Method for improving enzyme digestion yield of insulin analogue and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a method for treating insulin analogues through sequential enzyme digestion so as to improve the enzyme digestion yield. The method comprises the step of carrying out specific enzyme digestion on the proinsulin analogue by using recombinant double-base amino acid endopeptidase (Kex-2 enzyme) and recombinant trypsin in sequence, and comprises the following steps: firstly, adding the Kex-2 enzyme to perform enzyme digestion on double bases between Linker-A chains to generate an intermediate peptide 1, and then adding the recombinant trypsin to cut a guide peptide and a connecting peptide to obtain a target product. According to the method, the enzyme digestion yield can be remarkably increased by 25% or above, meanwhile, miscut impurities generated by single recombinant trypsin enzyme digestion are avoided, and the method is suitable for efficient enzyme digestion in insulin analogue production.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for increasing the yield of insulin analogues by enzymatic digestion and its application. Background Technology

[0002] Recombinant DNA technology has enabled the large-scale production of insulin analogs. Typically, proinsulin analogs (containing a leader chain, B chain, linker chain, and A chain) are first expressed, then the leader and linker chains are removed by enzymatic digestion, and the product is purified into mature insulin. Proinsulin analogs are precursor proteins that require enzymatic digestion to convert into the target product during insulin analog production. Insulin analog enzymatic digestion technology is a key process in the biopharmaceutical field, primarily used to efficiently and specifically convert insulin analog precursors (such as proinsulin analogs or long-acting analog precursors) into biologically active insulin analog molecules (such as detemir insulin, human insulin, glargine insulin, lispro insulin, degludec insulin, and icotinin). Existing enzymatic digestion methods often use single or mixed enzymes (most commonly recombinant trypsin and recombinant carboxypeptidase B) to perform digestion at specific sites. Recombinant trypsin selectively cleaves the linker chain-A chain junction in proinsulin analogs, requiring the removal of C-terminal arginine / lysine by carboxypeptidase B; carboxypeptidase B removes residual C-terminal basic amino acids after trypsin digestion, avoiding interference with insulin analog activity. Modern processes also employ the combined use of trypsin and lysine endonuclease to achieve single-step conversion, thereby obtaining active products with structures identical to or modified from natural insulin. However, the simultaneous addition of two enzymes for digestion leads to problems such as low digestion efficiency, severe mis-digestion, and low yield. For example, traditional recombinant trypsin digestion may result in various mis-digested impurities, affecting the yield and purity of the digested product. Therefore, a more efficient and controllable digestion method is needed to improve yield and purity. Summary of the Invention

[0003] The purpose of this invention is to provide a sequential enzymatic digestion method that improves the yield of insulin analogues by optimizing the digestion steps and conditions. The digestion method involves adjusting the pH to 7.5–9.5, adding 10–50 U / g of Kex-2 enzyme and 1856–5776 U / g of recombinant trypsin to the target protein, stirring at 20–35°C, and carrying out the digestion reaction to obtain the digestion reaction solution.

[0004] The technical solution of this invention is as follows: A method for increasing the yield of insulin analogue proenzyme digestion, wherein the insulin analogue has a Leader-B chain-Linker-A chain structure, with the A chain and B chain linked by a disulfide bond, the disulfide bond linking point being the amino acid sequence B7-SS-A7; B 19 -SSA20 A6-SSA 11 The amino acid sequence of the B chain is shown in SEQ ID NO.1, the linker amino acid chain is shown in SEQ ID NO.2 or SEQ ID NO.6, the amino acid sequence of the A chain is shown in SEQ ID NO.3, and the leader is selected from SEQ ID NO.4 or SEQ ID NO.5.

[0005] The target product is a B-chain-A-chain.

[0006] Chain B, SEQ ID NO.1: FVNQHLCGSHLVEALYLVCGERGFFYTPK.

[0007] Linker, SEQ ID NO.2: EAEDLQVGQVELGGGPGAGSLQKR or SEQ ID NO.3: EAEDLQVGQKR or SEQ ID NO.4: EAEDLQKR.

[0008] A chain, SEQ ID NO.5: GIVEQCCTSICSLYQLENYCN.

[0009] Leader, SEQ ID NO.6: MRNSAR or SEQ ID NO.7: MSRLNAR or SEQ ID NO.8: MRLNSAR or SEQ ID NO.9: MRASAR.

[0010] The method includes the following steps: a) Use the Kex-2 enzyme to cleave the insulin analogue proto-peptide, cutting the double base (KR) between the Linker-A chains to form intermediate peptide 1. b) The product of step a) was sequentially cleaved using recombinant trypsin, first cleaving the C-terminal arginine (R) base of the leader at the junction with the B chain, and then cleaving the B chain. 29 The enzymatic cleavage reaction is terminated at the linker site where the C-terminal lysine (K) base is linked to the linker, yielding the target product. Further, step a) involves taking insulin analogs, adjusting the pH to 7.5-9.5, stirring at 20-35℃, adding KeX-2 enzyme for digestion; detecting intermediate peptide 1 of the digestion product, and terminating KeX-2 digestion with 50mM EDTA when the peak area of ​​insulin analogs in intermediate peptide 1 is ≤5.0%.

[0011] Furthermore, a) the enzyme digestion time does not exceed 8 hours.

[0012] Furthermore, the KeX-2 enzyme is added to the system at a ratio of 10~50 U / g of the target protein.

[0013] Further, step b) involves adding recombinant trypsin to step a) and continuing enzymatic digestion.

[0014] Furthermore, b) the recombinant trypsin digestion time does not exceed 6 hours.

[0015] Furthermore, the recombinant trypsin is added to the system at a ratio of 1856~5776 U / g of the target protein.

[0016] Further, in step b), the enzymatic digestion reaction is terminated by using RP-HPLC to detect the digestion product. When the purity of the target product is ≥50.0% and the peak area ratio of intermediate peptide 1 is ≤5.0%, the pH is adjusted to 5.0~5.5 with phosphate to terminate the digestion and obtain the target product.

[0017] The beneficial effects of this invention are: The method of this invention can significantly increase the enzyme digestion yield by more than 25%, while avoiding miscutting impurities generated by single recombinant trypsin digestion, and is suitable for high-efficiency enzyme digestion in insulin analog production. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the proinsulin sequence and its restriction enzyme sites.

[0019] Figure 2 RP-HPLC chromatogram of proinsulin analogue.

[0020] Figure 3 RP-HPLC chromatogram of insulin analogue prodrug digested with Kex-2 enzyme.

[0021] Figure 4 RP-HPLC chromatogram of insulin analogue proto-analyte digested with recombinant trypsin.

[0022] Figure 5 RP-HPLC chromatogram of proinsulin analogue.

[0023] Figure 6 RP-HPLC chromatogram of insulin analogue prodrug digested with Kex-2 enzyme.

[0024] Figure 7 RP-HPLC chromatogram of insulin analogue proto-analyte digested with recombinant trypsin. Detailed Implementation

[0025] The method of the present invention will be described in detail below through examples, but the present invention is not limited thereto.

[0026] Example 1

[0027] a) Take the original insulin analog sample, adjust the pH to 8.0~9.0 under suitable buffer conditions, stir at 20~35℃, and perform enzymatic digestion. Use KeX-2 enzyme (addition ratio of 35U / g target protein) for ≤8h, and take samples every 2h for RP-HPLC detection; when the peak area of ​​the original insulin analog in intermediate peptide 1 is ≤5.0%, terminate KeX-2 enzyme digestion with 50mM EDTA; b) Recombinant trypsin (added at a ratio of 1976 U / g target protein) was used to sequentially digest the product of step a); digestion time ≤ 6 h; samples were taken every 2 h for RP-HPLC analysis; The purity of the target product was determined by RP-HPLC to be ≥50.0%; at the same time, the peak area ratio of intermediate peptide 1 was ≤5.0%. The enzyme digestion was terminated by adjusting the pH to 5.0~5.5 with phosphate.

[0028] RP-HPLC detection method: Mobile phase A was 25% phosphate buffer, pH 8.0, 10% acetonitrile, and 0.1M sodium chloride; Mobile phase B was 50% acetonitrile; Elution gradient was 40-48% B phase for 25 min; 48-100% B phase for 10 min; 100% B phase for 15 min; and 100-40% B phase for 10 min; Silica-based C18 column, 4.6 mm * 250 mm, flow rate 1 cm / min.

[0029] The sequential enzyme digestion of this invention is shown in the figure. Figure 1 This shows the cleavage sites of Kex-2 enzyme and pancreatic enzyme. Specifically, the original insulin analog sample has a Leader-B chain-Linker-A chain structure, with the A and B chains linked by disulfide bonds. The disulfide bond link is: B7-SS-A7; B 19 -SSA 20 A6-SSA 11 , The amino acid sequence of the B chain is shown in SEQ ID NO.1: FVNQHLCGSHLVEALYLVCGERGFFYTPK.

[0030] The amino acid linkage of the linker chain is shown in SEQ ID NO.2: EAEDLQVGQVELGGGPGAGSLQKR.

[0031] The amino acid sequence of chain A is shown in SEQ ID NO.5: GIVEQCCTSICSLYQLENYCN.

[0032] The amino acid sequence of the leader is shown in SEQ ID NO.6: MRNSAR.

[0033] The target product is a B-chain-A-chain.

[0034] The results are as follows Figures 2-4 The high-performance liquid chromatograms during the enzymatic digestion process show the changes in product retention time at each step. The results show that the peak position of proinsulin is at 14.187 min; the peak position of intermediate peptide 1 is at 11.619 min; and the peak position of the target product is at 17.793 min.

[0035] The method in this embodiment can increase the enzyme digestion yield by 29.3% and avoid the B-type enzymes produced by digestion with recombinant trypsin alone. 22 Dislocation shearing (dislocation rate of approximately 17%) generates related impurities and increases the original conversion rate (approximately 12%).

[0036] Example 2

[0037] a) Take the original insulin analog sample, adjust the pH to 7.5~9.5 under suitable buffer conditions, stir at 20~35℃, and perform enzymatic digestion. Use KeX-2 enzyme (addition ratio of 20U / g target protein) for ≤4h, and take samples every 1h for RP-HPLC detection; when the peak area of ​​the original insulin analog in intermediate peptide 1 is ≤5.0%, terminate KeX-2 enzyme digestion with 100mM EDTA; b) Recombinant trypsin (added at a ratio of 3890 U / g target protein) was used to sequentially digest the product of step a); digestion time ≤ 4 h; samples were taken every 1 h for RP-HPLC analysis; The termination conditions were: target product purity ≥ 50.0%; peak area percentage of intermediate peptide 1 ≤ 5.0% as detected by RP-HPLC; and pH adjusted to 5.0-5.5 with phosphate to terminate enzymatic digestion. The RP-HPLC detection method was the same as in Example 1. The sequential enzyme digestion of this invention is shown below. Figure 1 This shows the cleavage sites of Kex-2 enzyme and pancreatic enzyme. Specifically, the original insulin analog sample has a Leader-B chain-Linker-A chain structure, with the A and B chains linked by disulfide bonds. The disulfide bond link is: B7-SS-A7; B 19 - SSA 20 A6-SSA 11 The amino acid sequence of the B chain is shown in SEQ ID NO.1, the linker amino acid link is shown in SEQ ID NO.3, the amino acid sequence of the A chain is shown in SEQ ID NO.5, and the leader amino acid link is shown in SEQ ID NO.7.

[0038] SEQ ID NO.1: FVNQHLCGSHLVEALYLVCGERGFFYTPK SEQ ID NO.3: EAEDLQVGQKR SEQ ID NO.5:GIVEQCCTSICSLYQLENYCN SEQ ID NO.7: MSRLNAR The target product is a B chain: FVNQHLCGSHLVEALYLVCGERGFFYTPK-A chain: GIVEQCCTSICSLYQLENYCN (the disulfide bond link is: B7-SS-A7; B 19 -SSA 20 A6-SSA 11 ).

[0039] The results are as follows Figures 5-7 The high-performance liquid chromatograms during the enzymatic digestion process show the changes in product retention time at each step. The results show that the peak position of proinsulin is at 13.565 min; the peak position of intermediate peptide 1 is at 11.179 min; and the peak position of the target product is at 19.632 min.

[0040] The method described in this embodiment can increase the enzyme digestion yield by approximately 25.9%, avoiding the B-type enzymes produced by digestion with recombinant trypsin alone. 22 Dislocation shearing (dislocation rate of approximately 15%) generates related impurities and increases the original conversion rate (approximately 10%).

[0041] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for improving the yield of insulin analogue proenzyme cleavage, characterized in that, The insulin analogue is a Leader-B chain-Linker-A chain structure, A chain and B chain are connected by disulfide bond, and the disulfide bond connection is: B7-S-S-A7; B 19 -S-S-A 20 ; A6-S-S-A 11 ; the B-chain amino acid sequence is shown as SEQ ID NO. 1, the Linker-chain amino acid sequence is shown as SEQ ID NO. 2 or SEQ ID NO. 3 or SEQ ID NO. 4, the A-chain amino acid sequence is shown as SEQ ID NO. 5, and the Leader is selected from SEQ ID NO. 6 or SEQ ID NO. 7 or SEQ ID NO. 8 or SEQ ID NO. 9; the target product is B-chain-A-chain the method comprises the following steps: a) cleaving the insulin analogue proenzyme with Kex-2 enzyme to cut the Leader and the A-chain, so that the insulin analogue proenzyme forms an intermediate peptide 1; b) Sequential cleavage of the product of step a) using recombinant trypsin, first cleaving the C-terminal arginine base of the Leader, then the C-terminal base of the B 29 and the C-terminal base of the Linker, and finally the C-terminal lysine base of the B strand-Linker junction, terminating the cleavage reaction, to obtain the target product.

2. The method of claim 1, wherein, Step a) is to take the insulin analogue, adjust the pH to 7.5-9.5, stir at 20-35℃, and add KeX-2 enzyme for enzyme cleavage; detect the intermediate peptide 1 in the enzyme cleavage product, when the peak area of the insulin analogue in the intermediate peptide 1 is ≤5.0%, stop the Kex-2 enzyme cleavage with 50mM EDTA.

3. The method of claim 1, wherein, a) The enzyme cleavage time is not more than 8h.

4. The method of claim 1, wherein, The enzyme addition amount of KeX-2 enzyme in the system is 10-50U / g of target protein.

5. The method of claim 1, wherein, b) is to add recombinant trypsin for enzyme cleavage.

6. The method of claim 1, wherein, b) The recombinant trypsin enzyme cleavage time is not more than 6h.

7. The method of claim 1, wherein, b) The enzyme addition amount of recombinant trypsin in the system is 1856-5776U per target protein.

8. The method of claim 1, wherein, b) The termination of enzyme cleavage reaction is to use RP-HPLC detection to meet the target product purity ≥50.0% and the peak area of intermediate peptide 1 ≤5.0%, then adjust the pH to 5.0-5.5 with phosphoric acid to terminate the enzyme cleavage, and obtain the target product.

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