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biopharmaceuticals biochemistry and biotecnology - Walsh G.

Walsh G. biopharmaceuticals biochemistry and biotecnology - John Wiley & Sons, 2003. - 572 p.
ISBN 0-470-84327-6
Download (direct link): biochemistryandbiotechnology2003.pdf
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Streptokinase
Streptokinase is an extracellular bacterial protein produced by several strains of Streptococcus haemolyticus group C. It displays a molecular mass in the region of 48 kDa and an isoelectric point of 4.7. Its ability to induce lysis of blood clots was first demonstrated in 1933. Early therapeutic preparations administered to patients often caused immunological and other complications, usually prompted by impurities present in these products. Chromatographic purification (particularly using gel filtration and ion-exchange columns), overcame many of these initial difficulties. Modern chromatographically pure streptokinase preparations are usually supplied in freeze-dried form. These preparations often contain albumin as an excipient. The albumin prevents flocculation of the active ingredient upon its reconstitution.
Streptokinase is a widespreadly employed thrombolytic agent. It is administered to treat a variety of thrombo-embolic disorders, including:
• pulmonary embolism (blockage of the pulmonary artery by an embolism), which can cause acute heart failure and sudden death (the pulmonary artery carries blood from the heart to the lungs for oxygenation);
• deep-vein thrombosis (thrombus formation in deep veins, usually in the legs);
• arterial occlusions (obstruction of an artery);
• acute myocardial infarction.
Streptokinase induces its thrombolytic effect by binding specifically and tightly to plasminogen. This induces a conformational change in the plasminogen molecule, which renders it proteolytically active. In this way, the streptokinase-plasminogen complex catalyses the proteolytic conversion of plasminogen to active plasmin.
As a bacterial protein, streptokinase is viewed by the human immune system as an antigenic substance. In some cases, its administration has elicited allergic responses, ranging from mild rashes to more serious anaphylactic shock (anaphylactic shock represents an extreme and generalized allergic response, characterized by swelling, constriction of the bronchioles, circulatory collapse and heart failure).
Another disadvantage of streptokinase administration is the associated increased risk of haemorrhage. Streptokinase-activated plasminogen is capable of lysing not only clot-associated fibrin, but also free plasma fibrinogen. This can result in low serum fibrinogen levels and hence compromise haemostatic ability. It should not, for example, be administered to patients suffering from coagulation disorders or bleeding conditions such as ulcers. Despite such potenital clinical complications, careful administration of streptokinase has saved countless thousands of lives.
386 BIOPHARMACEUTICALS
Urokinase
The ability of some components of human urine to dissolve fibrin clots was first noted in 1885, but it was not until the 1950s that the active substance was isolated and named ‘urokinase’.
Urokinase is a serine protease produced by the kidney and is found in both the plasma and urine. It is capable of proteolytically converting plasminogen into plasmin. Two variants of the enzyme have been isolated: a 54 kDa species and a lower molecular mass (33 kDa) variant. The lower molecular mass form appears to be derived from the higher molecular mass moiety by proteolytic processing. Both forms exhibit enzymatic activity against plasminogen.
Urokinase is used clinically under the same circumstances as streptokinase and, because of its human origin, adverse immunological responses are less likely. Following acute medical events such as pulmonary embolism, the product is normally administered to the patient at initial high doses (by infusion) for several minutes. This is followed by hourly i.v. injections for up to 12 h.
Urokinase utilized medically is generally purified directly from human urine. It binds to a range of adsorbants, such as silica gel and, especially, kaolin (hydrated aluminium silicate), which can be used to initially concentrate and partially purify the product. It may also be concentrated and partially purified by precipitation using sodium chloride, ammonium sulphate or ethanol as precipitants.
Various chromatographic techniques may be utilized to further purify urokinase. Commonly employed methods include anion (DEAE-based) exchange chromatography, gel filtration on Sephadex G-100 and chromatography on hydroxyapatite columns. Urokinase is a relatively stable molecule. It remains active subsequent to incubation at 60°C for several hours, or brief incubation at pHs as low as 1.0 or as high as 10.0.
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.
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