Two naturally occurring peptides that induce chondrogenesis in culture have been purified to apparent homogeneity. These cartilage-inducing factors (CIF-A and CIF-B) were isolated from bovine demineralized bone by dissociative extraction, gel filtration, cation-exchange chromatography, and reversed-phase HPLC. CIF-A and CIF-B at concentrations of 1-10 ng/ml each induce embryonic rat mesenchymal cells in culture to assume a cartilage morphology and synthesize cartilage-specific proteoglycan and type II collagen. The amino acid compositions of CIF-A and CIF-B are similar but not identical. Both factors have an apparent Mr of 26,000, as determined by NaDodSO4/PAGE. In the presence of 2-mercaptoethanol, both are converted to species of about one-half that Mr, indicating that they are dimers of identical or very similar chains.When fragments of demineralized bone are implanted subcutaneously or intramuscularly, they elicit a biological response that has all the elements of endochondral bone formation (1). These include the attraction, proliferation, and subsequent induction of mesenchymal cells into cartilage cells and matrix which are then replaced by bone (2). Similar findings have been obtained with a powder prepared from a 4 M guanidine HCl (Gdn HCl) extract of demineralized bone particles, suggesting that the response involves a hormonelike factor or group of factors (3, 4).The early chondrogenic induction and cartilage formation of this response can be elicited in vitro (5). By using an ELISA for cartilage markers, the chondrogenic activity of soluble factors can be assayed in a rapid, highly sensitive, and quantitative manner (4). Treatment of embryonic rat mesenchymal cells, isolated from muscle and embedded in agarose gel, with an extract from demineralized bone powder induces the synthesis of cartilage-specific proteoglycan and type II collagen. Using this assay, we have isolated, purified, and characterized two bovine demineralized-bone proteins that are potent inducers of chondrogenesis in vitro and that may be involved in the osteogenic response to demineralized bone in vivo. MATERIALS AND METHODSPreparation of Demineralized Bone Powder. Bovine metatarsal bone was obtained fresh from the slaughterhouse and transported on dry ice. Bones were cleaned of all marrow and periosteum, broken into fragments, and pulverized in a liquid-nitrogen-cooled mill. The pulverized bone was washed twice for 15 min with deionized water (10 ml/g). The bone was then washed overnight with the same volume of 0.01 M HCl at 4°C and defatted as described elsewhere (4). The bone powder was demineralized for 16 hr in 0.5 M HCl (25 ml/g) at 40C. The acid was decanted, and the demineralized bone powder was washed several times with cold deionized water until the wash reached a pH >4. The excess water was removed on a suction filter.Extraction of Chondrogenic Factors. Demineralized bone powder was extracted with 4 M Gdn HCl/1 mM N-ethylmaleimide/10 mM EDTA, pH 6.8, (3.3 ml/g) for 16 hr at 40C.The suspension was suction-filtered...

UCOE vectors contain non-tissue specific chromatin-opening-elements that permit rapid expression of a protein in anintegration independent manner. Efficient expression can bederived from a single copy of an integrated gene site resulting ina higher percentage of cells expressing the marker gene in theselected pool in comparison to standard non-UCOE containingvectors. This, in combination with the utilization of a serum-free, suspension adapted parent cell line allows for rapidproduction of large quantities of protein in a short period oftime. Utilizing this system more than 300 mg of a recombinantantibody has been produced in less than 1 month from transfectionpools in shake flask. Selected subclones have been scaled intosmall bioreactors in less than 2 months, producing significantquantities of monoclonal antibody using a protocol generic for theparent cell line. The increased efficiency obtained with the UCOEvector reduces the number of transfectants which need to bescreened in order to obtain high productivity subclones.Transfection of a standard host cell line, preadapted to grow in alarge-scale setting, allows for rapid cell line developmentdecreasing the transition time from research into development andmanufacturing. Alternatively, the traditional approach of using aparent cell line which requires serum-free and suspensionadaptation after transfection further increases the need forscreening a large number of subclones, because many of thesubclones will not be able to grow under conditions that allowlarge-scale protein production. The use of a preadapted cell linecan reduce the time required to develop a cell line from months toweeks.