Method for the isolation and purification of vitamin K-dependent proteins

A method for the separation of vitamin K-dependent proteins from non-vitamin K-dependent accompanying proteins is described wherein the method is characterized in that at least anion exchange chromatography and optionally affinity chromatography is carried out as well. The method is suitable especially for the purification of Factor II, VII, IX, X as well as Protein S, Protein C and Protein Z. With the aid of the method according to the invention a vitamin K-dependent protein is obtained which is present at a purity of 95%.

Aside from the naturally occurring proteins, it is possible today, with the 
aid of gene technology methods, to produce mammalian proteins by 
recombinant techniques, especially human proteins. For this purpose, host 
cells transformed or transfected with foreign DNA are cultured, wherein in 
the case of eucaryotic host cells the recombinantly produced protein is 
released in soluble form into the cell culture medium (R. G. Werner and W. 
Berthold, Arzneim.-Forsch. Drug Res. 38, 422-428 (1988)). However, since 
the host cells also release other proteins into the cell culture medium in 
addition to the desired recombinantly produced protein, it is necessary to 
enrich and/or isolate the desired protein in one or more purification 
steps. Concerning this, methods are needed which effectively and 
selectively permit the isolation of recombinant proteins from the cell 
culture medium. 
As a rule, physical and chemical properties of the proteins are used for 
the purification of recombinant proteins. Such properties are the size of 
the proteins, the natural charge of the surface, their hydrophilicity or 
solubility. Additional purification methods concern the binding with other 
molecules, such as for example antibodies. The same applies for the 
purification of proteins from natural sources. Here as well, based on the 
physical and chemical properties of a protein, a separation of the same 
occurs from the remaining accompanying proteins. 
Vitamin K-dependent proteins were purified so far according to such methods 
as protein precipitation, ion exchange chromatography and gel filtration 
M. Bertina and J. J. Veltkamp, Haemostasis and Thrombosis, (Ed. A. L. 
Bloom and D. P. Thomas, Churchill Livingstone, New York, 1987, 116-130). 
B. Dahlb ack (Biochem. J. 209, 837-846 (1983)) describes a purification 
method for Protein S (PS). After a barium citrate precipitation, PS is 
isolated on DEAE-Sephacel. J. Malm et al., (Eur. J. Biochem. 187, 737-743 
(1990)) purified recombinant PS (rPS) by affinity chromatography on a 
monoclonal antibody. Factor IX (FaIX) was isolated by B. Osterud and R. 
Flengsrud (Biochem. J. 145, 469-474 (1975)) by barium sulfate 
precipitation and ion exchange chromatography on cellulose. Monoclonal 
antibodies were used by H. C. Kim et al., (Sem. Hematol. 28(3) Suppl. 6, 
15-19 (1991) for the purification of FaIX. 
U.S. Pat. No. 5,055,557 describes a method for the purification and 
concentration of vitamin K-dependent proteins from plasma as well as 
recombinantly produced proteins with the aid of a immunoadsorbent by using 
a monoclonal antibody. 
In the above named methods attention must be paid to the fact that the 
properties of many proteins do not appreciably differ from each other 
which makes their fractionation more difficult. Therefore, it is necessary 
to apply combinations of precise, inter-coordinated purification steps in 
order to take advantage optimally of differences in the properties of the 
proteins. 
A number of proteins naturally occurring in human and animal blood can bind 
divalent cations. Such proteins are synthesized in a vitamin K-dependent 
process in the cell wherein cation binding sites arise by the conversion 
of glutamic acid (Glu) to gamma carboxy glutamic acid (Gla). These cation 
binding sites can then be saturated by calcium ions (Ca.sup.2+). Beside 
calcium ions, these cation binding sites are also capable, as a rule, of 
binding divalent strontium or barium (B. Furie and B. C. Furie, Cell 53, 
505-518 (1988)). 
Many of these vitamin K-dependent proteins are components of the blood 
plasma and play an important role in homeostasis. The gamma carboxy 
glutamic acid groups in these vitamin K-dependent proteins are found in 
the structurally homologous N-terminal Gla regions (A. Tulinsky, Thromb. 
Haemost. 66, 16-31 (1991). Among these calcium ion binding proteins with 
homologous Gla regions are, among others, Protein S (PS), Protein C, 
Factor IX (FaIX), Factor II, Factor VII, Factor X and Protein Z. 
As a consequence of the binding of calcium ions these proteins demonstrate 
definitively altered properties in comparison to non-calcium binding 
proteins. This difference is exploited in EP 363 126 for the purification 
of proteins. EP 363 126 describes a method for the isolation of vitamin 
K-dependent proteins which are obtained recombinantly. Thereby, the 
divalent cations are completely removed by addition of a chelator 
component to the culture medium before the actual protein isolation, and 
the protein is thereby capable to bind to an ion exchange resin such as 
Mono Q. Furthermore, the protein is eluted from an anion exchanger by 
addition of NaCl and Ca.sup.2+ ions. Thereby, a 58% enriched product 
(Protein C) was obtained. The impurities (42%) constitute such proteins 
which are also released from the ion exchanger at the applied salt 
concentration (0.15M). In a further purification step, the obtained 
protein-cation complex is adsorbed on a column on which immobilized EDTA 
is bound, washed, and the protein eluted. The proteins in the eluent are 
bound to an in-line ion exchanger and eluted with a salt gradient. After 
addition of CaCl.sub.2 in the eluent, the protein-cation complex is 
adsorbed on a hydrophobic column and the desired protein is eluted by EDTA 
buffer. 
The disadvantage of the method described in EP 363 126 is that a widespread 
conformation change of the vitamin K-dependent protein occurs through the 
multiple removal and addition of calcium ions which leads to changes of 
the properties of the protein (A. E. Johnson et al., Biol. Chem. 258, 
5554-5560 (1983); J. Stenflo, J. Biol. Chem. 251, 355-363 (1976)). In 
contrast to this, a process is made available with the method according to 
the invention which does not possess these disadvantages because no 
process step for the removal of divalent metal ions, for example by 
chelators, is necessary during the entire purification process. 
The advantage of the present invention is that the original conformation 
and stability of the vitamin K-dependent protein is preserved during the 
purification. 
EP 354 354 describes a method for the enrichment of the blood clotting 
factors II, VII, IX and X. The above named prothrombin-complex factors are 
isolated by adsorption on an matrix carrying alpha hydroxyl amino groups, 
wherein especially Factor IX is strongly bound. As a function of the ion 
strength, the Factors II, VII and X are previously eluted. The addition of 
calcium ions do not play a roll in this method. 
EP 317 376 describes a method for the separation of a Factor IX-containing 
human plasma fraction wherein cryoprecipitate is at first 
chromatographically prepurified, then a separation by anion exchange 
chromatography and selective elution through a buffer with increasing 
ionic strength is performed, and finally affinity chromatography on 
Heparin-Sepharose with selective elution is carried out. This method also 
does not consider the altered protein properties of Factor IX in the 
presence or absence of calcium ions. 
The object of the present invention is to make available a method for the 
separation of vitamin K-dependent and calcium ion binding proteins from 
non-vitamin K-dependent accompanying proteins from solutions without 
requiring the necessity of removal of calcium ions and a possible 
denaturation of proteins associated with it. This method, which can be 
carried out with enriched protein solutions from natural sources as well 
as with cell culture supernatants of recombinant protein production, 
should be simple and lead to highly pure vitamin K-dependent proteins. 
The above object is achieved by the present invention by providing methods 
of separating a vitamin K-dependent protein from non-vitamin K-dependent 
accompanying proteins, and thus can be used for isolating vitamin 
K-dependent and non-vitamin K-dependent proteins. The vitamin K-dependent 
proteins separated according to the invention include factor II, factor 
VII, factor IX, factor X, protein S, protein C and protein Z. The proteins 
can be obtained with the aid of gene technology methods, and can be 
isolated from the culture supernatant of transformed or transfected cells. 
The separation of vitamin K-dependent proteins from non-vitamin K-dependent 
accompanying proteins from a protein-containing solution is characterized 
by adding calcium ions to the solution and then bringing the 
calcium-containing solution into contact with an anion exchanger under 
conditions in which the accompanying non-vitamin K-dependent proteins, but 
not the vitamin K-dependent protein, are adsorbed. The resulting solution 
containing the vitamin K-dependent protein can be brought into contact 
with an anion exchanger under conditions in which the vitamin-K dependent 
protein is adsorbed. The vitamin K-dependent protein can be washed, and 
eluted, and optionally the ionic strength of the eluent can be reduced. 
The calcium employed according to the method can be provided by adding 
CaCl.sub.2 at a minimum concentration of 1.0 mM. Preferably, the protein 
containing solution has a pH value between 4 and 9. 
Optionally, any non-adsorbed vitamin K-dependent protein is adsorbed on an 
affinity substrate and selectively eluted. Appropriate affinity substrates 
include heparin binding substrate materials, heparin-sepharose and heparin 
agarose. Preferably, when employing an affinity substrate, the ionic 
strength of the protein containing solution after elution is reduced to 
0.15 to 0.2M. 
The steps of the method can be carried out in any combination or order. In 
one embodiment, the separation step is carried out with the same anion 
exchanger, preferably with the aid of chromatography. The anion exchanger 
can have hyperdiffusion properties, and can include Q-Hyper D.RTM.. 
Optionally, the anion exchanger can possess a tentacle structure. The 
invention also comprises the highly pure vitamin K-dependent proteins 
isolated with the aid of these methods. 
For example cell culture supernatants as well as cell culture medium 
processed in another way, supernatants from tissue cultures or protein 
solutions from natural sources which comprise vitamin K-dependent proteins 
are first chromatographed over an anion exchanger in the method according 
to the invention. A specific removal of divalent cations from the protein 
solution before the chromatography step is not required in contrast to the 
method described in EP 363 126. Rather, it was surprisingly determined 
that by the addition of small amounts of calcium ions at low salt 
concentration no binding of the vitamin K-dependent protein, yet binding 
of non-vitamin K-dependent proteins, occurs on the anion exchanger. For 
the first time, a new type of anion exchanger with hyperdiffusion 
properties, for example Q-Hyper D.RTM. (Sepracor) was used in the method 
according to the invention for the separation of vitamin K-dependent and 
non-vitamin K-dependent proteins. Anion exchange chromatography is 
followed then by affinity chromatography--as long as a further protein 
purification is desired. 
According to the invention, the vitamin K dependent protein solution can be 
enriched by an additional protein concentration step without previous 
removal of divalent cations from the protein solution, subsequently 
filtered through an anion exchanger, and optionally chromatographed over 
an affinity column as well. 
According to a preferred alternative the protein concentrate enriched in a 
prepurification step (1st step) is filtered through an anion exchanger 
(2nd step) and then directly applied on an affinity column. The preferred 
combination is characterized as follows: 
(a) A solution comprising a vitamin K-dependent protein is brought into 
contact with anion exchanger wherein the vitamin K-dependent protein 
adsorbs on the exchanger and subsequently is eluted with increasing salt 
concentration; after that the salt concentration of the eluent is reduced; 
(b) calcium ions are added to the eluent from (a) and this is again brought 
into contact with anion exchanger under conditions in which the 
accompanying proteins, yet not the vitamin K-dependent protein, is 
adsorbed; 
(c) the non-adsorbed vitamin K-dependent protein from (b) is adsorbed on an 
affinity substrate and selectively eluted. 
The above named preferred combination of method steps is more closely 
illustrated as follows: 
For the protein concentration step (1st step) the solution which comprises 
a recombinant or natural vitamin K-dependent protein is brought in contact 
with an anion exchanger. This can occur simply by mixing or guiding over a 
column. Moreover, the vitamin K-dependent protein is found together with 
the accompanying proteins in a salt solution of lower concentration 
(minimal salt concentration). Under these conditions, the vitamin 
K-dependent protein is bound on the anion exchanger together with a number 
of other proteins. By increasing the salt concentration (ionic strength) 
vitamin K-dependent protein is thus hereinafter selectively released from 
the exchanger. 
Cell culture supernatants comprise, as a rule, a dye which indicates the pH 
status. This dye, such as Phenol Red, clouds the clear protein solution, 
binds strongly to usual anion exchangers such as Q Sepharose Fast Flow 
(Pharmacia) or MacroPrep.RTM. (Bio-Rad), and adsorbs the light at the wave 
length of protein determination, i.e. 280 nm. This leads to an obstruction 
of the purification of proteins from cell culture medium. In order to make 
an improvement here, cell culture supernatants according to the invention 
were, for the first time, filtered over an anion exchanger with 
hyperdiffusion properties (Q-Hyper D.RTM., Sepracor). Thereby, it was 
shown that the unspecific binding of the dye Phenol Red is reduced to a 
minimum by using Q-Hyper D as an anion exchanger. The dye is already 
eluted from the anion exchanger at salt concentrations under the minimal 
concentration. In this way the eluted proteins could be better detected 
during the chromatography. Moreover, a disturbing competitive reaction 
between the proteins and the dye on the ion exchanger is avoided to a 
large extent. 
The further anion exchange chromatography (2nd step) consists in the fact 
that the ionic strength of the solution comprising the vitamin K-dependent 
protein is reduced to a value under the minimal salt concentration by 
dialysis or dilution with salt-free buffer and small amounts of calcium 
ions are added. Then the protein solution is again brought in contact with 
the anion exchanger and/or filtered through this. Thereby, it was 
surprisingly determined that vitamin K-dependent proteins do not bind on 
the anion exchanger and the non-vitamin K-dependent accompanying proteins 
adsorb on the substrate. This is a result of the different charge of the 
proteins owing to the binding of Ca ions. The vitamin K-dependent protein, 
now further purified from the contaminating proteins of the preliminary 
stage, are thereby directly obtained (without further salt dependent 
elution). 
In many cases of protein purification, a combination of a prepurification 
step (1st step) with a purification on an anion exchanger (2nd step) is 
sufficient. Thereby, the second purification step is all the more 
effective, the less the vitamin K-dependent protein to be separated is 
contaminated with accompanying proteins. 
In the second purification step on the anion exchanger, the vitamin 
K-dependent protein is not bound on the anion exchanger by the selection 
of a certain salt concentration below the minimal salt concentration of 
the band width and by the addition of calcium ions. 
By the exploitation of the calcium binding properties of the vitamin 
K-dependent proteins with Gla-region, these are separated from the 
accompanying proteins in that, at certain ionic strengths, only the 
accompanying proteins are bound on the anion exchanger, however, the 
vitamin K-dependent proteins pass through the anion exchanger without 
binding to it. Therefore, aside from vitamin K-dependent proteins from 
natural sources, the method according to the invention is particularly 
suitable for the purification of recombinant vitamin K-dependent proteins 
with Gla-region, such as for example Protein C, Factor IX, Factor II, 
Factor VII, Protein S and Protein Z. 
For the above named steps 1 and 2, it is preferred to used the same anion 
exchanger, wherein particularly good results are obtained when the anion 
exchange chromatography occurs on a column. 
In the in-line method (3rd step), namely the affinity chromatography, a 
binding of the vitamin K-dependent protein on the affinity matrix occurs. 
In this step, the vitamin K-dependent protein is adsorbed on the affinity 
matrix. In this case, the prepurification step connected in series to the 
anion exchanger proves to be particularly advantageous because by this a 
number of contaminating proteins were removed. This fosters the elution of 
the vitamin K-dependent protein from the affinity matrix and allows the 
isolation of the desired protein in high purity. 
The method according to the invention can include a virus inactivation step 
known to the person skilled in the art from the prior art which 
encompasses the treatment of the vitamin K-dependent protein solutions 
with physical-chemical or chemical methods. For this purpose, the 
treatment in the presence of antiviral substances, optionally combined 
with a radiation or heat treatment are considered. According to the 
present invention, the virus inactivation can occur before the separation 
of vitamin K-dependent proteins from non-vitamin K-dependent accompanying 
proteins. 
The method according to the invention can consist of a combination of the 
purification steps 1, 2 and 3 which can be carried out in any order. 
Thereby, however, the method of separating vitamin K-dependent proteins 
from non-vitamin K-dependent accompanying proteins from a 
protein-containing solution is characterized by adding calcium ions to the 
solution and then bringing the calcium-containing solution into contact 
with an anion exchanger under conditions in which the accompanying 
non-vitamin K-dependent proteins, but not the vitamin K-dependent protein, 
are adsorbed, is always obligatory.

The following Examples demonstrate the purification according to the 
invention of Protein S, Factor IX and Factor II 
EXAMPLE 1 
a) Purification of rPS by Anion Exchange Chromatography 
In the following Example, a quaternary amino-type anion exchanger with 
hyperdiffusion properties (Q-Hyper D, Sepracor) was used. 
Materials: 
Column: Q-Hyper D, Sepracor; 2 cm.times.4 cm. 
Buffer A: 20 mM Bis-Tris/HCl, pH 7,0. 
Buffer B: 20 mM Bis-Tris/HCl, pH 7,0, 0.18M NaCl. 
Buffer C: 20 mM Bis-Tris/HCl, pH 7,0, 0.4M NaCl. 
Buffer D: 20 mM Bis-Tris/HCl, pH 7,0, 1 mM NaCl. 
Recombinant protein (rPS) was isolated, based on usual laboratory methods, 
after infection of Veto cells (Monkey kidney cells) with vaccinia virus by 
cell culture technology. Vero/vaccinia expression systems and cell culture 
conditions are comprehensively described in F. G. Falkner et al, 
Thrombosis and Haemostasis, 68, 119-124 (1992) and N. Barrett et al., AIDS 
Res. Hum. Retrov., 5, 159-171 (1989); F. Dorner and N. Barrett, AIDS 
Vaccine Research and Clinical Trials, (Ed. S. D. Putney and D. P. 
Bolognesi), Marcel Dekker, Inc., New York (1990). The expression of rPS 
occurs in commercially available, synthetic DMEM medium. After cell 
culture, the culture supernatant was isolated by centrifugation and spiked 
with the synthetic protease inhibitor Pefabloc.RTM. SC, Pentapharm, to 0.1 
mMol/l. 
The column was regenerated corresponding to the instructions of the 
manufacturer and equilibrated with Buffer A. Subsequently, 485 ml of cell 
culture supernatant, which contained recombinant Protein S, were applied 
with a speed of 10 ml/min on the column. The material not bound to the 
column was removed with the same flow speed by washing with Buffer A. 
After that, the column was first eluted with Buffer B and subsequently 
with Buffer C. Subsequent elution occurred with Buffer D. The protein 
adsorption was followed during the chromatography in the normal manner at 
280 nm. After execution of the chromatography, the protein concentration 
was determined by means of the Bradford method (M. M. Bradford, Anal. 
Biochem. 72, 248-254 (1976). The content of Protein S was determined by 
means of a commercial ELISA system (Asserachrome Protein S, Boehringer 
Mannheim) as well as by means of a clotting test (Protein S clotting test, 
Boehringer Mannheim). 
It was found that almost all rPS was bound to the matrix. rPS was eluted 
from the anion exchanger in 0.4M NaCl (Buffer C). 
By using the above named anion exchanger, the dye Bromophenol Red, commonly 
contained in cell culture medium, was already eluted from the column at a 
salt concentration of 0.18M which substantially fostered the subsequent 
isolation of rPS at 0.4M NaCl. This constitutes an advantage compared to 
the use of other anion exchangers. 
The essential results of the purification of rPS on the anion exchanger 
(1st step) are summarized in FIG. 1 and Table 1. By the purification 
described in Example 1, the amount of 3% rPS antigen to the total protein 
of the cell culture medium was increased to 8% rPS antigen to protein in 
the 0,4M NaCl fraction. The specific activity increased 25-fold. 
TABLE 1 
______________________________________ 
vol- Protein S specific 
umes protein antigen activity 
activity 
sample (ml) (mg/ml) (mU/ml) 
(mU/ml) 
(U/mg) 
______________________________________ 
cell 485 0.108 137 11 0.1 
supernatant 
unbound 560 0.05 4 0 0 
fraction 
0.18M NaCl 
60 0.014 4 0 0 
0.4M NaCl 
14 0.537 1700 1358 2.5 
1M NaCl 20 0.54 4 0 0 
______________________________________ 
b) Purification of rPS by Adsorption of Accompanying Proteins by Means of 
Anion Exchange Chromatography with Addition of Calcium Ions (2nd Step). 
The same anion exchange type was used as described under 1.a). 
Materials: 
Column: Q-Hyper D, Sepracor; 1 cm.times.4 cm. 
Instrument: Pharmacia FPLC LCC-500. 
Buffer A: 20 mM Bis-Tris/HCl, pH 7.0. 
Buffer B: 20 mM Bis-Tris/HCl, pH 7.0, 0.15M NaCl, 10 mM CaCl.sub.2. 
Buffer C: 20 mM Bis-Tris/HCl, pH 7.0, 1M NaCl. 
The column was regenerated corresponding to the instructions of the 
manufacturer and equilibrated with Buffer B. Recombinant Protein S, which 
was isolated from cell culture supernatant as described in Example 1.a), 
was diluted 2.5-fold with Buffer A such that the concentration of NaCl 
lied under 0.18M. CaCl.sub.2 with a concentration of 10 mM was added. 
Subsequently, the protein mixture was applied on the column and the 
unbound protein was washed out of the column with Buffer B. Bound protein 
was eluted by means of Buffer C. The course of the chromatography was 
followed as described in Example 1.a) and the respective protein 
concentration was determined. 
The results show that rPS passed through the column unimpeded, whereas the 
predominant majority of the further proteins remained stuck on the column. 
These contaminating proteins were then eluted with 1M NaCl. The essential 
results of this experiment are summarized in FIG. 2, FIGS. 3A and 3B and 
Table 2. 
Through the purification described in Example 1.b), the antigen content of 
rPS was increased 12-fold in relationship to the other proteins. The 
specific activity increased 14-fold. The denaturing electrophoretic 
analysis (U.K. Laemmli, Nature 227, 680-685 (1970)) demonstrated (FIGS. 3A 
and 3B) that by the purification described in Example 1.b), rPS was 
isolated at more than 95% purity. 
TABLE 2 
______________________________________ 
vol- Protein S specific 
umes protein antigen activity 
activity 
sample (ml) (mg/ml) (mU/ml) 
(mU/ml) 
(U/mg) 
______________________________________ 
preparation 
34 0.215 680 543 2.5 
from 
Example 1a 
unbound 34 0.014 600 520 37 
fraction 
1.0M NaCl 
10 0.332 83 0 0 
______________________________________ 
EXAMPLE 2 
a) Purification of rFaIX by Anion Exchange Chromatography. 
In the following Example, a quaternary amino-type anion exchanger with 
hyperdiffusion properties (Q-Hyper D, Sepracor) was used. 
Materials: 
Column: Q-Hyper D, Sepracor; 2 cm.times.4 cm. 
Instrument: Pharmacia FPLC LCC-500. 
Buffer A: 20 mM Bis-Tris/HCl, pH 5.5. 
Buffer B: 20 mM Bis-Tris/HCl, pH 5.5, 0.18M NaCl. 
Buffer C: 20 mM Bis-Tris/HCl, pH 5.5, 0.3M NaCl. 
Buffer D: 20 mM Bis-Tris/HCl, pH 5.5, 1.0M NaCl. 
Recombinant Factor IX (rFIX) was obtained in an analogous manner as for rPS 
(Example 1.a). 
The column was regenerated corresponding to the instructions of the 
manufacturer and equilibrated with Buffer A. Subsequently, 1997 ml of cell 
culture supernatant, which contained recombinant Factor IX, were applied 
with a speed of 10 ml/min on the column. The material not bound to the 
column was removed with the same flow speed by washing with Buffer A. 
After that, the column was first washed with Buffer B and subsequently 
with Buffer C. Subsequent elution occurred with Buffer D. 
The protein adsorption was followed during the chromatography in the usual 
manner at 280 nm. After ending the chromatography, the protein 
concentration was determined by means of the Bradford method (M. Bradford, 
Anal. Biochem. 72, 248-254 (1976). The content of Factor IX was determined 
by means of a commercial clotting test (Factor IX clotting, Immuno). 
The results show that almost all of the rFaIX was bound to the anion 
exchanger. rFaIX was eluted from the anion exchanger in 0.3M NaCl. 
Analogous results were also obtained by using other quaternary amino anion 
exchangers. On the other hand, by using Q-Hyper D, the dye Bromophenol 
Red, normaly contained in cell culture medium, was already eluted from the 
column at a salt concentration under 0.3M which substantially fostered the 
subsequent isolation of rFaIX at 0.3M NaCl. Other anion exchangers do not 
have this advantageous property. 
The essential results of the purification of rFaIX on the anion exchanger 
are summarized in FIG. 4 and Table 3. By the purification described in 
Example 2.a, rFaIX was enriched 20-fold; the specific activity increased 
by 12-fold. 
TABLE 3 
______________________________________ 
Factor IX specific 
volumes 
protein activity activity 
sample (ml) (mg/ml) (mU/ml) (U/mg) 
______________________________________ 
cell 1997 0.178 100 0.56 
supernatant 
unbound 2100 0.091 0 0 
fraction 
0.18M NaCl 
94 0.382 162 0.41 
0.3M NaCl 94 0.300 2099 6.86 
1M NaCl 93 0.09 32 0.35 
______________________________________ 
b) Purification of rFaIX by Adsorption of Accompanying Proteins by Means of 
Anion Exchange Chromatography with Addition of Calcium Ions. 
Q-Hyper D served as an anion exchanger as it was also used in Example 2.a). 
Materials: 
Column: Q-Hyper D, Sepracor; 2 cm.times.4 cm. 
Instrument: Pharmacia FPLC LCC-500. 
Buffer A: 20 mM Bis-Tris/HCl, pH 7.4. 
Buffer B: 20 mM Bis-Tris/HCl, pH 7.4, 0.15M NaCl, 10 mM CaCl.sub.2. 
Buffer C: 20 mM Bis-Tris/HCl, pH 7.4, 1.0M NaCl. 
The column was regenerated corresponding to the instructions of the 
manufacturer and equilibrated with Buffer A. 85 ml of recombinant Factor 
IX, as it was obtained in Example 2.a), was diluted 2-fold with Buffer A 
such that the concentration of NaCl was reduced to 0.15M. CaCl.sub.2 was 
added to 10 mM. Subsequently, the protein mixture was applied and the 
unbound protein was washed out of the column with Buffer B. Protein bound 
on the column was eluted by means of Buffer C. The course of the 
chromatography was followed as described in Example 2.a). The protein 
concentrations and enzyme activities were determined. 
The results show that rFaIX passed through the column unimpeded, whereas 
the predominant majority of the contaminanting proteins remained stuck on 
the column. These contaminating proteins were then eluted with 1M NaCl. 
The essential results are summarized in FIG. 5, FIGS. 6A and 6B and Table 
4. Through the purification described in Example 2.b), the specific 
activity of rFaIX increased by 4-fold. The denaturing electrophoretic 
analysis according to Laemmli, demonstrated (FIGS. 6A and 6B) that by the 
purification described in Example 2.b), rFaIX was isolated at more than 
80% purity. 
TABLE 4 
______________________________________ 
Factor IX specific 
volumes 
protein activity activity 
sample (ml) (mg/ml) (mU/ml) (U/mg) 
______________________________________ 
preparation 
170 0.153 1040 6.7 
from Example 2a 
unbound 200 0.04 1076 27.5 
fraction 
1M NaCl 20 0.45 0 0 
______________________________________ 
c) Purification of rFaIX by Affinity Chromatography 
In the following, rFaIX, as obtained as in Example 2.a), was purified by 
affinity chromatography. Heparin-Sepharose was used as the affinity 
matrix. 
Materials: 
Column: HiTrap.RTM. Heparin, Pharmacia; 5 ml. 
Instrument: Pharmacia FPLC LCC-500. 
Buffer A: 50 mM Tris, 20 mM sodium citrate, pH 7.4. 
Buffer B: 50 mM Tris, 20 mM sodium citrate, pH 7.4, 0.15M NaCl. 
Buffer C: 50 mM Tris, 20 mM sodium citrate, pH 7.4, 0.3M NaCl. 
Buffer D: 50 mM Tris, 20 mM sodium citrate, pH 7.4, 1M NaCl. 
The column was regenerated corresponding to the instructions of the 
manufacturer and equilibrated with Buffer A. 78 ml of rFaIX, as it was 
obtained in Example 2.a), was applied on the column and the unbound 
protein was washed out of the column with Buffer A. Subsequently, the 
column was eluted with Buffer B, thereafter with Buffer C. The subsequent 
elution occurred with Buffer D. The course of the chromatography was 
followed as described in Example 2.a); the protein concentrations and 
enzyme activities were determined. 
The results show that rFaIX was completely bound on the column, and first 
eluted from this by 0.3M NaCl. In this method however, further proteins 
were also simultaneously eluted with rFaIX and therewith not separated 
from rFaIX. The specific activity or rFaIX was only increased by 4.5-fold 
through method represented in Example 2.c). 
The essential results are summarized in FIG. 7 and Table 5. 
TABLE 5 
______________________________________ 
Factor IX specific 
volumes 
protein activity activity 
sample (ml) (mg/ml) (mU/ml) (U/mg) 
______________________________________ 
preparation 
78 0.121 788 6.5 
from Example 2a 
unbound 90 0.038 0 0 
fraction 
0.15M NaCl 10 0.10 0 0 
0.3M NaCl 22 0.053 1572 29.6 
______________________________________ 
EXAMPLE 3 
Purification of rFaIX by Direct Coupling of Anion Exchange Chromatography 
and Affinity Chromatography. 
In the following Example, rFaIX, as obtained in Example 2.a), was purified 
in such a way that it was first applied on an anion exchanger and 
thereafter on an affinity chromatography column. Q-Hyper D served as the 
anion exchanger; this is an anion exchanger of the amino type with 
hyperdiffusion properties. Heparin-Sepharose (HiTrap.RTM. Heparin, 
Pharmacia) was used as the affinity matrix. The Q-Hyper D column was 
attached in direct series before the Heparin-Sepharose column. Only a 
flexible tube connection restricted to the minimum, optionally with a 
valve, was mounted between the columns. 
Materials: 
Column 1: Q-Hyper D.RTM., Sepracor; 2 cm.times.4 cm. 
Column 2: HiTrap.RTM. Heparin, Pharmacia; 5 ml. 
Instrument: Pharmacia FPLC LCC-500. 
Buffer A: 20 mM Tris/HCl, pH 7.4. 
Buffer B: 20 mM Tris/HCl, pH 7.4, 150 mM NaCl, 10 mM CaCl.sub.2. 
Buffer C: 20 mM Tris/HCl, pH 7.4, 1.0M NaCl. 
Buffer D: 50 mM Tris, 20 mM sodium citrate, pH 7.4, 0.15M NaCl. 
Buffer E: 50 mM Tris, 20 mM sodium citrate, pH 7.4, 0.3M NaCl. 
Buffer F: 50 mM Tris, 20 mM sodium citrate, pH 7.4, 1M NaCl. 
The outlet of the column with the anion exchanger was directly connected 
with the inlet of the affinity chromatography column by a flexible tube 
connection such that the stream of liquid first ran through the column 1 
and subsequently directly through column 2. 
The columns were regenerated corresponding to the instructions of the 
manufacturer and equilibrated with Buffer B. 70 ml rFaIX, which was 
isolated from cell culture supernatant as described in Example 2.a), was 
diluted to 2-fold with Buffer A such that the concentration of NaCl was 
reduced to 0.15M. CaCl.sub.2 was added until a concentration of 10 mM. 
Subsequently, the protein mixture was applied through the column 1 
(Q-Hyper D.RTM.) at 2.5 ml/min and directly transferred on column 2 
(Heparin-Sepharose), wherein unbound protein was washed from the columns 
with Buffer B. Thereafter, column 1 was disconnected from the affinity 
coulmn and the stream of liquid was directly applied on column 2. In 
analogy to the method represented in Example 2.c), unbound protein from 
the heparin column was removed by washing with Buffer D. The elution of 
the heparin column ensued with Buffer E. The subsequent elution was 
carried out with Buffer F. Analogous results were obtained when, after the 
application of the sample, the Q-Hyper D.RTM. column was not removed from 
the heparin column but instead the liquid stream for the elution of the 
heparin column was lead directly on the heparin column through an 
interconnected valve. 
The proteins bound on the column 1 (Q-Hyper D.RTM.) were eluted by Buffer 
C. 
The course of the chromatography was followed as described in Example 2.a); 
the protein concentrations and enzyme activities were determined. 
The results show that by the application of the sample through the 
introduced Q-Hyper D.RTM. column, the rFaIX passes through this unimpeded 
and is completely absorbed on the heparin column placed thereafter. rFaIX 
was eluted from the latter by 0.3M NaCl. 
The essential results are summarized in FIG. 8, FIG. 9 and Table 6. 
rFaIX is enriched 8.4-fold by the purification described in Example 3; the 
specific activity is increased by 8.8-fold. The denaturing electrophoretic 
analysis (according to Laemmli) showed (FIG. 9) that rFaIX is isolated at 
more than 95% purity by the purification described in Example 3. 
In Example 3, the purification of rFaIX was carried out by a direct 
combination of the methods from Examples 2.a),b) and c) wherein the same 
starting material was employed. In comparison to the purification from 
Example 2.c) however, a higher enrichment and a higher specific activity 
were obtained through a combination of anion exchange chromatography and 
affinity chromatography. Through the combination of both chromatography 
methods, such contaminating proteins which unfavorably influence the 
separation process on the affinity substrate are obviously separated 
before hand, whereby a higher specificity and selectivity of the affinity 
substrate results. 
TABLE 6 
______________________________________ 
Factor IX specific 
volumes 
protein activity activity 
sample (ml) (mg/ml) (mU/ml) (U/mg) 
______________________________________ 
preparation 
148 0.0196 123 6.3 
from Example 2a 
unbound 160 0.01 0 0 
fraction 
0.15M NaCl 32 0.01 0 0 
0.3M NaCl 10 0.019 1041 54.8 
1M NaCl 30 0.015 0 0 
______________________________________ 
EXAMPLE 4 
Purification of rFaII by Anion Exchange Chromatography 
In the following Example, a quaternary amino-type anion exchanger with 
tentacle structure (Fraktogel EMD TMAE 650 M, Merck) was used. 
Materials: 
Column: Fraktogel EMD TMAE 650 M, Merck, 1.6 cm.times.5 cm. 
Instrument: Pharmacia FPLC LCC-500 system. 
Buffer A: 50 mM Tris/HCl, pH 7.4. 
Buffer B: 50 mM Tris/HCl, pH 7.4, 200 mM NaCl. 
Buffer C: 50 mM Tris/HCl, pH 7.4, 300 mM NaCl. 
Buffer D: 50 mM Tris/HCl, pH 7.4, 1M NaCl. 
Recombinant Factor II was isolated based on the usual laboratory methods of 
cell culture technology (Falkner et al, Thrombosis and Haemostasis, 68, 
119-124 (1992)). 
The expression of rFaII ensued in commercially obtainable synthetic DMEM 
medium. Cell-free culture supernatant was obtained by centrifugation. 
The column was regenerated corresponding to the instruction of the 
manufacturer and equilibrated with Buffer A. Subsequently, 200 ml of cell 
culture supernatant, which contained recombinant Factor II, were applied 
on the column with a speed of 4 ml/min. Material not bound to the column 
was removed by washing with Buffer A at the same flow rate. Hereinafter, 
the column was first washed with Buffer B and subsequently with Buffer C. 
Then, subsequent elution was with Buffer D. Protein adsorption was 
followed in the usual way at 280 nm during the chromatography. After the 
chromatography, the protein concentration was determined by means of the 
Bradford method. The content of Factor II was determined with the aid of a 
clotting test (Thrombinzeit, Immuno AG). 
It was found that almost the entire rFaII was bound on the anion exchange 
gel. rFaII was eluted from the anion exchanger by 0.3M NaCl. In 
addition--in contrast to other amino-type anion exchangers, as for example 
MacroPrep.RTM. (Bio-Rad) or Q-Sepharose Fast Flow (Pharmacia)--the dye, 
Bromophenol Red, commonly contained in the cell culture supernatant, was 
already eluted from the column at a salt concentration of 0.2M NaCl, which 
substantially fostered the subsequent isolation of rFaII at 0.3M NaCl. 
The essential results of the purification of rFaII on the anion exchanger 
are summarized in FIG. 10 and Table 7. By the purification described in 
Example 4, the specific activity of rFaII increased by 3.5-fold. 
TABLE 7 
______________________________________ 
Factor II specific 
volumes 
protein activity activity 
sample (ml) (mg/ml) (mU/ml) (U/mg) 
______________________________________ 
cell medium 
200 0.45 50 0.1 
unbound 200 0.177 2 0.01 
fraction 
0.2M NaCl 40 0.4 6 0.015 
0.3M NaCl 45 0.36 150 0.42 
0.5M NaCl 32 0.25 0 0 
______________________________________ 
b) Purification of rFaII by Adsorption of Accompanying Proteins by Means of 
Anion Exchange Chromatography with Addition of Calcium Ions. 
Fraktogel EMD TMAE 650 M (Merck) served as an anion exchange resin. 
Materials: 
Column: Fraktogel EMD TMAE 650M, 1.6 cm.times.5 cm. 
Instrument: Pharmacia FPLC LCC-500. 
Buffer A: 50 mM Tris/HCl, pH 7.4. 
Buffer B: 50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 10 mM CaCl.sub.2. 
Buffer C: 50 mM Tris/HCl, pH 7.4, 1.0 mM NaCl. 
The column was regenerated corresponding to the instructions of the 
manufacturer and equilibrated with Buffer B. Recombinant Factor II, as it 
was isolated from cell culture supernatant as described in Example 4a), 
was diluted 2-fold with Buffer A such that the concentration of NaCl 
totaled less than 0.2M. CaCl.sub.2 was added until a concentration of 10 
mM. Subsequently, the protein mixture was applied on the column and the 
unbound protein was washed out of the column with Buffer B. Bound protein 
was eluted by means of Buffer C. 
The course of the chromatography was followed as in the preceding example 
and the protein concentrations were determined. The results show that 
rFaII passed through the column unimpeded, whereas the predominant 
majority of the contaminating proteins remained stuck on the column. These 
proteins were then eluted with 1M NaCl. 
The essential results are summarized in FIG. 11, FIGS. 12A and 12B and 
Table 8. Through the purification described in Example 4.a) and b), the 
specific activity of rFaII was increased by 11-fold. 
The denaturing electrophoretic analysis according to Laemmli, demonstrated 
(FIG. 9) that by the purification described in Example 4a and b, rFaII was 
isolated at more than 95% purity. 
TABLE 8 
______________________________________ 
Factor II specific 
volumes 
protein activity activity 
sample (ml) (mg/ml) (mU/ml) (U/mg) 
______________________________________ 
preparation 
80 0.18 75 0.41 
from Example 4a 
unbound 80 0.015 75 4.6 
fraction 
1.0M NaCl 15 0.62 0 0 
______________________________________