Purification of biologically active peptides from milk

Details Purification of biologically active peptides from milk Purification of biologically active peptides from milk

1996-05-13

2001-07-31

Celsa, Bennett (Department: 1627)

Chemistry: natural resins or derivatives; peptides or proteins;

Proteins, i.e., more than 100 amino acid residues

Separation or purification

C530S412000, C530S413000, C530S416000, C530S832000, C435S069100, C800S281000

Reexamination Certificate

active

06268487

ABSTRACT:

BACKGROUND OF THE INVENTION
This invention relates generally to an improved method for purifying components of interest from milk. More specifically, it provides a method for obtaining peptides from raw whole milk, without prior processing to remove fats, lipids or particulate matter, by use of tangential flow filtration, preferably through a closed-loop continuous extraction system.
Milk from domestic animals has been used as a source of proteins and other products for the food and pharmaceutical industries for many years, and a variety of techniques are known for isolating these products. Milk is a colloidal suspension composed primarily of fats, lactose and proteins in water. Among ruminants and laboratory animals, milk contains an average of 30 to 140 grams of protein per liter, or about 4-17% by weight, depending on the species. The bulk of these proteins are caseins, which are complexed with calcium and phosphate in supramolecular structures known as micelles. The other major class of milk proteins is whey proteins, predominantly comprised of beta-lactoglobulin and alpha-lactalbumin, but also including lactoferrin, immunoglobulins, and serum albumin.
Milk proteins usually are isolated by a combination of processes. Raw milk first is fractionated to remove fats, for example, by skimming, centrifugation, sedimentation (H. E. Swaisgood, Developments in Dairy Chemistry, I: Chemistry of Milk Protein, Applied Science Publishers, NY, 1982), acid precipitation (U.S. Pat. No. 4,644,056) or enzymatic coagulation with rennin or chymotrypsin (Swaisgood, ibid.). Next, the major milk proteins may be fractionated into either a clear solution or a bulk precipitate from which the specific protein of interest may be readily purified.
Even recent improvements in milk protein isolation require a first process for removing fats and lipids, followed by filtration to recover components of the approximate size of the protein of interest. For example, French Patent No. 2487642 describes the isolation of milk proteins from skim milk or whey by membrane ultrafiltration in combination with exclusion chromatography or ion exchange chromatography. Whey is first produced by removing the casein by coagulation with rennet or lactic acid. U.S. Pat. No. 4,485,040 describes the isolation of an alpha-lactoglobulin-enriched product in the retentate from whey by two sequential ultrafiltration steps. U.S. Pat. No. 4,644,056 provides a method for purifying immunoglobulin from milk or colostrum by acid precipitation at pH 4.0-5.5, and sequential cross-flow filtration first on a membrane with 0.1-1.2 micrometer pore size to clarify the product pool and then on a membrane with a separation limit of 5-80 kd to concentrate it.
Similarly, U.S. Pat. No. 4,897,465 teaches the concentration and enrichment of a protein such as immunoglobulin from blood serum, egg yolks or whey by sequential ultrafiltration on metallic oxide membranes with a pH shift. Filtration is carried out first at a pH below the isoelectric point (pI) of the selected protein to remove bulk contaminants from the protein retentate, and next at a pH above the pI of the selected protein to retain impurities and pass the selected protein to the permeate. A different filtration concentration method is taught by European Patent No. EP 467 482 B1 in which defatted skim milk is reduced to pH 3-4, below the pI of the milk proteins, to solubilize both casein and whey proteins. Three successive rounds of ultrafiltration or diafiltration then concentrate the proteins to form a retentate containing 15-20% solids of which 90% is protein.
Alternatively, British Patent Application No. 2179947 discloses the isolation of lactoferrin from whey by ultrafiltration to concentrate the sample, followed by weak cation exchange chromatography at approximately a neutral pH. No measure of purity is reported. In PCT Publication No. WO 95/22258, a protein such as lactoferrin is recovered from milk that has been adjusted to high ionic strength by the addition of concentrated salt, followed by cation exchange chromatography.
In all of these methods, milk or a fraction thereof is first treated to remove fats, lipids, and other particulate matter that would foul filtration membranes or chromatography media. The initial fractions thus produced may consist of casein, whey, or total milk protein, from which the protein of interest is then isolated. However, these techniques present significant disadvantages, including the requirement for large and expensive batch and/or continuous centrifuges, low yields due to protein loss by entrapment during precipitation, and loss of biological activity of the protein of interest by precipitation methods requiring low pH. These limitations may be tolerated for relatively inexpensive proteins present in very large amounts and used as commodities in foodstuffs, e.g., in the production of cheese. However, they become a significant economic disincentive if the protein represents a small fraction of total milk protein, represents an expensive pharmaceutical, or consists of an enzyme or other therapeutically active protein that must retain its biological activity.
All of these conditions would obtain, for example, in the purification of a pharmaceutical protein from the milk of transgenic animals. Exogenous protein expression levels generally range from less than 1 to 10 or more grams per liter, depending on the protein and the species. In a product with a potential annual market value of, for example, $100 million, every 1% loss represents $1 million.
Methods are known in the art for expressing exogenous proteins at commercially feasible levels in the milk of transgenic animals. Commercial production of a wide range of proteins in the milk of transgenic livestock is now under development (A. J. Clark, et al.,
Trends in Biotechnology,
5:20-24, 1987; A. J. Clark,
Journal of Cellular Biochemistry
49:121-127, 1992; W. Bawden et al.,
Biotechnology and Genetic Engineering Reviews,
12:89-137, 1994; N. S. Rudolph,
Genetic Engineering News,
15:8-9, 1995). Exogenous peptides, and in particular human peptides, may be produced in milk at relatively high concentrations and in large volumes, providing continuous high-level output of normally processed peptides that are easily harvested from a renewable resource. Purification of these valuable proteins by conventional processes is subject to the yield and activity losses described above. For example, A. J. Clark et al. reported recovery of anti-hemophilic Factor IX of approximately 2.0-2.5% by acid precipitation of casein from milk obtained from transgenic ewes, for a loss of approximately 98% (A. J. Clark et al.,
Biotechnology
7:487-492, 1989). J. Denman et al. reported recovery of a long-acting variant of tissue plasminogen activator of about 25%, or a loss of 75%, by acid precipitation of milk caseins from transgenic goats (J. Denman et al.,
Biotechnology
9:839-843, 1991).
PCT Patent Publication No. WO 94/19935 discloses a method of isolating a biologically active protein from whole milk by stabilizing the solubility of total milk proteins with a positively charged agent such as arginine, imidazole or Bis-Tris. This treatment forms a clarified solution from which the protein may be isolated, e.g., by filtration through membranes that otherwise would become clogged by precipitated proteins. The concentration of the additive is high, on the order of 1-3 molar. In some cases, it may be preferable to minimize the large required amounts of particularly expensive agents, such as arginine, which in any case must be removed in subsequent purification steps. This method also requires a first centrifugation step to remove milk fat.
What is disclosed herein is an improvement over methods known in the art for isolating soluble milk components. The present invention reduces losses in yield by providing mild conditions that preserve biological activity in an efficient and cost-effective method suitable for large-scale production.
SUMMARY OF THE INVENTION
The present invention provides a method for isolating a soluble

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