Modern Media for Protein Separation and Purification

Transcription

1 Modern Media for Protein Separation and Purification 现代蛋白分离纯化技术 Jan-Christer Janson Department of Physical & Analytical Chemistry Surface Biotechnology Uppsala University Uppsala, Sweden General Requirements 基本要求 Design parameters to be controlled 需要控制的设计参数 Base matrix 基架 Ligand 配基 S Mechanical stability Chromatographic mode Selectivity Capacity Chemical stability 机械稳定性 Chemical stability Efficiency/Resolution Surface properties Pressure/flow properties 色谱模式 化学稳定性 选择性 柱效/分辨率 表面特性 载样量 化学稳定性 压力/流速特性 1

2 The Ligand 配基 The type of ligand determines the mode of chromatography. There is an optimal window for the ligand density for different applications. 配基的类型决定色谱的模式 有根据应用不同优化配基密度的优化窗口 HIC Reversed phase Ion exchange Affinity (Hydrophobicity) 疏水相互作用 (Hydrophobicity) 疏水相互作用 (Charge) 电荷 (Biorecognition) 生物识别 S Comparison of gel media 凝胶介质的比较 Agarose or synthetic polymer? 琼脂糖或合成的多聚物 Synthetic polymer 合成的多聚物 Medium porosity 5-7 % 中等孔度5 7 Sintered microbead structure 烧结的微珠结构 High rigidity 高硬度 Fragile 易碎的 2

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6 Monodisperse media 颗粒大小均一的填料 MiniBeads 3 m MonoBeads 1 m Micro-purification 微量纯化 Polishing 精细纯化 SURCE m Polishing 精细纯化 SURCE 3 3 m Intermediate steps 中度纯化 Agarose Gel Formation A polysaccharide composed of alternating galactose and anhydrogalactose residues, derived from red algae growing in tropical/subtropical oceans. Gracilaria verrucosa Agarose dissolves in boiling water and forms strong gels on cooling Porous agarose gel 6

7 Agarose Beads for Chromatography Preparation of Sepharose type of media by emulsification of an aqueous agarose solution in an oil phase. Additional chemical cross-linking Covalently cross-linked gel Sepharose type of bead 1 m to 2 m Why agarose for protein chromatography? 为何琼脂糖适合蛋白质色谱 High porosity 9-96 % 高孔度9-96% pen structure with connected rods 纤维连接成的开放结构 High connectivity 高连结性 High surface area 高表面积 Stable to NaH CIP 对在位的氢氧化钠洗涤稳定 Low non-specific adsorption 低非特异性吸附 Elastic 有弹性 7

8 Macro structure difference between agarose gel media and cross-linked dextran, SephadexTM. Agarose is a linear polysaccharide obtained from red sea weeds such as Gelidium and Gracilaria species. The unit is the disaccharide agarobiose. 8

9 Agarose melts at about 1ºC and solidifies at about 45 ºC. Upon cooling the polymer aggregates into double helix fiber structures. Pore radius as a function of agar(ose) gel concentration. 9

10 Transmission electron micrograph of a 4% agarose gel. Scanning electron micrograph of a 2% agarose gel. 1

11 Cross-linking of agarose by epichlorohydrin gives rise to glyceryl bridges between the individual galactan polymer chains. 由环氧氯丙烷交联的琼脂糖在半乳糖和聚合物链间形成甘油基桥 Flow velocity (cm/h) Sepharose 4 Fast Flow HF Agarose 35Agarose (MabSelect) New High Flow Pressure (bar) 11

12 Synthesis of the New High Flow agarose 高流速琼脂糖的合成 H CH 2H H H Neat agarose in solution. Chemical modification in solution. 化学修饰 R CH2H R H Modified agarose in solution. 修饰的琼脂糖 Mixing of modified agaroses in solution followed by gel setting and cross-linking. 混合修饰的琼脂糖 形成凝胶并交联 The New High Flow Agarose Concept 高流速琼脂糖概念 Significantly improved pressure/flow properties with maintained control over the porosity 保持孔度控制的同时 压力/流速特性明显提高 Sepharose 4 Fast Flow (High porosity 高孔度) MPa Sepharose 6 Fast Flow (Medium porosity 中等孔度) 8. New High Flow agarose 高流速琼脂糖 (High porosity 高孔度) Increasing flow rate 流速加快 12

13 Time line for the development of agarose gel based media for protein separation 用于蛋白质分离的琼脂糖凝胶介质的发展时间表 THE DEVELPMENT F AGARSE BASED MEDIA FR PRTEIN SEPARATIN STREAMLINE Sepharose Big Beads CNBr-Sepharose Sepharose Sepharose CL Sepharose FF Superose Past state Sepharose HP Superdex Present state Past conditions Sepharose XL New High Flow Agarose is used in Capto S, Q Capto MMC, Adhere MabSelect MabSelect SuRe 2 Future state Present conditions How to increase the binding capacity of chromatographic gel media? 如何提高色谱凝胶介质的载样量 13

14 Dextran as a surface extender 表面延伸剂 葡聚糖 - Increased equilibrium capacity 提高平衡容量 - Increased mass transfer 提高传质阻力 Unmodified agarose base matrix Dextran modified agarose base matrix 未修饰的琼脂糖基架 葡聚糖修饰的琼脂糖基架 Schematic drawing not at scale 示意图 不是按比例的 现代离子交换剂的示意结构 14

15 Capto Q and Capto S Strong ion exchanger S Ligand 配基 Sulphoethyl 磺酸乙烷 强离子交换剂 基 S3 + N Base matrix High flow agarose 9 µm (average) 基架 高流速琼脂糖 9 µm (平均) Surface extender Animal free dextran 表面延伸剂 葡聚糖 H Q Ligand 配基 Quaternary ammonium 季胺 15

16 Salt tolerant ion exchange media 耐受盐的离子交换介质 Most traditional IEC media do not bind proteins at high ionic strengths. 大多数传统的离子交换介质在高离子强度下不结合蛋白质 Hydrogen bond Hydrophobic Ionic etc Traditional ion exchange chromatography 传统的离子交换色谱 Multi-modal ion exchange chromatography 多模式离子交换色谱 Multimodal cation exchanger 多模式阳离子交换剂 Capto MMC + Base matrix High flow agarose 75 µm (average) 基架 高流速琼脂糖 75 µm 平均 MMC Ligand 配基 Multimodal weak cation 多模式弱阳离子 Multiple types of interactions 多种类型相互作用 ionic interaction 离子相互作用 hydrophobic interaction 疏水相互作用 hydrogen bonding 氢键 16

17 Effect of salt on binding capacity 盐对蛋白质结合能力的影响 4 QB (mg BSA/ml) [NaCl] mm Multimodal anion exchanger 多模式阴离子交换剂 Capto adhere H H + Base matrix Capto High flow agarose 75 µm (average) TM 基架 CaptoTM 高流速琼脂糖 75 µm (平均) + N H N-Benzyl-N-methylethanolamine N-苯甲基-N-甲基乙醇胺 Multimodal strong anion 多模式强阳离子交换剂 Multiple types of interactions 多种类型相互作用 ionic interaction 离子相互作用 hydrophobic interaction 疏水相互作用 17

18 Highly productive two step procedure for the purification of monoclonal antibodies 两步法纯化单克隆抗体 Cell culture 细胞培养 Cell removal 去除细胞 MabSelect SuRe Virus Inactivation & Filtration 病毒灭活和过滤 Capto adhere Pool for Final Filtration UF/DF Recombinant protein modifications Proteolytic cleavage Incorrect reading frame 2HN Aggregation S Heterogeneity in N or C terminus Misfolding, random disulfide bridges M C C ther modifications - glycosylation - phosphorylation - acylation CH N xidation of Methionine Deamidation of Asparagine and Glutamine These trace contaminants often require high resolution steps! 18

19 How to achieve the high resolution required in the polishing step? Aim in polishing: 1. Remove trace contaminants or closely related substances 2. btain high purity and yield 3. High throughput Introduction of Capto ImpRes Extension of the Capto platform into polishing: combining high resolution with high throughput. First products: Capto SP ImpRes and Capto Q ImpRes Strong cation/anion exchanger for late intermediate purification and polishing with high productivity. 19

20 Polishing step was the next bottleneck Sepharose Fast Flow platform updated with more rigid media for capture. Demands on media with high resolution and high throughput. Extension into polishing next step. Combining high-flow agarose technique with small bead size MabSelect Xtra MabSelect, MabSelect SuRe Sepharose 4 Fast Flow 8 Capto ImpRes Sepharose 6 Fast Flow Sepharose Big Beads Capto Q, S, DEAE Sepharose High Performance 4 Capto adhere,mmc * Not a complete map Average particle size [µm] Sepharose FF technology High flow agarose technology Characteristics of Capto SP ImpRes Matrix Ion exchange type Charged group Total ionic capacity Particle size (d5v) Flow velocity Dynamic binding capacity ph stability short term long term Working temperature Chemical stability Avoid High-flow agarose Strong cation, SP -CH2CH2CH2S mmol H+/ml medium µm Approx. 4 cm/h (<4 bar or.4 MPa) or approx. 3 cm/h (<3 bar or.3 MPa). Measured in a packed bed, 1 m diameter column, 2 cm bed height at 2 C using process buffers with the same viscosity as water > 95 mg BSA/ml medium >7 mg lysozyme/ml medium C to 3 C All commonly used aqueous buffers incl. 1 M NaH, 8 M urea, 6 M guanidine hydrochloride, 3% isopropanol, and 7% ethanol xidizing agents, cationic detergents 2

21 Characteristics of Capto Q ImpRes Matrix Ion exchange type Charged group Total ionic capacity Particle size (d5v) Flow velocity High-flow agarose Strong anion, Q -CH2N+(CH3) mmol Cl-/ml medium µm Approx. 4 cm/h (<4 bar or.4 MPa) or approx. 3 cm/h (<3 bar or.3 MPa). Measured in a packed bed, 1 m diameter column, 2 cm bed height at 2 C using process buffers with the same viscosity as water > 55 mg BSA/ml medium > 48 mg ß-lactoglobulin/ml medium Dynamic binding Capacity ph stability short term long term Working temperature Chemical stability Avoid C to 3 C all commonly used aqueous buffers incl. 1 M NaH, 8 M urea, 6 M guanidine hydrochloride, 3% isopropanol, and 7% ethanol xidizing agents, anionic detergents GE Healthcare cation exchangers High resolution: Capto SP ImpRes (36 44 μm) 13okt29SP2:1_UV1_28nm 13okt29SP2:1_Cond 13okt29SP2:1_Conc 13okt29SP2:1_Inject Capto SP ImpRes, SP Sepharose High Performance and SURCE 3S 13okt29SP2:1_Logbook mau Intermediate resolution: 1 SP Sepharose Fast Flow 8 6 High capacity: 4 Capto S and SP Sepharose XL SP Sepharose HP (34 μm) 13okt29SP1:1_UV1_28nm 13okt29SP1:1_Cond 13okt29SP1:1_Conc 13okt29SP1:1_Inject 5. ml SURCE 3S (3 μm) 16okt9SP2:1_UV1_28nm 13okt29SP1:1_Logbook mau mau okt9SP2:1_Cond 16okt9SP2:1_Conc 16okt9SP2:1_Inject Capto S (9 μm) SP Sepharose FF (9 μm) 16okt9SP2:1_Logbook SP Seph FF art177291lot366911:1_uv1_28nm SP Seph FF art177291lot366911:1_inject SP Seph FF art177291lot366911:1_cond SP Seph FF art177291lot366911:1_logbook 13okt29SP4:1_UV1_28nm SP Seph FF art177291lot366911:1_conc 13okt29SP4:1_Cond 13okt29SP4:1_Conc 13okt29SP4:1_Inject 13okt29SP4:1_Logbook mau mau ml ml ml ml 21

22 GE Healthcare anion exchangers High resolution: Capto Q ImpRes (36-44 μm) Q2B342:1_UV1_28nm Q2B342:1_Cond Q2B342:1_Conc Q2B342:1_Inject Capto Q ImpRes, Q Sepharose High Performance, and SURCE 3Q Q2B342:1_Logbook mau 5 4 Intermediate resolution: Q Sepharose Fast Flow 3 High capacity: 2 Capto Q and Q Sepharose XL Q Sepharose HP (34 μm) Q Seph HP lot14744:1_uv1_28nm Q Seph HP lot14744:1_logbook Q Seph HP lot14744:1_cond Q Seph HP lot14744:1_conc 5. ml SURCE 3Q (3 μm) Q Seph HP lot14744:1_inject mau Source 3 Q lot :1_UV1_28nm Source 3 Q lot :1_Logbook Source 3 Q lot :1_Cond Source 3 Q lot :1_Conc Q Sepharose FF (9 μm) Source 3 Q lot :1_Inject mau 6 5 Q Seph FF lot :1_UV1_28nm Q Seph FF lot :1_Logbook Q Seph FF lot :1_Cond Q Seph FF lot :1_Conc Capto Q (9 μm) Q Seph FF lot :1_Inject Capto Q art :1_uv1_28nm Capto Q art :1_logbook mau mau Capto Q art :1_cond Capto Q art :1_conc Capto Q art :1_inject ml ml ml ml High flow velocities high throughput Capto ImpRes Based on high-flow agarose technique used for all Capto products. High flow velocities for high throughput also at large scale. Two-fold increased pressure/flow properties compared with Sepharose High Performance. Pressure/flow specification: ~3 cm/h in 1 m diameter column, 2 cm bed height, below 3 bar Capto SP ImpRes and SP Sepharose High Performance in AxiChrom 3, 2 cm bed height 22

23 Thank you very much for your attention! 23