INCORPORATION BY REFERENCE

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, was created on Dec. 2, 2021, is named 180244SEQLISTING-4.txt, and is 7,268 bytes in size.

TECHNICAL FIELD

The present disclosure introduces methods for producing highly purified recombinant human insulin (RHI) and compositions thereof. The highly purified RHI is suitable for use as an active pharmaceutical ingredient (API) for RHI drug products.

BACKGROUND

Human insulin is a hormone that regulates glucose metabolism. The human insulin protein consists of two polypeptide chains—an A-chain of 21 amino acids (SEQ ID NO: 1) and a B-chain of 30 amino acids (SEQ ID NO: 2) for a total of 51 amino acids. The A and B polypeptide chains are linked by disulfide bonds between A-chain cysteines paired with a respective B-chain cysteine. The disulfide bonds and corresponding cysteines (Cys) are CysA7-CysB7, CysA20-CysB19, and CysA6-CysA11. Recombinant human insulin is indicated as a therapeutic composition for patients with diabetes. Diabetes mellitus is characterized by elevated blood glucose levels as a result of insulin deficiency and/or increased hepatic glucose production. Recombinant human insulin may be administered to a patient by subcutaneous or intravenous injection.

The basic techniques of synthesizing recombinant human insulin API using recombinant DNA technology are generally known in the art. But the technical challenge of implementing these basic techniques into a robust production/synthesis process remains a difficult technical challenge because of the different types and amounts of impurities that are produced during the production process of recombinant human insulin API. Also, different pharmaceutical companies may produce different impurities during the recombinant human insulin production/synthesis because different companies may use different production process strategies, different starting materials such as different sequences of the single-chain precursor (SCP), different expression host cells for gene expression, among other factors. Throughout this disclosure, “human insulin” or “insulin” may be used interchangeably and synonymously with “recombinant human insulin” or “RHI.”

The RHI drug products currently on the market contain certain level of impurities. The typical level of total impurities (not including the related substance desamido AsnA21-human insulin (“desamido AsnA21-RHI”), as specified by the United States Pharmacopeia (USP) for RHI) is in the range of about 1-2% (w/w). Some of these total impurities are generated in the finished RHI drug product (e.g. solution), which may increase as a function of time, and some of these total impurities exist in the API. Therefore, an RHI API with less impurity will provide a basis for RHI drug products with less impurity. Accordingly, an RHI API with high purity is needed in the art. This disclosure is directed at a process to generate RHI API with a Total Impurity (not including the related substance desamido AsnA21-RHI, as specified by USP) of not more than 0.8% (w/w) based on the total weight of the RHI API, hereinafter referred to as “highly purified recombinant human insulin API,” or “highly purified RHI API.”

In addition, certain impurities may be difficult to remove, such as acetylated lysine at position 31 of the B-chain (LysineB31, LysB31, or KB31) of the RHI, denoted as “Impurity C” or “impurity C” herein. In particular, prior to the processing of SCP to RHI, the impurity C precursor can be as high as about 2-3% (w/w) in the SCP batch. An example of an impurity C precursor is acetylated LysC1in the SCP, which may have been developed during the synthesis of SCP. The synthesis of the SCP, consisting of A-Chain, B-Chain, C-peptide, and leading sequence, could be performed using the master host cell (i.e.E. coli) through the fermentation process developed by Ferring International Center S. A. (Ferring). See, e.g., Ferring's European Patent No.

EP0871474B1, entitled “Generation of Human Insulin,” filed Dec. 29, 1994, and granted Mar. 1, 2007. The present disclosure seeks to continue and significantly improve upon such work by Ferring, such as significantly reducing total impurities (not including the related substance desamido AsnA21-RHI, as specified by USP) and impurity C in order to produce highly purified RHI for use as an API.

Even after the application of several purification actions during the processing of the SCP to RHI, impurity C can still be as high as 0.7% (w/w) as an impurity in the final purified RHI API composition. Therefore, a technical solution to reducing the amount of impurity C during the production/synthesis of RHI is needed in order to produce highly purified RHI API. Accordingly, the present disclosure solves this technical problem by presenting methods for producing RHI API having a purity of 99.0% (w/w) or greater, a total impurity (not including the related substance desamido AsnA21-RHI) of 0.8% or less, and impurity C of 0.1% (w/w) or less, based on the total weight of the RHI API. The present disclosure also presents compositions of RHI API having a purity of 99.0% (w/w) or greater, a total impurity (not including the related substance desamido AsnA21-RHI) of 0.8% or less, and impurity C of 0.1% (w/w) or less, based on the total weight of the RHI API. Additionally, the present disclosure introduces compositions of, and methods for producing RHI API having a purity of 99.0% (w/w) or greater, a total impurity (not including the related substance desamido AsnA21-RHI) of 0.8% or less, impurity C of 0.1% (w/w) or less, and impurity E of 0.2% (w/w) or less, based on the total weight of the RHI API.

SUMMARY

Exemplary embodiments of the present disclosure address at least the above problems and/or disadvantages and advance the art by providing at least the advantages described below. Additional objects, advantages, and salient features of exemplary embodiments of the present disclosure will become apparent to those skilled in the art from the following detailed description, which, taken in conjunction with the annexed drawings, discloses exemplary embodiments of the present disclosure.

Methods for Producing Highly Purified RHI API

First, the present disclosure provides methods for producing highly purified recombinant human insulin (RHI) active pharmaceutical ingredient (API) having a purity of 99.0% (w/w) or more, a Total Impurity of 0.8% (w/w) or less, and an impurity C of 0.1% (w/w) or less, based on the total weight of the RHI API. The Total Impurity does not include desamido AsnA21-RHI, and impurity C is an acetylated LysB31-RHI.

In a preferred embodiment, the methods introduced in this present disclosure produce highly purified RHI API, in solid form, having a purity of 99.0% (w/w) or more, a Total Impurity of 0.8% (w/w) or less, and an impurity C of 0.1% (w/w) or less, based on the total weight of the highly purified RHI API in a solid form. In other embodiments, the disclosed methods produce RHI API, in a liquid form, having a purity of 99.0% (w/w) or more, a Total Impurity of 0.8% (w/w) or less, and an impurity C of 0.1% (w/w) or less, based on the total weight of the RHI API in the liquid form. Additionally, the disclosed methods produce RHI API having a purity of 99.0% (w/w) or greater, a total impurity (not including the related substance desamido AsnA21-RHI) of 0.8% or less, impurity C of 0.1% (w/w) or less, and impurity E of 0.2% (w/w) or less, based on the total weight of the RHI API, in a solid form or a liquid form.

As defined herein, “highly purified recombinant human insulin API” or “highly purified RHI API” means recombinant human insulin API (solid form or liquid form) with a Total Impurity of 0.8% (w/w) or less based on the total weight of the RHI API. “Total Impurity” or “Total Impurities” means all impurities in the RHI API except for the related substance desamido AsnA21-RHI. For brevity herein, RHI API having a Total Impurity of 0.8% (w/w) or less based on the total weight of the RHI API, is deemed “highly purified RHI API.” For example, RHI API having a purity of 99.0% (w/w) or more, a Total Impurity of 0.8% (w/w) or less, and an impurity C of 0.1% (w/w) or less, based on the total weight of the RHI API, is deemed “highly purified RHI API.” As will be described herein, the highly purified RHI API is the highly purified RHI API produced using the methods introduced in this present disclosure. The USP guidance on RHI specifies that the total impurities of RHI do not include the related substance desamido AsnA21-RHI.

In some embodiments, the methods for producing highly purified RHI API comprise:

protecting two Lysine residues on a single-chain precursor (SCP) using a Lysine protecting group, wherein the SCP has a formula of [(Leader Peptide)-(B-Chain)-(C-peptide)-(A-Chain)], wherein the C-peptide is a short peptide consisting of ArgC2-LysC1(SEQ ID NO: 3), wherein the two Lysine residues is a first Lysine residue at a first residue of the C-peptide (C1) of the SCP (LysC1), and a second Lysine residue at residue 28 of the B-chain (B28) of the SCP (LysB28);

enzyme-cleaving the SCP to produce an ArgC2-LysC1-RHI;

applying a first column purification action to separate the Leader Peptide from the ArgC2-LysC1-RHI to generate ArgC2-LysC1-RHI;

enzyme-cleaving a half of the C-peptide in the ArgC2-LysC1-RHI to produce LysC1-RHI and an Impurity C, wherein the Impurity C is acetylated LysB31-RHI;

de-protecting the two Lysine residues in the LysC1-RHI;

applying a second column purification action to separate one or more impurities from the LysC1-RHI;

applying a third column purification action to the LysC1-RHI to reduce an amount of the Impurity C in the LysC1-RHI to 0.1% (w/w) or less based on the total weight of the LysC1-RHI;

enzyme-cleaving the LysC1-RHI to produce LysC1and the RHI; applying a fourth column purification action to separate one or more impurities from the RHI to produce highly purified RHI for use as an API; and

crystallizing the highly purified RHI API to produce highly purified RHI API in a solid form;

wherein the method produces highly purified RHI API, in a solid form, having a purity of 99.0% (w/w) or more, a total impurity of 0.8% (w/w) or less, wherein the total impurity does not include desamido AsnA21-RHI, and the Impurity C of 0.1% (w/w) or less, and wherein w/w denotes weight by weight and is based on the total weight of the highly purified RHI API in the solid form.

In other embodiments, the methods produce highly purified RHI API, in a solid form, having a purity of 99.3% (w/w) or more, a Total Impurity of 0.5% (w/w) or less, and impurity C of 0.1% (w/w) or less, all based on the total weight of the highly purified RHI API in the solid form. The purity and impurity percentages (w/w) of the highly purified RHI API in the solid form can be assessed shortly after the highly purified RHI API is crystallized into the solid form, such as within 6, 12, 24, 48, or 72 hours after the highly purified RHI API is crystallized into the solid form.

In still other embodiments, the methods can produce highly purified RHI API in a liquid form, having a purity of 99.3% (w/w) or more, a Total Impurity of 0.5% (w/w) or less, and impurity C of 0.1% (w/w) or less, all based on the total weight of the highly purified RHI API in the liquid form. The purity and impurity percentages (w/w) of the highly purified RHI API in the liquid form can be assessed shortly after the final purification action, such as within 6, 12, 24, 48, or 72 hours after the final purification action. Action111in method100is an exemplary embodiment of such final purification action. In some embodiments of the methods of the present disclosure, the enzyme-cleaving of the SCP to produce the ArgC2-LysC1-RHI comprises applying trypsin to the ArgC2-LysC1-RHI. In some embodiments of the methods, the step of enzyme-cleaving the ArgC2-LysC1-RHI to produce LysC1-RHI and an impurity C comprises applying carboxypeptidase B (CPB) to the ArgC2-LysC1-RHI. In some embodiments of the methods, the step of enzyme-cleaving the LysC1-RHI to produce RHI also comprises applying CPB to the LysC1-RHI.

In some embodiments of the methods, the first column purification action utilizes anion-exchange column chromatography. In some embodiments of the methods, the second column purification action utilizes anion-exchange column chromatography. In other embodiments of the methods, the third column purification action utilizes RP-HPLC for reducing the amount of the impurity C in the LysC1-RHI to 0.1% (w/w) or less on the total weight of the LysC1-RHI, which in turn, enables the production of highly purified RHI API having impurity C of 0.1% (w/w) or less based on the total weight of the highly purified RHI API, as described herein. In other embodiments of the methods, the fourth column purification action utilizes RP-HPLC.

In some embodiments, the third column purification action and the fourth column purification action each utilizes RP-HPLC. In some embodiments, the third column purification action and the fourth column purification action each utilizes RP-HPLC having a C18 column. In other embodiments, the third column purification action and the fourth column purification action each utilizes RP-HPLC having a C8 column.

In some embodiments of the methods, the third column purification action that reduces the amount of the impurity C in the LysC1-RHI to 0.1% (w/w) or less, comprises applying reverse-phase high performance liquid chromatography (RP-HPLC) utilizing a C18 column to separate the impurity C from the LysC1-RHI. In other embodiments of the methods, this third column purification action further includes utilizing an aqueous solution comprising (NH4)2SO4, isopropyl alcohol (IPA), or a combination thereof in the RP-HPLC. In still other embodiments of the methods, this third column purification action further includes applying a pH of about 2.5 to about 3.0 in the RP-HPLC. In some embodiments of the methods, this third column purification action utilizes RP-HPLC having a preparation column, such as a preparation column having a length of about 250 mm and an internal diameter of about 10.0 mm. In other embodiments of the methods, this third column purification action further utilizes RP-HPLC having a semi-preparation column, such as a preparation column having a length of about 250 mm and an internal diameter of about 21.2 mm.

In some embodiments, the methods further comprise refolding the SCP using a refolding buffer. In other embodiments, the methods further comprise, optionally, removing the trypsin using aprotinin.

In some embodiments, the one or more impurities separated by the second column purification action include at least ArgC2. In some embodiments, the one or more impurities separated by the fourth column purification action include at least LysC1. At each column purification action, the one or more impurities separated may include those commonly known in the art as well as the impurities identified herein, including the impurities identified in the Tables 1-6. Note that the separation of impurities may include the type of impurities, the amount of the impurities, or the combination thereof.

In other embodiments, the method for producing highly purified RHI API comprises:

protecting at least two Lysine residues on a single-chain precursor (SCP) using a Lysine protecting group, wherein the SCP has a formula of [(Leader Peptide)-(B-Chain)-(C-peptide)-(A-Chain)], wherein the C-peptide comprises at least ArgC2-LysC1wherein the Lysine residues comprise a first Lysine residue at a first residue of the C-peptide (C1) of the SCP (LysC1), and a second Lysine residue at residue 28 of the B-chain (B28) of the SCP (LysB28);

enzyme-cleaving the ArgC2-LysC1-RHI to produce LysC1-RHI and an Impurity C, wherein the Impurity C is acetylated LysB31-RHI;

de-protecting the Lysine residues in LysC1-RHI;

applying a plurality of column purification actions to produce highly purified RHI for use as an API, wherein at least one of the column purification actions reduces an amount of the Impurity C in the LysC1-RHI to 0.1% (w/w) or less based on the total weight of the LysC1-RHI;

enzyme-cleaving the LysC1-RHI to produce RHI; and

crystallizing the highly purified RHI API to produce highly purified RHI API in a solid form;

wherein the method produces highly purified RHI API, in a solid form, having a purity of 99.0% (w/w) or more, a total impurity of 0.8% (w/w) or less, wherein the total impurity does not include desamido AsnA21-RHI, and the impurity C of 0.1% (w/w) or less, and wherein w/w denotes weight by weight and is based on the total weight of the highly purified RHI API in the solid form.

In still other embodiments, the method for producing highly purified recombinant human insulin (RHI) comprises:

protecting at least two Lysine residues on a single-chain precursor (SCP) using a Lysine protecting group, wherein the SCP has a formula of [(Leader Peptide)-(B-Chain)-(C-peptide)-(A-Chain)], wherein the C-peptide comprises at least ArgC2-LysC1, wherein the Lysine residues comprise a first Lysine residue at a first residue of the C-peptide (C1) of the SCP (LysC1), and a second Lysine residue at residue 28 of the B-chain (B28) of the SCP (LysB28);

enzyme-cleaving the ArgC2-LysC1-RHI to produce LysC1-RHI and an Impurity C, wherein the Impurity C is acetylated LysB31-RHI;

de-protecting the Lysine residues in LysC1-RHI;

applying a plurality of column purification actions to produce highly purified RHI for use as an API, wherein at least one of the column purification actions reduces an amount of the Impurity C in the LysC1-RHI to 0.1% (w/w) or less based on the total weight of the LysC1-RHI; and

enzyme-cleaving the LysC1-RHI to produce RHI;

wherein the method produces highly purified RHI API, in a liquid form, having a purity of 99.0% (w/w) or more, a total impurity of 0.8% (w/w) or less, wherein the total impurity does not include desamido AsnA21-RHI, and the impurity C of 0.1% (w/w) or less, and wherein w/w denotes weight by weight and is based on the total weight of the highly purified RHI API in the liquid form.

In some embodiments, the plurality of column purification actions include:

a first column purification action to separate at least the Leader Peptide Sequence from the ArgC2-LysC1-RHI, wherein the first column purification action occurs after the step of enzyme-cleaving the SCP to produce the ArgC2-LysC1-RHI but before the step of enzyme-cleaving the ArgC2-LysC1-RHI to produce the LysC1-RHI and the Impurity C;

a second column purification action to separate at least an ArgC2from the LysC1-RHI, wherein the second purification action separates one or more impurities after the step of de-protecting the Lysine residues in the LysC1-RHI;

a third column purification action, which is the column purification action that reduces the amount of the Impurity C to 0.1% (w/w) or less in the LysC1-RHI, and this third column purification action occurs after the step of de-protecting the Lysine residues in the LysC1-RHI but before the step of enzyme-cleaving of the LysC1-RHI to produce the RHI; and

a fourth column purification action to separate at least LysC1from the RHI, wherein the fourth column purification action occurs after the step of cleaving the LysC1-RHI.

Additionally, the disclosed method further reduces an amount of impurity E to 0.2% (w/w) or less, the impurity E is ThrB30deletion-RHI, thereby producing highly purified RHI API, in a solid form or a liquid form, having a purity of 99.0% (w/w), Total Impurity of 0.8% (w/w) or less, impurity C of 0.1% (w/w) or less, and impurity E of 0.2% (w/w) or less, based on the total weight of the highly purified RHI API in the solid form or the liquid form, respectively. In still other embodiments, the method produces highly purified RHI API, in solid form or liquid form, having a purity of 99.3% (w/w) or more, a Total Impurity of 0.5% (w/w) or less, an impurity C of 0.1% (w/w) or less, and an impurity E of 0.2% (w/w) or less, and wherein w/w denotes weight by weight of the API and is based on the total weight of the highly purified RHI API, in solid form or liquid form, respectively.

In some embodiments, the disclosed method reduces the amount of impurity C to 0.1% (w/w) or less based on the total weight of the highly purified RHI API, including but not limited to, 0.09% (w/w) or less, 0.08% (w/w) or less, 0.07% (w/w) or less, 0.06% (w/w) or less, 0.05% (w/w) or less, 0.04% (w/w) or less, 0.03% (w/w) or less, 0.02% (w/w) or less, 0.01% (w/w) or less, or not detected. In other embodiments, the Impurity C is acetylated LysB31-RHI. In still other embodiments, the Impurity C comprises acetylated LysB31-RHI.

In other embodiments, the disclosed method reduces an amount of impurity E to 0.25% (w/w) or less based on the total weight of the highly purified RHI API, including but not limited to, 0.24% (w/w) or less, 0.23% (w/w) or less, 0.22% (w/w) or less, 0.21% (w/w) or less, 0.20% (w/w) or less, 0.19% (w/w) or less, 0.18% (w/w) or less, 0.17% (w/w) or less, 0.16% (w/w) or less, 0.15% (w/w) or less, 0.14% (w/w) or less, 0.13% (w/w) or less, 0.12% (w/w) or less, 0.11% (w/w) or less, 0.10% (w/w) or less, 0.09% (w/w) or less, 0.05% (w/w) or less, 0.04% (w/w) or less, 0.03% (w/w) or less, 0.02% (w/w) or less, 0.01% (w/w) or less, or not detected.

Compositions of Highly Purified RHI API

Second, the present disclosure also provides compositions for active pharmaceutical ingredient (API) comprising highly purified recombinant human insulin (RHI), in solid form or liquid form, having a purity of 99.0% (w/w) or more, a Total Impurity of 0.8% (w/w) or less, and an impurity C of 0.1% (w/w) or less, and wherein w/w denotes weight by weight of the API and is based on the total weight of the highly purified RHI API, in solid form or liquid form, respectively. As disclosed herein, the Total Impurity does not include the related substance desamido AsnA21-RHI−, and impurity C is acetylated LysB31-RHI.

In some embodiments of the compositions, the highly purified RHI API, in solid form or liquid form, has a purity of 99.3% (w/w) or more, a Total Impurity of 0.5% (w/w) or less, and an impurity C of 0.1% (w/w) or less, and wherein w/w denotes weight by weight of the API and is based on the total weight of the highly purified RHI API, in solid form or liquid form, respectively.

In still other embodiments of the compositions, the highly purified RHI API, in solid form or liquid form, has a purity of 99.0% (w/w) or more, a Total Impurity of 0.8% (w/w) or less, an impurity C of 0.1% (w/w) or less, and an impurity E of 0.2% (w/w) or less, and wherein w/w denotes weight by weight of the API and is based on the total weight of the highly purified RHI API, in solid form or liquid form, respectively. In other embodiments of the compositions, the highly purified RHI API, in solid form or liquid form, has a purity of 99.3% (w/w) or more, a Total Impurity of 0.5% (w/w) or less, an impurity C of 0.1% (w/w) or less, and an impurity E of 0.2% (w/w) or less, and wherein w/w denotes weight by weight of the API and is based on the total weight of the highly purified RHI API, in solid form or liquid form, respectively.

In some embodiments, the API composition is in a solid form. The purity and impurity percentages (w/w) of the highly purified solid RHI API can be assessed shortly after the highly purified liquid RHI API is crystallized into solid form, such as within 6, 12, 24, 48, or 72 hours after the highly purified liquid RHI API is crystallized into solid form. In other embodiments, the highly purified RHI API composition is in a liquid form, such as in an aqueous form. The purity and impurity percentages (w/w) of the highly purified liquid RHI API can be assessed shortly after the final purification action, such as within 6, 12, 24, 48, or 72 hours after the final purification action. An exemplary embodiment of a final purification action is action111in method100. Exemplary embodiments of these highly purified RHI API compositions are provided in Examples 1-8 and these API compositions can be produced using method100. Disclosed compositions of highly purified RHI API can be for RHI drug products, such as RHI aqueous solutions for subcutaneous or intravenous injection.

In still other embodiments of the compositions, the highly purified RHI API has an amount of impurity C of 0.1% (w/w) or less based on the total weight of the highly purified RHI API, including but not limited to, 0.09% (w/w) or less, 0.08% (w/w) or less, 0.07% (w/w) or less, 0.06% (w/w) or less, 0.05% (w/w) or less, 0.04% (w/w) or less, 0.03% (w/w) or less, 0.02% (w/w) or less, 0.01% (w/w) or less, or not detected.

In other embodiments of the compositions, the highly purified RHI API has an amount of impurity E of 0.25% (w/w) or less based on the total weight of the highly purified RHI API, including but not limited to, 0.24% (w/w) or less, 0.23% (w/w) or less, 0.22% (w/w) or less, 0.21% (w/w) or less, 0.20% (w/w) or less, 0.19% (w/w) or less, 0.18% (w/w) or less, 0.17% (w/w) or less, 0.16% (w/w) or less, 0.15% (w/w) or less, 0.14% (w/w) or less, 0.13% (w/w) or less, 0.12% (w/w) or less, 0.11% (w/w) or less, 0.10% (w/w) or less, 0.09% (w/w) or less, 0.05% (w/w) or less, 0.04% (w/w) or less, 0.03% (w/w) or less, 0.02% (w/w) or less, 0.01% (w/w) or less, or not detected.

