Recombinant plasma proteins

Vox Sanguinis (2011) 100, 68–83 ª 2010 The Author(s) Vox Sanguinis ª 2010 International Society of Blood Transfusion DOI: 10.1111/j.1423-0410.2010.01384.x

REVIEW

Recombinant plasma proteins T. Burnouf Human Protein Process Sciences (HPPS), Lille, France

Received: 20 May 2010, revised 8 July 2010, accepted 12 July 2010

For almost 50 years, the fractionation of human plasma has been the sole possible source of a wide range of therapeutic proteins–such as coagulation factors, anticoagulants, immunoglobulins, and albumin – essential to the treatment of serious congenital or acquired bleeding or immunological diseases. In the last 20 years, the application of recombinant technologies to mammalian cell cultures has made possible – although with some limitations in productivity, costs, and immunogenic risks – to produce and commercialize complex plasma glycoproteins for human therapeutic applications and has opened the way to the development of new molecular entities. Today, the advanced exploration of alternative cell lines and enhanced cell culture systems, as well as the development of alternative expression technologies, such as transgenic animals, is opening a new era in the production of the full range of recombinant plasma protein therapeutics. In this review, we examine the achievements and ongoing challenges of the recombinant DNA technology as a platform for the production of plasma proteins and the advantages and limitations of such products compared to fractionated plasma proteins. Key words: plasma, proteins, recombinant.

Introduction The development of a plasma fractionation process in the 1940s [1] has been a milestone in human health care by opening the gate to industrial production of human protein therapeutics. Plasma fractionation has evolved into a sophisticated industry [2] with over 50 fractionators worldwide producing a range of up to 20 different protein biologicals from 20 to 25 million litres of plasma annually [3,4]. Plasma-derived (PD) products are invaluable for treating bleeding, immunological, and metabolic disorders associated with congenital or acquired protein deficiencies or for compensating blood losses, thereby contributing to improving and saving the life of many patients. Unfortunately, before the late 1980s, their use has led to numerous viral transmissions at a time when the plasma industry lacked scientific assessment, manufacturing processes did not include dedicated viral reduction treatments, donor selection, and donation testing were not as sophisticated Correspondence: Thierry Burnouf, HPPS, Human Protein Process Sciences, Lille, France E-mail: [email protected]

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and rigorous as now, and the regulatory oversight was weak. Risks of viral transmissions, inability to meet product demand, and perceived high cost of PD-protein therapeutics have built objective scientific and economical interests towards the development of recombinant human (rh) plasma proteins. Perspectives of producing by genetic engineering technologies have emerged in the 1980s when Factor IX (FIX) and FVIII gene cloning and expression in mammalian cell culture systems moved from the laboratory to concrete industrial reality [5,6], with the prospect of reproducing the complex human post-translational modifications required for proper protein folding and functionality. After the pioneering commercial introduction of rhFVIII and rhFIX, many other genetically engineered therapeutic plasma proteins have been developed. Recombinant DNA technology is now an established technology platform. In this paper, we examine the technological approaches available for producing recombinant plasma proteins, review the products licensed or under development compared to the PD-counterparts, discuss benefits and limits, and look at expected new developments in the field. More detailed information can be found in previous reviews [7,8].

Recombinant plasma proteins 69

Expression systems Recombinant protein therapeutics encompass cytokines, growth factors, vaccines, hormones, enzymes, coagulation factors, anticoagulants, albumin, and monoclonal antibodies (mAbs) [9]. Whereas microbial cell systems (bacteria or yeast [10]) are used to produce 40% (and 60% in sales value) of licensed recombinant pharmaceuticals, such microbial organisms were, until recently, unsuitable for achieving complex post-translational modifications or intricate folding of most plasma proteins. Expression systems for plasma proteins are selected based upon structural characteristics (e.g. molecular mass [MM] and glycosylation pattern), intended clinical applications (e.g. prophylaxis), and mode of administration. Cost-effectiveness requires expression levels competitive with PD-proteins that often serve as market benchmarks. Large proteins nowadays are usually expressed in mammalian animal cells equipped with the cellular machinery able to perform glycosylation close to or similar to human-type N- and O-linked. Transgenic animals secreting recombinant proteins in milk are generating interest because of their high productivity, and human cell lines for a possible, still controversial ability to deliver fully human glycosylation patterns [9].

Escherichia coli Escherichia coli systems offer genetic flexibility and are commonly used for expressing simple polypeptides such as insulin, human growth hormone, interferon-a, and PDGFBB [11]. Expression of complex functional high MM plasma glycoproteins has been unsuccessful. Indeed, essential post-translational modifications cannot be achieved by E. coli, which affects protein folding and disulphide bonds formation and leads to aggregation, loss of activity, as well as reduced stability in vitro and rapid protein clearance from blood [12]. For instance, E. coli-expressed alpha 1antitrypsin (AAT) showed reduced stability in vitro and altered pharmacokinetic properties [13].

Yeasts Yeast production systems (Saccharomyces cerevisiae and the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha) are successfully used to express therapeutic blood proteins, such as interferon-a and PDGF-BB, with a MM < 60 kDa [12]. Yeasts have well-characterized genetic make-up, are relatively easy to manipulate, grow fast, and do not produce endotoxins [14]. Proteins can be expressed intracellularly or be secreted. Protease-deficient strains limit risks of proteolytic degradation. Yeasts can perform early-stage N-glycosylations in the endoplasmic reticulum, but complex oligosaccharides synthesis and glycan

maturation by terminal sialylation in the Golgi apparatus could not be achieved, until recently [9,10], resulting in incorrect glycosylation patterns and glycoprotein heterogeneity, rapid clearance from the blood stream, and/or immunogenic risks. New strains that better modulate complex glycosylation may lead to some progress [10,15]. Expression of proteins at levels of 1–10 g/l and more has been reported [9]. Successful large-scale production of albumin, a non-glycosylated plasma protein, has been achieved [16], as discussed in a following section.

Filamentous fungi Filamentous fungi may perform glycosylation patterns resembling those of mammals [17] and avoid hypermannosylation problems known for yeast. At present, only a very few human genes have been expressed in filamentous fungi, and plasma proteins are unlikely to be produced in this system for the foreseeable future.

Insect cells Insect cells hosting the baculovirus expression vector system can do many post-translational modifications, such as N- and O-glycosylation or disulphide bond formation, and achieve protein folding [12]. With an appropriate gene promoter (polyhedrin), high protein expression may be achieved [18,19]. However, glycosylation mechanisms of heterologous proteins by insect cells are still largely undescribed because of lack of several enzymes crucial for human-type N-glycan synthesis or sialylation and risks of addition of non-human sugars. Current insect cell systems are believed unsuitable for the production of complex human plasma glycoproteins [13].

Mammalian cells Mammalian host cells, primarily Chinese hamster ovary (CHO) and baby hamster kidney cells (BHK-21), are the expression systems of choice for most recombinant plasma protein therapeutics as they can perform complex posttranslational modifications [9,20–23] and secrete proteins in the media in their authentic form [23], and the CHO cell line is known to be free of viral adventitious agents. Drawbacks have included low cell density (approximately 5 million cells/ml) of CHO or BHK systems [7], lack of reproducibility, and potential risk of contamination by infectious agents when processes require exogenous animal proteins such as serum. Use of animal-derived-componentfree media supplemented with plant hydrolysates or chemically defined media is the standard of the new generation of recombinant proteins expressed in mammalian cells [23]. More stringent clone selection techniques, enhanced

2010 The Author(s) Vox Sanguinis 2010 International Society of Blood Transfusion, Vox Sanguinis (2011) 100, 68–83

70 T. Burnouf

density (12–14 million cells/ml), use of high expression constructs [7], and improved medium formulation may increase productivity substantially and reduce the cost of rh-proteins produced so far at low expression levels.

Transgenic animals With the transfer of mammalian genes into the genetic material of animals, transgenic animals (mice, rats, sheep, goats, and pigs) are interesting ‘bioreactors’ for recombinant plasma proteins. Difficulties encompass construction of expression vectors, predictable expression of the transgene, potential deleterious effects of the recombinant proteins on the mammary gland function or on the animals themselves, and achievement of complete human posttranslational modifications [24]. Cell densities close to 1 billion/ml in mammary tissues may allow secretion levels of 2–20 g/l [25,26]. While the small amount of milk produced by transgenic mice restricts their use to proof of concept, other animals have higher productivity, from 1Æ5 l/lactation/rabbit (allowing annual commercial scale of up to 1 kg) up to 10 000 l/year/cow [26]. A major hurdle remains the risk of immune responses as the complex posttranslational modifications (c-carboxylation, b-hydroxylation; N- and O-linked glycosylation; phosphorylation; and sulphation) achieved may be species- and tissuespecific. Purification of recombinant proteins from milk is relatively straightforward. Careful animal selection, veterinary controls, and dedicated treatments are needed to reduce viral and prion risks [24]. The whole production chain should comply with regulatory GMP guidelines. The recent licence of antithrombin (AT) from the milk of transgenic goats demonstrates the technical and commercial feasibility [27]. Transgenic animal herds producing other human plasma proteins [AAT, C1 esterase inhibitor (C1-inh), fibrinogen, albumin], and mAbs have been created, and products are under development.

Transgenic plants Transgenic plants can perform post-translational, in particular N-glycan, modifications [28], but the Golgi apparatus is unable to synthesize some sugar structures and terminal sialic acid (mutants may alleviate some of these limits) and addition of non-human sugars (such as xylose) that may create immunogenicity issues. Ensuring GMP of transgenic plants in fields or greenhouses [29] is problematic, and the regulatory framework is only emerging [30]. In vitro cell cultures are preferred for the optimal control of growth conditions, batch-to-batch consistency, and containment [29,31]. Plants are believed free of viruses and prions pathogenic to humans and are cost-effective expression systems. No plasma proteins from transgenic plant have been

licensed yet, but therapeutic mAbs are under development [32].

Coagulation factors Table 1 presents the regulatory status and expression systems of recombinant plasma proteins, already licensed or at an advanced stage of development.

Factor IX FIX deficency (haemophilia B) occurs in about 1/30 000 male births. FIX has a MM of about 58 kDa, comprises 415 amino acids (AA), is secreted in the liver as a single-chain molecule, and circulates in plasma at about 5 mg/l. Complex post-translational modifications during FIX synthesis are essential to its functionality. Gamma carboxylation allows interaction with phospholipid surfaces and ensures activity [7]. Patients with severe haemophilia B (FIX < 0Æ01 IU/ml) on prophylatic treatment to prevent spontaneous bleeding episodes receive FIX at 25–40 IU FIX/kg twice a week. Most patients in developing countries are treated, usually when bleeding episodes occur, using either plasma, cryo-poor plasma, prothrombin complex (PCC), or PD-FIX. PD-PCC, which has been available for decades, contains several vitamin-K-dependent coagulation factors (FII, IX, and X and, for some, FVII) as well as protein C and protein S. FIX concentration is close to 25 IU/ml and specific activity as low as 0Æ5–1 IU/mg because of protein contaminants, some of which linked to thrombogenic risks. High-purity PD-FIX is at 50–100 IU/ml and 150–200 IU/mg. Viral safety is ensured by careful donor screening, plasma donation testing, robust viral reduction treatments, primarily solvent-detergent, and 15- to 20 -nm nanofiltration [2]; 300–350 IU of PCC or 200–300 IU of single FIX can be prepared per litre of plasma. To date, about 10–30% of the plasma available for fractionation is probably used to produce either PCC or single FIX. Both are produced by methods compatible with the extraction of major bulk proteins (including IgG). The plasma fractionation industry could manufacture more PD-FIX but existing capacity is used to produce more economically important intravenous immunoglobulin G (IVIG). The FIX gene was the first of coagulation factors to be cloned [6]. Development of functional rh-FIX in BHK cells required proper gamma carboxylation. Co-expression of furin resulted in a several fold increase in the proteolytic processing of rFIX, and the addition of vitamin-K to the cell culture improved active rh-FIX expression. Risk of activation had to be carefully controlled. Optimal growth conditions and use of specific strains allowed expression 30 times that in plasma [7,33]. Rh-FIX is expressed in a serumfree medium, purification achieved without immunoaffinity



Table 1 Recombinant plasma proteins (licensed or under advanced development) Expression system

Regulatory status

Mammalian cells Animals

Protein Coagulation factors FIX (Benefix) FIX FL-FVIII (Recombinate) FL-FVIII (Kogenate/Helixate) FL-FVIII plasma/albumin free (Advate) BDD-FVIII (ReFacto) BDD-FVIII plasma/albumin free (Xyntha) BDD-FVIII (Greengene) BDD-FVIII FVIIa (Novoseven) FVIIa Thrombin VWF VWF Fibrinogen FXIII Protease inhibitor/anticoagulant Activated PC AT AAT AAT C1-Inh Oncotic protein Albumin

CHO

BHK

Micro-organisms Human

Bacteria

Yeast

HEK

Escherichia coli

Pichia/ Saccharomyces

Transgenic animals

X

Year licensed in EU (or other countries)

Advanced development

1997 X

X

X 1992 2000

X X

2004

X X

1999 2008 (USA) 2009 (Korea)

X X

X [136]

X

1996 X

X X [64] X [66] X [26,73] X X X Xb Xb X X [16]

X 2008a (USA) – – – – 2002 2006 – – –

X X X X

X [137]

2008 (Japan)c

Immunoglobulins (see Tables 2 and 3) FL, full length; BDD, B-domain-deleted; AAT, alpha 1-antitrypsin; PC, protein C; AT, antithrombin; BHK, baby hamster kidney; CHO, Chinese hamster ovary; VWF, von Willebrand factor. a Topical use. b Abandonned. c Withdrawn from the market in Japan in 2009

techniques, and no stabilizer of human origin needed for formulation. Rh-FIX specific activity is 150–300 IU/mg. Although all rh-FIX forms exhibit normal procoagulant activity, only 60% are gamma-carboxylated at all 12 glutamic acid residues. In addition, two other possibly functionally important post-translational modifications, sulfation of tyrosine 155 and phosphorylation of serine 158, are limited in rh-FIX. Rh-FIX exhibits a normal plasma

half-life, but 30% reduction in recovery compared to PDFIX requiring an adjustment of dosing after market introduction [34]. Clinical use demonstrated the safety and haemostatic effect of rh-FIX [35,36]. Recently, the FIX gene could be expressed in the mammary glands of transgenic mice [37]. Secretion of coagulant active FIX at 3% of the total soluble proteins has been achieved, opening up the prospect of commercial production in transgenic animals.


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Expression of up to 16 ng/g fresh weight of a rh-FIX exhibiting in vitro clotting activity was recently obtained in the fruits of tomato plants transformed by Agrobacterium tumefaciens [38]. Expression systems with higher rh-FIX productivity would be needed to improve supply and costeffectiveness of haemophilia B treatment worldwide. The relatively small size of the FIX market and the absence of market segments/indications other than haemophilia B may rebut investors’ interest and restrict the number of new rhFIX developed, underlining the importance of a continuous supply of PD-FIX for the foreseeable future.

Factor VIII FVIII deficiency (haemophilia A) occurs in 1/5000–10 000 male births. FVIII is synthesized in the liver and circulates in plasma at about 200 lg/l as an inactive procofactor essentially fully bound to its chaperone molecule, von Willebrand factor (VWF), that protects against proteolysis. FVIII is a complex non-covalent multidomain heterodimer synthesized as a 300-kDa precursor protein which, after Nterminal processing and glycosylation, comprises 2332 AA assembled into six structural domains, organized in a heavy chain (A1-A2-B domains) and a light chain (A3-C1C2 domains) [39,40]. The A domains contain clusters of aspartic acid and glutamic acid residues (acidic regions). The B-domain is encoded by a single large exon and is highly glycosylated. The C domains exhibit homology to proteins that bind glycoconjugates and negatively charged phospholipids. Small acidic regions a1 and a2 in the C-terminal part of A1 and A2, and a3 in the N-terminal of A3 are required for optimal coagulation function, but not the B-domain. Patients with haemophilia A need long-life substitutive intravenous (IV) FVIII therapy. In wealthier countries, prophylaxis is preferred and requires FVIII infusions at 25–40 IU/kg three times a week, for a total of ca. 150 000 IU FVIII/year for severe haemophilia, at a cost close to €200 000. In the developing world, patients are usually treated on-demand with cryoprecipitate, PD-FVIII, or plasma, usually at hospitals. In the last 40 years, several PD-FVIII product generations have been produced from single-donor or pooled cryoprecipitate (5 IU FVIII/ml; 0Æ2– 0Æ4 IU FVIII/mg) to chromatographically purified high-purity preparations (100 IU FVIII/ml; > 50–150 IU/mg) [2]. PD-FVIII production does not compete with the simultaneous production of immunoglobulins or albumin from the same pool of plasma; in fact, only fibrinogen or VWF concentrate production interferes with FVIII processing in some, but not all [2], procedures. Until robust viral inactivation treatments, such as S/D, pasteurization, dry-heat, and nanofiltration virtually eliminated viral risks, pooled PD-FVIII products frequently resulted in infection with HIV and HCV infection [41].

To date, rh-FVIII remains the largest and most complex protein produced by recombinant DNA technology. Cloning of the FVIII gene [39,40] led to the development and production of the first generation of rh-FVIII products using CHO or BHK cell expression systems [42]. The CHO cells presented relatively low proteolytic activity and exhibited a good viral safety profile, and selected strains enabled the amplification of genes of interest [7]. Owing to large size and complexity, the expression level of rh-FVIII was two to three orders of magnitude lower than other recombinant proteins produced in mammalian cell lines [43]. First-generation products were obtained by inserting the full-sequence human FVIII cDNA into expression vectors, yielding ‘full-length’ rh-FVIII. FVIII expression was initially low in part because the molecule remained associated with the membrane of the CHO cells and was eventually degraded [7]. As absence of the Bdomain does not compromise FVIII procoagulant activity [4], its deletion from the construct enhanced the expression of rh-FVIII as much as 20-fold [12] and led to the development of a second-generation B-domain-deleted rh-FVIII that was developed with an albumin-free formulation. B-domain deletion led to discrepancies in assessing FVIII potency in vitro and in vivo, requiring assay adjustments. The range of rh-FVIII concentrates now includes several preparations of full-length rh-FVIII formulated without PD-albumin or without the need for plasma proteins (but recombinant insulin) in the cell culture media (third-generation products). Co-expression of VWF with FVIII has been, among others, a contributing factor for stabilization in serum-free medium [23]. Purification processes include dedicated viral reduction treatments, including S/D and nanofiltration. Haemostatic efficacy of the various rh-FVIII on the market is equivalent to that of PD-FVIII. Significant progress in the reduction in viral risks of PD-FVIII makes the safety advantages of rh-FVIII, obvious at the time of their launch, a rather theoretical discussion now. Measures to control the potential risks from prions require careful monitoring [44]. The promises of ample supply of safe and cost-effective FVIII to meet global needs, in part through recombinant DNA technology, have not been delivered yet. With current plasma fractionation technologies, only 100–200 IU FVIII/l plasma is obtained. Most of the good-quality plasma collected in the world is used to prepare PD-FVIII, and FVIII production volume is unlikely to increase substantially. There is a lack of interest in developing alternative high-yield PD-FVIII fractionation processes as those would impact other plasma products, whereas R&D efforts target IVIG yield improvements. Economical interest in PD-FVIII is decreasing as wealthy market with premium price is switching to rh-FVIII. Additional PD-FVIII may become available from new producers in emerging countries [45,46], but the volume of plasma involved (a total of < 1 million litres) makes impact on



global supply limited. Increasing the FVIII availability worldwide may come from the development of improved rh-FVIII, using more productive cell lines or new FVIII genetic constructs [47], the production of biosimilar rhFVIII in developing countries, and the development of long-acting FVIII with extended circulation half-life [48]. These developments should address the increasing evidence – not scientifically surprising [49], of a possibly higher immunogenicity of current rh-FVIII compared to at least some PDFVIII products [50–53], in particular those rich in VWF (VWFAg/FVIIIAg ratio close to 0Æ5 or above) [50,54]. The CANAL cohort study did not find evidence of higher immunogenicity of rh-FVIII compared to a range of PD-FVIII concentrates that had a low (< 0Æ01 IU VWFAg per IU of FVIIIAg) or very low (> 0Æ01) VWF content [55].

Factor VIIa The development of anti-FVIII inhibitors is a serious complication of haemophilia A treatment that occurs at a rate of about 10–30% [50,56]. Several human PD-products (PCC and activated PCC) containing both zymogens and activated coagulation factors, including FVII and activated FVII (FVIIa), as well as porcine FVIII, have been used, with varying success, to achieve haemostasis in patients with inhibitors [56]. The development of rh-FVIIa as a haemostatic agent relies on the idea that IV administration of exogenous FVIIa enhances thrombin generation on the platelet surface at the site of injury, independently of the presence of FVIII/FIX, resulting in rapid thrombin generation and formation of a tight fibrin haemostatic plug [57]. Although PD-FVIIa is considered difficult to produce, a large-scale process, yielding 40% recovery and a FVIIa with specific activity of about 40 U/lg, has been described [58]. Rh-FVII has been expressed in BHK cells and shown to have identical AA sequence to human FVII but under-gammacarboxylation. Rh-FVII is autoactivated during purification via a physiological cleavage event [7]. The product has shown benefits in haemophilia with inhibitors (at a suggested dosing of 90–120 lg/kg/dose every 2–3 h, although 270–400 lg/kg has also been used) but clinical monitoring in this indication remains difficult and risks of thromboenbolism should be monitored. Rh-FVII is also licensed in some countries for acquired haemophilia, FVII congenital deficiency, and Glanzmann’s thrombasthenia and other indications continue to be evaluated [59]. The introduction of rh-FVIIa has been a milestone in the field of recombinant plasma proteins but one drawback remains its very high cost. Considering the expanding indications of a molecule presented as a universal haemostatic agent, biosimilar FVIIa, targeting higher productivity, may be developed. Work to develop a FVIIa in the milk of transgenic rabbits is underway (unpublished data).

VWF VWF is a very large (> 20 000 kDa) multimeric glycoprotein made by the assembly of N-glycosylated monomers of 2050 AA present in plasma in a few mg/l. VWF dimers form multimers by crosslinking of cysteine residues through Cterminal disulphide bond formation. Post-translational modifications lead to multimerization and removal of a propeptide. VWF is produced constitutively in endothelium, megakaryocytes, and subendothelial connective tissue. The large multimers exhibit highest functionality. Hereditary or acquired defects cause von Willebrand disease (VWD) characterized by mild to severe bleeding episodes of the skin and mucous membranes and are classified into three main types (I, II, and III). Treatment depends upon the nature of the abnormality and the severity of the symptoms. The more severe cases of VWD require substitutive therapy using VWF products [60]. PD-VWF concentrates are produced from cryoprecipitate by processes intended to limit proteolytic degradation of the haemostatically active high-molecular-weight (HMW) multimers. Most PD-products that contain both FVIII and VWF are of relatively low specific activity, with a high ratio of VWF (e.g. 2Æ4 IU VWF:RCo/ FVIII IU) [61]. Other products, chromatographically purified, contain high-purity FVIII and VWF [62], or VWF only [63]. A combined rhVWF/rh-FVIII complex expressed in CHO is under development. Rh-vWF with preserved HMW multimers is secreted into protein-free medium. During production, un-processed rh-VWF is exposed to r-furin to remove its pro-peptide. Rh-VWF, purified by chromatography, is claimed to exhibit homogeneous multimer distribution with ultra-HMW multimers resembling that of platelet/endothelial-stored VWF [7], to have normal glycosylation pattern, to bind FVIII and collagen, and to promote platelet adhesion under shear stress [64]. In a VWF-deficient animal models, rh-VWF corrected VWF concentration, reduced blood loss, and stabilized endogenous FVIII. Half-life was prolonged compared to that of PD-VWF preparations [65]. This therapeutic approach may also help extend FVIII half-life. The preparation is under phase 1 clinical trials in type 3 VWD (http:// clinicaltrials.gov/ct2/show/NCT00816660). Work is also underway to express VWF in transgenic swine. Transgenic rh-VWF has an AA sequence similar to that of human VWF and is expressed in the milk at levels 28- to 56-folds greater than in human plasma [66], thereby opening perspectives for a cost-effective production.

Fibrinogen Fibrinogen is a complex 340-kDa glycoprotein synthesized mainly in the liver, circulating at 1Æ5–3 g/l in plasma, with a half-life of 3–5 days, and converted into fibrin by


74 T. Burnouf

thrombin. The two sets of three polypeptides (Aa, Bb, and c chains, with 610, 461, and 411 AA residues, respectively) are linked by 29 disulphide bonds (17 interchain and 12 intrachain bonds) [67]. The Bb and c chains are N-glycosylated, not the Aa chain [68]. Afibrinogenemia (< 0Æ2 g/l of plasma) and dysfibrogenemia (alteration of functionality) affect about 1 in 1–2 millions, congenital hypofibrinogenemia (0Æ2–0Æ8 g/l) being less frequent. Acquired deficiency occurs during disseminated intravascular coagulation (DIC) and is an early event in seriously bleeding patients. IV substitutive therapy relies on PD-fibrinogen concentrates, cryoprecipitate, or plasma. Fibrinogen is also a constituent of fibrin glue (fibrin sealant) used topically as haemostatics or tissue sealants [69,70]. Fibrinogen for IV or topical use has been available for decades in Europe. Fibrinogen is extracted from cryoprecipitate or Cohn fraction I at a recovery up to about 1 g per litre. Most fractionation procedures compete with Factor VIII, explaining small production level globally. Viral inactivation treatments by S/D or heat have been implemented in the 1980s [2]. Single-donor non-virally inactivated cryoprecipitate is largely used in many countries as fibrinogen source [71]. Engineered BHK or CHO cells express biologically active rh-fibrinogen but low expression level constitutes a barrier to commercialization. Expression in protease A-deficient P. pastoris has been reported; the various subunits are expressed, assembled, and secreted to form a complete molecule clottable by thrombin, suggesting application as an haemostatic agent [72]. Transgenic fibrinogen can be expressed in mice at 200 lg/ml [73] and in larger animals (goats or cows) to improve productivity [26]. Fully assembled rh-fibrinogen can be secreted at 0Æ1–5 g/l levels in the mammary gland, but some unassembled rh-fibrinogen chains are also secreted [74]. Rh-fibrinogen from transgenic goats has been granted Orphan Drug Status by the US FDA for substitutive IV therapy and could also be considered for use as fibrin glue and gauze dressing component. So far, PD-fibrinogen for substitutive therapy or fibrin sealants has been available only in limited countries. The plasma fractionation industry appears unlikely to increase fibrinogen production dramatically in the forthcoming future. Recombinant technology is a logical approach to help meet the clinical demand if a safe and cost-effective rh-fibrinogen can be brought to the market.

Thrombin Prothrombin is a 72-kDa vitamin-K-dependent serine protease, synthesized primarily in the liver, and present in plasma at about 300 ng/ml. Its conversion to thrombin requires calcium, phospholipids, and factors Xa/Va. Thrombin plays a key role in cleaving fibrinogen into fibrin, leading to the formation of blood clots, exhibits proteolytic

activities towards numerous plasma proteins [75], and is a key activator of platelets. Thrombin can be used clinically as a stand-alone topical product to minimize blood oozing during surgery, as a surgical haemostatic/sealing agent combined with haemostatic sponges or fibrinogen, or as platelet activator for the preparation of platelet gels [76]. Purified thrombin can be readily prepared from plasma by CaCl2 activation of prothrombin (present in PCC or a by-product of FIX) chromatographic isolation, and viral reduction treatments [77]; 20–40 IU of thrombin can be generated per iu of prothrombin [77]. Concentrates are marketed in Europe as components of fibrin sealants [70], rather than stand-alone products. Topical human thrombin was licensed in the USA in 2007. Before that, only bovine thrombin was available [78], exposing patients to potential immunological side-effects [79] and prion transmission [80]. Commercial activation devices of human plasma have also been developed to generate 50–60 IU/ml of thrombin in 30 min [81]. The first rh-thrombin approved in the USA in 2008 [76] is produced from a prethrombin-1 precursor obtained from CHO cell culture [82]. The clinical studies indicate good haemostatic efficacy and tolerance. Development of non-neutralizing antibodies has been found so far in about 1–2% of patients [83,84]. The scientific and commercial rationale underlying the development of rh-thrombin lies likely in the need to replace bovine thrombin. Why human PD-thrombin as a stand-alone product has not been developed more aggressively by plasma fractionators remains surprising as production is straightforward and compatible with that of major plasma products. Respective market share of rhthrombin, PD-thrombin, and bovine thrombin will likely be based on cost and viral/immunological safety considerations [78].

Factors V, XI, and XIII FXI deficiency occurs in 1 of 100 000 (more frequent in Ashkenazi jews), and FV or FXIII deficiency affects one in about 1–5 million. In the absence of PD-FV concentrate, substitutive therapy relies on plasma infusion. PD-FXI and PD-FXIII concentrates are available in some countries [2]; elsewhere deficient patients are treated by plasma or cryoprecipitate, respectively. The long half-life of FXIII (8–12 days) facilitates prophylaxis using cryoprecipitate. FXIII deficiency is also associated with wound-healing disorders, and the PD-protein is included in some fibrin glue formulation [70]. A rh-FXIII has been developed in S. cerevisiae as a non-glycosylated FXIII A2 homodimer that showed a mean half-life of 8Æ5 days and apparently normal functionality in healthy volunteers and FXIII-deficient adults. However, animal studies in cynomolgus monkeys using supraphysiological doses led to coagulopathy and death [7].



There do not seem to be active R&D activities towards the development of commercial rh-FV or rh-FXI.

Protease inhibitors and anticoagulation factors Alpha 1-antitrypsin AAT is the most abundant (1Æ1–2Æ8 g/l) serine protease inhibitor (serpin) in plasma, synthesized mainly by hepatocytes, with a 4–5 days half-life. The 52-kDa single polypeptide chain of 394 AA residues has 12% of its MM represented by complex carbohydrates (3 N-linked glycans; almost exclusively bi-antennary and tri-antennary structures) [13]. Severe AAT deficiency predisposes to pulmonary emphysema, as well as juvenile cirrhosis, hepatocellular carcinoma, and panniculitis [85]. Standard treatment of emphysema requires weekly IV infusion of 60 mg AAT/kg to reach circulating level intending to slow disease progression [86]. Treatment by inhalation of nebulized powder or aerosolized solution is under study. Only 4% of individuals with AAT deficiency are identified, and few are receiving treatment [13]. PD-concentrates manufactured at 0Æ1–0Æ6 g/l recovery by a handful of fractionators have been available for many years [2]. Insufficient plasma supply and modest recovery (as production interferes with that of albumin) makes the fractionation industry unable to meet the assumed global clinical demand. Recombinant technology offers an alternative production approach. However, in many cellular systems [13], lack of, or aberrant, glycosylation affects AAT folding leading to impaired activity or rapid IV clearance. A yeast-derived topical rh-AAT has been considered for the treatment of atopic dermatitis [87]. Production of rh-AAT has been achieved in transgenic sheep but the project stopped. Aerosolized rh-AAT from transgenic sheep milk induced an antibody response to sheep AAT and a1-antichymotrypsin in patients with cystic fibrosis [88] evidencing the difficulties of this approach.

Antithrombin AT is a 58-kDa single-chain glycoprotein of 432 AA, with three disulphide bonds and four potential glycosylation sites, produced by the liver, circulating at ca. 0Æ12 mg/ml of plasma, and with a half-life of about 3 days. It exhibits multiple functionality as anticoagulant, anti-inflammatory, anti-angiogenic, and antitumour agent [89]. Congenital AT deficiency (functional levels < 50% of normal) that affects 1/2000–5000 individuals may lead to thrombophilias and venous thromboembolism in high-risk episodes. AT substitutive therapy is initiated in high-risk situations to increase AT activity to ‡ 120% initially and then ‡ 80% of normal levels [90]. High-dose AT (without heparin) has been used in

intensive care during septic episodes [91]. PD-AT concentrates, available in Europe since the 1980s, are produced from cryo-poor plasma (or Fraction IV-1) by immobilized heparin-affinity chromatography combined with viral reduction treatments (pasteurization, S/D, 15 -nm nanofiltration) [2]. Only a small portion of the plasma available worldwide is fractionated into AT, making PD-concentrates reserved to a few countries. rh-AT expression has been obtained in CHO cells [92]. However, rh-AT from the transgenic goat milk has been licensed recently to prevent venous thromboembolic events during surgery of hereditary deficient patients [93]. Treatment of DIC associated with severe sepsis is being explored [26]. Expression level of rh-AT can reach 2 g/l (more than 10 times that in plasma) and purification recovery over 50% [26]. Manufacturing processes from transgenic goats should be validated for their capacity to remove viruses and prions, and the goats should be certified free of scrapie [26]. Transgenic rh-AT exhibits a pharmacokinetics (faster loss to the liver and vessels) different from that of PD-AT in a rabbit model [94] and differs in glycosylation [26]. Approval of transgenic rh-AT illustrates the potentials and cost-effectiveness of this manufacturing platform for glycoproteins required in relatively large amount.

C1-inhibitor C1-inh, an acute-phase 105-kDa serpin comprised of 478 AA, circulates in plasma at about 0Æ25 g/l, is synthesized by several cells including hepatocytes, and has a half-life of 28 h in non-deficient individuals (67–72 h in deficient patients) [95]. Sugars represent close to 50% of the MM with 13 carbohydrate groups, most being located at the N-terminal region. Sialic acids are important for normal in vivo halflife [96]. C1-Inh inhibits the complement system, preventing spontaneous activation and the generation of vasoactive peptides (bradykinin or anaphylatoxins), and is a potent inhibitor of plasma kallikrein, FXIa, and FXIIa. Deficiency (approximately 1/30 000 individuals) induces subcutaneous and submucosal swellings in the respiratory and gastrointestinal tracts [96] at an average of seven times per year. PDC1-Inh was made available over 20 years ago to treat hereditary angioedema [2]. C1-inh is captured from cryo-poor plasma by anion-exchange chromatography using processes that interfere little with that of other plasma products [2]. Only a few PD-C1-Inh concentrates are available commercially, and only a fraction of the plasma fractionated worldwide is used to produce C1-Inh. Presence of 13 putative glycosylation site would create difficulties in producing this molecule by expression in mammalian cells. rh-C1-Inh has been expressed in transgenic rabbits’ milk. Efficacy in treating angioedema has been evaluated in two randomized controlled studies [97]. Marketing authorization is being seeked. Interest in other pathological conditions may be


76 T. Burnouf

evaluated (sepsis, acute myocardial infarction, cytokineinduced vascular leak syndrome, organ transplant) [95].

Activated protein C Processes to manufacture protein C (PC) from plasma have been developed and PD-PC concentrates commercialized in Europe for many years for the treatment of patients with deficiency [2,98]. The plasma fractionation industry, however, failed to develop an activated PC (aPC), although thrombin activation of PC could readily be used to prepare stable aPC [99]. Rh-aPC has been successfully expressed in human embryonic kidney 293 (HEK293) cells. The product was shown to significantly reduce mortality in patients with severe sepsis but may be associated with an increased risk of bleeding [100].

Albumin Albumin, a 66Æ5-kDa globular non-glysosylated singlechain 585 AA protein synthesized by the liver, is the most abundant protein in human plasma (35–40 g/l). It contains 35 cysteinyl residues (17 disulphide bridges and one free sulfhydryl group) leading to complex folding. Albumin regulates the blood volume by maintaining the oncotic pressure and is a carrier for low-molecular-weight hydrophobic molecules (e.g. hormones). Therapeutic applications include volume replacement at doses of several grams. Albumin is also used as excipient in the formulation of various pharmaceutical products [101]. Most PD-albumin is produced by ethanol fractionation [2] at a recovery of 23– 27 g/l and a purity close to 98–99%. Viral inactivation relies on a terminal pasteurization, and the product has established viral safety [102]. Several steps during production of albumin have been shown experimentally to contribute to a significant removal of prions [44]. Most plasma in the world is fractionated into albumin, yielding about 500 tons yearly. Rh-albumin expression has been achieved using various micro-organisms that provide a productivity higher than mammalian cells. Saccharomyces cerevisiae and P. pastoris fermentation processes have been upscaled to commercialize rh-albumin for transfusion or as an excipient [103]. Under optimized culture conditions, stable production of 1Æ4 g/l or more was reported [104,105] and high purity, with minimal contamination with yeast proteins and undetectable cell wall components, was obtained [106]. Rh-albumin had lower amounts of palmitic and stearic acids [107] and less structural heterogeneity than PDalbumin [108] but conformation appears identical without added neoantigenicity [105,107]. rh-albumin was reported safe and efficacious in various clinical conditions (haemorrhagic shock, cirrhosis with ascites, etc.) [103] without signs of allergic events [109–111]. However, marketing

authorization of the P. pastoris rh-albumin, delivered in October 2007 in Japan, was withdrawn in early 2009 when falsification of preclinical data has been discovered (http://www.mt-pharma.co.jp/e/release/nr/2009/pdf/e_medway 090324.pdf). Production of albumin from transgenic cows, with expression levels of 1–2 up to 40 g/l compatible with realistic production scale, has been reported [112]. Challenges include the ability to produce by tons a highly purified product at the price level of a commodity.

Immunoglobulin G Immunoglobulin G (IgG) represents a group apart within the therapeutic plasma proteins. mAbs cannot replace PD-IgG for most indications and, by contrast to the other proteins described earlier, are not intended for replacement therapy. Mabs should, therefore, not be regarded as bio-similars to PD-IgG, and their mechanism of action is thought to be distinct [113,114]. However, mAbs targeting inflammatory and neurological diseases and infections are discussed in the following sections since some of these indications are currently treated by PD-IgG.

PD-immunoglobulin G Polyvalent IVIG from human plasma pools containing millions of functionally active IgG molecules is used to protect immune-deficient patients against recurrent infections [115]. IVIG is also a first-line therapy against various immune-mediated inflammatory, rheumatic, neurological disorders, as reviewed recently [116]. Therapeutic benefit and mechanism of action in corticoresistant polymyositis, psoriasis, psoriatic arthritis (PsA), rheumatic diseases [such as juvenile idiopathic arthritis (JIA) and Sjo¨gren’s syndrome], and multiple sclerosis (MS) are under evaluation [117–119]. Hyperimmune IgG containing clinically important antibodies are used to treat or prevent specific viral and bacterial. They contain many types of antibodies, only a minute fraction with the intended neutralizing activity [120]. Anti-D IgG prevents haemotytic anaemia of the newborn. Phase III clinical studies are ongoing to evaluate the benefits of IVIG (that contain auto-antibodies against the beta-amyloid peptide) in slowing down Alzheimer disease [121]. The manufacturing methods of IVIG have been reviewed recently [122]. With a production capacity of 87Æ5 tons worldwide (based on 25 million l of plasma fractionated at a recovery of 3Æ5 g/l), the supply does not meet global demand.

Monoclonal antibodies Initially using mice hybridoma production systems, the mAb technology has moved, through chimeric and



humanized constructs, to the production of fully human mAbs, thereby reducing immunogenic mouse components and immunogenenicity [113,123]. Twenty mAbs were licensed in 2010, representing a market expected to reach $20 billion in 2010 [124], about ten times that of IVIG. Over 150 mAbs are under clinical trials [123]. They are used for malignancies and transplant rejection, but also increasingly for autoimmune and infectious diseases, thereby overlapping, or complementing, PD-IgG usage.

Autoimmune inflammatory disorders Table 2 presents the 11 main mAbs developed to treat immune-mediated inflammatory disorders. Their cellular targets are well identified and classified into four groups (see Table 2) [113]. Four mAbs (Adalimumab; Golimumab; Infliximab; Certolizumab) are directed against TNF-a, a cytokine playing a central role in several inflammatory reactions leading to rheumatoid arthritis (RA), ankylosing spondylitis (AS), Crohn disease, JIA, PsA, and psoriasis. TNF-a inhibitors appear ineffective in Sjo¨gren syndrome, in several forms of vasculitis, and in MS [113]. Tocilizumab is directed against IL-6 receptor, thus blocking the activity

of a regulatory cytokine involved in T- and B-cell activation and various functions relevant to RA pathogenesis. It is used for treating RA in combination with methotrexate [113]. Ustekinumab, directed against IL-12 and IL-23, is used against moderate to severe plaque psoriasis and is investigated for PsA and Crohn disease but appears uneffective against MS [125]. Other mAbs are directed against T cells, as autoreactive T cells play a key role in the immunedriven inflammatory responses in patients with RA, Crohn disease, PsA, and psoriasis. Daclizumab and basiliximab are directed against CD25, the protein a component of the IL-2 receptor. Blocking of IL-2 binding in T and B cells inhibits their activation and the development of an immune response and induces anergy. Both, initially approved against transplant rejection, have been proposed for the treatment of auto-immune disorders. Daclizumab is in phase II trials in MS [113,126]. In 2006, a humanized anti-CD28 mAb (TGN1412) intended, among other indications, to treat RA was stopped after inducing severe systemic inflammatory response in six volunteers during Phase 1 clinical trials [127]. B-cell inhibitors contribute to the immune response in various autoimmune diseases

Table 2 Examples of mAb for immunomodulation and inflammatory/rheumatic diseases [118,138]a

INN name Cytokine inhibitors Adalimumab Golimumab Infliximab Certolizumab pegol Tocilizumab Ustekinumab T-cell inhibitors Daclizumab

Basiliximab TGN1412 B-cell inhibitors Rituximab

Therapeutic targets (licensed or expected)

Regulatory status

Species

Target

Licensed Licensed Licensed Licensed Licensed Licensed

Human IgG1 Human IgG1 Chimeric IgG1 Human Fab fragment Humanized IgG1 Human IgG1

TNF alpha TNF alpha TNF alpha TNF alpha IL-6 IL-12 & IL-23

RA; AS; Crohn disease; JIA; PsA; psoriasis

Licensed for acute organ rejection (Withdrawn from EU for commercial reasons) (licensed for acute organ rejection)

Humanized IgG1

CD25 (IL-2Ra receptor)

MS (clinical studies)

Chimeric IgG1

Withdrawn from developmentb

Humanized

CD-25 (IL-2Ra receptor) CD-28

Potential indications in inflammatory disorders RA

Licensed EU & FDA

Chimeric IgG1

CD-20

RA; Systemic Lupus Erythematus; primary Sjögren syndrome; ITP; chronic inflammatory demyelinating polyneuropathy; vasculitis

Humanized IgG4 Humanized IgG1

a4b1 integrin CD11a

MS and Crohn’s disease Skin psoriasis

Cell adhesion/migration inhibitors Natalizumab Licensed Efalizumab Withdrawnc

RA Plaque psoriasis

AS, ankylosing spondylitis; ITP, Idiopathic thrombocytopenic purpura; JIA, juvenile idiopathic arthritis; MS, multiple sclerosis; PsA, psoriatic arthritis; RA, rheumatoid arthritis. a For specifically approved or considered indications in EU or USA see : http://www.ema.europa.eu/htms/human/epar/x.htm or http://clinicaltrials.gov. b Severe inflammatory reactions in six volunteers during phase 1 clinical trial. c Episodes of progressive multifocal leukoencephalopathy.


78 T. Burnouf

(e.g. production of pathologic autoantibodies and proinflammatory cytokines, antigen presentation to T cells). Anti-CD20 mAb Rituximab is of substantial benefits in auto-immune diseases, is approved for the treatment of RA [128], and has been successfully tested in phase II trials in MS [126]. Use for systemic lupus erythematus (SLE), primary Sjo¨gren syndrome, ITP, chronic inflammatory demyelinating polyneuropathy, and vasculitis is investigated but three cases of progressive multifocal leukoencephalopathy (PML) have been observed in patients with SLE [113]. Two humanized IgG1 mAbs against CD22 and CD52 are under studies for the treatment of SLE, Sjo¨gren syndrome, and RA [118]. Two licensed mAbs are directed against integrins, the adhesion molecules located on T lymphocytes, that can interact with ligands on endothelial cells and trigger various pathological effects. Natalizumab is directed against a4b1, an integrin involved in MS pathogenesis that facilitates the migration of lymphocytes into the site of disease. Through various mechanisms, Natalizumab reduces T cell– mediated inflammation and improved MS but was associated with the development of PML. Efalizumab, directed against the cell adhesion molecule CD11a, a subunit of lymphocyte function–associated antigen, was found to improve skin psoriasis but was stopped after development of PML in several patients. Additional mAb under clinical development are likely to provide treatment options in various rheumatoid, inflammatory, and neurological disorders. Combining mAbs with IVIG may lead to more effective therapy and dosage for not-yet-proven, off-label indications of IVIG [118].

Infections Antigenic differences between host and pathogens should make mAb development for infectious diseases easier than for other indications, but the multiplicity of the antigens is a difficulty. One mAb preparation consists of one type of immunoglobulin lacking, by contrast to polyclonals,

variability with regard to epitope and isotype of a pathogen [129], with the risk that the targeted epitope may not be conserved on all pathogenic strains, and inducing a selective pressure of mutated not-neutralized resistant agents [120]. This may be counterbalanced by mAbs against conserved areas of viral particles or using mixtures of mAbs against various epitopes. Table 3 give examples of mAbs developed to treat infections. Only one, Palivizumab, is licensed for the prevention of RSV in high-risk infants and immunocompromised adults [129]. Emerging infections (West Nile, corona, flu, or Dengue viruses) and potential bioterrorism threats generate interest in anti-infective mAbs [130]. Several mAbs, directed or not against viral antigens, are in development to treat pathologies associated with various viral agents (e.g. HIV, HBV, HCV, CMV). Two mAbs are under clinical trials to control HBV and HCV infections during liver transplantation but are not directed against the viruses. Bavituximab is specific for phosphatidylserine, a phospholipid exposed on membranes of damaged cells, and the other, MDX-1106, for PD-1, an inhibitory T-cell costimulation receptor [129]. Combinations of mouse or human mAb against the rabies virus (RV) are also under development [120,131]. In an animal model, a human mAb cocktail that recognize nonoverlapping, non-competing epitopes was found equivalent to human PD-rabies immunoglobulin [120]. If cost-effective and efficacious and if the regulatory and licensing issues for approval of mAb cocktails are overcome [129], these products may complement human or equine PD-IgG in rabies postexposure prophylaxis. Bapineuzumab, an antibody to the beta-amyloid plaques, is being clinically tested for the treatment of Alzheimer’s disease [132]. mAb targeting infectious diseases treated by PD-products or emerging pathogens may become available as long as economic feasibility, market need, or urgency are sufficient to justify development cost [129]. Production of rh-polyclonal IgG by immunizing animals transgenic for human

INN name

Regulatory status

Species

Inhibition target

Target

Palivizumab Motavizumab CCR5mAb004 PRO 140 3 mAb cocktail F105 Ibalizumab Sevirumab Bavituximab MDX1106 CL-184 MGAWN1

Licensed Clinical studies Clinical studies Clinical studies Clinical studies Clinical studies Clinical studies Clinical studies Clinical studies Clinical studies Clinical studies Clinical studies

Humanized IgG1 Humanized IgG1 Human Humanized

Glycoprotein F Glycoprotein F CCR5 CCR5

RSV prevention

Human Humanized Human Chimeric Human Cocktail Humanized

GP120 viral protein CD4 Env glycoprotein H Phosphatidylserine PD-1 Envelope glycoprotein

Table 3 Example of mAbs licensed or under development against viral diseases [129]

HIV

CMV HCV HCV Rabies WNV

mAb, monoclonal antibodies.



immunoglobulin genes is a new developing approach of interest [133]. In addition, a recent study in mice has demonstrated the efficacy of a transgenic plant-produced mAb against West Nile Virus (WNV) several days after exposure, providing a proof of concept of efficacy [134].

Conclusion A now mature biotech industry has demonstrated its ability to market an increasing range of therapeutic plasma proteins, including complex coagulation factors. However, the hope for abundant, affordable, and safe plasma proteins for all patients worldwide has not been reached yet. Plasma fractionation and biotech industry complement each other in supplying to the developed world an extended range of quality products each with their advantages and limitations. The advantages of PD-proteins include demonstrated efficacy and low immunogenicity conferred by the human plasma origin. One of their limitations remains the risk of contamination by bloodborne pathogenic agents, although the enormous progress in donors surveillance, donation testing and infrastructure, pathogen reduction technologies, and regulatory oversight should be recognized. Plasma is a source of multiple therapeutic proteins but its drawbacks include an insufficient supply worldwide and its high cost that impose marketing a diversified product portfolio to ensure economical balance. Consequently, collecting marginal plasma volume for increasing IVIG supply is currently unsustainable. Focused on IVIG recovery and prion removal studies, fractionators have made only timid attempts to develop new PD-proteins and explore new indications. Established fractionators are reluctant to implement new high-yield technologies affecting already licensed products. Meanwhile, patients in developing countries cannot obtain enough safe and affordable products, and potential new fractionators are confronted to various barriers to entry (capital requirements, quality plasma supply, skilled manpower, and regulatory barriers).

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The advantages of rh-plasma proteins include the unlimited and relatively low cost of the source material. Pathogen safety is linked to control of the cell lines, use of confined production systems, and avoidance of extraneous protein materials. Manufacturing methods of proteins from transgenic animals should follow an approach similar to that used for PD-proteins. Immunogenicity of complex rhplasma glycoproteins expressed in non-human/non-physiological systems remains a significant risk and brings uncertainty for product development and clinical studies. Manufacturing processes with improved performance and proteins with enhanced therapeutic properties or half-life are likely to be developed [135]. Transgenic animals (and possibly yeast systems for simple or non-glycosylated proteins) present interest by their expression levels that can surpass that of human plasma and mammalian cell cultures, opening an avenue for the development of more abundant, more affordable rh-plasma proteins. In addition, development of the biotech industry in emerging countries is likely to bring about new rh-products in the next 10 years. In conclusion, if, historically, the risk of viral transmission by PD-products has been the medical (and politically-correct) justification put forward for the development of rh-plasma proteins, today the need for ample supply of these products at affordable cost, without jeopardizing quality, appears to be an objective driving force for the biotech industry, as long as it will be compatible with the financial returns expected by companies behind their development. Finally, it can be expected that PD and rhproteins because of their respective advantages will both play a role in human therapeutics in the foreseeable future in both developed and developing countries.

Acknowledgements Sincere thanks are expressed to Herve´ Broly, PhD and Frank Mueller, PhD for careful reading of the manuscript and valuable expert comments.

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