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1.
Hydrophobic charge induction chromatography is a recently developed method for protein separation based on the use of dual-mode ligands. They are designed in such a way so as to combine a molecular interaction supported by a mild hydrophobic association effect in the absence of salts. When environmental pH is changed, the ligand becomes ionically charged resulting into the desorption of the protein. This method is applied to the separation of antibodies from ascite fluids and culture supernatants from hybridomas cultured in the presence of fetal bovine serum or in protein free environment. Typically adsorption from cell culture supernatants is accomplished without any pH or ionic strength adjustment; the column is then washed with a typical buffer to eliminate protein impurities. Antibodies are then desorbed using acetate buffer, pH 4. Antibody binding capacity is in the range of 30 mg per ml of resin at 10% breakthrough. Antibody purity varies according to the initial feed stock and can reach values higher than 90% in a single pass. One example of antibody purification process involving hydrophobic charge induction chromatography as a capture step followed by a polishing phase with DEAE Ceramic HyperD is described. Longevity and ligand leakage are compatible with large-scale applications. 相似文献
2.
Girot P Averty E Flayeux I Boschetti E 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2004,808(1):25-33
The report describes the use of 2-mercapto-5-benzimidazolesulfonic acid (MBISA) as a ligand for the separation of antibodies by chromatography. The ligand shows a relatively specific adsorption property for antibodies from very crude biologicals at pH 5.0-5.5. At this pH range most of other proteins do not interact with the resin especially when the ionic strength is similar to physiological conditions. Several characterization studies are described such as antibody adsorption in different conditions of ionic strength, pH and temperature. These properties are advantageously used to selectively capture antibodies from very crude feed stocks without dilution or addition of lyotropic salts. Demonstration was made that the adsorption mechanism is neither based on ion exchange nor on hydrophobic associations, but rather as an assembly of a variety of properties of the ligand itself. Binding capacity in the described conditions ranges between 25 and 30 mg/mL of resin. The sorbent does not co-adsorb albumin (Alb) and seems compatible with a large variety of feedstocks. Quantitative antibody desorption occurs when the pH is raised above 8.5. The final purity of the antibody depends on the nature of the feedstock, and can reach levels of purity as high as 98%. Even with very crude biological liquids such as ascites fluids, cell culture supernatants and Chon fraction II + III from human plasma fractionation where the number of protein impurities is particularly large, immunoglobumins G (IgG) were separated at high purity level in a single step. 相似文献
3.
Costa SR Bonifácio MJ Queiroz JA Passarinha LA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2011,879(19):1704-1706
In the last years, chromatographic supports with amino acids as immobilized ligands (AAILs) were been used successfully for isolation of several biomolecules, such as proteins. In this context and based on specific properties of human soluble cathecol-O-methyltransferase (hSCOMT), we screened and analyzed the effect of experimental conditions, such as pH and ionic strength manipulation for hSCOMT adsorption, over six different AAIL commercial supports. Typically, the proteins adsorption on AAIL chromatographic supports is around their pI. While hSCOMT isoelectric point is around 5.5, this parameter leads us to design new adsorption strategies with several acid buffers for the chromatographic process. In terms of the ionic strength manipulation strategy, the results suggest that the AAILs-hSCOMT interaction is strongly affected by the intrinsic hSCOMT hydrophobic domains. On the other hand, the interaction mechanism of hSCOMT on amino acid resins appears to be highly dependent on the binding pH. Consequently the retention mechanism of the target enzyme on the AAILs can be as either in typical hydrophobic or ionic chromatographic supports, so long as selecting various mobile phases and separation conditions. In spite of these mixed-mode interactions and operation strategies, the elution of interferent's proteins from recombinant host can be achieved only with suitable adjusts in pH mobile phase set point. This lead to a new approach in biochromatographic COMT retention, while possess a higher specificity than other chromatographic methods reported in literature. 相似文献
4.
The adsorption equilibria of bovine serum albumin (BSA), gamma-globulin, and lysozyme to three kinds of Cibacron blue 3GA (CB)-modified agarose gels, 6% agarose gel-coated steel heads (6AS), Sepharose CL-6B, and a home-made 4% agarose gel (4AB), were studied. We show that ionic strength has irregular effects on BSA adsorption to the CB-modified affinity gels by affecting the interactions between the negatively charged protein and CB as well as CB and the support matrix. At low salt concentrations, the increase in ionic strength decreases the electrostatic repulsion between negatively charged BSA and the negatively charged gel surfaces, thus resulting in the increase of BSA adsorption. This tendency depends on the pore size of the solid matrix, CB coupling density, and the net negative charges of proteins (or aqueous - phase pH value). Sepharose gel has larger average pore size, so the electrostatic repulsion-effected protein exclusion from the small gel pores is observed only for the affinity adsorbent with high CB coupling density (15.4 micromol/mL) at very low ionic strength (NaCl concentration below 0.05 M in 10 mM Tris-HCl buffer, pH 7.5). However, because CB-6AS and CB-4AB have a smaller pore size, the electrostatic exclusion effect can be found at NaCl concentrations of up to 0.2 M. The electrostatic exclusion effect is even found for CB-6AS with a CB density as low as 2.38 micromol/mL. Moreover, the electrostatic exclusion effect decreases with decreasing aqueous-phase pH due to the decrease of the net negative charges of the protein. For gamma-globulin and lysozyme with higher isoelectric points than BSA, the electrostatic exclusion effect is not observed. At higher ionic strength, protein adsorption to the CB-modified adsorbents decreases with increasing ionic strength. It is concluded that the hydrophobic interaction between CB molecules and the support matrix increases with increasing ionic strength, leading to the decrease of ligand density accessible to proteins, and then the decrease of protein adsorption. Thus, due to the hybrid effect of electrostatic and hydrophobic interactions, in most cases studied there exists a salt concentration to maximize BSA adsorption. 相似文献
5.
Brenac V Ravault V Santambien P Boschetti E 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,818(1):61-66
A synthetic ligand called 2-mercapto-5-benzimidazolesulfonic acid has been successfully used for the specific chromatographic capture of antibodies from a cell culture supernatant. Adsorption occurred at physiological ionic strength and pH range between 5.0 and 6.0, with some binding capacity variations within this pH range: antibody uptake increased when the pH decreased. With very dilute feedstocks, as was the case with the cell culture supernatant under investigation, it was found that the pH had to be slightly lowered to get a good antibody sorption capacity. To optimize separation conditions, a preliminary study was made using ProteinChip Arrays that displayed the same chemical functionalities as the resin. Arrays were analyzed using SELDI-MS. By this mean, it was possible to cross-over simultaneously different pH conditions at the adsorption and the desorption steps. Best conditions were implemented for preparative separation using regular lab-scale columns. At pH 5.2, antibody adsorption was not complete, while at pH 5.0 the antibody was entirely captured. pH 9 was selected at elution, rather than pH 8.0 or 10.0, and resulted in a complete desorption of antibodies from the column. Benefits of the prediction of separation conditions of antibodies on MBI beads using SELDI-MS were a significant reduction in analysis time and in sample volume. This was possible because the separation of IgG on the chip surface did mimic very well the separation on beads. 相似文献
6.
The recovery of lactoferrin and lactoperoxidase from sweet whey was studied using colloidal gas aphrons (CGAs), which are surfactant-stabilized microbubbles (10-100 microm). CGAs are generated by intense stirring (8000 rpm for 10 min) of the anionic surfactant AOT (sodium bis-2-ethylhexyl sulfosuccinate). A volume of CGAs (10-30 mL) is mixed with a given volume of whey (1-10 mL), and the mixture is allowed to separate into two phases: the aphron (top) phase and the liquid (bottom) phase. Each of the phases is analyzed by SDS-PAGE and surfactant colorimetric assay. A statistical experimental design has been developed to assess the effect of different process parameters including pH, ionic strength, the concentration of surfactant in the CGAs generating solution, the volume of CGAs and the volume of whey on separation efficiency. As expected pH, ionic strength and the volume of whey (i.e. the amount of total protein in the starting material) are the main factors influencing the partitioning of the Lf.Lp fraction into the aphron phase. Moreover, it has been demonstrated that best separation performance was achieved at pH = 4 and ionic strength = 0.1 mol/L i.e., with conditions favoring electrostatic interactions between target proteins and CGAs (recovery was 90% and the concentration of lactoferrin and lactoperoxidase in the aphron phase was 25 times higher than that in the liquid phase), whereas conditions favoring hydrophobic interactions (pH close to pI and high ionic strength) led to lower performance. However, under these conditions, as confirmed by zeta potential measurements, the adsorption of both target proteins and contaminant proteins is favored. Thus, low selectivity is achieved at all of the studied conditions. These results confirm the initial hypothesis that CGAs act as ion exchangers and that the selectivity of the process can be manipulated by changing main operating parameters such as type of surfactant, pH and ionic strength. 相似文献
7.
Mixed‐mode chromatography (MMC) is an interesting technique for challenging protein separation processes which typically combines adsorption mechanisms of ion exchange (IEC) and hydrophobic interaction chromatography (HIC). Adsorption equilibria in MMC depend on multiple parameters but systematic studies on their influence are scarce. In the present work, the influence of the pH value and ionic strengths up to 3000 mM of four technically relevant salts (sodium chloride, sodium sulfate, ammonium chloride, and ammonium sulfate) on the lysozyme adsorption on the mixed‐mode resin Toyopearl MX‐Trp‐650M was studied systematically at 25℃. Equilibrium adsorption isotherms at pH 5.0 and 6.0 were measured and compared to experimental data at pH 7.0 from previous work. For all pH values, an exponential decay of the lysozyme loading with increasing ionic strength was observed. The influence of the pH value was found to depend significantly on the ionic strength with the strongest influence at low ionic strengths where increasing pH values lead to decreasing lysozyme loadings. Furthermore, a mathematical model that describes the influence of salts and the pH value on the adsorption of lysozyme in MMC is presented. The model enables predicting adsorption isotherms of lysozyme on Toyopearl MX‐Trp‐650M for a broad range of technically relevant conditions. 相似文献
8.
The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength. 相似文献
9.
The effects of charged residues in peptide antigens on the binding characteristics of polyclonal antipeptide antibodies were studied using immunoadsorbents prepared by coupling the antibodies to CNBr-activated Sepharose 4B. Among the antipeptide antibodies, an antibody to the peptide without charged residues showed the most stable interaction with the peptide to the changes in pH. Conversely, the binding affinity of antibodies to the pep-tides with histidine residues having a unique pKa value of 6.0 decreased steeply with pH at around 6.0. The binding affinity of an antibody to the peptide with many charged residues decreased steeply with an increase in the ionic strength (adjusted by NaCl). Since circular dichroism (CD) spectrum measurements indicate that these peptides show disordered structures in the pH range of adsorption measurement, the dependence of peptide-antibody interaction on environmental conditions is attributed to the characteristics of side chains of the peptides. These results indicate that the dependence of the binding affinity of antipeptide antibodies on pH and the ionic strength is dominantly affected by the number and the pKa values of charged residues in the peptides. 相似文献
10.
Boschetti E 《Trends in biotechnology》2002,20(8):333-337
Hydrophobic charge induction chromatography using 4-mercapto-ethyl-pyridine as the ligand is an effective method for the separation of antibodies from a variety of feedstocks. Antibodies are adsorbed in physiological conditions without preliminary concentration. Desorption occurs when the pH is lowered, thus inducing an ionic charge of the same sign to the ligand and the antibody. Antibody capture conditions are compatible with crude samples in terms of pH, conductivity, binding capacity and expression level. The final purity of the antibody is feedstock dependent, but can reach levels of purity as high as 98%. Examples of antibody separation are given and ligand structure information discussed. 相似文献
11.
Phase diagram of mixed monolayers of stearic acid and dimyristoylphosphatidylcholine. Effect of the acid ionization 总被引:1,自引:0,他引:1
The aim of this work is to study the phase diagram of mixed monolayers composed of dimyristoylphosphatidylcholine (DMPC) and stearic acid (SA) at different ionic strength and bulk pH of the aqueous subphase. In this way, the effect of ionization of SA on the interaction and thus on phase separation with the DMPC matrix can be analyzed. To this purpose, we first determined the ionization state of pure SA monolayers as a function of the bulk subphase pH. The SA monolayers are nearly fully ionized at pH 10 and essentially neutral at pH 4 and the mixture of DMPC and SA was studied at those two pHs. We found that the DMPC-enriched phase admits more SA if the SA monolayer is in a liquid-expanded state, which is highly related to the acid ionization state, and thus to the bulk pH and ionic strength. At pH 4 the molecules hardly mix while at pH 10 the mixed monolayer with DMPC can admit between 30 and 100% of SA (depending on the lateral pressure) before phase separation is established. The addition of calcium ions to the subphase has a condensing effect on SA monolayers at all pHs and the solubility of SA in the DMPC matrix does not depend on the bulk pH in these conditions. The observed phase diagrams are independent on the manner in which the state of the mixed film is reached and may thus be considered states of apparent equilibrium. 相似文献
12.
A quantitative understanding of how proteins interact with hydrophobic charge induction chromatographic resins is provided. Selectivity on this mode of chromatography for monoclonal antibodies as compared to other model proteins is probed by means of a linear retention vs pH plot. The pH-dependent adsorption behavior on this mode of chromatography for a hydrophobic, charged solute is described by taking into account the equilibrium between a hydrophobic, charged solute and an ionizable, heterocyclic ligand. By analogy, an equation that is seen to adequately describe macromolecular retention under linear conditions over a range of pH is developed. A preparative, nonlinear isotherm that can capture both pH and salt concentration dependency for proteins is proposed by using an exponentially modified Langmuir isotherm model. This model is seen to successfully simulate adsorption isotherms for a variety of proteins over a range of pHs and mobile phase salt concentrations. Finally, the widely differing retention characteristics of two monoclonal antibodies are used to derive two different strategies for improving separations on this mode of chromatography. A better understanding of protein binding to this class of resins is seen as an important step to future exploitation of this mode of chromatography for industrial scale purification of proteins. 相似文献
13.
Zwitterionic carboxybetaine (CB) has unique dual functionality for ligand immobilization on a nonfouling background. The properties of CB groups depend on their spacer groups between the positive quaternary amine groups and the negative carboxyl groups and environmental factors (e.g., ionic strengths and pH values). In this work, five polycarboxybetaines were prepared, including one polycarboxybetaine methacrylate (polyCBMA) and four polycarboxybetaine acrylamides (polyCBAAs) with different spacer groups. The polymers were grafted from a gold surface covered with initiators using surface-initiated atom transfer radical polymerization. Fibrinogen adsorption was measured as a function of ionic strengths and pH values using surface plasmon resonance sensors. The responsive protein adsorption on four polyCBAAs was mapped out. Results show that most of these surfaces exhibit high protein resistance in a wide range of ionic strengths and are more effective than zwitterionic self-assembled monolayers. Although protein adsorption tends to increase at low ionic strength and low pH value, it is still very low for polycarboxybetaines with a methylene, an ethylene, or a propylene spacer group but is more evident for polyCBAA with a longer spacer group (i.e., a pentene group). The response to ionic strengths and pH values can be attributed to the antipolyelectrolyte and protonation/deprotonation properties of polycarboxybetaines, respectively. Both of these properties are related to the spacer groups of CBs. 相似文献
14.
A F Sikorski 《Acta biochimica Polonica》1988,35(1):19-27
Purified spectrin was found to interact strongly with hydrophobic agaroses such as Phenyl- or Octyl-Sepharose, in the presence of EDTA. From the complexes formed spectrin was eluted with ethylene glycol but not with low ionic strength solutions. The binding capacity of spectrin increased with increasing ionic strength of the equilibration buffer and showed but little dependence on its pH value. The fragments obtained by proteolysis of spectrin carried out under mild conditions were also found to bind strongly to phenyl-agarose, and were eluted with ethylene glycol. The fractions eluted with ethylene glycol contained two closely related polypeptides of Mr 65,000 and 60,000. 相似文献
15.
Buffers of different pH and ionic strength were employed as coating buffers for antigen adsorption to microtitre plates. Their efficiency for coating plates with rinderpest virus (RPV) and foot-and-mouth disease virus (FMDV) antigens was studied by ELISA with polyclonal and monoclonal antibody preparations. While the adsorption and detection of RPV antigen with polyclonal antiserum was highly dependent on the ionic strength and pH of coating buffer, adsorption of antigenically active FMDV antigen was relatively unaffected by the buffering conditions. Both antigens were adsorbed optimally in 0.01 M phosphate buffer, pH 8.0. When monoclonal antibodies were used to detect antigen, there was a greater degree of dependence on the coating buffer than that found with polyclonal antisera. Moreover, when they were used to detect antigen adsorbed under several buffering conditions, monoclonal antibodies showed a variety of preferred buffers. The usefulness of this differential reactivity in distinguishing epitope specificity is demonstrated. 相似文献
16.
Hydrophobic ligands were introduced onto agarose beads, and the adsorption capacity of the beads was measured. The adsorption capacity increased with increase in the carbon number of the ligand, ionic strength of the buffer solution, and temperature. Crude alpha-amylase was purified with these hydrophobic adsorbents and the breakthrough and elution curves were estimated based on the mass transfer theory. Under strongly hydrophobic conditions, impurities contained in crude feeds and the lack of uniformity of packing caused by aggregation of beads affected adsorption and elution behaviors. 相似文献
17.
The electrophoresis mobilities of native calf thymus DNA adsorbed on the charged solid particles were measured by a micro-electrophoretic method as functions of pII, ionic strength, and DNA concentration. The mobility data confirm the adsorption of DNA both on the positively charged alumina and negatively charged resin particles at wide range of pH and ionic strength. The mobility data also indicate significant DNA adsorption by negatively charged glass in the acidic range of pH. The electrophoretic mobilities of DNA adsorbed on different substrate particles under identical conditions do not differ widely, indicating the major role of the adsorbed DNA rather than the covered substrate in controlling the charge behavior of the particle. The mobilities of the adsorbed DNA at salt pH are of a comparable order of magnitude to those for the dissolved DNA in solution. The mobility of the adsorbed heat-denatured and alkali-denatured DNA is lower than that of the native adsorbed DNA under identical conditions of pH and ionic strength. 相似文献
18.
Endotoxin, a major pyrogen of concern to the biological industry, is a lipopolysaccharide containing a highly hydrophobic region, lipid A, in its structure. The effect of hydrophobic interaction on endotoxin adsorption from an aqueous solution was studied by covalently bonding aminoalkyl groups with varying hydrocarbon lengths to a cellulose and acrylic composite matrix. The amount of endotoxin adsorbed on the matrix increased with the increasing length of alkyl groups, demonstrating the contribution of hydrophobic interaction between endotoxin and the solid matrix. Both the hydrophobic and the charge interaction prove to be effective for endotoxin adsorption, and a synergistic effect from the dual chemical forces is achievable under specified conditions. The effect of solvent, pH and salts on endotoxin adsorption provides further evidence for the importance of hydrophobic force as a means of removing endotoxin from aqueous solutions. 相似文献
19.
Yuebiao Sheng Wei Wang P Chen 《Protein science : a publication of the Protein Society》2010,19(9):1639-1648
Protein adsorption on a surface plays an important role in biomaterial science and medicine. It is strongly related to the interaction between the protein residues and the surface. Here we report all‐atom molecular dynamics simulations of the adsorption of an ionic complementary peptide, EAK16‐II, to the hydrophobic highly ordered pyrolytic graphite surface. We find that, the hydrophobic interaction is the main force to govern the adsorption, and the peptide interchain electrostatic interaction affects the adsorption rate. Under neutral pH condition, the interchain electrostatic attraction facilitates the adsorption, whereas under acidic and basic conditions, because of the protonation and deprotonation of glutamic acid and lysine residues, respectively, the resulting electrostatic repulsion slows down the adsorption. We also found that under basic condition, during the adsorption peptide Chain II will be up against a choice to adsorb to the surface through the hydrophobic interaction or to form a temporary hydrophobic core with the deposited peptide Chain I. These results provide a basis for understanding some of the fundamental interactions governing peptide adsorption on the surface, which can shed new light on novel applications, such as the design of implant devices and drug delivery materials. 相似文献
20.
Wimonrat Phottraithip Dong-Qiang Lin Fei Shi Shan-Jing Yao 《Biotechnology and Bioprocess Engineering》2013,18(6):1169-1175
New hydrophobic charge-induction chromatography (HCIC) resin coupled with 2-mercaptoimidazole (MI) as the ligand was prepared and used to purify IgG from porcine plasma. The cellulose matrix was activated by divinylsulfone (DVS), and was then coupled with MI as the functional ligand. The reaction conditions were optimized as pH: 11, volume ratio of DVS to matrix: 1.0, reaction time: 4 h. The ligand density reached about 100 μmol/mL gel. The adsorption isotherms of porcine IgG was determined at different pH values, and high saturated adsorption capacities of 78.02 mg IgG/mL gel were found at pH 8. The adsorption of IgG showed a typical pHdependent property of HCIC, and the adsorption capacity decreased significantly in acidic conditions. The prepared resin was used to separate IgG from porcine plasma. After precipitating the fibrinogen by salting-out, the supernatant was loaded onto the column at pH 7 and the elution pH was optimized. The results indicated that acidic elution pH was necessary to recover the IgG. The purity of IgG in the elution fractions was in the range of 81 ~ 90%, which demonstrated that HCIC with the new ligand showed the excited separation performs and is a potential effective technique to purify IgG from the complex feedstock. 相似文献