Biopharmaceutical 12. Recombinant blood products and therapeutic enzymes

1. Biopharmaceutical12. Recombinant blood products andtherapeutic enzymes Dr. Tarek El-Bashiti Assoc. Prof. of Biotechnology

2. Introduction • Blood and blood products constitute a major group of traditional biologics. • The main components of blood are the red and white blood cells, along with platelets and the plasma in which these cellular elements are suspended. • Whole blood remains in routine therapeutic use, as do red blood cell • and platelet concentrates.

3. A variety of therapeutically important blood proteins also continue to be purified from plasma. • These include various clotting factors and immunoglobulins. • We focus in this chapter upon blood proteins/blood-related proteins produced by genetic engineering. • These include recombinant coagulation factors, anticoagulants (such as hirudin) and thrombolytics (such as tPA). • Also,there are a number of recombinant enzymes that have found therapeutic application.

4. Haemostasis: • Blood plays various vital roles within the body and it is not surprising that a number of processes have evolved capable of effectively maintaining haemostasis (the rapid arrest of blood loss upon vascular damage, in order to maintain a relatively constant blood volume). • In humans, three main mechanisms underline the haemostatic process: • 1- The congregation and clumping of blood platelets at the site of vascular injury, thus effectively plugging the site of blood leakage. • 2- Localized constriction of the blood vessel, which minimizes further blood flow through the area.

5. 3- Induction of the blood coagulation cascade. • This culminates in the conversion of a soluble • serum protein, fibrinogen, into insoluble fibrin. • Fibrin monomers then aggregate at the site of damage, thus forming a clot (thrombus) to seal it off. • These mechanisms are effective in dealing with small vessel injuries (e.g. capillaries and arterioles), although they are ineffective when the damage relates to large veins/arteries.

6. The coagulation pathway: • At least 12 distinct factors participate in the coagulation cascade, along with several macromolecular cofactors. • The clotting factors are all designated by Roman numerals (Table 12.1) and, with the exception of factor IV, all are proteins. • Most factors are proteolytic zymogens, which become sequentially activated. • An activated factor is indicated by inclusion of a subscript ‘a’ (e.g. factor XIIa is activated factor XII). • Although the final steps of the blood clotting cascade are identical, the initial steps can occur via two distinct pathways: extrinsic and intrinsic.

8. Both pathways are initiated when specific clotting proteins make contact with specific surface molecules exposed only upon damage to a blood vessel. • Clotting occurs much more rapidly when initiated via the extrinsic pathway. • Two coagulation factors function uniquely in the extrinsic pathway: factor III (tissue factor) and factor VII. • Tissue factor is an integral membrane protein present in a wide variety of tissue types (particularly lung and brain). • This protein is exposed to blood constituents only upon rupture of a blood vessel, and it initiates the extrinsic coagulation cascade at the site of damage as described below.

9. Factor VII contains a number of γ carboxyglutamate residues (as do factors II, IX and X), which play an essential role in facilitating their binding of Ca2+ ions. • The initial events initiating the extrinsic pathway entail the interaction of factor VII with Ca2+ and tissue factor. • In this associated form, factor VII becomes proteolytically active. • It displays both binding affinity for, and catalytic activity against, factor X. • It thus activates factor X by proteolytic processing, and factor Xa, which initiates the terminal stages of clot formation, remains attached to the tissue factor– Ca2+ complex at the site of damage.

11. This ensures that clot formation only occurs at the point where it is needed (Figure 12.1). • The initial steps of the intrinsic pathway are somewhat more complicated. • This system requires the presence of clotting factors VIII, IX, XI and XII, all of which, except for factor VIII, are endo-acting proteases. • As in the case of the extrinsic pathway, the intrinsic pathway is triggered upon exposure of the clotting factors to proteins present on the surface of body tissue exposed by vascular injury. • These protein binding/activation sites probably include collagen.

12. Additional protein constituents of the intrinsic cascade include prekallikrein, an 88 kDa protein zymogen of the protease kallikrein, and high molecular mass kininogen, a 150 kDa plasma glycoprotein that serves as an accessory factor. • The intrinsic pathway appears to be initiated when factor XII is activated by contact with surface proteins exposed at the site of damage. • High molecular mass kininogen also appears to form part of this initial activating complex (Figure 12.2). • Factor XIIa can proteolytically cleave and, hence, activate two substrates:

14. prekallikrein, yielding kallikrein (which, in turn, can directly activate more XII to XIIa). • factor XI, forming XIa. • Factor XIa, in turn, activates factor IX. • Factor IXa then promotes the activation of factor X, but only when it (i.e. IXa) is associated with factor VIIIa. • Factor VIIIa is formed by the direct action of thrombin on factor VIII. • The thrombin will be present at this stage because of prior activation of the intrinsic pathway.

15. Terminal steps of coagulation pathway: • Both intrinsic and extrinsic pathways generate activated factor X. • This protease, in turn, catalyses the proteolytic conversion of prothrombin (factor II) into thrombin (IIa). • Thrombin, in turn, catalyses the proteolytic conversion of fibrinogen (I) into fibrin (Ia). • Individual fibrin molecules aggregate to form a soft clot. • Factor XIIIa catalyses the formation of covalent crosslinks between individual fibrin molecules, forming a hard clot (Figures 12.3 and 12.4). • Prothrombin (factor II) is a 582 amino acid, 72.5 kDa glycoprotein, which represents the circulating zymogen of thrombin (IIa).

16. It contains up to six γ-carboxyglutamate residues towards its N-terminal end, via which it binds several Ca2+ ions. • Binding of Ca2+ facilitates prothrombin binding to factor Xa at the site of vascular injury. • The factor Xa complex then proteolytically cleaves prothrombin at two sites (arg274–thr275 and arg323–ile324), yielding active thrombin and an inactive polypeptide fragment (Figure 12.5). • Fibrinogen (factor I) is a large (340 kDa) glycoprotein consisting of two identical tri- polypeptide units, α, β and γ. • Its overall structural composition may thus be represented as (α β γ)2.

20. The N-terminal regions of the α and β fibrinogen chains are rich in charged amino acids, which, via charge repulsion, play an important role in preventing aggregation of individual fibrinogen molecules. • Thrombin, which catalyses the proteolytic activation of fibrinogen, hydrolyses these N-terminal peptides. • This renders individual fibrin molecules more conducive to aggregation, therefore promoting soft clot formation. • The soft clot is stabilized by the subsequent introduction of covalent cross-linkages between individual participating fibrin molecules. • This reaction is catalysed by factor XIIIa.

21. Clotting disorders: • Genetic defects characterized by: • lack of expression or • an altered amino acid sequence of any clotting factor can have serious clinical consequences. • In order to promote effective clotting, both intrinsic and extrinsic coagulation pathways must be functional, and the inhibition of even one of these pathways will result in severely retarded coagulation ability. • The result is usually occurrence of spontaneous bruising and prolonged haemorrhage, which can be fatal. • With the exception of tissue factor and Ca2, defects in all other clotting factors have been characterized.

22. Up to 90 per cent of these, however, relate to a deficiency in factor VIII, and much of the remainder is due to a deficiency in factor IX. • Such clotting disorders are generally treated by ongoing administration of whole blood or, more usually, concentrates of the relevant coagulation factor purified from whole blood. • This entails significant risk of accidental transmission of blood-borne disease, particularly hepatitis and AIDS. • In turn, this has hastened the development of blood coagulation factors produced by genetic engineering, several of which are now approved for general medical use (Table 12.2).

24. Factor VIII and haemophilia: • Haemophilia A (classical haemophilia, often simply termed haemophilia) is an X-linked recessive disorder caused by a deficiency of factor VIII. • Von Willebrand disease is a related disorder, also caused by a defect in the factor VIII complex, as discussed below. • Intact factor VIII, as usually purified from the blood, consists of two distinct gene products: • factor VIII and (multiple copies of) von Willebrand’s factor (vWF; Figure 12.6). • This complex displays a molecular mass ranging from 1 to 2 MDa, of which up to 15 per cent is carbohydrate.

26. The fully intact factor VIII complex is required to enhance the rate of activation of factor IX of the intrinsic system. • The factor VIII polypeptide portion of the factor VIII complex is coded for by an unusually long gene (289 kb). • Transcription and processing of the mRNA generates a shorter, mature, mRNA that codes for a 300 kDa protein. • Upon its synthesis, this polypeptide precursor is subsequently proteolytically processed, with removal of a significant portion of its mid region. • This yields two fragments: an amino terminal 90 kDa polypeptide and an 80 kDa carboxyl terminal polypeptide.

27. These associate non-covalently (a process requiring Ca2 ions) to produce mature factor VIII (sometimes called factor VIII:C). • This mature factor VIII is then released into the plasma where it associates with multiple copies of vWF forming the biologically active factor VIII complex. • vWF stabilizes factor VIII in plasma (particularly against proteolytic degradation). • It also can associate with platelets at the site of vascular damage and, hence, presumably plays a role in docking the factor VIII complex in an appropriate position where it can participate in the coagulation cascade.

28. Persons suffering from haemophilia A exhibit markedly reduced levels (or the complete absence) of factor VIII complex in their blood. • This is due to the lack of production of factor VIII:C. • Persons suffering from (the rarer) von Willebrand’s disease lack both components of mature factor VIII complex (Figure 12.6). • Persons completely devoid of it (or expressing levels below 1 per cent of normal values) will experience frequent, severe and often spontaneous bouts of bleeding.

29. Persons expressing 5 per cent or above of the normal complex levels experience less severe clinical symptoms. • Treatment normally entails administration of factor VIII complex purified from donated blood. • More recently, recombinant forms of the product have also become available. • Therapeutic regimens can require product administration on a weekly basis, for life. • About 1 in 10 000 males are born with a defect in the factor VIII complex and there are approximately 25 000 haemophiliacs currently resident in the USA.

30. Production of factor VIII: • Native factor VIII is traditionally purified from blood donations first screened for evidence of the presence of viruses such as hepatitis B and HIV. • A variety of fractionation procedures (initially mainly precipitation procedures) have been used to produce a factor VIII product. • The final product is filter sterilized and filled into its finished product containers. • The product is then freeze-dried and the containers are subsequently sealed under vacuum, or are flushed with an inert gas (e.g. N2) before sealing. • No preservative is added.

31. The freeze-dried product is then stored below 8 C until shortly before its use. • Although earlier factor VIII preparations were relatively crude (i.e. contained lower levels of various other plasma proteins), many of the modern preparations are chromatographically purified to a high degree. • The use of immunoaffinity chromatography has become widespread in this regard since 1988 (Figure 12.7). • The extreme bioselectivity of this method can yield a single-step purification factor of several thousand-fold.

33. Although fractionation can reduce very significantly the likelihood of pathogen transmission, it cannot entirely eliminate this possibility. • Blood-derived factor VIII products, including those prepared by immunoaffinity chromatography, generally undergo further processing steps designed to remove/inactivate any virus present. • The raw material is often heated for up to 10 h at 60°C or treated with solvent or dilute detergent prior to chromatography. • Recombinant factor VIII is also often treated with dilute detergent in an effort to inactivate any viral particles potentially present.

34. Production of recombinant factor VIII (Table 12.2) has ended dependence on blood as the only source of this product, and eliminated the possibility of transmitting blood-borne diseases specifically derived from infected blood. • In the past, over 60 per cent of haemophiliacs were likely to be accidentally infected via contaminated products at some stage of their life. • Several companies have expressed the cDNA coding for human factor VIII:C in a variety of eukaryotic production systems (human VIII:C contains 25 potential glycosylation sites).

35. CHO cells and BHK cell lines have been most commonly used, in addition to other cell lines, such as various mouse carcinoma cell lines. • The recombinant factor VIII product generally contains only VIII:C (i.e. is devoid of vWF). • However, both clinical and preclinical studies have shown that administration of this product to patients suffering from haemophilia A is equally as effective as administering blood-derived factor VIII complex. • The recombinant VIII:C product appears to bind plasma vWF with equal affinity to native VIII:C upon its injection into the patient’s circulatory system.

36. Animal and human pharmacokinetic data reveal no significant difference between the properties of recombinant and native products. • Some patients, particularly those suffering from severe haemophilia A (i.e. those naturally producing little or no VIII:C), will mount an immune response against injected factor VIII:C whatever its source. • The production of anti-factor VIII:C antibodies renders necessary administration of higher therapeutic doses of the product. • In severe cases, the product may even become ineffective. • Several approaches may be adopted in order to circumvent this problem.

37. These include: • 1. Exchange transfusion of whole blood. • This will transiently decrease circulating anti-factor VIII:C antibodies. • 2. Direct administration of factor Xa, thus bypassing the non-functional step in the coagulation cascade (Figures 12.2 and 12.3). • 3. Administration of high levels of a mixture of clotting factors II, VII, IX and X, which works effectively in 50 per cent of treated patients. • 4. Administration of factor VIIa, as discussed subsequently.

38. 5. Administration of porcine factor VIII, which may or may not cross-react with the antibodies raised against human factor VIIIa. (However, the immune system will soon begin to produce antibodies against the porcine clotting factor). • 6. Administration of factor VIII, with concurrent administration of immunosuppressive agents. • Owing to the frequency of product administration, the purification procedure for recombinant factor VIII:C must be particularly stringent. • Unlike the situation pertaining when the product is purified from human blood, any contaminant present in the final product will be non-human and, hence, immunogenic.

39. Sources of such contaminants would include: • 1. host cell proteins; • 2. animal cell culture medium; • 3. antibody leaked from the immunoaffinity column. • Emphasis is placed not only upon ensuring the absence of contaminant proteins, but also other potential contaminants, particularly DNA. (Host cell-line-derived DNA could harbour oncogenes) • Researchers are also attempting to develop modified forms of VIII:C (by site-directed mutagenesis) that display additional desirable characteristics. • Particularly attractive in this regard would be the development of a product exhibiting an extended circulatory half-life.

40. This could reduce the frequency of injections required by haemophilia A sufferers. • However, any alteration of the primary sequence of the molecule carries with it the strong possibility of rendering the resultant mutant immunogenic. • Individuals who display a deficiency of factor IX develop haemophilia B, also known as Christmas disease. • Although its clinical consequences are very similar to that of a deficiency of factor VIII, its general incidence in the population is far lower. • Persons suffering from haemophilia B are treated by i.v. administration of a concentrate of factor IX.

41. This was traditionally obtained by fractionation of human blood. • Recombinant factor IX is now also produced in genetically engineered CHO cells (Table 12.2). • Factor IX obtained from blood donations is normally only partially pure. In addition to factor IX, the product contains lower levels of factors II, VII and X and has also been used to treat bleeding disorders caused by a lack of these factors. • Factor IX may also be purified by immunoaffinity chromatography, using immobilized anti- IX murine monoclonals.

42. Some 5–25 per cent of individuals suffering from haemophilia A develop anti-factor VIII antibodies, and 3–6 per cent of haemophilia B sufferers develop anti-factor IX antibodies. • This complicates treatment of these conditions and, as mentioned previously, one approach to their treatment is direct administration of factor VIIa. • The therapeutic rationale is that factor VIIa could directly activate the final common steps of the coagulation cascade, independently of either factor VIII or IX (Figure 12.1). • Factor VIIa forms a complex with tissue factor that, in the presence of phospholipids and Ca2, activates factor X.

43. A recombinant form of factor VIIa (called ‘NovoSeven’ or ‘eptacog alfa-activated’) is marketed by Novo-Nordisk (Table 12.2). • The recombinant molecule is produced in a BHK cell line, and the final product differs only slightly (in its glycosylation pattern only) from the native molecule. • Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. • It is initially produced as an unactivated, single-chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor VIIa complex.

44. After sterilization by filtration, the final product is aseptically filled into its final product containers, and freeze-dried. • A (very rare) genetic deficiency in the production of factor XIII also results in impaired clotting efficacy in affected persons. • In this case, covalent links that normally characterize transformation of a soft clot into a hard clot are not formed. • Factor XIII preparations, partially purified from human blood, are used to treat individuals with this condition; to date, no recombinant version of the product has been commercialized.

45. Anticoagulants: • Although blood clot formation is essential to maintaining haemostasis, inappropriate clotting can give rise to serious, sometimes fatal medical conditions. • The formation of a blood clot (a thrombus) often occurs inappropriately within diseased blood vessels. • This partially or completely obstructs the flow of blood (and hence oxygen) to the tissues normally served by that blood vessel. • Thrombus formation in a coronary artery (the arteries that supply the heart muscle itself with oxygen and nutrients) is termed coronary thrombosis.

46. This results in a heart attack, characterized by the death (infarction) of oxygen-deprived heart muscle; hence the term myocardial infarction. • The development of a thrombus in a vessel supplying blood to the brain can result in development of a stroke. • In addition, a thrombus (or part thereof) that has formed at a particular site in the vascular system may become detached. • After travelling through the blood, this may lodge in another blood vessel, obstructing blood flow at that point. • This process, which can also give rise to heart attacks or strokes, is termed embolism.

47. Anticoagulants are substances that can prevent blood from clotting and, hence, are of therapeutic use in cases where a high risk of coagulation is diagnosed. • They are often administered to patients with coronary heart disease and to patients who have experienced a heart attack or stroke (in an effort to prevent recurrent episodes). • The major anticoagulants used for therapeutic purposes are listed in Table 12.3. • Heparin is a carbohydrate-based (glycosaminoglycan) anticoagulant associated with many tissues.

49. Upon release into the bloodstream, heparin binds to and thereby activates an additional plasma protein, namely antithrombin. • The heparin–antithrombin complex then binds a number of activated clotting factors (including IIa, IXa, Xa, XIa and XIIa), thereby inactivating them. • The heparin now disassociates from the complex and combines with another antithrombin molecule, thereby initiating another turn of this inhibitory cycle. • Heparin was originally extracted from liver (hence its name), but commercial preparations are now obtained by extraction from beef lung or porcine gastric mucosa.

50. Although the product has proven to be an effective (and relatively inexpensive) anticoagulant, it does suffer from a number of clinical disadvantages, including the need for a cofactor (antithrombin III) and poorly predictable dose responses. • Despite such disadvantages, however, heparin still enjoys widespread clinical use. • The vitamin K antimetabolites dicoumarol and warfarin are related coumarin-based anticoagulants which, unlike heparin, may be administered orally. • These compounds induce their anticoagulant effect by preventing the vitamin K-dependent γ carboxylation of certain blood factors, specifically factors II, VII, IX and X.