Modified milk growth factor

Details Modified milk growth factor Modified milk growth factor

1996-09-30

2001-02-27

Lankford, Jr., Leon B. (Department: 1651)

Chemistry: molecular biology and microbiology

Animal cell, per se ; composition thereof; process of...

Method of culturing cells in suspension

C426S656000, C435S325000, C514S002600, C530S350000, C530S412000

Reexamination Certificate

active

06194208

ABSTRACT:

This invention relates to the modification of growth factors in milk or milk extracts and to improved methods for the extraction of such growth factors from milk or milk products and their use in cell culture and pharmaceutical applications.
Animal cells are grown in culture to provide a number of pharmaceutical, diagnostic and veterinary products including human vaccines, lymphokines, hormones, monoclonal antibodies, other pharmaceutically active protein products, veterinary hormones and for research and development and diagnostic purposes.
The growth of animal cells requires a defined isotonic medium that contains salts, nutrients, lipid precursors, nucleic acid precursors, vitamins and amino acids that are formulated to mimic the medium that would normally bathe those cells in vivo. Examples in common use include Eagle's Minimal Essential Medium, Dulbecco's modified Eagle's Minimal Essential Medium (DMEM), Medium 199, RPMI 1640 medium and Ham's F12 Medium. However, virtually no animal cells will grow in such media, but require the co-addition of serum. Fetal bovine serum is frequently used as it is more effective than serum obtained from post-natal animals and it contains only minimal concentrations of immunoglobulins which otherwise could have undesirable effects.
The supply of fetal bovine serum is limited by the number of pregnant cows slaughtered. It also has undesirable lot-to-lot variations and may include toxins. Particular concern surrounds its use for the eventual production of recombinant proteins and other pharmaceuticals for human use because the serum may also contain viruses that are harmful to humans and may be carried through a purification protocol that yields the desired product. Principally for these reasons, extensive efforts have been directed towards the replacement of serum by pure ingredients. Examples of such ingredients are growth factors, hormones and cell attachment factors. Unfortunately, the requirements of each cell type being grown are different and are difficult to establish. Frequently it has not proved possible to achieve equivalent growth properties or equivalent yields of cell products with “serum-free” media as can be obtained with medium containing fetal bovine serum.
The limited availability of fetal bovine serum, its lot-to-lot variability, its resultant considerable cost as well as the deficiencies of “serum-free” media described above have prompted the investigation of other biological fluids as potential replacements in cell culture media. Some progress has been reported in the prior art with bovine milk and bovine colostrum as evidenced by the following selected reports: M. Klagsbrun, Proc. Natl. Acad. Sci. USA 75, 5057 (1978); M. Klagsbrun et al. J. Supramol. Struct. 11, 349 (1979); M. Klagsbrun, J. Cell Biol. 84, 808 (1980); K. S. Steimer et al. J. Cell Biol. 88, 294 (1981); A. Sereni et al. Cell Biol. Intl. Rep. 5, 339 (1981); A. Khar Experientia 39, 534 (1983); O. Damerdji et al. Biotech. Tech. 2, 253 (1988); O. T. Ramirez et al. Lait 70, 313 (1990); H. Ichiba et al. Biol. Neonate. 61, 47 (1992); R. Pakkanen et al. Appl. Microbiol. Biotechnol. 37, 451 (1992). These and other publications alluded to herein are incorporated by reference.
The prior art also includes U.S. Pat. No. 4,440,860 to M. Klagsbrun which describes “compositions and methods for promoting cell growth featuring, in one aspect, cell culture media containing milk or colostrum and fibronectin; fibronectin is preferably pre-coated onto the culture substrate” and Japan Patent JP 59166879 to Morinaga “A culture medium for cell incubation—containing milk or milk components”. Ultrafiltrates of milk whey have also been used to support the growth of cultured cells, as in European Patent A0219372 to G. Linden et al. “Fractions de lait, Procedee d'obtention de ces fractions et milleux de culturo cellulaires renfermant ces fractions” and application WO 90106357 to G. Linden et al. “Supplements of animal cell culture media based on products derived from the milk industry”. An active fraction from colostrum obtained by hydroxyapatite chromatography has been reported in application WO 92/00014 by B. Quinque et al. “Method for treating colostrum thereby obtained, and a cellular medium containing same” and by cation exchange chromatography in Australian Patent 645589 to F. J. Ballard et al. “Growth-promoting agent”. The prior art also includes Australian Patent 598205 to F. J. Ballard et al. “Growth Factor” and European Patent 03 13515 to R. R. Burk et al. “A polypeptide growth factor from milk” in which individual growth factors have been isolated from colostrum or milk in pure form.
This progress has led to the development of formulations that support the growth of some cell types but usually require protein concentrations in the medium that approach those in more general use with fetal bovine serum.
It is accordingly an object of the present invention to overcome or at least alleviate one or more of the deficiencies related to the prior art.
According to the present invention there is provided a process for preparing a plurality of modified milk growth factors which process includes providing
a milk product extract including
a plurality of milk growth factors having basic to approximately neutral isoelectric points; and
a source of acid,
subjecting the milk product extract to transient acidification utilising the acid source; and
isolating a plurality of modified milk growth factors from the transiently acidified milk product extract.
Preferably the isolation step includes subjecting the transiently acidified milk product extract to a purification step to remove inactive proteins.
By “transient acidification” is meant acidification for a period sufficient to enhance the growth promoting activity of the factors whilst avoiding any significant degradation of the factors.
The present invention further provides a growth promoting composition exhibiting enhanced growth stimulating activity including a plurality of modified milk growth factors having isoelectric points above approximately 6.0 and molecular weights in the range of approximately 5000 to 30,000, the milk growth factors being modified by transient acidification.
Preferably the modified milk growth factors have isoelectric points between approximately 6.0 and 10.5.
The team “milk” as used herein refers to lactational secretions of human or animal origin.
The term “modified milk growth factor” as used herein refers to growth factors of milk that exhibit enhanced growth promoting activity compared to the growth promoting activity of the same growth factors in their occurring state or when extracted from milk, a milk product or a milk product extract.
The term “milk product” as used herein refers to a derivative from human or animal milk in which the fat and/or protein constituents thereof are reduced. Examples of milk product include milk whey, skim milk, cheese whey and acid (casein) whey. Preferably the milk product is cheese whey.
The term “milk product extract” as used herein refers to an extract of milk product in which the salt and/or main protein constituents thereof are reduced. Examples of milk product extracts are ultrafiltrates of milk products or milk products that have been subjected to adsorption and to elution from chromatography matrices. Preferably the milk product extract is cheese whey that has been subjected to cation exchange chromatography by the method described in Australian Patent 645,589.
Preferably the modified growth factor are obtained by acidifying milk product extract containing growth factors having basic to approximately neutral isoelectric points. Preferably inactive proteins are removed from the modified milk growth factors in accordance with the invention. Molecular sieve chromatography or ultrafiltration under acidic conditions may be used to remove inactive proteins. Molecular sieve chromatography is particularly preferred as it removes higher molecular weight proteins such as growth factor binding proteins that would otherwise recombine with certain growt

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