Fermentation medium and its application in the preparation of teriparatide

Abstract

The present invention provides a fermentation medium and its application in the preparation of teriparatide, belonging to the field of biopharmaceutical technology. The fermentation medium comprises the following components: yeast peptone, yeast extract powder, dipotassium hydrogen phosphate trihydrate, potassium dihydrogen phosphate, ammonium sulfate, magnesium sulfate heptahydrate, glycerol, trace element solution, defoamer, and the solvent is pure water. The trace element solution added to the fermentation medium of the present invention controls the type and amount of trace elements to obtain a medium that is more conducive to the expression of inclusion bodies. In addition, the strategy of starting at a fixed time point and adjusting the feed flow rate is adopted during the fermentation process, thereby improving the stability of fermentation production.

Description

Technical Field

[0001] The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a fermentation medium and an application thereof in the preparation of teriparatide. Background Art

[0002] Osteoporosis (OP) is a metabolic bone disease caused by bone loss and bone mass reduction due to various reasons, leading to increased bone brittleness and even fractures. Osteoporosis can be divided into primary and secondary types. Type I primary osteoporosis occurs in postmenopausal women, and type II primary osteoporosis occurs in the elderly. The risk of fractures caused by osteoporosis seriously affects the patient's mind, body and quality of life. Osteoporosis often has no significant clinical symptoms in the early stages, so early prevention and treatment are of great significance. Currently, the drugs commonly used in clinical practice are mainly three categories of drugs: those that promote bone mineralization, those that resist bone resorption, and those that enhance bone synthesis.

[0003] The molecular formula of teriparatide is C 181 H 291 N 55 O 51 S2, with a molecular weight of 4117.72 and a theoretical isoelectric point of 8.29, is a parathyroid hormone (PTH) derivative. It is a biologically active 1-34 amino acid fragment of the N-terminal region of 84 amino acids of endogenous human parathyroid hormone. Its immunological and biological properties are exactly the same as those of endogenous parathyroid hormone.

[0004] Teriparatide is an anti-osteoporosis drug that helps regulate calcium metabolism and promote bone formation. It promotes new bone formation by increasing the activity and number of osteoblasts, improving bone strength, effectively reducing the risk of vertebral and non-spinal fractures, improving back pain, and improving mobility. The drug promotes the growth of new bone, while other osteoporosis drugs increase bone density by inhibiting bone resorption or decomposition.

[0005] Teriparatide is generally prepared by fermentation with genetically engineered bacteria. How to increase the yield of teriparatide by fermentation has gradually become a hot topic.

[0006] The use of E. coli for recombinant expression of teriparatide has been reported in some literature and patents. For example, in the authorized patent CN110938151B, Feng Qiang et al. constructed pET28a-HSTP / BL21 (DE3) engineering bacteria, used TB medium containing 50 μg / ml kanamycin, cultured in a 60L fermentation volume for 5 hours and induced with IPTG for 3 hours. The average wet strains of the three batches of fermentation were 66.7, 65.4 and 66.7 g / L, and the average inclusion body yields were 11.6, 9.3 and 8.3 g / L, respectively.

[0007] Attempts have also been made to increase the expression of recombinant protein products by increasing the oxygen supply and achieving high-density fermentation. In Chinese patent CN111378027B, Wen Liangzhu et al. attempted to use high-density fermentation to recombinantly express the precursor of semaglutide (similar in size to teriparatide). The engineered bacteria also used BL21 (DE3) host bacteria to express the target polypeptide in the form of inclusion bodies. This study used 10g / L glucose, 30g / L yeast powder, 4g / L potassium dihydrogen phosphate, 4g / L disodium hydrogen phosphate, 6g / L ammonium sulfate, 0.02g / L calcium chloride dihydrate, and 10g / L magnesium sulfate heptahydrate as fermentation medium, and 20g / L yeast powder and 30g / L glucose as feed medium; the temperature was controlled at 34°C, pH 6.9, and dissolved oxygen was above 30%. After 6 hours of IPTG induction, this expression method was used to increase the wet bacterial weight of the fermentation to 145g / L. In another Chinese patent CN 104592381 A, Wen Liangzhu et al. attempted to use high-density fermentation to recombinantly express liraglutide (similar in size to teriparatide). The engineered bacteria also used BL21 (DE3) as the host bacteria to express the target polypeptide in the form of inclusion bodies. This study used glucose 10g / L; yeast powder: 30g / L; disodium hydrogen phosphate 4g / L; potassium dihydrogen phosphate 4g / L; ammonium sulfate 6g / L; magnesium sulfate heptahydrate 10g / L; calcium chloride dihydrate 0.02g / L; VB1 trace as the fermentation medium, yeast powder: 20g / L, glucose: 30g / L as the feed medium; after 6h of feeding, the temperature was controlled at 30℃, pH 7.0, dissolved oxygen 30%, and IPTG was used for induction for 6 hours. This expression method was used to increase the wet bacterial weight of the fermentation to 178g / L, and the wet weight of the inclusion body was 30g / L of the fermentation liquid.

[0008] There are also reports on designs for adapting to high-density fermentation of teriparatide through equipment modification. For example, in Chinese patent CN219174485 U, Deng Quanyu et al. improved the stirring effect during teriparatide fermentation by optimizing the stirring paddle, but the patent did not disclose the specific process of high-density fermentation of teriparatide and the expression level of teriparatide inclusion bodies.

[0009] However, the yield of teriparatide inclusion bodies prepared by the existing methods in Escherichia coli high-density fermentation is low and cannot better meet the needs. Therefore, it is urgent to develop a fermentation medium and fermentation method that can increase the yield of teriparatide in Escherichia coli high-density fermentation. Summary of the invention

[0010] Based on the deficiencies in the prior art, the present invention optimizes the fermentation conditions of teriparatide to obtain a fermentation medium, which can increase the yield of teriparatide in high-density fermentation of Escherichia coli.

[0011] The fermentation medium of the present invention can increase the yield of inclusion bodies of teriparatide in high-density fermentation of Escherichia coli to 40-45 g / L, and the yield of teriparatide after purification of the inclusion bodies can reach up to 1754.8 mg / L.

[0012] In order to achieve the above object, the present invention adopts the following technical solutions:

[0013] In one aspect, the present invention provides a fermentation medium, the fermentation medium comprising the following components: 12-20 g / L yeast peptone, 4-10 g / L yeast extract powder, 3-5 g / L dipotassium hydrogen phosphate trihydrate, 0.5-2 g / L potassium dihydrogen phosphate, 0.5-3 g / L ammonium sulfate, 0.5-2 g / L magnesium sulfate heptahydrate, 3-10 g / L glycerol, 1-3 ml / L trace element solution, 0.1-0.5 g / L defoamer, the solvent is pure water, and the pH is adjusted to 6.8-7.2 with 2 mol / L sodium hydroxide solution;

[0014] The trace element solution contains nickel sulfate, titanium tetrachloride and ferrous chromite.

[0015] Preferably, the concentrations of nickel sulfate, titanium tetrachloride and ferrous chromite are 1-3 g / L, 0.5-2 g / L and 10-20 g / L respectively.

[0016] More preferably, the trace element solution further comprises the following components: disodium ethylenediaminetetraacetic acid dihydrate, manganese sulfate, sodium molybdate dihydrate, cobalt chloride, zinc sulfate, copper sulfate and boric acid, and the solvent is pure water.

[0017] Further preferably, the trace element solution also includes the following components: 20-30 g / L disodium ethylenediaminetetraacetic acid dihydrate, 8-10 g / L manganese sulfate, 0.5-1.5 g / L sodium molybdate dihydrate, 16-20 g / L cobalt chloride, 6-10 g / L zinc sulfate, 4-6 g / L copper sulfate and 2-4 g / L boric acid, and the solvent is pure water.

[0018] As some preferred embodiments, the trace element solution includes the following components: 20-30 g / L disodium ethylenediaminetetraacetic acid dihydrate, 8-10 g / L manganese sulfate, 10-20 g / L ferrous chromite, 1-3 g / L nickel sulfate, 16-20 g / L cobalt chloride, 0.5-2 g / L titanium tetrachloride, 6-10 g / L zinc sulfate, 4-6 g / L copper sulfate, 0.5-1.5 g / L sodium molybdate dihydrate, 2-4 g / L boric acid, and the solvent is pure water.

[0019] Preferably, the fermentation medium comprises the following components: 15-18 g / L yeast peptone, 5-8 g / L yeast extract powder, 4-5 g / L potassium dihydrogen phosphate trihydrate, 1-1.5 g / L potassium dihydrogen phosphate, 1-2 g / L ammonium sulfate, 1-1.5 g / L magnesium sulfate heptahydrate, 5-8 g / L glycerol, 1.5-2.5 ml / L trace element solution, 0.2-0.4 g / L defoamer, the solvent is pure water, and the pH is adjusted to 6.8-7.2 with 2 mol / L sodium hydroxide solution;

[0020] The trace element solution comprises the following components: 22-28 g / L of disodium ethylenediaminetetraacetate dihydrate, 9-10 g / L of manganese sulfate, 12-18 g / L of ferrous chromite, 1.5-2.5 g / L of nickel sulfate, 17-19 g / L of cobalt chloride, 1-1.5 g / L of titanium tetrachloride, 7-9 g / L of zinc sulfate, 4.5-5.5 g / L of copper sulfate, 1-1.5 g / L of sodium molybdate dihydrate, 2.5-3.5 g / L of boric acid, and the solvent is pure water.

[0021] As a preferred embodiment, the fermentation medium comprises the following components: 20 g / L yeast peptone, 10 g / L yeast extract powder, 5 g / L dipotassium hydrogen phosphate trihydrate, 2 g / L potassium dihydrogen phosphate, 3 g / L ammonium sulfate, 2 g / L magnesium sulfate heptahydrate, 10 g / L glycerol, 3 ml / L trace element solution, 0.5 g / L defoamer, the solvent is pure water, and the pH is adjusted to 7.2 with 2 mol / L sodium hydroxide solution;

[0022] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0023] As another preferred embodiment, the fermentation medium comprises the following components: 12 g / L yeast peptone, 4 g / L yeast extract powder, 3 g / L potassium dihydrogen phosphate trihydrate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 3 g / L glycerol, 1 ml / L trace element solution, 0.1 g / L defoamer, the solvent is pure water, and the pH is adjusted to 6.8 with 2 mol / L sodium hydroxide solution;

[0024] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0025] The defoamer is selected from one or more of an organosilicon defoamer, a polymer defoamer, and a biological defoamer (i.e., a fermentation-specific defoamer), and is preferably a biological defoamer (i.e., a fermentation-specific defoamer).

[0026] On the other hand, the present invention also provides the use of the above fermentation medium in the preparation of teriparatide.

[0027] In another aspect, the present invention also provides a method for preparing teriparatide inclusion bodies using the above fermentation medium, comprising the following steps:

[0028] (1) firstly preparing the other components except the trace element solution into a culture medium A, then filtering the trace element solution and adding it into the culture medium A to obtain a fermentation culture medium; then inoculating the seed liquid into the culture medium to carry out fermentation;

[0029] (2) When the OD600 of the fermentation liquid is ≥60, an inducer is added and the temperature is lowered. After the induction expression is induced for 5-7 hours, the fermentation ends and the fermentation liquid is obtained. First, the fermentation liquid is centrifuged to collect the bacterial precipitate, and the bacterial precipitate is resuspended, crushed, and centrifuged to obtain inclusion bodies.

[0030] As some preferred embodiments, the filtration in step (1) is performed using a 0.22 micron filter membrane.

[0031] The inoculation amount of the seed solution in step (1) is 5-10 v / v%, the initial culture conditions are 37° C., pH 6.7-7.3, and dissolved oxygen ≥ 30%.

[0032] The dissolved oxygen content is controlled by the stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1-1.5 vvm.

[0033] During the fermentation process described in step (1), feed needs to be added continuously, and the feed flow rate needs to be gradually increased as the density of the microbial cells increases.

[0034] The specific variable speed feeding scheme is as follows: start feeding at 2.5 ml / h·L after 1 hour of cultivation -1 After 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 When OD600 ≥ 30, the feeding rate is increased to 40 ml / h·L -1 .

[0035] The feed comprises the following components: 180-220 g / L yeast peptone, 80-120 g / L yeast extract powder, 350-400 g / L glycerol, 1-2 g / L defoamer, and the solvent is pure water;

[0036] As a preferred embodiment, the feed comprises the following components: 220 g / L yeast peptone, 120 g / L yeast extract powder, 400 g / L glycerol, 2 g / L defoaming agent, and the solvent is pure water.

[0037] The defoamer is a biological defoamer.

[0038] The inducing agent in step (2) is IPTG, and the amount of the inducing agent added is 0.1-0.5 mmol / L; the cooling temperature is 30°C.

[0039] The resuspension operation described in step (3) is: adding a lysis buffer to the bacterial precipitate and then resuspending to obtain a resuspension; the mass volume ratio of the bacterial precipitate to the lysis buffer is 1kg:5-20L.

[0040] The bacterium-breaking buffer comprises the following components: 4-8 g / L of tris(hydroxymethyl)aminomethane, 0.2-0.5 g / L of disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0041] As a preferred embodiment, the bacteria-breaking buffer comprises the following components: 8 g / L tris(hydroxymethyl)aminomethane, 0.5 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0042] As another preferred embodiment, the bacteria-breaking buffer comprises the following components: 4 g / L tris(hydroxymethyl)aminomethane, 0.2 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0043] Compared with the prior art, the beneficial effects of this application are:

[0044] 1. The high-density fermentation medium of teriparatide of the present invention has clear components and a stable source, which is more conducive to ensuring product quality in the production of biological drugs;

[0045] 2. The composition of the trace element solution added to the teriparatide high-density fermentation medium of the present invention is more conducive to the expression of inclusion bodies;

[0046] 3. The high-density fermentation method of teriparatide described in the present invention adopts a strategy of starting at a fixed time point and adjusting the feed flow rate, which is more conducive to the stability of fermentation production.

[0047] 4. The high-density fermentation method of teriparatide described in the present invention adopts a low-temperature induction method, which is more conducive to reducing the side effects of the inducer on bacterial growth in the fermentation environment and improving the expression rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a schematic diagram of the construction of the recombinant plasmid in Basic Example 1. DETAILED DESCRIPTION

[0049] The principles and features of the present invention are described below in conjunction with embodiments. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0050] The implementation of the present invention is based on the following basic embodiments, however, the basic embodiments are only used to assist in demonstrating the technical effects of the invention rather than to limit the technical solutions.

[0051] Basic Example 1: Construction of recombinant engineering bacteria:

[0052] The gene sequence (shown in SEQ ID No.2) encoding the fusion protein of PTH (1-34) and propeptide (SEQ ID No.1) was inserted into the expression vector pET28a. A DDDDK amino acid sequence was inserted between the propeptide sequence and the PTH (1-34) sequence for enterokinase cleavage and removal of the propeptide during purification, and a linker composed of GGGS repeating fragments increased the flexibility near the restriction site, reduced the influence of the propeptide on the enterokinase binding cleavage site, and improved the cleavage efficiency. The recombinant plasmid was amplified by the Gibson cloning method. The primers for the amplified sequences were the plasmid backbone forward primer (SEQ ID No.3) A1: 5'-GGTATATCTCCTTCTTAAAGTTAAACAAAA-3'; the reverse primer (SEQ ID No.4) A2: 5'-TAACAAAGCCCGAAAGGAAGCTGAGTTGGC-3'. Insert fragment forward primer (SEQ ID No.5) A3: 5'-GCTTTGTTAAAAGTTATGCACATCCTGCAG-3'; reverse primer (SEQ ID No.6) A4: 5'-TATACCATGAAAACCTTTGAAATTCTGAAA-3'. The amplified fragment was digested with T5 exonuclease. The digested plasmid backbone and insert sequence were mixed and incubated with Taq DNA ligase and transformed into Escherichia coli (E.coliBL21(DE3)) and screened on kanamycin plates to obtain recombinant engineered bacteria.

[0053] SEQ ID No.1:

[0054] MKTFEILKHLQADAIVLFMKVHNFHWNVKGTDFFNVHKATEEIYEEFADMFDDLAERIVQLGHHPLVTLSEAIKLTRVKEETKTSFHSKGGGSGGGSGGGSDDDDKSVSEIQLMHNLGKHLNSMERVEWLRKKLQDVHNF

[0055] SEQ ID No.2:

[0056] TTAAAAGTTATGCACATCCTGCAGTTTTTTGCGCAGCCATTCCACGCGTTCCATGCTGTTCAGATGTTTGCCCAGGTTATGCATCAGCTGAATTTCGCTCACGCTTTTATCATCATCATCGCTGCCGCCGCCGCTGCCGCCGCCGCTGCCGCCGCCTTTGCTATGAAAGCTGGTTTTGGTTTCTTTCTTCACGCGGGTCAGTTTAATCGCT TCGCTCAGGGTCACCAGCGGATGATGGCCCAGCTGCACAATGCGTTCCGCCAGATCATCAAACATATCCGCAAATTCTTCATAAATTTCTTCGGTCGCTTTATGCACGTTAAAAAAATCGGTGCCTTTCACGTTCCAATGAAAGTTATGCACTTTCATAAACAGCACAATCGCATCCGCCTGCAGATGTTTCAGAATTTCAAAGGTTTTCAT

[0057] Basic Example 2: Preparation of Seed Solution:

[0058] The recombinant engineered bacteria prepared in Basic Example 1 were inoculated into a culture medium containing LB 50 μg / ml of kanamycin and cultured at 37°C in a shaking incubator (220 rpm) until the OD 600 At about 1-2 hours, IPTG solution with a final concentration of 50 μg / ml was added. After continuing to culture for 6 hours, 300 OD bacterial solution was centrifuged at 12000g for 5 minutes to collect the bacteria, and the bacteria were resuspended with 20 μl of pure water, and then 20 μl of reducing sample buffer was added, placed in a 100°C metal bath for 5 minutes, and 10 μl of sample was loaded for SDS-PAGE electrophoresis. Electrophoresis conditions: 4.5% concentrated gel 120V electrophoresis for 15 minutes, 15% separation gel 180V electrophoresis for 45 minutes. After the electrophoresis, the gel was stained with Coomassie brilliant blue R250 and decolorized with decolorizing solution (acetic acid: ethanol: water -1:2:7), and the expression of teriparatide was detected on the BIO-RAD gel imaging system, and strains with high expression levels were selected as seeds for preservation.

[0059] The above seeds were inoculated into 2YT medium and cultured at 35°C and 220 rpm in a shaking incubator until OD 600 End the culture when the OD is 4.5-6.0 and dilute it with 2YT medium to OD 6004.5 The seed solution for fermentation is obtained. The seed solution for fermentation in the examples and comparative examples of the present invention is prepared according to this method.

[0060] Basic Example 3: The specific steps of obtaining the teriparatide stock solution after denaturation, enzyme digestion, impurity precipitation, cation exchange chromatography, reverse phase chromatography and concentration of the inclusion bodies are as follows:

[0061] (1) Inclusion body denaturation

[0062] The prepared inclusion bodies were denatured and dissolved using 8 mol / L urea solution, with the mass volume ratio of the inclusion bodies to the solution being about 1:9, and the solution was stirred until it became clear to obtain a denatured solution;

[0063] (2) Fusion protease cleavage

[0064] Add a CaCl2 solution with a final concentration of 2 mmol / L and enterokinase with a titer corresponding to the amount of inclusion bodies (1 U enterokinase per mg of inclusion bodies) to the denaturing solution of step (1), stir evenly, let stand at 15°C for 6 h, add an EDTA solution with a final concentration of 0.02 mol / L to stop the enzyme digestion, and obtain an enzyme digestion solution;

[0065] (3) Protein impurity precipitation

[0066] Adding polyethyleneimine at a final concentration of 0.1% (M / V) to the digestion solution of step (2) to precipitate most of the host protein and nucleic acid protein, standing at 4°C for 30 minutes, and centrifuging at 12000g for 30 minutes to collect the supernatant;

[0067] (4) Cation exchange chromatography

[0068] The supernatant of the centrifugation in step (3) was filtered through a 0.2 μm filter membrane and loaded with a sample. Impurities such as propeptides were removed by cation exchange chromatography. Q Sepharose SP was used as the filler of the cation exchange chromatography column. The column volume was 25 L, the loading flow rate was 120 cm / h, the equilibrium buffer A solution was 0.01 mol / L pH 6.0 phosphate buffer, and the equilibrium buffer B solution was 0.01 mol / L pH 6.0 phosphate + 1 mol / L NaCl buffer;

[0069] (5) Reverse Phase Chromatography

[0070] The eluent in step (4) was subjected to chromatography to remove impurities with different polarity from teriparatide; C18-100 was used as the filler for reverse phase chromatography. The column volume was 25 L, the sample flow rate was 160 cm / h, the buffer A solution was 20 mmol / L sodium dihydrogen phosphate solution: acetonitrile (90:10) (pH 4.0), and the buffer B solution was 20 mmol / L sodium dihydrogen phosphate solution: acetonitrile (50:50) (pH 4.0);

[0071] (6) Concentration

[0072] The eluate in step (5) was concentrated by cation exchange chromatography to achieve a purity of teriparatide stock solution of more than 95%. Q Sepharose SP was used as the filler of the cation exchange chromatography column, with a column volume of 25 L, a sample loading flow rate of 100 cm / h, and a 0.01 mol / L pH 6.0 phosphate buffer solution A and a 0.01 mol / L pH 6.0 phosphate + 1 mol / L NaCl buffer solution B.

[0073] The following are examples directly related to the invention. If no specific conditions are specified in the examples, the conventional conditions or the conditions recommended by the manufacturer are followed. If the manufacturer of the reagents used is not specified, they are all conventional products that can be purchased commercially. The defoamer used in the following examples was purchased from Yantai Hengxin Chemical Technology Co., Ltd., and the model number is THIX-298 high-efficiency fermentation special defoamer.

[0074] Example 1 A fermentation medium and its application in the preparation of teriparatide

[0075] The recombinant engineered bacteria prepared in Basic Example 1 were fermented and induced to express to obtain teriparatide inclusion bodies; the fermentation was carried out using high-density fermentation, specifically as follows:

[0076] The fermentation medium comprises the following components: 12 g / L yeast peptone, 4 g / L yeast extract powder, 3 g / L potassium dihydrogen phosphate trihydrate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 3 g / L glycerol, 1 ml / L trace element solution, 0.1 g / L defoamer, the solvent is pure water, and the pH is adjusted to 6.95 with 2 mol / L sodium hydroxide solution;

[0077] The trace element solution comprises the following components: 20 g / L disodium ethylenediaminetetraacetate dihydrate, 8 g / L manganese sulfate, 10 g / L ferrous chromite, 1 g / L nickel sulfate, 16 g / L cobalt chloride, 0.5 g / L titanium tetrachloride, 6 g / L zinc sulfate, 4 g / L copper sulfate, 0.5 g / L sodium molybdate dihydrate, 2 g / L boric acid, and the solvent is pure water.

[0078] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0079] (1) First, the other components except the trace element solution were prepared into medium A, and the trace element solution was filtered through a 0.22 μm filter membrane and then added to the medium A to obtain a fermentation medium. Then, the seed solution (OD 6004.5) inoculating the culture medium for fermentation, with an inoculation amount of 5% (v / v), initial culture conditions of 37°C, pH 7.0, and controlling dissolved oxygen ≥ 30% by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1 vvm;

[0080] The fermentation process requires variable speed feeding, specifically: feeding starts at 2.5 ml / h·L after 1 hour of cultivation -1 , after 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 , when OD 600 When the feed rate is ≥30, it is increased to 40ml / h·L -1 ;

[0081] (2) When the fermentation liquid OD 600 When the pH value was ≥60, the inducer IPTG was added in an amount of 0.1 mmol / L, the fermentation control temperature was lowered to 30°C, and the fermentation was terminated after 5 hours of induction expression to obtain a fermentation broth; the fermentation broth was centrifuged to collect the bacterial precipitate, the bacterial body was resuspended in a lysis buffer (1 kg of bacterial body was added to 10 L of lysis buffer) to obtain a resuspension, the resuspension was crushed by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body, and the inclusion body was deformed, digested, precipitated, cation exchange chromatography, reverse phase chromatography and concentrated to obtain the teriparatide stock solution.

[0082] The feed in step (1) comprises the following components: 180 g / L yeast peptone, 80 g / L yeast extract powder, 350 g / L glycerol, 1 g / L defoaming agent, and the solvent is pure water.

[0083] The bacteriolysis buffer described in step (2) comprises the following components: 4 g / L tris(hydroxymethyl)aminomethane, 0.2 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0084] The inclusion body yield calculated by weighing was 0.243 g / g 菌体 , 40.1g / L 发酵液 .

[0085] The yield of teriparatide was calculated to be 40 mg / g after the purified stock solution was tested. 包涵体 , 1604.0mg / L 发酵液 .

[0086] Example 2 A fermentation medium and its application in the preparation of teriparatide

[0087] The recombinant engineered bacteria prepared in Basic Example 1 were fermented and induced to express to obtain teriparatide inclusion bodies; the fermentation was carried out using high-density fermentation, specifically as follows:

[0088] The fermentation medium comprises the following components: 20 g / L yeast peptone, 10 g / L yeast extract powder, 5 g / L potassium dihydrogen phosphate trihydrate, 2 g / L potassium dihydrogen phosphate, 3 g / L ammonium sulfate, 2 g / L magnesium sulfate heptahydrate, 10 g / L glycerol, 3 ml / L trace element solution, 0.5 g / L defoamer, the solvent is pure water, and the pH is adjusted to 7.1 with 2 mol / L sodium hydroxide solution;

[0089] The trace element solution comprises the following components: 20 g / L disodium ethylenediaminetetraacetate dihydrate, 8 g / L manganese sulfate, 10 g / L ferrous chromite, 1 g / L nickel sulfate, 16 g / L cobalt chloride, 0.5 g / L titanium tetrachloride, 6 g / L zinc sulfate, 4 g / L copper sulfate, 0.5 g / L sodium molybdate dihydrate, 2 g / L boric acid, and the solvent is pure water.

[0090] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0091] (1) First, the other components except the trace element solution were prepared into medium A, and the trace element solution was filtered through a 0.22 μm filter membrane and then added to the medium A to obtain a fermentation medium. Then, the seed solution (OD 600 4.5) inoculating the culture medium for fermentation, with an inoculation amount of 6% (v / v), initial culture conditions of 37°C, pH 7.0, and controlling dissolved oxygen ≥ 30% by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1 vvm;

[0092] The fermentation process requires variable speed feeding, specifically: feeding starts at 2.5 ml / h·L after 1 hour of cultivation -1 , after 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 , when OD 600 When the feed rate is ≥30, it is increased to 40ml / h·L -1 ;

[0093] (2) When the fermentation liquid OD 600 When the pH value was ≥60, the inducer IPTG was added in an amount of 0.2 mmol / L, the fermentation control temperature was lowered to 30°C, and the fermentation was terminated after 5 hours of induction expression to obtain a fermentation broth; the fermentation broth was centrifuged to collect the bacterial precipitate, the bacterial body was resuspended in a lysis buffer (1 kg of bacterial body was added to 10 L of lysis buffer) to obtain a resuspension, the resuspension was crushed by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body, and the inclusion body was deformed, digested, precipitated, cation exchange chromatography, reverse phase chromatography and concentrated to obtain the teriparatide stock solution.

[0094] The feed in step (1) comprises the following components: 180 g / L yeast peptone, 80 g / L yeast extract powder, 350 g / L glycerol, 1 g / L defoaming agent, and the solvent is pure water.

[0095] The bacteriolysis buffer described in step (2) comprises the following components: 4 g / L tris(hydroxymethyl)aminomethane, 0.2 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0096] The inclusion body yield calculated by weighing was 0.241 g / g 菌体 , 40.5g / L 发酵液 .

[0097] The yield of teriparatide was calculated to be 38 mg / g after the purified stock solution was tested. 包涵体 , 1539.0mg / L 发酵液 .

[0098] Example 3 A fermentation medium and its application in the preparation of teriparatide

[0099] The recombinant engineered bacteria prepared in Basic Example 1 were fermented and induced to express to obtain teriparatide inclusion bodies; the fermentation was carried out using high-density fermentation, specifically as follows:

[0100] The fermentation medium comprises the following components: 12 g / L yeast peptone, 4 g / L yeast extract powder, 3 g / L potassium dihydrogen phosphate trihydrate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 3 g / L glycerol, 1 ml / L trace element solution, 0.1 g / L defoamer, the solvent is pure water, and the pH is adjusted to 6.8 with 2 mol / L sodium hydroxide solution;

[0101] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0102] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0103] (1) First, the other components except the trace element solution were prepared into medium A, and the trace element solution was filtered through a 0.22 μm filter membrane and then added to the medium A to obtain a fermentation medium. Then, the seed solution (OD 600 4.5) inoculating the culture medium for fermentation, with an inoculation amount of 7% (v / v), initial culture conditions of 37°C, pH 7.0, and controlling dissolved oxygen ≥ 30% by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1 vvm;

[0104] The fermentation process requires variable speed feeding, specifically: feeding starts at 2.5 ml / h·L after 1 hour of cultivation -1 , after 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 , when OD 600 When the feed rate is ≥30, it is increased to 40ml / h·L -1 ;

[0105] (2) When the fermentation liquid OD 600 When the pH value was ≥60, the inducer IPTG was added in an amount of 0.2 mmol / L, the fermentation control temperature was lowered to 30°C, and the fermentation was terminated after 5 hours of induction expression to obtain a fermentation broth; the fermentation broth was centrifuged to collect the bacterial precipitate, the bacterial body was resuspended in a lysis buffer (1 kg of bacterial body was added to 10 L of lysis buffer) to obtain a resuspension, the resuspension was crushed by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body, and the inclusion body was deformed, digested, precipitated, cation exchange chromatography, reverse phase chromatography and concentrated to obtain the teriparatide stock solution.

[0106] The feed in step (1) comprises the following components: 220 g / L yeast peptone, 120 g / L yeast extract powder, 400 g / L glycerol, 2 g / L defoaming agent, and the solvent is pure water.

[0107] The bacteriolysis buffer described in step (2) comprises the following components: 4 g / L tris(hydroxymethyl)aminomethane, 0.2 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0108] The inclusion body yield calculated by weighing was 0.259 g / g 菌体 , 43.6g / L 发酵液 .

[0109] The yield of teriparatide was calculated to be 40 mg / g after the purified stock solution was tested. 包涵体 , 1744.0mg / L 发酵液 .

[0110] Example 4 A fermentation medium and its application in the preparation of teriparatide

[0111] The recombinant engineered bacteria prepared in Basic Example 1 were fermented and induced to express to obtain teriparatide inclusion bodies; the fermentation was carried out using high-density fermentation, specifically as follows:

[0112] The fermentation medium comprises the following components: 20 g / L yeast peptone, 10 g / L yeast extract powder, 5 g / L potassium dihydrogen phosphate trihydrate, 2 g / L potassium dihydrogen phosphate, 3 g / L ammonium sulfate, 2 g / L magnesium sulfate heptahydrate, 10 g / L glycerol, 3 ml / L trace element solution, 0.5 g / L defoamer, the solvent is pure water, and the pH is adjusted to 7.2 with 2 mol / L sodium hydroxide solution;

[0113] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0114] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0115] (1) First, the other components except the trace element solution were prepared into medium A, and the trace element solution was filtered through a 0.22 μm filter membrane and then added to the medium A to obtain a fermentation medium. Then, the seed solution (OD 600 4.5) inoculating the culture medium for fermentation, with an inoculation amount of 6% (v / v), initial culture conditions of 37°C, pH 7.0, and controlling dissolved oxygen ≥ 30% by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1.2 vvm;

[0116] The fermentation process requires variable speed feeding, specifically: feeding starts at 2.5 ml / h·L after 1 hour of cultivation -1 , after 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 , when OD 600 When the feed rate is ≥30, it is increased to 40ml / h·L -1 ;

[0117] (2) When the fermentation liquid OD 600 When the pH value was ≥60, the inducer IPTG was added in an amount of 0.3 mmol / L, the fermentation control temperature was lowered to 30°C, and the fermentation was terminated after 5 hours of induction expression to obtain a fermentation broth; the fermentation broth was centrifuged to collect the bacterial precipitate, the bacterial body was resuspended in a lysis buffer (1 kg of bacterial body was added to 5 L of lysis buffer) to obtain a resuspension, the resuspension was crushed by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body, and the inclusion body was deformed, digested, precipitated, cation exchange chromatography, reverse phase chromatography and concentrated to obtain the teriparatide stock solution.

[0118] The feed in step (1) comprises the following components: 220 g / L yeast peptone, 120 g / L yeast extract powder, 400 g / L glycerol, 2 g / L defoaming agent, and the solvent is pure water.

[0119] The bacteriolysis buffer described in step (2) comprises the following components: 8 g / L tris(hydroxymethyl)aminomethane, 0.5 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0120] The inclusion body yield calculated by weighing was 0.279 g / g 菌体 , 44.1g / L 发酵液 .

[0121] The yield of teriparatide was calculated to be 39 mg / g after the purified stock solution was tested. 包涵体 , 1719.9mg / L 发酵液 .

[0122] Example 5 A fermentation medium and its application in the preparation of teriparatide

[0123] The recombinant engineered bacteria prepared in Basic Example 1 were fermented and induced to express to obtain teriparatide inclusion bodies; the fermentation was carried out using high-density fermentation, specifically as follows:

[0124] The fermentation medium comprises the following components: 12 g / L yeast peptone, 4 g / L yeast extract powder, 3 g / L potassium dihydrogen phosphate trihydrate, 0.5 g / L potassium dihydrogen phosphate, 0.5 g / L ammonium sulfate, 0.5 g / L magnesium sulfate heptahydrate, 3 g / L glycerol, 1 ml / L trace element solution, 0.1 g / L defoamer, the solvent is pure water, and the pH is adjusted to 6.8 with 2 mol / L sodium hydroxide solution;

[0125] The trace element solution comprises the following components: 20 g / L disodium ethylenediaminetetraacetate dihydrate, 8 g / L manganese sulfate, 10 g / L ferrous chromite, 1 g / L nickel sulfate, 16 g / L cobalt chloride, 0.5 g / L titanium tetrachloride, 6 g / L zinc sulfate, 4 g / L copper sulfate, 0.5 g / L sodium molybdate dihydrate, 2 g / L boric acid, and the solvent is pure water.

[0126] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0127] (1) First, the other components except the trace element solution were prepared into medium A, and the trace element solution was filtered through a 0.22 μm filter membrane and then added to the medium A to obtain a fermentation medium. Then, the seed solution (OD 600 4.5) inoculating the culture medium for fermentation, with an inoculation amount of 8% (v / v), initial culture conditions of 37°C, pH 7.0, and controlling dissolved oxygen ≥ 30% by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1.5 vvm;

[0128] The fermentation process requires variable speed feeding, specifically: feeding starts at 2.5 ml / h·L after 1 hour of cultivation -1 , after 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 , when OD 600 When the feed rate is ≥30, it is increased to 40ml / h·L -1 ;

[0129] (2) When the fermentation liquid OD 600 When the pH value was ≥60, the inducer IPTG was added in an amount of 0.4 mmol / L, the fermentation control temperature was lowered to 30°C, and the fermentation was terminated after 5 hours of induction expression to obtain a fermentation broth; the fermentation broth was centrifuged to collect the bacterial precipitate, the bacterial body was resuspended in a lysis buffer (1 kg of bacterial body was added to 20 L of lysis buffer) to obtain a resuspension, the resuspension was crushed by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body, and the inclusion body was deformed, digested, precipitated, cation exchange chromatography, reverse phase chromatography and concentrated to obtain the teriparatide stock solution.

[0130] The feed in step (1) comprises the following components: 220 g / L yeast peptone, 120 g / L yeast extract powder, 400 g / L glycerol, 2 g / L defoaming agent, and the solvent is pure water.

[0131] The bacteriolysis buffer described in step (2) comprises the following components: 8 g / L tris(hydroxymethyl)aminomethane, 0.5 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0132] The inclusion body yield calculated by weighing was 0.252 g / g 菌体 , 42.9g / L 发酵液 .

[0133] The yield of teriparatide was calculated to be 38 mg / g after the purified stock solution was tested. 包涵体 , 1630.2mg / L 发酵液 .

[0134] Example 6 A fermentation medium and its application in the preparation of teriparatide

[0135] The recombinant engineered bacteria prepared in Basic Example 1 were fermented and induced to express to obtain teriparatide inclusion bodies; the fermentation was carried out using high-density fermentation, specifically as follows:

[0136] The fermentation medium comprises the following components: 20 g / L yeast peptone, 10 g / L yeast extract powder, 5 g / L potassium dihydrogen phosphate trihydrate, 2 g / L potassium dihydrogen phosphate, 3 g / L ammonium sulfate, 2 g / L magnesium sulfate heptahydrate, 10 g / L glycerol, 3 ml / L trace element solution, 0.5 g / L defoamer, the solvent is pure water, and the pH is adjusted to 7.2 with 2 mol / L sodium hydroxide solution;

[0137] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0138] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0139] (1) First, the other components except the trace element solution were prepared into medium A, and the trace element solution was filtered through a 0.22 μm filter membrane and then added to the medium A to obtain a fermentation medium. Then, the seed solution (OD 600 4.5) inoculating the culture medium for fermentation, with an inoculation amount of 5% (v / v), initial culture conditions of 37°C, pH 7.0, and controlling dissolved oxygen ≥ 30% by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1 vvm;

[0140] The fermentation process requires variable speed feeding, specifically: feeding starts at 2.5 ml / h·L after 1 hour of cultivation -1 , after 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 , when OD 600 When the feed rate is ≥30, it is increased to 40ml / h·L -1 ;

[0141] (2) When the fermentation liquid OD 600 When the pH value was ≥60, the inducer IPTG was added in an amount of 0.5 mmol / L, the fermentation control temperature was lowered to 30°C, and the fermentation was terminated after 5 hours of induction expression to obtain a fermentation broth; the fermentation broth was centrifuged to collect the bacterial precipitate, the bacterial body was resuspended in a lysis buffer (1 kg of bacterial body was added to 10 L of lysis buffer) to obtain a resuspension, the resuspension was crushed by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body, and the inclusion body was deformed, digested, precipitated, cation exchange chromatography, reverse phase chromatography and concentrated to obtain the teriparatide stock solution.

[0142] The feed in step (1) comprises the following components: 180 g / L yeast peptone, 80 g / L yeast extract powder, 350 g / L glycerol, 1 g / L defoaming agent, and the solvent is pure water.

[0143] The bacteriolysis buffer described in step (2) comprises the following components: 8 g / L tris(hydroxymethyl)aminomethane, 0.5 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0144] The inclusion body yield calculated by weighing was 0.251 g / g 菌体 , 41.6g / L 发酵液 .

[0145] The yield of teriparatide was calculated to be 37 mg / g after the purified stock solution was tested. 包涵体 , 1539.2mg / L 发酵液 .

[0146] Example 7 A fermentation medium and its application in the preparation of teriparatide

[0147] The recombinant engineered bacteria prepared in Basic Example 1 were fermented and induced to express to obtain teriparatide inclusion bodies; the fermentation was carried out using high-density fermentation, specifically as follows:

[0148] The fermentation medium comprises the following components: 16 g / L yeast peptone, 7 g / L yeast extract powder, 4 g / L potassium dihydrogen phosphate trihydrate, 1.5 g / L potassium dihydrogen phosphate, 2 g / L ammonium sulfate, 1.5 g / L magnesium sulfate heptahydrate, 7 g / L glycerol, 2 ml / L trace element solution, 0.3 g / L defoamer, the solvent is pure water, and the pH is adjusted to 7.2 with 2 mol / L sodium hydroxide solution;

[0149] The trace element solution comprises the following components: 25 g / L disodium ethylenediaminetetraacetate dihydrate, 9 g / L manganese sulfate, 15 g / L ferrous chromite, 2 g / L nickel sulfate, 18 g / L cobalt chloride, 1 g / L titanium tetrachloride, 8 g / L zinc sulfate, 5 g / L copper sulfate, 1 g / L sodium molybdate dihydrate, 3 g / L boric acid, and the solvent is pure water.

[0150] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0151] (1) First, the other components except the trace element solution were prepared into medium A, and the trace element solution was filtered through a 0.22 μm filter membrane and then added to the medium A to obtain a fermentation medium. Then, the seed solution (OD 600 4.5) inoculating the culture medium for fermentation, with an inoculation amount of 5% (v / v), initial culture conditions of 37°C, pH 7.0, and controlling dissolved oxygen ≥ 30% by stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1 vvm;

[0152] The fermentation process requires variable speed feeding, specifically: feeding starts at 2.5 ml / h·L after 1 hour of cultivation -1 , after 1.5 hours, it increases to 5 ml / h·L -1 , then increase the feeding rate by 5 ml / h·L every 30 minutes -1 , up to 35ml / h·L -1 , when OD 600 When the feed rate is ≥30, it is increased to 40ml / h·L -1 ;

[0153] (2) When the fermentation liquid OD 600 When the pH value was ≥60, the inducer IPTG was added in an amount of 0.5 mmol / L, the fermentation control temperature was lowered to 30°C, and the fermentation was terminated after 5 hours of induction expression to obtain a fermentation broth; the fermentation broth was centrifuged to collect the bacterial precipitate, the bacterial body was resuspended in a lysis buffer (1 kg of bacterial body was added to 10 L of lysis buffer) to obtain a resuspension, the resuspension was crushed by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body, and the inclusion body was deformed, digested, precipitated, cation exchange chromatography, reverse phase chromatography and concentrated to obtain the teriparatide stock solution.

[0154] The feed in step (1) comprises the following components: 200 g / L yeast peptone, 100 g / L yeast extract powder, 375 g / L glycerol, 1.5 g / L defoaming agent, and the solvent is pure water.

[0155] The bacteriolysis buffer described in step (2) comprises the following components: 6 g / L tris(hydroxymethyl)aminomethane, 0.4 g / L disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

[0156] The inclusion body yield calculated by weighing was 0.268 g / g 菌体 , 42.8g / L 发酵液 .

[0157] The yield of teriparatide was calculated to be 41 mg / g after the purified stock solution was tested. 包涵体 , 1754.8mg / L 发酵液 .

[0158] Comparative Example 1 A fermentation medium and its application in the preparation of teriparatide

[0159] The difference from Example 4 is that no trace element solution is added, and the contents of other components and the preparation method are the same as those in Example 4.

[0160] The inclusion body yield calculated by weighing was 0.152 g / g 菌体 , 21.1g / L 发酵液 .

[0161] The yield of teriparatide was calculated to be 38 mg / g after the purified stock solution was tested. 包涵体 , 801.8mg / L 发酵液 .

[0162] Comparative Example 2 A fermentation medium and its application in the preparation of teriparatide

[0163] The difference from Example 4 is that the induction temperature is 37°C, and the contents of other components and the preparation method are the same as those in Example 4. Specifically:

[0164] When the OD600 of the fermentation liquid was ≥60, the inducer IPTG was added in an amount of 0.3 mmol / L, and the fermentation temperature was maintained at 37°C. After 5 hours of induction, the fermentation was terminated to obtain the fermentation liquid; the fermentation liquid was centrifuged to collect the bacterial precipitate, the bacteria were resuspended in a lysis buffer (1 kg of bacteria were added to 10 L of lysis buffer) to obtain a resuspension, the resuspension was broken by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected as the inclusion body.

[0165] The inclusion body yield calculated by weighing was 0.174 g / g 菌体 , 26.8g / L 发酵液 .

[0166] The yield of teriparatide was calculated to be 37 mg / g after the purified stock solution was tested. 包涵体 , 991.6mg / L 发酵液 .

[0167] Comparative Example 3 A fermentation medium and its application in the preparation of teriparatide

[0168] The difference from Example 4 is that: no trace element solution is added, the feed is fed at a constant rate during the fermentation process, and the induction temperature is 37°C. The contents of other components and the preparation method are the same as those in Example 4. Specifically:

[0169] The method for preparing teriparatide inclusion bodies comprises the following steps:

[0170] (1) The seed liquid (OD6004.5) was inoculated into the culture medium for fermentation, with an inoculum size of 5% (v / v), an initial culture condition of 37°C, a pH of 7.0, and the dissolved oxygen was controlled to be ≥30% by the stirring speed, aeration volume and oxygen, wherein the total aeration volume of air and oxygen was 1 vvm;

[0171] The fermentation process requires variable speed feeding, specifically: feeding starts at 20 ml / h·L-1 after 1 hour of cultivation, and this flow rate is used until the end of fermentation;

[0172] (2) When the OD600 of the fermentation liquid was ≥60, the inducer IPTG was added at a dosage of 0.1 mmol / L, and the fermentation temperature was maintained at 37°C. After 5 hours of induction, the fermentation was terminated to obtain the fermentation liquid; the fermentation liquid was centrifuged to collect the bacterial precipitate, and the bacterial cells were resuspended in a lysis buffer (1 kg of bacterial cells were added to 5 L of lysis buffer) to obtain a resuspension, the resuspension was broken by a high-pressure homogenizer and centrifuged again, and the centrifugal precipitate was collected, which was the inclusion body.

[0173] The inclusion body yield calculated by weighing was 0.166 g / g 菌体 , 23.7g / L 发酵液 .

[0174] The yield of teriparatide was calculated to be 40 mg / g after the purified stock solution was tested. 包涵体 , 948.0mg / L 发酵液 .

[0175] Comparative Example 4 A fermentation medium and its application in the preparation of teriparatide

[0176] The difference from Example 4 is that: no trace element solution is added, the feed is fed at a constant rate during the fermentation process, and the contents of other components and the preparation method are the same as those in Example 4. Specifically:

[0177] (1) The seed liquid (OD6004.5) was inoculated into the culture medium for fermentation, with an inoculum amount of 10% (v / v), an initial culture condition of 37°C, a pH of 7.0, and the dissolved oxygen was controlled to be ≥30% by the stirring speed, aeration volume and oxygen, wherein the total aeration volume of air and oxygen was 1.5 vvm;

[0178] The fermentation process requires variable speed feeding, specifically: feeding at 20 ml / h·L-1 begins after 1 hour of cultivation, and continues at this flow rate until the end of fermentation.

[0179] The inclusion body yield calculated by weighing was 0.168 g / g 菌体 , 25.9g / L 发酵液 .

[0180] The yield of teriparatide was calculated to be 38 mg / g after the purified stock solution was tested. 包涵体 , 984.2mg / L 发酵液 .

[0181] Comparative Example 5 A fermentation medium and its application in the preparation of teriparatide

[0182] The difference from Example 4 is that ferrous chromite is not contained, 32 g / L of ferrous sulfate heptahydrate is added, and the contents of other components and the preparation method are the same as those of Example 4. That is:

[0183] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetic acid dihydrate, 10 g / L manganese sulfate, 32 g / L ferrous sulfate heptahydrate, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0184] The inclusion body yield calculated by weighing was 0.231 g / g 菌体 , 36.2g / L 发酵液 .

[0185] The yield of teriparatide was calculated to be 39 mg / g after the purified stock solution was tested. 包涵体 , 1411.8mg / L 发酵液 .

[0186] Comparative Example 6 A fermentation medium and its application in the preparation of teriparatide

[0187] The difference from Example 4 is that titanium tetrachloride is not contained, and the contents of other components and the preparation method are the same as those of Example 4. That is:

[0188] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0189] The inclusion body yield calculated by weighing was 0.232 g / g 菌体 , 36.9g / L 发酵液 .

[0190] The yield of teriparatide was calculated to be 38 mg / g after the purified stock solution was tested. 包涵体 , 1402.2mg / L 发酵液 .

[0191] Comparative Example 7 A fermentation medium and its application in the preparation of teriparatide

[0192] The difference from Example 4 is that nickel sulfate is not contained, and the contents of other components and the preparation method are the same as those of Example 4. That is:

[0193] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetic acid dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 20 g / L cobalt chloride, 2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0194] The inclusion body yield calculated by weighing was 0.241 g / g 菌体 , 37.6g / L 发酵液 .

[0195] The yield of teriparatide was calculated to be 40 mg / g after the purified stock solution was tested. 包涵体 , 1504.0mg / L 发酵液 .

[0196] Comparative Example 8 A fermentation medium and its application in the preparation of teriparatide

[0197] The difference from Example 4 is that nickel sulfate and titanium tetrachloride are not contained, and the contents of other components and the preparation method are the same as those of Example 4. That is:

[0198] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 20 g / L cobalt chloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0199] The inclusion body yield calculated by weighing was 0.226 g / g 菌体 , 34.8g / L 发酵液 .

[0200] The yield of teriparatide was calculated to be 37 mg / g after the purified stock solution was tested. 包涵体 , 1287.6mg / L 发酵液 .

[0201] Comparative Example 9 A fermentation medium and its application in the preparation of teriparatide

[0202] The difference from Example 4 is that 4.8 g / L nickel sulfate and 0.2 g / L titanium tetrachloride are added, and the contents of other components and the preparation method are the same as those in Example 4. That is:

[0203] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 20 g / L ferrous chromite, 4.8 g / L nickel sulfate, 20 g / L cobalt chloride, 0.2 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, and the solvent is pure water.

[0204] The inclusion body yield calculated by weighing was 0.233 g / g 菌体 , 36.4g / L 发酵液 .

[0205] The yield of teriparatide was calculated to be 38 mg / g after the purified stock solution was tested. 包涵体, 1383.2mg / L 发酵液 .

[0206] Comparative Example 10 A fermentation medium and its application in the preparation of teriparatide

[0207] The difference from Example 4 is that 2 g / L of ferrous chromite, 3.3 g / L of titanium tetrachloride, and 30 g / L of ferrous sulfate heptahydrate are added, and the contents of other components and the preparation method are the same as those in Example 4. That is:

[0208] The trace element solution comprises the following components: 30 g / L disodium ethylenediaminetetraacetate dihydrate, 10 g / L manganese sulfate, 2 g / L ferrous chromite, 3 g / L nickel sulfate, 20 g / L cobalt chloride, 3.3 g / L titanium tetrachloride, 10 g / L zinc sulfate, 6 g / L copper sulfate, 1.5 g / L sodium molybdate dihydrate, 4 g / L boric acid, 30 g / L ferrous sulfate heptahydrate, and the solvent is pure water.

[0209] The inclusion body yield calculated by weighing was 0.227 g / g 菌体 , 34.5g / L 发酵液 .

[0210] The yield of teriparatide was calculated to be 38 mg / g after the purified stock solution was tested. 包涵体 , 1311.0mg / L 发酵液 .

[0211] Comparative Example 11 A fermentation medium and its application in the preparation of teriparatide

[0212] The difference from Example 4 is that the trace element solution comprises the following components: 3.2 g / L ferrous sulfate, 1 g / L manganous chloride, 1.5 g / L cobalt nitrate, 1 g / L calcium chloride, 0.25 g / L cupric chloride, 0.4 g / L zinc sulfate, 0.3 g / L boric acid and 3 g / L sodium molybdate, the solvent is 1 mol / L hydrochloric acid, and the contents of other components and the preparation method are the same as those in Example 4.

[0213] The inclusion body yield calculated by weighing was 0.199 g / g 菌体 , 28.9g / L 发酵液 .

[0214] The yield of teriparatide was calculated to be 39 mg / g after the purified stock solution was tested. 包涵体 , 1127.1mg / L 发酵液 .

[0215] Performance data:

[0216] 1. The detection method of the content of teriparatide stock solution is:

[0217] The RP-HPLC method was used to detect the content of teriparatide in the stock solution by comparison with the standard (China Food and Drug Inspection Institute, 0.975 mg / bottle, 10000 IU / mg). The chromatographic conditions were carried out in accordance with the general rule "0512 High Performance Liquid Chromatography" of the 2020 edition of the Pharmacopoeia of the People's Republic of China. The obtained teriparatide stock solution and the control sample were prepared with 0.2 mol / L sulfate buffer (pH 2.3): acetonitrile solution (75:25) to prepare a solution containing about 0.25 mg of teriparatide per 1 ml. 20 μl of the sample to be tested and the control sample were accurately measured and injected into the liquid chromatograph. The peak time of the sample to be tested and the control sample should be consistent. The teriparatide content (mg / ml) was calculated by the external standard method, and the teriparatide mass was calculated from the volume of the purified stock solution. According to the mass of the teriparatide inclusion body used in the stock solution, the teriparatide fermentation yield (mg / L) was calculated.

[0218] 2. The detection method of the activity of teriparatide stock solution is:

[0219] (1) Reagents: cell line URM-106 (ATCC); basal medium DMEM; complete medium consisting of basal medium + 10% total volume of bovine serum + penicillin at a final concentration of 100 U / ml + streptomycin at a final concentration of 100 μg / ml; diluent consisting of basal medium + 3-isobutyl-1-methylxanthine at a final concentration of 1 mmol / L + 0.1% bovine serum albumin; lysate provided by the cAMP detection kit.

[0220] (2) Cell treatment: Digest and count the UMR106 cells in the logarithmic growth phase and dilute them to 2*10 with complete culture medium. 5 100μl per well, spread to 96-well cell culture plate, culture in 37℃, 5% CO2 incubator for 18-24h; discard the liquid in the culture plate the next day, add 100μl basal culture medium to each well and culture in 37℃, 5% CO2 incubator for 1h; discard the liquid in the cell culture plate, add different starting concentrations of test samples, 100μl / well, double well, culture in 37℃, 5% CO2 incubator for 20min, then take out the 96-well plate, discard the liquid in the cell culture plate, add PBS 200μl / well, wash, discard the liquid in the plate, wash twice; add lysis solution 100μl / well, culture in 37℃, 5% CO2 incubator for 60min. After shaking the cell culture plate for 5min, centrifuge at 4000rpm / min for 5min.

[0221] (3) cAMP detection

[0222] Add 50μl / well of primary antibody solution, seal the plate, and place on a micro-oscillator for 1h at room temperature. Discard the liquid in the well, add 400μl / well of washing solution, let it stand for 30s, shake off the liquid, pat dry on absorbent paper, and repeat 4 times. First add 50μl / well of cAMP coupling compound solution, then add the collected samples to be tested and teriparatide standard to the ELISA plate at 100μl / well, and place on a micro-oscillator for 2h at room temperature. Add 200μl / well of substrate solution and place at room temperature in the dark for 30min. Finally, add 100μl / well of stop solution, and measure the absorbance (OD) value at 450nm with 570nm as the reference wavelength. Use the ELISA software for four-parameter regression calculation.

[0223] (4) Calculation of teriparatide activity

[0224] Calculate the half-effective concentration (EC 50 ), and the activity of teriparatide was calculated as follows:

[0225]

[0226] The results of the inclusion body yield and the teriparatide yield after purification in each example and comparative example are shown in Table 1.

[0227] Table 1

[0228]

[0229]

[0230] According to the test results in Table 1 above, it can be seen that the fermentation medium provided in Examples 1-5 of the present invention is used to prepare teriparatide inclusion bodies, and the obtained inclusion body yield and teriparatide yield are both high; while in the comparative example, changing the type and content of elements in the trace element solution or omitting the trace element solution, or changing the feeding method during the fermentation process will lead to a decrease in the yield of inclusion bodies and teriparatide.

[0231] The biological activity of the teriparatide stock solutions obtained in Example 1, Example 4, and Comparative Examples 1 and 5 was tested, and the test data are shown in Table 2 below.

[0232] Table 2

[0233]

[0234] According to the test data, different inclusion body expression levels (21.1-44.1g / L 发酵液 ) and teriparatide production levels (801.8-1719.9 mg / L 发酵液), the biological activity (specific activity) of the products is similar, with the specific activity being 88.83-97.70% of the national standard, and both meet the requirements of the United States Pharmacopoeia for the specific activity of teriparatide stock solution (60-120% of the specific activity of the standard).

[0235] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, a person skilled in the art can still modify or make equivalent substitutions to the specific implementation schemes of the present invention. Any modifications or equivalent substitutions that do not depart from the spirit and scope of the present invention are within the scope of protection of the claims of the present invention.

Claims

1. A fermentation medium, characterized in that: The invention comprises the following components: 12-20 g / L yeast peptone, 4-10 g / L yeast extract powder, 3-5 g / L potassium dihydrogen phosphate trihydrate, 0.5-2 g / L potassium dihydrogen phosphate, 0.5-3 g / L ammonium sulfate, 0.5-2 g / L magnesium sulfate heptahydrate, 3-10 g / L glycerol, 1-3 ml / L trace element solution, 0.1-0.5 g / L defoamer, the solvent is pure water, and the pH value is adjusted to 6.8-7.2 with 2 mol / L sodium hydroxide solution; The trace element solution contains nickel sulfate, titanium tetrachloride and ferrous chromite, and the concentrations thereof are 1-3 g / L, 0.5-2 g / L and 10-20 g / L respectively.

2. The fermentation medium according to claim 1, characterized in that: The trace element solution also includes the following components: disodium ethylenediaminetetraacetate dihydrate, manganese sulfate, sodium molybdate dihydrate, cobalt chloride, zinc sulfate, copper sulfate and boric acid, and the solvent is pure water.

3. The fermentation medium according to claim 2, characterized in that: The trace element solution also includes components with the following concentrations: 20-30 g / L disodium ethylenediaminetetraacetic acid dihydrate, 8-10 g / L manganese sulfate, 0.5-1.5 g / L sodium molybdate dihydrate, 16-20 g / L cobalt chloride, 6-10 g / L zinc sulfate, 4-6 g / L copper sulfate and 2-4 g / L boric acid, and the solvent is pure water.

4. Use of the fermentation medium according to claim 1 in the preparation of teriparatide by fermentation with Escherichia coli.

5. A method for preparing teriparatide inclusion bodies by using the fermentation medium of claim 1 in Escherichia coli, characterized in that: The following steps are involved: (1) First, the other components except the trace element solution are prepared into a culture medium A, and then the trace element solution is filtered and added to the culture medium A to obtain a fermentation medium; then the seed liquid is inoculated into the culture medium for fermentation; (2) When the OD600 of the fermentation liquid is ≥60, an inducer is added and the temperature is lowered. After 5-7 hours of induction, the fermentation ends and the fermentation liquid is obtained. First, the fermentation liquid is centrifuged to collect the bacterial precipitate. The bacterial precipitate is resuspended, crushed, and centrifuged to obtain crude inclusion bodies.

6. The method according to claim 5, characterized in that: The inoculation amount of the seed solution in step (1) is 5-10 v / v%, the initial culture conditions are 37° C., pH 6.7-7.3, and dissolved oxygen ≥ 30%.

7. The method according to claim 6, characterized in that: The dissolved oxygen content is controlled by the stirring speed, ventilation volume and oxygen, wherein the total ventilation volume of air and oxygen is 1-1.5 vvm.

8. The method according to claim 5, characterized in that: During the fermentation process described in step (1), feed needs to be added continuously, and the feed flow rate needs to be gradually increased as the density of the microbial cells increases.

9. The method according to claim 8, characterized in that: The feeding method is variable speed feeding, and the variable speed feeding scheme is as follows: feeding starts after 1 hour of cultivation at 2.5 ml / h•L -1 , after 1.5 hours it increases to 5 ml / h•L -1 , then increase the feeding rate by 5 ml / h•L every 30 minutes -1 , up to 35 ml / h•L -1 When OD600 ≥ 30, the feeding rate is increased to 40 ml / h•L -1 .

10. The method according to claim 8, characterized in that: The feed comprises the following components: 180-220 g / L yeast peptone, 80-120 g / L yeast extract powder, 350-400 g / L glycerol, 1-2 g / L defoaming agent, and the solvent is pure water.

11. The method according to claim 5, characterized in that: The inducer in step (2) is IPTG, and the amount of the inducer added is 0.1-0.5 mmol / L.

12. The method according to claim 5, characterized in that: The cooling temperature in step (2) is 30°C.

13. The method according to claim 5, characterized in that: The resuspension operation in step (2) is as follows: adding a lysis buffer to the bacterial precipitate and then resuspending to obtain a resuspension; the mass volume ratio of the bacterial precipitate to the lysis buffer is 1 kg: 5-20 L.

14. The method according to claim 13, characterized in that: The bacterium-breaking buffer comprises the following components: 4-8 g / L of tris(hydroxymethyl)aminomethane, 0.2-0.5 g / L of disodium ethylenediaminetetraacetic acid dihydrate, and the solvent is pure water.

Images