Gradient Elution Ion-exchange Chromatography Research Articles

An accelerated approach for mechanistic model based prediction of linear gradient elution ion-exchange chromatography of proteins

With growing demands for therapeutic monoclonal antibodies, in silico downstream process development based on mechanistic modeling of chromatography separation process is being increasingly used for process optimization and process characterization. Application of mechanistic modeling in biopharmaceutical industry has been sparse due to the significant investment of time and resources that are required for performing model calibration. Mechanistic modeling of the chromatography process involves a large number of mass transport and binding parameters and their initial input values are required for simulations. These input values of column parameters can be easily obtained either from experiments or from empirical correlations available in literature. On the other hand, obtaining the model input valves for binding kinetic parameters is usually a cumbersome process as it involves performing batch experiments which are not only tedious but also require significant quantities of purely isolated main product and its related impurities, which is challenging as the product related impurities are typically present in smaller quantities and hence are difficult to obtain as pure species. In the present work, a mechanistic model that is based on the general rate model coupled with extended Langmuir binding model has been used for prediction of linear gradient elution peaks of monoclonal antibody on cation exchanger chromatography. The present work describes an accelerated approach for obtaining the input values for binding kinetic parameters in the extended Langmuir binding model from the two Yamamoto coefficient A and B values obtained by Yamamoto method directly from the model calibration linear gradient elution runs of different gradient slopes and at low to moderate protein loadings. The equations that can relate the two coefficients to the extended Langmuir model equation binding kinetic parameters were derived. Therefore, once A and B are determined, the binding kinetic parameter values were determined straightforward, thereby avoiding the problem of multiple solutions for the model parameters. The estimated binding parameters were successfully validated from isocratic elution experiments performed at low loading. What we demonstrate is that the proposed approach allows us to estimate binding kinetic parameters in a significantly more efficient and accelerated manner than presently used approaches, thereby accelerating development and implementation of mechanistic modeling for process chromatography.

Determination of water-soluble forms of oxalic and formic acids in soils by ion chromatography

Carboxylic acids (CA) play an important role in the chemical composition origin of soils and migration of elements. The content of these acids and their salts is one of the important characteristics for agrochemical, ecological, ameliorative and hygienic assessment of soils. The aim of the article is to determine water-soluble forms of same carboxylic acids — (oxalic and formic acids) in soils by ion chromatography with gradient elution. For the separation and determination of water-soluble carboxylic acids we used reagent-free gradient elution ion-exchange chromatography ICS-2000 (Dionex, USA), the model solutions of oxalate and formate ions, and leachates from soils of the Kola Peninsula.The optimal gradient program was established for separation and detection of oxalate and formate ions in water solutions by ion chromatography. A stability indicating method was developed for the simultaneous determination of water-soluble organic acids in soils. The method has shown high detection limits such as 0.03 mg/L for oxalate ion and 0.02 mg/L for formate ion. High signal reproducibility was achieved in wide range of intensities which correspond to the following ion concentrations: from 0.04 mg/g to 10 mg/L (formate), from 0.1 mg/g to 25 mg/L (oxalate). The concentration of formate and oxalate ions in soil samples is from 0.04 to 0.9 mg/L and 0.45 to 17 mg/L respectively.

Determination of pharmaceutically related compounds by suppressed ion chromatography: IV. Interfacing ion chromatography with universal detectors

This work forms the final part of a study investigating gradient elution ion-exchange chromatography of pharmaceutically relevant compounds, aiming at achieving complementary selectivity to reversed-phase HPLC. In this study the coupling of three universal detectors (electro-spray ionisation mass spectrometer (ESI-MS); corona charged aerosol detector (CAD); and evaporative light scattering detector (ELSD)) to suppressed IC using complex elution profiles with potassium hydroxide eluents is demonstrated. The non-volatile ions were removed from the eluent by the suppressor prior to detection, thus allowing a stable detector response, especially with the prototype electrolytic suppressor. The detector response for ten weakly anionic pharmaceuticals followed the expected models and the limits of detection obtained were not compromised by the use of a suppressor, yielding values below 50ng/mL with MS, low to sub μg/mL levels with CAD and 2–20μg/mL with ELSD (25μL injections). When coupled to MS and CAD, the prototype electrolytic suppressor showed percentage relative standard deviations (%RSDs) in peak areas of 0.4–2.5% on average, compared to the chemical suppressor which yielded 1.5–3 fold higher %RSD values for the test analytes. The prototype electrolytic suppressor also generally exhibited wider linear response ranges than the chemical suppressor.

Determination of pharmaceutically related compounds by suppressed ion chromatography: I. Effects of organic solvent on suppressor performance

This overall study aims to investigate gradient elution ion-exchange chromatography of pharmaceutically relevant compounds using universal nebulisation detectors, such as evaporative light scattering detection (ELSD). Addition of organic solvents to the eluent is necessary to minimise hydrophobic adsorption on the polymeric stationary phase and improve solubility of analytes. It is also necessary to de-salt the eluent prior to detection, and in this work, ion chromatography suppressors were used for this step. Such suppressors have been designed for aqueous eluents, so the purpose of the present study was to investigate the effects of methanol and acetonitrile on suppressor performance. Chemical and electrolytic suppressors were evaluated for baseline drift, noise and efficiency of suppression using aqueous/organic eluents containing up to 40% (v/v) methanol or acetonitrile. Chemical suppression of aqueous/organic eluents showed minimal noise levels, uniform low baseline and low gradient drift. Electrolytic suppression gave good performance, but with higher baseline conductivity levels and baseline drift than chemical suppression. The elevated baseline was found not to be caused by incomplete suppression of the eluent, but was attributed to chemical reactions involving the organic solvents and facilitated by high electric currents and heat generation. It was demonstrated that suppressed ion-exchange separation using a complex KOH elution profile could be coupled with ELSD, with the suppressor effectively de-salting the eluent, producing a stable baseline. Finally, complementary separation selectivity was demonstrated using a set of pharmaceutically related organic acids separated by reversed-phase and ion-exchange methods.

Rational Design Calculation Method for Stepwise Elution Chromatography of Proteins

Although linear gradient elution ion-exchange chromatography is best suited for very fine separation of proteins, it is not commonly employed for the actual protein purification process. Instead, stepwise elution is usually used. However, it is difficult to design an efficient stepwise elution process as small changes in the buffer salt concentration often affect the retention volume and the peak width significantly. In this paper, a design calculation method for stepwise elution proposed previously was modified and extended. This method uses the data obtained by linear gradient elution experiments. The efficiency of the process was examined on the basis of the peak width and the separation time. The advantages using convection-aided chromatography media (monolithic columns) were investigated.

Apparent absorption of eight micronutrients and phytic acid from vegetarian meals in ileostomized human volunteers

Objectives Apparent absorption of eight micronutrients and degradation of phytic acid were studied in human subjects who underwent ileostomy. The prominent factors affecting micronutrient absorption from vegetarian Indian meals ( n = 11) were identified. Methods Levels of β-carotene, ascorbic acid, riboflavin, and thiamine in food and ileostomy contents were estimated by spectrophotometry and spectrofluorometry. Contents of zinc, iron, copper, and manganese were estimated by atomic absorption spectrometry and that of phytic acid by gradient elution ion exchange chromatography. Statistical analyses were done with SPSS 10.0. Results Absorption of β-carotene, ascorbic acid, riboflavin, and thiamine was 63% to 75.6%. There was a negative non-significant trend in values of β-carotene absorption with increased intake of β-carotene ( r = − 0.51, P > 0.1) and iron ( r = −0.67, P = 0.1) but a positive significant trend with riboflavin intakes ( r = 0.84, P = 0.018). Percentage of absorption of ascorbic acid showed weak positive associations with intakes of riboflavin ( r = 0.71) and ascorbic acid ( r = 0.5). Percentage of absorption of ascorbic acid was positively correlated with percentage of absorption of β-carotene ( r = 0.80, P < 0.05), iron, and riboflavin ( r = 0.64, P = 0.086), indicating some common influencing factors. Percentages of absorption for zinc (20.2), iron (9.9), and copper (17.6) was comparable with those reported for soy protein-based, high phytate diets. Pattern of phytic acid in the meals and output indicated partial degradation and absorption (34%). Conclusions For vegetarian Indian meals, apparent absorptions of β-carotene and ascorbic acid were 76% and 73.5% and of riboflavin and thiamine was 63%. Zinc, copper, and iron showed a lower absorption (10% to 20%).

Theoretical background of short chromatographic layers

Although linear salt gradient elution ion-exchange chromatography (IEC) of proteins is commonly carried out with relatively short columns, it is still not clear how the column length affects the separation performance and the economics of the process. The separation performance can be adjusted by changing a combination of the column length, the gradient slope and the flow velocity. The same resolution can be obtained with a given column length with different combinations of the gradient slope and the flow velocity. This results in different separation time and elution volume at the same resolution. Based on our previous model, a method for determining the separation time and the elution volume relationship for the same resolution (iso-resolution curve) was developed. The effect of the column length and the mass transfer rate on the iso-resolution curve was examined. A long column and/or high mass transfer rate results in lesser elution volume. The resolution data with porous bead packed columns and monolithic columns were in good agreement with the calculated iso-resolution curves. Although the elution volume can be reduced with increasing column length, the pressure drop limits govern the optimum conditions.

Plate height determination for gradient elution chromatography of proteins

A method for determining the plate height HETP from the elution curve obtained by the linear gradient elution (LGE) ion-exchange chromatography (IEC) of proteins is presented. The method was developed on the basis of the numerical solutions of a chromatography model which considers the zone sharpening and the distribution coefficient as a function of the salt concentration. The plate height HETP is determined from the peak width and the salt concentration at which the peak is eluted in LGE. The method was applied to the experimental results with various ion-exchange chromatography media. A calculation example based onthe present method is presented to show how the chromatographic and operating parameters should be tuned to obtain a desired resolution. A simplified calculation procedure for the peak profile is also described. (c) 1995 John Wiley & Sons, Inc.

Characterization of the influence of displacing salts on retention in gradient elution ion-exchange chromatography of proteins and peptides

It has been shown earlier that the choice of displacing salt has a large effect on the retention in ion-exchange chromatography of proteins and peptides. The influence of different displacing salts cannot be predicted or quantitatively explained, owing to the current lack of an adequate theoretical framework. In this work a general characterization is made by using a considerable number of proteins and peptides and all displacing salts found feasible. Principal component analysis is used to interpret the large amount of data that is generated. The results of the analysis indicate that most of the retention variations are due to non-specific effects and can be explained by changes in the apparent gradient slope, i.e., the increase in elution strength per unit volume, and the elution strength of the starting buffer. This differs from the interpretation given earlier, where the selectivity changes were attributed to specific effects of the salts. However, as it is impossible to test all existing proteins and peptides, specific effects are still possible, but they might be less common than previously considered.

Purification and sequence determination of canine relaxin.

Relaxin immunological activity has been observed in the plasma of pregnant bitches, and preliminary studies in our laboratory indicated that the highest relaxin concentrations were found in placentas. Therefore, canine placentas were collected at term and also from spay and relaxin was purified by methods developed for equine relaxin. Tissue was prepared by homogenization and purification on a C18 column. The preparation was further purified by stepwise elution ion-exchange chromatography, gel filtration, and gradient elution ion-exchange chromatography. One predominant peak in relaxin immunoactivity was collected. Canine relaxin was found to be larger than either porcine or equine relaxin as determined by SDS-PAGE. It migrated faster under reducing conditions, indicating a subunit structure. Purified canine relaxin was used for tracer and standard in a canine radioimmunoassay (RIA) using an antiporcine relaxin antibody. Concentrations of relaxin immunoactivity using the canine assay were up to 300-fold higher in placental preparations than those measured in the porcine relaxin assay. Sequence analysis of canine relaxin revealed a structure similar to other relaxins in the presence and placement of cystine residues.

Interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry for analysis of proteins

Molecular weights (MWs) of different proteins were determined by interfacing gradient elution ion-exchange chromatography and low-angle laser light-scattering photometry (IEC-LALLS). A high-performance strong cation-exchange column was used to elute proteins using fast (5 min) and conventional (15-30 min) gradients. The eluted proteins were characterized on-line by determining their MWs using LALLS. The specific refractive index (RI) increment (d n/d c) and the RI of the solvent used over the gradient range were determined off-line and used to calculate the absolute weight-average MWs. Four proteins, ribonuclease A, α-chymotrypsinogen A, trypsinogen and β-lactoglobulin A (β-LACT) were studied. Accurate MWs were obtained for all the proteins using fast and conventional gradients, except for β-LACT, which aggregated as a function of the gradient employed. The degree of aggregation of β-LACT increased as the rapidity of the gradient was increased over a fixed gradient range. This study indicated that it is possible to separate and characterize proteins rapidly using IEC-LALLS.

Purification and reconstitution studies of the nucleoside transporter from pig erythrocytes

The pig erythrocyte nucleoside transporter has been identified as a band 4.5 polypeptide ( M r 64 000) on the basis of photoaffinity labelling experiments with the nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). This protein was purified 140-fold by treatment of haemoglobin-free erythrocytes ‘ghosts’ with EDTA (pH 11.2) to remove extrinsic proteins, extraction of the protein-depleted membranes with n- octyl- glucoside and subsequent gradient-elution ion-exchange chromatography on DEAE-cellulose. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified material revealed the presence of only two detectable protein bands, one which co-migrated with the radiolabelled NBMPR-binding protein, and a lower molecular weight species with an M r of 43 000. The latter protein may be a degradation product of the band 3 anion-exchange transporter. The overall purification of the NBMPR-binding protein with respect to the M r 64 000 band was 350-fold. Reversible NBMPR-binding to the partially-purified band 4.5 preparation was saturable (apparent K d 7.2 nM). Adjustment of the chromatography conditions to allow elution of the NBMPR-binding protein along with the majority of solubilised membrane phospholipid reduced the apparent K d value to 3.0 nM. Purification of reversible NBMPR-binding activity during ion-exchange chromatography was paralleled by an increase in the specific activity of nitrobenzylthioguanosine (NBTGR) -sensitive uridine transport as assayed in proteoliposomes reconstituted by a freeze-thaw-sonication procedure.

Resolution of proteins in linear gradient elution ion-exchange and hydrophobic interaction chromatography

The following equation was derived for the resolution, R s, of proteins in linear gradient elution ion-exchange and hydrophobic interaction chromatography: R s ∝ [(column length · molecular diffusivity)/(slope of the gradient · column gel volume · linear mobile phase velocity · particle diameter 2)] 1 2 . Experimental results obtained under a wide range of experimental conditions (column length, 1–30 cm; particle diameter, 37–92 μm; linear mobile phase velocity, 0.2–3.5 cm/min; temperature, 15–35°C) have shown that this relationship is valid except for very short columns with a shallow slope of the gradient and for low flow-rates. The relationship is very useful for scaling-up the linear gradient elution of proteins from data obtained with small columns.

Purification of β-galactosidase by large-scale gradient elution ion-exchange chromatography

Purification of β-galactosidase by large-scale gradient elution ion-exchange chromatography

An improved buffer system for use in single-column gradient-elution ion-exchange chromatography of amino acids

An improved buffer system for use in single-column gradient-elution ion-exchange chromatography of amino acids

Urinary Metabolic Profiles for Choosing Test Animals for Chronic Toxicity Studies: Application to Naproxen

Gradient elution ion-exchange chromatography was employed to determine urinary metabolic profiles of naproxen in test animals and humans. Retention molarities were found to be very reproducible, and the quantitation and column recovery were good. The profiles add another dimension to the basic information required to pick the most appropriate test animal for the chronic toxicological evaluation of a new drug. Based on the profiles, the mini pig and rat appeared to be the animals of choice for the chronic toxicological evaluation of naproxen. Gradient elution ion-exchange chromatography was employed to determine urinary metabolic profiles of naproxen in test animals and humans. Retention molarities were found to be very reproducible, and the quantitation and column recovery were good. The profiles add another dimension to the basic information required to pick the most appropriate test animal for the chronic toxicological evaluation of a new drug. Based on the profiles, the mini pig and rat appeared to be the animals of choice for the chronic toxicological evaluation of naproxen.

Deuterium Isotope Effect in Enzymatic Transamination of L-Deuterio-phenylalanine to Deuterio-phenylpyruvic Acid

L-Deuterio-phenylalanine was isolated by gradient elution ion-exchange chromatography of the ionic fraction obtained from the cell wall hydrolysate of algae grown in culture medium containing 99.6% deuterium oxide. The enzyme system present in rat brain extract was used for determining the rate of transamination of L-deuterio-phenylalanine and its protio analog. The phenylpyruvate formed was determined spectrophotometrically at 300nm. using the arsenate-catalyzed enol-borate assay procedure. Transamination rates were determined in media containing verying concentrations of amino acid and α-ketoglutarate. Kinetic determination of maximum velocity was conducted through graphical analysis of the data. The deuterium isotope effect, expressed as the VH/VD value, was 1.64.

Isolation and partial characterization of an elastase-associated acidic endopeptidase and its interaction with elastase at low ionic strength

An acidic endopeptidase has been isolated from dried preparations of swine pancrease by column chromatography on DEAE cellulose. The yield of acidic endopeptidase from 100 g of pancreas powder was 210 mg. The product was found to be electrophoretically homogeneous and produced a single peak in the analytical ultracentrifuge. The sedimentation coefficient (s 0 20,w) , diffusion coefficient (D 20,w) , apparent molecular weight, electrophoretic mobility, E 1 cm 1% at 280 mμ and pH optimum were respectively, 3.13, 8.7 · 10 −7 cm 2 · sec −1 , 35 300, −5.9 · 10 −5 cm 2 · V −1 · sec −1 , 14.6 and 10.7. The enzyme was inhibited by diisopropyl phosphorofluoridate, by swine serum and to a lesser extent by human serum. It was not “activated” by 1 · 10 −3 M FeCl 2 , MnCl 2, MgCl 2, CaCl 2 or CoCl 2 or by 1 · 10 −3 M ZnSO 4 ; however, the activity of the enzyme was increased by 1 · 10 −3 M cysteine. The similarity of the acidic endopeptidase to proteolytic enzymes described by other investigators is discussed. Gradient elution ion-exchange chromatography of water insoluble elastase euglobulin produces two major water soluble components, elastase and the acidic endopeptidase. When distilled water solutions of the purified enzymes were mixed complex formation occurred (precipitation) between the basic protein elastase and the acidic endopeptidase. The formation of the euglobulin when acid extracts of swine pancreas are dialyzed against distilled water is thus explained. Complex formation was maximal at low ionic strength when the enzymes were oppositely charged and was minimal below the isoelectric point of the acidic endopeptidase (∼ pH 4.1) or above the isoelectric point of elastase (pH 9.5). The reaction was essentially abolished at I 0.011. The equivalence point for the reaction was 2.8 mg of elastase  1 mg of acidic endopeptidase. One mole of acidic endopeptidase is capable of binding approximately four moles of elastase. Although trypsin, another basic protein, was precipitated by the acidic endopeptidase the data suggest a preferential reaction of the latter enzyme with elastase.

An automated ion-exchange method for the determination of sodium monofluorophosphate in dentifrices

Sodium monofluorophosphate can be accurately analyzed in dental cream by an AutoAnalyzer method. The monofluorophosphate is separated from other phosphate species by gradient elution ion-exchange chromatography. The column effluent is fed to an AutoAnalyzer unit which continuously hydrolyzes the phosphates to orthophosphate and develops a color (molybdenum blue) proportional to the phosphate level. The method is useful for the analysis of large numbers of samples.

Incorporation of L-carbamyl-C14-aspartate into acid-soluble pyrimidine nucleotides of mouse brain.

SummaryAcid-soluble pyrimidine nucleotides were separated by gradient elution ion-exchange chromatography after intracranial injection of L-carbamyl-C14-aspartate into mice. Radioactivity was associated with orotic acid, UMP, UDP, UTP, UDPAG, UDPG, CTP, CDP, CMP and CDP-ethanolamine; this shows that brain tissue is capable of synthesizing these pyrimidine nucleotides from simple precursors. Specific activities of isolated pyrimidine nucleotides indicate that biosynthesis of pyrimidine nucleotides by brain tissue occurs in a manner similar to that in rat liver and bacterial extracts. Comparison of specific activities of CTP, CDP and CMP suggests that uridine to cytidine nucleotide conversion takes place between UTP and CTP in mouse brain, probably by a reaction similar to that shown by others in extracts of E. coli. Distribution of radioactivity was the same in brains of mice previously infected with Theiler's encephalomyelitis virus and in noninfected brains.