丙球蛋白制作过程

合集下载

静脉注射纯度级别人血浆丙种球蛋白(IgG)纯化流程

A chromatographic method forthe production of a humanimmunoglobulin G solution forintravenous useK. Tanaka, E. Sawatani, Divisão de Produção e Desenvolvimento Industrial,E.M. Shigueoka, Fundação Pró-Sangue Hemocentro de São Paulo, São Paulo, SP, BrasilT.C.X.B. Campos,H.C. Nakao, G.A. Dias,R.K. Fujita and F. ArashiroAbstractCorrespondence Immunoglobulin G (IgG) of excellent quality for intravenous use was Key wordsK. Tanaka obtained from the cryosupernatant of human plasma by a chromato-•Immunoglobulin G Divisão de Produção e productiongraphic method based on a mixture of ion-exchange, DEAE-SepharoseDesenvolvimento Industrial •Hemoderivate productionFF and arginine Sepharose 4B affinity chromatography and a finalFundação Pró-Sangue Hemocentro •Intravenous gammapurification step by Sephacryl S-300 HR gel filtration. The yield of 10de São Pauloglobulin productionexperimental batches produced was 3.5 g IgG per liter of plasma. AAv. Enéas C. Aguiar, 155, 1º andar•Industrial chromatography05403-000 São Paulo, SP solvent/detergent combination of 1% Tri (n-butyl) phosphate and 1%•Downstream processTriton X-100 was used to inactivate lipid-coated viruses. Analysis of the final product (5% liquid IgG) based on the mean for 10 batches Publication supported by FAPESP.showed 94% monomers, 5.5% dimers and 0.5% polymers and aggregates.Anticomplementary activity was 0.3 CH50/mg IgG and prekallikreinactivator levels were less than 5 IU/ml. Stability at 37o C for 30 days in theliquid state was satisfactory. IgG was stored in flasks (2.5Received April 28, 1998 Accepted August 18, 1998 g/flask) at 4 to 8o C. All the characteristics of the product wereconsistent with the requirements of the 1997 Pharmacopée Européenne.Introduction or purification by ion-exchange and gel filtrationchromatography (4,5). The IgG iso-Commercially available liquid or lyo-lated from human plasma in the 1940’s and philizedimmunoglobulins G (IgG) are pro-1950’s by the method of fractionation with duced from pooledhuman plasma from a cold ethanol of Cohn and Oncley (2,6,7) was large number of donors,usually more than suitable for intramuscular use and could not one thousand, so that a widevariety of anti-be administered intravenously because of bodies will be present in the product (1). undesirable effects due to the modifications Several production processes are employed, in the IgG molecule, such as aggregates re-most of them based on the method of Cohn sulting from the fractionation process with (2), i.e., fractionation with cold ethanol, with ethanol and other agents which increased its polyethyleneglycol (PEG) precipitation (3) anticomplement activity (8,9).Thus, the major goal of IgG producers was to develop methods that would guarantee intravenous tolerance, eliminating or preventing the aggregation of molecules without affecting the activity of the antibodies present in the IgG preparation. The first process for the production of intravenous(iv) IgG was based on treatment of IgG with a quantity of pepsin (10) or plasmin (11) that caused enzymatic cleavage of IgG molecules. This process, corresponding to the preparation of first-generation iv IgG, is today considered to be obsolete (9).Specific structural changes of IgG were introduced in the enzymatic methods to re-duce anticomplementary activity. The prod-ucts thus obtained usually have reduced in vivo survival times and their continuous use may cause an antigen response depending on the enzyme used (12). The second-gen-eration iv IgG consisted of chemically modi-fied preparations with more or less impaired Fc-related effector functions (9). From 1975 to 1989, the chemical modification of the protein was probably the most successful approach to the preparation of iv IgG (12). The reagents used for this modification range from ß-propiolactone (13) to reducing/alkylating agents (12,14), reducing/amidating agents (15) and reducing/sulfonating agents (16).Today, all of these preparations are being replaced with third-generation products con-taining intact IgG molecules which retain effector functions. The latest generation in-cludes preparations that are free of comple-ment-activating aggregates thanks to the use of small or trace amounts of pepsin, pH 4.0, PEG and bulk adsorption with ion-exchange gel (9). This development was encouraged by the fact that the chromatographic method provides a safe and effective iv IgG product meeting the 1982 requirements of the World Health Organization Committee (17). The choice of a third-generation preparation which exhibits all the functions of the IgG molecule will be determined on the basis of safety in terms of viral transmission and absence of contaminants. Furthermore, the preparation should contain a normal distri-bution of IgG subclass molecules and have a half-life after infusion (9) which is in the physiological range of 21 to 36 days. Material and MethodsProduction equipmentPharmacia liquid chromatography equip-ment was employed using a Bio Process controller (Uppsala, Sweden). The following columns were used: step 1, desalting, gel filtration on Sephadex G-25, 1 column, mo-del BPSS 400/600 (60 cm in height by 40 cm in diameter). Step 2, anion-exchange DEAE-Sepharose FF, 1 column, model PS-370/15 (15 cm in height and 37 cm in diameter). Step 3, affinity gel Arginine Sepharose 4B (40%) + anion-exchange DEAE-Sepharose FF (60%), 1 column, model PS-370/15 (15 cm in height by 37 cm in diameter) and cation-exchange CM-Sepharose FF, 1 column, model Index 200/500 (15 cm in height and 20 cm in diameter). Step 4, cation-exchange CM-Sepharose FF, 1 column, model Index 200/500. Step 5, gel filtration on Sephacryl S-300, 1 column, model BPG 200/ 950 (95 cm in height and 20 cm in diameter).Other instruments used were a tangential flow ultrafiltration Pellicon cassette system and a filtration system with a stainless steel sanitary filter holder, 293 mm in diameter (Millipore, Bedford, MA, USA), and a con-tinuous flow centrifuge model AG BKA-6 (Westphalia Separator, Oeld, Germany). Details about the buffer solutions are given in Table 1.Preparation of immunoglobulin G Approximately 1200 human plasma bagsstored at -30o C were thawed at a temperature of 2 to 4o C in order to form a 200-l pool. Thawed plasma was centrifuged at 2o C toBraz J Med Biol Res 31(11) 1998obtain a cryoprecipitate to be used for the production of factor VIII. The supernatant of the cryoprecipitate was cleared by filtration through a 30-S depth filter (Zeta Plus, Cuno, Meriden, CT, USA) and desalted with coarse Sephadex G-25 filtration gel on a BPSS 400/ 600 column using 5 mM sodium acetate as elution solution. The pH was adjusted to 5.2 with 1 M CH 3CO 2H and the protein solution (410 l) was allowed to stand overnight in a cold chamber at 4oC for euglobulin precipi tation. On the following day the preparation was centrifuged at 4oC to remove the pre cipitated euglobulins and the supernatant was cleared by filtration. The pH was readjusted to 5.2 with 1 M CH 3CO 2H and conductivity was adjusted to 1.4 mS/cm with NaCl. The sample was applied to a PS-370/ 15 column containing DEAE-Sepharose FF in order to separate gamma-globulin (unadsorbed) from other plasma proteins such as albumin, etc.The gamma-globulin fraction was submitted to chromatography for the preparation of IgG as described below. The albumin fraction was further purified by a chromatographic method (18). The pH of the gamma-globulin fraction (480 l) was adjusted to pH6.0 with 1 M NaOH and conductivity was adjusted to 1.4 mS/cm. The fraction was then applied to two columns, PS-370/15 and Index 200/500, coupled in series. The first column was packed with a mixture of two gels, 40% arginine Sepharose 4B and 60% DEAE-Sepharose FF, and the second with 8 l of CM-Sepharose FF gel.Arginine + DEAE-Sepharose and CM-Sepharose FF Equilibration 20 mM sodium acetate, pH 6.0, and conductivity 1.4 mS/cm Elution of arginine + DEAE-Sepharose FF 0.5 M sodium acetate, pH 7.0, and conductivity >20 mS/cm Elution of CM-Sepharose FF 1.0 M NaCl, pH 7.0 Cleaning and storage 0.5 M NaOH and 10 mM NaOHCM-Sepharose FF (after virusinactivation) Equilibration 10 mMglycine, pH 7.0 Elution 0.1 M glycine + 0.15 M NaCl, pH 9.0 Cleaning and storage 0.5 M NaOH and 10 mM NaOHSephacryl S-300 HR 1st Equilibration 0.1 M sodium acetate + 50 mM NaCl, pH 6.0 2nd Equilibration 0.15 M NaCl, pH 6.0 Elution with2nd equilibration buffer Cleaning and storage 0.5 M NaOH and 10 mM NaOHTable 1 - Buffer solutions used. Sephadex G-25 C Equilibration 5 mM sodium acetate, pH 7.0, and conductivity 0.37 mS/cmElutionwith equilibration buffer Cleaning and storage 0.5 M NaOH and 10 mM NaOHDEAE-Sepharose FFEquilibration 20 mM sodium acetate, pH 5.2, and conductivity 1.4 mS/cm1st Elution with equilibration buffer2nd Elution25 mM sodium acetate, pH 4.5, and conductivity 1.75 mS/cmThe proteins not of interest for the presentstudy, IgM, IgA, transferrin, etc, wereadsorbed to the first column. IgG wasadsorbed to the second column and eluted andconcentrated to 5% (w/v) using the-Thawing 4o CCryoprecipitate-CentrifugationCryosupernatantDesalting on Sephadex G-25 CSephadex fraction -Euglobulin precipitation at pH 5.2 -Kept overnight at 4o C -Centrifugation -Filtration SupernatantAlbumin fraction -Purification Unadsorbed + Gamma-globulin 60% DEAE-Sepharose FF 40% Arginine-Sepharose 4 BUnadsorbed protein fractionCM-Sepharose FFIgG -Concentration to 5% (w/ v) -S/D Virus inactivationCM-Sepharose FFIgG without S/D-Enzyme treatment (0.1 mg pepsin/g of IgG, pH 4) -Temperature 37o C/12 h -pH adjustment to 6.0 with 1.0 M NaOHSephacryl S-300IgG without enzyme or polymers-Concentration to 7% (w/v) -Formulation-Sterilization by filtrationFigure 1 - Flow diagram for the production of liquid intravenous IgG.Pellicon Cassette System 30,000 NMWL. The pH of the IgG solution (16 l) was adjusted to 5.5 and the material was submitted to viral inactivation with a solvent/detergent combination (19), i.e., 1% Tri (n-butyl) phosphate and 1% Triton X-100 at 35o C for 10 h. After viral inactivation, the solvent/deter-gent combination was removed by ion-ex-change chromatography on CM-Sepharose FF in an Index 200/500 column containing 8 l of gel. The IgG solution was concentrated to 7%, pepsin was added (0.1 mg/g protein), pH 4.0, and the solution was heated at 37o C for 10 h. The preparation was cooled to 20o C and then applied to a BPG 200/950 column containing 20 l of Sephacryl S-300 HR fil tration gel to remove the aggregated IgG molecules and pepsin. The eluted IgG solution was concentrated to 6.5% and then formulated as follows: the pH was adjusted to5.0 with 1 N HCl, the conductivity to 9 to 10 mS/cm with solid NaCl, and 7.5% (w/v) maltose and 0.1 M glycine were added as stabilizers (12,20). The material was steril-ized by filtration through a 0.22-µm mem-brane (Millipore) and the final product (14 l), containing 5% protein (w/v), was bottled in 50-ml type I (neutral) flasks, with 2.5 g IgG per flask and was stored at 4 to 8o C (see Figure 1).Analytical methodsThe characteristics of normal liquid 5% IgG for intravenous use were evaluated by the methods described in the Pharmacopée Européenne (1) and in Regulation No. 2.419 of 12/17/1996 of the Brazilian Ministry of Health.The immunochemical tests were carried out by cellulose acetate electrophoresis, by the micro-Ouchterlony method and by im-munoelectrophoresis using anti-total human protein and anti-animal protein antisera of domestic species such as horses, cows and sheep. Protein concentration was determined by the biuret method, pH was measured by Braz J Med Biol Res 31(11) 1998diluting to 1% in a 0.9% sodium chloride solution, and anti-A and anti-B hemaggluti-nins were determined by the indirect Coombs method. The presence of other plasma proteins such as IgA and IgM and of the IgG subclasses IgG1, IgG2, IgG3 and IgG4 was determined by radial immunodiffusion on Bindarid plates (The Binding Site Inc., San Diego, CA, USA).Anticomplementary activity was deter-mined by the method of Mayer using guinea pig complement and sheep red cells. The prekallikrein activator was determined using the S2302 chromogenic substrate of Chro-mogenix (Mölndal, Sweden).The biological safety of the product was evaluated by tests for the detection of anti-HIV, anti-HTLV-1 and 2, anti-HCV, and HB antigens at the Serology Laboratory of Fundação Pró-Sangue Hemocentro de São Paulo. Sterility was evaluated by the Steritest membrane method (Sterility Testing System, Millipore). Pyrogenicity and toxicity were tested at Medlab, São Paulo, Brazil.The distribution of monomers, dimers and polymers was evaluated by HPLC, and anti-polio I, II and III, anti-measles and anti-herpes activities were determined at Instituto Adolfo Lutz, São Paulo, Brazil. Anti-rubeola, anti-CMV and anti-streptolysin O activities were determined at the Immunology Laboratory of IAMSPE, São Paulo, Brazil. Stability was evaluated by incubating the preparation at 57o C for 4 h and observing the presence of jelling.Results and DiscussionThe size distribution of the product, evaluated by HPLC, indicated 94% monomers, 5.5% dimers and 0.5% polymers, corresponding to standard values. The profile of the IgG molecule did not show any alterations when the IgG preparation was incubated at 37o C for one month; the anticomplementary activ ity was 0.3 CH50/mg IgG, in agreement with the specifications that determine a value of less than 1 CH50/mg IgG, and the functional activity of Fc was unchanged, presenting an index of 125 to 128% of Fc, meeting the recommendationof Pharmacopée Européenne, whichrequires >60%. On the basis of these evaluations,we concluded that the product contained intactmolecules. The subclasses determined were IgG1= 65.6%, IgG2 = 28.7%, IgG3 = 4.2%, and IgG4= 1.4%, with no significant variations comparedto normal plasma. For prekallikrein activator(PKA) determination we used a reference PKAfrom the FDA and the S2302 chromogenicsubstrate of Chromogenix. Values of less than 5IU/ml 5% (w/v) IgG were obtained, well belowthe specification limit of 35 IU/ml of PKA. Theremaining data which characterize the productare presented in Tables 2 and 3, and celluloseacetateTable 2 - Characteristics of liquid intravenous IgG.N = 10. ND, Not detected. *Pharmacopée Européenne(1).Table 1 - Buffer solutions used.Sephadex G-25 CEquilibration 5 mM sodium acetate, pH 7.0, and conductivity 0.37mS/cmElution with equilibration bufferCleaning and storage 0.5 M NaOH and 10 mM NaOHDEAE-Sepharose FFEquilibration 20 mM sodium acetate, pH 5.2, and conductivity 1.4mS/cm1st Elution with equilibration buffer2nd Elution 25 mM sodium acetate, pH 4.5, and conductivity 1.75mS/cm3rd Elution 0.15 M sodium acetate, pH 4.0, and conductivity 8.0mS/cmCleaning and storage 0.5 M NaOH and 10 mM NaOHAnalysis Results Specifications*Protein concentration 5.0 ±0.2% >90 to <110%pH determination 4.5-5.0 4.0-7.4PKA determination <5.0 IU/ml <35 IU/mlPurity (electrophoresis) >99% >95%IgA ND -IgM ND -Anti-A hemagglutinin ≤1:8 <1:64Anti-B hemagglutinin ≤1:8 <1:64 Anticomplementaryactivity<0.3 CH50/mg IgG <1 CH50/mg IgGAluminum <20 µg/ml -Fc function 125-128% >60%electrophoresis and immunoelectrophoresis data are shown in Figures 2 and 3, respectively.We describe the preparation of intravenous 5% immunoglobulins in the liquid state from a pool of human plasma from 1200 donors by the chromatographic method. The analysesperformed for quality control showed that the IgG met international specifications. In thestability test involving heat-Table 3 - Antibodies detected in liquid intravenous IgG.N = 10.Antibody Method Titer and/or uniting to 57o C for 4 h there was no jelling, and in the quarantine test involving incubation in an oven at 37o C for 4 weeks, again there were no alterations in the IgG molecule.Viral inactivation was performed with a solvent/detergent system (19), i.e., 1% Tri (n-butyl) phosphate and 1% Triton X-100, pH 5.5, at 35o C for 10 h, which was then removed by the ion-exchange gel, CM-Sepharose FF. The protein aggregates gener ated during the process were removed by gel filtration chromatography through Sephacryl S-300 HR. Thus, the product obtained consisted mainly of the monomer, presenting only trace amounts of polymers, consistent with low anticomplementary activity, i.e.,less than 1 CH50/mg IgG.Anti-polio I Neutralization test 1:256 Anti-polio II Neutralization test 1:128 Of the several stabilizing agents tested,Anti-polio III Neutralization test 1:64 glucose, sucrose and maltose, the last one at Anti-measles Hemagglutination inhibition 1:64a 7.5% concentration, and 0.1 M glycine, pH Anti-rubeola ELISA 756 IU/ml5.0 (12,20), were found to be the most ap Anti-herpes Immunofluorescence 1:128 Anti-cytomegalovirus ELISA 898 IU/ml propriate. The IgG solution was limpid andAnti-streptolysin O Nephelometry 716 IU/mltransparent with no detectable molecular al-terations even when stored for more than 2years at 5 to 8o C.On the basis of in vitro and in vivo laboratorytests, we conclude that the product fullysatisfies all the requirements of thePharmacopée Européenne (1), as well as thenorms of the Brazilian Ministry of Health(Regulation No. 2419 of December 17, 1996).We are awaiting the results of clinical testscurrently underway to liberate the productfor use.Acknowledgments009 009Figure 2 - Cellulose acetate electrophoresis. a, Plasma pool (25 µl of 10 g/l); b, immunoglo-The authors wish to thank Dr. Hiroyoshi bulin G (25 µl of 30 g/l). Protein was detected with Ponceau S and the data are reported asIto from the Japanese Red Cross, Chitose, percent of total densitometer units.Hokkaido, Dr. Komei Ohashi fromKaketsuken, Kumamoto, and Dr. KentaroNakamura from Green Cross, Osaka, forFigure 3 - Immunoelectrophoretheir helpful suggestions and the opportunitysis. a , Plasma pool (2 µl of 10 g/ l); b , immunoglobulin G (2 µl ofof training at theirPlasmaFractionation a30 g/l). Protein was detected Plants inJapan, and Dr. Erica Kitahara, withlight green stain.bPharmacia Biotech, forsupplying the reagents.Braz J Med Biol Res 31(11) 199814. Pappenhagen A, Lundblad J & Schroeder D (1975). Pharmaceutical compositionscomprising intravenously injectable modified serum globulin, its production and use. US Patent No. 3,903,262.15. Schmidtberger R (1978). Amidated immune globulins and process for preparing them. US Patent No. 4,118,379.16. Masuho Y, Tomibes S, Matsuzawa K & Ohtdu A (1977). Development of an intravenous gamma-globulin with Fc activities.I. Preparation and characterization of S-sulfonated human gamma-globulin. VoxSanguinis , 32: 175-181.17. WHO Expert Committee on Biological Standardization (1982). Report of an informal meeting on intravenous immunoglobulins (human), Geneva.18. Tanaka K, Sawatani E, Nakao HC, Dias GA & Arashiro F (1996). An alternative column chromatographic process for the productionof human albumin. Brazilian Journal of Medical and Biological Research , 29: 185-191.19. Horowitz B, Wieb ME, Lippin A & Stryker H (1985). Inactivation of viruses in labile blood derivatives. Transfusion , 25: 516522. 20. McCue JP, Hein RH & Tendold R (1986). Three generations of immunoglobulin G preparations for clinical use. Reviews of InfectiousDiseases , 8 (Suppl 4): 374-381.Table 1 - Buffer solutions used. Sephadex G-25 C Equilibration 5 mM sodium acetate, pH 7.0, and conductivity 0.37 mS/cmElutionwith equilibration buffer Cleaning and storage 0.5 M NaOH and 10 mM NaOHDEAE-Sepharose FFEquilibration 20 mM sodium acetate, pH 5.2, and conductivity 1.4 mS/cm1st Elution with equilibration buffer2nd Elution 25 mM sodium acetate, pH 4.5, and conductivity 1.75 mS/cm3rd Elution0.15 M sodium acetate, pH 4.0, and conductivity 8.0 mS/cmCleaning and storage 0.5 M NaOH and 10 mM NaOH AnalysisResults Specifications* Protein concentration 5.0 ± 0.2% >90 to <110% pH determination 4.5-5.0 4.0-7.4 PKA determination <5.0 IU/ml <35 IU/ml Purity (electrophoresis) >99% >95% IgA ND - IgMND - Anti-A hemagglutinin ≤1:8 <1:64 Anti-B hemagglutinin ≤1:8<1:64Anticomplementary activity <0.3 CH 50/mg IgG <1 CH 50/mg IgG Aluminum <20 µg/ml - Fc function125-128%>60%Molecular distributionMonomers 94.0% mono + dimers = 90%Dimers5.5%A PRIVATE BRAZILIAN BIOTECHNOLOGY COMPANYLEADER IN THE BRAZILIAN INSULINMARKETDO YOU KNOW OUR COMPANY?If you don't know us or, if you already know us, did you realize we do all that? Call us! It will be a pleasure toserve you. BIOBRÁS S.A. Fax: 55-31-335-79-88。

丙种球蛋白的电泳制备(圆盘电泳)

• 实验室中常用到的电泳方法（以介质分类）
• 纸电泳 • 醋酸纤维膜电泳琼脂糖凝胶电泳 • 凝胶电泳聚丙烯酰胺凝胶聚丙烯酰胺凝胶电泳
• 聚丙烯酰胺凝胶是由单体丙烯酰胺（Acr）和交联剂N，N甲叉双丙烯酰胺（Bis）在加速剂和催化剂的作用下聚合，并联结成三维网状结构的凝胶，以此凝胶为支持物的电泳称为聚丙烯酰胺凝胶电泳（PAGE）。 • 类型：垂直板电泳、圆盘电泳、等点聚焦电泳、双向电泳等。（本实训中选择的是圆盘电泳） • 作用：用于分离蛋白质和寡核苷酸。 • 作用原理：聚丙烯酰胺凝胶为网状结构，具有分子筛效应。
制备丙种球蛋白的方法
• 丙种球蛋白的两种分离制备的方法 • 电泳法（如：聚丙烯酰胺凝胶电泳法） • 定义：利用溶液中带有不同量的电荷的阳离子或阴离子，在外加电场中以不同的迁移速度向电极移动，而达到分离目的的分析方法。 • 层析法（如：SephadexG-50) • 定义：是一种利用混合物中诸组分在两相间的分配原理以获得分离的方法。
操作步骤
1.储备液配置 A液：取1mol/L HCl 48ml,Tris 36.6g,TEMED 0.24ml,混匀。用稀得 HCl调节pH至8.9，加去离子水至100ml.浑浊可过滤。 B液：取1mol/L HCl 24 ml,Tris 3.0g,TEMED 0.24ml，混匀。用稀得 HCl调节pH至6.7，加去离子水至50ml,浑浊可过滤。 C液：取Acr. 14.0 g,Bis. 0.37g,加去离子水溶解后定溶于50ml，过滤。 D液：取Acr. 14.0 g,Bis. 0.37 g,加去离子水溶解后定容至50ml,过滤。 E液：40%蔗糖。 F液：新配制的0.14%过硫酸铵。以上储备液可以提前配制，冰箱保存备用。其中A、B、C、D液可用2个月，F液可用一周。

丙种球蛋白的制备综述

丙种球蛋白的电泳制备
—预习方案
指导老师：韦平和彭佳平
组别：第二组
成员：张婉月臧赢徐洁严梦迎
陈孝颜
丙种球蛋白概念
• 中文简称：“丙球” 英文名称：γ-globulin、 gamma globulin 别名：免疫血清球蛋白，普通免疫球蛋白，人血丙种球蛋白，丙种球蛋白，静脉注射用人免疫球蛋白（pH4）丙种球蛋白预防传染性肝炎，预防麻疹等病毒性疾病感染，治疗先天性丙种球蛋白缺乏症，与抗生素合并使用，可提高对某些严重细菌性和病毒性疾病感染的疗效。注：由于人血中的免疫球蛋白大多数为丙种球蛋白（γ-球蛋白），有时丙种球蛋白也被混称为 “免疫球蛋白”(immunoglobulin) 。
• 垂直平板： 1多个样品在同一块凝胶上，电泳条件一致－－便于利用各种鉴定方法，直接比较各种样品区带，保证结果的准确可靠 2可以进行双向电泳 3电泳时热量容易消散，凝胶结果便于照相和易于制成干胶圆盘电泳： 1、缺点是由于聚合效应，凝胶的长度和直径有细微差别，即使同一样品在两个凝胶柱上以同样的条件电泳，其结果也会不同 2、但是研究工作中还会用到圆盘电泳，例如测定放射性标记的样品测定蛋白质的生物活性；测定蛋白质分离的最适pH，凝胶浓度；双向电泳中第一相的分离。
电泳
• 作用：用于分离蛋白质和寡核苷酸。原理聚丙烯酰胺凝胶为网状结构，具有分子筛效应。它有两种形式：非变性聚丙烯酰胺凝胶及SDS-聚丙烯酰胺凝胶（SDSPAGE）；非变性聚丙烯酰胺凝胶，在电泳的过程中，蛋白质能够保持完整状态，并依据蛋白质的分子量大小、蛋白质的形状及其所附带的电荷量而逐渐呈梯度分开。
实验步骤
• （1）溶液的配制 a) a贮液为49.5%T,3%C b) b贮液为49.5%T,6%C。 c) 胶缓冲液为3molTris d) 0.3g/LSDS用HCl调pH值至8145 e) 阳极缓冲液为0.2molTris f) 用HCl调pH值至8.9。 g) 阴极缓冲液为0.1molTris h) 0.1molTricine i) 0.13g/LSDS用HCl 调pH值至8125。 j) 样品缓冲液为3molTris(pH8.45) k) 0.8gSDS l) 0.3molDTT m) 少许溴酚蓝,定容至 10ml。 n) 胶的制备:按文献分别配制分离胶、间隙胶和浓缩胶,依次灌胶。 • (2)样品处理 • 去除血清Байду номын сангаас蛋白后的样品与2倍体积 U9缓冲液 (9molUrea,2%CHAPS,1%DTT) 混匀,400～600r/min冰浴振荡30min 变性。取变性后样品与样品缓冲液等体积混匀,60℃水浴加热5min。 • (3)电泳 • 内槽装阴极缓冲液,外槽用阳极缓冲液恒压电泳,先40V约1.5h,当样品进入分离胶时,电压升至60V约1.5h。

丙种球蛋白的制备方案

实验背景
丙种球蛋白预防传染性肝炎，预防麻疹等病毒性疾病感染，治疗先天性丙种球蛋白缺乏症，与抗生素合并使用，可提高对某些严重细菌性和病毒性疾病感染的疗效。
预防传染性肝炎，如乙肝、甲肝等。用于麻疹、水痘、带状疱疹等病毒感染和菌感染的防治。用于哮喘、过敏性鼻炎等内源性过敏性疾病与抗生素合并使用，可提高对某些严重细菌性和病毒性疾病感染的疗效。主要治疗先天性丙种球蛋白缺乏症和免疫缺陷病。
2. 待蒸馏水流至柱面平时，(注意，不可使柱面干掉)，马上沿层析柱内壁小心加4滴样品于柱床面，注意尽量不使床面搅动，马上加入1—2倍于样晶体积的蒸馏水，反复两次后，可加大量蒸馏水连续洗脱，这样可以使样品稀释最小。 3．样品一旦加入后马上用量筒或刻度离心管收集流出液，柱床上不断加蒸馏水，待红色(血红蛋白)流出一半时记录毫升数，此为血红蛋白Ve也为Vo。继续收集流出液，待黄色(核黄素)流出一半时记录体积，此为核黄素Ve。
1、实验器材层析柱（玻璃柱）、铁架台 2、实验材料样品液：0.05%核黄素(VitB2)与6%丙种球蛋白等量混合置4oC保存。
3、实验试剂： (1) 洗脱液(pH7.2 ;0.1mol/L磷酸盐缓冲液）:取0.1mol/L磷酸氢二钠720ml和 0.1mol/L磷酸二氢钠280ml,混匀。
实验操作步骤
1 、取直径0.8—1.2cm，长度为25cm的层析柱，在上口箍橡皮筋，出口处装上乳胶管，柱内加注蒸馏水2—3ml，排出乳胶管内的气泡，抬高乳胶管防止柱内的蒸馏水排空。自顶部缓缓加入经膨胀的葡聚糖G-50悬液。待底部凝胶沉积2—3cm时将乳胶管放低，仍继续加入上述悬液至凝胶层积至18cm 高度即可。操作过程中，应防止气泡与分层现象的发生。让凝胶自然下沉，使表层平整。待凝胶柱层稳定后，以洗脱液(蒸馏水)洗柱5分钟，调节流速至 4滴每分钟。

项目二丙种球蛋白的分离制备(刘蒋巍)

它的流经体积Ve是全部外水体积加上内水体积的一
部分，即 Ve=VO+KdVi 式中Kd称作“排阻系数”或“分配系数”。它反映了物质分子进入凝胶颗粒的程度。 Kd=(Ve-VO)／Vi 当Kd=1时，Ve=VO+Vi为全渗入。当Kd=0时，Ve=VO为全排阻。当0<Kd<1时，Ve=VO+KdVi为部分排阻。如Kd>t，则说明该物质分子与凝胶有吸附作用。
将含有三种不同分子量物质的混合样品用某种规格的凝胶柱
进行分离。首先将样品小心自柱顶端加入，洗脱，以分步收集器收集洗脱液，测定每管物质浓度，然后以洗脱体积为横坐标，各物质浓度为纵坐标即得如下的洗脱曲线：由图可见最先流出的物质是，A，它的分子量最大，大于该种凝胶的排阻限(A物质分子的直径大于凝胶的孔径)，完全不能进入颗粒内部，只能从颗粒间隙流过，称“全排阻”。其流经体积最小，与外水体积VO相等。最后流出的物质是C，它的分子量最小，小于该种凝胶的渗入限，其分子可以自由进出凝胶颗粒，这叫“全渗入”。流经体积是外水体积VO与内水体积Vi的和。而物质B的分子量介于排阻限与渗入限之间，其分子能够部分地进入凝胶颗粒之中，不能全部地不受限制的通过，这叫做“部分排阻”或“部分渗入”。
这三种物质的分离过程可见示意图如下：
Vi也可通过计算求出：
Vi=aWr a=凝胶千重
Wi=每克干凝胶吸水毫升数
Vt=Vo+Vi+Vg Vg=凝胶骨架体积 Vt=可通过测定层析柱内径及高度计算得出： Vt=1／4D2h
下表为交联葡聚糖凝胶的种类和规格：
基本操作仪器介绍
项目二丙种球蛋白层析分离
【实验内容】丙种球蛋白凝胶层析分离【实验目的】1.学习和掌握分子筛凝胶层析法的工作原理和基本操作技术。2．学会使用SephadexG－ 50分离丙种球蛋白。

丙种球蛋白的制备_Sephadex G-50分离制备丙种球蛋白

Sephadex G-50的性质
干粒直径：粗粒100~300中粒50~150细粒20~80 极细10~40 吸水量：5.0±0.3mL/g干凝胶膨胀体积：9~11mL/g 分离肽或球状蛋白：1500~30000 浸泡时间：20℃需3h;100℃需1h
名词解释
丙种球蛋白：按其来源不同可分为两种，一种是从健康人静脉血中提取制成的人血丙种球蛋白，另一种是从健康人胎盘血中提取制成的人胎盘血丙种球蛋白。二者作用相同，后者价格低于前者，且用量多于前者，但目前已停产。有些人盲目地、错误地认为丙种球蛋白是营养药、是补针，于是不管病情轻重、有病无病地滥用，结果会导致不良反应
（2）聚丙烯先胺凝胶：聚丙烯先凝胶是由丙烯酰胺经 N,N-次甲基双丙烯3）琼脂糖凝胶: 琼脂糖凝胶不需用化学交联剂聚合，主要依靠琼脂糖链间的次级键（如氢键）形成网状结构，其结构的疏密则由琼脂糖的浓度来控制。葡聚糖凝胶的稳定性较差，但分离范围广，由血清巨球蛋白到亚细胞颗粒都可以得到分离。
平整胶面待凝胶柱层稳定后以洗脱液蒸馏水洗柱5分钟调节流速至4滴min加样待蒸馏水流至柱面平时注意不可使柱面干掉马上沿层析柱内壁小心加4滴样品于柱床面注意尽量不使床面搅动马上加入12倍样品体积的蒸馏水反复两次可加大量蒸馏水洗脱这样可使样品稀释最小洗脱样品一旦加入后马上用量筒或刻度离心管收集流出液床柱上不断加蒸馏水待红色血红蛋白流出一半时记录体积此时血红蛋白ve也为v0继续收集流出液待黄色核黄素流出一半时记录体积此时核黄素ve用蒸馏水反复加在柱床上面洗柱10分钟然后将主柱内凝胶回收在凝胶瓶内样品收集与测定样品一旦加入后马上用量筒或刻度离心管收集流出液柱床上不断加蒸馏水待红色血红蛋白流出一半时记录毫升数此为血红蛋白ve也为vo
Thank you !

人血丙种球蛋白制造及检定规程

人血丙种球蛋白制造及检定规程本品系由乙型肝炎疫苗免疫健康人的血浆或血清经低温乙醇法提取的免疫球蛋白制剂。

含两种球蛋白90%以上。

用于常见病毒性感染的被动免疫，主要用于预防麻疹和传染性肝炎。

1 制造1.1制造要求与《人血白蛋白（低温乙醇法）制造及检定规程》中1.1项规定相同。

1.2制造工艺1.2.1采用低温乙醇法。

可含适宜稳定剂（如含甘氨酸，应为0.3或0.4mol/L）。

1.2.2分批每批制品最少应由1000名以上健康献血员的血浆（清）混合制成。

同一制造工艺同一空中溶解稀释的制品作为一批，不同滤器除菌过滤或不同机柜冻干的制品应分为亚批。

1.2.3半成品检定液体制剂于除菌过滤后应做理化检查（残余乙醇含量≤0.03%）及热原质试验，并按亚批抽样做无菌试验。

直接分装时应留样做上述实验。

1.2.4 冻干半成品经检定合格后及时分装，并按《人血白蛋白（低温乙醇法）制造及检定规程》1。

2。

5项规定进行冻干，但制品温度不得超过35℃。

1.3剂型与规格1.3.1剂型分为液体及冻干两种。

1.3.2规格液体制剂蛋白质浓度为10%。

液体和冻干制剂每瓶（支）蛋白质装量均为150mg、300mg。

1.4制品重滤和再制与《人血白蛋白（低温乙醇法）制造及检定规程》中1.4项相同。

2 成品检定2.1抽样每批成品应抽样做全面检定，不同机柜冻干的制品应分别抽样做无菌试验及水分测定。

2.2物理检查2.2.1外观冻干制剂应为白色或灰白色的疏松体，无融化迹象。

液体制剂和冻干制剂溶解后溶液应为接近无色，可带乳光，或淡黄色澄明液体，不应含有异物、混浊或摇不散的沉淀。

2.2.2冻干制剂溶解时间冻干制剂加入20～25℃标示量的灭菌注射用水后，应在15分钟内完全溶解。

2.2.3热稳定性试验液体制剂置57±0.5℃水浴保温4小时后，应无凝胶化或絮状物。

2.3化学检定按《生物制品化学检定规程》进行。

2.3.1水分冻干制剂的水分含量应≤3%（g/g）。

丙种球蛋白的制备

聚丙烯酰胺分类：聚丙烯酰胺（PAM）为水溶性高分子聚合物，不溶于大多数有机溶剂，具有良好的絮凝性，可以降低液体之间的磨擦阻力。按离子特性分可分为：非离子阴离子阳离子两性型
聚丙烯酰胺凝胶特点：

凝胶透明，有弹性，机械性能好；化学性能稳定，与被分离物不起化学反应；对PH和温度变化较稳定，几乎无吸附和电渗作用；样品用量少，灵敏度可达10-6g; 凝胶孔径可调节；分辨率高。
丙种球蛋白的制备
任务一：丙种球蛋白分离制备
聚丙烯酰胺凝胶电泳
丙种球蛋白预防传染性肝炎，预防麻
疹等病毒性疾病感染，治疗先天性丙种球蛋白缺乏症，与抗生素合并使用，可提高对某些严重细菌性和病毒性疾病感染的疗效。

性状冻干制剂应为白色或灰白色的疏松体，液体制剂和冻干制剂溶解后，溶液应为接近无色或淡黄色的澄明液体，微带乳光。但不应含有异物或摇不散的沉淀。

(5)固定与染色为防止凝胶柱内已分离成分的扩散，需要进行固定。剥出的凝胶柱只要浸泡在7%乙酸或12.5%三氯乙酸的水溶液中几分钟就可达到蛋白带固定的效果。
电泳后蛋白质区带的检测最常用的方法是用染料和生物大分子结合形成有色的复合物。选用染料通常应考虑以下要求： A)必须与大分子结合以形成一个不溶性的，有色的，紧密的复合物，但不结合到凝胶中和支持膜上，以便从凝胶中除去，否则背景会影响蛋白带的辨别和定量扫描; B)染料必须容易溶解在对大分子没有影响的溶剂中，以利于背景的脱色; C)选用高吸光系数的染料有利于提高定量测定的灵敏度; D)选用能与大分子有专一性结合的染料，并在结合后能产生不同的颜色，可以提高检测的选择性。
(2)样品的准备

凝胶层析分离丙种球蛋白与核黄素实验流程

凝胶层析分离丙种球蛋白与核黄素实验流程下载温馨提示:该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。

文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor.I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copy excerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!丙种球蛋白与核黄素的凝胶层析分离实验流程详解一、实验目的：本实验旨在通过凝胶层析法，有效分离并纯化血液中的丙种球蛋白（γ-球蛋白）和核黄素（维生素B2），以了解该方法在生物大分子分离中的应用。

凝胶层析分离丙种球蛋白与核黄素实验流程

凝胶层析分离丙种球蛋白与核黄素实验流程下载温馨提示:该文档是我店铺精心编制而成，希望大家下载以后，能够帮助大家解决实际的问题。

文档下载后可定制随意修改，请根据实际需要进行相应的调整和使用，谢谢!并且，本店铺为大家提供各种各样类型的实用资料，如教育随笔、日记赏析、句子摘抄、古诗大全、经典美文、话题作文、工作总结、词语解析、文案摘录、其他资料等等，如想了解不同资料格式和写法，敬请关注!Download tips: This document is carefully compiled by theeditor.I hope that after you download them,they can help yousolve practical problems. The document can be customized andmodified after downloading,please adjust and use it according toactual needs, thank you!In addition, our shop provides you with various types ofpractical materials,such as educational essays, diaryappreciation,sentence excerpts,ancient poems,classic articles,topic composition,work summary,word parsing,copy excerpts,other materials and so on,want to know different data formats andwriting methods,please pay attention!丙种球蛋白与核黄素的凝胶层析分离实验流程详解凝胶层析，又称为分子筛色谱，是一种广泛应用的生化分离技术。