Urokinase, purification

Affinity chromatography of mammalian phospho-fructokinase on immobilized adenine nucleotides Studies of the effect of the chain-length on the adsorption of albumin removal of albumin from sera Purification of commercial preparations of urokinase Purification of Solanum tuberosum agglutinin (sta-lectin)... 

After its purification, sterile filtration and aseptic filling, human urokinase is normally freeze-dried. Because of its heat stability, the final product may also be heated to 60 °C for up to 10 h in an effort to inactivate any undetected viral particles present. The product utilized clinically contains both molecular mass forms, with the higher molecular mass moiety predominating. Urokinase can also be produced by techniques of animal cell culture utilizing human kidney cells or by recombinant DNA technology. 

Bansal, V. and Roychoudhury, P. 2006. Production and purification of urokinase a comprehensive review. Protein Expression and Purification 45(1), 1-14. 

Fig. 3. Overview of purification sequence for the nonrecombinant tissue plasminogen activator (t-PA) which also contains urokinase plasminogen activator (u-PA). Serum-free culture conditional media is from normal human cell line. The temperature for all steps, except for size-exclusion chromatography...

Kumar A, Bansal V, Andersson J, Roychoudhury PK, Mattiasson B (2006), Super-macroporous cryogel matrix for integrated protein isolation - immobilized metal affinity chromatographic purification of urokinase from cell culture broth of a human kidney cell line, J. Chromatogr. A 1103 35-42. 

Nielsen LS, Kellerman GM, Behrendt N, Picone R, Dano K, Blasi F. A 55,000-60,000 Mr receptor protein for urokinase-type plasminogen activator. Identification in human tumor cell lines and partial purification. J Biol Chem 1988 263(5) 2358—2363. 

Hou KC and Zaniewski R. Purification of urokinase by combined cation exchanger and affinity chromatographic cartridges. J. Chromatogr. B 1990 525 297-306. 

Stump DC, Lijnen HR, Collen D (1986) Purification and characterization of single-chain urokinase-type plasminogen activator from human cell cultures. J Biol Chem 261(3) 1274-1278... 

Ultimately, the cost of immunosorbent isolation will depend on the entire process and must be evaluated against alternative processes. Consider, as an example, the costs and decisions involved in the purification of urokinase. One course of drug therapy consists of 33 mg of urokinase (4,000,000 CTA units). At the hospital pharmacy the drug costs for one course of treatment are currently 3,000 (9), or 91,000/gram. There are approximately 76,000 patients in the U.S. that could be treated with urokinase therapy each year requiring an annual production of approximately 2,500 g. We have selected a monoclonal antibody that has allowed the purification of urokinase from urine, tissue culture media, and bacterial culture media in a single step with 85% retention of urokinase activity (6). This monoclonal antibody was immobilized at 2 gL l with an immobilization yield of 0.8 and a cycle half number of 300 cycles. The urokinase capacity for the first cycle would be 1.2 gL l of immunosorbent. 

New techniques are continuing to be developed which can be expected to be used in future specialized industrial applications. Multi-segmented columns have been demonstrated for the preparative purification of urokinase. Begovichandcoworkers havedevelopedatechnique for continuous spiral cylinder purifications which allow separation of the... 

Winkler ME, Blaber M, Bennett GL et al. (1985) Purification and characterization of recombinant urokinase from Escherichia coli. Bio/Technology 3 990-1000 Wisdom GB (1976) Enzyme-immunoassay. Qin Chem 22 1243—1255... 

Amino-2-naph thamidine (37) Affinity chromatographic purification of urokinase 342 ... 

---