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Original article © Schattauer 2012 Hämophilie/VWS BAX 855, a PEGylated rFVIII product with prolonged half-life Development, functional and structural characterisation P. L. Turecek1; M. J. Bossard2; M. Graninger1; H. Gritsch1; W. Höllriegl1; M. Kaliwoda1; P. Matthiessen1; A. Mitterer1; E.-M. Muchitsch1; M. Purtscher1; H. Rottensteiner1; A. Schiviz1; G. Schrenk1; J. Siekmann1; K. Varadi1; T. Riley2; H. J. Ehrlich1; H. P. Schwarz; F. Scheiflinger1 1Baxter Innovations GmbH, Vienna, Austria; 2Nektar Therapeutics, Huntsville, AL, USA Keywords Recombinant factor VIII, PEG, haemophilia, substitution therapy Summary 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 ADVATETM based on the ADVATETM 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, Correspondence to: Peter L. Turecek Baxter Innovations GmbH, Vienna, Austria E-mail: [email protected] 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 ADVATETM. 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 ADVATETM 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. BAX 855, ein PEGyliertes rekombinantes FVIIIProdukt mit verlängerter Halbwertszeit – Entwicklung sowie funktionelle und strukturelle Charakterisierung Hämostaseologie 2012; 32 (Suppl 1): S29–S38 received: April 10, 2012; accepted: July 2, 2012 Factor VIII (FVIII) is the key protein catalyst of the intrinsic coagulation pathway. Haemophilia A is an X-linked recessive, congenital bleeding disorder caused by deficient or defective coagulation FVIII leading to bleeding episodes in joints and certain tissues. FVIII concentrates (either plasma derived or recombinant) are used by haemophilia A patients to provide a hae- mostatic FVIII level sufficient to treat and prevent bleeding episodes. The Medical and Scientific Advisory Council (MASAC) of the National Hemophilia Foundation considers prophylactic therapy with FVIII to be the optimal treatment for haemophilia A patients without inhibitors (1, 2). A number of clinical studies have investigated the effects of pro- S29 phylactic regimens in patients diagnosed with severe (3–10) haemophilia A (11–13). These studies have so far convincingly demonstrated that when begun at a young age, prophylaxis reduces the number of bleeding episodes and prevents haemophilic arthropathy, (3) thereby decreasing the rate of disability and resulting in lower, long-term health care costs for patients (7, 12). On the opposite, the discontinuation of prophylactic therapy and suboptimal adherence to the prophylactic regimen compromises the clinical outcome leading to further arthropathy (5, 14). In addition, the early initiation of prophylaxis may have a protective effect against inhibitor development, the most serious complication associated with FVIII treatment (8, 9). Although the importance of adherence to prophylactic regimens for haemophilia A treatment is well established, frequency of infusions required, due to the short circulating FVIII half-life of 12 to 14 hours, still poses a challenge to patient compliance (15, 16). On average, 2–3 infusions a week are needed to maintain a FVIII level of at least 1% of normal to effectively prevent or reduce spontaneous bleeding episodes. The need for regular venous access, the required time and the increased caregiver support are significant burdens, causing patients, especially when moving from adolescence to adulthood, to switch to an on-demand, acute treatment regimen. A longer-acting FVIII concentrate would reduce the frequency of infusions, thus improving convenience and increasing the compliance with improved clinical outcomes. Modification with polyethylene glycol (PEGylation) is a method to improve the pharmacokinetic profile and prolong half-life and circulation of therapeutic proteins by increasing size and thus reduction Hämostaseologie 4a/2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. S30 P. L. Turecek et al.: BAX 855 Hämophilie/VWS of renal clearance (17, 18). The clearance of FVIII occurs through interaction with lowdensity lipoprotein receptor related protein (LRP), primarily in the liver (19–21). Also FVIII has a molecular weight of 280 kDa and is thus too large to be cleared by the kidney. Chemical modification of FVIII by PEG polymers might achieve a longeracting FVIII due to reduced binding of the PEG conjugated FVIII to LRP (22, 23). Methods and results Manufacturing process BAX 855 is a PEGylated form of Baxter’s recombinant FVIII (rFVIII) product based on the ADVATETM manufacturing process. The conjugation process for preparing BAX 855 uses proprietary stable PEGy- Tab. 1 lation from Nektar Therapeutics. Similar technology has been successfully employed for marketed and licensed PEGylated drug products and drugs in clinical use. The PEGylation process used to produce BAX 855 is based on the reaction of an activated PEG reagent with accessible amino groups on the FVIII protein. The reaction settings were optimised such that mainly the ε-amino groups of the lysine residues are modified. The manufacturing process for BAX 855 comprises several steps, including chromatographic purification and concentration of the conjugate leading to the preformulated bulk drug substance (BDS). The reaction and purification conditions had been selected to remove the excess of free PEG reagent as well as highly PEGylated rFVIII and non PEGylated rFVIII and resulted in a conjugate of an average molar ratio between PEG and protein of approxi- mately 2 (mol/mol). Formulation of the BDS includes a filling and lyophilization step to obtain the final drug product. The process is suited to manufacture BAX 855 in gram scale and showed a excellent batch to batch consistency. Biochemical and structural characterization BAX 855 was characterised by a number of analytical methods, focusing on the elucidation of the primary structure, posttranslational modifications, PEGylation site distribution and three-dimensional structure (씰Tab. 1). 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 Methods and tests used for biochemical, structural and functional characterization of the BAX 855 drug product assay physicochemical characterization method rationale primary structure peptide mapping, tryptic digest, LC-MS analysis of peptides determination of amino acid sequence, post translational modifications glycosylation analysis N-glycans enzymatic release, HPLC with fluorescence detection analysis of N-linked oligosaccharides protein composition and PEGylation analysis native RP-HPLC composition of the conjugate from free FVIII subunits and conjugated subunits thrombin digestion combined with two-dimensional RP-HPLC further differentiation of the FVIII fragments generated by thrombin digestion regarding the PEGylation of these fragments degree of PEGylation of thrombin fragments definition of the extent of PEGylation of individual thrombin fragments reducing SDS-PAGE and Western blot analysis banding pattern of BAX 855, identity by Western blot comparative 2D-PAGE analysis biological activity comparison of the changes in the spot pattern introduced by PEGylation higher order structure protein analysis by FTIR and DLS assessment of three-dimensional structure similarities functionality assessment of the kinetics of the assembly of FIXa-FVIII complex FIXa cofactor activity time course of thrombin-mediated activation determination of the rate of activation and inactivation of and inactivation BAX 855 by thrombin binding of BAX 855 to TGA (thrombin generation assay) overall haemostatic potency of BAX 855 APC-mediated FVIII and FVIIIa inactivation measurement of the rate of inactivation of untreated or thrombin-activated BAX 855 by activated protein C (APC). von Willbrand factor (VWF) kinetics of the binding to VWF the LRP receptor kinetics of the binding to LRP phospholipids kinetics of the binding to phospholipids Hämostaseologie 4a/2012 © Schattauer 2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. and detected with on-line electrospray ionization mass spectrometry. Assuming a mean modification degree of about 2 (mol PEG/mol FVIII) expected changes to the amino acid composition will not be detectable with the currently used technology (acid hydrolysis, HPLC separation of labeled amino acids). Nevertheless, peptide mapping with mass spectrometry analysis of the separated peptide fragments was performed to confirm the amino acid composition of critical segments of the rFVIII protein sequence (N-termini, C-termini, critical ligand binding and activation regions). Due to the specific PEGylation approach, no relevant decrease of peptide intensity was observed for a single site upon PEGylation. 씰Figure 1 Fig. 1 Structural integrity of BAX 855: tryptic peptide map: HPLC chromatograms show good consistency and only minimal differences between BAX 855 samples and ADVATETM. Fig. 2 BAX 855: PEG-distribution screening and PEGylation degree: HPLC separation of domain fragments generated by thrombin was used to compare several BAX 855 batches and ADVATETM. This method is sensitive for the extent of PEGylation. PEGylation degree measured by MALDI-TOF mass spectrometry (MS) was found consistent between preclinical and clinical BDS and FDP batches: 2.0 ± 0.3 (n = 15). PEGylation site analysis was performed by 2D thrombin digestion HPLC – MS. The results indicate good consistency between all batches. © Schattauer 2012 Hämostaseologie 4a/2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. S31 Hämophilie/VWS P. L. Turecek et al.: BAX 855 Hämophilie/VWS S32 P. L. Turecek et al.: BAX 855 shows the peptide maps after tryptic digestion of 4 batches of BAX 855 in comparison to 2 batches of unmodified rFVIII (ADVATETM). The peak pattern of unmodified rFVIII and BAX 855 is very similar demonstrating the structural integrity of the FVIII polypeptide chain that is retained after PEGylation. Due to the PEGylation approach, PEG is attached on different sites on FVIII in BAX 855. As a consequence of the low extent of modification at the individual sites, no changes in the peptide map can be observed when comparing to rFVIII in the ADVATETM product. The comparison of the four batches of BAX 855 shows good consistency of the chromatographic pattern indicating a highly reproducible PEGylation process that does not result in batch specific differences. The good reproducibility of the PEGylation process was recently confirmed by investigation of different batches of PEGylated rFVIII by a complimentary method. As described by Monetti et al. (24) two-dimensional electrophoresis using the 2D-DIGE imaging system showed a complete overlap of electrophoretic spots separated by the two-dimensional separ- Fig. 3 In vitro structural analysis of BAX 855: domain structure analysed by Western Blot: PEGylation resulted in a shift of the FVIII protein bands to higher MW regions for any of the FVIII domains investigated with different polyclonal and domain specific monoclonal anti-FVIII antibodies, reflecting a homogenous PEGylation of rFVIII. Fig. 4 Thrombin generation (TG) of BAX 855 and unmodified rFVIII (ADVATE in human FVIII deficient plasma a) Thrombin generation of a batch of rFVIII (ADVATETM) spiked into FVIII deficient plasma b) Thrombin generation of a batch of BAX 855 spiked into FVIII deficient plasma TM) c) Comparison of peak thrombin levels of unmodified rFVIII ADVATETM with preclinical and clinical batches of BAX 855 as a function of FVIII activity spiked into FVIII deficient plasma. BAX 855 corrects the impaired thrombin generation of a FVIII deficient plasma in a concentration-dependent manner. The response is comparable to the unmodified rFVIII (ADVATETM). Good consistency was demonstrated for several batches of BAX 855. Hämostaseologie 4a/2012 © Schattauer 2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. ation technique when two independent batches of PEG-rFVIII produced by the manufacturing method used for BAX 855 were compared. Carbohydrate analysis was performed by separating PNGaseF-released N-glycans using Dionex technology. Protein-bound oligosaccharides were determined by nor- mal 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 Fig. 5 FIXa cofactor activity of BAX 855 a) Assay principle of the FXa generation cofactor activity assay. The assay mimics the physiological tenase complex by incubation of FVIII with FIXa and FX. FXa activity is measured by a chromogenic substrate. The assay is performed with and without pre-activation by thrombin. b) Time-dependent FXa generation comparing unmodified rFVIII with preclinical and clinical batches of BAX 855 without pre-activation by thrombin. and detailed analysis of the consistency of PEGylation was investigated by limited proteolysis of BAX 855 with thrombin. 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 c) Time-dependent FXa generation comparing unmodified rFVIII with preclinical and clinical batches of BAX 855 after pre-activation by thrombin. Similar FXa generation characteristics were found for BAX 855 and unmodified rFVIII (ADVATETM). Good consistency for several BAX 855 batches from different production scales was demonstrated. Fig. 6 Thrombin susceptibility of BAX 855: Thrombin activation and inactivation of unmodified rFVIII in comparison to BAX 855 by 0.5 nmol/l thrombin (a) and 0.1 nmol/l thrombin (b). A similar FVIII inactivation profile of BAX 855 and unmodified rFVIII (ADVATETM) was seen. Activation and inactivation of FVIII were dependent on the thrombin concentration used. Good consistency was found for several BAX 855 batches from different production stages. © Schattauer 2012 Hämostaseologie 4a/2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. S33 Hämophilie/VWS P. L. Turecek et al.: BAX 855 Hämophilie/VWS S34 P. L. Turecek et al.: BAX 855 composition and PEGylation (씰Fig. 2). 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 (씰Fig. 3). 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 indicative for good consistency of the manufacturing process (data not shown). Fig. 7 APC-mediated FVIII inactivation of BAX 855: FVIII was incubated with activated protein C and FVIII activity was measured over time by the chromogenic FVIII assay. Samples without activated protein C were kept as process controls (dotted lines). ADVATETM and BAX 855 showed a time-dependent inactivation by activated human protein C. Inactivation rates of BAX 855 were similar to unmodified rFVIII. Functional characterization BAX 855 was functionally characterised in vitro and its features were compared with those of the unmodified parent rFVIII by all relevant methods that are currently available to assess the functionality of a FVIII molecule (씰Tab. 1). The overall haemostatic potency of BAX 855 was measured using a thrombin generation assay. Similar to unmodified rFVIII, BAX 855 corrected the impaired thrombin generation of the FVIII deficient plasma in a concentration-dependent manner (씰Fig. 4). The role of FVIII within the tenase complex was determined by measuring the kinetics of FXa generation with a FIXa-cofactor activity assay (25), using either untreated or thrombin activated BAX 855. Comparison of the kinetic parameters and the maximum FXa generated revealed similar characteristics for BAX 855 and unmodified rFVIII (씰Fig. 5). BAX 855 fully retained its ability to be activated and inactivated by thrombin (26) (씰Fig. 6). The susceptibility of BAX 855 to activated protein C (APC) inactivation (27, 28) was also similar for BAX 855 and unmodified rFVIII (씰Fig. 7). The binding affinities for VWF were similar for unmodified rFVIII and BAX 855 (씰Tab. 2). Also the binding characteristics of BAX 855 to synthetic phospholipids were comparable to unmodified rFVIII (29) (씰Fig. 8, 씰Tab. 2). The kinetics of association and dissociation and the association and dissociation con- stants were found similar for rFVIII ADVATETM and BAX 855. The results shown in 씰Table 2 indicated that all association and dissociation rates were within the same magnitude confirming that there was no relevant difference. In contrast, the binding capacity of BAX 855 to the low-density lipoprotein-receptor-related protein (LRP) clearance receptor was much lower than that of the unmodified rFVIII (씰Fig. 9). In vivo studies A comprehensive study program to investigate safety and efficacy of the BAX 855 study drug was performed with a large variety of Tab. 2 BAX 855 interaction with VWF and phospholipids In a surface plasmon resonance spectroscopic analysis using the Biacore instrument FVIII binding to rVWF was investigated. Kinetics of the VWF-FVIII complex formation (association and dissociation; ka and kd) and equilibrium dissociation constant (KD) were similar between BAX 855 and ADVATETM. For comparison, the normal range of KD for the VWF-FVIII interaction published previously was found between 0.2–0.9 nM (33). Data confirm the retained ability of BAX 855 to be stabilised by its natural chaperon protein VWF. interaction with rFVIII mean ka (1/Ms) kd (1/s) KA (1/M) KD* (nM) von Willebrand factor ADVATETM (starting material for PEGylation) n=4 3.45E+05 2.06E-04 1.67E+09 0.61 BAX 855 preclinic n=4 3.30E+05 2.58E-04 1.27E+09 0.82 clinic n=2 3.14E+05 2.48E-04 1.27E+09 0.81 ADVATETM (starting material for PEGylation) n=4 1.25E+06 1.54E-03 8.16E+08 1.2 BAX 855 preclinic n=3 1.74E+06 2.77E-03 6.10E+08 1.7 clinic n=5 2.03E+06 2.63E-03 7.31E+08 1.4 phospholipids Hämostaseologie 4a/2012 © Schattauer 2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. S35 Hämophilie/VWS P. L. Turecek et al.: BAX 855 Fig. 9 BAX 855 interaction with LRP clearance receptor: By surface plasmon spectroscopy binding of BAX 855 in comparison to unmodified rFVIII was determined both for preclinical and clinical batches of BAX 855. The unmodified and PEGylated FVIII products were measured for their interaction with immobilised LRP1 receptor. The results indicated a substantially reduced binding by about 50%. While binding capacity was reduced binding affinity remained similar for ADVATETM and BAX 855 (ADVATETM: KD 60.5 nmol/l vs. BAX 855 preclinical batches: KD 66.1 nmol/l and BAX 855 clinical batches: KD 63.7 nmol/l KD). Fig. 8 Assay principle to determine the interaction of BAX 855 with phospholipids: Test set-up was surface plasmon resonance spectroscopic analysis using the Biacore instrument. On a senor chip phospholipid vesicles consisting of phosphatidyl choline (PC) and phosphatidyl serine (PS) at a molar ratio of 60:40 were immobilised, then a FVIII containing sample was injected. The association of FVIII binding to phospholipid and the dissociation were determined and the kinetic parameters of FVIII-phospholipid interaction were calculated. test models (씰Tab. 3). This program took advantage of standard animal models, but also used FVIII deficient dogs and knockout mice. PK and haemostatic efficacy studies in different species display longer survival and longer action of BAX 855 compared to ADVATETM. A murine FVIII knock out model closely mimicking haemophilia A as described recently (30, 31) was used as one model. Mice were treated with two different batches of BAX 855 (lot A, lot B) and one batch of ADVATETM. The test items were administered as an intravenous bolus injection. Mice received 200 IU/kg body weight rFVIII of either BAX 855 or ADVATETM. Blood samples were obtained by cardiac puncture of mice at different time points from 5min-48h after dosing. Citrated plas- Tab. 3 Nonclinical in vivo safety studies for BAX 855 study species tail tip bleeding FVIII ko mouse carotid occlusion FVIII ko mouse thrombogenic potential rabbit cardiovascular effects (telemetry) macaque pharmacokinetics FVIII ko mouse single dose rat two doses ADME repeated dose toxicity macaque rat and single dose macaque (escalating dose) including TK rat macaque comparative immunogenicity in vitro human whole blood human plasma in vivo © Schattauer 2012 ● E17 FVIII k.o. – human MHC-class II (HLADR15) transgenic mice – mice on a Balb/c background – mice on a C57BL/6 background – human FVIII transgenic mice macaque Hämostaseologie 4a/2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. P. L. Turecek et al.: BAX 855 Hämophilie/VWS S36 Fig. 10 Pharmacokinetics of BAX 855 and rFVIII (ADVATETM) in FVIII ko mice Fig. 11 Pharmacokinetics of BAX 855 in macaques – single dose treatment: The graphs show dose adjusted PK curves adjusted to a dose of 100 IU FVIII/kg body weight. Fig. 12 Pharmacokinetics of BAX 855 in macaques – repeated dosing ma samples were analysed for FVIII activity (chromogenic assay). FVIII levels above the limit of quantification were measurable after ADVATETM administration up to 18 hours post infusion. In contrast, measurable FVIII levels were found even 32 hours after injection of BAX 855 indicating the longer halflife. Results are shown in 씰Figure 10. Area under the curve (AUC0-tlast, the area under the concentration vs. time curve from 0 to the last measured time point) and mean residence time (MRT) were measured to compare the pharmacokinetic behaviour of BAX 855 and ADVATETM. The AUC for ADVATETM was almost doubled for BAX 855: 0.0797 compared to ADVATETM: 0.0421. The mean residence time for BAX 855 was found at 7.9 hours compared to 4.9 hours for ADVATETM. BAX 855 in comparison to ADVATETM was also tested in another pharmacokinetic study in haemophilic dogs (Queen’s University, Kingston, Ontario; principle investigator Dr. D. Lillicarp). In this study a similar improvement of pharmacokinetic of BAX 855 compared to ADVATETM was found (data not shown). As a non-haemophilic model, macaques were used to determine the PK of BAX 855 in comparison to ADVATETM. Here BAX 855 was given in different doses between 350 and 480 IU FVIII/ kg in comparison to 320 to 870 IU / ADVATETM. The result is shown in 씰Figure 11 as a pool of data from three independent studies. The curves demonstrate FVIII levels above the limit of quantitation as determined by the chromogenic assay for 24 hours for ADVATETM but 72 hours for BAX 855. The limit of quantitation in this model is defined by the level of endogenous FVIII in coagulant normal animals. The pharmacokinetic analysis gave dose adjusted AUC (IU/ml : IU/kg) of BAX 855 ranging from 0.16 to 0.50 compared to ADVATETM (range 0.06 to 0.27). The mean residence time in the macaques for BAX 855 was calculated at 15.4 hours while ADVATETM had a mean residence time of 10.7 hours. Macaques were also used to test the pharmacokinetics of BAX 855 in comparison to ADVATETM upon repeated dosing. BAX 855 was given at a dose of 370 IU/kg bodyweight on day 1 and day 8, while rFVIII ADVATETM was dosed at a dose of 317 IU/kg on day 1, 3 and 5. Hämostaseologie 4a/2012 © Schattauer 2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. 씰Figure 12 shows the FVIII plasma levels measurable above the FVIII background of the coagulant normal macaques. The curves indicate that one dose of BAX 855 would replace 3 doses of ADVATETM under the assumption that at day 8 the same trough level would be reached by both dosing regimes. The detailed PK analysis showed an improvement of the mean residence time of about 1.8-fold compared to ADVATETM at the first dose of BAX 855 and 2.1-fold at the second dose of BAX 855. BAX 855 in comparison to ADVATETM was also tested in two efficacy models of haemophilia A. Efficacy of BAX 855 was again evaluated in haemophilia A mice using a tailtip bleeding model (TTBM) and a carotid occlusion model (COM). The COM is based and was further developed from a vascular injury model as established by Furie and Furie (32). Both models used the same strain of FVIII knock-out mice as described above. The first model is the classic tail tip bleeding model. BAX 855 was tested at a dose of 200IU/kg rFVIII. ADVATETM served as the active control item, and buffer as the negative control item. Mice received intravenous treatment with BAX 855 or ADVATETM 12–40 h or with buffer 5–15min before the experiment. In the TTBM (n = 16) the tip of the tail was cut off and total blood loss [mg] was assessed over 60 min. In the COM study (n = 10) the left carotid artery was exposed and the endothelium was denuded by topical application of FeCl3. Time to occlusion [min] was assessed. Buffer-treated animals had a median blood loss of 951 mg in the TTBM and did not show vessel occlusion within 30 min in the COM. In the TTBM, a prophylactic effect of ADVATETM could be observed for up to 18 h (121 mg). Prophylactic efficacy after treatment with BAX 855 could be observed for up to 40h (30 h: 73 mg; 40 h: 436 mg) (씰Fig. 13). In the COM, treatment with ADVATETM 12 h before the experiment shortened the time to occlusion to 3.8 min. With BAX 855, a similar efficacy could be observed for up to 24 h after administration (5.2 min) also indicating an approx. 2-fold longer-lasting efficacy of BAX 855 in comparison to unmodified rFVIII (씰Fig. 14). Fig. 13 Primary pharmacodynamics of BAX 855 compared to ADVATETM: tail tip bleed in FVIII ko mice Fig. 14 Primary pharmacodynamics of BAX 855 compared to ADVATETM in carotid occlusion model in FVIII ko mice Conclusion Baxter has developed a long-acting FVIII concentrate, PEGylated recombinant human factor VIII (BAX 855). BAX 855 is a novel long-acting FVIII product using state of the art PEGylation technology. The rFVIII derivative can be manufactured reproducibly without changes to the protein structure characteristic for a fully functional FVIII molecule. Consequently; the functional properties of BAX 855 are fully retained, indicating that PEGylation as performed by Baxter does not have any impact on the haemostatic function of rFVIII in vitro and in vivo. Functionality and im- S37 Hämophilie/VWS P. L. Turecek et al.: BAX 855 provement of pharmacodynamic and pharmacokinetic properties was demonstrated in relevant animal models for human haemophilia A. Based on the favourable pre-clinical pharmacokinetic and efficacy data and the lack of safety concerns in animal studies (data not shown) a phase 1 clinical study in patients with haemophilia A was initiated. Acknowledgements We thank Manuela Leibrecht for editing the manuscript, and Sabine Mandl for preparing figures. Conflict of interest The authors are full time employees of either Baxter Innovations GmbH or Nektar Therapeutics. © Schattauer 2012 Hämostaseologie 4a/2012 Downloaded from www.haemostaseologie-online.com on 2017-06-17 | IP: 88.99.165.207 For personal or educational use only. No other uses without permission. All rights reserved. S38 P. L. Turecek et al.: BAX 855 Hämophilie/VWS References 1. 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