Note: Descriptions are shown in the official language in which they were submitted.
~, WO 94/11525 214 ~ 21 2 PCr/DK93/00375
Improved recombinant production of proteins
having factor VIII:C activlty.
TECHNICA~ FIELD ~ .
~: :
This invention relates to a method for effecting an increased
expression o~ recombinant proteins, especially proteins having
5 Factor VIII:C activity.
: : ,
BACKGROUND OF THE INVENTION : ~ :
Hemophilia A is an X-chromosome-linKed~inherited disease~which
a~flicts 1-2 males per 10l000. The desease is caused by an
absence of deficiency of Factor VIII:~C.~Factor VIII:C lS a very
10 large glycoprotein (native Mr;330 K~ 360 K),~which is present
in plasma at extremely low concentrations~.; It is a necessary
j element in the proteolytic cascade ~which~ converts soluble
1` ~ibrinogen to insoluble fibrin, forming~;a clot to prevent blood
¦ loss from traumatized tissue. In the bloodstream, it is found~
1 15 in noncovalent assoaiation with von~ ~illebrand factor (vWF)j
which acts as a stabilizing carrier protein.~Faator~ VIII:C~is
very susceptible to aleavage by`thrombin~ plasmin, activated
protein C, and other serine proteases.~It is generally isolated
from~plasma or plasma products as a series~of~related polypep-~
20 tides ranging from Mr l~0 K-40 K with predominant species f~Mr
92 K and Mr 80~ K-77 K:. This aomplex;pattern has made~ the~
analysis of the stru:cture of active Factor;VIII:C;~Very di~ffi-
cult.
Factor VIII:C and the related polypeptides have been described25 by P. ~Rotb~at et~al,~Biochemistry (~1985)~24:42g4-4300;~ G.~A.
Vehar et al, Nature~ (1984) 312:~33~7-342~; J.J.~ ~Toole et~al,
Nature ~(1984) 31~2;:342-347;~and~N.A~.~Truett et~al,~ DNA (1985)
` 4:333-349. E. Orr ~;~et al,~ ~Nol;ecular Genetlcs of_ Clottin
Factors, p. 54, s~321.~;The sequence~;has been~reporte~ by J.J.
30 Toole~et al, supra; W~ Wood et~al,`;Nature~(1984) 3~12:330-33~;
l.
! WO94/11525 ~1 ~ 921 2 PCT/DK93/0037~ ~
and M.A. Truett et al, supra. The full-length protein contains
three repeats of~one sequence (I), and two repeats of a second
sequence (III). A third, heavily glycosylated sequence (II) is
present between the second and third I repeats, and is ap-
S parently cleaved proteolytically;to form~the Mr 92 K and Mr 80K polypeptides. The first two I ~repeats~form the A domain,
while the third I repeat and~the two~ repeats form ~the C
domain. The II sequence forms the~ B~domain. Thus, the full-
length protein has the structure Il-I2~ I3~ III2 (A-B-C),~
lO while the Mr 92 K and Mr 80 K polypeptides (A and C) have the
structures Il-I2 and I3~ -III2, respectively. R.L. Burke et
al, J Biol Chem (1986), have~shown by expression o~f the 92 K
and 80 K polypeptides that both peptides are necessary ~for
Factor VIII:C activity. ~ ~
15 Factor VIII:C has historically been isolated from blood in a
concentrated form for~ therapéutic~ treatment of hemophilia.
However, concerns regarding transmission~of HIV~ and~ other
blood-borne diseases have stimulated~activity to provide
alternative supplies of Factor VIII:C. It is~of~substantial
0 interest to be able to supply composit~ons having Factor VIII:C
activity without concerns as to the transmission viral diseases
associated with the native Factor VIII:C.
The recombinant proteins having Factor~VIII:C activity which
are prepared accord-ing~to the~present invention may~be~full
25 length Factor`VIII:C corresponding~to the~prote;in~isolated~;from
plasma, or a derivative thereof having~ the~capability~of
normalizing the insufficient blood clotting caused by deficien-
cy of Factor VIII:C. The derivatives of Factor VIII:C may be ~ ~y
shortened sing~le~chain~ forms;~ or~derivatives comprising~two
; ~ 30 chains. Even fragmen~s of Factor VIII:C~which may not~per se
show coagulant activity,~but~which~may~be~used in~the treatment ~;~
of haemophi1iàcs~e.g.~ for saturàtion~of~antLbodies against Fac~
tor~VIII:C present~in inhibitor patients.~
The prote-ns produced in accordance w1th he present inven~ion~ ~,
WO94/11525 2l 4 921 2 PCT~DK93~003~5
show homology with all or a part of the natural Factor VIII:C
molecule. .
The preparation of rec~mbinant proteins having Factor VIII:C
activity by recombinant techniques has inter alia been dis- ;"
5 closed in a num~er of publications. Thus, European Patent .
Application No. 160 457 and International Patent Application
No. WO 86/01961 disclose the production of full len~th Factor
~III:C, and European Patent Application No.;EP 150 735, Inter-
national Patent Application No..WO 86/06101, European Patent
10 Application No. EP 232 112, International Patent Application
No. WO 87/04187, International Patent:Application No. W0 87/
07144, International Patent: Application ~No. ~ WO 88/00381, .
European Patent. Application No. EP 251~843, European Patent
Application No. EP 253 455, U.S. Patent No. 4.980.456, European
15 Patent Application No. EP 294 910, European Patent Application
No. EP 265 778, European Patent Application No. EP 303 540,
International Patent ~pplication No. WO 91/07490, and Inter-
national Patent Application No. W0 91/09122 disclose recombi-
nant expression of shortened single chain forms or subunits of
20 Factor VIII:C or co-expression of~ sub~hits for the production
of complexes showing coagulant activity or binding affinity to
antibodies inhibiting Factor VIII:C. : :
Expre~sion of recombinant full-length human Factor VIII:C LS
usually low and the molecure is unstable due to proteolysis. ~: :
25 The derivatives of Factor VIII:C in the: form~ of shortened i~
~;single chain forms or derivati~es compris~ing two .chains have
also been successfully :produced by recombinant~ techniques.
~lthough these derivatives normally are:expressed in a higher
:yield ~han full-length Factor;VIII:C, there ls still a desire
30 for increasing the~level of express~ion.~
~:: : It is described in:W0 87/04187 and~EP:251 843 that expression
of Factor VIII:C in:the:~presence of~von Willebrand Factor (vWFj
or phospholipide increases the~ expression of Factor VIII:C.~In .
WOg4/11525 21 4 321 2 PCT/DK93/00375
EP 441 6~5 it is disclosed to express Factor VIII:C or an
analogue thereof in the presence of a cationic or anionic
polymer, preferably a polysaccharide which most preferred is in
a sulphatizes form. Among other additives heparin was tested in
5 an amount of lO to 80 IU/ml. The addition of heparin only
"showed a very limited to no effect" on the expression of
Factor VIII delta 2 having a deletion of~amino acids 771 to
1666, as disc}osed in EP 303 540 as compared~to serum free
medium. The expression level is slightly increased with
10 increased concentration of heparin.~ However,~ the expression
level is far below the e~pression level in the presence of
serum or vWF.
,
DISCLOSURE OF THE INVENTION
The present invention relates to a ~method~ for effectlng an
15 inareased expression of recombinant proteins having Factor
VIII: C activity in a host cell being able to express said
protein comprising culturing said host i a cell` growth medium
comprising heparin in a very low concen~ration below~lOIU/ml so
as to express said protein.
:
20 DBTAI~ED DESCRIPTION~OF THE INVENTION
It has surprisingly been found that the addition of hepar~in in
small amounts below lOIU heparin/ml increases the expression~of
recombinant proteins showing ~Factor VIII:C activity and
stabilizes the product to an extent leading to an increased
25 yi~ld of more than 50%. The stabilizing~effect of heparin is
indicated through the observed reduction of the activation of
the 92 kD subunit by proteases.~ Such addition may,~according;to
the invention, be made;~to cell growth medium comprising serum,~
preferably in the form of fetal~or new born calf serum, and to
30 serum free medium comprlsing~lipoprotein vWF or phospholipid
or other additional constituents used for increasing the
: ~ : i
21~9212
WO94/11525 PCT/DK93/00375
expression in serum free media in order to obtain the improved
expression.
The lipoprotein used in the method of the invention may e.g. be
lipoproteins as describad in EP 254076. Such lipoproteins are
5 commercially available under the Trade Mark EX-CYTE. The
lipoprotein may also be isolated from egg yolk, for example by
the method described in Immunological Communications 9 (5),
475-493 (1'~80).
,.
Phospholipids used in the method of the Lnvention may e.g. be
l0 such phospholipids as described in~WO87/04187.
Other additional constituents used for increasing the
expression in serumfree media may e.g. be an egg yolk fraction
being free of lipoprotein and lipids.
According to a preferred aspect of the invention, heparin is
15 added to a concentration of from 0.5 to 8 IU/ml, a concentra-
tion of from 1 to 2 IU/ml being most preferred.
¦ The addition of heparin to a cell growth medium comprising
:
serum is a preferred aspect of the invention giving rise to a
considerable increase of the Factor VIII:C level.
1 : :
20 Still more preferred in the addition of heparin to a cell
growth medium comprising serum and further added lipoprotein
giving rise to an extremely high level of Factor VIII:C on
expression in suspension.
i
Ac~ording to another preferred aspect of the invention, serum
25 free cell culture medium is used in order to avoid the addition - ~;
of constituents which might give rise to~transference of blood ~-borne diseases, such medium being supplemented with lipopro- ~3
tein, von Willebrand Factor, phospholipid, or a combination~of
two or more of these. ~ ;
.~ .
i~
:: :
.
WO94/11525 2 ~ 2 PCT/DK93/00375 ~ ¦
In accordance with a more preferred aspect of the invention
lipoprotein and heparin are added to a serum free medium. In a 1 ~"
preferred embodiment about 5% of lipoprotein fraction and about , ~;
2IU of heparin are added giving rise to a very high expression
5 of Fator VIII:C in the term of expressed Factor VII~:C ac-
tivity,
According to another preferred aspect of the invention, heparin
is added to a growth medium for culturing a host cell for
expressing a complex of the 92kD and 80/77kD subunits of Factor
lO VIII:C. For such cultivation heparin does not only increase the
level of expression of;the i~dividual subunits of Factor
VI}I:C, but also increases the degree of complex formation and
stabilizes the produced complex and thus, the yield of product
showing coagulant activity. ~ I;
.,
lS The term "A domain" refers to that portion of human Factor
VIII:C which constitutes the Mr~92 K protein~subunit or the
Factor VIII:C heavy chain (FVIII-HC). The A domain contains
from about 740 to about 760 amino acids,~and is found at the N-
terminus of the native human Facto~ VII}:C. Of particular
20 interest is an N-terminal chain having the entire se~uence to
the thrombin cleavage site at~Arg740-Ser741.
.
The term "B domain" refers to that portion of native human
Factor VIII:C which is generally removed by intracellular
cleavage, and which is heavily glycosylated in human plasma and
25 when expressed in mammalian cells such as COS7, CHO and BHK
cells. The B domain contains an N-terminal sequence, which
.,
allows cleavage of the A domain from the B~domaln by thrombin.
The B domain also has a~C-termlna1 processing site~which allows
cleavage of the~C domain from the A-B precursor~by an enzyme
30 located in the Golgi apparatus~of~the~mammalian cell. ~`t
'
~ The term "C domain" refers~ to;that portion of native human t`
1~;Factor VIII:C which constitutes ~t~he C-terminus of the ful1
~length protein,~and ~is~ F1ea~;-d intracel1ularly to form the
~ WO94/11525 2 1 4 9 2 t Z PCT/DK93/00375
Factor VIII:C light chain (FVIII-LC). The light chain will have
an amino acld sequence substantially the same as the amino acid
sequence of the C-terminus of a Factor VIII:C polypeptide,
usually at`least about 80%, more usually at least about 90% of
5 the Factor VIII:C Mr 80 K chain, particularly beginning with
amino acid 1640, preferably at about amino acid 1649, +l0 amino
acids, more particularly +1 amino acid, and continuing to at
least about amino acid~ 2300, usually 23l0, +lO amino acids,
preferably 2325, +5 amino acids, ;more preferably to the
l0 terminal amino acid (2332). Usually, the light chain will have
at least about 85%, more usually at least 95%, of the III1-III2
domains, desirably the I3-IIIl-III2~dom`a~ins.
~ ,
The term "co-expressing" as used~hereinirefers to simultaneous
expression of an A domain polypeptide (92 K)land a C domain
lS polypeptide (80 K) within the sam~e; host cell. The poly- !:
nucleotide sequences encoding the A and C domains~may be on the
same or on different expression cassettes or plasmids. Co-ex-
pression of the A and C domains permits proper~folding to oc-
cur, which in turn provides an A-C complex having activity and
20 efficiency of secretion.
The term "production medium" as~used herein ~refers to any
medium suit~ble for culturing host;cells,~ and includes media
suitable for obtaining expression of~ recombinant products
whether or not actual cell "growth"~ occurs. Production~media
2S generally include nutrients and a metabolizable energy s~ource
in an aqueous solution~. If desired, production media may~ also
include a compound which induces expression of the recombinant
polypeptides of the invention. Selection of such an inducing
compound~depends upon the promoter selected to control expres-
; ~ 30 sion. Other typical additives include ~selection compounds
(i.e., drugs or other~chemicals added~to the media to insure
;~ ~ that only transformed~ host~cells sUrYiVé in the medium) and ~7
serum, such as ~eea1 bovine~serum (F~5).
; The term "serum-f~ree medium";is a solution which has been sup-
~`
WO94/11525 PCT/DK93/00375 ~ ~
21~9~,12 8
ple~ented to such an extent that the necessary trace factors
present in seru= need not be added in the form of serum. The
serum ~ree medium may be a synthetical medium not comprising
components isolated from animal tissues or body fluids. There
5 are many suitable cell growth media available from commercial
sources.
The term "IU" as used herein in connection with heparin is
defined by standardization against the 4th International
Standard for Heparin (code-labelled 82/502) prepared by the
lO National Institute for Biology and Standard and Control. London
UK.
The term "homology" as used herein mqans identity or sub-
stantial similarity between two polynucleotides or two~poly-
peptides. Homology is determined on the basis of the nucleotide
15 or amino acid sequence of the polynucleotide or polypeptide. In
general terms, usually not more than lO, more usually not more
than S number~, preferably not more than about~l number% of the
amino acids in the chains will differ from the amino acids
naturally present in the Factor VIII:C A and C domains. Par-
20 ticuIarly, not more than a~out 5%, more usually not more than
about 1% will be nonconservative substitutions. Conservative
substitutions include~
Gly ~ Ala; ~ Lys ~ Arg;
Val ~ Ile ~ ~eu; ~ Asn ~ Gln; and
Asp ~ G1u; ~ Phe ~ Trp ~ Tyr.
Nonconservative changes are generally substitutions of one of
;j the above amino acids with an amino acid from a~different group
(e.g., substituting Asn for GIu), or substituting Cys, Met, t
His, or Pro for any of the above amino acids.~ J
~ 30 The specific activity of a protein complex~prepared according
;~ to the invention may~be determined by;~m~eans known in the art,
as described below (e.g~ by uslng the commercial~y available
Coatest assay).
~ t
. ~ :
: ~3
~ WO94/11525 2 1 4 9 2 1 2 PCT/DK93/0037s
The structural genes typlcally incIude a leader sequence codingfor the signal peptide which directs the polypeptide into the
lumen of the endoplasmic reticulum for processing and matura-
tion. OptionalIy included are ~additional sequences encoding
5 propeptides which are processed post-translationally by
endopeptidases, where the endopeptidases~cleave a peptide bond,
removing the propeptide to generate the mature polypeptide. The
signal peptide may be the naturally occurring one, particularly
for the N-terminal peptide, or may be;any signal~peptide~which
10 provides for the processing and maturation~o;f the polypeptides.
Various mammalian host cells may be employed in which the
regulatory sequences~and rep~ication system~are functional.
Such cells include COS7 cells, Chinese ~hamster ovary (CHO)
cells, mouse kidney cells, hamster~kidnèy cells, HeLa cells,
lS HepG2 cells, or the like, e.g VE~O cells, W-138 or MDCK cell
llnes.
The expressed product can be purified by affinity chromato-
graphy using antibodies, particularly monocl~onal antibodies
~ directed against the FVIII-~C or FVII~HC, chromatography~, e.~g.
1~ 20 HPLC, electrophoresis, or;extraction.
~ The subject method provides for production of a complex o~f the~
¦~ active chains (92` X and 80 K) which has Factor VIII:C activity.
Production is evidenced by conditioned~;~media~as described~in~
the experimental~section, which~will ~have at ~least about 1
25 usually at least about 5~U/mL,~ more usually ~at least about i0
U/mL of Factor VIII:C activity;in~the Coatest assay.
The proteins having Factor~VIII:C;activ~ity~produced according~
I to the invention are~ primarily intende~d~ far treatment~of~
hemophilia¢s and~ patien~s~suffering ~from other~ conditions ;
30 involving blood clotting~disorders.~The~;subject proteins may be
administered ~in ~physiologically~acceptable carrier, such~as
water, saline, phosphate buffered saline, and citrate buffered
sal1ne,~at concentratlons~in the~range of about`10-200 U~mL.
WO94/11525 21 a 92~ 2 PCT/Dh93/00375
' 10
See U.S. Patent Nos. 3,631,018: 3,652,530, and 4,069,216 for
method~ of administration and amounts. Other conventional addi-
tives may also be included. They also have a variety of uses as
immunogens for the production of antibodies,~for isolation of
5 von Willebrand ~actor by affinity chromatography and in
diagnostic assays for Factor VIII:C.
:
BRIEF DE8CRIPTION OF THB DRAWING8
The invention is described with reference to the drawings on
which ~ :
l0 Fig. l shows a titration of the heparin effect on level of
Factor VIII:C units in suspension culture, and
Fig. ~ shows a qel illustrating;the stabilizing effect of
heparin on Factor VIII-HC
: ~ : ;
`
The examples presented below are provide~ as:a further guide to
15 the practitioner o~ ordinary skill in the art, and are:not to
be construed as limiting the invention in any:way. ~
: ~ :: :
!
EXPERIMENTAL PART ` ;~
: : : :
: ~ .
MATERIALS AND METHODS
Heparin from DAK, Denmark, was us~ed:for~all exp~eriments. ~
20 Lipoprotein was isolated~ from~:egg~ yolk ;in the form of a .-!
~:fraction being rich in lipoproteln. The fractionation was
carried out as disclosed in:detail in Example 6.
:
i:PEG 6000 fram Merck, Catalogue No.~ 807491 was used for the
~ W094~11525 21 4 9 212; PCT/DK93/00375
11
fractionation of egg yolk.
PBS used for the fractionation~of~ egg yolk was made by dis-
solving 8.0 g of NaCl, 0.2 g of KCl,~1.15 g of Na2HP04 and 0.2
g of KH2P04 in deionized water, adjusting the pH to 7.3 by
5 adding lM HCl/NaOH and adding deionized water ad l liter. All
chemicals were from Merck. ;
.
` ~ :
: ~ :
EXAMPLE 1
PROVIDING CELL LINES Co-EXPRESSING~FACTOR VIII:C HE~VY~ CHAIN~
AND LIGHT CHAIN ` ~ `
lO Transfection-procedure
The DHFR CHO cell line~M44 (G.~ Urlaub~ et~al~., Som Cell MQ1 !
Genet (1986) l2:555_566)`was first transfeoted~with the plasmid
~ pCMF8-80AT: In this plasmid the CMV promoter (described;~in
¦~ exampIe 7 of W09l/07490) transcribes the;FVIII-LC cDNA derived
15~rom pSVF:8-80AT (described in example~6~of WO9l/07490) land
` d`ownstream`is placed the Ad-M~P/dhfr~`casette;~erived from pAd- ~
DHFR (described in example 4 of W09l/07490).~The transfection `
method used was the polybrene method~of W~ C~aney et al.~ (Som
Cell Mol Genet (198~6) l2:237-244)~. By;selection of DHFR' cells~
~` 20`(DMEM;+ 10% DFCS)~several FVIII-LC producers were~isolatedi one
of these;was designatedl~llW.
In order to introduce~ FVIII-HC in~ llW the ~cell line~ was
I
cotransfected with~the p~lasmid pPR78 ~this plasmid~is an analog~
to pCMVF8-80AT, but harbours instead of the~FVI~I-LC cDNA the
25 FVIII-NC cDNA de`rived from~pCMVF8~-~9~2R~described in example~8 of I ~ '
WO9l/07490~) and pSV2-neo~(P.J. Southern and~P.~Berg, J~ol Appl ~-
~ ; Genet (1982)~ l:327-341)~ The transfection~method used was the
;~ modified~calcium~phosphate~proaedure~o~f;C.~Chen and H. Okayama ~ ~1
(Mol ~Cell Biol~(198~7)~;7~ 274;5-275`2~)~. Transfectants ~were
3~0~isolated~in medium~containing 700~ Geneticin (G41B Sulphate,~ ~-
Gibco)~ p~er~ml.;Cel1s ~rom the~primary pool were subcloned by
WO94/11~25 ~ PCT/DK93/~0375 ~ ~
~1~3~2
12
the limited dilution method and the individual clones were
tested ~or expression fo active FVIII. In this way several
FVIII:C producing cell l~ines wère isolated and two of these
were designated "45"~and "57"j respectively.
5 The cells selected in this way on the basi~s of the expression
level were seeded into T-flasks or spinners~for cultivation in
the absence of heparln or in the presence of heparin in various
concentrations. ~
; The description of transfectlon referred to in WC9l/07490 is
~0 hereby incorporated by reference, inaluding the reference to
the plasmide pSVF8-92, pSVF8-80~and pSYF8-200 deposited under
the accession number ATCC 40222, ATCC 40223 and ATCC 40190,
respectively.
'
E~AMPLE 2 ; ~ ~ ~
15 CULTURING CELL LINES CO-EXPRESSING~FACTOR VIII:C HEAVY CHAIN
AND LIGHT CHAIN ~ ~
The cell line designated "45" co-expressing Factor VIII:C Heavy
Chain and Light Chain was cultivated in suspension at 37 C in
cell factories in~DMEM + l0~ dialysed fetal calf serum ln the
20 conventional manner, and cells were harvested by trypsinization
and resuspended in l00 ml TECHNE spinners at a ~density of 2
million cells per ml in DMEMI~Gibco 074-90024T) supplemented
with ll0 mg/l Na-pyruvate, 150 mg/l l-proline, 3.7 g/l NaCHO3,
1.4 g/l tryptose phosphate,~5 mg/l insulin, 0.5 gJl 6-amino-
25 hexanoic acid and 2% NBS heat inactivated at 5~C for 30minutes. The cultures were incubated at~27C. Samples~ were
taken over a period~of g days. Var~ious amounts of heparin were
added. ;~
The r-sults;of the titratlon of~the heparin effec~is shown in
30 Figure~1. It is seen that the~optimal concentration of heparin
~ WO94/l1525 2 1 4 9 2 1 2 PCT/DK93/00375
in this type of culture is 1-2 IU/ml where an increase of the
level of expressed Factor VIII:C units by 50% is seen. The
effect of heparin i5 clearly less pronounced in a concentration
of 5 and 10 IU/ml.
`
5 EXAMPLE 3
~:
The isolated cell line designated "57" co-expressing Factor
VIII:C Heavy Chain and Light Chain was cultivated in t-flasks
in DMEM + 10% dialysed fetal caIf serum in the conventional
manner to confluence. Confluent t-flasks were shifted to the
10 same medium as in Example 2, and incubated at the same tempera-
ture in the presence and absence of heparin. The cultures were
fed fresh medium with 3 days intervals. Samples were taken for
FVIII assays prior to media change.
Table 1 shows the increased FVIII:C ~levels obtained from t-
15 flask cultures when heparin is added in low concentration ~2IU/ml). The increase is to a level of 113~to 138% of the FVIII
level of the control without heparin a~dition as a function of
the cultivation time in the presence~;of heparin. ~
~:
:
Table
'
20 Increased yield of Factor VIII:C in T-flask culture _in the
resence of 2 IU/mI heparin ~ ~
~ , ,
~axvest FVIII:C ; ~ FVIII:C ~ Yield`
on day U/ml ~U/ml on hep. add.
;~ + hep- hep~ ~ ~ln % of yield
~ ~ of control
! . ,
'
'`~ ` : ` : '
iy ~
`f ~
,'~ :
!~ .. .
WO94/11525 PCI/DK93/00375~ ,
2 1 2 14 :~
~ ~ -- -- 8-.2- - 113 -
7 22.3 17.5 128
0 23.5 ~ 138
' ~ ~ ~ ~,
~`
;
-
`
~ WO94/11SZS 2 1 1 g 2 1 2 PCT/DK93/00375
-
EXAMP~E 4
The isolated cell lines designated "45" and "57" co-expressing
Factor VIII:C Heavy Chain ~and Light Chain~were cultivated in
suspension as explained above~ 1n the presence and absence of
5 heparin.
As compared to ExampIe 3, the increase in~;FVIII level is~even
more pronounced`when the cells~are cultured~in suspension. As
seen from tables 2 and 3, the increase`in~FVIII~level caused by
addition~of heparin ~is ~120~ to 193% of the~ control, where no
lO heparin is added.~
Tables 2 and 3 also indicate that the increase of the Factor
VIII:C level is ~in~excess of~the ~increased~ expression of~the
Factor VIII:LC and Factor VIII:HC ~when co-expression in the
presence of heparin.
lS Ta~le 2
Increased Yield of Factor VIII~:C and of Factor V II:LC~ and
Factor VIII:HC when cultivatinq cell li~ne~"45" in suspension~
culture in;the~Dresence o~ 2~IU/ml~hepar~in.
; . :
Time ~ FVIII:C ~ ~ FV~ LC ~ ; FVIII:HC ¦~
` 20 ~ days yield~in % ;yield~in;%~; yield in %;~ ~ ~
I ~ of yield of of yield~of ~ of yield of 1
`control control control ~ 1
:: o ~ :; ~ : ~ :; : ~ : ~
120 ~ ; 97~ ~ 129
S ~ ~ 137 ~ ~ 101~ ~ 128
~ ~ 157 ~ ~ 108~ ; ~ 132
' :: ' ; ~ ~ ~ ' :
WO94/11525 ` PCT/DK93/00375 ~ ,~
21 4 9 2~ 2 16
Increased Yield of Factor VIII:C and of Factor VIII:LC and ~
Facto~ VIII:HC when cultivatinq cell llne "57" in sus~ension in L
the ~resence o~ 2 IU/ml heparin ~
. . _ ____ _ _
S Sample on FVIII:C ~ FVIII:LC FVIII:HC
day yield in % yield in % yield in %
of yield of of yield of of yield of
control control control
_ . . .,
3 118; 106 : 128
4 127 126 128
148 134 86
~ i62
: ~ :: : : :
~ '.'
:
'i :
1 i
;;' '~
~ WO94/11525 2 1 ~ 9 2 1 2 PCT/DK93/00375
17
EX~MPLE 5
-- "
STABILIZING EFFECT OF HEPARIN ON FACTOR VIII:C HEAVY CHAIN
DURING COEXPRESSION OF FACTOR VIII:C HEAVY CHAIN AND FACTOR
VIII:C LIGHT CHAIN ,
I~.
5 3.5 cm's dishes were seeded with cell line "45" to confluency
in the following media: DMEM + 2% NCS + 5 mg/l insulin + 1.4
g/l tryptose phosphate ~TP) and in the~same medium also added
2 U/ml heparin. After incubation over night at~37C, the dishes
were incubated at ~7C for five days for adaptation. After two
l0 washes each dish was labeled with 80 ~Ci 355-methionine for 20
hours in the following methionine-free media: `
,
l~ DMEM+2% NCS~Smg/l insulin+l.4 g/l TP
2. DMEM+2% NCS~5 mg/l insulin~l.4 g/l TP+2 IU/ml heparin
After the labelling period the media were collected for immuno-
15 precipitation with a dog polyclonal~ antiserum to human FVIII;
this antiserum binds both the complex of the s~bunits and free
heavy and light chains of FVIII. The precipitated samples were 1,
loaded on a lO~ SDS gel. The resul~ting exposure is shown in
Fig. 2. The 92 kD HC and the 80 kD LC doublet are seen in all
20 lanes. Comparing lanes l and 2 it is seen tha~ the amounts of
the 50/43 kD bands oriqinating~ from the HC are much more~
pronounced in lane l than in lane 2, indicating that heparin
has suppressed the activating cleavage of the heavy chain and
hence, a greater fraction of~the subunit-complex is found as
, 25 the more stable; complex of the 92 kD and 80 kD subunits.
EXAMP1E 6
¦~ Increased Yield of Factor VIII:C in T-~flash Culture i Serum
Free Medium Containing Heparin and Lipoprote~in.
: : ~ ~ ~
" ~ " ~ "~ " ~ ~ "~ " ~ _ ";, ~ " . ; ~ -,, " ~
~ ; l` `
W094/11525 PCT/DK93/00375 ~ ~
~14~212 18
PREPARATION OF EGG YOLK LIPOPRQTEIN FRA~TION ~`
lOO ml egg yolk and 200 ml PBS were stirred~for 30 min. PEG
6000 was added to 3.5%,~stirred for 60 min. and centrifuged at
3000 RPM for 30 min. ~;
! .
5 The sediment was dissolved in 200 ml PBS, stirred for 30 min.
and spun down at lO,OOO RPM for 20 min.
The sediment was redissolved in 200 ml ~ M NaCl and stirred
overnight at 40 C.~The mixture was~then~centrifuged at lO,OOo
RPM for 20 min. and the~supernàtant was sterilised by filtering
lO through 0.2 ~ pore size filter.
:.
This lipoprotein fraction~was used in the below culturing.
. ~
The ceIl line designated "57" co-expressing Factor VIII:C Heavy
Chain and Factor VIII:C Light Chain was cultured;in T-80 flasks
in serum containing medium. After reaching~confluency tha cells
15 were adapted to production conditions~for~three days. Produc-
tion~medium and conditions were describ~ed-in~Example 3, except
that there was no addition of- serum and;the ~basal medium was
DMEM/F-12. 5% addition~ of; the egg yolk~fraction containing
lipoprotein was tested alone and together with 1, 2 and 5;~IU
20 heparin,~and for comparison the same~medium without lipoprotein
was tested using~ 2 and 5 IU heparin.~Serum~ free medium
without lipoprotein~and heparin was~used as~control.~;The medium
was changed and~samples~were taken on day 2~and day 4 and
~; ~ assayed for FVIII:,C (coa~ activity. ~ ~
:
25~In samples containing lipoprotein ~(egg ~yolk fraction)~ the
FVIII:C activity increased 60-70`%~when~adding 1~IU/ml heparin,~
80-lOO~when adding~2~IU/ml and 6-32~ when~adding~5 IU/ml. The
` ~ addition ~o~ heparin; alone did~ not~increase the~FVIII:C ac-~
tivity. Thus,~the combined~effeot of~add~ition~of~lipoprotein
30 and~heparin is a more than;additlve effect of the two separate
components~
~14:9212 I:
WO94/l1525 PCT/DK93~00375
19 ' ` " '
Sample No. Medium ~ FVIII-C U/ml FVIII:C U/ml
Additions ~ Day 2: Day 4
. .,, .. . . . _ . ~ ,.,
1. 5% LP 4.21 6.14 -~
. : _ _ i.
2 5~ LP + 1 7.17 : 9.70
IU/mI heparin : : ___
. ~ _ ,~ _
3 5% LP ~ 2 : 8 43 11.10
IU/ml heparin ; : ~ :
~, . . . _ _, .
4 5~ LP ~ 5: 4.47 ~ ~ 8.12
IU/ml heparin .
. . . ~ , ._
None 0.39 1.43
_. _ . .
6 1 IU/ml 1.20 : 1.32
heparin :
_ _ _ _ _ , =
7 2 IU/ml 0.78~ 1.64
: heparin : ~ :
. _ ~ ~ .
: 8 5 IU/ml 0.9g ~ 2.02 :
heparin ~ ~:
10 LP = Lipoprotein, e~g yolk fraction.;
: :
' ~ .3
WO94/11525 PCT~DK93/00375 ~
214~212 ` 20
EXAMPLE 7
Increased Yield of Factor VIII:C in T-flash ~ulture i;Serum r '`~
~ree Medium Containing Heparin and an egg yolk fraction free of
lipoproteins.
5 :B~a~31P~L9~1~E LIPOPROTEIN FREE EGG~YOLK PROTEIN FRACTION
l00 ml egg yolk and 200 ml PBS were stirred for 30 min. PEG
6000 was added to 3.S%, stirred for~60 min.~ and centrifuged at
3000 RPM for 30 min.
1'he supernatant is the lipoproteinfree egg yoIk protein
l0 fraction (SUP 0).
,'~
`:
This proteinfraction was used in the below~culturing.
The cell line designated "57"~co-expressing Factor VIIT:C Heavy
Chain and Factor VIII:C Light Chain was cultured in T-80 flasks
in serum containing medium. After reaching confluency the cells
15 were adapted to produ`ction conditions for three days. Produc-
tion medium and conditions were descr~bed~in Example 3, except
that there was no addition of serum and;the basal medium was
~MEM/F-12. A 5% addition of the egg yolkprotein fraction was
tested alone and~ together with ~ 5 and~;lO IU heparin. The
20 medium was changed and samples were taken on day 2 and~day 4
and assayed for FVIII:C (coa) activity. ~
In samples containing SUP O the~ FVIII:C ~activity increased
markedly on addition of heparin giving an increased level of
Factor VIII:C activity. This effect is reduced when adding as
25 much as lO IU heparin.
:
WO 94/11525 21~ 9 212 PCr/DK93/00375
21
_ -- _ .
5 % sup O Heparin FVIII: C FVIII: C
IU/ml U/ml . U/ml
day 2 day 4
. . -_ _ - _ _ .
+ 0 4.7 11.2
_ _ 6 . 7 ~_ _ 17 . 6
_ _ _
+ 5 7.0 17.3
_ ,.
+ 10 5.0 6.6
l .-- . ,
-
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