Method for obtaining recombinant human brain-derived neurotrophic factor

Abstract

The invention relates to a human proBDNF mutant protein and a method for recombinantly producing a human brain-derived neurotrophic factor (BDNF) with biological activity.

Description

Technical Field

[0001] The present invention relates to human proBDNF mutant protein and a method for recombinantly producing human brain-derived neurotrophic factor. Existing technology

[0002] Neurotrophins are a family of growth factors that are crucial for regulating the maintenance, differentiation, and survival of neurons. In particular, neurotrophin signaling regulates a diverse set of neural processes, including differentiation, neurite outgrowth, axon pruning, apoptosis, and cell survival during development, adulthood, and after nervous system injury.

[0003] Neurotrophins mediate these effects by binding to different receptors: tropomyosin-related kinase (Trk) receptor tyrosine kinase, p75 neurotrophin receptor and sortilin family members. The human neurotrophin family consists of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and neurotrophin-4 (NT-4). Each of these structurally related neurotrophins is synthesized as a precursor before proteolytic cleavage to produce mature neurotrophins (Huang E. J. et al., Annu. Rev. Biochem. 2003; 72: 609-642).

[0004] Mature human BDNF has 119 amino acid residues (SEQ ID NO: 1, Gray K. et al., FEBS letters. 2008; 582(6): 907-10), which represents the functional domain of the protein, where three disulfide bonds are located (Robinson RC et al., Protein Science. 1999; 8(12): 2589-97). The BDNF protein gene is located on the short arm of human chromosome 11 (Tian F. et al., Amino acids. 2010; 38(4): 1067-74).

[0005] BDNF binds to at least two cell surface receptors that are responsive to this growth factor, TrkB and p75. This signaling molecule has been well documented to have the ability to regulate neuronal plasticity, cell growth, proliferation, cell survival, and long-term memory. In particular, it has been shown to act on certain neurons in the central and peripheral nervous systems, helping to support the survival of existing neurons and promoting the growth and differentiation of new neurons and synapses. In the brain, it is active in the hippocampus, cortex, and basal forebrain—areas crucial for learning, memory, and higher thinking. BDNF is also expressed in the retina, kidney, prostate, motor neurons, and skeletal muscle, and is also present in saliva.

[0006] Currently, BDNF administered as a therapeutic molecule is usually recombinantly produced.

[0007] To proceed to clinical development, recombinant human BDNF (rhBDNF) must comply with current regulations for the production of clinically acceptable therapeutic products. In particular, it is necessary to produce recombinant human BDNF characterized by sufficiently high purity and a consistent composition and activity profile.

[0008] Therefore, an optimal method for producing recombinant human BDNF for therapeutic use must ensure the production of a molecule that meets these requirements and is suitable for large-scale implementation.

[0009] Some methods for obtaining and / or producing rhBDNF have been developed, but they are characterized by several drawbacks, have complex protocols and do not produce a protein with suitable characteristics for therapeutic use.

[0010] Therefore, there is a felt need to develop improved, simple methods for producing biologically active recombinant human BDNF, which ensure high purity of the final product and satisfactory and reproducible biological activity. Summary of the Invention

[0011] The present invention relates to a human proBDNF mutant protein having the amino acid sequence of SEQ ID NO: 3, wherein X1, X2 and X3 are selected from non-basic amino acids and histidine, and X4 is selected from arginine and lysine.

[0012] Another object of the present invention is a nucleic acid encoding a human proBDNF mutant protein as defined above.

[0013] Another object of the present invention is a method for producing human BDNF, comprising the following steps:

[0014] (i) providing a solution of human proBDNF mutant protein as defined above;

[0015] (ii) purifying the human proBDNF mutein from the solution provided in step (i), thereby obtaining a solution of purified human proBDNF mutein; and

[0016] (iii) cleaving the human proBDNF mutant protein in the solution to obtain human BDNF.

[0017] Another object of the present invention is human BDNF obtained or obtainable by the method described above. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Shown are concentration-response (0.23 to 9 μg / mL) proliferation curves of C6 cells incubated with BDNF for 48 hours.

[0019] Figure 2 Figure (A) shows the following electrophoresis analysis results according to Example 9: SeeBlue Plus2 prestained standard (protein molecular weight marker, MK), rhBDNF (reference standard, RS), T0 load (TA1), fraction 1 (TA2), fraction 2 (TA3), fraction 3 (TA4), fraction 4 (TA5), fraction 5 (TA6), flow-through (TA7), and SeeBlue Plus2 prestained standard (protein molecular weight marker, MK); Figure (B) shows the following electrophoresis analysis results according to Example 9: SeeBlue Plus2 prestained standard (protein molecular weight marker, MK), rhBDNF (reference standard, RS), fraction 6 (TA8), fraction 7 (TA9), fraction 8 (TA10), fraction 9 (TA11), fraction 10 diluted at 1:50 (TA12), fraction 10 diluted at 1:20 (TA13), empty lane (TA4). Lane, EL), SeeBlue Plus2 prestained standard (protein molecular weight marker, MK).

[0020] definition

[0021] The term "human wild-type BDNF" or "human BDNF" refers to the human form of the protein BDNF having the amino acid sequence of SEQ ID NO: 1; optionally, the human BDNF may be "recombinant human BDNF" (rhBDNF), i.e., the "human BDNF" is recombinantly produced.

[0022] The term "human wild-type proBDNF" or "human proBDNF" refers to the precursor of human BDNF having the amino acid sequence of SEQ ID NO: 2; optionally, the human proBDNF may be "recombinant human proBDNF" (rh-proBDNF), ie, the "human proBDNF" is recombinantly produced.

[0023] The term "human proBDNF mutant protein" or "human proBDNF mutant" refers to human proBDNF containing one or more amino acid substitutions; optionally, the human proBDNF mutant can be a "recombinant human proBDNF mutant" (rh-proBDNF mutant), that is, the "human proBDNF mutant" is recombinantly produced.

[0024] The term "correctly folded" is used in this article:

[0025] - When referring to human wild-type BDNF / human BDNF, it is intended that human wild-type BDNF has a three-dimensional structure corresponding to the three-dimensional structure of native biologically active human BDNF.

[0026] - when referring to a human proBDNF mutein, means a human proBDNF mutein having a three-dimensional structure that results in the formation of correctly folded human BDNF after cleavage of the pro-sequence with a serine protease.

[0027] The term "non-basic amino acid" refers to any amino acid other than a basic amino acid. DETAILED DESCRIPTION

[0028] As will be explained in more detail in the experimental part, the present inventors have developed a method for the recombinant production of human brain-derived neurotrophic factor (rhBDNF) that is superior to the methods disclosed in the prior art because it is simple and allows obtaining biologically active rhBDNF in high purity and yield.

[0029] This is achieved by expressing human proBDNF mutein as a BDNF precursor as described below, and by using specific process parameters and materials as discussed below.

[0030] Therefore, a first object of the present invention is a human proBDNF mutant protein having the amino acid sequence of SEQ ID NO: 3:

[0031]

[0032] wherein X1 at position 108, X2 at position 109, and X3 at position 110 are selected from non-basic amino acids and histidine, and X4 at position 111 is selected from arginine and lysine.

[0033] Preferably, X1, X2 and X3 are independently selected from alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and histidine (SEQ ID NO: 5).

[0034] More preferably, X1, X2 and X3 are independently selected from valine, alanine, glycine, serine, threonine, tyrosine, asparagine, aspartic acid, glutamine, glutamic acid, methionine and histidine (SEQ ID NO: 6).

[0035] In one preferred embodiment, X1 and X3 are independently selected from alanine and valine (SEQ ID NO: 7); in another preferred embodiment, X2 is selected from valine and serine (SEQ ID NO: 8); in another preferred embodiment, X4 is arginine (SEQ ID NO: 9).

[0036] According to a preferred embodiment, X1 and X3 are independently selected from alanine and valine, X2 is selected from valine and serine, and X4 is arginine (SEQ ID NO: 10).

[0037] A particularly preferred proBDNF mutein according to the invention is the mutein of SEQ ID NO: 4, which corresponds to the mutein having the amino acid sequence of SEQ ID NO: 3, wherein X1 is valine, X2 is serine, X3 is alanine, and X4 is arginine.

[0038] Another object of the present invention is a nucleic acid encoding a human proBDNF mutein as described herein.

[0039] Another object of the present invention is a method for recombinantly producing human BDNF, comprising:

[0040] (i) providing a solution of a human proBDNF mutant protein according to the first object of the present invention,

[0041] (ii) purifying the human proBDNF mutant protein from the solution provided in step (i) to obtain a solution of purified human proBDNF mutant protein, and

[0042] (iii) cleaving the human proBDNF mutant protein in the solution to obtain human BDNF, more particularly recombinant human BDNF.

[0043] Preferably, the method for recombinantly producing human BDNF comprises:

[0044] (i) providing a solution of the human proBDNF mutant protein according to the first object of the present invention by expressing the human proBDNF mutant protein according to the first object of the present invention in host cells cultured in a liquid culture medium, and isolating the human proBDNF mutant protein from the host cells, wherein the human proBDNF mutant protein is correctly folded;

[0045] (ii) purifying the human proBDNF mutein from the solution provided in step (i) by chromatography, preferably using an eluent solution comprising urea, more preferably an eluent solution comprising citric acid and urea, thereby obtaining a solution of purified human proBDNF mutein; and

[0046] (iii) cleaving the prosequence of the human proBDNF mutant protein in the solution to obtain biologically active human BDNF, more particularly recombinant human BDNF; preferably, before the cleavage, the pH of the solution obtained in step (ii) is adjusted to a pH value of 5.8 to 9.

[0047] Preferably, step (i) of the method for recombinantly producing human BDNF comprises the following steps: a) providing a nucleic acid encoding a human proBDNF mutant protein according to the first object of the present invention;

[0048] b) introducing the nucleic acid into an expression vector;

[0049] c) introducing the expression vector into a host cell;

[0050] d) culturing the host cell in a suitable liquid culture medium, wherein the host cell expresses the human proBDNF mutant protein according to the first object of the present invention in the form of inclusion bodies;

[0051] e) lysing the host cells and isolating the human proBDNF mutant protein inclusion bodies produced by the host cells in step d);

[0052] f) dissolving the inclusion bodies in a denaturing solution to obtain a denatured human proBDNF mutant protein solution;

[0053] g) diluting the denatured human proBDNF mutant protein solution into a refolding solution so that the denatured human proBDNF mutant protein presents a correctly folded conformation, thereby obtaining a correctly folded human proBDNF mutant protein solution.

[0054] Preferably, the nucleic acid provided in step a) is DNA.

[0055] Preferably, the nucleic acid provided in step a) has a codon-optimized sequence, more preferably a codon-optimized DNA sequence, for efficient expression in a host cell, preferably in Escherichia coli (E. coli).

[0056] Preferably, the expression vector of step b) is selected from pET28a, pET28b, pET28c, pBR322, pMAL, pUC19 and all derivatives, more preferably, it is pET28a or pET28b or pET28c. However, all suitable expression vectors known in the art can be used for this purpose.

[0057] Preferably, the host cell of step c) is a prokaryotic host cell.

[0058] Suitable prokaryotic host cells that can be used according to the present invention are well known in the art and include, but are not limited to, prokaryotic cells, such as bacteria, e.g., Escherichia coli, Bacillus sp., and Salmonella, which can be transformed with, for example, plasmid DNA, recombinant phage DNA, or cosmid DNA expression vectors containing the nucleic acids of the present invention; kits for such expression systems are commercially available.

[0059] According to a preferred embodiment, the host cell of step c) is E. coli, preferably selected from E. coli BL21 (DE3), JM 108 / 109 (K12), JM106, JM83 and TBI. Any other E. coli strain suitable for expressing recombinant proteins can be used.

[0060] In step c), the expression vector is introduced into the host cell by any method known in the art. Preferably, according to the present invention, the expression vector is introduced into the host cell by temperature shock, wherein the expression vector is transformed into the host cell, preferably Escherichia coli.

[0061] Preferably, in step d), the suitable liquid culture medium in which the host cells are grown is Terrific Broth (TB).

[0062] Preferably, in step d), the liquid culture medium contains kanamycin, preferably at a concentration of 30 μg / ml.

[0063] Preferably, in step d), the liquid culture medium contains a compound that induces the expression of the recombinant protein, preferably isopropyl β-d-1-thiogalactopyranoside (IPTG).

[0064] Preferably, in step d), when the optical density measured at 600 nm reaches 1 (OD 600 =1), a compound that induces recombinant protein expression is added, preferably isopropyl β-d-1-thiogalactopyranoside (IPTG).

[0065] Preferably, the inclusion bodies obtained in step d) may also contain proteins other than the human proBDNF mutant protein of the present invention.

[0066] Preferably, the inclusion bodies contain at least 60%, at least 70%, at least 80% or at least 90% by weight of the human proBDNF mutein according to the invention, based on the total amount of protein present in the inclusion bodies.

[0067] In step e), lysis of the host cells can be performed by conventional methods, such as high pressure homogenization, sonication or lysozyme (Rudolph, R., et al. (1997); Folding proteins. In: Creighton, TE (ed.): Protein Function: A Practical Approach. Oxford University Press, pp. 57-99).

[0068] Preferably, in step e), lysing the host cells is performed by high pressure homogenisation in a lysis buffer. Preferably, the lysis buffer comprises, preferably consists of, 0.1 M Tris buffer at pH 7 containing 0.025 M EDTA.

[0069] Preferably, in step e), the separation of human proBDNF mutein inclusion bodies is performed by sequential centrifugation of the host cell lysate.

[0070] Preferably, in step f), the denaturing solution comprises the following components:

[0071] i. Guanidine hydrochloride (Guanidinium-HCl), 1 to 8 M, preferably 3 to 6 M, more preferably 4 M, cysteine, 1 to 100 mM, preferably 5 mM,

[0072] ii. Tris, 0.001 to 1 M, preferably 0.1 M,

[0073] iii. EDTA, 1 to 50 mM, preferably 10 mM,

[0074] The pH of the denaturing solution is 7 to 10, preferably 8.

[0075] After solubilization of the human proBDNF mutant protein, the protein is refolded to obtain correctly folded human proBDNF mutant protein. For the refolding process, it is important to minimize competing reactions of misfolding and aggregation. To prevent aggregation, refolding is performed at very low protein concentrations (approximately 250 μg / ml) because at high protein concentrations, protein aggregation predominates.

[0076] Preferably, in step g), the refolding solution comprises:

[0077] i. a molecular chaperone, preferably arginine, preferably at a concentration of 0.5 to 1.0 M, more preferably 0.75 M,

[0078] ii. a metal chelator, preferably EDTA, preferably at a concentration of 1 to 10 mM, more preferably 5 mM,

[0079] iii. a redox shuffling system, preferably selected from a combination of L-cystine and L-cysteine ​​and a combination of oxidized glutathione and reduced glutathione, more preferably selected from a combination of 1 mM L-cystine and 5 mM L-cysteine ​​and a combination of 1 mM GSSG (oxidized glutathione) and 5 mM GSH (reduced glutathione);

[0080] And the pH of the refolding solution is 8 to 11.

[0081] Alternative redox rearrangement systems may be used, such as Cystamin / Cysteamin.

[0082] Preferably, 360 ml of the solution of denatured human proBDNF mutein are diluted into 6 L of the refolding solution as disclosed above. Preferably, the concentration of guanidine in the final solution obtained after dilution in the refolding solution is at most 0.3 M.

[0083] According to the present invention, molecular chaperones are compounds that promote protein folding. Such compounds are known to those skilled in the art. They can aid folding in a variety of ways. According to the present invention, arginine is a preferred molecular chaperone. Arginine destabilizes incorrectly folded intermediates, causing them to at least partially unfold (from thermodynamic dead ends) and thus allow them to fold correctly again.

[0084] Preferably, step g) further comprises the step of adjusting the pH of the final solution in which the human proBDNF mutein is refolded to a value of 8.5 to 9.5, more preferably to a value of 8.8 to 9.2, even more preferably to a value of 9.1.

[0085] By carrying out the method according to the invention with denaturation and subsequent refolding, an aqueous solution of correctly folded human proBDNF mutein is obtained.

[0086] Preferably, in step (ii) of the method for recombinantly producing human BDNF, the solution of purified human proBDNF mutant protein obtained contains urea, more preferably, contains citric acid and urea.

[0087] Preferably, the concentration of urea in the solution is less than 2 M, more preferably 1 M. Preferably, the concentration of citric acid in the solution is 5 mM to 100 mM, more preferably 50 mM. Preferably, the pH of the solution is 3 to 5.5, preferably pH 4.

[0088] Preferably, step (ii) comprises purifying the human proBDNF mutein from the solution provided in step (i) by chromatographic purification.

[0089] More preferably, in the chromatographic purification, an eluent solution comprising urea is used, more preferably an eluent solution comprising citric acid and urea. According to this embodiment, the eluate obtained in the chromatographic purification can be used directly in step (iii).

[0090] Preferably, the concentration of urea in the eluent solution is less than 2 M, more preferably 1 M. Preferably, the concentration of citric acid in the eluent solution is 5 mM to 100 mM, more preferably 50 mM. Preferably, the pH of the eluent solution is 3 to 5.5, preferably pH 4.

[0091] Preferably, the chromatographic purification is performed by mixed mode chromatography.

[0092] In one embodiment, the pH of the solution provided in step (i) is adjusted to 8 before loading onto the chromatography column.

[0093] In an alternative embodiment, the pH of the solution provided in step (i) is not altered before loading onto the chromatography column and is therefore between 8 and 11, preferably between 8.5 and 9.5, more preferably between 8.8 and 9.2, even more preferably 9.1.

[0094] The most preferred column for the chromatographic purification in step ii) is a column with a synthetic affinity ligand, preferably 4-mercapto-ethyl-pyridine (MEP Hypercell; Sartorius). The advantages of this medium are that binding is independent of ionic strength, no salt accumulation is required, and higher flow rates are possible to speed up the process. In addition, elution is performed by pH change.

[0095] Other mixed-mode material columns are known and can be used. Examples include, but are not limited to, MEP (Sartorius; affinity ligand: 4-mercaptoethylpyridine), HEA (Sartorius; affinity ligand: hexylamino), PPA (Sartorius, affinity ligand: phenylpropylamino), MBI (Sartorius; affinity ligand: 2-mercapto-5-benzimidazole sulfonic acid), Capto MMC (GEHC), Capto adhere (GEHC; affinity ligand: N-benzyl-N-methylethanolamine), CHT hydroxyapatite (BioRad), and CHT fluoroapatite. MEP, HEA, PPA, and MBI columns have hydrophobic binding, while Capto MMC is a cation exchanger with mixed-mode functionality, and Capto adhere is an anion exchanger with mixed-mode functionality. BioRad columns are ion exchange columns with a hydrophobic component.

[0096] Preferably, in step (iii) of the method for recombinantly producing human BDNF, the cleavage comprises adjusting the pH of the solution of the purified human proBDNF mutein obtained in step (ii) to a pH value of 5.8 to 9, more preferably a pH value of 5.8 to 8, more preferably a pH value of 5.8 to 7, more preferably a pH value of 5.8 to 6.5, and even more preferably a pH value of 6, and cleaving the human proBDNF mutein in the solution to obtain biologically active human BDNF, more particularly recombinant human BDNF.

[0097] Preferably, the cleavage of the human proBDNF mutant protein is performed by a trypsin-like protease, more preferably by trypsin.

[0098] Proteases with trypsin-like substrate specificity cleave proteins without digesting the active portion of the protein molecule. Trypsin-like proteases cleave peptide bonds following positively charged amino acids (e.g., arginine or lysine). Preferably, trypsin is used to cleave the pro-sequence, but other proteases can be used instead. It should be noted that cleavage is not limited to trypsin itself, but can also involve other proteases with trypsin-like substrates. Generally, the ratio of human proBDNF mutein to trypsin (or other proteases) is appropriately adjusted so that properly folded mature human BDNF (more specifically, recombinant human BDNF) will not be cleaved by the protease. In contrast, denatured protein and folding intermediates expose sequences that are susceptible to protease attack.

[0099] Preferably, for cleavage of human proBDNF mutein into human BDNF (more particularly, recombinant human BDNF), the ratio of trypsin-like protease (preferably trypsin) to human proBDNF mutein is 1:20,000 to 1:30,000 w / w, more preferably 1:23,000 to 1:27,000 w / w, and most preferably 1:25,000 w / w. In a preferred embodiment, cleavage is performed at room temperature for at least 12 hours, preferably for more than 13 hours, more preferably for a period of 12 to 22 hours, even more preferably for 13 to 17 hours, and most preferably for 15 or 16 hours.

[0100] Preferably, step (iii) further comprises the step of inhibiting the activity of a trypsin-like protease.

[0101] Preferably, the inhibiting step is performed by adjusting the pH of the solution of human BDNF obtained in step (iii) to a value of 4.5 to 5.5, preferably 5.

[0102] Preferably, the inhibition step is performed by diluting the solution obtained in step (iii) with a solution of arginine, citric acid and urea to obtain a final solution having a pH of 4.5 to 5.5 and an arginine concentration of 0.1 M to 0.2 M, more preferably a concentration of 0.125 M. Preferably, the solution comprises: 1 M arginine, 50 mM citric acid and 1 M urea and is added to the solution of step (iii) at a dilution ratio of 1 / 8 to obtain a pH drop of about 5.2 and a final arginine concentration of 0.125 M. Under these conditions, arginine has the following advantages:

[0103] i) naturally lowers pH, ii) acts as a molecular chaperone and prevents protein aggregation, iii) inhibits trypsin activity as a substrate competitor, and iv) when present, is compatible with the subsequent chromatography method of step (iv) for purifying the human BDNF.

[0104] According to a preferred embodiment, the method of the present invention further comprises the step (iv): purifying the human BDNF obtained in the step (iii).

[0105] Preferably, the purification in step (iv) comprises separating trypsin and trypsin-digested product-related impurities from BDNF.

[0106] Preferably, the purification also reduces host cell protein (HCP), endotoxin and DNA impurities.

[0107] Any method known in the art for protein purification may be used in step (iv).

[0108] Preferably, said purification in step (iv) comprises one or more chromatographic purification steps.

[0109] Preferably, a Sepharose column is used in the one or more chromatographic purification steps, more preferably a SP Sepharose High Performance, Phenyl Sepharose 6 Fast Flow or Q Sepharose Fast Flow column.

[0110] Preferably, step (iv) comprises chromatographic purification of human BDNF on an SP Sepharose High Performance column, more preferably, followed by further purification on a Phenyl Sepharose Fast Flow low substitution column.

[0111] The purity of the final product BDNF produced from the human proBDNF mutein can be analyzed by SDS-PAGE, RP-HPLC, SE-HPLC, IEX-HPLC, RP-UPLC, SE-UPLC, and IEX-UPLC.

[0112] Preferably, the purity of human BDNF (more particularly recombinant human BDNF) obtained by the method according to the present invention as measured by RP-UPLC is at least 94%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%.

[0113] Such high purity levels are achievable due to specific features of the process according to the invention identified by the inventors.

[0114] The present inventors have discovered that, for the production of human BDNF (more particularly, recombinant human BDNF), cleavage of the prosequence from the human proBDNF mutein according to the present invention by a trypsin-like protease has a higher specificity than cleavage of the prosequence from wild-type proBDNF. Thus, this method allows for the efficient production of correctly folded, full-length BDNF (more particularly, recombinant human BDNF), reduces the production of incorrectly cleaved byproducts, and allows for efficient and easier purification of the protein.

[0115] In this regard, Example 9 highlights the drawbacks and difficulties of cleaving wild-type proBDNF by trypsin-like proteases, which results in a very low yield and quality of correctly digested mature full-length BDNF (more particularly, recombinant human BDNF), which is associated with an unexpectedly high number of incorrectly digested and over-digested BDNF forms.

[0116] Furthermore, as will be described in more detail in the experimental section below, the present inventors have found that it is advantageous to use a solution comprising urea as cleavage buffer, since under this condition the protein is correctly cleaved and no precipitation is observed.

[0117] Furthermore, the present inventors have discovered that when the purification of step (ii) is performed by chromatographic purification using a solution containing urea and citric acid as an eluent, both step (iii) of cleaving the human proBDNF mutant protein and step (iv) of purifying the resulting BDNF can be performed in the same solution eluted from the chromatographic column of step (ii), simply by adjusting the pH to the desired value for the specific process step by adding an acid or base to the solution containing citric acid and urea. This represents a considerable advantage, as the operational flow is simplified and the process requires the use of less material, which allows for facilitating further purification steps and obtaining a highly pure final product. In particular, the use of an eluent containing citric acid and urea in step (ii) avoids the need for buffer exchange by tangential flow filtration at the end of the purification step and prior to the cleavage step. Furthermore, the combination of citric acid and urea not only allows for easy modification of the solution pH, but also prevents protein aggregation and, unexpectedly, does not interfere with trypsin activity.

[0118] Another object of the present invention is the use of a human proBDNF mutein according to any of the above embodiments of the first object of the invention in a method for producing human BDNF, more particularly recombinant human BDNF.

[0119] Another object of the present invention is human BDNF (more particularly recombinant human BDNF) obtainable by or through the method according to the invention.

[0120] Another object of the present invention is human BDNF (more particularly recombinant human BDNF) having a purity of at least 94%, preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, even more preferably at least 99%, as measured by RP-UPLC.

[0121] Another object of the present invention is a human BDNF (more particularly, recombinant human BDNF) composition comprising a peptide impurity having the following sequence:

[0122]

[0123] or

[0124]

[0125] The amount of the peptide impurities is equal to or lower than 5%, preferably the amount is equal to or lower than 4%.

[0126] Another object of the present invention is a pharmaceutical composition comprising human BDNF (more particularly recombinant human BDNF) as described above and at least one pharmaceutically acceptable ingredient.

[0127] In one embodiment, the pharmaceutically acceptable ingredients can be diluents, carriers, fillers, salts, buffers, stabilizers, penetration enhancers. Techniques for formulating the pharmaceutical compositions of the present invention can be found in standard textbooks, such as "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, latest edition.

[0128] The present invention will be further described in the following examples, which do not limit the scope of the invention as defined in the claims.

[0129] Example

[0130] Example 1 - Cloning DNA encoding proBDNF mutant protein into expression vector and inserting into E. coli

[0131] A synthetic gene corresponding to SEQ ID NO: 4 of the human proBDNF mutant protein was prepared in which the triplet codons were optimized for their expression in Escherichia coli. For cloning, the initiating methionine residue (CC AT GG), and an XhoI restriction site was inserted after the last stop codon.

[0132] After digestion by NcoI and XhoI restriction enzymes, the corresponding DNA fragment encoding proBDNF ORF was introduced into the pET28a expression vector (Novagen) cut with the same enzymes.

[0133] The obtained expression vector was transformed into E. coli BL21 (DE3) strain by temperature shock according to the supplier's instructions (BL21 (DE3) Chemi Competent cells ref 156-3003; BioRad).

[0134] Example 2 - Fermentation and induction of protein expression

[0135] A single colony of the recombinant E. coli strain was inoculated into 30 ml of Terrific Broth (TB) medium (Sigma Aldrich) containing 30 μg / ml kanamycin, which was then incubated overnight at 37° C. with stirring. The next day, 300 ml of fresh TB containing 30 μg / ml kanamycin was inoculated with 3 ml of the overnight culture, and when the OD 600nm When the C value reached 1, 1 mM isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to the culture medium to induce the expression of the recombinant protein and the culture was continued for another 3 hours.

[0136] To monitor the overexpression of the target protein during the fermentation process, samples were analyzed by SDS-PAGE before and after induction.

[0137] Since human proBDNF mutant protein has three intrachain disulfide bridges, the protein accumulates in the cytoplasm of cells in the form of inclusion bodies (iB).

[0138] Example 3 - Isolation and solubilization of inclusion bodies

[0139] Cells were harvested by centrifugation, resuspended in lysis buffer (0.1 M tris, 0.025 M EDTA, pH 7.0) at 1.5 weight / volume, and homogenized for four cycles at 800 bar using a NS1001L2K Niro Soavi high pressure homogenizer. The homogenate obtained was diluted 1:0.5 with a Brij 35 (60 g / L) formulation and stirred for 30 minutes.

[0140] The pellet corresponding to the inclusion bodies was isolated by centrifugation (8500 rpm, Sorval GS3 rotor) and washed twice with Tris 0.1 M pH 7.0 at 0.4 to 1 weight / volume. The mixture was centrifuged (7500 rpm, Sorval GS3 rotor) and the pellet was dissolved in a guanidine solution to a final concentration of 4 M. In particular, the following guanidine solution was used:

[0141] i. Guanidine hydrochloride 4M, cysteine ​​5mM,

[0142] ii. Tris 0.1M,

[0143] iii. EDTA 10mM,

[0144] iv.pH 8.0

[0145] Example 4 - Refolding of rh-proBDNF mutein

[0146] To prepare correctly folded human proBDNF muteins according to the present invention (i.e., rh-proBDNF muteins), 360 mL of the dissolved material obtained according to Example 3 was diluted into 6 L of a refolding solution having the following composition: 0.75 M arginine-HCl, 5 mM EDTA, 5 mM cysteine-HCl, and 1 mM cystine, and maintained at a temperature of 0 to 10° C., wherein the human proBDNF mutein exhibits a correctly folded conformation. After dilution into the refolding solution, the guanidine concentration was at most 0.3 M. The pH of the refolding solution was adjusted to 9.1 with sodium hydroxide.

[0147] The performance of the refolding reaction was analyzed by RP-HPLC and estimated to be 55% of the initial solubilized protein.

[0148] Example 5 - Purification of human proBDNF mutant protein from refolding solution

[0149] To purify human proBDNF mutant protein from the refolded solution, hydrophobic induced charge chromatography was performed using a column with the synthetic affinity ligand 4-mercaptoethylpyridine (MEP). Prior to loading the solution with the refolded protein, the column was equilibrated with five column volumes of 0.75 M arginine-HCl, 5 mM EDTA, pH 9.1.

[0150] Two different experimental setups, designated 5a and 5b, were tested for elution of protein from the column and are summarized below.

[0151] Setup 5a → In this setup, the refolded human proBDNF mutein was loaded onto a MEP column after filtration, the column was washed with 0.1 M Tris pH 8.0, and the protein was eluted in 0.75 M arginine-HCl, 0.05 M acetate buffer, pH 4.0. The eluted fraction containing the protein was then buffer-exchanged by tangential flow filtration on a 10 kDa cutoff cassette, followed by six consecutive diafiltrations with 0.025 M sodium phosphate pH 6.5 to remove arginine and facilitate subsequent enzymatic cleavage of the human proBDNF mutein, as discussed in the Examples below.

[0152] • Setup 5b→ In this setup, refolded human proBDNF mutein was directly loaded in refolding solution at pH 9.1, the column was washed with Tris buffer, pH 8.0, and the protein was eluted with a solution containing 50 mM citric acid (pH 4.0) and 1 M urea.

[0153] Without being bound by theory or principle, the present inventors found that setup 5b, in which human proBDNF muteins were eluted with a solution containing citric acid and urea, was superior to setup 5a, in which human proBDNF muteins were eluted in an arginine buffer and then the arginine buffer was exchanged for a phosphate buffer, for reasons that will be explained in the following Examples.

[0154] Example 6 - Cleavage of human proBDNF mutant protein to obtain mature rhBDNF

[0155] Once eluted from the MEP column, the purified human proBDNF mutein is cleaved to form mature BDNF (i.e., rhBDNF). Trypsin is a preferred enzyme for cleaving human proBDNF mutein to obtain mature BDNF because it cleaves after arginine (R) or lysine (K) and is commercially available in GMP grade. Therefore, the purified protein solution obtained according to Example 5 was incubated with trypsin. The pH of the protein solution was adjusted to a value of 6.5 or 6.0 (see Table 1 below) to facilitate enzymatic cleavage of the human proBDNF mutein by trypsin.

[0156] The inventors tested several experimental setups for the cleavage step, designated 6a to 6g, the conditions of which are summarized in Table 1 below and discussed below.

[0157] Table 1

[0158]

[0159] To purify human proBDNF mutein and subsequently cleave it, the present inventors first tried purification setup 5a described in Example 5, followed by each of the cleavage setups 6a to 6e described in Table 1 .

[0160] When cutting setting 6a was used, the formation of a large amount of precipitate was observed as the digestion progressed during the incubation period.

[0161] Without being bound by theory or principle, the inventors hypothesize that the precipitate is due to peptides produced by trypsin activity that aggregate as digestion progresses. As dissolved material aggregates and precipitates, the resulting soluble rhBDNF is carried along with it due to a carry-over phenomenon.

[0162] Since trypsin cleaves after lysine (K) and arginine (R) residues, observing the pro-protein sequence, all of the following peptides can be generated during digestion.

[0163] 1. MAPMK (SEQ ID NO: 11)

[0164] 2.EANIRR (SEQ ID NO: 12)

[0165] 3. GQGGLAYPGVR (SEQ ID NO: 13)

[0166] 4. THGTLESVNGPKK (SEQ ID NO: 14)

[0167] 5.AGSR (SEQ ID NO: 15)

[0168] 6.GLTSLADTFEHVIEELLDEDQKK(SEQ ID NO:16)

[0169] 7VR

[0170] 8. PNEENNK (SEQ ID NO: 17)

[0171] 9. DADLYTSRR (SEQ ID NO: 18)

[0172] 10.VMLSSQVPLEPPLLFLLEEYK(SEQ ID NO:19)

[0173] 11.NYLDAANMSMVSAR(SEQ ID NO:20)

[0174] In addition, an additional hexapeptide corresponding to the first six amino acids of BDNF with the following sequence can be generated during digestion: HSDPAR (SEQ ID NO: 24). Indeed, this peptide, which carries an arginine at its carboxyl terminus, is accessible during trypsin digestion, and its cleavage generates a truncated form of BDNF, hereinafter designated "hyperdigested," with the following sequence:

[0175]

[0176] As it has been shown that the first seven N-terminal amino acids of BDNF (HSDPARR, SEQ ID NO: 21) are responsible for the major interaction between BDNF and its receptor TrkB, it can be assumed that this truncated form of BDNF is inactive. Therefore, during trypsin cleavage, it is necessary not only to control the precipitation of the product, but also to control the generation of over-digested BDNF forms, which need to be removed by successive chromatography together with the peptides corresponding to the pro part digestion.

[0177] In an attempt to maintain the protein in solution, after performing purification setup 5a described in Example 5, each of the cleavage setups 6b to 6e reported in Table 1 above was tested instead of cleavage setup 6a. These setups involve adding various substances to the protein solution obtained after procedure 5a in an attempt to maintain the protein in solution as digestion proceeds. However, as enzymatic digestion proceeds, the protein precipitates even in the presence of these additives.

[0178] The present inventors then decided to add urea (1 M) to the cleavage buffer.

[0179] Without being bound by theory or rationale, the inventors hypothesize that urea may act as a disaggregating agent and prevent the protein from precipitating from the solution. However, the use of urea may also be detrimental to the production of the desired BDNF because it may denature the proBDNF mutant protein and make new cleavage sites previously buried within the refolded protein accessible.

[0180] Surprisingly, the present inventors have found that when urea is added to the cleavage buffer, the protein is correctly cleaved in only two hours and no precipitation is observed.

[0181] Based on these results, it was decided that the preferred cleavage setup involved the addition of 1 M urea to the cleavage buffer.

[0182] Then, to further simplify the process, the inventors tested a different purification setup, namely setup 5b described in Example 5 above, followed by cleavage setup 6f described in Table 1 above.

[0183] Without being bound by theory or principle, the inventors have found that the combination of the conditions of purification setup 5b and cleavage setup 6f is advantageous as it leads to a significant simplification of the operational flow of the method according to the invention.

[0184] In fact, the setup 5b disclosed in Example 5, in which human proBDNF mutein was eluted with a solution containing citric acid and urea, was superior to the setup 5a, in which human proBDNF mutein was eluted in arginine buffer and then the arginine buffer was exchanged for phosphate buffer, due to the following.

[0185] When using setup 5b instead of setup 5a, the eluate containing purified human proBDNF mutant protein eluted from the MEP column can be used directly as a buffer solution for subsequent trypsin cleavage of human proBDNF mutant protein from mature BDNF and for continuous ion exchange chromatography to purify mature BDNF. This is achieved by adding acid or base to the solution containing citric acid to adjust the pH to the value required in the specific process step. This is possible because citric acid has three pKa values ​​at 25°C (5.21, 4.28, and 2.92) and can therefore cover the entire required buffer range in this method, from pH 4.0 for elution from MEP to pH 6.0 or 6.5 for enzymatic digestion.

[0186] The presence of urea in the elution solution of Set 5b is also advantageous not only because it prevents aggregation and precipitation during the subsequent human proBDNF mutein cleavage step, but also because it prevents the use of arginine during the human proBDNF mutein purification step, thereby avoiding the need to perform tangential flow filtration to prepare the protein solution for trypsin digestion, as in Set 5a. While arginine is known to help maintain proteins in solution and can therefore be used to counteract the natural tendency of proBDNF to aggregate, it is also a substrate for trypsin and must therefore be removed prior to cleavage of the human proBDNF mutein into BDNF. The removal of arginine complicates the process flow, a drawback addressed by the use of the eluent according to the present invention, which does not involve the use of arginine. The omission of arginine during the human proBDNF mutein purification step also improves the efficiency of subsequent trypsin cleavage of the human proBDNF mutein. Consequently, less trypsin needs to be used, significantly simplifying the final purification of the resulting mature BDNF.

[0187] Based on all of the above, the combination of citric acid and urea is the preferred solution for eluting the MEP column, performing the enzymatic digestion, and loading the digested material onto another Cation Exchange Chromatography (CEX) column, all in a compatible buffer.

[0188] Finally, the parameters of cutting setting 6f were slightly modified to increase the digestion time, thereby allowing for some in-process control. Cutting setting 6g was therefore tested. In this particular setting, the pH of the solution was adjusted to 6.0, at which the activity of trypsin was lower than at 6.5, and the trypsin / proBDNF mutant protein ratio was changed from 1 / 7500 to 1 / 25000. The temperature was maintained at 22°C for all reaction times.

[0189] The trypsin / proBDNF mutant protein ratio is surprisingly very low. Since trypsin needs to be removed from the final product, having a lower amount of trypsin is advantageous and leads to a simplification of the purification process. In fact, the isoelectric point of trypsin is 10.5, which means that it will bind to the SP Sepharose column that is used to purify BDNF from the fragments produced during digestion after cleavage (see Example 7 below). Therefore, the less trypsin used during the cleavage step, the better, both in terms of achieving higher cleavage specificity and easier purification of the mature BDNF formed.

[0190] Different digestion times (i.e., 13 hours, 14 hours, 15 hours, 16 hours, and 17 hours) were tested for cleavage of human proBDNF mutein. These digestion times were long enough to allow for the introduction of some in-process controls. The results are reported in Table 2 below.

[0191] Table 2

[0192]

[0193] The reaction temperature was maintained at 22°C for all digestion times used.

[0194] As can be seen, all digestion times tested (i.e., 13 hours and above) resulted in satisfactory yields of BDNF from human proBDNF muteins, with minimal formation of overdigested forms. In particular, human proBDNF muteins were almost completely gone by 15 hours (2.56% of the initial human proBDNF mutein remaining), with approximately 38% of overdigested forms remaining, an amount that could be removed by continuous chromatography. These parameters were also effective for 16 hours, allowing ample time for processing the solution.

[0195] Based on these results, purification setting 5b and subsequent cleavage setting 6g were selected as preferred conditions for purification of refolded human proBDNF mutein and subsequent cleavage to mature BDNF, respectively.

[0196] Example 7 - Purification of active rhBDNF

[0197] After the trypsin digestion was completed, the pH of the cleavage solution containing BDNF (i.e., rhBDNF) was adjusted to 5.0 by adding a solution of 1 M arginine, 50 mM citric acid, and 1 M urea at a ratio of 1 / 8 to inhibit the activity of trypsin, and then the solution was loaded onto an SP Sepharose high performance column to deplete trypsin, cleavage byproducts, and other impurities. The solution was filtered before loading to protect the column.

[0198] The column was pre-equilibrated with 50 mM citric acid, 1 M urea pH 5.0. The column was then washed with 25 mM phosphate buffer pH 6.5 and the protein was subsequently eluted in the same buffer using a step gradient of increasing salt concentration.

[0199] During elution, the different peaks were fractionated, with fractions 9 and 10 corresponding to the main peak. All fractions were then analyzed by RP-UPLC and the results are reported in Table 3 below.

[0200] Table 3

[0201]

[0202] The results showed that BDNF protein eluted in a major peak, with the percentage of the over-digested form decreasing from 33% present in the load material to approximately 10%.

[0203] Fractions 9 and 10 were combined, and the resulting solution was adjusted to a 2 M NaCl concentration.

[0204] At this point, the solution was filtered and loaded onto a Phenyl Sepharose Fast Flow low substitution column. The column was pre-equilibrated with phosphate buffer pH 6.5, 2 M NaCl, and the protein was eluted by decreasing the salt concentration.

[0205] The protein was eluted as a single fraction corresponding to 5 column volumes.

[0206] Analysis of this fraction determined the purity of the protein to be approximately 94% w / w; this fraction also contained product-related impurities, primarily oxidized forms of rhBDNF, approximately 1% w / w, and the over-digested form of BDNF described above (SEQ ID NO: 23), approximately 5% w / w.

[0207] Example 8 - Testing for the biological activity of rhBDNF

[0208] The biological activity of recombinant human BDNF obtained according to the method disclosed above was tested by using rat C6 glioma cells expressing high levels of the BDNF TrkB receptor (Xiong J. et al., Oncology Letters 2015; 10: 223-227). It has been shown that mature BDNF promotes C6 cell growth and survival in a dose-dependent manner through this receptor (Xiong J. et al., Oncology Reports 2013; 30 (6): 2719-2724). C6 cells were plated in 96-well microplates at a density of 5000 cells / well and treated with rhBDNF at concentrations ranging from 0.23 to 9 μg / mL. After incubation for 48 hours, CellTiter Aqueous single solution cell proliferation assay (CellTiter AQ ueous One Solution Cell Proliferation Assay) (MTS assay) was used to determine the biological activity of rhBDNF from the number of viable C6 cells.

[0209] EC was determined by using GraphPad statistical software for rhBDNF produced according to the method disclosed above. 50 (corresponding to the concentration of rhBDNF required to induce 50% cell proliferation), and an average value of approximately 3 μg / mL (0.11 μM) was obtained ( Figure 1 ).

[0210] Example 9 - Production of full-length rhBDNF by the method according to the invention starting from wild-type human proBDNF Analysis of yield and purity.

[0211] The inventors also analyzed the yield and purity of correctly folded mature full-length rhBDNF obtained by the method of the present invention starting from human wild-type proBDNF instead of the human proBDNF mutein according to the present invention.

[0212] In particular, synthetic genes corresponding to human wild-type proBDNF of SEQ ID NO: 2 were prepared in which the codon triplets were optimized for their expression in E. coli.

[0213] The protocol reported in Example 1 was followed: DNA encoding wild-type proBDNF was cloned into an expression vector and subsequently inserted into E. coli.

[0214] The fermentation and induction of protein expression, isolation and solubilization of inclusion bodies and the refolding steps of human wild-type proBDNF were performed according to the protocols reported in Examples 2, 3 and 4, respectively.

[0215] Purification of human wild-type proBDNF was performed according to setup 5b of the protocol reported in Example 5.

[0216] The purified human wild-type proBDNF thus obtained was cleaved by trypsin to obtain human mature full-length BDNF.

[0217] Specifically, 735 ml of purified human wild-type proBDNF, corresponding to approximately 400 mg of proBDNF (and corresponding to a proBDNF concentration of approximately 550 μg / mL), were digested with trypsin (trypsin ratio 1 / 25.000) at pH 6.0 and 20°C.

[0218] After 16 hours of digestion, the digested material was analyzed by RP-UPLC and the results are reported in Table 4 below.

[0219]

[0220] Table 4: RP-UPLC analysis of digested material.

[0221] As shown in Table 4 reported above, the over-digested forms accounted for approximately 14% of the properly digested mature rhBDNF. Additionally, the purity of the BDNF was less than 50% (ie, 48.78).

[0222] Digestion was stopped by adding a solution of 1 M arginine, 50 mM citric acid, and 1 M urea to the digested material, followed by incubation at room temperature for 1 hour and then filtration.

[0223] The digested solution thus obtained contained a total of approximately 200 mg of rhBDNF, which was a mixture of apparently correctly digested mature full-length rhBDNF forms, hyper- and hypo-digested rhBDNF forms, and was loaded onto an SP Sepharose High Performance column equilibrated with 50 mM citric acid, 1 M urea pH 5.0.

[0224] After loading the digested solution, the column was washed with 25 mM phosphate buffer (pH 6.5), and then the rhBDNF forms were eluted in the same buffer using a step gradient of increasing salt concentration.

[0225] During elution, different peaks were fractionated, and the fractions thus obtained were subsequently analyzed by RP-UPLC.

[0226] The RP-UPLC results are reported in Table 5 below.

[0227]

[0228] Table 5: RP-UPLC analysis of fractions 1 to 10; T0 corresponds to the analysis of the sample at the beginning of its loading onto the column.

[0229] The results showed that only less than 25 mg (ie, 24.48 mg) of rhBDNF was recovered in fractions 5 to 8, which corresponded to approximately 13% of the rhBDNF (200 mg) contained in the digested solution loaded on the SP column.

[0230] Considering in particular fractions 6 and 7, where correctly digested mature full-length rhBDNF was primarily expected to elute, only approximately 12 mg of rhBDNF was detected; in addition, an unexpectedly high percentage of over-digested BDNF forms was detected.

[0231] Fractions 1 to 10 were then subjected to SDS-polyacrylamide (SDS-PAGE) gel analysis to obtain high-resolution separation of the rhBDNF forms contained therein.

[0232] Prepare SDS-PAGE gels according to the following protocol.

[0233] SDS-PAGE analysis was performed under reducing conditions. Samples were mixed with NuPAGE 4× LDS sample buffer (Invitrogen) and NuPAGE 10× reducing agent (Invitrogen) and denatured continuously at 100°C for 10 minutes before loading onto the gel. Approximately 2.33 μg of rhBDNF reference standard (homemade, Dompé) was loaded onto the gel, while for the analytical test samples (fractions 1 to 10), the loading amount depended on their respective measured concentrations. NuPAGE Novex 4% to 12%, Bis-Tris polyacrylamide gels (Invitrogen) were assembled in an XCell Surelock Mini Cell apparatus (Invitrogen), and the inner chamber was filled with 200 mL of 1× running buffer supplemented with an antioxidant (NuPAGE Antioxidant (Invitrogen)), while the outer chamber was filled with approximately 300 mL of 1× running buffer. 1× running buffer was prepared from NuPAGE 20× MES SDS running buffer stock solution (Invitrogen). The gel was run at a constant voltage of 200 V for 35 minutes; afterward, the gel was fixed and stained with a NOVEX Colloidal Blue Staining Kit (Invitrogen).

[0234] Figure 2 A and 2B report the results of electrophoresis analysis based on the following gel lanes:

[0235]

[0236]

[0237] As expected, fractions 1 to 5 contained primarily underdigested rhBDNF forms (which are higher molecular weight species relative to mature full-length rhBDNF), while fractions 8 and 9 contained primarily overdigested rhBDNF forms (which are lower molecular weight species relative to mature full-length rhBDNF).

[0238] As expected, mature full-length rhBDNF was observed in fractions 6 and 7; however, these bands were heavily contaminated with over-digested rhBDNF forms with almost the same intensity as mature full-length rhBDNF.

[0239] The material eluting at a higher salt concentration in fraction 10 corresponds to a large amount of protein, as this band produced a strong signal even after a 50-fold dilution. The inventors noted that this band was also contaminated with over-digested forms of rhBDNF.

[0240] To further characterize the eluted material, fractions 6, 7, and 10 described above were analyzed by mass spectrometry according to the following protocol.

[0241] All samples were injected and analyzed "as received" on a HPLC-MS Orbitrap Fusion system (ThermoFisher).

[0242] The chromatographic conditions used for the analysis are reported below:

[0243] -Analytical column: Acquity Premier Protein C4 100×2.1 mm, 1.7 μm (Waters); - eluent (mobile phase): A = H2O 0.05% TFA; B = CH3CN 0.05% TFA; - flow rate: 0.3 mL / min;

[0244] - Injection volume: 2 / 20 μL;

[0245] - Running time: 13 minutes;

[0246] - Column temperature: 60°C;

[0247] - Sample temperature: 5°C;

[0248] -Detection: MS (full scan);

[0249] - Typical retention time: rhBDNF = 6.8 minutes;

[0250] -gradient:

[0251] Time (minutes) Flow rate (mL / min) ％A ％B 0 0.3 80 20 1 0.3 80 20 6 0.3 60 60 6.1 0.3 30 70 8 0.3 30 70 8.1 0.3 80 20 13 0.3 80 20

[0252] The mass spectrometry conditions used for sample analysis are reported below:

[0253]

[0254]

[0255] The mass spectrometry results of fraction 6 are reported in Table 6 below.

[0256]

[0257] Table 6: Mass spectrometry results of fraction 6.

[0258] The results showed that fraction 6 contained correctly digested mature full-length rhBDNF (27.84%), as expected, but it was heavily contaminated with over-digested rhBDNF forms (31.87%) and contained trace amounts of a form with an additional arginine residue at the NH2-terminus of the protein (rhR-BDNF, SEQ ID NO: 25).

[0259] The mass spectrometry results of fraction 7 are reported in Table 7 below.

[0260]

[0261] Table 7: Mass spectrometry results of fraction 7.

[0262] As expected, the results for fraction 7 also showed correctly digested mature full-length rhBDNF (30.22%), as observed in fraction 6, but this fraction was heavily contaminated with over-digested rhBDNF forms (34.06%). In fraction 7, trace amounts of a form with an additional arginine residue at the NH2-terminus of the protein (rhR-BDNF, SEQ ID NO: 25) were also observed.

[0263] The mass spectrometry results of fraction 10 are reported in Table 8 below.

[0264]

[0265] Table 8: Mass spectrometry results of fraction 10.

[0266] The results showed that less than 9% of the digested material in fraction 10 corresponded to properly digested full-length rhBDNF. In fact, this fraction contained mainly rhBDNF forms with three additional amino acids (VRR-BDNF, SEQ ID NO: 26, 25.82%) and one additional arginine (R-BDNF, SEQ ID NO: 25, 26.72%) at the NH2-terminus of the protein; in addition, fraction 10 contained 13% of over-digested rhBDNF forms.

[0267] In view of the above, the results show that carrying out the method according to the invention starting from wild-type human proBDNF instead of the proBDNF mutein according to the invention results in a very low yield of correctly digested mature full-length rhBDNF and unexpectedly high amounts of impurities, i.e. incorrectly digested rhBDNF forms and over-digested BDNF forms (SEQ ID NO: 23).

[0268] Due to said unexpectedly high amount of impurities, it was almost impossible to purify the correct full-length mature recombinant human BDNF starting from wild-type human pro-BDNF by performing the method according to the invention.

Claims

1. A human proBDNF mutant protein having the following amino acid sequence: wherein X1, X2 and X3 are selected from non-basic amino acids and histidine, and X4 is selected from arginine and lysine.

2. The human proBDNF mutant protein according to claim 1, wherein X1, X2 and X3 are independently selected from alanine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine and histidine; (SEQ ID NO: 5), preferably selected from valine, alanine, glycine, serine, threonine, tyrosine, asparagine, aspartic acid, glutamine, glutamic acid, methionine and histidine (SEQ ID NO: 6). 3 . The human proBDNF mutant protein according to claim 1 , wherein X1 and X3 are independently selected from alanine and valine; X2 is selected from valine and serine; and X4 is arginine (SEQ ID NO: 10). 4 . The human proBDNF mutein according to claim 1 , wherein X1 is valine, X2 is serine, X3 is alanine and X4 is arginine (SEQ ID NO: 4). 5 . A nucleic acid encoding the human proBDNF mutant protein according to claim 1 .

6. A method for producing human BDNF, comprising the following steps: (i) providing a solution of a human proBDNF mutein as defined in any one of claims 1 to 4; (ii) purifying the human proBDNF mutant protein from the solution provided in step (i) to obtain a solution of purified human proBDNF mutant protein, and (iii) cleaving the human proBDNF mutant protein in the solution to obtain human BDNF.

7. The method of claim 6, wherein step (i) comprises the following steps: a) providing a nucleic acid encoding a human proBDNF mutant protein as defined in any one of claims 1 to 4; b) introducing the nucleic acid into an expression vector; c) introducing the expression vector into a host cell; d) culturing the host cell in a suitable liquid culture medium, wherein the host cell expresses the human proBDNF mutant protein in the form of inclusion bodies; e) lysing the host cells and isolating the human proBDNF mutant protein inclusion bodies produced by the host cells in step d); f) dissolving the inclusion bodies in a denaturing solution to obtain a denatured human proBDNF mutant protein solution; g) diluting the denatured human proBDNF mutein solution into a refolding solution so that the denatured human proBDNF mutein presents a correctly folded conformation, thereby obtaining a correctly folded human proBDNF mutein solution.

8. The method according to claim 7, wherein in step f), the denaturing solution comprises the following components: i. Guanidine hydrochloride, 1 to 8 M, preferably 3 to 6 M, more preferably 4 M, cysteine, 1 to 100 mM, preferably 5 mM, ii. Tris, 0.001 to 1 M, preferably 0.1 M, iii. EDTA, 1 to 50 mM, preferably 10 mM, The pH of the denaturing solution is 7 to 10, preferably 8.

9. The method according to claim 7 or 8, wherein in step g), the refolding solution comprises: i. a molecular chaperone, preferably arginine, preferably at a concentration of 0.5 to 1.0 M, more preferably 0.75 M, ii. a metal chelator, preferably EDTA, preferably at a concentration of 1 to 10 mM, more preferably 5 mM, iii. a redox rearrangement system, preferably selected from a combination of L-cystine and L-cysteine ​​and a combination of oxidized glutathione and reduced glutathione, more preferably selected from a combination of 1 mM L-cystine and 5 mM L-cysteine ​​and a combination of 1 mM GSSG (oxidized glutathione) and 5 mM GSH (reduced glutathione); And the pH of the refolding solution is 8 to 11. 10 . The method according to claim 6 , wherein the solution of purified human proBDNF mutein obtained in step (ii) comprises urea, preferably citric acid and urea.

11. The method according to any one of claims 6 to 10, wherein step (ii) comprises purifying the human proBDNF mutein from the solution provided in step (i) by chromatographic purification.

12. The method according to claim 11, wherein in the chromatographic purification, an eluent solution comprising urea, preferably an eluent solution comprising citric acid and urea is used.

13. The method according to any one of claims 6 to 12, wherein in step (iii), the cleavage comprises adjusting the pH of the solution of the purified human proBDNF mutein obtained in step (ii) to a pH value of 5.8 to 9, and cleaving the human proBDNF mutein in the solution to obtain human BDNF.

14. The method according to any one of claims 6 to 13, wherein in step (iii), the cleavage is performed by a trypsin-like protease. 15 . The method according to claim 14 , wherein the ratio of trypsin-like protease to human proBDNF mutant protein is 1:20,000 to 1:30,000 w / w, preferably 1:23,000 to 1:27,000 w / w, more preferably the ratio is 1:25,000 w / w. 16 . The method according to claim 6 , further comprising step (iv): purifying the human BDNF obtained in step (iii).

Images