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JEuropgisches Patentamt 
European Patent Office ® Publication number: 0^21976 



A2 

Office europ6en des brevets 



® 



EUROPEAN PATENT APPLICATION 



@ Appnca«onnu.ber:8430032.. @ ■-^'•'f/A6?K35??2. A61K37/02 

@ Date of filing : 1 D.01 .84 



@ Priority: 12.04.83 US 484286 


@ 


Applicant: COLLAGEN CORPORATION, 2500 Faber 
Place, Palo Alto, Caltlornla 94303 (US) 


® Date of publication of application: 17.10^ 
Bulletin 84/42 


® 


inventor: Seyedin, Saeld, 9 Sutter CreeK Lane, Mountain 
View California 94043 (US) 
Inventor: Thomas, Thomas, 249 Santa Rita, Palo Alto 
Caltfomla 94301 (US) 


@ Designated Contracting States : AT BE CH DE FR GB IT 
ULU NLSE 


@ 


Representative: Harrison, David Christopher at al, 
MEWBURN ELUS & CO 2/3 Curaltor Streeti Undon 
EC4A1BQ (QB) 



@ Partially purlflBd osteogenic factor and process for preparing same from demlneranzed bone or an osteosarcoma, 

@ A process for partially purifying an osteogenic factor 

from deminerallzed bone particles or osteosarcomas In 

which nonfibrous proteins are extracted from the 

demineralized bone or osteosarcomas with a dissociative 

extractant for nonfibrous proteins, such as urea or guanl- 

dine hydrochloride, the extracted nonfibrous proteins are 

fractionated by anion exchange chromatography using 

DEAE cellulose at pH 7. the fraction not adsorbed by the 

DEAE cellulose is further fractionated by cation exchange 
d chromatography using CM cellulose at pH 4.8, the fraction 
jtf adsorbed by the CM cellulose is eluted therefrom and the 

partially purified osteogenic factor is recovered from the 
^ eluate as a < 30 000 dalton protein isolate. 

r* 
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d 



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ACTORUM AG 



0121976 



PARTIALLY PURIFIED OSTEOGENIC FACTOR 
AND PROCESS FOR PREPARING SAME FROM 
DEMINERALIZED BONE OR AN OSTEOSARCOMA 



5 

The invention is in the fields of protein 
chemistry and osteoplasty- More particularly the 
invention relates to a partially purified extract from 
demineralized bone or an osteosarcoma that exhibits 
10 cartilage and bone induction activity. 

Prior efforts to isolate and characterize the 
chemical or chemicals present in bone or osteosarcomas 
that induce osteogenesis have been reported. US Pat No. 

15 4,294,753 describes the partial isolation of a bone 

inducing agent, called "bone morphogenetic protein," from 
demineralized bone. Bone morphogenetic protein was not 
fully characterized and was obtained as a crude bone or 
dentin tissue extract. The process by which the crude 

20 protein was obtained involved: demineralizing bone 

tissue; extracting the factor from the demineralized bone 
with an aqueous solution of a neutral salt and either, 
urea or* guanidine? and removing the urea or guanidine 
from the extract to precipitate the crude protein. 

25 Ion exchange purification of crude bone 

morphogenetic protein is reported by Urist, M. R.r et al, 
Clin Orthopaedics and Related Research (1982) 162 ;219" 
232. The crude protein was redissolved and 
reprecipitated in 6 M and 1 M aqueous urea, respectively. 

30 The final precipitate was taken up in 6 M urea and 

chromatographed on CM-cellulose (a cationic exchanger) at 



0121976 



pH 4,8. The unadsorbed fraction was further 
chromatographed on DBAE-cellulose (an anion exchanger) at 
pH 7.2. Bloassay of the adsorbed and unadsorbed 
fractions from both separations showed that only the 
5 unadsorbed fractions from each separation exhibited 
osteoinductive activity. High molecular weight proteins 
were separated from the DEAE unadsorbed fraction by gel 
filtration. 

A principal object of the present invention is 
0 to provide a partially purified osteogenic factor that is 
either different from or purer than the factor reported 
by Orist, et al. 



The osteogenic factor of the Invention is 
present in vertebrate bone and osteosarcomas and has the 
following characteristics: 

(a) it is a nonfibrous protein; 

(b) it is unadsorbed by diethylaminoethyl 
cellulose anion exchanger at a pH of about 7.0; 

(c) it is adsorbed by carboxymethyl cellulose 
cation exchanger at a pH of about 4.8; and 

(d) it has a molecular weight below about 
30,000 daltons. 

^ The process for partially purifying this 
osteogenic factor from particulate demineralized bone or 
osteosarcomas comprises extracting nonfibrous proteins 
from demineralized bone or an osteosarcoma with a liquid 
dissociative nonfibrous protein extractant and purifying 
the factor from the extract by ion exchange 
chromatography and is characterized in that: 

(a) the extract is first contacted with an 
anion exchanger at a pH of about 6.8 to about 7.2; 



0121976 



(b) the unadsorbed fraction from the anion 
exchanger is contacted with a cation exchanger at a pH of 
about 4.5 to about 5.2; 

(c) the adsorbed fraction from the cation 

5 exchanger is elutedj and 

(d) materials having a molecular weight below 
about 30,000 daltons are isolated from the eluate. 



In the drawings: 
10 Figure 1 is a graph of the optical densities of 

eluate fractions from the preparative ion exchange of 
the example; infra ; and 

Figure 2 is a graph of the optical densities of 
the gel filtration fractions of the example, infra . 

15 

The bone or osteosarcoma that is used in the 
invention may be collected from a variety of mammalian 
sources. Homogeneic and xenogeneic sources may be used, 
osteosarcomas may be induced in laboratory animals. 

20 Bovine and porcine bone, preferably long bone, will 
normally be used because of its availability. The 
surface of the bone is first cleaned by physically 
removing periosteum by scraping or brushing. The bone is 
then ftagmented or pulverized into small pieces and the 

25 fragments are washed with a very mild aqueous acid, e.g., 
0.01 N HCl, with agitation to remove any water soluble 
materials remaining on the fragments. The washing is 
preferably carried out at reduced temperatures, usually 
about O'C to 18»C, preferably about 5«C, with frequent 

30 changing of the wash medium. The fragments are then 



0121976 



dried, and defatted by extraction with one or more 
lipophilic solvents, such as ethanol ethylacetate and 
diethylether , to remove lipids. 

The principal mineral component of bone is 
5 calcium phosphate. The term "calcium phosphate" as used 
herein is intended to encompass the numerous calcium- 
phosphorus complexes and compounds present in bone such 
as the various polycalcium phosphates, hydroxyapatite, 
chlorapatite, and the like. Calcium phosphate usually 

10 constitutes about 80% of the mineral content of bone. 
Other mineral components of bone include calcium 
carbonate, calcium fluoride, calcium chloride, and 
magnesium phosphate. These minerals are normally soluble 
in dilute mineral and organic acids and such acids may be 

15 used to demineralize bone without digesting the protein 
components of the bone. The concentration of the acid 
used to demineralize the bone will usually be between 
0.1 M to 1.0 M. Hydrochloric acid at a concentration of 
0.5 M is preferred. The bone will normally be contacted 

20 with the acid for one hour to several days at 

temperatures ranging from about 0**C to about 25*^0. 
Agitation will facilitate extraction of the minerals from 
the bone- After the extraction is complete the bone is 
separated from the acid such as by sedimentation, 

25 filtration or other conventional solid-liquid separation 
techni<iues and the resulting demineralized, non-digested 
bone is washed with water to remove adsorbed acid. 

Following demineralization noncollagenous pro- 
teins (including proteoglycans) are extracted from the 

30 demineralized bone by contacting the bone with an aqueous 
liquid extractant that dissociates ionic bonds and solu- 
bilizes the noncollagenous proteins in the bone. This 
extractant is sometimes referred to herein as a "disso- 
ciative nonfibrous protein extractant." The extraction 



-6- 



0121976 



is done under conditions that inhibit digestion or 
denaturation of the extracted proteins. Examples of 
dissociative extractants that may be used are guanidine 
hydrochloride (at least about 4 M), urea (8 M) plus salt 
5 (1 M), and sodium dodecyl sulfate (at least about 1% by 
volume), A preferred extractant is 4 M guanidine 
hydrocholoride^ pH 6.8. The extractants will usually be 
mixed with the bone with mild agitation at bone 
extractant ratios (v/w) of at least about 10:1, usually 
10 about 15:1 to 20:1. The extraction is preferably carried 
out at reduced temperatures in the presence of a protein 
inhibitor to reduce the likelihood of digestion or 
denaturation of the extracted protein. The 
temperature will usually be in the range of O'^C and 20**C, 
15 preferably 0**C and 5**C. Examples of protease inhibitors 
that may be included in the extraction medium are 
phenylmethylsulf onyl fluoride, N-ethyl maleimide, 
benzamidine, and 6-aminohexanoic acid- The pH of the 
extraction medium will depend upon the particular 
20 extractant that is used. The bone will usually be 

contacted with the extraction medium for about 16 to 24 
hr. After the extraction is complete the undissolved 
bone is separated from the extract by conventional solid- 
liquid separation techniques such as centr if ugation or 
25 filtration. Further work up of the extract by repeated 
precipin:ation or dialysis may be carried out if desired. 

The next step in the purification of the 
osteogenic factor is preparative ion exchange 
chromatography. The chromatography is carried out in two 
30 substeps: (1) fractionation on an anion exchanger 

followed by (2) fractionation on a cation exchanger. In 
the anion exchange fractionation the noncollagenous 
protein is contacted with an anion exchanger at a pH of 
about 6.8 to 7.2, preferably about 7.0. Cellulosic ion 



0121976 



exchangers and ion exchange gels derived from cross- 
linked dextran or polyacrylamide are examples of anion 
exchangers that may be used. Cellulosic anion exchangers 
are preferred. Diethylaminoethyl is a preferred 
5 functional anion exchange group. The anion exchange 
fractionation may be carried out in a column or batch 
operation. The noncollagenous proteins are in an aqueous 
solution containing one of the above-described 
extractants (to keep the proteins in solution) and 

10 preferably also an effective amount of a protease 
inhibitor when contacted with equilibrated exchanger. 
The sblution is buffered to the desired pH. The contact 
time should be sufficient to allow the exchange to reach 
equilibrium. In a batch operation the unadsorbed 

15 fraction of the extract may be separated from the anion 
exchange material by centrifuging or filtering. In 
either instance, the unadsorbed fraction of the extract 
contains the desired osteogenic factor and is collected 
or isolated and further fractionated on the cationic 

20 exchanger. 

The cation exchange fractionation is effected 
by contacting the unadsorbed fraction from the anion 
exchange substep with a cation exchanger at a pH of about 
4.5 to about 5.2, preferably about 4.8. Examples of 

25 cation exchangers that may be used are the available . 
cellulose cation exchangers and cation exchange gels 
derived from polyacrylamide and cross-linked dextran. 
Cellulosic cation exchangers are preferred. 
Carboxymethyl is a preferred cation exchange functional 

30 group. Before being contacted with the cation exchanger 
the buffers in the unadsorbed fraction of the anion 
exchange step are removed from the fraction such as by 
dialysis. The proteins in the unadsorbed fraction are 
redissolved, if necessary, and the solution is buffered 



0121976 



to the desired pH for the cation exchange. The solution 
is then contacted with equilibrated cation exchanger 
again for a time sufficient to let the exchange reach 
equilibrium. In the cation exchange fractionation, the 
5 fraction of the solution that is adsorbed by the cation 
exchanger contains the desired osteogenic factor and 
must be recovered from the cation exchanger. When the 
exchange is carried out batchwise the exchanger is first 
separated from the unadsorbed fraction of the solution. 
10 The exchanger is then washed with starting buffer to 
remove any residual unadsorbed material. 

Following washing of the cation exchanger 
noncollagenous protein adsorbed thereon is recovered by 
elution with an elutant of appropriate pH or ionic 
15 strength. Stepwise or gradient elution may be used to 
obtain differential elution of the adsorbed proteins if 
desired. When carboxymethyl cellulose is used as a 
cation exchanger in the process the osteogenic factor may 
be eluted from the exchanger using a buffer having an 
20 ionic strength in the range of about 10 to about 400 mM. 
The final step in the purification is 
fractionating the eluate by molecular weight to recover 
the ? 30K dalton fraction of the eluate. This 
fractionation may be done by gel filtration or other 
25 conventional separation techniques. Partially purifi.ed 
factor'may be recovered from the ^ 30K dalton fraction of 
the eluate by conventional procedures such as dialysis 
and lyophilization. The partially purified material may 
be implanted in mammals with or without a carrier to 
30 induce bone formation at the implant site. 

The following example further illustrates the 
invention process and the partially purified osteogenic 
factor produced thereby. This example is not intended to 
limit the invention in any manner. 



-8- 0121976 



Preparation of Deroineralized Bone 

Bovine metatarsal bone obtained fresh from a 
slaughterhouse and frozen at -70*C was used. The bone was 
cleaned of periosteum, broken into fragments smaller than 
5 1 cm in diameter and pulverized in a large mill. The 
pulverized bone was washed overnight in 0.01 N HCl at 
4''C. It was then defatted by 3 washes of approximately 
20 rain duration with 3 volumes of ethanol and the same 
with diethylether. The defatted bone powder was 
10 demineralized n 0.5 N HCl at 4^C. The acid was removed 
and the demineralized bone powder was washed twice with 
10 volumes of water. 

Extraction of Noncollagenous Proteins 

Demineralized bone powder was extracted with 10 

15 volumes of 4 M guanidine-HCl, 15 mM ethylene- 
diaminetetraacetic acid (EDTA) pH 6.5, 1 mM 
phenylmethylsulfonyl fluoride (PMSP) , and 10 mM N- 
ethylmaleimide (NEM) for 16 hr at 4*»C. The suspension 
was centrifuged at 10,000 rpm for 30 min. The pellet was 

20 washed with another five volumes of 4 M guanidine 

solution and centrifuged as above. • Soluble fractions 
were combined and concentrated at least five volumes by 
ultrafiltration using an Aroicon ultrafiltration (lOK) 
unit. The concentrate was dialyzed employing the same 

25 unit ag*ainst a solution containing 6 M urea, 1 mM PMSF, 
1 mM NEM, 20 mM Na phosphate (pH 7.0) and 50 mM NaCl or 
dialyzed against water and lyophilized. 

Preparative Ion Exchange 

An anion exchanger, diethylaminoethyl (DEAE)- 
30 cellulose (Whatman), equilibrated with 6 M urea, 1 mM 
PMSF, 1 mM NEM, 20 mM Na phosphate (pH 7.0), and 50 mM 
NaCl was used in the first step of the ion exchange 



0121976 

purification of the extract. The soluble extract or 
lyophilized extract was equilibrated or dissolved in 
DEAE-cellulose equilibration buffer and mixed with the 
exchanger and stirred gently over a period of one or two 
5 hr. The unadsorbed materials were further fractionated 
by cation exchange chromatography using carboxymethyl 
(CM)-cellulose, which had been equilibrated with a 6 M 
urea, 10 mM NaCl, 1 mM PMSF, 1 mM NEM, 50 mM Na acetate, 
pH 4.8. The unadsorbed material was mixed with the OH- 
IO cellulose and the slurry was used to pack a 50 cc column. 
The column was washed with 10 mM NaCl in the above 
buffer. Adsorbed proteins were eluted using a 50 mM to 
400 mM NaCl gradient in the same buffer. Fractions were 
collected and then combined based on their adsorbance and 
15 electrophoretic profiles. Optical density (OD) readings 
at 280 nm of the fractions were made. Figure 1 is a plot 
of those readings. Fractions 23-32 were combined, 
dialyzed against water and lyophilized. 

Gel Filtration 

20 The combined fraction was then fractionated on 

a Sephacryl S-200 column equilibrated in 6 M urea, 10 mM 
NaCl, 10 mM EDTA, pH 7 (alternatively, 4 M guanidine, 
0.02% NaN3f 10 mM EDTA, pH 6.8 may be used). OD readings 
at 280 nm of the fractions were made. Figure 2 is a plot 

25 of those readings. Fractions 45-53 were combined and 
constitute the S-200 low molecular weight (_30K dalton) 
protein fraction. This fraction was dialyzed against 
0.05 N NH3HCO3 and lyophilized. 

The osteoinductive activities of various 

30 fractions reported in the above example were assayed by 
their ability to cause rat muscle fibroblasts in agarose 
to synthesize type II collagen and cartilage 



-10 



0121976 



proteoglycans. The materials and methods used in the 
assay were as follows: 

Muscle tissue was aseptically dissected from 
the upper limbs of 19 day old Sprague Dawley rat fetuses. 
5 The tissues were minced and cultured in Eagle's minimum 
essential medium (MEM) with 10% fetal calf serum and 50 D 
penicillin, 50 g streptomycin/ml media. Cellular 
outgrowth usually reached confluency within one week. 
Cells were trypsinized at confluency, split 1:2 and used 

10 for experimentaion within the first three passages. 

Cells were embedded in agarose gels by the 
method described in Cell (1982) 30:215-224. Briefly, 1.0 
ml of 1% high melting agarose (Bio-Rad, #162-0100) was 
added to each 35 mm well and allowed to gel on a level 

15 surface at room temperature. Low melting agarose, 2%, 
(Bio-Rad, #162-0017) equilibrated to SS'^C was mixed with 
an equal volume of 2 X concentrated Ham's F-12 nutrient 
mixture and held at 38^. The agarose/F-12 solution was 
then mixed with an equal volume of cell suspension and 

20 1.5 ml was added to each well. When firm, gels were 
overlaid with 3.0 ml of media, cultured at 37**C in 5% 
C02' 95% air and fed twice weekly. Media for cell 
suspension and feeding consisted of Ham's F-12 with 10% 
heat-inactivated fetal calf serum, 0.5% chick embryo 

25 extract (optional) penicillin/streptomycin and 

suppleAented with the test fraction. Agarose solutions 
were sterilized by autoclaving at 121**C for 15 min prior 
to use. The final cell concentration was 3 x 10^ 
cells/35 mm well. Cultures were done in duplicate. 

30 After 14-21 days of culture, gels were fixed 

and stained according to the method described in 
J Cell Biol (1970) 45:434-438. Gels were briefly rinsed 
two times in phosphate buffered saline (PBS), fixed in 
40% formalin, rinsed with distilled water stained with 



-II- 



0121976 



0.5% toluidine blue in 25% acetone and washed overnight 
with distilled water. 

After fixation, a portion of each gel was 
removed for histological processing. Gels were 
5 dehydrated, cleared and embedded in paraffin. Four to 
six Vm sections were taken perpendicular and parallel to 
the surface of the gel. Sections were stained with 
toluidine blue or safranin O. Proteoglycan matrix 
deposition was determined by metachromatic staining 

10 (purple with toluidine blue, orange with safranin O). 

Proteoglycans and collagen were extracted from 
the gels treated with extract for 3, 7, 14 days by 
homogenization in 2 volumes of 6 M guanidine-HCl, 0.075 M 
sodium acetate, 20 mM EDTA, 2 mM PMSF, 10 mM NEM, pH 5.8 

15 followed by shaking at 4*C for 18 hr. Agarose was 
removed by centr if ugation (15K rpm, 30 min). The 
supernatant was then dialyzed against 10 volumes of water 
at 4®C containing 0.2 mM PMSF (final guanidine HCl 
concentration '^0.05 M). Samples were lyophilized and 

20 resuspended in 1/10 volume water (final guanidine HCl 
concentration '^0.5 M). 

Proteoglycans and type II collagen were then 
measured by the enzyme-linked immunosorbent assay (ELISA) 
technique essentially as described by Rennard, et al, 

25 Anal Biochem (1980) 104:205-214. Antisera to cartilage 
proteoglycans and type II collagen were raised in rabbits 
using standard techniques. Both proteoglycan and type II 
collagen for immunization were isolated from Swarm rat 
chondrosarcomas. Antisera to type II collagen was 

30 affinity purified using a cyanogen bromide Sepharose 
column. 

For ELISA, microtiter wells were coated with 
200 yl of the appropriate antigen in 20 mM carbonate 
buffer, pH 9.6, at 4'*C overnight (100-1000 ng/well). 



- 12- 



0121976 



Wells were washed 3 times with 0.9% NaCl containing 0.05% 
Tween 20 surfactant to remove unbound antigen. 
Proteoglycans and type II collagen were measured by 
inhibition ELISA. Sample aliquots (10-100 yl) were 
5 incubated in PBS containing 1 mg/ml bovine serum albumin 
and 0.05% Tween 20 surfactant with the appropriate 
antisera (1:500 affinity purified type II or 1:2000 
proteoglycan) for 2 hr at room temperature. Two 
hundred yl of incubation mixture was then added to 

10 antigen-coated plates for 45-60 roin at room temperature. 
After washing 3 times with PBS-Tween 20 surfactant, goat 
anti-rabbit IgG conjugated to horseradish peroxidase 
(Tago, 1:200) was then added for one hr at room 
temperature. Unbound second antibody was then removed 

15 with PBS-Tween 20 surfactant and substrate 

(o-phenylenediamine, 1 mg/ml in 0.1 M citrate, pH 4.6) was 
added for 30-60 min. Reactions were stopped by addition 
of 50 yl 2 M sulfuric acid. Spectrophotometric readings 
(492 nm) of the reaction media were done and compared to 

20 standard curves to determine whether production of type 
II collagen and proteoglycan had been induced. The 
results of these assays (+ indicates induction, 
- indicates no induction) are tabulated below. 
Fraction Type II Collagen Proteoglycans 

25 Control - - ' 

Extract' + + 
DBAE adsorbed 

DEAE unadsorbed + + 

CH adsorbed + + 
30 CM unadsorbed 

Sephacryl S-200 

(-r30K dalton fraction) + + , 



Sephacryl S-200 

(>30K dalton fraction) 



0121976 



Claims 

1. A process for partially purifying an 
osteogenic factor from particulate demineralized bone or 
an osteosarcoma comprising extracting nonfibrous proteins 

5 from the demineralized bone or osteosarcoma with a liquid 
dissociative nonfibrous protein extractant and separating 
the osteogenic factor from the extract by ion exchange 
chromatography characterized in that: 

(a) the extract is first contacted with ah 
lOanion exchanger at a pH of about 6.8 to about 7.2; 

(b) the unadsorbed fraction from the anion 
exchanger is contacted with a cation exchanger at a pH of 
about 4.5 to about 5.2; 

(c) the adsorbed fraction from the cation 
15 exchanger is eluted; and 

d) nonfibrous protein having a molecular 
weight below about 30,000 daltons are isolated from the 
eluate. 

2. The process of claim 1 further 

20 characterized in that steps (a) through (c) are each 
carried out in the presence of a protease inhibitor. 

3. The process of claim 1 further 
charact^erized in that the anion exchanger and the cation 
exchanger are cellulosic ion exchangers. 

25 4. The process of claim 3 further 

characterized in that the anion exchanger is 
diethylaminoethyl cellulose and the cation exchanger is 
carboxymethyl cellulose; step (a) is carried out at a pH 
of about 7.0; and step (b) is carried out at a pH of 

30 about 4.8. 



13 



0121976 

5. The process of claim 1 further 
characterized in that step (d) is carried out by gel 
filtration. 

6. The process of claim 2 further 

5 characterized in that the extractant is removed from the 
extract, the nonfibrous proteins in the extracts are 
dissolved in 6 mH urea for contacting with the anion 
exchanger, the anion exchanger is diethylaminoethyl 
cellulose, the pH of step (a) is about 7.0, and cation 
iO exchanger is carboxymethyl cellulose, and the pH of step 
(c) is about 4.8« 

7. Partially purified osteogenic factor 
prepared by the process of claim 1. 

8. Partially "purified osteogenic factor 
15 present in vertebrate bone characterized in that it: 

(a) is nonfibrous; 

(b) is unadsorbed by diethylaminoethyl 
cellulose anion exchanger at a pH of about 7.0; 

(c) is adsorbed by carboxymethyl cellulose 
20 cation exchanger at a pH of about 4.8; and 

(d) has a molecular weight below about 30,000 

daltons. 



14 



0121976 



0.6- 




0 10 ZO 30 40 50 
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