded sorbents, similar to those used for HPLC, are widely used with solvent-modified fluids containing additives to suppress undesirable solute-sorbent interactions that lead to poor peak shapes. Composition programming is the most useful approach to gradient elution separations since solvent-modified fluids have low compressibility. Packed column SFC is most useful for the separation of mixtures usually separated by normal-phase HPLC. Compared to normal-phase HPLC it offers faster separations, higher efficiencies, faster column re-equilibration, and a wider range of experimental variables for optimization. Packed column SFC is being increasingly selected for the analytical and preparative separation of racemic mixtures using enantiomer-selective sorbents.     

Composite fluorine polymer-containing sorbents for isolation and purification of biopolymers

Composite fluoropolymer-containing sorbents based on porous silicas were synthesized for the isolation and purification of biopolymers under nondenaturing conditions. Examples of the application of these sorbents in the separation of various mixtures of peptides and proteins and purification of nucleic acids from various sources (plasmid DNA and DNA from nucleated human blood cells) using the cartridge, column, and batch (sorption in a stirred volume) methods are presented. It was shown that the sorbents can be used in laboratory practice because they are selective to nucleic acids (DNA and RNA) and proteins. These materials combine the mechanical properties of the inorganic matrix with the specific sorption properties of the polymer phase and exhibit enhanced stability to alkaline hydrolysis. Alternative methods of preparing sorbents containing polytetrafluoroethylene, polytrifluorostyrene, and polyfluorobutadiene are described. By the example of polyfluorobutadiene-containing sorbents, a completely new method for obtaining fluorinated polymer phases was developed: the polymer phase was preliminarily formed on the surface of porous disperse carriers and was fluorinated with xenon difluoride.     

High performance exclusion microcolumn chromatography of proteins and peptides

A new method of determination of molecular mass of proteins and peptides has been developed, basing on the microcolumn exclusion chromatography on non-modified silica sorbents in trifluoroacetic acid. It is shown that in this eluent the proteins and peptides adopt the random-coil conformation and are not hydrolyzed for three days at room temperature.     

Effect of the matrix structure and concentration of polymer-immobilized Ni2+-iminodiacetic acid complexes on retention of IgG1

Terpolymer bead particles (100-350 microm in diameter) were prepared by suspension radical polymerization from methacrylate esters [2,3-epoxypropyl methacrylate (GMA), 2-(2-hydroxyethoxy)ethyl methacrylate (DEGMA) and ethylene dimethacrylate (EDMA)] and subsequently derivatized affording iminodiacetic acid (IDA) chelating sorbents. The sorbents differed in pore volumes (0-0.7 cm3/g) and specific surface areas (0.03-9.8 m2/g) of their matrices as well as in the amounts of immobilized Ni2+-IDA complexes (0.03-1.58 mmol/g). The binding of imidazole was studied by frontal chromatography to evaluate the accessibility of Ni2+-IDA complexes. It was found that an increase in the bonded imidazole content with increasing immobilized Ni2+-IDA concentration was strongly dependent on the matrix morphology. A higher pore volume of the matrix significantly improved the utilizability of Ni2+-IDA complexes for imidazole binding. The performance of the sorbents based on two porous matrices with immobilized Ni2+-IDA concentration (0.1-1.58 mmol/g) differing in pore size distributions was compared in immobilized metal affinity chromatography (IMAC) of monoclonal mouse immunoglobulin IgG1 specific against human choriogonadotropic hormone (GTH-spec IgG1). The results have shown that sorbents based on matrix with large pores (up to 20 microm in diameter) exhibited high protein binding capacities. The GTH-spec IgG1 (Mw=158,000) was eluted from all the sorbents in its native form as was confirmed by MALDI-TOF.     

Effect of the Compositions of Sample and Polymer Sorbents on the Extraction of Volatile Compounds by Solid-Phase Microextraction

A comparative study was performed by solid phase microextraction and capillary gas chromatography to establish the ability of four polymer sorbents of different compositions to extract and concentrate volatile organic compounds from the gas phase above an aqueous solution. All polymer sorbents sorbed nonpolar monoterpene hydrocarbons via a cooperative mechanism with almost equal and high efficiency. Sorbents based on polymethyl disiloxane and its mixture with divinylbenzene were more effective in extracting acetates and sesquiterpenes. As the concentration of these compounds in the gas phase increased, their binding by sorbents decreased. It was found that the determination of polar compounds depended on the presence of a solvent in the system. Compounds that are highly soluble in water (alcohols, ketones, etc.) had low coefficients of distribution between gas and water phases. Consequently, their sorption to any of the polymer sorbents was negligible. In the absence of the solvent, the degree of their extraction from the gas phase above the sample was high. It was shown that the actual composition of compounds in the initial mixture of essential oils could significantly differ from their composition in the gas phase. This method is convenient and informative for the purpose of studying the composition of volatile compounds in the gas phase that determine the flavor of the product.     

Solid-phase precipitation and extraction, a new separation process applied to the isolation of synthetic peptides.

A new method for separation and purification is described. The process, referred to as solid-phase precipitation and extraction (SPPE), was developed and applied to postcleavage isolation of synthetic peptides. The technique uses normal approaches of chromatography and solid-phase extraction sorbents with a precipitation or drying procedure so that the sorbent becomes a support matrix for thin-film deposition of the compounds of interest. This procedure causes precipitated compounds of interest to be trapped on the large surface area or in the pores of the matrix so that by-products and impurities can be removed by strong wash solvents. In application to solid-phase peptide synthesis chemistry, by-products from the cleavage and deprotection are selectively extracted from the crude sample mixture under mild conditions. In comparison to the ether precipitation method used in peptide chemistry, the SPPE process provides isolated products that are 14-17% (w/w) higher purity.