WO2014122653A1

WO2014122653A1 - Process for preparing insulin

Info

Publication number: WO2014122653A1
Application number: PCT/IL2014/050131
Authority: WO
Inventors: Jacob Hartman; Lixin Jiang; Tamar Richter; Shmuel Hess
Original assignee: Valin Technologies Ltd.
Priority date: 2013-02-07
Filing date: 2014-02-06
Publication date: 2014-08-14

Abstract

The present invention relates to an improved process for converting an insulin precursor into an active insulin compound, preferably a long-acting insulin. Specifically, this invention provides a process wherein lysine residues in an Arg-Arg precursor insulin are reversibly blocked thus enabling cleavage of the reversibly blocked Arg-Arg precursor insulin by trypsin.

Description

PROCESS FOR PREPARING INSULIN

FIELD OF INVENTION

[001] The present invention relates to an improved process for converting an insulin precursor into a long-acting active insulin compound.

BACKGROUND OF THE INVENTION

[002] Insulin is a pancreatic hormone involved in the regulation of blood-glucose concentrations. For example, human, porcine, and bovine insulin, insulin analogues and mixed insulins are given to patients with insulin-dependent diabetes mellitus to control their blood- glucose concentrations.

[003] Mature insulin is a 2 chain peptide hormone containing 51 amino acids. The A-chain is composed of 21 amino acids and the B-chain is composed of 30 amino acids. The molecule contains 2 interchain disulfide bonds and 1 intrachain disulfide bond within the A-chain. In vivo, the hormone is first synthesized as a long precursor molecule, preproinsulin, which is sequentially processed into proinsulin and insulin. The first processing step is the proteolyic removal of the prepeptide during the transfer of the nascent chain through the membranes of the endoplasmic reticulum. Then, proinsulin folds with concomitant oxidation of the disulfide bonds and is transported to the Golgi apparatus were it is packaged into secretory vesicles. Here it is further processed by specific proteases to form mature insulin. The C-chain (C peptide) which connects the B and A chains in proinsulin is excised by this proteolytic cleavage. The C peptide is 35 amino acids long and comprises 2 pairs of basic amino acids at its amino and carboxy termini, ArgArg and LysArg, respectively (Steiner, 1984). It was assumed that the C peptide which bridges both insulin chains, aids in appropriate folding and disulfide bond formation. In fact, its omission or replacement by shorter synthetic peptide bridge, of one to few amino acids long, still allows proper insulin generation from an insulin-like precursor.

[004] Until the 1980's insulin was isolated from bovine and porcine pancreatic preparations. Early studies have demonstrated that insulin can be produced in vitro by combining the A and B chains in their S-sulfonated forms or by spontaneous reoxidation of reduced proinsulin. Insulin could subsequently be recovered by treatment with trypsin and carboxypeptidase B. However, these methods were not practical for large scale production. Later, semi- synthetic human insulin has become available from porcine insulin by transpeptidation (for example, U.S. Pat. No. 4,343,898) with trypsin and threonine which replaced B30 alanine, the only difference between porcine and human insulins.

[005] For example EP 87,238 provides a transpeptidation reaction performed in a solvent system comprising between about 75% and 97% (v/v) of at least one non-aqueous reaction miscible solvent including at least about 50% (vol/vol) butane- 1,4-diol. Transpeptidation process performed by using a L-specific serine carboxypeptidase enzyme is disclosed in U.S. Pat. No. 4,579,820.

SUMMARY OF THE INVENTION

[006] In one embodiment, the present invention provides a process for preparing an insulin, comprising the steps of: (a) reversibly blocking lysine residues in an Arg-Arg precursor insulin, comprising contacting the Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride at pH 6-11 ; and (b) cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin of step (a) with trypsin, thereby, preparing an insulin.

[007] In one embodiment, the present invention further provides a process for preparing an insulin, comprising the steps of: exposing an Arg-Arg precursor insulin to pH values of 11.0- 11.5; (b) reversibly blocking lysine residues in the Arg-Arg precursor insulin, comprising contacting the Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride at pH 6-11 ; and (c) cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin of step (b) with trypsin, thereby, preparing an insulin.

[008] In one embodiment, the present invention further provides a process for preparing an insulin, comprising the steps of: (a) reversibly blocking lysine residues in an Arg-Arg precursor insulin, comprising contacting the Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride at pH 9; (b) cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin of step (a) with trypsin; and (c) adjusting the pH of the insulin of step (b) to pH 6-10 (step (c)), thereby, preparing an insulin.

[009] In one embodiment, the processes of the invention further include insulin purification procedures such as: microfiltration, ion-exchange chromatography, reverse phase high pressure liquid chromatography (RP-HPLC), or any combination thereof. [010] In one embodiment, the processes of the invention further include insulin crystallization, comprising contacting the insulin with Zn⁺⁺ in citrate-ethanol (pH 6.4).

BRIEF DESCRIPTION OF THE DRAWINGS

[Oi l] Figures 1 is a scheme describing the process of making insulin of the invention from an Arg-Arg insulin precursor.

[012] Figure 2 is the Insulin-ArgArg Production Scheme.

[013] Figure 3 is a graph of RP-HPLC analysis of purified Insulin-ArgArg compared to comercial Lantus. The peak preceding insulin- ArgArg is the preservative m-cresol.

[014] Figure 4 is the nucleotide and corresponding amino acid sequence of the recombinant human insulin-analogue precursor expression cassette of the invention. The B chain amino acids are boxed. The A chain amino acids are boxed. The first 64 amino acids (codes for partial sequence of Cu/Zn- superoxide dismutase) which precede the insulin-analogue sequence and the Arg Arg bridge are indicated in bold. RBS - Ribosomal binding site.

[015] Figure 5 is a graph showing the mean group ± SD blood glucose values in Sprague- Dawley male rats subjected to diabetes induction by STZ and subsequently treated with daily injections (see arrows) of the indicated test materials. The upper panel is a normalized presentation to the blood glucose level of the vehicle control.

[016] Figure 6 is the amino acid sequences of the insulin produced by the methods described herein.

[017] Figure 7 shows graphs of blood glucose levels (mM) over time (hours) in diabetic beagle dogs after administration of fast acting Insulin, (Humulin R), long acting Insulin (Lantus) and a long acting insulin, Insulin-ArgArg (Insulin RR), in a randomized cross over study.

DETAILED DESCRIPTION OF THE INVENTION

[018] In one embodiment, the present invention provides an improved process for preparing insulin from an insulin precursor. In another embodiment, the present invention provides an improved process for preparing insulin from an Arg-Arg insulin precursor. In another embodiment, in the improved process of the present invention an Arg-Arg insulin precursor is directly dissolved and folded in an alkaline pH and then subjected to trypsin treatment, thus generating insulin in a simple, improved, way. In another embodiment, the utilization of an Arg- Arg insulin precursor according to the process of the invention results in authentic human insulin or its long acting B31 Arg-B32Arg variant. In another embodiment, the improved process of the present invention includes: reversibly blocking Lys residues in an Arg-Arg insulin precursor with citraconic anhydride and treating the reversibly blocked Arg-Arg insulin precursor with trypsin thus cleaving off the leader peptide and the "C-chain" junction. In another embodiment, the improved process of the present invention includes: reversibly blocking Lys residues in an Arg-Arg insulin precursor with citraconic anhydride and treating the reversibly blocked Arg-Arg insulin precursor with trypsin thus cleaving off the leader peptide and the "C- chain" junction, specifically preferentially following the second Arg residue (see Fig. 1).

[019] In another embodiment, the present process overcomes the disadvantages of the state of the art by providing limited trypsin cleavage sites within the precursor insulin. In another embodiment, the present process is based on reversibly blocking undesired trypsin cleavage sites within the precursor insulin. In another embodiment, reversibly blocking undesired trypsin cleavage sites within the precursor insulin overcomes the disadvantages of the state of the art.

[020] In another embodiment, all lysine residues in the precursor protein are reversibly blocked by reaction with excess citraconic anhydride at pH 9, which is maintained by automatic titration. In another embodiment, remaining reagent is hydrolyzed to citraconic acid. In another embodiment, remaining reagent is quenched by addition of 5 mM ethanol amine. In another embodiment, the acyl derivatives of the insulin precursor acquire an extra negative charge of -7 which can be readily analyzed on urea-PAGE.

[021] In another embodiment, the insulin of the present invention (the product of the process described herein) is insulin-ArgArg with a pi value of about 6.9. In another embodiment, the insulin-ArgArg of the invention has a long in vivo activity profile. In another embodiment, the insulin-ArgArg of the invention has a very long in vivo activity profile. In another embodiment, the insulin-ArgArg of the invention has depot-like characteristics since it precipitates following subcutaneous injection. In another embodiment, the insulin-ArgArg of the invention slowly dissolves into the circulation. [022] In another embodiment, the present invention provides an improved process for preparing insulin from an Arg-Arg insulin precursor, comprising the steps of: (a) reversibly blocking lysine residues in the Arg-Arg precursor insulin, comprising contacting the Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride; and (b) cleaving the Arg-Arg precursor insulin, comprising reacting the Arg-Arg precursor insulin of step (a) with trypsin.

[023] In another embodiment, the improved process for preparing insulin from an Arg-Arg insulin precursor further includes exposing Arg-Arg precursor insulin to pH values of 11.0-11.5 prior to step (a). In another embodiment, exposing Arg-Arg precursor insulin to pH values of 11.0-11.5 prior to step (a) results in optimal folding. In another embodiment, at the end of the folding step the pH is reduced to 9 with HC1.

[024] In another embodiment, the improved process for preparing insulin from an Arg-Arg insulin precursor further includes step (c), wherein step (c) includes adjusting the pH of the insulin obtained in step (b) to pH 2.8. In another embodiment, the improved process for preparing insulin from an Arg-Arg insulin precursor further includes step (c), wherein step (c) includes adjusting the pH of the insulin obtained in step (b) to pH 2.8.

[025] In another embodiment, the improved process for preparing insulin from an Arg-Arg insulin precursor further includes step (d). In another embodiment, step (d) follows step (c). In another embodiment, step (d) includes purifying the insulin. In another embodiment, purifying is performed by microfiltering. In another embodiment, purifying is performed by ion-exchange and mix mode chromatography. In another embodiment, purifying is performed by reverse phase high pressure liquid chromatography (RP-HPLC).

[026] In another embodiment, the improved process for preparing insulin from an Arg-Arg insulin precursor further includes the step of contacting the insulin resulting from step (d) with zinc salts.

[027] In another embodiment, the improved process for preparing insulin from an Arg-Arg insulin precursor further includes crystallizing the insulin step (e). In another embodiment, step (e) follows step (d). In another embodiment, step (e) follows step (c). In another embodiment, step (e) follows step (b). In another embodiment, crystallizing insulin is contacting the insulin with Zn⁺⁺. In another embodiment, crystallizing insulin is contacting the insulin with Zn⁺⁺ in citrate-ethanol. In another embodiment, crystallizing insulin is contacting the insulin with Zn⁺⁺ in citrate-ethanol (pH 6.4).

[028] In another embodiment, the precursor insulin of the invention is isolated from disrupted bacteria as inclusion bodies. In another embodiment, the precursor insulin of the invention dissolves and folds readily at alkaline pH. In another embodiment, insulin is generated by specific trypsin cleavage of the bridging Arg residues while reversibly blocking all Lys residues with citraconic anhydride. In another embodiment, this reagent avidly reacts with amino groups at about pH 9, but the acyl derivatives are completely hydrolyzed at acidic pH. In another embodiment, following ion-exchange chromatography and mix mode chromatography, the purified insulin- ArgArg variant is further purified by RP-HPLC and formulated as long acting insulin. In another embodiment, a final purification step removes insulin-like molecules and other minor contaminants by preparative reverse phase (RP) HPLC. In another embodiment, insulin is recovered, by crystallization with Zn++ in citrate-ethanol (pH 6.4), and vacuum dried. Arg-Arg insulin precursor

[029] In another embodiment, an Arg-Arg insulin precursor is a hybrid protein comprising part of Cu/ZnSOD. In another embodiment, the N-terminal sequence of an Arg-Arg insulin precursor is fused by an Arg residue to a proinsulin-like moiety in which ArgArg bridges the B and A chains of insulin. [030] In another embodiment, an Arg-Arg insulin precursor comprises the following amino acid sequence: RFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVEQCCTSICSLYQLENYCG (SEQ ID NO: 1). In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by any length from 5 to 150 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by any length from 10 to 120 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by any length from 20 to 100 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by any length from 10 to 100 amino acids. In another embodiment, the preceding sequence is not restricted to any particular sequence.

[031] In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by at least 5 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by at least 10 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by at least 20 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by at least 30 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by at least 50 amino acids. In another embodiment, an Arg-Arg insulin precursor comprises the amino acid sequence of SEQ ID NO: 1 preceded (at the amino terminus) by at least 80 amino acids.

[032] In another embodiment, an Arg-Arg insulin precursor comprises the following amino acid sequence:

MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGS TSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVEQCCTSICSLYQLENYCG

(SEQ ID NO: 2).

[033] In another embodiment, an Arg-Arg insulin precursor is encoded by any DNA molecule encoding an Arg-Arg insulin precursor amino acid sequence of the invention. In another embodiment, a DNA molecule encoding an Arg-Arg insulin precursor encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, a DNA molecule encoding an Arg-Arg insulin precursor encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and at least additional 5 amino acids which precede the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, a DNA molecule encoding an Arg- Arg insulin precursor encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and at least additional 10 amino acids which precede the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, a DNA molecule encoding an Arg-Arg insulin precursor encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and at least additional 20 amino acids which precede the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, a DNA molecule encoding an Arg-Arg insulin precursor encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and at least additional 30 amino acids which precede the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, a DNA molecule encoding an Arg-Arg insulin precursor encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 and at least additional 50 amino acids which precede the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In another embodiment, a DNA molecule encoding an Arg-Arg insulin precursor encodes the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2and at least additional 80 amino acids which precede the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. [034] In another embodiment, an Arg-Arg insulin precursor is encoded by a DNA molecule comprising the following nucleic acid sequence:

ATGGCAACCAAAGCAGTTAGCGTTCTGAAAGGTGATGGTCCGGTTCAGGGCATTAT TAATTTTGAACAGAAAGAAAGCAATGGTCCGGTTAAAGTTTGGGGTAGCATTAAAG GTCTGACCGAAGGTCTGCATGGTTTTCATGTTCATGAATTTGGCGATAATACCGCAG GTAGCACCAGCGCAGGCCCGCGTTTTGTTAATCAGCATCTGTGTGGT AGCCATCTGG TTGAAGCACTGTATCTGGTTTGTGGTGAACGCGGTTTTTTTTATACCCCGAAAACCC GTCGTGGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAA ATTATTGCGGCTAA (SEQ ID NO: 3).

[035] In another embodiment, an insulin produced by the methods or processes of the invention comprises two chains: (1) chain A comprising the following amino acid sequence: GIVEQCCTSICSLYQLENYCG (SEQ ID NO: 4); and (2) chain B comprising the following amino acid sequence: F VNQHLCGS HL VE AL YL VCGERGFFYTPKTRR (SEQ ID NO: 5).

[036] In another embodiment, insulin of the invention is insulin produced by the process described herein. In another embodiment, insulin of the invention is human insulin. In another embodiment, insulin of the invention is a mammal insulin. In another embodiment, insulin of the invention is farm animal insulin. In another embodiment, insulin of the invention is an insulin analog or an insulin derivative.

[037] In another embodiment, an insulin analog is an analog of naturally occurring insulin, which differs by substitution of at least one naturally occurring amino acid residue with other amino acid residues and/or addition/removal of at least one amino acid residue from the corresponding, otherwise identical, naturally occurring insulin. In another embodiment, the added and/or replaced amino acid residues can also be those which do not occur naturally. In another embodiment, insulin analogs are described in EP 0 214 826, EP 0 375 437 and EP 0 678 522 which are incorporated herein by reference in their entirety. [038] In another embodiment, an insulin derivative is a derivative of naturally occurring insulin or an insulin analog which is obtained by chemical modification. In another embodiment, the chemical modification consists in the addition of one or more specific chemical groups to one or more amino acids.

[039] In another embodiment, insulin of the invention is an insulin analog is selected from the group consisting of LysB28ProB29 human insulin, B28 Asp human insulin, human insulin comprising proline in position B28 substituted by Leu, Asp, Ala, Val or Lys with or without Lys in position B29 Lys substituted by Pro; AlaB26 human insulin; des(B28-B30) human insulin; des(B27) human insulin and des(B30) human insulin. In another embodiment, insulin analog is insulin glulisine. In another embodiment, insulin analog is insulin glargine. [040] In another embodiment, contacting Arg-Arg precursor insulin with citraconic anhydride is at pH 7-11. In another embodiment, contacting Arg-Arg precursor insulin with citraconic anhydride is at pH 8-10. In another embodiment, contacting Arg-Arg precursor insulin with citraconic anhydride is at pH 8.5-9.5. In another embodiment contacting, Arg-Arg precursor insulin with citraconic anhydride is at pH 9. In another embodiment, contacting is contacting for at least 2 minutes (mins). In another embodiment, contacting is contacting for at least 5 minutes (mins). In another embodiment, contacting is contacting for at least 10 minutes (mins). In another embodiment, contacting is contacting for at least 15 minutes (mins). In another embodiment, contacting is contacting for at least 20 minutes (mins). In another embodiment, contacting is contacting for at least 25 minutes (mins). In another embodiment, contacting is contacting for at least 30 minutes (mins).

[041] In another embodiment, citraconylation is carried out in a buffer devoid of free amino group. In another embodiment, citraconylation is carried out in carbonate, phosphate or borate buffer. In another embodiment, the pH of the reaction is maintained at 6 -10. In another embodiment, the pH of the reaction is maintained at 7 -9.5. In another embodiment, 3 fold excess of citraconic anhydride are used over the estimated insulin in the solution on the basis of absorbance at 280 nm. In another embodiment, reaction time is less than 1 hour at room temperature. In another embodiment, reaction time is 20-60 minutes at room temperature.

[042] In another embodiment, trypsin is porcine trypsin. In another embodiment, trypsin is TPCK treated. In another embodiment, trypsin is used at a 1 :6000-10000 ratio to the estimated insulin amount in the solution on the basis of absorbance at 280 nm. In another embodiment, trypsin is used at a 1 :6000-9000 ratio to the estimated insulin amount in the solution on the basis of absorbance at 280 nm. In another embodiment, trypsin is used at a 1 :7500 ratio to the estimated insulin amount in the solution on the basis of absorbance at 280 nm. In another embodiment, the reaction is run for 0.5-2 hrs at room temperature. In another embodiment, the reaction is optimal in the presence of 5-20 mM Ca++, preferably 10 mM Ca++. In another embodiment, the reaction is run for 1 hr at room temperature (22 ± 3°C). In another embodiment, reaction time is dependent on enzyme concentration, temperature and pH.

[043] In another embodiment, folding takes place at a pH range of 9-13. In another embodiment, folding takes place at a pH range of 10-12. In another embodiment, folding takes place at a pH range of 11-11.5.

[044] In another embodiment, pH adjustments are done either with HC1 or NaOH.

[045] In another embodiment, crystallizing of insulin is performed in the presence of Zn⁺⁺ in citrate-ethanol at pH 4-8. In another embodiment, crystallizing of insulin is performed in the presence of Zn⁺⁺ in citrate-ethanol at pH 6-7. In another embodiment, crystallizing of insulin is performed in the presence of Zn⁺⁺ in citrate-ethanol at pH 6.2-6.6. In another embodiment, crystallizing of insulin is performed in the presence of Zn⁺⁺ in citrate-ethanol at pH 6.4.

[046] In another embodiment, contacting Arg-Arg precursor insulin with citraconic anhydride is at 16 -40°C. In another embodiment, contacting Arg-Arg precursor insulin with citraconic anhydride is at 20 -35°C. In another embodiment, contacting Arg-Arg precursor insulin with citraconic anhydride is at 19-25°C.

[047] In another embodiment, reacting the Arg-Arg precursor insulin of step (a) with trypsin is at about pH 8. In another embodiment, reacting the Arg-Arg precursor insulin of step (a) with trypsin is at about pH 6-10. In another embodiment, reacting the Arg-Arg precursor insulin of step (a) with trypsin is at about pH 7.5-8.5. In another embodiment, reacting the Arg-Arg precursor insulin of step (a) with trypsin is at about pH 9.

[048] In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is at 16 -40°C. In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is at 20 -40°C. In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is at t 22+3 °C. In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is at room temperature.

[049] In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is for at least 30 mins. In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is for at least one hour. In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is for at least 2 hours. In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is for at least 3 hours. In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is for at least 4 hours.

[050] In another embodiment, reacting Arg-Arg precursor insulin of step (a) with trypsin is for 1-4 hours. In another embodiment, the duration of the reaction is easily determined by one of skill in the art and depends, inter-alia, on the temperature, pH, and the concentration of trypsin. In another embodiment, the duration of the reaction, the temperature, and the pH of the trypsin reaction mixture is determined by one of skill in the art according the specific trypsin that is utilized (the specific kinetics and specificities). [051] In some embodiments, "Arg-Arg precursor insulin" or "insulin" as used herein encompasses native proteins (either degradation products, synthetically synthesized proteins or recombinant proteins) and peptidomimetics (typically, synthetically synthesized proteins), as well as peptoids and semipeptoids which are protein analogs, which have, in some embodiments, modifications rendering the proteins even more stable while in a body or more capable of penetrating into cells.

[052] In some embodiments, further modifications according to the invention include, but are not limited to N-terminus modification, C-terminus modification, peptide bond modification, including, but not limited to, CH2-NH, CH2-S, CH2-S=0, 0=C-NH, CH2-0, CH2-CH2, S=C- NH, CH=CH or CF=CH, backbone modifications, and residue modification. Methods for preparing peptidomimetic compounds are well known in the art and are specified, for example, in Quantitative Drug Design, C.A. Ramsden Gd., Chapter 17.2, F. Choplin Pergamon Press (1992), which is incorporated by reference as if fully set forth herein. Further details in this respect are provided hereinunder.

[053] In some embodiments, peptide bonds (-CO-NH-) within the peptide are substituted. In some embodiments, the peptide bonds are substituted by N-methylated bonds (-N(CH3)-CO-). In some embodiments, the peptide bonds are substituted by ester bonds (-C(R)H-C-O-O-C(R)- N-). In some embodiments, the peptide bonds are substituted by ketomethylen bonds (-CO-CH2- ). In some embodiments, the peptide bonds are substituted by a-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl, e.g., methyl, carba bonds (-CH2-NH-). In some embodiments, the peptide bonds are substituted by hydroxyethylene bonds (-CH(OH)-CH2-). In some embodiments, the peptide bonds are substituted by thioamide bonds (-CS-NH-). In some embodiments, the peptide bonds are substituted by olefinic double bonds (-CH=CH-). In some embodiments, the peptide bonds are substituted by retro amide bonds (-NH-CO-). In some embodiments, the peptide bonds are substituted by peptide derivatives (-N(R)-CH2-CO-), wherein R is the "normal" side chain, naturally presented on the carbon atom. In some embodiments, these modifications occur at any of the bonds along the peptide chain and even at several (2-3 bonds) at the same time.

[054] In some embodiments, natural aromatic amino acids of the peptide such as Trp, Tyr and Phe, are substituted for synthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine (Nol), ring-methylated derivatives of Phe, halogenated derivatives of Phe or o-methyl-Tyr. In some embodiments, the peptides of the present invention include one or more modified amino acid or one or more non-amino acid monomers (e.g. fatty acid, complex carbohydrates etc).

[055] In one embodiment, "amino acid" is understood to include the 20 naturally occurring amino acid; those amino acid often modified post-translationally in vivo, including, for example, hydroxyproline, phosphoserine and phosphothreonine; and other unusual amino acid including, but not limited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine, nor-leucine and ornithine. In one embodiment, "amino acid" includes both D- and L-amino acid.

[056] In some embodiments, recombinant protein techniques are used to generate the "Arg-Arg precursor insulin" of the present invention. In some embodiments, recombinant protein techniques are used for generation of relatively long peptides (e.g., longer than 18-25 amino acid). In some embodiments, recombinant protein techniques are used for the generation of large amounts of the "Arg-Arg precursor insulin" of the present invention. In some embodiments, recombinant techniques are described by Bitter et al., (1987) Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods in Enzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsu et al. (1987) EMBO J. 6:307-311 , Coruzzi et al. (1984) EMBO J. 3 :1671 -1680 and Brogli et al, (1984) Science 224:838-843, Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 421-463.

[057] In another embodiment, "Arg-Arg precursor insulin" of the present invention are synthesized using a polynucleotide encoding a protein of the present invention. In some embodiments, the polynucleotide encoding an Arg-Arg precursor insulin of the present invention is ligated into an expression vector, comprising a transcriptional control of a cis-regulatory sequence (e.g., promoter sequence). In some embodiments, the cis-regulatory sequence is suitable for directing constitutive expression of the Arg-Arg precursor insulin of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing specific expression of the Arg-Arg precursor insulin of the present invention. In some embodiments, the cis-regulatory sequence is suitable for directing inducible expression of an Arg-Arg precursor insulin of the present invention.

[058] In one embodiment, the phrase "a polynucleotide" refers to a single or double stranded nucleic acid sequence which be isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and/or a composite polynucleotide sequences (e.g., a combination of the above).

[059] In one embodiment, "genomic polynucleotide sequence" refers to a sequence derived (isolated) from a chromosome and thus it represents a contiguous portion of a chromosome.

[060] In one embodiment, "composite polynucleotide sequence" refers to a sequence, which is at least partially complementary and at least partially genomic. In one embodiment, a composite sequence can include some exonal sequences required to encode an Arg-Arg precursor insulin of the present invention, as well as some intronic sequences interposing there between. In one embodiment, the intronic sequences can be of any source, including of other genes, and typically will include conserved splicing signal sequences. In one embodiment, intronic sequences include cis acting expression regulatory elements. [061] In some embodiments, polynucleotides of the present invention are prepared using PCR techniques, or any other method or procedure known to one skilled in the art. In some embodiments, the procedure involves the ligation of two different DNA sequences (See, for example, "Current Protocols in Molecular Biology", eds. Ausubel et al., John Wiley & Sons, 1992).

[062] In one embodiment, polynucleotides of the present invention are inserted into expression vectors (i.e., a nucleic acid construct) to enable expression of the recombinant protein. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in prokaryotes. In one embodiment, the expression vector of the present invention includes additional sequences which render this vector suitable for replication and integration in eukaryotes. In one embodiment, the expression vector of the present invention includes a shuttle vector which renders this vector suitable for replication and integration in both prokaryotes and eukaryotes. In some embodiments, cloning vectors comprise transcription and translation initiation sequences (e.g., promoters, enhances) and transcription and translation terminators.

[063] In one embodiment, a variety of prokaryotic or eukaryotic cells can be used as host- expression systems to express the Arg-Arg precursor insulin of the present invention. In some embodiments, these include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing an Arg-Arg precursor insulin coding sequence; yeast transformed with recombinant yeast expression vectors containing an Arg-Arg precursor insulin coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing an Arg-Arg precursor insulin coding sequence.

[064] In some embodiments, non-bacterial expression systems are used (e.g. mammalian expression systems such as CHO cells) to express an Arg-Arg precursor insulin of the present invention. In one embodiment, the expression vector used to express polynucleotides of the present invention in mammalian cells is pCI-DHFR vector comprising a CMV promoter and a neomycin resistance gene.

[065] In some embodiments, bacterial systems such as Escherichia Coli BL-21 are utilized. In one embodiment, large quantities of protein are desired. In one embodiment, vectors that direct the expression of high levels of the protein product, possibly as a fusion with a hydrophobic signal sequence, which directs the expressed product into the periplasm, or into inclusion bodies of the bacteria or the culture medium where the protein product is readily purified are desired. In one embodiment, certain fusion protein engineered with a specific cleavage site to aid in recovery of the protein. In one embodiment, vectors adaptable to such manipulation include, but are not limited to, the pET series of E. coli expression vectors [Studier et al., Methods in Enzymol. 185:60-89 (1990)].

[066] In another embodiment, the present invention provides the use of recombinant DNA processes that allow precursors of insulin or insulin analogs, in particular human proinsulin or proinsulin which has an amino acid sequence and/or amino acid chain length differing from human insulin to be prepared in microorganisms. In another embodiment, proinsulin (synonymous with the precursor insulin of the invention) prepared from genetically modified Escherichia coli cells does not have any correctly bonded cystine bridges. In another embodiment, a process for obtaining insulin using E. coli is described in EP 0 055 945 which is incorporated herein by reference in its entirety.

[067] In one embodiment, yeast expression systems are used. In one embodiment, a number of vectors containing constitutive or inducible promoters can be used in yeast as disclosed in U.S. Pat. Application. No: 5,932,447. In another embodiment, vectors which promote integration of foreign DNA sequences into the yeast chromosome are used.

[068] In one embodiment, various methods can be used to introduce the expression vector of the present invention into cells. Such methods are generally described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Springs Harbor Laboratory, New York (1989, 1992), in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md. (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich. (1995), Vega et al, Gene Targeting, CRC Press, Ann Arbor Mich. (1995), Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4 (6): 504-512, 1986] and include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and 5,487,992 for positive-negative selection methods.

[069] In some embodiments, transformed cells are cultured under effective conditions, which allow for the expression of high amounts of an Arg-Arg precursor insulin. In some embodiments, effective culture conditions include, but are not limited to, effective media, bioreactor, temperature, pH and oxygen conditions that permit protein production. In one embodiment, an effective medium refers to any medium in which a cell is cultured to produce an Arg-Arg precursor insulin of the present invention. In some embodiments, a medium typically includes an aqueous solution having assimilable carbon, nitrogen and phosphate sources, and appropriate salts, minerals, metals and other nutrients, such as vitamins. In some embodiments, cells of the present invention can be cultured in conventional fermentation bioreactors, shake flasks, test tubes, microtiter dishes and petri plates. In some embodiments, culturing is carried out at a temperature, pH and oxygen content appropriate for a recombinant cell/bacterium. In some embodiments, culturing conditions are within the expertise of one of ordinary skill in the art.

[070] In one embodiment, following a predetermined time in culture, recovery of an Arg-Arg precursor insulin is effected.

[071] In one embodiment, the phrase "recovering the an Arg-Arg precursor insulin " used herein refers to collecting the whole fermentation medium containing an Arg-Arg precursor insulin and need not imply additional steps of separation or purification.

[072] In one embodiment, purification methods are utilized to purify an Arg-Arg precursor insulin or insulin of the present invention. Purification methods include a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, mixed mode chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

[073] In one embodiment, an Arg-Arg precursor insulin or insulin of the present invention is retrieved in "substantially pure" form.

[074] In one embodiment, the phrase "substantially pure" refers to a purity that allows for the effective use of the protein in the applications described herein such as for treating diabetes.

[075] In some embodiments, an Arg-Arg precursor insulin is synthesized and purified; its therapeutic efficacy can be assayed either in vivo or in vitro. [076] In some embodiments, Insulin of the invention may be formulated in peroral or oral compositions and in some embodiments, comprise liquid solutions, emulsions, suspensions, and the like. In some embodiments, pharmaceutically- acceptable carriers suitable for preparation of such compositions are well known in the art. In some embodiments, liquid oral compositions comprise from about 0.001% to about 0.933% of insulin, or in another embodiment, from about 0.01 % to about 10 %.

[077] In some embodiments, compositions for use in the methods of this invention comprise solutions or emulsions, which in some embodiments are aqueous solutions or emulsions comprising a safe and effective amount of insulin and optionally, other compounds, intended for topical intranasal administration.

[078] In one embodiment, injectables of the invention are formulated in aqueous solutions. In one embodiment, injectables of the invention are formulated in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. In some embodiments, for transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[079] In one embodiment, the preparations described herein are formulated for parenteral administration, e.g., by bolus injection or continuous infusion. In some embodiments, formulations for injection are presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. In some embodiments, compositions are suspensions, solutions or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[080] The compositions also comprise, in some embodiments, preservatives, such as phenol, m- cresol, metaprazol, benzalkonium chloride and thimerosal and the like; chelating agents, such as edetate sodium and others; buffers such as phosphate, citrate and acetate; tonicity agents such as sodium chloride, potassium chloride, glycerin, mannitol and others; antioxidants such as ascorbic acid, acetylcystine, sodium metabisulfote and others; aromatic agents; viscosity adjustors, such as polymers, including cellulose and derivatives thereof; and polyvinyl alcohol and acid and bases to adjust the pH of these aqueous compositions as needed. The compositions also comprise, in some embodiments, local anesthetics or other actives. The compositions can be used as sprays, mists, drops, and the like. [081] Additional objects, advantages, and novel features of the present invention will become apparent to one ordinarily skilled in the art upon examination of the following examples, which are not intended to be limiting. Additionally, each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples.

EXAMPLES

[082] Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al, "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801 ,531 ; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533 ; 3,996,345 ; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281 ,521 ; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are provided throughout this document.

EXAMPLE 1

Process for efficiently producing insulin

Expression clone

[083] The nucleotide sequence coding for the insulin precursor protein was optimized to improve expression in E. coli by GeneOptimizer® Software. The synthetic gene was then assembled from synthetic oligonucleotides and cloned into pET24a (Novagen) using Ndel and Blpl restriction enzyme sites. E. coli strain BL21 (DE3) cells were transfected with the above clone. The gene was transcribed under the control of T7 promoter and lac operator. Transcription termination was controlled by T7 terminator and translation initiation was promoted by the pET24 ribosome binding site. Expression of the 12.7 kD fusion protein used as insulin precursor was induced by IPTG resulting in production of very high levels of insulin precursor. This precursor contains part of Cu/Zn SOD as the amino terminal portion fused by an Arg residue to a proinsulin-like moiety in which Arg-Arg bridges the B and A chains of insulin. The expression cassette of the insulin precursor - nucleotide and corresponding amino acid sequence - is shown in Figure 4.

Growth condition and accumulation of Insulin precursor

[084] Fermentation was carried out in minimal salt medium supplemented with yeast extract, glucose, ammonia and oxygen at 37°C until cell concentration reaches an OD660 ~ 50. For induction, IPTG was added and low glucose levels were tightly maintained and controlled for 6 more hours until culture density of OD₆6o ~ 120 was achieved. Very high levels of insulin precursor accumulated as intracellular inclusion bodies (IB's). Cells were harvested by ultrafiltration using microporous hollow fiber cartridges and washed with saline. Recovery

[085] IB's were recovered following disruption of bacterial cells by high shear mixing and high-pressure homogenization at 850 bars. IB's were collected by continuous centrifugation and the precipitate was washed twice in a carbonate buffer.

Dissolution and folding

[086] IB's were suspended in NaHC(¾ buffer and dissolved by brief exposure to pH 12. The solution was then adjusted to pH 11.5, clarified by centrifugation and was further incubated at 23 °C for a total of 6 hours from start of the alkali treatment. It should be pointed out that the pH is an important parameter for optimal folding which is optimal at pH values of 11.0-11.5. At the end of the folding period the pH is reduced to 9 with HC1.

Modification by citraconic anhydride and trypsin cleavage

[087] All lysine residues in the precursor protein are reversibly blocked by reaction with excess citraconic anhydride at pH 9, which is maintained by automatic titration. Remaining reagent is either hydrolyzed to citraconic acid or quenched by addition of 5 mM ethanol amine. The acyl derivatives of the insulin precursor acquire an extra negative charge of -7 which can be readily analyzed on urea- PAGE. The protein is then treated with dilute trypsin at 23 °C for 1 - 2 hours with 10 mM Ca++, generating citraconylated insulin- ArgArg. At the end of the reaction period trypsin is inactivated by reducing the pH to 2.0 and incubation for about 16 hours at room temperature is carried on to completely remove the citraconyl residues.

Purification

[088] The solution is clarified by centrifugation and microfiltration and chromatographed on SP Sepharose column in citrate, pH 3.0. The eluted pool in 30% isopropanol was adjusted to pH 9.0 and treated with aprotinin to inactivate any residual trypsin and chromatographed on mixed mode PPA HyperCell column. The insulin- ArgArg enriched main pool was eluted at pH 3.0 and precipitated with Zn⁺⁺ at pH 7.0.

The precipitate was collected by TFF. The concentrated slurry was dissolved under acidic conditions with EDTA, and insulin-ArgArg was further purified by preparative reverse phase HPLC and crystallized. Insulin crystals were collected by filtration, washed and vacuum dried and bulk material was stored frozen at -20°C. Figure 3 shows the HPLC chromatogram of highly purified insulin-ArgArg preparation. EXAMPLE 2

The efficiently produced insulin is active in- ivo

[089] In order to assess the potential treatment effects of the long acting insulin analogues that were generated in example 1 , insulin- ArgArg and Lantus were tested in streptozotocin (STZ)- induced diabetic rats (Junod et al, 1969) by determination of their blood glucose levels at various time points post dosing. Young Sprague-Dawley male rats about 9 weeks old were subjected to single intraperitoneal injection of 60 mg STZ per kilogram body weight. Five days post induction of diabetes most animals showed blood glucose values higher than 300 mg/dL. These animals were administered the test materials corresponding to 6 IU/kg by subcutaneous injections on 3 consecutive days. The study consisted of 3 experimental groups, 6 animals in each group, treated with the 2 different insulins indicated above and with a vehicle as control. Blood glucose was determined before treatment and 1, 4, 8 and 24 hours following each dosing day. As can be seen in Fig. 5 both test materials have comparable activity profile in vivo.

EXAMPLE 3

The efficiently produced insulin is active as a long acting insulin in-vivo in dogs

[090] The effect of various insulin preparations on lowering blood glucose levels was tested in alloxan-induced diabetic beagle dogs. Eight (8) healthy spayed female dogs at the age of 2 - 5 years weighing 10-17 kilograms, were treated by intravenous injection of alloxan at 50 mg per kg body weight. Three (3) days after treatment all dogs developed a reproducible experimental diabetic state manifested by hyperglycemia and other characteristic symptoms (Watanabe et al, The pathological features of alloxan diabetes in beagle dogs, J Toxicol Pathol, 2004, 17, 187- 195).

[091] Insulin-ArgArg ("Insulin RR") prepared as described above was compared to two commercial insulin preparations, namely, Humulin R (Elli Lily) and Lantus (Sanofi-Aventis). The 3 preparations and a vehicle control were tested by randomized cross over study in the 8 diabetic beagle dogs.

[092] The various insulin preparations, as well as the control, were administered once daily on two consecutive days. A subcutaneous bolus of vehicle control and insulin preparation of 0.4 IU per kg body weight per day was injected into the dorsal aspect of the neck. Whole blood samples for rapid glucose measurements (Glucotrend 2, Roche) were collected throughout the day and glucose determination was carried-out and recorded within 2 minutes after each sampling.

[093] The results shown in Figure 7, demonstrate that in contrast to the fast acting soluble insulin (Humulin R), both Lantus and insulin- ArgArg have a comparable long activity profile in vivo in diabetic beagle dogs.

Claims

CLAIMS What is claimed is:

1. A process for preparing an insulin, comprising the steps of:

(a) reversibly blocking lysine residues in an Arg-Arg precursor insulin comprising the amino acid sequence of SEQ ID NO: 1 , comprising contacting said Arg-Arg precursor insulin with a solution comprising a buffer and citraconic anhydride at pH 6-10; and

(b) partially cleaving said Arg-Arg precursor insulin, comprising reacting said Arg-Arg precursor insulin of step (a) with trypsin, thereby, preparing an insulin.

2. The process claim 1 , wherein said contacting said Arg-Arg precursor insulin with citraconic anhydride is at pH 9.

3. The process claim 1 , wherein said contacting said Arg-Arg precursor insulin with citraconic anhydride is at room temperature for 2 hours.

4. The process claim 1, wherein said reacting said Arg-Arg precursor insulin of step (a) with trypsin is at pH values of 6.0-11.5.

5. The process claim 1 , wherein said reacting Arg-Arg precursor insulin of step (a) with trypsin is at room temperature.

6. The process claim 5, wherein said reacting said Arg-Arg precursor insulin of step (a) with trypsin is for at least 0.5 hours.

7. The process claim 5, wherein said reacting said Arg-Arg precursor insulin of step (a) with trypsin is for 1 -4 hours.

8. The process claim 1, wherein said Arg-Arg precursor insulin is exposed to pH values of 10.0-11.5 prior to step (a).

9. The process claim 1, further comprising adjusting the pH of said insulin of step (b) to pH 9 (step (c)).

10. The process claim 9, further comprising purifying said insulin (step (d)).

11. The process claim 10, wherein said purifying is performed by microfiltering.

12. The process claim 10, wherein said purifying is performed by ion-exchange chromatography, reverse phase high pressure liquid chromatography (RP-HPLC), or both.

13. The process claim 10, further comprising crystallizing said insulin, comprising contacting said insulin of step (d) with Zn⁺⁺ in citrate-ethanol.

14. The process claim 1 , wherein said insulin is long-acting insulin.

15. The process claim 1 , wherein at least 50% of said Arg-Arg precursor insulin is converted to said insulin.

16. The process claim 1, wherein said precursor insulin is obtained from a bacterial inclusion body.

17. The process claim 1 , wherein said solution is free of amines.

18. The process claim 1 , wherein said buffer is carbonate buffer.

19. The process claim 1 , wherein said Arg-Arg precursor insulin comprising the amino acid sequence of SEQ ID NO: 1 is an Arg-Arg precursor insulin consisting the amino acid sequence of SEQ ID NO: 2.