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WORLD INT€LL€CTUAL PROPERTY ORGANIZATION 
International Bureau 




PCT 

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) 



(51) International Patent Classification ^ : 
C07G 7/00 



Al 



(11) International Publication Number: 
(43) International Publication Date 



WO 85/ 04173 

26 September 1985 (26.09.85) 



(21) International Application Number: PCT/US 85/00446 

(22) International Filing Date: 18 March 1985 (18.03.85) 



(31) Priority Application Number: 591.505 

(32) Priority Date: ' 20 March 1984 (20.03,84) 

(33) Priority Country : US 

(71)(72) Applicants and Inventors: CAPLAN, Arnold, I. [US/ 
US]; 1300 Oak Ridge Drive, Cleveland, OH 44121 
(US). SYFTESTAD, Glenn, T. [US/US]; 3660 War- 
rensville Center Road, Shaker Heights, OH 44122 
(US). 

(74) Agent: HEINKE, Lowell, L.; Watts, Hoffmann, Fisher 
& Heinke Co., 1805 The East Ohio Building, Cleve- 
land, OH 44114-2889 (US). 



(81) Designated States: AT (European patent), AU, BE (Eu- 
ropean patent), BR, CH (European patent). DE (Eu- 
ropean patent), FR (European patent), GB (Euro- 
pean patent), JP, LU (European patent), NL (Euro- 
pean patent), SE (European patent). 



Published 

With international search report. 
With amended claims. 



(54) Title: BONE PROTEIN PURIFICATION PROCESS 



(57) Abstract 

A process of extracting and purifying a bone protein capable of stimulating chondrogenic expression in undifferen- 
tiated cells in culture. The purification process is monitored at various stages by bioassaying the bone protein for chon- 
drogenic activity in embryonic limb bud mesenchymal cell cultures. 



FOR THE PURPOSES Of INFORMATION ONLY 



Codes used to identify States party to the PCX on the front pages of pamphlets pubUshing international appU- 
cations under the PCX. 



AT Austria 

AU Australia 

BB Barbados 

BE Belgium 

BG Bulgaria 

BR Brazil 

CF Central African Republic 

CG Congo 

CH Switzerland 

CM Cameroon 

DE Germany, Federal Republic of 

DK Denmark 

FI Fmland 

FR France 



GA Gabon 

GB United Kingdom 

HU Hungary 

rr Italy 

JP Japan 

KP Democratic People's Republic 

of Korea 

KR Repub lie of Kor«a 

LI Liechtenstein 

LK Sri Lanka 

LU Luxembourg 

MC Monaco 

MG Madagascar 

ML Mali 



MR Mauritania 

MW Malawi 

NL Netherlands 

NO Norway 

RO Romania 

SD Sudan 

S£ Sweden 

^N Senegal 

SU Soviet Union 

TD Chad 

TG Togo 

US United States of America 



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Description 
Bone Protein Purification Process 

Technical Field 

5 This invention relates generally to a bone protein 

purification process, and more specif ic ally to a process 
for extracting and purifying soluble bone protein capable 
of stimulating chondrogenesis. 
Background Art 

10 Bone matrix is known to contain a number of pro- 

teins which influence the behavior of various cell types. 
Some bone matrix proteins stimulate or inhibit the rep- 
lication of bone cells (Farley et al Biochem . , 21 ; 3508- 
3513, 1982; Sampath et al. Experimental Cell Res . 142: 

15 460-464, 1982, and Puzas et al, Proc . Soc. Exp . Bio. 
and Med. 166 ; 113-122, 1981). Other bone matrix pro- 
teins stimulate collagen synthesis in bone cells (Canalis 
et al, Science 210 ; 1021-1023, 1980) . Bone matrix, 
proteins such as Alpha2HS glycoprotein, osteonectin, 

20 and Type 1 collagen are chemotatic factors for monocytes 
and macrophages (Malone et al, J. Cell Bio . 92: 227- 
230, 1982; Minkin et al. Metabolic Bone Disease and 
Related Res. 2 : 363-369, 1981) . 

Cartilage, but not bone, will form in pieces of 

25 muscle grown directly upon demineralized bone matrix. 

Demineralized bone matrix or bone matrix gelatin implan- 
ted in muscle pouches in vivo or implanted in diffusion 
chambers in muscle pouches in vivo is capable of recrui- 
ting native mesenchymal cells and inducing bone forma- 

30 tion (Urist et al . Arch . Surg . 112 ; -612-621, 1977; 
Nogami et al, Clin, Orthopaedics 103 ; 235-247, 1977). 

U.S. Patent No. 4,294,753 discloses a process for 
obtaining a water-insoluble bone raorphogenic protein 
(BMP) whose action is analogous to bone matrix gelatin 



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in that it stimulates cartilage and bone formation when 
implanted in a muscle pouch in vivo' . 
Disclosure of the Invention 

This invention provides a novel process for obtaining 
5. a soluble purified bone protein that causes undifferentiated 
cells to differentiate in culture. The product of the 
invention has potential human use in enhancing the rate 
of bone ingrowth into limb protheses, thereby elim- 
inating the use of artificial cements. It also has 

10 potential human use in stimulating or enhancing the 
regeneration of damaged or diseased skeletal tissue, 
including periodontal defects* 

This application is related to copending application 
Ser. No. 591. 4A0 (Watts, Hoffmann, Fisher & Heinke 

15 Dkt. 9-722) and Ser. No. 628.168 (Watts, Hoffmann, 

Fisher & Heinke Dkt. 9-749) which disclose processes or 
techniques for delivering the soluble bone protein to 
anatomical sites. The disclosures of both of said copen- 
ding applications are incorporated herein by reference. 

20 In the process of the invention each step of the 

purification process is combined with a bioassay that 
identifies the way in which the bone protein influenc-es 
cells by its ability to stimulate cartilage formation 
in cultured cells, such as embryonic mesenchymal cells. 

25 Chick embryo limb bud mesenchyme cells, for example, 
are capable of differentiating in culture into either 
cartilage or bone or connective tissue fibroblasts. 
The emergence of one of these cell types is dependent 
upon plating density and nutrient medium composition. 

30 Since cultured mesenchymal cells will form a predictable 
number of chondrocytes when grown under specific con- 
ditions, this in vitro system can be utilized as a 
bioassay for substances which enhance or inhibit the 
limb mesenchyme-to-chondrocyte transition (Caplan, Exp . 

35 Cell Res. 62: 341-355, 1970). A limb mesenchymal cell 



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10 



system is, therefore / ideal for identifying the desired 
protein found in bone in that the purification process 
can be focused on those fractions with the desired modu- 
lating activity. 

In a preferred embodiment, the invention provides 
a process of purifying a mixture of bone matrix protein 
to obtain a protein capable of enhancing chondrogenesis 
which includes the steps of fractionating the mixture 
of bone matrix protein a plurality of times, bioassaying 
all fractions in undifferentiated cells at the conclusion 
of each fractionating step in order to identify the 
fractions having the greatest cell differentiating ac- 
tivity, and using only those identified fractions having 
the greatest cell differentiating activity in the next 
succeeding fractionating step. 

In an especially preferred embodiment, the invention 
provides a process of purifying a mixture of bone matrix 
protein to obtain a 30 to 32K dalton protein which includes 
the steps of preparing a guanidinium chloride extract 
20 of demineralized, defatted bone, dialyzing the extracted 
mixture of bone matrix protein until it is substantially 
salt-free, separating the water soluble retentate from 
the water insoluble precipitate, absorbing the water 
soluble retentate with an anionic exchanger and dasorbing 
25 by eluting with a substantially linear salt gradient, 
bioassaying fractions eluted by the salt gradient in 
cultured undifferentiated cells to identify fractions 
having the greatest chondrogenic activity, passing only 
those identified fractions having the greatest chondro- 
20 genie activity over a molecular sieve, bioassaying frac- 
tions passed over the molecular sieve in cultured undif- 
ferentiated cells to identify fractions having the greatest 
chondrogenic activity, repeating the ste-ps of passing 
over a molecular sieve and bioassaying to identify frac- 
35 tions, passing only those identified fractions having 



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the greatest chondrogenic activity over lectin coupled 
gelr collecting the eluate from the lectin coupled gel 
in one fraction and passing the fraction over a molecular 
sieve to isolate a single 30 to 32K dalton protein. 

5 Other features and a fuller understanding of the 

invention will be had from the following detailed des- 
cription of a best mode. 
Best Mode for Carrying Out the Invention 

The following example illustrates the invention 

10 and describes the process of extracting and purifying 

from bone a soluble protein capable of stimulating chon- 
drogenesis. 

Diaphyseal cortical bone shaft from beef femurs 
were cut into 2-3mm thickness rings and demineralized 

15 for 7 days in 0.6M hydrochloric acid at 4°C. The acid 
was decanted and the bone matrix washed in distilled 
water overnight at 4®C. The matrix was defatted by a 2 
hour extraction in chloroform-methanol (1:1) . The sol- 
vent was decanted and the matrix air dried overnight. 

20 The matrix was extracted in 4M guanidiniura chloride for 
three days at 4°C. In alternate procedures or examples 
of the invention, the matrix has been extracted with IM 
NaCl for five days at 37**C. The resultant solvent- 
protein mixture -was dialyzed at 4^C in 12,000 to 14,000 

25 molecular weight pore size tubing against step wise 

decreasing ionic strength buffers, first against 0.5M 
NaCl in 50mM Tris, pH 7, then 0.15M NaCl in ^OmM Tris, 
pH 7; and finally against distilled water until the 
dialysate was chloride free. A cold water- insoluble 

30 precipitate which formed during dialysis was discarded. 
The cold water soluble components in the r^tentate were 
lyophilized. 

The lyophilized water soluble retentate was further 
purified by resuspension in 50mM Tris buffer, pH 8.^ 
35 and absorbed on a DEAE - Sephacyl anionic exchange column 



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(35 X l.Scm). The column was first eluted with Tris 
buffer (70ml) to collect unbound protein and then with 
a linear salt gradient of 0.1 to l.OM NaCl (1.1 ml/min; 
total gradient volume = 250ml). Tubes containing 1.0ml 

5 of eluent were collected and pooled into 6 fractions 
and dialyzed against cold distilled water. Fraction 
VI/ desorbed between 0.6 to 1.0 molar NaCl (Tube numbers 
270-320) contain the chondrogenic activity. This protein 
fraction is hereinafter called Protein Avi* 

10 Protein Ayj was resuspended in 4M guanidinium 

chloride and was passed through a Sepharose CL-6B molec- 
ular sieve column (100 x 0.5cm) equilibrated with 4M 
guanidinium chloride. 0.6ml fractions were collected 
(total volume = 50ml). The effluent protein concentra- 

15 tion was monitored on a Gilson recording spectrophotometer 
at 280nm. Three broad protein-peaks were observed and 
the individual collection tubes corresponding to each 
peak were pooled, dialyzed against cold wat^r and lyophi- 
lized. Fractions corresponding to the second peak were 

20 active (Tube numbers 31-53) . This protein fraction is 
hereinafter called Protein Bij. 

Protein Bji was then rechromatograph-ed through the 
same column. The fraction containing the greatest bio- 
logical activity (Tube numbers 42-53) were dialyzed and 

25 lyophilized. This protein is hereinafter called Protein 

Cm- 

The lyophilized Protein Cm was resuspended in 
l.OM NaCl in Tris buffer, pH 7.0 and passed through a 
Sepharose-Conconavalin A column (lOcm x .5) equilibrated 
30 with 1.0 NaCl. (Total Volume = 15ml). The conconavalin 
- A bound only contaminating glycoproteins. The active 
factor passed through the column and the eluate was 
collected in one fraction, dialyzed, and lyophilized. 
This protein is hereinafter called Protein Di. 



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Protein Dj, which contained 3 prominent protein 
components as assessed by polyacrylamide gel electropho- 
^ res is / was re-cycled through a Sepharose CL-6B column 
to isolate a single 30-32k dalton component with in 

5 vitro chondrogenic stimulating activity. This protein, 
which is referred to as Protein Ei, has been found 
effective in stimulating chondrogenesis in undifferen- 
tiated cells. 

The bioassay for chondrogenic activity in each of 

10 the purification steps above utilized a cell culture 
system previously reported by Caplan, Exp . Cell Res . 
62:341-348 (1970). 

The lyophilized water-soluble proteins from each 
purification step were resuspended in warm water (37**C 

15 to 45**C) and added to serum supplemented nutrient medium 
(Eagle's Minimum Essential Medium plus 5% chick embryo 
extract plus 3% fetal calf serum plus 7% horse serum) 
at decreasingly smaller doses depending upon the degree 
of purity. For the least pure protein. Protein Avi, 

20 maximal chondrogenic activity was detected at Lowry 

protein concentrations ranging from 40-60ug/ml; and for 
Protein E^ at 1 to 5ug/ml. 

A similar increase in limb bud cell chondrogenesis 
was observed when cultures were maintained in serum- 

25 free medium composed of the following defined substances 
a basal medium containing Ham's F-12 and Dulbecco's 
modified Eagles to which is added insulin (5ug/ml), 
transferrin (5ug/ml) , hydrocortisone (lOOitM) and 0.1%, 
bovine serum albumen. The quantity of soluble bone 

30 protein necessary to produce a significant stimulation 

in cartilage formation was approximately 0.125 to O.lOO 
times that required in similar cultures grown in serum 
supplemented medium. 

1 ml of nutrient solution containing 2.0 to 2.5 x 

35 106 enzymatically isolated embryonic (HH stage 23-24) 



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7 

chick limb bud mesenchymal cells was plated onto 35mm 
tissue culture dishes. 5ug of Protein was added to 
the culture dishes 18 to 24 hours after plating the 
cells. The cells were incubated at 37**C in 5% CO2 for 

5 7-8 days. The chondrogenic effect was, documented by 
visual observation of living cultures using a phase 
contact inverted microscope , by Toludine Blue staining 
of fixed day 8 cultures and by radioactive precursor 
uptake into cartilage-specific proteoglycans. 

10 A 7 day exposure to Protein stimulated undif- 

ferentiated limb bud mesenchyme to form cartilage in a 
dose dependent manner. 

In 35mm plates, the reaction had the following 
characteristics: 

15 

1. An initial seeding density of approximately 2x10^ 
♦ cells was necessary to observe the chondrogenic 

response. 

20 2. A maximal chondrogenic response was observed when 
cultured mesenchyme were exposed to Protein E^ 
during the interval between 0.5-2.5 days following 
plating. The stimulation of chondrogenesis was 
slight if exposure to the protein was later than 

25 2.5 days after plating. 

3. A maximal chondrogenic response was observed when 
cultured mesenchyme were exposed to Protein Ei for 
seven continuous days. Exposure times of 1-2 days 
30 resulted in only a slight increase in chondro- 

genesis (i.e., 1.5-2 times the ^^S-SO^^ incorpora- 
tion) . 



4. 

35 



The appearance of morphologically recognizable 
chondrocytes occur ed on days 5-6 and chondrocytes 



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8 

continued to develop so that over 90% of the culture 
dish was covered with cartilage by day 8. This 
represented a maximum response and correlated with 
a 4-5 fold increase in cell-lay-er associated 35s- 
5 SO4 uptake/ug DNA and an intensely metachromatic 

Toludine Blue staining pattern when compared to 
untreated cells. 

Modifications of the above invention and materials 
10 and procedures employed therein which are obvious to 
persons of skill in the art are intended to be within 
the scope of the following claims. 



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9 

Claims 

1. A process of purifying a mixture of bone matrix 
protein to obtain protein capable of enhancing chondro- 

5 genesis comprising the steps of: 

a) fractionating the mixture a plurality of 

times; 

b) bioassaying all fractions in undifferen- 
tiated cells at the conclusion of each fractionating 

10 procedure in order to identify the particular fractions 

having the greatest desired cell differentiating activity; 
and 

c) using only those identified fractions 
having the greatest cell differentiating activity in 

15 the next succeeding fractionating step. 

2. The process of Claim 1 wherein the undifferen- 
tiated cells are cultured cells. 



20 3. The process of Claim 1 wherein the undifferen- 

tiated cells are cultured embryonic cells. 

4. The process of Claim 3 wherein the undif- 
ferentiated cells are enzymatically isolated embryonic 

25 (HH stage 23-24) chick limb bud mesenchymal cells. 

5. The process of Claim 1 wherein said fractionating 
is carried out to obtain a single 30 to 32K dalton protein. 

30 6. The process of Claim 1 or Claim 2 wherein 

each bioassaying step comprises preparing nutrient media, 
each medium containing protein from one fraction, the 
concentration of protein ^eing dependent upon the degree 
of purity; incubating undifferentiated cells with sai-d 

35 nutrient media; and, monitoring said cells for differen- 
tiation activity. 



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10 



7. The process of Claim 6 wherein the nutrient 
medium is a serum-free medium composed of a basal medium, 
insulin, transferrin, hydrocortisone and bovine serum 
albumen. 

5 

8. The process of Claim 1 or Claim 2 wherein the 
first fractionating procedure comprises of: 

a) demineralizing bone tissue; 

b) extracting a protein mixture from said 
10 demineralized bone tissue in a solubilizing solution; 

and , 

c) separating the protein mixture from the 
solubilizing solution. 

15 9. The process of Claim 1 or Claim 2 wherein the 

second fractionating procedure comprises the steps of: 

a) absorbing the protein mixture on an anion 
exchange resin; 

b) desorbing the protein mixture with a 

20 salt concentration gradient to obtain a number of frac- 
tions; and, 

c) separating the proteins of the fractions 
having the greatest cell differentiating activity from 
the eluate. 

25 

10. The process of Claim 1 or Claim 2 wherein the 
third fractioning procedure comprises the steps of: 

a) passing the proteins through a mol«ecular 
sieve column to obtain a number of fractions; 
30 b) monitoring the protein concentration in 

said fractions; 

c) separating the proteins of the fractions 
having the greatest cell differentiating activity from 
the eluate; and. 



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11 



d) repeating steps a) - c) a selected number 

of times. 

11. The process of Claim 1 or Claim 2 wherein the 
5 fourth fractioning procedure comprises the steps of:* 

a) passing the protein through a lectin 
coupled gel, said lectin being capable of selectively 
binding contaminating glycoproteins; and^ 

b) separating the isolated desired protein 
10 from the eluate. 

12. The process of Claim 1 or Claim 2 wherein the 
final fractioning procedure comprises the steps of: 

a) passing the proteins through a molecular 
15 sieve column to obtain a number o'f fractions; 

b) assessing the protein components by electro- 
phoresis; and 

c) repeating steps a) - b) a selected number 

of times. 

20 

13. A process of isolating soluble bone protein 
capable of stimulating cartilage growth comprising the 
steps of: 

a) demineralizing bone tissue, extracting 

25 protein from the deraineralized bone tissue in a solubil- 
izing solution and separating the protein from the sol- 
ubilizing solution; 

b) resuspending the separated protein; 

c) absorbing the resuspension on an anionic 

30 resin; 

d) desorbing the protein with a salt gradient 
to obtain a number of fractions; 

e) bioassaying said fractions in undiff>eren- 
tiated cells to identify chondrogenic activity ther-eof; 



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12 

f) separating protein of the most biologically 
active fractions from the eluate; 

g) resuspending the protein from step f) in 
a suitable buffer; 

5 h) passing the buffered protein through a 

molecular sieve and monitoring the protein content of 
the fractions passing through the sieve; 

i) bioassaying selected fractions in undif- 
ferentiated embryonic cells and selecting those fractions 
10 with greatest chondrogenic activity; 

3) repeating steps f) through 1) a selected 
number of times; 

k) resuspending and purifying a selected 
protein from step k) • 

15 

14. A process of purifying a mixture of bone matrix 
proteins to obtain a 30 to 32K dalton protein comprising 
the steps of: 

a) preparing a guanadinium chloride extract 
20 of demineralized/ defatted -bone; 

b) dialyzing the extracted mixture of bone 
protein until it is substantially salt-free; 

c) separating the water soluble retentate 
from the water soluble precipitate; 

25 <3) absorbing the water soluble retentate 

with an anionic exchanger and desorbing by eluting with 
a substantially linear salt gradient; 

e) bioassaying fractions eluted by the salt 
gradient in undifferentiated cells to identify fractions 

30 having the greatest chondrogenic activity; 

f) passing only those identified fractions 
having the greatest chondrogenic activity over a molecular 
sieve; 



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13 

g) bioassaying fractions passed over the 
molecular sieve in undifferentiated cells to identify 
fractions having the greatest chondrogenic activity; 

h) repeating steps f) and g) ? 

5 i) passing only those identified fractions 

having the greatest chondrogenic activity over lectin 

coupled gel; 

j) collecting the eluate from step i) in 

one fraction; and 
10 k) passing the fraction from step j) over a 

molecular sieve to isolate a single 30 to 32K dalton 

protein. 

15. The purified protein produced by the process 
15 of Claim 1. 

16. The purified protein produced by the process 
of Claim 2. 

20 17. The purified protein produced by the process 

of Claim 3. 

18. The purified protein produced by the process 
of Claim 13. 

25 

19. The purified protein produced by the process 
of Claim 14. 



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AMENDED CLAIMS 

(received by the International Bureau on 03 July 1985 (03.07,85); 
(5 pages) • 

1-13. (Cancelled) 

14o (Amended) A process of purifying bone matrix 
proteins to obtain a cold-water-soluble 30 to 32k dalton 
protein capable of stimulating cartilage formation in 
mesenchymal-like cells comprising the steps of: 

a) preparing a guanadinium chloride extract 
of demineralized, defatted bone; 

b) dialyzing the guanidinium chloride soluble 
extract against decreasing ionic strength buffers down 

to water until it is substantially salt-free; 

c) separating the cold-water-soluble proteins 
from the cold-water-insoluble proteins present in the 
retentate; 

d) adsorbing the cold-water-soluble proteins 
in the retentate with an anionic exchanger at about pH 
8.0 and desorbing by eluting with a substantially linear 
salt gradient; 

e) assaying fractions eluted by the salt 
gradient in undifferentiated mesenchymal-like cell cultures 
to identify fractions having the greatest chondrogenic 
activity; 

f) passing only those identified fractions 
having the greatest chondrogenic activity over a molecular 
sieve column; 

g) assaying fractions passed, over the molecular 
sieve in undifferentiated mesenchymal-like cells to 
identify fractions having the greatest chondrogenic 
activity; 

h) repeating steps f ) and g) ; 

i) passing only those identified protein 
fractions having the greatest chondrogenic activity 
over concanavalin-A coupled gel; 



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1 5 

j) collecting the unbound protein from step 
i) in one fraction; and 

k) passing the unbound protein from step j) 
over a molecular sieve column to isolate a single 30 to 
32k dalton protein. 

15. - 18. (Cancelled) 

19. The purified protein produced by the process 
of Claim 14. 

20. (New) A process of purifying bone matrix 
proteins to obtain a cold-water-soluble 3 0 to 32k dalton 
protein capable of stimulating cartilage formation in 
embryonic cells comprising the steps of: 

a) preparing. a guanadinium chloride extract 
of demineralized f defatted bone; 

b) dialyzing the guanidinium chloride soluble 
extract against decreasing ionic strength buffers down 

to water until is is substantially salt-free; 

c) separating the cold-water-soluble proteins 
from the cold-water-insoluble proteins present in the 
retentate; 

d) absorbing the cold-water-soluble proteins 
in the retentate with an anionic exchanger at about pH 

8 and desorbing by eluting with a substantially liiiear 
salt gradient; 

e) assaying fractions eluted by the salt 
gradient in undifferentiated embryonic limb bud mesenchymal 
cell culture to identify fractions having the greatest 
chondrogenic activity; 

f) passing only those identified fractions 
having the greatest chondrogenic activity over a molecular 
sieve column; 



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15 

g) assaying fractions passed over the molecular 
sieve in undifferentiated embryonic limb bud cells to 
identify fractions having the greatest chondrogenic 
activity; 

h) repeating steps f) and g) ; 

i) passing only those identified protein 
fractions having the greatest chondrogenic activity 
over concanavalin-A coupled gel; 

j) collecting the unbound protein from step 
i) in one fraction; and 

k) passing the unbound protein from step j) 
over a molecular sieve column to isolate a single 30 to 
32k dalton protein* 

21. (New) A process of purifying a defatted, 
demineralized guanidinium or sodium chloride extract of 
bone .matrix to obtain a cold-water-soluble 30 to 32k 
dalton protein capable of stimulating cartilage formation 
comprising the steps of: 

a) dialyzing the extract at I-IO^^C first 
against 0.5M guanidinium chlorider then against Tris 
buffered 0,15M NaCl solution at about pH 7, and then 
against cold distilled water until the extract is sub- 
stantially free of chloride ion thereby forming a cold- 
water-insoluble precipitate and a retentate containing 
cold-water-soluble proteins; 

b) adsorbing the retentate containing cold- 
water-soluble proteins with a DEAE-Sephacyl anionic 
exchanger buffered at about pH 8.0; 

c) eluting unbound cold-water -soluble proteins 
with an eluate buffered at about pH 8.0; 

d) desorbing the bound cold-water-soluble 
proteins with a substantially linear salt gradient of 
from about 0.1 to about l^OM salt; 



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I 7 

e) collecting the cold-water-soluble proteins 
desorbed between about 0,6 to l.OM salt; 

f) passing the desorbed cold-water-soluble 
proteins through a Sepharose CL-6B molecular sieve column 
equiliboated in IM salt solution at about pH 7,0 to 
obtain three cold-water-soluble protein fractions; 

g) passing the second fraction of cold- 
water-soluble protein over conconavalin-A sepharose in 
about l.OM salt to remove conconavalin-A binding protein; 
and 

h) passing the concanavalin-A unbound protein 
through a Sepharose <;l-6B molecular sieve column to 
obtain a single 30-32k dalton component. 

22. (New) The highly purified protein extracted 
from bone matrix by the process of Claim 14 having the 
following characteristics: 

a) molecular weight of 30-32k daltons as 
assessed by SDS-PA<3 electrophoresis under reducing and 
non-reducing conditions; 

b) solubility in substantially pure water 
at temperatures at least as low as 4**C; 

c) affinity for anionic exchangers at about 

pH 8.0; 

d) non-affinity for *concanavalin-A; and 

e) activity as a stimulator of chondrogenesis 
in undifferentiated cells. 

23. (New) The highly purif iad protein extracted 
from bone matrix by the process of Claim 20 having the 
following characteristics: 

a) molecular weight of 30-32k daltons as 
assessed by SDS-PAG electrophoresi-s under reducing and 
non-reducing conditions; 



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18 



PCT/US85/00446 



b) solubility in substantially pure water 
at temperatures at least as low as 4*'C; 

c) affinity for anionic exchangers at about 

pH 8,0; 

d) non-affinity for concanavalin-A; and 

e) activity as a stimulator of chondrogenesis 
in undifferentiated cells. 

24. (New) The highly purified protein extracted 
from bone matrix by the process of Claim 21 having the 
following characteristics: 

a) molecular weight of 30-32k daltons as 
assessed by SDS-PAG electrophoresis under reducing and 
non-reducing conditions; 

b) solubility in substantially pure water 
at temperatures at least as low as 4*'C; 

c) . affinity for anionic exchangers at about 

pH 8.0; 

d) non-affinity for concanavalin-A; and 

e) activity as a stimulator of chondrogenesis 
in undifferentiated cells. 

25. (New) A highly purified protein extracted 
from bone matrix having the following characteristics: 

a) molecular weight of 30-32k daltons as 
assessed by SDS-PAG electrophoresis under reducing and 
non-reducing conditions; 

b) solubility in substantially pure water 
at temperatures at least as low as 4**C; 

c) affinity for anionic exchangers at about 

pH 8.0; 

d) non-affinity for concanavalin-A; and 

e) activity as a stimulator of chondrogenesis 
in undifferentiated cells. 



INTERNATIONAL SEARCH REPORT 

International Application NopCj/USS 5/00446 



I, CLA SSIFICATION OF SUBJECT MATTER <lf several classification symbols apply, Indicate all)' 
According to International Patent Classification (IPC) or to both National Classification and IPC ^ 



IPC C07G 7/00 



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Classification Symbols 



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Doeumentatlon Searched other than Minimum Documentation 
to the Extent that such Documents are Included In the Fields Searched » 



III. DOCUMENTS CONSIDEWED TO BE RELEVANT '« 



Catesory • | Citation ot Document. '« with Indication, where appropriate, of the relevant passages » 



Y 
Y 



URIST, SCIENCE I5O: 893-899 (1965) 

r.-/ATA ET AL CLIN . ORTHO RELATED RES . 84: 
257-274 (1974) 

URIST ET AL PROC . NATL . ACAD SCI , t^, 
70: 3511-3515 (1973) 

URIST ET AL 3. THEOR. BIOL. 38: 155-1^8 
(1973) 

HAI>IAr.-;URA ET AL CLIN . ORTHO . RELATED RES . 
148: 231-292 (I98O) 

SEYEDI.N ET AL f. CELL BIOLOGY, 97: 950-953 
(1983) 

URIST, CARTILAGE, D5VSL0FT.ISKT . DIFFSRSI\^T - 
lATION AND GROV/TH (3.K. HALL, ED), VOL. 2, 
! i^i- . 2-8ST1983T 
AI\ASTASSIADES ET AL CALCIF. TISS RES . 26: 
173-179 (1978) 

TSRHINE ET AL PROC . NATL. ACAD. SCI. USA . 
81: 2213-2217 (1984) 

URIST ET AL ARCH . SURG. 112; 6I2-6I9 (1977) 



Relevant to Claim No. >• 



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1-19 

1-19 

1-19 

1-19 

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• Special categories of cited documents: 

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

considered to be of particular relevance 
"E" earlier document but published on or after the international 

filing 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 

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



"T" later document published after the ^"^^^ 

or Driority date and not in conflict with the appllcat on but 
dted 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 

"Y" document of particular relevance;, the claimed invention 
cannot be conSdered to Involve an '"ventive step when the 
document is combined with one or more other such docu- 
mentrsuch combination being obvious lo a person skilled 
in the art. 

"A" document member of the same patent family 



IV. CERTIFICATION 

Date of the Actual Completion of the International Search « 



18 APRIL 1985 



International Searching Authority i 



ISA/US 



Date of Mailing of this International Search Report a 

2 9 APR 1985. 



Sionalure.olAuthoriJedOfflterM ' 

HO;VARD E. SCHAIN 



Form PCT/ISA/210 (second sheet) <Oetober 1981) 



International Application No. 

PnT/TTS8^/00^46 



III. DOCUMENTS CONSIOEREO TO BE RELEVANT (CONTINUED FROM THE SECOND SHEET) 


Category* 


Citation of Oocumentt with indication, where appropriate, of the relevant passages 


Relevant to Claim No is 


Y 


SAI.1PATH ET AL EXP. CELL RES. 142: if 60-464 
(1982) 


.1-19 


Y . 


rurnaN et al t^ETA^CLic soke disease and 

RELATED RES. 2: 363-369 (1981) 


1-19 


A 


JIALONS ET AL T. CELL BIO. 92; 227-230 (1982) 


1-19 


Y 


PUZAS ST AL PROG. SOC. EXP. BIO. AND RISD. 
166: 113-122 (1981) 


1-19 


Y 


FARLEY ET AL BIOCHH!-! ^1: 3S02-3507 (1982) 


1-19 


Y 


i;gga:.:i st al cliit. crtho. aitd related res. 
115: 268-273 (1976) 


1-19 


J. 


CAIIALIS et al SCmiCS 210 : 1021-1023 (1980) 


1-19 




■oS, a, ^f29^f',753 ru3jjISn±.D Ij OClCBER 19al 

urist 


1-19 


J*. 


Us, A, 4 , ^•3^1- , 09'-l' i-USHbhSD 2o I!£,ERIjARX Ivo**- 

SEYEDIN ET AL 


1-19 


X P 


US, A, 4,455,256 PUBLISHED I9 1984 

URIST 


1-19 


Y 


URIST ET AL PROC. ITATL. ACAD'. SCI. USA. 76: 
I82S-I832 (1979) 


1-19 


Y 


TERI-ilMZ ET AL COLL. 26: 99-105 (1981) 


1-19 


Y. 


ri.-ii-iAi.»UAA iiT AL C- i-ZK . OR iHO . RELATUD -.ib .■ 
153: 232-240 (I93O) 


1-19 


X 


iJii-Lor iii HJj riitfLr . axjcl'J. iCl . UoA, ol: 

371-375 (Ji\r-:UARY 1934) 


1-19 


Y 


?RCC. see. EXP. BIO. AND LTED. 162: 48-53 
(1979) URIST ST AL 


1-19 


V 

I 


173: 19^-199 (1933) 


1-19 


1 


l^iLi-iXrlii ijj. AJj o • JjXO. Oiijiin ^^OS JLU*4'\J :>-"iU*rUO 


l-±9 


Y 


COKGVER ET AL CHEKISTRY AND BIOLOGY 0? 
KIRSPuALIZED CCNKSCTIVS TISSUES (VEIS ED) , 

597-606 (1981) 


1-19 


Y 


URIST ET Ai CLIN. GRTHO. RELATED RES. 162: 
219-232 (1982) 


1-19 


Y 


CAPLAIJ, EXP. CELL RES. 62: 3^1-355 (1970) 

■ 

• . ■ ■ 


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Form PCT'lSA/aip (extra sheet) (October 1381) 



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