PROCESS FOR PRODUCTION OF SOLUBLE RECOMBINANT PEPTIDES

Abstract

The present invention provided the techniques and recipes for enhancing recombinant peptide production in microorganism like E. coli, Saccharomyces cerevisiae, Pichia pastoris and Bacillus subtilis. The designs of fusion protein with a polypeptide and high cell density fermentation process to over express the peptides are given. Methods for separation of polypeptides from fusion protein and methods for isolation and purification of peptides are mentioned. This invention also provides an uncomplicated and unique purification processes for manufacturing of Teriparatide, Liraglutide precursor and Semaglutide precursor with purifies of >98%.

Claims

1: A synthetic oligonucleotide sequence encoding the peptides with STU/STE fusion tag consisting of a protease recognition sites, wherein the STU and STE sequence code is as given below: TABLE-US-00015 STU1: MHHHHHHMLYESVSGEIKSTFSWLMLHQQRNQHARLIP VELYMSDKMQIFVKTLTGKTITLEVESSDTIDNVKSKI QDKEGIPPDQQRLIFAGKQLEDGRTLSDYNIQKESTLH LVLRLRGGHAEGTFTSDVSSYLEGQAAKEFIAWLVRGR G STU2: MHHHHHHMLYESVSGEIKSTFSWLMLHKTLTGKTITLE VESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGR TLSDYNIQKESTLHLVLRLRGGHAEGTFTSDVSSYLEG QAAKEFIAWLVRGRG STU3: MHHHHHHMLYESVSGEIRRKKRRKRKRKTLTGKTITLE VESSDTIDNVKSKIQDKEGIPPDQQRLIFAGKQLEDGR TLSDYNIQKESTLHLVLRLRGGHAEGTFTSDVSSYLEG QAAKEFIAWLVRGRG STE1: MHHHHHHMLYESVSGEIKSTFSWLMLHQQRNQHARLIP VELYMSDKMQIFVKTLTGKTITLEVESSDDDDKHAEGT FTSDVSSYLEGQAAKEFIAWLVRGRG STE2: MHHHHHHMLYESVSGEIKSTFSWLMLHDDDDKHAEGTF TSDVSSYLEGQAAKEFIAWLVRGRG STE3: MHHHHHHMLYESVSGEIRRKKRRKRKRDDDDKHAEGTF TSDVSSYLEGQAAKEFIAWLVRGRG.

2: The peptides as referred in claim 1, wherein the peptides relate to Teriparatide, Liraglutide and Semaglutide precursor.

3: The peptides as referred in claim 1, wherein the fusion tag is a soluble form which contains 90 to 130 nucleic acids, wherein the unfolding of protein does not take place and hence refolding is not required.

4: A process for preparation of peptides using recombinant microorganism which involves a) cloning of gene sequence encoding the peptides with STU/S fusion tag consists of a protease recognition site, b) expression and isolation of fusion protein, and c) fusion protein cleavage, isolation and purification of peptide.

5. (canceled)

6: The process as claimed in claim 4, wherein the process comprises expressing the peptides as a fusion protein and then the fusion tag is removed by an enzymatic digestion process.

7: The process as claimed in claim 4 wherein the process comprises production of recombinant peptides of the peptides in microorganism which is E. coli, Saccharomyces cerevisiae, Pichia pastoris and Bacillus subtilis.

8: The process as claimed in claim 4, wherein the process comprises production of recombinant peptides in E. coli with HCDM3 medium and optimized process parameters during fermentation.

9: The process as claimed in claim 4, wherein the isolation of fusion protein comprises usage of 100 to 1000 kDa of cross flow filters for clarification of cell lysate or fermentation broth and usage of 1-8 molar urea in lysate or cell suspension or in broth.

10: The process as claimed in claim 4, wherein the cleavage process of fusion protein comprises the enzyme and substrate ratios of 1:5 to 1:100, wherein the enzymes are enterokinase, ubiquitin hydrolase and the substrates are STE/STU fusion proteins.

11: The process as claimed in claim 4, wherein the process comprises unique purification process for Teriparatide, Liraglutide and Semaglutide precursor by using protein precipitation, ion exchange chromatography and RP-HPLC methods.

12. (canceled)

13: The process as claimed in claim 4, wherein the cleavage process of fusion protein is carried out at a temperature in the range from 15-35° C.

14. (canceled)

15: The process as claimed in claim 4, wherein the process carried out by ultra filtration (100 to 1000 kDa) and cell lysate or broth suspension using buffer contains 1 to 8M urea.

16. (canceled)

17. (canceled)

Description

BRIEF DESCRIPTION OF DRAWINGS

[0027] FIG. 1 Shows amino acid sequence of STU solubility tag followed peptide sequence, (Three different STU tags were tried for protein expression and all the three has shown similar expression titers)

[0028] FIG. 2 Shows amino acid sequence of STE solubility tag followed peptide sequence, (Three different STE tags were tried for protein expression and all the three has shown similar expression titers)

[0029] FIG. 3 Shows expression cassette comprising solubility tag with peptide sequence,

[0030] FIG. 4 Shows highest protein expression after 6 h of induction, and

[0031] FIG. 5 Shows the flow chart of final purification process.

DETAILED DESCRIPTION OF INVENTION

[0032] In describing the embodiments of the invention, specific terminology is resorted for the sake of clarity. However, it is not intended that the invention be limited to the specific terms so selected and it is to be understood that each specific term includes all technical equivalents that operate in a similar manner to accomplish a similar purpose.

[0033] Embodiments herein and the various features and advantageous details thereof are explained more fully with reference to the non-limiting embodiments that are illustrated in the accompanying figures and detailed in the following description. The examples used herein are intended merely to facilitate an understanding of ways in which the embodiments may be practiced and to further enable those skilled in the art to practice the embodiments herein. Accordingly, the examples should not be construed as limiting the scope of the embodiments herein.

[0034] The present invention is related to a novel process for production of recombinant peptides in prokaryotic expression system. In present invention all the natural peptides are expressed as a fusion protein and then the fusion tag is removed by an enzymatic digestion process. According to the present invention, natural peptides can be produced more efficiently when compared with prior art processes.

[0035] Present invention also provides an optimized high yielding fermentation media for the expression of natural peptides like Teriparatide, Liraglutide and Semaglutide precursor in microorganism.

[0036] This invention also provides a unique purification process for Teriparatide, Liraglutide and Semaglutide precursor by using protein precipitation, ion exchange chromatography and RP-HPLC methods. The process yields more than 95% purity of the peptides.

[0037] The microorganism used herein may be selected from B. subtilis, E. coli, P. pastoris and S. cerevisiae.

[0038] The fusion protein of the present invention is soluble form which contains 90-130 amino acids. With the fusion tag used in the present invention the unfolding of protein does not take place and hence refolding is not required. In view of this, affinity chromatography is avoided for purification; instead the purification can be carried out using simple purification techniques like precipitations, ultra filtrations and conventional ion exchange chromatography techniques. This is a significant change over the prior-art which reduces the overall cost of production.

[0039] The isolation of fusion protein comprises usage of 100 to 1000 kDa of cross flow filters for clarification of cell lysate or fermentation broth and usage of 1-8 molar urea in lysate or cell suspension or in broth.

[0040] The cleavage process of fusion protein comprises the enzyme and substrate ratios of 1:5 to 1:100, wherein the enzymes are enterokinase, ubiquitin hydrolase and the substrates are STE/STU fusion proteins

[0041] The cleavage process of fusion protein is carried out at a temperature in the range from 15-35° C. at pH in the range from 5-9, wherein the process carried out by ultra filtration (100 to 1000 kDa) and cell lysate or broth suspension using buffer containing 1 to 8M urea. The simultaneous steps of cleavage and precipitation to isolate peptides at temperature in the range from 0-10° C. at pH in the range from 4-7.

[0042] The representative example Liraglutide, Teriparatide and Semaglutide used herein are intended merely to facilitate an understanding of ways in which the embodiments of the invention may be practiced and to further enable those skills in the art to practice the embodiments herein. Accordingly, this example should not be construed as limiting the scope of the embodiments herein.

[0043] According to the present invention, the purity of the peptides will be in range of 95% to 99% when measured by using RP-HPLC (with the purities of Teriparatide >98%, Liraglutide precursor >95% and Semaglutide precursor >95%.

[0044] Based on the microorganism used for expression, the location of the fusion protein expression and the protein isolation process will change. Cleavage of fusion protein is same irrespective of organism and the peptide. It only differs in conditions of cleavage and ratio of enzyme and substrate.

[0045] Further, the isolation and purification of the final peptide will change with respect to the end peptide.

Stage 1: Clone Development

1. Cloning and Over Expression of Peptides in Microorganism Like Bacteria and Yeast

[0046] For the over expression of peptides in the selected microorganism, following two cloning strategies were employed with fusion tags either in an expression vector or chromosomal insertions.

[0047] 1. STU solubility tag followed by peptide (STU)

[0048] 2. STE solubility tag followed by peptide (STE)

2. Cloning and Expression of Peptide with Solubility Tag STU/STE in E. coli.

[0049] a. Cloning of Synthetic Gene Construct (Peptide) with STU/STE Fusion Tag [0050] The synthetic gene construct of peptide with STU/STE fusion tag was obtained as a synthetic construct cloned in pUC57 cloning vector. The synthetic construct was transformed into E. coli DH5α and the desired fragment was isolated by restriction digestion of the plasmid DNA with the restriction enzymes Nde1, Xho1, BamHI and SacI.

[0051] b. Sub-Cloning of Peptide with STU/STE Fusion Tag into Expression Vector [0052] The expression vector (pET24/pET28) was digested with Nde1, Xho1, BamHI and SacI for cohesive end ligation. The digested plasmid and the above isolated STU-peptide/STE-peptide insert were ligated in a molar ratio of 3:1. The ligated product (pET24/28: STU-peptide/STE-peptide) was transformed into E. coli DH5α by heat shock method and transformants were selected by antibiotic selection marker (Kanamycin (50 μg/ml)).

[0053] c. Characterization of Recombinant Clone of E. coli DH5α Containing Recombinant Expression Vector (pET24/28STU-Peptide/STE-Peptide) [0054] Positive colonies were initially characterized by PCR amplification (colony PCR method) using T7 primers and finally characterized by restriction enzyme digestion (Nde1, Xho1, BamHI and SacI).

[0055] d. Expression Studies of Peptide in E. coli Expression Hosts [0056] For expression studies, the characterized recombinant expression plasmid (pET24/28 STU-peptide/STE-peptide) was transformed into E. coli expression host BL21 (DE3) by heat shock method and the transformants were selected using kanamycin as a selection marker.
To check expression of peptide with STU/STE tag, one colony from each plate is inoculated into LB medium and grown at 37° C. When the culture reached to mid-log phase (O.D of 0.6 to 0.7), the cells were induced with 1 mM IPTG. After induction, 1 ml of sample was collected from both un-induced and induced cultures at 0, 1, 2 and 3 hrs to check the expression on SDS gel.

[0057] Expression of the peptide with STU/STE tag was observed in all the constructs which were designed. Each one of them was designed keeping in mind of the different methods employed for the removal of solubility tag. The STU/STE tag can be cleaved by the addition of ubiquitin hydrolase/Enterokinase enzyme respectively.

[0058] When the cleavage efficacy of enterokinase and ubiquitin hydrolase was tested, STU and STE fusion peptides, both the tags were found to be the best choice. The cleavage efficiency of enterokinase is high comparing with ubiquitin hydrolase.

Stage 2: Fermentation Process Development

[0059] Comparison Recombinant Protein Expression and Titers with Different Fermentation Medium:
A total of seven different media compositions and process parameters were tested for protein expression and titers. Media 7 (HCDM 3) was found to be the best media for E. coli growth and fusion protein expression

Media 1: Luria Bertani Broth

Composition:

[0060]

TABLE-US-00001 Component g/L Tryptone 10.0 Yeast extract 5.0 Sodium chloride 5.0

OD.SUB.600.: 4-6

[0061] Protein titer: 100-150 mg/L
Media 2: Terrific Broth with Glucose

Composition:

[0062]

TABLE-US-00002 Component g/L Tryptone 12 Yeast extract 24 Glucose 20 Dipotassium hydrogen phosphate 9.4 Potassium dihydrogen phosphate 2.2

OD.SUB.600.: 12-15

[0063] Protein titer: 250-350 mg/L

LB and Terrific Broth Media:

[0064] LB, Terrific broth-glucose and Terrific broth-glycerol were tested for cell growth and protein expression. The growth and protein expressions are observed less in LB, Terrific broth-glucose compared to Terrific broth glycerol. Since the growth and protein expressions are better in Terrific broth-glycerol, feeding was applied to increase the expression levels and cell growth.
Media 3: Terrific Broth with Glycerol

Composition:

[0065]

TABLE-US-00003 Component g/L Yeast extract 24 Glucose Glycerol 5 Dipotassium hydrogen phosphate 9.4 Potassium dihydrogen phosphate 2.2

OD.SUB.600.: 15-20

[0066] Protein titer: 400-500 mg/L
Media 4: Terrific Broth with Feed

Composition:

[0067]

TABLE-US-00004 Component g/L Tryptone 12 Yeast extract 24 Glycerol 5 Dipotassium hydrogen phosphate 9.4 Potassium dihydrogen phosphate 2.2

Feed Composition:

[0068]

TABLE-US-00005 (g/L) Feed A Glucose 500 Thiamine 1 ml Trace elements 2 ml Feed B Tryptone 12 g Yeast extract 24 g Trace element solution FeSO.sub.47H.sub.2O 10 ZnSO.sub.47H.sub.2O 2.25 CuSO.sub.45H.sub.2O 1.0 MnSO.sub.4H.sub.2O 0.5 Na.sub.2B.sub.4O.sub.7 10 H.sub.2O 0.23 CaCl.sub.2 2.0 (NH.sub.4).sub.6Mo.sub.7O.sub.24H.sub.2O 0.1

OD.SUB.600.: 25-30

[0069] Protein titer: 1.0-1.5 g/L
Terrific broth with feed: In order to increase the cell density the tank medium was given glucose at basal concentration and further fed with concentrated glucose and yeast extract to meet the C:N ratio requirement. There was an increase in cell growth (25-30 O.D) and protein expression (1-1.5 g/L) with feed addition.

Media 5: HCDM 1

Composition:

Tank Media:

[0070]

TABLE-US-00006 Component g/L Potassium dihydrogen phosphate 2.2 Ammonium sulphate 4.5 Citric acid 1.0 Glucose 20 MgSO.sub.4 130 ml (70 mg/ml concentration) Cacl.sub.2 5 ml (80 mg/ml concentration) Trace metal solution 10 ml Thiamine Hcl 2 ml (100 mg/ml concentration)

Trace Metal Solution:

[0071]

TABLE-US-00007 Component mg/10 ml Volume (ml) EDTA 500 5 FeSO.sub.4 500 10 ZnSO.sub.4 500 2 MnSO.sub.4 500 2 CoCl.sub.2 500 0.2 CuSO.sub.4 500 0.1 Na.sub.2MoO.sub.4 500 0.2 H.sub.3BO.sub.3 500 0.1

Feed Medium Composition:

[0072]

TABLE-US-00008 Component g/L Glucose 180 gm Tryptone 50 gm MgSO.sub.4 57.5 ml (70 mg/ml concentration) Cacl.sub.2 25 ml (80 mg/ml concentration) Trace metal solution 6 ml Thiamine Hcl 0.2 ml (100 mg/ml concentration) FeSO.sub.4 500 mg Trace metal solutions 50 ml
All the components were added and made up to volume of 1 liter.

OD.SUB.600.: 40-50

[0073] Protein titer: 2.0-2.5 g/L

Media 6: HCDM 2

Composition:

[0074]

TABLE-US-00009 Media Component g/L Tryptone 20 Yeast extract 10 Sodium chloride 5 Dextrose 5 Potassium chloride 1 Magnesium sulphate 2 Magnesium chloride 2

Feed Medium: 2 L

[0075]

TABLE-US-00010 Media Component g/L Yeast Extract 150 Dextrose Anhydrous 500

OD.SUB.600.: 65-70

[0076] Protein titer: 3.0-4.0 g/L

Media 7: HCDM 3

Composition:

Production Media:

[0077]

TABLE-US-00011 Media Component g/L Tryptone 20 Yeast extract 10 Sodium chloride 5 Dextrose 5 Potassium chloride 1 Magnesium sulphate 2 Magnesium chloride 2
Feed Medium: 2 L (for 7.2 liters)

TABLE-US-00012 Media Component g/L Yeast Extract 150 Lactose 250 MgSO.sub.4 1.5 (NH.sub.4).sub.2 SO.sub.4 1.5

OD.SUB.600.: 80-90

[0078] Protein titer: 5-6 g/L

Results:

High Cell Density Media:

[0079] Three different fermentation Media were used to increase the cell density and protein expression, OD.sub.600 40-50, 50-60 and 70-80 were achieved with HCDM 1, HCDM 2 and HCDM 3 respectively, whereas the protein titers are 2-2.5 g/L, 3-4 g/L and 5-6 g/L. HCDM 3 medium has shown better results than HCDM 1 and HCDM 2.

Conclusion:

[0080] Based on the above experimental data, HCDM 3 media and below process parameters were freezed as optimum conditions for peptide production.
Seed media: LB broth
Seed age: 7 to 10 hours
OD600: 1.7 to 2.5 (mid log phase)
Fermentation process:

Media: HCDM 3

[0081] Batch phase duration: 6-9 hours
Fed batch duration: 7-10 hours

Harvest OD.SUB.600.: 80 to 90

Production Media for 10 Liters

[0082] Production media: 7.2 ltrs

TABLE-US-00013 Media Component g/L Tryptone 20 Yeast extract 10 Sodium chloride Dextrose 5 Potassium chloride 1 Magnesium sulphate 2 Magnesium chloride 2

Feed Medium: 2 L

[0083]

TABLE-US-00014 Media Component g/L Yeast Extract 150 Lactose 250 MgSO.sub.4 1.5 (NH.sub.4).sub.2 SO.sub.4 1.5

Stage 3: Purification Process Development

1. Isolation and Purification of Fusion Protein:

Trial 1:

[0084] 1. 10% cell suspension was prepared in 20 mM Sodium phosphate buffer, 8M urea pH 7.2 and passed through high pressure homogenizer at 800 bar pressure for cell lysis, repeated the passages till the suspension OD.sub.600 decreases to <5. [0085] 2. 0.6μ Cross flow filtration was done for cell lysate, Fusion peptide was collected in filtrate and the retentate contained cell debris and other impurities. 90% of the fusion peptide was recovered in filtrate with purity of around 40-50%. [0086] 3. Filtrate was loaded on affinity chromatography (Ni-Sepharose) and eluted the fusion protein using 250 mM imidazole with a purity of >80% [0087] 4. The above purified fusion protein was applied for enzymatic cleavage to separation the peptide from fusion tag.

Trial 2:

[0088] 1. 10% cell suspension was prepared in 20 mM Tris buffer, 6M urea pH 8.5 and passed through high pressure homogenizer at 800 bar pressure for cell lysis, repeated the passages till the suspension OD.sub.600 decreases to <5. [0089] 2. 0.2μ Cross flow filtration was applied for cell lysate, Fusion peptide was collected in filtrate and the retentate contained cell debris and other impurities. 90% of the fusion peptide was recovered in filtrate with purity of around 50-60%. [0090] 3. Filtrate was loaded on affinity chromatography (Ni-Sepharose) and eluted the fusion protein using 250 mM imidazole with a purity of >80% [0091] 4. The above purified fusion protein was applied for enzymatic cleavage to separation the peptide from fusion tag.

Trial 3:

[0092] 1. 10% cell suspension was prepared in 20 mM Tris buffer pH 8.5 and passed through high pressure homogenizer at 800 bar pressure for cell lysis, repeated the passages till the suspension OD.sub.600 decreases to <5. [0093] 2. 2M urea was added to the cell lysate, dissolved and then passed through 750 kDa Cross flow filtration, Fusion peptide was collected in filtrate and the retentate contained cell debris and other impurities. 90% of the fusion peptide was recovered in filtrate with purity of around 90%. [0094] 3. The above purified fusion protein was applied for enzymatic cleavage to separate the peptide from fusion tag.

Conclusion:

[0095] Based on the above three trial run results, in trial 3 got the best recovery and purity

2. Fusion Protein Digestion

[0096] Digestion with Light Chine Enterokinase:
The enzyme enterokinase cuts STE fusion protein exactly at the peptide part to release peptide. Enterokinase enzyme was added to the purified fusion protein at pH 8.0, 2 mM CaCl.sub.2 and incubated at 35° C. under mild stirring and the percentage of cleavage was tested by RP-HPLC. ≥90% cleavage was recorded in 5 hours duration at 30° C. under mild stirring.
Digestion with Ubiquitin Hydrolase:
The enzyme ubiquitin hydrolase cuts STU fusion protein exactly at the peptide part to release peptide. Ubiquitin hydrolase enzyme was added to purified fusion protein at pH 8.0 and incubated at 25° C. under mild stirring and the percentage of cleavage was tested by RP-HPLC. ≥90% cleavage was recorded in 3 hours duration at 30° C. under mild stirring.
The enzyme and substrate ratios were tried for 1:5 to 1:100 for both the enzymes enterokinase and yeast ubiquitin hydrolase. In both the cases 1:15 was found to be optimum.

3. Isolation and Purification of Peptide:

[0097] In both the strategies (STU and STE) the cloning, fermentation, purification and digestion of fusion protein are similar to all natural peptides.

EXAMPLES

Example 1: Isolation and Purification of Liraglutide Precursor

[0098] 1. 3.0M sodium chloride was added to the digestion mixture and the pH of the sample was reduced to 7.0 using diluted HCl. [0099] 2. Centrifuged the sample to remove separated fusion tag and fusion protein from the separated peptide. Fusion tag and fusion protein were come in pellet and 90% of the peptide was in supernatant. [0100] 3. Centrifuged supernatant was further adjusted the pH to 4.0 using diluted HCl and collected the pellet for peptide. [0101] 4. Peptide pellet was dissolved in 20 mM Tris pH 8.5 buffer and tested for purity by RP-HPLC and it was 90-95% pure. It was used for liraglutide preparation.

Example 2: Isolation and Purification of and Purification of Teriparatide

[0102] 1. 3.5M sodium chloride was added to the digestion mixture and the pH of the sample was reduced to 7.0 using diluted acetic acid. [0103] 2. Centrifuged the sample to remove separated fusion tag and fusion protein from the separated peptide. Fusion tag and fusion protein were come in pellet and 90% of the peptide was in supernatant. [0104] 3. Centrifuged supernatant was further adjusted the pH to 5.5 using diluted acetic acid and collected the pellet for peptide. [0105] 4. Peptide pellet was dissolved in 10 mM Sodium acetate pH 4.0 buffer and tested for purity by RP-HPLC and it was 90-95% pure. It was further purified for Teriparatide API preparation.

Example 3: Isolation and Purification of and Purification of Semaglutide Precursor

[0106] 1. 3.5M sodium chloride was added to the digestion mixture and the pH of the sample was reduced to 7.5 using diluted HCl. [0107] 2. Centrifuged the sample to remove separated fusion tag and fusion protein from the separated peptide. Fusion tag and fusion protein were come in pellet and 90% of the peptide was in supernatant. [0108] 3. Centrifuged supernatant was further adjusted the pH to 4.5 using diluted HCl and collected the pellet for peptide. [0109] 4. Peptide pellet was dissolved in 10 mM Tris pH 8.5 buffer and tested for purity by RP-HPLC and it was 90-95% pure. It was further used for semaglutide preparation.