Modelling Affinity Chromatography

Abstract

Affinity chromatography plays a significant role in the separation and purification of biologically active macromolecules in laboratory and large-scale applications. There is a need for models which could be used to predict accurately the dynamic behavior of affinity chromatography separations, in order to permit the design, optimization, control, and process scale-up of affinity chromatography systems. Furthermore, the construction and use of such models will contribute to a better fundamental understanding of the physicochemical and biospecific mechanisms involved in affinity chromatography processes. The parameters of the models should be obtainable by using information from a small number of experiments.

This work reviews the modeling of affinity chromatography, and presents general models that could be used to describe the dynamic behavior of the adsorption, wash, and elution stages of affinity chromatography systems. Certain model structures, modeling approaches and operational strategies for systems having porous or nonporous adsorbent particles are also suggested, and experiments are proposed whose data are necessary for parameter estimation and model discrimination studies in affinity chromatography.

Particular emphasis is given to :he modeling of the intrinsic mechanisms of intraparticle diffusion, adsorption, and desorption, because the intrinsic mechanisms are normally independent of the mode of operation (i.e., batch, fixed bed, fluidized bed, continuous countercurrent, or others).     

Application of a novel affinity adsorbent for the capture and purification of recombinant Factor VIII compounds

Recombinant Factor VIII (FVIII) therapies have been created to provide treatment for Hemophilia A, an inherited bleeding disorder caused by mutation in the FVIII gene. A major challenge in the purification of recombinant FVIII molecules is the development of an affinity chromatography step. Such a step must be highly specific and selective for the FVIII molecule, but also must be designed appropriately to ensure the FVIII molecule can be effectively recovered without resorting to harsh elution conditions which may be harmful to the product. Additionally, it is desirable to have affinity adsorbents designed to be reusable over a large number of column cycles while maintaining consistent purification performance. In this work, we describe the use of VIIISelect, a commercially available affinity adsorbent designed specifically for the purification of FVIII compounds. The VIIISelect adsorbent consists of a 13 kDa recombinant protein ligand attached to a cross-linked agarose base matrix. The structure of the recombinant ligand is based upon Camelid-derived single domain antibody fragments. The VIIISelect adsorbent is produced using a process free of animal-derived raw materials, which is a highly desirable attribute for adsorbents used in the purification processes of recombinant protein therapeutics. The VIIISelect adsorbent was used as the initial capture column to purify a FVIII compound directly from clarified cell culture fluid prior to further downstream purification. The purification performance of the VIIISelect was evaluated, which included measurement of the static binding capacity, dynamic binding capacity, product recovery, impurity clearance, and adsorbent reuse. Following laboratory-scale process development, the VIIISelect adsorbent was scaled up and used in the large scale manufacturing of a FVIII compound.     

吸附剂性能和操作参数对液相色谱流出峰不对称性的影响

色谱流出峰的拖尾现象普遍存在于制备和大型色谱分离过程中,它直接影响分离的产率和回收率．本文提出采用色谱流出峰形的不对称偏差度来表征色谱流出峰的拖尾程度;并基于液相制备色谱分离过程FAD－SMT模型及吸附速率理论,通过计算机模拟,定量分析了吸附剂性能和操作参数对色谱流出峰形不对称性的影响。结果表明：不仅是吸附剂的热力学和动力学性能（包括吸附相平衡关系、液固两相间的传质阻力）;而且柱的设计和吸附剂的装填状况（包括轴向扩散系数）,以及色谱分离的操作条件（进料时间、浓度和流速等）都直接影响色谱流出峰形的不对称性。随着吸附相平衡等温线的非线性程度增大,或者总传质系数的减小,色谱流出峰形的不对称偏差度明显增大;吸附剂吸附容量的减小也将引起色谱流出峰形的不对称偏差度的增加;色谱流出峰形的不对称偏差度与进科体积、浓度和流体线速,以及轴向扩散系数的增大成正比。     

Optimal design of batch and simulated moving bed chromatographic separation processes

Preparative chromatography, especially simulated moving bed (SMB) chromatography, is a key technology for the separation of fine chemicals on a production scale. Most of the design methods for batch and SMB processes proposed in the open literature deal with the optimisation of the operating conditions for a given chromatographic unit only. Therefore, a comparison of the process economy may lead to incorrect results. In this contribution, an effective strategy for the optimal choice of all process parameters (operation and design parameters) is proposed. The main idea of this strategy is to apply a detailed and experimentally validated process model and to reduce the number of influencing parameters by introducing and optimising dimensionless process parameters. It is shown that there is an infinite choice of design and operating parameters to achieve maximum productivity or minimum separation costs and not at the maximum pressure drop only. The detailed design of the chromatographic unit and the selection of the operating conditions can be adjusted by considering the availability of columns and packing materials. As the model system, the separation of a racemic mixture (EMD53986) on Chiralpak AD was investigated. After complete optimisation of a batch and a SMB unit, a real comparison of the process economy can be achieved. Finally, the influences of two different objective functions, productivity and specific separation cost, are analysed.     

Multi-objective optimisation strategy based on desirability functions used for chromatographic separation and quantification of l-proline and organic acids in vinegar

An optimisation strategy based on desirability functions together with experimental design has been used to optimise a chromatographic method applied to the separation and quantification of l-proline and seven organic acids in vinegar samples. Chromatographic problems often force to reach a compromise between different experimental variables in order to achieve the best chromatographic separation. The importance of the use of multi-objective optimisation methods lies in the ability to cope with this kind of problems. The quality of the multicriteria optimisation method was tested through the validation of the analytical parameters of the final chromatographic method developed. The versatility of this methodology allows to use it in other chromatographic applications resulting in a suitable adaptive procedure to solve new analytical problems. Furthermore, the determination of l-proline and organic acids in vinegar is useful for several industrial goals such as the correct monitoring of fermentation, for the study of nutrient needs at all times during the twofold fermentation process and for the detection of possible adulterations in the final product.     

Pore network modelling: determination of the dynamic profiles of the pore diffusivity and its effect on column performance as the loading of the solute in the adsorbed phase varies with time

A three-dimensional pore network model for diffusion in porous adsorbent particles was employed in a dynamic adsorption model that simulates the adsorption of a solute in porous particles packed in a chromatographic column. The solution of the combined model yielded the dynamic profiles of the pore diffusion coefficient of beta-galactosidase along the radius of porous ion-exchange particles and along the length of the column as the loading of the adsorbate molecules on the surface of the pores occurred, and, the dynamic adsorptive capacity of the chromatographic column as a function of the design and operational parameters of the chromatographic system. The pore size distribution of the porous adsorbent particles and the chemistry of the adsorption sites were unchanged in the simulations. It was found that for a given column length the dynamic profiles of the pore diffusion coefficient were influenced by: (i) the superficial fluid velocity in the column, (ii) the diameter of the adsorbent particles and (iii) the pore connectivity of the porous structure of the adsorbent particles. The effect of the magnitude of the pore connectivity on the dynamic profiles of the pore diffusion coefficient increased as the diameter of the adsorbent particles and the superficial fluid velocity in the column increased. The dynamic adsorptive capacity of the column increased as: (a) the particle diameter and the superficial fluid velocity in the column decreased, and (b) the column length and the pore connectivity increased. In preparative chromatography, it is desirable to obtain high throughputs within acceptable pressure gradients, and this may require the employment of larger diameter adsorbent particles. In such a case, longer column lengths satisfying acceptable pressure gradients with adsorbent particles having higher pore connectivity values could provide high dynamic adsorptive capacities. An alternative chromatographic system could be comprised of a long column packed with large particles which have fractal pores (fractal particles) that have high pore connectivities and which allow high intraparticle diffusional and convective flow mass transfer rates providing high throughputs and high dynamic adsorptive capacities. If large scale monoliths could be made to be reproducible and operationally stable, they could also offer an alternative mode of operation that could provide high throughputs and high dynamic adsorptive capacities.     

Affinity purification of proteins using expanded beds.

The use of expanded beds of affinity adsorbents for the purification of proteins from feedstocks containing whole or broken cells is described. It is demonstrated that such feedstocks can be applied to the bed without prior removal of particulate material by centrifugation or filtration thus showing considerable potential for this approach in simplifying downstream processing flow-sheets. A stable, expanded bed can be obtained using simple equipment adapted from that used for conventional packed bed adsorption and chromatography processes. Circulation and mixing of the adsorbent particles is minimal and liquid flow through the expanded bed shows characteristics similar to those of plug flow. Frontal analysis performed with the highly selective affinity system involving the adsorption of human polyclonal immunoglobulin G onto Protein A Sepharose Fast Flow indicate that the adsorption performance of the expanded bed is similar to that achieved when the same amount of adsorbent is used in a packed configuration at the same volumetric flow-rate. The adsorption performance of the expanded bed was not diminished when adsorption was carried out in the presence of intact yeast cells. Batch adsorption experiments also indicated that the adsorption characteristics of the affinity system were not greatly altered in the presence of cells in contrast to results from a less selective ion-exchange system. An expanded bed of Cibacron Blue Sepharose Fast Flow was used to purify phosphofructokinase from feedstock of disrupted yeast prepared by high pressure homogenisation without the need for prior removal of particulate material. The potential for the use of expanded beds in large scale purification systems is discussed.     

Retention process in reversed phase TLC systems with polar bonded stationary phases

The retention of a solute in RP chromatography is a very complex process which depends on many factors. Therefore, the study of the influence of a mobile phase modifier concentration on the retention in different reversed phase chromatographic systems is very important for understanding the rules governing retention and mechanisms of substance separation in a chromatographic process. Composition changes and the nature of mobile phases enable tuning of the separated analytes' retention over a wide range of retention parameters and optimization of the chromatographic process as well. Optimization of the chromatographic process can be achieved by several different methods; one of them is the so-called interpretative strategy. The key approach adopted in this strategy is the implementation of adequate retention models that couple the retention of solute with the composition of a mixed mobile phase. The use of chemically bonded stationary phases composed of partially non-bonded silica matrix and organic ligands bonded to its surface in everyday chromatography practice leads to questions of the correct definition of the retention model and the dominant retention mechanism in such chromatographic systems. The retention model for an accurate prediction of retention factor as a function of modifier concentration and the heterogeneity of the adsorbent surface should be taken into consideration. In this work the influence of mobile-phase composition on the retention of sixteen model substances such as phenols, quinolines, and anilines used as test analytes in different RP-TLC systems with CN-, NH2-, and Diol-silica polar bonded stationary phases has been studied. The aim of this study is to compare the performance of three valuable retention models assumed as the partition, adsorption/partition, and adsorption mechanism of retention. All the models were verified for different RP-TLC systems by three statistical criteria. The results of investigations presented in this work demonstrate that the best agreement between the experimental and calculated Rf values was obtained by the use of new-generation retention models, which assume heterogeneity of adsorbent surface. The results reported here show that heterogeneity of the adsorbent surface may be important in analysis of the elution process in liquid chromatography. Consideration of the goodness of fit for the experimental data to the examined retention models is in conformity with the adsorption mechanism of retention on all polar bonded stationary phases in most eluent systems for most investigated compounds.     

Comparison among three anion exchange chromatographic supports to capture erythropoietin from cell culture supernatant Lourdes

Affinity and ion exchange conventional chromatography have been used to capture erythropoietin (EPO) from mammalian cell culture supernatant. Currently, chromatographic adsorbent perfusion is available, however a limited number of applications have been found in the literature. In this work, three anion exchange chromatographic supports (gel, membrane and monolithic) were evaluated in the capture step of the recombinant erythropoietin purification process. The influences of load and flow rate on each support performance were analyzed. Also the purity of the EPO molecules was determined. A productivity analysis, as a decision tool for larger scale implementation, was done. As a conclusion, the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity. However, the monolithic column admits high operating velocity, showing the highest adsorption capacity and productivity.     

Generic method for systematic phase selection and method development of biochromatographic processes. Part I. Selection of a suitable cation-exchanger for the purification of a pharmaceutical protein

Even if the first protein therapeutics are now for more than 20 years on the market the selection of suitable adsorbents for the preparative downstream processing (DSP) of these biomolecules as well as the method development towards process conditions are still based mainly on 'trial and error'. Therefore, theses processes are not perfectly efficient, but indeed very time consuming and laborious. In this study a novel systematic method is introduced to find a suitable adsorbent (not necessarily the best one) with appropriate separation parameters for a specific separation with reduced effort. Following this strategy, the adsorbents must first be packed into columns under preparative conditions and then characterized completely with regard to, e.g. pressure drop, k'-values, plate heights (HETP curves), selectivity and capacity by using test substances, which are similar in their characteristics (molecular mass, size, charge distribution, hydrophobicity) to the target proteins. With the database once determined, a preselection of most suitable adsorbents including separation parameters is made regarding chromatographic and also economical properties. After this, preparative experiments must be conducted with a reduced number of adsorbents to figure out the individual influence of side components. This approach is demonstrated for the separation of an exemplary industrial protein mixture using cation-exchange chromatography (CEX). Characterization of different weak CEX-adsorbents is illustrated. After comparing these phases with each other, a first preselection and a prediction of suitable adsorbents is made. In the following preparative separation conditions (load, velocity, gradient) are determined for the preparative separations using the database and results of some additional experiments. The final comparison of separation performance in preparative scale confirms this selection and so the applicability of the new method.     

Modeling the effects of column packing quality and residence time changes on protein monomer/aggregate separation

The packing quality of chromatography columns used for the purification of protein therapeutics is routinely monitored to ensure consistent and reproducible performance. In this work, we used established chromatography models to determine the effect of column packing quality and fluid residence time on the separation of protein therapeutic monomer and aggregate species using a hydrophobic interaction chromatography adsorbent (Phenyl Sepharose Fast Flow). The relationship between the number of theoretical plates, fluid residence time, and column separation performance was quantified using modeling simulations. The simulations showed the separation depended on both the fluid residence time and the number of theoretical plates. However, when the number of theoretical plates was increased to ≥150, the simulations predicted that the separation performance of the column was not significantly improved. The approach described here could be used as a method to quantify acceptable height equivalent of a theoretical plate values for columns, and serve as a tool to understand how column packing quality impacts a given chromatographic separation prior to column scale-up, as well as during the monitoring of column lifetime in the manufacturing of large scale protein therapeutics.     

Novel column-based protein refolding strategy using dye-ligand affinity chromatography based on macroporous biomaterial

A novel column-based chromatographic protein refolding strategy was developed using dye-ligand affinity chromatography (DLAC) based on macroporous biomaterial. Chitosan–silica (CS–silica) biomaterial with macroporous surface was used as the supporting matrix for the preparation of the DLAC material. The dye-ligand Cibacron Blue F3GA (CBF) was selected as affinity handle and could be covalently immobilized to form dye-ligand affinity adsorbent (CBF–CS–silica) using the reactivity of NH₂ on CS–silica biomaterial. After the model protein catalase was denatured with 6 mol/L urea, the denaturant could be rapidly removed and catalase could be successfully refolded as facilitated by the adsorption of CBF–CS–silica. The urea denaturation process and the elute condition for the chromatographic refolding were optimized by measuring tryptophan fluorescence and activity of catalase. The refolding performance of the proposed DLAC was compared with dilution refolding. The protein concentration during the proposed chromatographic refolding increased by a factor of 20 without reducing the yield achieved as compared to dilution refolding. The column-based protein refolding strategy based on dye-ligand affinity chromatography with porous biomaterial being matrix possessed potential in chromatographic refolding of protein.     

Application of Mixed-Mode Oasis MCX Adsorbent for Chromatographic Separation of Selenomethionine from Antarctic Krill After Enzymatic Digestion

The present communication describes the novel application of mixed-mode adsorbents for bifunctional chromatography. Oasis MCX mixed-mode adsorbent (Waters GmbH, Eschborn, Germany) shows a high binding capacity for Se-Met. The adsorbed selenoamino acid is easily eluted by pH change and addition of organic solvent. Determined adsorption isotherm parameters are suitable for its analytical and preparative chromatographic isolation. A chromatographic column packed with the Oasis MCX adsorbent, designed for a single use solid phase extraction, was successfully applied for bifunctional chromatographic separation of Se-Met from hydrolysed Antarctic krill samples with 85 ± 5% recovery. The MCX-packed column was used for more than 60 runs without any change in its performance.     

Ion chromatography with inductively coupled plasma mass spectrometry,a powerful analytical tool for complex matrices estimation of Pt and Pd in environmental samples

A new method for palladium and platinum direct determination in environmental samples is proposed by coupling ion chromatography with quadrupole inductively coupled plasma MS. In order to optimise Pd and Pt separation and to minimise interference from matrix in real samples, several anionic and cationic stationary phases have been compared at different mobile phase compositions. In particular, the effect of acidity and of the addition of oxalic acid to the eluent on separation and detection performance has been studied, and the anion-exchange column AG11 turned out to be more suitable. After chromatographic and mass spectrometer parameter optimisation, several potential interferences and the main quality parameters of the method, according to the Eurachem-CITAC recommendations, were evaluated: the detection limit for Pt was 5 ng l(-1) while the value for Pd was 230 ng l(-1). The method was successfully employed in the determination of platinum group elements in urban road dust and atmospheric particulates and the complete absence of matrix spectral interferences was demonstrated.     

Mechanism and kinetics of protein transport in chromatographic media studied by confocal laser scanning microscopy. Part II. Impact on chromatographic separations

The impact of different transport mechanism on chromatographic performance was studied by confocal laser scanning microscopy (CLSM) for solutions containing bovine serum albumin (BSA) and monoclonal IgG 2a under different solid- and fluid-phase conditions. During this investigation, a clear influence of the uptake mechanism on the affinity of the respective proteins for the different adsorbents and thus separation performance of the chromatographic process could be observed. For the system SP Sepharose Fast Flow at pH 4.5 pore diffusion could be ascribed to be the dominant transport mechanism for both proteins and the adsorption profiles resembled a pattern similar to that described by the 'shrinking core' model. Under these conditions a significantly higher affinity towards the adsorbent was found for BSA when compared to IgG 2a. With changing fluid- and solid-phase conditions, however, a change of the transport mode for IgG 2a could be detected. While the exact mechanism is still unresolved it could be concluded that both occurrence and magnitude of the now governing transport mechanism depended on protein properties and interaction with the adsorbent surface. For the system SP Sepharose XL at pH 5.0 both parameters leading to the change in IgG 2a uptake were combined resulting in a clear change of the system affinity towards the IgG 2a molecule, while BSA adsorption was restricted to the most outer shell of the sorbent.     

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.

     

分析测试新成果(160~168)金属吸附剂用于去除血清蛋白及核苷检测

利用多糖与金属离子复合制备了一种高效的蛋白质吸附剂.海藻酸钠和羧甲基纤维素钠是两种富含羟基和羧基的多糖, 具有较强的金属亲和性.将其用钙离子交联后制备成金属-多糖复合材料, 进一步修饰铜离子, 得到蛋白质吸附剂.吸附剂对富含组氨酸的牛血红蛋白的吸附量可以达到33 g/g, 对少量组氨酸的牛血清白蛋白的吸附量也可以达到9.8 g/g.蛋白质吸附剂对人血血清进行两次吸附后, 可以去除其中98%的蛋白, 能够满足人血血清中核苷类物质的直接色谱进样检测.     

3种阴离子交换色谱固定相捕获细胞培养上清液中红细胞生成素的效果比较（英文）

Affinity and ion exchange conventional chromatography have been used to capture erythropoietin(EPO)from mammalian cell culture supernatant.Currently,chromatographic adsorbent perfusion is available,however a limited number of applications have been found in the literature.In this work,three anion exchange chromatographic supports(gel,membrane and monolithic)were evaluated in the capture step of the recombinant erythropoietin purification process.The influences of load and flow rate on each support performance were analyzed.Also the purity of the EPO molecules was determined.A productivity analysis,as a decision tool for larger scale implementation,was done.As a conclusion,the evaluated supports are technically suitable to capture EPO with adequate recovery and good purity.However,the monolithic column admits high operating velocity,showing the highest adsorption capacity and productivity.     

Adsorption in a stratified column bed packed with porous particles having partially fractal structures and a distribution of particle diameters

Stratified column bed systems whose sections are formed by packing adsorbent particles with a partially fractal structure are proposed and studied. The simulation results clearly show that the breakthrough times and the shape of the breakthrough curves obtained from stratified column beds are significantly larger and sharper than those obtained from conventional columns. The stratified column beds provide, to the designer and user of chromatographic column systems, more degrees of freedom with respect to the number of parameters and variables that could be controlled in the design, construction, and operation of efficient chromatographic adsorption systems. Furthermore, the results suggest that the stratified column beds could provide a higher dynamic adsorptive capacity than conventional columns when it is required to increase the column throughput.