Search

In the present study, the feasibility of immunoaffinity chromatography (IAC) as a purification technology for the analysis of bioactive components in Traditional Chinese Medicine (TCM) was evaluated. IAC was used to analyze hesperidin (HP) and narirutin (NR) in TCM preparations containing Citri reticulatae Pericarpium (CRP, Chenpi in Chinese). An IAC column for the specific extraction and enrichment of HP and NR from TCM preparations containing CRP was developed and characterized. After HP reacted with carbonyl diimidazole and coupled to protein, it was used to immune mice for the generation of antibody. Through cell fusion, cloning and screening, monoclonal antibody was obtained. The IAC column was constructed by covalently coupling specific monoclonal antibody against HP and NR to CNBr-activated Sepharose 4B and packed into a common solid phase extraction cartridge. The extraction conditions including loading, washing and eluting, as well as flow rate for the extraction of HP and NR were optimized. Under optimal conditions, the maximum capacity, extraction recovery rate and stability of IAC column was also characterized. Results revealed that the maximum capacity of IAC column for HP and NR was approximately 16 μg and the relative binding capacity per 1 mL of the column volume was 27 μg. The extraction recovery rate of IAC column for HP and NR at three spiked levels was in the range of 94.05–109.15%. After the repeated application for 5 times, no significant loss of specific recognition was observed. Using high performance liquid chromatography (HPLC) as an effective analytic tool, HP and NR could be successfully separated via IAC column without the inference from impurities, suggesting that the extraction of HP and NR using the prepared IAC column is feasible. The application of IAC can solve the problem of quantitative analysis due to severe interference or low content. Furthermore, pretreatment methods in different matrixes can be unified. The IAC purification procedure can be used as an alternative effective analytical method for the pretreatment of bioactive components in TCM. [ABSTRACT FROM AUTHOR]

The total alkaloids from Nitraria sibirica leaves have been confirmed to exhibit significant protective effects against inflammatory renal injury, hypertension and albuminuria in angiotensin II-salt hypertension. In the present study, a separation method of pH-zone-refining counter-current chromatography was established for separation of the alkaloids from N. sibirica . The separation was performed with a solvent system of MtBE- n -BuOH-H 2 O (2:2:5, v / v ) at a flow rate of 2.0 mL/min. And 15 mM triethylamine (TEA) was added to the upper organic phase, while 10 mM hydrochloric acid was added to the lower aqueous phase. As a result, a new alkaloid, schobemine (5.6 mg), and a known alkaloid, nitraramine (5.0 mg), together with fractions A and B were obtained from the total alkaloids of N. sibirica . The fractions A and B were further purified by means of pH-zone-refining counter-current chromatography with solvent systems of n -hexane- n -BuOH-H 2 O (1.5:3.5:5, v / v ) and (2:3:5, v / v ), respectively. TEA (10 mM) was added to the upper phase, and 10 mM of HCl was added to the lower phase in above two solvent systems, respectively. As a result, a known alkaloid, schoberidine (5.0 mg), and a new alkaloid, schoberimine (3.0 mg) were obtained from fractions A and B, respectively. The purities of the compounds were measured by HPLC–ELSD, and their structures were identified by ESI–MS, 1D and 2D NMR. [ABSTRACT FROM AUTHOR]

Abstract: In this study, resin adsorption as a means to separate and purify baicalin and wogonoside from extracts of Scutellaria baicalensis was investigated. Among the ten tested resins, the non-polar resin HPD-100 offered the best adsorption and desorption properties. Langmuir and Freundlich isotherms were used to describe the interactions between solutes and resin at different temperatures, and the equilibrium experimental data were well fitted to the two isotherms. Column packed with HPD-100 resin was used to perform dynamic adsorption and desorption tests to optimize the separation process. After one round treatment with HPD-100 resin, the contents of baicalin and wogonoside were 3.6-fold and 12.0-fold increased with recovery yields of 85.7% and 65.6%, respectively. In addition, a laboratory preparative-scale separation was carried out under the final conditions. The results showed that the preparative separation of baicalin and wogonoside can be easily and efficiently achieved via adsorption and desorption on HPD-100 resin. The developed method is a promising basis for large-scale preparation of baicalin and wogonoside from S. baicalensis extracts. [Copyright &y& Elsevier]

Abstract: Fusion M13 phage with disulfide constrained heptapeptide, C-WSFFSNI-C, inserted into the minor coat protein (gpIII), has been selected in the current study as ligand in direct purification of hepatitis B core antigen (HBcAg) from unclarified Escherichia coli (E. coli) feedstock. The selected fusion phage showed strong association with the surface of the core particle. In the present study, this fusion M13 phage was immobilized onto Streamline base matrix via epoxy activation and used as adsorbent to capture HBcAg from crude E. coli homogenate. The maximum binding capacity for the adsorbent was 3.76mg/mL with equilibrium coefficient of 1.83mg/mL. Due to the slow uptake rate of HBcAg by M13 phage-immobilized adsorbents, a modified EBAC operation with recirculation of feedstock into the expanded bed has been investigated in this study. The introduction of feedstock recirculation has led to an 18% increase in yield; however, the purity of the eluted product was reduced by 15% compared with typical EBAC operation. The level of antigenicity exhibited by the core particles purified by both EBAC operations employed in the present study was comparable to that purified using sucrose ultracentrifugation. [Copyright &y& Elsevier]

Abstract: Functionalized magnetic particles are emerging as a reliable and convenient technique in the purification of biomacromolecules (proteins and nucleic acids) and cell separation. In this study, we used novel solid nickel ferromagnetic particles coated with Protein A for the affinity purification of antibody. The study demonstrated that IgG can be purified from undiluted mouse serum in as few as 5min using Protein A-coated nickel particles. Further, protein crosslinking was shown to stabilize the Protein A on the nickel particle surfaces to minimize Protein A leaching during the affinity purification and elution of IgG. The separation procedure is gentle, scalable, automatable, efficient and economical. By modifying the functional groups of amino acids in the protein coating, crosslinked nickel particles can be used not only for protein affinity purification but for other biological sample preparation and chromatographic applications as well. Methods proposed and tested in this study can be easily modified for small and medium scale antibody purification in lab and pre-clinical research. [Copyright &y& Elsevier]

Abstract: A microwave-assisted extraction (MAE) method is presented for the extraction of xanthones, α-mangostin and γ-mangostin from Garcinia mangostana. The MAE conditions including extraction temperature, liquid/solid ratio, extraction time and concentration of ethanol were optimized with an orthogonal test, and 5g sample was extracted with the optimized conditions. The crude extraction of MAE was successfully isolated and purified by high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of petroleum ether–ethyl acetate–methanol–water (0.8:0.8:1:0.6, v/v) in one-step separation. The separation yielded 75mg of α-mangostin at 98.5% purity, and 16mg of γ-mangostin at 98.1% purity from 360mg crude extract of G. mangostana in less than 7h. The purity of the two xanthones was determined by HPLC. Their structures were further identified by ESI-MS, 1H NMR and 13C NMR. [Copyright &y& Elsevier]

Abstract: Given an existing demand to establish a process of tetanus vaccine production in a way that allows its complete validation and standardization, this paper focuses on tetanus toxoid purification step. More precisely, we were looking at a possibility to replace the widely used ammonium-sulphate precipitation by a chromatographic method. Based on the tetanus toxin''s biochemical characteristics, we have decided to examine the possibility of tetanus toxoid purification by hydrophobic chromatography, and by chromatographic techniques based on interaction with immobilized metal ions, i.e. chelating chromatography and immobilized metal affinity chromatography. We used samples obtained from differently fragmented crude tetanus toxins by formaldehyde treatment (assigned as TTd-A and TTd-B) as starting material for tetanus toxoid purification. Obtained results imply that purification of tetanus toxoid by hydrophobic chromatography represents a good alternative to ammonium-sulphate precipitation. Tetanus toxoid preparations obtained by hydrophobic chromatography were similar to those obtained by ammonium-sulphate precipitation in respect to yield, purity and immunogenicity. In addition, their immunogenicity was similar to standard tetanus toxoid preparation (NIBSC, Potters Bar, UK). Furthermore, the characteristics of crude tetanus toxin preparations had the lowest impact on the final purification product when hydrophobic chromatography was the applied method of tetanus toxoid purification. On the other hand, purifications of tetanus toxoid by chelating chromatography or immobilized metal affinity chromatography generally resulted in a very low yield due to not satisfactory tetanus toxoid binding to the column, and immunogenicity of the obtained tetanus toxoid-containing preparations was poor. [Copyright &y& Elsevier]

Abstract: Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L9 (3)3. The optimum extraction conditions were determined as follows: extraction temperature 120°C, static extraction time 7min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300mg of crude extract, 51.2mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and 1H NMR. [ABSTRACT FROM AUTHOR]

Abstract: The adsorbent ω-aminodecyl-agarose was evaluated as to its feasibility for the adsorption of human serum and plasma proteins, aiming at the purification of immunoglobulin G (IgG). The contribution of electrostatic and hydrophobic interactions (mixed-mode) and the effects of buffer system on the adsorption of serum proteins were also studied. The adsorption isotherm parameters of human serum albumin (HSA) and IgG were evaluated, pointing to the existence of cooperative effects in the process. A positive (n =2.30±0.38) and negative cooperativity (n =0.63±0.12) were observed for IgG and HSA binding, respectively. High purity IgG was obtained (based on total protein concentration and nephelometric analysis of HSA, transferrin, and immunoglobulins A, G, and M) with a 75% recovery in Hepes 25mmolL−1 pH 6.8 feeding human serum. These results indicate that the use of ω-aminodecyl-agarose is a potential technique for purification of IgG from human serum. [Copyright &y& Elsevier]

Abstract: Purification of target proteins from a crude biological mixture containing proteins, peptides and other biomolecules is the chromatographic challenge. Mixed mode chromatography offers additional selectivities to improve the overall productivity of commercial bioprocesses with novel chromatographic sorbents being introduced to overcome the problem. HEA HyperCel™ (n-hexyl amine) and PPA HyperCel™ (phenyl propyl amine) are industry scalable mixed mode chromatography sorbents where both hydrophobic and electrostatic interactions are predominant. Our study focuses on understanding the underlying mechanism of interaction of protein with the sorbent. Parameters like buffer conditions, pH and temperature were tuned to study the adsorption and desorption conditions of the protein. Dynamic binding capacity of HEA HyperCel™ and PPA HyperCel™ sorbents was studied with human IgG as a model protein. Our study shows that, in HEA the interaction of IgG to the sorbent is predominantly hydrophobic as the binding is enhanced (50–60mg/ml of sorbent) by presence of salt in buffer and increase in temperature. Binding capacity of PPA is 50–60mg/ml of sorbent irrespective of temperature effect and/or the presence of salt. The chromatographic experiments show that the interaction could be hydrophobic or ionic or some charge transfer mechanism depending upon the buffer conditions. [Copyright &y& Elsevier]

Abstract: Polyamine precipitation conditions for removing host cell protein impurities from the cell culture fluid containing monoclonal antibody were studied. We examined the impact of polyamine concentration, size, structure, cell culture fluid pH and ionic strength. A 96-well microtiter plate based high throughput screening method was developed and used for evaluating different polyamines. Polyallylamine, polyvinylamine, branched polyethyleneimine and poly(dimethylamine-co-epichlorohydrin-ethylenediamine) were identified as efficient precipitants in removing host cell protein impurities. Leveraging from the screening results, we incorporated a polyamine precipitation step into a monoclonal antibody purification process to replace the Protein A chromatography step. The optimization of the overall purification process was performed by taking the mechanisms of both precipitation and chromatographic separation into account. The precipitation-containing process removed a similar amount of process-related impurities, including host cell proteins, DNA, insulin and gentamicin and maintained similar product quality in respect of size and charge variants to chromatography based purification. Overall recovery yield was comparable to the typical Protein A affinity chromatography based antibody purification process. [Copyright &y& Elsevier]

Abstract: Water-soluble salvianolic acid A (Sal A) and salvianolic acid B (Sal B) were successfully isolated and purified from the crude extract of Salvia miltiorrhiza by high-speed counter-current chromatography (HSCCC). The solvent system was n-hexane–ethyl acetate–methanol–water (3:6:6:10, v/v/v/v). 4.27mg of Sal A and 32.09mg of Sal B were obtained from 260mg of the crude sample. The purities of Sal A and Sal B were 96.67% and 97.43%, respectively. Their structures were identified by 1H NMR and 13C NMR. Antioxidant activities of Sal A and Sal B were also evaluated and compared by the methods of 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay and 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid (ABTS +) radical cation decolourisation assay. Both Sal A and Sal B showed high radical scavenging activities with their EC50 values being 1.43±0.09 and 1.81±0.01μg/ml in DPPH radical method. The ABTS results showed that Sal A and Sal B exhibited high total antioxidant activities, their EC50 values were 1.35±0.00 and 1.43±0.01μg/ml, respectively. [Copyright &y& Elsevier]

Abstract: High-speed counter-current chromatography methods, combined with resin chromatography were applied to the separation and purification of flavonoid glycosides from the Chinese medicinal herb, Radix Astragali. Five flavonoid glycosides, namely calycosin-7-O-β-d-glucoside, ononin, (6aR, 11aR)-9,10-dimethoxypterocarpan-3-O-β-d-glucoside, (3R)-2′-hydroxy-3′,4′-dimethoxyisoflavan-7-O-β-d-glucoside and calycosin-7-O-β-d-glucoside-6′′-O-acetate, were obtained. Among them, calycosin-7-O-β-d-glucoside-6′′-O-acetate was preparatively separated from Radix Astragali for the first time. Their structures were identified by ESI–MS, 1H NMR, 13C NMR, and 2D NMR. [Copyright &y& Elsevier]

Abstract: Chromatography is deemed the most promising technology for large-scale purification of viral vectors. The authors have previously shown that heparin affinity chromatography could be successfully employed for the purification of VSV-G pseudotyped Moloney murine leukemia virus (MoMLV)-derived vectors giving excellent results in terms of recovery of active particles, reproducibility and selectivity. In this study, the authors examined whether the ability of retrovirus particles to specifically bind to heparin ligands is restricted to VSV-G pseudotypes produced by 293-based packaging cells. It is shown that VSV-G deficient retrovirus particles are captured by a heparin chromatography column as efficiently as VSV-G containing particles. Most strikingly, RD114 pseudotyped retrovirus particles derived from a HT1080-based cell line were found to bind heparin with the same affinity as 293-derived VSV-G pseudotypes. RD114 pseudotyped retrovirus particles were successfully isolated using heparin affinity chromatography obtaining good recoveries of functional particles (43%). These results indicate that heparin affinity chromatography can be extended to the purification of retroviral vectors produced by different packaging cell lines independently of the Env-protein used for pseudotyping. [Copyright &y& Elsevier]

Counter-current chromatography (CCC) using a cross-axis coil planet centrifuge (X-axis CPC) was applied to the purification of glucosyltransferase (GTF) from a cell-lysate of cariogenic bacteria. The purification was performed using an aqueous polymer two-phase system composed of 4.4% (w/w) polyethylene glycol (PEG) 8000–6% (w/w) dextran T500 containing 10 mM phosphate buffer at pH 9.2 by eluting the upper phase (UP) at 1.0 ml/min. The bacterial GTF in the cell-lysate of Streptococcus mutans was selectively retained in the dextran-rich lower stationary phase. The column contents were diluted and subjected to hydroxyapatite (HA) chromatography to remove the polymers from the GTF. Fractions eluted with 500 mM potassium phosphate buffer were analyzed by GTF enzymatic activity as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS–PAGE). The GTF purity in the final product was increased about 87 times as that in the cell-lysate with a good recovery rate of about 79% through this purification process. [Copyright &y& Elsevier]

By selecting the different combination schemes, a simple, fast and highly efficient method for separation and purification of nerve growth factor (NGF) from venom of Chinese cobra is reported in this paper. This purification process consists of a two-step chromatographic separation on DEAE-Sepharose F.F. anion-exchange medium followed by a Sephadex G-50 gel filtration. On reducing and non-reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), the nerve growth factor obtained with this process proved to be homogeneous and its molecular weight was separately estimated to be approximately 14.5 and 29.0 kD, which was consistent with that reported in literature; and on high performance size-exclusion chromatography and reversed-phase chromatography, its purity was about 99%. The yield of this purification method was 0.51% and the nerve growth factor obtained had the activity of eliciting neurite outgrowth from chick embryonic dorsal root ganglia. The optimum concentration of nerve growth factor was 5–100 ng/ml and the minimal concentration eliciting neurite outgrowth from chick embryonic dorsal root ganglia was 5.0 ng/ml. [Copyright &y& Elsevier]

We described a novel purification method for a recombinant glucocorticoid receptor (GR) in detail. The purification procedure consists of sequential chromatographies using common ion-exchange columns (Mono Q and Mono S). This procedure is based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH. The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors. [Copyright &y& Elsevier]

Optimized procedures for the affinity purification of soybean agglutinin (SBA) from soybean flour, and its further immobilization, were developed. Lectin purification on galactosyl-Sepharose yielded 44.5±3.5 mg of pure SBA/50 g of flour. To prepare SBA adsorbents, the lectin was immobilized onto 1-cyano-4-(dimethylamino)pyridinium tetrafluoroborate (CDAP) activated Sepharose with high yields (77%). Feasibility of the use of this improved SBA adsorbent for affinity purification of Streptococcus pneumoniae capsular polysaccharides from strain 14 (CPS-14) at laboratory scale was demonstrated. Using SBA-Sepharose adsorbent (7.0 mg lectin per ml), amounts of 6.3 mg of pure CPS-14 per cycle were produced, the adsorbent being reused up to four times without loss of capacity. [Copyright &y& Elsevier]

Recombinant DNA-derived proteins and, in particular, human pituitary hormones, are increasingly used for research, diagnostic and therapeutic purposes. This trend has demanded new synthetic approaches and improved purification techniques. The type and sequence of the purification steps have to be selected in accordance with the cloning and protein expression strategy, the host organism and cellular localization of the protein of interest, with a view to producing the desired product at a required purity, biological activity and acceptable cost. This review article describes and analyzes the main synthetic and purification strategies that have been used for the production of recombinant human growth hormone, prolactin, thyrotropin, luteinizing hormone and follicle-stimulating hormone, giving special consideration to the few published downstream processes utilized by the biotechnology industry. Practically all types of prokaryotic and eukaryotic organisms utilized for this purpose are also reviewed. [Copyright &y& Elsevier]

Protein C deficiency (inherited and acquired) has a relatively high incidence rate in the general population worldwide. For many years, protein C deficient patients have been treated with fresh frozen plasma, prothrombin complex concentrates, heparin or oral anticoagulants, which all have clinical drawbacks. We report the production process of a highly purified human protein C concentrate from 1500 l of cryo-poor plasma by a four-step chromatographic procedure. After DEAE-Sephadex adsorption, protein C was separated from clotting factors II, VII and IX by DEAE-Sepharose FF and further purified, using a new strategy, by an on-line chromatographic system combining DMAE-Fractogel and heparin-Sepharose CL-6B. In addition, the product was treated against viral risks by solvent-detergent and nanofiltration on 15-nm membranes. The protein C concentrate was essentially free of other vitamin K-dependent proteins. Proteolytic activity was undetectable. Neither activated protein C, prekallikrein activator, nor activated vitamin K-dependent clotting factors were found resulting in good stability of the protein C activity. In vitro and in vivo animal tests did not reveal any sign of potential thrombogenicity. The final freeze-dried product had a mean protein C concentration of 58 IU/ml and a mean specific activity of 215 IU/mg protein, corresponding to over 12 000-fold purification from plasma. Therefore, this concentrate appears to be of potential benefit for the treatment of protein C deficiency. [Copyright &y& Elsevier]

The aim of this work was to test a recycling method for imidazole used in immobilized metal affinity chromatography (IMAC) as eluent for recombinant histidine-tag (His-tag) protein. After evaluating two supports, the method was optimized with a mixture of bovine serum albumin, sodium chloride and imidazole. Recycling was performed with an eluate fraction from IMAC of His-tag enhanced green fluorescent protein produced in our laboratory and pure imidazole was recovered in water and was analyzed after being freeze-dried. The imidazole was then reused as eluent in IMAC without any modification in its structure or behavior. This procedure can be used for large-scale chromatography. [Copyright &y& Elsevier]

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential. [Copyright &y& Elsevier]

Experiments were carried out to evaluate the fractionation of proteins and peptides according to mass. Model mixtures were separated by either reversed-phase or ion-exchange chromatography with mass spectrometry-compatible mobile phase additives. Fraction collection was triggered by the mass/charge ratio of each one of the components of the mixture. Chromatography was additionally monitored with a UV–Vis detector in order to compare the new technique with generally accepted in separations. The results indicated that adequate purification is achieved by this new technique. Fraction collection triggered by changes in the mass/charge ratio reduces sample handling and analysis time. This study demonstrates the utility of mass-directed fractionation of peptides and proteins when mass spectrometry-compatible mobile phase additives are used. [Copyright &y& Elsevier]

• Robust and fast HPLC-UV method for quantification of anidulafungin and micafungin. • SPE improved the signal-to-noise ratios of chromatograms. • Higher sensitivity allows for quantification of lower concentrations in body fluids. • Appropriate approach for pharmacokinetic studies with critically ill patients. The echinocandins anidulafungin (ANID) and micafungin (MICA) are recommended for treatment of invasive Candida infections. As target-site concentrations of antimicrobial agents are crucial for eradication of pathogens, we established and validated high-performance liquid chromatography-UV detection (HPLC-UV) assays for quantification of ANID and MICA in human plasma, ascites fluid, pleural effusion, and in cerebrospinal fluid (CSF). Sample pre-purification was performed by protein precipitation with acetonitrile followed by solid phase extraction. For both assays, intra- and interday precision, and accuracy fulfilled the requirements for bioanalytical methods issued by the European Medicine Agency (EMA). The lower limit of quantification was 0.01 mg/L for both drugs. At 25 °C, ANID and MICA concentrations declined by up to 70% within 24 h. Concentrations remained stable over 24 h at 4 °C and over four weeks at −80 °C. In conclusion, the developed methods are fit for the assessment of target-site pharmacokinetics of ANID and MICA in clinical studies. [ABSTRACT FROM AUTHOR]