ALBERT

Description: 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.

Description: Abstract 4356 Baxter and Nektar have developed a longer acting recombinant FVIII (BAX 855), which is manufactured by coupling stable PEG using Nektar technology to Baxter’s full-length rFVIII bulk drug substance from the protein-free ADVATE™ manufacturing process. BAX 855 was characterized by a number of analytical methods, focusing on the elucidation of the primary structure, posttranslational modifications, PEGylation site distribution and three-dimensional structure. The primary structure of BAX 855 was investigated using a peptide mapping approach. Samples were digested with trypsin and the resulting peptides were separated by reversed phase chromatography and detected with on-line electrospray ionization mass spectrometry. This approach resulted in a sequence coverage of 94% with good consistency demonstrated between different BAX 855 batches. Protein-bound oligosaccharides were determined by normal phase HPLC of fluorescence labeled N-glycans released by PNGase F treatment. The composition of the N-linked oligosaccharides showed a similar pattern between BAX 855 and unmodified rFVIII, confirming that the N-glycosylation pattern remained intact during the PEGylation process. PEGylation site distribution and detailed analysis of the consistency of PEGylation was investigated by activating BAX 855 with thrombin. The resulting PEGylated and non-PEGylated fragments were separated using a RP-HPLC approach and the bound PEG was measured for each thrombin fragment. The distribution of PEG among the different thrombin fragments of rFVIII was shown to be consistent between several BAX 855 batches. Moreover, RP-HPLC of native BAX 855 showed reproducible subunit composition and PEGylation. SDS-PAGE and Western blot analysis of BAX 855 revealed changes in the electrophoretic pattern due to PEGylation without the appearance of any relevant degradation products. The random PEGylation of rFVIII was shown using antibodies with different epitope specificities. Dynamic light scattering and Fourier-transformed infrared spectroscopy (FTIR) were used to monitor the consistency of three dimensional structures. The mean hydrodynamic diameter of BAX 855 was between 30 and 40 nm, which is a characteristic size for a ∼300-kDa protein. Several BAX 855 batches showed almost overlapping FTIR absorbance spectra, especially in the conformationally susceptible amide I and amide II peak at 1650 and 1550 cm−1, respectively, indicative for good consistency of the manufacturing process. In summary, BAX 855, a PEGylated rFVIII derivative, can be manufactured reproducibly without changes to the protein structure characteristic for a fully functional FVIII molecule. Disclosures: Kaliwoda: Baxter Innovations GmbH: Employment. Graninger:Baxter Innovations GmbH: Employment. Matthiessen:Baxter Innovations GmbH: Employment. Schrenk:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Description: Abstract 4653 Human coagulation factor VII (FVII) is a vitamin-K-dependent protein with a molecular weight of 50 kDa. Activation of FVII occurs by cleavage of the arginine 152 – isoleucine 153 peptide bond resulting in two disulfide-linked peptide chains. The therapeutic utility of rFVIIa is based on its capacity to trigger hemostasis independently from factor VIII and factor IX and thus even in the presence of factor VIII or factor IX inhibitors. Baxter has developed a recombinant FVIIa (rFVIIa) that is synthesized by a genetically engineered Chinese hamster ovary (CHO) cell line. No materials of human or animal origin are employed in the manufacture, purification, or formulation of the final product, thus reducing the risk of transmission of adventitious agents. The growth medium is a chemically defined medium, and the downstream process does not use monoclonal antibodies for the purification of rFVIIa. The rFVIIa drug candidate was functionally characterized in vitro and its features were compared with those of a commercially available rFVIIa. The overall hemostatic potency of rFVIIa was assessed by its FVIII bypassing activity in a human FVIII-deficient plasma with high-titer inhibitor by measuring the kinetics of thrombin generation and fibrin formation. The thrombin-generating capacity of the rFVIIa drug candidate was similar to that of commercial rFVIIa. The time and rate of fibrin formation was measured by thromboelastography, where a dose-dependent normalization of the impaired clotting times and fibrin formation was observed. A similar FXa-generating potency was found for rFVIIa and commercial rFVIIa when measured on the surface of TF-expressing fibroblasts, suggesting a full capability to bind to TF-bearing cells and trigger hemostasis on their surfaces. rFVIIa could be inactivated by anti-thrombin III-heparin in solution with relipidated TF with no relevant difference to the comparator product. TF pathway inhibitor effectively inhibited FXa generation in a cell-based activity assay, with a similar IC50 for rFVIIa and the comparator. In summary, the functional characteristics of the rFVIIa drug candidate were very similar to those of commercially available rFVIIa. Disclosures: Turecek: Baxter Innovations GmbH: Employment. Varadi:Baxter Innovations GmbH: Employment. Vejda:Baxter Innovations GmbH: Employment. Boehm:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Reiter:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Mundt:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment.

Description: Abstract 4362 Baxter and Nektar have developed BAX 855, a PEGylated form of Baxter’s recombinant FVIII (rFVIII) product based on the ADVATE™ manufacturing process. The conjugation process for preparing BAX 855 uses proprietary stable PEGylation from Nektar Therapeutics. Similar Nektar technology has been successfully employed for marked and licensed PEGylated drug products and drugs in clinical use. The manufacturing process for BAX 855 comprises several steps, including chromatographic purification on a MacroCap SP resin, a strong cation exchanger, which allows the fractionation of species with different PEGylation degrees and concentration of the conjugate collected by an ultra- / diafiltration step leading to the pre-formulated bulk drug substance (BDS). Final formulation of the BDS includes a filling and lyophilization step to obtain the final drug product. The process described is suited to manufacture BAX 855 in gram scale and showed a good batch to batch consistency, ensuring an equivalent product quality for each batch. BAX 855 manufactured by this process has a specific activity similar to that of rFVIII in ADVATE™ and PEGylation degrees in the narrow range of 2 to 3 mols PEG / mol rFVIII. SDS-PAGE and Western blot analysis of BAX 855 confirm the stable PEGylation and demonstrate an increase in the molecular weight of the various FVIII domains. PK studies in different species display longer survival of BAX 855 compared to ADVATE™. Disclosures: Siekmann: Baxter Innovations GmbH: Employment. Martin:Baxter Innovations GmbH: Employment. Zöchling:Baxter Innovations GmbH: Employment. Kellerer:Baxter Innovations GmbH: Employment. Rottensteiner:Baxter Innovations GmbH: Employment. Mitterer:Baxter Innovations GmbH: Employment. Bossard:Nektar Therapeutics: Employment. Phillips:Nektar Therapeutics: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment.

Description: Abstract 2236 Baxter has developed a human recombinant (r) ADAMTS13 for treatment of patients with hereditary TTP. The protein is produced in a CHO cell-line using serum and protein-free fermentation technology. The purification process does not use immune-affinity chromatography, and includes two viral reduction steps. The final drug product is formulated without proteins of animal or human origin. Here we characterize preclinical and clinical lots of rADAMTS13 with respect to their structural properties. The molecular weight of rADAMTS13 was determined by MALDI TOF as ∼173 kDa. SDS-PAGE showed one band at ∼190 kDa; no additional distinct protein bands corresponding to other proteins or product related degradation products were visible in the gels. Investigation of the primary structure of rADAMTS13 by peptide mapping revealed a sequence coverage of 98% compared to the theoretical amino acid sequence, and no peptides from the signal or propeptide. Peptide mapping was also used to determine posttranslational modifications of rADAMTS13. Seven O-glycosylation sites were identified in addition to the predicted N-glycosylation, O-fucosylation and C-mannosylation sites. The relative monosaccharide and N-linked oligosaccharides composition was consistent in all preclinical and clinical rADAMTS13 batches. The main N-glycan structure found on rADAMTS13 is a biantennary, core fucosylated complex type oligosaccharide with one or two sialic acids. Quantification of sialic acid determination showed consistent amounts of sialic acid on the terminal ends of the glycans in preclinical and clinical rADAMTS13 samples. N-glycolylneuraminic acid, a potentially immunogenic variant of sialic acid, was

Description: Post-translational glycosylation determines the pharmacodynamic and pharmacokinetic properties of therapeutic proteins. While desialylation of FVIII/VWF (asialo-FVIII/VWF) has a significantly reduced half-life compared to normal FVIII/VWF complex (Sodetz et al 1977), we introduced additional sialic acids on recombinant (r) FVIII to obtain the opposite: prolonged survival of rFVIII in the circulation. Additional sialic acid residues were introduced to rFVIII by covalently binding polysialic acid (PSA) to full length (FL) rFVIII. The resulting drug candidate BAX 826, polysialylated human rFVIII, is manufactured from octocog alfa which is expressed in Chinese Hamster Ovary Cells by a plasma/albumin free cell culture method and is the active substance in Baxalta´s licensed product ADVATE. The manufacturing process for BAX 826 comprises several steps, starting with the bulk drug substance (BDS) of the ADVATE process which is subjected to polysialylation, i.e. covalent attachment of PSA of an average molecular weight of 20 kDa to rFVIII. Polysialylation is followed by a sequence of chromatographic purification steps and concentration of the conjugate by an ultra-/diafiltration step leading to the pre-formulated BDS. Final formulation of the BDS includes a filling and lyophilization step to obtain the final drug product. The process described is suited to manufacture BAX 826 in large scale and showed a good batch to batch consistency, ensuring an equivalent product quality for each batch. BAX 826 was extensively structurally and functionally characterized. The methods used included reducing and non-reducing peptide mapping to determine amino acid sequence and post translational modifications, qualitative analysis of modification sites, SDS-PAGE and Western blot analysis, assessment of three dimensional structure similarities of BAX 826 and rFVIII, ADVATE, by Fourier-transformed infrared spectroscopy (FTIR), dynamic light scattering (DLS) and circular dichroism (CD). Functional characterization was performed by assessment of the kinetics of the assembly and activity of FIXa-FVIII (tenase) complex, determination of the rate of activation and inactivation of BAX 826 by thrombin, determination of overall hemostatic potency by a thrombin generation assay, measurement of the rate of inactivation of untreated or thrombin-activated BAX 826 by activated protein C, measurement of kinetics of the binding of BAX 826 to von Willebrand factor (VWF), to phospholipids, and to low-density lipoprotein (LDL)-receptor-related protein 1 (LRP1) by surface plasmon resonance spectroscopy. While BAX 826 retained full hemostatic functionality of FVIII as a co-factor of the tenase complex, and can thus be considered a fully active FVIII molecule, it was found to have a reduced binding to VWF and to LRP1. Together with the conception that VWF as FVIII's chaperone dictates the maximally achievable terminal half-life extension for FVIII reduced binding to VWF and FVIII's major clearance receptor LRP1likely explains the prolonged pharmacokinetic (PK) properties when comparing BAX 826 with unmodified FL rFVIII in animal models. In summary, BAX 826, a polysialylated rFVIII derivative, can be manufactured reproducibly without relevant changes to the protein structure characteristic for a functional FVIII molecule with high specific activity. Disclosures Turecek: Baxalta Innovations GmbH: Employment. Siekmann:Baxalta Innovations GmbH: Employment. Mitterer:Baxalta Innovations GmH: Employment. Graninger:Baxalta Innovations GmbH: Employment. Schrenk:Baxalta Innovations GmbH: Employment. Matthiessen:Baxalta Innovations GmbH: Employment. Rottensteiner:Baxalta Innovations GmbH: Employment. Hoellriegl:Baxalta Innovations GmbH: Employment. Putz:Baxalta Innovations GmbH: Employment. Schwarz:Baxalta Innovations GmbH: Employment. Scheiflinger:Baxalta Innovations GmbH: Employment.

Description: Recombinant factor IX (rFIX) expressed in Chinese hamster ovary (CHO) cells has been shown to be safe and effective in clinical studies, but differs in pharmacokinetics from plasma-derived FIX (pdFIX). In clinical studies, CHO-derived rFIX had a 30–50 % lower in-vivo recovery when compared to pdFIX, whereas mean residence time and terminal half-life did not differ between preparations. Although rFIX shows high similarity to pdFIX in structure and function, differences in glycosylation and gamma-carboxylation degree can be detected. Moreover, although experimental proof has yet to be published, the lower degree of phosphorylation of amino acid serine 155, and the lower degree of sulfation of tyrosine 158 have been hypothesized to be causative for the lower in-vivo recovery of rFIX. These two modifications occur at less than 20 % for the tyrosine-sulfation and at less than 1 % for the serine phosphorylation in rFIX, whereas pdFIX has both modifications to more than 90 % completed. We identified human HEK293 cells to perform rFIX phosphorylation and sulfation to a higher extent than CHO cells. A rFIX-producing cell line derived from HEK293 cells was generated by stable transfection, and was adapted to suspension culture conditions to allow lab-scale fermentation. rFIX was produced and purified from a single fermentation run using two different down-stream process schemes: the first was able to enrich high-phosphorylated and -sulfated rFIX; the second to purify total rFIX from the supernatant at high yield. For pharmacokinetic comparison, these HEK293 materials, CHO-derived rFIX, and a pdFIX preparation were formulated in the same buffer. Determination of phosphorylation and sulfation by mass spectrometry showed a phosphorylation and sulfation degree of 50 % plus a 20 % single modification (phosphorylation or sulfation) for the HEK293-material purified by the modification enrichment method versus 15 % for both modifications plus a 15 % single modification for the material purified by the high-yield protocol. The values for CHO-derived rFIX and pdFIX were similar to those in the literature. Oligosaccharide mapping revealed glycosylation differences among CHO-, HEK293-, and pdFIX preparations, but high similarity between both HEK293-derived materials. We compared the pharmacokinetics of the various FIX preparations in FIX-knock-out mice. In-vivo recovery and area under the curve were statistically significantly higher for the high phosphorylated and sulfated HEK293-material than for total rFIX derived from HEK293 cells. However, these two parameters were lower for both HEK293-derived rFIX preparations than for CHO-derived rFIX, and lower for CHO-derived rFIX than for pdFIX. This may be due to glycosylation differences between these FIX preparations. Mean residence times and terminal half-lives were similar for all preparations. In summary, these findings emphasize that the degree of rFIX-sulfation and -phosphorylation influences the pharmacokinetic properties of rFIX.

Description: Recombinant VWF was co-expressed with recombinant FVIII in CHO cells. In order to obtain fully processed, mature rVWF, rVWF was exposed to recombinant CHO-cell-derived furin for VWF pro-peptide removal. Fermentation for both rVWF and furin and downstream processing were performed under serum-free conditions. Mature rVWF was highly purified by a series of conventional chromatography steps to a purity of 〉 95%. rVWF was formulated in a protein-free buffer. Several large scale batches of rVWF were produced and characterized in comparison to Humate-P, Wilfactin and a highly purified plasma-derived (pd)VWF, an experimental pd VWF product obtained from cryoprecipitate. Residual pro-VWF levels were approximately 2% of total VWF antigen compared with 0.12, 0.05 and 0.13 for the rVWF product, experimental pd VWF, Humate-P and Wilfactin, respectively. VWF pro-peptide levels were approximately 0.5 pmol per unit VWF antigen, compared with 0.6 and 0.9 for Humate-P and Wilfactin, respectively; in the experimental pd VWF pro-peptide was below the limit of detection. Residual FVIII content was approximately 0.02 units (chromogenic assay) per unit VWF antigen, compared with 0.4 units in Humate-P, and 0.04 units in Wilfactin, and ≤ 0.01 in the experimental pd VWF product. Ristocetin cofactor activity was around 0.7 VWF:RCo units per unit antigen for all rVWF and pd VWF products tested. Collagen-binding activity was also similar between rVWF and pd VWF controls (around 0.8 – 0.9 units per unit). FVIII-binding capacity of rVWF was compared by setting the mean pd VWF as 100% and was found to also be ± 100%. The VWF affinity for FVIII was similar in rVWF and pd VWF (Biacore). rVWF showed high molecular weight multimers in low resolution agarose gels, with a pre-dominance in the high molecular weight range. In contrast to pd VWF, rVWF showed homogenous multimer bands without triplet structure formation due to the non-exposure to ADAMTS13 during the manufacturing process. The pharmacokinetic properties of rVWF were compared with pd VWF in VWF knock-out mice. The half-life or rVWF in VWD mice was 4.3 and 3.5 hours for 2 batches investigated, which seems longer than the half-lives obtained for pd VWF, 2.9 hours for Humate-P and 2.2 hours for a highly purified pd VWF control. Both VWF preparations stabilized endogenous murine FVIII which rose over time to the levels observed in normal C57BI/6J mice. Overall, these results showed that the properties of mature, furin-processed rVWF in vitro and in vivo are equivalent to those of plasma-derived VWF.