WO2025099208A1

WO2025099208A1 - Recombinant insulin precursor

Info

Publication number: WO2025099208A1
Application number: PCT/EP2024/081603
Authority: WO
Inventors: Pedro A. MOURA; Isaac Read MASTERS; Allison D. ORTIGOSA
Original assignee: Intervet International BV; Intervet Inc
Current assignee: Intervet International BV; Intervet Inc
Priority date: 2023-11-10
Filing date: 2024-11-08
Publication date: 2025-05-15
Anticipated expiration: 2026-05-10

Abstract

The invention relates to recombinant insulin precursors. To improve the recombinant production of insulin, a recombinant insulin precursor according to the formula L-B-C-A is provided, wherein A is an A-chain of insulin, B is a 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

RECOMBINANT INSULIN PRECURSOR

Field of the invention

The invention relates to the field of recombinant insulin precursors to maximize expression, yield and final purity of a recombinant insulin precursor.

Background

Insulin is part of the insulin superfamily consisting of insulins, insulin-related polypeptides and insulin-like growth factor. These polypeptides are not only found in vertebrates, but also in fungi and protists. In fact, the molecular origins go at least as far back as the simplest unicellular eukaryotes.

The mature insulin found in mammals is a heterodimer consisting of two polypeptide chains, an A-chain and a B-chain, which are linked together by two intermolecular disulfide bonds In human insulin. The intermolecular disulfide bonds form 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 mass of around 5900 Da and consists of 51-53 amino acids in total, with minor variations between 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, with the only difference being in residue 30 of the B- chain, where human insulin contains a threonine and porcine insulin contains an alanine.

In most vertebrates, insulin is produced as an inactive insulin precursor called preproinsulin in the beta cells of the islets of Langerhans in the pancreas. Preproinsulin is the initial translational product of insulin mRNA. As shown in Fig. 1, preproinsulin is comprised of an N-terminal signal peptide or leader peptide (L-peptide), and the A-chain and the B-chain linked together by a C- chain or connecting peptide (C-peptide). While the A-chain and B-chain are highly conserved across species, the C-peptide shows more sequence variation and can vary in length, e.g., between 26 and 38 residues. Preproinsulin is co-translationally transported into the endoplasmic reticulum, where the L-peptide is removed to produce the inactive insulin precursor called proinsulin. While folding, the three disulfide bonds are formed and the C-peptide is removed, thus resulting in mature insulin. The mature insulin is then packed in granules as hexamers and can be secreted upon stimulation of the beta cell.nu Insulin secretion occurs when the glucose levels in the blood rise, and large amounts of glucose are taken up and metabolized by the beta cells. It may 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 by sympathetic activation of the nervous system. Small amounts of insulin are however secreted continuously, even when fasting. Secreted insulin binds specific receptors on the outer membranes of target cells of liver, muscle, and adipose tissue which causes the translocation of glucose transporters to the cell membrane from within the cell allowing glucose to be taken up by the cell. Insulin also promotes lipogenesis, glycogenesis and protein synthesis for skeletal muscle and fat tissues through the kinase tyrosine receptor pathway.

When insulin does not or can no longer properly regulate blood glucose levels, diabetes mellitus (or in short diabetes) can develop. Type I diabetes is characterized by the destruction of the beta cells. Therefore, less or no insulin is produced, leading to an insulin deficiency. In most cases, type I diabetes is due to an auto-immune response and a T-cell mediated attack of the beta cells. Type II diabetes is characterized by insulin resistance, which may be combined with relatively reduced insulin secretion. Type II is the most common type of diabetes, and primarily due to lifestyle factors and genetics. Treatment of type II diabetes is therefore directed at a change in lifestyle, the addition of medication aimed at improving insulin sensitivity or reducing glucose production by the liver, and through the administration of insulin. On the other hand, type I diabetes is primarily inherited and is treated through the administration of insulin.

Already in 1910 it was proposed that diabetes was due to one hormone lacking from the pancreas. In 1921, this missing hormone was identified as insulin, and since then, insulin has been used in treating diabetes. Initially, insulin that was isolated from pigs and cows was used, but the invention of DNA cloning led to the first recombinant insulin being produced in Escherichia coli bacteria in 1978.

Nowadays, E. coli is still a widely used expression system for the production of recombinant insulin. Generally, the bacteria are transformed with a vector encoding for an inactive precursor molecule of insulin and the cells grown in fermenters. After induction, the precursor is expressed and typically accumulates inside the bacteria as insoluble inclusion bodies. After the cells are harvested, they are lysed to obtain the inclusion bodies predominantly comprising the precursor. The inclusion bodies are subsequently isolated and solubilized, and the precursor is refolded into its native structure where the disulfide bonds are formed. Next, the folded precursor is purified to remove host cell impurities and misfolded forms of the molecule. Finally, the precursor is digested with trypsin and/or carboxypeptidase B to cleave the N-terminal L-peptide and the C- peptide resulting in mature recombinant insulin. As a part of the recombinant manufacturing process, additional steps of purifying the mature insulin will be needed to reach the desired product quality.

The number of people diagnosed with diabetes rose from 108 million in 1980 to 537 million in 2021 worldwide, and this number is expected to keep rising. Furthermore, animals can suffer from diabetes, it being most common in dogs and cats. Like in humans, the prevalence of diabetes is increasing in animals. To meet the demand of insulin for treatment in both humans and animals, the production process of recombinant insulin is continuously being improved to reduce the cost, increase the effectiveness and to increase yields.

WO 2001/049742 describes the synthesis in Saccharomyces cerevisiae of a precursor of human insulin comprising C-peptides containing at least one aromatic amino acid residue Phe, Trp, or Tyr and generally not longer than 15 amino acids. These precursors resulted in a higher production yield as compared to precursors having a native C-peptide.

WO 2001/025278 describes precursors of human insulin comprising C-peptides ranging between 2 and 50 amino acids in length which improve the production yield of the precursor.

Min et al., 2011, J. Biotechnol. 151 :350-356 describes an improved expression, folding and enzyme reaction rate of recombinant human insulin when certain L-peptides were selected for the precursor of human insulin.

WO 2017/040363 describes a process for obtaining insulin with correctly formed disulfide bonds.

However, there is still a need to improve the recombinant production of insulin even further.

Summary of the invention

In a first aspect of the invention, a recombinant insulin precursor according to the formula L-B- C-A is provided, wherein:

A is an A-chain of insulin,

B is a B-chain of insulin,

C is a C-peptide connecting the A-chain and 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 and/or wherein L is an L-peptide comprising an amino acid sequence according to SEQ ID NO: 2. Another aspect of the invention is a DNA sequence encoding the recombinant insulin precursor according to the invention and/or embodiments thereof, preferably wherein the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 9 and/or wherein the L-peptide is encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 10.

It was found that the recombinant insulin precursor according to the invention provides for a desirable yield, such as an improved yield of insulin.

The yield (e.g., as provided as fermentation titer, e.g., in mmol/L or g/L) of one or more recombinant insulin precursors according to the invention was compared to 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 is substantially according to an insulin precursor described in WO 2017/040363 (see, Figure 2 of WO 2017/040363). A difference between SEQ ID NO:5 and the insulin precursor shown in WO 2017/040363 comprises that the reference recombinant insulin precursor of SEQ ID NO: 5 comprises an additional methionine (M) residue at the N-terminal position compared to the precursor disclosed in WO 2017/040363. This methionine residue is cleaved off during fermentation in Escherichia coli by methionine aminopeptidase. WO 2017/040363 describes a process for obtaining insulin with correctly formed disulfide bonds using an insulin precursor. The cleavage of the additional methionine residue at the N-terminal position of the insulin precursor of W02017/040363 is already completed before initiating the process described in W02017/040363. Further, the reference recombinant insulin precursor according to SEQ ID NO:

5 comprises at residue B30 of the B-chain an alanine (A), whereas the amino acid sequence of Figure 2 of WO 2017/040363 comprises a threonine (T) at residue B30 of the B-chain.

The recombinant insulin precursor comprising an amino acid sequence according to SEQ ID NO:

6 is a recombinant insulin precursor according to the invention and/or embodiments thereof and contains the A-chain and B-chain of insulin comprising an amino acid sequence according to SEQ ID NO: 3 and 4, respectively, and the reference L-peptide from the reference recombinant insulin precursor (SEQ ID NO: 5) which is substantially similar as the L-peptide disclosed in W02017/040363; SEQ ID NO: 5 further including an N-terminal methionine residue. In contrast to reference recombinant insulin precursor (SEQ ID NO: 5), the recombinant insulin precursor according to the invention of SEQ ID NO: 6 contains a novel recombinant C-peptide according to SEQ ID NO: 1. Surprisingly, the fermentation titer (in mmol/L) of the insulin precursor according to SEQ ID NO: 6 was higher than the fermentation titer (in mmol/L) of the reference recombinant insulin precursor according to SEQ ID NO: 5. As shown in Table 1 of Example 1, the fermentation titer (in mmol/L) of the insulin precursor according to SEQ ID NO: 6 was about 43% higher than the fermentation titer (in mmol/L) of the reference insulin precursor according to SEQ ID NO: 5 when the precursors were expressed in host cells under the same fermentation conditions. Thus, replacing the native human C-peptide of the reference precursor by the C- peptide according to SEQ ID NO: 1 significantly increases the fermentation titer of the precursor. Another recombinant insulin precursor according to the invention and/or embodiments thereof is a recombinant insulin precursor comprising an amino acid sequence according to SEQ ID NO: 7. Said recombinant insulin precursor contains the A-chain and B-chain of insulin comprising an amino acid sequence 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 the human C-peptide. The fermentation titer (in mmol/L) of the insulin precursor according to SEQ ID NO: 7 was higher than the fermentation titer (in mmol/L) of the reference recombinant insulin precursor (SEQ ID NO: 5). As shown in Table 1 of Example 1, the fermentation titer (in mmol/L) of the insulin precursor according to SEQ ID NO: 7 was about 39% higher than the fermentation titer (in mmol/L) of the reference recombinant insulin precursor (SEQ ID NO: 5). Thus, replacing the native human L-peptide of the reference precursor by the L-peptide according to SEQ ID NO: 2 increases the fermentation titer of the insulin precursor.

Another recombinant insulin precursor according to the invention and/or embodiments thereof is a recombinant insulin precursor comprising an amino acid sequence according to SEQ ID NO: 8 and contains the A-chain and B-chain of insulin comprising an amino acid sequence according to SEQ ID NO: 3 and 4, respectively, and the C-peptide according to SEQ ID NO: 1. In addition to this, SEQ ID NO: 8 contains a L-peptide according to SEQ ID NO: 2. The fermentation titer (in mmol/L) of the insulin precursor according to SEQ ID NO: 8 was even higher than the fermentation titer (in mmol/L) of the insulin precursor according to either SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7. As shown in Table 1 of Example 1, the fermentation titer (in mmol/L) of the insulin precursor according to SEQ ID NO: 8 was even about 118% higher than the fermentation titer (in mmol/L) of the reference recombinant insulin precursor (SEQ ID NO: 5). Thus, replacing the native human C-peptide by the C-peptide according to SEQ ID NO: 1 and additionally the reference L-peptide by the L-peptide according to SEQ ID NO: 2 increases the fermentation titer of the insulin precursor in a synergistic manner.

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

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

A further aspect relates to method of producing recombinant insulin, wherein a recombinant insulin precursor according to the invention is expressed, wherein the method comprises: a) expressing the recombinant insulin precursor through fermentation of a microorganism, b) lysing the microorganism to obtain the recombinant insulin precursor, c) refolding the recombinant insulin precursor, and d) cleaving the L-peptide and the C-peptide of the recombinant insulin precursor by an enzymatic reaction to obtain recombinant insulin.

A further aspect relates to use of a recombinant insulin precursor according to the invention for the production of recombinant insulin.

A final aspect relates to a composition comprising recombinant insulin, wherein the composition comprises a detectable amount of a C-peptide and/or L-peptide as provided herein.

Description of the Figures

Fig. 1 shows a diagram of a preproinsulin molecule, a proinsulin molecule, and an insulin molecule.

Fig. 2 shows a liquid chromatography-mass spectrometry profile of a mature insulin molecule resulting from expression of a DNA construct encoding asparagine residues by the codon “AAC” in A. coli.

Fig. 3 shows a liquid chromatography-mass spectrometry profile of a mature insulin molecule resulting from expression of a DNA construct encoding serine residues by the codon “TCT” in E. coli.

Definitions

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

The term “Peptide” refers to a short sequence of amino acids, typically 2-50 amino acids long. The term “C-peptide” refers to the connecting peptide or connection moiety “C” of the B-C-A sequence of an insulin precursor molecule. The C-peptide connects the C-terminus of the B-chain and the N-terminus of the A-chain of insulin.

The term “L-peptide” refers to the leader peptide which is present as an N-terminal leader sequence on the precursor form of the insulin protein. The L-peptide of naturally occurring insulin allows the protein to translocate into the endoplasmic reticulum. The L-peptide is normally cleaved off during this translocation process.

The term “Precursor” refers to an immature form of a protein, which still is to be processed before obtaining the final mature form of the protein. In the case of insulin, the precursor still comprises the C-peptide and/or the L-peptide. The C-peptide and the L-peptide should be cleaved off to yield mature insulin. The term “Proinsulin” refers to an immature form of insulin consisting of a B-chain peptide, which is fused to the N-terminus of a C-peptide which in turn is fused at its C-terminus to the N- terminus of an A-chain peptide. Proinsulin is a type of insulin precursor.

The term “Preproinsulin” refers to an immature form of insulin consisting of a N-terminal L- peptide which is fused at its C-terminus to the N-terminus of a B-chain peptide, which is fused to the N-terminus of a C-peptide which in turn is fused at its C-terminus to the N-terminus of an A- chain peptide. Preproinsulin thus is represented by the formula L-B-C-A. Preproinsulin is a type of insulin precursor.

The term “vector” herein refers to a construct used to introduce a nucleic acid, e.g., DNA, into a host cell. After the vector is introduced into the host cell, the nucleic acid, e.g., DNA, carried by the vector can then be expressed by the host cell, resulting in the production of a protein encoded by the nucleic acid, e.g., DNA. The vector can for example be a virus, but is typically a plasmid, such as a pET plasmid or a pGEX plasmid.

The term “host cell” herein refers to a cell used to express a recombinant insulin precursor. Typically, the host cell is a microorganism such as a yeast cell or a bacterium. Suitable host cells are Escherichia coli (E. coll) and Saccharomyces cerevisiae (5. cerevisiae).

The term “culture of host cells” refers to a population of host cells grown under controlled conditions, such as in a medium comprising one or more nutrients and having a pH suitable for the particular host cell and in a temperature and a O2 and/or CO2 concentration appropriate for the propagation of the particular host cell.

The term “fermentation titer” refers to the concentration of a molecule of interest in a solution. Here, the fermentation titer is the concentration of the recombinant insulin precursor in the medium used to grow the host cell expressing the precursor. The fermentation titer can for example be expressed in g/mL or mmol/L (mM) and can be, e.g., used as a measure of yield of a protein.

As used herein, one amino acid sequence is 100% "identical" or has 100% “sequence identity” to a second amino acid sequence when the amino acid residues of both sequences, when aligned, are identical, when determined by using the computer program " BLAST®" by selecting subprogram: "Global Alignment” using standard settings, that can be found at https://blast.ncbi.nlm.nih.gov/Blast.cgi. Accordingly, an amino acid sequence (or nucleic acid sequence) can be 50% "identical" to a second amino acid sequence (or second nucleic acid sequence) when 50% of the amino acid residues of the two amino acid sequences (or nucleic acids of the two nucleic acid sequences) are identical when determined by using the computer program "BLAST®" by selecting sub-program: "Global Alignment” using standard settings. The sequence comparison is performed over a contiguous block of amino acid residues comprised by a given protein, e.g., a protein, or a portion of the polypeptide being compared. In a particular embodiment, selected substitutions, deletions, or insertions that either do or do not alter the correspondence between the two amino acid sequences are also encompassed. Similarly, a nucleic acid sequence is 100% "identical" or has 100% “sequence identity” to a second nucleic acid sequence when the nucleic acid residues of both sequences, when aligned, are identical, when determined by using the computer program "BLAST®" by selecting sub-program: "Global Alignment", that can be found at https://blast.ncbi.nlm.nih.gov/Blast.cgi and using standard settings.

Detailed description of the invention

The invention is defined herein and in the accompanying claims. Subject-matter which is not encompassed by the scope of the claims does not form part of the present claimed invention. It is contemplated that any product, method, use, or composition described herein can be implemented with respect to any other product, method, use or composition described herein. Embodiments disclosed in the context of products, methods, uses or compositions of the invention may be employed with respect to any other product, method, use, or composition described herein. Thus, an embodiment pertaining to one product, method, use or composition may be applied to other products, methods, uses or compositions of the invention as well.

Amino acid sequences

The invention is related to a recombinant insulin precursor according to the formula L-B-C-A. Herein, L represents an N-terminal L-peptide, B represents a B-chain of insulin, C represents a C-peptide connecting the A-chain and the B-chain and A represents an A-chain of insulin.

C is a connecting peptide (or C-peptide) connecting 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 between species, the C-peptide shows more variation in both sequence and length. The native human C- peptide is, for example, 31 residues long, while the native porcine C-peptide is 29 residues long and the native bovine C-peptide is 26 residues long.

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

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

To facilitate cleaving the C-peptide, e.g., by using trypsin, after the expression of a recombinant insulin precursor in a microorganism, it is preferred that the C-terminal residue of the B-peptide and/or C-peptide comprises an amino acid residue suitable for enabling the cleaving of the peptide, such a one or more arginine residues.

Hence, 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 consisting of an amino acid sequence that has 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 consists of an amino acid sequence according to SEQ ID NO: 1. It was found that a recombinant insulin precursor comprising the C-peptide as provided herein resulted in desirable fermentation titers of the insulin precursor.

L is an N-terminal leader peptide (or L-peptide) connected to the N-terminal end of the B-chain. The L-peptide in naturally occurring human insulin has a length of 24 residues. When insulin is expressed in a mammalian cell, the L-peptide acts as a signal peptide to transport preproinsulin to the endoplasmic reticulum, where the L-peptide is cleaved off. However, when recombinant insulin is expressed in a microorganism, the L-peptide serves to protect the recombinant insulin from internal degradation and/or modification of the recombinant insulin during synthesis in the microorganism, and to improve protein expression of the recombinant insulin precursor in the microorganism. When recombinant insulin is expressed in a microorganism, the L-peptide is cleaved off after expression of the recombinant insulin precursor. To facilitate cleaving the L- peptide, e.g., by using trypsin or a trypsin-like agent, after the expression of a recombinant insulin precursor in a microorganism, it is preferred that the C-terminal residue of the L-peptide comprises an amino acid residue suitable for enabling the cleaving of the peptide, such as one or more arginine residues.

Preferably, the L-peptide comprises 10-25 amino acid residues. More preferably, the L-peptide comprises 12-24 amino acid residues, and even more preferably comprises 15-20 residues. In one preferred embodiment the L-peptide according to the invention consists of 16 amino acid residues (novel recombinant L-peptide; SEQ ID NO: 2). To obtain a relatively high fermentation titer of the recombinant insulin precursor upon expression of the recombinant insulin precursor in a microorganism, the L-peptide provided herein preferably has a net positive charge at pH 6.0. In other words, the L-peptide has a pl > 6.0. Preferably, the L-peptide has a pl > 6.5, and more preferably a pl > 7.0. The amino acids lysine, arginine and histidine have a pl of > 7.5. Thus, preferably the L-peptide 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 chosen 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 chosen from lysine, arginine, and histidine. Hence, 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 consisting of an amino acid sequence that has 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 consists of an amino acid sequence according to SEQ ID NO: 2. It was found that a recombinant insulin precursor comprising the L-peptide as provided herein resulted in desirable fermentation titers of the insulin precursor.

It was further found that a recombinant insulin precursor having an amino acid sequence of SEQ ID NO: 8 resulted in particular desirable fermentation titers of the insulin precursor. Therefore in one embodiment recombinant insulin precursor according to the 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 an amino acid sequence according to SEQ ID NO: 1 and the L-peptide consists of an amino acid sequence according to SEQ ID NO: 2.

Insulin is a highly conserved protein, with only minor variations between different animal species. For example, the A-chain and B-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 Bl- B30).

A is an A-chain of insulin and can be any kind of A-chain, such as an A-chain of human, sheep, canine, feline, porcine or bovine insulin. In a preferred embodiment, A is derived from an A- chain of porcine insulin. The A-chain of porcine insulin corresponds to the A-chain of human insulin. The A-chain may comprise an amino acid sequence according to SEQ ID NO: 3, while bovine A-chain differs from the human A-chain in only two amino acids. Human and porcine insulin contain a threonine and isoleucine residue at residue positions A8 and A10 (referring to the 8^th and 10^th amino acid residue, e.g., according to numbering in SEQ ID NO: 3), respectively, while bovine insulin contains an alanine and valine residue at the respective positions. In a preferred embodiment the A-chain is derived from an A-chain of porcine insulin.

Furthermore, 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 example, the substitution of the asparagine residue at position A21 of regular human insulin by glycine insulin glargine, a slow acting type of insulin remaining at relatively low levels in the blood for a time span of 24 hours upon subcutaneous injection. This modification in an insulin protein is also encompassed by the current invention.

Thus, in an embodiment of the invention and/or embodiments 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 can be modified by e.g., amino acid substitution, deletion or insertion at a position selected from Al, A2, A3, A4, A5, A8, A9, A10, A12, A13, A14, A15, A16, A17, A18, A19 and/or A21 to arrive at the A-chain comprised in the recombinant insulin precursor. In an embodiment of the invention and/or embodiments 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 skilled person is aware of the amino acid sequences of suitable A-chains of insulin that can be used in the herein provided invention.

Additionally, amino acids of the A-chain of the recombinant insulin precursor can undergo post- translational modifications 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), either directly or via a linker, or deamidation of asparagine residues. In an embodiment of the invention and/or embodiments thereof 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 of the A-chain of the recombinant insulin precursor is/are post- translationally modified.

B is a B-chain of insulin and can be any kind of B-chain, such as a B-chain of human, sheep, canine, feline, porcine or bovine insulin. In a preferred embodiment, the B-chain is derived from a B-chain of porcine insulin. Like the A-chain, the B-chain of insulin is highly conserved between animal species. Porcine insulin and bovine insulin only differ from residue human insulin at residue B30, where human insulin contains a threonine residue, while porcine and bovine insulin contain an alanine residue. Substitution of amino acids of the B-chain of insulin may 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 the proline residue at position B28 of human insulin for lysine and the lysine residue at position B29 of human insulin for proline results in fast acting insulin causing a blood insulin spike within 30 minutes after subcutaneous injection and being degraded within 4 hours from injection.

The B-chain as disclosed and provided herein according to SEQ ID NO: 4 comprises the two arginine residues at the C-terminus of the B-chain, i.e., at position B31 and B32. It is understood that the current invention also encompasses B-chains of insulin that do not comprise said two arginine residues.

Thus, in an embodiment of the invention and/or embodiments 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 can be modified by e.g., amino acid substitution, deletion or insertion at a position selected from Bl, B2, B3, B4, B5, B9, B10, B13, B14, B15, B16, B17, B18, B20, B21, B22, B23, B26, B27, B28, B29, and/or B30 to arrive at the B-chain as comprised in the recombinant insulin precursor. In an embodiment of the invention and/or embodiments thereof, the B-chain according to SEQ ID NO: 4 is modified on at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 residue positions. A skilled person is aware of the amino acid sequences of suitable B-chains of insulin that can be used in the herein provided invention.

The amino acids of the B-chain of the recombinant insulin precursor can undergo similar post- translational modifications as previously described for the amino acids of the A-chain of the recombinant insulin precursor. In an embodiment of the invention and/or embodiments 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 the recombinant insulin precursor is/are post-translationally modified.

The A-chain and the B-chain of the recombinant insulin precursor can be derived from different species. The A-chain may for example be an A-chain from human insulin, while the B-chain is a B-chain from porcine insulin, or vice versa. Further combinations of A-chains and B-chains of different species are also encompassed herein.

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. In a further embodiment of the invention and/or embodiments 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 with a sequence identity of 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% 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.

The recombinant insulin precursor comprising an amino acid sequence according to SEQ ID NO: 6, 7 or 8 contain an A-chain according to SEQ ID NO: 3 and a B-chain according to SEQ ID NO: 4. It is contemplated herein that a skilled person can derive the A-chain and/or B-chain of the recombinant insulin precursor from different species (e.g., human, ruminant or pet) and provide for a recombinant insulin precursor comprising a C-peptide and/or L-peptide according to the invention further comprising an A-chain and/or the B-chain from different species. Hence, the invention also provides for recombinant insulin precursors that comprise a sequence identity of 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% with the recombinant insulin precursor according to SEQ ID NO: 6, 7 and/or 8, or that comprise the C-peptide according to SEQ ID NO: 1 and the L-peptide according to SEQ ID NO: 2, and that comprise an A-chain and/or B-chain from different species. Furthermore, a protein comprising an amino acid sequence according to SEQ ID NO: 6 comprises the C-peptide according to SEQ ID NO: 1 and the reference L-peptide of W02017/040363 further comprising a N-terminal methionine. The protein comprising an amino acid sequence according to SEQ ID NO: 7 comprises the L-peptide according to SEQ ID NO: 2 and a modified C-peptide derived from the sequence of the human C-peptide. The protein comprising an amino acid sequence according to SEQ ID NO: 8 comprises both the C-peptide according to SEQ ID NO: 1 and the L-peptide according to SEQ ID NO: 2. As is herein exemplified, the expression of the recombinant insulin precursors that comprise amino acid sequence according to SEQ ID NO: 6, 7 or 8 resulted in a higher fermentation titer of the recombinant insulin precursor than the reference recombinant insulin precursor according to SEQ ID NO: 5 (derived from W02017/040363, wherein the amino acid sequence of SEQ ID NO: 5 further contains a N-terminal methionine).

DNA sequences

In another aspect the invention relates to a DNA sequence encoding a recombinant insulin precursor according to the formula L-B-C-A, wherein:

A is an A-chain of insulin,

B is a 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 A-chain, the B-chain, the C-peptide and the L-peptide as disclosed herein may be encoded by any DNA sequence resulting in an amino acid sequence forming said A-chain of insulin, a B- chain of insulin, a C-peptide, preferably according to SEQ ID NO: 1, and/or an L-peptide, preferably according to SEQ ID NO: 2, respectively.

In one embodiment the invention relates to a DNA sequence encoding a recombinant insulin precursor according to the formula L-B-C-A, wherein:

A is an A-chain of insulin,

B is a 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 and/or L is 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.

It is known that similar codons are transcribed with varying efficiency in different organisms. Since the recombinant insulin precursor is mostly produced in a microorganism the DNA sequence in some preferred embodiments of the invention is codon optimized for expression in a microorganism. The optimization can for example be achieved using software, such as GeneOptimizer and DNA2.0.

The DNA sequence of the gene encoding native insulin, such as native human or native porcine insulin, including the A-chain, the B-chain, the C-peptide has been described in the art, for example by Bell, G., Pictet, R., Rutter, ffl. et al. Sequence of the human insulin gene. Nature 284, 26-32 (1980) or by 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. A skilled person hence is familiar with DNA sequences encoding native insulin, or any one of native A-chain, native B-chain, native C-peptide, or native L-peptide, as found in various mammals. An example of a DNA sequence for a native human C- peptide

Provided herein is a DNA sequence for expressing a recombinant insulin precursor according to the formula L-B-C-A, wherein:

A is an A-chain of insulin, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 11; B is a B -chain of insulin, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 12;

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

L is an N-terminal L-peptide, wherein the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 9 and/or wherein the L-peptide is encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 10.

Preferably, the A-chain of the recombinant insulin precursor is encoded by a DNA sequence according to SEQ ID NO: 11, or a DNA sequence having, with increasing preference, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with SEQ ID NO: 11. Preferably, the B-chain of the recombinant insulin precursor is encoded by a DNA sequence according to SEQ ID NO: 12, or a DNA sequence having, with increasing preference, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with SEQ ID NO: 12. Preferably, the C-peptide of the recombinant insulin precursor is encoded by a DNA sequence according to SEQ ID NO: 9, or a DNA sequence having, with increasing preference, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with SEQ ID NO: 9. Preferably, the L- peptide of the recombinant insulin precursor is encoded by a DNA sequence according to SEQ ID NO: 10, or a DNA sequence having, with increasing preference, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with SEQ ID NO: 10.

Insulin is a mammalian protein which is not normally expressed by the microorganism typically used as host cells to produce recombinant insulin. While typically one amino acid is encoded for by different codons, the codon preference differs between mammalian cells and microorganisms, and expressing a mammalian DNA sequence in a microorganism typically results in relatively low yields of the encoded peptide or protein. Codon optimizing the DNA sequence for expression in a microorganism improves the yield of the recombinant precursor and the fidelity of the expression.

The recombinant insulin or mature insulin molecule produced upon expression of a DNA sequence by a microorganism and subsequent digestion of the molecule to remove the L-peptide and the C-peptide can be analyzed using analysis techniques known in the art, such as liquid chromatography-mass spectrometry (LC-MS).

As shown in Fig. 2, the peaks of the LC-MS profile at between 21 and 23 seconds (indicated by arrows) are strongly reduced when the codon “ AAC” was used (instead of “ AAT”) to encode the asparagine residues present at residue positions Al 8 and A21 of the A-chain of the insulin molecule when the DNA sequence encoding the recombinant insulin precursor was expressed in E. coli. This indicates an improved fidelity of expression and reduction of amino acid misincorporation. Thus, in an embodiment of the invention and/or embodiments thereof, an asparagine residue of the A-chain of the recombinant insulin precursor is encoded by the codon “AAC”. Preferably, the asparagine residues at residue positions A18 and A21 of the A-chain of insulin are encoded 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 encoded by the codon “AAC”.

As shown in Fig. 3 the peaks of the LC-MS profile at between 20.5 and 21.5 seconds are strongly reduced when the codon “TCT” was used (instead of “AGC”) to encode the serine residues present at residue positions A9 and A12 of the A-chain of the insulin molecule when the DNA sequence encoding the recombinant insulin precursor was expressed in E. coli. Again, this indicates an improved fidelity of expression and reduction in amino acid misincorporation. Thus, in an embodiment of the invention and/or embodiments thereof, a serine residue of the A-chain of the recombinant insulin precursor is encoded by the codon “TCT”. Preferably, the serine residues at residue positions A9 and A12 of the A-chain of insulin are encoded by the codon “TCT”. When more than one serine residue is present in the A-chain of the recombinant insulin precursor, for example 2, 3, 4 or all serine residues of the A-chain of the recombinant insulin precursor are encoded by the codon “TCT”.

In some embodiments of the invention and/or embodiments thereof one or more asparagine residue of the A-chain of the recombinant insulin precursor are encoded by the codon “AAC” and one or more serine residue of the A-chain of the recombinant insulin precursor is encoded by the codon “TCT”. In one preferred embodiment, the A-chain comprises that the asparagine residues at residue positions Al 8 and A21 of the A-chain of insulin are encoded by the codon “AAC” and the serine residues at residue positions A9 and A12 of the A-chain of insulin are encoded by the codon “TCT”, such as is exemplified in the amino acid sequence of the A-chain SEQ ID NO: 3 and the corresponding DNA sequence SEQ ID NO: 11.

Since the amino acid sequence of the A-chain is located on the C-terminal end of the amino acid sequence of the recombinant insulin precursor, the DNA sequence encoding the A-chain and/or recombinant insulin precursor according to the invention may further comprise a stop codon sequence. A skilled person is aware of the function and nucleic acid sequences of such stop codons, which thus are encompassed herein. A stop codon as disclosed herein may, in a non- limiting example, be “TAA” and can be situated after nucleic acid residue 63 of SEQ ID NO: 11 (thus, “TAA” will be nucleic acid residues 64 - 66 in said SEQ ID NO: 11).

In a further embodiment the herein disclosed DNA sequences have been optimized for expression in a microorganism, preferably E. coli. It is understood that codon optimization may be used for optimizing the DNA sequences for expression in other microorganisms other than E. coli. Hence, it is understood that such optimized DNA sequences, preferably any one or more of the DNA sequences of SEQ ID NO: 9 - 15 are also encompassed by the current disclosure.

In an embodiment of the invention and/or embodiments thereof, the DNA sequence encoding for the recombinant insulin precursor according to the 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 invention is encoded by a DNA sequence having, with increasing preference, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% sequence identity with SEQ ID NO: 13, 14 or 15. In one preferred embodiment the recombinant insulin precursor according to the invention is encoded by a DNA sequence of SEQ ID NO: 13, 14 or 15.

The DNA sequence encoding the recombinant insulin precursor can be included in a vector for expressing the recombinant insulin precursor. Therefore, the invention relates in another aspect to a vector comprising a DNA sequence for expressing a recombinant insulin precursor as provided herein. In some embodiments there is provided for a vector comprising a DNA sequence for expressing a recombinant insulin precursor according to the formula L-B-C-A, wherein:

A is an A-chain of insulin, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 11;

B is a B -chain of insulin, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 12;

C is a C-peptide connecting the A-chain and the B -chain, preferably wherein the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 9; and L is an N-terminal L-peptide, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 10.

The DNA sequence is typically cloned downstream of and operatively linked to a promoter sequence, such as a T7 promoter or tac promoter. The DNA sequence can be cloned into the vector by cutting the vector open using restriction enzymes recognizing restriction sites present in the vector, such as Xhol, Bam, Ndel, Bgll, Hindlll. A DNA sequence flanked by nucleic acid sequences compatible with the respective restriction site can then be inserted in the vector, and the vector closed using the enzyme ligase. Typically, the vector comprises one or more antibiotic resistance genes conferring resistance against e.g., kanamycin or ampicillin, to facilitate selection of a host cell that has taken up the vector after transformation. The promoter can be an inducible promoter that is for example under the control of LacI, thus preventing expression of the DNA sequence until induction by e.g., isopropyl P-d-1 -thiogalactopyranoside (IPTG).

Preferably, the vector is a plasmid. More preferably, the vector is a plasmid comprising a multiple cloning site. A multiple cloning site contains several restriction sites. In this way, the choice of possible restriction enzymes that can be used to insert the DNA into the vector is broadened.

The vector encoding the recombinant insulin precursor can be expressed in a host cell. Therefore, the invention relates in another aspect to a host cell comprising a DNA sequence or comprising a vector comprising a DNA sequence for expressing a recombinant insulin precursor as provided herein. Hence, in one embodiment there is provided for a host cell comprising a DNA sequence according to the formula L-B-C-A, wherein:

Typically, the host cell is a microorganism, such as a bacterium or a yeast cell. In an embodiment of the 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.

To produce increased quantities of the recombinant insulin precursor, the recombinant insulin precursor can be expressed in a population of host cells to obtain the insulin precursor. Therefore, the invention relates in another aspect to a culture of host cells comprising a DNA sequence for expressing a recombinant insulin precursor according to the invention.

Typically, the conditions under which the culture of host cells is grown are controlled. The host cells are for example grown in a medium comprising one or more nutrients and having a pH suitable for the particular host cell and in a temperature and at a O2 and/or CO2 concentration appropriate for the propagation of the particular host cell. Methods for producing insulin

Methods and processes for production of insulin or insulin analogs generally include several steps. Similar processes of producing mature insulin are known to a skilled person and have been used previously, such as in W02017/040363A1. The recombinant insulin precursor can be used in methods of producing recombinant (mature) insulin. Therefore, the invention relates in another aspect to a method of producing recombinant (mature) insulin, wherein a recombinant insulin precursor according to the formula L-B-C-A is expressed, wherein:

C is a C-peptide connecting the A-chain and the B -chain, preferably wherein the C-peptide is encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 9; and L is an N-terminal L-peptide, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 10; the method 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 the C-peptide of the recombinant insulin precursor by an enzymatic reaction to obtain recombinant insulin.

The expressing of the recombinant insulin precursor in a microorganism may be through using methods known in the art, such as fermentation of a host cell, preferably wherein the host cell is a microorganism.

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

In some embodiments the method further comprises a step for purifying the recombinant insulin obtained by the production method disclosed herein. A skilled person is aware of suitable methods for purification of the recombinant insulin.

In embodiment of the invention and/or embodiments thereof, the microorganism is Escherichia coli or Saccharomyces cerevisiae. In another aspect, the invention relates to a use of a recombinant insulin precursor as provided herein in the production of recombinant insulin.

Finally, the 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.

In some embodiments the C-peptide and/or the L-peptide may be connected to the A-chain and/or the B-chain of an insulin molecule. This can for example occur when the insulin precursor is not fully digested. Alternatively, the C-peptide and/or the L-peptide may be present in the insulin composition in a free form. This can for example occur when the insulin precursor is fully digested, but the purification of the digested insulin is suboptimal. Thus, in an embodiment, an insulin composition comprises a detectable amount of a C-peptide, an L-peptide, or of both a C- peptide and an L-peptide.

The presence of the C-peptide and/or the L-peptide can be detected using standard analytical techniques, such as liquid chromatography-tandem mass spectroscopy (LC-MS/MS). For example, Foulon et al., 2022, JMSACL, 25: 19-26 describes a method of quantifying insulin and the C-peptide by LC-MS/MS without the use of antibodies, using serum samples. The skilled person will however know to apply similar methods on a sample obtained from an insulin composition.

In an embodiment of the invention and/or embodiments thereof, the composition comprising insulin comprises the C-peptide and/or the L-peptide in a concentration of < 10 pg/mL, preferably less than 9 pg/mL, < 8 pg/mL, < 7.5 pg/mL, < 7 pg/mL, < 6 pg/mL, < 5 pg/mL, < 4 pg/mL, < 3 pg/mL, < 2.5 pg/mL, < 2 pg/mL, < 1 pg/mL, < 0.5 pg/mL, < 0.25 pg/mL or < 0.1 pg/mL. For example, the composition comprising insulin comprises the C-peptide according to SEQ ID NO: 1 and/or the L-peptide according to SEQ ID NO: 2 in a concentration of 1-5000 ng/mL, such as 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 the C-peptide according to SEQ ID NO: 1 and/or the L-peptide according to SEQ ID NO: 2 or < 3 ppm of the C-peptide according to SEQ ID NO: 1 and/or the L-peptide according to SEQ ID NO: 2, or < 3000 ng/ml of the C-peptide and/or the L-peptide. The concentration of the C-peptide and the concentration of the L-peptide can therein be independent from each other. Another embodiment of the invention is a recombinant insulin composition produced by the above methods and a pharmaceutically acceptable carrier. Another embodiment of the invention is a method of treating diabetes comprising administering to an animal in need a therapeutic amount of the above recombinant insulin composition. Another embodiment is a recombinant insulin composition for use in the treatment of diabetes. The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications, such as specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein.

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

It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

Having now generally described the invention, the same will be more readily understood through reference to the following example which are provided by way of illustration and are not intended to be limiting to the present invention. Further aspects and embodiments will be apparent to those skilled in the art.

Examples

Example 1

Production of insulin precursor and insulin

Codon optimization

The DNA sequences encoding the insulin precursors according to SEQ ID NO: 5-8 were codon optimized for expression in E. coli using GeneART and DNA2.0, resulting in DNA sequences according to SEQ ID NO: 13-15.

Furthermore, incorporation of the codon AAC for all asparagine residues and the codon TCT for all serine residues reduced the forming of impurities (amino acid misincorporations) after digestion as shown in Fig. 2 and 3, respectively.

Construction of the plasmids pET29 vector as the expression vector was cleaved with restriction enzymes Ndel and Xhol, and electrophoresed on 1% agarose gel to isolate a DNA segment of 5.2 kb.

A DNA sequence according to SEQ ID NO: 13, 14 or 15 flanked by an Ndel restriction enzyme recognition site and a Xhol restriction enzyme restriction site was then joined with the cleaved vector using T4 DNA ligase to form the plasmid comprising the DNA sequence according to SEQ ID NO: 13, 14, or 15.

E. coli BL21(DE3) was transformed by the heat shock method and streaked on an agar plate. After incubation, the transformed cells resistant to kanamycin were selected. The plasmid DNA was isolated from an individual transformant, and it was confirmed that the desired DNA had been properly inserted using an analysis by restriction enzyme cleavage.

Production of the precursor

Competent E. coli ITP3 cells were transformed by the heat shock method with a plasmid comprising the DNA sequence according to SEQ ID NO: 13, 14, or 15 in order to produce an insulin precursor according to SEQ ID NO: 5, 6, 7 or 8, respectively. SEQ ID NO: 5 corresponds substantially to the insulin precursor described in W02017/040363, except that compared to the sequence of W02017/040363 SEQ ID NO: 5 further comprises a methionine residue present at the first position (N-terminal position) of SEQ ID NO: 5.

Transformed E. coli ITP3 cells were then grown for 18 h at 37 °C in shaking flasks containing 800 ml LB broth supplemented with 100 pg/L kanamycin until the cell density reach about 2.1 (A600 nm). After cultivation, 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 level of each fused proinsulin was quantitated using ChemiDoc.

Cells were harvested by centrifugation at approximately 7000 rpm for 20 min and wet cell weights were measured by electronic scale (Sartorius, Germany). The pelleted cells were suspended in 20 ml of 50 mM Tris-HCl (pH 7.5), 10 mM EDTA, 0.02% lysozyme, lysed by sonication and centrifuged at approximately 10,000 rpm for 20 min.

The pellet containing the inclusion bodies (IBs) comprising insulin precursor was washed by resuspending in 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 2 M urea buffer, followed by centrifugation at 10,000 rpm for 20 min. Finally, the pellet was washed twice with deionized water.

Table 1 shows the fermentation titer of the insulin precursors according to SEQ ID NO: 5-8 produced upon expression of DNA sequences according to SEQ ID NO: 13-15, respectively, in E. coli. The N-terminal L-peptide is underlined, and the C-peptide is indicated in bold. The recombinant insulin precursor according to SEQ ID NO: 6 contains the A-chain and B-chain of insulin according to SEQ ID NO: 3 and 4, respectively, and the reference L-peptide of W02017/040363 including the N-terminal methionine residue for fermentation. In contrast to SEQ ID NO: 5, SEQ ID NO: 6 contains a C-peptide according to SEQ ID NO: 1. Surprisingly, the fermentation titer of the insulin precursor according to SEQ ID NO: 6 was higher than the fermentation titer of the reference insulin precursor according to SEQ ID NO: 5. As shown in Table 1 of Example 1, the fermentation titer in mmol/L of the insulin precursor according to SEQ ID NO: 6 was about 43% higher than the fermentation titer in mmol/L of the reference insulin precursor according to SEQ ID NO: 5 when the precursors were expressed in host cells under the same fermentation conditions. Thus, replacing the native human C-peptide of the reference precursor according by the C-peptide according to SEQ ID NO: 1 significantly increases the fermentation titer of the precursor.

Furthermore, the recombinant insulin precursor according to SEQ ID NO: 7 contains the A-chain and B-chain of insulin according to SEQ ID NO: 3 and 4, respectively, in combination with a L- peptide according to SEQ ID NO: 2. Furthermore, SEQ ID NO: 7 contains a modified C-peptide derived from the sequence of the human C-peptide. As shown in Table 1 of Example 1, the fermentation titer in mmol/L of the insulin precursor according to SEQ ID NO: 7 was about 39% higher than the fermentation titer of the reference insulin precursor according to SEQ ID NO: 5 and comprising the reference L-peptide and native human C-peptide under the same fermentation conditions.

Like 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 to this, SEQ ID NO: 8 contains the L-peptide according to SEQ ID NO: 2. The fermentation titer of the insulin precursor according to SEQ ID NO: 8 was even higher than the fermentation titer of the insulin precursor according to either SEQ ID NO: 5 or SEQ ID NO: 6. As shown in Table 1 of Example 1, the fermentation titer in mmol/L of the insulin precursor according to SEQ ID NO: 8 was even about 118% higher than the fermentation titer in mmol/L of the reference insulin precursor according to SEQ ID NO: 5. Thus, replacing the native human C-peptide by the C-peptide according to SEQ ID NO: 1 and additionally the reference L-peptide by the L-peptide according to SEQ ID NO: 2 increases the fermentation titer of the insulin precursor in a synergistic manner. Table 1. Fermentation titers (in both g/L and in mmol/L) of recombinant insulin precursors expressed in E. coli. The respective L-peptides are underlined, and the respective C-peptides are indicated in bold.

Solubilization of inclusion bodies

An inclusion body protein slurry comprising insulin precursor at a concentration of about 30 g/L in water is provided. After charging the IBs into the water, ethanolamine is added as a neat liquid to a final concentration of about 315 mM followed by the addition of 8 M urea to a final concentration of about 4.0 M. Finally, a 1 M aqueous dithiothreitol (DTT) solution is added to target a concentration of about 2.5 mM, and the mixture is agitated for a minimum of 30 minutes to complete the IB solubilization.

Refolding the precursor

The insulin precursor is diluted approximately 10-fold to a target insulin precursor concentration of about 1.6 g/L. First, the target amount of refold diluent solution (lOmM Ethanolamine, 10% (v/v) Hexylene Glycol) is prepared, then a certain amount of IM cystamine dihydrochloride (e.g., about 384 pM) is added and mixed until homogeneous. Next, the solubilized protein solution is transferred into the refold diluent solution with a minimal level of agitation needed to ensure solution mixing while maintaining a solution temnerature of 10°C (± 2°C). The refold reaction is agitated at 10°C until the rate of conversion to correctly folded insulin precursor falls below 5% per hour, as measured by the POROS HPLC assay, at which point the reaction is quenched. After completion of the refold, the reaction is stopped/slowed down by acidifying the refold solution, with 2N hydrochloric acid, to a pH of about 9.2 at 10°C. The temperature is maintained at 10°C during pH adjustment.

Anion exchange (AEX) chromatography

Cellular debris and precipitated Host Cell Protein (HCP) is separated out prior to loading on the AEX column. In preparation for the clarification, the temperature of the post-refold solution is increased from 10°C to 20°C over a span of 60 - 120 minutes. The clarification is performed via two banks of depth filters and one bank of 0.22 pm membrane filters, all in series. Depth filters consist of CUNO EXT 60ZA05A filters (bank #1, 56 m² total area) in series with CUNO EXT 90ZA08A filters (bank #2, also 56 m²). Additionally in series with the depth filters are 0.22 pm filters (9 m² total area). Both banks of depth filters are flushed together with 54 L/m² of water, or approximately 3000 L, prior to use. Once the depth filters have been flushed, the 0.22 pm filters are flushed (>20 L/m²) by flowing water through depth filter banks #1 and #2 and then through the 0.22 pm filters to drain due to equipment restrictions. An air-displacement of the flush water on the depth filters is performed before introducing product. The filters are operated at first under constant flux and then, as the filters start fouling, the flow rate is reduced to prevent the pressure from exceeding 40 psig. After the post refold solution has been completely processed, a recovery water chase is performed (25-30 L/m²) to maximize recovery of correctly folded precursor into a clarified post-refold solution.

The pH of the clarified post-refold solution is adjusted to about 9.4 with sodium hydroxide and it is diluted with water, if necessary, to achieve a conductivity of less than 2.5 mS/cm. The resulting solution is loaded onto a column packed with DEAE Sepharose Fast Flow, which has been equilibrated with an equilibration solution comprising about 50 mM sodium borate and 2.5 mM sodium chloride at pH 9.4. Flow rates during the loading and subsequent elution steps are adjusted to maintain a residence time of about five to seven minutes, and a loading factor of about 23 g of the correctly folded insulin precursor per L of column resin is used. After loading, the column is washed with about five column volumes (CV) of equilibration solution (50 mM sodium borate, 2.5 mM sodium chloride at pH 9.4), and then with about six CV of elution solution (about 50 mM sodium borate and 160 mM sodium chloride at pH 9.0). The main peak observed during elution with the 160 mM sodium chloride elution solution is collected to provide a post-AEX pool that contains the correctly folded insulin precursor.

Citraconylation and tryptic digestion The purpose of the citraconylation step is to decrease the generation of mis-cleavages produced during the tryptic digest step. Citraconylation is accomplished through the reaction of citraconic anhydride with the insulin precursor under basic conditions. The citraconic anhydride reacts with any primary amine, thus “blocking” the N-terminus as well as all the lysine residues found in the molecule. In this “protected” state the molecule proceeds through the tryptic digest step, where recombinant porcine trypsin is added to cleave the N-terminal L-peptide and the internal C- peptide from the insulin precursor molecule. After digestion is complete, the protein is deprotected via acid hydrolysis to yield the desired insulin product for further purification via subsequent downstream processing.

Prior to beginning the protection reaction, the concentration of correctly folded insulin precursor in the post-AEX pool is determined. Next, three bolus shots of neat citraconic anhydride are added to the AEX pool at room temperature to achieve the correct ratio of anhydride to correctly folded insulin precursor. After the anhydride addition is complete, the pH is adjusted to 8.5 (if needed) with HC1 or NaOH solution and the protection reaction is allowed to proceed for 2 ± 0.25 hours.

After the protection reaction is complete, the pH is checked and, if it has changed, titrated back to 8.5 using an HC1 or NaOH solution. The trypsin solution is then added to achieve a ratio of trypsin to correctly folded insulin precursor of about 1 : 11,000 by mass. Digestion proceeds for 12 ± 1 hours. After digestion is complete, the reaction is stopped by first adding acetic acid to achieve a pool concentration of 150 mM acetate, and then adjusting the pH of the reaction pool to pH 2.4 using HC1. The deprotection reaction (in a volume of approximately 4100 L) then continues for 4 ± 0.25 hours to produce correctly folded insulin.

Table 2: The amino acid sequences of SEQ ID NO: 1-8 and the corresponding DNA sequences of SEQ ID NO: 9-15. Sequence 5 discloses the amino acid sequence of a in insulin precursor comprising the reference L-peptide and the native human C-peptide. In sequences 6 8 and 13 - 15 the respective amino acid sequences of the L-peptides and the DNA sequences encoding the same are underlined, and the respective amino acid sequences of the C-peptides and the DNA sequences encoding the same are indicated in bold.

Claims

WHAT IS CLAIMED IS:

1. A recombinant insulin precursor according to the formula L-B-C-A, wherein A is an A-chain of insulin,

B is a 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.

2. A recombinant insulin precursor according to the formula L-B-C-A, wherein A is an A-chain of insulin,

B is a 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 SEQ ID NO: 2 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 2.

3. The recombinant insulin precursor according to claim 1 or 2, 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, and L comprises 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.

4. The recombinant insulin precursor according to any one of the preceding claims, the C- peptide consists of an amino acid sequence according to SEQ ID NO: 1 and the L-peptide consists of an amino acid sequence according to SEQ ID NO: 2.

5. The recombinant insulin precursor according to any of the preceding claims, wherein the A-chain comprises an amino acid sequence according to SEQ ID NO: 3 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 3.

6. The recombinant insulin precursor according to any of the preceding claims, wherein the B-chain comprises an amino acid sequence according to SEQ ID NO: 4 or an amino acid sequence having at least 60% sequence identity with SEQ ID NO: 4.

7. The recombinant insulin precursor according to any of the preceding claims, wherein the A-chain is derived from porcine insulin and/or the B-chain is derived from porcine insulin.

8. The recombinant insulin precursor according to any of the preceding claims, wherein the recombinant insulin precursor comprises an amino acid sequence according to SEQ ID NO: 6, 7 or 8 or a sequence having at least 60% sequence identity with SEQ ID NO: 6, 7 and/or 8.

9. The recombinant insulin precursor according to any of the preceding claims, wherein the recombinant insulin precursor consists of an amino acid sequence according to SEQ ID NO: 6, 7 or 8.

10. A DNA sequence encoding a recombinant insulin precursor according to the formula L- B-C-A, wherein

A is an A-chain of insulin,

B is a 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 and/or L is 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.

11. The DNA sequence according to claim 8, wherein the DNA sequence is codon optimized for expression in a microorganism.

12. The DNA sequence according to claim 9, wherein the DNA sequence is codon optimized for expression in E. coli.

13. The DNA sequence according to any one of claims 10 - 12, wherein

B is a B-chain of insulin, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 12;

C is a C-peptide connecting the A-chain and the B-chain, preferably wherein the C- peptide is encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 9; and

L is an N-terminal L-peptide, preferably encoded by a DNA sequence having at least 60% sequence identity with SEQ ID NO: 10.

14. A vector comprising a DNA sequence according to any one of claims 10 - 13.

15. A host cell comprising a DNA sequence according to any one of claims 10 - 13 or a vector according to claim 14.

16. The host cell according to claim 14, wherein the host cell is an E. coli cell and/or a S. cerevisiae cell.

17. A method of producing recombinant insulin, wherein a recombinant insulin precursor according to the formula L-B-C-A is expressed, wherein

A is an A-chain of insulin,

B is a 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 and/or L is 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, and the method comprises: 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 the C-peptide of the recombinant insulin precursor by an enzymatic reaction to obtain recombinant insulin.

18. The method according to claim 17, wherein step a) is preceded by a step of cloning a DNA sequence encoding the recombinant insulin precursor into a vector.

19. The method according to claim 17 or 18, wherein the method further comprises a step of purifying the recombinant insulin.

20. Use of a recombinant insulin precursor according to any one of claims 1 - 9 for the production of recombinant insulin.

21. A composition comprising recombinant insulin, wherein the composition comprises a detectable amount of a 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.

22. A recombinant insulin composition comprising a recombinant insulin produced by any of the methods of claim 17-19 and a pharmaceutically acceptable carrier.

23. A method of treating diabetes comprising administering to an animal in need a therapeutic amount of the recombinant insulin composition of claim 22.

24. The recombinant insulin composition of claim 22 for use in the treatment of diabetes.