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WORLD INTELLECTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification 6 : 
C07K 1/18 



Al 



(11) Internationa) Publication Number: WO 99/31120 

(43) International Publication Date: 24 June 1999 (24.06.99) 



(21) International Application Number: PCT/US98/26208 

(22) International Filing Date: 10 December 1998 (10.12.98) 



(30) Priority Data: 

08/989,543 



12 December 1997 (12.12.97) US 



(71) Applicant: GENETICS INSTITUTE, INC. [US/US]; 87 Cam- 

bridgePark Drive, Cambridge, MA 02140 (US). 

(72) Inventors: FOSTER, Barry; 1 1 Chestnut Hill Road, Chelms- 

ford, MA 01 824 (US). GERMAIN, Bonnie; 5 Kimball Lane, 
Webster, NH 03303 (US). HAMMER STONE, Karen; 15 
Daisy Lane, Tyngsboro, MA 01879 (US). 



(74) Agent: GYURE, Barbara, A.; Genetics Institute, Inc., 
CambridgePark Drive, Cambridge, MA 02140 (US). 



87 



(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR, 
BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE, 
GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, 
LC, LK, LR, LS, LT, LU. LV, MD, MG, MK, MN, MW, 
MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, 
TM, TR, TT, UA, UG, UZ, VN, YU, ZW, ARIPO patent 
(GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian patent 
(AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European patent 
(AT, BE, CH, CY, DE, DK, ES, Fl, FR, GB, GR, IE, IT, 
LU, MC, NL, PT. SE), OAPI patent (BF, BJ, CF, CG, CI, 
CM. GA, GN, GW, ML, MR, NE, SN, TD, TG). 



Published 

With international search report. 

Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 
amendments. 



(54) Title: NOVEL TGF-BETA PROTEIN PURIFICATION METHODS 
(57) Abstract 

Method of purifying TGF-/? superfamily proteins, including osteogenic proteins, such as bone morphogenetic proteins (BMPs), are 
disclosed. 



FOR THE PURPOSES OF INFORMATION ONLY 
Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT. 



AL 


Albania 


ES 


Spain 


LS 


Lesotho 


Si 


Slovenia 


AM 


Armenia 


Fl 


Finland 


LT 


Lithuania 


SK 


Slovakia 


AT 


Austria 


FR 


France 


LU 


Luxembourg 


SN 


Senegal 


AU 


Australia 


GA 


Gabon 


LV 


Latvia 


sz 


Swaziland 


AZ 


Azerbaijan 


GB 


United Kingdom 


MC 


Monaco 


TD 


Chad 


BA 


Bosnia and Herzegovina 


GE 


Georgia 


MD 


Republic of Moldova 


TG 


Togo 


BB 


Barbados 


GH 


Ghana 


MG 


Madagascar 


TJ 


Tajikistan 


BE 


Belgium 


GN 


Guinea 


MK 


The former Yugoslav 


TM 


Turkmenistan 


BF 


Burkina Faso 


GR 


Greece 




Republic of Macedonia 


TR 


Turkey 


BG 


Bulgaria 


HU 


Hungary 


ML 


Mali 


TT 


Trinidad and Tobago 


BJ 


Benin 


IE 


Ireland 


MN 


Mongolia 


UA 


Ukraine 


BR 


Brazil 


IL 


Israel 


MR 


Mauritania 


UG 


Uganda 


BY 


Belarus 


IS 


Iceland 


MW 


Malawi 


US 


United States of America 


CA 


Canada 


IT 


Italy 


MX 


Mexico 


UZ 


Uzbekistan 


CF 


Central African Republic 


JP 


Japan 


NE 


Niger 


VN 


Viet Nam 


CG 


Congo 


KE 


Kenya 


NL 


Netherlands 


YU 


Yugoslavia 


CH 


Switzerland 


KG 


Kyrgyzstan 


NO 


Norway 


ZW 


Zimbabwe 


CI 


Cote d*l voire 


KP 


Democratic People's 


NZ 


New Zealand 






CM 


Cameroon 




Republic of Korea 


PL 


Poland 






CN 


China 


KR 


Republic of Korea 


PT 


Portugal 






CU 


Cuba 


KZ 


Kazakstan 


RO 


Romania 






CZ 


Czech Republic 


LC 


Saint Lucia 


RU 


Russian Federation 






DE 


Germany 


U 


Liechtenstein 


SD 


Sudan 






DK 


Denmark 


LK 


Sri Lanka 


SE 


Sweden 






EE 


Estonia 


LR 


Liberia 


SG 


Singapore 







WO 99/31120 



PCT/US98/26208 



NOVEL TGF-p PROTEIN PURIFICATION METHODS 

FIELD OF INVENTION 

This present invention relates generally to novel protein recovery and purification methods for the 
transfonning growth factor-P (TGF-P) superfamily of proteins. More particularly, this invention relates 
to novel methods of purification of such proteins, including bone morphogeneuc proteins (BMPs). 

BACKGROUND OF THE INVENTION 

The transforming growth factor-P superfamily of proteins, including the BMPs and other 
osteogenic proteins may be produced in cultures (e.g. yeast, E. coli, and mammalian cells) transformed 
with an expression vector containing the corresponding DNA. The cloning and expression of the 
transforming growth factor-P superfamily of proteins, including the bone morphogenetic proteins (also 
termed osteogenic proteins), have previously been described. See, for example, United States Patents 
4,877,864; 5,108,922; 5,013,649; 5,116,738; 5,106,748; 5,187,076; 5,141,905; 5,688,678; 5,661,007; 
5,637,480; 5,639,638; 5,658,882; and 5,635,372. Other compositions which may also be useful include 
Vgr-2, and any of the growth and differentiation factors (GDFs), including those described in PCT 
publications W094/1 5965; W094/1 5949; WO95/01 801 ; WO95/01 802; W094/21 68 1 ; W094/1 5966; and 
others. Also useful in the present invention may be BP, disclosed in WO94/01557; and MP52, disclosed 
in PCT publication WO93/16099. The disclosures of all of the above referenced publications are hereby 
incorporated by reference. 

The use of suitably transformed host cells allows for the recombinant production of high levels 
of protein. For proteins which are secreted from the host cell, purification of the protein of interest 
generally involves isolation and purification from the host cell culture medium. Typically, the culture 
medium contains selected nutrients (e.g., vitamins, amino acids, co-factors, minerals) and can contain 
additional growth factors/supplements, including insulin and possibly additional exogenous proteins. In 
addition, the conditioned medium often contains not only the secreted protein of interest, but also 
significant quantities of additional secreted host cell proteins and other host cell substances (e.g. nucleic 
acids, membrane vesicles). Thus, even though it is expressed at high levels, the product of interest may 
represent only a minority of all proteins present in the conditioned medium. Not unexpectedly, proteins 
secreted by transformed host cells may possess characteristics quite similar to those of the product of 
interest (e.g. charge, molecular size, amino acid composition), thereby placing significant burden on the 
process used for purification. Certain purification conditions which are effective in avoiding denaturation 
of the product of interest are ineffective at distinguishing minor differences between secreted proteins, 
thereby making it extraordinarily difficult to separate the product of interest from all other host cell proteins 
present. 



WO 99/31120 PCT/US98/26208 

In addition to the unwanted secreted host cell proteins described above, conditioned medium may 
also contain products derived from the heterologously-expressed gene encoding 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, gamma carboxylation, or other 
5 modifications potentially necessary for biological activity (such as processing of precursor forms). In 
addition, proteolytically-degraded forms of the product of interest may be present in conditioned medium 
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, hydrophobic interaction 
chromatography, and size exclusion chromatography do not provide the extent of resolution necessary to 

10 distinguish the product of interest from the undesired forms of the product. To take full advantage of minor 
differences between the desired product and contaminants (e.g. small charge differences, small differences 
in molecular size), the use of strong denaturants is often required. Such denaturants, however, can lead to 
loss of biological activity, expression of neo-antigenic sites, and can potentially enhance chemical 
decomposition of selected post-translational modifications. 

15 Typically, researchers have used combinations of traditional chromatographic techniques to purify 

desired products. Often, such techniques are insufficient 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 interacts specifically. Following appropriate washing, the desired product can be eluted by 

20 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 are often ineffective, and must be used in conjunction with 
other affinity resins and/or traditional separation techniques. Unfortunately, using multiple steps to achieve 
greater resolution can also result in unacceptably low yields. Even high resolution affinity chromatography 

25 steps (e.g., immunoaffinity purification using an immobilized monoclonal antibody) may not afford 
sufficient resolution of the desired product from other components present in the culture medium due to 
common sites of interaction. For example, where an epitope which is present on the product of interest, 
is also present in a proteolytically-degraded form of the product or a precursor form of the desired product, 
both will compete for the same site. 

30 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 separate the desired product 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. For example, certain proteins 



2 



WO 99/31 120 PCT/US98/26208 

once expressed and secreted actually "bind-back" to the host cell and remain recalcitramly associated with 
the host cell making purification without concomittant denaturation virtually impossible. 

Accordingly, there continues to exist a need in the art for protein purification methods that 
effectively overcome all of these difficulties. 

5 

BRIEF SUMMARY OF THE INVENTION 

The methods of the present invention are directed to protein purification comprising the steps of 
applying cell culture medium to a heparin orheparin-like resin, elution with salt to displace the protein of 
interest, applying the first eluate to a hydrophobic interaction resin, eluting with decreased ionic strength 

10 or with non polar solvents to minimize hydrophobic interactions, and then optionally applying the second 
eluate to an anion exchange resin, used in the non-adsorptive mode, and used in tandem with a cation 
exchange resin and eluted with salt. Also, optionally, this third eluate is diafiltrated and/or concentrated, 
using, e.g., a spiral-wound membrane cartridge or other suitable device. 

More specifically, conditioned medium containing cell culture is filtered through a filter and 

15 loaded onto a Cellufine Sulfate chromatography column. Suitable 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 Sulfon, Fractogel EMD S0 3 , and Fractogel-EMD COO, with the preferred being 
Matrex Cellufine Sulfate. The column is washed and then eluted to collect, e.g., BMP. A suitable first 

20 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 washing without eluting BMP and include for 
example 5 mM to 600 mM salt, and preferably is 50 mM Tris, 500 mM sodium chloride. The first eluant 
comprises 50 mM Tris, 0,5 M NaCl, 0.5 M arginine; suitable concentration ranges are those which are 

25 effective in eluting BMP, including for example a solution containing a buffering agent at pH about 8.0, 
such as Tris, in the range of 5 to 100 mM, preferably approximately 50 mM, a salt such as NaCl in the 
range of 200 to 1000 mM, preferably 500 mM, and arginine in the range of 0 to 1000 mM, preferably 500 
mM. 

This first eluate is applied to a Butyl Sepharose column which is washed with a suitable second 
30 wash which comprises a salt solution such as sodium chloride, ammonium sulfate, 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 washing the column, 
without eluting BMP, and include for example 750 mM to 1 250 mM salt, and preferably is 50 mM Tris, 
1000 mM sodium chloride. The second eluant is one which is sufficient to -elute the protein of interest, 
35 for example one which comprises 50 mM Tris, 0.5 M arginine, 20% propylene glycol; suitable 



3 



WO 99/31120 PCT/US98/26208 

concentration ranges are those which are effective in eluting BMP, and include for example a solution 
containing a buffering agent at pH about 7.0, such as Tris, or its equivalent, in the concentration range of 
5 to 1 00 mM, preferably approximately 50 mM, arginine, or its equivalent, in the range of 250 mM to 1 000 
mM, preferably approximately 500 mM, and a nonpolar solvent, such as propylene glycol, or its 
5 equivalent, in the range of 1 0% to 50%, preferably approximately 20%. 

The eluate of the Butyl Sepharose column (referred to herein as the second eluate) is optionally 
pumped through a DEAE anion exchange resin; the unbound flow-through is pumped into a Carboxy 
Sulfon cation exchange resin connected in tandem to the DEAE resin. The DEAE and Carboxy Sulfon 
columns are washed, disconnected, and then the Carboxy Sulfon column is eluted with salt to collect BMP 

10 (referred to herein as the third eluate). Suitable anion exchange resins include those resins having a 
positively charged group such as diethyleaminoethane (DEAE), polyethyleneimine (PEI), and quarternary 
aminoethane (QAE) and include Q-Sepharose Fast Flow, DEAE-Sepharose Fast Flow, POROS-Q, 
Fractogel-TMAE, Fractogel-DMAE, QAE-Toyopearl, and DEAE-Toyopearl with the preferred resin being 
DEAE-Toyopearl (Tosohaas). Suitable cation exchange resins include those having a negatively charged 

15 group such as heparin, sulfated esters of cellulose, sulfylpropyl (SP), carboxyl, and carboxy methyl and 
include Matrex Cellufine Sulfate,SP-Sepharose Fast Flow, Mono S, Resource-S, Source S, Carboxy 
Sulfon, Fractogel EMD-S0 3 , and Fractogel-EMD COO, with the preferred being Carboxy Sulfon. A 
suitable third wash comprises a salt solution such as sodium chloride, potassium chloride, sodium sulphate, 
or ammonium sulfate, and may contain a suitable buffering agent and optionally arginine. Suitable 

20 concentration ranges are those which are effective in washing without eluting BMP and include for 
example 0 mM to 250 mM salt, and preferably is 50 mM potassium phosphate, 250 mM arginine. The 
third eluant comprises 50 mM potassium phosphate, 400mM NaCl, and 500 mM arginine; suitable 
concentration ranges are those which are effective in eluting BMP, including for example a solution 
containing a buffering agent at pH about 7.5, such as potassium phosphate, in the range of 5 to 300 mM, 

25 preferably approximately 50 mM, a salt such as NaCl in the range of 200 to 1000 mM, preferably 400 mM 
or higher, and arginine in the range of 0 to 1000 mM, preferably 500 mM. 

Optionally, a spiral-wound membrane cartridge is used to exchange the Carboxy Sulfon elution 
buffer into a suitable formulation buffer. Immediately after this diafiltration step, the BMP may be 
concentrated to *2.4 absorbance unitsymL (at 280 nm) using the spiral-wound cartridge, if necessary. The 

30 concentrated BMP is then filtered, sampled, labeled, and stored frozen at -80°C. 

The effectiveness of the process in purifying BMP is demonstrated by SDS-PAGE analysis. After 
the Cellufine Sulfate step, BMP is clearly visible as two major bands in the 15-20 kd region on a reduced 
gel, although other contaminating proteins are still present. These protein contaminants are largely 
removed by the Butyl Sepharose and are further separated by the DEAE/Carboxy Sulfon step. 



4 



WO 99/31 120 PCT/US98/26208 

Also provided by the present invention are purified BMP compositions produced by the methods 
of the invention. 

DETAILED DESCRIPTION OF THE INVENTION 

As used herein, the term "BMP" includes, but is not limited to proteins of the transforming growth 
5 factor-P superfamily of proteins, including the BMPs, isolated from a variety of tissue sources (including 
but not limited to epidermis, tendon, bone, cartilage, blood, fetal tissue, neuronal tissue, liver, ligament, 
muscle, pancreas, lung, heart, spleen, kidney), from transformed cell lines, and recombinantly produced 
proteins isolated from host cell culture medium or microbial sources (including, but not limited to 
fermentation broth, E.coli lysate, yeast lysate, and the like). 

10 As used herein, the terms "heparin" resin and "heparin-like" resin are used interchangeably, and 

include but are not limited to, resins containing an immobilized negatively charged moiety such as heparin, 
sulfated esters of cellulose, sulfylpropyl (SP), carboxyl, and carboxy methyl and includes Fractogel-EMD- 
S0 3 , Carboxy Sulfon, Fractogel-EMD-COO, Heparin-Sepharose, Matrex Cellufine Sulfate and equivalents 
thereof, with Matrex Cellufine Sulfate presently most preferred. 

15 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 (5 mM salt) to high 
(600 mM salt), with 500 mM sodium chloride presently preferred. 

As used herein, the term "first eluant" includes, but is not limited to, solutions composed of a 

20 buffering agent (e.g. Tris) at a concentration of approximately 50 mM, salt (e.g. NaCl) at a concentration 
which is sufficient for elution from the resin (e.g. approximately 500 mM), at about pH 8.0, and about 500 
mM arginine; suitable concentration ranges are those which are effective in eluting BMP, including for 
example a solution containing a buffering agent at pH about 8.0, such as Tris, in the range of 5 to 100 mM, 
preferably approximately 50 mM, a salt such as NaCl in the range of 200 to 1000 mM, preferably 500 mM, 

25 and arginine in the range of 0 to 1 000 mM, preferably 500 mM. 

As used herein, the term "Butyl Sepharose-like" includes, but is not limited to Butyl Sepharose 
4B, Butyl Sepharose Fast Flow, Butyl-Toyopearl, and other hydrophobic interaction media including 
Phenyl Sepharose Fast Flow, Phenyl Toyopearl, Phenyl Fractogel, Butyl Fractogel, and suitable 
equivalents, with Butyl Sepharose 4B presently being most preferred. 

30 As used herein, the "second wash" can be any salt solution and includes, for example, sodium 

chloride, potassium chloride, sodium sulphate, ammonium sulfate, sodium phosphate, or potassium 
phosphate, and can be suitably buffered. Typically, concentrations range from low (750 mM salt) to high 
(1250 mM salt), with 50 mM Tris, 1000 mM sodium chloride presently preferred. 

As used herein, the term "second eluant" includes, but is not limited to, solutions comprising a 

35 buffering agent (e.g. Tris) at a concentration of approximately 5 to 100 mM, preferably 50 mM, a 



5 



WO 99/31120 



PCT/US98/26208 



solubility-promoting agent (e.g. arginine, urea, or other equivalent chaotropic agent), preferably arginine 
at a concentration range of approximately 250 mM to 1000 mM, preferably approximately 500 mM, and 
a nonpolar solvent (e.g. propylene glycol, ethylene glycol, glycerol and equivalents) at a concentration 
sufficient to disrupt the interaction of BMP with the Butyl Sepharose, at approximately pH 7.0, at a 
5 concentration range of approximately 10% to 50% and preferably propylene glycol, or its equivalent, at 
approximately 20%. As used in this process, the second eluant is preferably compatible with the 
subsequent process step, including dilution or diafiltration prior to loading into the next step. 

As used herein, the term "anion exchange resin" includes, but is not limited to, resins having a 
positively charged moiety (at neutral pH), such as diethyleaminoethane (DEAE), polyethyleneimine (PEI), 

10 and quaternary aminoethane (QAE) and includes, for example, Q-Sepharose Fast Flow (Pharmacia), 
DEAE-Sepharose Fast Flow, DEAE-Toyopearl, QAE-Toyopearl, POROS-Q, Fractogel-DMAE, Fractogel 
EMD-TMAE, Matrex Cellufine DEAE, and the like, with DEAE presently preferred. 

As used herein, the term "cation exchange resin" includes, but is not limited to, resins having a 
negatively charged group such as heparin, sulfated esters of cellulose, sulfylpropyl <SP), carboxyl, and 

15 carboxy methyl, and include Matrex Cellufine Sulfate, SP-Sepharose Fast Flow, Mono S, Resource-S, 
Source S, Carboxy Sulfon, Fractogel EMD-S0 3 , and Fractogel-EMD COO, with the presently preferred 
being Carboxy Sulfon. 

As used herein, the term "third wash" can be any salt solution and includes, for example, sodium 
chloride, potassium chloride, sodium sulphate, or ammonium sulfate, and can be suitably buffered (e.g. 

20 Tris, phosphate, or sulfate), and optionally can contain arginine. Typically, salt concentrations range from 
low (0 mM salt) to high (250 mM salt), with 0 mM sodium chloride presently preferred. The presently 
preferred ''third wash' v comprises about 50 mM phosphate buffer and about 250 mM arginine. 

As used herein, the term "third eluant" includes, but is not limited to, solutions comprising a 
buffering agent (e.g. Tris, phosphate, or sulfate) at a concentration range of approximately 5 to 100 mM, 

25 preferably 50 mM, a solubility-promoting agent (e.g. arginine, urea, or other chaotropic agents), preferably 
arginine at a concentration range of 0 to 1000 mM, preferably a concentration of approximately 500 mM, 
and salt (e.g. sodium chloride, potassium chloride) at a concentration sufficient to disrupt interaction of 
BMP with the resin (e.g. in the range of 200 to 1000 mM, and preferably, approximately 400 mM or 
higher). 

30 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 skilled in the art that numerous variations and 
modifications are possible and that such modifications are within the present invention. For example, the 
order can be re-configured if desired and steps can be omitted. 



6 



WO 99/31120 



PCT/US98/26208 



10 



15 



20 



FIGURE 1 
Overview of Purification Process 

Filtered Conditioned Medium 

i 



Matrex Cellufine Sulfate 
Chromatography 



i 



Butyl Sepharose Chromatography 



DEAE/Carboxy Sulfon Chromatography 



Viresolve 



j. 



Ultrafiltration 
and Diafiltration 



BMP 



Genes encoding recombinant osteogenic proteins may be expressed in mammalian cell lines such 
as CHO (Chinese Hamster Ovary), COS, BHK, Balb/c 3T3, 293, and similar cell lines known in the art. 

25 The mammalian cells may be grown in any suitable medium known in the art. Suitable ^cell culture media 
may contain amino acids vitamins, inorganic salts, glucose, sodium pyruvate, thioctic acid, linoleic acid 
hydrocortisone, putrescine, recombinant insulin, dextran sulfate, and methotrexate. For example, a suitable 
medium is a DME/F12 (50:50)-based cell culture medium supplemented with hydrocortisone, putrescine, 
recombinant insulin, dextran sulfate and methotrexate. Other media, such as a-MEM, Dulbecco's MEM, 

30 RPMI 1 640, may also be suitable, with suitable supplements as may be necessary. (Freshney, R.L, Culture 
of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York (1983)). The cells may be 
grown in the presence or absence of a serum supplement such as fetal bovine serum (FBS). The cells may 
be grown in monolayer or suspension culture, and additionally may be grown in large production scale 
batches. Incorporated by reference are the disclosures of WO 95/12664 (<3I 5217-PCT) relating to 



7 



- WO 99/31 120 PCT/US98/26208 

methods and nutrient media useful for adapting mammalian cell lines to culture densities, and of pending 
USSN 08/481 ,774 (GI 5233) relating to a cell culture medium for production of dimeric proteins. 

Any cell capable of producing a protein of the TGF-P superfamily of proteins may be used in the 
method of the present invention. Transformed CHO cells are the preferred host cells used to produce an 
5 osteogenic protein, such as BMPs, particularly BMP-2, in accordance with the present invention. The cell 
growth medium may be supplemented with FBS to improve the growth of transformed CHO cells in 
culture. If it is desired to add FBS, concentrations of FBS as low as 0.5% (v/v) may be added. However, 
addition of animal-origin proteins always presents the risk of harboring viruses and other deleterious 
agents. The addition of FBS is not necessary for the practice of the present invention. Serum-free media 
10 are preferred for use in producing recombinant osteogenic proteins in accordance with the present 
invention. 

CHO cells are known to release lipids, carbohydrates, nucleic acids and C-type (defective 
retroviral-like) particles into conditioned media. Therefore, the capacity of a purification process to 
remove and/or inactivate host derived contaminants which may be present is an important aspect of the 

15 process. CHO cell protein removal is confirmed by intentionally mixing radiolabeled CHO cell protein 
with load material and quantifying the reduction at each step. A reduction factor for host cell protein 
contaminants at each step of purification, and overall, is estimated by introducing concentrations of CHO 
cell protein which are higher than that expected during normal production. 

The C-type particles present in CHO cells have never been demonstrated to be infectious. 

20 However, removal or inactivation of these particles during the purification process is still considered 
desirable. A consensus set of viruses are used to estimate removal/inactivation potential of the purification 
steps. These viruses have been chosen to represent different size ranges and types (e.g., enveloped/non- 
enveloped, DNA containing/RNA containing). Included is a murine retrovirus (Murine Xenotropic 
Leukemia virus) and others that are human pathogens for which CHO cells are permissive (Parafluenza 

25 3 and Retrovirus 3). Simian virus 40 is also included to investigate a more resistant virus. In these studies, 
virus is introduced into the process at each chromatographic step and the removal/inactivation determined. 

Most media components are small chemicals, including salts, amino acids and sugars that do not 
generally co-purify with the protein of interest over chromatographic columns and are generally not 
retained by a diafiltration membrane. However, large polymers, such as dextran sulfate and polyvinyl 

30 alcohol, which are useful media additives, may specifically interact with the product of interest and often 
do co-purify. These components must therefore be purified away from the protein of interest. For 
example, one dextran sulfate useful in the media for producing recombinant proteins such as BMP has a 
molecular weight of 5,000 and sulfur content 1 8% (Sigma catalogue # D-7037). Another dextran sulfate 
has a molecular weight of 500,000 and a sulfur content of 17% (Pharmacia). Incorporated herein by 

35 reference is USPN 5,5 1 6,654 (Gl 5 1 80A), which relates to a method of protein production wherein dextran 



WO 99/31120 PCT/US98/26208 
sulfate is added to the culture medium. In accordance with the present invention, dextran sulfate may be 
added to the growth medium at a range of concentrations of from about 1 to about 500 ng/mL, preferably 
about 200 Mg/mL dextran sulfate. 

Methotrexate and other selectable markers, which are often used in small volume in the early 
production of cell cultures, may be toxic. Their removal from the protein preparation is an important step 
(e .g. f the Matrex Cullufine Sulfate Step which provides a 3,540-fold removal) of the purification process 
(see Table 8). 

Expression of an osteogenic protein, such as BMP-2, can be achieved by inserting a suitable gene 
into an expression vector, inserting this vector into a mammalian cell, and selecting for cells which express 
the osteogenic protein. For example, vectors encoding BMP-2 are described in United States Patent 
5,013,649, the contents of which are incorporated herein by reference. 

The yield of recombinant osteogenic protein, such as BMP-2, from mammalian cells which express 
the BMP-2 gene may be measured by known methods such as radioactively labeling cells with [ 35 S]- 
methionine and analyzing secreted proteins by polyacrylamide gel electrophoresis (PAGE) and 
autoradiography. For measurement of BMP-2 expression from production-scale batches, the amount of 
functional BMP-2 secreted can be quantitated by bioassay or chromatographic assay methodologies.. Any 
appropriate bioassay may be used, for example, assay of induction of alkaline phosphatase activity in a 
BMP-2-responsive cell line, or assay of ectopic bone formation in a mammal such as rat, rabbit, cat or dog. 
Any chromatographic assay method which separates the product of interest form contaminants may be 
used, including RP-HPLC. 

While the examples below describe the present invention being carried out with a cell line which 
encodes BMP-2, these examples are not limiting. The present invention may also be used with similar 
results for other protein members of the transforming growth factor beta superfamily, particularly the bone 
morphogenetic proteins, including BMP-1 through BMP-15. Osteogenic proteins of the BMP family are 
a promising development in the bone and cartilage field. The BMP family of proteins includes BMPs 1 
through 15, and proteins which are encoded by DNA sequences which hybridize thereto under stringent 
conditions. The following examples illustrate practice of the invention. These examples are for illustrative 
purposes only and are not intended in any way to limit the scope of the invention claimed. Example 1 
describes the heparin/heparin-like affinity step; Example 2 relates to the hydrophobic interaction 
chromatography step; Example 3 describes the purification steps using tandem anion-cation exchange on 
Matrex Cellufine Sulfate; Example 4 relates to further purification using the diafiltration/concentration 
step; and Example 5 describes the purity testing. 



WO 99/31 120 PCT/US98/26208 

EXAMPLES 

EXAMPLE 1: HEPAWN/HEPARIN-LIKE AFFINITY STEP 

Upon secretion from the host ceil, the secreted protein is positively charged and binds tightly to 
the outer surface of the host cell which is negatively charged. A preferred way to disrupt this binding to 
the outside of the host cell, without destroying the protein of interest and/or without disruption and further 
leakage of the host cell contents into the medium, is to add dextran sulfate to the culture medium. 
Although this has the desired effect of disrupting the interaction with the host cell, it creates another 
problem, namely the binding of the protein of interest to the dextran sulfate which further complicates the 
purification process. 

Surprisingly, it has been found that a heparin or heparin-like resin will effectively compete with 
with dextran sulfate for binding to BMP so that such resin can be effectively employed to separate the 
BMP from the dextran sulfate. 

Matrex Cellufine Sulfate (Amicon) is used as an affinity matrix for purification of rhBMP-2 from 
conditioned medium. This resin is composed of spheroidal cellulose beads functionalized with sulfate 
esters and functions as a heparin analog for purification of heparin-binding proteins. The resin efficiently 
competes with dextran sulfate present in cell culture medium for binding to rh BMP-2 at pH 8.0. Elution 
of the bound rhBMP-2 is achieved by using 0.5M L-arginine added to 50mM TRIS plus 0.5M NaCl. 

Cellufine Sulfate, or an equivalent chromatography column, is the first step in the purification of 
BMP. Conditioned medium is filtered and titrated to pH 8.0 ± 0.2. The titrated material is loaded onto 
an equilibrated Cellufine Sulfate column at a linear flow rate of £ 3 cm/min. The column is then washed 
(50mM TRIS, 0.5M NaCl, pH 8.0) and may be reverse eluted (50mM TRIS, 0.5M NaCl, 0.5M L-arginine- 
HC1, pH 8.0). The column eluate is collected as a single eluting peak, approximately one column volume. 
Suitable operating parameters are described in Table 1 . 



10 



WO 99/31120 



PCT/US98/26208 



Table 1 



Operating Parameters for Cellufine Sulfate Column Step 


Purification Procedures 


Parameter 


Target Range 


All Procedures 


Pressure 


<; 20 psig 


Equilibration 


Flow Rate 


*180 cm/hr 




pH 


8.0 ± 0.2 




Conductivity 


<; 33.5 mS/cm 


Titration 


Volume 


50-125 mL titrant/L cell culture 






medium 




pH 


8.0 ±0.2 




Conductivity 


5-20 mS/cm 


Load 


Flow Rate 


<> 180 cm/hr 


Wash 


Volume 


< 1 5 column volumes 


Elution 


Flow Rate 


< 180 cm/hr 



10 

EXAMPLE 2: HYDROPHOBIC INTERACTION CHROMATOGRAPHY STEP 

The heparin-like step effectively removes various species of protein contaminants, methotrexate, 
dextran sulfate and DNA. Still present in the first eluate, along with the protein of interest, are various 

15 forms of the BMP at various stages of proteolytic processing, including the higher molecular weight 
precursor forms (approximately 1 10 KD and 80 KD on non-reducing SDS-PAGE analysis) and the desired 
product (15KD - 20 KD on reducing SDS-PAGE analysis). Because these various species differ only 
slightly in hydrophobicity, it is difficult to purify these species from each other by conventional methods. 
Surprisingly, it has been found that the Butyl Sepharose allows for separation of the various forms of 

20 BMP-2 by an unconventional use of displacement chromatography. The precursor species of BMP-2 
compete with the desired product for binding to the resin. Due to the slight differences in hydrophobicity, 
the desired product, which is more hydrophobic, binds more tightly to the resin; this allows the other 
species to be removed in the column load and wash. 

Butyl Sepharose 4B (Pharmacia) is a resin used for purification of BMP in a hydrophobic 

25 interaction chromatography (H1C) mode. This resin is composed of butylamine coupled to CNBr-activated 
Sepharose 4B. Butyl Sepharose, or an equivalent column, is the second step of the present invention. 
Proteins are bound to HIC resins at high conductivities, which promote hydrophobic interactions. "High 
conductivity" is a minimum value of about 50 mS/cm. Elution is accomplished by decreasing ionic 
strength and/or by addition of non-polar solvents to minimize hydrophobic interactions. Decreased ionic 



11 



. WO 99/31120 PCT/US98/26208 

strength is defined as decreasing the conductivity to a value of below about 20 mS/cm. Non-polar solvents 
include propylene glycol, ethylene glycol, glycerol, and equivalents thereof. 

The Matrex Cellufine Sulfate elution peak is adjusted to pH 7.0 ± 0.2 and 1000 mM NaCl with 
200 mM MES, 4000 mM NaCl, 500 mM L-arginine HC1, pH 6.8. MES is 2-[N-morpholino]ethane 
sulfonic acid. This material is then loaded onto an equilibrated Butyl Sepharose, or an equivalent column, 
at a flow rate of s 30 cm/hr. The column is then washed (50mM TRIS, 1000 mM NaCl, 500 mM L- 
arginine-HCl, pH 7.0) and bound rhBMP-2 is optionally reverse eluted with 50mM TRIS, 20% propylene 
glycol, 500 mM L-arginine- HC1, pH 7.0. The column eluate is collected as a single elution peak, 
approximately 1.5 column volumes. Suitable column operating parameters are detailed in Table 2. 



Table 2 



Operating ] 


Parameters for Butyl Sepharose Column Step 


Purification Procedures 


Parameter 


Target Range 


All Procedures 


Pressure 


*7psig 


Equilibration 


Flow Rate 
pH 

Conductivity 


^30 cm/hr 
7.0 ± 0.2 
50-70 mS/cm 


Titration 


pH 

Conductivity 


7.0 ± 0.2 
60-70 mS/cm 


Load 


Flow Rate 


< 30 cm/hr 


Wash 


Flow Rate 
Volume 


s 30 cm/hr 

2.5-2.75 column volumes 


Elution 


Flow Rate 


< 15 cm/hr 



EXAMPLE 3: TANDEM ANION-CATION EXCHANGE 

The Butyl Sepharose step shows removal of CHO protein contaminants, DNA, and BMP related 
species, other than the defined product. It has been found that inclusion of an anion exchange 
chromatography step results in increased removal of DNA, and other non-proteinaceous contaminants. 
An additional cation exchange chromatography step provides further removal of CHO protein 
contaminants and concentration of BMP. 

Toyopearl-DEAE (TosoHaas) is a weak anion exchange resin that binds negatively-charged 
proteins and other contaminants on the basis of their charge. It is used in the nonadsorptive mode for 
purification of BMP, such that it does not bind to the resin, but negatively charged contaminants are able 
to bind to the DEAE resin. 



12 



WO 99/31120 



PCT/US98/26208 



Carboxy Sulfon (J.T. Baker, Inc.) is a silica-based matrix functionaiized with mixed weak and 
strong cation exchange groups (i.e., carboxy and sulfone groups). BMP binds to Carboxy Sulfon via 
charge interactions and is eluted by disruption of these interactions using buffers with increased ionic 
strength. 

The final chromatographic step in the purification of BMP is composed of these two ion-exchange 
columns, or their equivalents, operated in tandem: Toyopearl-DEAE anion exchange column (or its 
equivalent) followed by Carboxy Sulfon cation exchange column (or its equivalent). The inlet to the 
DEAE/Carboxy Sulfon system enters the DEAE column first. The outlet of the DEAE column is then 
plumbed to the inlet of the Carboxy Sulfon column. 

The Butyl Sepharose peak is diluted with 9-1 1 volumes of (50mM potassium phosphate, 0.25M 
L-arginine-HCl, pH 7.6). This solution is pumped through the DEAE column and onto the Carboxy 
Sulfon column at a flow rate of £ 300 cm/hr. The columns are then washed (50mM potassium phosphate, 
0.25M L-arginine-HCl, pH 7.6). The two columns are disconnected and the BMP bound to the Carboxy 
Sulfon column is eluted with 50mM potassium phosphate, 0.5M L-arginine-HCl, 0.4M NaCl, pH 7.5. The 
column eluate is collected as a single eluting peak, approximately 1 column volume. Suitable operating 
parameters for these columns are detailed in Table 3. 



Table 3 



Operating Parameters for DEAE/Carboxy Sulfon Column Step 


Purification Procedures 


Parameter 


Target Range 


All Procedures 


Pressure 


s 20 psig 


Charge Resin 


Flow Rate 


<; 300 cm/hr 




Volume 


s 1 column volume 


Equilibration 


Flow Rate 


< 300 cm/hr 




LAL 


< 4 EU/mL 




pH 


7.6 ±0.2 




Conductivity 


10-18 mS/cm 


Dilution 


pH 


7.6 + 0.2 




Conductivity 


10-18 mS/cm 


Load 


Flow Rate 


<; 300 cm/hr 


Wash 


Flow Rate 


* 300 cm/hr 




Volume 


* 6 column volumes 


Elution 


Flow Rate 


< 120 cm/hr 



13 



WO 99/31 120 PCT/US98/26208 
EXAMPLE 4: DIAFILTRATION/CONCENTRATION STEP 

This is an optional "finishing" step. Tangential flow filtration is used for buffer exchange and 
concentration of protein solutions. Membrane of specified molecular weight cut-offs are used to retain 
large molecular weight components (*.g., BMPs) while lower molecular weight components (e.g., salts) 
are removed. By continuously adding new buffer to the retentate, at a similar rate that solution is passing 
through the filter, the original buffer components will gradually be diluted away. In this continuous 
diafiltration fashion, replacement of 5 retentate volumes of a new buffer will effectively replace * 98% of 
the original buffer. Without addition of new buffer, a protein solution is concentrated without altering the 
buffer composition. 

The final step in the purification process involves exchange of the Carboxy Sulfon elution buffer 
into an appropriate formulation buffer for BMP. This is followed by concentration of the material to *2.4 
absorbance units (at 280 nm), as necessary. This step may be performed using a spiral-wound 1 0,000 MW 
cut-off membrane, or equivalent. The Carboxy sulfon eluate is placed in a clean, autoclaved and sealed 
vessel. The material in the vessel is then pumped across the membrane, at a positive transmembrane 
pressure, and recirculated back into the vessel. The positive transmembrane pressure forces low-molecular 
weight solutes through the membrane. Buffer solution (0.01 M L-histidine, 0.5M L-arginine-HCl or other 
suitable buffer solution) enters the vessel at approximately the same rate as material flows through the 
membrane, thereby diluting out the Caiboxy Sulfon elution buffer. This diafiltration process is continued 
until at least 5 volumes of buffer solution have flowed into the vessel. 

After the diafiltration is complete, the valve that allows buffer solution to enter the vessel is closed. 
To concentrate BMP, the system pump is restarted and material is filtered until a concentration of *2.4 
absoitance units (at 280 nm) is obtained. The BMP buffer is pumped out of the vessel, through a 0.2 \xm 
filter, and into appropriately sized Teflon bottles. The material is sampled, weighed, labeled, and stored 
at -80°C. Suitable operating parameters for this process step are detailed in Table 4. 



14 



WO 99/31120 



PCT/US98/26208 



Table 4 



Operating Parameters for Diafiltration/Concentration Step 


Purification Procedures 


Parameter 


Target Range 


All Procedures 


Inlet Pressure 
Permeate Flow Rate 


20-30 psig 
30-70 mL/min 


Equilibration 


Retentate Flow Rate 

LAL 

pH 

Conductivity 


700-1000 mL/min 
<4EU/mL 
6.5 ±0.2 
20-30 mS/cm 


Diafiltration 


Permeate pH 
Permeate Conductivity 
Permeate Volume 


6.5 ±0.2 

20-30 mS/cm 

* 5 times load volume 



EXAMPLE 5: PURITY TESTING 

Studies investigating the effectiveness of tne above purification process for removing DNA, 
10 viruses, dextran sulfate and methotrexate were performed with the results described in the tables below; 



Table 5 

DNA Removal Studies 



Purification Process Step 


Fold Removal 


Log Removal 


Matrex Cellufme Sulfate 


251 


2.40 


Butyl Sepharose 


1122 


3.05 


DEAE/Carboxy Sulfon 


15 


1.18 


Overall 




6.63 


Table 6 

MuLV Virus Removal/Inactivation Studies 


Purification Process Step 


Fold Removal 


Log Removal 


Matrex Cellufme Sulfate 


4677 


3.67 


Butyl Sepharose 


275 


2.44 


DEAE/Carboxy Sulfon 


1950 


3.29 


Overall 




9.4 



15 



WO 99/31120 



PCT/US98/26208 



Table 7 

Dextran Sulfate Removal Studies 



Purification Process Step 


Fold Removal 


Log Removal 


Matrex Cellufine Sulfate 


1318 


3.12 


Butyl Sepharose 


141 


2.15 


DEAE/Carboxy Sulfon 


67.6 


1.83 


Overall 




7.10 


Table 8 

Methotrexate Removal Studies 


Purification Process Step 


Fold Removal 


Log Removal 


Matrex Cellufine Sulfate 


3540 


4.55 



While the above examples are not limiting, it can be seen that the above process results in a high- 
fold removal of potential contaminants, including DNA, virus, dextran sulfate and methotrexate, from 
20 recombinant osteogenic protein produced from transfected CHO cells. 

While the present method of the invention is exemplified by purification of recombinantly 
produced BMP from transformed host cells, the method is also amenable to purification of BMP naturally 
occurring within a cell and can be used to purify proteins from solution or from various tissue types, cell 
homogenates, cell culture supernatants, or isolated cellular sub-fractions. While the present invention has 
25 been described in terms of specific methods and compositions, it is understood that variations and 
modifications will occur to those skilled in 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 
30 cover all such equivalent variations which come within the scope of the invention as claimed. 



16 



WO 99/31 120 PCT/US98/26208 

WHAT IS CLAIMED: 

1 . A method for purification of a TGF-P superfamily protein in a solution comprising the 
steps of: 

applying said solution to a heparin-like resin, 

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

applying said first eluate to a Butyl Sepharose-like resin, 

eluting said Butyl Sepharose-like resin with a second eluant to form a second eluate 
containing said TGF-P superfamily protein. 

2. The method of claim 1 , further comprising the steps of: 
applying said second eluate to an ion exchange resin, and 

eluting said ion exchange resin with a third eluant to form a third eluate. 

3. The method of claim 2, wherein said ion exchange resin is a resin selected from the group 
consisting of an anion exchange resin and a cation exchange resin. 

4. 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 caiboxy methyl. 

5. The method of claim 4 wherein said heparin-like resin is Matrex Cellufine Sulfate. 

6. The method of claim 1 , wherein said first eluant comprises a salt. 

7. The method of claim 6, wherein said first eluant comprises 50 mM Tris, 0.5 M NaCl, 0.5 
M L-arginine. 

8. The method of claim 1 , wherein said Butyl Sepharose-like resin is a member selected from 
the group consisting of Butyl Sepharose 4B, Butyl Sepharose Fast Flow, and Butyl-Toyopearl. 

9. The method of claim 8, wherein said Butyl Sepharose-like resin is Butyl Sepharose 4B. 

10. The method of claim 1, wherein said second eluant comprises a buffering agent, a 
chaotropic agent, and a non-polar solvent. 

11. The method of claim 10, wherein said second eluant is about 50 mM Tris, 500 mM 
arginine, and 20% propylene glycol. 

1 2. The method of claim 3, 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 aminoethane (QAE). 

13. The method of claim 12, wherein said anion exchange resin is DEAE. 

14. The method of claim 3, wherein said cation exchange has a negatively xharged group 
which is a member selected from the group consisting of as heparin, sulfated esters of xylulose, 
sulfylpropyl (SP), carboxyl, and carboxy methyl. 

15. The method of claim 14,wherein said cation exchange resin is Carboxy Sulfon. 



17 



WO 99/31 120 PCT/US98/26208 

1 6. The method of claim 1 , wherein said third eluam comprises a buffering agent, a solubility- 
promoting agent, and a salt. 

1 7. The method of claim 1 6, wherein said third eluam comprises about 50 mM Tris, 500 mM 
arginine, and 400 mM sodium chloride. 

1 8. The method of claim 1 , wherein said TGF-p superfamily protein is a BMP. 

1 9. The method of claim 1 8, wherein said BMP is BMP-2. 

20. A BMP produced by the method of claim 1 8. 

21 . A method for purification of BMP-2 in a solution comprising the steps of: 
applying said solution to a Cellufine Sulfate resin, 

eluting said Cellufine Sulfate resin with a first eluam to form a first eluate, 
applying said first eluate to a Butyl Sepharose 4B resin, 

eluting said Butyl Sepharose 4B resin with a second eluam to form a second eluate, 
containing said BMP-2. 

22. The method of claim 21 further comprising the steps of: 

applying said second eluate containing said BMP-2, to a DEAE resin, 
washing said DEAE resin to form a third wash, 
applying said wash to a Carboxy Sulfon resin, and 

eluting said Caiboxy Sulfon resin with a third eluam to form a third eluate containing said 

BMP-2. 

23. The method of claim 22, wherein: 

said first eluam comprises about 50 mM Tris, 500 mM NaCl, and 500 mM arginine; 
said second eluam comprises about 50 mM Tris, 500 mM arginine, and 20% propylene 

glycol; 

said third wash comprises about 50 mM potassium phosphate and 250 mM arginine; and 
said third eluam comprises about 50 mM Tris, 500 mM arginine, and 400 mM sodium 
chloride. 



18 



INTERNATIONAL SEARCH REPORT 


/national Application No 

PCT/US 98/26208 


A. CLASSIFICATION OF SUBJECT MATTER 

IPC 6 C07K1/18 




According to International Patent Classification (IPC) or to both national classification and IPC 




B. FIELDS SEARCHED 


Minimum documentation searched (classification system followed by classification symbols) 

IPC 6 C07K 



Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched 



Electronic data base consulted during the international search (name of data base and. where practical, search terms used) 



C. DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 1 



Citation of document, with indication, where appropriate, of the retevanl passages 



Relevant to claim No. 



WO 96 40883 A (GENETICS INST) 

19 December 1996 

see the whole document 

WO 93 09229 A (GENETICS INST) 13 May 1993 

see abstract; example 4 

US 4 828 990 A (HIGASHI NAOKI ET AL) 
9 May 1989 

see the whole document 

US 5 618 924 A (WANG ELIZABETH A ET AL) 

8 April 1997 

see abstract; example 7 

-/-- 



1-7,10, 
12-23 



1-9, 
16-23 



1-4,6, 

8-10, 

12-15,21 



1,6, 
18-21 



| X| Further documents are listed in the continuation of box C. 


|X j Patent family members are listed in annex. 


° Special categories of cited documents : 

"A* document defining the general state of the art which ts not 
considered to be of particular relevance 

"E" eartier document but published on or after the international 
fiDng date 

"L" document which may throw doubts on priority claim* s> or 
which is cited to establish the publication date of another 
citation or other special reason (as specified) 

"O" document referring to an oral disclosure, use. exhibition or 
other means 

V document published prior to the international filing date but 
later than the priority date claimed 


T* later document published after the international filing date 
or priority date and not in conflict with the application but 
cited to understand the principle or theory underlying the 
invention 

*X" document of particular relevance; the claimed invention 
cannot be considered novel or cannot be considered to 
involve an inventive step when the document is taken alone 

"Y" document of particular relevance; the claimed invention 
cannot be considered to involve an inventive step when the 
document is combined with one or more other such docu- 
ments, such combination being obvious to a person skilled 
in the art 

document member of the same patent family 


Date of the actual completion ot the international search 


Date of mailing ot the international search report 


13 April 1999 


27/04/1999 


Name and mailing address of the ISA 

European Patent Office, P.B. 5618 Patentlaan 2 
NL-2260 HV Rijswijk 
Tel. (+31-70) 340-2040. Tx. 31 651 epo nt, 
Fax: (+31-70) 340-3016 


Authorized officer 

Knehr, M 



Form PCT/ISA£10 (second sheet) (July 1092) 



page 1 of 2 



INTERNATIONAL SEARCH REPORT 



r national Application No 

PCT/US 98/26208 



C.(Continuatfon) DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 0 Citation of document, with rndicatioawhere appropriate, of the relevant passages 



Relevant to claim No. 



SHODA A ET AL.: "Presence of high 
molecular weight forms of BMP-2 1n early 
xenopus embryos" 
GROWTH FACTORS, 

vol. 8, 1993, pages 165-172, XP002099665 
see the whole document 

US 5 639 638 A (CELESTE ANTHONY J ET AL) 
17 June 1997 

see abstract; claims 8-10; example 8 

EP 0 741 187 A (HOFFMANN LA ROCHE) 

6 November 1996 

see the whole document 

WO 96 38570 A (GENETICS INST) 

5 December 1996 

see the whole document 

US 5 631 142 A (WANG ELIZABETH A ET AL) 
20 Hay 1997 

* see especially example 1 * 
see the whole document 



Form PCT/ISA/210 (continuation of second sheet) (July 1992) 



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WO 9640883 



19-12-1996 



US 
AU 
CA 
EP 



5714583 A 
5754396 A 
2220501 A 
0832200 A 



WO 9309229 



13-05-1993 



AU 
AU 
EP 
JP 
MX 
US 



674500 B 
3062292 A 
0612348 
7500968 
9206315 
5866364 



A 
T 
A 
A 



US 5618924 



08-04-1997 



AT 
AU 
AU 
DE 
DE 
DK 
DK 
EP 
EP 
GR 
IE 
IL 
JP 
JP 
JP 
JP 
JP 
KR 
MX 
PT 
U0 
US 
US 
US 
US 
US 
US 
US 
US 
US 

us 

•us 

NO 
NO 
US 
US 



141928 T 

613314 B 
7783587 A 
3751887 
3751887 
53497 

106288 
0313578 A 
0688869 A 

871028 
75881 
83003 
2729222 
6298800 
10070989 
2500241 
2713715 B 
9705583 B 

170919 B 

85225 A,B 
8800205 
5543394 
5631142 
5013649 
5459047 
5166058 
5635373 
5849880 
5187076 
5116738 
5366875 
5108922 

963788 

963789 
5106748 
5141905 



D 
T 
A 
A 



A 
B 
A 
B 
A 
A 
T 



A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 
A 



03-02-1998 
30-12-1996 
19-12-1996 
01-04-1998 



02-01-1997 
07-06-1993 
31-08-1994 
02-02-1995 

01- 05-1993 

02- 02-1999 



US 4828990 


A 


09-05-1989 


AU 


598455 B 


28-06-1990 








AU 


5304886 A 


29-07-1986 








DK 


407386 A 


27-08-1986 








EP 


0227834 A 


08-07-1987 








FI 


863378 A 


21-08-1986 








UO 


8604067 A 


17-07-1986 








JP 


7024596 B 


22-03-1995 



15- 09 

01- 08 
29-01 

02- 10 
06-03 
09-05 
28-04 

03- 05 

27- 12 
11-01 

24- 09- 
31-07 
18-03- 

25- 10- 

17- 03- 
01-02- 

16- 02- 

18- 04- 
22-09- 
01-08- 

14- 01- 

06- 08- 

20- 05- 

07- 05- 

17- 10- 

24- 11- 
03-06- 

15- 12- 

16- 02- 

26- 05- 
22-11- 

28- 04- 

17- 02- 
17-02- 

21- 04- 

25- 08- 



-1996 
1991 
-1988 
1996 
1997 
1997 
1988 
1989 
1995 
1988 
1997 
1995 
1998 
1994 
1998 
1990 
1998 
1997 
1993 
1987 
1988 
1996 
1997 
1991 
1995 
1992 
1997 
1998 
1993 
1992 
1994 
1992 
1988 
19B8 
1992 
1992 



US 5639638 



17-06-1997 



US 



5700911 A 



23-12-1997 



Foim PCT/lSA/210 (patent famly annex) (July 1092) 



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EP 
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JP 
NO 
WO 



678582 B 
6910594 A 
9406715 
0698094 

955419 
9501304 

954492 
9426892 



A 
A 
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T 
A 
A 



EP 0741187 A 06-11-1996 



W0 9638570 A 05-12-1996 



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Publication 
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05- 06-1997 
12-12-1994 

06- 02-1996 
28-02-1996 
08-01-1996 
10-02-1997 
08-11-1995 
24-11-1994 



All 

AU 


688210 


n 
D 


05-03-1998 


AM 
AU 


r 1 ft*Tftft.£ 
5197896 


A 

A 


14-11-1996 




100558 


* 
A 


31-03-1997 


BR 


9602166 


A 


13-01-1998 


CA 


2175298 


A 


06-11-1996 


LN 


IIP 

1157290 


A 

A 


20-08-1997 


LZ 


9601297 


A 


15-01-1997 


DE 


"7 A 1 1 ft ^ 

741187 


T 


30-04-1997 


ES 


2093593 


T 


01-01-1QQ7 


GR 


96300075 


T 


31-12-1996 


LID 

HR 


ftf ft ft 1 1 

960213 


A 


31-10-1997 


HU 


9601120 


A 


28-11-1996 


ID 
JP 


ft ft ft ft n 

9003098 


A 


07-01-1997 


NO 


ftf 1 "7 ft 

961796 


A 


06-11-1996 


in 

NZ 


o ft ^ a £ e 

286466 


A 


25-03-1998 


NZ 


O 1 AftP T 

314957 


* 

A 


27-05-1998 


D 1 

PL 


314051 


A 


12-11-1996 


ob 


49337 


A 


18-05-1998 


CI/ 


f^ft ft ^ 
56996 


A 


09-04-1997 


US 


C 7£ ft t Oft 

5760189 


A 

A 


02-06-1998 


A 1 1 

AU 


5537896 


A 

A 


18-12-1996 


r a 
LA 


ZZZ0447 


A 

A 


05-12-1996 


CD 

tr 


ftftftOOX *5 

0828842 


A 


18-03-1998 


1 IC 

Ub 


5166058 


A 

A 


24-11-1992 


IIC 

Uo 


5013649 


A 


ft^ ft!" » ftA4 

07-05-1991 


IIC 
UO 


5543394 


A 

A 


06-08-1996 


1 IC 

Ho 


5459047 


A 

A 


■% —J 4ft 4 ftA*> 

17-10-1992 


IIC 

Ub 


5635373 


A 

A 


no ft^ ^ 
03-06-1997 


1 IC 


5849880 


A 


15-12-1998 


IIC 
UO 


518/07O 


A 


If ftft iftnft 

16-02-1993 


1 IC 

Uo 


5366875 


A 


22-11-1994 


AT 


141928 


T 


15-09-1996 


AU 


613314 


B 


01-08-1991 


AU 


7783587 


A 


29-01-1988 


DE 


3751887 


D 


02-10-1996 


DE 


3751887 


T 


06-03-1997 


DK 


53497 


A 


09-05-1997 


OK 


106288 


A 


28-04-1988 


EP 


0313578 


A 


U3-05-1989 


EP 


0688869 


A 


27-12-1995 


GR 


871028 


A 


11-01-1988 


IE 


75881 


B 


24-09-1997 


IL 


83003 


A 


31-07-1995 


JP 


2729222 


B 


18-03-1998 


JP 


6298800 


A 


25-10-1994 


JP 


10070989 


A 


17-03-1998 


JP 


2500241 


T 


01-02-1990 



Form PCT/tSA/21 0 (patent family anmx) (July 1992) 



page 2 of 3 



INTERNATIONAL SEARCH REPORT 

Information on patent family members 



rnatJonal Application No 

PCT/US 98/26208 



Patent document 
cited in search report 



Publication 
date 



US 5631142 



Patent family 
member(s) 



Publication 
date 



Jp 


2713715 B 


16-02- 


-1998 


KR 


9705583 B 


18-04- 


1997 


MX 


170919 B 


22-09- 


-1993 


PT 


85225 A,B 


01-08- 


■1987 


WO 


8800205 A 


14-01- 


1991 


US 


5618924 A 


08-04- 


1997 


US 


5116738 A 


26-05- 


1992 


US 


5108922 A 


28-04- 


1992 


NO 


963788 A 


17-02- 


1988 


NO 


963789 A 


17-02- 


1988 


US 


5106748 A 


21-04- 


1992 


US 


5141905 A 


25-08- 


1992 



Form PCT/1SA/210 (patent tamfly annex) (Jdy \992) 



page 3 of 3 



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