Preparation of bioactive compounds

Abstract

The present invention provides a method for preparing a bioactive complex, the method comprising dissolving a mixture of a polypeptide component in the form of a powdered complex, such as alpha-lactalbumin or a fragment thereof, and oleic acid or a pharmaceutically acceptable salt thereof, which is also in solid form, in an aqueous solution containing a mixture of at least two salts, preferably three salts; wherein the method is carried out at a moderate temperature. Since the preparation process does not require a large amount of heating, the operation is simple and effective.

Description

[0001] The industry has great interest in the production of complexes involving partially unfolded proteins and lipids. These proteins may have very different properties, especially biological properties, compared to the corresponding proteins in the fully folded state. The acquisition of new beneficial functions after partial protein unfolding and fatty acid binding is a remarkable phenomenon and may reflect an important general approach to functional diversification by changing the conformational state of proteins and related ligands. Therefore, in addition to the alternative splicing of mRNA transcripts, post-translational modifications and tertiary structural changes of specific domains, the partial unfolding of original natural proteins is gradually considered to be a mechanism for generating functional diversity. This may be due to the response of cells to unfolded proteins and lipid cofactors, which determines their variable properties. However, this response may be different in, for example, tumor cells, which means that they may have therapeutic potential. In order to form a stable part, the unfolded protein is often modified in some way, and in particular can be combined with cofactors such as fatty acid cofactors. The complex formed in this way may be stable, and thus generate new treatment options.

[0002] HAMLET (human α-lactalbumin lethal to tumor cells) is an example of a new family of tumor-killing molecules with remarkable properties. HAMLET is formed from partially unfolded α-lactalbumin and, together with oleic acid, forms the complete form. HAMLET was discovered by chance during studies of the ability of human milk to prevent bacterial binding to cells. Early in vitro experiments showed that HAMLET has broad antitumor activity with high tumor selectivity; subsequent therapeutic studies confirmed HAMLET's tumoricidal activity and relative selectivity for tumor tissue in vivo. In placebo-controlled clinical studies, topical application of HAMLET can eliminate skin papillomas or reduce their size; and in patients with bladder cancer, topical instillation of HAMLET can lead to rapid tumor cell death, but not to the death of healthy tissue surrounding the tumor. Recently, the therapeutic efficacy of HAMLET in a bladder cancer model was demonstrated in a murine bladder cancer model, where HAMLET treatment delayed tumor progression and led to improved rat survival in a rat glioblastoma xenograft model without evidence of cell death in healthy brain tissue. Thus, HAMLET appears to recognize a conserved death pathway in tumor cells, thereby distinguishing them from healthy, differentiated cells.

[0003] Other complexes using horse lysozyme and oleic acid have also been shown to cause cell death (Vukojevic et al. Langmuir, 2010, 26(18) 14782-14787), suggesting that different, unfolded proteins can be cytotoxic when coupled to appropriate cofactors.

[0004] Other work has focused on the use of peptide fragments of these proteins which may also be utilized (see for example EP-B-2643010 and co-pending UK Patent Application No. 1621752.3).

[0005] Classically, these types of complexes are prepared according to methods such as those described by Svensson et al. in Proc Natl Acad Sci U. SA 97, 4221-4226, 2000. Native α-lactalbumin is purified from human milk by hydrophobic interaction chromatography. The protein is unfolded by EDTA, ion exchange chromatography is performed on a matrix pretreated with oleic acid, and eluted with high salt (specifically 1 M NaCl) to obtain a biologically active complex. This type of method has been used to produce other biologically active complexes from bovine α-lactalbumin, including BAMLET; and complexes formed by recombinant forms of α-lactalbumin, particularly complexes without cysteine ​​residues as described in WO 2010 / 079362.

[0006] WO2010 / 131010 describes an alternative preparation of such a biologically active complex. In this reference, BAMLET is prepared in a single-phase system, in which α-lactalbumin is reconstituted in phosphate-buffered saline (PBS) and sodium oleate is added. The resulting mixture is then heated to a temperature of 60° C. or higher to obtain the active complex. This method has the advantage of being easy to implement and can even be implemented in situ in a clinical situation with the aid of a kit.

[0007] In other references, biologically active complexes are prepared for use by dissolving previously lyophilized complexes in PBS (see, for example, WO 2010 / 079362).

[0008] It is therefore obvious that this complex relies on the presence of salts in its production. Phosphate buffered saline (PBS) as used previously comprises a mixture of at least three salts, sometimes four salts. Among them are sodium chloride, disodium hydrogen phosphate and potassium dihydrogen phosphate, and in some cases potassium chloride.

[0009] Applicants have studied the effects of the salt mixture used to prepare the compound and surprisingly found that the precise identity of the salts used to prepare the complex affects the activity of the product. This suggests that the products are distinguishable and therefore each product with a specific salt balance is a unique product. SUMMARY OF THE INVENTION

[0011] The present invention provides a method for preparing a biologically active complex, which comprises dissolving a mixture of a polypeptide component in powder form and oleic acid or a pharmaceutically acceptable salt thereof, also in solid form, in an aqueous solution comprising at least two salts, wherein the first salt is sodium chloride or potassium chloride and the second salt is disodium hydrogen phosphate or potassium dihydrogen phosphate, wherein, in particular, the method is carried out at moderate temperature.

[0012] As used herein, the expression "moderate temperature" refers to a temperature of up to 50°C, such as 0-50°C, such as 10-40°C, more preferably 15-25°C, such as at ambient temperature. Such temperatures are generally below the "melting temperature" at which polypeptides unfold or denature. However, the applicant has found that they are still able to form biologically active complexes under these salt conditions.

[0013] Applicants have found that by the simple dissolution method of the present invention, an active complex can be prepared that exhibits a clear dose-dependent response. Although the mixture can be heated, for example to 50°C, such as to 40°C, to achieve rapid dissolution, if there is a suitable salt balance in the aqueous solvent, there is no need to heat the solution extensively, such as boiling as described. Therefore, in a particular embodiment, the method is carried out at ambient temperature.

[0014] Agitation (e.g. vortexing) may aid dissolution. If desired, the solution may be filtered through a sterile filter at this stage. Suitable filters include polyethersulfone membranes (PES) or NML cellulose acetate membrane.

[0015] Any such stirring process will be carried out for a period of time to ensure that the components are dissolved in the salt solution. Although the specific time may depend on a variety of factors, such as the specific properties of the polypeptide used and the temperature at which the mixture is maintained, the time will generally be short, such as not more than 10 minutes, such as 1-5 minutes, such as about 2 minutes.

[0016] In a specific embodiment, the solvent further comprises a third salt, which is sodium dihydrogen phosphate or dipotassium hydrogen phosphate, in particular dipotassium hydrogen phosphate. Such a mixture is present in a conventional PBS solution.

[0017] Therefore, the method is easily prepared in a variety of manufacturing and non-manufacturing environments.

[0018] The term "polypeptide" as used in the present invention encompasses proteins and peptides including long peptides.

[0019] Suitable "polypeptide components" for use in the methods of the invention include naturally occurring proteins, in particular α-lactalbumin, lysozyme or other proteins with membrane perturbing activity, recombinant proteins and in particular variants of said naturally occurring proteins, said variants lacking intramolecular chemical bonds, such as variants by mutation of cysteine ​​residues, or in particular fragments of any of these proteins, in particular peptides of up to 50 amino acids.

[0020] The term "variant" refers to a protein or polypeptide that has a similar biological function but has an amino acid sequence that differs from the primary sequence from which it is derived, in that one or more amino acids in the sequence from which it is derived are replaced by other amino acids. Amino acid substitutions may be considered "conservative," where an amino acid is replaced by a different amino acid with broadly similar properties. Non-conservative substitutions are replacements of an amino acid with an amino acid of a different type.

[0021] A "conservative substitution" refers to the substitution of one amino acid with another amino acid of the same class, where class is defined as follows:

[0022]

[0023] As is well known to those skilled in the art, altering the primary structure of a peptide by conservative substitutions may not significantly alter the activity of the peptide, because the side chains of the amino acids inserted into the sequence may form similar bonds and contacts as the original side chains of the substituted amino acids, even when the substitutions are located in critical regions that determine the conformation of the peptide.

[0024] Non-conservative substitutions may be made as long as the function of the DNA binding domain polypeptide is not disrupted.

[0025] In broad terms, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide.

[0026] It is well within the routine capabilities of those skilled in the art to determine the effect of any substitution (and indeed, any amino acid deletion or insertion), who can readily determine whether a variant polypeptide retains the essential properties and activity of a basic protein. For example, when determining whether a variant of a polypeptide is within the scope of the present invention, the skilled person will determine whether the complex comprising the variant retains the biological activity (e.g., tumor cell death) of the complex composed of the native protein in its unfolded form. The polypeptide has at least 60%, preferably at least 70%, more preferably at least 80%, and even more preferably 90%, 95%, 96%, 97%, 98%, 99% or 100% of the biological activity of the native protein.

[0027] The polypeptide variant may comprise or consist essentially of an amino acid sequence that is at least 70% identical to the amino acid sequence of a native protein, such as at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 96%, 97%, 98% or 99%, such as an α-lactalbumin or lysozyme sequence.

[0028] The level of sequence identity is suitably determined by using the BLASTP computer program, with the natural protein sequence as the base sequence. This means that the natural protein sequence forms the sequence for which the percent identity is determined. The BLAST software is publicly available from http: / / blast.ncbi.nlm.nih.gov / Blast.cgi (available for access before May 10, 2017).

[0029] In a specific embodiment, the polypeptide component is a peptide having no more than 50 amino acids, and in particular may have 10-45 amino acids. Such complexes are easier to prepare and have lower raw material costs. For example, conventional methods for producing peptides can be used to prepare peptides. Due to the small molecular weight, the complex formed is easier to handle and formulate for administration.

[0030] The complex is suitably derived from a naturally occurring protein or a variant thereof. Suitable proteins are those active in such a complex, for example alpha-lactalbumin, beta-lactoglobulin or lysozyme, but may also be derived from any membrane perturbing protein.

[0031] Membrane perturbations are proteins that have the ability to interact with the cell membrane interface, particularly causing disruptive effects, such as forming tube channels on the cell membrane. Typically, the protein will be embedded in the cell membrane. Such protein examples include coat complexes, such as COPI, COPII (e.g., SAR 1), HOPS / CORVET, SEA (Sehl binding), and clathrin complex BAR domain proteins such as endorphins, and ESCRT complexes, including Snf7 domain subunits.

[0032] In particular, the peptide is derived from an alpha-helical domain of a naturally occurring protein as described above. The alpha-helical domain of said protein is well known in the art or can be determined using routine methods.

[0033] In some embodiments, when the α-helical domain comprises cysteine ​​residues, they can be modified to different amino acid residues, such as alanine residues, to avoid intermolecular disulfide bonds.

[0034] In a particular embodiment, the peptide is a fragment of alpha-lactalbumin, in particular a fragment of the alpha domain of alpha-lactalbumin. In a specific embodiment, the peptide comprises the amino acids of alpha 1 (residues 1-40) or alpha 2 (residues 81-123) of human alpha-lactalbumin, or similar regions of other alpha-lactalbumins such as bovine alpha-lactalbumin.

[0035] The peptide suitably does not contain components that cause folding, and thus suitably lacks amino acids that cause intramolecular bonding, such as cysteine ​​residues. In particular, when the peptide is derived from a naturally occurring protein, any cysteine ​​residues are substituted with other amino acids, such as alanine.

[0036] Thus, in a specific embodiment, the complex comprises the amino acids of human α-lactalbumin α1 (residues 1-40) or α2 (residues 81-123), wherein the cysteine ​​is replaced by another amino acid, such as alanine, to prevent any intramolecular bonding.

[0037] Therefore, the sequence of the peptide may be shown as SEQ ID NO 1 or SEQ ID NO 2

[0038] KQFTKXELSQLLKD IDGYGGIALPELIXTM FHTSGYDTQA(SEQ ID NO 1)

[0039] LDDDITDDIMXAKKILDIKGIDYWLAHKALXTEKLEQWLXEKL(SEQ ID NO 2)

[0040] Where X is an amino acid other than cysteine

[0041] A specific example of such a sequence is the following SEQ ID NO 3 or SEQ ID NO 4:

[0042] KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQA(SEQ ID NO 3)

[0043] LDDDITDDIMAAKKILDIKGIDYWLAHKALATEKLEQWLAEKL (SEQ ID NO 4).

[0044] In some cases, the peptide chain corresponding to SEQ ID NO 1 can be deleted, for example, by omitting the terminal alanine residue, to generate a peptide chain as shown in SEQ ID NO 6, wherein SEQ ID NO 7 is a special example.

[0045] KQFTKXELSQLLKD IDGYGGIALPELIXTMFHTSGYDTQ(SEQ ID NO 6)

[0046] KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ(SEQ ID NO 7)

[0047] Such peptides are novel and together with the biologically active complexes comprising them constitute a further aspect of the invention.

[0048] Other peptides may also be used in complexes, and suitability may be tested by determining whether the complex with a fatty acid salt is active, such as killing cells using the methods described below.

[0049] In another embodiment, the peptide is derived from the COPII protein family such as SAR1. A specific example of such a peptide is the peptide chain corresponding to SEQ ID NO 5.

[0050] MAGWDIFGWFRDVLASLGLWNKH (SEQ ID NO 5).

[0051] In another embodiment, the polypeptide component is a naturally occurring protein or a synthetic form thereof, in particular alpha-lactalbumin, such as human, bovine, ovine, camel or goat alpha-lactalbumin. In particular, the protein is bovine lactalbumin.

[0052] As used herein, the term "biological activity" means that the complex has a biological activity different from or higher than that of the individual components. In particular, the complex is capable of inducing cell death, in particular selectively inducing cell death of tumor cells, and / or has a bactericidal or antiviral effect that the natural protein (including, for example, monomeric α-lactalbumin forms) does not have, although it may also achieve other therapeutic effects.

[0053] In particular, the oleic acid used in the method of the present invention is of the formula CH3(CH2)7CH=CH(CH2)7COOH or CH3(CH2)7CH=CH(CH2)7COOH. - of C18:1 oleic acid.

[0054] In a specific embodiment, the method uses a pharmaceutically acceptable salt of oleate. Suitable pharmaceutically acceptable salts can be understood according to common knowledge in the art.

[0055] The use of salts, especially water-soluble salts of oleic acid, fatty acids or lipids, means that the preparation process is simpler because they can be formed into aqueous solutions for use in, for example, ion exchange columns, etc. Suitable water-soluble salts are alkali metal or alkaline earth metal salts, such as sodium or potassium salts.

[0056] It has also been found that salts, especially oleates, such as sodium oleate, may have some intrinsic tumor killing effects. Therefore, including them in the complex may enhance the relevant biological activity of the complex.

[0057] In a specific embodiment, the first salt used in the method of the present invention is sodium chloride.

[0058] In another specific embodiment, the second salt used in the method of the present invention is disodium hydrogen phosphate.

[0059] In another specific embodiment, the third salt used in the method of the present invention is potassium dihydrogen phosphate.

[0060] The first salt used in the method of the present invention is suitably in a ratio of 8:1 to 1:1, such as 5:1 to 2:1, especially 4:1 to 3.5:1. If a third salt is present, the ratio of the first salt to the third salt is 20:1 to 5:1, such as 15:1 to 10:1, such as 12.5:1 to 11.5:1.

[0061] In a specific embodiment, the ratio of the first salt to the second salt to the third salt is 13-12:4-3:1.

[0062] In the method of the present invention, the appropriate ratio of oleic acid or oleate to peptide in the mixture is in the range of 20: 1 to 1: 1, but preferably oleate is in excess, for example, the ratio of oleate to peptide is about 5: 1. The mixing temperature is 0-50°C, usually at ambient temperature and pressure.

[0063] If desired, the product of the method of the invention can be solidified, for example by lyophilization, for storage or formulation. Thereafter, it can be reconstituted using, in particular, sterile water. This step is particularly suitable for situations where the polypeptide is a peptide rather than a protein. Applicants have found that when subjected to procedures such as lyophilization, proteins can be restored to their natural folded state.

[0064] This natural folding problem can be alleviated by stabilizing the polypeptide in an unfolded state, for example by lowering the pH of the solution to, for example, below 4, or adding a calcium chelator such as EDTA to the solvent during preparation. In a second aspect, the present invention provides a complex obtainable by the method of the first aspect.

[0065] Thus, the complexes of the second aspect of the invention may be formulated into useful pharmaceutical compositions by mixing them with a pharmaceutically acceptable carrier in a conventional manner. Such compositions constitute the third aspect of the invention.

[0066] The composition of the third aspect of the present invention is a suitable pharmaceutical composition suitable for topical use, such as cream, ointment, gel or aqueous or oily solution or suspension. These may include pharmaceutically recognized available carriers, fillers and / or emergency methods.

[0067] Topical solutions or creams suitably contain an emulsifier for the protein complex and a diluent or cream base.

[0068] The daily dose of the complex varies according to the characteristics of clinical practice and depends on the patient, the nature of the disease to be treated, etc. Usually, 2-200 mg / dose of the biologically active complex is used per administration.

[0069] In another aspect of the present invention, there is provided a method of treating cancer, the method comprising administering the biologically active compound as described above to a patient in need thereof.

[0070] In particular, the complex can be used to treat cancers such as human skin papilloma, human bladder cancer and glioblastoma. In the latter case, the compound can be administered by infusion according to methods known in the art.

[0071] The present invention further provides a biologically active complex as defined above for use in therapy, in particular for use in the treatment of cancer.

[0072] The complex can also be used to prevent cancer, in particular gastrointestinal cancer, as described, for example, in WO2014 / 023976. In this case, the complex can be combined with a food, such as a dairy product such as yogurt, for use as a health product. Compositions of this type form another aspect of the present invention.

[0073] Throughout the description and claims of this specification, the words "comprise" and "include" and variations thereof, such as the nouns "include" and "comprising", mean "including but not limited to", and do not exclude other components, integers or steps. In addition, unless the context indicates otherwise, singular nouns include their plural forms: in particular, where the indefinite article is used, the specification should be understood as contemplating plurality as well as singularity, unless the context indicates otherwise.

[0074] Preferred features of each aspect of the invention may be as described in combination with any other aspect. Within the scope of the present application, the present application shall state that the various aspects, embodiments, examples and alternatives set forth in the preceding paragraphs, claims and / or the following description and drawings, in particular their individual features, may be used independently or in combination. That is, unless the features are incompatible with each other, all embodiments and / or features of any embodiment may be combined in any manner and / or combination. BRIEF DESCRIPTION OF THE DRAWINGS

[0075] The present invention will now be described in detail with reference to the following examples, in which

[0076] Figure 1 Results from studies using a range of complexes with biological activity against tumor cells using ATP Lite, PrestoBlue, and Tryphan Blue are shown;

[0077] Figure 2 A comparison of similar results obtained with filtered and unfiltered solutions is shown;

[0078] Figure 3 (A) Schematic diagram and (B) photograph showing the preparation of a complex comprising bovine α-lactalbumin;

[0079] Figure 4 The results of studies of ATP Lite, PrestoBlue, and TryphanBlue on tumor cells to which the resulting solutions were applied are shown.

[0080] Detailed description

[0081] Example 1

[0082] Preparation of bioavailable complexes

[0083] A series of bioavailable complexes were prepared using the peptide sequence shown in SEQ ID NO 7:

[0084] Ac-KQFTKAELSQLLKDIDGYGGIALPELIATMFHTSGYDTQ-OH(SEQ ID NO 7)

[0085] It is a variant fragment of human α-lactalbumin.

[0086] The peptide (700 μM) in lyophilized form was added to the tubes along with sodium oleate flakes (3.5 mM). Each tube was then reconstituted with the required volume of either:

[0087] 1) PBS (NaCl 6.8g / L;), Na2HPO 4× 2H2O (4.8 g / L); and KH2PO4 (1.3 g / L) (pH 7.2)

[0088] 2) NaCl solution (116 mM) (pH 7.01)

[0089] 3) Na2HPO4 solution (31 mM) (pH 8.6)

[0090] 4) KH2PO4 solution (9.56 mM) (pH 4.6)

[0091] 5) A mixture of (2) and (4) (pH 4.63)

[0092] 6) A mixture of (2) and (3) (pH 8.37)

[0093] 7) A mixture of (3) and (4) (pH 7.29)

[0094] Each mixture was vortexed until the solution was clear.

[0095] The obtained complex was then lyophilized. The lyophilization conditions were a pressure below 1.2 mbar and a temperature below -55°C.

[0096] Each tube was stored at -20°C or lower and reconstituted by adding 30 ml of sterile water before use.

[0097] Example 2

[0098] Determination of cell death

[0099] Human lung cancer cells (A549, ATCC) were cultured in RPM1-1640 medium containing nonessential amino acids (1:100), 1 mM sodium pyruvate, 50 μg / ml gentamicin, and 5-10% fetal calf serum (FCS) at 37°C and 5% carbon dioxide. For cell death experiments, cells were plated in 96-well plates (2 × 10 4 / well, Tecan Group Ltd) were grown overnight. The cells were incubated with the bioactive complex obtained in Example 1 at a dose equivalent to 7, 21 or 35 μM peptide in serum-free RPMI-1640 at 37°C. FCS was added after 1 hour. Three hours after peptide-oleic acid treatment, cell death was quantified by the following three biochemical methods, including 1) using luminescence-based ATPlite TM Kit (Perkin Elmer) to estimate intracellular ATP levels, 2) PrestoBlue fluorescent staining (Invitrogen, A13262), and 3) Tryphan Blue exclusion assay. Fluorescence and luminescence were measured using a microplate reader (Infinite F200, Tecan).

[0100] The results are as follows Figure 1 The complex prepared in PBS was highly active and triggered cell death in a dose-dependent manner ( Figure 1 A). Those complex solutions made with only PBS salt ( Figure 1 B) showed a significant loss of activity. Figure 1As shown in C, the above mixture (6) maintained a reasonable level of tumor cell cytotoxic activity in a dose-dependent manner.

[0101] Example 3

[0102] Effect of Filtration on the Inventive Method

[0103] The method of Example 1 was repeated twice using PBS solution (1), but in this case each solution was passed through chemically different filters, namely polyethersulfone membrane (12846445, VWR) and NML cellulose acetate membrane filter (60810103, Sartorius). The biological efficacy of the product was tested according to the method described in Example 2 and compared with the unfiltered product. The results are shown in Figure 2 shown.

[0104] No significant differences in the bioactivity of the complexes were observed between the measurements of total cellular ATP levels, PrestoBlue staining, and Tryphan Blue exclusion assays.

[0105] Example 4

[0106] BAMLET Production

[0107] Lyophilized bovine α-lactalbumin (700 μM) was added to the tube along with sodium oleate flakes (3.5 mM). Phosphate buffered saline (1 ml) was then added to the tube and vortexed for 1-2 minutes at room temperature. A clear solution ( Figure 3 A and 3B).

[0108] The resulting solutions were tested using the assay described in Example 2. The results are shown in Figure 4 Clearly, the solution was biologically active and killed A549 lung cancer cells in a dose-dependent manner. However, this effect was short-lived because lyophilization of the complex removed the activity. SEQUENCE LISTING <110> Hamlet Pharmaceuticals AB <120> Preparation of biologically active compounds <130> P19114250WP <150> GB 1707715.7 <151> 2017-05-14 <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Mutant protein fragment <220> <221> MISC_FEATURE <222> (6) <223> Any amino acid except Cys <220> <221> MISC_FEATURE <222> (28)..(28) <223> Any amino acid except Cys <400> 1 Lys Gln Phe Thr Lys Xaa Glu Leu Ser Gln Leu Leu Lys Asp Ile Asp 1 5 10 15 Gly Tyr Gly Gly Ile Ala Leu Pro Glu Leu Ile Xaa Thr Met Phe His 20 25 30 Thr Ser Gly Tyr Asp Thr Gln Ala 35 40 <210> 2 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Mutant protein fragment <220> <221> MISC_FEATURE <222> (11)..(11) <223> Any amino acid except Cys <220> <221> MISC_FEATURE <222> (31)..(31) <223> Any amino acid except Cys <220> <221> MISC_FEATURE <222> (40)..(40) <223> Any amino acid except Cys <400> 2 Leu Asp Asp Asp Ile Thr Asp Asp Ile Met Xaa Ala Lys Lys Ile Leu 1 5 10 15 Asp Ile Lys Gly Ile Asp Tyr Trp Leu Ala His Lys Ala Leu Xaa Thr 20 25 30 Glu Lys Leu Glu Gln Trp Leu Xaa Glu Lys Leu 35 40 <210> 3 <211> 40 <212> PRT <213> Artificial Sequence <220> <223> Mutant protein fragment <400> 3 Lys Gln Phe Thr Lys Ala Glu Leu Ser Gln Leu Leu Lys Asp Ile Asp 1 5 10 15 Gly Tyr Gly Gly Ile Ala Leu Pro Glu Leu Ile Ala Thr Met Phe His 20 25 30 Thr Ser Gly Tyr Asp Thr Gln Ala 35 40 <210> 4 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> Mutant protein fragment <400> 4 Leu Asp Asp Asp Ile Thr Asp Asp Ile Met Ala Ala Lys Lys Ile Leu 1 5 10 15 Asp Ile Lys Gly Ile Asp Tyr Trp Leu Ala His Lys Ala Leu Ala Thr 20 25 30 Glu Lys Leu Glu Gln Trp Leu Ala Glu Lys Leu 35 40 <210> 5 <211> twenty three <212> PRT <213> Artificial Sequence <220> <223> Mutant protein fragment <400> 5 Met Ala Gly Trp Asp Ile Phe Gly Trp Phe Arg Asp Val Leu Ala Ser 1 5 10 15 Leu Gly Leu Trp Asn Lys His 20 <210> 6 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Mutant protein fragment <220> <221> MISC_FEATURE <222> (6) <223> Any amino acid except Cys <220> <221> MISC_FEATURE <222> (28)..(28) <223> Any amino acid except Cys <400> 6 Lys Gln Phe Thr Lys Xaa Glu Leu Ser Gln Leu Leu Lys Asp Ile Asp 1 5 10 15 Gly Tyr Gly Gly Ile Ala Leu Pro Glu Leu Ile Xaa Thr Met Phe His 20 25 30 Thr Ser Gly Tyr Asp Thr Gln 35 <210> 7 <211> 39 <212> PRT <213> Artificial Sequence <220> <223> Mutant protein fragment <400> 7 Lys Gln Phe Thr Lys Ala Glu Leu Ser Gln Leu Leu Lys Asp Ile Asp 1 5 10 15 Gly Tyr Gly Gly Ile Ala Leu Pro Glu Leu Ile Ala Thr Met Phe His 20 25 30 Thr Ser Gly Tyr Asp Thr Gln 35

Claims

1. A method for preparing a biologically active complex, the method comprising dissolving a mixture of a polypeptide component in powder form and oleic acid or a pharmaceutically acceptable salt thereof which is also in solid form in an aqueous solvent comprising at least two salts, wherein the first salt is sodium chloride or potassium chloride and the second salt is disodium hydrogen phosphate or potassium dihydrogen phosphate, wherein the method is carried out at 15-25° C.; wherein the polypeptide component is bovine α-lactalbumin or a fragment thereof.

2. The method of claim 1, wherein the aqueous solvent further comprises a third salt, which is sodium dihydrogen phosphate or potassium dihydrogen phosphate.

3. The method of claim 1, further comprising filtering the formed solution.

4. The method of claim 1, further comprising removing the solvent and obtaining the complex in a solid form.

5. The method of claim 4, wherein the solvent is removed by lyophilization.

6. The method of claim 1, wherein the bovine α-lactalbumin fragment is a fragment of at most 50 amino acids.

7. The method of claim 6, wherein the bovine α-lactalbumin fragment comprises an α-helical domain of bovine α-lactalbumin.

8. The method of claim 1, wherein the first salt is sodium chloride.

9. The method of claim 1, wherein the second salt is disodium hydrogen phosphate.

10. The method of claim 2, wherein the third salt is potassium dihydrogen phosphate.

11. The method of claim 4 or claim 5, wherein the complex is reconstituted with sterile water before use.

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