Method for producing human analog insulin and its derivatives in mammalian cells

Abstract

The present disclosure provides a method for producing a functional recombinant protein having its native conformation in a mammalian host cell culture, wherein the recombinant protein is an insulin analog and / or derivative thereof.

Description

[Technical field]

[0001] cross reference

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 308,913, filed February 10, 2022, the entire contents of which are incorporated herein by reference.

[0002] Description of electronically submitted XML files

[0002] The contents of the electronic sequence listing (IBTS_001_01WO_SeqList_ST26.xml; size: 32,369 bytes; and creation date: February 6, 2023) are incorporated herein by reference in their entirety.

[0003] Technical Field

[0003] The present disclosure relates generally to methods for producing functional recombinant proteins in their native conformation. The present invention is particularly useful for producing proteins that lend themselves to post-translational modifications such as trimming, proper folding and glycosylation. [Background technology]

[0004] Related Field Description

[0004] Insulin is an important polypeptide hormone produced by the pancreatic β-cells of the islets of Langerhans and is necessary to maintain blood glucose homeostasis in mammals, including humans, and other vertebrates. In healthy individuals, elevated blood glucose levels stimulate the pancreatic β-cells to secrete insulin. The insulin polypeptide then binds to specific receptors in muscle, liver, and adipose tissue, resulting in increased glucose uptake by these target tissues, increased metabolism, and decreased hepatic glucose production. The cumulative effect of these reactions is responsible for maintaining blood glucose concentrations at a constant level.

[0005]

[0005] Approximately 177 million people worldwide suffer from diabetes. This includes approximately 17 million type I diabetes patients, for whom replacement of deficient endocrine insulin is currently the only available treatment, without which complications such as ketoacidosis, coma, and even death within weeks may occur. Type I diabetes patients treated with suboptimal doses of insulin develop secondary diseases such as ketoacidosis, blindness, heart attacks, and kidney failure. Despite great advances in transplantation and cell therapy, a cure for type 1 diabetes has yet to be found. Type II diabetes, in contrast to type I diabetes, has a relative rather than an absolute deficiency of insulin, but in many cases, especially in advanced stages, treatment with insulin therapy is unavoidable and is the most preferred treatment. Furthermore, estimates suggest that the number of individuals diagnosed with diabetes will double over the next 25 years to approximately 300 million (Kjeldsen, T. et al., 2001, Biotechnol. Gen. Eng. Rev. 18:89-121). However, the use of insulin therapy is associated with high costs, with the average cost of insulin in the United States increasing annually. It is therefore highly desirable to be able to cost-effectively produce human insulin in quantities that can satisfy the expected increase in the world's insulin demand.

[0006] Since the development of recombinant DNA technology, many methods have been described for the production of insulin, precursors, and analogs thereof in genetically engineered host cells. Numerous production systems for recombinant proteins are now available, ranging from bacteria, yeast, and insect cells to plant and mammalian cells. However, existing production systems and methods for recombinant insulin production suffer from various problems. Prokaryotic production systems routinely used for the recombinant production of insulin analogs, such as Escherichia coli (Frank et al., 1981, in Peptides: Proceedings of the 7th International Conference on Insulin and Its Related Substances, vol. 133, no. 103, 1997), have not been successfully used for the recombinant production of insulin analogs. thAmerican Peptide Chemistry Symposium (Rich & Gross, eds.), Pierce Chemical Co., Rockford. Ill pp 729-739; Chan et al., 1981, Proc Natl. Acad. Sci. USA 78: 5401-5404), Saccharomyces cerevisiae (Thim et al., 1986, Proc. Natl. Acad. Sci. USA 83: 6766-6770), etc., are unable to confer correct folding of the expressed polypeptide or form the disulfide bond linking the A and B chains in mature insulin. Eukaryotic systems, although offering improvements, still have some drawbacks. For example, hypermannosylation in yeast strains affects the ability of yeast to properly express glycoproteins. Moreover, hypermannosylation often even provokes immune reactions when the therapeutic proteins prepared in this way are administered to patients.Furthermore, since secretion signals in yeast are different from mammalian signals, it becomes more difficult to transport mammalian proteins, such as human polypeptides, out of the cell, which results in problems with their continuous production and / or isolation.

[0007]

[0007] Wang et al. (Biotechnol. Bioeng., 2001, 73:74-79) have shown that yeasts such as Pichia pastoris are suitable for insulin production. However, one of the main drawbacks of this system is the post-translational modification of the resulting protein, which subsequently exists as an impurity in the final product and is difficult to purify. In addition, the enzymatic steps utilized in existing production systems are time-consuming, costly, and introduce additional impurities that must be removed in further downstream processing steps, such as expensive chromatography steps.

[0008]

[0008] Mammalian cells are widely used for protein production due to their ability to carry out extensive post-translational modifications. However, recombinant production of glycoproteins or proteins containing at least two (different) subunits in mammalian cells remains a challenge. Summary of the Invention [Problem to be solved by the invention]

[0009]

[0009] Given the complex, high cost and low yields of existing production systems and methods for recombinant production and purification of biologically active insulin and / or insulin analogs, there is a need for alternative methods for recombinant expression of functional insulin and / or insulin analogs with native conformation. The embodiments disclosed herein address these needs and provide other related advantages. [Means for solving the problem]

[0010] overview

[0010] Provided herein are methods and compositions for producing functional recombinant proteins in their native conformation in a host cell culture. The disclosed methods include transforming a host cell with at least one expression vector to co-express a protein precursor (i.e., a proprotein or propeptide) and at least one agent (e.g., a protease) for processing the protein precursor into a functional protein by post-translational modification (e.g., enzymatic cleavage). Non-limiting examples of proteins that can be produced by the disclosed methods include insulin, amylin, gastrin, ghrelin, glucagon, somatostatin, α-MSH, ACTH, β-endorphin, or other peptide hormones. In some embodiments, the host cell is a mammalian cell selected from the group consisting of HEK293 cells, CHO cells, COS, and HeLa cells. In other embodiments, the host cell is selected from the group consisting of algae and yeast.

[0011]

[0011] According to the present disclosure, the recombinant protein comprises mature insulin or insulin analogues. In particular, the new and improved method of the present disclosure relates to a process for preparing at least one insulin analogue and / or insulin derivative, which provides the advantage of reducing the number of steps required to obtain at least one insulin analogue and / or insulin derivative while at the same time increasing the yield and purity of the desired product. The method disclosed herein relates to a highly efficient process for the production of recombinant human insulin analogues and / or derivatives thereof, which further provides the advantage of reducing and / or eliminating the presence of undesired cleavage by-products and eliminating multiple time-consuming and expensive purification steps.

[0012]

[0012] In some aspects, the present disclosure provides improved methods for the production of insulin analogs or derivatives thereof. In certain aspects, the present disclosure provides methods for the production of insulin analogs or derivatives thereof in mammalian host cells. Advantageously, the methods and compositions according to the present disclosure can take advantage of the more evolved ability of the mammalian cell host machinery to read, fold, and cleave expressed recombinant insulin analogs in vivo. Furthermore, the methods and compositions of the present disclosure optimize and simplify the process for producing functional insulin and / or insulin analogs by using agents exogenously introduced into the mammalian cell host that allow for in vivo processing of the recombinant insulin analogs, thereby allowing for the production of mature insulin analogs without additional steps of digestion, processing, and purification. The optimized methods and compositions of the present disclosure provide cost savings in terms of materials, equipment, and time, as well as improved yields due to reduced protein loss compared to current / existing production methods.

[0013]

[0013] Accordingly, the present disclosure provides a method for producing a functional recombinant protein in its native conformation, including an insulin analog and / or derivative thereof, in a mammalian host cell. In certain aspects, the method includes providing a competent mammalian host cell. According to the present disclosure, the method includes transforming the competent mammalian host cell with an expression vector suitable for expression in the mammalian host cell. The expression vector includes a heterologous nucleic acid sequence encoding at least one precursor peptide of human insulin analog or derivative thereof and / or modified human proinsulin peptide. In certain aspects, the method includes culturing a colony of transformed mammalian host cells in a suitable growth medium under conditions suitable for expression and secretion of at least one precursor peptide of human insulin analog or derivative thereof and / or modified human proinsulin peptide; and harvesting or recovering the secreted insulin analog and / or derivative thereof. In certain aspects, recovering the secreted peptide includes recovering the processed mature peptide from the growth medium. In any of the embodiments disclosed herein, the secreted insulin analog and / or derivative thereof is a functional insulin analog and / or derivative thereof in its native conformation, which is capable of binding to an insulin receptor peptide.

[0014]

[0014] The present disclosure provides an expression vector comprising, as operably linked components, in the 5' to 3' direction of transcription: (i) a nucleic acid sequence capable of controlling expression in a mammalian host cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of an insulin analog or derivative thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide; and (v) a nucleic acid sequence encoding one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof. The agent for processing the modified proinsulin into a functional insulin analog or derivative thereof is selected from the group consisting of prohormone convertase 1 / 3 (PC1 / 3), PC2, and carboxypeptidase E (CPE). In some embodiments, the agent for processing the modified proinsulin into a functional insulin lispro or derivative thereof comprises PC1 / 3, CPE, and optionally PC2. In another embodiment, the agent for processing modified proinsulin into functional insulin glargine or a derivative thereof comprises PC1 / 3 and optionally PC2.

[0015]

[0015] The present disclosure provides a first and a second expression vector. In some aspects, the first expression vector comprises, as operably linked components, in the 5' to 3' direction of transcription: (i) a nucleic acid sequence capable of controlling expression in a mammalian cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of an insulin analog or derivative thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; and (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide. In another aspect, the second expression vector comprises, as operably linked components, in the 5' to 3' direction of transcription: (i) a nucleic acid sequence capable of controlling expression in a mammalian cell; and (ii) a nucleic acid sequence encoding one or more agents for processing modified proinsulin into a functional insulin analog or derivative thereof. The agent for processing modified proinsulin into a functional insulin analog or derivative thereof is selected from the group consisting of PC1 / 3, PC2, and CPE. In some embodiments, the agent for processing modified proinsulin and / or proinsulin analogues into functional insulin lispro or derivatives thereof comprises PC1 / 3, CPE, and optionally PC2. In another embodiment, the agent for processing modified proinsulin into functional insulin glargine or derivatives thereof comprises PC1 / 3, and optionally PC2.

[0016]

[0016] The present disclosure provides heterologous nucleic acid sequences encoding at least one human insulin analog precursor peptide and / or modified proinsulin peptide, wherein the heterologous nucleic acid sequence is prepared by modifying the proinsulin gene by substitution of codons at appropriate sites in the native human proinsulin gene. In certain aspects, the substitution of codons encodes desired amino acid residue substitutions. Non-limiting representative examples of desired amino acid substitutions are B28Lys, B29Pro human insulin, B28Asp human insulin, A21Gly human insulin, B31Arg, B32Arg human insulin, B31Leu, B32Ala human insulin; B31Leu, B32Ala, desB30 human insulin, B31Phe, B32Leu human insulin, and B31Phe, B32Leu, desB30 human insulin.). According to the present disclosure, the heterologous nucleic acid is prepared by synthesizing the entire DNA sequence encoding at least one human insulin analog.

[0017]

[0017] In a particular aspect, the heterologous nucleic acid encoding at least one modified proinsulin peptide and / or precursor peptide of human insulin analog or derivative thereof encodes the B chain of human insulin or an analog thereof, the A chain of human insulin or an analog thereof, and a C peptide linking the B chain and the A chain.

[0018] According to the present disclosure, the heterologous nucleic acid sequence encoding the precursor peptide of at least one human insulin analog or derivative thereof further comprises a nucleic acid sequence encoding one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof. In certain embodiments, the one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof comprise PC1 / 3, PC2, CPE, and a blocking agent effective to block endogenous CPE activity. In certain embodiments, the mammalian host cell is modified to express one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof.

[0019]

[0019] The present disclosure provides a method comprising transforming a competent mammalian host cell with a first expression vector comprising a heterologous nucleic acid sequence encoding at least one precursor peptide of human insulin analog or derivative thereof and / or modified human proinsulin peptide, and a second expression vector comprising a heterologous nucleic acid sequence encoding one or more agents for processing the modified proinsulin into functional insulin. In certain aspects, the one or more agents for processing the modified proinsulin into functional insulin comprise an endoprotease, carboxypeptidase E, and a blocking agent effective to block endogenous carboxypeptidase E activity. In one aspect, the endoprotease comprises PC1 / 3 and PC2. In certain aspects, the mammalian host cell is modified to express one or more agents for processing the modified proinsulin into functional insulin analog or derivative thereof.

[0020]

[0020] In certain aspects, the heterologous nucleic acid sequence encodes a peptide having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0021]

[0021] In certain embodiments, the heterologous nucleic acid sequence encodes a peptide having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0022] According to the present disclosure, the heterologous nucleic acid further encodes one or more agents for processing the modified proinsulin into functional insulin. In some embodiments, the one or more agents have at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In certain embodiments, the one or more agents have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0023]

[0023] A heterologous nucleic acid sequence encoding at least one insulin analog or derivative thereof is inserted into a suitable expression vector, which, when transferred into a suitable host organism, expresses the desired product, which is secreted from the host cell and recovered from the culture broth.

[0024]

[0024] In certain aspects, the disclosure provides a polynucleotide encoding a peptide comprising modified human proinsulin or a human proinsulin analog or derivative thereof; and one or more agents for processing the modified proinsulin into functional insulin. In certain aspects, the heterologous nucleic acid sequence encoding the peptide comprising modified human proinsulin or a human proinsulin analog or derivative thereof has at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some aspects, the modified human proinsulin or human proinsulin analog or derivative thereof encoded by the peptide has the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In certain aspects, the polynucleotide further comprises a heterologous nucleic acid sequence encoding one or more agents for processing the modified proinsulin into functional insulin. In certain embodiments, the polynucleotide further comprises a heterologous nucleic acid sequence encoding a blocking agent effective to block endogenous carboxypeptidase E activity. The one or more agents for processing modified proinsulin into functional insulin include one or more endoproteases and carboxypeptidase E. The one or more endoproteases are selected from PC1 / 3, PC2. In some embodiments, the one or more agents have at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In certain embodiments, the one or more agents have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0025]

[0025] The present disclosure also relates to an expression vector and a modified mammalian host cell comprising a polynucleotide encoding a peptide comprising modified human proinsulin or a human proinsulin analog or derivative thereof, and at least one agent for processing the modified proinsulin into a functional insulin. In some aspects, the mammalian host cell comprises a genetic modification to promote the formation of a stable disulfide bond in the cytoplasm to process the modified proinsulin into a functional insulin. In certain aspects, the present disclosure also provides a human cell comprising an expression vector encoding a peptide comprising modified human proinsulin or a human insulin analog or derivative thereof. In certain aspects, the present disclosure also provides a human cell comprising an expression vector encoding a peptide comprising modified human proinsulin or a human proinsulin analog or derivative thereof, and at least one agent for processing the modified proinsulin into a functional insulin.

[0026] In some aspects, the disclosure provides a method for producing an insulin analog and / or derivative thereof comprising producing a mammalian host cell that expresses and secretes insulin, comprising transforming the mammalian host cell with an expression vector that co-expresses a modified proinsulin polypeptide and one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof. In another aspect, the disclosure provides a method for producing an insulin analog and / or derivative thereof comprising producing a competent mammalian host cell that makes and secretes insulin, comprising transforming the host cell with a first expression vector that expresses a proinsulin polypeptide and a second expression vector that expresses one or more agents for processing the proinsulin and / or proinsulin analog into a functional insulin analog or derivative thereof.

[0027]

[0027] In some aspects, the disclosure provides a method for producing an ultrafast-acting insulin analog and / or derivative thereof, comprising transforming a mammalian host cell with a recombinant expression vector comprising: (i) a nucleic acid sequence capable of controlling expression in a mammalian host cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of the insulin analog or derivative thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide; (v) a nucleic acid sequence encoding PC1 / 3; (vi) a nucleic acid sequence encoding CPE; and (vii) optionally, a nucleic acid sequence encoding PC2. In some aspects, the ultrafast-acting insulin analog is insulin lispro.

[0028] In some aspects, the disclosure provides a method for producing a long-acting insulin analogue and / or derivatives thereof, the method comprising transforming a mammalian host cell with a recombinant expression vector comprising: (i) a nucleic acid sequence capable of controlling expression in a mammalian host cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of the insulin analogue or derivatives thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide; (v) a nucleic acid sequence encoding PC1 / 3; and (vi) optionally, a nucleic acid sequence encoding PC2. In some aspects, the long-acting insulin analogue is insulin glargine.

[0029] In some aspects, the method for producing long-acting insulin analogs and / or derivatives thereof further comprises an optional step of blocking endogenous carboxypeptidase E (CPE) activity. In some aspects, the step of blocking endogenous carboxypeptidase E activity comprises introducing a deletion or mutation into endogenous CPE, resulting in reduced or eliminated expression or activity. In some aspects, the host cell may be a CRISPR-modified host cell in which CPE activity is reduced or eliminated. Such a host cell may be produced, for example, by reducing or eliminating the expression of CPE using CRISPR, or by allowing the expression of catalytically inactive CPE. For example, the serine at position 202 of the sequence set forth in SEQ ID NO: 9 may be mutated, for example, to proline. In some aspects, the reduction or elimination of CPE activity may be conditional or inducible (e.g., in the form of a Cre-dependent construct). In some aspects, blocking endogenous carboxypeptidase E activity comprises co-expressing in the mammalian host cell a mutant CPE having at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9. In some aspects, the nucleic acid sequence encoding the mutant CPE is included in an expression vector that co-expresses the modified proinsulin polypeptide. In another aspect, the nucleic acid sequence encoding the mutant CPE is included in a separate expression vector. In some aspects, the disclosure provides a host cell expressing a catalytically inactive CPE, wherein the CPE binds to a substrate but does not cleave a peptide bond, wherein the catalytically inactive CPE has at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9.In some embodiments, the step of blocking endogenous CPE activity comprises adding a blocking agent selected from the group consisting of dopamine quinine, dopamine, norepinephrine, epinephrine, potato carboxypeptidase inhibitor (PCI), a 9-mer peptide called CPI-2KR, and a peptide encoding a decoy arginine sequence. The decoy arginine peptide of the present disclosure comprises a polymer having an amino acid sequence that is 3 amino acids long and has at least 70% amino acid sequence identity to the C-terminal extension of the B chain of a long-acting insulin, e.g., the sequence set forth in SEQ ID NO: 15. The decoy arginine peptide is configured to mimic the C-terminal extension of the B chain of a long-acting insulin and bind to endogenous CPE, thereby preventing endogenous CPE from removing the C-terminal extension of the B chain.

[0030] In some aspects, the disclosure relates to a method for obtaining a purified biologically active heterologous recombinant protein expressed in a mammalian expression system. In certain aspects, the method comprises culturing a host cell transformed with at least one vector comprising a heterologous nucleic acid sequence encoding a peptide having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, under a growth medium and conditions suitable for expression of the recombinant protein. In certain aspects, the method comprises culturing a host cell transformed with at least one vector comprising a nucleotide sequence encoding a peptide having an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. In certain aspects, the vector comprising the heterologous nucleic acid encoding the peptide further encodes one or more agents for processing the heterologous protein into a mature functional protein. In some embodiments, the one or more agents have at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8. In certain embodiments, the one or more agents have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.

[0031]

[0031] In certain aspects, the method includes recovering or harvesting the expressed recombinant protein, where recovering the recombinant protein includes separating the protein from the culture medium to produce a recovered recombinant protein preparation. In some aspects, the method further includes purifying the recovered recombinant protein preparation by contacting the recovered recombinant protein preparation with a chromatography matrix to remove at least one associated impurity. The purification is performed by applying a polar organic buffer solvent into an aqueous phase containing an organic acid buffer and precipitating the eluted protein. In any of the aspects disclosed herein, the mature functional recombinant protein is a human insulin analog and / or derivative thereof. In any of the aspects disclosed herein, the recombinant human insulin comprises an A chain having an amino acid sequence set forth in SEQ ID NO: 10 and a B chain having an amino acid sequence set forth in SEQ ID NO: 11. In another aspect, the recombinant human insulin comprises an A chain having an amino acid sequence set forth in SEQ ID NO: 12 and a B chain having an amino acid sequence set forth in SEQ ID NO: 13. In yet another embodiment, the recombinant human insulin comprises an A chain having the amino acid sequence set forth in SEQ ID NO: 14, and a B chain having the amino acid sequence set forth in SEQ ID NO: 14. In a particular embodiment, the recombinant human insulin comprises an A chain having an amino acid sequence at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 10, and a B chain having an amino acid sequence at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO: 11.In certain embodiments, the recombinant human insulin comprises an A chain having an amino acid sequence at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:12, and a B chain having an amino acid sequence at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:13. In certain embodiments, the recombinant human insulin comprises an A chain having an amino acid sequence at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:14, and a B chain having an amino acid sequence at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence set forth in SEQ ID NO:15.

[0032] According to the present disclosure, the yield of purified insulin analogues and / or derivatives thereof is 75%-100%. According to another aspect of the present disclosure, the yield of purified insulin analogues and / or derivatives thereof is 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.

[0033]

[0033] The present disclosure provides an integrated and continuous process for producing recombinant human insulin analog and / or derivatives thereof, comprising: (i) culturing recombinant human insulin analog-secreting mammalian cells in a perfusion bioreactor containing a liquid culture medium under conditions that permit the cells to secrete the recombinant insulin analog and / or derivatives thereof into the culture medium, wherein a substantially cell-free volume of culture medium is continuously or periodically removed from the perfusion bioreactor and fed to a first cyclic countercurrent chromatography system (PCCS1); and (ii) capturing the recombinant insulin analog and / or derivatives thereof from the culture medium using PCCS1. (iii) purifying the recombinant insulin analogue and / or derivatives thereof using PCCS2, wherein the purification is carried out using a resin of PCCS2 that has a different chemical structure compared to the resin of PCCS2 used to carry out the polishing, wherein the eluate from PCCS2 is insulin analogue and / or derivatives thereof; the above process is integrated and is carried out continuously from the culture step to the eluate from PCCS2 that is insulin analogue and / or derivatives thereof. In certain embodiments, the recombinant human insulin analog-secreting mammalian cell is transformed with at least one vector comprising a heterologous nucleic acid sequence encoding a peptide having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4. In certain embodiments, the recombinant human insulin analog-secreting mammalian cell is transformed with at least one vector comprising a heterologous nucleic acid sequence encoding a peptide comprising the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.

[0034]

[0034] Human insulin analogs and derivatives thereof according to the present disclosure are useful in the treatment of conditions that are sensitive to insulin. Thus, human insulin analogs and derivatives thereof may be used in the treatment of type 1 diabetes, type 2 diabetes, and hyperglycemia, such as that sometimes found in, for example, those with severe injuries and those who have undergone major surgery.

[0035]

[0035] In another aspect, the present disclosure relates to a pharmaceutical formulation comprising a human insulin analog in combination with suitable pharma- ceutically acceptable adjuvants and additives, such as one or more agents suitable for stabilization, preservation, or isotonicity.

[0036]

[0036] Thus, in yet another aspect, the present disclosure provides a method for treating diabetes in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of a human insulin analog obtained by the method disclosed herein.

[0037]

[0037] Other features and advantages of the present disclosure will be readily apparent from the following detailed description. However, since various changes and modifications within the spirit and scope of the present disclosure will be readily apparent to those skilled in the art from this detailed description, it should be understood that the detailed description and specific examples are provided by way of example only, while showing preferred embodiments.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0038] The embodiments disclosed herein will become more fully apparent from the following description and appended claims, taken in conjunction with the accompanying drawings. [Brief description of the drawings]

[0039] [Figure 1]

[0039] We outline the history of insulin production and also provide a schematic of the synthesis strategy for insulin analogs according to the methods of the present disclosure. Procedural details are described in Examples 1-3. [Figure 2A]

[0040] 1 shows processing sites for the production of mature insulin analogues in mammalian cells. [Figure 2B]

[0040] Processing sites for the production of mature insulin analogues in mammalian cells are shown. [Diagram 3]

[0041] We explain insulin synthesis in pancreatic β cells. [Figure 4]

[0042] Small scale production (50 ml) of lispro and glargine harvested from 20x concentrated supernatant of HEK cells transformed with vectors containing heterologous nucleic acid sequences encoding the respective insulin analogues and an agent (Furin) required for processing the recombinant protein into the mature functional insulin analogue is shown. [Diagram 5]

[0043] In the case of the production of the lispro insulin analog, the agents utilized by the human body (PC1 / 3, PC2, carboxypeptidase E) have been shown to be superior to furin in cleaving the α, β, and c chains of recombinant insulin to produce mature insulin. [Figure 6]

[0044] In the case of the production of glargine insulin analogues, the agents utilized by the human body (PC1 / 3, PC2, and mutant carboxypeptidase E) have been shown to be superior to furin in cleaving the α, β, and c chains of recombinant insulin to produce mature insulin. [Figure 7A]

[0045] 1 shows mass spectrometry analysis of insulin analog lispro (Scn-lispro) produced using PC1 and CPE (non-reduced). [Figure 7B]

[0045] Mass spectrometry analysis of insulin analog lispro (Scn-lispro) produced using PC1 and CPE (reduced). [Figure 7C]

[0045] Mass spectrometry analysis of insulin analog lispro (Scn-lispro) produced using PC1 (non-reduced). [Figure 7D]

[0045] Mass spectrometry analysis of insulin analog lispro (Scn-lispro) produced using PC1 (reduced). [Figure 8A]

[0046] 1 shows mass spectrometry of the insulin analogue glargine produced by the method of the disclosure using PC1 and mutant CPE (non-reduced). [Figure 8B]

[0046] Mass spectrometry analysis of insulin analogue glargine produced by the method of the present disclosure using PC1 and mutant CPE (reduced). [Figure 8C]

[0046] Mass spectrometry of the insulin analogue glargine produced by the method of the present disclosure using PC1 (non-reduced). [Figure 8D]

[0046] Mass spectrometry of insulin analogue glargine produced by the method of the present disclosure using PC1 (reduced). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0040] Detailed Description

[0047] It will be readily understood that the embodiments generally described herein are exemplary. The following description of various embodiments is not intended to limit the scope of the present disclosure, but is merely representative of various embodiments. Furthermore, those skilled in the art may change the order of steps or operations of specific methods disclosed herein without departing from the scope of the present disclosure. In other words, the order or use of specific steps or operations may be changed, unless a specific order of steps or operations is required for proper operation of the embodiment.

[0041]

[0048] Before describing the disclosure in more detail, it is believed to be helpful to an understanding of the disclosure to provide definitions of certain terms used herein. Additional definitions are set forth throughout the disclosure.

[0042]

[0049] Unless otherwise defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where possible, all patents, applications, published applications, and other publications, including nucleic acid and polypeptide sequences from GenBank, SwissPro, and other databases, referred to in this disclosure are incorporated by reference in their entirety.

[0043]

[0050] In this disclosure, unless otherwise indicated, any concentration range, percentage range, ratio range, or integer range is understood to include any integer value within the stated range, and fractions thereof, where appropriate (such as tenths and hundredths of integers). Additionally, numerical ranges recited herein for physical characteristics such as polymer subunits, size, or thickness should be understood to include any integer value within the stated range, unless otherwise indicated.

[0044]

[0051] The term "about" as used herein means 10% greater or less than the stated value or range of values, but is not intended to specify a value or range of values ​​only in this broad definition. Each value or range of values ​​preceded by the term "about" is also intended to encompass the embodiment of the absolute value or range of values ​​stated.

[0045]

[0052] As used herein, the terms "a" and "an" should be understood to mean "one or more" of the listed components. The use of the alternative terms (e.g., "or") should be understood to mean either one, both, or any combination. As used herein, the terms "include," "have," and "comprise" are used interchangeably and these terms and variations thereof are intended to be construed as open ended.

[0046]

[0053] "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not be present, and that the description includes instances when the element, component, event, or circumstance is present and instances when it is not present.

[0047]

[0054] As used herein, the terms "prodrug" and / or "proinsulin" and / or "precursor peptide" are defined as any compound that undergoes chemical modification prior to exerting its pharmacological effect(s).

[0048]

[0055] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those that are encoded by the genetic code, as well as those that are subsequently modified, such as, for example, hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an α-carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to compounds that have a structure that is different from the general chemical structure of amino acids, but that function in a manner similar to naturally occurring amino acids.

[0049]

[0056] Additionally, "amino acid" encompasses molecules that contain both amino and carboxyl functional groups, where the amino and carboxylate groups are attached to the same carbon (the alpha carbon). The alpha carbon may optionally bear one or two further organic substituents. For purposes of this disclosure, when an amino acid is designated without specifying the stereochemistry, it is intended to encompass either the L- or D-form of the amino acid, or a racemic mixture. However, when an amino acid is designated by its three-letter code and includes a superscript number, the D-form of the amino acid is designated by including a lower case d before the three-letter code and superscript number (e.g., dLys). -1 ), where the name without the lowercase d (e.g., Lys -1 ) is intended to designate the native L-form of the amino acid. In this nomenclature, the inclusion of a superscript number indicates the position of the amino acid in the insulin analog sequence, where amino acids located within the insulin analog sequence are designated by positive superscript numbers numbered consecutively from the N-terminus. Additional amino acids linked to the insulin analog peptide, either at the N-terminus or via side chains, are designated by numbers starting at 0 and increasing in negative integer value away from the insulin analog sequence. For example, the position of an amino acid within a dipeptide prodrug linked to the N-terminus of an insulin analog is designated by aa -1 -aa 0 - named insulin analogues, where aa 0 represents the carboxyl terminal amino acid of the dipeptide, and aa -1 represents the amino terminal amino acid of the dipeptide.

[0050]

[0057] As used herein, "protein," "polypeptide," or "peptide" refers to a polymer of amino acid residues. Protein applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to non-naturally occurring amino acid polymers.

[0051]

[0058] As used herein, an "effective" or "therapeutically effective" amount of an insulin analog refers to a non-toxic but sufficient amount of insulin analog to provide a desired effect. For example, one desired effect may be the prevention or treatment of hyperglycemia. An "effective" amount may vary from subject to subject, depending on the individual's age and general condition, method of administration, etc. Thus, it is not always possible to specify an exact "effective amount." However, the appropriate "effective" amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation.

[0052]

[0059] The term "nucleic acid sequence" as used herein refers to a sequence of nucleoside or nucleotide monomers composed of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences that include non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present disclosure may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine.

[0053]

[0060] As used herein, unless otherwise indicated, a sequence identity of a stated percentage preceded by "at least about" includes the stated percentage plus or minus 20% thereof, as well as all integer and non-integer percentages greater than the specified percentage. Thus, "at least about 85%" identity to a reference sequence (e.g., any one of SEQ ID NOs: 1-4) includes about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference sequence, as well as any non-integer percentage between two integer percentages (e.g., 92.5%, 99.1%, etc.).

[0054]

[0061] The terms "nucleic acid sequence encoding insulin" and "nucleic acid sequence encoding an insulin analog" or "nucleic acid sequence encoding an insulin polypeptide or modified insulin peptide" or "proinsulin" may be used interchangeably herein and refer to any nucleic acid sequence encoding an insulin polypeptide, including but not limited to the proinsulin polypeptides listed in Table 2 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4), as well as any mammalian insulin polypeptide, and any nucleic acid sequence encoding proinsulin, preproinsulin peptides, and their analogs and / or derivatives. The term "agents for processing" refers to agents necessary for processing proinsulin into mature functional insulin, including but not limited to any nucleic acid sequence encoding the polypeptides listed in Table 3 (SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8).

[0055]

[0062] In vivo, human insulin polypeptide is produced as a single polypeptide chain precursor of 110 amino acids, i.e. preproinsulin, containing a 24 amino acid presequence located at the N-terminus, which is cleaved immediately after completion of the biosynthesis of the chain to give proinsulin (Steiner, DF 2000. J. Ped. Endocrinol. Metab. 13:229-239). Proinsulin has the structure BCA, with the C-peptide chain connecting the C-terminal amino acid of the B chain and the N-terminal amino acid residue of the A chain. During hormone packaging for secretion, the C-peptide is cleaved and removed by the prohormone convertases PC2 and PC1 / PC3 (Steiner, DF 2000. J. Ped. Endocrinol. Metab. 13:229-239) to obtain mature or active human insulin, a 51 amino acid protein consisting of two polypeptide chains, A (21 amino acids long) and B (30 amino acids long), linked by two interchain disulfide bonds. The A and B chains are linked by two disulfide bridges between the A7 and B7, and A20 and B19 cis residues, respectively. In addition, biologically active insulin molecules have an intramolecular (intrachain) disulfide bridge between Cys residues at positions A6 and A11.

[0056]

[0063] Nucleic acid sequences encoding insulin polypeptides or peptides further include any nucleic acid sequence that (i) encodes a polypeptide substantially identical to an insulin polypeptide sequence described herein; or (ii) hybridizes under at least moderately stringent hybridization conditions to any of the nucleic acid sequences described herein, or that, except for synonymous codon usage, can hybridize under at least moderately stringent conditions to it.

[0057]

[0064] The term "proinsulin" as used herein is a protein of the formula: A is the A chain of insulin or a functional derivative thereof; B is the B chain of insulin or a functional derivative thereof having an .ε.-amino group; and C is the connecting peptide of proinsulin. Preferably, proinsulin is the A chain of human insulin, the B chain of human insulin, and C is the native connecting peptide. When proinsulin is in the native sequence, it has three free amino groups: phenylalanine (1) (α.-amino group), lysine (29) (.ε.-amino group), and lysine (64) (ε.-amino group). As used herein, "proinsulin" refers to the insulin polypeptide and includes the connecting peptide or "C peptide" that connects the B and A insulin polypeptide chains. In native human insulin, the C peptide is a 31 amino acid residue polypeptide chain that connects residues B30 and A1. The term "preproinsulin" refers to a proinsulin molecule that additionally contains an N-terminal signal sequence that directs translation to occur on the ER ribosome.

[0058]

[0065] The term "insulin analog" as used herein refers to a protein exhibiting insulin activity of the formula AB: A is insulin chain A (α) or a functional derivative of insulin chain A; B is insulin chain B (β) or a functional derivative of insulin chain B having an ε-amino group, where at least one of A or B contains an amino acid modification of the native sequence. Table 1 shows the sequences of the A and B chains of exemplary insulin analogs.

[0059]

[0066] In this disclosure, whenever the term insulin is used in the plural or in a generic sense, it is intended to encompass both naturally occurring insulin, as well as insulin analogues and derivatives thereof.

[0060]

[0067] As used herein, "insulin polypeptide" refers to a compound having a molecular structure similar to that of human insulin, including disulfide bridges between Cys.sup.A7 and Cys.sup.B7, and between Cys.sup.A20 and Cys.sup.B19, and an intramolecular disulfide bridge between Cys.sup.A6 and Cys.sup.A11, and having insulin activity.

[0061]

[0068] Insulin peptides include, but are not limited to, insulin, human; insulin, porcine; IGF-1, human; insulin-like growth factor II (69-84); proinsulin-like growth factor II (68-102), human; insulin-like growth factor II (105-128), human; [AspB28] insulin, human; [LysB28] insulin, human; [LeuB28] insulin, human; [Va1B28] insulin, human; [AlaB28] insulin, human; [AspB28, ProB29] insulin, human; [LysB28, ProB29] insulin, human; [LeuB28, ProB29] insulin, human; [Va1B28, ProB29] insulin, human; [AlaB28, ProB29] insulin, human; [GlyA21] insulin, human; GlnB3] insulin, human; [AlaA21] insulin, human; [AlaA21 Gln.sup.B3] insulin, human; [GlnB3] insulin, human; [GlnB30] insulin, human; [GIyA21 GIuB30] insulin, human; [GlyA21 GlnB3 GluB30] insulin, human; [G1nB3 GIuB30] insulin, human; B22B30 insulin, human; B23B30 insulin, human; B25B30 insulin, human; B26B30 insulin, human; B27B30 insulin, human; B29B30 insulin, human; human insulin A chain, and human insulin B chain.

[0062]

[0069] As used herein, the terms "precursor peptide of human insulin analog" and "modified proinsulin polypeptide", which may be used interchangeably herein, refer to any precursor peptide of insulin polypeptide, including the insulin polypeptides and / or peptides listed in Table 1, as well as polypeptide molecules that comprise a sequence of amino acid residues that are: (i) substantially identical to the amino acid sequence constituting any insulin polypeptide and / or insulin peptide described herein, or (ii) capable of hybridizing under at least moderately stringent conditions with any nucleic acid sequence encoding insulin described herein, or capable of hybridizing under at least moderately stringent conditions with any nucleic acid sequence encoding insulin described herein, except for synonymous codon usage. The terms insulin and insulin polypeptide include proinsulin polypeptides and mini-insulin polypeptides, analogs and derivatives thereof. Insulin analog polypeptides are preferably of human origin.

[0063]

[0070] The present disclosure contemplates certain derivatives or further substitutions of insulin analogues. Thus, it is possible to derivatize one or more of the functional groups of the amino acid residues. Examples of such derivatives are the conversion of acidic groups in the insulin molecule to ester or amide groups, the conversion of alcohol groups to alkoxy groups or vice versa, and selective deamidation, as known per se. As an example, A21Asn can be deamidated to A21Asp by hydrolysis in an acidic medium, or B3Asn can be deamidated to B3Asp in a neutral medium.

[0064]

[0071] Furthermore, it is also possible to modify the insulin analogues of the present invention by adding or removing amino acid residues at the N-terminus or C-terminus. The insulin analogues of the present disclosure may have up to four amino acid residues deleted at the N-terminus of the B chain and up to five amino acid residues deleted at the C-terminus of the B chain without significantly affecting the overall properties of the insulin analogue. An example of such a modified insulin analogue is an insulin analogue with a deleted B1Phe or B30Thr amino acid residue.

[0065]

[0072] Naturally occurring amino acid residues can also be added to one or more termini of the polypeptide chain, provided that they do not significantly affect the overall properties and effects of insulin.

[0066]

[0073] Such deletions or additions at the termini of the polypeptide chain of an insulin analogue of the invention can be carried out in vitro on an insulin analogue having an amino acid substitution according to the present disclosure, or alternatively, the gene of an insulin analogue according to the present disclosure can be modified by adding or removing, respectively, codons corresponding to the additional or deleted amino acid residues at the termini of the polypeptide chain.

[0067]

[0074] As used herein, the term "modified polynucleotide" refers to a polynucleotide sequence that has been altered to contain at least one mutation to encode a "modified" protein. In some instances, the term "polynucleotide" is used without "modified", but this does not exclude modified polynucleotide embodiments.

[0068]

[0075] As used herein, the terms "protease" and "proteolytic activity" refer to a protein or peptide that exhibits the ability to hydrolyze peptides or substrates having peptide bonds.

[0069]

[0076] The term "oligonucleotide" is used for a nucleic acid molecule, DNA (or RNA), less than 100 nucleotides in length.

[0070]

[0077] "Transformation" means introducing DNA into an organism, i.e., a host organism, so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integration.

[0071]

[0078] Depending on the host cell used, transformation is carried out using standard techniques appropriate to such cells. General aspects of mammalian cell host system transformation are described by Axel in U.S. Patent No. 4,399,216, issued August 16, 1983.

[0072]

[0079] "Cells" or "cell cultures" or "recombinant host cells" or "host cells" are often used interchangeably, as will be clear from the context. These terms include the immediate subject cell that expresses the desired protein of the invention, and, of course, its progeny. It is understood that not all progeny will be completely identical to the parent cell, due to chance mutations or differences in environment. However, such modified progeny are included in these terms, so long as the progeny retain the characteristics associated with those imparted to the originally transformed cell.

[0073]

[0080] The term "expression" and the verb "express" refer to the transcription of a DNA sequence and / or the translation of the transcribed mRNA in a host organism (thereby producing a preprotein) and therefore do not include post-translational processes.

[0074]

[0081] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. The term "expression vector" includes a plasmid, cosmid, or phage capable of synthesizing a protein of interest encoded by each recombinant gene carried by the vector. Preferred vectors are those capable of autonomous replication and / or expression of nucleic acids to which they are linked. In the present specification, "plasmid" and "vector" are used interchangeably, as the plasmid is the most commonly used form of vector. Furthermore, the present invention is intended to include other forms of expression vectors which serve equivalent functions and which subsequently become known in the art.

[0075]

[0082] A "promoter" is a regulatory nucleotide sequence that stimulates transcription. These terms are understood by those skilled in the art of genetic engineering. Like a promoter, a "promoter element" also stimulates transcription, but constitutes a subfragment of a larger promoter sequence.

[0076]

[0083] The term "operably linked" refers to the association of two or more nucleic acid segments on a vector so that the function of one is affected by the function of the other. For example, a promoter is operably linked to a coding sequence, i.e., a nucleotide sequence encoding a protein or preprotein, when it is capable of affecting the expression of the coding sequence, i.e., when the coding sequence is under the transcriptional control of the promoter.

[0077]

[0084] The term "post-translational processing" or "post-translational modification" refers to a modification step to which a preprotein or a preproprotein is subjected to in order to obtain a mature protein within an intracellular or extracellular compartment.

[0078]

[0085] A "signal peptide" refers to a cleavable signal sequence of amino acids present in the preprotein or preproprotein form of a secretable protein. Proteins that are transported through a cell membrane, i.e., "secreted," generally have an N-terminal sequence, typically about 15-30 amino acids long, that is rich in hydrophobic amino acids. In some cases, during passage through the membrane, the signal sequence is cleaved by a signal peptidase (Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. (eds), Molecular Biology of the Cell, fourth edition, 2002, Garland Science Publishing). Many sources of signal peptides are known to those of skill in the art, including, for example, the amino acid sequence of the α-factor signal peptide from Saccharomyces cerevisiae. In general, the preprotein N-terminus of essentially any secreted protein is a potential source of a signal peptide suitable for use in the present disclosure. The signal peptide may also be bipartite, comprising two signal peptides that direct the preprotein to a first and a second cellular compartment, which are cleaved stepwise during the secretory pathway.

[0079]

[0086] Preproteins with an N-terminal signal peptide are directed to enter the "secretory pathway", which includes post-translational processing steps that ultimately result in the secretion of the protein. Glycosylation and disulfide bond formation are steps that are part of the secretory pathway prior to secretion.

[0080]

[0087] The term "substantially identical" means that two polypeptide sequences are preferably at least 75% identical, more preferably at least 85% identical, and most preferably at least 95% identical, e.g., 96%, 97%, 98% or 99% identical. To determine the percentage of identity between two polypeptide sequences, the amino acid sequences of the two sequences of interest are preferably aligned using the Clustal W algorithm (Thompson, JD, Higgins DG, Gibson TJ, 1994, Nucleic Adds Res. 22 (22): 4673-4680) in conjunction with the BLOSUM 62 scoring matrix (Henikoff S. and Henikoff JG, 1992, Proc. Natl. Acad. Sci. USA 89: 10915-10919) with a gap opening penalty of 10 and a gap extension penalty of 0.1 to obtain the highest level of correspondence between the two sequences, where at least 50% of the entire length of one of the sequences is involved in the alignment. Another method that can be used to align sequences is the Clustal Algorithm of Smith and Waterman (Adv. Appl. Math., 2003), to obtain the highest degree of correspondence between the two sequences and to determine the number of identical amino acids between the two sequences. The alignment method of Needleman and Wunsch (J. Mol. Biol., 1970, 48: 443), revised by Roberts, J. Mol. Biol., 1981, 2: 482). Other methods for calculating the percentage of identity between two amino acid sequences are generally known and include, for example, those described by Carillo and Lipton (SIAM J. Applied Math., 1988, 48: 1073) and those described in Computational Molecular Biology, Lesk, ed Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, a computer program will be used for such calculations.Computer programs that can be used in this regard include, but are not limited to, GCG (Devereux et al., Nucleic Adds Res., 1984, 12:387), BLASTP, BLASTN and FASTA (Altschul et al., J. Molec. Biol., 1990:215:403).

[0081]

[0088] By "at least moderately stringent hybridization conditions" is meant that conditions are selected that promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization can occur to all or part of the nucleic acid sequence molecule. The hybridization portion is typically at least 15 (e.g., 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrid, is determined by its Tm, which is a function of sodium ion concentration and temperature in a sodium-containing buffer (Tm=81.5°C-16.6(Log10[Na+])+0.41(%(G+C)-600 / 1), or a similar formula). Thus, the parameters of the washing conditions that determine hybrid stability are sodium ion concentration and temperature. For further guidance regarding hybridization conditions, see Current Protocols in Molecular Biology, John Wiley & Sons, NY, 1989, 6.3.1.-6.3.6 and in: Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.

[0082] [Table 1]

[0083]

[0089] The term "chimeric" as used herein with respect to nucleic acid sequences refers to at least two linked nucleic acid sequences that are not linked in nature. Chimeric nucleic acid sequences include linked nucleic acid sequences of different natural origin. Chimeric nucleic acid sequences may also include nucleic acid sequences of the same natural origin, provided that they are not linked in nature. For example, a nucleic acid sequence that constitutes a promoter obtained from a particular cell type may be linked to a nucleic acid sequence that codes for a polypeptide obtained from that same cell type, but is usually not linked to the nucleic acid sequence that constitutes the promoter. Chimeric nucleic acid sequences also include nucleic acid sequences that include any natural nucleic acid sequence linked to any non-natural nucleic acid sequence.

[0084]

[0090] Disclosed herein is a novel method for the recombinant production of insulin and / or insulin analogs or derivatives thereof in mammalian cells. The method described herein describes a specific combination of conditions and reagents that results in increased recombinant insulin and / or insulin analog expression as soluble protein in host cells and does not require extraction from inclusion bodies or in vitro refolding or disulfide shuffling steps. One advantage provided by the method disclosed herein is that shorter and simplified protein extraction and purification schemes can be used due to the increased production of properly folded and processed insulin and / or insulin analog proteins in the soluble fraction of the host cells.

[0085]

[0091] FIG. 1 depicts the history of insulin production and also outlines a four-step synthesis strategy for insulin analogs according to the disclosed method. Recombinant human insulin first entered human clinical trials in 1980. At that time, the A and B chains of the insulin molecule were produced separately in Escherichia coli and then joined by chemical techniques (Frank et al., 1981). Since 1986, another recombinant method has been used. A human gene encoding proinsulin containing the A and B chains is inserted into Saccharomyces cerevisiae (Thim et al., 1986) or Pichia pastoris (Wang et al., 2001). The linking peptide is enzymatically cleaved from the proinsulin to form human insulin. However, all of these procedures suffer from lower than desired yields and higher costs. The present disclosure relates to improved procedures for the production of insulin in mammalian cells. According to the present disclosure, A-chain, B-chain and C-peptide are co-expressed in a vector in a mammalian cell to allow proper folding and secretion of insulin from the cell. The A-chain and B-chain make up the insulin molecule, and the C-peptide is the connecting peptide that joins the two chains. Including the C-peptide of proinsulin in a vector expression system can ensure efficient and rapid reading and translation of the amino acid sequence, proper folding and stability of the insulin molecule, reduced protein loss, and improved insulin production. When proinsulin is cleaved by a protease, the A-chain and B-chain are separated from the C-peptide, allowing insulin to be formed and released. Adding a protease such as PC1 / 3, PC2 and / or CPE to the cell by co-expression in the same or a separate vector ensures proper cleavage and / or trimming to produce fully functional insulin. In some embodiments, a vector encoding proinsulin including A-chain and B-chain and C-peptide, and one or more agents that modify proinsulin into functional insulin, is introduced into a mammalian cell line.In another embodiment, two vectors are co-introduced into a mammalian cell line, one encoding proinsulin containing the A and B chains and the C peptide, and the other encoding one or more agents that modify proinsulin into functional insulin, and fully functional insulin is secreted from the cells and harvested without lysing the cells.

[0086]

[0092] The method of the present application has important advantages over the prior art. The insulin producing cells of the present disclosure are capable of continuously secreting functional insulin. Continuous secretion of fully functional insulin allows for immediate capture and purification, enabling a closed loop system that fully automates the process. According to one exemplary non-limiting embodiment, an integrated unit is provided that includes a perfusion bioreactor and a multi-column chromatograph with continuous flow of materials throughout to maintain the insulin producing cells and capture the secreted insulin. The bioreactor is perfused at a set rate and its effluent is fed directly to the purification train, thus achieving periodic or continuous insulin collection over a similar or extended time frame to meet product demand. Details of the continuous manufacturing process are described in Chiang, et al, Biotechnology and Bioengineering. 2019 and Lute et al, Biotechnol Progress. 2020, the contents of each of which are expressly incorporated herein by reference for all purposes. The insulin production procedure of the present disclosure is a continuous process, as opposed to the intermittent production process of the current technology. Continuous manufacturing allows for shorter processing times, less manual intervention, simple and rapid adjustment of production volume to demand, and reduced facility size through the use of smaller or fewer tanks, bioreactors, and columns. For example, the size of the facility can be reduced to the size of a living room as opposed to a huge factory hall by using one tank instead of multiple tanks. With the technology of the present invention, the production period of insulin is significantly reduced from several months to 1-4 weeks.

[0087]

[0093] In certain aspects, the disclosure provides recombinant nucleic acids encoding insulin analogs, expression vectors comprising the insulin analogs, host cells comprising the variant nucleic acids and / or expression vectors, and methods for producing the variant proteins.

[0088]

[0094] Thus, the present disclosure relates to a process or method for producing insulin and / or insulin analogues in mammalian host cells, which avoids expensive and time-consuming downstream purification and processing steps to provide higher yields of insulin and / or insulin analogues, thereby reducing costs and making insulin, insulin analogues and / or derivatives thereof more affordable.

[0089]

[0095] Biosynthesis of insulin through its precursors preproinsulin and proinsulin is one of the key processes that ensures the production of sufficient insulin in pancreatic β-cells. See Steiner D., Chan, S. & Rubenstein, A. (2000) in Handbook of Physiology, The Endocrine System, eds.Jefferson, L. & Cherrington, A. (Oxford Univ. Press, New York), Vol. II, pp. 49-77, Goodge KA & Hutton, JC (2000) Semin.Cell.Dev.Biol.11, 235-242. Efficient conversion of proinsulin to insulin requires cleavage of the linking segment that connects the B and A chains at both junctions, releasing insulin and C-peptide. These products are normally stored in mature secretory granules (>95% of all insulin-related material) awaiting secretion in response to glucose and other stimuli. As shown in Figure 2A, the first processing cleavage occurs at the junction of the B and A chains, between residues 32 and 33 (R↓E) and residues 65 and 66 (R↓G), respectively. Recognition of each of these sites by the neuroendocrine convertases PC1 / 3 (SPC3) and PC2 (SPC2) (Seidah NG & Chretien, M. (1999) Brain Res.848, 45-62, Zhou A., Webb, G., Zhu, X. & Steiner, DF (1999) J. Biol.Chem.274, 20745-20748) requires interactions with 4-6 residues upstream and at least 2 residues downstream, i.e., residues 27-34 and 60-67, which bracket these two sites in human proinsulin. 31 -R 32 Insulin extended at the C-terminus of the B chain by 64 -R 65The initial cleavage product, consisting of a C-peptide extended at the C-terminus by and then trimmed by removal of these basic residues by carboxypeptidase E (CPE) (L. Fricker L. (1988) Annu. Rev. Physiol. 50, 309-321. pmid:2897826), yields the mature β-cell secretory product. These convertases and CPE act primarily within the mature dense-core granules of the regulated secretory pathway.

[0090]

[0096] FIG. 2B illustrates the structures of some insulin analogues. Insulin lispro and insulin aspart are ultrafast-acting analogues with reduced self-association as a result of protein engineering. In insulin lispro, the lysine-proline (Lys-Pro) sequence at the insulin B-chain end is inverted, resulting in steric hindrance and reduced ability to self-associate. Insulin aspart incorporates an amino acid change (Pro B28 to aspartic acid Asp), which also creates charge repulsion and steric hindrance due to local conformational changes at the carboxyl end of the B-chain. Insulin glargine is a long-acting insulin that contains two additional arginine molecules (Arg B31 and Arg B32) at the end of the B-chain to modify the isoelectric point. Glycine substitution of A21 (A-chain) was performed to stabilize the molecule. Insulin detemir is another long-acting insulin that contains acylation of the ε-amino group of Lys B29. Acylation promotes the reversible binding of insulin to albumin, thereby slowing its absorption from subcutaneous tissue and transport across the capillary endothelium of skeletal muscle.

[0091]

[0097] In previous insulin production methods, A and B chains were produced by transfecting vectors encoding them separately into a suitable host cell. The two chains are then purified, mixed and linked by a disulfide bond via a reduction-oxidation reaction before further purification. The purification step is achieved by chromatography or separation, a technique that exploits the differences in the charge, size and affinity of the molecules for water. Procedures used include ion exchange columns, reversed-phase high performance liquid chromatography and gel filtration chromatography columns. Alternatively, biosynthetic human insulin and its analogs are produced by transformation of single-chain fusion proteins. Methods for producing these drugs are generally described in many patents and scientific publications and are based on the overexpression of genes encoding preproinsulin, a hybrid polypeptide consisting of a leader protein and proinsulin, i.e., insulin B chain (or a derivative thereof), a linker peptide and insulin A chain (or a derivative thereof). Once the fusion protein is isolated, the next production step is the removal of the leader protein and the linker peptide, followed by isolation of the pure hormone.

[0092]

[0098] In certain aspects, the disclosure provides methods for producing recombinant functional human insulin analog or derivatives thereof by culturing a host cell comprising a heterologous nucleic acid sequence encoding a precursor peptide of human insulin analog and / or derivative thereof or a modified proinsulin peptide. In some aspects, the nucleic acid sequence encoding the precursor peptide of human insulin analog or derivative thereof further encodes one or more agents for processing the modified proinsulin or derivative thereof into a functional insulin analog or derivative thereof. In some aspects, the precursor of human insulin analog or derivative thereof comprises the B chain of human insulin or analog thereof, the A chain of human insulin or analog thereof, and a C peptide linking the B chain and the A chain.

[0093]

[0099] A preferred heterologous nucleic acid encodes a proinsulin polypeptide. In certain embodiments, the heterologous nucleic acid sequence encodes a modified proinsulin polypeptide having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4 (Table 2). In some embodiments, the heterologous nucleic acid sequence encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some embodiments, the modified proinsulin polypeptide comprises an insulin A chain, an insulin B chain, and a C peptide linking the A chain and the B chain. In some embodiments, the insulin A chain comprises a sequence having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO: 9, 11 or 13. In some embodiments, the insulin B chain comprises a sequence having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO: 11, 13 or 15.

[0094] [Table 2]

[0095]

[0100] In certain embodiments, the one or more agents for processing modified proinsulin into functional insulin analogs or derivatives thereof include one or more endoproteases and carboxypeptidase E. In some embodiments, the endoproteases include prohormone convertase 1 / 3 (PC1 / 3) and prohormone convertase 2 (PC2). The carboxypeptidase enzyme may be any carboxypeptidase enzyme capable of efficiently removing the C-terminal extension of the B chain. A suitable enzyme is carboxypeptidase Y enzyme (CPY) or a variant thereof. In certain embodiments, the mammalian host cell is modified to express one or more agents for processing modified proinsulin into functional insulin analogs or derivatives thereof. In some embodiments, one or more agents have at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In certain embodiments, one or more agents have the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0096] [Table 3]

[0097] [Table 4]

[0098]

[0101] According to the present disclosure, nucleic acids encoding insulin analogs of interest are isolated, cloned, and often modified using recombinant methods for use, including but not limited to, for protein expression purposes or during the generation of variants, derivatives, expression cassettes, or other sequences derived from insulin polypeptides.

[0099]

[0102] In some embodiments, the sequences encoding the polypeptides of the invention are operably linked to a heterologous promoter, such as a strong constitutive promoter that drives expression in many mammalian cell types, including, but not limited to, the adenovirus major late promoter, the human cytomegalovirus immediate early promoter, the SV40 and Rous sarcoma virus promoters, and the mouse 3-phosphoglycerate kinase promoter, EF1α.

[0100]

[0103] Nucleic acid sequences encoding insulin analogs or derivatives thereof that may be used according to the methods and compositions provided herein may be any nucleic acid sequence encoding an insulin analog polypeptide, such as any proinsulin and preproinsulin and / or their respective analog forms. In certain aspects, the nucleic acid may contain one or more substitutions, additions, deletions, or insertions. Due to the redundancy of the genetic code, nucleic acid variants may or may not affect the amino acid sequence. Exemplary nucleic acid sequences encoding insulin analogs, proinsulin and preproinsulin and / or their respective analog forms are well known in the art and are generally readily available from a variety of mammalian sources, including humans (Bell, GI et al., 1980, Nature 284:26-32). Alternative methods for isolating other nucleic acid sequences encoding insulin polypeptides may be used, or novel sequences may be found and used according to the present disclosure. In a preferred aspect, the nucleic acid sequence encoding an insulin analog or derivative thereof is human insulin. Nucleic acid sequences encoding an insulin analogue and / or its insulin precursor analogue or derivative may be of genomic or cDNA origin, which can be obtained, for example, by preparing a genomic or cDNA library and screening for nucleic acid sequences encoding all or part of the polypeptide by hybridization with synthetic oligonucleotide probes, in accordance with standard techniques (see, for example, Sambrook, J, Fritsch, EF and Maniatis, T, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York, 1989).Nucleic acid sequences encoding insulin analogues and / or insulin analogue precursors or derivatives thereof can also be prepared synthetically by established standard methods, such as the phosphoramidite method described by Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, or the method described by Matthes et al., EMBO Journal 3 (1984), 801-805. Nucleic acid sequences can also be prepared by polymerase chain reaction using specific primers, for example as described in U.S. Pat. No. 4,683,202 or Saiki et al., Science 239 (1988), 487-491.

[0101]

[0104] Nucleotide sequences encoding insulin analogs containing non-naturally encoded amino acids can be synthesized based on the amino acid sequence of a parent polypeptide, including but not limited to those having the amino acid sequences shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and then altering the nucleotide sequence to result in the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue. The nucleotide sequence can be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotides may be prepared by chemical synthesis, including but not limited to the use of an oligonucleotide synthesizer, where oligonucleotides are designed based on the amino acid sequence of the desired polypeptide, preferably selecting codons that are favored in the host cell in which the recombinant polypeptide is to be produced.

[0102]

[0105] Numerous insulin analogs are known in the prior art (see, for example, U.S. Patent Nos. 5,461,031; 5,474,978; 5,164,366 and 5,008,241), which may be used in accordance with the present disclosure. Analogs that may be used herein include human insulin molecules in which amino acid residue 28 (B28) of the B chain is changed from its natural proline residue to aspartic acid, lysine, or isoleucine. In certain embodiments, the lysine residue at B29 is modified to proline. Additionally, the asparagine at A21 may be changed to alanine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, methionine, serine, threonine, tryptophan, tyrosine, or valine. Further examples of insulin analogs that may be used herein include human insulin with the B30 residue deleted (often referred to as "desB30" or "B(1-29)"); insulin "B(1-27)" with the last three amino acid residues deleted; insulin molecules with the B1 phenylalanine residue deleted; and analogs in which the A or B chains have N- or C-terminal extensions (e.g., the B chain can be extended at the N-terminus by the addition of two arginine residues). The present disclosure makes use of routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods used in the present invention include Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 1994)).

[0103]

[0106] General texts describing molecular biology techniques include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc, San Diego, Calif. (Berger), Sambrook et al., Molecular Cloning A Laboratory Manual (2nd Ed.), Vol.1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 1989 (“Sambrook”), and Current Protocols in Molecular Biology, FM Ausubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 1999) (“Ausubel”). These texts describe many other relevant topics, including but not limited to those related to mutagenesis, the use of vectors, promoters, and the generation of genes or polynucleotides containing selector codons to produce proteins containing unnatural amino acids, orthogonal tRNAs, orthogonal synthetases, and pairs thereof.

[0104]

[0107] The "operational elements" discussed herein include at least one promoter, at least one operator, at least one leader sequence, at least one Shine-Dalgarno sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for proper transcription and subsequent translation of the vector DNA. In particular, it is contemplated that such vectors include at least one origin of replication recognized by the host microorganism, together with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the synthetic DNA sequence. Furthermore, in one aspect, it is preferred that the vector includes certain DNA sequences that can function as regulators, and other DNA sequences that can code for regulatory proteins. These regulators, in one aspect, serve to prevent expression of the DNA sequence in the presence of certain environmental conditions and to allow transcription and subsequent expression of the protein encoded by the DNA sequence in the presence of other environmental conditions.

[0105]

[0108] In accordance with this, the nucleic acid sequence encoding at least one insulin analog or derivative thereof is linked to a promoter capable of controlling the expression of an insulin polypeptide in a mammalian host cell, or alternatively, the nucleic acid sequence encoding an insulin analog or derivative thereof and a nucleic acid sequence further encoding one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof are linked to a promoter capable of controlling the expression of an insulin polypeptide in a mammalian host cell.

[0106]

[0109] The promoter may be any DNA sequence that exhibits transcriptional activity in a selected host cell and is derived from a gene encoding a protein that is homologous or heterologous to the host cell, including mutant, truncated, and hybrid promoters. The promoter may be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. Thus, the present disclosure also provides a chimeric nucleic acid sequence that encodes an insulin analog or derivative thereof linked to a promoter capable of controlling expression in a mammalian host cell. Promoters that may be used herein are those generally recognized in the art and include any promoter capable of controlling expression of a polypeptide in a mammalian cell. Specific genetic elements that may enhance expression of an insulin analog polypeptide or derivative thereof may be used herein.

[0107]

[0110] In certain embodiments, a chimeric nucleic acid sequence encoding an insulin analog polypeptide or derivative thereof, comprising a promoter capable of controlling expression in a mammalian host cell, linked to a nucleic acid sequence encoding one or more agents for processing modified proinsulin into a functional insulin analog or derivative thereof, can be incorporated into a recombinant expression vector ensuring good expression in mammalian cells. In some embodiments, a chimeric nucleic acid sequence encoding a proinsulin analog polypeptide or derivative thereof, comprising a promoter capable of controlling expression in a mammalian host cell, linked to a nucleic acid sequence further encoding one or more agents for processing modified proinsulin into a functional insulin analog or derivative thereof, can be incorporated into a recombinant expression vector ensuring good expression in mammalian cells.

[0108]

[0111] In some aspects, a recombinant expression vector according to the present disclosure comprises a nucleic acid sequence encoding a cleavable linker linking a proinsulin polypeptide and an agent for processing the proinsulin into a functional insulin analog or derivative thereof. The cleavable linker may be a peptide, polypeptide, or a portion of a polypeptide that is cleaved after production of the protein or polypeptide. In particular, the cleavable linker is a self-cleavable, self-cleaving, self-cleaving peptide or linker, and these terms are used interchangeably herein. In one aspect, the cleavable linker comprises a 2A peptide. "2A" or "2A-like" sequences are part of a large family of peptides that can cause peptide bond skipping. In particular, it has been recently discovered that the mechanism of 2A-mediated "self-cleavage" is ribosome skipping the formation of a glycyl-prolyl peptide bond at the C-terminus of the 2A peptide. 2A-peptide-mediated cleavage begins post-translationally. Successful skipping and translation resumption results in two "truncated" proteins: the 2A upstream protein is bound to the entire 2A peptide except for a C-terminal proline, and the 2A downstream protein is bound to a single N-terminal proline. Several 2A peptides have been identified in picornaviruses, insect viruses, and type C rotaviruses. Examples of cleavable linkers according to the present disclosure include, but are not limited to, Porcine Teschovirus-1 2A (P2A), FMDV 2A (F2A); Equine Rhinitis A Virus (ERAV) 2A (E2A); and Thosea asigna Virus 2A (T2A), Cytoplasmic Polyhedrosis Virus 2A (BmCPV2A) and Flacherie Virus 2A (BmIFV2A), or combinations thereof, as described, for example, in Kim et al. (2011) PLoS ONE and Liu et al (2017) Sci Rep. 2017.

[0109]

[0112] The selection and construction of expression vectors containing the heterologous nucleic acid sequences disclosed herein and suitable for use in mammalian host cells are known in the art. Expression vectors usually contain a plasmid origin of DNA replication, a selectable marker (e.g., antibiotic selection marker or eGFP), and a promoter and transcription terminator separated by a multiple cloning site (expression cassette) and a DNA sequence encoding at least one ribosome binding site. Transcription of the heterologous nucleic acid (gene of interest, e.g., nucleic acid sequence encoding insulin analog polypeptide or its derivative) is usually controlled by a regulated promoter, which allows cell growth to be separated from product synthesis, resulting in higher yields than when the protein is constitutively expressed.

[0110]

[0113] The recombinant expression system is selected from eukaryotic hosts. In certain embodiments, the eukaryotic host comprises a mammalian cell. Commercial suppliers of mammalian cells used to express recombinant proteins also provide instructions on how to use the cells. The choice of expression system depends on the characteristics desired for the expressed polypeptide.

[0111]

[0114] The term "recombinant" as used herein to describe a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. The term "recombinant" as used herein with respect to a cell means a cell that can be or has been used as a recipient of a recombinant vector or other transferred DNA, including the progeny of the original transfected cell. It is understood that the progeny of a single parent cell may not be completely identical in morphology or in genomic or total DNA complement to the original parent cell due to accidental or deliberate mutation. Progeny of a parent cell that are sufficiently similar to the parent to be characterized by relevant characteristics, such as the presence of a nucleotide sequence encoding a desired polypeptide, are also considered progeny.

[0112]

[0115] Thus, the present disclosure includes a recombinant expression vector comprising, as operably linked components, in the 5' to 3' transcriptional direction: (i) a nucleic acid sequence capable of controlling expression in a mammalian cell; and (ii) a nucleic acid sequence encoding a proinsulin polypeptide or derivative thereof, wherein the expression vector is suitable for expression in a mammalian host cell. Thus, the present disclosure also includes a recombinant expression vector comprising, as operably linked components, in the 5' to 3' transcriptional direction: (i) a nucleic acid sequence capable of controlling expression in a mammalian host cell; (ii) a nucleic acid sequence encoding at least one proinsulin polypeptide or derivative thereof; and (iii) a nucleic acid sequence encoding one or more agents for processing modified proinsulin into a functional insulin analog or derivative thereof, wherein the expression vector is suitable for expression in a mammalian host cell.

[0113]

[0116] A secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) may be provided in the expression vector to direct the insulin analog or derivative thereof into the secretory pathway of the host cell. The secretory signal sequence is joined in the correct reading frame with the DNA sequence encoding the proinsulin. The secretory signal sequence is generally located 5' of the DNA sequence encoding the peptide. The signal peptide may be a naturally occurring signal peptide, or a functional portion thereof, or may be a synthetic peptide. Thus, in some embodiments, the expression vector of the present disclosure further comprises a nucleic acid sequence encoding a signal peptide at the N-terminus of the nucleic acid sequence encoding the proinsulin polypeptide. The signal peptide of the present disclosure enables the cell to secrete the insulin molecule and is removed from the mature molecule during the secretion process. Table 4 lists some commonly used signal peptide sequences for efficient secretion of recombinant proteins expressed in mammalian cells.

[0114] [Table 5]

[0115]

[0117] In some embodiments, the expression vector of the present disclosure further comprises a nucleic acid sequence encoding an N-terminal fusion partner linked to the polypeptide in the 5' to 3' direction of transcription to generate a fusion protein. In certain embodiments, the fusion partner is present during translation but is cleaved by an intracellular protease such as Furin during protein export, thereby releasing the polypeptide of interest. Non-limiting examples of N-terminal fusion partners include human siderocalin (SCN), mouse siderocalin, chicken Ex-FABP, or quail Q83. SCN fusions promote enhanced protein folding and rapid protein expression. Details regarding the production of SCN fusion proteins are described in U.S. Patent No. 10,156,559, the contents of which are expressly incorporated herein by reference in their entirety for all purposes.

[0116]

[0118] Thus, in some aspects, the present disclosure provides an expression vector comprising, as operably linked components, in the 5' to 3' direction of transcription: (i) a nucleic acid sequence capable of controlling expression in a mammalian host cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of an insulin analog or derivative thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide; and (v) a nucleic acid sequence encoding one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof. In some aspects, the agent for processing the modified proinsulin into a functional insulin analog or derivative thereof is selected from the group consisting of prohormone convertase 1 / 3 (PC1 / 3), PC2, and carboxypeptidase E (CPE). In some aspects, the agent for processing the modified proinsulin into a functional insulin lispro or derivative thereof comprises PC1 / 3 and CPE, and optionally, PC2. In another embodiment, the agent for processing modified proinsulin into functional insulin glargine or a derivative thereof comprises PC1 / 3 and optionally PC2.

[0117]

[0119] In some aspects, the disclosure provides a first and a second expression vector. In some aspects, the first expression vector comprises, as operably linked components, in the 5' to 3' direction of transcription: (i) a nucleic acid sequence capable of controlling expression in a mammalian cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of an insulin analog or derivative thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; and (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide. In another aspect, the second expression vector comprises, as operably linked components, in the 5' to 3' direction of transcription: (i) a nucleic acid sequence capable of controlling expression in a mammalian cell; and (ii) a nucleic acid sequence encoding one or more agents for processing modified proinsulin into a functional insulin analog or derivative thereof. In some aspects, the agent for processing modified proinsulin into a functional insulin analog or derivative thereof is selected from the group consisting of PC1 / 3, PC2, and CPE. In some embodiments, the agent for processing modified proinsulin and / or proinsulin analogs into functional insulin lispro or derivatives thereof comprises PC1 / 3 and CPE, and optionally PC2. In another embodiment, the agent for processing modified proinsulin into functional insulin glargine or derivatives thereof comprises PC1 / 3, and optionally PC2.

[0118]

[0120] In some aspects, the disclosure provides methods for producing insulin analogs and / or derivatives thereof, comprising producing a competent mammalian host cell that expresses and secretes insulin, comprising transforming the host cell with an expression vector that co-expresses a modified proinsulin polypeptide and one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof. In another aspect, the disclosure provides methods for producing insulin analogs and / or derivatives thereof, comprising producing a competent mammalian host cell that expresses and secretes insulin, comprising transforming the host cell with a first expression vector that expresses a modified proinsulin polypeptide and a second expression vector that expresses one or more agents for processing the modified proinsulin into a functional insulin analog or derivative thereof.

[0119]

[0121] In some aspects, the disclosure provides a method of producing an ultra fast-acting insulin analog and / or derivatives thereof, the method comprising transforming a mammalian host cell with an expression vector comprising: (i) a nucleic acid sequence capable of controlling expression in a mammalian host cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of the insulin analog or derivatives thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide; (v) a nucleic acid sequence encoding PC1 / 3; (vi) a nucleic acid sequence encoding CPE; and (vii) optionally, a nucleic acid sequence encoding PC2. In some aspects, the ultra fast-acting insulin analog is insulin lispro.

[0120]

[0122] In some aspects, the disclosure provides a method for producing a long-acting insulin analogue and / or derivatives thereof, comprising transforming a mammalian host cell with a recombinant expression vector comprising: (i) a nucleic acid sequence capable of controlling expression in a mammalian host cell; (ii) a nucleic acid sequence encoding a signal peptide for efficient secretion of the insulin analogue or derivatives thereof; (iii) optionally, a nucleic acid sequence encoding an N-terminal fusion partner; (iv) a nucleic acid sequence encoding a modified proinsulin polypeptide; (v) a nucleic acid sequence encoding PC1 / 3; and (vi) optionally, a nucleic acid sequence encoding PC2. In some aspects, the long-acting insulin analogue is insulin glargine.

[0121]

[0123] For the production of long-acting insulins such as insulin glargine, which contains two additional arginine molecules (Arg B31 and Arg B32) at the end of the B chain, it may be necessary to block endogenous carboxypeptidase E activity to maintain the two terminal arginines. Thus, the present disclosure provides a method for producing long-acting insulin analogs and / or derivatives thereof, further comprising an optional step of blocking endogenous carboxypeptidase E (CPE) activity. In some aspects, the step of blocking endogenous carboxypeptidase E activity comprises introducing a deletion or mutation into endogenous CPE that results in reduced or eliminated expression or activity. In some aspects, the host cell may be a CRISPR-modified host cell with reduced or eliminated CPE activity. Such a host cell may be produced, for example, by using CRISPR to reduce or eliminate expression of CPE or by allowing expression of catalytically inactive CPE. For example, the serine at position 202 of the sequence set forth in SEQ ID NO: 9 may be mutated, for example, to a proline. In some aspects, the reduction or elimination of CPE activity may be conditional or inducible (e.g., in the form of a Cre-dependent construct). In some aspects, the step of blocking endogenous carboxypeptidase E activity comprises co-expressing in a mammalian host cell a mutant CPE having at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9. In some aspects, the nucleic acid sequence encoding the mutant CPE is included in an expression vector that co-expresses the modified proinsulin polypeptide. In another aspect, the nucleic acid sequence encoding the mutant CPE is included in a separate expression vector. In some aspects, the disclosure provides a host cell expressing a catalytically inactive CPE, where the CPE binds to a substrate but does not cleave a peptide bond, wherein the catalytically inactive CPE has at least one mutation at a position selected from the group consisting of 72H, and 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9.In some embodiments, the step of blocking endogenous CPE activity comprises adding a blocking agent selected from the group consisting of dopamine quinine, dopamine, norepinephrine, epinephrine, potato carboxypeptidase inhibitor (PCI), a 9-mer peptide called CPI-2KR, and a peptide encoding a decoy arginine sequence. The decoy arginine peptide of the present disclosure comprises a polymer having an amino acid sequence that is 3 amino acids long and has at least 70% amino acid sequence identity to the C-terminal extension of the B chain of a long-acting insulin, e.g., the sequence set forth in SEQ ID NO: 15. The decoy arginine peptide is configured to mimic the C-terminal extension of the B chain of a long-acting insulin and bind to endogenous CPE, thereby preventing endogenous CPE from removing the C-terminal extension of the B chain.

[0122]

[0124] In some aspects, the disclosure provides a mutant CPE or a polynucleotide encoding the mutant CPE, wherein the mutant CPE is catalytically inactive. In some aspects, the mutant CPE binds to a protein but does not hydrolyze the protein. In some aspects, the mutant CPE has at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9.

[0123]

[0125] The term "suitable for expression in a mammalian host cell" means that the recombinant expression vector comprises a chimeric nucleic acid sequence of the present disclosure linked to the genetic elements necessary to achieve expression in a mammalian cell. Expression of recombinant proteins is often difficult outside of the native host. For example, codon usage bias has been observed to vary among different species of bacteria (Sharp et al., 2005, Nucl.Acids.Res.33:1141-1153). Overexpression of recombinant proteins can be difficult even in the native host. In certain aspects of the invention, the nucleic acid introduced into the host cell can be codon-optimized to enhance protein expression. Codon optimization refers to the modification of codons in a gene or coding region of a nucleic acid for transformation of an organism to reflect the typical codon usage of the host organism without changing the polypeptide that the DNA encodes.

[0124]

[0126] In this regard, genetic elements that may be included in an expression vector include transcription termination regions, polyadenylation signals, and translational enhancer sequences, one or more nucleic acid sequences encoding marker genes, one or more origins of replication, etc. In certain embodiments, the recombinant expression vector may further include a DNA sequence that enables the vector to replicate in a mammalian host cell. In some embodiments, the expression vector further includes genetic elements necessary for integration of the vector or a portion thereof into the nuclear genome of a mammalian host cell.

[0125]

[0127] The recombinant vector may be an autonomously replicating vector, i.e. a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, e.g. a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means to ensure autonomous replication. Alternatively, the vector may be one that, upon introduction into a host cell, is integrated into the genome and replicated together with the chromosome into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used. The vector may be a linear or closed circular plasmid, and preferably contains elements that allow stable integration of the vector into the genome of the host cell, or autonomous replication of the vector within the cell independent of the genome. In certain aspects, the recombinant expression vector is capable of replicating in a mammalian host cell. Recombinant vectors suitable for the introduction of nucleic acid sequences into a host cell include mammalian expression systems, e.g. lentivirus-based vectors, pHEK293 Ultra expression vector, and pEF1α-IRES.

[0126]

[0128] A host cell is genetically engineered (including but not limited to, transformed, transduced or transfected) with a polynucleotide of the present disclosure or a construct comprising a polynucleotide of the present disclosure (such as but not limited to, a vector of the present disclosure, which may be a cloning vector or an expression vector). For example, the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derivatized are operably linked to gene expression control elements functional in the desired host cell. The vector may be, for example, in the form of a plasmid, cosmid, phage, bacteria, virus, naked polynucleotide, or conjugated polynucleotide. Vectors are introduced into cells and / or microorganisms by standard methods, including electroporation (Fromm et al., Proc.Natl. Acad.Sci. USA 82, 5824 (1985)), infection with viral vectors, high velocity ballistic penetration by small particles carrying nucleic acid either within the matrix of small beads or particles or on their surface (Klein et al., Nature 327, 70-73 (1987)). Suitable techniques for in vitro nucleic acid introduction into cells include the use of liposomes, microinjection, cell fusion, DEAE-dextran, calcium phosphate precipitation, etc. In vivo gene transfer techniques include, but are not limited to, transfection with viral (typically retroviral) vectors, viral coat protein-liposome mediated transfection [Dzau et al., Trends in Biotechnology 11:205-210(1993)]. In some situations, it may be desirable to provide the nucleic acid source with an agent that targets the target cell, such as a cell surface membrane protein or an antibody specific for the target cell, or a ligand for a receptor on the target cell.Where liposomes are used, proteins that bind to cell surface membrane proteins associated with endocytosis may be used for targeting and / or to facilitate uptake, such as capsid proteins or fragments thereof with tropism for specific cell types, antibodies against proteins that undergo internalization in the circulation, proteins that target intracellular localization and increase intracellular half-life.

[0127]

[0129] In certain aspects, the host cell provided in the method disclosed herein is a mammalian host cell. The mammalian host cell into which the nucleic acid sequence and / or recombinant expression vector is introduced can be any mammalian cell capable of expressing insulin precursors and / or insulin analogs, including, but not limited to, HEK293, CHO, COS and HeLa cells. The present disclosure also provides a host cell selected from algae and yeast. The host cell provided in the method disclosed herein can include a genetic modification that allows for the formation of stable disulfide bonds in the cytoplasm.

[0128]

[0130] The recombinant expression vectors, nucleic acid sequences and chimeric nucleic acid sequences of the present disclosure can be prepared according to methods well known to those skilled in the art of molecular biology. The procedures used to link and insert the DNA sequence encoding the insulin product, optionally a sequence encoding one or more agents for processing modified proinsulin into functional insulin analogs or derivatives thereof, a promoter and optionally a terminator and / or secretion signal sequence, respectively, into a suitable vector containing the information necessary for replication are well known to those skilled in the art (see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY, 1989). Such preparation typically requires, but is not limited to, the bacterial species Escherichia coli as an intermediate cloning host. The preparation of E. coli vectors as well as mammalian transformation vectors can be achieved using known techniques such as restriction digestion, ligation, gel electrophoresis, DNA sequencing, polymerase chain reaction (PCR) and other methods. These methods allow the linking of the nucleic acid sequences and polypeptides to which the present disclosure pertains. A wide variety of cloning vectors are available to carry out the steps required to prepare a recombinant expression vector. In a particular embodiment, the above method comprises In-Fusion cloning. In-Fusion cloning is a highly efficient, ligation-independent cloning method based on annealing of the complementary ends of a cloning insert and a linearized cloning vector. This method ensures easy, single-step, directional cloning of any gene of interest into any vector at any locus. In-Fusion constructs are seamless and allow continuity of the translational reading frame without interfering "scar" sequences.

[0129]

[0131] Typically, these cloning vectors contain a marker that allows for the selection of transformed cells. Nucleic acid sequences can be introduced into these vectors, and the vectors can be introduced into E. coli grown in an appropriate medium. The recombinant expression vector can be easily recovered from the cells upon harvesting and lysis of the cells. Furthermore, general guidance on the preparation of recombinant vectors can be found, for example, in Sambrook et al., Molecular Cloning, a Laboratory Manual, Cold Spring Harbor Laboratory Press, 1989, Vol. 3.

[0130]

[0132] Marker genes that can be used according to the present disclosure include all genes that allow transformed cells to be distinguished from non-transformed cells, including all selectable and screenable marker genes. Marker genes can be, for example, resistance markers such as antibiotic resistance markers against kanamycin, ampicillin, G418, bleomycin, hygromycin, or resistance or resistance markers against chemical agents that allow the selection of traits by chemical means. When linked closely to the nucleic acid sequence encoding an insulin polypeptide, the resistance marker can be used to maintain selection pressure on a population of plant cells or plants that have not lost the nucleic acid sequence encoding an insulin analog polypeptide or its derivative. Screenable markers that can be employed to identify transformants by visual inspection include 3-glucuronidase (GUS) and green fluorescent protein (GFP and eGFP).

[0131]

[0133] It will be appreciated that vectors can be constructed by first preparing a DNA construct containing the entire DNA sequence encoding the insulin precursor of the present disclosure, optionally modified proinsulin, and one or more agents for processing the insulin precursor into a functional insulin analog or derivative thereof; and then inserting this fragment into a suitable expression vector, or by sequentially inserting DNA fragments containing the genetic information for the individual elements (e.g., signal, propeptide, modified C peptide, A and B chains, optionally agents for processing proinsulin into a functional insulin analog or derivative thereof, etc.) followed by ligation.

[0132]

[0134] After the host organism is selected, the vector is transferred into the host organism using methods well known to those skilled in the art. An example of such a method is described in Advanced Bacterial Genetics by RW Davis et. al., Cold Spring Harbor Press, Cold Spring Harbor, NY, (1980), which is expressly incorporated herein by reference. In one aspect, transformation is preferably performed at low temperature, and temperature regulation is contemplated as a means of regulating gene expression through the use of operational elements as defined.

[0133]

[0135] The engineered host cells can be cultured in conventional nutrient media, modified as appropriate for tasks such as, for example, screening steps, activating promoters, or selecting transformants. These cells can optionally be cultured into transgenic organisms. Other useful references for cell isolation and culture (e.g., subsequent nucleic acid isolation) include, but are not limited to, Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. The mammalian host cells are cultured under conditions appropriate for the expression of the recombinant insulin analog and / or its derivatives. These conditions are generally specific to the host organism and are readily determined by the skilled artisan in light of the published literature on growth conditions for such organisms, e.g., Bergey's Manual of Determinative Bacteriology, 8th Ed., Williams & Wilkins Company, Baltimore, Md., which are expressly incorporated herein by reference.

[0134]

[0136] Examples of suitable mammalian host cells are known to those of skill in the art. Such host cells can be Chinese Hamster Ovary (CHO) cells, (e.g., CHO-K1; ATCC CCL-61), Green Monkey cells (COS) (e.g., COS 1 (ATCC CRL-1650), COS 7 (ATCC CRL-1651)); mouse cells (e.g., NS / O), baby hamster kidney (BHK) cell lines (e.g., ATCC CRL-1632 or ATCC CRL-10), and human cells (e.g., HEK 293 (ATCC CRL-1573)). These and other cell lines are available from public depositories such as the American Type Culture Collection, Rockville, Md. To improve glycosylation of insulin polypeptides, mammalian host cells can be modified to express a sialyltransferase, such as a 2,6-sialyltransferase, e.g., as described in U.S. Pat. No. 5,047,335, incorporated herein by reference.

[0135]

[0137] In certain aspects, the present disclosure provides recombinant mammalian cells comprising a polynucleotide sequence encoding a desired insulin analog and / or derivative thereof. In certain aspects, the polynucleotide sequence further encodes at least one agent for processing modified proinsulin into functional insulin. An expression vector comprising such a polynucleotide sequence is introduced into a host cell such that the vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector.

[0136]

[0138] In certain aspects, the method further comprises culturing a mammalian host cell transformed with a recombinant vector of the present disclosure under conditions suitable for expression of the transfected recombinant vector. The expressed recombinant product is then isolated and purified or recovered from the culture broth. In certain aspects, the method disclosed herein further comprises purifying the recovered expression product from the culture broth.

[0137]

[0139] In some embodiments, the step of purifying the recovered expression product comprises subjecting the recovered expression product to a reverse-phase HPLC column, eluting the sample containing the insulin analogue and / or derivative thereof with an organic solvent under conditions that allow the compound to bind to the resin, and washing the organic solvent from the resin with an aqueous buffer. Effective practice of the invention requires individualizing the appropriate combination of chromatography matrix used, pH value, and ionic strength of the buffer for efficient purification.

[0138]

[0140] The production of large amounts of mature insulin or insulin analogues in mammalian cells can significantly reduce the number of downstream purification steps required to produce insulin or insulin analogue products of sufficient purity for pharmaceutical use. US Patent No. 4,916,212 discloses a method for producing insulin in yeast cells, in which the insulin precursor is converted to human insulin in two steps: transpeptidation, which converts the single-chain insulin precursor B(1-29)-Ala-Ala-Lys-A(1-21) to an ester of human insulin, and then hydrolysis of the insulin ester to human insulin. Each conversion step requires an initial separation step followed by at least one purification step. Thus, at least six additional steps are required to produce mature insulin, including at least one enzymatic conversion.

[0139]

[0141] No enzymatic cleavage is carried out to 100% cleavage, leaving behind uncleaved or partially cleaved impurities that must be efficiently removed for pharmaceutical products. Therefore, each cleavage step is followed by at least one isolation or purification step, typically chromatographic purification by exchange chromatography, gel filtration chromatography, affinity chromatography, etc.

[0140]

[0142] Methods for small- or large-scale fermentation can also be used for protein expression, including, but not limited to, fermentors, shake flasks, fluidized bed bioreactors, hollow fiber bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Each of these methods can be performed in batch, fed-batch, or continuous mode processes.

[0141]

[0143] The human insulin polypeptide of the present invention can be recovered using methods generally standard in the art. For example, the culture medium can be centrifuged and / or filtered to remove cellular debris. The supernatant can be concentrated or diluted to a desired volume, or diafiltered into a suitable buffer to condition the preparation for further purification. Further purification of the insulin polypeptide of the present invention includes separating deamidated and clipped forms of the insulin polypeptide variants from the intact form.

[0142]

[0144] For purification of the insulin polypeptides of the present invention, any of the following exemplary procedures can be used: affinity chromatography; anion or cation exchange chromatography (using, but not limited to, DEAE SEPHAROSE); chromatography on silica; high performance liquid chromatography (HPLC); reversed-phase HPLC; gel filtration (using, but not limited to, SEPHADEX G-75); hydrophobic interaction chromatography; size exclusion chromatography; metal chelate chromatography; ultrafiltration / diafiltration; ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; displacement chromatography; electrophoretic methods (such as, but not limited to, preparative electrophoretic methods), differential solubility (such as, but not limited to, ammonium sulfate precipitation), SDS-PAGE, or extraction.

[0143]

[0145] Since the chromatography column materials used on a commercial scale are very expensive, reducing the number of such chromatography steps has a large impact on production economics. In addition, fewer downstream conversion and purification steps further improve production economics, as the amount of labor and time spent on the process is reduced. Advantageously, the disclosed methods and compositions provide the ability to produce, recover, and purify mature functional insulin or analogs thereof in high yields directly from the culture broth, thereby requiring fewer downstream process steps to produce a product of sufficient purity for pharmaceutical use.

[0144]

[0146] In some aspects, the disclosure relates to a method for obtaining a purified biologically active heterologous recombinant protein expressed in a mammalian expression system. In certain aspects, the method comprises culturing a host cell transformed with at least one vector comprising a heterologous nucleic acid sequence encoding a peptide having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In certain aspects, the method comprises culturing a host cell transformed with at least one vector comprising a nucleotide sequence encoding a peptide having an amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In certain aspects, the vector comprising the heterologous nucleic acid encoding the peptide further encodes an agent for processing the heterologous protein into a mature functional protein.

[0145]

[0147] In certain aspects, the method further comprises recovering the expressed heterologous protein, wherein recovering the protein comprises isolating the protein from the host cell to produce a recovered protein preparation. In some aspects, the method further comprises purifying the recovered protein preparation by contacting the recovered protein preparation with a chromatography matrix to remove at least one associated impurity. Purification is performed by applying a polar organic buffer solvent into an aqueous phase comprising an organic acid buffer to precipitate the eluted protein. In any of the aspects disclosed herein, the mature functional protein is a human insulin analog and / or a derivative thereof.

[0146]

[0148] According to one embodiment of the present disclosure, the yield of purified insulin analogues and / or derivatives thereof is 75%-100%. According to another embodiment of the present disclosure, the yield of purified insulin analogues and / or derivatives thereof is 75%-80%, 80%-85%, 85%-90%, 90%-95%, or 95%-100%.

[0147]

[0149] In certain aspects, the present disclosure provides an integrated and continuous process for producing recombinant human insulin analog and / or derivatives thereof, the process comprising: (i) culturing recombinant human insulin analog-secreting mammalian cells in a perfusion bioreactor containing a liquid medium under conditions that allow the cells to secrete the recombinant insulin analog and / or derivatives thereof into the medium, wherein a substantially cell-free volume of medium is continuously or periodically removed from the perfusion bioreactor and fed to a first cyclic countercurrent chromatography system (PCCS1); and (ii) capturing the recombinant insulin analog and / or derivatives thereof from the medium using PCCS1. (iii) purifying the recombinant insulin analogue and / or derivatives thereof using PCCS2, wherein the eluate of PCCS1 containing the recombinant insulin analogue and / or derivatives thereof is continuously fed to a second cyclic counter-current chromatography system (PCCS2); and (iv) purifying the recombinant insulin analogue and / or derivatives thereof using PCCS2, wherein the purification is carried out using a resin of PCCS2 which has a different chemical structure compared to the resin of PCCS2 used to carry out the polishing, wherein the eluate from PCCS2 is insulin analogue and / or derivatives thereof; the above process is integrated and is carried out continuously from the culture step to the eluate from PCCS2 which is insulin analogue and / or derivatives thereof. In certain embodiments, recombinant human insulin analog-secreting mammalian cells are transformed with at least one vector comprising a heterologous nucleic acid sequence encoding a peptide having at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In certain embodiments, recombinant human insulin analog-secreting mammalian cells are transformed with at least one vector comprising a heterologous nucleic acid sequence encoding a peptide comprising the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.In certain embodiments, at least one vector comprising a heterologous nucleic acid sequence further encodes at least one agent for processing modified proinsulin into functional insulin. In alternative embodiments, the recombinant human insulin analog-secreting mammalian cell is further transformed with a second vector comprising a heterologous nucleic acid sequence encoding at least one agent for processing modified proinsulin into functional insulin. In some embodiments, at least one agent encoded by the heterologous nucleic acid sequence has at least about 85% (i.e., at least about 85%, 86%, 87%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8. In certain embodiments, at least one agent encoded by the heterologous nucleic acid sequence has the amino acid sequence set forth in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.

[0148]

[0150] Pharmaceutical insulin formulations can be prepared from purified insulin and / or purified insulin analogs, and such formulations can be used to treat diabetes. In general, purified insulin is mixed with a sufficient amount of a pharma- ceutically acceptable carrier or diluent to exert a therapeutically useful effect without undesirable side effects in the patient being treated. To formulate an insulin composition, a weight fraction of insulin is dissolved, suspended, dispersed, or otherwise mixed in a selected carrier or diluent at an effective concentration such that the condition being treated is improved. Pharmaceutical insulin formulations are preferably formulated for single administration. Therapeutically effective doses for parenteral administration of human insulin are well known to those skilled in the art. When insulin analogs are used, or other modes of delivery are used, therapeutically effective doses can be readily empirically determined by those skilled in the art using known testing protocols or by extrapolation of in vivo or in vitro test data. However, it is understood that concentrations and dosages can vary depending on the severity of the condition to be alleviated. It is further understood that for any particular subject, a particular dosage regimen can be adjusted over time according to the individual judgment of the person administering or supervising the administration of the formulation.

[0149]

[0151] Pharmaceutical solutions or suspensions may contain sterile diluents such as water, lactose, sucrose, dicalcium phosphate, or carboxymethylcellulose. Carriers that can be used to form solutions or suspensions include water, saline, water dextrose, glycerol, glycol, and ethanol. If necessary, pharmaceutical compositions can also contain non-toxic auxiliary substances such as wetting agents, emulsifying agents, solubilizing agents, antibacterial agents such as benzyl alcohol and methyl parabens, antioxidants such as ascorbic acid and sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid (EDTA), zinc ions, pH buffers such as sodium hydroxide, hydrochloric acid, acetate, citrate, or phosphate buffers, and combinations thereof.

[0150]

[0152] The final formulation of the insulin preparation will generally depend on the insulin delivery mode. The insulin prepared according to the present disclosure can be delivered in any desired mode; however, parenteral delivery forms are considered to be the most likely delivery mode to be used. The insulin analogs produced by the method according to the present disclosure can be used to treat conditions that are sensitive to insulin. Thus, insulin analogs can be used to treat type 1 diabetes, type 2 diabetes, and hyperglycemia, such as that sometimes found in, for example, severely injured people and those who have undergone major surgery. If convenient, the insulin analogs can be used in mixtures with other types of insulin, for example insulin analogs with a more rapid onset of action. Examples of such insulin analogs are described, for example, in European patent applications with publication numbers EP214826, EP375437 and EP383472.

[0151]

[0153] Pharmaceutical compositions comprising the insulin analogues of the present disclosure can be used to treat conditions that are sensitive to insulin. Thus, insulin analogues can be used to treat type 1 diabetes, type 2 diabetes, and hyperglycemia, such as that sometimes found in people with severe injuries and those who have undergone major surgery. The optimal dosage level for any patient may depend on a variety of factors, including the effectiveness of the insulin analogue used, the age, weight, physical activity, and diet of the patient, possible combinations with other drugs, and the severity of the condition to be treated. It is recommended that the daily dosage of insulin derivatives be determined for each individual patient by those skilled in the art in a similar manner to known insulin compositions. EXAMPLES

[0152] Working Example Example 1: Transfection or transduction of HEK293 cells

[0154] Procedures for introducing recombinant expression vectors into mammalian host cells (e.g., HEK293) are well known in the art. Transformation of host cells can be carried out by a number of procedures well known to those skilled in the art, including non-viral methods or methods mediated by viral vectors.

[0153]

[0155] For non-viral transfection, 10,000–15,000 HEK293 cells were plated per well of a 24-well plate in 0.5 ml of complete growth medium 12–24 h prior to transfection. Cells were washed with PBS 1×, and 0.5 ml of fresh growth medium was added to each well. Transfection complexes were prepared by mixing 40 μl of serum-free medium, 4.5 μl of transfection reagent, and 500 ng of expression vector (referring to the final volume including growth medium). Transfection complexes were incubated for 15–30 min at room temperature (RT). 2 μl of complex condensation reagent was added. Complex condensation reagent increases transfection efficiency by reducing the size of transfection complexes; however, it may increase cytotoxicity. After incubation, the prepared transfection complexes were added to 0.5 ml of complete growth medium per well of washed HEK293 cells. The cells were incubated with the transfection complexes at 37°C in a humidified CO2 incubator. Successfully transfected cells were selected using an appropriate marker, for example by cell sorting if a fluorescent marker was used, or by antibiotic resistance marker or culture. A fraction of successfully transfected cells was assayed for expression of the target gene 48-72 hours after transfection.

[0154]

[0156] For viral transduction, 4.5 x 10 6293T cells were split into 10 cm dishes (9 ml) the day before transfection. On the day of transfection, total plasmid DNA (ug) in 500ul was diluted. Transfer vectors contain viral packaging (psPAX2): viral envelope (pMD2G) in a 4:2:1 ratio (6:3:1.5ug, respectively). 42ul of PEI (1ug / uL) was added to the diluted DNA and mixed immediately by pipetting up and down / vortexing. The amount of PEI used is based on a 4:1 ratio of PEI (ug):total DNA (ug). The DNA / PEI mixture was incubated at RT for 10-15 minutes, after which 500ul of DNA / PEI mixture was added to each plate and incubated overnight. Media was aspirated and cells were washed once with 8ml of pre-warmed PBS. The cells were then overlaid with 9-10ml of pre-warmed transfection medium (phenol red-free DMEM supplemented with 3-4% FBS and glutamine) and incubated for 18-24 hours. The viral supernatant was then collected, filtered using 0.22μm, and stored at 4°C. Optionally, the viral supernatant was aliquoted into 50ml conical tubes, spun at 8500g overnight at 4°C, the medium carefully aspirated (pellet may be loose), and resuspended at 100x in HBSS. The virus can be aliquoted and stored indefinitely at -80°C. HEK293 cells were plated and infected at 50-80% confluence. The virus was thawed in a water bath. Polybrene was added to the viral supernatant at a final concentration of 6ug / ml, and the viral supernatant / polybrene mix was filtered through a 0.45uM syringe filter. The cells were washed with 2 ml of fresh DMEM complete medium and 2 ml of viral supernatant / polybrene mix was added. The cells were spun at 1800 rpm (750 x g) for 1 hour and then incubated at 37 degrees overnight. All media was then aspirated from the wells and the cells were washed with 1x PBS. 2 ml of fresh DMEM complete medium was added to the cells. The cells were cultured for a further 24 hours before being trypsinized and analyzed by FACS.

[0155] Example 2 - Preparation of Human Analog Insulin 50 / 500ml in a Shaker

[0157] HEK293 cells suitably transfected with insulin analogue constructs were cultured using appropriate growth medium and culture conditions, at 0.5×10 6 The cells were grown to 100 cells / mL. Fresh medium was added every 3-4 days as needed. The expressed insulin analogues secreted into the growth medium were harvested after 1-4 weeks. Briefly, the medium was collected and filtered through a 0.22um filter. SDS Page gel to confirm sufficient protein production.

[0156] Example 3 - Purification

[0158] Insulin analogs were precipitated from the growth medium in the following way: zinc chloride solution (18%) was added to the glargine insulin to a final concentration of 0.1%. The medium containing glargine was adjusted to pH 6.1 and the medium containing lispro was adjusted to pH 12.1, incubated at 4°C for 16 hours, and then centrifuged at 20,000×g for 30 minutes. The supernatant was removed and the insulin-containing precipitate was collected. The glargine-containing pellet was resolved for ion exchange chromatography with sample buffer (7M urea, 0.25M acetic acid, pH 2.5) and lispro (7M urea, 0.25M acetic acid, pH 5.5).

[0157]

[0159] Insulin was purified from the sample buffer using a cationic column packed with Sp Sepharose Fast Flow resin (GE Healthcare Bio-Sciences, USA) on an XK 16 column (GE Healthcare Bio-Sciences, USA) on an AKTA avant (GE Healthcare Bio-Sciences). The insulin-containing buffer was loaded at a flow rate of 1 ml / min onto a 50 ml SP Sepharose column equilibrated with 10 CV (column volume) of equilibration buffer (7 M urea, 0.25 M acetic acid, pH 2.5 / 5.5) at a flow rate of 5 ml / min. After washing the column with 10 CV elution buffer A (7 M urea and 0.25 M acetic acid, pH 2.5 / 5.5) at a flow rate of 5 ml / min, the bound insulin was eluted by application of a linear gradient (0–1 M NaCl) with 6 CV elution buffer A and B (7 M urea, 0.25 M acetic acid, 1 M sodium chloride, pH 2.5 / 5.5) at a flow rate of 5 ml / min. The eluent was monitored at 280 nm and each peak was collected in a fraction tube. The collected fractions were analyzed by HPLC using a Protein & Peptide C4 analytical column.

[0158] Example 4 - Preparative High Performance Liquid Chromatography (Prep-HPLC):

[0160] Fractions collected by cation exchange chromatography with insulin purity >60% were pooled based on the purity of HPLC analysis. Prep-HPLC was performed on an Agilent 1200 system (USA) equipped with a C8 prep HT column (21.2 mm × 150 mm, 5 μm particle size) (Agilent Technologies, USA). Solvent A was prepared with 0.25 M acetic acid and 15% acetonitrile (ACN), and solvent B was prepared with 0.25 M acetic acid and 45% ACN. The column was equilibrated with 10 CV of solvent A at a flow rate of 3 ml / min, and the collected protein was loaded onto the column at a flow rate of 1 ml / min. After washing with 10 CV of solvent A at a flow rate of 3 ml / min, the bound protein was eluted by applying a linear gradient (0% to 70% solution B) with 6 CV of solvent A and B at a flow rate of 3 ml / min. The eluent was monitored at 280 nm, and each peak was collected.

[0159]

[0161] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent documents referenced herein and / or described in the Application Data Sheets are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified, if necessary, to employ concepts from the various patents, applications, and publications to provide further embodiments.

[0160]

[0162] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but rather to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. A method for producing a functional recombinant protein in its native conformation in a host cell using a human propeptide, wherein the human propeptide is (a) transforming the host cell with an expression vector, the expression vector comprising a heterologous nucleic acid having a sequence encoding the human propeptide; (b) growing the host cells recombinantly expressing the propeptide and at least one agent for processing the propeptide by post-translational modification under conditions sufficient to allow maturation and secretion of the protein; and (c) continuously harvesting the secreted protein from the growth medium. wherein the host cell is a mammalian cell.

2. The method described in claim 1, wherein the protein is selected from the group consisting of insulin, insulin analogs, amylin, gastrin, ghrelin, glucagon, somatostatin, α-MSH, ACTH, and β-endorphin.

3. The method described in claim 1, wherein the mammalian cells are selected from the group consisting of HEK293 cells, CHO cells, COS cells, and HeLa cells.

4. The expression vector further comprises a heterologous nucleic acid sequence encoding the agent; the method further comprises co-transforming the host cell with a second expression vector comprising a heterologous nucleic acid sequence encoding the agent; the host cell comprises a heterologous nucleic acid sequence encoding the agent; The method of claim 1.

5. The method of claim 1, wherein the agent is a protease.

6. The method described in claim 5, wherein the protease is selected from an endoprotease and a carboxypeptidase.

7. The method described in claim 6, wherein the endoprotease is selected from the group consisting of prohormone convertase 1 / 3 (PC1 / 3) and PC2, and the carboxypeptidase is carboxypeptidase E (CPE).

8. The method described in claim 1, wherein the agent comprises a sequence set forth in any of SEQ ID NOs: 5 to 8.

9. The method described in claim 1, wherein the peptide comprises the sequence set forth in SEQ ID NO: 1; and the agent comprises PC1 / 3 and CPE, or the agent comprises PC1 / 3, CPE and PC2.

10. The method described in claim 1, wherein the propeptide comprises the sequence set forth in SEQ ID NO: 3; and the agent comprises PC1 / 3, or the agent comprises PC1 / 3 and PC2.

11. The method of claim 1, further comprising a step of blocking endogenous carboxypeptidase E activity in the host cell.

12. (a) the blocking step comprises introducing a deletion or mutation into the endogenous CPE at position 202 of the sequence set forth in SEQ ID NO:9 by CRISPR-based homologous recombination into the host cell; (b) the blocking step comprises co-expressing in the host cell a mutant CPE having at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9; (c) the blocking step comprises adding a blocking agent selected from the group consisting of quinine, dopamine, norepinephrine, epinephrine, potato carboxypeptidase inhibitor (PCI), a 9-mer peptide called CPI-2KR, and a peptide encoding a decoy arginine sequence; (d) the host cell comprises a mutant CPE having at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9; or It is a combination of these, The method of claim 11.

13. The method described in claim 12, wherein the blocking step includes introducing an S202P mutation into the endogenous CPE of sequence number 9.

14. A polynucleotide comprising: (a) a human proinsulin polypeptide analog comprising the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; and (b) at least one agent for processing the human proinsulin polypeptide analog into a functional insulin analog, wherein the at least one agent is selected from an endoprotease and a carboxypeptidase. A polynucleotide encoding

15. The polynucleotide described in claim 14, wherein the at least one drug is an endoprotease selected from the group consisting of PC1 / 3 and PC2.

16. A polynucleotide described in claim 15, wherein PC1 / 3 comprises the sequence set forth in SEQ ID NO: 5, PC2 comprises the sequence set forth in SEQ ID NO: 6, or a combination thereof.

17. The polynucleotide described in claim 14, wherein at least one of the drugs is carboxypeptidase E (CPE).

18. The polynucleotide described in claim 17, wherein the CPE comprises the sequence set forth in SEQ ID NO: 7 or SEQ ID NO:

8.

19. The polynucleotide described in claim 14, wherein the human proinsulin polypeptide analog comprises the sequence set forth in SEQ ID NO: 1; and the at least one drug comprises PC1 / 3 and CPE, or the at least one drug comprises PC1 / 3, PC2 and CPE.

20. The polynucleotide described in claim 14, wherein the human proinsulin polypeptide analog comprises the sequence set forth in SEQ ID NO: 3; and the at least one drug comprises PC1 / 3, or the at least one drug comprises PC1 / 3 and PC2.

21. The polynucleotide described in claim 14, wherein the polynucleotide further encodes a blocking agent configured to block endogenous carboxypeptidase E activity.

22. The polynucleotide described in claim 21, wherein the blocking agent comprises a mutant CPE having at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:

9.

23. A modified host cell comprising the polynucleotide described in claim 14.

24. A modified mammalian host cell comprising a mutant CPE gene encoding a mutant CPE, The expression and / or activity of endogenous CPE is reduced or eliminated; or the mutant CPE is catalytically inactive; Modified mammalian host cells.

25. The modified mammalian host cell described in claim 24, wherein the mutant CPE comprises at least one mutation at a position selected from the group consisting of 72H, 75E, 147R, 192H, 202S, 243Y, and 296E of the sequence set forth in SEQ ID NO:9.

Images