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1、刘毅MILLIPORE CHINABIOPROCESS DIVISION,超滤原理及应用,Who is Millipore?密理博简介,创建于1954年,总部位于美国麻省波士顿，世界最早的膜生产商之一。雇员超过6000人专注于生物/制药及生命科学领域分离纯化设备的领先者拥有超过6500种产品2006年销售额达到15亿美元,讲座主要内容,Millipore公司简介超滤技术原理与简单优化膜的结构和功能为分离工艺选择合适的膜超滤系统介绍切向流超滤的操作超滤技术在生物工程领域的应用,Who is Millipore?密理博简介,创建于1954年,总部位于美国麻省 波士顿，世界最早的膜生产商之一。雇员超
2、过5800人专注于生物/制药及生命科学领域分离纯化设备的领先者拥有超过6500种产品2006年销售额达到15亿美元,Millipore全球有7个符合ISO9000标准的制造工厂,密理博产品范围:从实验室研发到生物制药的分离纯化,细胞培养及环境监测,Bioprocess57%,Life Science23%,Lab Water20%,小针剂型产品,大输液产品,滴眼剂,生物制药,澄清和预过滤系统最优化的预过滤配置，从实验室到放大生产,MILLISTAK+传统的“大饼”型式,MILLISTAK+先进的Pod形式,Lab Plant,切向流超滤系统超滤系统、盒式膜包、膜从小试到生产,Lab Plant
3、,Pellicon 2 超滤膜实现了完全线性放大。,层析系统控制系统、层析柱、层析介质从小试到生产,Vantage L实验室级层析柱,Prosep亲和层析填料 Quickscale层析柱,Lab Plant,除菌过滤产品及除病毒过滤产品过滤芯，Disposable囊式过滤器、膜片从小试到生产,实验室用各种除菌，除病毒过滤器,Facility用呼吸器，空气过滤器,料液用囊式过滤器,炮筒式滤芯,第四代完整性测试仪,Lab Plant,全面的微生物监控解决方案,Equinox无菌检验系统 M Air T空气微生物检测仪 M Air T Compressed压缩空气取样系统 Milliflex-Plu
4、s液体过滤系统,新的整合，新的技术-Celliance,新的整合，新的技术-NovAseptic,NovaSeptum取样装置,新的整合，新的技术-Mobius,无菌工艺:已经有超过800亿支无菌产品使用我公司的Durapore产品（2005）,Millipore Products in MAb Processes Millipore的产品在重组单抗生产中的应用,Millipore TFF systems are used in 9 of the top 10 selling monoclonals.在目前上市销售的单抗前10位产品中有9个应用了Millipore TFF系统。,Millipo
5、re的产品在血制品生产中的应用,产物albumin,hemoglobin(64kD)factor VIII&factor IXimmunoglobins(antibodies)fibrinogen,plasminogen客户Bayer:Kogenate(rFVIII)Baxter:Recombinate(fFVIII)Wyeth:Benefix(rFIX)Biopure:(hemoglobin)Abbott:Abbokinase(urokinase),迅速发展的疫苗行业,3 vaccines in 125 years,1800,1925,1990,2002,Number of Vaccines
6、,10,20,30,40,16 vaccines in 65 years,13 Vaccines in 13 years,SmallpoxRabiesTyphoid,TuberculosisDiptheria,Pertussis,InfluenzaTetanus,Yellow Fever,Rubella,Measles,etc.,Hib,Hepatitis A,B,MeningococcusPneumococcal,Mening C,aPertussis,.,HillermanSalk,PasteurEhrlich,我们的部分客户Aventis-Pasteur,Pfizer,Bayer,Sol
7、vay,Wyeth,Merck,GSK等,密理博中国,1987年在中国开始设立办事处2000年在上海设立贸易有限公司在香港,上海,北京,广州,成都,沈阳等地都有办事处目前在中国的雇员超过100名（销售，维修，技术支持，工艺开发，系统工程师）密理博公司的服务领域包括：1.制药及生物工程领域 2.生命科学研究领域,Millipore 的技术人员队伍：,技术开发支持PDS工艺开发专员（process development specialist)工艺技术解决方案-加速上市！系统设计/制造服务高级系统设计工程师针对特殊工艺要求的设计专业的售后服务售后服务工程师(service engineer)验证支
8、持服务验证实验室（海外）应用开发实验室（上海）验证服务专员工艺验证服务（过滤工艺）质量验证服务（微生物控制）,产业规模,从实验室研发到大规模生产的顺利放大,实验规模,How to get from here to there?,Millipore 您的战略合作伙伴,膜分离过程,什么是超滤?,超滤的应用范围,A 除热原应用：Depyrogenation:缓冲液/动物细胞培养基的热原去除生物样品分离纯化终产物除热原中药除热原样品透析/缓冲液转换 B 生物样品的分离纯化/浓缩应用：Bioproduct Separation/Purification/Concentration:新药研发，小规模制备样
9、品单抗，基因工程表达产物的分离疫苗，活性要求高的生物样品分离除杂质超滤分离，浓缩的应用处理珍贵的生物大分子样品,C 生物工艺的终产物的透析缓冲液更换：Bioproduct Diafiltration Or Buffer exchange:生物样品（培养/发酵表达产物，生物提取产物）的透析样品上层析柱前或洗脱后的缓冲液更换，及透析微滤实验：（0.1/0.22/0.45/0.65um)D Micro-filtration Application:发酵菌体的浓缩收集/更换缓冲液/洗濯（E.coli,Yeast 等）菌体破碎后，澄清过滤 细胞培养液的澄清/减菌中药提取液的澄清分离微滤之后，便可直接上层
10、析柱！,超滤的应用范围,滤膜孔径分布,0.1 kD-2.0 kD,反渗透 纳滤 超滤 微滤,0.1-0.65 m,1 kD 1 000 kD0.001-0.10 um,过滤方式,普通过滤(NFF)滤芯形式或“死过滤”流向是垂直于过滤介质的所有的液体全部透过过滤介质颗粒被截留在过滤膜内部或表面,切向流过滤(TFF)交叉流动过滤流向是切向(平行)于过滤膜表面的一小部分液体透过过滤介质截留的颗粒从膜的表面被”扫除”,普通过滤(死端过滤),切向流过滤(错流过滤),切向流速,透过流速,溶液浓度 Cb,膜表面,膜表面浓度 Cw,料液浓度 Cb,膜表面,膜表面浓度 Cw,切向流过滤(TFF),膜表面,常规过
11、滤(NFF),超滤系统模型,Basic TFF System Layout超滤系统示意图,Permeate Flow透出液,Retentate Flow回流液,Membrane膜,Inlet Pressure进口压,Retentate Pressure回流压,Permeate Pressure透过压,Mixed Tank混合罐,CROSSFLOW,Bulk Concentration Cb溶液浓度,Wall Concentration Cw膜表面浓度,Feed Flow,Pump,Flush,Mixed,Diafiltration Buffer Exchange透析,缓冲溶液置换,Retent
12、ate Valve回流阀,Heat Exchanger热交换器,%Passage=透过率,permeate,x 100,%Retention=100-%Passage 截留率,基本超滤术语,Retentate 回流液Permeate 透过液%Passage 透过率%Retention 截留率,体积/升,面积/平方米,时间/小时,Litres/(Square Metre x Hour)or LMH 升/(平方米X小时),Flux:通量,基本超滤术语,Typical Fluxes Millipore超滤/微滤膜典型通量,Start,Finish,Volumetric Concentration F
13、actor 体积浓缩因子,基本超滤术语,Yield=product recovered at end of process 收率Minus losses 损失最小化loss to permeate/retentate 透过液/回流液中的损失adsorption to membrane 膜吸附unrecoverable product(hold up)无法回收的产物(死体积),Finish,基本超滤术语,Transmembrane Pressure跨膜压Pressure Drop压降,P=PF-PR=k.Q,基本超滤操作参数,Optimal crossflow is 3 l/min-increa
14、sing crossflow beyond this value doesnt improve flux significantly.,超滤参数优化-切向流速,Find Optimal Crossflow at Cinitial 优化的流速,Flux vs.TMP and Crossflow(Q),TMP,psi,Flux,lmh,超滤参数优化-TMP,Find Optimal TMP at one Crossflow 优化的TMP,Flux vs.TMP(at one Q),polarized,optimal TMP,unpolarized,0,10,20,30,40,50,60,70,80
15、,90,TMP,psi,0,10,20,30,40,50,60,Flux,lmh,膜的结构和功能,Regenerated Cellulose Composite 30 kD Membrane at 2014x,摘要,膜的结构和材质微孔过滤(MF)超滤(UF)反渗透/纳滤(RO/NF)膜的关键特性分离工艺膜的选择,膜的材质,微孔过滤膜聚醚砜Polyethersulfone再生纤维素Regenerated Cellulose聚偏二氟乙烯PVDF-polyvinylidene fluoride聚碳酸酯Polycarbonate尼龙Nylon超滤膜聚醚砜Polyethersulfone 再生纤维素Re
16、generated Cellulose聚偏二氟乙烯PVDF-polyvinylidene fluoride,膜的材质,物理性质可压性弹性韧性化学性质pH溶剂 化学兼容性热敏性质 温度,Membrane Functionality膜的功能,Capture mechanisms捕获机理Size Exclusion大小排除Surface capture/Entrapment表面捕获/内部捕获Adsorption吸附Particles are adsorbed into the membrane structure颗粒被吸附在膜上Porosity开孔率Defined by%of membrane ma
17、de up by the pores定义墨上开孔的比率The higher the porosity the higher the permeability高开孔率高通透率,Membrane Structure:UF Cross-sectional SEMs膜结构,Void Free BiomaxPES membranes无缺陷的聚砜膜Biomax,Conventional PES membranes with finger voids手指状的传统聚砜膜,PES Membranes聚砜膜,Regenerated Cellulose再生纤维素,Conventional Regenerated C
18、ellulose传统再生纤维素,Composite Regenerated Cellulose Ultracel复合再生纤维素 Ultracel,Membrane Structure:UF Cross-sectional SEMs膜结构:,降低成本-产物是最重要的因素!,Characteristics of UF Membran超滤膜的特性,Ultrafiltration Membranes超滤膜参数,Operating Pressure 操作压力0-100 psi(0-6.9 bar)Tighter pore structure requires higher pressures to ov
19、ercome the resistance of the membrane 紧密的膜孔结构可在高压下也可获得膜的截留率Pore Rating 孔径Based on retention of Globular protein markers for nominal pore size ratings相对孔径基于球状蛋白截留率的标定Single solute testing most common通常是单一溶液测试Mixed solute testing is more realistic but newer混合溶液测试更现实,但是较新Structure 结构Cast Membrane铸造膜Thi
20、n skin薄皮Asymmetric不对称Finger voids or void-free 手指状或无缺陷Non-composite or composite非复合或复合 Integrity Testing完整性测试Diffusion(Testing only detects gross defects)气体扩散,孔径定义-葡聚糖截留曲线,膜孔径选择与产物损失,截流率导致的产物损失受以下操作参数影响体积浓缩因子透析体积对于在回流液中的产物损失计算%Product Loss to filtrate=100*1-e(R-1)*(ln VCF+N)35倍的余量,孔径选择与产物损失产物在回流液,Ty
21、pical Membrane Retention Characteristics典型的膜截留特征,Selecting a Membrane for a Separation如何为分离工艺选择合适的膜?,Objective of the separation分离的目的Characteristics of the fluid and the solute 液体和溶质的性质Concentrations-initial and final浓度-初始和最后Stability/solubility 稳定性/可溶性Effective pore size有效孔径Processing sensitivity 过
22、程的敏感性Potential hazards 潜在的风险,Defining the Separation Objective 明确分离的目的,What is the product solution/suspension to be filtered?待过滤的产品溶液/悬浮液是什么?name,composition,properties,stability.名字.组成,性质,稳定性What must be removed from the product during filtration?过滤中什么必须被除去?solvent,other solutes,insolubles,and thei
23、r characteristics溶剂.其他溶液,不溶物和他们的性质What are the concentrations of product,solvent,impurities?产品,溶剂,杂质的浓度是多少?At what temperature should filtration be conducted?在多少温度下操作?,How much feed volume to be filtered?进料液有多少体积被滤过?What is the desired final product concentration,or product solution volume?最后产品的浓度或产
24、品溶液的体积要求是多少?Will the retentate need to be diafiltered?If so,what is the quantitative objective of Diafiltration?截留物质是否需要透析?如果是,透析的数量目标是什么?How much time is available for processing a single batch?处理一个批次需要多久?,Defining the Separation Objective 明确分离的目的,Understand the solvent了解溶剂pHIonic strength 离子强度Visc
25、osity 粘性Processing Conditions 过程条件Prefiltration/Pretreatment 预过滤/预处理Temperature 温度Time/Frequency 时间/频率Hardware restrictions 硬件限制Cleaning/Sanitizing chemical selection清洗/消毒化学物质的选择Personnel Restrictions人员的限制,Characteristics of the fluid and the solute 液体和溶质的性质,Some Membrane Selection Questions膜材料选择的问题
26、,截留孔径Solute size/characteristics 溶质的大小/性质Membrane performance 膜的表现溶剂的化学兼容性Solvent type/concentration溶剂的类型/浓度清洗化学物质相关的问题Compatibility/Exposure/Validation化学兼容性/暴露/验证膜的粘联/吸附性质Initial&final protein concentration is important初始&最后蛋白的浓度最重要产物的回收率/截留率Remember UF membranes are not totally retentive超滤膜不是全部截留E
27、stablish acceptable standards first 首先建立可接受的标准,Guidelines for Choosing a Polymeric Membrane选择膜材料的指导建议,Compare two types of UF membranes比较两种类型的超滤膜Cellulose(low protein binding,solvent resistant)纤维素(低蛋白吸附,耐有机溶剂)Polyethersulfone(broad pH range,oxidant resistant)聚醚砜(广泛的PH范围,抗氧化)Recommend Cellulose Membr
28、anes for:纤维素膜推荐用于:Solutions containing organic solvents溶液含有有机溶剂Dilute protein solutions(initial concentration 20g/l)浓缩蛋白浓度达20G/LProlonged exposure to NaOH or oxidants like chlorine延长NAOH或氧化剂如氯的耐受,膜的选择-Pellicon平板膜与中空纤维对比,Millipore推荐您在生物/制药领域超滤应用时使用Pellicon 板膜!,Approximate Protein Adsorption of Differ
29、ent Membrane Materials不同膜材质对蛋白的吸附,Membrane Material,Protein Adsorption(ug/cm2),Hydrophilized PVDF(Durapore,Viresolve),1-10,Regenerated Cellulose(PL series),1-35,Cellulose Acetate,40-50,Modified(low protein binding)Polyethersulfones(Biomax),10-125,Unmodified Polyethersulfones(PT series),150-200,Nylon
30、&other Polyamides,130-200,Sources:.May 1987 pp 110-114,C.Samy.Nov.1992 pp 38-48,Millipore in-house tests,Membrane Selection Polymer膜的选择-材料,Principle-the membrane should have minimal interaction with the solute原则-膜和溶质间有最小的相互作用select a membrane known to have low interaction 选择一种有最小相互作用的膜Benefit is low
31、 adsorption 低吸附的益处more consistent flux 更快的流速more consistent rejection/passage更高的截留率/通透量greater potential yield 更高的回收率easier cleaning 易于清洗Understand 必须了解basic solute characteristics 基本的溶质性质manufacturers product information产品生产信息solute concentration溶质浓度concentration factor 浓缩因子,Membrane Selection Rete
32、ntion膜的选择-截留,Principle-the membrane should be chosen to either pass or retain the solute原则-膜被选择因为溶质可通过膜或者被截留Clarification:the selected solute passes through the membrane and into the filtrate澄清:该溶质可以透过膜而进入滤出液Concentration:the solute remains in the retentate浓缩:溶质被截留Diafiltration:the low molecular wei
33、ght solutes pass through the membrane while the solute of interest remains in the retentate透析:小分子物质透过膜,而目标 大分子被截留Tools&Information 工具&信息Solute molecular weight 溶质的分子量大小Solute structure 溶质结构,Relative Flux for Different UF Devices各超滤设备的流通量,Conclusion总结,Tips要点Choose the membrane carefully仔细选择膜Select th
34、e membrane based on the separation process 在分离过程的基础上选择膜If retention is maximized and adsorption minimized you gain the greatest yields.如果截留率最大,吸附最低,那么你将获得最大的回收率,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试Buffer Condition缓冲液的条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 获得产
35、品CIP(SIP)在线清洗Water Flux 水通量Storage 保存,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试Buffer Condition缓冲液的条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 获得产品CIP(SIP)在线清洗Water Flux 水通量Storage 保存,Operation:Flushing操作:冲洗,Flushing is done to remove storage or cleaning solution 冲洗是为了
36、去除保存液或清洗剂Flushing fluid(water)can be recirculated back to tank or directed to drain冲洗液(水)可循环流回罐中或直接排掉Tips:技巧Use clean water 使用干净的水Flushing directly to drain is more efficient 直接排放的冲洗更有效率Flushing with warm water can help 温水冲洗效果较好,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试Buffer Condition缓冲液的
37、条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 获得产品CIP(SIP)在线清洗Water Flux 水通量Storage 保存,Operation:Integrity Testing操作:完整性测试,Why Integrity Test?为什么进行完整性测试?If filter is not integral,your separation will not be performed properly and product may be lost如果膜不是完整的,分离将不会表现良好而且产品可能会丢失May b
38、e required to satisfy regulatory concerns需要满足法规的要求Principle of Integrity Testing完整性测试的原理Wet the membrane completely with water用水完全润湿膜Measure air flow through the wetted membrane at low pressure(5-30 PSI)在低压下(5-30PSI)测量空气通过湿润膜的体积,Operation:Integrity Testing操作:完整性测试,Procedure步骤Clean if necessary 如果必要的
39、话清洗Flush Membranes to thoroughly wet 冲洗膜达到完全润湿Drain Membranes 排掉多余水Pressurize Feed/Retentate Side with Air 在进口/回流口用空气加压Allow Air Flow to Stabilize 空气流动稳定Measure Air Flow 测量空气的流量Capture Air in Inverted CylinderMass Flow Meter 用倒置的量筒测量空气的体积,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试Buffer Con
40、dition缓冲液的条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 获得产品CIP(SIP)在线清洗Water Flux 水通量Storage 保存,Operation:Buffer Conditioning操作:缓冲溶液的条件,What is Buffer Conditioning?什么是缓冲溶液的条件?Wetting the membrane with a buffer that is compatible with the feed solution湿润膜的缓冲溶液和进料的溶剂是兼容的Prevents an
41、y solutes from being precipitated or denatured when they contact the membrane当它们接触膜时,防止任何溶剂沉淀或变性How do I Condition the Membranes?我们怎样使膜达到要求的情况?Circulate an appropriate buffer through the membranes under pressure.在一定的压力下,选择一种适当的缓冲溶液来循环洗膜,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试Buffer Condi
42、tion缓冲液的条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 获得产品CIP(SIP)在线清洗Water Flux 水通量Storage 保存,Operation:Processing操作:工艺,Types of Processing处理的类型Concentration 浓缩Diafiltration 透析Procedures 步骤Fill tank with process fluid 用料液将罐注满Start pump and adjust system to recommended flows/press
43、ures 启动泵然后调节系统到建议的流速/压力Concentrate and diafilter per SOPs 在每个SOP下浓缩和透析Add diafiltration fluid where required在要求的地方加入透析液,Concentration浓缩,Concentration浓缩,Concentration浓缩,Concentration浓缩,Concentration浓缩,Concentration浓缩,Concentration浓缩,Concentration浓缩,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试B
44、uffer Condition缓冲液的条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 获得产品CIP(SIP)在线清洗Water Flux 水通量Storage 保存,Operation:Diafiltration操作:透析,Diafiltration is the addition of a fluid(buffer)to the feed tank during a UF operation while permeate is being removed from the system透析是当透出液被从系统中
45、去除,在料液罐中加入另外一种液体(缓冲溶液)In clarification applications it is done to improve removal of passing species在澄清的应用中,它是用来提高透出物的透出效率的In concentration applications it is done to change the buffer 在浓缩的应用中,它是用来置换缓冲溶液的,Diafiltration透析,Diafiltration透析,Diafiltration透析,Diafiltration透析,Diafiltration透析,Diafiltration透析
46、,Diafiltration透析,Diafiltration透析,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试Buffer Condition缓冲液的条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 回收产物CIP(SIP)在线清洗Water Flux 水通量Storage 保存,Operation:Product Recovery操作:获得产品,Product in Permeate 产品在透过液中Diafiltration washes product
47、through membrane 通过膜透析回收产品Product in Retentate 产品在回流液中Gravity drain 自重排放Pressure“blow-down”with drain 用压力”吹干”排空Plug flow rinse 活塞流冲出Put buffer in tank and push fluid out of system用缓冲溶液将料液顶出系统 Recirculated rinse 循环洗出Drain and then add buffer to tank 排干后将缓冲溶液加入罐中Recirculate buffer and collect 循环缓冲溶液然后
48、收集回收,Basic Operation超滤的基本操作,Flush 冲洗Integrity Test 完整性测试Buffer Condition缓冲液的条件Process-Conc.工艺-浓缩/澄清Process Diafiltration工艺-透析Recover Product 获得产品CIP(SIP)在线清洗(灭菌)Water Flux 水通量Storage 保存,Operation:Cleaning(CIP)操作:清洗(CIP),Why Clean?为什么要清洗?To remove product residue from the system 为了去除系统中产品的残余prevents
49、cross-contamination of batches防止批次之间的交叉污染To remove bioburden 去除生物负荷kill bacteria,mold,viruses 杀死细菌.霉菌,病毒To remove endotoxin 去除内毒素To restore membrane permeability 恢复膜的通透性ensure reproducible filtration from run to run保证每次运行后可重复过滤,Operation:Cleaning操作:清洗,Cleaning Agents清洗剂Must effectively remove proces
50、s material left in system必须有效地去除系统中料液的残留Must be compatible with materials of construction-membrane,module,system hardware必须与膜,膜具,系统硬件兼容Should be able to validate removal of cleaning agent from the system从系统中清洗剂去除可以被验证Commonly used agents通常使用的介质NaOH at 0.1-1.0 NNaOCl(bleach)at 200-500 ppm active chlo
