High-performance affinity monolith chromatography for chiral separation and determination of enzyme kinetic constants

A new kind of immobilized human serum albumin (HSA) column was developed by using the sub-micron skeletal polymer monolith based on poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) [poly(GMA-EDMA)] as the support of high-performance affinity chromatography. Using the epoxide functional groups presented in GMA, the HSA immobilization procedure was performed by two different means. The affinity columns were successfully adopted for the chiral separation of d,l-amino acids (AAs). Then this method was shown to be applicable to the quantitative analysis of d-tryptophan, with a linear range between 12.0 μM and 979.0 μM, and a correlation coefficient above 0.99. Furthermore, it was used for the analysis of urine sample. This assay is demonstrated to be facile and relatively rapid. So it allows us to measure the enzyme catalytic activity in the incubation of d,l-AAs with d-AA oxidase and to study the kinetics of the enzyme reaction. It implied that the affinity monolithic columns can be a useful tool for studying DAAO enzyme reaction and investigating the potential enzyme mechanism requirement among chiral conversion.     

Affinity monolith chromatography: A review of general principles and recent developments

Affinity monolith chromatography (AMC) is a liquid chromatographic technique that utilizes a monolithic support with a biological ligand or related binding agent to isolate, enrich, or detect a target analyte in a complex matrix. The target-specific interaction exhibited by the binding agents makes AMC attractive for the separation or detection of a wide range of compounds. This article will review the basic principles of AMC and recent developments in this field. The supports used in AMC will be discussed, including organic, inorganic, hybrid, carbohydrate, and cryogel monoliths. Schemes for attaching binding agents to these monoliths will be examined as well, such as covalent immobilization, biospecific adsorption, entrapment, molecular imprinting, and coordination methods. An overview will then be given of binding agents that have recently been used in AMC, along with their applications. These applications will include bioaffinity chromatography, immunoaffinity chromatography, immobilized metal-ion affinity chromatography, and dye-ligand or biomimetic affinity chromatography. The use of AMC in chiral separations and biointeraction studies will also be discussed.     

混合抗体的亲和色谱分离策略

乳腺生物反应器可以高效表达重组人单克隆抗体,但是目标产品与乳液原料中的牛抗体性质、结构非常类似,分离难度很大。本文对牛抗体和重组人抗体的种属差异进行了分析,并在此基础上制定了新型分离策略,采取Protein A亲和色谱和免疫亲和色谱来解决混合抗体的分离问题,并讨论了色谱洗脱模式对分离效果的影响。结果表明,Protein A亲和色谱结合梯度洗脱可以有效地纯化得到混合抗体,但是难以彻底分离重组人抗体和牛抗体;相比之下,使用Protein A亲和色谱结合置换色谱模式可以更加高效地分离混合抗体,最终可以得到纯度高达95%以上的重组人抗体,回收率可达95%以上。免疫亲和色谱同样可以有效地分离纯化重组单克隆抗体,且其通用性更强,可以应用于任何动物乳腺表达重组人抗体的分离纯化中。     

Screening of inhibitors for influenza A virus using high-performance affinity chromatography and combinatorial peptide libraries

The affinity inhibitor of fusion peptide of influenza A virus has been studied using a combination of high-performance affinity chromatography (HPAC) and combinatorial peptide libraries. Fusion peptide (FP) (1-11) of influenza A virus was used as the affinity ligand and immobilized onto the poly(glycidyl methacrylate) (PGMA) beads. Positional scanning peptide libraries based on antisense peptide strategy and extended peptide libraries were designed and synthesized. The screening was carried out at acidic pH (5.5) in order to imitate the environment of virus fusion. A hendecapeptide FHRKKGRGKHK was identified to have a strong affinity to the FP (1-11). The dissociation constant of the complex of the hendecapeptide and the FP (1-11) is 3.10 x 10(-6) mol l(-1) in a physiological buffer condition. The polypeptide has a fairly inhibitory effect on three different strains of influenza A virus H1N1 subtype.     

Microchip extraction of catecholamines using a boronic acid functional affinity monolith

A novel solid phase extraction microchip with a boronic acid functional affinity monolithic disc was developed in this work. Vinyl phenylboronic acid–ethylene glycol dimethacrylate co-polymer monoliths, which have pore sizes up to 20 μm, were investigated for extraction of catecholamines using adsorption and desorption studies in a batch system. Desorption yields of greater than 90% were achieved for catecholamines at pH 3 and below. Monolithic discs were then formed in chambers in borofloat glass microfluidic chips using in situ UV polymerization. Adsorption on the monolithic discs was performed via electrokinetic flow, with catecholamines determined via laser-induced native fluorescence (LINF) detection following electrokinetic elution. Microchips containing the boronic acid functional polymer discs worked well for extraction of catecholamines, providing greater than 100 fold concentration enrichment. This study demonstrated that a solid phase extraction microchip, containing an easily prepared monolith disc, will be useful for boronate affinity extraction of cis-diol containing compounds.     

Synthesis of silica-based benzeneboronic acid affinity materials and application as pre-column in coupled-column high-performance liquid chromatography

Three silica-based benzeneboronic acid affinity materials were synthesized by using an m-aminobenzeneboronic acid as the ligand and using three different spacer arms. Under high-pressure, three affinity pre-columns were packed with these materials and the retention of every affinity pre-column with 11 urinary nucleosides was studied. With different spacer arms of boronic acid-substituted silica materials, the absorption to vicinal alcohols (cis-diols) and stability under acidic elution conditions are of great difference. Coupled-column liquid chromatographic methods for the direct analysis of urinary nucleosides were respectively established. As a result, two of three affinity pre-columns showed good chromatographic property in the on-line analysis of urinary nucleosides. The coupled-column system including pre-column I is the best with excellent linearity (R² > 0.995), good recoveries (85.6–96.9%) and reproducibility (R.S.D.: 1.01–4.02%). The pre-column I could at least endure 150 repetitive injections of a 100 μL urinary sample.     

Characterization of metal chelate methacrylate monolithic disk for purification of polyclonal and monoclonal immunoglobulin G

Dynamic binding capacity (DBC) of commercial metal-chelate methacrylate monolith-convective interaction media (CIM) was performed with commercial human immunoglobulin G (IgG) (Cohn fraction II, III). Monoliths are an attractive stationary phase for purification of large biomolecules because they exhibit very low back pressure even at high flow rates and flow-unaffected binding properties. Adsorption of IgG onto CIM-IDA disk immobilized with Cu²⁺, Ni²⁺ and Zn²⁺ were studied with Tris-acetate (TA), phosphate-acetate (PA) and MMA (MES, MOPS and acetate) buffer systems at different flow rates. Adsorption and elution of IgG varied with different buffers and adsorption of IgG was maximum with MMA buffer. Adsorption of human IgG from Cohn fractions (II, III) was high when Cu²⁺ was used as ligand. CIM-IDA disk showed dynamic binding capacity in the range of 14–16 mg/ml with Cu²⁺ and 7–9 mg/ml with Ni²⁺ for human IgG with MMA buffer. In the case of CIM-IDA-Zn²⁺ column, the binding capacity was only about 0.5 mg/ml of support. Different desorption strategies like lowering of pH and increasing of competitive agent were also studied to achieve maximum recovery. Chromatographic runs with human serum and mouse ascites fluid were also carried out with metal chelate methacrylate monolithic disk and the results indicate the potential of this technique for polyclonal human IgG and monoclonal IgG purification from complex biological samples.     

Application of conjoint liquid chromatography with monolithic disks for the simultaneous determination of immunoglobulin G and other proteins present in a cell culture medium

The aim of this study was to develop a chromatographic method, as a substitute for enzyme-linked immunosorbent assays, for the rapid and simultaneous detection of IgG, insulin, and transferrin present in a cell culture medium. Conjoint liquid chromatography (conjoint LC) using monolithic disks was applied for this purpose. An anion-exchange disk was combined with a Protein G affinity disk in a preparative HPLC system. IgG bound to the Protein G disk, whereas transferrin and insulin were captured on the quaternary ammonium (QA) disk. Using this method, it was possible to simultaneously determine the concentrations of IgG, transferrin, and insulin in the cell culture medium. Thus, conjoint LC could be used for the rapid and simultaneous detection of different proteins present in a cell culture medium.     

Anion exchange silica monolith for capillary liquid chromatography

An anion exchange monolithic silica capillary column was prepared by surface modification of a hybrid monolithic silica capillary column prepared from a mixture of tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMS). The surface modification was carried out by on-column copolymerization of N-[3-(dimethylamino)propyl]acrylamide methyl chloride-quaternary salt (DMAPAA-Q) with 3-methacryloxypropyl moieties bonded as an anchor to the silica surface to form a strong anion exchange stationary phase. The columns were examined for their performance in liquid chromatography (LC) and capillary electrochromatography (CEC) separations of common anions. The ions were separated using 50 mM phosphate buffer at pH 6.6. Evaluation by LC produced an average of 30,000 theoretical plates (33 cm column length) for the inorganic anions and nucleotides. Evaluation by CEC, using the same buffer, produced enhanced chromatographic performance of up to ca. 90,000 theoretical plates and a theoretical plate height of ca. 4 μm. Although reduced efficiency was observed for inorganic anions that were retained a long time, the results of this study highlight the potential utility of the DMAPAA-Q stationary phase for anion separations. Figure Micro-LC performance evaluation of a strong anion exchange silica monolith column, 100H-MOP-DMAPAA-Q, 33 cm in length, with a mobile phase of 50 mM phosphate buffer, pH 2.8; linear velocity: u = 1.8 mm/s; UV-Vis detection at 254 nm. Sample solution (5 mg/mL of each component, 4 mL) was injected in split flow injection mode at a split ratio of ca. 1:1900 with a pump flow rate of 1.5 mL/min     

基于巯基-烯点击反应制备有机-无机杂化硼酸亲和整体柱用于糖蛋白的选择性富集

发展了以巯基-烯点击反应制备有机-无机杂化硼酸亲和整体柱的新方法。首先以四甲氧基硅烷(TMOS)和巯丙基三甲氧基硅烷(MPTMS)作为反应单体,采用溶胶-凝胶反应制备表面含巯基的硅胶整体柱。然后利用巯基-烯(thiol-ene)的点击反应在整体柱上修饰硼酸配基3-丙烯酰胺基苯硼酸(AAPBA),制成AAPBA-硅胶杂化亲和整体柱。对影响硼酸亲和整体柱性能的条件如TMOS与MPTMS的比例、聚乙二醇和甲醇的用量等进行了优化。并采用扫描电镜、红外光谱等分析仪器对整体柱形貌和机械稳定性能进行了表征。研究了AAPBA-硅胶杂化亲和整体柱的分离性能,结果表明,其在中性条件下对含有顺式二醇的生物小分子核苷具有良好的特异亲和能力,并已成功地应用于卵清蛋白、辣根过氧化物酶等糖蛋白的分离。基于巯基-烯反应的制备方法新颖、可靠,可用于制备多种不同类型的硼酸亲和整体柱,具有较大的应用前景。     

Monitoring of mouse immunoglobulin G by flow-injection analytical affinity chromatography

A flow-injection analytical affinity chromatographic (FIAAC) system was developed for the on-line monitoring of mouse immunoglobulin G (IgG). Protein A or anti-mouse IgG antibodies immobilized on oxirane beads were filled in a miniature column. The IgG-containing samples and standards were passed through the column and detected fluorimetrically after elution with citrate buffer (pH 3 or 2.5). The on-line monitoring of mouse IgG 2a during a 7-day cultivation of hybridoma cells in a perfusion reactor by FIAAC is presented. A chemical barrier was used to prevent contamination of the reactor from the FIAAC.     

蛋白质荧光标记中协同效应的高效液相色谱

以牛血清白蛋白和胰岛素为研究对象, 经1,5-I-AEDANS衍生后, 采用高效液相色谱(HPLC)进行分离分析, 发现两者的衍生存在明显的协同作用.     

High-performance liquid chromatography and pharmacokinetics of aceclofenac in rats

A simple and sensitive high-performance liquid chromatographic (HPLC) method was developed for quantification of aceclofenac in rat plasma. Ibuprofen was used as an internal standard (IS). The present method used protein precipitation for extraction of aceclofenac from rat plasma. Separation was carried out on reversed-phase C₁₈ column (250 mm × 4.6 mm, 5 μ) and the column effluent was monitored by UV detector at 282 nm. The mobile phase used was methanol-triethylamine (pH 7.0; 0.3% v/v in Milli-Q water) (60:40%, v/v) at a flow rate of 1.0 mL min⁻¹. This method was linear over the range of 50.0-3500.0 ng mL⁻¹ with regression coefficient greater than 0.99. The mean recovery of aceclofenac and IS were 84.62 ± 3.23 and 89.19 ± 1.57%, respectively and the method was found to be precise, accurate, and specific during the study. The method was successfully applied for pharmacokinetic study of aceclofenac in rats.     

高效液相色谱-荧光检测法分析麦类中麦角克列斯汀碱

提出了一种采用高效液相色谱-荧光检测法(HPLC-FLD)测定麦类样品中麦角克列斯汀碱的方法。麦类样品经V(乙腈)∶V(0.1 mol/L乙酸铵缓冲溶液)=1∶4提取,以C18小柱净化,C18色谱柱(4.6×250 mm,5μm)分离,V(水)∶V(乙腈)=3∶2作流动相,流速1.0 mL/min,以HPLC-FLD定量测定。标准工作溶液浓度在1.0~50.0μg/L范围内,与峰面积成良好的线性关系,线性相关系数0.9999,样品在10.0、50.0、250.0μg/kg添加水平的回收率为76%~85%,相对标准偏差(RSD)为6.6%~8.8%(n=8),方法检测限为5.0μg/kg(S/N10)。     

Performance of hexamer peptide ligands for affinity purification of immunoglobulin G from commercial cell culture media

Previous work has reported on the identification and characterization of the hexapeptide ligands HWRGWV, HYFKFD, and HFRRHL for the affinity capture of IgG through specific binding to its Fc fragment. This paper addresses issues related to the successful application of these ligands, on a commercial methacrylate chromatographic resin, for the purification of IgG from mammalian cell culture fluids. The concentrations of sodium chloride and sodium caprylate in the binding buffer were optimized to maximize the purity and yield of IgG upon elution. Screening of several regeneration conditions found that either 2M guanidine-HCl or a combination of 0.85% phosphoric acid followed by 2M urea resulted in complete recovery of the IgG adsorption capacity and that the column could be reused over many cycles. The hexapeptide ligands were used for the purification of humanized and chimeric monoclonal antibodies from two commercial CHO cell culture fluids. The chimeric MAb of IgG1 subclass was purified using the HWRGWV resin whereas the humanized MAb of IgG4 subclass was purified using the HWRGWV, HYFKFD and HFRRHL resins. The purities and yields obtained for both the MAbs were found to be higher than 94% and 85% respectively. These results compare well with the yields and purities obtained using Protein G columns. The residual DNA and host cell protein reduction obtained by the HWRGWV resin was in the range of 4 log reduction value (LRV) and 2 LRV respectively, comparable to those reported for Protein A resins. The dynamic binding capacity of all three peptide resins for the humanized monoclonal antibody was in the range of 20mg/mL.     

Understanding ligand–protein interactions in affinity membrane chromatography for antibody purification

Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand–ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.     

Rapid “one-pot” preparation of polymeric monolith via photo-initiated thiol-acrylate polymerization for capillary liquid chromatography

A facile approach was exploited for fast preparation of polymer-based monoliths in UV-transparent fused-silica capillaries via “one-pot” photo-initiated thiol-acrylate polymerization reaction of dipentaerythritolpenta-/hexaacrylate (DPEPA) and 1-octadecanethiol (ODT) in the presence of porogenic solvents (1-butanol and ethylene glycol). Due to relative insensitivity of oxygen inhibition in thiol-ene free-radical polymerization, the polymerization could be performed within 5 min. The effects of composition of prepolymerization solution on the morphology and permeability of poly(ODT-co-DPEPA) monoliths were investigated in detail by adjusting the content of monomer and binary porogen ratio. The physical properties of poly(ODT-co-DPEPA) monoliths were characterized by Fourier transform infrared spectroscopy (FT-IR), mercury intrusion porosimetry (MIP) and nitrogen adsorption/desorption measurement. The evaluation of chromatographic performance was carried out by capillary liquid chromatography (cLC). The results indicated that the poly(ODT-co-DPEPA) monolith was homogeneous and permeable, and also possessed a typical reversed-phase retention mechanism in cLC with high efficiency (∼75,000 N m⁻¹) for separation of alkylbenzenes. Eventually, the further separation of tryptic digest of proteins by cLC tandem mass spectrometry (cLC-MS/MS) demonstrated its potential in the analysis of biological samples.     

磺胺甲基异噁唑作配基的高效亲和色谱分离胰凝乳蛋白酶和胰蛋白酶

用磺胺甲基异噁唑作配基、大孔硅胶为基质的高效亲和色谱分离纯化胰凝乳蛋白酶及胰蛋白酶。20多种蛋白质和酶在该柱上无特异性吸附。磺胺甲基异噁唑在硅胶上的键合量为4．3mg／g；该亲和填料对胰凝乳蛋白酶吸附量为9．6mg／g；两者之间的亲和解离常数K1／M为1×103mol／L。