Blood Reviews (1997) 11, 169-177

0 1997 Harcourt Brace & Co. Ltd

Haemostasis and thrombosis

Recombinant coagulation factors

l? H. Roddie, C. A. Ludlam

The advent of technology that has allowed production of recombinant proteins on an industrial scale
has revolutionized haemophilia care. Recombinant coagulation factors, as opposed to their plasma-
derived counterparts, have a very low risk for transmission of infectious agents and their use should
eradicate the threat of infection from viruses such as hepatitis C and the human immunodeficiency
virus. This review outlines the manufacturing processesinvolved in the production of the
recombinant coagulation factors and the trials to date that have beenperformed to establish their
safety and efficacy.We also discusssome of the issuesinvolved in the change to useof recombinant
coagulation factors, such as viral safety and potential immunogenicity.

The development of recombinant coagulation factors
has been a major advance in haemophilia care promis-
ing almost complete safety of patients from the risk of
transfusion transmitted infections. Although plasma-
derived coagulation factor concentrates have a proven
record of efficacy, prior to the current viral inactiva-
tion procedures employed in their manufacture and
less rigorous exclusion of potentially infected donors
from the plasma pool, many haemophiliacs became
infected with human immunodeficiency virus (HIV)
and hepatitis C virus in the 1980s. At present,
although plasma-derived concentrates appear safe
from the point of view of HIV infection, concern
remains over the possibility of other viruses being
transmitted. This is because even with the dual viral
inactivation steps of solvent/detergent treatment and
heat treatment, human parvovirus B19 remains
detectable in concentrates and able to infect recipients.
In addition, hepatitis C virus transmission has been
P. H. Roddie MRCPath, C. A. Ludlam FRCPath, Department
Haematology, Royal Infirmary of Edinburgh, Lauriston Place,
Edinburgh EH3 9YW, UK. Tel: +44 (0)131 536 2122;
Fax: +44 (0)131 536 2145.
Correspondence to: Dr C. A. Ludlam
reported following the use of a monoclonal antibody-
purified plasma-derived factor VIII concentrate. At
the present time, two recombinant factor VIII prepa-
rations are licensed for clinical use in the treatment of
haemophilia A: Kogenate (Bayer) and Recombinate
(BaxtetYHyland). During clinical trials they have been
shown to be effective and safe with comparable phar-
macokinetic profiles to plasma-derived factor VIII
concentrates. A higher incidence of inhibitor develop-
ment has been found in comparison with retro-
spective trials of plasma-derived factor VIII, but
many of these inhibitors were low level and trans-
itory. A further recombinant factor VIII product,
Refacto (Pharmacia & Upjohn), that differs from the
previous two in that it lacks the B domain of the fac-
tor VIII molecule has reached the stage of phase
II/III trials and is not currently licensed. A recombi-
nant factor IX, BeneFix (Genetics Institute), has also
been manufactured and following clinical trials has
recently obtained its license for clinical use in the
USA. Recombinant FVIIa, NovoSeven (Novo
Nordisk), is a recombinant-derived factor VII that
of
undergoes activation to factor VIIa during the purifi-
cation process. It is licensed in Europe for use in the
treatment of patients with haemophilia A and B and
inhibitors and for patients with acquired haemophilia.

169
170 Blood Revienx
(B) 9okLhhcavychain .
H2N
Fig. 1 Factor VIII cDNA and protein structure. (A) cDNA for FVIII gene. (B) FVIII molecule in its inactive form circulates as a
heterodimer, the B domain being cleaved during proteolytic processing and activation.
MOLECULAR BIOLOGY OF FVIII
The gene for factor VIII was first cloned successfully
in 1984.l.’ At that time it was the largest gene to be
cloned with a sequence spanning almost 186 Kb and
comprising approximately 0.1% of the X chromo-
some. Factor VIII messenger RNA encodes a precur-
sor protein of 2351 amino acids whilst the mature
factor VIII protein is 2332 amino acids long. The
amino acid sequence shows an organization of three
structural domains that occur in the order
Al:A2:B:A3:Cl:C2 (Fig. 1). Factor VIII circulates in
the blood in the form of a heterodimer and is stabi-
lized by its interaction with a 50-fold excess of von
Willebrand factor. The B domain is cleaved during
proteolytic processing and activation and its function,
if any, is unknown.3
RECOMBINANT FACTOR VIII
Kogenate
Manufacturing process
rFVII1 (Kogenate) is produced by baby hamster kid-
ney (BHK) cells which have been transfected with the
cDNA for the human factor VIII gene. The cells are
cultured in bovine serum free medium and give high
yield expression of factor VIII with no requirement
for von Willebrand factor to stabilize the protein.
Fermentation takes place in deep tank fermenters
using a continuous, high cell density cultivation pro-
cess.4The recombinant factor VIII raw product from
the fermentation is processed through a multi-step
purification system resulting in a final product of high
purity and specific activity (Fig. 2). The purification
process includes ion exchange and immunoaffinity
ThmmVm cleavage sites
+
chromatography steps. Impurities which could con-
taminate the product, such as culture medium compo-
nents and murine immunoglobulin, are effectively
removed during the purification process. Within the
manufacturing process as a whole there is a three-
stage viral safety programme which includes: (i) char-
acterization of the cell bank to ensure it is virus free;
(ii) scrutiny of incoming biological raw materials and
(iii) process validation for removal and inactivation of
viruses. This includes heat treatment at 40°C for 8 h.
Human serum albumin is added as a stabilizer to the
purified product which is then filtered, bottled and
lyophilized.
Clinical trials
Following the successful completion of preclinical
trials in a variety of animal species to demonstrate
safety and efficacy and to assess its pharmacokinetic
profile, the first clinical trial of Kogenate in patients
with haemophilia A began in 1988. This was con-
ducted in three parts. The first part, corresponding to
a phase I trial, was concerned primarily with the
pharmacokinetics of Kogenate. The next two parts
addressed the issues of safety and efficacy.’ Phase I
testing employed a cross-over study design in 17
patients to compare the pharmacokinetics of
Kogenate with the plasma-derived factor VIII, Koate-
HS. No significant differences were observed. This
same group then continued with Kogenate given as
prophylaxis three times weekly over a 6-month period.
During this time there were no adverse events
reported and no factor VIII inhibitors or antibodies
to mouse or hamster proteins developed. Further
proof of safety and efficacy was sought in the phase
II/III style sections of the trial. Fifty-six previously
treated patients who had completed at least 5 months

Harvest
Cell separation
I exposure days. In the patients with severe haemophilia
Anion exchange chromatography
Virus inactivation
lmmunoabsorption chromatogmphy
Size exclusion chromatography
Anion exchange chromatography
Buffer exchange formdation
Sterile filtration
Lyophilization
Fig. 2 The purification process of recombinant
of home treatment with Kogenate were evaluable for
the purposes of this trial. The calculated mean annual
consumption of factor VIII was 64 000 units per
person. Eighty-two per cent of bleeding episodes
responded to a single infusion of recombinant factor
VIII whilst 12.2% of bleeding episodes required two
infusions. Adverse reactions were predominately mild
and included light-headedness, a metallic taste in the
mouth and localized rashes or discomfort at the infu-
sion site. The overall incidence of these was 0.75”/0.
Kogenate was also used to promote haemostasis dur-
ing major surgery and for the inpatient treatment of
serious haemorrhage on 32 occasions in 26 patients.
Haemostasis was judged to be excellent in all of these
cases. The above study demonstrated that Kogenate
was safe and efficacious but further investigation was
necessary to determine the incidence of inhibitor for-
mation. This was performed by means of a prospec-
tive trial in previously untreated patients. This is the
preferred population for this type of study as previ-
ously treated patients have already been exposed to
foreign protein from factor concentrates which may
have induced some degree of immune tolerance and
therefore could give a falsely low rate of inhibitor
development. In the Kogenate previously untreated
patient (PUP) study, following the first infusion of
Kogenate patients were monitored for the develop-
ment of factor VIII inhibitors by means of a Bethesda
assay performed at 3-monthly intervals.” Of the 101
patients who participated in the trial 64 had severe, 16
moderate and 21 mild haemophilia A. Mean follow-up
Recombinant coagulation factors 17 1
was of the duration of 4.2 years. A factor VIII
inhibitor level of > 0.5 BU was considered significant.
Overall incidence of inhibitor development in all
patients was 19.8% occurring at a median of 9
the inhibitor incidence was 28% (1 S/64). Of those who
developed an inhibitor just over half were high titre
(> 10 BU). Five of this group were treated with an
immune tolerance induction protocol using Kogenate
with good response. In the nine patients with low level
inhibitors all were continued with ‘on-demand’ treat-
ment with Kogenate and seven have spontaneously
lost their inhibitor whilst two continue to have low
level inhibitors.’
Recombinate
Munz4f&turing process
The manufacture of Recombinate differs from that for
Kogenate in that the cell lines used to produce the fac-
tor VIII are Chinese hamster ovary (CHO) cells and
these are transfected with cDNA for both human fac-
FVIII (Kogenate).
tor VIII and von Willebrand factor.8 The transfection
with cDNA for von Willebrand factor is necessary to
improve the yield and to stabilize the recombinant
factor VIII produced by the CHO cells. The von
Willebrand factor is removed during the purification
process. Within the fermentor cell growth takes place
through a ‘batch-refeed’ process as opposed to the
continuous high cell density cell cultivation employed
in the manufacture of Kogenate. Purification of the
recombinant factor VIII is performed along a number
of stages which include monoclonal immunoaffinity
column and ion-exchange separation steps. A similar
viral safety programme is undertaken to that used in
the manufacture of Kogenate except that there is no
viral inactivation step.
Cliniad trials
The first pilot study in patients of Recombinate was
performed in 1987 whereby its pharmacokinetic profile
was compared with that of a plasma-derived factor
VIII, Haemofil-T, in two haemophiliacs.’ Both patients
exhibited recovery and clearance curves for
Recombinate which were comparable with those of the
plasma-derived product. They were then continued on
home treatment with Recombinate and during this
period had excellent to good responses for the treat-
ment of bleeding episodes with no adverse events
reported. A phase II/III trial was then performed to
gather more pharmacokinetic data and to assesslong-
term safety and efficacy of the product. Sixty-six
previously treated patients with severe haemophilia A
were enrolled in a prospective trial and of these
172 Blood Reviews
55 completed at least 18 months of observation whilst
receiving treatment with Recombinate.” Good to excel-
lent haemostasis was achieved in the treatment of
bleeding episodes and no significant adverse events
occurred. Following on from this was a trial to assess
inhibitor formation in previously untreated patients.”
The rate of inhibitor development in 73 patients with
severe haemophilia A who had received at least one
infusion of recombinant factor VIII was 3 1.5% (23/73).
Five inhibitors were of high titre (BU > 10) and three
patients were treated with immune tolerance induction
regimes with Recombinate. Seventy-six per cent of all
inhibitors were identified within the first 10 exposure
days. In the 15 patients with low responding inhibitors
(0.5-10 BU) nine spontaneously lost their inhibitor and
all were being continued on Recombinate. This trial
also confirmed the findings of earlier studies in that
Recombinate appeared effective in treating bleeding
episodes and was associated with a very low incidence
of adverse events.
B domain deleted recombinant factor VIII
Studies on the function of factor VIII molecules pro-
duced by deleting various regions of the FVIII cDNA
showed that lack of the B domain did not affect the
functional activity of the factor VIII product. Also,
loss of the B domain led to enhanced expression of
factor VIII which was increased up to 5-fold com-
pared with the unaltered factor VIII cDNA. The fac-
tor VIII molecule so produced appears less prone to
proteolytic degradation and therefore no human albu-
min is needed for its stabilization.
Refacto
Manufacturing process
There is currently only one recombinant factor VIII
lacking the B domain, Refacto, that has been pro-
duced and is undergoing clinical trials. Its production
is achieved by transfecting CHO cells with factor VIII
cDNA lacking the majority of the gene coding for the
B domain. In the gene construct the major part of the
region is a sequence Ser-Glyn-Asn (SQN); the SQ in
the designated name rFVII1 SQ originates from the
amino acid sequence at the fusion site.” The rFVIII-
SQ is purified from the conditioned medium of CHO
cells by a series of steps which include immunoaffinity
chromatography, ion exchange chromatography and
size exclusion chromatography. A solvent/detergent
viral inactivation stage is included within the purifica-
tion process. The final product does not contain any
von Willebrand factor and albumin is not required for
its stabilization. Its specific activity is 15 000 iu/mg
protein.
Clinical trials
Clinical trials of Refacto commenced in 1993. In a
phase I/II study 36 previously treated patients with
severe haemophilia A participated in a trial where
they were split into three groups of 12 patients. One
group took part in a cross-over study to compare the
pharmacokinetic profile of Refacto with that of a
plasma-derived factor VIII, Octonativ-M.‘3 Most
pharmacokinetic parameters were similar for Refacto
and the plasma-derived factor VIII except that the
volume of distribution and clearance were slightly but
significantly higher after administration of Refacto.
This did not lead, however, to any significant differ-
ence in the clinically important factor VIII half-life.
Following on from this was a trial of long-term safety
and efficacy in previously treated patients.‘” Eighty-
seven patients are currently included in the above trial.
A total of 778 joint bleeds have been treated at a mean
dose of 30 iu/kg per injection. In 94% of joint bleeds
three or fewer injections were required to treat the
haemorrhage. Efficacy was reported as excellent or
good in 87% of cases. Adverse events were rare and
predominately mild with no serious adverse events
occurring that were considered to be related to the use
of Refacto. Refacto was also used to promote
haemostasis during surgery in 17 patients undergoing
22 procedures requiring at least 1 week of factor VIII
replacement. Haemostatic efficacy was judged to be
excellent in all cases. A study in previously untreated
patients to monitor for inhibitor development was
begun in October 1994. To date 72 patients with
severe haemophilia A have received at least one injec-
tion of Refacto. Ten (14%) patients have developed an
inhibitor of which four are high titre. However, as
mean time to inhibitor development was 9.5 days and
43 patients in the study had yet to have > 10 exposure
days the true inhibitor incidence is likely to be higher.
Recombinant factor VIII versus plasma-derived factor
VIII
There are a number of important issues which influ-
ence the decision to change from using plasma-derived
factor concentrates to using recombinant products
(Table 1). Most notable of these is the disastrous con-
sequences that arose through the transmission of viral
infections such as HIV by plasma-derived coagulation
factors to haemophiliacs. Exclusion of potentially
infected individuals from donating plasma and the
viral inactivation methods now used in the manufac-
ture of plasma-derived concentrates make these prod-
ucts very safe in terms of risk of HIV infection.
However, it appears likely that it will be impossible to
render them completely free from the risk of viral
transmission. The current recommendations from the
 Recombinant coagulation factors 173

Table 1 Issues involved in the decision to change
plasma-derived to recombinant FVIII
Reduced risk of transmission of infectious agents
Incidence of inhibitor development
Preservation of immune function
Cost implications
Consequences for plasma fractionation industry
Pressure from patients and their families
Potential unlimited supply
Committee for Proprietary Medicinal Products are
that plasma-derived factor VIII undergoes a dual viral
inactivation process. Human parvovirus B19, how-
ever, is resistant to both solvent/detergent treatment
and heating and is therefore capable of being trans-
mitted by plasma-derived concentrates and can cause
infection in those who have not previously been
exposed to the virus.‘5,‘h This infection, although usu-
ally mild, can have serious consequences in certain
groups of patients. Hepatitis A virus, being another
non-lipid enveloped virus, is resistant to solvent/
detergent treatment and an outbreak of hepatitis A in
haemophiliacs from several countries in continental
Europe, South Africa and North America was
attributed to a plasma-derived product that had
undergone this form of viral inactivation process.“.”
Of similar concern is the report of hepatitis C virus
transmitted by a monoclonal antibody-purified heat
treated plasma-derived factor VIII.19 These reports
taken in conjunction with the ever present threat of
infectious agents, particularly non-lipid coated
viruses, being transmitted by plasma-derived factor
concentrates provides probably the strongest argu-
ment in favour of moving to the use of recombinant
coagulation factors. It is important to remember,
however, that although recombinant factor VIII prod-
ucts carry a much higher margin of safety from the
point of view of transmission of infection there still
remains a small but definite potential risk. Potential
from sources of infectious agents contaminating the pro-
duction process are: (i) within the production cell line
itself; (ii) from the biological raw materials used in the
fermentation process, i.e. bovine insulin and; (iii) the
chromatographic steps used in the purification process
and in the manufacturing process. The production cell
line for both of the licensed recombinant factor VIII
products undergoes a screening programme for adven-
titious and endogenous viruses and in addition there is
a final viral removal phase used to treat the bulk factor
VIII product. Infections in production cell lines for
recombinant proteins have been reported and are
usually recognized by their cytopathic effect in the cell
line.‘OIf an infection occurred with a virus that did not
cause either a cytopathic effect or a failure of growth
of the cell line then it could potentially infect the factor
VIII product, illustrating the importance of continued
viral surveillance and in using a viral removal stage in
the manufacturing process. Human albumin is another
potential source of infection as it is used to stabilize
the final factor VlII product. It has an excellent safety
record with no reported incidences of viral infection in
recipients attributed to its use. However, there has been
a report of human parvovirus B19 DNA contami-
nating vials of both Kogenate and Recombinate which
was believed to have originated from the human albu-
min used for stabilization.” The authors of this report
conclude that although human parvovirus B19 DNA
was present in the recombinant factor VIII vials it was
not likely to be infective, but that efforts should be
made to use a non-plasma-derived stabilizer. Refacto
meets that criteria as no human albumin is required
in stabilization of the final factor VIII product and
therefore it is a safer product as regards potential viral
transmission. The recombinant factor IX, BeneFix, is
produced without using any human- or animal-derived
proteins and therefore has the highest margin of safety
of all the recombinant coagulation proteins currently
available (Table 2).

Table 2 Production characteristics of recombinant factors VIII and IX

Kogenate Recombinate Refacto BeneFix

Gene VIII VIII-VWF VIII-B domain-less IX-PACE

Cell line BHK CHO CHO CHO

Animal/human Human insulin Bovine albumin Human albumin Recombinant human

proteins used in Human albumin Bovine insulin Murine MoAb insulin
culture and Human transferrin Aprotinin
purification Murine MoAb Murine MoAb

Stabilizer Albumin Albumin None None

Virus inactivation 40°C 8 h None Solvent/detergent None

Specific activity 8-30 2-10 I5 000 200

unitslmg
174 Blood Reviews

Table 3 Results of inhibitor trials in previously untreated patients with severe haemophilia

Plasma derived Kogenate Recombinate Refract0

FVIII (de Bias?)

No. of patients with FVIII < 2% 48 64 73 72*

Mean no. of exposure days to 30 9 10 9.5
inhibitor development
Inhibitors
Total 11 (23%) 18 (28%) 23 (32%) 10 (14%)
< 10BU 2 (4%) 8 (13%) 15 (2lY”) 6 (8%)
> 10BU 9 (19%) 10 (15%) S(ll%) 4 (5%)
Spontaneous loss 0 (0%) 7 (19%) 9 (12%) -

* Forty-three patients in trial have < 10 exposure days.

Another important issue in the decision to use the two groups it is known that the level of CD4 posi-
recombinant factor VIII is its potential immunogen- tive lymphocytes is an important predictor of the risk
icity. This is in regard to its ability to stimulate the of developing AIDS. Therefore slowing the progressive
development of factor VIII inhibitors and in its effect decline in CD4 positive lymphocytes, that occurs in
on the immune system of patients receiving it long HIV infected haemophiliacs, by use of high-purity
term. The data from clinical trials of previously factor VIII products would be expected to confer a
untreated patients treated with Kogenate and significant clinical benefit. As the recombinant factor
Recombinate showed inhibitor rates in patients with VIII products are extremely pure it would be hoped
severe haemophilia A of 28% and 3 1.5% respectively. that they would have a minimal impact on immune
Many of these were low level inhibitors which function. A clinical trial performed in HIV seroposi-
developed within a short time of first exposure to tive and seronegative haemophiliacs treated with
recombinant factor VIII and often disappeared spon- Kogenate demonstrated a small but significant
taneously. However, these reported rates were much decrease in the number of CD4 positive lymphocytes
higher in comparison to previous reports on patients in the HIV seropositive individuals over the 35year
treated with intermediate-purity factor VIII concen- study period.24 However, this decline was slower in
trates. There had been concerns with the high-purity comparison to that reported in trials of patients
plasma-derived factor VIII that during the purifica- treated with intermediate-purity products. In the HIV
tion process neoantigen formation may take place and seronegative patients of 6 years of age or older the B,
thus lead to an increase in inhibitor rates. However, microglobulin levels, CD4 positive and CD8 positive
more recent prospective studies on previously lymphocyte counts did not change during the study
untreated patients treated with intermediate and low- period - further supportive evidence that Kogenate
purity concentrates have shown comparable rates of had no deleterious effects on immune function.
inhibitor developmenP (Table 3). It is likely that the An important factor that mitigates against the
higher reported incidence of inhibitors in the recom- immediate widespread use of recombinant factor VIII
binant factor VIII studies was due to the increased is its cost in comparison to the currently available
frequency of surveillance that these patients under- plasma-derived products. Changing patients from
went in comparison to earlier studies and that the their existing plasma-derived factor VIII to a recom-
recombinant products are no more antigenic in regard binant product will have major financial implications.
to inhibitor development than the plasma-derived In addition the move to treat children with prophylac-
concentrates. Continued long-term surveillance will tic factor VIII instead of on-demand treatment will
be necessary in order to definitively establish that this further contribute to this increased cost. As the
is the case but experience thus far is that the majority demand for recombinant factor VIII increases and
of inhibitors occur within 20 exposure days and there- along with it the scale of its production, in combina-
fore late inhibitor development is likely to be negligible. tion with increased competition between different
In terms of the effects on the immune system, it has manufacturers, the cost of recombinant factor VIII
been shown that the use of monoclonal antibody- should fall. In the short term, however, limited finan-
purified plasma-derived factor VIII concentrates cial resources will restrict the number of patients that
appears to halt the decline in numbers of CD4 posi- can be converted to recombinant factor VIII. In the
tive lymphocytes in HIV infected haemophiliacs in United Kingdom the recently published ‘Guidelines
comparison to those treated with intermediate-purity on therapeutic products to treat haemophilia’ pro-
products.23 Although within the context of the study duced by the UK Haemophilia Centre Directors
there was no difference in clinical outcome between Organisation Executive Committee recommend that

recombinant factor VIII is the treatment of choice for
all patients.25 However, they state that if prioritization
is necessary then patients least exposed to blood
products should be given first priority. The order of
priority they suggest in deciding who should be
treated with recombinant factor VIII is as follows:
HIV antibody-negative patients; previously untreated
patients > HCV-negative patients > HCV-positive
patients > HIV antibody-positive patients.
The financial implications of using recombinant
factor VIII are not solely confined to those purchasing
the product but will also have a considerable impact
on the current major producers of factor VIII, the
plasma fractionation industry. This industry has
always been driven by the need to produce sufficient
factor VIII to meet demand. The plasma fractiona-
tion companies by stopping their production of factor
VIII would save only a small amount in terms of
reduced manufacturing costs but would lose a large
amount of revenue which would probably have to be
borne by increasing the price of other plasma prod-
ucts such as albumin. However, an advantage of using
recombinant technology to produce factor VIII is that
the manufacturing process is not dependent on
obtaining sufficient amounts of plasma for fractiona-
tion and recombinant factor VIII could potentially be
produced in unlimited amounts.‘”
Haemophiliacs and their families are likely to
favour recombinant factor VIII for their treatment
because of its perceived absence of risk of transmis-
sion of infectious agents.
One of the technical considerations which needs to
be addressed when switching from using plasma-
derived factor VIII concentrates to recombinant
factor VIII products is how their potencies should be
assayed as it has been shown that the one-stage factor
VIII assay may underestimate the true potency of
recombinant factor VIII products.” This is especially
true for the B-domain deleted recombinant factor
VIII, Refacto. It is recommended that the chromo-
genic substrate assay method is used to determine
potency of the recombinant factor VIII products.
RECOMBINANT FACTOR IX
BeneFix
1Manufucturingprocess
The factor IX gene and cDNA sequence were cloned
in 1982.2R.2’CHO cells were subsequently transfected
with cDNA from the factor IX gene and were able to
express recombinant factor IX. Factor IX is a protein
which requires significant post-translational modifica-
tion in the form of gamma carboxylation of terminal
Recombinant coagulation factors 175
glutamic acid residues and propeptide cleavage in
order to become fully functional. The initial develop-
ment of recombinant factor IX was hampered by the
fact that these post-translational modifications were
incomplete and led to the production of a function-
ally deficient factor IX protein. These problems were
eventually overcome and the recombinant factor IX
now produced has full functional activity. Genetics
Institute manufacture BeneFix by co-transfecting the
CHO cell line with the cDNA from the factor IX gene
and with cDNA encoding an engineered form of the
protease PACE (paired basic amino acid cleaving
enzyme). PACE improves the processing efficiency of
the profactor IX produced by the CHO cell lines. The
purification procedure used to convert the raw prod-
uct to high-purity factor IX includes ultrafiltration-
diafiltration and immunoaffinity chromatography
stages. No human plasma- or animal-derived proteins
are used in the purification and albumin is not
required for stabilization of the final product.
Therefore the recombinant factor IX produced is vir-
tually free of any risk of transmission of human infec-
tious agents. The specific activity of the final product
is 270 u/mg.
Clinical trials
Preclinical animal trials have demonstrated that there
is comparable pharmacokinetic behaviour between
BeneFix and the high-purity plasma-derived factor IX,
Mononine.3” This was confirmed in phase I trials in 12
patients with haemophilia B patients using a cross-over
study design.” In addition, as part of the trial, markers
of activation of the coagulation system (prothrombin
fragments 1 + 2 and fibrinopeptide A) were monitored
and these did not become elevated following infusions
of BeneFix, evidence which suggests it is not poten-
tially thrombogenic. Phase II/III clinical trials to assess
safety and efficacy in previously treated patients were
then initiated. Forty-four patients with moderate or
severe haemophilia B were started on BeneFix for use
as home treatment. At the most recently reported eval-
uation a total of 693 bleeding episodes had been
treated. Eighty-two per cent of these required a single
infusion of BeneFix to arrest haemorrhage and in 84%
of the infusions given response was rated as excellent or
good.‘* There were no serious adverse events that were
deemed to have arisen as a consequence of the use of
BeneFix and in particular there were no thrombotic
events. A single patient developed a low titre inhibitor
to factor IX after 39 exposure days. This patient had
had previous heavy exposure to plasma-derived factor
IX. The peak inhibitor titre was 1 BU and it remained
detectable for 11 months before spontaneously resolv-
ing. In a trial of BeneFix use during surgery a total of
176 Blood Reviews

13 surgical procedures were performed (10 major, 3 factor VII is purified via a number of steps which
minor). A continuous infusion of BeneFix was given include ion exchange and immunoaffinity chromatog-
for three of the major surgical procedures. The raphy. Included within this purification process is a
response to treatment was excellent to good in all cases. solvent/detergent viral inactivation step. During ion
Clinical trials in previously untreated patients to study exchange chromatography the recombinant factor
in particular the incidence of inhibitor development VII undergoes autoactivation to the final product,
started in October 1995. recombinant factor VIIa. Nine of the ten glutamic
acid residues on the protein are fully gamma carboxy-
lated and the remaining one is partially gamma car-
RECOMBINANT FACTOR VIIA
boxylated. There is no requirement for any stabilizing
protein in the final product and in fact no human-
The development of recombinant factor VIIa was
derived proteins are used at any stage during the
motivated by the fact that there are a limited number
manufacturing process.
of therapeutic options available to treat haemophiliac
patients who have inhibitors. For many patients these
therapies may be ineffective or associated with an Clinical trials
unacceptable risk of side-effects. With high titre
Preclinical animal trials to assess potential thrombo-
inhibitors it is not usually possible to give sufficient
genicity did not demonstrate any evidence of systemic
quantities of factor VIII to overcome the inhibitor
activation of the coagulation cascade following infu-
and achieve measurable factor VIII levels. In these sit-
sion of recombinant factor VIIa.33 These were
uations although porcine factor VIII may be used
followed by clinical trials. The Compassionate Use
often the human factor VIII antibodies are cross-reac-
Programme started in 1988. Its purpose was to evalu-
tive with the porcine factor VIII making this therapy
ate the treatment of bleeding episodes with
similarly ineffective. Activated prothrombin complex
NovoSeven in patients with haemophilia and
concentrates work in around 60-70% of bleeding
inhibitors when all other therapeutic options had been
episodes. This product contains a variety of partially
exhausted or patients required essential surgery and
activated clotting factors, including factors IXa and
no alternative treatment was possible. In the early
Xa, and as such carries a risk of systemic activation of
stages of this study many patients were treated with 60
the coagulation system and thrombosis and therefore
yg/kg or less of NovoSeven but it became apparent
must be used with caution. Factor VIIa was chosen as
that a higher dose was required to ensure adequate
a potential bypassing agent because, in the absence of
dosing in all patients and it was therefore recom-
tissue factor, it has no proteolytic effect despite being
mended that the initial dose should be 90 yg/kg with
in an activated form and therefore does not have the
an interval between subsequent doses of 2-3 h. This
propensity to cause systemic activation of coagula-
recommendation was based upon the results of pre-
tion. Following administration it binds to tissue factor,
clinical and laboratory testing and from the experi-
the resulting complex being able to convert factor X to
ence of the clinical trials. It appears that in order to
factor Xa without any requirement for factors VIII or
achieve haemostasis levels of factor VII:coagulation
IX. Tissue factor is normally only expressed by dam-
activity (FVII:C) should be maintained at > 6 u/ml.‘”
aged endothelium localizing the action of the factor
The results of the Compassionate Use Programme are
VIIa to the site of tissue injury.
as follows: for the treatment of joint and muscle bleeds
Initially factor VIIa was produced by purification
global evaluation of response in 494 bleeding episodes
from normal plasma and was successfully used to
in 111 patients was judged to be excellent/effective in
treat bleeding episodes in haemophiliac patients with
79% of joint bleeds, 62% of muscle bleeds and 69% of
inhibitors. Because the concentration of factor VII in
tense muscle bleeds/compartment syndrome? Central
human plasma is very low (0.5 rig/l) large scale manu-
nervous system bleeds are one of the most serious
facture from human plasma would not have been fea-
and life threatening complications of haemophilia. In
sible and so attention turned to producing FVIIa by
22 episodes of central nervous system bleeding
means of recombinant technology.
in patients with haemophilia A and inhibitors
NovoSeven was effective in 83% of cases with only
NovoSeven one fatality.36 Results in the surgical setting are simi-
larly encouraging. NovoSeven was used to promote
Manufacturing process
haemostasis in 12 patients requiring essential or life
cDNA from the factor VII gene was transfected into saving surgery for a total of 13 surgical procedures. In
a BHK cell line. The culture medium containing one case a global evaluation of the end treatment
the secreted FVII is harvested and the recombinant result was not reported but in the other 12 cases the
 Recombinant coagulation factors 177

end result was considered excellent (11) or efficient (l).,’ 18. Normann A, Graff J, Gerritzen A et al. Detection of hepatitis
A virus RNA in commercially available factor VIII
There was no laboratory evidence of systemic activa- preparation. Lancet 1992; 340: 123221233.
tion of coagulation detected. A Home Treatment trial 19. Berntrop E, Nilsson IM, Ljung R, Wide11 A. Hepatitis C virus
(started in 1994) is underway which will evaluate the transmission by monoclonal antibody purified factor VIII
concentrate. Lancet 1990; 335: 1531-1532.
safety and efficacy of NovoSeven given to control 20. Rabenau H, Ohlinger V, Anderson J et al. Contamination of
joint, muscle and mucocutaneous bleeds in the home genetically engineered CHO cells by epizootic haemorrhagic
setting. Sixty patients have been enrolled and the aim disease virus (EHDV). Biologicals 1993: 21: 207.-214.
21. Eis-Hiibinger A, Sasowski U, Brackmann H, Kaiser R, Matz
is to accumulate 120 bleeding episodes.ix B, Schneweis K. Parvovirus B19 DNA is frequently present in
The Compassionate Use Programme has closed recombinant coagulation factor VIII products. Thromb
and NovoSeven is now licensed in Europe for the Haemost 1996; 786: 1118.
22. de Biasi R, Rocino A, Papa ML, Salerno E, Mastrullo L, De
treatment of bleeding in patients with haemophilia Blasi D. Incidence of factor VIII inhibitor development in
and inhibitors and in patients with acquired haemophilia A patients treated with less pure plasma derived
haemophilia due to factor VIII or IX inhibitors. concentrates. Thromb Haemost 1994; 71: 544547.
23. de Biasi R. Rocino A, Miraglia E, Mastrullo L. Quirino AA.
The impact of a very high purity factor VIII concentrate on
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