Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Separation or purification
Reexamination Certificate
1998-09-15
2001-01-23
Woodward, Michael P. (Department: 1654)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Separation or purification
C530S399000, C530S418000, C530S419000, C424S198100
Reexamination Certificate
active
06177550
ABSTRACT:
BRIEF SUMMARY OF THE INVENTION
The process of the invention for the extraction of a Growth Factor Complex is characterized in that water is added to natural and growth factors containing material, the pH value is adjusted to 2.5 to 3.2 by the addition of acid, the resulting precipitate is separated from the supernatant, the Growth Factor Complex in the aqueous solution is precipitated by the addition of an organic solvent miscible with water, separated from the liquid phase and dried and the powder obtained is, if desired, dialyzed.
BACKGROUND OF THE INVENTION
Growth factors are polypetides of particular interest since they possess specific biological properties. Because of their special properties, the growth factors per se and in particular in combination are promising compounds of medicaments and cosmetics. Until now, the extraction of growth factors was a very time- and cost-consuming process which yielded predominantly single growth factors.
According to patent specification EP 313 515, a process for the isolation of the Milk Growth Factor (MGF) is described, where the starting material is subject to one or more chromatographic techniques and, if required, to further purification steps. With this extensive extraction process, a single growth factor is obtained from milk. WO 95/29933 describes the manufacture of several growth factors from milk. For this laborious process, pasteurized whey which has been submitted to micro-filtration is used as starting material (details are described in Australian patent specification No. 645589). The protein containing material is then applied to a cation exchange resin, equilibrated with 50 mM sodium citrate buffer pH 6.5. After a wash procedure with the same buffer, the protein is eluted with 0.4 M NaCl, diafiltrated against water, concentrated by means of ultrafiltration and lyophilized. Further purification steps yield fractions which contain 10 mg lactalbumin per ml (the fractions each consist of 100 &mgr;l). The patent specification WO 95/26984 describes the manufacture of an insulin-like growth factor from cows milk by cation exchange chromatography and subsequent dialysis. JP 6279312 also describes the manufacture of an insulin-like growth factor by heating of milk or whey to a specific temperature, centrifuging and passing the supernatant through an 8 kDa ultrafilter.
In contrast to the prior art the present invention discloses a simplified and cheap process with a high yield: From 1 kg commercial milk powder, 350 g to 420 g of carbohydrates, in which the active Growth Factor Complex is contained, are isolated. This complex consists—as already mentioned hereinabove to the greater part of lactose. Using cow's milk as starting material, the protein proportion is about 5%. If desired, most of the lactose may be removed by dialysis. For therapeutic purposes, however, undialyzed lactose-containing material is advantageous since a correct and reproducible dosage of the biologically highly active factors can in this manner be managed considerably simpler.
The action of individual milk growth factors has been investigated in detail during the last 15 years. It has been shown inter alia that the biologically active polypeptides have physiological functions such as regulation, stimulation or inhibition of various cell functions (Am. J. Med. Sci., 1991, 301, 2, 124-132). Pittelkow M. R. (Advances in Dermatology, 1991, 7, 55-81) could demonstrate that some milk peptides act as natural mediators of cellular processes which are important for the maintenance of the dermis structure.
The biological activity of growth factors may be determined with the aid of the migration test and/or proliferation test. The migration test is performed according to B{umlaut over (u)}rk R. R. (Proc. Nat. Acad. Sci., 1973, 70, 2, 369-372): After cells (e.g. Balb/C-3T3-fibroblasts) have grown to a confluent monolayer, a 10 mm wide area is removed with a razor blade. By addition of milk growth factors the cell migration is increased as compared to the control. The proliferation test, also carried out on the Balb/C-3T3-fibroblasts revealed an increased cell growth upon treatment of fibroblasts with milk growth factors.
In the prior art, the use of single factors or a combination of two growth factors have been described. WO 95/29933 describes a growth factor abbreviated as GFE-2 that is produced from milk. This growth factor is used for wound treatment as well as for gastrointestinal disturbances, illnesses or ulcers. EP 367 447 describes the use of the Human Cell Transforming Growth Factor for growth stimulation of epithelial cells e.g. for treatment of wounds, ulcers, burns or for reepithelization after radiotherapy. FR 2472385 and FR 2533438 describe the cosmetic use of various isolated growth factors. JP 6040858 uses the Fibroblast Growth Factor in a hair tonic for prevention and treatment of alopecia. EP 313 515 describes the use of a milk growth factor in tooth paste, mouthwashes, cosmetic and nutritional preparations.
The present invention contains a natural set of growth factors that may act in concert. As disclosed in EP 313 515, various growth factors act synergistically when combined. In the patent specification mentioned, a milk growth factor is combined with the Epidermal Growth Factor (EGF) or the Transforming Growth Factor &agr; (TGF-&agr;) in order to achieve a better wound healing. The present invention has the advantage that the end product already contains of a combination of various growth factors.
DETAILED DESCRIPTION OF THE INVENTION
Natural and growth factors containing materials (in the following termed “starting materials”) may be the following: milk from mammals such as human milk, cow's milk, goat's milk, sheep's milk, mare's milk asf. and the corresponding wheys, bird's eggs such as hen's eggs, duck's eggs, ostrich's eggs asf., fish roe such as trout roe, sturgeon roe asf., blood from mammals such as human blood, beef blood, goat blood, sheep blood, horse blood asf., urine from mammals such as human urine, cattle urine, goat urine, sheep urine, horse urine asf., as well as bee's honey and plant seeds.
Preferred starting materials are human milk, cow's milk, hen's eggs, trout roe and beef blood. A particularly preferred starting material is skimmed cows milk or its whey obtained from cheese-making or a commercially available dried whey, which is obtained e.g. by spray drying. The process of the invention for the extraction of the Growth Factor Complex may be used on all examples stated herein as starting materials, and generally on all starting materials that contribute to growth or grow themselves (e.g. seeds). In general terms, the process of the invention may be used on any peptide- or protein-containing starting material.
To solid starting materials, water is added to bring them to a homogeneous solution before further treatment. Fat containing starting materials are defatted before treatment in a conventional manner, e.g. by skimming.
The extraction of the Growth Factor Complex according to the invention is described hereinafter; as an example that of the Growth Factor Complex from skimmed milk, where the solid dry substance is more concentrated than in unprocessed milk. Such starting materials with a high content of solid milk dry substance are manufactured in that natural milk is, after skimming, concentrated by evaporation of water under mild conditions or in that dried milk obtained from skimmed milk is mixed with a suitable amount of water. Skimmed milk containing at least 100 g/l of dry substance and especially dried milk from skimmed cow's milk are the preferred starting materials.
To adjust of the pH value of the solution containing the starting material to 2.5 to 3.2, generally non-oxidizing mineral acids and organic acids such as mono-, di- and tricarboxylic acids as well as hydroxy acids derived therefrom may be used. Suitable mineral acids are hydrochloric acid, sulphuric acid and phosphoric acid. Suitable organic acids are formic acid, acetic acid, propionic
Hofmann Dieter
Meyer Hans
Wasmer Hermann
Gupta Anish
IPR Institute for Pharmaceutical Research AG
Pennie & Edmonds LLP
Woodward Michael P.
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