Keywords
Recombinant von Willebrand factor - von Willebrand disease - hemophilia - substitution
therapy
Von Willebrand factor (VWF) is a multimeric adhesive glycoprotein which has a dual
function in hemostasis. It
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mediates platelet adhesion at sites of vascular injury, which is necessary for primary
hemostasis, and
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stabilizes factor VIII in the circulation.
The pathophysiological significance of these different biological functions is evident
in von Willebrand's disease (VWD), the most common hemorrhagic disorder, affecting
1% of the population. Approximately 1% of patients with VWD have severe VWF deficiency
resulting in defective platelet adhesion, secondary factor VIII (FVIII) deficiency,
and a prolonged bleeding time. These patients can only be treated effectively with
VWF concentrates [1], [2], [3], [4], [5].
A variety of plasma-derived concentrates (with or without FVIII) are available for
treating VWD. Development of a recombinant VWF (rVWF) manufactured and formulated
in the absence of animal or human plasma proteins will increase the choice of treatment
options, but also make VWD treatment independent of blood supply.
Methods
Plasma-derived VWF
Plasma-derived VWF (pdVWF), required as a control article for biochemical analysis,
functional characterization and in vivo studies was purified in-house from the cryoprecipitate
of pooled human plasma by a sequence of conventional chromatography steps and was
formulated as for rVWF. Two commercially available pdVWF products, Haemate HS (ZLB
Behring, Germany; lot 56966411A) and Wilfactin (LFB, Les Ulis, France; lot 33103–22238103)
were used in control experiments.
Biochemical methods
VWF : Ag ELISA was performed with a commercial polyclonal rabbit anti-human VWF antibody
(Dako, Glostrup, Denmark) using the single incubation multilayer immune technique
(SIMIT). VWF ristocetin cofactor (VWF : RCo) activity was measured with the BCS (Behring
Coagulation System) analyzer (Siemens, Marburg, Germany) according to the manufacturer’s
instructions using lyophilized von Willebrand reagent containing stabilized platelets
and ristocetin A (Siemens). The FVIII-binding capacity of VWF (VWF : FVIIIB) was measured
by an ELISA combined with a chromogenic assay (ECA) and by a Biacore 3000 system (Biacore,
Uppsala, Sweden) using a VWF-coated CM5 chip. SDS-polyacrylamide gel electrophoresis
(SDS-PAGE) was performed under reducing conditions using gradient (3–8%) Tris-acetate
gels followed by Coomassie staining. VWF multimer analysis was performed using low-
and high-resolution horizontal SDSagarose gel electrophoresis followed by immunostaining
with a polyclonal rabbit
anti-human VWF antibody [6], [7]. Carbohydrate analysis was performed by separating PNGaseF-released N-glycans using
Dionex technology. VWF-mediated platelet adhesion to collagen under shear stress was
determined using a parallel-plate perfusion chamber coated with fibrillar collagen
type 1.
Studies in VWF-deficient mice
Pharmacokinetic analysis was performed in VWF-deficient mice [8] by i.v. application of 200 U VWF : Ag/kg. Citrated plasma from five animals was
collected at each time point by heart puncture and analyzed for VWF : Ag (ELISA) and
FVIII activity (chromogenic assay). The half-life of VWF was calculated by a single
exponential fit between data points from 3 to 24 hours. The area under the curve for
the rise in endogenous FVIII was calculated and included baseline mouse FVIII.
Results
Up- and downstream processing
Recombinant VWF is co-expressed with recombinant FVIII in CHO cells. The material
obtained from column flow-through of the FVIII capture step, containing pro-VWF, is
the starting material for rVWF. It is an intermediate of the manufacturing process
of the commercial rFVIII product Advate [9]. CHO cells produce partially processed rVWF. Therefore to obtain fully processed,
mature rVWF, rVWF is exposed to recombinant CHO-cell-derived furin to remove the VWF
pro-peptide [10]. [Fig. 1a] depicts the characteristic molecular forms of CHO-cell expressed rVWF that contain
multimer species consisting of combinations of the homo- and hetero-polymers of mature
and pro-VWF. For example, dimeric forms alternatively consist of two mature monomers,
one mature and one pro-VWF monomer or two pro-VWF monomers, leading to the appearance
of a triplet structure. This structure had been seen with previous rVWF preparations
that were
obtained from CHO cells [11]. As previously described, exposure to rFurin in vitro converts the heterogeneous
multimer mixture of incompletely processed pro-VWF into one of highly homogeneous,
structurally intact multimers [12]. This maturation process is functionally important to FVIII binding as the FVIII-binding
site is located in the D'D3 domain at the N-terminus of the mature subunit [13]. In a surface plasmon resonance spectroscopic analysis using the Biacore instrument,
FVIII binding to rVWF was investigated and purified VWF, as expressed by CHO cells,
was compared with furin-matured rVWF. The FVIII-binding characteristics of furin-matured
rVWF were very similar to those of plasma-derived VWF while pro-rVWF preparations
showed a reduced FVIII-binding capacity [Fig. 1b].
Fig. 1
a) Pro-rVWF contains hetero- and homo-polymers of pro- and mature VWF (left lane), purified
rVWF is fully processed (right lane)
b) FVIII binding of rVWF is similar to that of pdVWF and several-fold higher than that
of pro-rVWF
Fermentation for both rVWF and furin and downstream processing are performed under
serum-free and protein-free conditions. After rVWF is captured by an ion-exchange
column it is fully processed to mature VWF by recombinant furin. After DNA removal,
rVWF is solvent-detergent (SD) treated and purified by two further chromatography
steps. The rVWF resulting from the downstream process has a purity of > 99% and is
thus almost pure VWF.
Recombinant VWF is formulated in a protein-free buffer. The goal of the product development
was to provide rVWF in an optimized ratio of VWF : RCo to FVIII : C to treat patients
with severe von Willebrand disease (type 3). Therefore, the two lyophilized drug products,
rVWF and rFVIII (Advate), are provided in individual vials, reconstituted separately
and finally combined to provide a complex of rVWF and rFVIII. The schematic manufacturing
process of rVWF is shown in [Fig. 2]
Structure and function
The purified rVWF final drug product was extensively characterized in a number of
preclinical studies with an array of state of the art biochemical and functional tests
for characterizing large glycoproteins. The tests are listed in [Tab. 1]. Some of the major findings from these studies are described.
The chromatographic purification in the absence of any adjuvant protein and the final
formulation results in rVWF which is a single band protein with a purity that comes
close to homogeneity. The level of process-related protein impurities and host cell
protein is well below the limits that are usually expected for recombinant proteins
derived from CHO cells [Fig. 3a]a. A major characteristic of furin-matured rVWF is the homogeneity of the multimer
distribution. VWF expressed by CHO cells has never been exposed to ADAMTS13. Multimer
analysis showed the presence of ultra-high molecular weight multimers [Fig. 3b]. High-resolution multimer analysis did not reveal typical satellite bands in rVWF
[Fig. 3c] which is evidence of rVWF’s non-exposure to limited proteolysis by ADAMTS13 similar
to endothelial or platelet-derived human VWF. When
rVWF is incubated with ADAMTS13, satellite bands are formed simultaneously with the
disappearance of high molecular weight multimers, resulting in a multimeric pattern
similar to pdVWF [14]. The FVIII-binding capacity and affinity of rVWF is comparable to that of VWF in
plasma.
Fig. 2 Production process of rVWF and rFVIII
Tab. 1 Methods and tests for biochemical, structural and functional characterization of
the rVWF drug product
|
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electron microscopy
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protein determination
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molecular integrity and protein composition by electrophoretic methods
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multimeric structure: SDS-agarose gel systems
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peptide mapping (primary sequence)
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RP-HPLC analysis (fragmentation)
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N-terminal sequence analysis
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carbohydrate pattern
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N- and O-glycans
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monosaccharides
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sialic acids
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potency
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susceptibility to ADAMTS13 cleavage
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FVIII binding capacity and affinity: static and under flow
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binding to platelets under shear forces
|
Carbohydrate analysis showed an intact glycosylation pattern typical for a fully glycosylated
human protein expressed in CHO cells. Similar glycosylation patterns were found for
rVWF and pdVWF, which was investigated as a control. Recombinant VWF has a slightly
higher proportion of sialilated tri- and tetra-antennary oligosaccharides than pdVWF,
indicative of intact N-glycosides. The main glycan structure found was a biantennary
di-sialilated N-glycan as also identified in pdVWF ([Fig. 4]). Accordingly, a significantly higher content of sialic acid was found on rVWF than
on plasma VWF by monosaccharide analysis. The main structure of O-glycans on rVWF
is the sialilated T-antigen as present in plasma (data not shown). There was no evidence
of ABO antigen structures on rVWF, which indicates that the glycosylation pattern
of rVWF is similar to that of platelet-derived rVWF (data not shown).
The specific activity of rVWF is higher than that of commercial plasma-derived VWF-FVIII
complex concentrates, reflecting the high purity of the product and the absence of
stabilizing proteins co-purified or added with plasma-derived VWF-containing products.
The plasma-derived VWF that was specifically prepared as a control article for rVWF
had a purity comparable to rVWF and also appeared as a single band in reducing SDS-PAGE
[Fig. 3a]. When comparing VWF : RCo with VWF : Ag, the specific activity was higher in rVWF
than in pdVWF. There was no difference between the highly purified plasma-derived
VWF control article and a commercial plasma-derived VWF-FVIII complex concentrate.
This reflects the high molecular weight multimers in rVWF, which bring its specific
activity close to the theoretical value of one RCo U per VWF : Ag [15], [16]. The specific activity that was
found for pdVWF was in line with published results for a commercial pdVWF-FVIII complex
concentrate, although a somewhat higher ratio of VWF : RCo to VWF : Ag was found previously
[17], [18]. Trace amounts of FVIII remain in the rVWF product due to the fact that it was purified
from a cell culture supernatant that contains both FVIII and VWF. The higher FVIII
content of the plasma-derived VWF-FVIII concentrate was found in the expected range.
FVIII-binding capacity could only be measured in rVWF and the highly purified pdVWF
control preparation, which both contain almost no FVIII ([Tab. 2]). Recombinant VWF can bind to collagen under static conditions and also promote
platelet adhesion to immobilized collagen under shear stress. The results are shown
in [Figure 5]. Five laboratory batches of rVWF were compared with three pdVWF products, two commercial
VWF-FVIII complex
concentrates and one highly purified VWF, used as a control preparation for the preclinical
study program. No relevant differences were seen in VWF-mediated platelet adhesion
between rVWF and pdVWF products.
In vivo characterization
A comprehensive study program to investigate safety and efficacy of the rVWF study
drug was performed with a large variety of test models. This program took advantage
of standard animal models, but also used von Willebrand-deficient pigs, dogs and knockout
mice and ADAMTS13 k.o. mice. The results from the pharmacokinetic study in VWF-deficient
mice are shown in [Figure 6]. When two batches of rVWF were infused into VWF knock-out mice, they showed a slightly
longer half-life than two pdVWF products (Haemate HS and the highly purified pdVWF
control preparation). When measuring FVIII levels in the VWF knock-out mice, rVWF
showed the characteristic secondary rise in endogenous FVIII levels in the VWF-deficient
animals demonstrating that human rVWF is able to stabilize endogenous murine FVIII.
A rise was also seen with the highly purified pdVWF control preparation but was difficult
to interpret when the plasma-derived VWF-FVIII concentrate was infused due to the
high amounts of
FVIII that were given simultaneously with VWF contained in the complex. The slightly
higher FVIII levels that were seen up to 24 hours after application in the animals
treated with rVWF while animals treated with pdVWF had returned to their starting
levels of FVIII also indicated the somewhat longer survival of rVWF in these animals.
Discussion, conclusion
Replacement therapy in patients with von Willebrand disease is currently limited to
plasma FVIII concentrates that also contain VWF, or purified VWF derived from human
plasma [19], [20]. So far no recombinant protein therapeutic containing VWF has been made available
for treatment. We used the well-established CHO cell line, which co-expresses rFVIII
and rVWF as a chaperon that facilitates and stabilizes secretion of FVIII and the
cell culture supernatant obtained from it as a source for the rVWF product. An intermediate
fraction from the rFVIII-PFM (Advate) [21], [22] manufacturing process was used as a starting material to manufacture a rVWF drug
product.
CHO cells do not sufficiently process pro-VWF into mature VWF and pro-peptide. Previous
attempts to obtain recombinant mature VWF used CHO cell clones that co-expressed recombinant
furin [23]. The process intermediate from the rFVIII-PFM manufacturing process contains a mixture
of pro- and mature VWF, therefore, we developed an in vitro processing procedure with
recombinant furin as the first downstream manufacturing step to obtain mature rVWF
[12], [24]. Following subsequent purification steps this drug product can be used in conjunction
with the rFVIII in Advate as a complex to treat patients with severe von Willebrand’s
disease (type 3) which is currently being tested in clinical trials.
Fig. 3 Electrophoresis of rVWF, proteins stained by Coomassie blue and agarose gel:
a) SDS-PAGE under reducing conditions reveals the molecular integrity and protein composition
of rVWF (experimental batches) compared with plasma-derived VWF (highly purified in-house
preparation and two commercially available VWF-FVIII complex concentrates): Recombinant
VWF shows single bands. Their migration is identical to that of the VWF bands in highly
purified plasma-derived VWF. Commercial plasma-derived VWF products contain other
proteins including albumin for stabilization.
b) Agarose gel (1%): rVWF contains high and ultra-large molecular weight multimers not
present in human plasma and pdVWF-FVIII concentrates (Haemate HS). rVWF batches from
large scale manufacturing are shown.
c) Agarose gel (2.5%): The absence of ADAMTS13-mediated proteolytic fragments is shown
by high resolution agarose electrophoresis.
Recombinant VWF is a homogenous recombinant protein with an intact multimer pattern
which it retains because it is never exposed to proteases that are able to degrade
it. In particular, rVWF is never exposed to ADAMTS13, which is the enzyme that by
limited proteolysis generates the characteristic degradation products seen as satellite
bands in agarose electrophoresis [25]. This makes rVWF similar to endothelial or platelet-derived human VWF. However,
the satellite bands are formed simultaneously with the disappearance of high molecular
weight multimers upon incubation with ADAMTS13 in the presence of urea under denaturating
conditions. The carbohydrate pattern of rVWF is similar to that of pdVWF with respect
to terminal sialic acid, indicating an intact glycosylation. Recombinant proteins
derived from CHO cells with their typical glycosylation pattern have been in therapeutic
use for the last two decades without any indication that the
glycosylation pattern impacts safety and efficacy of such recombinant proteins.
Fig. 4 Comparison of N-glycan pattern: rVWF (lower panel) and highly purified pdVWF (upper
panel) were analyzed for their N-terminal glycans by anionexchange chromatography.
rVWF shows a higher proportion of sialilated tri-and tetra-antennary oligosaccharides.
Tab. 2 Product characterization of rVWF compared with commercial plasma-derived VWF-FVIII
concentrate (Haemate HS) and a highly purified pdVWF produced as a reference preparation
for rVWF; for comparison previoulsy published results on Haemate: *Lethagen et al.
[17], n presumably = 1; **Budde et al. [18], n = 3; values in means ± SD where applicable; n.a.: not applicable
|
Product
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VWF:Ag protein (IU/mg)
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VWF:RCo protein (IU/mg)
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VWF:RCo VWF:Ag (IU/IU)
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FVIII:c VWF:RCo (IU/IU)
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VWF:FVIII binding capacity (%)
|
|
rVWF (n = 5)
|
114 ± 5
|
123 ± 24
|
1.09 ± 0.26
|
0.006 ± 0.003
|
108 ± 19
|
|
pdVWF (n = 1)
|
118
|
59
|
0.50
|
0.070
|
109
|
|
Haemate
|
17.9 ± 4.7
n = 12
|
8.0 ± 1.7
n = 7
|
0.51 ± 0.10
n = 12
|
0.67 ± 0.13
n = 13
|
n.a.
|
|
Haemate published data
|
16.5 *
|
15.0 *
|
0.91 *
0.84 **
|
0.49 *
0.35 **
|
----
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The specific activity, the calculated ratio between rVWF antigen content and RCo activity,
is close to 1 or even above, whereas the plasma-derived sample has a much lower value.
This is in accordance with the high multimer content of the rVWF preparation compared
with normal plasma VWF. The functional activity of rVWF shown by the binding capacity
for FVIII and VWF : RCo activity was found to be similar to or higher than that of
the in-house purified or commercial pdVWF products. The primary hemostatic activity
of rVWF was demonstrated by VWF-induced platelet adhesion under high shear stress.
Pharmacokinetics of rVWF and pdVWF are different in VWF-deficient mice: Recombinant
VWF has a slightly longer half-life than the pdVWF Haemate HS and a highly purified
pdVWF control. This is consistent with previous observations for another rVWF product
of substantially longer half-lives in dogs and pigs with severe von Willebrand deficiencies.
However, the rVWF product used in the
previous studies also contained heteropolymers of pro- and mature VWF and therefore
is not directly comparable to the current product [26], [27], [28], [29], [30], [31]. It remains to be demonstrated if similar differences will be seen in primate studies
and in the clinic. Preparations of rVWF stabilized endogenous murine FVIII in the
circulation of VWF-deficient mice. This secondary rise of endogenous FVIII levels
indicates that a clinically and therapeutically important feature of VWF has been
retained in the recombinant molecule.
Fig. 5 Concentration dependency of platelet adhesion to collagen fibrils under shear stress
(860 s-1) in the presence of increasing concentrations of VWF antigen
Fig. 6 Pharmacokinetics in VWF-deficient mice
a) rVWF and pdVWF
b) factor VIII levels over time upon i. v. application of rVWF and pdVWF
The similarity of rVWF’s in vitro and in vivo properties to those of pdVWF facilitated
the decision to develop the first recombinant entity that has the potential to treat
patients with VWD.
