Human alfa-fetoprotein: isolation and production of monoclonal antibodies.

Alfa-fetoprotein from human cord serum was purified in a single step by hydrophobic interaction chromatography on Phenyl Sepharose CL-4B with a final recovery of alfa-fetoprotein of about 90% and a purification factor of 900. The purified preparation was homogeneous on SDS-PAGE and native PAGE running with a relative molecular weight of 72,000. Monoclonal antibodies against this purified preparation were raised by hybridoma technology using Sp2/0 myeloma cells as a fusion partner. 50% of culture wells exhibited hybrid growth and 7% of these wells contained anti-AFP secreting hybrids. Positive hybrid cells were cloned twice by the limiting dilution method and 8 clones were obtained that secreted monoclonal antibodies. Five of these cell lines (3F6H10, 3F6H4, 3F6H1, 3F6G5 and 3F6G10) were selected at random for purification and characterization purposes. All 5 cell lines secreted monoclonal antibodies of IgG1 subclass which were purified by affinity chromatography on Protein A- Sepharose CL-4B column with a final recovery of 80% and a purification factor of about 13. The purified preparations were homogeneous on SDS-PAGE, native PAGE and IEF. The monoclonal antibodies were highly specific for human alfa-fetoprotein as determined by Western blotting. The affinity constants (K) of these Mab ranged from 10(6) to 10(9) l/mol.     

Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

Improved methods for prepurification and detection of staphylococcal enterotoxin B from cell-free culture filtrate

An improved ELISA method for the detection of Staphylococcal Enterotoxin B (SEB) in protein A preparations is presented. Fab fragments were obtained by digestion with papain of anti-SEB IgG bound to SEB immobilized on Sepharose 4B. Anti-SEB and peroxidase-labeled Fab fragments from secondary antibodies were successfully used in a modified ELISA of SEB in protein A preparations. SEB-Sepharose was used repeatedly for the production of anti-SEB Fab fragments by papain digestion without loss of affinity. In addition, for the purification of SEB from crude culture filtrates, an initial step utilizing a combined heat and pH treatment for the removal of significant amounts of contaminating proteins without losses of toxin activity is presented. This pretreatment step yielded positive effects in further downstream processing considering both shortened time and an increase in total recovery.     

药用级微生物谷氨酰胺转胺酶的纯化工艺研究

为了提高谷氨酰胺转胺酶的纯度和扩展在医药领域的应用,探索了一种适合工业化生产的、安全高效的微生物谷氨酰胺转胺酶纯化方法。轮枝链霉菌发酵后,经离心10 000 r/min 4℃除去菌体,调节发酵液电导率至4.1mS/cm和pH6.0后,以直线流速60cm/h通过SP Sepharose FF阳离子交换层析柱对目的蛋白高 选择性和高载量地捕获,再通过phenyl sepharose 6 FF（high sub）疏水层析柱进行精细纯化。纯化后经SDS-PAGE鉴定纯度达到95%以上,HPLC分析纯度> 99%。鲎试剂测定内毒素含量为0.013EU/ml,达到中国药典中血制品要求的低于0.15EU/ml标准。     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

Preparative purification of a recombinant protein by hydrophobic interaction chromatography: modulation of selectivity by the use of chaotropic additives

Development and implementation of a chaotropic wash step following protein loading on a hydrophobic interaction chromatographic (HIC) column is described for the purification of a recombinant protein. Various agents that reduce protein affinity in hydrophobic interaction chromatographic systems were screened for their utility in a wash step following protein loading on a Phenyl Fast Flow Sepharose HIC column. A combination of sodium thiocyanate, glycerol, and urea was selected as a suitable additive for the wash buffer that selectively eluted most of the major impurities present in the feed stream. Eluate purity, as monitored by reversed-phase chromatography and SDS-PAGE, was significantly increased by incorporation of this wash step in the purification process. Incorporation of this wash step on HIC enabled a reduction in the overall number of chromatographic steps in the downstream purification process for this recombinant protein, resulting in improved process yields and significant economic advantages.The effect of varying concentrations of each of the three wash additives on yield was studied. While the step yield decreased with an increase in concentration for urea and sodium thiocyanate, an optimum was observed with respect to glycerol concentration. The preferential interaction theory is employed to explain this effect.     

On-line casein micelle disruption for downstream purification of recombinant human myelin basic protein produced in the milk of transgenic cows

Downstream purification of a model recombinant protein (human myelin basic protein) from milk of transgenic cows is described. The recombinant protein was expressed as a His tagged fusion protein in the milk of transgenic cows and was found associated with the casein micellar phase. While difficulties in obtaining good recoveries were found when employing conventional micelle disruption procedures, direct capture using the cation exchanger SP Sepharose Big Beads? was found successful in the extraction of the recombinant protein. Early breakthrough suggested a slow release of the recombinant protein from the micelles and dictated micelle disruption in order to obtain good yields. A new approach for deconstruction of the calcium core of the casein micelles, employing the interaction between the micellar calcium and the active sites of the cation exchanger resin was developed. Milk samples were loaded to the column in aliquots with a column washing step after each aliquot. This sequential loading approach successfully liberated the recombinant protein from the micelles and was found superior to the conventional sample loading approach. It increased the recovery by more than 25%, reduced fouling due to milk components and improved the column hydrodynamic properties as compared to the conventional sample loading approach. Hardware and software modifications to the chromatography system were necessary in order to keep the whole process automated. A second purification step using a Ni²⁺ affinity column was used to isolate the recombinant protein at purity more than 90% and a recovery percentage of 78%.     

A single-step chromatographic method for the purification of proteins devoid of exposed histidine residues

The principle of the immobilized metal affinity chromatography (IMAC) is based on the differences in the affinity of proteins for metal ions bound in a 1:1 complex of iminodiacetic acid (IDA) immobilized on a chromatographic support. A single step purification was carried out for luteinizing hormone (LH) on Cu2+, Zn2+, Ni2+, and Co2+ IDA Sepharose affinity columns. Highly purified LH was obtained with a Cu2+ IDA Sepharose column. SDS-PAGE and Western blot analysis were done to confirm the purity of the hormone. Biological activity has been evaluated by radio-immunoassay. This method was found economically viable and suitable for the recovery of biologically active hormone.     

A Procedure for Human Pregnancy Zone Protein (and Human α2-Macroglobulin) Purification Using Hydrophobic Interaction Chromatography on Phenyl–Sepharose CL-4B Column

In the present work we describe a procedure for the purification of human pregnancy zone protein (PZP) from pooled late pregnancy plasma by using hydrophobic interaction chromatography (HIC) on a phenyl–Sepharose column. The HIC step allowed the complete isolation of haptoglobins and the partial separation of human α₂-macroglobulin (α₂-M) from a protein fraction containing PZP previously obtained by a DEAE-Sephacel chromatography. Pure and native PZP, with a recovery of nearly 25% and biological activity of protease-binding, was obtained by two definitive final steps consisting of zinc-chelate and size-filtration chromatographies. Moreover, we further present an alternative procedure for the purification of α₂-M from the same pregnancy plasma, based on the differential elution of PZP and α₂-M from the HIC. This purification step gave rise to a highly purified product with a recovery of 10%. This differential elution could be explained by differences in surface hydrophobicity observed between both proteins. In addition, considering the different hydrophobic properties exhibited by native PZP and PZP–protease complexes, HIC on phenyl–Sepharose column could also be used for separating both conformational states of PZP.     

Immobilized metal ion affinity chromatography in enzyme fractionation.

Ribitol dehydrogenase from Mycobacterium butyricum and alpha-mannosidase from Lupinus luteus seedlings were fractionated by the immobilized metal ion (Cu2+ or Zn2+) affinity chromatography (IMAC) on iminodiacetic acid coupled to Sepharose 6B. In a single step, ribitol dehydrogenase was purified 10-12 fold with the recovery above 80% when using Zn(2+)-Sepharose 6B as the sorbent and decreasing linear gradient of pH from 7 to 4. In the same conditions purification of alpha-mannosidase was less effective (2-3 fold, recovery 60-70%).     

Affinity recovery of Moloney Murine Leukaemia Virus

Lipid enveloped retroviruses such as Moloney Murine Leukaemia Virus (MoMuLV) are commonly used gene therapy vectors. Downstream processing protocols used for their purification are time consuming and a potentially generic, single step capture method for the recovery of retroviral particles is proposed that exploits streptavidin-biotin affinity chromatography. The ability of four conventional adsorbent solid phases, Fractogel, Sepharose, Magnespheres and STREAMLINE immobilised with streptavidin, to capture and recover biotinylated Moloney Murine Leukaemia Virus was studied. MoMuLV can be biotinylated whilst retaining infectivity and the biotinylated virus can be adsorbed to Streptavidin Magnespheres yielding a 2298-fold increase in titre. For optimal virus biotinylation purification using Fractogel streptavidin can yield a 1896-fold increase in cfu/mg of protein and a 1191-fold decrease in DNA/cfu. Infectious virus can be recovered from Fractogel streptavidin with a maximum recovery of 16.7%.     

Isolation of neutral proteinase of ribosomes (cathepsin R)

A method for isolation of cathepsin R from rat liver ribosomes allowing for a 264-fold increase of specific activity is described. The purification procedure includes enzyme extraction from ribosomes with 2-4 M LiCl and two-step affinity chromatography on Sepharose with immobilized soy bean trypsin inhibitor and trypsin-Sepharose.     

A single step procedure for purification of estradiol 17 beta-dehydrogenase from human placenta

A new and simple procedure for purification to homogeneity of the soluble estradiol 17 beta-dehydrogenase [E C.1.1.1.62] from human placenta has been developed. The purification is achieved in a single step by affinity-absorption of Cibachrome Blue F3G-A coupled to Sepharose and selective elution with NADP+. Homogeneous estradiol 17 beta-dehydrogenase has a specific activity of 7.2 units/mg. and has been purified 2400-fold with a 93% recovery.     

Snake venom phosphodiesterase: simple purification with Blue Sepharose and its application to poly(ADP-ribose) study.

A rapid method of purifying snake venom phosphodiesterase has been developed using Blue Sepharose or blue dextran/Sepharose as an affinity adsorbent. A sixty-fold purification of the enzyme from commercial preparations is achieved in a single step with a yield of 60%. The purified enzyme preparation is essentially free from phosphatase activities and exhibits a major protein band on SDS-polyacrylamide gel electrophoresis. Chain length analysis of poly(ADP-ribose) exemplifies the usefulness of this technique.     

Purification to Homogeneity of Biologically Active Human Interleukin 1

Human interleukin 1 (IL-1) produced by lipopolysaccharide-stimulated monocytes was purified to homogeneity with retention of biological activity. IL-1 was measured by its ability to enhance the proliferative response of thymocytes to phytohemagglutinin. The purification procedure included hydrophobic affinity chromatography on phenyl Sepharose, gel filtration through Ultrogel AcA54 and preparative isoelectric focusing. Both charged species of IL-1, pi 5.1 and 6.8 have a molecular weight of 14,500 as determined by SDS-polyacrylamide gel electrophoresis. The complete purification resulted in a recovery of approximately 0.01% of IL-1 protein and if protection against losses by denaturation and adsorption in the final purification step was provided by bovine serum albumin, approximately 11% of IL-1 activity can be recovered.     

Evaluation of CNBr, FMP and hydrazide resins for immunoaffinity purification of factor IX.

The American Red Cross has developed an immunoaffinity chromatography method to purify human coagulation Factor IX to high levels of purity for therapeutic treatment of hemophilia B. The resin currently used in this process is Sepharose CL2B, a cross-linked 2% agarose, which is activated with cyanogen bromide to immobilize an anti-Factor IX monoclonal antibody. This study evaluated two alternative resins and coupling chemistries, a synthetic polymer bead activated by 2-fluoro-1-methyl-pyridinium toluene 4-sulfonate (FMP) and a cross-linked 2% agarose bead with free hydrazide groups for site-specific coupling. The cyanogen bromide and FMP chemistries immobilize the monoclonal antibody in a random orientation. In hydrazide coupling, the monoclonal antibody is immobilized by the non-antigen-binding part of the molecule which, theoretically, should increase the amount of immobilized monoclonal antibody able to bind antigen. To examine this, the capacity of the resins to bind Factor IX and the purity and recovery of Factor IX eluted from the resins were measured. The FMP-activated resin exhibited the lowest capacity, binding only 2% of the Factor IX feed. Sepharose CL2B bound 87% of the loaded protein, while the hydrazide resin bound 43%. These results suggest that (a) hydrazide activation may be insufficient to orient monoclonal antibody and (b) other factors such as steric hindrances and diffusional resistances during immobilization may be important. Neither of the other resins tested demonstrated improved performance compared with cyanogen bromide-activated Sepharose CL2B for the immunoaffinity purification of Factor IX.     

Downstream processing of extracellular phytase from Aspergillus niger: Chromatography process vs. aqueous two phase extraction for its simultaneous partitioning and purification

The application of single step aqueous two-phase extraction (ATPE) for the downstream processing of phytase from Aspergillus niger NCIM 563, produced under solid state fermentation, has been studied and compared with the traditional multi-step procedure involving salt precipitation and column chromatography. High phytase recovery (98.5%) within a short time (3 h) and improved thermostability was attained by ATPE in comparison to 20% recovery in 96 h by chromatography process. The ATPE method, therefore, seems to be an interesting alternative for simultaneous partitioning and purification of phytase. The influence of system parameters; such as, phase forming salts, polymer molecular weight and system pH on the partitioning behavior of phytase was evaluated. The ATPE system consisting of combination of polyethylene glycol (PEG) 6000 and 8000 (10.5%) and sodium citrate (20.5%) resulted in one-sided partitioning of phytase in bottom phase with a purification factor of 2.5. This is the first report on phytase purification using liquid–liquid extraction and the results are likely to be beneficial in the poultry feed industry.     

Affinity chromatography of putrescine oxidase from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens.

Putrescine oxidase [EC 1.4.3.4], putrescine : oxygen oxidoreductase (deaminating) (flavin-containing), from Micrococcus rubens and spermidine dehydrogenase from Serratia marcescens were adsorbed on amine-Sepharose 4B in which one of the terminal amino groups of diamine or triamine was covalently bound to Sepharose 4B leaving the other terminal amino group(s) free. The affinities of these enzymes for the amine-Sepharose 4B increased on increasing the chain length of the methylene groups in the immobilized amines and fell upon addition of the substrate. The affinity of putrescine oxidase modified with 1-ethyl-3-(3-dimethylamino-propyl)-carbodiimide (EDC) was reduced in comparison with that of the native enzyme so far as 1,12-diaminododecane-Sepharose 4B was concerned. From these results, it can be concluded that the interactions between the enzyme and the amine-Sepharose result from specific affinities mediated through the active sites of the enzymes. It is suggested that spermidine dehydrogenase as well as putrescine oxidase has as anionic point and a hydrophobic region in the active site. On the basis of these results, the applicability of the enzyme affinities to purification procedures was examined. When partially purified enzymes were subjected to affinity chromatography, the following results were obtained. Putrescine oxidase gave a purification factor of 40-fold with about 100% recovery on a 1,12-diaminododecane-Sepharose column. In the case of spermidine dehydrogenase, the purification factor and recovery on a 1,8-diaminooctane-Sepharose column were about 1,200-fold and 86%, respectively. By introducing affinity chromatography as a purification step, each enzyme could be purified more simply and with higher recovery.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Purification of coagulation factor VIII using chromatographic methods. Direct chromatography of plasma in anion exchange resins

Coagulation factor VIII (FVIII) concentrates are used in the treatment of patients with Hemophilia A. Human FVIII was purified directly from plasma using anion exchange chromatography followed by gel filtration. Three Q-Sepharose resins were tested, resulting in 40% recovery of FVIII activity using Q-Sepharose XL resin, about 80% using Q-Sepharose Fast Flow and 70% using the Q-Sepharose Big Beads. The vitamin K-dependent coagulation factors co-eluted with FVIII from the anion exchange columns. In the second step of purification, when Sepharose 6FF was used, 70% of FVIII activity was recovered free from vitamin K-dependent factors.