ALBERT

Description: Purpose: This study summarizes the characteristics and virus safety profile of Wilate®, a new human plasma-derived, high purity, double virus inactivated VWF/FVIII concentrate. The manufacturing process comprises two chromatographic steps, ensuring high purity and preserving the integrity and functionality of the VWF/FVIII complex. The optimised solvent/detergent treatment (S/D) and terminal dry-heat treatment (PermaHeat) of the lyophilised product provide two effective and robust virus inactivation steps for enveloped (EV) and one step for non-enveloped viruses (NEV). Methods: Functional and structural properties of the VWF/FVIII concentrate were studied utilising state-of-the-art assays. The reduction factors for a panel of model viruses were determined in down-scale experiments valid for the respective manufacturing steps. Summary of results: The integrity of the VWF/FVIII concentrate was confirmed by all tests. The ratio between VWF:RCo and FVIII:C was close to that in plasma of blood donors. The VWF multimeric profile demonstrated 〉10 bands in all batches and the triplet structure was very well preserved. A high specific activity, on average 122 IU FVIII:C/mg total protein, was measured in 15 batches recently produced. SE–HPLC revealed a one-peak-product, representing the VWF/FVIII-complex. All viruses tested were rapidly inactivated by at least 4 log10. The PermaHeat treatment of the lyophilised product at +100°C for 2 hours, at a specified residual moisture of 0.7–1.6%, was efficient for the inactivation of both EV and NEV. Conclusion: Wilate® is a high purity, double virus inactivated concentrate containing functional VWF and FVIII in a ratio close to plasma of healthy subjects. The product has an excellent viral safety profile. The safe and effective treatment of patients with von Willebrand disease and haemophilia A was demonstrated in multiple clinical trials.

Description: Abstract 1298 Poster Board I-320 All current recombinant factor VIII products produced today are derived from the expression of FVIII in hamster cell lines. This is a report of the functional properties of a newly developed recombinant factor VIII concentrate produced in a human cell line. The possible advantage of using a human cell line for expression of recombinant factor VIII is that a human pattern of post-translational modifications (PTM), such as glycosylation, are obtained. These human like PTMs, may possibly lead to improved function and a reduced immunogenic product profile compared to recombinant factor VIII products from CHO or BHK cells that have hamster-specific PTM. Data are presented on the characteristics of the Human-cl rhFVIII, a B-domain-deleted factor VIII protein produced in HEK293F cells, and how they compare with those of other factor VIII products. Human-cl rhFVIII is highly pure (〉99.9% purity) with a high specific activity of 8000-11000 IU/mg protein, and a FVIII:C to FVIII:Ag ratio close to 1.0. This indicates that the entire factor VIII protein in the product is active. Human-cl rhFVIII showed no significant assay discrepancy between one-stage and chromogenic assay results. Because of the human glycosylation profile of the new Human-cl rhFVIII, there was an absence of the antigenic type of sialic acid, N-glycolylneuraminic acid as well as of antigenic Galα (1,3) Gal epitopes found in the non humane derived products. Another type of PTM of factor VIII is the sulfation of certain tyrosines that are crucial for the biological function e.g. the interaction with von Willebrand factor (vWF). Human-cl rhFVIII was found to be optimally sulfated when compared to endogenous human FVIII. In this context, it is of interest to note that Human cl-rhFVIII showed a 60% higher affinity in binding to vWF compared to other recombinant factor VIII products and the number of molecules in the product able to bind to vWF was significantly higher for Human-cl rhFVIII. Interaction with thrombin and capacity to generate thrombin were shown to be similar to those of plasma-derived factor VIII. The sensitivity to be inactivated by activated protein C was similar to all currently licensed rhFVIII products. The important properties of Human cell line derived recombinant factor VIII may be very promising for its use in future treatment of Haemophilia A. Disclosures Schwartz: Octapharma: Employment. Sandberg:Octapharma: Employment. Agerkvist:Octapharma: Employment. Kannicht:Octapharma: Employment. Ramström:Octapharma: Employment. Stenlund:Octapharma: Employment. Oswaldsson:Octapharma: Employment. Brunberg:Octapharma: Employment.

Description: Purpose: Wilate® is a novel, human plasma-derived, high purity, double virus inactivated coagulation factor concentrate. The safe and effective treatment of von Willebrand disease patients and haemophilia A has been demonstrated in clinical trials. The very high virus safety is achieved by an optimised solvent/detergent (S/D) method and terminal dry-heat treatment (PermaHeat) of the lyophilised product, not affecting the integrity of the proteins, which was investigated in this study. Methods: Analyses of PermaHeat treated (+100°C for 120 min) Wilate® samples with different residual moisture (RM) contents were performed in order to evaluate the upper RM limit in lyophilised samples. Samples with RM up to 7.3% were subjected to SDS-PAGE, immunoblotting and peptide mapping. Further investigations were based on FVIII:C activity testing and phospholipid binding properties, VWF multimer analysis, Ristocetin cofactor (VWF:RCo) activity and collagen binding (VWF:CB). Summary of results: The analyses confirmed that the structural and functional integrities of both VWF and FVIII were maintained during PermaHeat of Wilate® at +100°C for 120 minutes when the RM was kept at or below 1.8% during the heating procedure. Changes of protein-biochemical properties were detectable only for RM values exceeding 2%. Denaturation of samples occurred at RM of more than 3.5%. Conclusion: Beside S/D treatment, optimised PermaHeat treatment of the lyophilised VWF/FVIII product ensures the high viral safety margin of Wilate®. According to the results, the RM content during PermaHeat treatment was specified with a great safety margin to a maximum of 1.6%. This limit is carefully controlled in each vial before heating by RM testing utilising near infrared (NIR). At or below this RM no structural or functional changes of VWF and FVIII were detected after PermaHeat treatment.

Description: Abstract 3356 Objective: Multimeric glycoprotein von Willebrand factor (VWF) exhibits a unique triplet structure of individual oligomers, resulting from ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motifs 13) cleavage. The faster and slower migrating triplet bands of a given VWF multimer respectively have one shorter or longer N-terminal peptide sequence. Within this peptide sequence, the A1 domain regulates interaction of VWF with platelet glycoprotein (GP)Ib. Distribution of VWF triplet bands is significantly altered in some types of VWD, however, the impact of triplet structure on VWF function has not been investigated so far. Methods: Platelet-adhesive properties of two VWF preparations with similar multimeric distribution but different triplet composition obtained by size exclusion in addition to heparin affinity chromatography were investigated for differential functional activities. Preparation A was enriched in intermediate triplet bands, while preparation B predominantly contained larger triplet bands. Collagen- and GPIb-binding was determined by surface plasmon resonance (SPR). Platelet adhesion under flow was determined using flow-chamber models. Results: Binding studies revealed that preparation A displayed a reduced affinity for recombinant GPIb, but an unchanged affinity for collagen type III, when compared to preparation B. Under high-shear flow conditions, preparation A was less active in recruiting platelets to collagen type III. Furthermore, when added to blood from patients with von Willebrand disease (VWD), defective thrombus formation was less restored. Conclusion: Thus, VWF forms lacking larger size triplet bands appear to have a decreased potential to recruit platelets to collagen-bound VWF under arterial flow conditions. By implication, changes in triplet band distribution observed in patients with VWD may result in altered platelet adhesion at high-shear flow. Disclosures: Schwartz: Octapharma: Employment. Kannicht:Octapharma: Employment. Fuchs:octapharma: Employment. Kröning:octapharma: Employment. Solecka:Octapharma: Employment.

Description: Introduction: The Von Willebrand Factor (VWF), is a very large glycoprotein present in human plasma essential for normal thrombus formation at sites of vascular injury. Blood coagulation is initiated by exposure of vessel wall components, e.g. collagen, followed by platelet tethering, activation and adhesion leading to the formation of a stable clot. All steps of blood coagulation occur in flowing blood under various conditions depending on vessel size. It is important to assess the function of a VWF concentrate under physiologic conditions. Under static or low shear conditions, platelets can bind directly to collagen without assistance of VWF, while the VWF is essential for mediation of platelet adhesion under high flow occurring in the arterial circulation. It has been assumed that multimer size plays an important role in this binding and in subsequent platelet adhesion. In this study we evaluated the binding of VWF of different multimeric structures to collagen and determined VWF-mediated platelet binding under low to high shear rates in a flow chamber model. Methods: A flow-chamber coated with human collagen was developed to mimic physiological flow conditions. A high purity VWF/factor VIII (FVIII) concentrate (Wilate®) and two other VWF/FVIII concentrates were tested at shear rates of 400 1700 and 4000 s−1 reproducing shear rates occurring in veins, small arteries and capillaries. Collagen-bound VWF was characterized by antigen determination (VWF:Ag) and multimer (MM) analysis. Binding of labeled platelets was visualized by a fluorescence microscope and surface coverage was quantified. Results: All VWF MMs independent of MM size were found to bind to collagen even under high shear rates. The amount of collagen-bound VWF:Ag and VWF-mediated platelet adhesion at 1700 s−1 differed significantly between the tested concentrates, when equal VWF:Ag amounts were applied. Conclusion: Binding of VWF to collagen does not depend on VWF MM size in this model even when measured under high shear rates. The differences in collagen-bound VWF:Ag and VWF-mediated platelet binding do not seem to depend on the VWF MM distribution of the concentrates. Other structural features than VWF MM size may likely be caused by the differences seen in the binding levels of the different concentrates and should be further explored. Possible differences between VWF activity assays performed under static or flow conditions and their appropriate use for VWD diagnosis and quantification of in-vivo activity need to be further investigated.

Description: Background The multimeric plasma glycoprotein von Willebrand factor (VWF) is exceptionally rich in cysteine, and its structure is largely determined by inter- and intramolecular disulfide bonding. Additionally, VWF was shown to contain unpaired cysteine residues potentially affecting protein function. The significance of free thiols on the surface of plasmatic VWF has been confirmed previously with respect to platelet binding under pathologically high shear stress. Furthermore their potential involvement in functional VWF self-association occurring at elevated shear stress has been suggested. Aims Objective of the present study was to investigate whether free thiol groups of plasma VWF contribute to the physiological VWF function under high physiological arterial shear stress conditions. Furthermore, we aimed to elucidate possible underlying mechanisms involved in this regulation. Methods Free and accessible thiol groups of plasma-derived VWF were blocked with N-ethylmaleimide (NEM). Derivatization was followed by detailed structural and functional examination including multimer analysis (MMA) and Fourier transform infrared spectroscopy (FTIR). Functional differences between the NEM-derivatized sample and the control sample were detected using an in vitro flow chamber system with respect to VWF-mediated platelet adhesion to collagen. Interactions with collagen type III and platelet glycoprotein (GP)Ib receptor were investigated using surface plasmon resonance (SPR). Identification of accessible cysteine residues was accomplished using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation as well as mass spectrometry. Results Blocking free thiol groups provoked substantial loss of VWF activity with respect to platelet recruitment to collagen type III under flow. The lowered platelet adhesion to collagen type III was shown to be a combined effect of inhibition of (i) the initial VWF binding to collagen type III as well as (ii) VWF-platelet GPIb interaction. Free thiol groups were accessible for derivatization solely on the surface of coiled multimers. Domain incorporation studies revealed a high level of derivatization in VWF N- and C-terminus. This was in accordance with the mass spectrometric analysis, where 19 MPB-derivatized peptides, predominantly located at the N- and C-terminus, could be identified. Conclusion Blocking free thiol groups in VWF significantly impaired mediation of platelet adhesion under physiological shear stress conditions. This result suggests an essential functional role of free thiol groups in VWF regarding binding to subendothelial matrix as well as platelet recruitment. Disclosures: Solecka: Octapharma AG: Employment. Fuchs:Octapharma AG: Employment. Kannicht:Octapharma AG: Employment.

Description: Abstract 4354 Objective: The characteristic multimeric pattern of plasmatic VWF results from asymmetric cleavage by ADAMTS13. Regulation of VWF multimer distribution is critical for its physiological function. In human plasma, VWF multimer gels reveal species of various multimeric sizes with flanking satellite bands (triplets). The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Defects in VWF secretion, impaired assembly of multimers, or increased proteolysis can cause von Willebrand Disease (VWD). Distribution of VWF triplet bands is significantly altered in some plasma-derived VWF concentrates. The impact of triplet structure on VWF function has not been investigated so far. Methods: Four commercially available VWF concentrates were analyzed for ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by fluorescence resonance energy transfer (FRET) assay. Samples composed of different VWF triplet distribution but comparable multimers were obtained by heparin affinity chromatography. Platelet adhesion under flow was determined using a flow-chamber model. Results: VWF concentrates markedly differed with respect to their content of ADAMTS13 antigen and activity. A higher content of ADAMTS13 correlated with an altered triplet structure reflected by an increased presence of the faster migrating triplet band, indicating VWF proteolysis. VWF-mediated platelet adhesion under flow over time was increased using a VWF fraction predominantly containing the slower migrating triplet band. Conclusion: These findings suggest that an intact triplet structure has an impact on platelet adhesion at physiological high arterial shear rate conditions. The relevance of VWF N-terminal domains for platelet binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated. Disclosures: Fuchs: octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment. Kannicht:Octapharma: Employment. Schwartz:Octapharma: Employment.

Description: Abstract 1425 Introduction: The characteristic multimer pattern of plasmatic von Willebrand factor (VWF) results from asymmetric cleavage by the processing metalloprotease ADAMTS13 between Y1605/M1606 within the VWF A2 domain. In normal plasma, characteristic species of various multimeric sizes with flanking satellite bands (triplets) encircling the major band on VWF multimer gels are present. The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Even though the distribution of VWF satellite bands is significantly altered in some types of von Willebrand disease (VWD) and several commercial VWF concentrates, the impact of triplet structure on VWF function has not been investigated so far. Methods: Four commercially available VWF concentrates were analyzed with respect to ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by the fluorescence resonance energy transfer (FRET) assay. VWF zymogram gels were used to test for ADAMTS13 activity. Samples composed of different VWF triplet distribution but comparable VWF multimers were obtained by fractionation of plasmatic VWF using heparin affinity chromatography. VWF affinity to collagen was measured by surface plasmon resonance (SPR). Results: VWF concentrates markedly differed in their content of ADAMTS13 antigen and activity. A higher ADAMTS13 content correlated with an increased portion of the proteolyzed faster migrating VWF triplet band. The degree of VWF proteolysis, i.e. lack of an additional N-terminal fragment, correlated with a decreased collagen binding level measured by SPR. Conclusion: Proteolytic cleavage of N-terminal domains of VWF resulting in a higher content of faster migrating satellite bands affects the function of VWF. The impact of VWF N-terminal domains on collagen binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated. Disclosures: Schwartz: Octapharma: Employment. Fuchs:Octapharma: Employment. Kannicht:Octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment.