琼脂糖-DEAE离子交换介质的配基密度和孔径对BSA吸附的影响

离子交换色谱是蛋白质分离纯化的有效方法之一,配基密度和介质孔径是影响蛋白质吸附的关键因素。采用3种不同琼脂糖浓度的凝胶为基质,具有不同的平均孔径,分别偶联上阴离子交换配基DEAE,通过调控反应条件,包括反应温度、反应时间、碱浓度和DEAE浓度,得到了不同配基密度和介质孔径的系列DEAE离子交换介质。考察了牛血清白蛋白（BSA）的静态和动态吸附性能,发现随配基密度增加或介质孔径减小,BSA饱和吸附容量有所增大;对于吸附动力学,介质孔径显著影响有效扩散系数。结果表明,配基密度和介质孔径共同决定了蛋白质的吸附性能,介质孔径主导蛋白质的孔内扩散,而配基密度则影响配基-蛋白质间的相互作用。     

基于微孔过滤板的蛋白吸附高通量筛选方法

针对色谱分离过程优化,建立了基于微孔过滤板的蛋白吸附高通量筛选方法,用于介质初筛、吸附性能考察、吸附等温线和吸附动力学测定、吸附和洗脱条件优化等。首先优化了96孔过滤板的操作参数,以2种离子交换介质和2种混合模式介质为典型代表,采用微孔过滤板方法考察了不同介质和液相条件下牛血清白蛋白的吸附,得到结合载量分布图,确定了合适的蛋白吸附和解吸条件。进一步测定了4种介质在特定吸附条件下的吸附等温线和吸附动力学曲线,获得吸附相关参数。最后,采用微孔过滤板进行了洗脱条件优化,并与填充柱色谱分离进行比较,验证了方法的可靠性。结果表明,基于微孔过滤板的蛋白吸附高通量筛选是切实可行的,可以快速筛选色谱介质和液相,优化蛋白分离条件,具有资源消耗小、实验通量大、研发周期短、适用性广、稳定性高的特点,是蛋白色谱分离过程优化的一种新方法。     

离子交换纤维对低浓度二甲胺水溶液吸附性能研究

对离子交换纤维(IEF)吸附二甲胺(DMA)的特性进行了研究。等温吸附线研究表明,离子交换纤维对二甲胺的吸附符合Freundlich和Langmuir等温吸附方程。热力学研究表明,Freundlich模型可以很好地模拟二甲胺在离子交换纤维上的交换行为,交换反应的ΔG0,ΔH0,ΔS0,交换过程为自发的吸热过程。动力学研究表明,离子交换纤维对二甲胺的吸附以液膜扩散为主,其交换行为可采用Boyd液膜扩散方程描述。     

基于微孔过滤板的蛋白吸附高通量筛选方法

针对色谱分离过程优化,建立了基于微孔过滤板的蛋白吸附高通量筛选方法,用于介质初筛、吸附性能考察、吸附等温线和吸附动力学测定、吸附和洗脱条件优化等。首先优化了96孔过滤板的操作参数,以2种离子交换介质和2种混合模式介质为典型代表,采用微孔过滤板方法考察了不同介质和液相条件下牛血清白蛋白的吸附,得到结合载量分布图,确定了合适的蛋白吸附和解吸条件。进一步测定了4种介质在特定吸附条件下的吸附等温线和吸附动力学曲线,获得吸附相关参数。最后,采用微孔过滤板进行了洗脱条件优化,并与填充柱色谱分离进行比较,验证了方法的可靠性。结果表明,基于微孔过滤板的蛋白吸附高通量筛选是切实可行的,可以快速筛选色谱介质和液相,优化蛋白分离条件,具有资源消耗小、实验通量大、研发周期短、适用性广、稳定性高的特点,是蛋白色谱分离过程优化的一种新方法。     

聚锑酸离子交换剂对锶的吸附行为研究

研究了聚锑酸离子交换剂吸附0.01 mol/L HNO_3溶液中锶的热力学特征,通过吸附动力学实验,初步探讨了温度对吸附过程的影响.结果表明,聚锑酸对0.01mol/L HNO_3溶液中锶的吸附符合Freundlich 经验公式,表现为吸热的化学吸附过程.根据毛细孔扩散模型,由平衡数据和动力学曲线计算了锶在聚锑酸离子交换剂颗粒内液相有效扩散系数为8.18×10~(-9)cm~2/s(20℃)和1.33×10~(-8)cm~2/s(30℃).     

交变电场下离子交换色谱分离过程

以牛血清白蛋白在阴离子交换剂DEAE -SepharoseFastFlow上的吸附和解吸过程为例 ,考察了交变电场下离子交换色谱过程的吸附和洗脱过程及其传质特性 .结果表明 :在本文实验条件下 ,交变电场电流强度的变化对离子交换吸附平衡无显著影响 ,而外加电场在介质孔内所产生的电渗流可加速待分离组分进出固相介质的传递过程 ,显著提高色谱填充床的动态吸附容量 ,改善洗脱峰的特性     

共聚物接枝蛋白离子交换色谱介质制备及性能

高容量蛋白质色谱介质是色谱过程高效化的材料基础和重要前提。采用原子转移自由基聚合(ATRP)技术,以甲基丙烯酸3-磺酸丙酯钾和甲基丙烯酸甲酯(MMA)为单体化合物合成了多种无规共聚物接枝离子交换色谱介质,并对其蛋白质吸附性能进行研究。单体总浓度一定情况下共聚物接枝色谱介质孔道半径(r_(pore))随MMA浓度升高而增大,反映出接枝共聚物链渐趋塌陷的特征。蛋白质吸附结果表明,溶菌酶吸附容量取决于介质的离子交换容量;而抗体吸附容量则与r_(pore)及相应的聚合物层厚度变化密切相关。随着聚合物层厚度的增大,聚合物层对抗体的空间排阻作用增强,抗体吸附容量下降。此外,引入MMA优化共聚物分子链可显著提高蛋白质吸附量,在SEP-g S30/M30介质中抗体和溶菌酶的饱和吸附量分别达到237 mg·g~(-1)和380 mg·g~(-1)。无规共聚物接枝离子交换色谱介质孔道内聚合物层厚度和蛋白质吸附也受无机盐浓度调控。     

阴离子交换剂DEAE-Spherodex M的蛋白质吸附性能

通过间歇吸附实验,采用Langmuir等温方程和改进的平行扩散模型,研究了牛血清白蛋白和γ 球蛋白在阴离子交换剂DEAE SpherodexM中的静态和动态吸附行为,分析了吸附过程的热力学参数。研究发现,阴离子交换剂的吸附平衡是吸热反应,对于较宽分子尺寸范围的蛋白质,DEAE SpherodexM均具有较高的离子交换吸附速率。研究表明,DEAE SpherodexM具有较好的机械强度、化学和热稳定性、稳定的功能基、较高的交换容量和较快的离子交换速度,是一种较好的蛋白质分离介质。     

钩藤碱在离子交换纤维上的吸附动力学研究

采用静态吸附法考察钩藤碱在离子交换纤维上的吸附行为,并研究离子交换纤维对钩藤碱的吸附动力学。结果表明,钩藤碱在离子交换纤维上的吸附过程更好的符合Ho and McKay二级动力学方程,在吸附液初始浓度为100 mg/L,吸附温度323 K的条件下,二级动力学方程为t/q_t=0.001 2 t+0.006 5(R²=0.998);表观吸附活化能E_a为7.003 kJ/mol;速度控制步骤为液膜扩散控制,在吸附液初始浓度为60 mg/L,吸附温度303 K条件下,液膜扩散模型方程为-ln(1-q_t/q_e)=0.119 3 t(R²=0.998)。阳离子交换纤维可以吸附钩藤碱,并且具有良好的吸附性能。     

吸附密度对蛋白质在离子交换吸附剂中孔扩散系数的影响

通过间歇吸附动力学实验,采用孔扩散模型研究了牛血清白蛋白和γ-球蛋白在阴离子交换剂中的扩散行为,考察了蛋白质初浓度和平均吸附密度对孔扩散系数的影响.结果表明,随蛋白质初浓度和平均吸附密度增大,孔扩散系数均呈指数下降,且蛋白质分子尺寸越大下降趋势越明显.     

基于氨基酸的新型树枝状色谱置换剂的合成及性能

高效置换剂的设计与合成一直是置换色谱发展的重要方向。采用固相合成方法合成了新型树枝状聚电解质置换剂Phe-D和Leu-D,并与置换剂BAEE进行了对比研究。研究结果显示,置换剂的动态亲和性随着lgΔ值增大而降低。在lgΔ<0.845时,BAEE具有最高的动态亲和力;而随着lgΔ进一步升高,Phe-D展现出最强的动态亲和力,这种趋势一直维持至lgΔ值达到3.08。蛋白质的静态置换实验表明,新型置换剂对蛋白质具有更高的置换百分率。这种高置换率主要源于置换剂中伯胺基团数量的增加;同时,Phe-D分子中苯基的引入使模型蛋白的置换百分率提高了70%,展示了苯基对蛋白质置换的促进作用。     

Protein adsorption dynamics in cation-exchange chromatography quantitatively studied by confocal laser scanning microscopy

A self-contained research system based on the technique of confocal laser scanning microscopy (CLSM) was put up to quantitatively analyze the dynamics of protein adsorption to porous cation exchanger by mathematical modeling. Bovine serum albumin adsorption to the cation exchanger SP Sepharose FF was performed by batch adsorption and micro-flow cell in which protein concentration in single absorbent was visualized by CLSM. The effects of ionic strength and the protein concentration in liquid phase (50 mmol/L acetate buffer, pH 5.0) on the adsorption dynamics were examined. The intraparticle concentration profile data experimentally obtained from CLSM were quantitatively analyzed by three diffusive mass transfer models (i.e., pore diffusion, surface diffusion and Maxwell-Stefan models (MSM)) in virtue of the attenuation equation for the CLSM visualization developed earlier. The nuance between the model simulations and experimental results of the developing protein distribution in a single adsorbent particle could thus be found out. Without salt addition to the buffer, the adsorption isotherm was strongly favorable, and the pore diffusion model (PorDM) and MSM gave similarly good simulations of the experiments, whereas the surface diffusion model was unreasonable in the model presumption. Moreover, it was observed that the experimentally obtained adsorption front was relative flatter as compared with the calculated results from the PorDM, which implied the possible existence of surface diffusion. With increasing salt concentration, the simulations became to deviate from the experiments. Especially, when the salt concentration approached 50 mmol/L, all the three mass transfer models could hardly give good simulation of the experiment. This was considered due to the difference in adsorption behavior between the fluorescence labeled and unlabeled proteins therein.     

快速纯化在大肠杆菌中表达的增强型绿色荧光蛋白

As an excellent reporter molecule, enhanced green fluorescent protein （eGFP） was widely used for gene expression and regulation and was generally expressed in Escherichia coli strain. A rapid procedure consisting of ammonium sulfate precipitation, size exclusion chromatography, and anion exchange chromatography was developed for the purification of eGFP. Based on the proposed procedure, recombinant eGFP with an electrophoretic purity was achieved in combination with an overall yield of 66% and a purification factor of 17.9. The fluorescent spectrometry of purified eGFP and lysate from E. coli strain expressing eGFP exhibited the same wavelength of excitation and emission maxima, indicating that the purification procedure did not influence the construct and fluorescent characteristics of desired protein. The procedure mentioned was easy to scale up for the purification of large quantities of eGFP.     

制备型电动亲和色谱吸附过程传质动力学

建立了描述电动亲和色谱分离过程吸附操作的传质方程 ,研究了人血清白蛋白 (HSA)和BlueSepharoseFastFlow (Blue)体系中Blue介质的电渗特性及其影响因素 ;测定了Blue对HSA的吸附等温线以及在不同交变电场下的动态吸附曲线 ;由模型计算得到了吸附操作的传质系数及其与电场强度和交变频率的关系 ,实验结果和计算结果均表明 ,施加交变电场有助于提高传质系数 ,从而强化吸附过程的传质 .实验结果揭示了电渗流在强化色谱介质颗粒内传质的重要作用及其在发展大规模电动色谱技术中的良好应用前景     

Protein retention in dextran-grafted cation exchange chromatography: The influence of pHs,counterions and polymer structure

Polymer-grafted ion exchange adsorbents were of great interest for the development of high-performance protein chromatography in biopharmaceutical and related fields. In this work, protein retention was systematically investigated in ion exchange chromatography packed respectively with dextran-grafted cation exchange adsorbents containing sulphopropyl(SP) ligand, SP Sepharose XL and Capto S, and non-grafted cation exchange adsorbent, SP Sepharose FF, using five proteins. With an increase of buffer p Hs, retention factors of proteins decreased among all the adsorbents, demonstrating the dominant role of electrostatic interaction for protein binding on cation exchange adsorbents. The evidences further revealed that the scattered positive charges on the surface of protein molecules, rather than net charge of protein molecule, determined protein retention on cation exchange adsorbent. Likely, counterions including NH~(4+), K~+, Na~+ and Mg~(2+) exhibited distinct influence on protein retention. It was well ascribed to solvent-mediated indirect ion–macromolecule interactions and direct ion–macromolecule interactions. Compared with SP Sepharose FF, polymer structure in dextran-grafted cation exchange adsorbents ultimately brought about different ligand distributions and smaller pore sizes, thereby regulating protein retention in cation exchange chromatography. By comparing the retention of myoglobin and β-lactoglobulin B in SP Sepharose XL and Capto S, we reasonably speculated that the enhancement of nonelectrostatic interaction caused by reducing the space arm length was a major reason for an increasing retention factor of myoglobin in Capto S. The results in this research help us understand adsorption mechanism of protein in polymer-grafted adsorbents and give scientific guidance for the development of chromatographic materials.     

Chemoenzymatic Preparation of Functional Click-Labeled Messenger RNA

Synthetic mRNAs are promising candidates for a new class of transformative drugs that provide genetic information for patients’ cells to develop their own cure. One key advancement to develop so-called druggable mRNAs was the preparation of chemically modified mRNAs, by replacing standard bases with modified bases, such as uridine with pseudouridine, which can ameliorate the immunogenic profile and translation efficiency of the mRNA. Thus the introduction of modified nucleobases was the foundation for the clinical use of such mRNAs. Herein we describe modular and simple methods to chemoenzymatically modify mRNA. Alkyne- and/or azide-modified nucleotides are enzymatically incorporated into mRNA and subsequently conjugated to fluorescent dyes using click chemistry. This allows visualization of the labeled mRNA inside cells. mRNA coding for the enhanced green fluorescent protein (eGFP) was chosen as a model system and the successful expression of eGFP demonstrated that our modified mRNA is accepted by the translation machinery.     

Theoretical evaluation of the performance characteristics of a combined hemodialysis/hemoperfusion system — I. Single pass flow

A mass transfer model with single pass flow is proposed for evaluating the performance characteristics of a combined system composed of a dialyzer for hemodialysis (HD) and a coated adsorbent packed cartridge for hemoperfusion (HP). In the dialyzer, the mass transfer equation is represented in terms of three dimensionless parameters, i.e., extraction ratio, number of transfer unit, and ratio of flow rates. For the hemoperfusion cartridge, the model equations are derived by formulating the following four processes: convective mass transfer in the bed, fluid phase mass transfer around a coated adsorbent particle, transport across a film of coated membrane, and diffusion within an adsorbent particle. The Freundlich isotherm model is used for the adsorption on adsorbent particles. Applications of the mathematical model to systems to be potentially used in clinical practice are discussed. Solutions for two different sequences of arrangement, i.e., HD-HP and HP-HD, are obtained and discussed in terms of their performance efficacies. Theoretical results demonstrate that the HD-HP arrangement will give better performance in most clinical encounters.     

间歇式吸附与连续式脱附耦合亲和层析法研究

用间歇式吸附和连续式脱附耦合的亲和层析法分离纯化了溶菌酶。采用Langmuir Freundlich方程描述等温吸附线 ,通过间歇式吸附实验结果得到了该方程的相关参数以及蛋白质在吸附剂孔内的扩散系数。以蛋白质回收率和吸附剂利用率为基准比较了间歇式吸附和连续式吸附在操作时间上的差异。结果表明在蛋白质回收率和吸附剂利用率完全相等的情况下 ,间歇式吸附所用时间远小于连续式吸附时间。     

变压吸附法回收环氧乙烷生产排放气中的乙烯

针对环氧乙烷生产装置中含乙烯尾气排放造成乙烯资源浪费的问题,提出采用变压吸附回收尾气中乙烯的方法。实验中测定了排放气中各种气体组分在普通活性炭和自制载铜吸附剂(NJ)上的吸附平衡;分别考察了气体组分C2H6、CO2、Ar、CH4、O2对NJ变压吸附混合气中乙烯的影响。实验中还测定了NJ用于工厂实际排放气体中变压吸附乙烯的性能,并初步考察了解吸性能。实验结果表明,除氧气有一定影响外,NJ吸附剂具有稳定的变压吸附特性,能达到环氧乙烷排放气中乙烯回收要求,具有广泛的工业应用前景。     

A 3-zone model for protein adsorption kinetics in expanded beds

The adsorption behavior of expanded beds is more complex than that of fixed beds, since the adsorbent particle size, local bed voidage and liquid axial dispersion will vary axially with expanded height. Models applicable to fixed beds maybe not adequately describe the hydrodynamic and adsorption behavior in expanded beds. In this paper, a 3-zone model is developed, in which the model equations are written for the bottom zone, middle zone, and top zone of the column, respectively, and the model parameters, such as the adsorbent particle diameter, bed voidage and liquid axial dispersion coefficient, are zonal values. In-bed breakthrough curves are predicted by the 3-zone model, and tested against literature data for lysozyme adsorption on Streamline SP in an expanded bed.Model parametric sensitivity is analyzed. The effects of film mass transfer resistance, liquid axial dispersion and adsorbent axial dispersion on the breakthrough curves are weaker than that of protein intraparticle diffusion resistance for stable expanded beds. Adsorbent particle size axial distribution and bed voidage axial variation significantly affect in-bed breakthrough curves, therefore, model parameters should not be assigned uniform values over the whole column; instead the model should account for the adsorbent particle size axial distribution and bed voidage axial variation.