JPS62255429A - Partially purified bone induction factor - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] 3. Amount of FI, small amount'■ (Industrial application field) FIELD OF THE INVENTION This invention relates to protein chemistry and bone formation.

In particular, it relates to a partially purified proteinaceous factor that promotes rapid bone growth.

BACKGROUND OF THE INVENTION It has been established that bone contains substances that can stimulate new tube formation when placed in contact with living systems (Urist,
M, I? ,, Cl1n 0rtho (
1968) 56: 37;5science (
1965) 150: 893: Reddi,
A, )1. , etal, Proc Natl
Acad Sci (USA) (1972) 6
9:1601).

Attempts have been made to purify whatever factor is responsible for this activity. Urist U.S. Patent No. 4.29
According to the disclosures of No. 4,753 and No. 4,455,256,
“Bovine morphogenetic protein (BMP) was extracted from demineralized bone using urea or guanidine hydrochloride and reprecipitated. ,
reported that an activity that is not adsorbed to carboxymethyl cellulose (CMC) at pH 4.8 occurs (lJris
L.

M, R,, Cl1n 0rtho Rel Res
(19B2) 162: 219).

Urist's latest report (Science (1983)
) 220: 680-685 and Pro
c Natl Acad 5science (
USA) (1984) 81: 371-375)
has a molecular weight of 17,500 Daltons and 18.500 Daltons.
Dalton's Bl'IP has been described.

U.S. Patent No. 4,434,094 describes the extraction of bone-derived proteins that apparently stimulate bone development by extraction with a chaotropic agent, separation on anion- and cation-exchange columns, and CMC at pH 4.8. By recovery of the activity from the adsorbed fraction.

It is reported that partial purification will be carried out. This new protein fraction was named osteogenic factor J (OF) and was characterized by having a molecular weight of less than about 30,000 Daltons and undergoing the purification steps described above.

Two proteins were purified to homogeneity from this protein. These two proteins are approximately 150 to 2
It is eluted from the CMC resin with a 00 mM NaCltJI gradient and has a molecular weight of approximately 26,000 Daltons. These proteins are co-owned European Patent Application Publication No. 85
-304348~6 (Published on January 22, 1986)
It is described in. Although these proteins are thought to be involved in bone formation, they do not exhibit osteogenic activity by themselves.

The present application is present in the above CMC conjugate fraction and approximately 150 m
Elute from the resin with a NaCltl gradient below M. It is related to osteoinductive factors.

(Problems to be Solved by the Invention) The present invention provides partially purified bone i from demineralized bone (DMB).
A method for obtaining the rn'fL factor, and the factor prepared thereby.

The present invention relates to a graft material containing the factor and a method of inducing bone formation in a mammal using the factor.

(Means for solving the problems) The method for preparing a partially purified inducer of the present invention includes the following steps:
(a) extracting demineralized bone using a dissociative extractant that solubilizes non-fibrous proteins; , collecting a fraction containing proteins with a molecular weight of 10,000 to 30,000, (C) step fb
The active protein fraction was adsorbed onto a carboxymethyl cellulose cation exchanger at a pH of around 4.5 to 5.5, and the active protein fraction was eluted from the cation exchanger with a sodium chloride concentration gradient. The active protein fraction is eluted with a concentration gradient from about 10 mM to about 150 mM in the presence of a nonionic chaotropic agent.

The partially purified inducer of the present invention is characterized by being prepared by the above method.

The osteogenic graft material of the present invention is characterized in that its active ingredient is the above partially purified inducer.

The method of the present invention for inducing bone formation in a predetermined site of a living mammal includes injecting an osteogenic material into the site, and the method includes injecting an osteogenic material into the site, and the method includes injecting an osteogenic material into the site, and the method includes injecting an osteogenic material into the site, and the method includes injecting an osteogenic material into the site, and the method includes injecting an osteogenic material into the site. The natural source of the osteoinductive factor of the claimed invention is bone, dentin, osteosarcoma, or chondrosarcoma. The claim that human, monkey, bovine and rat bone-attracting π-proteins are non-species specific in their ability to form endochondral bone within xenografts (Sampatli).

T., K., et al., Proc Natl A
cad Sci (USA) (1983)'80:
6591), it is believed that the factors of the claimed invention have been highly conserved among mammalian species (i.e., factors from different mammalian species have substantially homologous amino acid sequences). However, it may be altered by the addition, deletion, or substitution of one or more amino acid residues in place, without adversely affecting the non-species-specific osteoinductive activity of the molecule. Will).

In this regard, as used herein, the terms "substantially equivalent" and "substantially homologous" refer to the terms "substantially equivalent" and "substantially homologous," regardless of the species, in which the factors have the same amino acid composition as the factors described in the Examples. 2. If the amino acid composition or sequence is similar but different, and the difference does not adversely affect the non-species-specific endochondral osteoinductive activity.

shall mean. Accordingly, such factors can be obtained from cells or tissues derived from various mammals. As a source of factors prepared by purification from natural sources, porcine or bovine long tubes are convenient because they are readily available.

Various firsts! The I11-wing method is possible, but basically.

The bone is first cleaned by mechanical or abrasive techniques, crushed and further washed, preferably with a dilute aqueous acid solution, preferably at low temperature, and then defatted by extraction with a lipophilic solvent such as ether or ethyl acetate. The bone is then denuded to remove various forms of calcium phosphate, usually by extraction with stronger acids. These techniques are understood in the art3 and are described, for example, in U.S. Pat.
, 434,094. The resulting preparation, demineralized bone, is the starting material for the claimed osteogenic factor preparation.

The initial extraction removes non-fibrous (ie, non-collagenous) proteins from demineralized bone. This is guanidine hydrochloride (about 4 moles or more), urea (8 moles)
Plus salt or sodium dodecyl sulfate (about 1
% or more) or other chaotropic agents as known in the art (Termin
e, et al, JRiot Che+n (19
80) 255:9760-9772: and 5age
raand Hascall, J Biol C.
hem (1969) 244 near 7-87and
2384-2396).

Extraction is preferably carried out at a lower temperature in the presence of a protease inhibitor to reduce the possibility that the extracted protein will be degraded or denatured. Examples of protease inhibitors include:

Phenylmethylsulfonyl fluoride (I'MSF)
Sodium azide, N-ethylmaleimide (NEM)
.

benzamidine, and 6-aminohexanoic acid.

The pH of the medium depends on the extractant selected. The extraction process usually lasts on the order of about 4 hours to 1 day.

After extraction, the extractant may be removed by suitable means such as dialysis against water, optionally preceded by concentration by ultrafiltration. Salt too. Removal may be performed using controlled electrophoretic single molecule sieving, or any other means known in the art. It is also preferred to maintain low temperatures during this step to minimize protein denaturation. Alternatively, the extractant chaotropic agent need not be removed; rather, the solution may simply be concentrated, for example by ultrafiltration.

Extracts dissolved or redissolved in chaotropic agents are
Purity is increased by gel filtration to obtain fractions with molecular weights less than about 30,000 Daltons. Gel sizing is performed using standard techniques, but is preferably carried out on a Sephacryl column at room temperature (10 to 25°C).

The low molecular weight fraction is then subjected to ion exchange chromatography using a CMC with a pll of about 4.5 to 5.2, preferably about 4.8, in the presence of a non-ionic chaotropic agent such as 6M urea. . Other cation exchangers may be used, including those derived from polyacrylamide and cross-linked dextran. However, cellulose cation exchangers are preferred. As in any ion exchange operation, it is of course necessary to remove competing ions from the solution before applying it to the column. The factor is adsorbed onto the column and eluted using a salt gradient ranging from about 10 mM to about 150 mM.

10mM to 150mM Na on cation exchange column
To further purify the Cl fraction, R['-+1PL
c or may be subjected to constant gel electrophoresis.

The presence of the factor in the 10mM to 150mM NaCl fraction is confirmed by an in vivo osteoinduction assay detailed below.

(Example) The following example shows an example of a purification method applied to a specific sample, and the present invention is not limited thereto.

A. Preparation of self-isolated tubes 1 Fresh bovine metatarsal bones were newly obtained from a slaughterhouse and transported on dry ice. The bone is cleaned to remove bone marrow and non-skeletal tissue and broken down into pieces smaller than 1 cm in diameter.

Then, it was crushed in a mill at 4°C. The crushed bones were washed twice with 9.477 g/kg of bone for approximately 15 minutes each and then with 0.0 IN IIcI overnight at 4°C. Wash the washed bones 3 x 3 volumes of ethanol.

Subsequently, it was degreased using 3×3 volumes of diethyl ether. Each wash was performed for 20 minutes, all at room temperature.

The obtained defatted bone powder was then diluted with 0.5N llCl (25
It/kg defatted bone) was demineralized at 4°C.

The acid was decanted and the resulting DMB was washed until the wash pH was above 4, and the DMB was dried by suction filtration.

B. Extraction of non-collagen proteins The DMB prepared in section A was added at a concentration of 3.31 4/kg
M guanidine hydrochloride, 10mM ethylenediaminetetraacetic acid (EDTA) (pH 6,8), 1mM
Extracted with PMSF and 10mM NEM for 16 hours.

Suction filter the suspended liquid. The insoluble material was then extracted again for 4 hours. The soluble fractions were combined and subjected to Amicon ultrafiltration (I
The concentrate was dialyzed over 4 days against 6 changes of 35 volumes of chilled deionized water and then lyophilized.

All operations in this section were performed at 4°C. However, the freeze-drying was carried out under standard freeze-drying conditions.The extract of Section B was redissolved in 4M guanidine hydrochloride.

4M guanidine hydrochloride, 0.02% sodium azide.

and 10 mM EDTA (pH (6,8)). The fractions were analyzed by absorbance at 280 nm and one fraction was combined as shown in Figure 1. Fraction F2, which constitutes the low molecular weight (LMW, 10.000 to 30.000 daltons) protein fraction with maximum activity, was dialyzed against 6 changes of 180 volumes of deionized water and frozen. All operations except lyophilization and dialysis (4°C) were performed at room temperature.

D, fraction F2 of ion exchange chromatography section 0, 6M urea, 10mM NaCl
, 1mM NEM.

and 501 dissolved in sodium acetate (pH 4,8),
Centrifugation was performed at 10,000 rpm for 5 minutes. The supernatant was transferred to C+'+52 (commercially available CM
C) Separated by column. The binding protein was dissolved in the same buffer from 10mM to 400mM NaCl? Using H degree gradient, total volume 350ml, flow rate 2” 1ml/
eluted from the column in time. (Three major fractions, named Jl-1, CM-2 and CL3, were collected as shown in Figure 2. Each fraction was dialyzed for 4 days against 6 changes of 110 volumes of deionized water. It was lyophilized. All the above operations were performed at room temperature except for dialysis (4°C).

E, RP-IPLC The freeze-dried fractions CM-2 and CM-3 of section D were each dissolved in 0.1% trifluoroacetic acid (TFΔ), and a portion of the solution was applied to a Vydac C18RP-11 PLC column (4
, 6ur ID X25cIn) and 0.1
1mj using %TFA? /min for 5 minutes. The elution solvent was a concentration gradient of 0% to 60% acetonitrile in 0.1% TFA.

The speed was 2%/min. Two peaks were obtained: peak A at about 29.5 minutes and peak B at about 31.3 minutes.

The presence of desired proteins in the fractions during purification was determined in vitro for proteoglycan (PG) production.

Confirmed by test. Identification was done by ELISA (enz
yme-1 inked immunosorbent
(assay).

The assay is based on an agarose gel culture model using fibula muscle cells isolated from rat fetuses. It tests the ability of a sample to induce cartilage-specific PG production. Dan vitr.

The correlation between cartilage induction and bone formation of the U.
Din.

S, et al, J Cell Biol (1
983) 97: 1950-1953.

Muscle tissue was aseptically removed from the upper limbs of 19-day-old Sprague Dawley rat fetuses, minced tissue was minced, and it was treated with Eagle's minimum supplemented with 10% fetal bovine serum (FBS) and 50 units/ffl1 penicillin or 50 μg/thl streptomycin. Essential medium (M
E) A cell culture was prepared by culturing in 1). Cells proliferated and reached confluence within 2 usually one week. Therefore, cells were trypsinized, split 1:2 and used for experiments within the first three passages.

Cells were placed in agarose gel cultures containing either control media or test samples. The procedure is basically Beny
a, et al, Ce1l (1982) 30
: According to 215. Briefly, monolayers of cells were obtained by trypsinization, counted on a hemocytometer, and resuspended at twice the final cell concentration in medium with or without the protein fraction to be tested. The control medium is Hamus F12/
Dulbecco's minimal essential medium (DMIEM) or CMR
L 1066 (manufactured by Gypco), each containing 10% FBS and antibiotics. 0. The test protein fraction in OIN HCL was diluted directly with 1% low melting point agarose (Bio-Rad, #162-0017) in F12 to give the desired concentration of test protein diluted in 8 equal volumes of 1.

And 0.15m of diluted liquid 0.2m72 7! 1% high melting point agarose (manufactured by BioRa 7D, #162)
-0100) into the 17th dragon well. The resulting culture was incubated at 37°C for 5 minutes and cooled at 4°C for 10 minutes.

Then 1 mff of the corresponding medium (control or test protein) was overlaid. Cells were then exposed to 5% COz, 95
% air, followed by complete replacement with control medium every 3-4 days. After 7 days, the culture was frozen and kept at -80°C before assay.
I stored it.

Thaw the culture at 4°C and add 50mM sodium acetate.

13mM EDTA, 6mM NEM and 3mM
Homogenized in 4M guanidine hydrochloride containing MPMSF at pH 5.8.

and by stirring and extracting overnight at 4 °C.

Culture assays were performed. 25. OOOXg, 11″
Centrifuge for 400 minutes at
, 2M NaCl.

Dialysis was performed overnight at 4°C against 50mM Tris (pH 7,4).

The supernatant was purified by Leonard, et al.
Proteoglycans were assayed by ELrSA as described in Biochem (1980) 10 Rainbow: 205 and US Patent No. 4,434,094.

Briefly, for [:LISA, antiserum against cartilage PG was raised in rabbits using standard techniques.

This is hyaluronic acid or PG extracted from rat bone.
showed no cross-reactivity. Purified proteoglycan from Swarm rat chondrosarcoma tissue (Seyedin+
S., et al., supra) was used as a standard antigen. The dialyzed sample was treated with 0.05% Tween 20 and 1/m7! 1 in phosphate buffered saline (PBS) (pH 7,2) containing bovine serum albumin (BSA):
It was diluted to 1 (v/v) and used for assay. Goat anti-rabbit IgG conjugated to horseradish peroxidase
(manufactured by Tabo) was used as a secondary antibody using 0-phenylenediamine as a substrate.

Three CM-bound fractions (CMC-8-1, CMC-
8-2° and CMC-8-3), and the protein of peak A in term E (named CIF-A).

Shown in FIGS. 3(a) and (bl).

For reconstitution, the protein prepared by the above method is
Bone collagen powder (BCP, freeze-dried solid in Section C)
and Collagen in Solution (CIS, registered trademark ZyG from Collagen Coborection, Palo Aldo, California)
(available from EN A) in a weight ratio of 9:1. CIS contains 10% natural soluble bovine skin collagen in proportions according to its in vitro chondrogenic activity. Figure 4 schematically shows the procedure.
The details of the composition of the reconstructed (1υ material) are shown in the table of FIG. 5. According to this method.

All reconstituted samples had approximately the same number of units of chondrogenic activity (approximately 1000 units/100 R-DBP). Double the dose for CIF-A only.

CMC-8-1 was also reconstituted at two different dosages.

The protein content of CMC-8-1 was determined by biuret analysis and H at 220 nm for all other samples.
OE was determined by integrating the HPLC peak in comparison to the peak of a known bovine serum albumin standard. For pure CIF-A and CIF-B, H
The r'LC fraction was added directly to the crs solution and then mixed with BCP. A control sample consisting of BCP/CIS carrier was also prepared under the same conditions.

The osteoinductive capacity of H. paioafuseini samples was assayed by its ability to induce endochondral bone formation in the subcutaneous tissue of male and female Sprague-Dawley rats. The sample was mixed with 2 volumes of double-distilled sterile water (ν
/v4''), mix thoroughly and fill into an LCC syringe.

It was cut and weighed. All samples were implanted into the ventral thorax on each side of the animal. Explants were removed after 14 and 28 days and biochemically and histologically evaluated.

knee JIV costume Explants were removed after 14 and 28 days.

The cells were fixed in 10% neutral formalin for 26 hours and then embedded in paraffin for histological evaluation.

Four to six micron sections were made from the embedded tissue and treated with hematoxylin-eosin (-Melon Cytology).

Sequential staining was performed with either safronin (proteoglycan) and Gomori trichrome (collagen).

Length - Raw■Ne Hinoki Sada The 14-day rod explant tissue was divided into two parts and the wet weight was measured.

Freeze at -80°C until processing. Samples were first extracted and assayed for alkaline phosphatase activity and subsequently extracted and assayed for cartilage-specific proteoglycans. Right armpit 28-day rod explants were extracted and assayed first for alkaline phosphatase and then for calcium. The extraction and testing procedures are described below.

J, 1. Proteoglycan A~ Cartilage proteoglycans were assayed by ELISA technique. The weight of the explanted tissue was measured immediately after removal.

Freeze at -70°C until extraction. As for extraction.

Thinly slice the explanted tissue in water-cold extraction buffer.

Homogenize twice for 30 seconds on the Tickmartis Schmeiser at the highest setting. The extraction buffer is 6M guanidine hydrochloride/75mM sodium acetate or 4M guanidine hydrochloride 150mM acetate, in each case containing 20n+M EDTA, 1mM PMSF and 10n+MNEM at pH 5.8. The buffer solution was used at a volume of 10:1 relative to the weight of the extracted explant tissue. The samples were then incubated overnight (20 hours) at 4°C. Sample next 12. OOOrpm, 4℃
Centrifuge for 1 hour at
Dialysis was performed overnight at 4°C against Tris, 200mM NaCl, (pH 7,4). For dialysis night, polystyrene microbrate (Flow Laboratory Lease,
Leonard,
et al, ArchBiochem 111-j2, 311-1)-(Kouni 1(-; Ni (1980) 2
07 2399 and 5eyedin, S.

et al., J Ceil Biol (1983)
97: EL as described in 1950.
I did an ISA. 5-eye mouth, S.

Antisera and proteoglycans were prepared from Swarm rat chondrosarcoma tissue as described in et al. (supra). Goat anti-rabbit IgG conjugated to horseradish peroxidase was used as the secondary antibody. The sample was 0.05% Tween 20 and 1rag/ll1
11n Chem
(1977) 81:1.

Mumu 1ken Uυ and Otsuchimu Bone formation was also assessed by calcium measurements.

The explanted tissue was cut into small pieces at 1:10 (m/
ν) in 0.5N+1CI to dissolve the ions. The sample was incubated for an additional 5 days at room temperature and 12
,000 rpm+ for 40 minutes. Calcium concentration in supernatant was determined by atomic adsorption (trace analysis laboratory).

Hayward, California).

Mumue dari uni” sphatase anal For alkaline phosphatase (AP) measurements.

Cut the explanted tissue into small pieces and add 10 volumes ffl (1/1
0) water-cooled 1.5M NaCl, 3mM Na1lCO
3, pH 7.5. The homogenized sample was then centrifuged at 12,000 rpm + 4°C for 50 min, and a portion of the supernatant was diluted 1:10 with cold distilled water. Iluggjns, et al.

Alkaline phosphatase was evaluated using polystyrene plates according to the method of L. L. (1961).

K9Mi! The table below summarizes some of the results of the 8 trials and the biochemical framework. The table is.

Figure 2 summarizes the histology and alkaline phosphatase activity of BCP/CSI combined with CMC fractionation.

From the above data, it appears that CMC-8-1 protein enhances canal m associated with CMC binding (total binding fraction), as reflected by the rate, quantity and quality of bone formation. I understand that. These experiments show that endochondral bone formation is influenced by the purity of the tube pile guide material and the protein distribution therein. These experiments further
Two proteins identified in European patent application 85.304848.6 function in the differentiation of cells involved in bone formation.

It also suggests the possibility of influencing the rate and relative amount of cartilage and bone formation.

Histological and biochemical data of 14-day and 28-day explants showed that reconstituted total CMC conjugates (total proteins bound to CMC) and CMC-8-1 was found to induce cartilage and bone formation in all implants. On the 14th day, cartilage formation was always higher in the R-CMCMC grafts and evenly distributed throughout the grafts.

Some new bone had formed around it. On the other hand, in R-CMC-B-1, the 14-day lily had only a small amount of cartilage, and many bones were already visible. On the 28th day, l? - The explant of the CMC conjugate still had cartilage. (The amount of cartilage and bone seemed to be about the same.) R-CMC-8-1, on the other hand, contained only traces of cartilage and well-developed bone with a fat-filled marrow cavity. there was. All of the latter explants appeared to be larger than one normally provided. Histological observations were confirmed by biochemical data. Both materials exhibited high levels of alkaline phosphatase activity. The calcium content at day 28 was very high for both materials (approximately 32 mg Ca/g wet tissue for R-CMC conjugates;
-8-1, it was about 39 mgCa/g wet tissue).

The claimed osteogenic materials can be used as active ingredients in osteogenic graft compositions for the repair, replacement, or augmentation of bone tissue in living mammals, including humans. An osteogenicly effective amount of the material will normally be formulated with a pharmacologically and physiologically acceptable solid carrier such as a BCP for implantation.

The weight ratio of active protein to carrier is typically 1
;50 to 1:1000. The graft is placed in place in the patient using conventional surgical techniques.

The claimed factors are also: It may also be useful for systemically treating bone defects such as osteoporosis and osteoporosis. For such treatments, a therapeutically effective amount of the protein is formulated with an injectable carrier and administered to the patient parenterally.

SUMMARY OF THE INVENTION A proteinaceous partially purified osteoinductive factor of 10,000 to 30,000 daltons is described. It is obtained from demineralized bovine bone and extracted with a chaotropic agent.

Gel filtration, cation exchange chromatography using carboxymethyl cellulose at pH 4.8, and NaCl from about 10 mM to about 150 mM. Obtained by gradient elution.

4. '' U on the surface Figure 1 shows the optical density (
It is a graph of absorbance (at 280 nm).

Figure 2 shows the optical density (280 nm
).

FIGS. 3(a) and 3(b) are graphs showing specific chondrogenic activity measured for proteins with term F described in the Examples.

FIG. 4 is a schematic diagram showing the reconfiguration procedure described in Example 0.

FIG. 5 is a table showing the reconstituted samples described in Section G and their specific chondrogenic activity determined by the assay in Section F.

that's all

Claims (1)

[Claims] 1. A method for preparing a partially purified osteoinductive factor, comprising the following steps: (a) treating demineralized bone with a chaotropic extractant that solubilizes non-fibrous proteins; b) subjecting the extract of step (a) to gel filtration to collect a fraction containing proteins with a molecular weight of 10,000 to 30,000; (c) converting the fraction of step (b) to a protein with a molecular weight of 4.5 to 5; p of about .5
(d) eluting the active protein fraction from the cation exchanger with a sodium chloride gradient; A method characterized by elution with a concentration gradient from about 10 mM to about 150 mM in the presence of an ionic chaotropic agent. 2. Partially purified osteoinductive factor, comprising the following steps: (a)
treating the demineralized bone with a chaotropic extractant that solubilizes non-fibrous proteins; (b) subjecting the extract of step (a) to gel filtration to obtain fractions containing proteins with molecular weights between 10,000 and 30,000; (c) collecting the fraction of step (b) with a p of about 4.5 to 5.5;
(d) eluting the active protein fraction from the cation exchanger with a sodium chloride gradient; A partially purified osteoinductive factor prepared by a method characterized by elution with a concentration gradient from about 10 mM to about 150 mM in the presence of an ionic chaotropic agent. 3. An osteogenic graft material, the active ingredient of which comprises the steps of: (a) treating demineralized bone with a chaotropic extractant that solubilizes non-fibrous proteins; (b) step (a) ) is subjected to gel filtration to collect a fraction containing proteins with a molecular weight of 10,000 to 30,000. (c) The fraction in step (b) has a p of about 4.5 to 5.5.
(d) eluting the active protein fraction from the cation exchanger with a sodium chloride gradient; An osteogenic graft material that is a partially purified osteoinductive factor prepared by a method characterized by elution in a concentration gradient from about 10 mM to about 150 mM in the presence of an ionic chaotropic agent. 4. A method for inducing bone formation in a predetermined site of a living mammal, the method comprising implanting an osteogenicly active material in the site, the material comprising: (a) infusing non-fibrous proteins; (b) subjecting the extract of step (a) to gel filtration to recover a fraction containing proteins with a molecular weight of 10,000 to 30,000; . (c) The fraction in step (b) has a p of about 4.5 to 5.5.
(d) eluting the active protein fraction from the cation exchanger with a sodium chloride gradient; A partially purified osteoinductive factor prepared by a method characterized in that it is eluted with a concentration gradient from about 10 mM to about 150 mM in the presence of an ionic chaotropic agent.