RU2717340C1 - Method of producing lactoferrin - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, specifically to a method of producing lactoferrin. Method of producing lactoferrin involves dissolving dry lactoferrin-containing preparation with purity of 90 % and less in 10 mM phosphate buffer pH 7.5 containing 0.35 M NaCl, further, filtering is carried out from undissolved suspension through filter 2.5 mcm, introduction onto column with cation-exchange sorbent, washing with 10 mM phosphate buffer pH 7.5, containing 0.35 M NaCl and 0.01 % Tween-20, for removal of lipopolysaccharide related to lactoferrin, lactoferrin elution in gradient NaCl 0.35–1.0 M, desalination by dialysis or diafiltration or gel filtration, microfiltration through 0.22 mcm filter and lyophilic drying.

EFFECT: method enables to obtain lactoferrin with purity of 99 %, which does not contain LPS.

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Description

The invention relates to biotechnology.

Commercial preparations of lactoferrin with a purity of 90% or less contain both milk proteins and LPS contamination with which lactoferrin forms a strong complex is possible. Of the milk proteins, impurities of lactoperoxidase, angiogenin and immunoglobulins are possible.

Due to the fact that lactoferrin is a multifunctional protein with antibacterial, antiviral, immunomodulatory activities, it can be used as a pharmaceutical basis for the creation of many drugs. The lactoferrin preparation obtained by the proposed method can be used as an active base as pharmaceuticals with a wide spectrum of action (for example, to restore the gastrointestinal mucosa in gastroenterological diseases, as an antitumor agent, as an antibacterial agent for various bacterial infections of the eyes and skin, for treatment of infectious diseases of the respiratory tract, in the prevention of osteoporosis), as well as dietary supplements, including in the nutrition of newborns.

A known method of producing lactoferrin from cow's milk, which consists in chromatography of lactoferrin from dairy raw materials in a column with a sorbent carboxymethyl cellulose balanced with 0.05 M sodium phosphate buffer, elution of lactoferrin with 0.05 M sodium phosphate buffer, ultrafiltration, desalination, lyophilization, and lyophilization which, according to the invention, raw cow's milk is used as raw milk, chromatography of lactoferrin from raw cow's milk is carried out in a column with a carboxymethylcell sorbent cellulose balanced with 0.05 M sodium phosphate buffer at pH 7.7, lactoferrin is eluted with 0.05 M sodium phosphate buffer, 0.1-1.2 M sodium chloride in the concentration gradient; lactoferrin solution fractions with optical are collected during elution with a density of 0.5 units at a wavelength of 280 nm and direct them to the second chromatography of lactoferrin in a second column with a sorbent carboxymethyl cellulose balanced with 0.05 M sodium phosphate buffer at pH 7.7, the lactoferrin is eluted from the second column with 0.05 M sodium phosphate buffer, in the concentration gradient of sodium chloride 0.1-1.2 M, when eluting again collected fractions of a solution of lactoferrin with an optical density of 0.5 units at a wavelength of 280 nm and direct them to ultrafiltration, desalination and lyophilization. The disadvantage of this method is the two-stage purification, as well as the possible contamination of LPS due to its sufficiently strong binding to lactoferrin. Also known is the technology for producing recombinant human lactoferrin from milk of goat producers (patent RU 2554742). The method includes ion exchange chromatography on a strong cation exchanger, elution of lactoferrin in a gradient of sodium chloride of 0.3-1.0 M, desalination, freeze drying. The method is characterized in that milk of transgenic goats is used as raw milk, chromatography is performed on a strong cation exchanger carrying sulfopropyl groups, sodium acetate buffer is used, stepwise washing of the sorbent with 20 mM sodium acetate buffer pH 7.0 containing 0.3 M is used sodium chloride and 10% ethanol to remove LPS bound to lactoferrin.

The scope of the invention.

The invention relates to the quality of lactoferrin and is aimed at the purification of commercial (industrial) preparations of lactoferrin containing 90% or less of lactoferrin in the sample. In this case, the removal of third-party milk proteins and the removal of LPS, which have an affinity for lactoferrin and are associated with them, worsen the quality of lactoferrin and affect its antibacterial activity. Contaminant proteins, in particular, include angiogenin, whose molecular weight is about 15 kDa, and the isoelectric point is 9.5, which is close to lactoferrin in properties, the angiogenin content, according to published data, is 1-8 mg / l of milk. Upon receipt of lactoferrin on an industrial scale, an increase in the angiogenin content by several times can occur, which can negatively affect the health of the consumer of the product. In addition, lactoperoxidase, which has a molecular weight of approx. 80 kDa, as well as lactoferrin, as well as milk immunoglobulins, can be contaminant proteins.

Therefore, the post-treatment of industrial preparations of lactoferrin, which are supposed to be used as a pharmaceutical substance, is preferred. It is known that the growth and development of the gastrointestinal tract of newborn animals fed with mother's milk is more intense than that of those fed with milk mixtures. A significant role in the growth and development of the newborn belongs to lactoferrin, therefore, its introduction into the diet of newborns is preferable. In Japan, Morinaga Milk Industry Co. Since 1986, Ltd has been producing baby food enriched with cow lactoferrin. The protective properties of LF against microbial and viral infections have been demonstrated in many scientific papers, and to date, several clinical trials of therapeutic drugs based on this protein are known. Nevertheless, our current knowledge allows us to determine LF not only as a key component of the primary protective system of the body, but also as a multifunctional regulatory factor that interacts and affects the microbial, viral or cellular components involved in the infection process.

It is known that lactoferrin of cow's milk exhibits bacteriostatic activity against bacteria. The strains of E. coli are most sensitive to its action. It has been shown that lactoferrin causes a change in the permeability of the outer membrane of certain gram-negative bacteria. Destabilization of the bacterial cell wall may result from nonspecific binding of calcium and magnesium to lactoferrin. Thus, lactoferrin is able to cleave lipopolysaccharide molecules (endotoxins) from the outer membrane of gram-negative bacteria, binding to them. Lactoferrin inhibition of bacterial sorption on non-biological and cell surfaces.

Microbiological adhesion and subsequent colonization and biofilm formation on non-biological surfaces, such as surgical and medical instruments, is a serious problem leading to various diseases and inflammations. The main approach to solving this problem is to create new composite materials that have minimal specificity for the sorption of microflora. In this regard, the possibility of using LF as a new component that protects the surface from microbiological adhesion looks very attractive. In a practical aspect, LF coating of eye lenses protects their surface from P. aeruginosa (Williams T. J., Schneider R. R and Willcox MD (2003) The effect of protein-coated contact lenses on the adhesion and viability of gram negative bacteria. Curr. Eye Res. 27: 227-235).

A number of inhibitory effects of lactoferrin on various parts of the immune and inflammatory reactions were noted. The inhibitory effect of lactoferrin is inversely related to the degree of saturation with iron.

The positive role of lactoferrin in the prevention of osteoporosis is noted (The molecular mechanism of the effect of lactferrin on bone formation. A critical review. P.E. Sadchikov, I.L. Goldman, S.V. Razin, A.D. Chernousov, L.I. Alekseeva, E. R. Sadchikova, Journal of Osteoporosis and Osteopathy, 2016, No. 3, pp. 12-64).

There is a patent (2465004 RU) "The use of the apo-form of human lactoferrin as an antihypoxant and stabilizer of hypoxia-inducible factor -1 alpha." In addition, there is evidence of a positive effect of lactoferrin in the prevention of Alzheimer's and Parkinson's.

Therefore, the post-treatment of industrial samples of lactoferrin for use as a pharmaceutical base is relevant.

Disclosure of Invention

The raw materials for the production of lactoferrin are various lactoferrin-containing preparations with a purity of 90% or less. These include commercial lactoferrin preparations, the purity of which varies greatly in protein content, and the LPS content is often not controlled.

Sorbents-cation exchangers that can be used according to this invention include sorbents containing carboxymethyl or sulfopropyl groups. The basis of these sorbents can be cellulose, dextran, agarose, polyacryl, etc.

The method of producing lactoferrin, which consists in dissolving a dry lactoferrin-containing preparation with a purity of 90% or less, in 10 mM phosphate buffer pH 7.5 containing 0.35 M NaCl, then filtering from insoluble suspension through a 2.5 μm filter, applying to a column with a cation exchange sorbent, washing with 10 mM phosphate buffer pH 7.5, containing 0.35 M NaCl and 0.01% tween-20, to remove lactoferrin-linked lipopolysaccharide, lactoferrin elution in a NaCl gradient of 0.35-1 , 0 M, desalination by dialysis or diafiltration or gel filtration, microfiles Removal through a 0.22 micron filter and freeze drying. The method allows to obtain lactoferrin with a purity of 99%, not containing LPS.

Claims (1)

The method of obtaining lactoferrin, which consists in dissolving a dry lactoferrin-containing preparation with a purity of 90% or less in 10 mM phosphate buffer pH 7.5 containing 0.35 M NaCl, then filtering from insoluble suspension through a 2.5 μm filter, applying on a column with a cation exchange sorbent, washing with 10 mM phosphate buffer pH 7.5 containing 0.35 M NaCl and 0.01% tween-20 to remove lactoferrin-linked lipopolysaccharide, lactoferrin elution in a NaCl gradient of 0.35-1, 0 M, desalination by dialysis or diafiltration or gel filtration, microfil tration through 0.22 .mu.m filter and freeze-drying.