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PCX 



WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




INTCRNATIONAL APPUCATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 
C12N 9/64 



Al 



(11) international Publication Number: 



WO 96/40883 



(43) 



IntemaUonal Publication Date: 19 Dcsccmber 1996 (19.12.96) 



(21) International Application Number: PCT/US96/07305 

(22) International FUlng Date: 21 May 1996 (21.05.96) 



(30) Priority Data: 
08/472.823 



(71) Applicant: GENETICS INSTTHm INC. [US/US]; 87 Cam- 
WeParic Drive. Cambridge. MA 02140 (US). 

(72) Inventors: FOSTER, W.. Bany. 11 Chesmut HiU Road, 
Chelmsfoid. MA 01824 (US). COSTIGAN. Robert. J.; 
7 Camelot Drive, Boxfoid. MA 01921 (US). BONAM. 
Duane; 4 Moreecioft Lane. Amesbury. MA 01913 (US). 
SWITZER, Mary. B.; 7 Hall Avenue. Andover. MA 01810 
(US). WALSH. Rochelle; 17 Laurelton Road. North 
Reading. MA 01864 (US). 

(74) Agent: MEINERT. M., C; Genetics Institute. Inf; 

Affaire. 87 CambridgcPark Drive, Cambridge, MA 02140 
(US). 



7 June 1995 (07.06.95) 



US 



(81) Desigmited States: AU. CA. JP, E^Pj?" P^^?| ^ftl^ ?f * 
CH. DE. DK. ES. Fl. FR. GB. GR, IE. IT, LU. MC, NL. 



PT, SE). 



Published 

With international search report 



(54) Title: NOVEL FACTOR DC PURIHCATION METHODS 



(57) Abstract 



P^vided by the p«sent invention a« novel methods of factor K protein .ecove-y and p^fication. n«king use of anion exchange 
chn,nS^Sy heSin'^ resins, hydroxyapatite and immobilized metal affinity chromatography. 



FOR THE PURPOSES OF INFORMATION ONLY 



Codes used to identify States 
applications under the PCT. 



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to the PCT on the front pages 



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beland 


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of K(nea 


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pamphlets publishing international 



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uz 


Uzbekistan 


VN 


Viet Nam 



wo 96/40883 



PCTAJS96/07305 



NOVEL FACTOR IX PURIHCATION METHODS 

* 5 FIELD OF INVENTION 

The present invention relates generally to novel protein recovery and purification 
methods and more specifically to novel methods for die recovery and purification of factor IX. 

10 BACKGROUND OF THE INVENTION 

The advent of recombinant technology now allows for the production of high levels of 
proteins within suitably transformed host cells. For secreted proteins, purification of the 
protein of interest involves isolation and purification from the host cell culture medium. 
Typically, the culture medium contains selected nutrients (e.g. vitamins, amino acids, cofactors, 
15 minerals,) and additional growth factors/supplements including insulin and possibly additional 
exogenous proteins. Conditioned medium contains not only the secreted product of interest, but 
also significant quantities of additional secreted host cell proteins and other substances (e.g. 
nucleic acids, membrane vesicles). Although expressed at high levels, the product of interest 
may represent a minority of all proteins present in conditioned medium. Not unexpectedly, 
20 proteins secreted by transformed host cells may possess characteristics quite different from 
those of the product of interest {e.g. charge, molecular size, amino acid composition). 
Similarly, selected secreted host cell proteins may exhibit properties very similar to those of 
the product of interest, thereby placing significant burden on the process used for purification. 
While developing a process for purification of a recombinant protein from conditioned medium, 
25 it is important that conditions used be limited with respect to denaturation of the product of 
interest (conditions which could be used to exploit minor differences between secreted proteins 
for major benefit to separation), thereby making it difficult to separate the produa of interest 
from all other host cell proteins present. 

In addition to secreted host cell proteins described above, conditioned medium may also 
30 contain products derived from the heterologously-expressed gene coding for the product of 
interest. These are not desirable for the final drug substance and include, for example, product 
forms lacking certain post-translational modifications such as glycosylation, sulfation, fiamma 
carboxylation, or other modification potentially necessary tor biological activity. In addition, 
proteolytically-degraded forms of the product of interest may be present in conditioned medium 
35 which also need to be removed during purification, but which very closely resemble the product 
of interest. Unfortunately, most approaches, such as ion exchange chromatography. 



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hydrophobic inieraaion chromatography, and size exclusion chromatography may not provide 
the extent of resolution of the product of interest necessary for use in human therapeutic 
situations from these undesired forms. To talce full advantage of minor differences between the 
desired product and contaminants {e.g. small charge differences, small differences in molecular 
5 size) the use of strong denaturants is often required. Such denaturants, however, can lead to 
loss of biological activity, expression of neoantigenic sites, and potentially enhance chemical 
decomposition of selected post-translational modifications. 

In addition to separating the product of interest from molecules with similar properties 
{e.g. modified forms of the expressed gene), it is also important to recognize the need to 
10 separate the desired produrt from components present in conditioned medium with which it 
specifically interacts. Where the protein of interest is positively charged, it will tend to bind 
to any negatively charged molecules present thereby making purification of the protein by 
traditional methods very difficult. 

Of general background interest to the present invention are the following. Yan, USPN 
15 4,981,952 (January 1, 1991) and Yan, era/. Bio/Technology 8:655 (July 1990) which disclosed 
the use of pseudo-affinity anion exchange chromatography for the purification of vitamin K- 
dependent proteins. Josic, et al. J. Chrom. 632:1 (1993) disclosed the use of heparin affinity 
chromatography to resolve factor IX from other vitamin K-dependent proteins. Suomela, 
TTiromb. Res. 7:101 (1975); Suomela, Eur. J. Bio. Chem. 71:145 (1976); and Suomela, 
20 Thrombos. Haemostis. 35:21 1 (1976) described the use of hydroxyapatite in the separation of 
various clotting factors and factor IX plasma variants (based on charge differences due to 
variation in content of carbohydrate moieties, for example, sialic acid and galactose). 
However, Reekers, et al. Haemostasis 1 :2 (1972) demonstrated the inability of hydroxyapatite 
to separate factors II, VII and IX from each other and from other plasma proteins. Schwinn, 
25 et al. USPN 4,411,794 disclosed the partial purification of blood clotting factors using 
hydroxyapatite in the presence of calcium at a concentration of 50-200 mM. Feldman, et al. 
Biotech. Blood Proteins 227:63 (1993) and Roberts, et al. Vox Sang 67(suppl. 1): 69 (1994) 
disclosed the reduction of viral infectivity using acidification and copper-charged chelating 
Sepharose which resulted in low factor IX yields from human plasma. 
30 Typically, researchers have used combinations of traditional chromatographic 

techniques to purify desired products. Often times, such techniques are not sufficient for 
purification of a product to the level of purity and consistency desired for a human therapeutic 
product. Researchers have attempted to overcome this difficulty by use of affinity 
chromatography wherein a protein of interest is bound to an immobilized ligand with which it 



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interacts specifically. Following appropriate washing, the desired product can be eluted by 
disruption of the ligand-protein interaction, often resulting in a significantly more pure eluate. 
However, in the instance of separation of a desired product from modified forms present in 
conditioned medium, single step affinity chromatographic techniques may not be sufficient, and 

5 must be used in conjunction with other affinity resins and/or traditional separation techniques. 
Even high resolution affinity chromatography steps (e.g., immunoaffinity purification using an 
immobilized monoclonal antibody) may not afford sufficient resolution of the desired product 
from other components due to common sites of interaction {e.g., where an epitope which is 
present in the product of interest, is present as well in a proteolytically-degraded form of the 

10 product). 

Accordingly, there continues to exist a need in the an for protein purification methods 
that effectively overcome such difficulties. 

BRIEF SUMMARY OF THE INVENTION 
Provided by the present invention are methods for the purification of factor IX in a 
15 solution comprising the steps of applying the solution containing factor IX to an anion exchange 
resin, washing said anion exchange resin with a solution having a conductivity that is less than 
required to elute factor IX from the resin, eluting said anion exchange resin with a first eluant 
to form a first eluate, applying said eluate to a heparin or heparin-like (e.g., negatively charged 
matrix) resin, eluting said heparin or heparin-like resin with a second eluant to form a second 
20 eluate, applying said second eluate to an hydroxyapatite resin, and then eluting said 
hydroxyapatite resin with a third eluant to form a third eluate containing the purified factor IX. 
Optionally, the first eluate can be applied to an hydroxyapatite resin. As yet another option, 
the mediod comprises the further steps of applying the third eluate to an immobilized metal 
affinity resin, and then eluting the immobilized metal affinity resin with a fourth eluant to form 
25 a fourth eluate containing the purified faaor IX. According to the methods of the invention, 
the factor IX can be cither plasma-derived, expressed by cells in culture, or recombinantly 
produced as is known to one skilled in the art. Preferably, the first wash comprises a solution 
having a conductivity that is less than required to elute factor IX from the column and is 
generally greater than or equal to the conductivity of the load solution and of the first eluant 
30 buffer; this conductivity is sufficient to remove a substantial proportion of those contaminating 
proteins that would otherwise be present in the first eluate. A suitable first wash comprises a 
salt solution such as sodium chloride, potassium chloride, sodium sulphate, sodium phosphate, 
or potassium phosphate, and optionally, may contain a suitable buffering agent. Suitable 
concentration ranges are those which are effective in removing contaminants without eluting 



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factor IX and include for example 25 mM to 200 mM salt, and preferably is 200 mM sodium 
chloride. The first eluant comprises a divalent cation such as calcium, magnesium, manganese, 
strontium, zinc, cobalt, and nickel; suitable concentration ranges are those which are effective 
in eluting factor IX, including for example a solution containing a buffering agent at pH about 

5 8.0 such as Tris, in the range of 5 to 100 mM, preferably approximately 50 mM, a salt such 
as NaCI in the range of 50 to 250 mM. preferably 100 mM. and calcium chloride in the range 
of 5 to 20 mM, preferably approximately 10 mM. 

Suitable anion exchange resins include those resins having a positively charged group 
such as diethyleaminoethane (DEAE), polyethyleneimine (PEl), and quaternary aminoethane 

10 (QAE) and include Q-Sepharose Fast Flow, DEAE-Sepharose Fast Flow, POROS-Q, Fractogel- 
TMAE, Fractogel-DMAE, and QAE-Toyopearl, with the preferred resin being Q-Sepharose 

Fast Flow (Pharmacia). 

The second eluant can be a suitable salt in buffer, such as Tris with sodium chloride 
and potassium chloride, with 50mM TRIS, 0.50M NaCl, pH 8.0 being preferred. Suitable 
15 heparin or heparin-like resins include those resins having a negatively charged group such as 
heparin, sulfated esters of cellulose, sulfylpropyl (SP), carboxyl, and carboxy methyl and 
include Matrex Cellufine Sulfate, Heparin Sepharose. Heparin Toyopearl, Carboxy Sulfbn, 
Fractogd EMD-SO,. and Fractogel-EMD COO, with the preferred being Matrex Cellufine 
Sulfote. 

20 The third eluam can be a salt, such as phosphate and sulphate, with 0.5M potassium 

phosphate, 0.2M NaCI, pH 7.2 preferred. Suitable hydroxyapatite resins include any 
containing calcium-phosphate such as ceraraic-Hydroxyapatite, Biogel HT, and others, with 
ceramic-HA preferred. The immobilized metal affinity resin can be one such as Fractogd- 
EMD-Chelate, Chelating-Sepharose. Matrex Cellufine Chelate, and POROS 20MC. with 
25 Fractogel EMD-Chelate currently preferred. The fourth eluant is a buffer solution containing 
a chelator such as imidazole, EDTA, EGTA, glycine, histidine, and Tris, with the preferred 
being 20 mM potassium phosphate, 15 mM imidazole, O.l M NaCI, pH 7.1. 

Also provided by the present Invention are factor IX compositions produced by the 
methods of the invention. The factor IX so produced has a specific activity in the range of 
30 240-400 U/mg, and Is optionally about 240 U/mg. 

DETAILED DESCRIPTION OF THE INVENTION 
As used herein, the term "factor IX" includes, but is not limited to factor IX isolated 
from plasma, transformed cell lines, and recombinantly produced faaor IX isolated from host 
cell culture medium. 



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As used herein, the term "anion exchange resin" includes, but is not limited to resins 
having a positively charged moiety (at neuual pH), such as diethylearainoethane (PEAE), 
polyethyleneimine (PEI), and quaternary aminoethane (QAE) and includes, for example, Q- 
Sepharose Fast Flow (Pharmacia), DEAE-Sepharose Fast Flow. DEAE-Toyopearl, QAE- 
5 Toyopearl, POROS-Q, Fractogel-DM AE, Fractogel EMD-TMAE, Matrex Cellufine DEAE and 
the like. 

As used herein, the term "first wash" includes, but is not limited to a solution having 
a conductivity that is less than required to elute factor IX from the anion exchange colunm and 
whose conductivity is generally greater than or equal to the conductivity of the load solution 
10 and of the conductivity of the first eluant; this conductivity is sufficient to remove a substantial 
proportion of tfiose contaminating proteins that would otherwise be present in the eluate. As 
one skilled in the art readily appreciates, the first wash can be any salt solution and includes, 
for example, sodium chloride, potassium chloride, sodium sulphate, sodium phosphate, or 
potassium phosphate, and can be suitably buffered. Typically, concentrations range from low 
15 (25 mM salt) to high (200 mM salt), with 200 mM sodium chloride presentiy preferred. 

As used herein, the term "first eluant" includes, but is not limited to: solutions 
composed of a buffering agent (e.g. Tris) at a concentration of approximately 0.05M, salt {e,g. 
NaCl) at a concentration which is not sufficient for elution from the resin in the absence of 
divalent cation i€,g. approximately 0.10M-0.20M), and divalent cation (e.g. CaCy at low 
20 concentrations of approximately O.OIM. at pH 8.0. The selection of buffer composition is 
compatible with the presence of divalent cation. Preferably, the Tirst eluant" has a lower 
conductivity than the "first wash". 

As used herein, the terms "heparin" resin and "heparin-like" r^in are used 
interchangeably, and include but are not limited to, resins containing an immobilized negatively 
25 charged moiety such as heparin, sulfated esters of cellulose, sulfylpropyl (SP). carboxyl. and 
carboxy metiiyl and includes Fractogel-EMD-SO,, Carboxy Sulfon, Fractogel-EMD-COO, 
Heparin-Sepharose, and Matrex Cellufine Sulfate. 

As used herein, the term "second eluant" includes, but is not limited to: solutions 
composed of a buffering agent (e.g. Tris) at a concentration of approximately 0.05M, and salt 
30 {e.g. NaCl, KCl, Na,S04) at a concentration sufficient to disrupt the interaction of faaor IX 
with the negatively-charged resin support {e.g. 0.50 M) at approximately pH 8.0. As used in 
this process, the second eluant should be compatible with the subsequent process step, i.e., 
hydroxyapatite. 



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As used herein, the term "hydroxyapatite column" includes, but is not limited to: 
calcium phosphate gel supports including for example, BioGel-HT, and Ceramic- 
hydroxyapatite. 

As used herein, the term "third eluant" (and "second phosphate buffer") includes, but 
5 is not limited to: solutions composed of a buffering agent {e.g, phosphate or sulfate) at 
concentrations sufficient to disrupt interaction of factor IX with the resin (e.g. approximately 
0,20 M or higher) and salt (e.g. NaCi, KCl) present at concentrations sufficient to minimize 
charge-interactions of the factor IX with the hydroxyapatite resin, at approximately neutral pH 
(pH 7.2); the term "first phosphate buffer" includes but is not limited to solutions composed 
10 of a buffering agent (e.g., phosphate or sulfate) at concentrations sufficient to remove inactive 
forms of factor IX from the hydroxy-apatite resin. 

As used herein, the term "immobilized metal affinity resin" GMAC) includes, but is 
not limited to: resins containing an immobilized functional moiety (e.g. iminodiacetic acid) 
capable of binding and coordinating multivalent cations including Chelating-Sepharose, 
15 Fractogel-EMD-Chelate, POROS 20MC, and Matrex Cellufine Chelate. The bound metal ion 
can be selected from several possible choices including but not limited to copper, nickel, 
cadmium, cobalt, iron, zinc, or strontium. 

The term "fourth eluant" (also termed "displacer") includes but is not limited to, any 
compound which will displace bound factor IX from the IMAC resin support, while minimizing 
20 displacement of the immobilized metal ion from the resin support, and includes but is not 
limited to such compounds as glycine, histidine, tris, imidazole, EDTA, EGTA, and the like. 
As one skilled in the art readily appreciates, the appropriate concentration of displacer will vary 
according to binding affinity and can be ascertained by experimental evaluation of conditions. 
Typically, concentrations range from low (e.g. 5-15 mM displacer) to high (e.g. 100-200mM 
25 displacer). 

Reference to factor IX specific activity of "U/mg" includes but is not limited to: 
biological activity determined in the in vitro (APTT) clotting assay using pooled plasma or 
isolated, purified factor IX as standard. The concentration of protein can be determined by any 
of several appropriately validated methods including SEC, RP-HPLC, dye-based assays (e.g., 
30 Bradford, Lowry) or absorbance at 280 nm. Factor IX activity is determined according to the 
method of Pittman, D., et al , Blood 7P:389-397 (1992) utilizing factor IX-deficient plasma. 

Figure 1 provides an overview of the process. While the order of the steps set forth 
is the presently preferred embodiment, it will be appreciated by one skUled in the art that the 
order can be re-configured if desired and that steps can be omitted. 



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PCT/US96/0730S 



10 



15 



According to the present invention, cells are first removed from conditioned medium, 
e.g. by microfiitration (MF) utilizing tangential flow filtration membranes with pore size of 
approximately 0.6^m. Optionally, cell-free conditioned medium is prepared for purification by 
filtering through a 0.45/im depth filter. The cell-free conditioned medium can then be 
concentrated by ultrafiltration, if desired, followed by diafiltration into an appropriate buffer 
for loading onto the first chromatographic step. Alternatively, the cell-free conditioned medium 
may be loaded directly onto the first chromatography column equilibrated in an appropriate 
buffer. 

HGUREl 

Overview of factor IX Purification Process 

PROCESS STEP 
Filtered Conditioned Medium 



Ultrafiltration 
and Diafiltration 



20 



Q-Sepharose FF Pseudoaffinity 
Anion Exchange Chromatography 



Matrex Cellufine Sulfate 
Chromatography 



25 



Ceramic-Hydroxyapatite 
Chromatography 



Chelate-EMD-Cuai) 
Affinity Chromatography 



30 



Ultrafiltration 
and Diafiltration 



Factor IX 



7 



PCT/US96/07305 

WO 96/40883 

The initial process step, UF/DF#1, entails concentration of the cell-free factor IX- 
containing conditioned media by ultrafiltration, followed by diafUtration. Although not 
required for binding of factor IX to the first chromatography column, this step is effective in 
removing small-molecular-weight cell culture media components. Sudi components may bind 
5 to the mitial chromatography column, thereby decreasing the capacity of the column for factor 
IX. UF/DF#1 is used to exchange the factor IX into an appropriate buffer solution for 
subsequent processing. 

In the fu^t chromatography step, anion-exchange on Q-Sepharose Fast Flow (FF) 
(Pharmacia), the factor IX Is captured and purified from host-cell components present in the 
10 UF/DF #1 concentrated pool. The Q-Sepharose FF column adsorbs the factor IX protein, and 
contaminating host-cell proteins with isoelectric points greater than the operating pH are 
removed from the process stream by flowing through the colunm. The colunm to which factor 
IX is adsorbed is then washed prior to eiution to remove loosely-bound contaminants and adjust 
the conductivity of the buffer in preparation for eiution. 
15 Typically, bound proteins are eluted from Q-Sepharose FF by increasing the ionic 

strength of the buffer. The faaor IX purification process, however, employs this resin in a 
pseudo-affinity anion-exchange mode in which active factor IX is eluted by addition of e.g., 
calcium chloride to the buffer. This divalent cation results in eiution of active forms of factor 
K from the resin. Some less active forms of factor IX may also elute from the Q-Sepharose 
20 FF colunm with this eiution buffer. Selected inactive forms of factor IX and other, 
contaminating host-cell proteins remain bound to the column. The Q-Sepharose FF st^ 
achieves a significant increase in the purity of the factor IX. 

In the second chromatography step, the Q-Sepharose FF eiution pool is loaded directly, 
without dilution, onto the Matrex Cellufine Sulfate column. The factor IX adsorbs to the 
25 column, while other, contaminating proteins (e.g., soluble PACE and other host-cell proteins 
present in the Q-Sepharose FF eluate) are removed from the process stream by flowing through 
the column. The column is washed with a low-ionic-strength buffer to remove all non-binding 
proteins. The factor IX is eluted by an increase in the ionic strength of the buffer, using salt 
(e.g., sodium chloride). 

30 Further removal of inaaive factor IX forms is obtained during the third 

chromatography step, Ceramic-HA column chromatography. The pH of the Matrex G^llufine 
Sulfate eiution pool is adjusted to approximately 7.5, and the eiution pool is then loaded 
directly onto the Ceramic-HA column. The factor IX is adsorbed by the column. The 
Ceramic-HA column is washed with buffer to remove loosely bound contaminants, followed 



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by a wash with 50 mM potassium phosphate, 0.185 M NaCl (pH 7.2) to remove more tightly 
bound contaminants, including inactive forms of factor IX. Bound, active fiactor IX is eluted 
in a step-wise manner using a solution containing a higher concentration of potassium phosphate 
{e.g. 200mM or greater, pH 7.2) as the eluant. 
5 The fourth chromatography step, Fractogel EMD-Chelate -CuOI) chromatography, 

removes low levels of contaminating host-cell proteins still present in the product stream. The 
Ceramic-HA elution pool is loaded directly onto the Fractogel EMD-Chelate -CuGI) colunm. 
Factor IX and a number of contaminating proteins are adsorbed to the column. Purified aaive 
factor IX is eluted from the column by low concentrations of imidazole {e.g. approximately 
10 15mM) in the buffer, and the residual, contaminating host-cell proteins are removed from the 
product stream by remaining bound to the colunui. 

Finally, the Fractogel EMD-Chelate -CuOI) elution pool is concentrated by 
ultrafiltration, followed by diafiltration (UF/DF#2) into a buffer identical to a formulation 
buffer except that it does not contain polysorbate 80. A suitable formulation buffer comprises 
15 histidine, glycine, sucrose, and polysorbate-80 optionally at i OmM, 260mM, 1 % , and 0.005 % , 
respectively. Upon completion of the diafiltration, factor IX Is concentrated to achieve a target 
concentration. The product pool is removed from the UF/DF 2, apparatus and formulated by 
addition of polysorbate 80 to a target concentration of 0.005% . The factor IX drug substance 
is then filtered (0.2 pLin), sampled, labeled, and stored frozen at approximately -80 "C. The 
20 last process step, UF/DF#2, is effective in concentrating and diafiltering the purified factor IX 
drug substance without significant protein denaturation or loss. SDS-PAGE analysis (reduced 
and nonreduced) is one mediod used to evaluate overall process performance. Each step 
provides greater than %0% to 100% yield and the average overall yield of factor IX is about 
51%. The overall process yield is determined from the dotting activity entering the 
25 purification process and the total clotting activity in the factor IX drug substance (excluding 
material removed as in-process samples and retains). 

The following examples illustrate practice of the invention. These examples are for 
illusuative purposes only and are not intended in any way to limit the scope of the invention 
claimed. 

30 Example 1 describes concentration of protein by ultrafiltration/diafiltration; Example 

2 relates to purification of factor IX by pseudo-affinity anion-exchange chromatography on Q- 
sepharose fast flow; Example 3 describes purification of faaor IX by chromatography on 
Matrex Cellufine Sulfate; Example 4 relates to purification of protein with hydroxyapatiie 
chromatography; Example 5 describes purification of protein by inmiobilized metal affinity 



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chromatography; and Example 6 relates to concentration and formulation of protein by 
uitrafiltration/diafiltration. 

Example 1 - Concentration of Protein by Ultrafiltration/Diafiltration 

Optionally, uitrafiltration/diafiltration (UF/DF#1) can be performed to concentrate and 

5 to buffer-exchange the cell-free conditioned medium using tangential-flow membrane filtration. 
The membrane used in the tangential-flow device serves as a selectively permeable filter that 
separates substances on the basis of molecular weight. Solution components of high molecular 
weight, such as factor IX, are retained by the membrane, and components of low molecular 
weight, such as inorganic salts and buffer components, pass ireely through the porous 

10 membrane structure and are removed in the permeate. 

When buffer is drawn from the tangential-flow device at a rate faster than that at which 
replacement buffer is added to the retentate, the protein solution is concentrated. When 
replacement buffer is added to the ungentiai-flow retentate at a rate approximately equal to the 
rate at which the buffer is drawn through the membrane, the initial buffer is continuously 

15 diluted (protein diafiltration). Under these conditions, compounds of low molecular weight are 
readily exchanged and the protein concentration remains constant. The addition of five 
retentate volumes of buffer results in a theoretical replacement of ^99% of the initial buffer. 

Before use, the UF/DF#1 system is equilibrated with 50 mM TRIS, 150 mM NaCl, 
pH 7.5. The cell-free conditioned medium is concentrated approximately 20-fold relative to 

20 the initial volume of the cell-free conditioned medium. The concentrated cell-free conditioned 
medium is then diafiltered into the buffer. The diafiltration is complete when at least five 
retentate volumes of the buffer have passed through the membrane, resulting in a theoretical 
removal of ^99% of salts and other low-molecular-weight components present in the cell-free 
conditioned medium. 

25 Once diafiltration has been completed, the retentate is concentrated if necessary. The 

equipment is then flushed with sufficient buffer to recover residual factor IX product from the 
reservoir and tubing. The pool is then pumped out of the UF/DF vessel and filtered through 
an autoclaved 0.2-;im filter into a clean vessel. The UF/DF#1 pool is stored at 2 to 8 **C until 
it is further processed. 

30 Example 2- Purification of factor IX by Pseudo-Affinity Anion-Exchange 
Chromatography on Q-Sepharose Fast Flow 

Q-Sepharose Fast Flow (FF) (Pharmacia) is a strong anion-exchange resin composed 
of a cross-linked agarose matrix that is covalently derivatized with a quaternary amine ^roup 
35 through a short linker. Acidic proteins (such as factor IX) and other polyionic substances with 



10 



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a net negative charge at the pH of operation bind to Q-Sepharose FF via charge interactions. 
Typically, bound components are differentially eluted from Q-Sepharose FF by disruption of 
these charge interactions with solutions of increased conductivity. However, the factor IX 
purification process employs the Q-Sepharose FF resin in a pseudo-affinity mode. Factor IX 
5 is eiuted from the column using a solution containing low-concentration (10 mM) calcium 
chloride. The inclusion of calcium ions in the elution buffer causes a conformational change 
in the factor IX that results in elution from the resin. 

QrSepharose FF is used to capture factor IX from UF/DF^l retentate; to remove 
uncharged and basic contaminants from the process stream (in the unbound fraaion during 
10 loading of the column); to separate factor IX from acidic proteins (which bind to the resin but 
are riot eluted by addition of calcium chloride to the buffer), including inactive forms of faaor 
IX; and to deliver a concentrated factor IX process stream into the subsequent purification 
process chromatography step, Matrex Cellufine Sulfate. 

Optionally, all chromatography operations for this step are performed at 2** to 8** C. 
15 The Q-Sepharose FF column is first charged with 50 mM TRIS, 2 M NaCl, pH 8.0, followed 
by equilibration with 50 mM TRIS, 150 mM NaCI, pH 8.0. The UF/DFiS^l retentate is loaded 
onto the Q-Sepharose FF column, and the column is then washed with 50 mM TRIS, 200 mM 
NaCl, pH 8.0. This first wash ensures that the entire load has passed through the column and 
that non-adsorbing impurities in the load, as well as contaminants that are loosely bound to the 
20 resin, have been washed from the system. The column is then washed with 50 mM TRIS, 100 
mM NaCl, pH 8.0 to lower the conductivity in preparation for elution. 

Factor IX is eluted from the column with 50 mM TRIS, 100 mM NaCl, 10 mM CaClj, 
pH 8.0, and the eluted product is collected as a single peak. The Q-Sepharose FF eluate is 
sampled and stored at 2 to 8 ^'C until it undergoes further processing. Optionally, the column 
25 can be regenerated and reused. 

Example 3 - Purification of factor IX by Chromatography on Matrex Cellufine Sulfate 

Matrex Cellufine Sulfate is composed of spheroidal cellulose beads derivatized with 
sulfate esters. It can be used as an immobilized heparin analogue for affinity-purification of 
proteins containing heparin-binding domains. It can also be used for cation-exchange 
30 chromatography because of its negatively charged sulfate functions. Basic proteins, other 
polyionic substances with a net positive charge at the pH of operation, and heparin-binding 



11 



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PCT/US96/07305 



proteins bind to the resin and are eluted with solutions of increasing ionic strength. The 
Matrex Cellufine Sulfate resin is used in the factor IX purification process for removal of host- 
ceil proteins other than factor IX in the Q-Sepharose FF elution pool and, optionally, to 
provide appropriate buffer conditions for loading the hydroxyapatite column. 
5 Optionally, all chromatography operations for this step are carried out at 2 to 8 •C. 

In preparation for the load step, the Matrex Cellufine Sulfate colunm is equilibrated with 50 
mM TWS, pH 8.0. The Q-Sepharose FF elution pool is loaded directly onto the equilibrated 
Matrex Cellufine Sulfate column, the column is washed with 50 mM TRIS. 150 mM NaCl, 10 
mM CaCl^, pH 8.0 to ensure that all of the load has passed through the column and that weakly 
10 bound impurities are removed from the system. Next, the colunm can be washed to remove 
calcium ions prior to elution. 

After the wash steps have been completed, the Matrex Cellufine Sulfate column is 
eluted with 50 mM TRIS, 500 mM NaCl, pH 8.0, and the eluate collected as a single UV- 
absorbing elution pool. The Matrex Cellufine Sulfate elution pool is sampled and stored at 2** 
15 to 8* C until it is further processed. Optionally, the column can be regenerated and reused. 
Example 4 - Purification of Protein with Hydroxyapatite Chromatography 

Ceramic-Hydroxyapatite (Cwamic-HA) is a synthetic form of calcium phosphate 
consisting of spheroidal macroporous particles with high mechanical strength. Ceramic-HA 
separates proteins with a wide range of charges and isoelectric points largely on the basis of 
20 charge interactions. Factor IX is an acidic protein that binds to Ceramic-HA at approximately 
neutral pH. Typically, acidic proteins are eluted from Ceramic-HA by the addition of 
phosphate to the buffer solution. The concentration of phosphate required for elution varies, 
depending upon the properties of the molecule of interest, thereby allowing differential elution 
of bound proteins. Ceramic-HA is used in the factor IX purification process to remove inactive 
25 factor IX, and other contaminants, in the Matrex Cellufine Sulfate elution pool and to exchange 
the eluate buffer to one compatible with the final chromatographic step. Because the final 
chromatography step is immobilized metal affinity chromatography, the buffers used for elution 
of Ceramic-HA are selected to be compatible with IMAC. This avoids an in-process 
diafiltration or other buffer exchange procedure. Buffers such as Tris, glycine, histidine are not 
30 compatible with IMAC because of disruption of the metal ion-immobilized ligand interaction. 
Elution of the Ceramic-HA column with phosphate buffers avoids such complications. 

In preparation for loading, the Ceramic-HA column is equilibrated with 50 mM TRIS, 
500 mM NaCl, pH 7.5. The Matrex Cellufine Sulfate elution pool is titrated to pH 7.5 with 
dilute HCl and loaded directly onto the Ceramic-HA column. Upon completion of loading, the 



12 



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PCT/US96/07305 



column is washed with buffer (0.5 M NaCI, 50 mM TRIS, pH 7.5) to ensure that all of the 
load has passed through the column and that loosely bound contaminants are removed from the 
column. Next, the column is washed with 50 mM K^HPO*, 185 mM NaCl, pH 7.2 to remove 
inactive forms of factor IX from the process stream. 
5 Upon completion of the wash steps, the bound factor IX is eluted with 500 mM 

KJIP04. 200 mM NaCI, pH 7.2, and the factor IX eluate is collected as a single UV-absorbing 
eiution pool. The eluate pool is sampled and stored at 2 to 8 until it undergoes further 
processing. Optionally, the column can be regenerated and reused. 
Example 5 - Purification of Protein by immobilized Metal Affinity Chromatography 
10 Fractogel-EMD-Chelate is composed of a methacrylate polymer derivatized with 

iminodiacetic fiinctional groups to which transition-state metal ions can be bound. In 
preparation for use in the purification process, the resin is charged with copper ions using a 
solution of copper sulfate. Proteins capable of interacting with the immobilized copper ions 
are retained on the colunm, and non-intwacting contaminants pass through in the unbound 
15 frartion. Bound proteins are eluted from the resin using solutions containing imidazole. A 
Fractogel-EMD-Chelate-Cuai) step can be used in the protein purification process to remove 
from the process stream contaminants that do not bind to the immobilized metal ion or that 
require higher concentrations of imidazole for eiution than those required by factor IX. The 
term IMAC (immobilized metal affinity chromatography) is also used to denote this 
20 chromatography step. 

In preparation for loading, the uncharged (no immobilized metal ion) Fractogel-EMD- 
Chelate column is washed with 100 mM acetic acid, 500 mM NaCI, pH 4.0 and is 
subsequently charged with 200 mM CuSO., 500 mM NaCI. Loosely bound copper ions are 
removed by washing the charged resin with 100 mM acetic acid, 500 mM NaCI. pH 4.0, 
25 followed by 200 mM imidazole, 500 mM NaCI, pH 7.1. The Fractogel-EMD-Chelate-Cuai) 
resin is then equilibrated in 200 mM K.HPO4, 200 mM NaCI, pH 7.1 (Equilibration V). The 
Ceramic-HA eiution pool is loaded directly onto the equilibrated Fraaogel-EMD-Chelate-Cu(n) 
colunm. 

Upon completion of loading, the column is washed with equilibration buffer to ensure 
30 that all of the load has passed through die column. Factor IX bound to the resin is eluted using 
20 mM K2HPO4, 15 mM imidazole, 100 mM NaCI, pH 7.1. The Fractogel-EMD-Chelate- 
Cu(n) eluate is collected as a single UV-absorbing pool. After collection, the eiution pool is 
diluted with 20 mL of 500 mM EDTA, pH 8.0 per liter of column eluate. The diluted eiution 
pool is stored at room temperature until it undergoes further processing. 



13 



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PCTAJS96/07305 



Example 6 - Concentration and Formulation of Protein by 
Ultranitration/Dianitration #2 

To transfer the factor IX to a buffer of choice, a combination ultrafiltration/diafiltration 
5 step is used. Tangential-flow UF/DF is a non-chromatographic separation method that can be 
used to concentrate and buffer-exchange substances in solution, A feedstrcam is directed 
parallel to the surface of a selectively permeable membrane, and pressure is applied to the 
retentate side of the membrane outlet to effect transpon of water and solutes at the membrane 
surface on the basis of their relative permeability. Under these circumstances, low-molecular- 
10 weight feedstream components pass freely through the membrane pores into the permeate 
fraction, and higher-molecular-weight substances {e.g., factor IX) are retained by the membrane 
and constitute the retentate fraction. In this manner, water and buffer salts can be removed 
from die Fractogel-EMD-Chelate-Cu(II) eiution pool, and the factor DC can be concentrated to 
a target concentration. 

15 Optionally, a spiral-wound cartridge component of the tangential-flow system is first 

equilibrated whh 10 mM histidine, 260 mM glycine, 1% sucrose, pH 6.8. The Fractogel- 
EMD-Chelate-CuGI) eiution pool (previously diluted with 500 mM EDTA) is then transfened 
to the stainless-steel retentate pressure vessel of the tangential-flow apparatus In preparation for 
protein concentration. 

20 After the transfer is completed, the retentate solution is pumped continuously from the 

pressure vessel through the spiral-wound cartridge and back to the pressure vessel under a n^ 
positive transmembrane pressure. The volume of the retentate is monitored continuously during 
this operation by measuring the permeate fraction volume using a graduated collection vessel. 
When the target retentate volume is reached, the retentate pool is diafiltered into the 

25 buffer of choice. During this operation, diafiltration buffer is pumped into the pressure vessel 
at the same rate at which permeate flows from the system, thereby maintaining a constant 
retentate volume. 

After completion of the diafiltration step, the retentate fraction is concentrated to a 
target volume using ultrafiltration. The outflow fraction from the retentate pressure vessel is 
30 stopped, and the retentate fraction in the spiral-wound cartridge is flushed into the retentate 
pressure vessel with a target volume of die buffer of choice. The concentrated, diafiltered 
factor IX product is recovered from the pressure vessel by pumping into tared pool bottles. 

The factor DC product pool is diluted with 10 mM histidine, 260 mM glycine, 1% 
sucrose, 1% polysorbate 80, pH 6.8 to a final concentration of 0.005% polysorbate 80. The 
35 product is then mixed thoroughly and filtered through a 0.2-Mm filter (previously equilibrated 



14 



wo 96/40883 



PCTAJS96/07305 



in 10 mM histidine, 260 mM glycine, \% sucrose, 0.005% polysorbate 80, pH 6.8 into 
depyrogenated Teflon bottles. The protein is then sampled, labeled, frozen quickly in liquid 
nitrogen, and stored at -80" C 

While the present method of the invention is exemplified by purification of 

5 recombinantly-produced factor IX from transformed host cells, the method is also amenable to 
purification of factor IX naturally occurring within a cell and can be used to purify proteins 
from solution or from plasma, cell homogehates, cell culture supematants, or isolated cellular 
sub-fractions. While the present invention has been described in terms of specific methods and 
compositions, it is understood that variations and modifications will occur to those skilled in 

10 the art upon consideration of the present invention. 

Numerous modifications and variations in the invention as described in the above 
illustrative examples are expected to occur to those skilled in the an and, consequently, only 
such limitations as appear in the appended claims should be placed thereon. Accordingly, it 
is intended in the appended claims to cover all such equivalent variations which come within 

15 the scope of the invention as claimed. 



15 



wo 96/40883 



PCT/US96/07305 



WHAT IS CLAIMED: 

1 . A method for purification of factor IX in a solution comprising the steps of: 
applying said solution to an anion exchange resin, 

eluting said anion exchange resin with a first eluant to form a first eluate, 
applying said eluate to a heparin-like resin. 

eluting said heparin-like resin with a second eluant to form a second eluate, 

applying said second eluate to an hydroxyapaiite resin, and 

eluting said hydroxyapatite resin with a third eluant to form a third eluate. 

2. The method of claim 1, further comprising the step of: 

washing said anion exchange resin with a first wash prior to eluting with said first 

eluant. 

3. The method of claim 2, wherein said first wash comprises a solution selected from the 
group consisting of: sodium chloride, potassium chloride, sodium sulphate, sodium phosphate, 
or potassium phosphate. 

4. The method of claim 3, wherein said first wash is 200 mM sodium chloride, 

5. the method of claim 1, further comprising the steps of: 

applying said third eluate to an immobilized metal affinity resin, and 

eluting said immobilized metal affinity resin with a fourth eluant to form a fourth eluate 

6. The method of claim 1, wherein said first eluant comprises a divalent cation selected 
from the group consisting of: calcium, magnesium, manganese, strontium, zinc, cobalt, and 
nickel. 

7. The method of claim 6, wherein said first eluant is 10 mM calcium. 

8. The method of claim 1, wherein said anion exchange resin has a positively charged 
group which is a member selected from the group consisting of: diethyleaminoethane (DEAE), 
polyethyleneimine (PEI), and quaternary aminoeUiane (QAE). 

9. The method of claim 8, wherein said anion exchange resin is Q-Sepharose Fast Flow. 

10. The method of claim 1 , wherein said heparin-like resin has a negatively charged group 
which is a member selected from the group consisting of heparin, sulphated esters of cellulose, 
sulfylpropyl (SP), carboxyl, and carboxy mediyl. 

11. The method of claim 10 wherein said heparin-like resin is Matrex Cellufine Sulfate. 

12. The method of claim 1, wherein said second eluant is a member selected firom the 
group consisting sodium chloride and potassium chloride. 

13. The method of claim 12, wherein said second eluant is 50mM Tris, 0.50M NaCl, 
pH 8.0. 



16 



PCT/US96/07305 

96/40883 



14. The method of claim 1, wherein said third eluant is a member selected from the group 
consisting of phosphate and sulphate. 

15. The method of claim 14, wherein said third eluant is 0.5M potassium phosphate, 0.2M 
NaCl, pH 7.2, 

16. The method of claim I , wherein said hydroxyapatite resin is member selected from the 
group consisting of ceramic-hydroxyapatite and BioGel-HT. 

17. The method of claim 16, wherein said hydroxyapatite resin is ceramic-hyroxyapatite. 

18. The method of claim 5, wherein said inmiobilized metal affinity resin is a member 
selected from the group consisting of Fractogei-EMD-Chelate, Chelating-Sepharose, Matrex 
Cellufme CHidate, and POROS 20MC. 

19. The method of claim 5, wherein said immobilized metal affinity resin is Fractogel 
EMD-Chelate. 

20. The method of claim 5, wherein said fourth eluant is a displacer. 

21 . The method of claim 20, wherein said displacer is a member selected from the group 
consisting of imidazole, EDTA, EGTA, glycine, histidine, and Tris. 

22. A method for purification of faaor IX in a solution comprising the steps of: 
applying said solution to an anion exchange resin, 

eluting said anion exchange resin with a first eluant to fom a first eluate, 

applying said first eluate to an hydroxyapatite resin, and 

eluting said hydroxyapatite resin with a third eluant to form a third eluate. 

23. The method of claim 22, further comprising the step of: 

washing said anion exchange resin with a first wash prior to eluting with said first 
eluant. 

24. The method of claim 22, further comprising the steps of: 
applying said third eluate to an immobilized metal affinity resin, and 

eluting said immobilized metal affinity resin with a fourth eluant to form a fourth 
eluate. 

25. A method for purification of factor IX in a solution comprising the steps of: 
applying said solution to an anion exchange resin, 

washing said anion exchange resin with a first wash, and 

eluting said anion exhange resin with a first eluant to form a first eluate, 

wherein said first eluant has a conduaivity lower than the higher of said first 
wash conductivity and of said solution conductivity. 



17 



wo 96/40883 



PCT/US96/07305 



26. The method of claim 25, wherein said first wash comprises a solution selected from 
the group consisting of: sodium chloride, potassium chloride, sodium sulphate, sodium 
phosphate, or potassium phosphate. 

27. The method of claim 26, wherein said first wash is 200 mM sodium chloride. 

28. A method for purification for fartor IX in a solution comprising the steps of: 
applying said solution to an hydroxyapatite resin, 

washing said hydroxyapaptite resin with a first phosphate buffer to remove inactive 
forms of factor IX, 

eluting said hydroxyapatite resin with a second phosphate buffer, wherein said second 
phosphate buffer has a higher phosphate concentration than said first phosphate buffer. 

29. The method of claim 28, wherein said first phosphate buffer is a sulphate buffer. 

30. The method of claim 28, wherein said first phosphate buffer is 50 mM potassium 
phosphate, 0.185 M NaCl, pH 7.2 and said second phosphate buffer is 0.5 M potassium 
phosphate, 0.2 M NaCI, pH 7.2. 

31. A factor IX produced by the method of claim 1. 

32. A factor IX produced by the method of claim 5. 

33. A factor IX produced by the method of claim 22. 

34. A factor IX produced by the method of claim 24. 

35. A factor IX produced by the method of claim 25. 

36. A factor IX produced by the method of claim 28. 

37. A factor IX having a specific activity of 240-400 U/mg. 

38. The factor K of claim 37, wherein said specific activity is about 240 U/mg. 



18 



INTERNATIONAL SEARCH REPORT , ,^^^^anHc 

PCT/US 96/07305 



I A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C12N9/64 



AccDidiBg to InWMtioMl P«tent Qasifiaition QPC) or to both natonal daaifiottion and IPC 



Minimum <tocuma.t.tionf.ardi«i (daaifieation io«t«n> Wlowtd by d«ssifi<ation .yraboH) 

IPC 6 C12N 



Documentation waidied 



^er than miniroum docummt.tieo to ttie extent ttat iud> doeumentj arc included in the ttdds acardkcd 



Beclroittc 



data base consulted during the intemalional search (name of d«» base and. t»hcre pracHcal. .eardi tern, wed) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Cakgoiy* 



OtaUan of document, wiOi indication, where appropriate, of the rdevant passages 



Rdevant to daim No. 



X 

Y 



EP.A.0 363 126 (LILLY CO ELI) 11 April 
1990 

see the whole document 



THROMBOSIS RESEARCH. 

vol. 49, no. 2, 1988, 

pages 277-286, XP002013371 

A. BENNY ET AL: "Comparative separation 

of human plasma proteins on polysulphated 

chromatographic media" 

see the whole document 



25-27. 
35,37.38 
1-24, 
31-34 

37,38 



1-21,31, 
32 



p^j Further documents are listed in the oontinuatian of box C. 



m 



Patmt family members are listed in annex. 



* Special categories of dtcd documents : 

'A* document defining the general state of the art which is not 

considered to be of partcidar relevance 
'E' earlier document but puWidwd on or after die international 
filing date 

'V document which may ftrow doubte on priwi^ 

which is oted to establish die pufUicabon date of another 
dtation or other special reason (as qiedfied) 
•O* document leferhng to an oral disdosure, use, cadiibition or 
other means 

'V documem published prior to the international filing date but 
later than the priority date daimed 



-r Uter document puWidied after the international filing d^ 
or priority date and not in confiirt with the application but 
dted to understand dte prindple or theory underlying the 
invention 

*X' document of particular relevance; the daimed invention 
cannot be considered novd or cannot be conadered to 
involve an inventive step when the document is taken alone 

•Y' document of paiticidar relevance; the daimed invention 
cannot be considered to involve an inventive stq) whenthe 
document is combined with one or more other such docu- 
ments, such combination being obvious to a persaa doUed 
in the arc 

documem meotfier of the same patent family 



Date of the actual completion of the btenutional search 

20 Septeniber 1996 



Date of mailing of the tntematiottal search report 



Name and mailing address of the ISA 

European Patent Oflice. SSI 8 Patendaan 2 

NL- 2280HVRijswijk 

Td. (+31-70) 340-2040, Tx. 31 651 epo id. 
Fax: (4 31-70) 34O-30I6 



Authorized officer 



Van der Schaal, C 



Femi PCT/ISA/MO (leeend rtieti) (July IWJ) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



I int iooal Application No 

PCT/US 96/07305 



I C(&mlinu.tio n) DOCUMENTS CONSIDERED TO BE RELEVANT 

■c«eg«y- I Oialionofdocunw*»i»iiMBc.li(m.wlitr.w™pri«««.o'*«^ 

JOURNAL OF CHROMATOGRAPHY, 
vol. 632, 1993, AMSTERDAM NL, 
pages 1-10, XP002O13372 
D. JOSIC ET AL: "Isolation of plasma 
proteins from the clotting cascade by 
heparin affinity chromatography" 
see the whole document 



HAEMOSTASIS, 

vol. 1, 1972, 

pages 2-22, XP08O6O2120 

P. REEKEERS AND H. HEMKER: "Purification 

of blood coagulation factors II, VII, IX, 

X from bovine citrated plasma" 

cited in the application 

see the whole document 



EUROPEAN JOURNAL OF BIOCHEMISTRY, 
vol. 71, 1976, 

pages 145-154. XPO00602032 ^ 

H. SUOMELA: "Human coagulation factor IX 

see the whole document 

BIOTECHNOLOGY OF BLOOD PROTEINS, 

vol. 227, 1993. 

pages 63-68. XP000602055 

P. FELDMAN ET AL: "Preparation of a high 

purity factor IX concentrate using metal 

chelate affinity chromatography" 

cited in the application 

see the whole document 



EP.A.0 617 049 (SCLAVO SPA ;AIMA DERIVATI 
SPA (IT)) 28 September 1994 
see the whole document 



|X 
Y 

P.X 



W0.A.89 05650 (CENTRAL BLOOD LAB 
AUTHORITY) 29 June 1989 
see the whole document 



BLOOD. ^ ,„„ 

vol. 86, no. 10, November 1995, 

page 870a XP002013377 

W. FOSTER ET AL: "Development of a 

process for purification of reconijinant 

human factor IX" 

see abstract 3468 



I Relevant to claim No. 

25-27. 
35,37,38 



1-24, 
31-34 

25-30, 
35-38 



1-21.31. 

32 

1-4. 

6-17, 

25-31. 

35-38 

5.18-21, 

32 

25-27, 
35.37,38 



5,18-21. 
24,32,34 

25-27. 
35.37.38 
5,18-21. 
24,32,34 

37,38 

5,18-21, 
24.32.34 

1-21. 

25-32, 

35-38 



fma PCTASA/aO (cdbiIubmIoii of uamt ikcM) (July IWJ) 



page 2 of 2 



INTERNATIONAL SEARCH REPORT 

^nfarmation on patent family monbcrs 



I [ntr onal Application No 

PCT/US 96/07305 



Patent document 
cited in search report 

EP-A-e363126 



Publication 
date 

11^04^90 



Patent family 
member(s) 



US-A- 
AT-T- 
AU-B- 
AU-A- 
CA-A- 
DE-D- 
DE-T- 
ES-T- 
lE-B- 
JP-A- 



4981952 
106406 
635222 
4251989 
1314011 
68915675 
68915675 
2054019 
63765 
2208180 



EP-A-0617049 
WO-A-8905650 



28- 09-94 

29- 05-89 



US-A- 



5378365 



AU-A- 
DE-A- 
DE-T- 
EP-A- 
JP-B- 
JP-T- 
NO-B- 
US-A- 



3032989 
3881895 
3881895 
0391974 
6000064 
3501085 
178882 
5445958 



Publication 
date 

01- 0^91 

15-06-94 
18-03-93 
12-04-90 

02- 03-93 

07- 07-94 
20-10-94 
01-08-94 
14-06-95 

08- 08-90 



03-01-95 



19-07-89 
22-07-93 
02-12-93 

17- 10-90 
05-01-94 
14-03-91 

18- 03-96 
29-08-95 



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