Development of a plasma- and albumin-free recombinant von Willebrand factor.

Baxter has developed a recombinant therapy for treating von Willebrand's disease. Recombinant VWF is co-expressed with the rFVIII in CHO cells used to produce the rFVIII product Advate. This rVWF is used as a drug component for a rVWF-rFVIII complex drug product. CHO cells produce partially processed and partially un-processed rVWF still containing the pro-peptide. In order to make a consistent preparation containing mature and processed rVWF only rVWF is exposed to recombinant furin to remove the pro-peptide. Recombinant VWF and furin are produced under serum- and protein-free conditions. It is highly purified by a series of chromatographic steps and formulated in a protein-free buffer and has a homogeneous multimer distribution. The specific activity is higher in rVWF than in commercial plasma-derived VWF-FVIII complex products. SDS agarose electrophoretic analysis shows the presence of ultra-high molecular weight multimers. The FVIII-binding capacity and affinity of rVWF to FVIII is comparable to VWF in plasma. Carbohydrate analysis shows an intact glycosylation pattern. Recombinant VWF binds to collagen and promotes platelet adhesion under shear stress. It stabilizes endogenous FVIII in VWF-deficient knock-out mice as seen by a secondary rise in murine FVIII.

Recombinant von Willebrand factor: preclinical development.

Von Willebrand factor (vWF) is a multimeric glycoprotein (GP) that attracts platelets to the site of vascular injury, mediates platelet-platelet interaction, and stabilizes factor VIII (FVIII) in the circulation. Quantitative and qualitative defects of vWF result in von Willebrand disease (vWD), manifested by modest to severe bleeding episodes. Substitution therapy, with plasma-derived FVIII/vWF complex concentrates, is used for patients suffering the more severe forms of vWD. Efficacy of these preparations is often unsatisfactory because inadvertent proteolytic degradation during the manufacturing process causes them to lack the hemostatically most active high-molecular-weight multimers. In contrast, recombinant vWF (r-vWF), which is constitutively expressed at high yields in Chinese hamster ovary (CHO) cells and secreted into the conditioned medium under perfusion fermentation in "protein-free" medium, has high-molecular-weight multimers of extraordinary structural integrity. Functional analysis has shown that r-vWF promotes ristocetin cofactor-mediated platelet aggregation, collagen interaction and FVIII binding, and platelet-collagen adhesion under shear stress. Infusing vWF-deficient animals with r-vWF corrected vWF concentration and reduced blood loss, subsequently stabilizing endogenous FVIII associated with the reduction of bleeding time. Compared with plasma-derived vWF preparations, r-vWF was found to have a prolonged half-life, further enhancing the potential value of r-vWF as a therapeutic agent for treating patients suffering from vWD.

[Development of a novel influenza vaccine derived from a continuous cell line]. / Entwicklung eines neuen, aus permanenten Zellen gewonnenen Grippe-Impfstoffes.

Influenza viruses for production are presently produced in embryonated hen"s eggs. This conventional standard methodology is extremely cumbersome; it requires millions of eggs and an extensive purification to reduce the amount of contaminating egg proteins and to minimise the risk of allergies against egg albumin. The shortage of eggs in a pandemic situation, the selection of egg-adapted variants and the presence of adventitious viruses has emphasised the necessity for production of Influenza vaccines on a well characterised stable cell line. Our established serum and protein free Vero cell technology has been successfully adapted to large scale production of a huge variety of Influenza virus strains. The production in 1200 liter fermenter cultures under serum free conditions gave antigen yields comparable to the conventional embryonated egg technology. The development of a rapid and efficient purification scheme resulted in a safe high purity vaccine which was at least as immunogenic as conventional egg-derived vaccines in a mouse model. Clinical trials in the UK, Poland and Austria demonstrated that the Vero cell derived influenza vaccine is well tolerated, safe and highly immunogenic in humans.

Development of a Vero cell-derived influenza whole virus vaccine.

Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and the presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The WHO-approved Vero cell line was used in serum-free culture to grow many influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter. A purification scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation.

Evidence for extracellular processing of pro-von Willebrand factor after infusion in animals with and without severe von Willebrand disease.

Although proteolytic processing of pro-von Willebrand factor (pro-vWF) resulting in free propeptide and mature vWF is known to be initiated intracellularly, vWF released from endothelial cells may contain a high proportion of incompletely processed pro-vWF. Because pro-vWF is only rarely detectable in normal human plasma, we investigated whether extracellular processing of pro-vWF is possible. A recombinant preparation (rpvWF) containing both pro-vWF and mature vWF subunits was infused into 2 pigs and 1 dog with severe von Willebrand disease, 2 mice with a targeted disruption of the vWF gene, and 2 healthy baboons. Total vWF antigen (vWF:Ag), free propeptide, and pro-vWF were measured using enzyme-linked immunosorbent assay techniques in blood samples drawn before and after infusion. vWF:Ag increased promptly. No pro-vWF could be detected when the first postinfusion sample was drawn after 30 minutes (pigs) or 60 minutes (mice), but pro-vWF was detectable for short periods when postinfusion samples were drawn after 15 minutes (dog) or 5 minutes (baboons). In contrast, free propeptide was increased at the first timepoint measured, suggesting that it was generated from the pro-vWF in the rpvWF preparation. vWF multimers were analyzed in the rpvWF preparation and in plasma samples drawn before and after infusion of rpvWF using ultra-high resolution 3% agarose gels to allow separation of homo- and hetero-forms of the vWF polymers. Within 30 minutes after infusion in the pigs, 1 hour in the dog and the mice, and within 2 hours in the baboons, the multimer pattern had changed to that typically seen in mature vWF. These data indicate that propeptide cleavage from unprocessed vWF can occur extracellularly in the circulation. The enzyme or enzymes responsible for this cleavage in plasma remain to be identified.

Preclinical evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.

Dutch Kooiker dogs with hereditary von Willebrand disease (vWD) have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of vWD in humans. We used this canine model of vWD to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with an rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 hours in one dog and 22.1 hours in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 hours, and LMW-rvWF, 9 hours. The in vivo recovery of vWF:Ag after administration of rvWF was 59, 64 and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78, 110 and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

A novel mammalian cell (Vero) derived influenza virus vaccine: development, characterization and industrial scale production.

Influenza virus for vaccine production are presently produced in embryonated chicken eggs. This conventional standard methodology is extremely cumbersome; it requires a huge amount of eggs and an extensive purification to reduce the amount of contaminating egg proteins and to minimize the risk of allergies against egg albumin. The shortage of eggs in a pandemic situation, the selection of egg-adapted variants and the presence of adventitious viruses has emphasized the necessity for production of influenza vaccines on a well characterized stable cell line. Our established Vero cell technology has been successfully adapted to large scale production of a variety of influenza virus strains. The production in 1200 litre fermenter cultures under serum-free conditions gave antigen yields comparable to the conventional embryonated egg technology. The development of a rapid and efficient purification scheme resulted in a safe high purity vaccine which was at least as immunogenic as conventional egg-derived vaccines in a mouse model. This vaccine has been shown to be safe and highly immunogenic in chimpanzees and to be capable of protecting ferrets against challenge with live virus. Clinical trials have now been initiated in the UK and Austria.

Development of a mammalian cell (Vero) derived candidate influenza virus vaccine.

Influenza vaccine production is dependent on the availability of embryonated hen eggs for virus growth. This is an extremely cumbersome system with many disadvantages with respect to selection of virus variants and presence of adventitious viruses. We have developed an alternative cell culture system which allows rapid production of large volumes of vaccine. The World Health Organisation (WHO) approved Vero cell line was used in serum-free culture to grow a multitude of influenza strains to high titre. This system could be scaled-up to allow vaccine production with a 1200 litre fermenter volume. A purification scheme was developed which resulted in a high purity whole virus vaccine. This was demonstrated to be at least as immunogenic as a conventional egg-derived preparation in a mouse model.

Evaluation of recombinant von Willebrand factor in a canine model of von Willebrand disease.

Dutch Kooiker dogs with hereditary von Willebrand disease have undetectable levels of von Willebrand factor (vWF), resulting in spontaneous haemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. We used this canine model of von Willebrand disease to study the in vivo effects of a new recombinant von Willebrand factor (rvWF) preparation that contained all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF) and with a plasma-derived factor VIII/vWF concentrate (pdvWF). Administration of rvWF in these vWF-deficient dogs resulted in a vWF:Ag half-life of 21.6 h in one dog and 22.1 h in a second dog. Administration of pdvWF resulted in a half-life for vWF:Ag of 7.7 h, and LMW-rvWF, 9 h. The in vivo recovery of vWF:Ag after administration of rvWF was 59%, 64% and 70% in three dogs, respectively; 33% after pdvWF, and 92% after LMW-rvWF. The in vivo recovery of ristocetin cofactor (RCoF) was 78%, 110% and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in FVIII. Although no effect was seen on bleeding time at the dosages used, the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

In vivo characterization of recombinant von Willebrand factor in dogs with von Willebrand disease.

Hereditary von Willebrand factor (vWF ) deficiency in Dutch Kooiker dogs, which have undetectable levels of vWF, causes spontaneous hemorrhage of mucosal surfaces similar to the clinical picture of von Willebrand disease in humans. Therefore, we used this canine model to study the in vivo effects of a new recombinant von Willebrand factor (rvWF ) preparation containing all species of vWF multimers compared with a rvWF fraction containing only low molecular weight multimers (LMW-rvWF ) and with a plasma-derived factor VIII/vWF concentrate (pdvWF ). In the vWF-deficient dogs, the half-life of vWF:Ag was 21.6 and 22.1 hours for rvWF, 7.7 hours for pdvWF, and 9 hours for LMW-rvWF; in vivo recovery of vWF:Ag was 59%, 64%, and 70% for rvWF, 33% for pdvWF and 92% for LMW-rvWF; in vivo recovery of RCoF was 78%, 110%, and 120% for rvWF, and 25% for pdvWF. Both rvWF and pdvWF caused increases in factor VIII, which were sustained even when vWF:Ag had decreased to nearly undetectable levels and only monomeric or dimeric species were detectable on agarose gels. At the dosages used, no effect was seen on bleeding time, but the rate of blood flow from cuticle wounds was reduced after a single bolus administration of rvWF. The rvWF was able to control a severe nose bleed in one dog.

Biochemical and functional characterization of recombinant von Willebrand factor produced on a large scale.

Recombinant von Willebrand factor (r-vWF) was produced in serum-free medium on a large scale in recombinant Chinese hamster ovary cells and was purified from fermentation supernatant by a combination of anion exchange chromatography and heparin affinity chromatography. Heparin affinity chromatography yielded r-vWF polymers of different degrees of multimerization. r-vWF was analysed by qualitative and quantitative functional analysis. We could show that while binding of r-vWF to platelets did not depend on multimerization of the molecule, ristocetin-induced platelet aggregation, binding to collagen and binding to heparin correlated directly with the extent of multimerization. Binding of recombinant coagulation factor VIII (r-FVIII) to r-vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The data indicated that binding of r-FVIII did not depend on r-vWF multimerization. Real-time biospecific interaction analysis suggested a potential stoichiometry of 2 to 2.5 r-vWF subunits per r-FVIII molecule. Kinetic analysis of the r-vWF-r-FVIII interaction gave a binding rate constant of 3 x 10(6) M-1 s-1 and an association constant of 2.5 x 10(9) M-1. Reaction of r-vWF with carbohydrate-specific lectins demonstrated that r-vWF contained a high proportion of N-glycans composed of mannose, galactose, glucose, N-acetylglucosamine and terminal sialic acid. Carbohydrate moities were covalently bound to the protein structure and were quantitatively removed from r-vWF only after protein denaturation. The results demonstrated that r-vWF produced on large scale under serum-free culture conditions exhibited qualitative and quantitative functional properties comparable to human plasma-derived vWF.

Differentiation between proteolytic activation and autocatalytic conversion of human prothrombin. Activation of recombinant human prothrombin and recombinant D419N-prothrombin by snake venoms from Echis carinatus and Oxyuranus scutellatus.

Recombinant human prothrombin (r-prothrombin) and recombinant mutant prothrombin with active site Asp419 substituted by Asn (D419N-prothrombin) were expressed in recombinant CHO cells, isolated and purified from the fermentation supernatant. The r-Prothrombin and D419N-prothrombin were digested by both Echis carinatus venom and Oxyuranus scutellatus venom. Prior to, during and after activation, generation of thrombin activity and the proteolytic degradation of the prothrombin polypeptide chain were analysed. Owing to the recombinant preparation and inactivity of D419N-prothrombin and its activation products, the proteolytic action of E.carinatus and O.scutellatus venoms could be studied without addition of thrombin inhibitor, without interference from autocatalytic digestion of prothrombin and in the absence of any other blood coagulation protease. The comparison between the activation of r-prothrombin and D419N-prothrombin by snake venoms permitted differentiation between proteolytic activation and autocatalytic conversion of prothrombin. Incubation of D419N-prothrombin with E.carinatus venom resulted in the generation of stable D419N-meizothrombin by hydrolysis of the peptide bond Arg320-Ile321. By contrast, O.scutellatus venom exhibited activity towards peptide bonds Arg320-Ile321 and Arg271-Thr272 and lower activity towards peptide bond Arg155-Ser156, thus converting D419-prothrombin into D419N-thrombin and also liberating Fragment-1, Fragment-2 and Fragment-1/2 activation peptide. Activation of r-prothrombin by E.carinatus and O.scutellatus venoms demonstrated the autocatalytic potential of prothrombin-derived molecules and indicated that meizothrombin hydrolysed the cleavage between Fragment-2 and thrombin A-chain in the meizothrombin molecule, but not in prothrombin, preferentially at position Arg284-Thr285. By contrast, both meizothrombin and thrombin exhibited no detectable activity towards peptide bond Arg320-Ile321 between thrombin A- and B-chain, although this site exhibits the optimum sequence for thrombin cleavage.

Effect of multimerization of human and recombinant von Willebrand factor on platelet aggregation, binding to collagen and binding of coagulation factor VIII.

The smallest circulating von Willebrand factor (vWF) molecule is a dimer composed of two identical subunits containing binding sites for heparin, collagen, platelet glycoproteins and coagulation factor VIII (FVIII). Interdimeric disulfide linking leads to multimers composed of up to 40 dimers. vWF serves as a carrier of FVIII and is required for normal interactions of platelets with the subendothelium of the injured vessel wall. Von Willebrand factor was purified from human plasma cryoprecipitate and fermentation supernatant of recombinant CHO cells by anion exchange chromatography. Heparin affinity chromatography was used to isolate vWF polymers of different degree of multimerization. Analysis of collagen binding and platelet aggregation revealed that these activities increase with increasing degree of multimerization of vWF. Binding of FVIII to vWF was studied by real-time biospecific interaction analysis and surface plasmon technology. The binding data showed that the binding of FVIII is independent of vWF multimerization. Using recombinant FVIII and recombinant vWF, real-time biospecific interaction analysis resulted in a potential stoichiometry of 2 to 2.5 vWF-subunits per bound FVIII molecule. The kinetic analysis of the vWF-FVIII interaction resulted in a binding rate constant of about 3 x 10(6) M-1 s-1 and an equilibrium dissociation constant of about 0.4 x 10(-9) M.

Rational design, recombinant preparation, and in vitro and in vivo characterization of human prothrombin-derived hirudin antagonists.

A mutant derivative of human prothrombin in which active site aspartate at position 419 is replaced by an asparagine (D419N-prothrombin) has been designed, expressed in recombinant Chinese hamster ovary cells, and purified to homogeneity. D419N-prothrombin was converted to the related molecules D419N-meizothrombin and D419N-thrombin by limited proteolysis by Echis carinatus and Oxyuranus scutellatus venom protease, respectively, and affinity-purified using an immobilized modified C-terminal hirudin-derived peptide. Neither D419N-thrombin nor D419N-meizothrombin exhibited thrombin activity. Titration resulted in no detection of the active site, but binding to the most specific thrombin inhibitor, hirudin, was conserved in both proteins. In vitro examinations showed that D419N-thrombin and D419N-meizothrombin bind to immobilized hirudin, neutralize hirudin in human blood plasma as well as in the purified system, and reactivate the thrombin-hirudin complex. Animal model studies confirmed that D419N-thrombin and D419N-meizothrombin act as hirudin antagonist in blood circulation without detectable effects on the coagulation system. Thus, both D419N-thrombin and D419N-meizothrombin combine for the first time hirudin-neutralizing properties with the advantages of recombinant production of human coagulation factors.

Immobilized hirudin and hirudin-based peptides used for the purification of recombinant human thrombin prepared from recombinant human prothrombin.

A simple and efficient activation-affinity purification system was developed to obtain thrombin from recombinant CHO cells expressing human prothrombin. In this method, a controllable process for the activation of recombinant prothrombin is directly coupled with a purification strategy for the recombinant thrombin generated. At a constant flow rate and with a contact time limited to few seconds, recombinant prothrombin was filtered through immobilized trypsin. In a closed flow system, the recombinant thrombin generated was filtered through newly designed thrombin-specific affinity gels. Hirudin, the most specific thrombin inhibitor, and hirudin-based peptides were covalently immobilized to Sepharose, thus creating thrombin-specific affinity gels that immediately absorb the thrombin generated from the activation mixture. Prothrombin and incompletely activated molecules did not bind to the affinity gel and were recirculated for a further activation cycle. Due to the specificity of the affinity gels for thrombin and the elimination of thrombin from the activation mixture, proteolytic degradation and autocatalytic inactivation of the recombinant thrombin was prevented. Recombinant thrombin was isolated from the hirudin-based affinity gels by chaotrope salt elution, resulting in high yields of highly pure, active thrombin. Affinity purification of thrombin was not deleteriously affected by contamination of the starting material with other proteins. Activation and affinity purification were equally effective for recombinant and human plasma-derived prothrombin as well as for human and recombinant thrombin.

Structural analysis of recombinant von Willebrand factor produced at industrial scale fermentation of transformed CHO cells co-expressing recombinant furin.

Thorough analysis of multimer composition and molecular structure of recombinant von Willebrand factor (r-vWF) produced by recombinant CHO cells demonstrated r-vWF to be more intact and less proteolytically degraded than plasma-derived vWF (pd-vWF) [B. Fischer et al. (1994) FEBS Lett. 351, 345-348]. In contrast to pd-vWF, r-vWF preparations consisted of pro-vWF (vWF containing covalently attached propeptide) as well as mature vWF subunits forming homo- and hetero-multimers. In order to ensure complete propeptide processing, a r-vWF-producing CHO cell clone was transfected with the cDNA of the human propeptide processing enzyme Furin. A r-vWF/r-Furin co-expressing cell clone was cultivated at industrial scale in high cell density perfusion fermenters. r-vWF obtained from these cells was fully processed. Analysis of r-vWF by multimer analysis revealed a multimer pattern equal in number of high molecular weight multimer to pd-vWF, but absence of satellite bands. Two-dimensional electrophoretic analysis of both the primary dimer and the complete multimer pattern of r-vWF showed that the recombinant coagulation factor was composed exclusively of intact and mature subunits. Since the triplet structure typical to pd-vWF is known to reflect proteolytic degradation, r-vWF thus exhibits an integrity far superior compared to pd-vWF.

Large-scale production and purification of a vaccinia recombinant-derived HIV-1 gp160 and analysis of its immunogenicity.

The human immunodeficiency virus (HIV-1) envelope gene was expressed in large-scale microcarrier cultures of Vero cells using a system involving coinfection with two recombinant vaccinia viruses. One recombinant contained the bacteriophage T7 RNA polymerase gene under control of a vaccinia virus promoter. The second contained the HIV-1 gp160 gene flanked by T7 promoter and termination sequences. The protein was expressed on the surface of infected cells, and it was shown to have a molecular weight of 160 kD and to react with gp41 and gp120 specific monoclonal antibodies. After purification by successive affinity and ion-exchange chromatography, the protein was demonstrated to be present in a particulate form with a diameter in the range of 15-30 nm. When injected into goats a high-titer gp160 specific antibody response was elicited and group-specific neutralizing activity could be demonstrated in vitro. The immunogenicity of the protein was also studied in conjunction with a number of adjuvant formulations, and the highest potency in mice was obtained using a preparation with 0.2% Al(OH)3 and 0.25% deoxycholate.