Download BAX 855, a PEGylated rFVIII product with prolonged half-life

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
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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
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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.
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P. L. Turecek et al.: BAX 855
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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.
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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.
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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
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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
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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.
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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
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S38
P. L. Turecek et al.: BAX 855
Hämophilie/VWS
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