Influence of ligand density on antibody binding capacity of cation-exchange adsorbents

Adsorption properties of a set of polymethacrylate-based cation exchangers designed for purification of monoclonal antibodies were investigated. The materials differed significantly in the density of sulphoisobutyl ligand groups. The ligand density had a pronounced effect on the static adsorption capacity of a polyclonal human immunoglobulin G. An optimal ligand density was observed at any pH and NaCl concentration tested when sharp optima were observed at low pH and ionic strength values. This was caused by effective clogging of pore mouth at high ligand densities. An anomalous effect of ionic strength was observed for the adsorbents with the high ligand density when the adsorption capacity increased with the addition of NaCl at low pH.     

Pore size distributions of cation-exchange adsorbents determined by inverse size-exclusion chromatography

The pore dimensions, pore size distributions, and phase ratios were determined for a set of cation-exchange adsorbents using inverse size-exclusion chromatography (ISEC). The adsorbents examined represent a diverse set of materials from Pharmacia, TosoHaas, BioSepra, and EM Industries, which are widely used for protein purification. The ISEC was carried out using dextran standards with relative molecular masses of 180-6,105,000. This technique provided a comparative characterization of the accessible internal pore surface area, as a function of solute size, for the adsorbents tested. Adsorbent preparation strategies in which polymers are generated in situ or grafted onto base materials were found to have significant effects on pore dimensions and phase ratios.     

Demonstration of simultaneous cation-exchange and reversed-phase mechanisms on a strong-acid cation-exchange column.

It is demonstrated in this report that a conventional strong-acid cation-exchange column can exhibit reversed-phase chromatographic behavior simultaneously with ion-exchange. Adjusting the pH to control cation retention has no effect on the retention of neutral organic analytes. Likewise, changes in the methanol content of the mobile phase to adjust organic analyte retention causes only a small decrease in retention of metal ions in the 0 to 10% (v/v) methanol range, and no significant effect beyond that. Linear calibration behavior of both metal cations and neutral organic analytes is found on this column over three-order of magnitude. Examples of simultaneous metal cation-neutral organic separations in both the isocratic and gradient modes are shown, with conductivity detection for the metal ions and UV for the organic analytes. An isocratic separation of metal ions and neutrals in a vitamin pill is also demonstrated.     

Separation of protein mixtures using pH-gradient cation-exchange chromatography

Historically, separation of a protein mixture after adsorption to a cation-exchange column is effected by alteration in ionic strength. An alternative separation method using pH induced gradient in the range of 4–7.5 was studied. A cation-exchange column with large particle beads containing excessive carboxyls was employed. A pH gradient across the column was generated by a step change at the column entrance using a non-retained buffer system. Consistency and accuracy of pH values in timed intervals were demonstrated in three different batches. In development of the application, we found a correlation coefficient of >0.9 between the elution pH values of six acidic proteins and their isoelectric points. One case study showed the resolution between a monoclonal antibody and non-retained protein species from a protein A column. Another case study showed the feasibility of separating polyethylene glycol conjugated protein from native protein.     

Relative retention of the fibroblast growth factors FGF-1 and FGF-2 on strong cation-exchange sorbents

The isocratic retention of two heparin-binding fibroblast growth factors, FGF-1 (acidic FGF) and FGF-2 (basic FGF), was compared on a set of six preparative strong cation-exchange adsorbents. The FGFs comprise a solute pair that are structurally equivalent, yet differ in protein parameters of potential importance in cation-exchange chromatography, such as isoelectric point, net charge, and the number and distribution of basic amino acids. The cation-exchange adsorbents comprise a diverse set of materials in common use for protein purification, with physical and chemical properties that have been characterized and described previously. Isocratic k' values for the two proteins obtained on each adsorbent at several different [NaCl] are compared with one another and with corresponding data for hen egg lysozyme, which is also strongly retained on cation-exchangers. Of the six adsorbents examined, three showed strong retention of both FGFs, with equivalent k' values for FGF-1 and FGF-2. Three others, which showed weaker overall retention for the FGF pair, showed much larger retention differences between FGF-1 and FGF-2. The trends in retention order among the stationary phases are very similar to those seen previously with other unrelated proteins. However, retention differences between the two FGFs, and between the FGFs and lysozyme, do not correlate well with simple charge properties such as net charge, indicating, as in some previous studies, the importance of local regions on the protein surface in determining retention. These observations are interpreted in terms of the structural features of the proteins and the physicochemical properties of the adsorbents.     

Separation of rat pancreatic secretory proteins by cation-exchange fast protein liquid chromatography.

Rat pancreatic secretory proteins were separated by an automated liquid chromatography system utilizing a Mono S cation-exchange column. Optimal resolution was obtained with a multistep salt and pH gradient (0.01-2 M LiCl, pH 5.3-63). A total of fourteen well-separated peaks, as well as several minor peaks, were detected by UV absorption. The main pancreatic enzymes were resolved (two amylases, two chymotrypsinogens, two trypsinogens, proelastase, lipase, prophospholipase A2, procarboxypeptidase A, procarboxypeptidase B, and ribonuclease). In addition, proteins without enzymic activity, such as lithostathine and pancreatitis-associated protein, were identified. Activation of proenzymes did not occur during the separation. At a flow-rate of 0.5 ml/min, ca. 250 micrograms to 5 mg of protein could be applied with equal resolution. The reproducibility of retention volumes and peak areas was high (less than 1% or 5% variation, respectively). When radiolabeled proteins were separated, a comparable pattern of peaks was obtained. The technique described is, therefore, not only useful for analytical and preparative separation of pancreatic proteins but can additionally serve for quantitative determination of the pancreatic isoenzyme pattern.     

High-speed protein purification by adsorptive cation-exchange hollow-fiber cartridges

Novel cation-exchange adsorptive membranes were assessed according to their protein adsorption capacity and permeation flowrate. Maximum static adsorption capacities for the three main egg-white proteins, lysozyme, ovoalbumin and conalbumin, were 140, 88 and 66 mg/ml, respectively. However, membranes showed an inverse relationship between permeation flowrate and static protein adsorption capacity. Two size cartridges (membrane volume of 0.42 and 3.5 ml) were built using the selected membrane. An adsorptive cross-flow cartridge was tested to recover and purify lysozyme from an egg-white solution. Breakthrough curves developed using a pure lysozyme solution showed a dynamic-to-static capacity ratio of 0.6, which was reduced to 0.4 during lysozyme recovery from egg-white solution in cross-flow mode. Total process cycle for the enzyme recovery and purification was in the range of 10–15 min for both cartridges. In both cases high-purity lysozyme (95%) was recovered with a productivity of 150 g/(l h) and no size-exclusion effect was detected.     

The special features of protein adsorption isotherms on silica adsorbents

The adsorption isotherms of hemoglobin, peroxidase, and β-galactosidase on silochrome and mesoporous and biporous silicas were comparatively studied. Adsorption developed in two stages, including fast “reversible” protein adsorption (equilibrium was reached in t ≤ 1–2 h) and a “slow stage” of irreversible binding in t ≫ 24 h (multipoint adsorption). The corresponding equilibrium constants were determined. The mechanism of unlimited linear association of peroxidase in the adsorption layer on the surface of silochrome was established.     

Unusual retention behaviour of trace element cations on a cation-exchange column in the presence of a colloid

Mono- and divalent cations of trace elements were not retained at all on a cation-exchange column in the presence of a colloid. When the colloid was dissolved, cations sorbed and eluted from the column in a normal way.     

Regularities in the retention of nonsubstituted polynuclear aromatic hydrocarbons in liquid chromatography on polar adsorbents

Summary The retention of polynuclear aromatic hydrocarbons (PAH) is studied in liquid chromatography, using columns packed with silica gel with a hydroxylated surface and the same silica gel with surface-bonded amine and nitrile groups. A linear relationship was found between retention and the number of π-electrons in the molecule, the size of the molecule and the different types of carbon atoms in the molecule. The different carbon atoms in a PAH molecule contribute differently to the retention and this contribution depends on the nature of the polar functional groups on the adsorbent's surface and on the nature and concentration of a polar modifier present in a non-polar eluent.     

Influence of acidic eluent for retention behaviors of common anions and cations by ion-exclusion/cation-exchange chromatography on a weakly acidic cation-exchange resin in the H+ -form

Influence of acidic eluent on retention behaviors of common anions and cations by ion-exclusion/cation-exchange chromatography (ion-exclusion/CEC) were investigated on a weakly acidic cation-exchange resin in the H(+)-form with conductivity. Sensitivities of analyte ions, especially weak acid anions (F(-) and HCOO(-)), were affected with degree of background conductivity level with pK(a1) (first dissociation constant) of acid in eluent. The retention behaviors of anions and cations were related to that of elution dip induced after eluting acid to separation column and injecting analyte sample. These results were largely dependent on the natures of acid as eluent. Through this study, succinic acid as the eluent was suitable for simultaneous separation of strong acid anions (SO(4)(2-), Cl(-), NO(3)(-) and I(-)), weak acid anions (F(-), HCOO(-) and CH(3)COO(-)), and cations (Na(+), K(+), NH(4)(+), Mg(2+) and Ca(2+)). The separation was achieved in 20 min under the optimum eluent condition, 20 mM succinic acid/2 mM 18-crown-6. Detection limits at S/N=3 ranged from 0.10 to 0.51 microM for strong acid anions, 0.20 to 5.04 microM for weak acid anions and 0.75 to 1.72 microM for cations. The relative standard deviations of peak areas in the repeated chromatographic runs (n=10) were in the range of 1.1-2.9% for anions and 1.8-4.5% for cations. This method was successfully applied to hot spring water containing strong acid anions, weak acid anions and cations, with satisfactory results.     

Hydroxyapatite-based immobilized metal affinity adsorbents for protein purification

The employment of metal ion-charged hydroxyapatite for the one-step purification of poly(His)-tagged recombinant proteins was investigated. Fe(III) showed the highest selectivity toward the poly(His)-tagged D-hydantoinase and the best operation stability. The optimal selectivity was observed in 20 mM pH 8.0 buffer containing 150 mM NaCl and 50 mM NaF. The adsorbed poly(His)-tagged enzyme could be quantitatively recovered from hydroxyapatite with 150 mM pH 8.0 phosphate buffer. The capacity of Fe(III)-loaded hydroxyapatite for poly(His)-tagged D-hydantoinase was 4.9 mg/g hydroxyapatite, comparable to commercial agarose-based Ni-NTA adsorbents. Under optimal conditions, a D-hydantoinase preparation with a purity above 95% from crude cellular lysate could be obtained with the one-step purification process employing Fe(III)-loaded hydroxyapatite. The application of Fe(III)-loaded hydroxyapatite for the purification of poly(His)-tagged N-acetyl-D-glucosamine 2-epimerase under denaturing conditions was also demonstrated. These results demonstrate that hydroxyapatite is a promising adsorbent for immobilized metal affinity chromatography.     

The retention of some open-chain diamines on a strong cation-exchange resin in ion chromatography as a function of their structure

This work reports the results of a study of the influence of the structure of some open-chain diamines on their retention in high-performance ion chromatography (HPIC). We examine a set of eight diamines, in which the N atoms are separated by two C atoms and are differently N- and C-alkyl substituted; four of these diamines have the same formula weight. In the other two sets, the diamines are different from each other, either in terms of the length of the alkyl chain between the N atoms or in terms of the length of the substituent(s) on the nitrogen atom(s). The mobile phase is a mixture of HClO4 0.100 mol/L, 0.160 < C(NaClO4) < 1.50 mol/L, and 0 < acetonitrile% < or = 15 (v/v). The amines are detected by integrated amperometry. Interpretation of experimental data shows that (a) elution occurs for both ionic and hydrophobic interactions and is dependent on all the components of the mobile phase, (b) the diamines are eluted in any case as dications, and (c) steric hindrance significantly influences elution times. Some examples of separation are proposed.     

Acid-base characteristics of carbon adsorbents,determined by potentiometric titration

For initial carbons of varied origin and carbons oxidized with various oxidizing agents, the continuous distribution function of surface groups with respect to the protonation constants was determined by acid-base potentiometric titration.Translated from Zhurnal Prikladnoi Khimii, Vol. 77, No. 8, 2004, pp. 1279–1283.Original Russian Text Copyright © 2004 by Puzii, Poddubnaya, Stavitskaya.     

Fundamental characteristics of synthetic adsorbents intended for industrial chromatographic separations

With the aim of obtaining comprehensive information on the selection of synthetic adsorbents for industrial applications, effect of pore and chemical structure of industrial-grade synthetic adsorbents on adsorption capacity of several pharmaceutical compounds was investigated. For relatively low molecular mass compounds, such as cephalexin, berberine chloride and tetracycline hydrochloride, surface area per unit volume of polystyrenic adsorbents dominated the equilibrium adsorption capacity. On the contrary, effect of pore size of the polystyrenic adsorbents on the equilibrium adsorption capacity was observed for relatively high molecular mass compounds, such as rifampicin, Vitamin B12 and insulin. Polystyrenic adsorbent with high surface area and small pore size showed small adsorption capacity for relatively high molecular mass compounds, whereas polystyrenic adsorbent with relatively small surface area but with large pore size showed large adsorption capacity. Effect of chemical structure on the equilibrium adsorption capacity of several pharmaceutical compounds was also studied among polystyrenic, modified polystyrenic and polymethacrylic adsorbents. The modified polystyrenic adsorbent showed larger adsorption capacity for all compounds tested in this study due to enhanced hydrophobicity. The polymethacrylic adsorbent possessed high adsorption capacity for rifampicin and insulin, but it showed lower adsorption capacity for the other compounds studied. This result may be attributed to hydrogen bonding playing major role for the adsorption of compounds on polymethacrylic adsorbent. Furthermore, column adsorption experiments were operated to estimate the effect of pore characteristics of the polystyrenic adsorbents on dynamic adsorption behavior, and it is found that both surface area and pore size of the polystyrenic adsorbents significantly affect the dynamic adsorption capacity as well as flow rate.     

High-throughput isotherm determination and thermodynamic modeling of protein adsorption on mixed mode adsorbents

The thermodynamic modeling of protein adsorption on mixed-mode adsorbents functionalized with ligands carrying both hydrophobic and electrostatic groups was undertaken. The developed mixed mode isotherm was fitted with protein adsorption data obtained for five different proteins on four different mixed mode adsorbents by 96-well microtitre plate high throughput batch experiments on a robotic workstation. The developed mixed mode isotherm was capable of describing the adsorption isotherms of all five proteins (having widely different molecular masses and iso-electric points) on the four mixed mode adsorbents and over a wide range of salt concentrations and solution pH, and provided a unique set of physically meaningful parameters for each resin-protein-pH combination. The model could capture the typically observed minimum in mixed mode protein adsorption and predict the precise salt concentration at which this minimum occurs. The possibility of predicting the salt concentration at which minimum protein binding occurs presents new opportunities for designing better elution strategies in mixed mode protein chromatography. Salt-protein interactions were shown to have important consequences on mixed mode protein adsorption when they occur. Finally, the mixed mode isotherm also gave very good fit with literature data of BSA adsorption on a different mixed mode adsorbent not examined in this study. Hence, the mixed mode isotherm formalism presented in this study can be used with any mixed mode adsorbent having the hydrophobic and electrostatic functional groups. It also provides the basis for detailed modeling and optimization of mixed mode chromatographic separation of proteins.     

Chromatographic behaviors of proteins on cation-exchange column

A weak cation-exchanger (XIDACE-WCX) has been synthesized by the indirect method. The chromatographic characteristics of the synthesized packing was studied in detail. The standard protein mixture and lysozyme from egg white were separated with the prepared chromatographic column. The chromatographic thermodynamics of proteins was studied in a wide temperature range. Thermodynamic parameters standard enthalpy change (deltaH0) and standard entropy change (deltaS0) and compensation temperature (beta) at protein denaturation were determined in the chromatographic system. By using obtained deltaS0, the conformational change of proteins was judged in the chromatographic process.The linear relationship between deltaH0 and deltaS0 can be used to identify the identity of the protein retention mechanism in the weak cation-exchange chromatography. The interaction between weak cation-exchanger and metal ions was investigated. Several metal chelate columns were prepared. The effects of introducing metal ion into the naked column on protein retention and the retention mechanism of proteins in the metal chalet affinity chromatography were discussed.     

Optimization of structural and energy characteristics of adsorbents for methane storage

Using numerical and analytical methods, a model for microporous carbon adsorbents with slit-shaped pores of different widths was developed. Such pores are formed during activation procedure by the removal of the hexagonal carbon layers burnt out in a graphite-like crystallites. Dubinin’s theory of volume filling of micropores was used to calculate methane adsorption equilibria on these model adsorbents. Isobaric dependences of methane adsorption on pore width, specific micropore volumes, and the specific surface were plotted in the range of pressures from 1 to 10 MPa. It was found that the isobaric adsorption curves had a maximum the position of which depends on both the structural-energy characteristics of the adsorbent and thermodynamic conditions chosen to operate the adsorption system. As pressure increased, the maximum of adsorption shifts to the porous systems with wider pores and larger micropore volume.