Safety of PEGylated recombinant human full-length coagulation factor VIII (BAX 855) in the overall context of PEG and PEG conjugates.

INTRODUCTION: BAX 855 is a PEGylated human full-length recombinant factor VIII (rFVIII) based on licensed rFVIII (ADVATE). The applied PEGylation technology has been optimized to retain functionality of the FVIII molecule, improve its pharmacokinetic properties and allow less frequent injections while maintaining efficacy. AIM: The aim of this study was to confirm that the excellent safety profile of ADVATE remains unchanged after PEGylation. METHODS: Non-clinical safety studies with BAX 855 and its respective unbound polyethylene glycol (PEG) were conducted in several species. The distribution of a single dose of radiolabelled BAX 855 was further investigated in rats. Publically available safety data on PEG alone and PEGylated biomolecules were summarized and reviewed for specific safety findings attributable to PEG or PEGylated biopharmaceuticals. RESULTS: Safety pharmacology studies in rabbits and macaques and repeated dose toxicity studies in rats and macaques identified no safety issues. Results of a distribution study in rats administered radiolabelled BAX 855 showed that radioactivity was completely excreted; urine was the major elimination route. A 28-day study in rats dosed with the unbound PEG constituent (PEG2ru20KCOOH) of BAX 855 showed no adverse or non-adverse effects. Safety data for PEG and PEG-protein conjugates indicate no safety concerns associated with PEG at clinically relevant dose levels. Although vacuolation of certain cell types has been reported in mammals, no such vacuolation was observed with BAX 855 or with the unbound PEG constituent. CONCLUSION: Non-clinical safety evaluation of PEG and BAX 855 identified no safety signals; the compound is now in clinical development for the treatment of patients with haemophilia A.

BAX 855, a PEGylated rFVIII product with prolonged half-life. Development, functional and structural characterisation.

A longer acting recombinant FVIII is expected to serve patients' demand for a more convenient prophylactic therapy. We have developed BAX 855, a PEGylated form of Baxter's rFVIII product ADVATE™ based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses a novel PEG reagent. The production process was adjusted to yield a rFVIII conjugate with a low PEGylation degree of about 2 moles PEG per FVIII molecule. This optimised modification degree resulted in an improved PK profile for rFVIII without compromising its specific activity. PEGylation sites were identified by employing various HPLC- and MS-based methods. These studies not only indicated that about 60% of the PEG chains are localised to the B-domain, which is cleaved off upon physiological activation during the coagulation process, but also demonstrated an excellent lot to lot consistency with regard to PEGylation site distribution. Detailed biochemical characterization further showed that PEGylated FVIII retained all the physiological functions of the FVIII molecule with the exception of binding to the LRP clearance receptor which was reduced for BAX 855 compared to ADVATE™. This might provide an explanation for the prolonged circulation time of BAX 855 as reduced receptor binding might slow-down clearance. Preclinical studies showed improved pharmacokinetic behaviour and clinically relevant prolonged efficacy compared to ADVATE™ without any signs of toxicity or elevated immunogenicity. The comprehensive preclinical data package formed the basis for approval of the phase 1 clinical study by European authorities which started in 2011.

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.

Comparison of blood smear microscopy to a rapid diagnostic test for in-vitro testing for P. falciparum malaria in Kenyan school children.

OBJECTIVE: To compare the diagnostic performance of microscopy using Giemsa-stained thick and thin blood smears to a rapid malaria dipstick test (RDT) in detecting P. falciparum malaria in Kenyan school children. DESIGN: Randomised, controlled feeding intervention trial from 1998-2001. SETTING: Rural Embu district, Kenya. The area is considered endemic for malaria, with four rainy seasons per year. Chloroquine resistance was estimated in 80% of patients. Children had a spleen rate of 45%. SUBJECTS: A sample of 515 rural Kenyan primary school children, aged 7-11 years, who were enrolled in a feeding intervention trial from 1998-2001. MAIN OUTCOME MEASURES: Percent positive and negative P. falciparum malaria status, sensitivity, specificity and positive and negative predictive values of RDT. RESULTS: For both years, the RDT yielded positive results of 30% in children compared to microscopy (17%). With microscopy as the "gold standard", RDT yielded a sensitivity of 81.3% in 1998 and 79.3% in 2000. Specificity was 81.6% in 1998 and 78.3% in 2000. Positive predictive value was 47.3% in 1998 and 42.6% in 2000, and negative predictive value was 95.6% in 1998 and 94.9% in 2000. CONCLUSION: Rapid diagnostic testing is a valuable tool for diagnosis and can shorten the interval for starting treatment, particularly where microscopy may not be feasible due to resource and distance limitations.

Improved flow cytometric method to enumerate residual cells: minimal linear detection limits for platelets, erythrocytes, and leukocytes.

Increasing demand for quality control of blood products requires more sensitive methods to enumerate residual cells. Presently, the reported threshold (in cells per microliter) is 400 for red blood cells, 30-500 for platelets, and 1 for leukocytes. To examine precision and linearity in enumerating residual platelets and red blood cells, EDTA-anticoagulated blood from healthy donors was serially diluted with serum, stained in TruCount tubes using a no-lyse/no-wash procedure and a monoclonal antibody cocktail against the CD42a (FL1) and glycophorin-A (FL2) epitopes, and analyzed by flow cytometry. Leukocyte counts were determined in separate tubes. Cell preparation and analysis were performed once for 20 blood samples each and 20 times using the same specimen. Acquisition from the same tube was performed separately for platelets (threshold on FL1) and red blood cells (threshold on FL2). Multiparameter analysis was used for data evaluation. Linear results were obtained for platelets per microliter between 3,410 and 5 and for red blood cells per microliter between 54,000 and 3. For the lower cell concentrations, the coefficient of variation was 16.7% for platelets and 10.9% for red blood cells. The presented method allows the distinction between physiologically intact and ghost red blood cells. The method represents a reliable, sensitive, and accurate approach to quantify platelets and red blood cells in diluted blood. It can be applied to enumerate residual cells in plasma products and meets the increasing demand for quality control in blood components.

von Willebrand disease in a pediatric-based population--comparison of type 1 diagnostic criteria and use of the PFA-100 and a von Willebrand factor/collagen-binding assay.

Definitive diagnosis of type 1 von Willebrand Disease (VWD) remains a problem. Provisional consensus guidelines for the diagnosis of definite and possible type 1 VWD were prepared by the Scientific Subcommittee on von Willebrand factor (VWF) of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) during the 1996 annual meeting for the specific purpose of further evaluation in retrospective and prospective studies by a Working Party on Diagnostic Criteria (1996 Annual Report of the SSC/ISTH Subcommittee on VWF). In the first phase of this study, we compared 2 definitions of type 1 VWD. each with 3 criteria: significant bleeding history, laboratory investigations, and family history. Using the ISTH consensus guidelines for type 1 VWD definition, significantly fewer patients were diagnosed with definite type 1 disease as compared to our "in house" Hospital for Sick Children (HSC) criteria (4 vs. 31). While we recognize that the provisional ISTH consensus guidelines were not intended for clinical use, we believe that the results of our studies are of interest and will assist in any future refinements to the ISTH guidelines. In the second phase of this study, we investigated the utility of 2 new tests, a laboratory screening test and a functional test, for VWD in our well characterized, pediatric-based population. The Platelet Function Analyzer (PFA-100) provides an in vitro measure of primary hemostasis under conditions of high shear, using disposable cartridges containing collagen and either epinephrine or ADP. All tested subjects with types 2 or 3 VWD had prolonged PFA-100 closure times (CTs) with both cartridge types (n = 17) and prolonged bleeding times (n = 14). In subjects with definite type 1 VWD, 20/24 (83%) had prolonged CTs with the collagen/ADP cartridge (19/24 (79%) with collagen/epinephrine), compared with 7/26 (27%) with prolonged bleeding times. In subjects with definite types 1, 2, or 3 VWD, collagen/ADP CTs were abnormal in 37/41 subjects, giving an overall sensitivity of 90%. With this high sensitivity, the PFA-100 is a better screening test for VWD than the bleeding time. We also tested a VWF collagen-binding assay (VWF:CBA) as a functional test for VWF, in comparison with the more routinely-used ristocetin cofactor assay (VWF:RC0). The VWF:CBA is based on an ELISA technique, which has the potential to be more reproducible than the VWF:RC0. We found that the VWF:CBA detected 43/49 (88%) subjects with definite types 1, 2, or 3 VWD, performing as well as the VWF:RC0, that detected 42/48 (88%). We also showed that, used in conjunction with VWF antigen levels, the VWF:CBA may be useful in classification of VWD subtypes.

Phospholipid-bound tissue factor modulates both thrombin generation and APC-mediated factor Va inactivation.

Hemostasis is initiated by tissue factor (TF) exposed on cellular phospholipid (PL) membranes, leading to thrombin generation. The binding of thrombin to thrombomodulin (TM), activates the protein C pathway, resulting in the inactivation of factors Va and VIIIa by activated protein C (APC) and a negative feedback effect on thrombin generation. A new assay system was developed for simultaneous measurement of thrombin and APC generation in defibrinated plasma induced by large unilamellar PL vesicles complexed with full-length recombinant TF (TF:PL). TF:PL preparations with a low TF concentration induced an initial rate of thrombin generation below 100 nM/min, and resulted in less thrombin formation in the presence of TM than in its absence. In contrast, TF:PL preparations with a high concentration of TF induced a higher rate of thrombin generation, and APC-mediated feedback inhibition did not occur, despite maximal APC generation. We used the same TF:PL surfaces to study factor Va inactivation by APC in a non-plasma reaction system, and found an inverse correlation between TF surface density and the rate of factor Va inactivation. This observation suggests a previously unrecognized hemostatic effect of TF, namely a non-enzymatic surface density-based inhibition of the anticoagulant effect of APC. In this model, high concentrations and surface density of TF exert complementary effects by promoting the regular procoagulant cascade and by inhibiting the protein C pathway, thereby maximizing hemostasis after vascular injury.

The determination of von Willebrand factor activity by collagen binding assay.

A new collagen binding assay has been developed for the determination of the functional activity of human von Willebrand factor based on the following principle: pepsin-digested type III collagen from human placenta was covalently immobilized on a microtitre plate. Binding of collagen to the microtitre plate was carried out in neutral phosphate buffer within 1 h. A collagen concentration of 3 micrograms mL-1 was sufficient to achieve optimal coating. After drying, the coated microtitre plates remained stable for months without losing their vWF-binding properties and could be incorporated into a ready-to-use kit. The suitability of various types of collagen for use in this assay was evaluated by simulating the binding of vWF to collagen immobilized on the microtitre plate by using surface plasmon resonance technology with collagen immobilized on a sensor chip. vWF was most effectively bound to pepsin-digested type III collagen from human placenta. The assay thus comprises the following steps: (1) serial dilutions of a vWF reference preparation and vWF-containing samples are prepared and bound to a microtitre plate which is precoated with collagen; (2) vWF is detected with a polyclonal antibody; (3) the substrate reaction is photometrically measured with an ELISA reader. This test is highly specific and sensitive for vWF (detection limit: 10 ng mL-1). The collagen binding activity measured corresponds to the degree of multimerization of vWF. The high sensitivity and the short time needed to carry it out may make it useful for clinical diagnosis and for the measurement of the functional activity of vWF in factor concentrates. In certain applications it may represent a suitable replacement for the ristocetin cofactor assay.

The pH dependence of the equilibrium constant KHyd for the hydrolysis of the Lys15-Ala16 reactive-site peptide bond in bovine pancreatic trypsin inhibitor (aprotinin).

The pH dependence of the equilibrium constant KHyd for the hydrolysis of the Lys15-Ala16 reactive-site peptide bond of the bovine pancreatic trypsin inhibitor (aprotinin) was investigated over the pH range 2.3-6.5. Solutions of aprotinin, modified aprotinin with the Lys15-Ala16 peptide bond cleaved and mixtures of both species were incubated with 10 mol% porcine beta-trypsin. The state of equilibrium was determined by analytical cation-exchange HPLC. The KHyd values obtained did not exactly obey the simple equation of Dobry et al. (1952), which had to be used in an extended form with two additional parameters for a satisfactory fit. The pH-independent equilibrium constant is 0.90 and the pK values of the Lys15 carboxyl group and of the Ala16 amino group are 3.10 and 8.22, respectively. The pK of an additional group is apparently perturbed by the peptide-bond hydrolysis. It is 4.60 in the native and 4.40 in the modified aprotinin.

Characterization and sequence determination of six aprotinin homologues from bovine lungs.

Six Kunitz inhibitors, which are dissimilar to aprotinin, can be isolated from bovine lungs. These homologues cannot be distinguished from aprotinin, in respect to their inhibitory specificity. They have, however, different amino-acid compositions and a different degree of basicity. The entire primary structures of these inhibitors were elucidated by automated Edman sequencing. Besides the known Glp-1-aprotinin another aprotinin homologue (des-Ala58-aprotinin) was isolated, which could result from a different proteolytic processing of the bovine aprotinin precursor. The other homologues can be denoted as aprotinin isoinhibitors, showing several amino-acid replacements compared to aprotinin and which also appear in the area of the contact region.

Pyroglutamyl-aprotinin, a new aprotinin homologue from bovine lungs--isolation, properties, sequence analysis and characterization using 1H nuclear magnetic resonance in solution.

A new Kunitz-inhibitor, which is different from aprotinin was extracted from bovine lungs with methanol, further purified by affinity chromatography on trypsin-Sepharose CL-6B and by repeated cation exchange chromatography on CM-Sephadex C-25. The inhibitor, which is less basic than aprotinin was characterized by polyacrylamide gel electrophoresis and ion-exchange HPLC. The N-terminus is blocked by pyroglutamic acid (Glu-1). After enzymatic removal of this residue with pyroglutamate aminopeptidase, complete identity with the primary structure of aprotinin was established by sequencing the inhibitor, which had been oxidized with performic acid, and by sequencing a tryptic fragment. The occurrence of the inhibitor, which can be denoted as pyroglutamyl-aprotinin or Glu-1-aprotinin, but which cannot be distinguished from aprotinin regarding its inhibitory specificity, is obviously the result of a different proteolytic processing of the bovine aprotinin precursor. By using CD and NMR-techniques it was shown that the N-terminus of the inhibitor is blocked, and that the conformation and the internal mobility correspond with those of aprotinin.

Note on wave propagation in a thin elastic tube containing a viscous fluid.

This paper deals with the theoretical and experimental investigation of the propagation of pressure waves through a straight thin-walled elastic tube, filled with a streaming Newtonian fluid. The tube used was of silicon rubber and measured 200 mm in length, 8 mm in internal diameter and 1 mm in wall thickness. The velocity profiles across the diameter of the tube were measured at the beginning, at the middle and the end of the elastic pipe and compared with the results of a mathematical-computational model. The test data agree fairly well with the calculated values.