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PubMed1991

Rapid purification of protein phosphatase 2A from mouse brain by microcystin-affinity chromatography.

Microcystin LR, which is a monocyclic heptapeptide containing two L-amino acids, leucine and arginine, is a new inhibitor of protein phosphatases 1 and 2A. Microcystin LR-affinity chromatography was used to purify protein phosphatase 2A as a holoenzyme. Five mg of microcystin LR were immobilized to ECH Sepharose 4B by the carbodiimide coupling reaction. Following DEAE-cellulose column chromatography, microcystin-affinity chromatography, as the second step in the procedure, resulted in purification of protein phosphatase 2A in a pure form. The enzyme isolated from mouse brain consisted of two regulatory subunits of 67 kDa and 58 kDa and a catalytic subunit of 41 kDa. Microcystin-affinity chromatography is useful for isolation of protein phosphatase 2A.

Animals↗
PubMed1986

Application of the stoichiometric displacement model of retention to anion-exchange chromatography of nucleic acids.

The stoichiometric displacement model has been refined in its application to anion-exchange chromatography. The revised stoichiometric displacement model has been shown to be valid for anion-exchange chromatography with respect to all particulars of the model tested. It has been shown that the use of displacing agent activity is not a necessary condition for valid application of the stoichiometric displacement model to anion-exchange chromatography. While the cation of the displacing salt can influence anion-exchange chromatography, the data indicate that the displacing anion is of primary importance. It has been shown that solutes with three-dimensional structure have Zn value to solute charge ratios less than unity, and that the stoichiometric displacement model may be useful as a probe of solute three-dimensional structure.

Adenine↗
PubMed1992

Multiple ligand applications in high-performance immunoaffinity chromatography.

A mixture of antibodies specific for albumin and transferrin, immobilized onto a single support, was used for the simultaneous extraction of albumin and transferrin from immunoglobulin G by high-performance immunoaffinity chromatography. The affinity column was coupled to a strong cation exchanger in order to monitor the success of the extraction and to demonstrate the compatibility of the two chromatographic modes. Coupling of non-affinity chromatography with multiple ligand affinity chromatography is discussed as an alternative to positive affinity chromatography for protein purification.

Chromatography, Affinity↗
PubMed1993

Evaluation of the capability of different chromatographic systems for the monitoring of thimerosal and its degradation products by high-performance liquid chromatography with amperometric detection.

Several liquid chromatographic systems using electrochemical detection on carbon electrodes were compared for the analysis of thimerosal and its degradation products, thiosalicylic acid and dithiodibenzoic acid. The studied separation systems included reversed-phase ion-suppressed chromatography, reversed-phase ion-pair chromatography and ion chromatography. Amperometry and coulometry were evaluated as electrochemical detection techniques. The best method for thimerosal determination in ophthalmic solutions in terms of selectivity and sensitivity was ion-pair chromatography.

Benzoates↗
PubMed1982

Purification of carcinoembryonic antigen by affinity chromatography with anti-alpha 1-acid glycoprotein.

Purification of radiolabeled carcinoembryonic antigen (CEA) preparations by affinity chromatography with anti-AG bound to Sepharose was attempted, since an immunological similarity between AG (alpha 1-acid glycoprotein) and a portion of CEA had been noted. When 125I-CEA was purified in this manner, the fraction which did not bind to the column showed decreased reactivity with either anti-AG or anti-CEA. The retained fraction showed enhanced reactivity with both anti-AG and anti-CEA. The yield of purified CEA increased when the CEA preparation was allowed to react with the anti-AG column overnight. Purification of CEA from tumor tissue was performed by affinity chromatography. A perchloric acid (PCA) extract from cancer tissue was mixed with antiserum against CEA to give an immune complex, and a CEA-reactive fraction obtained by PCA extraction. The CEA-reactive fraction was eluted from a Sephadex G-200 column, and final purification was by anti-AG chromatography. When purified CEA was applied to a Sephadex G-200 column with carrier protein after labeling with 125I, the eluted radioactivity was found only in the 180,000 dalton fraction. Almost all the radioactivity was precipitated from the labeled protein by either anti-AG or anti-CEA. Purification of CEA is possible by affinity chromatography with anti-AG bound to Sepharose.

Animals↗
PubMed1989

Analysis of albuterol in human plasma based on immunoaffinity chromatographic clean-up combined with high-performance liquid chromatography with fluorimetric detection.

A method combining immunoaffinity chromatography with high-performance liquid chromatography was developed for the determination of albuterol in human plasma. The immunoaffinity chromatography, based on the specific interaction of albuterol with the immobilized antibody raised against it, was used as a clean-up step. Albuterol eluted from this immunochemical solid-phase clean-up step was analysed by reversed-phase high-performance liquid chromatography with fluorimetric detection. The performance of the assay was validated on six normal volunteers after a 4-mg oral dose of albuterol, which gave a peak plasma concentration in the range 6.67-15.31 ng/ml at 3-4 h after the dose. Plasma levels (0.79-1.56 ng/ml) of albuterol could be detected up to 24 h after the dose.

Albuterol↗
PubMed1992

Determination of D- and L-amino acids in mouse kidney by high-performance liquid chromatography.

A method for the enantiomeric analysis of amino acids of mammalian tissues is described. An excellent resolution of D- and L-enantiomers of common protein amino acids was achieved by employing a combination of thin-layer chromatography and high-performance liquid chromatography. D-Enantiomers and L-enantiomers of glutamate, aspartate, glutamine, asparagine, serine, threonine, alanine, proline, tyrosine, valine, methionine, isoleucine, leucine, phenylalanine and histidine, as well as glycine were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide. The amino acid diastereomers were separated by two-dimensional thin-layer chromatography. Each amino acid diastereomer was then analysed by reversed-phase high-performance liquid chromatography for the resolution of D- and L-enantiomers. Very sharp peaks were obtained using a conventional octadecylsilyl-bonded column, and the possibility of analysing these amino acids (except tyrosine and histidine) in subnanomole amounts was indicated. The method was used to demonstrate the presence of D-enantiomers of alanine, proline and serine in mouse kidney.

Alanine↗
PubMed1996

Direct analysis of mannitol, lactulose and glucose in urine samples by high-performance anion-exchange chromatography with pulse amperometric detection. Clinical evaluation of intestinal permeability in human immunodeficiency virus infection.

Clinically, the ratio of lactulose/mannitol excretion in urine after administration of these non-metabolized sugars has been used to evaluate the extent of malabsorption and intestinal permeability disruption in several infections and nutritional diseases, including human immunodeficiency virus (HIV) infection. A range of methodologies have been reported to determine the lactulose/mannitol ratio, including enzymatic assay, gas-liquid chromatography (GC), thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Most published methods involve tedious sample preparations, rendering them unsuitable for routine or automated clinical laboratory testing. We describe in this paper a method in which weak anion-exchange high-performance liquid chromatography in conjunction with a pulsed amperometric detector was used. It requires very simple sample preparation and avoids interference by other components present in the urine. The linear range of determination for mannitol, lactulose and glucose are up to 10 nmol, in a single injection. The limits of detection are 8, 12, 47 and 52 pmol, respectively, for mannitol, glucose, lactose and lactulose. The separation and quantification using this method are highly reproducible, yielding standard errors of less than 2.5% for retention times and less than 3.5% for quantitation. The ratios of lactulose/mannitol recovery in controls and in HIV-infected subjects with and without diarrhea showed striking differences, which are in close agreement with the published results derived with similar HPLC methods.

Calibration↗
PubMed2004

Factorial screening of antibody purification processes using three chromatography steps without protein A.

Protein A affinity chromatography is often employed as a capture step to meet the purity, yield, and throughput requirements for pharmaceutical antibody purification. However, a trade-off exists between step performance and price. Protein A resin removes 99.9% of feed stream impurities; however, its price is significantly greater than those of non-affinity media. With many therapeutic indications for antibodies requiring high doses and/or chronic administration, the consideration of process economics is critical. We have systematically evaluated the purification performance of cation-exchange, anion-exchange, hydroxyapatite, hydrophobic interaction, hydrophobic charge induction, and small-molecule ligand resins in each step of a three-step chromatographic purification process for a CHO-derived monoclonal antibody. Host cell proteins were removed to less-than-detectable for three processes (cation-exchange-anion-exchange-hydrophobic interaction chromatography, cation-exchange-anion-exchange-mixed cation-exchange chromatography, and cation-exchange-mixed cation-exchange-anion-exchange chromatography). The order of the process steps affected purification performance significantly.

Animals↗
PubMed2006

Semi-automated peak trapping recycle chromatography instrument for peak purity investigations.

A peak trapping recycle chromatography system has been developed and optimized for peak purity assessment of active pharmaceutical ingredients analyzed by high performance liquid chromatography (HPLC). After being analyzed using a reversed phase analytical column, peaks of interest are trapped and are subsequently introduced to a recycle chromatography system. In addition to the increased effective length afforded the recycling system, the small selectivity difference between the analytical and recycling methods help separate potential impurities under the main peak. For more difficult to separate components, the increased efficiency of recycle chromatography provides the necessary resolution. Over 227,000 theoretical plates have been obtained in the recycle dimension for some compounds. The sensitivity of the system fell short of the target (0.1%), but it did show sensitivity (0.5%) comparable to other peak purity techniques commonly used in the pharmaceutical industry. The recovery and repeatability have also been shown to be adequate for peak purity assessment. The system has also been automated using a Visual Basic macro, simplifying the interface allowing it to be used as an open access instrument.

Automation↗
PubMed2006

Direct comparison between ion-exchange chromatography and aqueous two-phase processes for the partial purification of penicillin acylase produced by E. coli.

A direct comparison of a chromatography and an aqueous two-phase system (ATPS) processes for the partial purification of penicillin acylase (PA) produced by a recombinant strain of E. coli, was performed. An established chromatography process for the recovery of PA was selected as a model system and characterised for comparison with a developed ATPS prototype process. PEG-phosphate systems were selected for the recovery of PA over PEG-citrate systems, since higher enzyme recovery and increased purity was obtained. ATPS proved to be suitable to process highly concentrated disrupted extract (35%, w/w) and maintain a high top phase enzyme recovery. In the direct comparison of the processes, the superiority of the ATPS approach was highlighted since a reduction of the number of unit operations from 7 to 4 was achieved. An outline economic analysis based on the cost of separation agent of the processes favour the ATPS process, in which a gross cost reduction of 37% (from 0.47 to 0.30 US dollars) was achieved. Such result was obtained considering a potential re-use of up to 100 times of the resin used in the chromatography process. Additionally, by assuming that all the unit operations are equivalent in investment and operating cost, further reduction of approximately 43% of the respectively involved cost can be obtained when the ATPS process is used. Overall, the proposed ATPS process comprising of PEG1450-phosphate, tie-line length (TLL) of 48.5% (w/w), volume ratio (Vr) of 1.0, pH of 7.0 and 35% (w/w) PA crude extract loaded into the system proved to be more efficient, recovering 97% of PA at the top phase (PEG rich phase) with a purity factor of 3.5. It is clear that the results reported herein raise the consideration for the potential substitution of the chromatography process for PA recovery from E. coli as a first step for the development of an optimised and economic process with evident commercial application.

Chromatography, Ion Exchange↗
PubMed2005

Investigation of the retention/pH profile of zwitterionic fluoroquinolones in reversed-phase and ion-interaction high performance liquid chromatography.

The retention/pH profiles of three fluoroquinolones, ofloxacin, norfloxacin and ciprofloxacin, was investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC) and reversed-phase ion-interaction chromatography (RP-IIC), using an octadecylsilane stationary phase and acetonitrile as organic modifier. Sodium hexanesulphonate and tetrabutylammonium hydroxide were used as sources of counter ions in ion-interaction chromatography. The retention/pH profiles under in RP-HPLC were compared to the corresponding lipophilicity/pH profiles. Despite the rather hydrophilic nature of the three fluoroquinolones positive retention factors were obtained while there was a shift of the retention maximum towards more acidic pH values. This behavior was attributed mainly to non-hydrophobic silanophilic interactions with the silanized silica gel material of the stationary phase. In ion-interaction chromatography the effect of counter ions over a broad pH range was found to be ruled rather by the ion pair formation in the mobile phase which led to a drastic decrease in retention as a consequence of the disruption of the zwitterionic structure and thereupon the deliberation of a net charge in the molecules. At pH values at which zwitterionic structure was not favored both the ion-exchange and ion pair formation mechanisms were assumed to contribute to the retention.

Acetonitriles↗
PubMed1975

Application of over-run thin-layer and gas-liquid chromatography in the separation of closely related C19O2 steroids.

An improved method for the separation of epimeric C19O2 steroids and their related allylic alcohols is described. In this method, the steroids are first separated by over-run thin-layer chromatography, and the unresolved groups are further analysed as free or as trimethylsilyl ether derivatives by gas-liquid chromatography. The behaviour of twenty-one C19O2 steroids was investigated by thin-layer chromatography in four systems and by gas-liquid chromatography in four liquid phases. All steroid pairs of similar polarity were resolved by the combination of these two fractionation procedures.

17-Ketosteroids↗
PubMed1988

Rapid high-performance affinity chromatography on micropellicular sorbents.

Short columns (30 x 4.6 mm I.D.), packed with 2-micron fluid-impervious silica microspheres with surface-bound Protein A or a lectin were used for fast separation and quantitation of immunoglobulins and glycoproteins by biospecific interaction chromatography. With stepwise elution, the total analysis time including column reequilibration did not exceed 3 min. In the assay of IgG with a stepwise change in pH best results were obtained with citrate buffer, which facilitated not only fast but also very sensitive analysis. The calibration curve was linear in the range 0.5-40 micrograms of human IgG. By using morpholinoethanesulfonic acid-4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-acetic acid buffer with a linear decrease in pH from 6.0 to 4.0 and an increase in magnesium chloride concentration to 200 mM for elution, the subclasses of human IgG were separated at 40 degrees C above pH 4.0 in 3 min. Micropellicular concanavalin A and wheat germ agglutinin were used for rapid affinity chromatography of horseradish peroxidase and fetuin, respectively. The results suggest that micropellicular affinity sorbents afford fast and sensitive high-performance liquid chromatographic analysis by biospecific interaction chromatography. Although developed primarily for rapid analysis, the micropellicular Protein A exhibited unexpectedly high adsorption capacity (e.g., 4.5 mg human IgG per ml of wet bed volume). This suggests that such columns could be employed in preparative protein chromatography as well.

Chromatography, Affinity↗
PubMed2001

Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts.

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.

Chromatography, Affinity↗
PubMed1988

Identification of the more complex triacylglycerols in bovine milk fat by gas chromatography-mass spectrometry using polar capillary columns.

The fourth most volatile 2.5% molecular distillate of butteroil obtained by redistillation of the most volatile 10% cut was examined by gas chromatography on a polar capillary column (RSL-300) with electron impact and chemical ionization mass spectrometry. For this purpose the distillate was first freed from the acetyldiacylglycerols by thin-layer chromatography on plain silica gel and the remainder resolved into long and short chain length saturates, cis- and trans-monoenes, dienes and trienes by thin-layer chromatography on silver nitrate-silica gel. The order of gas chromatographic elution was established for more than 100 major and minor species making up the bulk of the molecular distillate. The results were used to derive the quantitative composition of the triacylglycerol species making up the various peaks obtained by polar capillary column gas chromatography of the total molecular distillate, which closely resembles the lower half of the molecular mass distribution of whole bovine milk fat.

Animals↗
PubMed1985

Separation of flavivirus membrane and capsid proteins by multistep high-performance liquid chromatography optimized by immunological monitoring.

Complete separation of the three structural proteins, E, C and M, of an enveloped virus (tick-borne encephalitis virus) was achieved by means of a two-step high-performance liquid chromatography (HPLC) technique in less than 1 h. The hydrophobically associated membrane proteins E and M were successfully separated by high-performance gel permeation chromatography (TSK-3000 SW column) in the presence of sodium dodecyl sulphate (SDS), whereas the separation of M and C as well as desalting and removal of SDS was achieved by subsequent reversed-phase chromatography on a C3 column. With regard to further characterization by peptide mapping, analysis of the amino acid composition and aminoterminal sequencing, the second step was performed with volatile buffer systems. Quality control of the separation was achieved by a combination of HPLC with a highly sensitive dot immunoassay by the use of polyclonal as well as monoclonal antibodies. This method proved extremely sensitive and revealed strong tailing effects and cross-contaminations of peaks well-separated in reversed-phase chromatography, which were neither apparent in the absorbance curve at 214 nm nor in the analysis by SDS-polyacrylamide gel electrophoresis. By visualization of the peak-tailing effect, the chromatographic conditions could be modified in order to achieve an optimum separation of proteins.

Antibodies, Monoclonal↗
PubMed1985

General method for the separation of cyanogen bromide digests of proteins by high-performance liquid chromatography. Rabbit skeletal troponin I.

In this study, we have demonstrated the necessity for a combination of size-exclusion, ion-exchange and reversed-phase high-performance liquid-chromatography to resolve completely a protein digest. Our approach minimizes the number of steps, and the column order provides the maximum information about the properties of the fragments. The order is: (1) size-exclusion (Bio-Rad TSK-250 column), (2) strong cation-exchange (Synchropak S300 column), and finally (3) reversed-phase chromatography (Ultrapore C3). It was desirable for the first step of the procedure to be size-exclusion chromatography to produce the least number of fractions. The volatile eluent used in size-exclusion eliminated the need for subsequent sample desalting. Volatile buffers were not necessary for the ion-exchange chromatography, since the fractions were both desalted and purified in the final reversed-phase step. All column effluents were compatible with absorbance measurements at 210 nm to provide maximum sensitivity for peptide detection. The results obtained in this study strongly suggest that the combined use of three methods of separation, which utilize different selectivities (size, charge, hydrophobicity), can provide excellent resolving power for peptide separations. We believe this fast, efficient procedure should be generally applicable to other protein digests.

Animals↗