Food or edible material: processes – compositions – and products – Products per se – or processes of preparing or treating... – Basic ingredient lacteal derived other than butter...
Reexamination Certificate
1999-06-11
2001-02-06
Hendricks, Keith D. (Department: 1761)
Food or edible material: processes, compositions, and products
Products per se, or processes of preparing or treating...
Basic ingredient lacteal derived other than butter...
C426S657000, C530S361000
Reexamination Certificate
active
06183803
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method of processing milk involving forming a solid phase containing reformed casein micelles and a liquid phase that is substantially free of caseins and casein micelles. The invention also relates to isolating a biologically active target material, such as a recombinant protein, from milk without significant loss in yield. The present invention can be used, among other things, to prepare food products, including baby formula and fat substitutes.
BACKGROUND OF THE INVENTION
Recombinant DNA technology permits the introduction of a heterologous gene or cDNA into a host cell genome. The heterologous gene is transcribed and translated by the host cell machinery, providing an expression system for production of heterologous protein. Various host cells are used for this purpose, including bacteria, yeast, and cultured insect and mammalian cells.
The choice of an expression system is governed by the characteristics of the heterologous protein and the particular production demands. Bacterial expression systems produce high concentrations of recombinant proteins, but fail to provide processing (i.e., proper folding and post-translational modification) necessary to the impart biological activity to many eukaryotic recombinant proteins. Cultured eukaryotic cells provide the proper processing to ensure production of proteins in their native form, but fail to produce them in high concentrations. Instead, cultured eukaryotic systems generally produce only very low concentrations of recombinant protein at a prohibitive cost. U.S. Pat. No. 4,873,316.
In recent years, transgenic animals have been recognized to provide an advantageous alternative to traditional cell culture expression systems, serving as living bioreactors for the production of recombinant protein. Like bacterial expression systems, transgenic animals can produce high concentrations of recombinant product at a relatively low cost. Like eukaryotic expression systems, transgenic animals provide the processing needed to ensure biologically active recombinant eukaryotic proteins. The methods for producing transgenic animals include microinjection of the recombinant DNA construct into the original genome (U.S. Pat. Nos. 5,880,327 and 5,639,940) and, more recently, cloning technologies (
Transgenic Animals For Production of Proteins
, Genetic Eng. News, May 1999). The offspring of these transgenic animals inherit the transgene in normal Mendelian fashion.
Expression of recombinant proteins can be targeted to a particular tissue, such as the mammary gland. U.S. Pat. Nos. 5,565,362 and 5,589,604. Mammary expression provides a highly efficient system for the synthesis and secretion of large quantities of recombinant proteins. Expression of numerous biologically active human proteins in transgenic milk has been reported, including human lactoferrin (Strijker et al.,
Expression of Human Lactoferrin in Milk of Transgenic Animals
, in Harnessing Biotechnology for the 21st Century, 38-41 (Ladish, M. R. and Bose, A., eds., 1992), protein C (Morcol et al., The Porcine Mammary Gland as a Bioreactor for Complex Proteins, Annals New York Acad. Sci. 721, 218-233 (1994)), tissue-type plasminogen activator (tPA) (Ebert et al., (1991) Bio/Technology 9: 853-837); Gordon et al., (1987) Bio/Technology 5: 1183-1187); Denman et al., Bio/Technology 9: 839-843), and alpha-1-antitripsin (&agr;1-AT) (Wright et al., Bio/Technology 9: 830-834).
While showing great promise, production of recombinant protein in transgenic milk is presently limited by the difficulties involved in recovering the recombinant product. (Velander et al.,
Expression of Human Protein C in Transgenic Swine
, in Harnessing Biotechnology for the 21t Century, 34-37 (Ladisch, M. R. and Bose, A., 1992). Milk is a complex secretion of well over 50 discrete proteins, some present in very high concentrations. The sheer quantity of protein naturally present in milk provides a significant obstacle to industrial-scale isolation of recombinant products and the required high degree of purity. Caseins are the most abundant of these milk proteins, accounting (in various forms) for over 50% of the protein in various animal milks (and about 70% of cow milk), and present in the tens of grams per liter. (Groves and Farrel, (1995) Biochim. Et. Biophys. Acta 844: 105-112). Bovine milk, for example, contains abut 10 g/l each of &agr;S1- and &bgr;-casein, and 3/gl each of &agr;S2- and k-casein. (Strijker et al.) Most of the caseins in milk aggregate with calcium in the form of colloidal micelles, ranging from about 0.1 to about 0.5 &mgr;m in diameter.
Known separating milk into three fractions (casein, whey and cream) using a combination of physical separation techniques (i.e., filtration and sedimentation), and/or acid precipitation. (U.S. Pat. No. 4,519,945); (Groves and Farrel); (Denman et al.). Conventional separation methods are undesirable, however, when used as the initial step in a method for isolating a target material from milk. More particularly, conventional methods produce very low yields, because much of the target material becomes entrapped in the casein precipitate fraction. Acid precipitation, moreover, produces a significant loss in biological activity of many target materials. The disadvantages of using conventional methods as the initial step in an isolation strategy is evident in reports of purification of recombinant tPA from transgenic goat milk (only 25% of overall yield, mostly due to a greater than 50% loss of activity during the initial acid precipitation step) (Denman et al.) and recombinant factor IX from transgenic ewe milk (2-2.5%) (Clark et al.(1989) Bio/Technology 9: 487-492). See alson Drahan et al.,
A Scalable Method for the Purification of Recombinant Human Protein C from the Milk of Transgenic Swine
, in Advances in Bioprocess Engineering.
Known methods of casein removal are similarly disadvantageous in other industrial contexts. For example, casein micelles are commonly separated from milk to produce a whey fraction enriched in alpha-lactalbumin (&agr;-La), which is then used in the preparation of humanized milk products (e.g., baby formula). Yet, conventional casein removal methods produce an acidic pH in the starting whey fraction that undesirably lowers the &agr;-La to beta-lactoglobulin (&bgr;-Lg) ratio during the subsequent defiltration steps. U.S. Pat. No. 5,420,249. Casein micelles are also useful as fat substitutes, and commercially available caseins (e.g., acid caseins, alkali metal caseinates) are used for this purpose. U.S. Pat. No. 5,173,322. Yet, acid precipitation of milk destroys the micelle structure, so that commercially available caseins must be reconstructed as micelles to provide the desired fat substitute.
An important need remains, therefore, for a means of processing milk to produce a solid phase containing casein micelles, and a liquid phase that is substantially casein-free. More particularly, a need remains for an efficient means of isolating a biologically active target material from milk at high yields. The method of the present invention fully satisfies that need.
SUMMARY OF THE INVENTION
The present invention relates to a method of processing milk comprising casein micelles and whey proteins. The method involves an initial step wherein a chelating agent is added to the milk sample under conditions such that the casein micelles are wholly or partially deconstructed, forming a clarified milk serum containing caseins and whey proteins, but substantially no intact casein micelles. The clarified milk serum is then contacted with insoluble divalent cation salts, particularly calcium phosphate particles, under conditions such that at least some of the casein micelles reform as aggregates of casein around divalent cation cores. The reformed casein micelles precipitate forming a solid phase containing reformed casein micelles and a liquid phase containing whey proteins but substantially no caseins or casein micelles.
In one embodiment of the present invention, the milk contains a target material. Ac
Bell Steve J. D.
Morcol Tulin
BioSante Pharmaceuticals, Inc.
Gray Bruce D.
Hendricks Keith D.
Kilpatrick & Stockton LLP
Mallatt Kristin D.
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