Method to produce recombinant insulin glargine

FIELD: biotechnologies.

SUBSTANCE: method includes a stage of yeast cultivation, transformed by a vector containing a DNA sequence, determined by the formula X-B-Y-A, coding the precursor of insulin glargine, where X is a sequence of leader peptide, containing at least one amino acid. B is a B1-B30 sequence of amino acids of B-chain of the insulin glargine molecule. Y is a linker peptide containing at least two amino acids. A is an A1-A21 sequence of amino acids of an A-chain of a molecule insulin glargine, a stage of extraction of an expressing precursor of insulin glargine, a stage of crystallisation of the extracted precursor of insulin glargine, a stage of completion of fermentative conversion of insulin glargine precursor crystals at pH from 8 to 10 in presence of tripsin or tripsin-like ferment and water soluble organic dissolvents at the ratio from 40% to 60% of the reaction mix with formation of insulin glargine, containing at least one related admixture. Then the stage of insulin glargine treatment by reverse phase highly efficient liquid chromatography is carried out on a chromatographic matrix, using a polar organic buffer dissolvent in a water phase, containing a buffer based on organic acid, in which the matrix is first balanced with 10% acetonitrile in 250 mM of acetic acid with further elution of insulin glargine in the specified acetonitrile. Then the matrix is again balanced with 10% acetonitrile in the buffer on the basis of organic acid in concentration from 20 mM to 200 mM at pH from 3 to 8.5 with subsequent elution of insulin glargine in the specified acetonitrile, and further repeatedly the matrix is balanced with 6% ethanol in the buffer on the basis of organic acid in concentration from 10 mM to 50 mM with subsequent elution of the specified insulin glargine in the specified ethanol. Further the treated insulin glargine is deposited by means of addition of the buffer on the basis of citric acid and zinc chloride at pH from 6 to 8.

EFFECT: invention makes it possible to produce insulin glargine with high purity and low content of glycolised admixtures.

12 cl, 9 dwg, 8 tbl, 9 ex

 

The technical field to which the invention relates.

The invention relates to a method of separation and/or purification of the impurities that give the purified product of a heterologous protein, free from related impurities or almost minimal amounts of data glycosylated impurities. More specifically the invention relates to the identification of glycosylated forms of insulin analogues, such as impurities of glargine; characterized by mostexpensive systems on the basis of yeast, such as Pichia pastoris. The invention also relates to methods used for cloning of the gene encoding the protein insulin glargine;; insertions of the corresponding gene in a suitable yeast host; obtaining a culture of the recombinant strain, stimulation of the expression of the heterologous polypeptide, its secretion and purification after fermentation and the corresponding enzymatic conversion.

The level of technology

Recombinant forms of insulin, analogs and/or derivatives of insulin from various microbial expressing systems. Currently organisms such as E.coli, S.cerevisiae, use for commercial production of recombinant human insulin and its derivatives. Due to a number of shortcomings of these systems, such as low levels of expression, difficulties subsequent cleaning and the like, preferably using METI otravnych yeast Pichia pastoris as an expression system, protein. Expressing the system provides a number of advantages, such as high expression level, easy processing, low cost production, high culture density (US 6800606).

Yeast expressing systems are popular because they are easy to grow, they develop very quickly and scale; however, some yeast expressing systems give unstable results, and it is sometimes difficult to achieve high outputs. One of the yeast expressing systems, which demonstrates the high potential, is the methylotrophic Pichia pastoris. Compared with other eukaryotic expressing systems Pichia gives many advantages, because it does not have the problem of endotoxin associated with bacteria, or viruses, characteristic of proteins from cultures of animal cells (see Cino, Am Biotech Lab, May 1999). The speed of the productive growth of Pichia makes it easily scalable for large-scale production, although scaling problems include pH control, limiting the oxygen limitation of nutrients, temperature and other security issues (see Gottschalk, 2003, BioProcess IntI 1(4):54-61; Cino Am Biotech Lab, may 1999).

Although expressing systems on the basis of yeast, such as Pichia pastoris, is associated with various advantages, one of the main drawbacks of the Anna system is a post-translational modification of the resulting proteins, which are then present as impurities in the final product, which is difficult to clean. Although known for a number of posttranslational modifications of proteins, the most common form of post-translational modification is glycosylation (see Hart G.W, Glycosylation, Curr. Opin. Cell. Biol 1992; 4:1017). Glycosylation can be either N-linked or O-linked depending on expressing system (see Gemmill TR et al., Overview of N - and O - linked oligosaccharide structures found in various yeast species (for an Overview of N - and O-linked oligosaccharide structures found in various yeast), Biochemica et Biophysica Acta, 1999; 1426:227). Glycosylation affects the stability of the conformation of the protein, immunogenicity, rate of excretion, protection from proteolysis, and enhances protein solubility (see Walsh G, Biopharmaceutical benchmarks 2006, Nature Biotechnology, 2006; 24:769).

Despite great strides in the improvement of the biotechnological production, there are no General solutions for each protein. Method for the production of a specific therapeutic protein requires new and innovative solutions to problems that may be specific for a given protein or protein family. Similarly successful versions of commercial applications are often based on the combination of specific properties of the protein or family of proteins and methods of preparation, used for production is as given protein or family of proteins as pharmaceutical products.

The present invention relates to the identification of different glycoform insulin analogues, more specifically insulin glargine; through chemical methods of identification associated with the methods of mass spectrometry, such as electrospray ionization and ionization by matrix laser desorption. Thus the invention is to enable selective purification of the product from the above-mentioned impurities using optimized further cleaning methods associated with a deeper understanding of the nature of the impurities present in the final product. The final product thus purified, will be substantially free from impurities, described in this invention.

US 4444683 and related applications disclose glycosylated insulin, in which the glucose or mannose that are associated with insulin via spacer elements group derived from dicarboxylic acids, anhydrides of acids or phenylamino or their combinations.

WO 90/10645, in particular, reveals the glycosylated insulin containing one or more monosaccharide groups or one or more oligosaccharide groups containing up to three sugar groups. Described monopolizirovany or triglyceridemia insulin in positions A1, B1 or B.

WO 99/52934 declares the method of separation of glycosylated proteins from deglycosylation Belk is in by khromatograficheskoi processing solution, containing glycosylated and deglycosylated proteins, containing CA++ eluent and obtain fractions containing deglycosylated proteins, and this fraction is substantially free from glycosylated proteins.

Glycoform of glargine; not disclosed in any of the materials corresponding to the prior art. In the invention discuss ways to clean insulin analogues such as insulin glargine with reduced levels of data characterized glycosylated impurities, to obtain a product of glargine; 100% purity after fermentation with expressing systems based on yeast.

Disclosure of inventions

The main purpose of the present invention is to develop a method of obtaining a purified, biologically active heterologous protein, recombinante expressed in yeast expressing the system, characterized in that the purified protein is free from or contains very low amount of glycosylated products.

Another objective of the present invention is to develop a method of obtaining a purified, biologically active heterologous protein, recombinante expressed in the cell host.

Another objective of the present invention is to develop a method of obtaining the shelled biologically active heterologous protein.

Another objective of the present invention to provide a purified biologically active product of the heterologous protein with a purity of at least 96%.

Another objective of the present invention to provide a purified biologically active product of the heterologous protein with a purity that lies in the range of 97-100%.

Another objective of the present invention to provide a purified insulin glargine; with a purity of at least 96%.

Another objective of the present invention to provide a purified insulin glargine; containing less than 1% of the glycosylated impurities.

Another objective of the present invention to provide a purified insulin glargine; free of glycosylated impurities.

Another objective of the present invention to provide a purified insulin glargine; free of glycosylated impurities, and the glycosylated form of the protein can be monopolizirovannoi, triglycineselenate or poliglecaprone.

Accordingly, the present invention relates: to a method for producing a purified, biologically active heterologous protein, recombinante expressed in yeast expressing the system, characterized in that the purified protein is free or contains almost the minimum number is and glycosylated products; the method of obtaining a purified, biologically active heterologous protein, recombinante expressed in the cell host, and the method comprises the stage of: a) culturing host cells transformed by a vector containing a DNA sequence defined by formula I that encodes a heterologous protein under conditions suitable for expression of the protein; (b) the allocation of expressed proteins, including the selection of the specified protein from host cells to obtain a drug selected protein; (C) processing of protein extracted at the stage (b), at the stage of crystallization; (d) conducting stage enzymatic conversion in the presence of trypsin or enzyme such as trypsin; (e) cleaning of the specified protein containing at least one related impurity, including contacting the above-mentioned mixture of proteins with a chromatographic matrix, and cleaning is performed using a polar organic buffer solvent in the aqueous phase, containing the buffer based on organic acid; and (f) precipitation lirovannomu protein; a method for producing a purified, biologically active heterologous protein according to claim 9, which includes: a) inoculation aqueous fermentation medium by transformants strain of yeast carrying expressing vector that directs the expression of the indicated protein; and (b) Viridian the e of the transformed strain in a fermentation medium under conditions efficient expression of the indicated protein; purified, biologically active heterologous protein with a purity of at least 96%; purified biologically active product of the heterologous protein with a purity that lies in the range of 97-100%; purified insulin to glargine; with a purity of at least 96%; purified insulin glargine; the containing less than 1% of the glycosylated impurities; purified insulin glargine; the free of glycosylated impurities; and a purified insulin to glargine; and glycosylated form of the protein can be monopolizirovannoi, triglycineselenate or poliglecaprone.

The present invention relates to a method of obtaining a purified, biologically active heterologous protein, recombinante expressed in yeast expressing the system, characterized in that the purified protein-free or contain very low quantities of glycosylated products.

In another embodiment of the present invention, DNA encoding a heterologous protein, represented by formula I

X-In-Y-A,

in which

X is a sequence of the leader peptide containing at least one amino acid;

In represents the amino acid sequence of the b-chain of the insulin molecule, its production is s or analogues;

Y represents a linker peptide containing at least two amino acids;

And represents the amino acid sequence of the a-chain of the insulin molecule, its derivatives or analogs, and a - and b-chain may be modified by substitution, deletion and/or additions of amino acids.

In another embodiment of the present invention the linker peptide may be any sequence that contains at least two amino acids, provided that the first two amino acids are "RR".

In yet another embodiment, the present invention specified protein defined by SEQ ID:1 or SEQ ID:2.

In yet another embodiment of the present invention a host cell yeast expressing system selected from Pichia sp.

In yet another embodiment of the present invention a host cell yeast expressing system selected from the group comprising Pichia pastoris, Pichia methanolica, Saccharomyces cerevisiae, Schisosaccharomyces pombe, Yarrovia lipolitica, Hansenula polymorpha, Kluyveromyces lactis.

In yet another embodiment, the present invention glycosylated form of the protein can be monopolizirovannoi, triglycineselenate or poliglecaprone.

In yet another embodiment, the present invention indicated purified protein contains less than 1% of the glycosylated note is this.

The present invention also relates to a method for obtaining a purified, biologically active heterologous protein, recombinante expressed in the cell host, and the method comprises the stages:

a) culturing host cells transformed by a vector containing a DNA sequence defined by formula I that encodes a heterologous protein under conditions suitable for expression of the protein;

b) allocation of expressed proteins, including Department-specified protein from the host cells with the aim of obtaining the drug selected protein;

c) effects on isolated protein corresponding to stage (b), for stage crystallization;

d) stage enzymatic conversion in the presence of trypsin or enzyme such as trypsin;

e) purification of the specified protein containing at least one related impurity, involving contacting the above-mentioned mixture of proteins with a chromatographic matrix, and cleaning is performed with the use of polar organic buffer solvent in the aqueous phase, containing the buffer based on organic acids, and

f) deposition lirovannomu protein.

In another embodiment of the present invention, DNA encoding a heterologous protein, represented by formula I

X-B-Y-A

in which

X performance is to place a sequence of the leader peptide, containing at least one amino acid;

In represents the amino acid sequence of the b-chain of the insulin molecule, its derivatives or analogs;

Y represents a linker peptide containing at least two amino acids;

And represents the amino acid sequence of the a-chain of the insulin molecule, its derivatives or analogs, and a - and b-chain may be modified by substitution, deletion and/or additions of amino acids.

In yet another embodiment of the present invention the linker peptide may be any sequence that contains at least two amino acids, provided that the first two amino acids are "RR".

In yet another embodiment of the present invention, the gene defined by SEQ ID 1 or 2, cloned in reading frame with the signal peptide.

In yet another embodiment of the present invention, the gene defined by SEQ ID 1 or 2, cloned in reading frame with the signal peptide mat-or.

In yet another embodiment of the present invention a host cell selected Pichia sp.

In yet another embodiment of the present invention a host cell selected from the group consisting of Pichia pastoris, Pichia methanolica, Saccharomyces cerevisiae, Schisosaccharomyces pombe, Yarrovia lipolitica, Hansenula polymorpha, Kluyveromyces lactis.

In yet another embodiment, realized is I of the present invention a host cell is a Pichia pastoris.

In yet another embodiment of the present invention, the strain-the owner is a GS115.

The present invention also relates to a method for obtaining a purified, biologically active heterologous protein, which provides:

a) inoculation aqueous fermentation medium by transformants strain of yeast carrying expressing vector that directs the expression of the indicated protein, and

b) growing the transformed strain in a fermentation medium under conditions effective for expression of the indicated protein.

The present invention also relates to a method for obtaining a purified, biologically active heterologous protein, and the expressed protein is recovered from fermentation broth using ion-exchange chromatography.

In yet another embodiment of the present invention the method includes the stage of crystallization of the protein by addition of zinc chloride and phenol.

The present invention also relates to a method for obtaining a purified, biologically active heterologous protein, and this method provides for the stage enzymatic conversion is carried out in the presence of trypsin miscible with water and organic solvent.

In yet another embodiment, the present invention miscible with water, the solvent content of inorganic fillers selected from the group including DMSO (dimethylsulfoxide), DMF (dimethylformamide), ethanol, acetone, acetonitrile, ethyl acetate, or mixtures thereof.

In yet another embodiment of the present invention the method provides for purification stage RP-HPLC (reversed-phase HPLC) of a mixture of proteins containing at least one related impurity, by contacting the above-mentioned mixture of proteins with a chromatographic matrix resin, and cleaning is performed using a polar organic buffer solvent in the aqueous phase, containing the buffer based on organic acids.

In yet another embodiment of the present invention the polar buffer solvent represents acetonitrile.

In yet another embodiment of the present invention the buffer is an organic acid selected from the group consisting of citric acid, acetic acid, boric acid, formic acid, hydrochloric acid and phosphoric acid.

The present invention also relates to the product purified, biologically active heterologous protein obtained according to any of the preceding claims, with a purity of at least 96%.

The present invention also relates to a method of obtaining the product purified, biologically active heterologous protein with a purity of lying in INTA the shaft 97-100%.

In yet another embodiment, the present invention is proposed purified insulin glargine with a purity of at least 96%.

In yet another embodiment, the present invention is proposed purified insulin glargine containing less than 1% of the glycosylated impurities.

In yet another embodiment, the present invention purified insulin glargine does not contain glycosylated impurities.

In yet another embodiment, the present invention is proposed purified insulin glargine, with the glycosylated form of the protein can be monopolizirovannoi, triglycineselenate or poliglecaprone.

Now made detailed reference to the currently preferred embodiments of the invention, which, together with the following examples serve to explain the principles of the invention.

The following examples are given to aid in understanding the invention, but they are not intended and should not be construed as limiting its scope in any way. The examples do not include detailed descriptions of the accepted methods used to construct vectors, the insertion of genes encoding polypeptides, data vectors, or the introduction of the resulting plasmids in the hosts. In addition, the examples do not include detailed descriptions of PR is accepted ways used for the analysis of polypeptides produced by vector data systems of the host. These methods are well known to specialists in the field of technology and described in numerous publications, included as examples.

According to one aspect of the invention presents a method of obtaining a purified, biologically active heterologous protein, recombinante expressed in the cell-host, and the method comprises the stages:

a) culturing host cells transformed by a vector containing a DNA sequence defined SEQ ID 1 or 2 that encodes a heterologous protein under conditions suitable for expression of the protein;

b) allocation of expressed proteins, including Department-specified protein from the host cells with the aim of obtaining the drug selected protein;

c) effects on isolated protein corresponding to stage (b), for stage crystallization;

d) stage enzymatic conversion in the presence of trypsin or enzyme such as trypsin;

e) purification of the specified protein containing at least one related impurity, involving contacting the above-mentioned mixture of proteins with a chromatographic matrix, and cleaning is performed with the use of polar organic buffer solvent in the aqueous phase, containing the second buffer on the basis of organic acids,

f) deposition lirovannomu protein.

Definitions:

The terms "cell" or "cell culture" or "recombinant cell hosts" or "cell bosses" are often used interchangeably, as will be clear from the context. These terms include direct this cell that expresses the desired protein corresponding to the present invention, and, of course, their offspring. It should be understood that not all offspring identical to the parent cell due to random mutations or differences in the environment. However, this modified offspring included in these terms until such time as the offspring retains characteristics corresponding to those in the original transformed cell.

As used in this context, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. The term "expressing vector" includes plasmids, Comedy or phages, are able to synthesize the data of the proteins encoded by the corresponding recombinant genes transmitted vector. Preferred vectors are vectors capable of Autonomous replication and/or expression of nucleic acids with which they are associated. In this application, the terms "plasmid" and "vector" are used interchangeably, since plasma is IDA is the most commonly used form of vector. Moreover, the invention provides for the inclusion of these other forms of expressing the vectors which serve equivalent functions and which become known in the field of engineering after the appearance of this material.

Have in mind that the polypeptides are referred to in this context as possessing activity of insulin glargine; for example, represent insulin glargine, have an amino acid sequence with two changes in the structure of human insulin: the replacement of the amino acid glycine native asparagine at position A21 of the a-chain of human insulin and the addition of two molecules of arginine in the NH2-terminal part of the b-chain of human insulin, obtained by recombinant DNA technology. The primary action of any insulin, including insulin glargine, is the regulation of glucose metabolism. Insulin and its analogues reduce the levels of blood glucose by stimulating peripheral uptake of glucose, especially in skeletal muscle and fat, and by inhibiting the formation of glucose in the liver.

DNA encoding a heterologous protein, represented by formula I

X-In-Y-A

in which

X is a sequence of the leader peptide containing at least one amino acid;

In represents the amino acid sequence of the b-chain of the insulin molecule, its about svodnik or analogues;

Y represents a linker peptide containing at least two amino acids;

And represents the amino acid sequence of the a-chain of the insulin molecule, its derivatives or analogs, and a - and b-chain may be modified by substitution, deletion and/or additions of amino acids.

The term "C-peptide" or "linker peptide", as used in this context, includes all forms of C-peptide of insulin, including native or synthetic peptides. Data C-peptide of insulin can be a human peptides or can be isolated from other species and genera of animals, preferably mammals. Thus variations and modifications of the native C-peptide of insulin included as long as they remain active C-peptide of insulin. In engineering it is known that it is possible to modify the sequence of proteins or peptides while retaining their useful activity, and this can be achieved using methods that are standard in the field of technology and are widely described in the literature, for example, omnidirectional or site-directed mutagenesis, cleavage and ligation of nucleic acids, etc. Thus, functionally equivalent variants or derivatives of native sequences of the C-peptide of insulin can be easily obtained according to methods well known is applied in the field of technology and include sequences of peptides with functional, for example the biological activity of the native C-peptide of insulin. All of these analogs, variants, derivatives or fragments of the C-peptide of insulin, in particular, included in the scope of this invention and are United under the term "C-peptide of insulin".

The linker sequence may be any sequence that has at least two amino acids. The linker section may include from 2 to 25, 2 to 15, from 2 to 12 or from 2 to 10 amino acid residues, although the length is not critical and may be chosen for convenience or as needed.

The linker peptide may be any sequence that contains at least two amino acids, provided that the first two amino acids are "RR". Moreover, as a rule, will be borne in mind that in some cases it may be useful to obtain homologues of the protein insulin glargine; which are either agonists or antagonists only a subset of the biological activities of this protein. Thus, you can get specific biological effects by treatment with a homolog of limited function, and with fewer side effects.

In one embodiment, the nucleic acid corresponding to the invention, Cody is the duty to regulate the polypeptide, which is either an agonist or antagonist of the protein of human insulin glargine; and contains the amino acid sequence represented by SEQ ID No:2. Preferred nucleic acids encode a peptide having the activity of a protein insulin glargine; and at least 90% homologous, more preferably 95% homologous and most preferably 97% homologous amino acid sequence shown in SEQ ID No:2. Nucleic acids that encode agonistic or antagonistic forms of the protein insulin glargine; and have a homology of at least about 98-99% sequence shown in SEQ ID No:2, also included in the scope of the invention. Preferably, when the nucleic acid is a cDNA molecule that contains at least part of the nucleotide sequence that encodes a protein insulin glargine; shown in SEQ ID No.1.

The term "functional elements", as discussed in this context includes at least one promoter, at least one operator, at least one leader sequence, at least one Shine-dalgarno sequence, at least one terminator codon and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translationfree DNA. In particular, stipulate that these vectors will contain at least one origin of replication recognized by the microorganism-host, together with at least one selectable marker and at least one promoter sequence capable of initiating transcription of synthetic DNA sequences. In addition, preferably, when the vector in one embodiment, contains a number of DNA sequences capable of acting as regulators, and other DNA sequences can encode a regulatory protein. Data controllers in one of the embodiments serve to prevent expression of the DNA sequence under certain environmental conditions, and other environmental conditions enable transcription and subsequent expression of the protein encoded by the DNA sequence.

In addition, preferably, when there is an appropriate secretory leader sequence or vector, or at the 5'end of the DNA sequence. A leader sequence is in position, which allows a leader sequence directly adjacent to the initial part of the nucleotide sequence capable of directing the expression of inhibitor required protein without any disturbance signals therm the nation broadcast. The presence of the leader sequence is required in part on one or more of the following reasons:

1) the presence of the leader sequence may facilitate processing by the owner of the original product with the product of the Mature recombinant protein;

2) the presence of the leader sequence can facilitate the purification product of recombinant protein by protease inhibitor from the cytoplasm of cells;

3) the presence of the leader sequence may act on the ability of the recombinant protein product to meet your active structure, directing the protein from the cytoplasm of the cell.

The term "sequence regulation of transcription" is a generic term used throughout the description in relation to DNA sequences, such as initiation signals, enhancers, and promoters, which induce or control transcription of sequences encoding a protein with which they are functionally linked. In preferred embodiments, the implementation of the transcription of the gene recombinant insulin glargine; is under the control of a promoter sequence (or other sequence transcription regulation), which controls the expression of the recombinant gene in a cell type in which plan to carry out expression.

The term "controlling p is coherence" refers to DNA sequences, necessary for the expression of the operatively linked coding sequence in a particular organism, the host. Control sequences that are suitable for prokaryotes, for example, include a promoter, optionally a sequence operator, the binding site of the ribosome and possibly other, as yet poorly understood sequence. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

The term "transformation" means the introduction of DNA into an organism so that the DNA is replicated, either as an extrachromosomal element or by integration into the chromosome. Depending on the host cell transformation carried out using standard methods appropriate for data cells. The main aspects of the transformation system of the mammalian cell hosts described Axel in U.S. patent No.4399216, issued August 16, 1983, Transformation in yeast, as a rule, carried out according to the method described in the articles of Van Solingen, P., et al., J. Bad, 130: 946 (1977) and Hsiao, .L, et al., Proc. Natl. Acad. Sci. (USA) 76: 3829 (1979).

Recombinant expressing the system chosen from prokaryotic and eukaryotic hosts. Eukaryotic hosts include yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris, mammalian cells or plant cells. Bacterial and eukaryotic cell is available for competent professionals in the field of engineering in a number of different sources, including commercial sources, for example, the American type culture collection (ATSS; Rockville, Md.). Commercial sources of cells used for expression of recombinant proteins, are also instructions for using the cells. The choice of expressing the system depends on the properties required for the expressed polypeptide.

Therefore, the aim of the present invention is expressing the system or the cassette is functional in a cell isolated from a yeast selected from the group consisting of a strain of Pichia, especially selected from the group consisting of Pichia pastoris, Pichia methanolica and Schizosaccharomyces pombe, and enabling the expression of the desired polypeptide encoding fragments of its protein placed under the control of elements necessary for its expression.

The term "recombinant"as used in this context to describe a protein or polypeptide means a polypeptide formed by expression of recombinant polynucleotide. The term "recombinant"as used in this context in the cells means cells that can be or have been, used as recipients for recombinant vectors or other transfer DNA, and include the progeny of the original cell, which was transliterowany. It should be understood that the offspring of a single parent cell which may not be completely identical to the original parent on the morphology or the complement of genomic or total DNA due to random or directed mutation. The progeny of the parent cell, which is close enough to the parent, characterized by the relevant property, such as the presence of the nucleotide sequence that encodes the desired polypeptide is also considered the offspring.

"Gene of interest" (hereinafter GPI)is any sequence of nucleic acids, increased transcriptional expression of which is required. GUIs can encode a functional nucleic acid molecule (e.g., RNA, such as antisense RNA molecule) or, more typically, encodes a peptide, polypeptide or protein, increased the production of which is required. The vectors corresponding to the invention can be used for expression "heterologous" protein. As used in this context, the term "heterologous" refers to the sequence of the nucleic acid or polypeptide that originate from other species, or are significantly modified with respect to their original form if they originate from the same species. Moreover, modified or unmodified nucleic acid sequence or polypeptide, which are normally not expressed in the cell, is considered heterologous. The vectors corresponding to the invention can have one or more GUIs are entered in the same or different sites of insertions, etc is what every SIP functionally linked to a regulatory nucleic acid sequence, which enables the expression of GPI.

The term "cleanup" of the peptide of the composition containing the peptide and one or more impurities, means increasing, thus, the degree of purity of the peptide in the composition by reducing the content of at least one impurity from the composition of the peptides. More specifically, the cleaning method corresponding to the present invention, results in a biologically active heterologous protein, recombinante expressed in yeast expressing the system, characterized in that the purified protein-free or contain very low quantities of glycosylated products.

In addition to the glycosylated forms, using MALDI (m/Z: 6089) confirm the presence of non-polar impurities. The presence of this non-polar impurities varies in the interval 0-0,5% in the final product, which is produced during the processing of glargine;. The difference in mass between glargine; (m/z: 6063) and non-polar impurities (m/z: 6089) is 26 units. Chemical identity, based on molecular weight, indicates the probability of loss of water in one of the amino acid and acetylation.

The term "chromatography" refers to the manner in which the interest of the dissolved substance in the mixture is separated from other solutes in the mixture as a result of differences in speed to the mi individual soluble substances of the mixture pass through a stationary medium under the influence of the mobile phase, or in the processes of binding and elution.

The term "high-performance liquid chromatography," as used in this context, refers to a chromatographic procedure in which particles (stationary phase)used in the packing of the column, small (3 to 50 MK) and the correct form with minor deviations from the selected size. In this chromatography, typically use relatively high (approximately 3,45÷24,13 MPa (500 to 3500 psi)inlet pressure.

After selection of the host body, the vector is transferred into the body-master, using methods generally known to the ordinary expert in the field of technology. Examples of these methods can be found in the monograph R.W.Davis et. al. Advanced Bacterial Genetics (Advances in bacterial genetics), Cold Spring Harbor Press, Cold Spring Harbor, N.Y., (1980), which specifically included in this context by reference. In one embodiment, preferably, when the transformation occurs at low temperatures, and temperature regulation is considered as a means of regulation of gene expression through the use of functional elements, as shown.

Microorganisms-hosts are cultivated under conditions suitable for expression of the precursor of insulin glargine;. These conditions are usually specific to the host body, and an ordinary specialist in the field technicalism install them in the light of the published literature, related to the growth of these organisms, for example, monographs Bergey Manual of Determinative Bacteriology (Reference definition in bacteriology), 8th ed., Williams &Wilkins Company, Baltimore, Md., which specifically included in this context by reference.

Thus, the expression of the coding sequence functionally linked with regulatory sequences refers to a configuration in which the coding sequence can be expressed under the control of the data sequences and in which the associated DNA sequences follow each other and, in the case of a secretory leader, follow each other and are in the phase of reading.

According to one aspect of the invention, the precursor of insulin glargine; formula X-B-chain of glargine; [B1-B30]-Y-A-chain of glargine; [A1-A21], where X is a leader sequence, a chain is a sequence of b-chain of glargine; B1-B30, Y represents a linker peptide sequence between the B-chain and a-chain, A-chain is a chain of glargine;. The predecessor of GIP-And can be obtained by expression in expressing the system owner, not including bacteria other than E. coli, yeast such as Saccharomyces and Kluvyeromyces, techniques for which are known competent specialists in the field of machinery (see Methods in Enzymology Methods in Enzymology), t), and fungi, such as Aspergillus, Neurospora, and Trichoderma, techniques for which are known to the competent specialists in the field of technology, as described in the monograph Applied Molecular Genetics of Fungi (Applied molecular genetics of fungi), Ed. by Peberdy, Caten, Ogden and Bennett (Cambridge University Press, N.Y. 1991), many of which can be obtained in the American type culture collection, Rockville, Md., that routine use data experts in the field of technology. Preferably, when the strain-host selected from the group consisting of yeast, such as Saccharomyces cerevisiae, Schisosaccharomyces pombe, Yarrovia lipolitica, Hansenula polymorpha, Kluyveromyces lactis, Pichia methanolica and Pichia pastoris, which are able to Express high levels of recombinant protein. Even more preferably, when the strain-the owner is a Pichia pastoris.

In particular, the present invention provides a method of obtaining protein insulin glargine; including the process of fermentation, separation and purification process. The way envisaged in this context, includes the fermentation process, in which the above-mentioned protein produced in Pichia pastoris, having at least one sequence encoding this protein, which is integrated into the genome, and the fermentation process involves the fermentation of seed material for growing the host cells to the desired density of cells and the process of fermentation of education is receiving product, including periodic fermentation on the glycerol fermentation by induction with methanol and end products.

Then the predecessor of glargine; capture from fermentation broth using cation exchange chromatography. The process of allocation provided for in this context, provides linking predecessor glargine; and removing cells and cell debris, and the process of allocation provided for in this context, provides access 95%. Alternative competent specialist in the field of engineering can test and evaluate alternative ways of binding of the desired protein product and remove cells and cellular debris, including, but not limited to, various other chromatographic methods, centrifugation, filtration, differential precipitation and other methods that will be defined.

The predecessor of glargine; captured from the fermentation broth, then subjected to crystallization to remove staining and storage. Deposition of the precursor in crystalline form is conveniently carried out in aqueous media at pH lying in the range from 3.0 to 8.0, preferably about 4.0 to 6.0, most preferably at pH 4.0 to 5.0, and the protein concentration in the aqueous medium is in the range from 1 to 80 g/l, preferably from 2 to 50 g/l, most preferably from up to 14 g/L. The crystallization process can be performed at a temperature of from 2 to 30°C. the Crystals of the precursor can be distinguished from the supernatant or by centrifugation, decantation or filtration.

Crystallization predecessor glargine; GIP-A removes impurities brought from fermentation broth in the combined fractions obtained by elution from a cation exchange resin, as well as salt ammonium acetate, which is used for elution of the product by chromatography. Pure crystalline predecessor also helps to reduce the cost of subsequent stages and to increase the efficiency of the reaction. The crystalline form can be frozen and stored, which subsequently makes it stable for several days when stored at -20°C.

Glargine can be obtained from the predecessor glargine; by enzymatic conversion. The conversion is carried out in the presence of trypsin or enzymes such as trypsin, vegetable, animal or microbial nature. The reaction is carried out preferably in the presence of a miscible with water and organic solvents such as DMSO (dimethylsulfoxide), DMF (dimethylformamide), ethanol, acetone, acetonitrile or ethyl acetate, especially DMF and DMSO. The preferred ratio of organic solvent is about 0-65%, in particular approximately 40-60% of the reaction mixture.

Glargine receive the Ute by processing the predecessor GIP-A-trypsin. Leader (if present) will be derived from its predecessor by trypsin. The reaction of trypsin control so that the cleavage on the C-end "RR" C-peptide was maximum. However, the reaction can eliminate unwanted cleavage by trypsin in other centers cleavage of trypsin. Other centers trypsin cleavage between "RR" and-end-22 (Arg).

The rationale for the use of organic solvents should be installed in the solubility of the source materials, trends enzymes to denaturation and hydrolysis activity. As you may be aware of a competent specialist, you can also use a mixture of organic solvents. Adding organic solvents reduces the concentration of water in the reaction mixture, resulting in the prevention of hydrolysis of the product and, in addition, significantly increases the solubility of the products.

The concentration of the precursor, as a rule, is about 5-50 g/L. the Reaction is carried out at a pH of approximately 5 to 12, preferably pH of about 8-10. The reaction temperature is about 0-40°C., preferably about 2-25°C. Can be used trihydroxystilbene (TRIS) or other buffer systems with different values of ionic strength to maintain the desired pH. The response time of the variation is the duty to regulate, and it can be influenced by other reaction conditions. The reaction may continue until such time as the purity of the product is not begins to degrade due to hydrolysis of the product. It usually takes around 30 minutes to 24 hours and in most cases is approximately 4-10 hours.

The concentration of the enzyme is determined in dependence on substrate concentration and activity of the enzyme. For example, crystalline trypsin, commercially available, is used preferably in a concentration of about 10-100 mg/l of the reaction mixture.

The cleaning method includes processing the sample insulin glargine; at the stage of purification by reverse-phase chromatography, containing a matrix of resin to polymer based chromatographic conditions sufficient to obtain the peptide purity approximately 98-99%, preferably of a purity of 100%.

Thus, the formation of undesirable related to product impurities, which is cleaned by sequential reverse-phase chromatography to obtain the pure glargine;. Glycosylated (mono - and tri-) impurities can be detected only after the first stages of purification. Consequently, special attention is paid to cleaning glycosylated impurities on the 2nd stage of purification RP-HPLC. Glycosylated impurities generated during fermentation, completely is aracterized and ultimately clear from the final product to the maximum extent.

The invention provides, in a significant aspect of the method of purification protein, the method comprising the stage of processing of this protein on the column for reversed-phase chromatography and elution of the sample, which includes a peptide with an organic solvent under conditions that allow the compound to contact with the resin, and the laundering of the organic solvent from the resin aqueous buffer solution. Effective implementation of the present invention requires individualization correct combination of chromatographic matrix, intended for use, pH and ionic strength buffer for effective cleaning.

Any property chromatographic method plays an important role in obtaining the desired protein product. Suitable matrix used in the chromatographic column is C8 Daisogel.

According to one aspect of the invention the yield of the pure product insulin glargine; 75%-80%, according to another aspect of the invention the yield of the pure product insulin glargine; is 80%-85%, according to another aspect of the invention the yield of the pure product insulin glargine; 85%-90%, according to another aspect of the invention the yield of the pure product insulin glargine; 90%-95%, according to another further aspect of the invention the yield of the pure product of insul is on glargine; 95%-100%.

According to one aspect of the invention the purified product of insulin glargine; free or contain very low quantities of glycosylated products. According to one aspect of the invention the purity of the purified product of insulin glargine; is at least 96%, according to another aspect of the invention the purity of the purified product of insulin glargine; is at least 97%, according to another aspect of the invention the purity of the purified product of insulin glargine; is at least 98%, according to another aspect of the invention the purity of the purified product of insulin glargine; is at least 99%, according to another aspect of the invention the purity of the purified product of insulin glargine; is 100%.

Brief description of drawings

Figure 1: Graph of % output relative time (hour) trypsinization insulin glargine; and the reaction at different concentrations.

Figure 2: Analytical chromatogram of the reaction at various time points before and after treatment with the enzyme.

Figure 3: Profile of HPLC (high performance liquid chromatography) insulin glargine;.

Figure 4: Comparison of retention times of selected impurities in the final product.

Figure 5: Profile of reversed-phase HPLC of the final product insulin-glargine;.

6: Profile of exclusion WAS the final product insulin-glargine;.

7: Mass spectra obtained during electrospray-ionization of glargine;, monopolizirovannoi (mGIG) and triglyceridesanother (tGIG) insulin glargine;.

Fig: Mass spectrum obtained with ionization by matrix laser desorption monopolizirovannoi insulin glargine; (mGIG).

Figure 9: Mass spectrum obtained with ionization by matrix laser desorption triglyceridesanother of glargine; (tGIG).

The implementation of the invention

The invention also specified using the following examples. However, these examples should not be construed as limiting the scope of invention.

Examples

Example 1:

GIP-A is a predecessor of glargine; formula X-[B-chain of glargine; (B1-B30)]-Y-[A-chain of glargine; (A1-A21)], where X is a sequence of the leader peptide, b-chain is a sequence of b-chain of glargine; B1-B30, Y is a sequence of the linker peptide between the B-chain and a-chain, A-chain is a chain of glargine;. The sequence may not have a leader or another leader peptide. The linker peptide Y may be any of those shown in example RR, RRDADDR. Predecessor GIP-A can be obtained using any suitable expressing system such as Escherichia coli, Pichia pastoris, Saccharomyces cerevisiae cells, Cho (Chinese hamster ovary), etc.

resistant GIP-A clone in reading frame with the signal peptide mat-α expressing Pichia vector, pPIC9K. Strain-host Pichia pastoris GS115 transformed with recombinant plasmid to obtain a clone expressing the predecessor of glargine;. The secretory precursor is treated with trypsin to obtain glargine; and other related product impurities. Glargine purified using reverse-phase chromatography.

SEQ ID 1 is a sequence of predecessor glargine; having a given molecular weight 6045 and approximately a certain value pI 6,88. The sequence has 159 nucleotides and 53 amino acids.

SEQ ID 2 is a sequence for expression in Pichia pastoris with the desired molecular weight 6428,4 and approximately a certain value pI 7,78. The sequence has 171 nucleotide and 57 amino acids.

The predecessor of glargine; GIP-A secreted Pichia pastoris in the culture medium. The broth is centrifuged and the cells are separated from the supernatant. There are many opportunities available for capture predecessor GIP-A, including ion-exchange chromatography and hydrophobic chromatography. In this invention, the cation exchange chromatography and HIC (hydrophobic interaction chromatography) is used to associate GIP-A.

Example 2:

Construction of recombinant vector carrying the gene for the precursor of insulin glargine;

The predecessor of the Insa is in the glargine; (GIP-A) clone in reading frame with the signal peptide mat-α expressing vector Pichia pastoris, pPIC9K. The recombinant plasmid used for transformation of strain-host Pichia GS115, and the secretory GIP-A is a predecessor of insulin glargine; that is subjected to a subsequent purification process to obtain the final product.

Transformation of Pichia host recombinante vectors carrying the gene for the precursor of insulin glargine;:

Clone pPIC9K/IGP-A carrying recombinant plasmid DNA, cut with BgI II and used for transformation electrocompetent cells of P. pastoris GS115 (his4)-owners. The management of the company Invitrogen is the detailed Protocol of transformation.

Screening mnogokupolnyh of integrantes:

Approximately 2,000 transformants inoculant YPD broth in a 384-well tablets for micrometrology along with the appropriate controls. Tablets incubated at 30°C for 24 hours and then make prints on plates with YPD agar containing 0.5 mg/ml geneticin (G418). Select the 49 clones, the prints are then made on the cups 1, 2 and 3 mg/ml G418 for the second cycle of screening. Finally, select the seven clones resistant to 1-3 mg/ml G418, and use in studies of gene expression.

Confirmation of gene integration into the genome by PCR:

Genomic DNA from selected recombinant Pichia clones subjected to PCR using gene-specific primers to podtverzdeniye GIP-A gene.

Studies of the expression of the P. pastoris in small scale

Studies of the expression of P. pastoris on a small scale, carried out according to the Protocol presented in the management of the company Invitrogen. Briefly, clones grown at 30°C in BMGY followed by induction with methanol in BMMY at 24°C. the Induction with methanol is carried out in General within 3 days.

Example 3:

The method of fermentation

The method of fermentation of recombinant insulin-glargine; optimized at laboratory scale. Below is a brief description of the method.

Obtaining seed

Environment for seed contains the following ingredients: yeast as a nitrogen source, ammonium sulfate, glycerin, potassium dihydrophosphate, potassium phosphate and D-Biotin.

For inoculation of planting bulbs use one vial from the freezer. The bottles thaw to room temperature before inoculation in sterile conditions. The AMF inoculum is distributed in minimal glycerol medium (MGY) aseptic manner.

The number added to each flask, determined so that the initial OD (optical density) 600 in pre flask ranged from 0.1 to 0.2. Then the flask is incubated for 20-24 hours in a rocking chair at 30°C with a speed of 230 rpm

Loading of the fermenter

The fermentation method has two stages, the phase of growth (biomass accumulation) with sleduyushei phase induction. The product is produced and secreted into the broth during phase feed download methanol. The fermentation is carried out in a fermenter, sterilized in situ. Fermentation medium contains phosphoric acid, potassium sulfate, potassium hydroxide, magnesium sulfate, calcium sulfate, and glycerin. These chemical substances dissolved in drinking water in the tank fermentor and sterilized at a fixed point 121°C for a certain period. The original solution of salts, minerals and Biotin are prepared separately, sterilized by filtration and aseptically add to the fermenter after sterilization environment and summarize the pH to 5.0 with ammonium hydroxide.

Method of production/fermentation take. The fermenter inoculant 5% sowing AMF inoculum from the seed katalozhnyh Kolb. At the beginning of fermentation set the following parameters: pH 5.0, 30°C, DO2 30%.

Example 4:

Related transformation and purification method

The precursor of insulin glargine;, which is obtained by fermentation of Pichia pastoris, purified, under which is included in the purification method are as follows.

A column Packed matrix for fast flow SP Sepharose (GE Biosciences) balance 50 mm acetic acid. Cell-free supernatant with pH, increased to 3.8 using orthophosphoric acid load on the cation exchange column. Load to which the PMC is less than 50 g/L. The elution is performed with the use of ammonium acetate. The selection stage is 95% at a load of less than 50 g/l

Crystallization predecessor

The predecessor of glargine; captured from the fermentation broth using phase cation-exchange chromatography, and crystallized to remove staining and storage. Crystallization was carried out so that the concentration of the precursor in the beginning of crystallization is about 2-20 g/l, preferably 8-14 g/L. Crystallization was carried out by adding ZnCl2and phenol and then bringing the pH to 3.0 to 8.0, preferably of 3.5 to 5.5. The phenol may be added in concentrations of 0.1-0.5% of the amount collected by CIEX elution (cation exchange chromatography). 4% solution of ZnCl2can be added at a concentration of 3-15% of the volume harvested by CIEX elution. the pH can be increased using any alkali, preferably NaOH or TRIS. The crystallization process can be performed at a temperature of from 2 to 30°C, and the suspension is kept for some time, so that the crystals are completely formed. The crystals of the precursor can be separated from the supernatant or by centrifugation, or decantation.

Example 4A: take 463 ml of the obtained eluate (PE) (concentration predecessor of 13.8 g/l) and add 2,315 ml of phenol (0.5% volume PE) after proper thawing. Th is should add 57,875 ml of 4% solution of ZnCl 2(12.5% volume PE). the pH at this stage is 4,08, and adjusted to 4.8 by adding 420 ml of 2.5 N. NaOH. The original solution to support with slow stirring for 15 minutes and then transferred to cold storage (2-8°C), where they hold during the night. Then the whole mixture was centrifuged at 5000 rpm for 20 minutes in a centrifuge Beckman Coulter Avanti J-26 XP. Loss supernatant is 1,55%.

Example 4b: take 500 ml of the obtained eluate (concentration predecessor 2.9 g/l) and type of 0.625 ml of phenol (0.125% volume PE) after proper thawing. This is followed by adding 15,625 ml of 4% solution of ZnCl2(3,125% of PE). the pH at this stage is 4,08, and adjusted to 4.8 by the addition of 315 ml of 2.5 N. NaOH. The original solution to support with slow stirring for 15 minutes and then transferred to cold storage (2-8°C), where they hold for 5 hours. Then the supernatant is separated by centrifugation. Loss supernatant is 4%.

Optimized conditions of crystallization of the predecessor with the output:

The eluate obtained by elution with SP-Sepharose, cooled to 20°±5°C, add 5 ml (0.5% vol./about.) phenol per liter of the obtained eluate. The required amount of phenol added to the aliquot volume obtained during the elution, and ultimately add to the main tank for proper reconstitution. After the adding the phenol stirring is continued for some time before adding the next reagent for proper reconstitution. Then add 12.5% volume 40 g/l of zinc chloride (4% wt./about.) for the induction of crystallization. Then bring the pH to 4.8±0,1 using 2.5 N. NaOH (approximately 85-90% of the volume of SP-PE). This entire mixture is stirred for 30 minutes to ensure homogeneity and then maintain at 2-8°C for at least 8 hours prior to centrifugation.

Based on the loss of the supernatant, the output at this stage is approximately 90%.

Example 5:

Trypsinization

Glargine is obtained from the predecessor glargine; by enzymatic conversion. The conversion is carried out through the presence of trypsin or enzymes such as trypsin, vegetable, animal or microbial nature. Preferably, when the reaction is carried out in the presence of a miscible with water and organic solvents, such as DMSO, DMF, ethanol, acetone, acetonitrile, ethyl acetate, etc., especially DMF and DMSO. The preferred ratio of organic solvent is 0-65%, in particular approximately 40-60% of the reaction mixture.

The ratio of organic solvent is determined by the solubility of the source materials, trends enzymes to denaturation and hydrolysis activity. You can also use a mixture of organic solvents. Adding organic solvents reduces the concentration of water in the reaction mixture, resulting in R is the result, to prevent hydrolysis product, and also significantly increases the solubility of the products.

The concentration of the precursor, as a rule, is approximately 5 to 50 g/L. the Reaction is carried out at a pH of approximately 5 to 12, preferably pH of about 8-10. The reaction temperature is about 0-40°C., preferably about 2-25°C. Trihydroxystilbene (TRIS) or other buffer systems used for different values of ionic strength to maintain the desired pH. The reaction time varies and is affected by other reaction conditions. The reaction continues as long as the purity of the product is not begins to degrade due to hydrolysis of the product. It usually takes around 30 minutes to 24 hours and in most cases is approximately 4-10 hours.

The concentration of the enzyme is determined in dependence on substrate concentration and activity of the enzyme. For example, crystalline trypsin, commercially available, is used preferably in a concentration of about 10-100 mg/L.

Example 5A: 1 g predecessor glargine; dissolved in 1.5 ml of 1 M solution TRIS ml each of these solutions are selected in two test tubes, labeled as sample a and sample Century To the sample And add 3 ml of water and 166,6 mg of TRIS. The sample add 2 ml of DMF, 1 ml of water and 166,6 mg of TRIS. Each sample is divided into four parts by summing the N up to four different pH values. The reaction is carried out in all samples by adding bovine trypsin at a concentration of 50 μg/ml Investigated conditions result in table 1 as follows.

Table 1
SampleSolvent %pHThe concentration of TRIS (M)The concentration of trypsin (m kg/ml)The concentration of the precursor (mg/ml)
A10%9,20,55015,5
A28,2
A37,0
A46,2
B150%8,9
B28,0
B37,0
B46,3

All terms are for the product in different ratios. Sample B1 d is no better conversion at around 40%, while the conditions A1, A2, A3 and A4 give the level of conversion is less than 15%. The level of conversion in the glargine in other samples lies in the range from 15% to 40%.

Example 5b: 1.6 g predecessor glargine; dissolved in 2 ml of 1 M TRIS solution. 1 ml each of these solutions are selected in three test tubes labeled sample a, sample b and sample C. To the sample And add 1.8 ml of water, 1.2 ml of DMF and 161 mg of TRIS. To the sample In add 0.6 ml of water, 2.4 ml DMF and 161 mg of TRIS. The sample add 1 ml of water, 2.0 ml DMF and 161 mg of TRIS. Two samples, a and b, is divided into four parts, and the sample is in two parts by summing up different pH values. The reaction is carried out in all samples by adding bovine trypsin at a concentration of 50 μg/ml Investigated conditions result in table 2 as follows.

All terms are for the product in different ratios. Sample C1 and C2 gives the best conversion at around 40%, whereas the other terms give the level of conversion is less than 15%.

Example 5: 1.5 g predecessor glargine; dissolved in 2 ml of 1 M TRIS solution. 0.75 ml each of these solutions are selected in three test tubes labeled sample a, sample b and sample C. To the sample And add 0.75 ml of water, 1.5 ml of ethanol and 106 mg of TRIS. To the sample In add 0.75 ml of water, 1.5 ml DMF) and 106 mg of TRIS. To the sample With add 0.75 ml of water, 1.5 ml DMF) and 106 mg of TRIS. the BA sample, A and b, divided into two parts by summing up two different pH values. The sample was adjusted to a pH of 9.0. The reaction is carried out in all samples by adding bovine trypsin at a concentration of 50 μg/ml Investigated conditions result in table 3 as follows.

All terms are for the product in different ratios. Sample 1 gives the best conversion at around 40%, followed by B1 (~30%), whereas the other terms give the level of conversion is less than 15%.

Example 5d: 1.5 g predecessor glargine; dissolved in 2 ml of 1 M TRIS solution. Take 0.75 ml solution and add 0.75 ml of water, 1.5 ml of ethanol and 106 mg of TRIS. Then the pH was adjusted to 9.0. Then the solution is divided into two samples a and B. For sample And reaction was performed at room temperature (20-25°C), whereas for the sample In the reaction carried out at 2-8°C. the Reaction start adding bovine trypsin at a concentration of 50 m kg/ml

Both samples give a conversion rate of approximately 40%. The reaction at lower temperature (sample) takes longer, but shows a slightly higher output.

Example 5e: 1 g predecessor glargine; dissolved in 1 ml of 1 M TRIS solution. 0.75 ml each of these solutions are selected in two test tubes, labeled as sample a and sample Century To the sample And add 0.75 ml of water, 1.5 ml DMF and 130 mg of TRIS. It is brazzo In add 0.75 ml of water, 1.5 ml DMSO and 130 mg of TRIS. Two samples, a and b, divided into two parts after summarizing the pH to 8.7. The reaction is carried out in all four samples, the addition of bovine trypsin at a concentration of 50 μg/ml Reactions, each one aliquot of the samples a and b is carried out at 2-8°C. the Investigated conditions result in table 4 as follows.

All samples give similar levels of conversion (-40%). The reaction in the samples at room temperature is held for one hour, while the reaction in the cold room go more than 5-6 hours.

Example 5f: 2 g predecessor glargine; dissolved in a solution containing 0.5 g of TRIS, 2 ml water and 2 ml of DMF. From this solution get 6 samples is 3.08 ml, so that each of them contains 50% DMF and 0.5 M TRIS, at pH of 8.7. The concentration of the precursor varies among data samples from 10 g/l to 60 g/L. the Reaction start in each sample by adding 5 mg of trypsin/g predecessor. All samples are at 2-8°C during the reaction. The conditions result in table 5 as follows.

All samples show a conversion, but the samples with a lower concentration give a fast response. The condition is considered the best. The maximum yield obtained in each case, approximately 40-45%. The results are presented in figure 1.

Optimized the s terms of process yield and purity:

Example 5g:

The wet crystals predecessor stored at -20°C in a chamber deep freeze and take out before dissolution. These crystals dissolve putting them in a solution of TRIS (TRIS 50 wt%./mass. crystals of the precursor and water - 2 l/kg of crystals predecessor) in terms of mixing. For example, 1 kg of crystals of the precursor added to 2 l of TRIS solution containing 500 g of TRIS. This is followed by addition of DMF (2 l/kg of crystals predecessor). The entire mixture is completely solubilizers, and this solution is treated as the test solution. The resulting test solution to analyze the content of the product using the method C18 symmetric analytical RP-HPLC.

In accordance with the content of the product in the test solution prepare the control solution for the reaction of trypsinization so that the concentration of the recombinant precursor of insulin glargine; was 10 g/l of the reaction mixture. To get the control solution add the required amount of TRIS in water to maintain a final concentration of TRIS in the reaction mixture of 500 mm. Followed by the addition of DMF so that the final concentration of DMF was 50%. Then the temperature of this solution was lowered to 6°C±2°C and then adjusted the pH to 8.7 using glacial acetic acid (approximately 2% of the total) in the small is about mixing. This solution was treated as a control solution.

The reaction mixture is support at 6°C±2°C at very low speed agitators. After stirring 50 mg/l of trypsin to initiate the reaction. The enzyme reaction begins after trypsin well dissolved with stirring.

The enzymatic reaction is carried out at 6°C±2°C at very low speed agitators. The development of the reaction monitorium checking every hour the proportion of recombinant insulin glargine; and unreacted precursor, using the method C18 symmetric analytical RP-HPLC. The enzymatic reaction is usually completed in less than 10 hours. The reaction is stopped by bringing the pH of the reaction mixture to 5.0, using glacial acetic acid, when the proportion of unreacted precursor is less than 5%. The yield of the reaction is expected to be approximately 40% of recombinant insulin glargine; and the resulting purity is about 35-50%.

Analytical chromatogram of the reaction at different time points are presented in figure 2 and 3.

Example 6: Purification using RP-HPLC:

Example 6A:

The precursor of insulin glargine;, obtained by fermentation of Pichia pastoris, purified with the following stages are included in the process:

A column Packed matrix for fast is Otok SP Sepharose (GE Biosciences), balance 50 mm acetic acid. Cell-free supernatant brought to a pH of 3.8 using orthophosphoric acid load on the cation exchange column. The load on the column is less than 50 g/L. the Elution is performed with the use of ammonium acetate. The selection stage is 95% at a load of less than 50 g/L. the Amount obtained during the elution, crystallized. Crystals are used for trypsinization. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) 250 mm acetic acid (buffer A). The load at the end of trypsinization carried out with the dilution of 1:5 with water. A linear gradient of buffer used for elution purified of glargine; when 75% yield with a purity of 94.5% at a load of less than 10 g/l Column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) in 200 mm sodium acetate pH 5.0 (buffer A). Hold the load OF the eluate obtained at the previous stage, with this dilution to contain 10% acetonitrile. A linear gradient of buffer used for elution purified of glargine; at exit 80% with a purity of 98.5% and a content of glycosylated impurities 0,29%. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance of 6% ethanol (buffer) in 50 mm vinegar is th acid (buffer A). Hold the load OF the eluate obtained at the previous stage, with a dilution of 1:3 with water. A linear gradient of buffer used for elution purified of glargine; at exit 85% with a purity of 99.4% at a load of 35 g/l

Example 6b:

A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) 250 mm acetic acid (buffer A). The load at the end of trypsinization carried out with the dilution of 1:5 with water. A linear gradient of buffer used for elution purified of glargine; when 75% yield with a purity of 94.5% at a load of less than 10 g/l Column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) in 200 mm sodium acetate pH 5.0 (buffer A). Hold the load OF the eluate obtained at the previous stage, with this dilution, so that it contains 10% acetonitrile. A linear gradient of buffer used for elution purified of glargine; at exit 80% with a purity of 98.5% and a content of glycosylated impurities 0,29%. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance of 6% ethanol (buffer) 10 mm citric acid (buffer A). Hold the load OF the eluate obtained at the previous stage, with a dilution of 1:3 with water. A linear gradient of buffer used for elution of purified glargine is at the exit to 79.2% with a purity of 98.5% at a load of 35 g/l

Example 6C:

A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) 250 mm acetic acid (buffer A). The load at the end of trypsinization carried out with the dilution of 1:5 with water. A linear gradient of buffer used for elution purified of glargine; when 75% yield with a purity of 94.5% at a load of less than 10 g/l Column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) in 100 mm Tris+20 mm CaCl2, pH 8.5 (buffer A). Hold the load OF the eluate obtained at the previous stage, with this dilution, so that it contains 10% acetonitrile. A linear gradient of buffer used for elution purified of glargine; while 71% yield with a purity of 97.4% and a content of glycosylated impurities 0,22%. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance of 6% ethanol (buffer) 10 mm citric acid (buffer A). Hold the load OF the eluate obtained at the previous stage, with a dilution of 1:3 with water. A linear gradient of buffer used for elution purified of glargine; when the output 75,0% purity 98,0% at a load of 35 g/l

Example 6d:

A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) 250 mm acetic KIS the OTE (buffer A). The load at the end of trypsinization carried out with the dilution of 1:5 with water. A linear gradient of buffer used for elution purified of glargine; when 75% yield with a purity of 94.5% at a load of less than 10 g/l Column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) in 100 mm ammonium acetate, pH 5.0 (buffer A). Hold the load OF the eluate obtained at the previous stage, with this dilution, so that it contains 10% acetonitrile. A linear gradient of buffer used for elution purified of glargine; when the output 60,7% purity 98,0% and the content of glycosylated impurities of 0.91%. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance of 6% ethanol (buffer) 10 mm citric acid (buffer A). Hold the load OF the eluate obtained at the previous stage, with a dilution of 1:3 with water. A linear gradient of buffer used for elution purified of glargine; when the output 75,0% with a purity of 98.5% at a load of 35 g/l

Example 6E:

A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) 250 mm acetic acid (buffer A). The load at the end of trypsinization carried out with the dilution of 1:5 with water. A linear gradient of buffer used for elution purified of glargine; when in the course of 75% with a purity of 94.5% at a load of less than 10 g/L. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) in 20 mm perchloro acid, pH 3.0 (buffer A). Hold the load OF the eluate obtained at the previous stage, with this dilution, so that it contains 10% acetonitrile. A linear gradient of buffer used for elution purified of glargine; when the output is 37.4% with a purity of 96.2% and a content of glycosylated impurities of 0.28%. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance of 6% ethanol (buffer) 10 mm citric acid (buffer A). Hold the load OF the eluate obtained at the previous stage, with a dilution of 1:3 with water. A linear gradient of buffer used for elution purified of glargine; when the output 70,0% purity 97,0% at a load of 35 g/l

Example 6f:

A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) 250 mm acetic acid (buffer A). The load at the end of trypsinization carried out with the dilution of 1:5 with water. A linear gradient of buffer used for elution purified of glargine; when 75% yield with a purity of 94.5% at a load of less than 10 g/l Column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance 10% acetonitrile (buffer B) in 100 mm borate buffer, pH 8.5 (buffet is). Hold the load OF the eluate obtained at the previous stage, with this dilution, so that it contains 10% acetonitrile. A linear gradient of buffer used for elution purified of glargine; when the output 79,4% with a purity of 98.3% and a content of glycosylated impurities of 0.11%. A column Packed with resin to RP-HPLC (C8 Daisogel, pores 120 a, particle size 10 μm), balance of 6% ethanol (buffer) 10 mm citric acid (buffer A). Hold the load OF the eluate obtained at the previous stage, with a dilution of 1:3 with water. A linear gradient of buffer used for elution purified of glargine; when the output 75,0% with a purity of 98.8 per cent at a load of 35 g/l

Example 7:

The final precipitation:

Glargine, cleared at different stages of reversed-phase chromatography, precipitated by addition of a buffer based on citric acid and solution of ZnCl2and change of the pH value. (Buffer-based citric acid contains up to 15.4 g/l citric acid (anhydrous), 90 g/l of the buffer based on orthopaedically (anhydrous), pH adjusted to 6.3+0,1, phosphoric acid). The deposition is carried out at a pH lying in the range of 4.0 to 10.0, preferably from 6.0 to 8.0. Preferably, when the concentration of the product is 2-20 g/l After deposition, the product is separated from the clear supernatant either by centrifugation or decantation of the supernatant, without disturbing the underlying sediment. About the ADOC, thus obtained, washed with chilled water to remove the present unbound ions.

The output across the way is 85-90%, and the purity of the product is approximately 99%.

Example 8:

The final suspension is decanted after settling and suspension is aseptically transferred into trays installed for freeze drying. Then, the suspension is frozen to obtain a dry powder insulin glargine; that can be stored and used in the preparations of the next.

Example 9:

Isolation and characterization of glycosylated impurities:

Selection glycoform of glargine; active pharmaceutical ingredient, obtained in the laboratory:

Example 9a:

For characterization glycoform impurities mGIG (monopolizirovany insulin glargine) and tGIG (triglyceridemia insulin glargine) allocate prepreparation reversed-phase chromatography. A method for the identification of impurities is carried out on a chromatographic system Agilent 1100 (CA, USA) using column Prochrome 18 (250×8.0 mm). Solvents of the mobile phase is water with 0.1% TN (triperoxonane acid) (A) and 0.1% of TN in acetonitrile (B). Impurities of glargine; elute with a linear gradient: 0 min 25% B; 0-10 min 27% B; 10-15 min 29%; 15-21 min 30% B; 21-27 min 33% and 27-35 min 25% at a constant flow rate of 1.5 mlmin-1. The volume of injection under eribaum of 20 μl and the column temperature is maintained at 40°C. Eluate monitorium at a wavelength of 214 nm. Chromatographic peaks corresponding glycosylated impurities, analyze in real time using a mass spectrometer with an ion trap Agilent 1100 series LCMSD SL (Agilent Technologies, USA). The mass spectrometer operates in the mode ESI (electrospray ionization). The pressure of the sputtering gas is maintained at the level of 0.41 MPa (60 psi), the drying gas at the level of 12.0 l.min-1and the drying temperature is maintained at 350°C. the Mass spectrum obtained using electrospray ionization register as MS in the mass range 600-2200 m/z.

Peaks corresponding mGIG and tGIG, select manually. Because mGIG (1.0%) and tGIG (0,3%) observed in preparations at trace level, active pharmaceutical ingredient glargine (API)that corresponds to this invention is used at a concentration of 100 mgml-1for decontamination related method.

Characterization of the final purified product:

Example 9b:

Profiles OF HPLC and exclusion HPLC of the final product:

Profile OF HPLC and exclusion HPLC profile of the final protein product is presented in Figure 5 and 6.

Example 9c:

Mass spectra electrospray ionization of glargine;, monopolizirovannoi insulin glargine; (mGIG) and delicatelooking insulin glargine; (tGIG) presented on Fig.7, Fig and Fig.9, respectively.

SampleTimeSquareHeightWidthArea, %SymmetryDownload No.118,192190417RUB 399.40,96161000,881

Introduction monosaccharide level glargine reduces retention time due to the increase in polarity and increase in mass units 162 units; similarly, the introduction of three monosaccharides further increases the polarity and increases the weight on 486 units. Molecular weight glycoform obtained by deconvolution of multiple charged peaks in ESI-MS (table 6).

Table 6
Comparison of molecular weight, obtained by means of electrospray ionization and ionization by laser desorption using a matrix
Molecular weight
ESIMALDI
Insulin glargine6063,26063,4
mGIG6225,76225,6
tGIG6549,46549,8

Mass MALDI spectra obtained for mGIG and tGIG, show molecular weight 6225,6 and 6549,8, respectively. The molecular weight obtained in mGIG and tGIG, consistent with data obtained using ESI-MS. The difference in molecular mass between IG, mGIG and tGIG shows the ratio of 162 mass units, suggesting that glikana covalently attached to glargine;.

Fig. The mass spectrum obtained with ionization by matrix laser desorption monopolizirovannoi insulin glargine; (mGIG)

Experiments on recovery and alkylation is carried out to identify the circuit, which is glycosylation. Method standardizes using glargine as standard. 1.1 mg of glargine; dissolved in 500 μl of 8 M guanidine HCl, 0.1 M TRIS and 1 mm EDTA buffer (ethylenediaminetetraacetic acid)maintained at pH of 9.0. To the mixture add 10 ál of 1 M dithiothreitol (DTT). The contents mixed and incubated at 37°C for 2 hours. Inkubirovanie samples are transferred into the conditions of room temperature and add 10 ál of iodoacetamide. Education is s cover with aluminum foil to protect the glare incubated at 37°C for two hours and analyzed by LCMS (liquid chromatography-mass spectrometry). Similarly restore and alkylate mGIG and tGIG to identify glycosylation (table 7).

Example 9d:

The peptide mapping:

Glycoform of glargine; subjected to enzymatic decomposition to identify the position of the amino acids, which is glycosylation. 0.65 mg native of glargine; dissolved in 200 ál of 1 M TRIS (pH 9,0). To it add 50 ál of freshly prepared solution of 1 mgml-1the V 8 protease (Glu-C) and incubated at 37°C for two hours. Product trypsinogen decomposition IG, mGIG and tGIG separated on C18 column 250×4.6 mm, 5 micron, 300° (Waters; symmetry) at a flow rate of 0.8 mlmin-1. The column temperature is maintained at 40°C. Using the following gradient: 0-60 min, 5-80%, 60-65 min, 80-5% B. Solvent A=0.1% aqueous TN and solvent B=acetonitrile. Inject 20 ál of the sample and erwerbende peaks monitorium at a wavelength of 220 nm. The samples analyzed in real time on the instrument Agilent 1100 LCMS analysis of fragments GLU-C. Conduct a comparative study of IG, mGIG and tGIG to detect differences in retention time and molecular mass (table 8).

Summary of the invention

The aim of us is Vashego of the invention is to provide a method for obtaining and purification of recombinant protein which encodes a polypeptide displaying an immunological or biological activity of Mature insulin glargine;. It is shown that the purified protein has all the physiological, immunological and biochemical properties similar to Mature insulin to glargine;.

Another preferred aspect of the present invention is the characterization of glycosylated impurities of glargine; received in final product.

The final and most important objective of this invention to provide a protein insulin glargine; in pure form. The invention thus provides the possibility of large-scale receipt and subsequent treatment of insulin glargine;.

As a consequence, the object of the present invention consists of an improved method of producing insulin glargine; in pure form.

According to the first aspect of the invention a recombinant method of producing insulin glargine; includes:

(a) Obtaining a DNA sequence capable of controlling the formation of microorganism-host protein having the activity of insulin glargine;;

(b) Cloning the specified DNA sequence into a vector capable of transfer and replication in a microorganism host, and this vector contains the functional elements for DNA sequence;

(c) the Migration age is ora, containing the DNA sequence and functional elements, in a microorganism host, able to Express the protein insulin glargine;;

(d) Culturing the microorganism under conditions suitable for amplification of the vector and expression of the protein;

(e) Collecting the protein.

The second aspect of the invention describes a method of constructing a recombinant vector carrying a gene of interest, including the stage of cloning the gene predecessor insulin glargine; in reading frame with the signal peptide expressing in a suitable vector, transforming a host cell with the vector carrying the recombinant gene of interest, screening mnogokupolnyh of integrants, selection of recombinant clones with the successful integration of the precursor of insulin glargine; in the genome.

The third aspect of the invention relates to small-scale expression of the precursor of insulin glargine; for HPLC analysis to determine the level of expression of the desired protein of interest.

A fourth aspect of the invention relates to the fermentation process, including the stage of growth of recombinant cells in the medium for growth, and the cells represent the microorganism or cell culture transformed by expressing a vector containing DNA encoding the desired protein, which in this case is about the invention represents insulin glargine.

The fifth aspect of the invention is directed to a crystallization product of insulin glargine; obtained after fermentation.

The sixth aspect of the invention relates to a process of trypsinization. Glargine can be obtained from the predecessor glargine; by enzymatic conversion. The conversion is carried out in the presence of trypsin or trypsin-like enzymes from plant, animal or microbial nature.

A significant aspect of the present invention relates to the processes and procedures used to clean the selected protein having a biological activity similar to the activity of the natural protein insulin glargine; and, more specifically, to methods of using RP-HPLC or other chromatographic methods of separation of the active peptide substance from other substances that do not have this activity and, thus, they can be considered impurities.

The most preferred aspect of the invention relates to the identification of different glycoform insulin analogues, more specifically insulin glargine; using chemical methods associated with the techniques of mass spectrometry, such as electrospray ionization and ionization by matrix laser desorption intended for identification. Thus, the invention will provide selective purification of the product from the above-mentioned impurities through optimizirovannykh subsequent cleaning methods, due to a clearer understanding of the nature of the impurities present in the final product.

Additional objectives and advantages of the invention will be described in part in the description which follows and in part will be obvious from the description or can be ascertained from the practical implementation of the invention. Objectives and advantages may be realized and attained by using equipment and combinations specified in the attached claims. The present invention relates to a method of obtaining a purified, biologically active heterologous protein.

Accompanying drawings, which are included in this context and form part of this application, illustrate various features used in this invention, and, together with the description, serve to explain the principles of the invention.

1. A method of obtaining a recombinant insulin glargine; characterized by a purity of at least 96% and a content of glycosylated impurities at most 1%by expressing the system based on the yeast, including:
stage cultivation of yeast transformed with the vector containing the DNA sequence defined by the formula X-B-Y-A, encoding the precursor of insulin glargine; in which
X is a sequence of the leader peptide containing Myung is our least one amino acid,
Represents a B1-B30 the amino acid sequence of the b-chain molecules of insulin glargine;,
Y represents a linker peptide containing at least two amino acids,
But a A1-A21 the amino acid sequence of the a-chain molecules of insulin glargine;,
the extraction expressed predecessor insulin glargine; including the Department of the specified predecessor insulin glargine; from yeast with a drug selected predecessor insulin glargine;,
stage of crystallization selected predecessor insulin glargine;,
the stage of carrying out the enzymatic conversion of the crystals of the precursor of insulin glargine; at pH from about 8 to about 10 in the presence of trypsin or a trypsin-like enzyme and a water-soluble organic solvents in a ratio of from approximately 40% to approximately 60% of the reaction mixture with the formation of insulin glargine; containing at least one related impurity,
purification step RP-HPLC insulin glargine; containing at least one related impurity, comprising contacting the specified insulin glargine; with a chromatographic matrix, using polar organic buffer solvent in the aqueous phase, containing the buffer based on organic acids, to the Torah first balance the matrix approximately 10%acetonitrile in approximately 250 mm acetic acid, followed by elution of the specified insulin glargine; in the specified acetonitrile, then re-balance the matrix approximately 10%acetonitrile in a buffer based on organic acid in a concentration from about 20 mm to about 200 mm at a pH of from about 3 to about 8.5, with subsequent elution of the specified insulin glargine; specified in acetonitrile, and then re-balance the matrix approximately 6%ethanol in the buffer based on organic acid in a concentration from about 10 mm to about 50 mm, followed by elution of the specified insulin glargine; specified in ethanol, and
stage deposition of purified insulin glargine; with a purity of at least 96% and a content of glycosylated impurities at most 1% by adding a buffer based on citric acid and zinc chloride at a pH of from about 6 to about 8.

2. The method according to claim 1, wherein the linker peptide is a sequence containing at least two amino acids, provided that the first two amino acids are "RR".

3. The method according to claim 1, in which the precursor of insulin glargine; defined by SEQ ID No:1 or SEQ ID No:2, clone in reading frame with the signal peptide.

4. The method according to claim 3, in which the precursor of insulin glargine; defined by SEQ ID No:1 or SEQ ID No:2, clone in reading frame with the signal peptide mat-α.

. The method according to claim 1, in which the yeast are Pichia pastoris strain GS115.

6. The method according to claim 1, comprising a stage of inoculation aqueous fermentation medium by transformants strain of yeast carrying expressing vector that directs the expression of the indicated insulin glargine; and the stage of growing the transformed strain in a fermentation medium under conditions effective for expression of the indicated insulin glargine;.

7. The method according to claim 1, in which the precursor of insulin glargine; allocate using cation exchange chromatography.

8. The method according to claim 1, in which stage of crystallization of the precursor of insulin glargine; carried out by adding a 4% solution of zinc chloride and phenol in a concentration of from about 0.1% to about 0.5% at a pH of from about 3 to about 8, preferably a pH of from about 3.5 to about 5.5 and a temperature of approximately 2°to approximately 30°C.

9. The method of claim 1, in which stage of the enzymatic conversion is carried out in the presence of trypsin in the water-soluble organic solvent at a pH of from about 8 to about 10 and a temperature of approximately 2°to approximately 25°C.

10. The method according to claim 9, in which the water-soluble organic solvent selected from the group consisting of DMSO, DMF, ethanol, acetone, acetone the reel, ethyl acetate, or mixtures thereof.

11. The method according to claim 1, wherein the polar organic buffer solvent represents acetonitrile.

12. The method according to claim 1, wherein the buffer is an organic acid selected from the group comprising citric acid, acetic acid, boric acid, formic acid, hydrochloric acid and phosphoric acid.



 

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