超滤原理及应用讲座PPT.ppt

刘毅 millipore china bioprocess 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,bioprocess 57%,life science 23%,lab water 20%,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,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-plus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。,millipore的产品在血制品生产中的应用,产物 albumin, hemoglobin (64kd) factor viii & factor ix immunoglobins (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,10,20,30,40,16 vaccines in 65 years,13 vaccines in 13 years,smallpox rabies typhoid,tuberculosis diptheria, pertussis, influenza tetanus, yellow fever, rubella, measles, etc.,hib, hepatitis a,b, meningococcus pneumococcal,mening c, apertussis, .,hillerman salk,pasteur ehrlich,我们的部分客户 aventis-pasteur, pfizer, bayer, solvay,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 kd 0.001 - 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,retentate 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 factor 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-increasing 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,0,10,20,30,40,50,60,70,80,90,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regenerated 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 made 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 biomax pes 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 cellulose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 overcome 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 thin 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,typical 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 their 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 ph ionic strength 离子强度 viscosity 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,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 establish 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 membranes 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/l prolonged 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 different 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 & other polyamides,130 - 200,sources: a.m.pitt j.paren.sci.tech. may 1987 pp 110-114, c. samy pharm.tech.intl. 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 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 retention 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 weight 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 the 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 be 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 cylinder mass 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 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: 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 any 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 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: 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/pressures 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 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: 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,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 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 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 process 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 n naocl (bleach) at 200 - 500 ppm active chlorine phosphoric acid at 0.1 n 0.1n磷酸,operation: cleaning 操作:清洗,cleaning procedures清洗步骤 add cleaning (cip) solution to tank 将清洗液加入罐中 be careful to mix thoroughly注意完全混匀 operate system in recycle mode使系统循环运行 retentate and permeate directed back to tank 截留液和透出液直接流回罐中 cleaning flows and pressures usually similar to processing flows and pressures 清洗的流速和压力通常和处理过程中相似,operation: steaming 操作:蒸汽灭菌,why steam:为什么蒸汽灭菌? to insure sterility of system 为了保证系统无菌 system should be clean prior to steaming 系统在蒸汽灭菌前应先清洗 steam temperature蒸汽温度 typically 121 - 131c prostak specifications prostak参数 maximum tmp of 2 psi during steaming 蒸汽灭菌中最大tmp为2psi maximum temperature 133c 最高温度为133 rated for 20 cycles at 126 c 126可灭菌达20次,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: water flux 操作:水通量,what is water flux (nwp)?什么是水通量? a measure of the ability of water to pass through