生物分离工程-双语版

1、Chapter 2 Steps in bioseparation processes 1 Introduction Basically three steps in bioseparation process(1) Cell disruption (2) Initial fractionation(3) High-resolution fractionation Effective separation processes for genetic engineering:(1) Stable(2) Pure(3) Homogeneous (4) Active form(5) High yiel

2、d1 IntroductionConsidering protein properties for bioseparation(1) size(2) charge(3) biological affinity(4) hydrophobicity-solubilityProducts from mammalian cell techniques(1) enzyme(2) hormones(3) nonantibody immunoregulators(4) monoclonal antibodies(5) tumor specific antigens1 IntroductionProtein

3、folding (1) short ranged interaction ( secondary structure )(2) gradual combination and reshuffling (重新组合） (subdomains, domains, and subunit assemblies)Second generation biotechnological products(1) larger molecules(2) more complex(3) more glycosylate(4) more sulphydryl bonds1 IntroductionInitial bi

4、oseparation importance(1) filtration(2) centrifugation(3) chromatography Classified heuristics（论据，规则）(1) unit operation methods(2) design step sequence (3) species (components properties)(4) composition 1 IntroductionThree factors of designing bioseparation processes(1) purity(2) cost(3) marketMater

5、ial state and choice of separation methodsMaterial secreted ( ultrafiltration, centrifugation)Not secreted material (cell disruption, solid-liquid separation)Material in liquid ( ultrafiltration, adsorption)Material in solid (extraction into aqueous solution)New and words Protacte lawsuits 诉讼Deleter

6、ious 有害的，有毒的Vexing 烦恼Hierarchic 分等级Subdomain 亚域Domains 蛋白质结构的域Subunit 亚单位Endoplasmic reticulum 内质网Disulfide bond 二硫键Academia 学术界Delicate 脆弱的Flowchart 流程图Cytoplasmic 细胞质的Periplasmic 外周胞质Osmotic 渗透 urea 脲Guanidine 胍II Product excretion from the cell or cell disruption Excreted protein advantage(1) con

7、tamination level low(2) easy downstream processing steps(3) not require cell disruption(4) avoid handling viscous fluids and proteinase release(5) facility extraction of products (6) signal peptide determines secretionCell disruption (1) energy-intensive(强烈） and violent(2) without destroying interes

8、t protein(3) contain(兼容） process ( bacteria, yeast, mammalian, plant, and insect sources)(4) be controlled(5) be validatedProducts requiring cell disruptionVaccines (tetanus(破伤风）, meningitis（脑膜炎)Glucokinase（葡萄糖激酶）Glycerokinase （甘油激酶）Invertase （转化酶）Toxin (enterotoxin)Subcellular components (mitochond

9、rai, chloroplast)Intracellular constituents (DNA, RNA preparation, and virus-like particles)Recombinant insulinRecombinant growth hormoneProtein A and G.Ideal large scale cell disrupter(1) disrupt tough organisms(2) mechanism of cell disruption should be well understood(3) sterilizable(4) containabl

10、e（兼容） and validatable(5) amenable to being made automatic(6) continuous and compact（紧凑的）(7) economicalCell disruption goalObjective function (monetary（金钱） value of product)Small scale cell disruption(1) ultrasonic(2) freeze thawing (熔化）(3) force cells through very small orifices at high pressure(4)

11、glass or porcelain ball millsLarge scale cell disruptionHomogenizer Structure (positive displacement pump, pistons, nozzle)Mechanisms (1) Shear(2) Cavitation （气穴）(3) impingement (冲击）Condition(1) concentration (4-175 g dry wt/liter)(2) pressure (30-95 MPa)(3) passes number (5time)(4) modeled by equat

12、ion (5) wild-type E. coli cells more difficult to disrupt (heat induction at 42C)Ln(1/(1-R)=KNPDelineating mechanisms of cell disruption(1) facilitate controlling(2) validating(3) reproducible(4) better design and optimize equipment(5) Turbulent eddies (湍流涡旋） Responsible for cell disruption oscillat

13、e)(6) shear stress, turbulence by impingementEnzymatic lysis cell disruption(1) a lytic protease (dissolve the outer part of the cell wall(2) endo-(l-3) glucanase（葡聚糖酶） to disintergrate glucan net(3) source（酶的来源） and culture system kinetic analysisA understanding the systemB designing the small and

14、large scale systems C cytophaga （吞噬细胞）system exhibits high protease activity, higher conversion and higher lysisrate at longer timeD oerskovia systems high glucanase low protease activity, contain some inhibitorsE an initial lag in yeast lysis by both enzymeF simple two step model ( cell mass solubi

15、lized, protein hydrolyzed to peptides)G Mishaelis-Menten kinetic equation predict cell wall lysisH production of food grade single cell protein New words and vocabularyAmylase 淀粉酶Modulate 调节Interferons 干扰素Viscous fluids 粘性流体Signal peptide 信号肽Carboxypeptidase 羧肽酶Alkaline phosphatase 碱性磷酸酯酶Glyceraldeh

16、yde-3-phosphate dehydrogenase 甘油醛-3-磷酸脱氢酶Reduced nicotinamide adenine 还原型尼克酰胺腺嘌呤Dinucleotide 二核苷Reductase 还原酶Hydrophobic 疏水的Hydrophilic 亲水的New words and vocabularies (2)Pathogens 病源体Ball mills 球磨机Resonance 共鸣，回声Vibration 振动， 摆动Dissipate 分散Pinpointing 最佳点,针尖Oscillate 振荡Candida utilis 假丝酵母Extrusion 喷出

17、Sapphire 兰宝石Jewel 宝石Thermocouple 热电偶Sleeve 袖子，套Hydrodynamic 流体力学Hydroxylase enzyme 羟化酶Synergistic 增效，协同作用Glucan 葡聚糖Morphology III initial fractionationPrimary isolation step(1) adsorption(2) extraction(3) precipitation(4) distillation(5) filtration (6) solvent evaporationRequirementSignificant volum

18、e reductionInterest product in solution(1) gradually remove impurities(2) increase product concentration(3) chose methods depends on scale and organism(4) washing stages followed by dewatering stepYeasts easy to deal with(1) 10 m in diameter (2) easily flocculated(3) centrifugation (4) filtration(5)

19、 cross-flow filtrationAnalysis disrupted bakers yeast suspensions(1) viscosity(2) density(3) sedimentationMeaning (1) minimize degradation cell wall following rupture(2) minimize cell fragment proportion(3) restrict viscosity increase(4) correlation between lab and pilot scale centrifuges or industr

20、y(5) estimate separation costs at an early stageFirst crude separation or final product dewater steps (1) adsorption(2) precipitation(3) membrane separation A cross flow filtration B microfiltration C ultrafiltration(4) liquid-liquid extraction Aqueous two-phase partition(1) polymer + salt , polymer

21、 + polymer(2) easy to scale up(3) biocompatible(4) rapid (with centrifugation)(5) purification and concentration of biological product(6) cell particles and nucleic acids removed(7) large volume reductionA adsorption(1) purify protein(2) difficult to removing particulate using packed or fixed beds(3

22、) particulate entrapped in voids of the beds(4) excessive pressure drops and deformation of adsorbent(5) expanded bed(6) fluidized bedExpanded bed adsorption(1) eliminate a step for removal of cell or debris(2) scale up simple (similar to packed bed)(3) combine clarification, concentration and purif

23、ication(4) could be used for viscose fluid(5) high biomass content of feedstock(6) expanded bed stabilized by wide distribution of particle size用吸附剂分离的抗生素New words and vocabularyColloidal （胶状的）Glycan （聚糖）Lysate （溶解物）Aerosol（悬浮微粒,气溶胶）Particulate （微粒）Fluidized bed （沸腾床）Streptomycin （链霉素）Novobiocin （新生

24、霉素）Annexin V （膜联蛋白一类被钙离子活化后可与膜磷脂结合的蛋白）Scout （侦察）Washing （洗涤）Elution （洗脱）New words and vocabularyReverse phase high pressure liquid phase chromatography (反相HPLC）Circumvented （包围）Hybridoma （杂合细胞，(淋巴细胞)杂交瘤）Penicillin （青霉素）Tetracycline （四环素）Cephalosporin C （头孢菌素C）Divinyl benzene （二乙烯苯）Styrene （苯乙烯）B mem

25、brane separationFunction : Clarification and sterilizing Problems (1) Solid-liquid interface adsorption(2) significant surface area (500cm2)Membrane materials (1) hydroxyl-modified hydrophilic polyamide (2) hydrophilic polyvinylidene fluoride (PVDF) 聚偏二乙烯氟化物）(3) nylon-66Interest protein (1) insulin

26、(100% recovery)(2) bovine serum albumin (BSA) 98% recovery(3) immunoglobulinMinimizing protein loss(1) low-binging protein membrane(2) dilute protein solution(3) expensive (4) examine prefilters and final filtersRemove of virus(1) thermal inactivation(2) chemical treatment(3) denaturant (urea)(4) -p

27、ropiolactone（丙内酯）-UV light exposure(5) membrane filtration A base on size B reproducible C predictable D complete E validatable F high protein recovery New words and vocabularyInsulinBovine serum albumin （牛血清蛋白）Immunoglobulin （免疫球蛋白）Scale-down （按比例缩减）Discernible（可辩别的）Steady state（稳态）Asymptotically （

28、渐近的）Low-binding protein membrane-propiolactone UV light （-丙内酯-紫外光）Monotonically（无变化的，单调的）Platelet （血小板）ImmunodiseaseHydraulic permeability （水渗透性）Filtration resistance（过滤阻力）Pure water permeability (PWP) 纯水透过性Vocabularies Domain disulfide bonds, signal peptide, turbulent eddies Aqueous two-phase parti

29、tioning expanded bed adsorption fluidized bed packed bed reverse-phase high pressure liquid-phase chromatography floculate biocompatible scaled-down steady-state langmuir isothermQuestions 1 Which advantages secreted proteins?2 Which differences between Mammalian cell proteins and microorganism prot

30、eins ?3 Which methods disrupting cell in laboratory4 What is the most important in early part of bioseparation ?5 What advantage have expanded bed adsorption by comparing with packed bed ?6 How to remove virus or virus-like in protein solution ?Chapter 2 SecC UltrafiltrationExample： antibodies produ

31、ction(1) antibodies applicationA diagnosis B affinity ligand (cytokines and blood-clotting factors)(2) membrane material (cellulose triacetate )(3) use A 50-100-fold concentration B minimizing shear forces C tangential and frame ultrafiltration system D gel polarization （浓差极化） reduced F acceptable f

32、lux rateDenaturation during ultrafiltration(1) conformational change(2) instability(3) immunogenicity (免疫原性）Influencing ultrafiltration factorsA electrostatic interaction (attraction, repulsion)B Hydrophobicity New words and vocabularyMandate （命令，要求）Diagnostics 诊断学Cytokines 细胞因子Blood clotting factor

33、s （凝血因子）Tangential plate 切向板，错流过滤板Undermine（破坏）Integrity （完整）Tryptic -casein peptides -酪蛋白肽Antihypertensive （抗高血压的）Immunomodulatory 免疫调节剂Plasmin 血纤维蛋白溶解酶Transmission 输送Detrimental 有害的Interfacial tension 表面张力Emulsion liquid membrane 乳状液膜Carrier 载体Diffusivity 扩散率D cross-flow filtrationAffinity cross-f

34、low filtration(1) combining bioadsorption and membrane in a single step(2) escort supports A chemically and mechanically stable B possess a large specific area C Negligible nonspecific adsorption D easily modifiable to incorporate ligands(3) high protein loss in washing stepNew words and vocabularie

35、sAffinity cross-flow filtrationEscort 簇Supernatant 上清液Monomolecular layerInnermost 内心的，最里面的Langmiur isothermGlutaradehydeSchiff basesSodium borohydrideMicroparticles E Emulsion liquid membrane Penicillin G extraction by emulsion liquid membraneApplicationConcentrate weak acids, bases, metal ion and

36、biomaterial Penicillin extraction (1) Carrier : amine(2) pH adjuster Na2CO3 at pH 5-8 , penicillin G stable(3) 9 times concentrationF ExtractionInsulin purification process(1) extraction by ethanol(2) acidification at pH 2(3) neutralization with calcium carbonate(4) vacuum concentration(5) salt prec

37、ipitation(6) reprecipitation by isoelectric point(7) gel filtration chromatography or ion exchange chromatographyDifficult information available(1) effect of different process variables(2) sequencing of different subprocesses(3) scale level of operationNew words and vocabulariesEmulsion liquid membr

38、ane 乳化液膜Amine 胺Carrier 载体Pancreas 胰腺Hyperglycemia 高血糖Diabetes mellitus 糖尿病Porcine origin 猪器官Isoelectric point 等电点Reverse-phase chromatography 反相层析Scarce 缺乏的，不足的Proprietary 所有权G precipitation Applying high resolution technique at earlier stage (1) reduce volume(2) reduce chemical consumptionAffinity

39、precipitation using chitosan as a ligandPrinciple Soluble ligand binding to target protein lead to precipitate(1) aggregate(2) heterobifunctional ligandsIsolating trypsin with STI chitosan Precipitation factors(1) salt and cosolvents (supersaturation, seed particles , crystallization, agglomeration-

40、lactalbumin separationWhey components : lactose, proteins , salts and residual fat(1) 1:1 binding calcium ion (-1A)(2) conformational change (unfolding transitions, surface hydrophobicity)(3) selectively separating lactalbumin(-1A) and - lactoglubulin at pH isoelectric point and above 50 (4) nonspec

41、ific precipitationAffinity precipitation using ligand modified phospholipid as a ligand carrierPrinciple Soluble ligand binding target protein to form precipitateHeterobifunctional ligand (binding target molecule and facilitate precipitation of liand-target protein)Biotin ligand binding avidin(1) ra

42、pid binding(2) slow hydrophobic interaction between nonpolar alkyl side chains of phospholipids(3) high yield and purity of avidinDrawbacks(1) difficult to obtain stable and inexpensive bis-ligands, polyligands and ligand carriers(2) adsorption capacity low(3) nonspecific adsorption degree high Affi

43、nity precipitation mechanismTwo mechanism (1) primary effect Protein cross-linking and forming network with ligand(2) second effect Solubility decrease of ligand carrier due to pH , temperature, and salt concentrationSTI immobilized on PLS as an affinity agent for trypsin precipitation(1) material A

44、 artificial solution containing BSA and trypsin B a crude pancreatic extract(2) salt precipitated PLS due to osmotic shock (3) PLS redispersed in solution after removing salt by centrifuge(4) PLS had large specific surface area and ligand coupling capacity(5) lack of nonspecific adsorption(6) 91% tr

45、ypsin coprecipitated in artificial solution and 87% trypsin recovered in precipitate by 0.01 M NaOH(7) STI-PLS repeated three times(8) Purification factor was about six (9) loss of binding capacity (93.3%, 87.7%, 83.3%)(10) 80% activity recovery and specific activity with 1X104 U/mg(11) precipitate

46、and redisperse 10 times by added saltNew words and vocabulariesAffinity precipitation 亲和沉淀Heterobifunctional ligand 异种双功能试剂Chitosan 壳聚糖Oligomeric 寡聚体的Soybean trypsin inhibitor 大豆蛋白酶抑制剂Chymotrypsin 胰凝乳蛋白酶，糜蛋白酶Supersaturation 过饱和-lactalbumin 乳白蛋白Whey protein 乳清蛋白Mammary gland 乳腺Trytophan 脯氨酸Transition

47、s 过渡Turbidity 混浊Surfactant 表面活性剂Phospholipids 磷脂Avidin 生物素结合蛋白Biotin 生物素Dimyristoylphosphatidylethanolamine 二肉豆蔻酰磷酸乙醇胺Octaethylene glycol mono-n-dodecyl-ether 八乙烯醇单十二烷基醚Hastened 促进，加速Polymerized liposome 聚合脂质体Redispers 再分散Steric hindrance 空间位阻H chromatographic proceduresLarge scale purification(1) y

48、ield(2) purity(3) operation scale(4) chromatography (limited extent in initial step)Example 2.11 Staphylococcal enterotoxin B purification using chromatography(1) Two ion exchange steps (2) One gel filtration step(3) high linear flow rates at low pressure 5 kP(4) culture supernatant A (centrifugatio

49、n, sterile filtration, adsorption). B content 0.4mg/L C protein content 11mg/ml D purity 0.004% E volume 400 LPharmacia column 370 KS purification(1) protein content from 11 to1.5mg/ml(2) SEB content from 0.00042 to 0.12 mg/ml(3) purification factor 2000Biological molecules product standards(1) DNA

50、10 pg/dose(2) no viruses(3) pyrogen 300 endotoxins IU/doseExample 2.12Divinylbenzene-polystyrene resin separation (1) analysis of thermodynamics using HPLC(2) linear adsorption isotherm above 293-313K(3) temperature-dependent adsorption isotherm constants S,H(4) pore size diameter(5) polarity of res

51、in(6) chromatography is only a choice for industrial separationNew words and vocabulariesStaphylococcal enterotoxin 葡萄球菌外毒素Methicillin 甲氧苯青霉素Pharmacia columnDose 剂量Pyrogen 热原Miniscule 小字的Divinylbenzene polystyrene copolymer 苯乙烯-二乙烯苯共聚物Aspartame 门冬氨酰苯丙氨酸甲酯（比蔗糖甜200倍的甜味剂）Phenylalanine 苯丙氨酸Aspartic acid

52、 天门冬氨酸Asparagine 天门冬素，天冬酰胺酸Adsorption isotherm 吸附等温线Homologues 类似物，同族体IV high-resolution fractionationPrevious purification steps (1) volume reduction(2) clarification (3) adjustment of crude extractChromatography steps(1) cation exchange(2) anion exchange(3) affinity chromatography(4) hydrophobic c

53、hromatographyA chromatography proceduresAffinity chromatography(1) nonspecific adsorption(2) expensive labile ligand(3) difficulties of adsorbent sterilization(4) biomimetic ligand(5) natural ligand expensive and decreased activity when immobilized.(6) pseudo-ligand(7) ligand leakage and product con

54、taminationLarge scale purification (1) remove low levels of impurities(2) reduce pyrogen(3) exchange into buffer systems suitable for formulation(4) gel filtration chromatography has low productivitySEB:Staphylococcal enterotoxin 葡萄球菌外毒素Final products analysis (1) SDS(single band)(2) gel filtration

55、(single peak)Developing large-scale purification step(1) new media(2) equipment(3) optimizationUltrafast HPLC(1) instrumentation A selectivity B sensitivity(2) resin A rapid association and dissociation B material geometry play significant role(3) obtain quantitative product recovery estimation(4) visualize wide variety of host cell impurities Posttranslational modification ( deamidation, proteolysis and gl