BIOPHARMACEUTICALS BIOCHEMISTRY AND BIOTECHNOLOGY - PART 8 ppsx

major added excipient. The product, which displays a useful shelf-life of 2-years when stored at room temperature, is reconstituted with saline or water for injections immed iately prior to its i.v. administration. A second reco mbinant product (trade name Revasc, also produced in S. cerevisiae) has also been approved. Antithrombin Antithrombin, already mentioned in the context of heparin, is the most abundantly occurring natural inhibitor of coagulation. It is a single-chain 432 amino acid glycoprotein displaying four oligosaccharide side-chains and an approximate molecular mass of 58 kDa. It is present in plasma at concentrations of 150 mg/ml and is a potent inhibitor of thrombin (factor IIa) as well as factors IXa and Xa. It inhibits thrombin by binding directly to it in a 1:1 stoichiometric complex. Plasma-derived anti-thrombin (AT) concentrates have been used medically since the 1980s for the treatment of hereditary and acquired AT deficiency. Hereditary (genetic) deficiency is characterized by the presence of little/no na tive antithrombin activity in plasma and results in an increased risk of inappropriate blood clot/embolus formation. Acquired AT deficiency can be induced by drugs (e.g. heparin and oestrogens), liver disease (which causes decreas ed AT synthesis) or various other medical conditions. Recombinant AT ha s be en successfully expressed in engineered CHO cells. Commercial production via this route, however, is rendered unattractive due to high relative production costs and, to a lesse r extent, by the scale of production needed to satisfy market demand. Recombinant AT has been produced more economically in the milk of transgenic goats (Chapter 3) and this product is currently undergoing clinical trials (Figure 9.16). The recombinant product displays an identical amino acid sequence to that of native human AT, although its oligosaccharide composition does vary somewhat from the native protein. Ceprotin (human protein C concentrate) is an additional protein-based anticoagulant now approved for general medical use. Protein C is a 62 kDa glycopr otein synthesized in the liver but BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 379 Figure 9.16. Outline of the production and purification of antithrombin (AT) from the milk of transgenic goats. Purification achieves an overall product yield in excess of 50%, with a purity greater than 99% released into the blood as a circulating inactive zymogen. It is activated by thrombin in conjunction with another protein, thrombomodulin, and the activated form displays anti- coagulant acti vity. In vivo protein C plays an important role in controlling coagulation by preventing excessive clot formation. A range of genetic congenital deficiencies adversely affecting serum levels of functional protein C have been characterized. Sufferers generally display an increased risk of inapp ropriate venous thrombosis and Ceprotin has been app roved for the treatment of such individuals. An overview of its method of manufacture is provided in Figure 9.17. As the protein is sourced directly from pooled human plasma, it is more properly described as a product of pharmaceutical biotechnology, as opposed to a true biopharma- ceutical (Chapter 1). Thrombolytic agents The natural process of thrombosis functions to plug a damaged blood vessel, thus maintaining haemostasis until the damaged vessel can be repaired. Subsequent to this repair, the clot is removed via an enzymatic degradative process known as fibrinolysis. Fibrinolysis normally 380 BIOPHARMACEUTICALS Figure 9.17. Overview of the manufacture of Ceprotin. As the active ingredient is derived directly from pooled human plasma, particular emphasis is placed upon ensuring that the finished product is pathogen- free. Precautions entail the incorporation of two independent viral inactivation steps and high-resolution chromatographic purification. Additionally, extensive screening of plasma pool source material for blood- borne pathogens is undertaken. Viral screening is undertaken using a combination of immunoassay and PCR analysis for the presence of viral nucleic acid depends upon the serine protease plasmin, which is capable of degrading the fibrin strands present in the clot. In situations where inappropriate clot formation results in the blockage of a blood vessel, the tissue damage that ensues depends, to a point, upon how long the clot blocks blood flow. Rapid removal of the clot can often minimize the severity of tissue damage. Thus, several thrombolytic (clot-degrading) agents have found medical application (Table 9.10). The market for an effective thrombolytic agent is substantial. In the USA alone, it is estimated that 1.5 million people suffer acute myocardial infarction each year, while another 0.5 million suffer strokes. Tissue plasminogen activator (tPA) The natural thrombolytic process is illustrated in Figure 9.18. Plasmin is a protease which catalyses the proteolytic degradation of fibrin present in clots, thus effectively dissolving the clot. Plasmin is derived from plasminogen, its circulating zymogen. Plasm inogen is synthesized in, and released from, the kidneys. It is a single-chain 90 kDa glycoprotein, which is stabilized by several disulphide linkages. Tissue plasminogen activator (tPA, also known as fibrinokinase) represents the most important physiological activator of plasminogen. tPA is a 527 amino acid serine protease. It is synthesized predominantly in vascular endothelial cells (cells lining the inside of blood vessels) and displays five structural domains, each of which has a specific function (Table 9.11). tPA displays four potential glycosylation sites, three of which are normally glycosylated (residues 117, 184 and 448). The carbohydrate moieties play an important role in mediating hepatic uptake of tPA and hence its clearance from plasma. It is normally found in the blood in two forms; a single-chain polypeptide (type I tPA) and a two-chain structure (type II) proteolytically derived from the single chain structure. The two-chain form is the one predominantly associated with clots undergoing lysis, but both forms display fibrinolytic activity. Fibrin contains binding sites for both plasminogen and tPA, thus bringing these into close proximity. This facilitates direct activation of the plasminogen at the clot surface (Figure 9.18). This acti vation process is potentiated by the fact that binding of tPA to fibrin (a) enhances the subsequent binding of plasminogen and (b) increases tPA’s activity towards plasminogen by up to 600-fold. BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 381 Table 9.10. Thrombolytic agents approved for general medical use (r¼ recombinant, rh ¼ recombinant human) Product Company Activase (rh-tPA) Genentech Ecokinase (rtPA; differs from human tPA in that three of its five domains have been deleted) Galenus Mannheim Retavase (rtPA; see Ecokinase) Boehringer-Mannheim/Centocor Rapilysin (rtPA; see Ecokinase) Boehringer-Mannheim Tenecteplase (also marketed as Metalyse) (TNK-tPA, modified rtPA) Boehringer-Ingelheim TNKase (Tenecteplase; modified rtPA; see Tenecteplase) Genentech Streptokinase (produced by Streptokinase haemolyticus) Various Urokinase (extracted from human urine) Various Staphylokinase (extracted from Staphylococcus aureus and produced in various recombinant systems) Various Overall, therefore, activ ation of the thrombolytic cascade occurs exactly where it is needed — on the surface of the clot. This is important as the substrate specificity of plasmin is poor, and circulating plasmin displays the catalytic potential to proteolyse fibrinogen, factor V and factor VIII. Although soluble serum tPA displays a much reduced activity towards plasminogen, some free circulating plasmin is produced by this reaction. If uncontrolled, this could increa se the risk of subsequent haemorrhage. This scenario is usually averted, as circulating plasmin is rapidly 382 BIOPHARMACEUTICALS Figure 9.18. (a) The fibrinolytic system, in which tissue plasminogen activator (tPA) proteolytically converts the zymogen plasminogen into active plasmin, which in turn degrades the fibrin strands, thus dissolving the clot. tPA and plasminogen both bind to the surface of fibrin strands (b), thus ensuring rapid and efficient activation of the thrombolytic process neutralized by another plasma protein, a 2 -antiplasmin (a 2 -antiplasmin, a 70 kDa, single-chain glycoprotein, binds plasmin very tightly in a 1:1 complex). In contrast to free plasmin, plasmin present on a clot surface is very slowly inactivated by a 1 -antiplasmin. The thrombolytic system has thus evolved in a self-regulating fashion, which facilitates efficient clot degradation with minimal potential disruption to other elements of the haemostatic mechanism. First-generation tPA. Although tPA was first studied in the late 1940s, its extensive characterization was hampered by the low levels at which it is normally synthesized. Detailed studies were facilitated in the 1980s after the discovery that the Bowes melanoma cell line produces and secretes large quantities of this protein. This also facilitated its initial clinical appraisal. The tPA gene was cloned from the melanoma cell line in 1983, and this facilitated subsequent large-scale production in CHO cell lines by recombinant DNA technology. The tPA cDNA contains 2530 nucleotides and encodes a mature protein of 527 amino acids. The glycosylation pattern was similar, although not identical, to the native human molecule. A marketing licence for the product was first issued in the USA to Genentech in 1987 (under the trade name Activase). The therapeutic indication was for the treatment of acute myocardial infarction. The production process entails an initial (10 000 litre) fermentation step, during which the cu ltured CHO cells produce and secrete tPA into the fermentation medium. After removal of the cells by sub-micron filtration and initial concentration, the product is purified by a combination of several chromatographic steps. The final product has been shown to be greater than 99% pure by several analytical techniques, including HPLC, SDS–PAGE, tryptic mapping and N-terminal sequencing. Activase has proved effective in the early treatment of patients with acute myocardial infarction (i.e. those treated within 12 h after the first symptoms occur). Significantly increased rates of patient survival (as measured 1 day and 30 days after the initial event), are noted when tPA is administered in favour of streptokinase, a standard therapy (see later). tPA has thus established itself as a first-line option in the management of acute myocardial infarction. A therapeutic dose of 90–100 mg (often administered by infusion over 90 min), resul ts in a steady- state Activase concentration of 3–4 mg/l during that period. The product is, however, cleared rapidly by the liver, displaying a serum half-life of approximately 3 min. As is the case for most thrombolytic agents, the most significant risk associated with tPA administration is the possible induction of severe haemorrhage. Engineered tPA. Modified forms of tPA have also been generated in an effort to develop a product with an improved therapeutic profile (e.g. faster-acting or exhibiting a prolonged BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 383 Table 9.11. The five domains that constitute human tPA and the biological function of each domain tPA domain Function Finger domain (F domain) Promotes tPA binding to fibrin with high affinity Protease domain (P domain) Displays plasminogen-specific proteolytic activity Epidermal growth factor domain (EGF domain) Binds hepatic receptors thereby mediating hepatic clearance of tPA from blood Kringle-1 domain (K 1 domain) Associated with binding to the hepatic receptor Kringle-2 domain (K 2 domain) Facilitates stimulation of tPA’s proteolytic activity by fibrin plasma half-life). Reteplase is the international non-proprietary name given to one such modified human tPA produced in recombinant E. coli cells and is sold under the tradenames Ecokinase, Retavase and Rapilysin (Table 9.10). This product’s developm ent was based upon the generation of a synthetic nucleotide sequence encoding a shortened (355 amino acid) tPA molecule. This analogue contained only the tPA domains responsible for fibrin selectivity and catalytic activity. The nucleotide sequence was integrated into an expression vector subsequently introduced into E. coli (strain K12) by treatment with calcium chloride. The protein is expressed intracellularly where it accumulates in the form of an inclusion body. Due to the prokaryotic production system, the product is non-glycosylated. The final sterile freeze-dried product exhibits a 2 year shelf-life when stored at temperatures below 258C. An overview of the production process is presented in Figure 9.19. The lack of glycosylation as well as the absence of the EGF and K 1 domains (Table 9.11) confers an extended serum half-life upon the engineered molecule. Reteplase-based products display a serum half-life of up to 20 min, facilitating its administration as a single bolus injection 384 BIOPHARMACEUTICALS Figure 9.19. Production of Ecokinase, a modified tPA molecule which gained regulatory approval in Europe in 1996. The production cell line is recombinant E. coli K12, which harbours a nucleotide sequence coding for the shortened tPA molecule. The product accumulates intracellularly in the form of inclusion bodies as opposed to continuous infusion. Absence of the molecule’s F 1 domain also reduces the product’s fibrin-binding affinity. It is theorized that this may further enhance clot degradation, as it facilitates more extensive diffusion of the thrombolytic agent into the interior of the clot. Tenecteplase (also marketed under the tradename, Metalyse) is yet an additional engineered tPA now on the market. Produced in a CHO cell line, this glycosylated variant differs in sequence to native tPA by six amino acids (Thr 103 converted to Asn; Asn 117 converted to Gln and the Lys–His–Arg–Arg sequence at position 296–299 converted to Ala–Ala–Ala–Ala). Collectively, these modifications result in a prolonged plasma half-life (to 15–19 min), as well as an increased resistance to PAI-1 (plasminogen activator inhibitor-1, a natural tPA inhibitor). 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-exchan ge 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. BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 385 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 plasm a 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 pa rtially 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 util ized 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 608C 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 608C 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. Staphylokinase Staphylokinase is a protein produced by a number of strains of Staphylococcus aureus, which also displays therapeutic potenital as a thrombolytic agent. The protein has been purified from its natural source by a combination of ammonium sulphate precipitation and cation-exchange chromatography on CM cellulose. Affinity chromatography using plasmin or plasminogen immobilized to sepharose beads has also been used. The pure product is a 136 amino acid polypeptide displaying a molecular mass in the region of 16.5 kDa. Lower molecular mass derivatives lacking the first six or 10 NH 2 -terminal amino acids have also been characterized. All three appear to display similar thrombolytic activity in vitro at least. The staphylokinase gene has been cloned in E. coli, as well as various other recombinant systems. The protein is expressed intracellularly in E. coli at high levels, representing 10–15% of total cellular protein. It can be purified directly from the clarified cellular homogenate by a combination of ion-exchange and hydrophobic interaction chromatography. Although staphylokinase shows no significant homology with streptokinase, it induces a thrombolytic effect by a somewhat similar mechanism — it also forms a 1:1 stoichiometric complex with plasminogen. The proposed mechanism by which staphylokinase induces 386 BIOPHARMACEUTICALS plasminogen activation is outlined in Figure 9.20. Binding of the staphylokinase to plasminogen appears to initially yield an inactive staphylokinase–plasminogen complex. However, complex formation somehow induces subsequent proteolytic cleavage of the bound plasminogen, forming plasmin, which remains complexed to the staphylokinase. This complex (via the plasmin) then appears to catalyse the conversion of free plasminogen to plasmin, and may even accelerate the process of conversion of other staphylokinase–plasminogen complexes into staphylokinase–plasmin complexes. The net effect is generation of active plasmin, which BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 387 Figure 9.20. Schematic representation of the mechanism by which staphylokinase appears to activate the thrombolytic process via the generation of plasmin. See text for details displays a direct thrombolytic effect by degrading clot-based fibrin, as described previously (Figure 9.18). The serum protein a 2 -antiplasmin can inhibit the activated plasmin–staphylokinase complex. It appears that the a 2 -antiplasmin can interact with the active plasmin moiety of the complex, resulting in dissociation of staphylokinase, and consequent formation of an inactive plasmin– a 2 –antiplasmin complex. The thrombolytic ability of (recombinant) staphylokinase has been evaluated in initial clinical trials, with encouraging results; 80% of patients suffering from acute myocardial infarction who received staphylokinase responded positively (10 mg staphylokinase was administered by infusion over 30 min). The native molecule displays a relatively short serum half-life (6.3 min), although covalent attachment of polyethylene glycol (PEG) reduces the rate of serum clearance, hence effectively increasing the molecule’s half-life significantly. As with streptokinase, patients administered staphylokinase develop neutralizing antibodies. A number of engineered (domain- deleted) variants have been generated, which display significantly reduced immunogenicity. a 1 -Antitrypsin The respiratory tract is protected by a number of defence mechanisms which include: . particle removal in the nostril/nasopharynx; . particle expulsion (e.g. by coughing); . upward removal of substances via mucociliary transport; . presence in the lungs of immune cells, such as alveolar macrophages; . production/presence of soluble protective factors, including a 1 -antitrypsin, lysozyme, lactoferrin and interferon. Failure/ineffective functioning of one or more of these mechanisms can impair normal respiratory function, e.g. emphysema is a condition in which the alveoli of the lungs are damaged, which compromises the lung’s capacity to exchange gases, and breathlessness often results. This condition is often promoted by smoking, respiratory infections or a deficiency in the production of serum a 1 -antitrypsin. a 1 -Antitrypsin is a 394 amino acid, 52 kDa serum glycoprotein. It is synthesized in the liver and secreted into the blood, where it is normally present at concentrations of 2–4 g/l. It constitutes in excess of 90% of the a 1 -globulin fraction of blood. The a 1 -antitrypsin gene is located on chromosome 14. A number of a 1 -antitrypsin gene variants have been described. Their gene products can be distinguished by their differential mobility upon gel electrophoresis. The normal form is termed M, while point mutations in the gene have generated two major additional forms, S and Z. These mutations results in a greatly reduced level of synthesis and secretion into the blood of the mature a 1 -antitrypsin. Persons inheriting two copies of the Z gene, in particular, display greatly reduced levels of serum a 1 - antitrypsin activity. This is often associated with the development of emphysema (particularly in smokers). The co ndition may be treated by the administration of purified a 1 -antitrypsin. This protein constitutes the major serine protease inhibitor present in blood. It is a potent inhibitor of the protease elastase, and serves to protect the lung from proteolytic damage by inhibiting neutrophil elastase. The product is administered on an ongoing basis to sufferers, who receive up to 200 g of the inhibitor each year. It is normally prepared by limited fractionation of whole human blood, although the large quantities required by patients heightens the risk of accidental transmission of blood-borne pathogens. The a 1 -antitrypsin gene has been expressed in a number 388 BIOPHARMACEUTICALS [...]... fibrin-specific plasminogen activator with therapeutic potential? Blood 84 (3), 680 – 686 Collen, D (19 98) Staphylokinase: a potent, uniquely fibrin-selective thrombolytic agent Nature Med 4(3), 279– 284 Collins, R et al (1997) Drug therapy—aspirin, heparin and fibrinolytic therapy in suspected acute myocardial infarction N Engl J Med 336(12), 84 7 86 0 Datar, R et al (1993) Process economics of animal cell and. .. refocusing an elusive goal Br J Haematol 111(2), 387 –396 Wright, G et al (1991) High level expression of active human a-1-antitrypsin in the milk of transgenic sheep Bio/ Technology 9, 83 0 83 4 Anticoagulants and related substances Dodt, J (1995) Anti-coagulatory substances of bloodsucking animals: from hirudin to hirudin mimetics Angew Chem Int Ed Engl 34, 86 7 88 0 Eldora, A et al (1996) The role of the leech... and the total world market for glucocerebrosidase is estimated to be in the region of $200 million Cerezyme is produced in a CHO cell line harbouring the cDNA coding for human b-glucocerebrosidase The purified product is presented as a freeze-dried powder and also contains mannitol, sodium citrate, citric acid and polysorbate 80 as excipients It exhibits a shelf-life of 2 years when stored at 28C 88 C... in their binding to and uptake by the macrophages In this way, the ‘mannoseengineered’ enzyme is selectively targeted to the affected cells BLOOD PRODUCTS AND THERAPEUTIC ENZYMES 395 a-Galactosidase and urate oxidase Recombinant a-galactosidase and urate oxidase represent two additional biopharmaceuticals recently approved for general medical use a-Galactosidase is approved for long-term enzyme replacement... review of the scientific basis and mechanism of action of anticoagulant therapies Br J Anaesth 88 (6), 84 8 86 3 Thrombolytics Al-Buhairi, A & Jan, M (2002) Recombinant tissue plasminogen activator for acute ischemic stroke Saudi Med J 23(1), 13–19 Blasi, F (1999) The urokinase receptor A cell surface, regulated chemokine APMIS 107(1), 96–101 Castillo, P et al (1997) Cost-effectiveness of thrombolytic therapy... a 40 min infusion), the enzyme is taken up by various body cell types and directed to the lysosomes This cellular uptake and delivery process appears to be mediated by mannose-6-phosphate residues present in the oligosaccharide side-chains of the enzyme Mannose-6-phosphate receptors are found on the surface of various cell types and also intracellularly, associated with the Golgi complex, which then... 24(5), 287 –296 Conway, S & Littlewood, J (1997) rhDNase in cystic fibrosis Br J Hosp Med 57 (8) , 371–372 Conway, S & Watson, A (1997) Nebulized bronchodilators, corticosteroids and rhDNase in adult patients with cystic fibrosis Thorax 52(2), S64–S 68 Edgington, S (1993) Nuclease therapeutics in the clinic Bio/Technology 11, 580 – 582 James, E (1994) Superoxide dismutase Parasitol Today 10(12), 482 – 484 Mistry,... immunoglobulin G (IgG) Biopharmaceuticals: Biochemistry and Biotechnology, Second Edition by Gary Walsh John Wiley & Sons Ltd: ISBN 0 470 84 326 8 (ppc), ISBN 0 470 84 327 6 (pbk) 404 BIOPHARMACEUTICALS Antisera are generally produced by immunizing healthy animals (e.g horses) with appropriate antigen Small samples of blood are subsequently withdrawn from the animal on a regular basis and quantitatively analysed for... continuous human cell line and is also purified by a combination of five chromatographic purification steps, although it is marketed as a liquid solution Human a-galactosidase is a 100 kDa homodimeric glycoprotein Each 3 98 amino acid monomer displays a molecular mass of 45.3 kDa (excluding the glycocomponent) and is glycosylated at three positions (asparagines 1 08, 161 and 184 ) After administration (usually... growth of various leukaemias and other transformed cell lines PEG-coupled enzymes are often preferred, as they display an extended plasma half-life Although asparaginase therapy has proved effective, a number of side-effects have been associated with initiation of therapy These have included severe nausea, vomiting and diarrhoea, as well as compromised liver and kidney function Side-effects are probably due . glucocerebrosides, particularly in tissue- based macrophages. Clinical systems include enlargement and compromised function of these macrophage-containing tissues, particularly the liver and spleen,. human b-glucocerebrosidase. The purified product is presented as a freeze-dried powder and also contains mannitol, sodium citrate, citric acid and polysorbate 80 as excipients. It exhibits a shelf-life. stored at 28C 88 C. An integral part of the downstream processing process entails the modification of Cerezyme’s oligosaccharide comp onents. The native enzyme’s sugar side-chains are complex and, for