Method for producing factor VIII:C-type proteins

An improved method for producing Factor VIII:c-type proteins is disclosed which involves culturing mammalian cells which are capable of expressing the protein. In accordance with this invention the cells are cultured in a medium containing an effective amount of a substance comprising (a) von Willebrand Factor-type protein, (b) a phospholipid or phospholipid mixture, or a mixture of (a) and (b).

BACKGROUND OF THE INVENTION 
The Factor VIII complex has two distinct biologic functions: coagulant 
activity and a role in primary hemostasis. The analysis of Factor VIII 
deficiency diseases, classic hemophilia and von Willebrand's disease, have 
contributed to the understanding that Factor VIII is a complex of two 
components. The Factor VIII:c procoagulant protein (antihemophilic factor) 
and the Factor VIII related antigen (von Willebrand factor,VWF) are under 
separate genetic control, have distinct biochemical and immunologic 
properties, and have unique and essential physiologic functions. 
The Factor VIII:c molecule is an important regulatory protein in the blood 
coagulation cascade. After activation by thrombin, it accelerates the rate 
of Factor X activation by Factor IXa, eventually leading to the formation 
of the fibrin clot. Deficiency of Factor VIII:c (classic hemophilia) is an 
X-linked chromosomal disorder that has been a major source of hemorrhagic 
morbidity and mortality in affected males. Treatment usually consists of 
frequent transfusions with blood products. The latter has resulted in a 
high incidence of infectious complications (such as various forms of 
hepatitis and acquired immunodeficiency disease) in the hemophiliac 
population. 
The VWF molecule is an adhesive glycoprotein that plays a central role in 
platelet agglutination. It serves as a carrier for Factor VIII:c in plasma 
and facilitates platelet-vessel wall interactions. Discrete domains of VWF 
which bind to platelet receptor sites on glycoprotein 1b and on the 
glycoprotein IIb-IIIa complex, as well as binding sites on collagen have 
been noted. VWF is made up of multiple, probably identical, subunits each 
of about 230,000 daltons. VWF is synthesized in endothelial cells and 
megakaryocytes. In the plasma it exists as high molecular weight multimers 
ranging from 5.times.105 to 10.sup.7 daltons. Von Willebrand Factor 
contains 5-6% complex carbohydrate, which appears important in the 
molecule's ability to bind platelets. A variety of abnormalities in VWF 
activity can result in Von Willebrand's disease. The disorder is generally 
inherited in autosomal dominant fashion and may affect as many as one in 
2000 individuals. Mild forms of the disease frequently go undiagnosed, 
whereas severely affected patients may require frequent blood product 
support with its associated risks. 
Recently, the isolation of the genes for both Factor VIII:c and Von 
Willebrand Factor have made possible the production of recombinant factor 
VIII:c and VWF preparations, respectively, which are essentially free of 
contaminating viruses (Toole et al., 1984, Nature 312:342; Wood et al., 
1984, Nature 312:330; Lynch et al., 1985, Cell 41:49-56; Ginsberg et al., 
1985, Science 228:1401-1406). The production of Factor VIII:c or analogs 
thereof through recombinant DNA technology has been achieved utilizing 
mammalian cells transfected or transformed with appropriate expression 
vectors containing DNA encoding Factor VIII:c or the analogs thereof. 
Primary concerns for the synthesis of recombinant Factor VIII:c-type 
proteins include (i) the yield of recombinant protein obtainable from the 
culture medium, (ii) the stability of the recombinant protein so produced, 
(iii) the efficiency and cost of purification of the protein and (iv) the 
overall cost of producing purified recombinant protein. 
For best results, we have heretofore typically cultured cells producing 
FVIII:c-type protein in media containing mammalian serum, e.g. 
conventional preparations of fetal bovine serum, in amounts of about 10% 
serum by volume relative to total media volume. (For the sake of 
simplicity, all serum concentrations hereinafter are expressed as a volume 
% of total media.) We have found that in the absence of serum supplements 
both the yield and stability of the recombinant FVIII:c suffer 
significantly. However, the cost of serum and the added inconvenience and 
expense in purification resulting from the addition of serum to the media 
rendered the use of serum an undesireable necessity and the wide scale use 
of recombinant FVIII:c a commercially less attractive alternative to 
natural FVIII:c purified from plasma. Interestingly, despite the great 
excitement in the medical and pharmaceutical communities over the clinical 
potential of recombinant FVIII:c, we are aware of no reports heretofore of 
the above-mentioned serum dependence, its biochemical basis or methods to 
circumvent it. 
After extensive experimental modifications of media for FVIII:c-producing 
cells we have surprisingly found a method for producing higher yields of 
stable FVIII:c-type proteins (as described hereinafter) even when using 
media containing reduced amounts of serum (e.g., semi-defined media, 
containing .about.1% serum) or essentially serum-free media (defined 
media). We have found that host cells producing FVIII:c-type proteins 
produce recoverable, stable FVIII:c-type proteins in semi-defined and 
defined media in yields at least comparable, and in some cases superior, 
to those obtained in the presence of 10% serum if the semi-defined or 
defined media contains a suitable amount of a hydrophobic substance such 
as VWF or certain phospholipids. We have further found that FVIII:c-type 
proteins produced in semi-defined or defined media lacking such 
supplements typically exhibit dramatic instability and are recoverable in 
extremely low yield if at all. 
Evidence to date suggests either that VWF may have a stabilizing effect in 
vivo on the Factor VIII:c in plasma, or that the VWF can ellicit the in 
vivo release from storage depots or stimulate the in vivo synthesis and/or 
secretion of Factor VIII:c (Weiss, H. J. et al., 1977,J. Clin. Invest. 60: 
390-404). It has also been suggested that thrombin-activated Factor VIII 
(derived from natural, human FVIII) is stabilized by phospholipids, 
presumably with respect to thrombin-mediated degradation. See Andersson 
and Brown, 1981, Biochem. J. 200:161-167. However, to our knowledge there 
is no suggestion in the prior art as to any possible effect(s) of VWF or 
phospholipid on the in vitro production of Factor VIII:c-type proteins 
(where, for example, thrombin is substantially absent) or any suggestion 
of media supplements for the production of Factor VIII:c comprising VWF or 
a VWF-type protein and/or phospholipids either substantially free from the 
complex mixture of components present in mammalian serum or in 
concentrations higher than afforded by 10% serum supplements in accordance 
with this invention. It should be noted that mammalian serum contains VWF. 
As a point of reference, conventional media for mammalian cells which 
contains about 10% serum, contains about lug VWF/ml media. 
SUMMARY OF THE INVENTION 
This invention concerns an improved method for the production of Factor 
VIII:c-type proteins. 
"Factor VIII:c-type" proteins, as the term is used herein, means proteins 
exhibiting Factor VIII:c-type procoagulant activity. Factor VIII:c-type 
proteins within the ambit of this invention are encoded for by DNA 
sequences capable of hybridizing to DNA encoding Factor VIII:c under 
conditions that avoid hybridization to non-Factor VIII:c genes, e.g., 
under conditions equivalent to 65.degree. C. in 5.times.SSC 
(1.times.SSC=150 mM NaCl/0.15M Na citrate). In addition to natural 
mammalian, e.g. human, Factor VIII:c, Factor VIII:c-type proteins include, 
for example, proteins which contain deletions of one or more amino acids 
between the 90 Kd and 69 Kd cleavage sites with respect to native Factor 
VIII:c, as described in greater detail in International Application No. 
PCT/US86/00774, published 23 October 1986. Factor VIII:c-type proteins 
also include Factor VIII:c analogs containing deletion(s) of one or more 
amino acids between the 50/40 cleavage site and the 69 Kd cleavage site 
which may be produced by methods analogous to those disclosed in 
PCT/US86/00774. Factor VIII:c-type proteins further include analogs (with 
or without deletions as mentioned above) wherein one or more of the 
cleavage sites spanning arginine residues at positions 226, 336, 562, 740, 
776, 1313, 1648 or 1721 have been rendered resistant to proteolytic 
cleavage, e.g., by replacement of one or more amino acids with different 
amino acids by conventional site-directed mutagenesis of the cDNA to be 
expressed. Factor VIII:c-type proteins thus include natural Factor VIII:c, 
"recombinant" Factor VIII:c and analogs thereof having procoagulant 
activity, and non-recombinant Factor VIII:c or analogs thereof produced by 
cell lines derived from cells which produce the protein. 
The method of this invention thus utilizes mammalian cells which contain 
DNA encoding a Factor VIII:c-type protein and which are capable of 
expressing the protein. In accordance with the method of this invention 
the cells are cultured in media containing an effective amount of a 
stabilizing substance for a Factor VIII:c-type protein. Such substances 
include: (i) a VWF-type protein; (ii) a stabilizing phospholipid or 
phospholipid mixture; and (iii) mixtures containing a VWF-type protein and 
phospholipid(s). Although termed "stabilizing substances", it should be 
understood that VWF-type proteins may possess, in addition to a 
stabilizing effect on Factor VIII:c-type proteins, other effects which 
result in higher levels of synthesis and/or export of the Factor 
VIII:c-type proteins from the producing cells. VWF-type proteins include 
truncated or otherwise modified analogs of von Willebrand Factor which are 
encoded by a cDNA capable of hybridizing under to a cDNA encoding a 
mammalian VWF under hybridization conditions such as those mentioned 
above, and which retain the ability to stabilize Factor VIII:c-type 
proteins or otherwise result in increased production and/or accumulation 
of the FVIII-type proteins in the culture media. 
For example, truncated forms of human VWF which may be used in the practice 
of this invention include (i) .DELTA.pro VWF, which lacks the "pro" 
sequence of VWF; (ii) .DELTA.mature VWF, which comprises the "pro" 
sequence without the mature sequence; and, (iii) VWF-5'-Sac, which 
comprises the sequence of pro-VWF from the N-terminus to the 5' Sac I 
restriction site and includes the "pro" portion of VWF as well as the 
first "D" domain of the mature sequence A full-length peptide sequence, 
nucleotide sequence and restriction map for VWF have been published. See, 
e.g. International Patent application Publication No. WO 86/06096 (Appln. 
No. PCT/US86/00760). With reference to that sequence, the "pro" portion 
spans amino acid positions 23 through Arg-763 and the "mature" protein 
spans amino acid positions 764 through 2813. A cDNA encoding .DELTA.pro 
VWF may be prepared by conventional loop-out mutagenesis using, for 
example, the full-length VWF cDNA present in the vectors described 
hereinafter and a synthetic loop-out oligonucleotide complementary to part 
or all of the VWF "pre" sequence and mature sequence but lacking the 
codons complementary to the "pro" sequence. A cDNA encoding .DELTA.mature 
VWF may be prepared by analogous methods or by making use of convenient 
restriction sites in the full length VWF cDNA to remove part or all of the 
mature sequence. Where only part of the mature sequence is thus removed, 
remaining cDNA regions encoding mature peptide sequence may be excised by 
conventional loop-out mutagenesis. Alternatively, .DELTA.mature VWF and 
VWF-5'-Sac may be produced by conventional mammalian expression of VWF 
cDNAs which have been mutagenized by conventional oligonucleotide-directed 
mutagenesis to insert a translational stop codon immediately 3' to the 
peptide sequence one wishes to produce. Other truncated or otherwise 
modified forms of VWF may also be prepared by analogous methods and may 
also be useful in the practice of this invention as may be readily 
determined by methods disclosed hereinafter (see e.g. the Examples which 
follow). In particular, it is contemplated that other truncated forms of 
VWF which contain one or more of the "D" domains (of which two are present 
in the "pro" portion) may also be useful in the practice of this 
invention. At present, .DELTA.pro VWF is preferred among truncated 
variants of VWF for use in accordance with this invention. 
Potential advantages of the use of these and similar truncated or otherwise 
modified forms of VWF include (i) imposing less stress on the producing 
cells by directing the synthesis, post-translational modification and 
export of significantly smaller proteins; (ii) decreased viscosity of the 
conditioned media due to the presence of smaller VWF-type proteins and the 
absence of higher molecular weight VWF-type multimers; and (iii) more 
facile purification of the FVIII-type protein from the truncated VWF-type 
protein rather than from the full-length VWF protein. 
VWF-type proteins may be readily produced and characterized, e.g. by 
conventional expression, preferably in mammalian cells, of cDNAs encoding 
the VWF-type protein. Such VWF-type proteins may then be tested for 
efficacy in the production of FVIII-type proteins by the methods described 
hereinafter, eg, in the Examples which follow. The cDNA may have been 
produced by mutagenesis in a random or site-specific manner. Mammalian VWF 
as well as VWF-type proteins are referred to herein simply as "VWF". 
One embodiment of this invention encompasses an improved method for 
producing Factor VIII-type proteins which comprises culturing mammalian 
cells capable of producing a Factor VIII-type protein in a medium to which 
exogenous VWF-type protein has been added. Exogenous VWF-type protein may 
thus be added to the medium, as is described in greater detail 
hereinafter, e.g., by virtue of prior conditioning of the medium by cells 
producing VWF-type protein, by coculturing cells producing VWF-type 
protein with cells producing a Factor VIII-type protein, by using cells 
genetically engineered to produce both VWF-type protein and a Factor 
VIII-type protein, or by adding exogenous VWF-type protein obtained, e.g. 
from conditioned medium. 
Preferred effective amounts of VWF-type protein generally range from about 
0.1-10 ug VWF/ml media, with .about.1-.about.3 ug/ml being more preferred 
and .about.2-18 3 ug/ml being especially preferred. It should be noted, 
however, that in cases where the exogenous VWF-type protein is added to 
the medium by using cells genetically engineered to produce both VWF-type 
protein and a Factor VIII-type protein, amounts at the lower end of the 
general range may be preferred. Lower media concentrations of the VWF-type 
protein may be useful in such methods since greater effective 
concentrations of the exogenous VWF-type protein may be available at the 
site of production and secretion into the medium of the Factor VIII-type 
protein despite the lower media concentration. Furthermore, the use of 
cells which produce both proteins is presently preferred to the 
co-culturing of different cells which individually produce one or the 
other protein. This is so because the latter method may introduce 
potential complications to the cell culture process such as variable 
growth rates of the different cells, and inherently results in a lower 
density for cells producing the FVIII-type protein (by virtue of the 
presence of the cells producing the VWF-type protein). 
One readily obtainable source of suitable phospholipids comprises 
commercially available dry milk preparations such as dried skim milk and 
low-fat skim milk. Such dried milk preparations may be added to the media 
in amounts ranging from about 0.01%-10% (weight of dry milk/volume of 
media). For optimal effect on Factor VIII production with minimal toxic 
effect on the cells, about 1%-3% dry milk is presently preferred. The dry 
milk preparations may be conveniently sterilized by first preparing a 10% 
aqueous suspension of the milk and autoclaving. Another readily obtainable 
source of suitable phospholipids is commercially available soybean 
lecithin, which may be added to the medium in accordance with this 
invention, preferably in liposome form. 
"Phospholipid" as the term is used herein means an ester of phosphoric acid 
containing one or two molecules of fatty acid, an alcohol and a 
nitrogenous base. Examples of such phospholipids include Cephalin, 
phosphatidyl serine: phosphatidyl choline mixtures, phosphatidyl inositol, 
phosphatidyl ethanolamine, soybean lecithin and mixtures thereof, with 
soybean lecithin being especially preferred. Other phospholipids useful in 
this method as well as effective and/or optimal concentrations and/or 
mixtures thereof may be readily identified by those skilled in the art 
using methods described in greater detail hereinafter. Presently preferred 
effective amounts of phospholipid or phospholipid mixtures comprise about 
1-1000 ug phospholipid or phospholipid mixture per ml of culture media, 
with concentrations greater than about 100 ug/ml being more preferred and 
concentrations between about 200-300 ug/ml being especially preferred. The 
utility of such phospholipid supplements is certainly surprising in view 
of the toxicity we have observed of such compositions on mammalian cells 
such as CHO cells. Indeed, when using phospholipid supplements in 
accordance with this invention, it is preferable to additionally include 
bovine serum albumin (BSA) in the medium to protect the cells from such 
toxicity. Suitable concentrations of BSA range from about 1 to about 10g 
BSA/1 medium, depending on such factors as the amount of phospholipid 
used, the hardiness of the cells, and the degree of toxicity observed in 
the absence of BSA. Additionally, it is presently preferred to add the 
phospholipid mixture to the culture media in the form of liposomes, 
preferably having a diameter of up to about 500 nm. Preferably the 
liposomes are unilamellar, although multilamellar liposomes may also be 
used. Most preferably the diameter of the liposomes is less than about 100 
nm. Furthermore, liposomes made by conventional methods from said 
phospholipids may be used, either in admixture with or containing the 
Factor VIII:c-type protein, as a carrier or vehicle for administering the 
protein to patients. Where dried milk is used as the source of 
phospholipids, the dried milk may be added directly (rather than in the 
form of liposomes) to the media. 
In one embodiment, cells producing VWF-type protein, such as cells suitably 
engineered to produce the VWF-type protein, are cultured in the medium to 
condition it with the VWF-type protein either prior to or simultaneously 
with the culturing of cells which produce a Factor VIII:c-type protein. 
Alternatively the recombinant VWF-type protein may be separately produced 
and added as an exogenous supplement to the media to be used for culturing 
the cells producing the Factor VIII:c-type protein. In another embodiment 
the cells which produce Factor VIII:c-type protein are suitably 
engineered, i.e. effectively transformed with transcription unit capable 
of directing the production of the VWF-type protein, such that the 
VWF-type protein and the Factor VIII:c-type protein are co-expressed by 
the same cells. In a further embodiment of this invention the media used 
for culturing the cells producing the Factor VIII:c-type protein contains 
both VWF-type protein, by virtue of one of the above-mentioned processes, 
and stabilizing phospholipid(s). In that case, it may be desirable to use 
reduced amounts of each component relative to the amounts used if used 
alone. By using appropriately supplemented defined media in accordance 
with this invention high levels of recoverable, stable Factor VIII:c-type 
activity are produced which may then be recovered and purified without the 
necessity for separation of serum components therefrom. The culture media 
used in this invention may additionally contain mammalian-derived serum, 
e.g. fetal bovine serum, preferably in amounts less than about 10%, more 
preferably in amounts less than about 5%, and even more preferably in 
amounts between 0 and 1%, although essentially serum-free media is 
especially preferred. Other conventional mammalian cell culture media 
supplements may also be added. 
It should be noted that in the practice of this invention, the FVIII-type 
protein so produced may be conveniently recovered from the culture medium 
into which it is secreted, and further purified, if desired, by any of a 
number of conventional procedures, including e.g. conventional 
chromatographic methods and immunoaffinity-based methods. 
This invention is illustrated in the following examples which set forth 
typical procedures demonstrating, among other things, the ability to 
overcome "serum" dependence in the production of recoverable, active 
Factor VIII:c-type proteins by using phospholipids, and/or VWF-type 
protein as media supplements. The examples are set forth to aid in an 
understanding of the invention but are not intended to, and should not be 
construed to, limit in any way the invention as set forth in the claims 
which follow thereafter.

EXAMPLE I 
Establishment of Chinese Hamster Ovary Cell Lines which Express Human 
Factor VIII:c 
The Factor VIII:c expression plasmid used in this Example was RxPy VIII-I 
which contains in clockwise order the polyomavirus enhancer, the first 
leader sequence of the adenovirus tripartite leader sequence, a Factor 
VIII:c transcription unit followed by a DHFR gene and SV40 polyadenylation 
signal, and a gene encoding tetracycline resistance. This plasmid was 
introduced into dihydrofolate reductase (DHFR) deficient Chinese hamster 
ovary cells by cotransformation with a DHFR expression plasmid and 
subsequent selection for cells that grow in the absence of added 
nucleotides. One particular pool of transformants designated lig 1 was 
subsequently grown in increasing concentrations of methotrexate (MTX) in 
order to amplify the DHFR and Factor VIII genes. The resultant cell line 
expressed high levels of Factor VIII activity as determined by either the 
ability to clot Factor VIII deficient plasma [Clotech (APPT) assay] or by 
the ability to generate Factor Xa in the presence of Factor IXa, 
phospholipid, calcium, and Factor X (Kabi Cotest assay). The ability of 
these CHO cells to produce Factor VIII:c is shown in Table I. The Factor 
VIII activity increased 10,000 fold with increasing levels of MTX 
resistance which correlated with the Factor VIII gene copy number. Other 
expression vectors may also be used in place of RxPy VIII-I so long as 
they are capable of directing the expression of Factor VIII:c or analogs 
thereof. Such vectors include, for example, pCVSVL2-VIII (ATCC No. 39813, 
see European Application No. 85202121.1) and pDGR-2 (ATCC No. 53100, see 
PCT/US86/00774-deletion analog). Other Factor VIII:c expression vectors 
containing, for example, the SalI fragment from pCVSVL2-VIII or pSP64-VIII 
(ATCC No. 39812) may be prepared using conventional expression vectors and 
techniques. The SalI fragment from either vector contains a DNA sequence 
encoding full-length Factor VIII:c. 
TABLE I 
______________________________________ 
Factor VIII Expression in Transfected and 
Amplified CHO cells 
Pool MTX (uM) mU/ml/day of VIII:c 
______________________________________ 
Lig 1 0.0 0.1 
0.02 11.5 
0.1 88.0 
1.0 288, 545* 
5.0 644, 875* 
20.0 1075 
______________________________________ 
*Represents samples from two independent assays 
Plasmids pAdD26SVpA(3) (Kaufman and Sharp, 1982, Mol. Cell. Biol.) and 
plasmid pRXPy-VIII-I were digested with Cla 1 and the resultant linearized 
DNA was ligated in vitro and coprecipitated with CaPO4 and used to 
transfect CHO DHFR deficient DUKX-BII cells. Cells which efficiently 
expressed DHFR would be expected to contain the enhancer element from 
pRXPyVIII-I associated with the DHFR gene from pAdD26SVpA(3). Results have 
been consistent with this hypothesis. Subsequent selection for increased 
DHFR expression by propagation of the cells in increasing concentrations 
of MTX results in cells which have amplified the Factor VIII gene and the 
DHFR gene. At each level of MTX selection, samples of the conditioned 
media (approximately 10.sup.6 cells/ml in alpha media supplemented with 
10% fetal bovine serum) were taken for Factor VIII:c activity assay 
determined by the Kabi Coatest method modified to obtain sensitivity 
better than 0.05 mU/ml. Comparable results were also obtained by the 
one-stage activated partial thromboplastin time (APTT) coagulation assay 
using Factor VIII:c deficient plasma. All samples exhibited thrombin 
activation of 30-50 fold. For thrombin activation the samples were 
pretreated 1-10 min with 0.2 units/ml thrombin at room temperature. 
EXAMPLE II 
Serum Dependence of Factor VIII:c Synthesis 
A subclone of the lig 1 CHO cells of Example I (Lig 1 2 A C subclone B10 in 
0.1 uM MTX at 80% confluence) which are rinsed and fed with media 
containing 10% FCS or defined media (serum free, containing: 5 mg/ml BSA, 
insulin, transferrin, selenium, hydrocortisone and putrescine) accumulate 
Factor VIII activity. The rate of appearance in defined media is roughly 
4-fold lower than in serum-containing media The 4-fold difference becomes 
larger as the cells are propagated in the absence of serum. This results 
from inefficient rinsing of the cells. The rate of Factor VIII:c 
appearance increases fairly linearly up to 24 hrs. which suggests the VIII 
is stable in the media. This result is similar to results obtained with 
COS cells at lower levels of VIII expression (Approx. 10 mU/ml/day). 
Cephalin, a mixture of phospholipids, can counteract at least part of the 
serum deficiency. A subclone of the CHO cells of Example I (Lig. 2 A pool 
in 20 uM MTX) were separately cultured with and without serum and rinsed 
after 4 hr. The two CHO cultures were then split again, and a portion of 
the serum.sup.+ and of the serum.sup.- CHO cells were supplemented with 
5 uM cephalin for an additional 2 hr. The other portion of the serum.sup.+ 
and serum.sup.- CHO cells were not supplemented with cephalin. Media was 
assayed at 6 hrs. and 25 hrs. and results shown below: 
______________________________________ 
Conditions FVIII:c Activity* 
Serum Cephalin 6 hr. 25 hr. 
______________________________________ 
+FCS +Ceph 594 1044 
-FCS +Ceph 408 514 
+FCS -Ceph 563 1492 
-FCS -Ceph 140 372 
______________________________________ 
The results suggest that cephalin alone can increase VIII activity in the 
absence of serum but that its effect is short lived (i.e. observed after 2 
hrs. but is diminished at 25 hrs.). In one experiment the concentration of 
cephalin was increased and there was no further increase in VIII activity. 
This indicated some component in the cephalin was not rate limiting. 
Part of the cephalin effect can be elicited by a simpler mixture of 
phospholipids or by single phospholipids. Cells (a subclone of the CHO 
cells of Example I, Lig 1 2 A 0.02 pool in 20 uM MTX) were fed with 10% 
fetal calf serum or serum free media for 24 hrs. and then either cephalin 
(5um) or a mixture (1:4) of phosphatidyl serine and phosphatidyl choline 
(PCPS) were added to serum free cultures. Results from media assayed after 
2 hrs. were: 
______________________________________ 
conditions FVIII:c Activity 
______________________________________ 
Serum Free 100 
Serum Free + Cephalin 
489 
Serum Free + PCPS 230 
10% FCS 613 
______________________________________ 
This result demonstrates that phospholipids alone can increase VIII 
activity in conditioned media. 
Analysis of the thrombin activation of VIII expressed in CHO cells growing 
under different conditions suggests that the presence of serum decreases 
the degree of thrombin activation. CHO cells (Lig 1 2 A pool in 20 uM MTX) 
were rinsed and fed with media containing 10% fetal calf serum or defined 
media (5 mg/ml BSA, transferrin, selenium, insulin, hydrocortisone, 
putrescine). 24 hrs. later either cephalin or 10% FCS was added and 2 hrs. 
later samples taken for assay and measure of thrombin activatibility: 
______________________________________ 
Assay at 26 hrs. 
Coagulation 
Assay 
Added at Cobas (fold 
Sample Media 
24 h mU/ml mU/ml activation) 
______________________________________ 
Defined media 
-- 315 300 20X 
Defined media 
5 uM cephalin 
752 1040 34X 
Defined media 
10% FCS 684 440 8.4X 
+ 10% FCS -- 1078 1200 10X 
+ 10% FCS 5 uM cephalin 
1154 1120 14X 
Defined media 
10% boiled 543 -- -- 
FCS 
______________________________________ 
These results suggest that the presence of serum increases the activity 
produced compared to serum free media but reduces the thrombin 
activatibility. In contrast, cephalin may compensate for the serum effect 
on increasing the activity of the VIII produced but does not reduce the 
thrombin activatibility. Thus, in serum free media, with the addition of 
cephalin 2 hrs. prior to harvest, CHO cells produce VIII at 1 unit/ml and 
this material exhibits a 34 fold thrombin activation. This experiment also 
demonstrates that 10% FCS added to serum free media 2 hrs. prior to assay 
can also increase the amounts of VIII activity. This ability was not 
diminished by boiling the serum 10 min prior to its addition. Thus, the 
serum factor required for VIII activity is heat stable. We conclude that 
the serum factor required for increasing VIII may comprise two components: 
a phospholipid and another, heat stable factor which may be required to 
stabilize the phospholipid. 
To determine whether the 10% serum was limiting for Factor VIII:c 
expression in the highly amplified CHO cell lines, we monitored the effect 
of increasing amounts of serum on the ability to elicit factor VIII:c 
activity in the cell line 10Al. 10A1 is a clone derived from selection of 
the Lig 1 pool for growth in 1 mM MTX. This experiment demonstrated the 
effect on Factor VIII activity of adding increasing amounts of fetal 
bovine serum to the 10A1 cells for 24 hrs. 50% serum yielded three-fold 
more activity in the 24 hr. conditioned media compared to 10% serum. Other 
results have indicated that the amount of active Factor VIII antigen is 
correspondingly increased when cells are propagated in 50% serum. Other 
cell lines, which express slightly lower levels of Factor VIII:c show less 
dramatic increases in Factor VIII:c activity upon growth in higher 
concentrations of serum. Thus there appears to be some limiting 
requirement for Factor VIII: expression in higher producing cells such as 
those which would be desirable for commercial-scale production of Factor 
VIII. 
Example III 
Serum Dependence of rFactorVIII:cSvnthesis in Suspension Cultures of CHO 
The following table illustrates the dependency of recombinant Factor VIII:c 
(rFVIII) activity on serum levels in culture. A relatively low rFVIII 
producer, clone 1E6, was grown in suspension culture for 3 to 4 days in 
medium containing various concentrations of fetal bovine serum (FBS). 
______________________________________ 
rVIII Activity 
Serum Concentration 
(mU/ml) after 
Average Productivity 
in Medium* 3-4 days (U/10.sup.6 cells/day) 
______________________________________ 
10% FBS 318 0.19 
5% FBS 100 0.03 
Defined* 4 1/4-0.01 
______________________________________ 
*RPMI 1640 was employed as basal medium for all of the above. The defined 
medium consists of insulin, 5 ug/l; transferrin, 5 ug/ml; selenium, 5 
ng/ml; hydrocortisone, 10.sup.-8 M, putrescene, 100 ng/ml; BSA, 5 mg/ml. 
The same serum dependence has been observed with other rFVIII secreting CHO 
cell lines. These results do not reflect genetic instability since 
original expression levels can be regained on addition of serum. 
We have found that addition of phospholipid to culture medium can replace 
the serum requirement, however relatively high concentrations of 
phospholipid are required (on the order of 10-20 fold higher than 
previously used with serum-containing media). With respect to the 
dependence of the recovery of FVIII activity on phospholipid 
concentration, we have found that supplementing defined media (DM) with 
240 ug soybean lecithin (SL)/ml media yielded (after 24 hrs) about twice 
as much FVIII activity as was obtained in DM containing 160 ug SL/ml and 
five times as much FVIII activity as was obtained in DM containing 80 ug 
SL/ml. Nonetheless, DM containing 80 ug SL/ml provided measurably more 
FVIII activity than DM containing 1% Fetal Bovine Serum(FBS)(semi-defined 
media), while the semi-defined media provided significantly more FVIII 
activity than did DM alone. Significantly, the level of rFVIII generated 
over a 24h period in defined medium in the presence of 240 ug SL/ml media 
is at least as high as that generated in DM supplemented with 10% FBS. 
Increasing the concentration of SL above 240ug/ml resulted in no further 
increase in rVIII activity in this experiment. Illustrative results of one 
experiment are shown below: 
______________________________________ 
Media rFVIII activity after 24 h 
______________________________________ 
Defined Media .about.40 mU/ml 
(DM) 
DM + Soybean lecithin (SL) 
.about.270 mU/ml 
(240 ug SL/ml media) 
Media containing .about.200 mU/ml 
Fetal bovine serum (FBS) 
(10% FBS) 
______________________________________ 
This data illustrates the increase in the rFVIII:c activity obtained with 
relatively high concentrations (e.g., 240 ug/ml) of soybean lecithin 
phospholipid in the absence of fetal bovine serum. CHO cells (1E6 in 0.1 
umolar MTX) were suspended at a concentration of 3.times.10.sup.5 cells/ml 
in defined medium containing 5 g/1 of bovine serum albumin either in the 
absence or presence of phospholipid or medium containing 10% FBS for 24 h 
at 37.degree. C. At the conclusion of the incubation samples of cell-free 
conditioned medium were assayed for rFVIII:c activity by a chromogenic 
assay. 
We have found that the addition of phospholipid in amounts up to about 320 
ug/ml media causes no marked changed in cell growth in either defined or 
semi-defined media. In one set of experiments we found that maximum rFVIII 
activity was obtained in the culture where 320 ug phospholipid/mL media 
was added. In semi-defined medium, maximum levels were obtained after 72 h 
where 240 ug/mL of soybean lecithin was added. These and other data 
illustrate that soybean lecithin added stepwise to cultures on days 0, 1, 
2 and 3 allowed production of rFVIII. The optimum concentrations were 
around 240 ug/mL in these and other experiments regardless of the method 
of preparation of the phospholipid. 
The cellular productivities in two different CHO cell lines from 
experiments similar to the above are shown below. 
______________________________________ 
Average Productivity 
Medium (u/10.sup.6 cells/day) 
(Cell Line - 1E6) 
______________________________________ 
10% FBS 0.19 
5% FBS 0.03 
Defined 1/4-0.01 
Defined + SL 
0.24 
Defined + 1% 
0.25 
FBS + SL 
______________________________________ 
Medium Average Productivity* 
(Cell Line - H9.05) 
______________________________________ 
10% FBS 0.43 
Defined +1% 
0.51 
FBS + SL 
______________________________________ 
*Units as above 
Thus, productivities of rFVIII from rCHO cells are at least equivalent in 
defined medium supplemented with phospholipid as in serum-containing 
medium. However, as illustrated by the data above, the bulk quantity of 
rFVIII produced in defined medium is less than in serum containing medium. 
This is due to the fact that cells grow more rapidly and to higher cell 
densities in serum-containing medium (rather than being more productive). 
On supplementation of defined medium with small quantities of serum (e.g. 
1%) cell growth is improved. Indeed, after a short period of adaption 
cells will grow almost as well in semi-defined medium as in 10% FBS 
supplemented medium. We have found that rCHO cell lines (e.g., our H9.05) 
grow to similar cell densities and are at least equally productive (in 
Factor VIII) in phospholipid supplemented semi-defined medium as in 10% 
FBS supplemented medium. 
Furthermore, we have also examined the physical nature of the soybean 
lecithin preparations. A size profile of a typical phospholipid 
preparation, where the soybean lecithin is suspended in saline and passed 
three times through a Manton-Gaulin homogenizer, then filtered through a 
0.2 um filter was obtained. The average mean size of the liposomes was 
around 100nm in diameter, suggesting that the majority of liposomes 
resulting from this process are small unilamellar vesicles (SUV's). The 
following experiment indicates that the size of the soybean lecithin 
liposomes may play an important role in the efficacy of the phospholipid, 
i.e., the ability of the SL to cause an increase in FVIII:c expression. 
A soybean lecithin preparation was constituted in saline but not 
homogenized. The preparation contained significantly less SUV's than a 
normal (i.e., with homogenization) preparation (only 44% of the liposomes 
were below 100nms vs 74% in a normal prep). It also contained a 
significant population of multilamellar vesicles (MV's) which were around 
500 nm's in diameter (30-40% of the total liposomes were MV's) which are 
present in only small quantities (usually&lt;5%) in normal preparations. The 
efficacy of this sample was reduced to about 60% of a normal sample 
indicating that the size of the liposomes may play an important role in 
the efficacy of the phospholipid. 
EXAMPLE IV 
Porcine VWF can act to elicit Factor VIII:c activity from CHO cell 
propagated in the absence of serum 
Lig 1 (20 uM MTX) cells obtained as in Example I were rinsed and fed 
defined media (alpha media containing insulin, transferrin, selenium, 
hydrocortisone, and putrescine, glutamine, and penicillin and 
streptomycin) added back with increasing concentrations of bovine serum 
albumin or with similar concentrations of ovalbumin Table II. Both 
proteins can act to elicit Factor VIII:c expression. However, when 
partially purified VWF is added back to media containing 5 g/1 bovine 
serum albumin, the Factor VIII:c activity increased four-fold to even 
greater than the levels obtained upon propagation of the cells in 10% 
fetal bovine serum. This dramatically demonstrates the ability of VWF to 
elicit Factor VIII:c activity in the absence of serum. This result has 
been duplicated with different preparations of porcine VWF and also with 
purified human VWF. 
In order to demonstrate that the ability to elicit factor VIII:c was due to 
VWF, the following experiment was performed. Cells which express Factor 
VIII:c were incubated in the presence of media containing serum derived 
from human VWF deficient plasma. Factor VIII:c activity in the CHO Lig 1 
cells incubated in VWF deficient serum was 25% the level compared to 
normal human serum. When the porcine VWF preparation was added back to the 
VWF deficient serum, the Factor VIII activity increased to the 10% fetal 
bovine serum value. The effect could be elicited with as little as 2.50 
ug/ml of VWF. See Table IIA. In another experiment, when the VWF 
concentration was decreased to 0.25 ug/ml, the activity was only 50% that 
of the 10% fetal bovine serum level. 
The CHO cell line 1E6 which had been adapted over a 2-3 month period to 
grow in the absence of serum, in defined medium, was used in the following 
experiment in order to demonstrate that exogeneous VWF could increase the 
level of rVIII expression in defined medium in the absence of even 
residual amounts of bovine VWF. The data presented below shows that 
supplementation of defined medium with approximately 1 microgram/mL of 
porcine VWF allows expression of rFVIII equivalent to the level obtained 
in 10% fetal bovine serum supplemented medium. Since these cells had been 
grown for more than 3 months in the absence of FBS no trace bovine VWF was 
present. Thus the observed increase in rFVIII:c levels was likely due to 
exogeneous VWF. 
______________________________________ 
Effect of VWF on rFVIII Expression in the Absence of Serum 
MEDIA rFVIII (mU/ml) 
______________________________________ 
Defined Media (DM) 
.about.40 
DM + VWF .about.190 
Media containing .about.200 
FBS (10%) 
______________________________________ 
This data illustrates the increase in the rFVIII activity by exogeneous, 
partially purified VWF (porcine) in the absence of fetal bovine serum. CHO 
cells (1E6 in 0.1 micromolar MTX) were suspended at a concentration of 
3.times.10E5 cells/mL in defined medium containing 5 g/L of bovine serum 
albumin either in the absence or presence of partially purified procine 
VWF (at approximately 1 microgram/mL) or medium containing 10% FBS for 24 
h at 37.degree. C. At the conclusion of the incubation samples of 
cell-free conditioned medium were assayed for rFVIII:c activity by 
chromagenic assay. 
TABLE II 
______________________________________ 
Factor VIII:c Expression in Defined Media with 
VWF Added back to Lig 1 Cells 
Defined Media + Units/ml/day 
______________________________________ 
Ovalbumin (g/l) 
0 0.164 
0.5 0.189 
1.0 0.215 
2.0 0.280 
5.0 0.290 
20.0 0.380 
5.0 
with procine VWF at 2.5 ug/ml 
1.200 
Bovine Serum Albumin 
(g/l) 
0 0.190 
0.5 0.320 
1.0 0.380 
2.0 0.375 
5.0 0.530 
20.0 0.490 
5.0 
with porcine VWF at 2.5 ug/ml 
1.350 
10% Fetal Bovine Serum 
0.978, 1.075 
______________________________________ 
TABLE IIA 
______________________________________ 
Effect of VWF on Factor VIII Production 
MEDIA Mu/ml/day 
______________________________________ 
10% fetal bovine serum 
1321 
defined media with 5 g/l 
342 
bovine serum albumin 
10% normal human serum 
937 
10% VWF deficient human serum 
246 
10% VWF deficient human serum 
with porcine VWF added back at: 
2.5 ug/ml 1124 
20 ug/ml 1397 
______________________________________ 
In order to examine the effect of added VWF on the amount of Factor VIII:c 
in the conditioned media, cells were labeled with a 1 hr. pulse of 35--S 
methionine and chased in either media containing 10% fetal bovine serum, 
10% VWF deficient human serum, or 10% VWF deficient human serum with 
porcine VWF added back. Results demonstrated that upon addition of VWF to 
VWF deficient serum, more Factor VIII:c (both the heavy 200 kDa and the 
light 76 kDa chains) was present in the media. No change in the 
intracellular synthesis of Factor VIII:c was observed. VWF addition to 10% 
fetal bovine serum resulted in no change in the level of Factor VIII:c in 
the conditioned media. These experiments indicate the VWF is necessary for 
the secretion and/or stability of Factor VIII:c. 
EXAMPLE V 
Expression of Human VWF in COS Cells 
The cloning of a partial segment of the human VWF cDNA has previously been 
reported (Ginsberg, et al. 1985, Science). Subsequent to that report, the 
full length VWF cDNA has been assembled and its sequence determined. The 
cloning, sequence and expression of VWF have been described in detail in 
International Application No. PCT/US86/00760, published on 23 October 
1986. We have inserted the full length cDNA clone into the expression 
vector pMT2 to produce pMT2-VWF (ATCC No. 67122). pMT2-VWF contains the 
adenovirus associated (VA) genes, SV40 origin of replication including the 
transcriptional enhancer, the adenovirus major late promoter including the 
adenovirus tripartite leader and a 5' splice site, a 3' splice site 
derived from an immunoglobulin gene, the VWF coding region, a non-coding 
DHFR insert, the SV40 early polyadenylation site, and the pBR322 sequences 
needed for propagation in E. coli. Details of this vector, which is a 
derivative of pQ2, are provided in Kaufman, Proc. Natl. Acad. Sci., USA 
82:689-693 (1985). pMT2-VWF DNA was then prepared for COS cell 
transfection by conventional methods. Sixty hours after DEAE dextran 
mediated transfection of COS cells, the cells were labelled with 35-S 
methionine and media and cell extracts were immunoprecipitated with a 
rabbit anti-human polyclonal antibody (Calbiochem) and precipitates 
analyzed by SDS reducing gel electrophoresis. Our results demonstrated a 
significant amount of VWF is synthesized in the transfected COS cells, the 
majority of its being secreted. In the conditioned media there is an 
approximately 260 kDa protein and a 220 kDa protein which resembles the 
completely processed form of VWF. Approximately 50% of the VWF synthesized 
is processed to the 200 kDa form. When analyzed for multimer formation by 
non-reducing gel electrophoresis, it was found the VWF was associated into 
multimers, but not of extremely high molecular weight like those seen in 
plasma. The multimers ranged up to 1 million daltons by a rough estimate. 
Analysis of the VWF antigen in the COS cell conditioned media indicated 
the presence of human VWF at 0.35 ug/ml. Other analyses have indicated 
that the VWF expressed in COS cells specifically binds both platelets and 
collagen. 
EXAMPLE VI 
Recombinant VWF can elicit the expression of human Factor VIII:c 
The VWF expression plasmid pMT2-VWF was transfected onto COS cells by DEAE 
dextran mediated transfection and 36 hours post-transfection, the media 
changed to serum free (DMEM lacking serum). 72 hours later the COS cell 
conditioned media was harvested and applied to the CHO Lig 1 cells (20 uM 
MTX resistant) which were previously rinsed with serum-free media (at 
10.sup.6 cells/ml). Twenty-four hours later the media was taken from the 
CHO cells and assayed for Factor VIII activity. The results are shown 
below and compared to Factor VIII:c activities from CHO cells propagated 
in 10% fetal bovine serum and in serum-free media for 24 hours. These 
results demonstrate the ability of rVWF to elicit Factor VIII from CHO 
cells. 
______________________________________ 
Media on CHO Lig 1 (20 uM MTX) mU/ml Factor VIII:c 
______________________________________ 
Conditioned media from mock 
141 
transfected COS cells 
Conditioned media from VWF 
423 
transfected COS cells* 
10% Fetal Bovine Serum 
950 
Serum-free media 30 
______________________________________ 
*The conditioned media in this experiment contained 0.3 ug/ml of human 
VWF. 
EXAMPLE VII 
Introduction, Expression, and Amplification of VWF Genes in CHO Cells which 
express Factor VIII:c 
For expression of VWF in Chinese hamster ovary (CHO) cells, a second 
expression vector, pMT2ADA-VWF (ATCC #67172), was used with a protocol of 
selection for cells over-expressing the enzyme adenosine deaminase to 
amplify the plasmid sequences (Kaufman et al., 1986, Proc. Natl. Acad. 
Sci. 83:3136; U.S. Ser. No. 619,801). A factor VlII:c expressing cell line 
which was cloned from ligl 2-a (from example I) in 1 mM MTX and designated 
10A1, was used as recipient for transfer of pMT2ADA-VWF. pMT2ADA-VWF was 
introduced into 10 A1 cells by protoplast fusion as described 
(Sandri-Goldin et al., 1981, Mol. Cell. Biol. 1:743). E. coli DH5 cells 
harboring pMT2ADA-VWF (DH5 was used to minimize homologous recombination 
and deletion of the VWF sequences) were grown in 50 ml of L-broth 
containing 50 ug/ml ampicillin to an A.sub.600 of 0.6. Chloramphenicol was 
added to 250 ug/ml and the culture incubated at 37.degree. C. for an 
additional 16 hrs, in order to amplify the plasmid copy number. A 
suspension of protoplasts was prepared as described (Sandri-Goldin et al., 
1981), added to 10A1 cells at a ratio of approximately 1-2.times.10.sup.4 
protoplasts/cell, and centrifuged onto the cells at 2000 rpm for 8 minutes 
in an IEC model K centrifuge. After centrifugation, the supernatant was 
removed by aspiration and 2 ml of polyethylene glycol solution (50g of PEG 
1450, Baker Chem. Co., in 50 ml of Dulbecco's modified medium) was added 
to each plate. Cells were centrifuged again at 2000 rpm for 90 seconds, 
the polyethylene glycol solution removed, and the plates rinsed 3 times in 
alpha medium containing 10% (v/v) fetal calf serum. Cells were then plated 
into tissue culture dishes in medium containing 100 ug/ml kanamycin, 10 
ug/ml each of penicillin and streptomycin, and 20 uM MTX. Two days later 
the cells were trpysinized and subcultured 1:15 into ADA selective media 
with 10% dialyzed fetal calf serum, 0.1 um deoxycoformycin, 10 ug/ml of 
penicillin and streptomycin, and in the presence and absence of 20 uM MTX. 
The ADA selective media (AAU) contained 1.1 mM adenosine, 10 ug/ml 
alanosine and 1 mM uridine. Subsequently it was shown that removal of the 
MTX selection at this stage resulted in a decrease in the factor VIII:c 
expression. Subsequently, the MTX has been left in the ADA selective 
media. 
It was possible to amplify the VWF gene by selection for growth in 
increasing concentrations of 2'-deoxycoformycin (dCF) in the presence of 
cytotoxic concentrations of adenosine. A pool (3-a) of transformants (6 
colonies) was prepared from 10A1 cells and selected for ADA in the 
presence of 20 uM MTX. The ADA selection mean was changed by sequentially 
increasing the concentration of 2'deoxycoformycin (steps of 0.1 uM, 0.5 
uM, 1.0 uM and 2.0 uM) in the presence of 20 uM MTX. At each step, the 
production of VWF and of factor VIII:c was measured after 24 hours in the 
presence of 10% fetal calf serum (FCS) or in defined media. The results 
are summarized below: 
______________________________________ 
Coxpression of VWF and FVIII:c in CHO cell lines 
Factor 
Selection VWF Antigen VIII:c 
Cell line 
dCF uM MTX uM ug/ml pg/cell 
uUnits/cell 
______________________________________ 
10A1 .38* 
(no VWF) 0.93** 
10A13a 0.1 20 0.07 0.1 
pool 
0.5 20 0.8 1.14 0.63* 
0.89** 
1.0 20 24 30 0.63* 
1.1** 
2.0 1000 7.4 24 1.4* 
1.5** 
______________________________________ 
*in defined media; **in media containing 10% Fetal calf serum; VWF antige 
was determined by an ELISA assay using affinitypurified rabbitanti-VWF 
antiserum (CalbiochemBehring, 782301), purified VWF antigen from normal 
human plasma pools to serve as standards and controls, and IgG isolated 
from CalbiochemBehring, 782301, and conjugated with alkaline phosphatase. 
Factor VIII:c activity was determined by the chromogenic assay described 
in Example I. 
These results demonstrate that VWF expression increased with increasing ADA 
selection. In addition, expression of factor VIII:c was not dependent on 
the presence of serum, as observed by line 10A13-a in 2 uM dCF and 1000 uM 
MTX which expresses 1.4 uUnits/cell/day of factor VIII:c in defined media. 
EXAMPLE VIII 
Fusion of CHO cells expressing Factor VIII:c and CHO cells expressing VWF 
The VWF gene has been introduced into CHO DHFR deficient cells (DUKX-B11, 
Chasin and Urlaub, 1980, Proc. Natl. Acad. Sci. 77:44216). Two approaches 
have been taken in order to obtain cells that express either MTX 
resistance or dCF resistance associated with VWF expression. Then either 
cell line can be subsequently used to fuse to other cells that express 
factor VIII:c with the ability to select for either MTX or dCF resistance. 
MTX Amplification in CHO DHFR deficient Cells 
Plasmid pMT2VWF and pAdD26SVpa(3) were mixed 10:1 and transfected by 
CaPO.sub.4 coprecipitation into CHO DUKX-B11 cells as described by Kaufman 
and Sharp (1982, J. Mol. Biol. 150:601). Cells were selected for the DHFR 
positive phenotype by growth in the absence of nucleosides and colonies 
pooled and selected for increasing MTX resistance. The results indicated 
that VWF expression increased with increasing MTX resistance and are 
depicted in the Table below: 
______________________________________ 
Selection ng/ml VWF 
______________________________________ 
0.02 uM MTX -- 
0.2 uM MTX 56 
1.0 uM MTX 91 
5.0 uM MTX 278 
______________________________________ 
dCF Selection for VWF in CHO DHFR Deficient Cells 
The plasmid pMT2ADA-VWF was introduced into CHO DUKX-B11 cells as described 
in Example VII and cells selected for growth in ADA selective alpha media 
with 4 uM xyl-A, 0.03 uM dCF, 10 ug/ml hypoxanthine, 10 ug/ml thymidine, 
and 10 ug/ml of penicillin and streptomycin. One clone PM5F was derived 
which expressed 3-5 pg of VWF/cell/day. This clone was subsequently used 
for fusion to factor VIII:c cell lines and as a recipient for the 
introduction of factor VIII:c genes. 
Fusion of Factor VIII:c-type and VWF Expressing Cel Lines 
The factor VIII:c-type expression plasmid pLA2 has been described (pLA2 
contains a transcription unit for a procoagulant B-domain 880 amino acid 
deletion mutant of FVIII:c, see International Application No. 
PCT/US86/00774). This plasmid has been introduced into DUKX-B11 CHO cells 
by protoplast fusion with selection for DHFR from the 3' region of the 
factor VIII-DHFR transcript (See PCT/US86/00774). A cell line was derived 
by selection for MTX resistance to 1.0 uM MTX and has been named LA3-5. 
This cell line expresses a deleted form of Factor VIII:c at 3-5 
uUnits/cell/day (in 10% fetal calf serum). This modified factor VIII:c 
also binds to and requires VWF for efficient synthesis. LA3-5 was fused to 
PM5F and hybrids were selected that expressed both the MTX resistance and 
dCF resistance phenotypes. 
For fusion, PM5F was treated with diethylepyrocarbonate (DEPC, 0.03% for 30 
minutes on ice) in order to kill the PM5F. These cells were then fused by 
polyethylene glycol-induced cell fusion to LA3-5: DEPC treated PM5F cells 
were centrifuged onto LA 3-5 (1.5.times.10.sup.6 cells) at 2000 rpm for 8 
minutes in an IEC model K centrifuge. After centrifugation, supernatant 
was removed and 2 ml of 50% PEG solution was added. PEG was left on for 45 
seconds and cells were washed thoroughly with serum free medium. Cells 
were left plated with medium containing serum for 48 hrs. and were then 
subcultured into selective medium containing 4 uM xyl-A, 0.03 uM dCF, in 
the presence of 10 ug/ml of each of the following: thymidine, 
hypoxanthinine, streptomycin, and penicillin. However, it was not 
necessary to include the thymidine and hypoxanthine. A pool of hybrids was 
obtained which expressed 0.73 pg/cell/day of VWF and 0.2--2.0 units/ml/day 
of the factor VIII:c-type protein. The pool was subsequently grown in the 
absence of thymidine and hypoxanthine in the presence of 0.5 uM MTX. These 
cells were cloned in alpha media with 4 uM xyl-A, 0.03 uM dCF, and 0.5 uM 
MTX to obtain the following clones: 
______________________________________ 
Coexpression in CHO Cells of VWF and a Factor VIII:c-type 
Protein 
VWF Expression Factor VIII:c-type Expression 
Clone (pg/cell) (uUnits/Cell-media) 
______________________________________ 
E6 16 2.8 - defined 
3.8 - 10% FCS 
B9 20 4.5 - defined 
5.1 - 10% FCS 
H6 34 7.7 - defined 
8.7 - 10% FCS 
G12 8. 10.5 defined 
11.8 10% FCS 
______________________________________ 
These results demonstrate the ability of the cell lines coexpressing VWF 
and the Factor VIII:c-type protein to produce high levels of the Factor 
VIII:c-type protein in defined media. 
EXAMPLE IX 
Introduction of Factor VIII:c-type Genes into Cells Expressing VWF 
A factor VIII:c deletion mutant of 907 amino acids has been constructed by 
heteroduplex mutagenesis (PCT/US86/00774) which directly fuses the 90 kDa 
cleavage site (at residue 740) to the 76 kDa cleavage site (at 1647). The 
resultant plasmid p90-76R has the appropriate Factor VIII:c-type 
transcription unit in pMT2. Protoplasts of E. coli HB101 harboring this 
plasmid were prepared and fused to the VWF expressing cell line PM5F as 
described in Example VIII. 48 hrs after recovery from protoplast fusion, 
the cells were subcultured into DHFR selection media (alpha media lacking 
nucleosides with 10% dialyzed fetal calf serum, 4 uM xyl-A, and 0.03 uM 
dCF. After two weeks, transformants were isolated and assayed for Factor 
VIII:c expression. Approximately 20% of the transformants which had arisen 
expressed both VWF and the Factor VIII:c deletion mutant. Results for one 
transformant are indicated below: 
______________________________________ 
Cell Line 
Factor VIII:c Activity 
Media 
______________________________________ 
F1 1.5 uUnits/cell (1375 mUnits/ml) 
def. media 
0.95 uUnits/cell (1330 mUnits/ml) 
10% FCS 
______________________________________ 
(with VWF = 1.69 ug/ml, 1.85 pg/cell) 
These results demonstrate the ability to select for the DHFR phenotype in 
the PM5F cell line and to coexpress a factor VIII:c-type protein and VWF 
in order to alleviate the serum dependence for factor VIII:c expression. 
EXAMPLE X 
Accumulation of Factor VIII:c-type Proteins in the presence and absence of 
VWF coexpression 
The accumulation of Factor VIII:c-type proteins over 3 days was determined 
by rinsing Factor VIII:c-type expressing cells and then adding back media 
containing 10% fetal calf serum (FCS) or defined media containing insulin, 
transferin, selenium, bovine serum albumin (5 g/l) as above in Example 
III. Factor VIII:c assays were then conducted 24, 48 and 72 hrs later. 
Results are shown below for four cell lines. The Chinese hamster Factor 
VIII:c expressing cell line 10A1 was described in Example VII. C6 is a 
subclone of the 10A13a pool which coexpresses human recombinant VWF and is 
selected in 1 mM MTX and 2.0 uM dCF from Example VII. The LA3-5 clone and 
the VWF coexpressing cell line G12 express a deleted form of Factor VIII:c 
and have been described in Example VIII. These results demonstrate the 
ability of the coexpressing cell lines to accumulate very high levels of 
Factor VIII:c-type proteins in either serum-containing or defined media 
compared to the original cell lines. 
______________________________________ 
Factor VIII Activity 
mUnits/ml Total 
Cell Line 
Media 24 hr 48 hr 72 hr uUnits/cell 
______________________________________ 
Wild-type Factor VIII:c: 
10Al FCS 736 558 414 0.3 
defined 309 117 70 0.06 
C6 FCS 796 2976 5170 5.9 
defined 531 1928 2980 3.2 
Deleted Factor VIII: 
LA3-5 FCS 1198 596 374 0.5 
defined 341 128 163 0.2 
G12 FCS 3527 5420 6380 22.0 
defined 3018 4400 4110 13.0 
______________________________________ 
EXAMPLE XI 
Similar if not improved results relative to those obtained in the preceding 
examples involving use of recombinant VWF should be obtained by those 
practicing this invention by substituting a VWF-type protein for the 
recombinant wild-type VWF. This may be readily accomplished by repeating 
the procedures using an expression vector directing the synthesis of a 
desired VWF-type protein instead of the wild-type VWF. Such vectors may be 
produced by any of the numerous procedures known in the art, or perhaps 
more conveniently, by conducting oligonucleotide-directed mutagenesis as 
desired upon the wt VWF-encoding vectors disclosed above.