Continuous purification of a clotting factor IX concentrate and continuous regeneration by preparative annular chromatography

Preparative continuous annular chromatography, a method to separate proteins in a truly continuous manner, was investigated in an industrial environment. Plasma-derived clotting factor IX concentrate was used as model protein. Separation of vitronectin, a common impurity in commercial available factor IX concentrates, from factor IX was studied and compared to conventional packed bed chromatography in batch mode. As sorbent, Toyopearl DEAE 650M was used. Regeneration was performed simultaneously with the purification of factor IX in continuous mode. All required parameters applied for preparative annular chromatography such as feed flow-rate and elution flow-rate were first estimated from experiments on conventional batch columns. Then preparative annular chromatography and conventional packed beds were compared regarding enrichment, purity and productivity. Three different process scenarios, the optimal batch process,the preparative annular chromatography process and the batch process equivalent to the preparative annular chromatography process were investigated. The productivity of the optimal batch process was higher than that of the preparative annular chromatography and batch process equivalent to the preparative annular chromatography process. Therefore the throughput could not be increased by the use of the continuous chromatographic system.     

溶质在气相色谱中进样量和保留值关系的方程

考虑到气相中溶质分子和其它成分在固定相上的竞争吸附作用,提出了一个描述溶质在气相色谱中进样量和保留值的关系式.由此方程可以获得两个描述色谱体系特征的重要参数:溶质和其它成分在固定相表面竞争吸附的热力学平衡常数Ka和单位体积固定相所能吸附溶质的量NmS.当其它参数给定时,Ka的大小直接决定溶质进样量与保留值关系式的性质.通过试验对此方程进行了初步验证.     

莠去津吸附的土壤淋溶柱色谱法研究

采用新发展的土壤淋溶柱色谱法,研究农药在水－土壤体系中的吸附。用土壤装填成液相色谱柱,以农药水溶液为流动相,以除草剂莠去津（２－氯－４－乙胺基－６－异丙胺基－ｓ－三嗪）为例,质量浓度从１９．７３μｇ／Ｌ至２９．６０ｍｇ／Ｌ,紫外检测器监测吸附平衡。吸附相中莠去津的质量浓度经甲醇淋洗液的测定值扣除柱内液相中的质量浓度后得到。对于极稀溶液,可采用在ＯＤＳ柱上富集－液相色谱台阶梯度法测定。莠去津在沙壤土上的吸附行为符合Ｆｒｅｕｎｄｌｉｃｈ方程式,其吸附常数Ｋｆ＝８４２．６ｍＬ／ｋｇ。     

Study of adsorption from solutions by frontal chromatography

Summary The automatic frontal chromatography installation was described. By this chromatographic method the adsorption isotherm of benzene from n-heptane solutions on hydroxylated surface of silica with various porosity has been determined. This investigation was performed at different flow rates of eluent and in a wide range of concentrations. The isotherm of adsorption obtained by this chromatographic method has been compared with the results of the static measurements. The coincidence of adsorption isotherms measured at the various flow rates are shown to be a criterion of proximity the chromatographic process to the equilibrium.     

Coating and fusing cell membranes onto a silica surface and their chromatographic characteristics

Summary A new cell membrane stationary phase (CMSP) consisting of porous silica coated with active cell membranes is presented for affinity chromatography. By immersing, silica into a suspension of cell membranes, the whole surface of silica was covered by the cell membranes due to the irreversible adsorption of silanol groups (Si−OH) on the silica surface and the self-fusion of the cell membranes. CMSP can be used directly as a chromatographic packing material without any additional chemical modification. The surface characteristics, enzymatic activity, and chromatographic behavior of CMSP were investigated. The results obtained from scanning electron microscope, surface energy spectrometer, enzyme assay, and liquid chromatography showed that the surface characteristics of CMSP were very different from that of normal and reversed stationary phases. CMSP was found to have the characteristics of both cell membrane activity and chromatographic separation. Moreover, CMSP, as a chiral stationary phase, could be used for the enantiomeric separation of (±) Bay-K8644. The capacity factor of some calcium antagonists on CMSP was found to have a good correlation with their pharmacological actions. It is concluded that CMSP may be used not only as a kind of packing material in bio-affinity chromatography, but also as a tool for studying the interactions between a drug and its receptor.     

Cation-exchange chromatographic isolation of the methionine-bound sulfur in casein hydrolysates for stable-isotope-ratio analysis

Summary A chromatographic method is described for the isolation of the methionine-bound sulfur in casein for stable-isotope-ratio analysis. Casein is cleaved by acid hydrolysis and the high-molecular-weight secondary products formed are removed by adsorption on octadecyl-silica. Methionine is separated from the bulk of the polar amino acids by cation-exchange chromatography and subsequently oxidized to methionine sulfone by performic and peracetic acid. In a further step the methionine is isolated from the accompanying nonpolar amino acids by cation-exchange chromatography with a mobile phase of very low eluent strength.     

Combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the direct extraction and purification of pseudohypericin and hypericin from St. John's wort (Hypericum perforatum L.)

St. John's wort has attracted particular attention because of its beneficial effects as an antidepressant, antiviral, and anticancer agent. A method for the combination of integrated expanded bed adsorption chromatography and countercurrent chromatography for the simultaneous extraction and purification of pseudohypericin and hypericin from the herb is presented in this paper. Firstly, the constituents were extracted and directly adsorbed by expanded bed adsorption chromatography under optimal conditions. The stepwise elution was then performed by expanded bed adsorption chromatography that enriched the targets with higher purities and recoveries compared to other methods. Secondly, the eluent fractions from expanded bed adsorption chromatography were further separated by two‐step high‐speed countercurrent chromatography. A two‐step high‐speed countercurrent chromatography method with a biphasic solvent system composed of n‐hexane/ethyl acetate/methanol/water with a volume ratio of 1:2:1:2 was performed by stepwise changing the flow rate of the mobile phase. Consequently, 5.6 mg of pseudohypericin and 2.2 mg of hypericin with purities of 95.5 and 95.0%, respectively, were successfully obtained from 40 mg of crude sample.     

Adsorption Effects Observed in Gel Permeation Chromatography of Thiouracil

Abstract

In gel permeation chromatography (GPC), several compounds deviate from the molecular volume/elution count relationship which is prepared using satured hydrocarbons. In this paper, this problem is investigated in detail using thiouracil in aqueous solution as a model chromatographic adsorbate. The concentration dependences of elution counts and peak heights prove the adsorption of thiouracil on Sephadex G-25 when water is the solvent. Thus to investigate further the mechanisms of adsorption responsable for the chromatographic behaviour, thiouracil-Sephadex interac—tions were investigated by studying equilibrium adsorption. Isotherms of type IV of BDDT classification were found which are typically associated with a weak adsorption such as physisorption, on a porous solid. The effect of water structure perturbants, ionic strength and pH on this adsorption was consistent with the-hypothesis that with water as a solvent both aromatic adsorption and electrostatic interaction are the determinants of the affinity of this gel for a thiouracil compound. This may be particularly useful since results of equilibrium adsorption isotherms are frequently used to develop liquid chromatographic theories.     

Determination of triton X-100 in plasma-derived coagulation factor VIII and factor IX products by reversed-phase high-performance liquid chromatography

Plasma protein pools are often virus-inactivated by the solvent-detergent method, using tri-n-butyl phosphate and Triton X-100, followed by removal and determination of these compounds. We used reversed-phase high-performance liquid chromatography for the determination of Triton X-100 in coagulation factor VIII and factor IX products, Octonativ-M and Nanotiv, respectively (Pharmacia, Stockholm, Sweden). The chromatographic system included a C18 silica column and a linear acetonitrile gradient. The advantage of this method is the low detection limit (0.3 microg/ml) combined with detection at 280 nm, which gives a more stable baseline and has less interference from other compounds. As compared to other methods, where shorter wavelengths are used.     

Automated chromatography of protected oligonucleotides with high separation efficiency

Summary During the total chemical synthesis of a structural gene for the human peptide hormone angiotensin II [1] some difficult problems in ion-exchange chromatography of protected oligodeoxyribonucleotides were solved using an adsorption and a chromatographic column in combination with an automatic gradient generator and gradienttermination controller. This arrangement allows a well-timed gradient termination and therefore the separation of substances with only a small difference in charge. On the other hand the automatic run of the chromatography of protected oligonucleotides reduces the time factor in oligonucleotide chemistry. This technique is applicable for ion-exchange chromatography in general.     

Application of monoliths for downstream processing of clotting factor IX

In this paper, the application of monolithic columns for downstream processing of different clotting factor IX concentrates is shown. Determination of basic chromatographic conditions as well as investigations on the regeneration of disk- and tube-shaped monolithic columns using human serum albumin as a model protein, were performed. Separation of factor IX and vitronectin, a possible impurity in commercial factor IX concentrates was accomplished using disk-shaped monolithic columns. These same applications were also carried out with identical results on up-scaled tube-shaped monolithic columns. Since these media allow very fast separations, this method can be successfully applied not only to an in-process control of the purification of factor IX but also to other biopolymers from human plasma. Besides, the same application on the up-scaled tube-shaped monolithic column was successfully carried out.     

HPLC study of the adsorption from solutions on two silicagels

Summary In this paper, we report and discuss the adsorption isotherms of hydro-organic mixtures and of compounds used as mobile phase in reversed phase and normal phase high performance liquid chromatography (RP-HPLC and NP-HPLC, respectively). This work is the first attempt to study the solid-liquid interface between two types of chromatographic silica surfaces and pure organic adsorbates from water and from organic eluents by HPLC. Frequently, among the dynamic techniques the method of choice for the measurement of an adsorption isotherm is frontal analysis. We suggest here the combination of the technique based on peak asymmetry calibration and peak profiles, which allows calculations directly from integration data. The group of systems studied permits the analysis of the intermolecular interactions on the silica surface. Particular attention was given to the system methanol-water and the measurement of the adsorption ofn-octanol from methanol on RP-silica was also carried out.     

Isolation of a peptide ligand for affinity purification of factor VIII using phage display

Polypeptides for use in affinity chromatography of factor VIII were identified using phage display technology. Phage libraries were designed to express polypeptide fusions containing five to seven residues flanked by two cysteines that form a disulfide bond. Individual bacteriophage were selected for the ability of these polypeptides to bind factor VIII, and then release the protein under mild elution conditions. Strong consensus sequences were observed that appear to be necessary for this reversible interaction. Chemically synthesized ligands identified by this screening were immobilized onto a chromatographic support and used for affinity purification of factor VIII from a complex feedstream. A chromatographic step was developed that provided a 10000-fold reduction in host cell proteins and DNA, while providing exceptional product recovery.     

Limited sample recovery in coupled methods of high-performance liquid chromatography of synthetic polymers

Complex polymer systems, which exhibit multiple distributions in their molecular parameters can be characterized by coupled liquid chromatographic methods. The latter combine entropic (exclusion) and enthalpic (interaction) retention mechanisms. However, recent experimental results suggest that some coupled liquid chromatographic methods may suffer from incomplete sample recovery. This refers, for example, to liquid chromatography under critical conditions of enthalpic interactions and to eluent gradient liquid chromatography. Sample recovery in both latter methods was investigated for selected model systems applying adsorption retention mechanism. Reduced sample recovery was confirmed for both methods. It was revealed that even very high final strength of mobile phase may be insufficient for complete elution of polymer samples in eluent gradient polymer liquid chromatography.     

Thermal desorption cold trap-injection in high-resolution gas chromatography: multivariate optimization of experimental conditions.

In studies of low concentrations of volatile compounds in air, the method of adsorption on porous polymers and determination by thermal desorption cold trap-injection high-resolution gas chromatography is finding increasing application. Factors considered important for injection and chromatographic separation of volatile compounds by this method were investigated with the use of multivariate techniques. For the amount injected on to the chromatographic column, the factors of main importance were found to be the temperature of the injection block, the thickness of the internal coating of the cold trap and the flow-rate. Strong interaction effects were noted. For the sharpness of the chromatographic peaks, the flow-rate was the most important factor.     

Calcium-modulated conformational affinity chromatography. Application to the purification of calmodulin and S100 proteins.

The purification of proteins by affinity chromatography is based on their highly specific interaction with an immobilized ligand followed by elution under conditions where their affinity towards the ligand is markedly reduced. Thus, a high-degree purification by a single chromatographic step is achieved. However, when several proteins in the crude mixture share affinity to a common immobilized ligand, they may not be resolved by affinity chromatography and subsequent "real" chromatographic purification steps may be required. It is shown that by using properly selected gradient elution conditions, the affinities of the various proteins towards the immobilized ligand may be gradually modulated and their separation may be achieved. This is exemplified by the isolation and separation of a group of Ca(2+)-activated proteins, Calmodulin, S100a and S100b, from bovine brain extract, using a melittin-Eupergit C affinity column which is developed with Ca(2+)-chelator gradients. As expected, separation of the three proteins into individual peaks, eluted in order of increasing affinity to the matrix, was obtained. Sigmoid selectivity curves calculated from the elution volumes under different elution conditions for each of the proteins were obtained, illustrating the chromatographic behaviour of the gradient affinity separation system.     

Novel method of purification of human leukocytic elastase using adsorption on a high-performance liquid chromatography gel permeation column

Neutrophil elastases are serine proteinases released during acute and chronic inflammatory states. We have developed a novel isolation method for neutrophil elastase, involving conventional gel chromatography followed by adsorption of protein at low ionic strength on a high-performance liquid chromatography gel permeation column. The bound elastase is then eluted by application of higher ionic strength. This adsorption step at low ionic strength, a step to be avoided in most purification methods, was used to advantage here to allow isolation of homogeneous material. This purification procedure should be useful for quick, simple bulk preparation of the enzyme.     

Pore size distributions of ion exchangers and relation to protein binding capacity

The pore structure of chromatographic media directly influences macromolecular transport and adsorption, and consequently separation resolution and loading capacity in chromatographic separations. The pore size distribution (PSD) is therefore a central structural characteristic of chromatographic materials and a critical determinant of chromatographic behavior. In this work the PSDs of a set of commercial anion exchangers were determined by inverse size-exclusion chromatography (ISEC). The PSDs were further utilized to develop relations to functional properties of adsorbents, such as intraparticle diffusivity, and static and dynamic binding capacities. We find that the detailed PSD is useful in semi-quantitative understanding of chromatographic behavior. However, more accurate prediction of column behavior requires more thorough knowledge of the pore structure, specifically the connectivity of the pore network, as well as improved understanding of the function of grafted resins.     

Purification and Preparation of Moenomycin A from Fermentation Broth by Multidimensional Chromatography

Based on various adsorption characteristics of resins, a novel method for purifying and preparing moenomycin A from fermentation broth was established by combining different chromatographic modes into a three-step preparative chromatography process. Fermentation broth of moenomycins was firstly prepurified by macroporous adsorbent XAD7HP to remove most strong polar impurities, then further purified by anion exchange resin FPA98Cl, and finally refined moenomycin A was obtained by use of semi-preparative reversed-phase chromatographic column packed with Chromtorex C8 silica gel. As the main indicators, purity and yield of moenomycin A were examined in order to optimise the chromatographic process for each step. Under optimized chromatographic conditions, the purity and total yield of moenomycin A were 95.0 and 22.2 %, and the biological potency of moenomycin A was 2232 U mg⁻¹, significantly higher than 1395 U mg⁻¹, which is the potency of the standard from Agriculture Ministry of China. Three-step preparative chromatographic mode could gradually and effectively remove impurities. The present method is practical, easy to be operated with less solvent consumption, and provides a new idea for the preparation of moenomycin A with high purity.