Highly purified recombinant human insulin (RHI) API and methods of producing the same
Methods are disclosed for producing highly purified recombinant human insulin (RHI) having a purity of 99.0% (w/w) or greater, a Total Impurity (not including the related substance desamido Asn A21 -RHI, as specified by USP) of 0.8% (w/w) or less, and an impurity C of 0.1% (w/w) or less. Also disclosed are API compositions of highly purified RHI having a purity of 99.0% (w/w) or greater, a Total Impurity of 0.8% (w/w) or less, and an impurity C of 0.1% (w/w) or less.
1. A method for producing highly purified recombinant human insulin (RHI) active pharmaceutical ingredient (API), the method comprising:
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 Arg C2 -Lys C1 , wherein the two Lysine residues are a first Lysine residue at a first residue of the C-peptide (C1) of the SCP (Lys C1 ), and a second Lysine residue at residue 28 of the B-chain (B28) of the SCP (Lys B28 );
enzyme-cleaving the SCP to produce an Arg C2 -Lys C1 -RHI;
applying a first column purification action to separate the Leader Peptide from the Arg C2 -Lys C1 -RHI to generate Arg C2 -Lys C1 -RHI;
enzyme-cleaving a half of the C-peptide in the Arg C2 -Lys C1 -RHI to produce Lys C1 -RHI and an Impurity C, wherein the Impurity C is acetylated Lys B31 -RHI;
de-protecting the two Lysine residues in the Lys C1 -RHI;
applying a second column purification action to separate one or more impurities from the Lys C1 -RHI;
applying a third column purification action to the Lys C1 -RHI to reduce an amount of the Impurity C in the Lys C1 -RHI to 0.1% (w/w) or less based on the total weight of the Lys C1 -RHI;
enzyme-cleaving the Lys C1 -RHI to produce Lys C1 and 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 Asn A21 -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.
2. The method of claim 1 , wherein the highly purified RHI API, in the solid form, has a purity of 99.3% (w/w) or more, the total impurity of 0.5% (w/w) or less, the total impurity does not include desamido Asn A21 -RHI, and the Impurity C of 0.1% (w/w) or less.
3. The method of claim 1 , wherein the step of enzyme-cleaving the half of the C-peptide in the Arg C2 -Lys C1 -RHI comprises applying carboxypeptidase B (CPB) to the Arg C2 -Lys C1 -RHI to produce Lys C1 -RHI.
4. The method of claim 3 , wherein the step of enzyme-cleaving the Arg C2 -Lys C1 -RHI comprises applying the CPB to the Arg C2 -Lys C1 -RHI at a pH of about 8.0 to about 10.0.
5. The method of claim 1 , wherein the third column purification action that reduces the amount of the Impurity C 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 Lys C1 -RHI.
6. The method of claim 5 , wherein the third column purification action that reduces the amount of the Impurity C to 0.1% (w/w) or less further comprises utilizing an aqueous solution comprising (NH 4 ) 2 SO 4 , isopropyl alcohol (IPA), or a combination thereof in the RP-HPLC.
7. The method of claim 5 , wherein the third column purification action that reduces the amount of the Impurity C to 0.1% (w/w) or less is carried out at a pH of about 2.5 to about 3.0.
8. The method of claim 5 , wherein the RP-HPLC utilizes a preparation column.
9. The method of claim 5 , wherein the RP-HPLC utilizes a semi-preparation column.
10. The method of claim 1 , wherein the first column purification action and the second column purification action each utilizes anion-exchange column chromatography.
11. The method of claim 1 , wherein the third column purification action and the fourth column purification action each utilizes RP-HPLC.
12. The method of claim 11 , wherein the third column purification action and the fourth column purification action each utilizes RP-HPLC having a C18 column.
13. The method of claim 11 , wherein the third column purification action and the fourth column purification action each utilizes RP-HPLC having a C8 column.
14. The method of claim 1 , wherein the Lysine protecting group comprises citraconic anhydride, tert-Butyloxycarbonyl (Boc), Benzyloxycarbonyl (Cbz, Z), Allyloxycarbonyl (Alloc), or a combination thereof.
15. The method of claim 1 , wherein the step of enzyme-cleaving the SCP to produce the Arg C2 -Lys C1 -RHI comprises applying trypsin to the SCP.
16. The method of claim 15 , further comprising removing the trypsin using aprotinin.
17. The method of claim 1 , wherein the step of enzyme-cleaving the Lys C1 -RHI to produce RHI comprises applying CPB.
18. The method of claim 1 , further comprising refolding the SCP using a refolding buffer.
19. The method of claim 1 , wherein the one or more impurities separated at the second column purification action comprise at least Arg C2 .
20. The method of claim 1 , wherein the one or more impurities separated at the fourth column purification action comprise at least Lys C1 .
21. The method of claim 1 , wherein the method further reduces an amount of impurity E to 0.2% (w/w) or less, wherein the impurity E is Thr B30 deletion-RHI, thereby producing highly purified RHI API 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 liquid form, respectively.
22. A method for producing highly purified recombinant human insulin (RHI), the method comprising:
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 Arg C2 -Lys C1 , wherein the Lysine residues comprise a first Lysine residue at a first residue of the C-peptide (C1) of the SCP (Lys C1 ), and a second Lysine residue at residue 28 of the B-chain (B28) of the SCP (Lys B28 );
enzyme-cleaving the Arg C2 -Lys C1 -RHI to produce Lys C1 -RHI and an Impurity C, wherein the Impurity C is acetylated Lys B31 -RHI;
de-protecting the Lysine residues in Lys C1 -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 Lys C1 -RHI to 0.1% (w/w) or less based on the total weight of the Lys C1 -RHI;
enzyme-cleaving the Lys C1 -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 Asn A21 -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.
23. The method of claim 22 , wherein the plurality of column purification actions comprise:
a first column purification action to separate at least the Leader Peptide from the Arg C2 -Lys C1 -RHI, wherein the first column purification action occurs after the step of enzyme-cleaving the SCP to produce the Arg C2 -Lys C1 -RHI but before the step of enzyme-cleaving the Arg C2 -Lys C1 -RHI to produce the Lys C1 -RHI and the Impurity C;
a second column purification action to separate at least Arg C2 from the Lys C1 -RHI, wherein the second purification action separates one or more impurities after the step of de-protecting the Lysine residues in the Lys C1 -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 Lys C1 -RHI, and this third column purification action occurs after the step of de-protecting the Lysine residues in the Lys C1 -RHI but before the step of enzyme-cleaving of the Lys C1 -RHI to produce the RHI; and
a fourth column purification action to separate at least Lys C1 from the RHI, wherein the fourth column purification action occurs after the step of enzyme-cleaving the Lys C1 -RHI.
24. The method of claim 22 , wherein the C-peptide comprises the Lys C1 , the Arg C2 , and an Arg as a last residue of the C-peptide that bonds to Gly A1 of the A-chain.
25. The method of claim 22 , wherein the method further reduces an amount of impurity E to 0.2% (w/w) or less, wherein the impurity E is Thr B30 deletion-RHI, thereby producing highly purified RHI API 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 liquid form, respectively.
26. A method for producing highly purified recombinant human insulin (RHI), the method comprising:
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 Arg C2 -Lys C1 , wherein the Lysine residues comprise a first Lysine residue at a first residue of the C-peptide (C1) of the SCP (Lys C1 ), and a second Lysine residue at residue 28 of the B-chain (B28) of the SCP (Lys B28 );
enzyme-cleaving the Arg C2 -Lys C1 -RHI to produce Lys C1 -RHI and an Impurity C, wherein the Impurity C is acetylated Lys B31 -RHI;
de-protecting the Lysine residues in Lys C1 -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 Lys C1 -RHI to 0.1% (w/w) or less based on the total weight of the Lys C1 -RHI; and
enzyme-cleaving the Lys C1 -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 Asn A21 -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.
27. The method of claim 26 , wherein the plurality of column purification actions comprise:
a first column purification action to separate at least the Leader Peptide from the Arg C2 -Lys C1 -RHI, wherein the first column purification action occurs after the step of enzyme-cleaving the SCP to produce the Arg C2 -Lys C1 -RHI but before the step of enzyme-cleaving the Arg C2 -Lys C1 -RHI to produce the Lys C1 -RHI and the Impurity C;
a second column purification action to separate at least Arg C2 from the Lys C1 -RHI, wherein the second purification action separates one or more impurities after the step of de-protecting the Lysine residues in the Lys C1 -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 Lys C1 -RHI, and this third column purification action occurs after the step of de-protecting the Lysine residues in the Lys C1 -RHI but before the step of enzyme-cleaving of the Lys C1 -RHI to produce the RHI; and
a fourth column purification action to separate at least Lys C1 from the RHI, wherein the fourth column purification action occurs after the step of enzyme-cleaving the Lys C1 -RHI.
28. The method of claim 26 , wherein the C-peptide comprises the Lys C1 , the Arg C2 , and an Arg as a last residue of the C-peptide that bonds to Gly A1 of the A-chain.
29. The method of claim 26 , wherein the method further reduces an amount of impurity E to 0.2% (w/w) or less, wherein the impurity E is Thr B30 deletion-RHI, thereby producing highly purified RHI API 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 liquid form, respectively.
30. The method of claim 29 , wherein at the step of enzyme-cleaving the Lys C1 -RHI to produce RHI, a water dilution of at least two times is applied at this step to further reduce the amount of impurity E to 0.2% (w/w) or less.
31. A composition comprising:
a highly purified recombinant human insulin (RHI) active pharmaceutical ingredient (API) comprising:
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 Asn A21 -RHI, and
an Impurity C of 0.1% (w/w) or less, wherein the Impurity C is acetylated Lys B31 -RHI,
wherein w/w denotes weight by weight and is based on the total weight of the highly purified RHI API.
32. The composition of claim 31 , wherein the highly purified RHI API is in a solid form.
33. The composition of claim 31 , wherein the highly purified RHI API is in a liquid form.
34. The composition of claim 31 , wherein the highly purified RHI API has a purity of 99.3% (w/w) or more, the total impurity is 0.5% (w/w) or less, the total impurity does not include desamido Asn A21 -RHI, and the Impurity C is 0.1% (w/w) or less, based on the total weight of the highly purified RHI API.
35. The composition of claim 31 , wherein the highly purified RHI API has a purity of 99.0% (w/w) or more, the total impurity is 0.8% (w/w) or less, the Impurity C is 0.1% (w/w) or less, and an impurity E is 0.2% (w/w) or less, based on the total weight of the highly purified RHI API, and wherein the impurity E is Thr B30 deletion-RHI.