Recombinant insulin precursor

Abstract

The present invention relates to a recombinant insulin precursor. To improve the recombinant production of insulin, a recombinant insulin precursor according to the chemical formula LBCA is provided, wherein A is the A-chain of insulin, B is the B-chain of insulin, C is a C-peptide connecting the A-chain and the B-chain, and L is an N-terminal L-peptide. The C-peptide comprises an amino acid sequence according to SEQ ID NO: 1 and / or the L-peptide comprises an amino acid sequence according to SEQ ID NO: 2.

Description

Technology Field

[0001] The present invention relates to the field of recombinant insulin precursors for maximizing the expression, yield, and final purity of recombinant insulin precursors. Background Technology

[0002] Insulin is part of the insulin superfamily, which consists of insulin, insulin-related polypeptides, and insulin-like growth factors. These polypeptides are found not only in vertebrates but also in fungi and protists. In fact, their molecular origins can be traced back to at least the simplest single-celled eukaryotes.

[0003] Mature insulin found in mammals is a heteromer composed of two polypeptide chains, the A-chain and the B-chain, connected by two intermolecular disulfide bonds in human insulin. Intermolecular disulfide bonds are formed between amino acid residue 7 of the A-chain and amino acid residue 7 of the B-chain, and between amino acid residue 20 of the A-chain and amino acid residue 19 of the B-chain. A third intramolecular disulfide bond is formed within the A-chain. In human insulin, this intramolecular disulfide bond is formed between amino acid residues 6 and 11 of the A-chain. Insulin has a molecular weight of approximately 5,900 Da and consists of a total of 51–53 amino acids, with slight variation among different animal species. For example, the A-chain and B-chain of human insulin consist of 21 and 30 amino acids, respectively. Porcine insulin is almost identical to human insulin, differing only in residue 30 of the B-chain, where human insulin contains threonine and porcine insulin contains alanine.

[0004] In most vertebrates, insulin is produced in the beta cells of the islets of Langerhans within the pancreas as an inactive insulin precursor called preproinsulin. Preproinsulin is the initial translation product of insulin mRNA. As shown in Fig. 1, preproinsulin consists of an A-chain and a B-chain linked together by an N-terminal signaling peptide or leader peptide (L-peptide) and a C-chain or linking peptide (C-peptide). While the A-chain and B-chain are highly conserved across species, the C-peptide exhibits greater sequence variation and can vary in length, for example, from 26 to 38 residues. Preproinsulin is transported to the endoplasmic reticulum simultaneously with translation, where the L-peptide is removed to produce an inactive insulin precursor referred to as proinsulin. During folding, three disulfide bonds are formed, and the C-peptide is removed to produce mature insulin. After that, mature insulin is packed into granules as a sarcoma and can be secreted upon stimulation of beta cells.

[0005] Insulin secretion occurs when blood glucose levels rise and large amounts of glucose are absorbed and metabolized by beta cells. This can also be stimulated by fatty acids, amino acids, hormones, and keto acids secreted by the gastrointestinal tract. On the other hand, insulin secretion is inhibited when blood glucose levels decrease by somatostatin and sympathetic activation of the nervous system. However, small amounts of insulin are continuously secreted even during fasting. Secreted insulin binds to specific receptors on the outer membranes of target cells in the liver, muscle, and adipose tissue, translocating glucose transporters from inside the cell to the cell membrane, thereby allowing glucose to be absorbed by the cell. Insulin also promotes lipogenesis, glycogen synthesis, and protein synthesis in skeletal muscle and adipose tissue through the kinase-tyrosine receptor pathway.

[0006] Diabetes mellitus (or diabetes for short) can develop when insulin fails to regulate blood glucose levels or can no longer regulate them adequately. Type I diabetes is characterized by the destruction of beta cells. Consequently, less or no insulin is produced, leading to insulin deficiency. In most cases, Type I diabetes is caused by autoimmune responses and T-cell-mediated attacks on beta cells. Type II diabetes is characterized by insulin resistance, which can be combined with relatively reduced insulin secretion. Type II is the most common type of diabetes and is primarily caused by lifestyle factors and genetics. Therefore, the treatment of Type II diabetes involves lifestyle changes, the addition of medications aimed at improving insulin sensitivity or reducing glucose production by the liver, and the administration of insulin. Meanwhile, Type I diabetes is primarily hereditary and is treated through the administration of insulin.

[0007] As early as 1910, it was suggested that diabetes was caused by a deficiency of a hormone from the pancreas. In 1921, this deficient hormone was identified as insulin, and since then, insulin has been used to treat diabetes. Initially, insulin isolated from pigs and cattle was used, but with the invention of DNA cloning, the first recombinant insulin was produced from Escherichia coli bacteria in 1978.

[0008] Today, E. coli remains a widely used expression system for the production of recombinant insulin. Typically, bacteria are transformed with a vector encoding an inactive precursor molecule of insulin, and the cells are cultured in a fermenter. After induction, the precursor is expressed and typically accumulates within the bacteria as insoluble inclusion bodies. After harvesting the cells, they are lysed to obtain inclusion bodies predominantly containing the precursor. The inclusion bodies are subsequently isolated and solubilized, and the precursor is refolded into its natural structure, where disulfide bonds are formed. The folded precursor is then purified to remove host cell impurities and misfolded molecules. Finally, the precursor is digested with trypsin and / or carboxypeptidase B to cleave the N-terminal L-peptide and C-peptide, thereby producing mature recombinant insulin. As part of the recombinant manufacturing process, an additional step of purifying the mature insulin will be required to achieve the desired product quality.

[0009] The number of people diagnosed with diabetes worldwide rose from 108 million in 1980 to 537 million in 2021, and this number is expected to continue rising. Additionally, animals can suffer from diabetes, which is most common in dogs and cats. As in humans, the prevalence of diabetes is increasing in animals. To meet the demand for insulin for treatment in both humans and animals, the production process for recombinant insulin is continuously being improved to reduce costs, increase efficacy, and increase yield.

[0010] WO 2001 / 049742 describes the synthesis of human insulin precursors from Saccharomyces cerevisiae containing at least one aromatic amino acid residue, Phe, Trp, or Tyr, and generally comprising a C-peptide of 15 amino acids or less in length. These precursors yielded higher production yields compared to precursors having natural C-peptides.

[0011] WO 2001 / 025278 describes a human insulin precursor comprising a C-peptide with a length range of 2 to 50 amino acids that improves the production yield of the precursor.

[0012] The literature [Min et al., 2011, J. Biotechnol. 151:350-356] describes improved expression, folding, and enzymatic reaction rates of recombinant human insulin when a specific L-peptide is selected for the precursor of human insulin.

[0013] WO 2017 / 040363 describes a method for obtaining insulin having a correctly formed disulfide bond.

[0014] However, there is still a need to significantly improve the recombinant production of insulin.

[0015] In a first aspect of the present invention, a recombinant insulin precursor according to the formula LBCA is provided, wherein:

[0016] A is the A-chain of insulin, and

[0017] B is the B-chain of insulin, and

[0018] C is a C-peptide that connects the A-chain and the B-chain, and

[0019] L is an N-terminal L-peptide, and

[0020] Here, C is a C-peptide containing the amino acid sequence according to SEQ ID NO: 1 and / or L is an L-peptide containing the amino acid sequence according to SEQ ID NO: 2.

[0021] Another aspect of the present invention is a DNA sequence encoding a recombinant insulin precursor and / or an embodiment thereof according to the present invention, wherein preferably, the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 9 and / or the L-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 10.

[0022] The recombinant insulin precursor according to the present invention has been found to provide a desirable yield, such as an improved yield of insulin.

[0023] The yield of one or more recombinant insulin precursors according to the present invention (e.g., expressed as fermentation potency, e.g., mmol / L or g / L) was compared with the yield of a reference recombinant insulin precursor (see SEQ ID NO: 5). The reference recombinant insulin precursor, consisting of the amino acid sequence according to SEQ ID NO: 5, substantially corresponds to the insulin precursor described in WO 2017 / 040363 (see FIG. 2 of WO 2017 / 040363). The difference between SEQ ID NO: 5 and the insulin precursor presented in WO 2017 / 040363 is that the reference recombinant insulin precursor of SEQ ID NO: 5 contains an additional methionine (M) residue at the N-terminal position compared to the precursor disclosed in WO 2017 / 040363. This methionine residue is cleaved by methionine aminopeptidase during fermentation in Escherichia coli. WO 2017 / 040363 describes a method for obtaining insulin having a precisely formed disulfide bond using an insulin precursor. The cleavage of an additional methionine residue at the N-terminal position of the insulin precursor of WO2017 / 040363 is already completed before initiating the method described in WO2017 / 040363. Additionally, the reference recombinant insulin precursor according to sequence identification number: 5 contains alanine (A) at residue B30 of the B-chain, whereas the amino acid sequence of FIG. 2 of WO 2017 / 040363 contains threonine (T) at residue B30 of the B-chain.

[0024] A recombinant insulin precursor comprising the amino acid sequence according to SEQ ID NO: 6 is a recombinant insulin precursor according to the present invention and / or embodiments thereof, comprising an A-chain and a B-chain of insulin comprising the amino acid sequences according to SEQ ID NO: 3 and 4, respectively, and a reference L-peptide from a reference recombinant insulin precursor (SEQ ID NO: 5) substantially similar to the L-peptide disclosed in WO2017 / 040363; SEQ ID NO: 5 further comprises an N-terminal methionine residue. Unlike the reference recombinant insulin precursor (SEQ ID NO: 5), the recombinant insulin precursor according to the present invention of SEQ ID NO: 6 contains a novel recombinant C-peptide according to SEQ ID NO: 1. Surprisingly, the fermentation potency (mmol / L) of the insulin precursor according to SEQ ID NO: 6 was higher than the fermentation potency (mmol / L) of the reference recombinant insulin precursor according to SEQ ID NO: 5. As shown in Table 1 of Example 1, when the precursors were expressed in host cells under the same fermentation conditions, the fermentation potency (mmol / L) of the insulin precursor according to sequence identification number: 6 was approximately 43% higher than the fermentation potency (mmol / L) of the reference insulin precursor according to sequence identification number: 5. Therefore, replacing the natural human C-peptide of the reference precursor with the C-peptide according to sequence identification number: 1 significantly increases the fermentation potency of the precursor.

[0025] Another recombinant insulin precursor according to the present invention and / or embodiments thereof is a recombinant insulin precursor comprising the amino acid sequence according to SEQ ID NO: 7. The recombinant insulin precursor contains the A-chain and B-chain of insulin, each comprising the amino acid sequences according to SEQ ID NO: 3 and 4, respectively, in combination with a novel recombinant L-peptide according to SEQ ID NO: 2 and a modified C-peptide derived from the amino acid sequence of a human C-peptide. The fermentation potency (mmol / L) of the insulin precursor according to SEQ ID NO: 7 was higher than the fermentation potency (mmol / L) of the reference recombinant insulin precursor (SEQ ID NO: 5). As shown in Table 1 of Example 1, the fermentation potency (mmol / L) of the insulin precursor according to SEQ ID NO: 7 was about 39% higher than the fermentation potency (mmol / L) of the reference recombinant insulin precursor (SEQ ID NO: 5). Therefore, replacing the natural human L-peptide of the reference precursor with the L-peptide according to sequence identification number: 2 increases the fermentation activity of the insulin precursor.

[0026] Another recombinant insulin precursor according to the present invention and / or embodiments thereof is a recombinant insulin precursor comprising the amino acid sequence according to SEQ ID NO: 8, and contains the A-chain and B-chain of insulin comprising the amino acid sequences according to SEQ ID NO: 3 and 4, respectively, and the C-peptide according to SEQ ID NO: 1. In addition, SEQ ID NO: 8 contains the L-peptide according to SEQ ID NO: 2. The fermentation potency (mmol / L) of the insulin precursor according to SEQ ID NO: 8 was much higher than the fermentation potency (mmol / L) of the insulin precursor according to SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. As shown in Table 1 of Example 1, the fermentation potency (mmol / L) of the insulin precursor according to SEQ ID NO: 8 was even about 118% higher than the fermentation potency (mmol / L) of the reference recombinant insulin precursor (SEQ ID NO: 5). Therefore, replacing the natural human C-peptide with the C-peptide according to sequence identification number: 1, and additionally replacing the reference L-peptide with the L-peptide according to sequence identification number: 2, increases the fermentation activity of the insulin precursor in a synergistic manner.

[0027] A further aspect of the present invention relates to a vector comprising a DNA sequence encoding a recombinant insulin precursor according to the present invention.

[0028] A further aspect of the present invention relates to a host cell (containing a vector) comprising a DNA sequence encoding a recombinant insulin precursor according to the present invention.

[0029] An additional aspect relates to a method for producing recombinant insulin, wherein a recombinant insulin precursor according to the present invention is expressed, and wherein the method

[0030] a) A step of expressing a recombinant insulin precursor through microbial fermentation,

[0031] b) a step of obtaining a recombinant insulin precursor by lysing microorganisms,

[0032] c) a step of refolding the recombinant insulin precursor and

[0033] d) includes the step of obtaining recombinant insulin by cleaving the L-peptide and C-peptide of the recombinant insulin precursor by an enzymatic reaction.

[0034] An additional aspect relates to the use of a recombinant insulin precursor according to the present invention for the production of recombinant insulin.

[0035] The final aspect relates to a composition comprising recombinant insulin, wherein the composition comprises detectable amounts of C-peptide and / or L-peptide as provided herein. Brief explanation of the drawing

[0036] Figure 1 shows a diagram of a pre-proinsulin molecule, a proinsulin molecule, and an insulin molecule. Figure 2 shows the liquid chromatography-mass spectrometry profile of a mature insulin molecule produced from the expression of a DNA construct encoding an asparagine residue by codon "AAC" in E. coli. Figure 3 shows the liquid chromatography-mass spectrometry profile of a mature insulin molecule produced from the expression of a DNA construct encoding a serine residue by codon "TCT" in E. coli. Specific details for implementing the invention

[0037] definition

[0038] For the purposes of the present invention, the following terms are defined below.

[0039] The term "peptide" refers to a short sequence of amino acids, typically 2 to 50 amino acids in length.

[0040] The term "C-peptide" refers to the linking peptide or linking moiety "C" of the BCA sequence of an insulin precursor molecule. The C-peptide links the C-terminus of the B-chain and the N-terminus of the A-chain of insulin.

[0041] The term "L-peptide" refers to the leader peptide present as an N-terminal leader sequence on the precursor form of the insulin protein. The L-peptide of naturally occurring insulin enables the protein to translocate into the endoplasmic reticulum. The L-peptide is normally cleaved during this translocation process.

[0042] The term "precursor" refers to the immature form of a protein that must still be processed before its final mature form is obtained. In the case of insulin, the precursor still contains C-peptides and / or L-peptides. To produce mature insulin, the C-peptides and L-peptides must be cleaved.

[0043] The term "proinsulin" refers to an immature form of insulin composed of a B-chain peptide fused to the N-terminus of a C-peptide, which in turn fuses its C-terminus to the N-terminus of an A-chain peptide. Proinsulin is a type of insulin precursor.

[0044] The term "preproinsulin" refers to an immature form of insulin composed of an N-terminal L-peptide, in which the L-peptide is fused at its C-terminus to the N-terminus of a B-chain peptide, the B-chain peptide is fused at the N-terminus of a C-peptide, and the C-peptide is fused at its C-terminus to the N-terminus of an A-chain peptide. Thus, preproinsulin is represented by the chemical formula LBCA. Preproinsulin is a type of insulin precursor.

[0045] In this document, the term "vector" refers to a construct used to introduce nucleic acids, such as DNA, into a host cell. After the vector is introduced into the host cell, the nucleic acid, such as DNA, contained within the vector may be expressed by the host cell to produce a protein encoded by the nucleic acid, such as DNA. The vector may be, for example, a virus, but is generally a plasmid, such as a pET plasmid or a pGEX plasmid.

[0046] In this document, the term "host cell" refers to a cell used to express a recombinant insulin precursor. Generally, the host cell is a microorganism, such as a yeast cell or a bacterium. Suitable host cells are Escherichia coli (E. coli) and Saccharomyces cerevisiae (S. cerevisiae).

[0047] The term “culture of host cells” refers to a population of host cells grown under controlled conditions, for example, in a medium containing one or more nutrients and having a pH suitable for specific host cells, and at a temperature and O2 and / or CO2 concentration suitable for the proliferation of specific host cells.

[0048] The term "fermentation potency" refers to the concentration of a molecule of interest in a solution. Here, fermentation potency is the concentration of a recombinant insulin precursor in the medium used to grow host cells expressing the precursor. Fermentation potency can be expressed, for example, in g / mL or mmol / L (mM), and can be used, for example, as a measure of protein yield.

[0049] As used herein, one amino acid sequence is 100% "identical" or has 100% "sequence identity" with a second amino acid sequence when the amino acid residues of the two sequences are aligned identically when determined using the "Global Alignment" sub-program of the computer program "BLAST®", which can be found at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi, with standard settings. Accordingly, an amino acid sequence (or nucleic acid sequence) may be 50% "identical" with a second amino acid sequence (or second nucleic acid sequence) when 50% of the amino acid residues of the two amino acid sequences (or the nucleic acids of the two nucleic acid sequences) are identical when determined using the "Global Alignment" sub-program of the computer program "BLAST®" with standard settings. Sequence comparison is performed on adjacent blocks of amino acid residues contained by a given protein, for example, a protein or a portion of the polypeptide being compared. In certain embodiments, selected substitutions, deletions, or insertions that alter or do not alter the correspondence between two amino acid sequences are also included. Similarly, one nucleic acid sequence has 100% "identical" or 100% "sequence identity" with a second nucleic acid sequence when the nucleic acid residues of the two sequences are aligned and determined using standard settings with the "Global Alignment" sub-program of the computer program "BLAST®", which can be found at https: / / blast.ncbi.nlm.nih.gov / Blast.cgi.

[0050] The present invention is defined in this application and the appended claims. Objects not included within the scope of the claims do not form part of the claimed invention.

[0051] Any product, method, use, or composition described herein is to be considered to be embodied in connection with any other product, method, use, or composition described herein. An embodiment disclosed in connection with a product, method, use, or composition of the present invention may be used in connection with any other product, method, use, or composition described herein. Accordingly, an embodiment relating to one product, method, use, or composition may also be applied to other products, methods, uses, or compositions of the present invention.

[0052] amino acid sequence

[0053] The present invention relates to a recombinant insulin precursor according to the chemical formula LBCA. Here, L represents an N-terminal L-peptide, B represents the B-chain of insulin, C represents a C-peptide connecting the A-chain and the B-chain, and A represents the A-chain of insulin.

[0054] C is a linking peptide (or C-peptide) that connects the N-terminus of the A-chain and the C-terminus of the B-chain of insulin. While the A-chain and B-chain are widely conserved across species, the C-peptide exhibits greater variation in both sequence and length. Natural human C-peptide is, for example, 31 residues long, natural porcine C-peptide is 29 residues long, and natural bovine C-peptide is 26 residues long.

[0055] To support the refolding process of the recombinant insulin precursor, the C-peptide of the recombinant insulin precursor contains 2 to 30 amino acid residues, preferably 8 to 20 residues, more preferably 10 to 15 amino acid residues.

[0056] Trypsin or trypsin-like enzymes catalyze the cleavage of protein amide bonds on the C-terminal side of arginine and lysine residues and are used for the conversion of a precisely folded precursor protein into a precisely folded insulin or insulin analog in this process. To facilitate the cleavage of the C-peptide using trypsin after the expression of a recombinant insulin precursor in microorganisms, it is preferable that the C-terminal residue of the C-peptide be an arginine residue. In one preferred embodiment, the C-peptide according to the present invention consists of 30 amino acid residues (modified C-peptide, SEQ ID NO: 7 and Table 2) or 11 amino acid residues (novel recombinant C-peptide; SEQ ID NO: 1).

[0057] After the expression of a recombinant insulin precursor in a microorganism, in order to facilitate the cleavage of the C-peptide by, for example, using trypsin, it is desirable that the C-terminal residues of the B-peptide and / or C-peptide contain amino acid residues suitable for enabling the cleavage of the peptide, such as one or more arginine residues.

[0058] Accordingly, in one preferred embodiment, the C-peptide is a C-peptide comprising an amino acid sequence according to SEQ ID NO: 1 or a C-peptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 1. In a more preferred embodiment, the C-peptide comprises an amino acid sequence according to SEQ ID NO: 1. A recombinant insulin precursor comprising a C-peptide as provided herein has been found to produce a desirable fermentation potency of the insulin precursor.

[0059] L is an N-terminal leader peptide (or L-peptide) connected to the N-terminal end of the B-chain. In naturally occurring human insulin, the L-peptide has a length of 24 residues. When insulin is expressed in mammalian cells, the L-peptide acts as a signal peptide that transports preproinsulin to the endoplasmic reticulum, where the L-peptide is cleaved. However, when recombinant insulin is expressed in microorganisms, the L-peptide serves to protect recombinant insulin from internal degradation and / or modification during synthesis in microorganisms and to improve the protein expression of the recombinant insulin precursor in microorganisms. When recombinant insulin is expressed in microorganisms, the L-peptide is cleaved after the expression of the recombinant insulin precursor. To facilitate the cleavage of the L-peptide after the expression of the recombinant insulin precursor in microorganisms, for example by using trypsin or a trypsin-like agonist, it is desirable that the C-terminal residue of the L-peptide contains amino acid residues suitable for enabling peptide cleavage, such as one or more arginine residues.

[0060] Preferably, the L-peptide comprises 10 to 25 amino acid residues. More preferably, the L-peptide comprises 12 to 24 amino acid residues, and even more preferably, comprises 15 to 20 residues. In one preferred embodiment, the L-peptide according to the present invention consists of 16 amino acid residues (novel recombinant L-peptide; sequence identification number: 2).

[0061] To obtain a relatively high fermentation potency of the recombinant insulin precursor upon expression in microorganisms, the L-peptide provided herein preferably has a net positive charge at pH 6.0. That is, the L-peptide has a pI > 6.0. Preferably, the L-peptide has a pI > 6.5, more preferably a pI > 7.0. The amino acids lysine, arginine, and histidine have a pI > 7.5. Accordingly, preferably, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, or at least 70% of the residues of the L-peptide are selected from lysine, arginine, and histidine. For example, the L-peptide contains 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 residues selected from lysine, arginine, and histidine.

[0062] Accordingly, in one preferred embodiment, the L-peptide is an L-peptide comprising an amino acid sequence according to SEQ ID NO: 2 or an L-peptide comprising an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 2. In a more preferred embodiment, the L-peptide comprises an amino acid sequence according to SEQ ID NO: 2. A recombinant insulin precursor comprising an L-peptide as provided herein has been found to produce a desirable fermentation potency of the insulin precursor.

[0063] It has been further revealed that a recombinant insulin precursor having the amino acid sequence of SEQ ID NO: 8 results in a particularly desirable fermentation potency of the insulin precursor. Accordingly, in one embodiment, the recombinant insulin precursor according to the present invention comprises a C-peptide comprising an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1, and an L-peptide comprising an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 2. Preferably, the C-peptide consists of the amino acid sequence according to SEQ ID NO: 1, and the L-peptide consists of the amino acid sequence according to SEQ ID NO: 2.

[0064] Insulin is a highly conserved protein with only slight variations among different animal species. For example, the A-chain of human insulin consists of 21 amino acids (numbered A1-A21), and the B-chain of human insulin consists of 30 amino acids (numbered B1-B30).

[0065] A is the A-chain of insulin and may be any type of A-chain, e.g., the A-chain of human, sheep, dog, cat, pig, or bovine insulin. In a preferred embodiment, A is derived from the A-chain of porcine insulin. The A-chain of porcine insulin corresponds to the A-chain of human insulin. The A-chain may contain the amino acid sequence according to SEQ ID NO: 3, while the bovine A-chain differs from the human A-chain by only two amino acids. Human and porcine insulin contain threonine and isoleucine residues at residue positions A8 and A10, respectively (e.g., referring to the 8th and 10th amino acid residues according to the numbering in SEQ ID NO: 3), whereas bovine insulin contains alanine and valine residues at the respective positions. In a preferred embodiment, the A-chain is derived from the A-chain of porcine insulin.

[0066] In addition, amino acid modifications have also been introduced to obtain different synthetic variants of insulin. Examples of insulin variants are insulin lispro and insulin glargine. An example of an amino acid modification to obtain synthetic insulin is, for instance, obtaining insulin glargine by substituting an asparagine residue at position A21 of regular human insulin with glycine; insulin glargine is a slow-acting type of insulin that remains at relatively low levels in the blood for a period of 24 hours upon subcutaneous injection. Such modifications in insulin proteins are also included in the present invention.

[0067] Accordingly, in one embodiment of the present invention and / or an embodiment thereof, the A-chain is an A-chain comprising an amino acid sequence according to SEQ ID NO: 3 or an A-chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 3. The A-chain according to SEQ ID NO: 3 may be modified by, for example, amino acid substitution, deletion, or insertion at a selected position from A1, A2, A3, A4, A5, A8, A9, A10, A12, A13, A14, A15, A16, A17, A18, A19, and / or A21 to obtain an A-chain included in the recombinant insulin precursor. In one embodiment of the present invention and / or an embodiment thereof, the A-chain according to SEQ ID NO: 3 is modified at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residue positions. A person skilled in the art knows the amino acid sequence of a suitable A-chain of insulin that can be used in the invention provided herein.

[0068] Additionally, amino acids of the A-chain of the recombinant insulin precursor may undergo post-translational modification, such as acetylation (conjugating an acetyl group to an amino acid), PEGylation (conjugating a polyethylene glycol (PEG) group to an amino acid), lipidation (conjugating a lipid to an amino acid), or glycosylation (conjugating a carbohydrate moiety to an amino acid), or deamidation of asparagine residues, either directly or through a linker. In one embodiment of the present invention and / or an embodiment thereof, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 amino acid residues of the A-chain of the recombinant insulin precursor are post-translationally modified.

[0069] B is the B-chain of insulin and can be any type of B-chain, such as the B-chain of human, sheep, dog, cat, pig, or bovine insulin. In a preferred embodiment, the B-chain is derived from the B-chain of porcine insulin. Like the A-chain, the B-chain of insulin is highly conserved among animal species. Porcine insulin and bovine insulin differ from human insulin only at residue B30, where human insulin contains a threonine residue, whereas porcine and bovine insulin contain an alanine residue.

[0070] Substitution of amino acids in the B-chain of insulin can affect the activity of insulin. For example, extending the B-chain with two arginine residues contributes to the slow-acting profile of insulin glargine. On the other hand, substituting a proline residue at position B28 of human insulin with lysine and substituting a lysine residue at position B29 of human insulin with proline produces a rapid-acting insulin that triggers a blood insulin spike within 30 minutes of subcutaneous injection and breaks down within 4 hours of injection.

[0071] The B-chain disclosed and provided herein according to sequence identification number 4 comprises two arginine residues at the C-terminus of the B-chain, i.e., at positions B31 and B32. The present invention is also understood to encompass a B-chain of insulin that does not comprise the two arginine residues.

[0072] Accordingly, in one embodiment of the present invention and / or an embodiment thereof, the B-chain is a B-chain comprising an amino acid sequence according to SEQ ID NO: 4 or a B-chain having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with SEQ ID NO: 4. The B-chain according to SEQ ID NO: 4 may be modified by amino acid substitution, deletion, or insertion at a selected position from B1, B2, B3, B4, B5, B9, B10, B13, B14, B15, B16, B17, B18, B20, B21, B22, B23, B26, B27, B28, B29, and / or B30 to obtain a B-chain such as that contained in, for example, a recombinant insulin precursor. In one embodiment of the present invention and / or an embodiment thereof, the B-chain according to SEQ ID NO: 4 is modified at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 residue positions. A person skilled in the art knows suitable amino acid sequences of the B-chain of insulin that can be used in the invention provided herein.

[0073] The amino acids of the B-chain of a recombinant insulin precursor may undergo post-translational modifications similar to those previously described for the amino acids of the A-chain of a recombinant insulin precursor. In one embodiment of the present invention and / or an embodiment thereof, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids of the B-chain of a recombinant insulin precursor are modified after translation.

[0074] The A-chain and B-chain of a recombinant insulin precursor may be derived from different species. The A-chain may be, for example, the A-chain from human insulin, while the B-chain may be the B-chain from porcine insulin or vice versa. Additional combinations of A-chains and B-chains from different species are also included herein.

[0075] Optionally, the recombinant insulin precursor may include one or more extension peptides and / or spacer peptides between the C-terminus of the leader peptide and the N-terminus of the B-chain peptide.

[0076] In a further embodiment of the present invention and / or an embodiment thereof, the recombinant insulin precursor comprises an amino acid sequence according to SEQ ID NO: 6, 7 or 8, or comprises an amino acid sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, or at least 99% sequence identity with the recombinant insulin precursor according to SEQ ID NO: 6, 7 and / or 8. Preferably, the recombinant insulin precursor is a protein having an amino acid sequence according to SEQ ID NO: 6, 7 or 8.

[0077] A recombinant insulin precursor comprising an amino acid sequence according to sequence identification number: 6, 7, or 8 comprises an A-chain according to sequence identification number: 3 and a B-chain according to sequence identification number: 4. It is considered herein that a person skilled in the art may derive the A-chain and / or B-chain of a recombinant insulin precursor from different species (e.g., humans, ruminants, or pets) and may provide a recombinant insulin precursor comprising a C-peptide and / or L-peptide according to the present invention further comprising the A-chain and / or B-chain from different species. Accordingly, the present invention also provides a recombinant insulin precursor comprising at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%, at least 99% sequence identity with the recombinant insulin precursor according to SEQ ID NO: 6, 7 and / or 8, or comprising a C-peptide according to SEQ ID NO: 1 and an L-peptide according to SEQ ID NO: 2, and comprising an A-chain and / or B-chain from different species.

[0078] Additionally, a protein containing the amino acid sequence according to sequence identification number: 6 contains a C-peptide according to sequence identification number: 1 and a reference L-peptide of WO2017 / 040363, and further contains N-terminal methionine. A protein containing the amino acid sequence according to sequence identification number: 7 contains an L-peptide according to sequence identification number: 2 and a modified C-peptide derived from the sequence of a human C-peptide. A protein containing the amino acid sequence according to sequence identification number: 8 contains both the C-peptide according to sequence identification number: 1 and the L-peptide according to sequence identification number: 2. As exemplified herein, the expression of a recombinant insulin precursor containing an amino acid sequence according to sequence identification number: 6, 7, or 8 resulted in a higher fermentation potency of the recombinant insulin precursor than that of the reference recombinant insulin precursor according to sequence identification number: 5 (derived from WO2017 / 040363, wherein the amino acid sequence of sequence identification number: 5 additionally contains N-terminal methionine).

[0079] DNA sequence

[0080] In another aspect, the present invention relates to a DNA sequence encoding a recombinant insulin precursor according to the chemical formula LBCA, wherein:

[0081] A is the A-chain of insulin, and

[0082] B is the B-chain of insulin, and

[0083] C is a C-peptide that connects the A-chain and the B-chain, and

[0084] L is an N-terminal L-peptide.

[0085] The A-chain, B-chain, C-peptide, and L-peptide disclosed herein may each be encoded by any DNA sequence that generates an amino acid sequence forming the A-chain of the insulin, the B-chain of the insulin, preferably the C-peptide according to sequence identification number: 1, and / or preferably the L-peptide according to sequence identification number: 2.

[0086] In one embodiment, the present invention relates to a DNA sequence encoding a recombinant insulin precursor according to the formula LBCA, wherein:

[0087] A is the A-chain of insulin, and

[0088] B is the B-chain of insulin, and

[0089] C is a C-peptide that connects the A-chain and the B-chain, and

[0090] L is an N-terminal L-peptide;

[0091] Here, C is a C-peptide containing an amino acid sequence according to sequence identification number: 1 or an amino acid sequence having at least 60% sequence identity with sequence identification number: 1, and / or L is an L-peptide containing an amino acid sequence according to sequence identification number: 2 or an amino acid sequence having at least 60% sequence identity with sequence identification number: 2.

[0092] It is known that similar codons are transcribed with varying efficiencies in different organisms. Since recombinant insulin precursors are mostly produced in microorganisms, in some preferred embodiments of the present invention, the DNA sequence is codon-optimized for expression in microorganisms. Optimization can be achieved, for example, using software such as GeneOptimizer and DNA2.0.

[0093] DNA sequences of genes encoding natural insulin, such as natural human or natural porcine insulin including A-chains, B-chains, and C-peptides, are described in the relevant field of technology, e.g., Bell, G., Pictet, R., Rutter, W. et al. [Sequence of the human insulin gene Nature 284, 26-32 (1980)] or Xu-Guang Han et al. [Cloning and characterization of porcine insulin gene, Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, Volume 129, Issue 1, 2001, Pages 87-95]. Therefore, a person skilled in the art is familiar with DNA sequences encoding natural insulin or any one of natural A-chains, natural B-chains, natural C-peptides, or natural L-peptides as found in various mammals. An example of a DNA sequence for natural human C-peptide.

[0094] A DNA sequence for expressing a recombinant insulin precursor according to the chemical formula LBCA is provided herein, wherein:

[0095] A is preferably the A-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 11;

[0096] B is preferably the B-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 12;

[0097] C is a C-peptide connecting the A-chain and the B-chain;

[0098] L is an N-terminal L-peptide, where the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 9 and / or the L-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 10.

[0099] Preferably, the A-chain of the recombinant insulin precursor is encoded by a DNA sequence according to sequence identification number: 11 or a DNA sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with sequence identification number: 11, and the higher the sequence identity, the more preferable it is. Preferably, the B-chain of the recombinant insulin precursor is encoded by a DNA sequence according to sequence identification number: 12 or a DNA sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with sequence identification number: 12, and the higher the sequence identity, the more preferable it is. Preferably, the C-peptide of the recombinant insulin precursor is encoded by a DNA sequence according to sequence identification number: 9 or a DNA sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with sequence identification number: 9, and the higher the sequence identity, the more preferable it is. Preferably, the L-peptide of the recombinant insulin precursor is encoded by a DNA sequence according to sequence identification number: 10 or a DNA sequence having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with sequence identification number: 10, and the higher the sequence identity, the more preferable it is.

[0100] Insulin is a mammalian protein that is not normally expressed by microorganisms commonly used as host cells for producing recombinant insulin. Although typically one amino acid is encoded by different codons, codon preferences differ between mammalian cells and microorganisms, and expressing mammalian DNA sequences in microorganisms generally results in a relatively low yield of the encoded peptide or protein. Codon optimization of DNA sequences for expression in microorganisms improves the yield of recombinant precursors and the fidelity of expression.

[0101] Recombinant insulin or mature insulin molecules produced during the expression of DNA sequences by microorganisms and subsequent digestion of molecules to remove L-peptides and C-peptides can be analyzed using analytical techniques known in the relevant art, such as liquid chromatography-mass spectrometry (LC-MS).

[0102] As shown in FIG. 2, when the DNA sequence encoding the recombinant insulin precursor is expressed in E. coli, the peak of the LC-MS profile at 21 to 23 seconds (indicated by the arrow) is strongly reduced when the codon "AAC" (instead of "AAT") is used to code for the asparagine residues present at residue positions A18 and A21 of the A-chain of the insulin molecule. This indicates improved expression accuracy and reduced amino acid miscontamination. Accordingly, in one embodiment of the present invention and / or an embodiment thereof, the asparagine residues of the A-chain of the recombinant insulin precursor are coded by the codon "AAC". Preferably, the asparagine residues at residue positions A18 and A21 of the A-chain of insulin are coded by the codon "AAC". When more than one asparagine residue is present in the A-chain of the recombinant insulin precursor, for example, 2, 3, 4, or all asparagine residues of the A-chain of the recombinant insulin precursor are coded by the codon "AAC".

[0103] As shown in FIG. 3, when the DNA sequence encoding a recombinant insulin precursor is expressed in E. coli, the peak of the LC-MS profile at 20.5 to 21.5 seconds is strongly reduced when the codon "TCT" (instead of "AGC") is used to code for serine residues present at residue positions A9 and A12 of the insulin molecule's A-chain. Likewise, this indicates improved expression accuracy and reduced amino acid miscontamination. Accordingly, in one embodiment of the present invention and / or an embodiment thereof, the serine residues of the A-chain of the recombinant insulin precursor are coded by the codon "TCT". Preferably, the serine residues at residue positions A9 and A12 of the insulin's A-chain are coded by the codon "TCT". Where more than one serine residue is present in the A-chain of the recombinant insulin precursor, for example, two, three, four, or all serine residues of the A-chain of the recombinant insulin precursor are coded by the codon "TCT".

[0104] In some embodiments of the present invention and / or embodiments thereof, one or more asparagine residues of the A-chain of the recombinant insulin precursor are coded by codon "AAC", and one or more serine residues of the A-chain of the recombinant insulin precursor are coded by codon "TCT". In one preferred embodiment, the A-chain comprises asparagine residues at residue positions A18 and A21 of the insulin A-chain being coded by codon "AAC", and serine residues at residue positions A9 and A12 of the insulin A-chain being coded by codon "TCT", as exemplified, for example, in the amino acid sequence of A-chain sequence identification number: 3 and the corresponding DNA sequence sequence identification number: 11.

[0105] Because the amino acid sequence of the A-chain is located at the C-terminal end of the amino acid sequence of the recombinant insulin precursor, the DNA sequence encoding the A-chain and / or the recombinant insulin precursor according to the present invention may additionally include a stop codon sequence. A person skilled in the art is aware of the function and nucleic acid sequence of such stop codons, and this is therefore incorporated herein. A stop codon as disclosed herein may, in non-limiting examples, be "TAA" and may be located after nucleic acid residue 63 of SEQ ID NO: 11 (thus, "TAA" would be nucleic acid residues 64–66 in SEQ ID NO: 11).

[0106] In additional embodiments, the DNA sequences disclosed herein have been optimized for expression in microorganisms, preferably E. coli. It is understood that codon optimization can be used to optimize DNA sequences for expression in microorganisms other than E. coli. Accordingly, it is understood that any one or more of such optimized DNA sequences, preferably the DNA sequences of sequence identification numbers: 9-15, are also covered by the present disclosure.

[0107] In one embodiment of the present invention and / or an embodiment thereof, a DNA sequence encoding a recombinant insulin precursor according to the present invention comprises a DNA sequence according to SEQ ID NO: 13, 14 or 15 or a DNA sequence having at least 60% sequence identity with SEQ ID NO: 13, 14 or 15. More preferably, the recombinant insulin precursor according to the present invention is encoded by a DNA sequence having at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence identity with SEQ ID NO: 13, 14 or 15, and higher sequence identity is more preferable. In one preferred embodiment, the recombinant insulin precursor according to the present invention is encoded by a DNA sequence of SEQ ID NO: 13, 14 or 15.

[0108] A DNA sequence encoding a recombinant insulin precursor may be included in a vector for expressing the recombinant insulin precursor. Accordingly, in another aspect, the present invention relates to a vector comprising a DNA sequence for expressing a recombinant insulin precursor as provided herein. In some embodiments, a vector comprising a DNA sequence for expressing a recombinant insulin precursor according to the formula LBCA is provided, wherein:

[0109] A is preferably the A-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 11;

[0110] B is preferably the B-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 12;

[0111] C is a C-peptide connecting the A-chain and the B-chain, and preferably, the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 9;

[0112] L is preferably an N-terminal L-peptide encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 10.

[0113] The DNA sequence is generally cloned downstream of a promoter sequence, such as a T7 promoter or a tac promoter, and operably linked to it. The DNA sequence can be cloned into the vector by open-cleaving the vector using a restriction enzyme that recognizes restriction sites present in the vector, such as XhoI, Bam, NdeI, BglI, and HindIII. Subsequently, the DNA sequence flanked with a nucleic acid sequence compatible with each restriction site can be inserted into the vector, and the vector can be closed using an enzymatic ligase. Generally, the vector contains one or more antibiotic resistance genes, for example, conferring resistance to kanamycin or ampicillin, to facilitate the selection of host cells that will absorb the vector after transformation. The promoter may be an inducible promoter that is under the control of, for example, LacI, and thus prevents the expression of the DNA sequence until induction by, for example, isopropyl β-d-1-thiogalactopyranoside (IPTG).

[0114] Preferably, the vector is a plasmid. More preferably, the vector is a plasmid containing multiple cloning sites. The multiple cloning sites contain multiple restriction sites. In this way, the selection of possible restriction enzymes that can be used to insert DNA into the vector is broadened.

[0115] A vector encoding a recombinant insulin precursor can be expressed in a host cell. Accordingly, in another aspect, the present invention relates to a host cell comprising a DNA sequence for expressing a recombinant insulin precursor as provided herein, or a vector comprising a DNA sequence. Accordingly, in one embodiment, a host cell comprising a DNA sequence according to the formula LBCA is provided, wherein:

[0116] A is preferably the A-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 11;

[0117] B is preferably the B-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 12;

[0118] C is a C-peptide connecting the A-chain and the B-chain, and preferably, the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 9;

[0119] L is preferably an N-terminal L-peptide encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 10.

[0120] Generally, the host cell is a microorganism, such as a bacterium or yeast cell. In embodiments of the present invention and / or embodiments thereof, the host cell is an Escherichia coli cell or a Saccharomyces cerevisiae cell. In one preferred embodiment, the host cell is an Escherichia coli cell.

[0121] To produce an increased amount of recombinant insulin precursor, the insulin precursor can be obtained by expressing the recombinant insulin precursor in a population of host cells. Accordingly, in another aspect, the present invention relates to a culture of host cells comprising a DNA sequence for expressing the recombinant insulin precursor according to the present invention.

[0122] Generally, the conditions under which a host cell culture grows are controlled. Host cells are grown, for example, in a medium containing one or more nutrients and having a pH suitable for the specific host cell, at a temperature and O2 and / or CO2 concentrations appropriate for the proliferation of the specific host cell.

[0123] How to produce insulin

[0124] Methods and processes for the production of insulin or insulin analogs generally involve several steps. Similar methods for producing mature insulin are known to those skilled in the art and have been previously used, as in WO2017 / 040363A1. Recombinant insulin precursors can be used in methods for producing recombinant (mature) insulin. Accordingly, in another aspect, the present invention relates to a method for producing recombinant (mature) insulin, which expresses a recombinant insulin precursor according to the formula LBCA, wherein:

[0125] A is preferably the A-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 11;

[0126] B is preferably the B-chain of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 12;

[0127] C is a C-peptide connecting the A-chain and the B-chain, and preferably, the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 9;

[0128] L is preferably an N-terminal L-peptide encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 10;

[0129] The method is:

[0130] a) A step of expressing a recombinant insulin precursor in a host cell,

[0131] b) a step of lysing host cells to obtain a recombinant insulin precursor,

[0132] c) a step of refolding the recombinant insulin precursor and,

[0133] d) includes the step of obtaining recombinant insulin by cleaving the L-peptide and C-peptide of the recombinant insulin precursor by an enzymatic reaction.

[0134] The expression of a recombinant insulin precursor in a microorganism can be achieved by using a method known in the relevant art, such as fermentation of a host cell, preferably wherein the host cell is a microorganism.

[0135] In some embodiments, a step of cloning a DNA sequence encoding a recombinant insulin precursor into a vector precedes step a) of a method for producing recombinant insulin as disclosed herein.

[0136] In some embodiments, the method further comprises the step of purifying the recombinant insulin obtained by the production method disclosed herein. A person skilled in the art knows a suitable method for purifying recombinant insulin.

[0137] In embodiments of the present invention and / or embodiments thereof, the microorganism is Escherichia coli or Saccharomyces cerevisiae.

[0138] In another aspect, the present invention relates to the use of a recombinant insulin precursor as provided herein in the production of recombinant insulin.

[0139] Finally, the present invention relates to a composition comprising insulin and a detectable amount of a C-peptide and / or an L-peptide as provided herein, preferably wherein the C-peptide comprises an amino acid sequence according to SEQ ID NO: 1 or a sequence having at least 60% sequence identity with SEQ ID NO: 1, and / or the L-peptide comprises an amino acid sequence according to SEQ ID NO: 2 or a sequence having at least 60% sequence identity with SEQ ID NO: 2.

[0140] In some embodiments, C-peptides and / or L-peptides may be attached to the A-chain and / or B-chain of the insulin molecule. This may occur, for example, when the insulin precursor is not completely digested. Alternatively, C-peptides and / or L-peptides may be present in the insulin composition in a free form. This may occur, for example, when the insulin precursor is completely digested but the purification of the digested insulin is suboptimal. Thus, in one embodiment, the insulin composition comprises detectable amounts of C-peptides, L-peptides, or both C-peptides and L-peptides.

[0141] The presence of C-peptide and / or L-peptide can be detected using standard analytical techniques, such as liquid chromatography-tandem mass spectrometry (LC-MS / MS). For example, the literature [Foulon et al., 2022, JMSACL, 25:19-26] describes a method for quantifying insulin and C-peptide by LC-MS / MS without the use of antibodies using serum samples. However, a person skilled in the art will know that a similar method is applied to samples obtained from insulin compositions.

[0142] In embodiments of the present invention and / or embodiments thereof, a composition comprising insulin contains C-peptide and / or L-peptide at a concentration of < 10 μg / mL, preferably less than 9 μg / mL, < 8 μg / mL, < 7.5 μg / mL, < 7 μg / mL, < 6 μg / mL, < 5 μg / mL, < 4 μg / mL, < 3 μg / mL, < 2.5 μg / mL, < 2 μg / mL, < 1 μg / mL, < 0.5 μg / mL, < 0.25 μg / mL, or < 0.1 μg / mL. For example, a composition containing insulin comprises a C-peptide according to SEQ ID NO: 1 and / or an L-peptide according to SEQ ID NO: 2 at a concentration of 1-5000 ng / mL, e.g., 50-4000 ng / mL, 100-3000 ng / mL, 250-2500 ng / mL, 500-2000 ng / mL, 750-1500 ng / mL, or 1000-1250 ng / mL. In an alternative embodiment, the insulin composition comprises < 10 ppm of a C-peptide according to SEQ ID NO: 1 and / or an L-peptide according to SEQ ID NO: 2, or < 3 ppm of a C-peptide according to SEQ ID NO: 1 and / or an L-peptide according to SEQ ID NO: 2, or < 3000 ng / ml of a C-peptide and / or L-peptide. The concentrations of C-peptide and L-peptide can be independent of each other within it.

[0143] Another embodiment of the present invention is a recombinant insulin composition produced by the above method and a pharmaceutically acceptable carrier.

[0144] Another embodiment of the present invention is a method for treating diabetes mellitus, comprising administering a therapeutic amount of the recombinant insulin composition to an animal requiring treatment for diabetes mellitus.

[0145] Another embodiment is a recombinant insulin composition for use in the treatment of diabetes mellitus.

[0146] The foregoing description of specific embodiments will sufficiently reveal the general nature of the invention, and others may easily modify and / or adapt various applications such as specific embodiments by applying knowledge within the art (including the contents of the references cited herein) without excessive experimentation and without departing from the general concept of the invention. Accordingly, such adaptations and modifications are intended to be within the meaning and scope of equivalents of the disclosed embodiments based on the teachings and guidelines set forth herein.

[0147] All references cited herein, including journal articles or abstracts, published or corresponding patent applications, patents, or any other references, are incorporated herein by reference in their entirety, including all data, tables, figures, and text presented in the cited references. Additionally, the entire contents of the references cited herein are also incorporated herein by reference in their entirety.

[0148] It should be understood that the terms or phrases in this specification are for descriptive purposes only and not restrictive, so that they may be interpreted by a person skilled in the art in light of the teachings and guidelines set forth herein in combination with the knowledge of a person skilled in the art.

[0149] Although the present invention has been described generally, it will be more easily understood by referring to the following examples, which are provided by way of example and are not intended to limit the invention. Further aspects and embodiments will be apparent to those skilled in the art.

[0150] Examples

[0151] Example 1

[0152] production of insulin precursors and insulin

[0153] Codon optimization

[0154] Using GeneART and DNA2.0, the DNA sequence encoding the insulin precursor according to sequence identification number: 5-8 was codon optimized for expression in E. coli, and the DNA sequence according to sequence identification number: 13-15 was generated.

[0155] In addition, the incorporation of codon AAC for all asparagine residues and codon TCT for all serine residues reduced the formation of impurities (amino acid mis-incorporation) after digestion, as shown in Figures 2 and 3, respectively.

[0156] Construction of plasmids

[0157] As an expression vector, the pET29 vector was cleaved with restriction enzymes Ndel and XhoI, and a 5.2 kb DNA fragment was isolated by electrophoresis on a 1% agarose gel.

[0158] Next, a DNA sequence according to sequence identification number: 13, 14, or 15, flanked with an NdeI restriction enzyme recognition site and an XhoI restriction enzyme restriction site, was ligated to a vector cut using T4 DNA ligase to form a plasmid containing the DNA sequence according to sequence identification number: 13, 14, or 15.

[0159] E. E. coli BL21(DE3) was transformed by heat shock and streaked onto agar plates. After incubation, kanamycin-resistant transformed cells were selected. Plasmid DNA was isolated from individual transformants, and it was confirmed that the desired DNA was properly inserted using restriction enzyme cleavage analysis.

[0160] Preparation of precursors

[0161] E. E. coli ITP3 cells were each transformed by a heat shock method into plasmids containing DNA sequences according to sequence identification numbers: 13, 14, or 15 to produce insulin precursors according to sequence identification numbers: 5, 6, 7, or 8. Sequence identification number: 5 corresponds substantially to the insulin precursor described in WO2017 / 040363, except that it additionally contains a methionine residue at the first position (N-terminal position) of sequence identification number: 5 when compared to the sequence of WO2017 / 040363.

[0162] Next, transformed E. coli ITP3 cells were grown in a shaking flask containing 800 ml LB broth supplemented with 100 μg / L kanamycin at 37°C for 18 hours until a cell density of approximately 2.1 (A600 nm) was reached. After culture, reduced SDS-PAGE analysis was performed on a precast 4-12% polyacrylamide gradient gel, and Invitrogen Simply Blue Safestain was used for staining. The expression levels of each fused proinsulin were quantified using ChemiDoc.

[0163] Cells were collected by centrifugation at approximately 7,000 rpm for 20 minutes, and wet cell weight was measured using an electronic balance (Sartorius, Germany). The pelleted cells were suspended in 20 ml of 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, and 0.02% lysozyme, lysed by sonication, and centrifuged at approximately 10,000 rpm for 20 minutes.

[0164] The pellet containing the inclusion body (IB) containing the insulin precursor was washed by resuspending it in 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, and 2 M urea buffer, and then centrifuged at 10,000 rpm for 20 minutes. Finally, the pellet was washed twice with deionized water.

[0165] Table 1 shows the fermentation titers of insulin precursors according to sequence identification numbers 5-8 produced upon expression of DNA sequences according to sequence identification numbers 13-15, respectively, in E. coli. N-terminal L-peptides are underlined, and C-peptides are shown in bold.

[0166] The recombinant insulin precursor according to sequence identification number: 6 contains the reference L-peptide of WO2017 / 040363, including the A-chain and B-chain of insulin according to sequence identification numbers: 3 and 4, respectively, and an N-terminal methionine residue for fermentation. Unlike sequence identification number: 5, sequence identification number: 6 contains the C-peptide according to sequence identification number: 1. Surprisingly, the fermentation potency of the insulin precursor according to sequence identification number: 6 was higher than that of the reference insulin precursor according to sequence identification number: 5. As shown in Table 1 of Example 1, when the precursor was expressed in host cells under the same fermentation conditions, the fermentation potency in mmol / L units of the insulin precursor according to sequence identification number: 6 was about 43% higher than the fermentation potency in mmol / L units of the reference insulin precursor according to sequence identification number: 5. Therefore, replacing the natural human C-peptide of the reference precursor with the C-peptide according to sequence identification number: 1 significantly increases the fermentation activity of the precursor.

[0167] Additionally, the recombinant insulin precursor according to sequence identification number: 7 contains the A-chain and B-chain of insulin according to sequence identification numbers: 3 and 4, respectively, in combination with the L-peptide according to sequence identification number: 2. Additionally, sequence identification number: 7 contains a modified C-peptide derived from the sequence of a human C-peptide. As shown in Table 1 of Example 1, the fermentation potency in mmol / L units of the insulin precursor according to sequence identification number: 7 was about 39% higher than the fermentation potency of the reference insulin precursor according to sequence identification number: 5, which contains the reference L-peptide and natural human C-peptide, under the same fermentation conditions.

[0168] Similar to the recombinant insulin precursor according to SEQ ID NO: 6, the recombinant insulin precursor according to SEQ ID NO: 8 contains the A-chain and B-chain of insulin according to SEQ ID NO: 3 and 4, respectively, in combination with the C-peptide according to SEQ ID NO: 1. In addition, SEQ ID NO: 8 contains the L-peptide according to SEQ ID NO: 2. The fermentation potency of the insulin precursor according to SEQ ID NO: 8 was much higher than that of the insulin precursor according to SEQ ID NO: 5 or SEQ ID NO: 6. As shown in Table 1 of Example 1, the fermentation potency (mmol / L) of the insulin precursor according to SEQ ID NO: 8 was even about 118% higher than that of the reference insulin precursor according to SEQ ID NO: 5 (mmol / L). Therefore, replacing the natural human C-peptide with the C-peptide according to sequence identification number: 1, and additionally replacing the reference L-peptide with the L-peptide according to sequence identification number: 2, increases the fermentation activity of the insulin precursor in a synergistic manner.

[0169] Table 1. I. Fermentation titers of recombinant insulin precursors expressed in E. coli (in both g / L and mmol / L units). Each L-peptide is underlined, and each C-peptide is in bold.

[0170]

[0171] Solubilization of the inclusion body

[0172] An inclusion body protein slurry containing an insulin precursor at a concentration of about 30 g / L in water is provided. After filling IB with water, ethanolamine is added as a pure liquid at a final concentration of about 315 mM, followed by the addition of 8 M urea at a final concentration of about 4.0 M. Finally, a 1 M aqueous dithiothreitol (DTT) solution is added to achieve a concentration of about 2.5 mM, and the mixture is stirred for at least 30 minutes to complete the solubilization of IB.

[0173] Refolding of precursors

[0174] The insulin precursor is diluted approximately 10-fold to a target insulin precursor concentration of about 1.6 g / L. First, a target amount of refolding diluent solution (10 mM ethanolamine, 10% (v / v) hexylene glycol) is prepared, then a certain amount of 1 M cystamine dihydrochloride (e.g., about 384 μM) is added and mixed until homogeneous. Subsequently, the solubilized protein solution is transferred to the refolding diluent solution while maintaining the solution temperature at 10°C (±2°C) and maintaining a minimum level of stirring to ensure solution mixing. The refolding reaction mixture is stirred at 10°C until the conversion rate to the correctly folded insulin precursor, as measured by the POROS HPLC assay, drops to less than 5% per hour, at which point the refolding reaction is quenched. After refolding is complete, the reaction is stopped / slowed down by acidifying the refolding solution to a pH of about 9.2 at 10°C using 2N hydrochloric acid. Maintain the temperature at 10℃ during pH adjustment.

[0175] Anion exchange (AEX) chromatography

[0176] Cell debris and precipitated host cell proteins (HCPs) are separated before loading onto the AEX column. In preparation for purification, the temperature of the solution was increased from 10°C to 20°C over a period of 60 to 120 minutes after refolding. Purification is performed using two banks of series-connected deep filters and one bank of 0.22 μm membrane filters. The deep filters are CUNO EXT 90ZA08A filters (Bank #2, total area 56 m²). 2 CUNO EXT 60ZA05A filter connected in series with ) (Bank #1, total area 56 m² 2 It consists of (identical to ). Additionally, a 0.22 μm filter (total area 9 m²) 2 ) exists in series with the deep filter. 54 L / m² before using the deep filters of the two banks. 2Flushing is performed with water or approximately 3000 L. When the deep filter is flushed, the 0.22 μm filter is flushed due to equipment limitations by draining water through deep filter banks #1 and #2, and then through the 0.22 μm filter (>20 L / m²). 2 Before introducing the product, the flush water in the deep filter is replaced with air. The filter is first operated under a constant flux, and then, if the filter begins to foul, the flow rate is reduced to prevent the pressure from exceeding 40 psig. After the post-refolding solution is completely treated, a recovery water chase (25-30 L / m³) is performed to maximize the recovery of the accurately folded precursor into the purified post-refolding solution. 2 Performs ).

[0177] After purification and refolding, the pH of the solution was adjusted to approximately 9.4 with sodium hydroxide and, if necessary, diluted with water to achieve a conductivity of less than 2.5 mS / cm. The resulting solution was loaded onto a column packed with DEAE Cepharose Fast Flow, equilibrated with an equilibrium solution containing approximately 50 mM sodium borate and 2.5 mM sodium chloride at pH 9.4. The flow rate during the loading and subsequent elution steps was adjusted to maintain a retention time of approximately 5 to 7 minutes, and a loading factor of approximately 23 g of precisely folded insulin precursor per L of column resin was used. After loading, the column was washed with approximately 5 column volumes (CV) of the equilibrium solution (50 mM sodium borate, 2.5 mM sodium chloride, pH 9.4), and then washed with approximately 6 CV of the elution solution (approx. 50 mM sodium borate and 160 mM sodium chloride, pH 9.0). Collect the major peaks observed during elution with a 160 mM sodium chloride elution solution to provide an AEX-post pool containing an accurately folded insulin precursor.

[0178] Citraconylation and trypsin digestion

[0179] The purpose of the citraconylation step is to reduce the formation of miscleavages generated during the trypsin digestion step. Citraconylation is achieved through the reaction of citraconic anhydride with an insulin precursor under basic conditions. Citracononic anhydride reacts with any primary amine to "block" all lysine residues found in the molecule, as well as the N-terminus. In this "protected" state, the molecule proceeds through the trypsin digestion step, where recombinant porcine trypsin is added to cleave the N-terminal L-peptide and the inner C-peptide from the insulin precursor molecule. After digestion is complete, the protein is deprotected through acid hydrolysis to produce the desired insulin product for further purification via subsequent downstream processing.

[0180] Before initiating the protective response, measure the concentration of accurately folded insulin precursors in the pool after AEX. Next, add three bolus shots of pure citraconic anhydride to the AEX pool at room temperature to achieve the correct ratio of anhydride to accurately folded insulin precursors. After the addition of anhydride is complete, adjust the pH to 8.5 (if necessary) using an HCl or NaOH solution and allow the protective response to proceed for 2 ± 0.25 hours.

[0181] After the protection reaction is complete, the pH is checked, and if the pH has changed, it is re-titrated to 8.5 using an HCl or NaOH solution. Subsequently, trypsin solution is added to achieve a ratio of approximately 1:11,000 of trypsin to accurately folded insulin precursor by mass. Digestion proceeds for 12 ± 1 hour. After digestion is complete, acetic acid is first added to achieve a pool concentration of 150 mM acetate, and the reaction is stopped by adjusting the pH of the reaction pool to pH 2.4 using HCl. Subsequently, the deprotection reaction (volume of approximately 4100 L) is continued for 4 ± 0.25 hours to produce accurately folded insulin.

[0182] Table 2: Amino acid sequences of sequence identification numbers: 1-8 and corresponding DNA sequences of sequence identification numbers: 9-15. Sequence identification number: 5 discloses the amino acid sequence of an insulin precursor containing a reference L-peptide and a natural human C-peptide. In sequence identification numbers: 6-8 and 13-15, the respective amino acid sequence of the L-peptide and the DNA sequence coding therefor are underlined, and the respective amino acid sequence of the C-peptide and the DNA sequence coding therefor are in bold.

[0183]

[0184]

Claims

Claim 1 A recombinant insulin precursor according to the chemical formula LBCA, wherein A is the A-chain of insulin, B is the B-chain of insulin, C is a C-peptide connecting the A-chain and the B-chain, and L is an N-terminal L-peptide, wherein C is a C-peptide comprising an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO:

1. Claim 2 A recombinant insulin precursor according to the chemical formula LBCA, wherein A is the A-chain of insulin, B is the B-chain of insulin, C is a C-peptide connecting the A-chain and the B-chain, and L is an N-terminal L-peptide, wherein L is an L-peptide comprising an amino acid sequence according to sequence identification number: 2 or an amino acid sequence having at least 60% sequence identity with sequence identification number:

2. Claim 3 A recombinant insulin precursor according to claim 1 or 2, wherein C is a C-peptide comprising an amino acid sequence according to sequence identification number: 1 or an amino acid sequence having at least 60% sequence identity with sequence identification number: 1, and L is an L-peptide comprising an amino acid sequence according to sequence identification number: 2 or an amino acid sequence having at least 60% sequence identity with sequence identification number:

2. Claim 4 A recombinant insulin precursor according to any one of claims 1 to 3, wherein the C-peptide is composed of the amino acid sequence according to sequence identification number: 1 and the L-peptide is composed of the amino acid sequence according to sequence identification number:

2. Claim 5 A recombinant insulin precursor according to any one of claims 1 to 4, wherein the A-chain comprises an amino acid sequence according to sequence identification number: 3 or an amino acid sequence having at least 60% sequence identity with sequence identification number:

3. Claim 6 A recombinant insulin precursor according to any one of claims 1 to 5, wherein the B-chain comprises an amino acid sequence according to sequence identification number: 4 or an amino acid sequence having at least 60% sequence identity with sequence identification number:

4. Claim 7 A recombinant insulin precursor according to any one of claims 1 to 6, wherein the A-chain is derived from porcine insulin and / or the B-chain is derived from porcine insulin. Claim 8 A recombinant insulin precursor according to any one of claims 1 to 7, comprising an amino acid sequence according to sequence identification number: 6, 7 or 8 or a sequence having at least 60% sequence identity with sequence identification number: 6, 7 and / or 8. Claim 9 A recombinant insulin precursor according to any one of claims 1 to 8, comprising an amino acid sequence according to sequence identification number: 6, 7 or 8. Claim 10 A DNA sequence encoding a recombinant insulin precursor according to the chemical formula LBCA, wherein A is the A-chain of insulin, B is the B-chain of insulin, C is a C-peptide connecting the A-chain and the B-chain, and L is an N-terminal L-peptide, wherein C is a C-peptide comprising an amino acid sequence according to sequence identification number: 1 or an amino acid sequence having at least 60% sequence identity with sequence identification number: 1, and / or L is an L-peptide comprising an amino acid sequence according to sequence identification number: 2 or an amino acid sequence having at least 60% sequence identity with sequence identification number:

2. Claim 11 In paragraph 8, a DNA sequence optimized for codon expression in microorganisms. Claim 12 In claim 9, a DNA sequence codon-optimized for expression in E. coli. Claim 13 A DNA sequence according to any one of claims 10 to 12, wherein A is preferably an A-strand of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 11; B is preferably a B-strand of insulin encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 12; C is a C-peptide connecting the A-strand and the B-strand, preferably the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with sequence identification number: 9; and L is preferably an N-terminal L-peptide encoded by a DNA sequence having at least 60% sequence identity with sequence identification number:

10. Claim 14 A vector comprising a DNA sequence according to any one of claims 10 to 13. Claim 15 A host cell comprising a DNA sequence according to any one of claims 10 to 13 or a vector according to claim 14. Claim 16 In paragraph 14, the host cell is a cell of E. coli and / or S. cerevisiae. Claim 17 A method for producing recombinant insulin, comprising: expressing a recombinant insulin precursor according to the chemical formula LBCA, wherein A is the A-chain of insulin, B is the B-chain of insulin, C is a C-peptide connecting the A-chain and the B-chain, and L is an N-terminal L-peptide, wherein C is a C-peptide comprising an amino acid sequence according to sequence identification number: 1 or an amino acid sequence having at least 60% sequence identity with sequence identification number: 1, and / or L is an L-peptide comprising an amino acid sequence according to sequence identification number: 2 or an amino acid sequence having at least 60% sequence identity with sequence identification number: 2; and comprising the steps of: a) expressing the recombinant insulin precursor in a host cell; b) lysing the host cell to obtain the recombinant insulin precursor; c) refolding the recombinant insulin precursor; and d) cleaving the L-peptide and C-peptide of the recombinant insulin precursor by an enzymatic reaction to obtain recombinant insulin. Claim 18 A method according to claim 17, wherein a step of cloning a DNA sequence encoding a recombinant insulin precursor into a vector precedes step a). Claim 19 A method according to claim 17 or 18, further comprising the step of purifying recombinant insulin. Claim 20 Use of a recombinant insulin precursor according to any one of claims 1 to 9 for the production of recombinant insulin. Claim 21 A composition comprising recombinant insulin, wherein the composition comprises detectable amounts of C-peptide and / or L-peptide, wherein the C-peptide comprises an amino acid sequence according to SEQ ID NO: 1 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 1, and / or the L-peptide comprises an amino acid sequence according to SEQ ID NO: 2 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO:

2. Claim 22 A recombinant insulin composition comprising recombinant insulin produced by the method of any one of claims 17 to 19 and a pharmaceutically acceptable carrier. Claim 23 A method for treating diabetes mellitus comprising administering a therapeutic dose of the recombinant insulin composition of claim 22 to an animal requiring treatment for diabetes mellitus. Claim 24 In paragraph 22, a recombinant insulin composition for use in the treatment of diabetes mellitus.

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