Proteins Separation and Purification by Expanded Bed Chromatography and Simulated Moving Bed Technology

Transcription

1 Proteins eparation and Purification by Expanded Bed hromatography and imulated Moing Bed Technology A issertation Presented to the UNIVERITY OF PORTO for the egree of octor in hemical Engineering by Ping Li uperisor: Professor Alirio E. Rodrigues LABORATÓRIO AOIAO Laboratory of eparation and Reaction Engineering epartment of hemical Engineering Faculty of Engineering, Uniersity of Porto, Portugal July, 006

2 Acknowledgements First of all, I want to thank my superisor, Professor Alirio E. Rodrigues, for his constant encouragement, support and guidance, for always challenging me to reach higher goals within my work, and for many insightful suggestions during the deelopment of the ideas in the thesis. To r. Xiu, my husband, an excellent collaborator in scientific research, for his constant encouragement and support. Also to my son for his understanding and independence in life and study when parent were busy themseles in work. I would like to express my gratitude to Faculdade de Engenharia da Uniersidade do Porto (FEUP), and to epartamento de Engenharia Química, for all support and facilities gien. To Laboratório de Engenharia de Reacção e eparação (LRE) headed by Professor Alirio E. Rodrigues, for all support and facilities gien. To Fundação para a iência e a Tecnologia, Lisboa, Portugal, for the Ph grant (FRH/B/676 /00). I am ery grateful to all professors of LRE, for friendship and scientific support wheneer I needed. To secretary usana Paula ruz for her friendship and support. I am also deeply grateful, for their help and collaboration, to Vera Mata, arlos Grande, imone aenati, Paula Gomes, Gandi Ganesh Kumar, Nabil Lamia, Eduardo Borges da ila, Eduardo Olieira, Yining Wang, Marta Otero, Kishore asari, Mirjana Mincea, Miriam Zabkoa, Michal Zabka, Viiana ila, Filipe V.. Lopes.

3 Abstract The study focuses on the adsorption kinetics for proteins separation and purification by expanded bed chromatography and salt gradient ion-exchange simulated moing bed technology. Mathematical models are deeloped for the protein adsorption kinetics in an expanded bed, and tested against the pilot scale experimental data: ) BA adsorption in an expanded bed, where a treamline 50 column was packed either with 00mL treamline EAE (classic ion exchanger) or with 00mL treamline irect T I (new type of ion exchanger with the multimodal ligand), carried out in this thesis. ) lysozyme adsorption in an expanded bed, where a treamline 50 column was packed with 400mL treamline P to test -zone model, data from the published paper of Bruce and hase (00). Then model parametric sensitiity is analyzed. The effects of protein intraparticle diffusion resistance, film mass transfer resistance, liquid axial dispersion, solid axial dispersion, particle size axial dispersion and bed oidage axial ariation, on breakthrough cures are ealuated for the expanded bed adsorption with treamline EAE, treamline P and treamline irect T I. The feasibility of capturing both BA and myoglobin by an expanded bed adsorption process is addressed, where a treamline 50 column is packed with 00mL treamline irect T I. Furthermore, two-component proteins ( BA/myoglobin) competitie adsorption and displacement on treamline irect T I are studied based on static batch experiments, frontal analysis and column displacement experiments. The design of the high-density matrix of adsorbent used in expanded beds can be improed by including a single heaier inert core material in the macroporous crossliked resin matrix, that is called pellicular adsorbent or inert core adsorbent The decrease of the intraparticle diffusion resistance by inert core adsorbents is quantitatiely estimated by the parameter / Θ. Furthermore, the theoretical analysis is gien to demonstrate the potential application of the inert core adsorbents in the fast, high-performance liquid chromatography (HPL) for the resolution of biological macromolecules as a result of the decrease of the intraparticle diffusion resistance. A real gradient MB model is used to analyze the performance of salt gradient ion-exchange MB for linear and nonlinear ion-exchange equilibrium isotherm of proteins. ome strategies will be discussed for the selection of salt gradient and the selection of flow rate in each section of salt gradient ion-exchange MB. The gradient MB configurations, open loop, closed loop and closed loop with a holding essel, are compared. Finally, we will present a comparison of the two strategies of modeling, equialent gradient TMB model and real gradient MB model, for the prediction of internal concentration profiles in gradient MB with open loop and closed loop.

4 REUMO Nesta tese é estudada a cinética de adsorção para separação e purificação de proteínas atraés da cromatografia de leito fluidizado e da cromatografia de troca iónica por gradiente salino em leito móel simulado (LM). Modelos matemáticos são desenolidos para predizer a cinética de adsorção de proteínas em leito fluidizado e os resultados são comparados aos dados experimentais obtidos em uma unidade piloto: ) Adsorção de albumina de soro boino (Boine erum Albumin-BA) em leito fluidizado, onde uma coluna treamline 50 é empacotada tanto com 00 ml do adsorente treamline EAE (resina de troca iónica clássica) como com 00 ml do adsorente treamline irect T I (noo tipo de resina com ligante multimodal elaborado neste trabalho). ) Adsorção de lisozima em leito fluidizado, onde uma coluna treamline 50 é empacotada com 400 ml do adsorente treamline P para testar o modelo de -zonas aos dados reportados por Bruce e hase (00). Analisa-se a sensibilidade paramétrica do modelo. ão analisados os efeitos da resistência à difusão intraparticular da proteína, resistência à transferência de massa no filme, dispersão axial tanto na fase líquida como na fase sólida, dispersão axial segundo o tamanho da partícula e porosidade, sobre as curas de ruptura de adsorção em leito fluidizado com treamline EAE, treamline P e treamline irect T I. É aaliada a possibilidade de capturar o BA e a mioglobina atraés de um processo de adsorção por leito fluidizado, onde uma coluna treamline 50 é empacotada com 00 ml do adsorente treamline irect T I. Ademais, a adsorção competitia e de deslocamento do sistema binário de proteínas (BA/mioglobina) são estudadas baseadas sobre experimentos de: batelada, cromatografia de análise frontal e de deslocamento. O projecto da matriz de alta densidade do adsorente usado nos leitos fluidizados pode ser melhorado pela inclusão de um material inerte mais pesado na rede macroporosa da matriz da resina, que é chamado adsorente pelicular ou adsorente de núcleo inerte. O decréscimo da resistência da difusão intraparticular no adsorente de núcleo inerte é quantitatiamente analisado pelo parâmetro /Θ. Além disso, análises teóricas são apresentadas para demonstrar o potencial de aplicação dos adsorentes de núcleo inerte na cromatografia líquida de alta performance (HPL high-performance liquid crhomatogrphy) para a resolução de macromoléculas uma ez que tal adsorente promoe um decréscimo da resistência de difusão intraparticular. Um modelo baseado na concepção intermitente de LM gradiente é usado para analisar o desempenho da unidade de leito móel simulado de permuta iónica por gradiente salino para isotermas de equilíbrio de troca iónica lineares e não-lineares das proteínas. Algumas estratégias são discutidas para a selecção do gradiente salino e do caudal em cada secção da unidade de LM de permuta iónica por gradiente salino. As configurações do LM gradiente: circuito fechado e aberto, circuito fechado com tanque de retenção, são comparadas. Finalmente, é apresentada uma comparação entre as duas estratégias de modelização modelo de leito móel erdadeiro gradiente equialente e modelo intermitente de leito móel simulado gradiente para a preisão dos perfis de concentração no LM gradiente de circuito aberto e de circuito fechado.

5 Résumé ette étude porte sur les cinétiques d adsorption de la séparation et la purification des protéines par chromatographie en lit fluidisé et par chromatographie d échange ionique par gradient salin en lit mobile simulé (LM). Les modèles mathématiques ont été déeloppés pour prédire la cinétique d adsorption de protéines en lit fluidisé et les résultats ont été comparés aux données expérimentales obtenues dans une unité pilote : ) Adsorption de l albumine de sérum boin (Boine erum Albumin - BA) en lit fluidisé, où une colonne treamline 50 est remplie soit aec 00 ml d adsorbant treamline EAE (résine d échange ionique classique) ou soit aec 00 ml de treamline irect T I (noueau type de résine aec ligand multimodal) ) Adsorption de lysozyme en lit fluidisé, où une colonne treamline 50 est empaquetée aec 400 ml d adsorbant treamline P pour tester le modèle de -zones aec les données reportées par Bruce et hase (00). La sensibilité paramétrique du modèle a été analysée. On a ainsi éalué les effets de différents paramètres sur les courbes de perçages de l adsorption en lit fluidisé aec treamline EAE, treamline P et treamline irect T I : résistance à la diffusion intraparticulaire de la protéine, résistance du transfert de matière dans le film, dispersion axiale aussi bien dans la phase liquide que dans la phase solide, dispersion axiale au nieau de la particule et enfin la ariation axiale des espaces ides du lit. Il a été confirmé la possibilité de capturer le BA et la myoglobine à traers un procédé d adsorption en lit fluidisé, où une colonne treamline 50 serait remplie aec 00 ml d adsorbant treamline irect T I. Par ailleurs, l adsorption compétitie et les déplacements du système binaire de protéines (BA/myoglobine) ont été étudiés en se reposant sur des d expériences de type batch, des chromatographies d analyse frontale et des expériences de déplacements. Le concept de la matrice de haute densité d adsorbant utilisé dans les lits fluidisés peut être amélioré par l intrusion d un matériau inerte plus lourd dans le réseau macroporeux de la matrice de résine, et appelé adsorbant au noyau inerte ou adsorbant peliculaire. La diminution de la résistance à la diffusion intraparticulaire par des adsorbants au cœur inerte est quantitatiement analysée par le paramètre l / Θ. e plus, des analyses théoriques sont présentées pour démontrer le potentiel d application des adsorbants aux noyaux inertes en chromatographie liquide de haute performance (HPL) pour la résolution de macromolécules biologiques par suite de la baisse de la résistance de la diffusion intraparticulaire. Un modèle basé sur le LM gradient est utilisé pour analyser la performance d une unité du lit mobile simulé à échange ionique par gradient salin pour des isothermes d équilibre d échange ionique linéaires ou non linéaires des protéines. Quelques stratégies seront mises en éidence et comparées pour le choix du gradient salin et la sélection du débit dans chaque section du LM à échange ionique par gradient salin. ifférentes configurations du LM gradient ont été comparées : circuit fermé ou ouert entre autre. Enfin, nous présenterons une comparaison des deux stratégies de modélisation mis en œure le modèle du lit mobile rai gradient équialent et le modèle du lit mobile simulé gradient pour la prédiction des profils de concentration dans un LM gradient aec circuit ouert ou fermé.

6 摘要本论文研究膨胀床和盐梯度离子交换模拟移动床技术分离和精制蛋白质 首先建立了数学模型 该模型用于描述膨胀床吸附蛋白质的动力学, 并用 下列的实验测定数据验证该数学模型. 实验测定牛血清蛋白在膨胀床内的吸附突破曲线 使用型号为 treamline50 膨胀床, 柱内分别填充 00 毫升型号为 treamline EAE 的第一代吸附剂和型号为 treamline irect T I 的第二代吸附剂. 溶菌酶在膨胀床内柱内吸附突破曲线 该实验数据摘自已发表的科技 论文 [Bruce and hase (00)] 型号为 treamline50 膨胀床填充 400 毫升型号为 treamline P 的第一代吸附剂 基于模型参数敏感性的分析, 探讨诸因素对膨胀床内突破曲线的影响, 诸因 素包括蛋白质在吸附剂内的扩散阻力 吸附剂外膜传质阻力 柱内液相轴向弥散 吸附剂的轴向弥散 吸附剂颗粒轴向分布和床层空隙率轴向变化 实验研究了牛血清蛋白和肌红蛋白在膨胀床内竞争吸附与解吸 使用型号为 treamline50 膨胀床, 并填充 00 毫升型号为 treamline irect T I 的第 二代吸附剂 同时间歇吸附实验 前锋分析 和柱子取代实验被用于分析血清蛋 白和肌红蛋白在型号为 treamline irect T I 的第二代吸附剂上竞争吸附和 取代的机理 使用高密度惰性芯吸附剂能提高膨胀床吸附蛋白质的效率, 因为该设计减少 了大分子蛋白质的颗粒内扩散阻力 蛋白质的颗粒内扩散阻力的减少量能够用模 型参数 / Θ 估算 基于线性拆分因子的导出式, 证明了采用惰性芯吸附剂填充的 色谱分离大分子蛋白质效果明显好于普通吸附剂填充的色谱分离效果 此外, 本 论文还给出了用于计算惰性芯吸附剂填充色谱的理论板当量 [ 等效 ] 高度线性解 析式 采用梯度 MB 数学模型分析盐梯度模拟移动床分离和精制蛋白质的性能 对 盐梯度选择和流速选择的策略进行了详细讨论 比较了循环与非循环的梯度模拟 移动床, 在循环梯度模拟移动床操作中, 加入一个固定体积的混合器可以减少梯 度的波动 比较了两种常用的数学模型, 梯度 MB 模型和等价梯度 TMB 模型, 讨 论了简单的等价梯度 TMB 模型的实用性

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8 Table of ontents List of Figures List of Tables VII XIII. Introduction. Releance and Motiation. Objecties and Outline References 5. Modeling separation of proteins by inert core adsorbent in a batch adsorber 7. Introduction 8. Mathematical model and analytical solutions 0.. Mathematical model 0.. Analytical solution for Henry isotherm 4.. Analytical solution for Rectangular isotherm 6. Results and discussion 6.. Effect of the model parameters on the time eolution of bulk concentration and uptake radial profiles 8.. Method for estimation of effectie pore diffusiity of protein and film mass transfer coefficient from the bulk concentration profile 4.4 onclusions 7 Notation 8 References 0 I

9 . Modeling breakthrough and elution cures in fixed bed of inert core adsorbents: analytical and approximate solutions 5. Introduction 6. Mathematical model and analytical and approximate solutions 8.. Analytical and approximate solutions for breakthrough cures 4... Analytical solution 4... Parabolic-profile approximate solution Quasi-lognormal distribution approximate solution 45.. Analytical and approximate solutions for peak elution cures Analytical and approximate solutions for irac input Analytical and approximate solutions for the generalized sample input mode 47.. eried equation of resolution R based on Q-LN approximation 49. Results and discussion 5.. Effect of model parameters on breakthrough cures 5.. Effect of model parameters on peak elution cures 55.. Resolution of two components by the fixed bed packed with inert core adsorbent 59.4 onclusions 6 Notation 6 References Analytical breakthrough and elution cures for inert core adsorbent with sorption kinetics Introduction Mathematical model and analytical and approximate solutions Analytical solution and parabolic-profile approximate solution for breakthrough cures Analytical solution 75 II

10 4... Parabolic-profile approximate solution Analytical and approximate solutions for peak elution cures Moments and height equialent to a theoretical plate Analytical solution for residence time distribution of an inert tracer with irac input Results and discussion Effect of model parameters on breakthrough cures Effect of model parameters on peak elution cures Theoretical plate number and the equialent height to a theoretical Plate (HETP) Residence time distribution onclusions 88 Notation 89 References A -zone model for protein adsorption kinetics in expanded beds Introduction Mathematical model Zone diision of the column Model deelopment Model parameters Numerical method 0 5. Results and discussion omparison between the experimental data and simulation results Parametric sensitiity analysis on the breakthrough cures Effect of intraparticle diffusion coefficient Effect of film mass transfer coefficient Effect of liquid axial dispersion Effect of adsorbent axial dispersion Effect of adsorbent particle size on the breakthrough cures III

11 5..4 Modified uniform model by taking account the adsorbent particle size and bed oidage axial dispersions 5.4 onclusions Notation 4 References 6 6. Experimental and modeling study of protein adsorption in expanded bed 9 6. Introduction 0 6. Experimental 6.. Equipment, adsorbents and model protein 6.. Batch adsorption experiments Residence time distribution experiments Experimental procedures for the whole expanded bed adsorption process 5 6. Mathematical model for the protein adsorption in expanded beds Results and discussion Adsorption isotherm and BA effectie pore diffusiity Bed expansion and liquid axial dispersion coefficient in expanded bed 6.4. BA protein breakthrough behaior in expanded beds packed with treamline irect T I and with treamline EAE omprehensie ealuations on the whole expanded-bed protein adsorption process with treamline EAE and with treamline T I onclusions 49 Notation 5 References 5 Appendix Expanded bed adsorption/desorption of proteins with treamline irect T I adsorbent Introduction Materials and methods 6 IV

12 7.. Equipment, treamline irect T I, model proteins Batch adsorption equilibrium isotherm experiments olumn displacement experiments Frontal analysis in a fixed bed Experimental procedures for the whole expanded bed adsorption Process Results and discussion ingle- and two-component BA/myoglobin adsorption isotherm Frontal analysis and column displacement measurements BA and myoglobin competitie adsorption/desorption in an expanded bed onclusions 79 References Proteins separation and purification by salt gradient ion-exchange simulated moing bed 8 8. Introduction Gradient MB strategies of modeling Formation of salt gradient in ion-exchange MB chromatography Model equations for the real gradient MB model Proteins and salt adsorption equilibrium isotherm on ion exchanger Model parameters and numerical method Results and discussion Proteins separation by salt gradient ion-exchange MB with linear ion-exchange equilibrium isotherm omparison of gradient MB configurations: open loop, closed loop and closed loop with a holding essel omparison of the two strategies of modeling, gradient MB/TMB model alt gradient ion-exchange MB with nonlinear ion exchange V

13 equilibrium isotherm of proteins Nonlinear ion exchange equilibrium isotherm of BA and myoglobin on Q-epharose FF anion exchanger Proteins separation and purification by salt gradient ion-exchange MB with nonlinear ion exchange equilibrium isotherm onclusions Notation References 4 Appendix 8 9. onclusions and suggestions for future work 9. onclusions 9. uggestions for future work 4 VI

14 List of Figures hapter Figure. Preparation and the Three tep Purification trategy for the high purify protein production hapter Figure. Batch adsorber and inert core adsorbent 0 Figure. Langmuir isotherm. 7 Figure. Effect of r on dimensionless bulk concentrations as a function of reduced time at different Bi for constant alue of N 9 Figure.4 Effect of r on dimensionless bulk concentrations as a function of reduced time at constant loading ratio L = ( r ) N for Bi = Figure.5 Uptake radial profiles within the adsorbent shell at arious times for Bi = 00 and r = Figure.6 omparison of analytical solution of ersus for rectangular isotherm and numerical solution for Langmuir isotherm with small alues of λ at different Bi for N = 0. 9 Figure.7 Verification of the numerical solution by comparison with analytical solution for Henry isotherm. 4 Figure.8 Estimation of effectie pore diffusiity, Pe, at high Bi alue for Langmuir isotherm. 6 Figure.9 Estimation of effectie pore diffusiity, Pe, at small Bi alue for Langmuir isotherm. 7 VII

15 hapter Figure. cheme of fixed-bed adsorber and inert core adsorbent 9 Figure. Typical elution cure. 49 Figure. Effect of ξ on Figure.4 Effect of Bi on y τ breakthrough cures. 5 y τ breakthrough cures for the column packed with inert core adsorbent and conentional adsorbent 54 Figure.5 Effect of Pe on y τ breakthrough cures for the column packed with inert core adsorbent and conentional adsorbent 55 Figure.6 Effect of ξ on Figure.7 Effect of Pe on y τ elution cures for θ = 0 56 y τ elution cures for the column packed with inert core adsorbent and conentional adsorbent 57 Figure.8 Effect of Bi on y τ elution cures for the column packed with inert core adsorbent and conentional adsorbent 57 Figure.9 Elution cures for different input modes at the column packed with inert core adsorbent adsorbent and conentional adsorbent 58 Figure.0 Influence of inert core radius on the relatie intraparticle diffusion resistance. 60 Figure. The resolution of two components at the column packed with inert core adsorbent and conentional adsorbent 6 hapter 4 Figure 4. cheme of fixed-bed adsorber and inert core adsorbent 7 Figure 4. Effect of ξ on Figure 4. Effects of Pe on Figure 4.4 Effects of Bi on y τ breakthrough cures. 8 y τ breakthrough cures for inert core adsorbent. 8 y τ breakthrough cures for inert core adsorbent. 8 Figure 4.5 Effect of ψ on y τ breakthrough cures for inert core adsorbent 84 VIII

16 Figure 4.6 Effect of ξ on Figure 4.7 Effect of Pe on Figure 4.8 Effect of Bi on Figure 4.9 Effect of ψ on y τ breakthrough cures for short column. 84 y τ elution cures for inert core adsorbent. 85 y τ elution cures for inert core adsorbent. 85 y τ elution cures for inert core adsorbent. 86 Figure 4.0 Effect of ξ on * HETP and N T under restricted adsorption /desorption rate 86 Figure 4. Effect of Bi on y τ residence time distribution for inert core adsorbent. 87 Figure 4. Effect of Pe on y τ residence time distribution for inert core adsorbent. 88 hapter 5 Figure 5. Expanded bed with three zones and flow configuration in Bruce and hase experimental system (00). 96 Figure 5. omparison among the in-bed experimental breakthrough cures and the simulation results. 0 Figure 5. Parametric sensitiity analysis of intraparticle diffusion coefficient to in-bed breakthrough cures in expanded bed. 06 Figure 5.4 Parametric sensitiity analysis of film mass transfer coefficient to in-bed breakthrough cures in expanded bed. 07 Figure 5.5 Parametric sensitiity analysis of liquid axial dispersion coefficient to in-bed breakthrough cures in expanded bed. 09 Figure 5.6 Parametric sensitiity analysis of adsorbent axial dispersion coefficient to in-bed breakthrough cures in expanded bed. 0 Figure 5.7 Effect of adsorbent particle size on in-bed breakthrough cures in expanded bed and comparison between the experimental data and the simulation results predicted by the modified uniform model. IX

17 Figure 5.8 treamline P adsorbent particle size axial distribution and bed oidage axial ariation in an expanded bed. hapter 6 Figure 6. BA adsorption isotherms on treamline EAE and on treamline direct T I at the room temperature. Figure 6. Estimation of BA effectie pore diffusiity in treamline EAE adsorbents and in treamline T I adsorbents in batch adsorber. Figure 6. Relationship between bed expansion degree and superficial liquid flow elocity in expanded beds packed with treamline EAE and with treamline T I. 4 Figure 6.4 Experimental data of RT cures are fitted by the analytical solution with the dirac input of acetone tracer at expanded beds packed with treamline EAE and packed with treamline direct T I. 5 Figure 6.5 Experimental and predicted BA breakthrough cure in expanded bed packed with treamline direct T I. 7 Figure 6.6 ontribution of each model parameter to the breakthrough cure in expanded bed packed with treamline direct T I. 9 Figure 6.7 Experimental and predicted BA breakthrough cure in expanded bed packed with treamline EAE. 4 Figure 6.8 Effect of the particle size axial distribution and bed oidage axial distribution on the breakthrough cures in expanded beds packed with treamline EAE. 4 Figure 6.9 Effluent cures of BA protein during adsorption, washing, and elution stages in expanded bed packed with treamline direct T I. 45 Figure 6.0 Effluent cures of BA protein during adsorption, washing, and elution stages in expanded bed packed with treamline EAE. 45 Figure 6. Effect of ionic strength of buffer on BA adsorption isotherms on treamline EAE and on treamline direct T I. 47 X

18 Figure 6. Effect of salt concentration in buffer on BA adsorption isotherms on treamline EAE and on treamline direct T I 48 hapter 7 Figure 7. Effect of ionic strength, salt concentration and ph alue in feedstocks on BA adsorption isotherm on treamline irect T I. 69 Figure 7. Effect of salt concentration on BA adsorption isotherm on treamline irect T I at 50mM acetate buffer (PH 7). 70 Figure 7. Myoglobin adsorption isotherm on streamline irect T I in 50mM acetate buffer with different ph alue. 70 Figure 7.4 BA and myoglobin competitie adsorption isotherm on treamline irect T I measured in static batch experiments at 50mM acetate buffer (ph 5). 7 Figure 7.5 isplacement adsorption between BA and myoglobin in a fixed bed packed with treamline irect T I. 7 Figure 7.6 Breakthrough cures in a fixed bed packed with treamline irect T I for single- and two-component BA/myoglobin adsorption system. 74 Figure 7.7 Effluent cures of BA and myoglobin during adsorption, wash and elution stages in expanded bed packed with treamline direct T I. 77 hapter 8 Figure 8. chematic diagram of a four-zone isocratic/gradient MB with closed loop 86 Figure 8. Operation modes for gradient MB. 89 Figure 8. Experimental conditions and configuration in salt gradient ion-exchange MB with open loop. 96 Figure 8.4 Transient concentration profiles before next switch in salt gradient ion -exchange MB with open loop in the first full cycle. 98 Figure 8.5 oncentration profiles at half switch time with different cycles in salt XI

19 gradient ion-exchange MB with open loop. 99 Figure 8.6 yclic steady state internal concentration profiles during a switch time interal in salt gradient ion-exchange MB with open loop. 0 Figure 8.7 yclic steady state internal concentration profiles during a switch time interal in salt gradient ion-exchange MB with open loop ( = 0. M and F = 0. M ). 0 Figure 8.8 imulation conditions and configurations of salt gradient ionexchange MB. 04 Figure 8.9 Internal concentration profiles at cycle steady state in salt gradient ion-exchange MB with open loop. 05 Figure 8.0 Internal concentration profiles at cycle steady state in salt gradient ion-exchange MB with closed loop. 06 Figure 8. Internal concentration profiles at cycle steady state in salt gradient ion-exchange MB with closed loop and a holding essel (V =0mL).07 Figure 8. Internal concentration profiles at the steady state in salt gradient ion-exchange MB with open loop and closed loop, respectiely, predicted by gradient TMB model. 09 Figure 8. Ion exchange equilibrium isotherms of BA and myoglobin on Q-epharose FF resin at room temperature.. Figure 8.4 Plots of log( q / ) ersus log( ) in the linear ion exchange equilibrium isotherm. Figure 8.5 BA and myoglobin breakthrough cures at arious Nal concentration Figure 8.6 Operating conditions and configuration in salt gradient ion-exchange MB with open loop. 9 Figure 8.7 BA and myoglobin separation with complete recoery of myoglobin in raffinate stream (ase ). 0 Figure 8.8 BA purification from myoglobin impurity without the recoery of myglobin (ase ). XII

20 List of tables Table. Langmuir equilibrium parameters for BA adsorption on B-6A inert core adsorbent (data from Zhang and un, 00) 7 Table 5. The model parameters used in the simulations based on the experimental data of Bruce and hase (00) 04 Table 5. Parametric sensitiity analysis to assess the impact of changes in P, k fk, Lk, and k on the breakthrough time at / 0 = in expanded beds. 08 Table 6. Experimental conditions and model parameters used for the simulation of the breakthrough cures in expanded beds. 40 Table 7. ome properties of treamline irect T I adsorbent. 64 Table 7. ome properties of BA and myoglobin. 64 Table 8. ome properties of Q-epharose-FF anion exchanger. 90 Table 8. eparation factor of BA to myoglobin by Q-epharose FF anion exchanger under linear adsorption equilibrium isotherm. 4 Table 8. ome constraints to the net fluxes for BA and myoglobin separation in salt gradient ion-exchange MB. 6 Table 8.4 Flow rate ratio range for the separation and purification of BA and myoglobin in salt gradient ion-exchange MB with open loop. 7 XIII

21 hapter Introduction.Introduction. Releance and Motiation Proteins a word deried from the Greek word Proteios, which means of the first rank was coined by Berzelius in 98 to stress the importance of this group of organic compounds. Proteins not only play an important action in biology, but also hae large potential applications in pharmaceuticals and therapeutics, food processing, textiles and leather goods, detergents, paper manufacturing. With the deelopment of molecular biology technologies, arious kinds of proteins can be prepared from upstream processes and from biological raw materials. Howeer, there exist arious proteins and contaminants in these source feedstocks, and the key issue is that proteins can be separated and purified efficiently from the sources materials, in order to reduce the production cost of the high purity protein. The deelopment of techniques and methods for proteins separation and purification has been an essential prerequisite for many of the adancements made in biotechnology. Most separation and purification protocols require more than one step to achiee the desired leel of protein purity. In Figure., a three step separation and purification strategy is presented by Amersham Biosciences, which included capture, intermediate separation and purification, and final polishing during a downstream protein separation and purification process. In the capture step the objecties are to isolate, concentrate and stabilize the target proteins. uring the intermediate separation and purification step the objecties are to remoe most of the bulk impurities, such as other proteins and nucleic acids, endotoxins and iruses. And in the polishing step most impurities hae already been remoed except for trace amounts or closely related substances. The objectie is to achiee

22 hapter Introduction final purity of protein. Figure. Preparation and the Three tep eparation and Purification trategy for the high purify protein production (Protein Purification Handbook from Amersham Biosciences). In the capture step, as primary recoery of proteins, the expanded bed adsorption (EBA) technology has been widely applied to capture proteins directly from crude unclarified source materials, such as, E. coli homogenate, yeast, fermentation, mammalian cell culture, milk, animal tissue extracts (hase, 994; Hjorth, 997). The expanded bed is designed in a way that the suspended adsorbent particles capture target protein molecules while cells, cell debris, particulate matter and contaminants pass through the column unhindered. After loading and washing, the bound proteins can be eluted by elution buffer and be concentrated in a small amount of elution solution, apart from the bulk impurities and contaminants in source materials. In the last decade, arious applications of EBA technology hae been reported from lab-scale to pilot-plant and large-scale production. uring the intermediate purification and final polishing steps, the techniques of the conentional elution chromatography hae been applied successfully. New challenge should be the application of simulated moing bed (MB) to the separation and purification of proteins. imulated moing bed (MB) chromatography is a continuous process, which for preparatie purposes can replace the discontinuous regime of elution chromatography.

23 hapter Introduction Furthermore, the countercurrent contact between fluid and solid phase used in MB chromatography maximizes the mass transfer driing force, leading to a significant reduction in mobile and stationary phase consumption when compared with elution chromatography (Broughton et al., 96; Juza et al., 000; Rodrigues et al., 004; Ruthen et al., 989). More significant improement is the deelopment of gradient MB technology for the separation and purification of proteins (Jensen et al., 000; Houwing et al., 00), where a step-wise gradient can be formed by introducing a solent mixture with a lower strength at the feed inlet port compared to the solent mixture introduced at the desorbent port, then the adsorbents hae a lower binding capacity to proteins in section I and section II to improe the desorption and hae a stronger binding capacity in section III, and IV to increase adsorption in MB chromatography (a four-zone MB). The solent consumption by gradient mode can be decreased significantly when compared with isocratic MB chromatography (Jensen et al, 000). Moreoer, when a gien feed is applied to gradient MB chromatography, the protein obtained from the extract stream can be enriched if protein has a medium or high solubility at the high solent strength, while the raffinate protein is not diluted at all.. Objecties and Outline The objecties of this thesis are the study of proteins adsorption kinetics in expanded bed and gradient MB chromatography. With specially designed adsorbents and columns, the adsorption behaior of the expanded bed is comparable to that of the fixed bed (hase, 994), but there still exists some differences between the expanded bed and the fixed bed. A mathematical model suitable to expanded bed adsorption process should be deeloped in order to efficiently optimize and design EBA process (Wright and Glasser, 00), which is one of our research objecties in this thesis. treamline direct T I is a new type of ion exchanger with multimodal functional group, which not only takes adantage of electrostatic interaction, but also takes adantage of hydrogen bond interaction and hydrophobic interactions to bind proteins in order to get a

24 hapter Introduction high binding capacity een in high ionic strength and salt concentration feedstocks (Johansson et al, 00). It will be a significant improement of EBA technology to capture proteins, such as BA and myoglobin, by an expanded bed packed with treamline irect T I adsorbents. In this thesis, relatie experimental researches will be reported. In an expanded bed, the adsorbents with a high-density matrix are used to form a stable expansion at high feed flow rates. Recently, some authors hae improed the design of the high-density matrix of adsorbent, by including a single heaier inert core material in the macroporous crossliked resin matrix, that is called pellicular adsorbent or inert core adsorbent (Palsson et al., 000; Theodossion et al., 00). The inert core adsorbents not only increase the particle density to form stable expansion at high feed flow rate in the expanded bed, but also reduce the protein diffusion resistance inside adsorbent due to shortening of the diffusion path, which made the adsorption behaior in EBA process more efficiently. In addition, the inert core adsorbents also are potential to use in the fast, high-performance liquid chromatography (HPL) due to reduce the intraparticle diffusion resistance for biological macromolecules (Kirkland et al., 000; Rodrigues, 997). In this thesis, some theoretical analysis about the protein adsorption kinetics on the inert core adsorbent will be addressed. The application of gradient MB technology to proteins separation and purification is more attractie. Up to now, the research is just underway, because of the expensie experimental research for practical proteins separation and purification by gradient MB chromatography; in published works, also authors used an equialent gradient TMB (true moing bed) model to simplify the simulation, and only linear adsorption was dealt with. In this thesis, we will begin our research with simulation, using a real gradient MB, to study the behaior of the separation of BA and myoglobin by salt gradient ion exchange MB chromatography. Outline of this thesis Protein adsorption behaiors on inert core adsorbents are studied theoretically in a batch adsorber (in hapter ) and in a fixed bed chromatography (in hapter and in 4

25 hapter Introduction hapter 4 where sorption kinetics is included). Mathematical models of EBA are deeloped in hapter 5. In hapter 6, protein adsorption in EBA is analyzed, and simulated results are tested against the experimental data of BA adsorption in a treamline 50 column packed with treamline EAE, and treamline irect T I, respectiely. In hapter 7, BA and myoglobin competitie adsorption/desorption in an expanded bed packed with streamline irect T I is studied experimentally, where treamline 50 column is packed with 00mL treamline irect T I adsorbents. In hapter 8, a detailed simulation, using a real gradient MB model, is carried out to study the behaior of the separation of BA and myoglobin by salt gradient ion exchange simulated moing bed chromatography. Finally, hapter 9 has the conclusions of this thesis and some suggestions for future work. References Broughton B and Gerhold G. (96). ontinuous sorption process employing fixed bed of sorbent and moing inlets and outlets. U.: Patent No.,985,589 hase HA Purification of proteins by adsorption chromatography in expanded beds. Trends Biotechnology : Hjorth R Expanded bed adsorption in industrial bioprocessing: Recent deelopments. Trends Biotechnology5: 0-5. Houwing J, an Hateren H, Billiet HAH, an der Wielen LAM. (00). Effect of salt gradients on the separation of dilute mixtures of proteins by ion-exchange in simulated moing beds. Journal of hromatography A,95, Jensen TB, Reijns TGP, Billiet HAH, an der Wielen LAM. (000). Noel simulated moing-bed method for reduced solent consumption. Journal of chromatography A, 87: Juza M, Mazzotti M, Morbidelli M. (000). imulated moing-bed chromatography and its application to chirotechnology. Trends in Biotechnology 8():

26 hapter Introduction Johansson BL., Belew M., Eriksson., Glad G., Lind O., Maloisel LJ., Norrman N. 00. Preparation and characterization of prototypes for multi-modal separation aimed for capture of positiely charged biomolecules at high-salt conditions. Journal of hromatography A 06: Kirkland, JJ., Truszkowski, FA., ilks, H. and Engel, G. (000). uperficially porous silica microspheres for fast high-performance liquid chromatography of macromolecules. Journal of hromatography A, 890, -. Palsson, E., Gustasson, PE. and Larsson, PO. (000). Pellicular expanded bed matrix suitable for high flow rates. Journal of hromatography A, 878, 7-5. Rodrigues, AE. (997). Permeable packings and perfusion chromatography in protein separation. Journal of chromatography B, 699: 47-6 Rodrigues AE, Pais L. (004). esign of MB chiral separations using the concept of separation olume. eparation cience and Technology,9(): Ruthen M, hing B. (989). ountercurrent and simulated countercurrent adsorption separation processes. hemical Engineering cience, 44(5): Theodossiou, I., Elsner, H., Thomas, ORT. and Hobley, TJ. (00). Fluidization and dispersion behaior of small high density pellicular expanded bed adsorbents. Journal of hromatography A, 964, Wright PR., Glasser BJ. 00. Modeling mass transfer and hydrodynamics in fluidized-bed adsorption of proteins. AIhE Journal, 47:

27 hapter Modeling separation of proteins by inert core adsorbent in batch adsorber. Modeling separation of proteins by inert core adsorbent in a batch adsorber * Adsorption/desorption kinetics of protein on the binding ligand of inert core adsorbent in a batch adsorber is analyzed theoretically for Langmuir isotherm coupled with the intraparticle diffusion and film mass transfer resistances. For the two limiting cases of Langmuir isotherm, there are analytical solutions. New analytical solutions are deried for Henry isotherm, and the analytical solution of shrinking core model is recommended for rectangular isotherm. The effects of the inert core radius, equilibrium constant, intraparticle diffusion and film mass transfer resistances on the time eolution of bulk concentration and particle radial profiles were inestigated. The applicable range of the analytical solution with rectangular isotherm is gien. A new method to estimate both film mass transfer coefficient, k f, and effectie pore diffusiity, Pe, from a single bulk concentration-time cure in batch adsorber is gien and tested with literature data for the adsorption of BA on B-6A inert core adsorbent. *This chapter is based on the paper by Li, P., Xiu, G. H. and Rodrigues, A. E., Modeling separation of proteins by inert core adsorbent in a batch adsorbent, hemical Engineering cience, 58, 6-7, 00. 7

28 hapter Modeling separation of proteins by inert core adsorbent in batch adsorber.. Introduction The process in which a target protein is directly captured from the crude feedstock, such as bacteria, yeast, and mammalian cell culture, is becoming important in biotechnology and pharmaceutical industry. Expanded-bed adsorption (EBA) is a single pass operation in which desired proteins are purified directly from crude, particulate containing feedstock, without the need for separate clarification, concentration and initial purification. The bed expansion increases bed oidage, which allows for unhindered passage of cells, cell debris and other particulates during application of crude feed to the column. Many successful processes using EBA hae been reported for protein purification directly from E. coli homogenate, yeast, fermentation, mammalian cell culture, milk, animal tissue extracts, etc. (Bertrand, ochet and artron, 998; Anspach et al., 999; Bruce and hase, 00; Ozyurt, Kirdar and Ulgen, 00; mith et al., 00). Recently, inert core adsorbents were deeloped to improe the separation performance of proteins in expanded bed (Griffith et al., 997; Voute et al., 999; Pai, Gondkar, and Lali, 000; Palsson, Gustasson, and Larsson, 000; Tong and un, 00; Jahanshahi et al., 00; Theodossiou et al., 00). For example, UpFront s FastLine adsorbents (UpFront hromatography A/, enmark) are based on highly cross-linked agarose beads with a central core of high density, e.g. glass or stainless steel; and ZA pellicular adsorbent matrix (Jahanshahi et al., 00) is a pellicular particle comprising a solid core (zirconia-silica) coated with a porous skin of 4% (w/) cross-linked agarose. These inert core adsorbents hae increased density by the incorporation of heaier inert core and are adequate for stable expansion at high flow rates in expanded bed. In addition, the protein diffusion resistance inside inert core adsorbent is effectiely reduced due to the shortening of the diffusion path. In the protein separation processes by elution chromatography, expanded-bed and simulated moing bed, the intraparticle diffusion resistance has significant influence on the operation performance of the process (Rodrigues, 997; Kirkland et al., 000); therefore, adopting this kind of binding ligand inert core adsorbent to decrease protein diffusion resistance will be attractie in proteins separation industry. Poroshell 00B-8 adsorbent (from Agilent technologies Inc., 00) is a 5µm particle with a thin layer of porous silica on a solid inert 8

29 hapter Modeling separation of proteins by inert core adsorbent in batch adsorber core, which has been used for fast, high-performance resolution proteins in liquid-chromatography (Kirkland et al., 000). Usually, the adsorption/desorption equilibrium and kinetics for the protein onto the adsorbent are ealuated in the batch absorber (Owen and hase, 999; Hunter and arta, 000; Zhang and un, 00); the measured model parameters (for example, adsorption equilibrium parameters and the effectie intraparticle diffusiity) are then used to predict the performance of industrial chromatography processes, such as elution chromatography, expanded-bed and simulated moing bed. Therefore, modeling separation of proteins by inert core adsorbent in a batch adsorber will be important, which is our objectie in this chapter. Although numerous models describing the kinetics of protein adsorption to porous particles in the batch adsorber hae been researched, their releance to the analysis of adsorption to inert core adsorbent is marginal due to their arious simplifying assumptions. For Langmuir isotherm, two limiting cases frequently occur in separation of proteins. One is the high faorable adsorption equilibrium or irreersible adsorption for the affinity adsorption with faorably selectie ligand on the adsorbent; the other one is the linear (Henry) adsorption equilibrium for the reersible adsorption of the low concentration protein from the diluted crude feedstock. For adsorption onto inert core adsorbent, analytical solutions were deried and recommended for linear (Henry) adsorption equilibrium and high faorable adsorption equilibrium (irreersible adsorption) where the intraparticle diffusion and film mass transfer resistances were both considered. We also deeloped the program package for the general mathematical model with orthogonal collocation method. A significant feature of this new model is that it furnishes an analytical solution to the problem for linear and rectangular adsorption isotherms, which is suitable in most cases for capturing proteins in dilute crude feedstocks. Intraparticle diffusiity measurements are usually performed ia batch uptake experiments. The experimental data of the bulk concentration profile are fitted with the simulation results of a suitable transport model to estimate the effectie intraparticle diffusiity. Although the stirring speed can be changed in batch adsorption experiment, the film mass transfer resistance is sometimes not negligible in many experimental systems; in 9

30 hapter Modeling separation of proteins by inert core adsorbent in batch adsorber those cases the estimation of the intraparticle diffusiity becomes more difficult. Geankopolis (98) gae a correlation to estimate a priori film mass transfer coefficient in stirred-tank experiments; such procedure was applied by Zhang and un (00) to estimate the intraparticle diffusiity of BA in B-6A inert core adsorbent from the bulk concentration profiles in batch adsorber. Another recommended method to determine both the intraparticle effectie diffusiity and the film mass transfer coefficient from a single bulk concentration-time cure in batch adosrber is the error maps technique (Kaguei, One, and Wakao, 989; Xiu et al., 99, 994). In this chapter, a new method is proposed on how to get both the intraparticle effectie diffusiity and the film mass transfer coefficient alues from a single bulk concentration-time cure in batch adsorber simultaneously from the slope of model dimensionless time τ ersus real experimental time t... Mathematical model and analytical solutions.. Mathematical model R P R Inert core hell Batch adsorber Inert core adsorbent Fig... Batch adsorber and inert core adsorbent The batch adsorber and the inert core adsorbent are shown in Fig... There are three steps inoled in the protein adsorption from the bulk solution into an adsorbent shell: (i) mass transfer from bulk liquid to the outer surface of the particles (film mass transfer resistance); (ii) diffusion through the pores of the adsorbent shell (intraparticle diffusion resistance) (Horstmann and hase, 989), and (iii) the protein binding to the pore-wall 0

31 hapter Modeling separation of proteins by inert core adsorbent in batch adsorber surface (surface-reaction resistance). Adsorption kinetic studies showed that the diffusion of proteins in the adsorbent matrix was the rate-controlling step during the protein adsorption process (Horstmann and hase, 989; kidmore, Horstmann, and hase, 990; hampluier and Kula, 99). In this work the liquid phase concentration of protein in the pore was assumed to be in equilibrium with the adsorbed phase concentration of protein at any radial position, and a pore diffusion model was employed to describe protein uptake kinetics. Intraparticle mass transfer occurs by diffusion in liquid-filled pores with a driing force expressed in terms of the pore fluid concentration gradient (Weaer and arta, 996; Wright, Muzzio, and Glasser, 998; Zhang and un, 00); a constant diffusiity was assumed. The other assumptions and simplifications are (handa and Rempel, 997, 999; Li, Xiu and Rodrigues, 00): () the adsorbent consists of an inert core, non-permeable to the protein solution; () the pore diffusion model will describe the protein transport in the outer shell layer; () the outer shell layer undergoes negligible swelling or shrinking during sorption. Mass balance of adsorbate for the liquid phase in batch adsorber: V L d dt i W A + ρ R p p k f [ i ( pi ) ] = 0 R= R p (.) where V L is the fluid olume in the adsorber, W A and ρ p are the mass and the apparent density of the adsorbent particle, respectiely, i is the adsorbate concentration in bulk phase, pi is the adsorbate concentration in adsorbent pore, R p is the radius of the adsorbent particle, R is the radial distance in adsorbent, t is the time, and k f is the film mass transfer coefficient. ρ [ R ρ + ( R R ) ρ ] R inert core of adsorbent, core of adsorbent. Mass balance in the adsorbent shell: p = p p, in which ρ is the density of the adsorbent shell, ρ is the density of R is the radius of the inert

32 hapter Modeling separation of proteins by inert core adsorbent in batch adsorber pi qˆ i pi + = pi ε + ρ pe ( R t t R R P ) (.) R R R where ε is porosity of the adsorbent shell, qˆ i is the amount adsorbed per kilogram of adsorbent shell, k f pe is the effectie pore diffusiity. The boundary conditions are: [ i ( pi ) ] pi R= R pe p R = (.) R= RP R pi R = R = 0 (.4) The initial conditions are: i i = i0, pi = 0, qˆ = qˆ i = 0 ( adsorption) ( ) ( desorption) i0 (.5) The adsorption isotherm is assumed to be of a Langmuir type q K m pi qˆ i = (.6) + K pi where q m and K are the maximum adsorbent capacity and equilibrium constant, respectiely. The following dimensionless ariables are introduced: pi c pi =, i0 i c i = (dimensionless fluid concentrations in pore and bulk phases) i0 qˆ i q i = (dimensionless adsorbed concentration in adsorbent shell) qˆ R P ( ) i0 R r = (dimensionless radial distance in adsorbent) t = (dimensionless contact time) pe τ ( + ξ m ) R pε ξ = qˆ is the capacity factor. where m ρ ( i0 ) ε i0 Model Eqs (.)-(.6) in dimensionless form become

33 hapter Modeling separation of proteins by inert core adsorbent in batch adsorber dc c i pi + N dτ r r= = 0 (.7) + ξ m dqi dc + ξ m pi c pi τ c = r pi c + r r pi ( r r ) (.8) c r pi r= = Bi [ c ( c ) ] i pi r= (.9) c r c c i i pi r = r =, cpi = 0, q i = 0 = 0 = ( adsorption) ( desorption) (.0) at τ = 0 (.) q i c pi = λ + λ ( ) c pi (.) where = ( + K ) λ is the dimensionless constant separation factor. i0 The other dimensionless model parameters are: k f R Bi = (Biot number) pe ( ) ε + ξ m W N = V ρ L p A (Loading ratio /( ) ) The loading ratio is the ratio of the number of moles retained in the adsorbent particle (pores + adsorbed) and the number of moles present initially in the bulk phase. The alue of the capacity factor ξ m is in the range 0 to r 4 0. In this paper, we take ξ m =.5 0 as an example from the literature (Zhang and un, 00). In fact, when ξ m 0, the term ( + ξ dq dc ) ( + ξ ) m i pi m in Eq. (.8) can be reduced to i dc pi dq. The model equations will be soled by orthogonal collocation, which transforms the system of partial differential equation (.8) into a system of ordinary differential equations of initial alue type. In this chapter, 0 radial collocation points for adsorbent were selected. These equations were integrated in the time domain using Gear s stiff ariable step integration routine.