WO2010131237A1 - Procédé de production d'un complexe de protéine globulaire biologiquement actif - Google Patents

Procédé de production d'un complexe de protéine globulaire biologiquement actif Download PDF

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WO2010131237A1
WO2010131237A1 PCT/IE2010/000034 IE2010000034W WO2010131237A1 WO 2010131237 A1 WO2010131237 A1 WO 2010131237A1 IE 2010000034 W IE2010000034 W IE 2010000034W WO 2010131237 A1 WO2010131237 A1 WO 2010131237A1
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globular protein
fatty acid
complex
lactalbumin
biologically active
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PCT/IE2010/000034
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English (en)
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Andre Brodkorb
Kamila Liskova
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Agriculture And Food Development Authority (Teagasc)
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the invention relates to a biologically active globular protein complex, especially a biologically active ⁇ -lactalbumin or ⁇ — lactoglobulin complex, and processes for the production thereof.
  • ⁇ -Lactalbumin is a major whey protein that has been found in milks of most mammals. It has a molecular weight of approx 14 kDa and consists of 123 amino acids in most species. It contains eight cysteines bound in four disulphide bridges. The crystal structure of ⁇ -lactalbumin was firstly determined for the baboon species in 1989 and two years later the structure of the human variant was resolved at 1.7A resolution. Human ⁇ -lactalbumin consists of one large helical domain and one smaller ⁇ -sheet domain. The two domains are connected by a calcium-binding loop, where four aspartates (Asp82, Asp84, Asp87 and Asp88) and Lys79 coordinate the Ca 2+ .
  • ⁇ -lactalbumin binds calcium with a high affinity and this has a strong effect on its structure and thermal stability. While the transition temperature of holo ⁇ - lactalbumin (calcium-bound) is about 66°C, apo ⁇ -lactalbumin (calcium-depleted form) is much less stable towards the thermal denaturation with the transition temperature around 38°C. At low pH ⁇ -lactalbumin forms a molten globule - a partially folded protein state with native-like secondary structure but dynamic tertiary structure.
  • a complex from human milk was discovered to induce apoptosis-like death selectively in tumor and immature cells.
  • the complex was obtained from acid precipitated casein, but molecular characterization of this complex revealed that it consisted of multimers of ⁇ -lactalbumin conserved in a molten globule state. Therefore, the complex was called MAL (Multimeric Alpha-Lactalbumin).
  • Native ⁇ - lactalbumin lacked cytotoxic activity, but later work showed that it can be converted to the cytotoxic complex by passing it through an ion-exchange chromatographic column preconditioned with a casein fraction (M. Svensson, Hakansson, Mossberg, Linse, & Svanborg, 2000).
  • HAMLET Human Alpha-lactalbumin Made LEthal to Tumor cells.
  • Homological complexes were prepared from bovine, equine, porcine and caprine ⁇ - lactalbumin - all with the cytotoxicity comparable to HAMLET but lower yields.
  • Oleic acid (Z-octadec-9-enoic acid) is a monounsaturated fatty acid found in large quantities in olive oil. It is also the most abundant fatty acid in human milk and second in cow milk.
  • chromatographic matrices can be used, oleic acid can be replaced by a number of different fatty acids and ⁇ -lactalbumin can be mixed with the fatty acid prior to the exposure to the chromatographic matrix.
  • the replacement of the chromatographic resin can result in higher possible load of ⁇ - lactalbumin, higher flow rate and more efficient conversion of ⁇ -lactalbumin to
  • HAMLET/BAMLET-like complexes were prepared by heating a mixture of ⁇ -lactalbumin and oleic acid (Kamijima et al., 2008). Oleic acid was added in 150-fold excess and after heating (50 or 60°C) the non-reacted amount was removed. The resulting complex had cytotoxicity comparable to HAMLET-BAMLET prepared via chromatography technique.
  • HAMLET/BAMLET-like complexes can be formed without contact with the ion-exchange resin.
  • the absence of the chromatographic step not only simplifies the process and makes it more suitable for industrial production, but it also contributes to clarity and controllability of the process environment.
  • the original three-phase system was reduced to two phase system: protein solution and water immiscible fatty acid.
  • the fatty acid still needs to cross the interface from organic to aqueous phase, where it can form HAMLET/BAMLET with ⁇ -lactalbumin.
  • Organic solvent has to be used (to solubilise the fatty acid) and the excess of fatty acid has to be removed afterwards.
  • the invention is based on the surprising finding that a biologically active globular protein complex can be generated by replacing the fatty acid with a water soluble salt of the fatty acid, for example replacement of oleic acid with sodium oleate or potassium oleate.
  • a water soluble form of the co-factor allows the reaction to be carried out in an aqueous phase, and obviates the need for working in two phases or with non-aqueous solvents, and the requirement for the use of chromatographic separation techniques.
  • a method of producing a biologically active globular protein complex comprising the step of contacting a globular protein- containing solution with an excess of a water soluble salt of a fatty acid in the presence of globular protein denaturing conditions.
  • the method of the invention results in a biologically active globular protein complex that has comparable biological activity to BAMLET produced using literature methods. Further, the process surprisingly allows the reaction to be carried out at considerably higher concentrations that the literature methods.
  • the process of the invention allows lOOg of product be generated using a reaction volume of IL; with the processes of the literature, reaction volumes of 33L (Kamihima et al.) and 700L (Knyazeva et al.) are required to generate lOOg of product.
  • the globular protein-containing solution comprises an aqueous solution of a globular protein or a mixture of globular proteins.
  • aqueous solution of a globular protein or a mixture of globular proteins examples would include solutions made from globular protein powder, and other sources of the globular protein, especially mammalian milk, preferably bovine milk, either in its natural form or in a pre-processed form (i.e skim milk).
  • the concentration of the globular protein- containing solution is typically from 0.1 g/L to 100g/L, suitably from 0.3g/L to 20g/L, and ideally from 3g/L to lOg/L (w/v).
  • excess of water soluble salt of fatty acid should be understood as meaning a molar excess of fatty acid salt to globular protein of at least 2:1, 3:1, 4:1, or 5:1.
  • the term means a molar excess of fatty acid salt to globular protein of from at least 5 : 1 , and ideally from 5 : 1 up to 20: 1.
  • globular protein denaturing conditions should be taken to mean conditions which cause the denaturation (full or partial unfolding) of the globular protein from its native conformation. Ideally, at least 5%, 10%, 15%, 20%, 30%, 40%, 50% unfolding should be achieved as part of the process.
  • suitable conditions include heat treatment, pressure treatment (static or dynamic pressure), or a combination of both (i.e. Ultra High Temperature - UHT treatment).
  • Conventional heat treatment usually involves heating the globular protein-containing solution to 50°C to 80°C, suitably 50°C to 70°C, ideally 55°C to 65°C for a suitable period of time.
  • the heating time required to achieve denaturation of the globular protein depends on a number of factors, including ionic strength, pH, the presence of other components, and the type and concentration of the globular protein solution; for example, at higher concentrations (i.e. lOg/L) shorter times are required (i.e. 30 minutes), whereas at lower concentrations (i.e. 3g/L) longer heating times are required (i.e. 60 minutes).
  • concentrations i.e. lOg/L
  • shorter times i.e. 30 minutes
  • at lower concentrations i.e. 3g/L
  • longer heating times i.e. 60 minutes.
  • a key element of the process of the invention is achieving full or partial unfolding of the globular protein, for example at least 5% unfolding as determined using the RP-HPLC techniques described herein, and that various means are available for achieving unfolding.
  • the exact method of achieving unfolding do not typically form part of the invention.
  • the process of the invention may involve a further step of removing excess fatty acid salt following formation of the globular protein complex.
  • Suitable methods of removal of excess fatty acid salt will be apparent to those skilled in the art, and include dialysis, ultrafiltration, and gel permeation.
  • the product is dehydrated for storage. Suitable methods of dehydration will be known to those skilled in the art, including freeze drying and spray drying.
  • the invention also relates to a biologically active globular protein complex obtainable by the method of the invention.
  • the globular protein is selected from ⁇ - lactalbumin, ⁇ -lactoglobulin, or lysozyme, or variants or fragments thereof.
  • the invention also relates to a biologically active globular protein complex comprising a denatured form of a globular protein (or a biologically active variant or fragment thereof) and a water soluble fatty acid salt.
  • the globular protein is selected from ⁇ -lactalbumin and ⁇ -lactoglobulin, especially mammalian ⁇ -lactalbumin and ⁇ -lactoglobulin.
  • globular protein should be taken to mean proteins with a spherical structure. Globular proteins, unlike their fibrous counterparts, are soluble in water as their hydrophobic amino acids and regions thereof are contained within the interior of the molecule whereas polar (hydrophilic) groups are bound outwards, allowing interactions with the aqueous solvents, which explains the molecule's solubility in water. Typical examples of globular proteins are the group of albumins and globulins such as the whey proteins ( ⁇ -lactalbumin or ⁇ -lactoglobulin etc), ovalbumin, hemoglobin, immunoglobulins, serum albumins and lysozyme etc.
  • Suitable variants include globular proteins in which the calcium binding domain is modified to disrupt calcium binding thereto, typically by modification, disruption or destruction of the calcium binding domains.
  • Various means for modifying the globular protein to disrupt calcium binding thereto are provided in the literature. For example, for human ⁇ -lactalbumin, site directed mutagenesis of the aspartic acid residue at position 87 to a non-acidic residue, in particular a non-polar or uncharged side chain, has been found to disrupt calcium binding activity.
  • Suitable variants include D87A and D87N.
  • a "variant" of a globular protein shall be taken to mean proteins having amino acid sequences which are substantially identical to wild-type globular protein.
  • the term should be taken to include proteins or polypeptides that are altered in respect of one or more amino acid residues.
  • alterations involve the insertion, addition, deletion and/or substitution of 5 or fewer amino acids, more preferably of 4 or fewer, even more preferably of 3 or fewer, most preferably of 1 or 2 amino acids only. Insertion, addition and substitution with natural and modified amino acids is envisaged.
  • the variant may have conservative amino acid changes, wherein the amino acid being introduced is similar structurally, chemically, or functionally to that being substituted.
  • variants which have been altered by substitution or deletion of catalytically-important residues or domains will be excluded from the term "variant". Details of such catalytically-important residues/domains will be well known to those skilled in the field of globular protein modelling and from the literature.
  • the variant will have at least 70% amino acid sequence homology, preferably at least 80% sequence homology, more preferably at least 90% sequence homology, and ideally at least 95%, 96%, 97%, 98% or 99% sequence homology with wild-type globular protein.
  • sequence homology comprises both sequence identity and similarity, i.e.
  • a polypeptide sequence that shares 70% amino acid homology with bovine ⁇ -lactalbumin is one in which any 70% of aligned residues are either identical to, or conservative substitutions of, the corresponding residues in wild- type bovine ⁇ -lactalbumin.
  • Specific variants included within the scope of the invention are the mutant ⁇ -lactalbumin protein disclosed in WP03/074547, especially variants in which the interface between the alpha and beta domains (residues 34-38 and 82-86 in human ⁇ -lactalbumin) is unmodified.
  • variant is also intended to include chemical derivatives of globular proteins, i.e. where one or more residues of the globular protein is chemically derivatized by reaction of a functional side group. Also included within the term variant are globular protein molecules in which naturally occurring amino acid residues are replaced with amino acid analogues.
  • Proteins and polypeptides (including variants and fragments thereof) of and for use in the invention may be generated wholly or partly by chemical synthesis or by expression from nucleic acid.
  • the proteins and peptides of and for use in the present invention can be readily prepared according to well-established, standard liquid or, preferably, solid-phase peptide synthesis methods known in the art (see, for example, J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Illinois (1984), in M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984).
  • a “fragment" of a globular protein means a contiguous stretch of amino acid residues of at least 5 amino acids, preferably at least 6 amino acids. Typically, the "fragment” will comprise at least 10, preferably at least 20, more preferably at least 30, and ideally at least 40 contiguous amino acids. In this regard, it would be a relatively straightforward task to make fragments of the globular protein and assess the cytotoxic or apoptotic activity of such fragments using the in-vitro models of cytotoxicity and apotosis described below.
  • fragments of globular proteins prteferably include the interface between the alpha and beta domains () of the protein. In human ⁇ -lactalbumin, this interface is provided by residues 34-38 and 82- 86.
  • globular protein as used herein should also be understood to include mixtures of globular proteins, for example ⁇ -lactalbumin and ⁇ -lactglobulin, ⁇ -lactalbumin and lysozyme, or lysozyme and ⁇ -lactglobulin.
  • the water soluble salt of a fatty acid may be a water soluble salt of any fatty acid, for example any of the fatty acids disclosed on Pages 47 to 51 of International Patent Application No: WO2008/138348.
  • a water soluble salt of a C16 to C20 fatty acid is employed.
  • a water soluble salt of a Cl 8 fatty acid is employed.
  • the fatty acid is oleic acid.
  • the fatty acid is in a cis conformation.
  • the fatty acid is undaturated, ideally mono- unsaturated.
  • the fatty acid is C18:l:9cis or C18:l:12cis.
  • the salt is an alkali metal salt of the fatty acid, for example, a sodium or potassium salt of the fatty acid may be employed (for example, sodium or potassium oleate).
  • a sodium or potassium salt of the fatty acid may be employed (for example, sodium or potassium oleate).
  • suitable water soluble salts of fatty acids will be known to those skilled in the art.
  • biologically active should be understood as meaning that the complex has cytotoxic or apoptotic activity towards tumour cells, for example, comparative cytotoxic or apoptotic activity to HAMLET made using literature techniques. Methods for assaying for cytotoxic or apoptotic activity are provided below. In one embodiment, the term should be taken to mean having an IC 50 of at least 60 ⁇ M against U937 tumor cells as determined in an in-vitro viability assay described herein.
  • the variant has a cytotoxicity (IC 50 ) of less than 60 ⁇ M, 50 ⁇ M, 40 ⁇ M or 30 ⁇ M. Cytotoxicity is measured using the technique detailed below.
  • the invention also relates to the biologically active globular protein complex of, or obtainable by the process of, the invention for use in treatment of a proliferative disease, for example cancer.
  • cancer should be taken to mean any cancer, including a cancer selected from the group consisting of: fibrosarcoma; myxosarcoma; liposarcoma; chondrosarcom; osteogenic sarcoma; chordoma; angiosarcoma; endotheliosarcoma; lymphangiosarcoma; lymphangioendotheliosarcoma; synovioma; mesothelioma; Ewing's tumor; leiomyosarcoma; rhabdomyosarcoma; colon carcinoma; pancreatic cancer; breast cancer; ovarian cancer; prostate cancer; squamous cell carcinoma; basal cell carcinoma; adenocarcinoma; sweat gland carcinoma; sebaceous gland carcinoma; papillary carcinoma; papillary adenocarcinomas; cystadenocarcinoma; medullary carcinoma; bronchogenic carcinoma; renal cell carcinoma; hepatoma; bile duct carcinoma; chori
  • the invention also relates to a medicament comprising a biologically active globular protein complex of, or obtainable by the process of, the invention.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a biologically active globular protein complex of, or obtainable by the process of, the invention in combination with a pharmaceutically acceptable carrier.
  • the term "pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals.
  • carrier refers to a diluent, adjuvant, escipient, or vehicle with which the therapeutic is administered.
  • Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, and sodium chloride
  • compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations.
  • compositions can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
  • Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences” by E. W. Martin.
  • Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the a suitable form for proper administration to the patient.
  • the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
  • compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
  • the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to, ease pain at the, site of the injection.
  • the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
  • the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline as the case may be.
  • the pharmaceutical composition may be a topical formulation.
  • creams emulsions
  • lotions emulsions
  • gels emulous liquids
  • formulations applied by spraying such as mist, aerosol or foam spraying.
  • a difference between these forms is their physical appearance and viscosity, which can be governed by the presence and amount of emulsifiers and viscosity adjusters present in the formulation.
  • Gels provide a particularly useful form of the invention. They are semisolid and liquid rich and form a suitable compatible formulation for application to wounds. They can be prepared with a range of viscosities.
  • Their formulations may contain solvents, emulsifiers, moisturizers, emollients, preservatives and other active ingredients that increase or enhance the efficacy of the final product.
  • solvents can contribute to the modulation of release rates.
  • the invention contemplates topical formulations designed for controlled-release of phenytoin to a wound surface.
  • Figure 1 FPLC chromatograms of BAMLET. NaCl gradient is in graphs represented by conductivity gradient. BAMLET is eluted with IM NaCl.
  • Figure 3 Peak of OA/SO in FTIR spectra of BAMLET samples. Spectra were vector normalized.
  • FIG. 4 Viability of U937 cells following supplementation with increasing concentrations of BAMLET (prepared by chromatographic method) samples for 24 h. Cell viability was determined using the MTS assay. Data are the means of three independent experiments, with standard errors represented by vertical bars. Legend: ⁇ - BAMLET with SO, - ⁇ - BAMLET with OA, -A- SO
  • Figure 5 FTIR spectra of BAMLET samples prepared by heating at 80°C for Ih. Spectra were vector normalized.
  • ⁇ -lactalbumin peak of SO in BAMLET samples is increasing in order 2 ⁇ SO, 5> ⁇ SO, 7.5 ⁇ SO, 1OxSO, 15 ⁇ SO, 2OxSO.
  • FIG. 7 Viability of U937 cells following supplementation with increasing concentrations of BAMLET (prepared in one phase with sodium oleate) samples for 24 h. Cell viability was determined using the MTS assay. Data are the means of three independent experiments, with standard errors represented by vertical bars. Samples 2 ⁇ SO (2 time molar excess of SO to ⁇ -lactalbumin) were not cytotoxic Legend: BAMLET samples prepared with molar excess of SO: ⁇ - 5 ⁇ SO, ⁇ - 7.5 ⁇ SO, -*- 10 ⁇ SO, ⁇ - 15 ⁇ SO, -*- 20 ⁇ SO
  • Figure 8 LD 50 values of BAMLET samples calculated from viability curves. Data are the means of three independent experiments, with standard deviations represented by vertical bars. Legend: D 60°C, Ih; ⁇ 6O 0 C, 3h; ⁇ 80°C, Ih
  • Figure 9 Native PAGE of BAMLET samples prepared in one-phase solution; Lane 1 : non-heated ⁇ -lactalbumin (control), other lanes: BAMLET samples prepared with molar excess of SO in ratio: line 2: 2 ⁇ SO, line 3: 5 ⁇ SO, line 4: 7.5> ⁇ SO, line 5:10 ⁇ SO, line 6: 15 ⁇ SO and line 7: 20 ⁇ SO.
  • Materials and Methods Materials ⁇ -Lactalbumin was purchased from Davisco Foods International, Inc., DEAE Trisacryl ® M matrix from Pall Corporation, the CellTiter 96 Aqueous One Solution Cell Proliferation Assay from Promega Corporation, and Bio-Rad Protein Assay from Bio-Rad Laboratories GmbH. All other chemicals and solutions were purchased from Sigma- Aldrich.
  • BAMLET preparation by the chromatographic method (partially comparative) ⁇ -Lactalbumin was reconstituted overnight in Milli-Q water at concentration 0.6% (w/v).
  • Denatured protein present in the commercial product was removed by isoelectric precipitation at pH 4.6, after which the solution was filtered through 0.45 ⁇ m filter.
  • IM Tris-HCl of pH 8.5 was added to solution to final concentration 1OmM and solid EDTA was added to final concentration 2mM.
  • Conditioning of the chromatographic column was performed with 140ml of 4.43mM solution of oleic acid (+2 % ethanol) or sodium oleate dissolved in buffer A (1OmM Tris-HCl, pH 8.5).
  • BAMLET 0.5% (w/w) ⁇ -lactalbumin was injected onto the preconditioned column.
  • the buffer gradient used for elution of BAMLET was 0% buffer B (1OmM Tris-HCl, IM NaCl, pH 8.5) for 1 CV, 15% B for 1.5 CV, 15% B for 2 CV, 100% B for 0.1 CV, 100% for 1 CV, 0% B for 0.1 CV, 0% B for 2 CV.
  • Collected BAMLET fractions were pooled and extensively dialyzed against distilled water prior to freeze-drying.
  • BAMLET samples were also prepared by heating mixture of ⁇ -lactalbumin and sodium oleate.
  • ⁇ -Lactalbumin was reconstituted in phosphate buffered saline (PBS) at 0.2ImM.
  • PBS phosphate buffered saline
  • Sodium oleate was added in molar ratio 2:1 (0.42mM), 5:l(1.07mM), 7.5:1 (1.6ImM), 10:1 (2.14mM), 15:1 (3.2ImM) and 20:1 (4.28mM).
  • Samples were heated at 6O 0 C for Ih and 3h, and at 80 0 C for Ih, after which samples were immediately cooled on ice.
  • the concentrations of native protein in heated samples were analysed by reversed-phase HPLC using a previously described method.
  • the process for producing BAMLET using -lactalbumin is substantially the same as that when -lactalbumin is used, with the exception that ⁇ -lactalbumin is employed at 0.2ImM, 3.78g/L, dissolved in MiIIiQ.
  • Human lymphoma cells U937 were used for cytotoxicity assays.
  • Cells were sub- cultured every 48h at a density of 1 x 10 5 in 10% Fetal Bovine Serum (FBS) supplemented RPMI 1640 media.
  • BAMLET samples were reconstituted in RPMI 1640 at concentration 7 gL "1 for 4 hours.
  • Sodium oleate was dissolved in RPMI 1640 at concentration 2.13 gL "1 . All solutions were filtered with 0.1 ⁇ m sterile filter; aliquots were made and stored at -80 0 C. For each assay a new aliquot was used.
  • cytotoxicity assays cells were seeded at density 1 * 10 5 in 5% FBS supplemented RPMI media, with final volume 100 ⁇ L in 96- well plates. Reagents were added to wells in the following order: 1) 50 ⁇ L of serum-free RPMI 1640; 2) appropriate volume of RPMI was replaced with volume of BAMLET in RPMI; 3) 50 ⁇ L of cells of density 2 x 10 5 in 10% FBS media. After 24-hour incubation, 20 ⁇ L of MTS reagent (CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega) was added to each well. Absorbance at 490 nm was recorded after 3h incubation with MTS reagent. Assays were performed in triplicates and three independent experiments were made. The precise protein concentration of BAMLET dissolved in RPMI media was determined using the Bio-Rad Protein Assay based on the method of Bradford (Bradford, 1976).
  • Intrinsic tryptophan fluorescence was measured using Cary Eclipse Fluorescence Spectrophotometer. ⁇ -Lactalbumin and BAMLET samples were prepared in Milli-Q ® water, with an absorbance 0.7 at 280 nm for each sample. The excitation wavelength was 280 nm and emission was scanned from 300 to 400 nm. Both excitation and emission slits were set up to 5nm at a temperature of 20°C and a scan rate 120 nmmin "
  • BAMLET samples produced by both chromatography and heating in solution with sodium oleate were prepared for analysis using VivaSpin 15R centrifuge columns of cut off 5kDa (Sartorius Stedim); samples were first extensively ultra filtered (by centrifugation) against Milli-Q ® to remove the buffer and salts present, and then concentrated.
  • FTIR measurements were carried out using a Bruker Tensor 27 instrument in transmission mode using a thermally controlled AquaSpecTM cell. Spectra were obtained at 20°C in Milli-Q ® as an average of 128 scans at a resolution of 4 cm "1 . Data were processed in OPUS 5.5 version program. After atmospheric compensation for CO 2 , H 2 O (g) and aqueous solution, all spectra were vector normalized.
  • BAMLET samples in RPMI solution were analysed by native polyacrylamide gel electrophoresis. Gels were run according to a modified method of Laemmli (Laemmli, 1970), whereby sodium dodecyl sulphate (SDS) was omitted from the gel and running buffer; and loading buffer contained neither SDS nor mercaptoethanol. Samples were not heated prior to loading on gel. Protein bands were stained with Coomassie blue.
  • BAMLET produced by the chromatographic method was produced on a chromatographic column using oleic acid (OA) or sodium oleate (SO) to condition the resin. Similar eluting profiles on both chromatograms ( Figure 1) indicated that BAMLET can be produced using SO, although the yield of BAMLET was significantly lower (p ⁇ 0.05). The mean values of yields (for five cycles) were 43.3% ⁇ 3.31 for OA and 28.1% ⁇ 7.02 in the case of SO.
  • Fluorescence intrinsic spectroscopy can provide information about the environment of the tryptophan residues and thus changes in the tertiary structure of the protein
  • BAMLET was prepared by heating of ⁇ -lactalbumin in the presence of variable amounts of SO in solution. Calcium was not removed from the solution; therefore, the work was carried out using holo ⁇ -lactalbumin.
  • ⁇ - lactalbumin denaturation was carried out using holo ⁇ -lactalbumin.
  • Ih The time of the heat-treatment was Ih, which was sufficient for complete irreversible denaturation, see Table 1 below.
  • Preparing a biologically active alpha-lactalbumin complex for treating cancer comprises converting the alpha-lactalbumin or its functional homolog to alpha- lactalbumin complex by mixing with a fatty acid or a lipid (pp. 142): NYA HAMLET PHARMA AB (NYAH-Non-standard).

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Abstract

La présente invention concerne un procédé de production d'un complexe de protéine globulaire biologiquement actif comprenant l'étape consistant à mettre un complexe contenant une protéine globulaire avec un excès de sel hydrosoluble d'un acide gras dans des conditions dénaturant les protéines globulaires. La protéine globulaire est choisie dans le groupe constitué par l'alpha-lactalbumine, la bêta-lactoglobuline et le lysozyme, et la solution contenant la protéine globulaire peut être du lait de mammifère, soit sous sa forme naturelle, soit sous une forme prétraitée (c'est-à-dire lait écrémé). Le sel hydrosoluble d'un acide gras est un sel hydrosoluble d'un acide gras en C16 à C20, par exemple l'acide oléique. L'invention concerne également un complexe de protéine globulaire biologiquement actif comprenant une forme dénaturée d'une protéine globulaire et d'un sel d'acide gras hydrosoluble.
PCT/IE2010/000034 2009-05-13 2010-05-13 Procédé de production d'un complexe de protéine globulaire biologiquement actif WO2010131237A1 (fr)

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WO2012069836A2 (fr) 2010-11-24 2012-05-31 Hamlet Pharma Ab Complexe biologiquement actif et sa préparation
WO2012069836A3 (fr) * 2010-11-24 2012-08-09 Hamlet Pharma Ab Complexe biologiquement actif et sa préparation
US9085643B2 (en) 2010-11-24 2015-07-21 Hamlet Pharma Ab Biologically active complex and its preparation
US9487561B2 (en) 2010-11-24 2016-11-08 Hamlet Pharma Ab Biologically active complex and its preparation
WO2014023976A1 (fr) * 2012-08-09 2014-02-13 Hamlet Pharma Ab Thérapie prophylactique et nutraceutique
US20150216945A1 (en) * 2012-08-09 2015-08-06 Hamlet Pharma Ab Prophylactic and nutraceutical therapy
EP3295953A1 (fr) * 2012-08-09 2018-03-21 HAMLET Pharma AB Complex content des protéines laitières et d'acides gras
US11103561B2 (en) 2012-08-09 2021-08-31 Hamlet Pharma Ab Prophylactic and nutraceutical therapy
US11865161B2 (en) 2012-08-09 2024-01-09 Hamlet Pharma Ab Prophylactic and nutraceutical therapy
CN105218658A (zh) * 2015-11-03 2016-01-06 禾朴乐生物医药(上海)有限公司 高活性牛alpha乳清蛋白肿瘤坏死因子的制备方法
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