Efficient expression and production process of recombinant human interleukin-1 receptor antagonist

Abstract

The invention discloses a high-efficiency expression and production process of a recombinant human interleukin-1 receptor antagonist. The expression method of the recombinant human interleukin-1 receptor antagonist comprises the following steps: (a) obtaining a host cell for expressing the recombinant human interleukin-1 receptor antagonist; (b) inoculating the host cells into a seed culture medium and activating; (c) inoculating the activated product in the step (b) into a fermentation culture medium and fermenting; and (d) inducing the host cell to express the recombinant human interleukin-1 receptor antagonist under the condition that an inducer is added when OD600 reaches 90-110.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of bioengineering, and particularly relates to a recombinant human interleukin-1 receptor antagonist, and more particularly to expression and purification of the recombinant human interleukin-1 receptor antagonist. BACKGROUND

[0002] The recombinant human interleukin-1 receptor antagonist specifically blocks the biological effects of interleukin-1 by binding to both type I and type II receptors for interleukin-1. Interleukin-1 receptor antagonist (IL-1Ra) is a natural cytokine in vivo that antagonizes IL-1. Its research has been increasingly valued internationally. The recombinant human interleukin-1 receptor antagonist (rhIL-1Ra) has been approved by FDA as a biological agent for clinical treatment of cytokines. Therefore, its production process has high commercial significance and value.

[0003] Currently, there are two methods for preparing the recombinant human interleukin-1 receptor antagonist, i.e. eukaryotic expression and prokaryotic expression. The eukaryotic expression has low yield and high cost. The prokaryotic expression is prone to produce a large amount of insoluble inclusion bodies.

[0004] Various methods for separating and purifying proteins mainly utilize the differences in various properties of proteins, including molecular size and shape, acid-base properties, solubility, adsorption properties, and biological affinity to other molecules. In the field of protein purification, it is generally believed that a combination of separation and purification methods with different separation principles must be used to take advantage of different physicochemical properties of proteins in order to obtain proteins with high purity. Currently, most methods for purifying the recombinant human interleukin-1 receptor antagonist adopt a 4-step chromatography method, which has a complex production process and high cost.

[0005] There is still an urgent need in the art for a production process for preparing and purifying the recombinant human interleukin-1 receptor antagonist with low cost, high yield, high purity, and high activity. SUMMARY

[0006] To solve the above technical problems, in one aspect, the present application provides an expression method of a recombinant human interleukin-1 receptor antagonist, which comprises:

[0007] (a) obtaining a host cell expressing the recombinant human interleukin-1 receptor antagonist;

[0008] (b) inoculating the host cell into a seed culture medium and activating;

[0009] (c) inoculating the activation product of step (b) into a fermentation culture medium and fermenting; and

[0010] (d) inducing the host cell to express the recombinant human interleukin-1 receptor antagonist under conditions comprising: adding an inducing agent when the OD600 reaches 90-110.

[0011] In another aspect, the present application provides a method for purifying a recombinant human interleukin-1 receptor antagonist, the method comprising:

[0012] (a) obtaining a sample containing the recombinant human interleukin-1 receptor antagonist;

[0013] (b) subjecting the sample to cation exchange chromatography to obtain an eluate;

[0014] (c) subjecting the eluate to anion exchange chromatography purification to obtain a flow-through; and

[0015] (d) subjecting the flow-through to dialysis and / or concentration.

[0016] In another aspect, the present application also provides a method for producing a recombinant human interleukin-1 receptor antagonist, the method comprising:

[0017] (a) obtaining a host cell expressing the recombinant human interleukin-1 receptor antagonist;

[0018] (b) inoculating the host cell into a seed culture medium and activating;

[0019] (c) inoculating the product of step (b) into a fermentation culture medium and fermenting;

[0020] (d) inducing the host cell to express the recombinant human interleukin-1 receptor antagonist under conditions comprising: adding an inducing agent when the OD600 reaches 90-110;

[0021] (e) obtaining a sample containing the recombinant human interleukin-1 receptor antagonist from the product of step (d);

[0022] (f) subjecting the sample to cation exchange chromatography to obtain an eluate;

[0023] (g) subjecting the eluate to anion exchange chromatography purification to obtain a flow-through; and

[0024] (h) subjecting the flow-through to dialysis and / or concentration. BRIEF DESCRIPTION OF DRAWINGS

[0025] The present application will be described in more detail by making reference to the accompanying drawings in which:

[0026] Figure 1 a constructional line drawing of the expression vector pET30a(+)-IL-1Ra of one embodiment of the present application;

[0027] Figure 2 SDS-PAGE pattern of sampling in the fermentation process of one embodiment of the application, wherein the lane information from left to right is as follows: 1. sample before induction; 2. sample of 1 hour induction; 3. sample of 2 hours induction; 4. sample of 3 hours induction; 5. sample of 4 hours induction; 6. sample of 5 hours induction; 7. sample of 6 hours induction; 8. sample of 7 hours induction; 9. sample of 8 hours induction; 10. molecular weight marker (X0520, Tian Gen Biochemical Technology (Beijing) Co., Ltd.); 11. reference substance (recombinant human interleukin-1 receptor antagonist control, catalog number: 20160901, You Sen Jian Heng Biomedical (Shanghai) Co., Ltd.);

[0028] Figure 3 SP chromatogram of one embodiment of the application, wherein: 1. UV280; 2. conductivity (Cond); 3. pH;

[0029] Figure 4 Q chromatogram of one embodiment of the application, wherein: 1. UV280; 2. conductivity (Cond); 3. pH;

[0030] Figure 5 SP chromatography SDS-PAGE loading and collection pattern of one embodiment of the application, wherein the lane information from left to right is as follows: 1. fermentation sample; 2. broken bacteria precipitate sample; 3. broken bacteria supernatant sample; 4. SP sample after loading; 5. SP chromatography first peak collection sample; 6. SP chromatography second peak collection sample; 7. SP chromatography second peak collection sample; 8. Q chromatography loading sample; 9. Q chromatography collection sample; 10. molecular weight marker (X0520, Tian Gen Biochemical Technology (Beijing) Co., Ltd.); 11. reference substance (recombinant human interleukin-1 receptor antagonist control, catalog number: 20160901, You Sen Jian Heng Biomedical (Shanghai) Co., Ltd.); 12. reference substance (recombinant human interleukin-1 receptor antagonist control, catalog number: 20160901, You Sen Jian Heng Biomedical (Shanghai) Co., Ltd.).

[0031] Figure 6 Q chromatography SDS-PAGE collection pattern of one embodiment of the application, wherein the lane information from left to right is as follows: 1. SP chromatography sample; 2. SP chromatography sample; 3. Q chromatography sample; 4. molecular weight marker (X0520, Tian Gen Biochemical Technology (Beijing) Co., Ltd.); 5. reference substance (recombinant human interleukin-1 receptor antagonist control, catalog number: 20160901, You Sen Jian Heng Biomedical (Shanghai) Co., Ltd.). DETAILED DESCRIPTION

[0032] This application relates to a method for expressing a recombinant human interleukin-1 receptor antagonist, the method comprising: (a) obtaining a host cell expressing the recombinant human interleukin-1 receptor antagonist. In one embodiment, the host cell comprises a prokaryotic cell. In one embodiment, the host cell comprises *Escherichia coli*. In one embodiment, the host cell is genetically modified to express the recombinant human interleukin-1 receptor antagonist.

[0033] The expression method of the recombinant human interleukin-1 receptor antagonist further includes: (b) seeding the host cells in a seed culture medium and activating them. In one embodiment, the activation conditions include: a temperature of 36-38°C, a culture time of 14-18 h, and a stirring speed of 200-240 rpm. In one embodiment, the temperature is about 37°C. In one embodiment, the culture time is about 16 h. In one embodiment, the stirring speed is about 220 rpm. In one embodiment, the seed culture medium contains sodium chloride and yeast extract. In one embodiment, the seed culture medium contains 8-12 g / L sodium chloride and 8-12 g / L yeast extract. In a preferred embodiment, the seed culture medium contains about 10 g / L sodium chloride and about 10 g / L yeast extract. In one embodiment, the OD600 after activation in step (b) is 2.0-6.0.

[0034] The expression method of the recombinant human interleukin-1 receptor antagonist further includes: (c) inoculating the activated product from step (b) into a fermentation medium and fermenting. In one embodiment, the fermentation includes pre-feed fermentation and post-feed fermentation. In one embodiment, the conditions for pre-feed fermentation include: a temperature of 36-38°C, a culture time of 0-2 h, a stirring speed of 200-400 rpm, and an air flow rate of 400-600 L / h. In one embodiment, the temperature is about 37°C. In one embodiment, the culture time is about 2 h. In one embodiment, the conditions for post-feed fermentation include: adding a feeding medium, a temperature of 36-38°C, a culture time of 2-10 h, a stirring speed of 400-800 rpm, and an air flow rate or a mixture of oxygen and air flow rate of 400-600 L / h. In one embodiment, the temperature is about 37°C. In one embodiment, the culture time is about 8 h. In one embodiment, the fermentation medium contains soybean peptone, yeast extract, glucose, sodium chloride, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate. In one embodiment, the fermentation medium comprises 8-12 g / L soybean peptone, 4-6 g / L yeast extract, 4-6 g / L glucose, 8-12 g / L sodium chloride, 2-3 g / L dipotassium hydrogen phosphate, and 0.5-1.5 g / L potassium dihydrogen phosphate. In a preferred embodiment, the fermentation medium comprises about 10 g / L soybean peptone, about 5 g / L yeast extract, about 5 g / L glucose, about 10 g / L sodium chloride, about 2.5 g / L dipotassium hydrogen phosphate, and about 1 g / L potassium dihydrogen phosphate. In one embodiment, the fed-batch medium comprises soybean peptone, yeast extract, and glucose. In one embodiment, the fed-batch medium comprises 15-25% soybean peptone, about 5-15% yeast extract, and about 40-60% glucose. In a preferred embodiment, the fed-batch medium comprises about 20% soybean peptone, about 10% yeast extract, and about 50% glucose. In one embodiment, fed-batch culture medium is added to maintain the following concentrations during fermentation: 140-160 g / L soybean peptone, 70-80 g / L yeast extract, and 120-130 g / L glucose. In a preferred embodiment, fed-batch culture medium is added to maintain the following concentrations during fermentation: about 150 g / L soybean peptone, about 75 g / L yeast extract, and about 125 g / L glucose. In one embodiment, the inoculum size in step (c) is 5-10%. In one embodiment, step (c) includes controlling the dissolved oxygen (DO) at 20%-60%. In one embodiment, step (c) includes controlling the fermenter pressure at 0.02-0.08 MPa. In one embodiment, step (c) includes controlling the fermentation pH at 6.7-7.3. In one embodiment, step (c) includes adding fed-batch culture medium when the OD600 reaches 8-10. In one embodiment, the feeding rate is 20-35 mL / L / h for 1-2 hours after adding the fed-batch culture medium.In one embodiment, the feeding rate is 40-60 mL / L / h 3-4 h after the addition of the feeding medium. In another embodiment, the feeding rate is 70-80 mL / L / h 5-10 h after the addition of the feeding medium. In one embodiment, step (c) includes introducing a mixture of oxygen and air when the OD600 reaches about 50.

[0035] The method for expressing the recombinant human interleukin-1 receptor antagonist further includes: (d) inducing the host cells to express the recombinant human interleukin-1 receptor antagonist, wherein the induction conditions include: adding an inducer when the OD600 reaches 90-110. In one embodiment, the induction conditions include: adding an inducer when the OD600 reaches approximately 100. In one embodiment, the induction conditions further include: a temperature of 36-38°C, a culture time of 2-8 h, a stirring speed of 700-800 rpm, and a mixed gas flow rate of oxygen and air of 400-600 L / h. In one embodiment, the temperature is approximately 37°C. In one embodiment, the culture time is approximately 4 h. In one embodiment, step (d) includes controlling the DO to 20%-60%. In one embodiment, step (d) includes controlling the fermenter pressure to 0.02-0.08 MPa. In one embodiment, step (d) includes controlling the fermentation pH to 6.7-7.3. In one embodiment, the inducer includes IPTG. In one embodiment, the concentration of the inducer is 0.05-0.2 mM. In a preferred embodiment, the concentration of the inducer is about 0.1 mM. In one embodiment, the feeding rate in step (d) is 60-80 mL / L / h.

[0036] This application also relates to a method for purifying a recombinant human interleukin-1 receptor antagonist, the method comprising: (a) obtaining a sample containing the recombinant human interleukin-1 receptor antagonist. In one embodiment, the sample is obtained by lysing and filtering host cells expressing the recombinant human interleukin-1 receptor antagonist. In one embodiment, the host cells are obtained by the expression method of the recombinant human interleukin-1 receptor antagonist according to any of the foregoing embodiments. In one embodiment, lysis comprises cell disruption. In a preferred embodiment, cell disruption comprises using a high-pressure homogenizer. In one embodiment, lysis is performed once or multiple times. In one embodiment, lysis is performed three times. In one embodiment, filtration comprises membrane filtration. In a preferred embodiment, membrane filtration comprises using a 0.45-micron filter membrane.

[0037] The purification method for the recombinant human interleukin-1 receptor antagonist further includes: (b) passing the sample through cation exchange chromatography to obtain an eluent. In one embodiment, the cation exchange chromatography includes cation exchange chromatography containing sulfopropyl or carboxymethyl groups as ligands. In a preferred embodiment, the cation exchange chromatography includes using an SP Bestrarose FF column. In one embodiment, the cation exchange chromatography includes equilibration with phosphate buffer before and after sample loading. In a preferred embodiment, the cation exchange chromatography includes equilibration with 10 mM PB, pH 5.9-6.1 before and after sample loading. In a preferred embodiment, the cation exchange chromatography includes elution with phosphate buffer containing sodium chloride and collection of the eluent. In a preferred embodiment, the cation exchange chromatography includes elution with 10 mM PB, 0.2 M NaCl, pH 5.9-6.1 and collection of the eluent.

[0038] The purification method of the recombinant human interleukin-1 receptor antagonist further includes: (c) purifying the eluent by anion exchange chromatography to obtain a flow-through. In one embodiment, the anion exchange chromatography includes anion exchange chromatography containing a quaternary ammonium group or an amino group as a ligand. In a preferred embodiment, the anion exchange chromatography includes a QBestrarose FF column. In one embodiment, the anion exchange chromatography includes equilibration with a phosphate buffer containing sodium chloride before and after sample loading, and collection of the flow-through. In a preferred embodiment, the anion exchange chromatography includes equilibration with 10 mM PB, 0.2 M NaCl, pH 5.9-6.1 before and after sample loading, and collection of the flow-through.

[0039] The purification method of the recombinant human interleukin-1 receptor antagonist further includes: (d) changing and / or concentrating the flow-through solution. In one embodiment, the change solution comprises 10 mM sodium citrate-citric acid, 8.1-8.3 g / L NaCl, 0.16-0.20 g / L EDTA, and pH 6.4-6.6. In a preferred embodiment, the change solution comprises 10 mM sodium citrate-citric acid, about 8.2 g / L NaCl, about 0.18 g / L EDTA, and pH about 6.5. In one embodiment, step (d) includes concentrating the flow-through solution to a protein concentration of 95-105 mg / ml. In one embodiment, the product of step (d) comprises a protective agent. In one embodiment, the protective agent comprises a surfactant. In a preferred embodiment, the surfactant comprises Tween-80. In one embodiment, the concentration of the protective agent is 0.5-5 g / L. In one preferred embodiment, the concentration of the protective agent is 1.0-1.1 g / L. In another preferred embodiment, the concentration of the protective agent is about 1.05 g / L.

[0040] This application also relates to a method for producing a recombinant human interleukin-1 receptor antagonist, the method comprising: (a) obtaining host cells expressing a recombinant human interleukin-1 receptor antagonist. This step includes step (a) described in any of the above-described methods for expressing a recombinant human interleukin-1 receptor antagonist.

[0041] The method for producing the recombinant human interleukin-1 receptor antagonist further includes: (b) inoculating the host cells into a seed culture medium and activating them. This step includes step (b) described in the expression method of the recombinant human interleukin-1 receptor antagonist according to any of the above embodiments.

[0042] The method for producing the recombinant human interleukin-1 receptor antagonist further includes: (c) inoculating the activated product from step (b) into a fermentation medium and fermenting it. This step includes step (c) as described in the expression method of the recombinant human interleukin-1 receptor antagonist of any of the above embodiments.

[0043] The method for producing the recombinant human interleukin-1 receptor antagonist further includes: (d) inducing the host cells to express the recombinant human interleukin-1 receptor antagonist, wherein the induction conditions include adding an inducer when the OD600 reaches 90-110. This step includes step (d) described in the expression method of the recombinant human interleukin-1 receptor antagonist according to any of the above embodiments.

[0044] The method for producing the recombinant human interleukin-1 receptor antagonist further includes: (e) obtaining a sample containing the recombinant human interleukin-1 receptor antagonist from the product of step (d). This step includes step (a) as described in the purification method for the recombinant human interleukin-1 receptor antagonist of any of the above embodiments.

[0045] The method for producing the recombinant human interleukin-1 receptor antagonist further includes: (f) passing the sample through cation exchange chromatography to obtain an eluent. This step includes step (b) described in the purification method for the recombinant human interleukin-1 receptor antagonist of any of the above embodiments.

[0046] The method for producing the recombinant human interleukin-1 receptor antagonist further includes: (g) purifying the eluent by anion exchange chromatography to obtain a flow-through solution. This step includes step (c) described in the purification method for the recombinant human interleukin-1 receptor antagonist of any of the above embodiments.

[0047] The method for producing the recombinant human interleukin-1 receptor antagonist further includes: (h) changing and / or concentrating the flow-through solution. This step includes step (d) described in the purification method for the recombinant human interleukin-1 receptor antagonist of any of the above embodiments.

[0048] In summary, compared with the prior art, the present invention has the following advantages:

[0049] 1. Compared with the prior art, the method of this application can prepare and purify recombinant human interleukin-1 receptor antagonists at a lower cost.

[0050] 2. Increase fermentation yield by increasing OD600 during induction.

[0051] 3. The recombinant protein products produced by this fermentation process have good solubility and no obvious inclusion bodies are formed.

[0052] 4. By simplifying the traditional 4-column chromatography method into a 2-column chromatography method with a specific packing material combination, the experimental cost and complexity are reduced, the oxidation and aggregation problems caused by complex processes are avoided, the activity is maintained, and the purity and yield are improved.

[0053] 5. By selecting appropriate packing material combinations, high purity, high yield, and low host cell protein residue were achieved.

[0054] Example

[0055] This application will be described in detail through the following exemplary embodiments. These embodiments are only intended to help those skilled in the art better understand the invention of this application. It should be noted that the spirit and scope of protection of this application are not limited to the following specific embodiments.

[0056] List of reagents and equipment used

[0057]

[0058]

[0059] Example 1: Fermentation Process

[0060] 1. Fermentation process

[0061] 1.1 Culture medium preparation and sterilization

[0062] 1.1.1 Preparation and sterilization of shake flask culture medium

[0063] The seed culture medium was prepared according to the seed culture medium formula. The water used for preparation was purified water. After preparation, the culture medium was sterilized in a sterilizer at 121°C for 20 minutes.

[0064] The seed culture medium formula is as follows:

[0065] Material name Content Sodium chloride 1％ Yeast extract 1％

[0066] 1.1.2 Preparation and sterilization of fermentation tank culture medium

[0067] The fermentation medium was prepared according to the fermentation medium formula. The water used for preparation was purified water. After preparation, sterilization was carried out using a sterilizer set at 115℃ for 20 minutes and online sterilization controlled at 121±1℃ for 30±1 minutes.

[0068] The fermentation medium formula is as follows:

[0069]

[0070]

[0071] 1.1.3 Preparation and sterilization of fed culture medium

[0072] The feed culture medium was prepared according to the feed culture medium formula. The water used for preparation was purified water. After preparation, the medium was sterilized at 115℃ for 20 min and 121℃ for 30 min, respectively.

[0073] The feed culture medium formula is as follows:

[0074] Material name Content Yeast extract 10％ Soy peptone 20％ Glucose (separately sterilized) 50％

[0075] 1.2 Working strains

[0076] 1.2.1 Seed Source

[0077] Take one working seed from the working seed bank (freezer at less than -60°C) and place it in a biosafety cabinet for 5-10 minutes to allow it to thaw naturally at room temperature.

[0078] Acquisition of genes

[0079] The human IL-1Ra gene was obtained by amplifying human liver cDNA using a template (Human Fetal Liver MATCHMAKER cDNALibrary, Clontech) and upstream and downstream primers (including restriction sites) designed according to the human IL-1Ra gene information provided on the NCBI website.

[0080] The primer sequences are as follows:

[0081] Upstream: 5'-GGAATTC CAT ATG (Nde I)CGACCCTCTGGGAGAAAATCC-3'

[0082] Note: From the 5' end, the sequence is: protective base (optimal protective base), Nde I restriction site, and primer sequence.

[0083] Downstream: 5'-CGC GGATCC (BamH I)TTACTCGTCCTCCTGGAAGTAGA-3'

[0084] Note: From the 5' end, the sequence is as follows: protective base (optimal protective base), BamHI restriction site, TTA, and primer sequence.

[0085] The PCR reaction conditions are as follows:

[0086]

[0087]

[0088] Gene nucleotide sequence:

[0089] The full-length IL-1Ra gene sequence (Genebank, M63099.1, GI: 186385, IL-1Ra) is 534 bp, and the CDS length is 534 bp, of which the first 75 bp of the CDS encodes a 25-aa signal peptide. Therefore, the sequence to be expressed should be 76-534, a total of 459 bp, encoding 152 aa.

[0090] Gene sequence (underlined portion is signal peptide):

[0091]

[0092] The target protein sequence of IL-1Ra obtained after transcription and translation has a total of 152 amino acids. Due to expression in E. coli, the protein has an extra start codon at the N-terminus for methionine (M), resulting in a total of 153 amino acids and a molecular weight of 17.3 kDa for the rhIL-1Ra protein. Its theoretical amino acid sequence is shown below.

[0093] Theoretical amino acid sequence of rhIL-1Ra:

[0094]

[0095] Construction of kanamycin resistance expression vector (pET30a(+)-IL-1Ra) and transformation of expression strains

[0096] (1) Transform DH5α competent cells (product number: CB101, Tiangen Biotech Co., Ltd.) with the purchased pET30a(+) vector. Pick a single colony from the plate and inoculate it into a test tube containing 5 ml LB medium. Incubate overnight at 37°C in a shaker. Take 500 μl of bacterial solution and add an equal volume of 60% glycerol to mix well to obtain a bacterial solution containing 30% glycerol. Store at -80°C.

[0097] (2) Take a small amount of the remaining bacterial culture from the above preservation and extract plasmid pET30a(+), and store it in a refrigerator at 2-8℃;

[0098] (3) Inoculate pTA2-IL-1Ra / DH5α glycerol bacteria into a test tube containing 5 ml of LB medium containing ampicillin, and culture overnight on a shaker at 37°C. Extract a small amount of plasmid.

[0099] (4) The pTA2-IL-1Ra and pET30a(+) plasmids were double-digested with Nde I and BamHI to obtain the digested pET30a(+) vector and human IL-1Ra gene fragment.

[0100] (5) Recover the pET30a(+) vector and the target gene fragment;

[0101] (6) Electrophoresis is used to determine the approximate concentrations of the carrier and the target fragment. Based on their respective concentrations, a ligation system is designed and a ligation reaction is carried out.

[0102] (7) The ligation product was transformed into DH5α and BL21(DE3) (Catalog No.: CB105, Tiangen Biotech Co., Ltd.); competent cells;

[0103] (8) Pick single colonies from the plate for PCR identification, and inoculate the correctly identified single colonies into LB medium and incubate overnight in a shaker at 37°C.

[0104] (9) Extract a small amount of plasmid for double enzyme digestion identification, and at the same time send a portion of bacterial culture for sequencing.

[0105] (10) Select a single colony and inoculate it into LB medium. Induce with IPTG and use SDS-PAGE electrophoresis to identify whether the target protein is expressed. Select strain 1 from the 4 strains and preserve it with glycerol to establish an original seed bank.

[0106] The characteristics of the expression vector are shown in Table 1; the construction route of the recombinant plasmid is shown in Table 1. Figure 1 .

[0107] Table 1: Characteristics of the expression vector pET-30a(+)

[0108]

[0109]

[0110] 1.2.2 Seed preparation

[0111] In a biosafety cabinet, kanamycin solution was added to the seed culture medium to achieve a final concentration of 100 mg / L. The thawed inoculum was then inoculated into a shake flask containing the seed culture medium, 200 μL / flask. The shake flask was placed in a shaker set at 37°C and 220 rpm for 16 hours.

[0112] 1.3 Fermentation tank cultivation

[0113] The primary seed culture was inoculated into a fermenter containing sterilized culture medium at an inoculation rate of 5%.

[0114] 1.3.1 Fermentation process conditions

[0115] 1.3.1.1 Fermentation temperature: 37℃

[0116] 1.3.1.2 Fermentation pH: 7.0

[0117] 1.3.1.3 Stirring speed: Initial speed 200 rpm, maintain 200-800 rpm during fermentation.

[0118] 1.3.1.4 Ventilation method: Initially maintain the ventilation rate at 400-600 L / h. First, adjust the rotation speed. Once the rotation speed reaches 800 rpm, increase the ventilation rate (or introduce pure oxygen) or tank pressure to maintain the dissolved oxygen (DO) in the fermentation broth (monitored by the dissolved oxygen probe on the fermenter) at 20%-60%.

[0119] 1.3.1.5 Feeding Control: Feeding begins when DO rises rapidly (accompanied by a rise in pH). Feeding control uses DO-STAT, maintaining DO levels between 20% and 60%. Feeding rates are shown in Table 2 below.

[0120] Table 2: Feeding Rate of 15L Fermentation Tank

[0121] Fermentation phase (h) Carbon source feed rate (ml / h) Nitrogen source feed rate (ml / h) 3 40-50 120-150 4 50-60 150-180 5 90-100 270-300 6 110-120 330-360 7 120-140 360-420 8 130-150 390-450 9 130-150 390-450 10 140-160 420-480 11 140-160 420-480

[0122] Induction phase (h) Carbon source feed rate (ml / h) Nitrogen source feed rate (ml / h) 1 120-140 360-420 2 120-130 360-390 3 110-120 330-360 4 110-120 330-360

[0123] 1.3.1.6 pH control: Based on the online pH display, add ammonia or phosphoric acid to control the pH to 7.0.

[0124] 1.3.1.7 Induction of Expression: Induction began when the OD600 of batches RF20221123, RF20230101, and RF20230202 was 100 (measured by a visible light spectrophotometer), and when the OD600 of batches F20091010, F20091104, and F20091208 was approximately 10-20. Sample retention was recorded as 0h (1 mL of fermentation broth was centrifuged at 10000 rpm for 5 min using a benchtop centrifuge, and the precipitate was retained). During the induction phase, solid samples were centrifuged every 1 hour and retained (two portions of each sample were retained and stored at 2-8℃), and the OD600 was measured until the end of fermentation.

[0125] 1.3.2 Monitoring items during fermentation

[0126] During fermentation, monitor each process parameter (temperature, pH, DO, aeration rate, tank pressure, etc.) every hour; perform aseptic sampling, measure OD600, and centrifuge and retain the samples.

[0127] 1.4 Collection and Preservation of Bacterial Cells

[0128] After fermentation was completed, fermentation was stopped, and the cells were transferred to a container. The cells were collected using a centrifuge and named FCB; centrifuged at 10,000 rpm. An appropriate amount of the centrifuged FCB cells was taken as the final fermentation sample for testing.

[0129] If the bacterial cells need to be preserved, they should be stored at -20°C for no more than 6 months.

[0130] SDS-PAGE spectra of samples taken during fermentation are as follows: Figure 2 As shown in Table 3, the comparison results of low-density and high-density fermentation data are presented. Different batches used the same fermentation process described above.

[0131] Table 3: Comparison of low-density and high-density fermentation data

[0132]

[0133]

[0134] Example 2: Purification Process

[0135] 1. Crude extraction process

[0136] 1.1 Preparation of lysis solution

[0137] Prepare the sterilization diluent according to the formula and pre-cool it for later use.

[0138] The formula for the sterilization diluent is as follows:

[0139]

[0140] 1.2 Cell lysis

[0141] Take the fermentation cells obtained in Example 1, dissolve the cells at a ratio of 1g:10mL for the lysing dilution solution, and stir to fully dissolve the cells.

[0142] 1.3 Disruption of bacterial culture

[0143] The bacterial cells were broken up using a high-pressure homogenizer at working pressures of 800 bar, 950 bar, and 950 bar respectively. The temperature of the broken-up solution was <30℃. The bacterial concentration was measured by sampling, and the OD600 was <4.

[0144] 1.4 Centrifugation of the disrupted bacterial culture

[0145] Connect the bacterial culture disruption tank to a continuous flow centrifuge. Collect the supernatant at 12,000 rpm and discard the precipitate. The temperature of the disrupted culture should be <30℃.

[0146] 1.5 Filtration of centrifuged supernatant

[0147] The supernatant from centrifugation was filtered through a 0.45 μm filter membrane. The filtrate was tested, and if OD600 < 1.0, the filtrate was collected as the sample for loading onto the SP column.

[0148] 1.6 Waste Disposal

[0149] Centrifuged sedimentation waste should be sterilized by steam at 121℃ for 30 minutes and then discarded.

[0150] 2. Purification Chromatography Process

[0151] 2.1 Preparation of purification buffer

[0152] Prepare the purification buffer solution according to the formulation of the purification chromatography buffer solution, and control the water temperature of the preparation water to be <25℃.

[0153] The buffer formulation for purification chromatography is as follows:

[0154]

[0155]

[0156] 2.2 Cation exchange SP (1.5L chromatography column)

[0157] Before and after use, the SP column should undergo CIP according to the purification column cleaning, disinfection, and regeneration procedures. The purification column cleaning, disinfection, and regeneration procedures are as follows:

[0158] (1) Washing with water

[0159] Purify the packing material by cleaning, disinfecting and regenerating the cation (SP bestarose FF) and anion (Q bestarose FF) solutions, and rinse with water for injection for 1-2 column volumes until the effluent conductivity is <1 mS / cm.

[0160] (2) Alkali washing

[0161] Pass 1–2 column volumes of 1 mol / L NaOH solution through the column until the pH of the effluent is >13; then let it stand for 1–2 hours. Rinse with 1–2 column volumes of water for injection until the conductivity of the effluent is <1 mS / cm.

[0162] (3) High-salt washing

[0163] Wash with 2 mol / L NaCl for 3–5 column volumes until the pH of the effluent is neutral. Finally, wash with water for injection for 1–2 column volumes until the conductivity of the effluent is <1 mS / cm. Once the endotoxin test is passed, it is ready for use.

[0164] (4) Washing

[0165] Purify the packing material by cleaning, disinfecting and regenerating the cation (SP bestarose FF) and anion (Q bestarose FF) solutions, and rinse with water for injection for 1-2 column volumes until the effluent conductivity is <1 mS / cm.

[0166] 2.2.1 Balance

[0167] Equilibrate with 10 mmol / L phosphate buffer, set the flow rate to 75 mL / min, and the volume to 2 CV until the baseline is stable.

[0168] 2.2.2 Sample loading

[0169] After centrifuging and filtering the lysate, adjust the pH of the sample to 6.0. Connect the pH-adjusted sample solution to the protein purification system and start loading the sample at a flow rate of 75 mL / min.

[0170] 2.2.3 Post-equilibrium

[0171] Wash the 2CV column with 0.01 mol / L phosphate buffer at a flow rate of 75 mL / min until the UV value drops to a stable level.

[0172] 2.2.4 Washing

[0173] The target protein was eluted with 0.01 mol / L phosphate + 0.2 mol / L sodium chloride buffer at a flow rate of 75 mL / min. The product peak was collected starting at 1500-1600 mAU (the third peak) using UV280 and ending at 300 mAU. After collection, the sample was transferred to 2-8℃ for storage. Protein concentration was measured using a micro spectrophotometer, and analyzed by SDS-PAGE, HPLC, and HCP. The SP chromatography chromatogram is shown below. Figure 3 As shown, the SDS-PAGE loading and collection patterns are as follows: Figure 5 As shown.

[0174] 2.3 Anion exchange Q (1.5L chromatography column)

[0175] Before and after use, Q column should undergo CIP according to the purification column cleaning, disinfection, and regeneration methods described above.

[0176] 2.3.1 Balance

[0177] Equilibrate with 0.01 mol / L phosphate + 0.2 mol / L sodium chloride buffer at a flow rate of 75 mL / min, equilibrate for 2 CVs, and allow the baseline to stabilize.

[0178] 2.3.2 Sample loading

[0179] Adjust the pH of the qualified sample on the SP1 column to 6.0, then connect the sample to the chromatography system and set the loading flow rate to 75 mL / min. Start collecting the flow-through when the UA280 reading rises (50-100 mAU); this is the target protein.

[0180] 2.3.3 Post-equilibrium

[0181] After sample loading, the Q-chromatographic column was washed with 0.01 mol / L phosphate + 0.2 mol / L sodium chloride buffer. UV280 collection was stopped at 50-100 mAU. After collection, the sample was transferred to 2-8℃ for storage until later use. The retained sample was used for protein concentration analysis (measured using a micro spectrophotometer), HPLC, endotoxin assay, and SDS-PAGE. The Q-chromatographic chromatogram is shown below. Figure 4 As shown, the SDS-PAGE collected spectrum is as follows: Figure 6 As shown.

[0182] 3. Protein Concentration Process

[0183] 3.1 Protein solution concentration

[0184] The protein solution was concentrated using an ultrafiltration device. Once the volume of the protein solution dropped to below 500 ml, a replacement solution was prepared to be added for replacement.

[0185] 3.2 Buffer solution displacement

[0186] Prepare the replacement solution according to the formula, add it in batches and continue to concentrate and replace until the volume of the replacement solution is about 5 times the initial replacement volume of the protein or the permeate conductivity is close to the replacement solution conductivity (±0.5mS / cm) and then stop the replacement.

[0187] The replacement fluid formula is as follows:

[0188]

[0189] 3.3 Protein Concentration

[0190] Continue to concentrate the protein solution, monitoring the protein concentration in real time, until the protein concentration reaches 95-105 mg / ml, then stop the protein concentration.

[0191] 3.4 Addition of excipients

[0192] Add polysorbate 80 according to the volume of the concentrated protein solution, using polysorbate 80 to achieve 1.05±0.05 g / L.

[0193] 3.5 Filtration and Sterilization

[0194] The protein solution was sterilized by using a 0.22 μm filter.

[0195] 4. Storage of the original solution

[0196] Store the stock solution in a refrigerator at 2–8°C for up to 6 months.

[0197] 5. Original purification process

[0198] The original purification process involved filtering the bacterial lysate and then performing a four-step chromatography (SP-Q-SP-Q) to obtain the stock solution. Details are as follows:

[0199] I. SP1 Chromatography

[0200] Column: SP Sepharose Fast Flow (17-0729-05, GE, USA), V = 10L

[0201] Sample: The supernatant from the above-mentioned ruptured centrifuge was filtered and then adjusted to pH 6.0.

[0202] Washing buffer: 10 mM PB, pH 6.0

[0203] Elution buffer: 10 mM PB, 0.2 M NaCl, pH 6.0

[0204] 1) Equilibration: Use wash buffer to equilibrate 5 CVs at a flow rate of 300-500 ml / min;

[0205] 2) Sample loading: Load the sample at a flow rate of 300-500 ml / min, and start collecting the flow-through peak after the UV280 absorption rises;

[0206] 3) Rinse: Wash with washing buffer at a flow rate of 300-500 ml / min until the UV280 baseline is stable;

[0207] 4) Elution: Use elution buffer to elute at a flow rate of 300-400 ml / min. The first small peak mainly consists of impurities and should not be collected. Collect the proteins in stages when the second peak appears.

[0208] II. Q1 Chromatography (Collapse Peak Transition)

[0209] Column: Q Sepharose Fast Flow (17-0510-05, GE, USA), V = 10L.

[0210] Samples: Peaks with host bacterial residual protein content <3% in SP1 were collected and merged.

[0211] Washing buffer: 10 mM PB, pH 6.0

[0212] 1) Equilibration: Use wash buffer to equilibrate 5 CVs at a flow rate of 300-500 ml / min;

[0213] 2) Sample loading: Load the sample at a flow rate of 300-500 ml / min, and start collecting the flow-through peak after the UV280 absorption rises;

[0214] 3) Rinse: Use washing buffer at a flow rate of 300-500 ml / min until the UV280 baseline is stable;

[0215] III. SP2 Chromatography

[0216] Column: SP Sepharose Fast Flow (17-0729-05, GE, USA), V = 10L

[0217] Sample: Q1 flow-through peak was diluted with washing buffer (10mM PB, pH 6.0) to a conductivity ≤5ms / cm, and the pH was adjusted to 6.0 at the same time.

[0218] Washing buffer: 10 mM PB, pH 6.0

[0219] Elution buffer: 20 mM Tris-HCl, pH 8.0

[0220] 1) Equilibration: Use wash buffer to equilibrate 5 CVs at a flow rate of 300-500 ml / min;

[0221] 2) Sample loading: Load the sample at a flow rate of 300-500 ml / min, and start collecting the flow-through peak after the UV280 absorption rises;

[0222] 3) Rinse: Wash with washing buffer at a flow rate of 300-500 ml / min until the UV280 baseline is stable;

[0223] 4) Elution: Elute with elution buffer at a flow rate of 300-400 ml / min, collect the peaks in steps, and send samples to detect the residual protein content of the host bacteria at the same time;

[0224] IV. Q2 Chromatography

[0225] Column: Q Sepharose Fast Flow (17-0510-05, GE, USA), V = 10L

[0226] Samples: Combine the collected peaks from SP2 containing <0.1% residual host bacterial protein, and simultaneously adjust the pH to 8.0. Wash buffer: 20 mM Tris-HCl, pH 8.0

[0227] Elution buffer 1: 10 mM citric acid, pH 6.5

[0228] Elution buffer 2: 10 mM citric acid, 0.1 M NaCl, pH 6.5

[0229] 1) Equilibration: Use wash buffer to equilibrate 5 CVs at a flow rate of 300-500 ml / min;

[0230] 2) Sample loading: Load the sample at a flow rate of 300-500 ml / min, and start collecting the flow-through peak after the UV280 absorption rises;

[0231] 3) Rinse: Rinse with washing buffer at a flow rate of 300-500 ml / min until the UV280 baseline is stable;

[0232] 4) Elution: First, wash with elution buffer 1 at a flow rate of 300-500 ml / min for 2-3 CV, then elute with elution buffer 2 at a flow rate of 300-400 ml / min and collect the protein peak;

[0233] V. Sterilization Filtration

[0234] In a clean bench, the protein collected in Q2 was sterilized and filtered through a 0.22μm membrane. The filtrate was collected using disposable sterile, pyrogen-free equipment and stored in a freezer at 2-8℃.

[0235] 6. Comparison

[0236] The current purification process involves filtering the lysed bacterial solution and then performing a two-step chromatography (SP-Q) to obtain the stock solution. Compared to the original purification process (4-column chromatography), the quantity and quality of the stock solution are significantly improved. When purifying with the same amount of wet bacteria, the stock solution yield is increased by approximately 53%, and the RP-HPLC purity is increased by approximately 1%.

[0237] Taking the purification of 100g of wet bacteria as an example, the 4-column chromatography method yielded an average of 2.99g of target protein after purification of 100g of bacterial cells, with an RP-HPLC purity of 97.80% (Table 4); the 2-column chromatography method yielded an average of 4.52g of target protein after purification of 100g of bacterial cells, with an RP-HPLC purity of 98.99% (Table 5).

[0238] Table 4.4 Column Chromatography

[0239]

[0240]

[0241] After purification, 100 grams of bacterial cells yielded an average of 2.99 grams of the target protein, with a RP-HPLC purity of 97.80%.

[0242] Table 5.2 Column Chromatography

[0243]

[0244] After purification, 100 grams of bacterial cells yielded an average of 4.52 grams of the target protein, with a RP-HPLC purity of 98.99%.

[0245] Remark:

[0246] *Protein quantity: refers to the amount of target protein obtained after each purification step;

[0247] *Purity: Detected by RP-HPLC;

[0248] *Yield: The average amount of target protein obtained per gram of wet bacterial cells.

[0249] Table 6 shows a comparison of different packing materials used in the SP step based on the purification method described above.

[0250] Table 6

[0251]

[0252] As shown in Table 6, SP / Q Bestarose FF packing material achieved the highest purity and the lowest residual host cell protein.

Claims

1. A method for expressing a recombinant human interleukin-1 receptor antagonist, the method comprising: (a) Obtaining host cells expressing recombinant human interleukin-1 receptor antagonists; (b) The host cells are inoculated into a seed culture medium and activated; (c) Inoculate the activated product from step (b) into a fermentation medium and ferment; and (d) Inducing the host cells to express a recombinant human interleukin-1 receptor antagonist, wherein the induction conditions include adding an inducer when the OD600 reaches 90-110.

2. A method for purifying a recombinant human interleukin-1 receptor antagonist, the method comprising: (a) Obtain a sample containing a recombinant human interleukin-1 receptor antagonist; (b) The sample is subjected to cation exchange chromatography to obtain an eluent; (c) The eluent is purified by anion exchange chromatography to obtain a flow-through solution; and (d) Replace and / or concentrate the flow-through fluid.

3. A method for producing a recombinant human interleukin-1 receptor antagonist, the method comprising: (a) Obtaining host cells expressing recombinant human interleukin-1 receptor antagonists; (b) The host cells are inoculated into a seed culture medium and activated; (c) Inoculate the activated product from step (b) into a fermentation medium and ferment; (d) Inducing the host cells to express a recombinant human interleukin-1 receptor antagonist, wherein the induction conditions include adding an inducer when the OD600 reaches 90-110; (e) Obtain a sample containing recombinant human interleukin-1 receptor antagonist from the product of step (d); (f) The sample is subjected to cation exchange chromatography to obtain an eluent; (g) The eluent is purified by anion exchange chromatography to obtain a flow-through solution; and (h) Replace and / or concentrate the flow-through fluid.

4. The method of claim 1 or 3, wherein the activation conditions in step (b) include: The temperature is 36-38℃, the incubation time is 14-18h, and the stirring speed is 200-240rpm; and / or the OD600 after activation is 2.0-6.0; The fermentation in step (c) includes pre-feeded fermentation and post-feeded fermentation. The pre-feeded fermentation conditions include: a temperature of 36-38℃, a culture time of 0-2 hours, a stirring speed of 200-400 rpm, and an air flow rate of 400-600 L / h. The post-feeded fermentation conditions include: adding fed culture medium, a temperature of 36-38℃, a culture time of 2-10 hours, a stirring speed of 400-800 rpm, and an air flow rate or a mixture of oxygen and air flow rate of 400-600 L / h; and / or The induction conditions described in step (d) also include: a temperature of 36-38℃, a culture time of 10-15h, a stirring speed of 700-800rpm, and a mixed gas flow rate of 400-600L / h for oxygen and air.

5. The method of claim 1 or 3, wherein the host cell comprises Escherichia coli; The seed culture medium contains sodium chloride and yeast powder, preferably 8-12 g / L sodium chloride and 8-12 g / L yeast powder; The fermentation medium comprises soybean peptone, yeast extract, glucose, sodium chloride, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate. Preferably, it comprises 8-12 g / L soybean peptone, 4-6 g / L yeast extract, 4-6 g / L glucose, 8-12 g / L sodium chloride, 2-3 g / L dipotassium hydrogen phosphate, and 0.5-1.5 g / L potassium dihydrogen phosphate; and / or The fed-batch culture medium contains soybean peptone, yeast extract and glucose. Preferably, the fed-batch culture medium is added to maintain the following concentrations during fermentation: 140-160 g / L soybean peptone, 70-80 g / L yeast extract and 120-130 g / L glucose.

6. The method of claim 1 or 3, wherein the inoculation amount in step (c) is 5-10%; and / or Steps (c) and / or (d) include controlling DO at 20%-60%, fermenter pressure at 0.02-0.08 MPa, and / or fermentation pH at 6.7-7.

3.

7. The method of claim 4, wherein step (c) comprises adding feeding medium when the OD600 reaches 8-10, preferably, the feeding rate is 20-35 mL / L / h for 1-2 hours after adding the feeding medium, 40-60 mL / L / h for 3-4 hours after adding the feeding medium, and 70-80 mL / L / h for 5-10 hours after adding the feeding medium; and / or Step (c) includes introducing a mixture of oxygen and air when the OD600 reaches 50.

8. The method of claim 1 or 3, wherein the inducer comprises IPTG, or preferably, the concentration of the inducer is 0.05-0.2 mM; and / or In step (d), the feeding rate is 60-80 mL / L / h.

9. The method of claim 2 or 3, wherein the sample is obtained by lysing and filtering host cells expressing a recombinant human interleukin-1 receptor antagonist, or preferably, the lysis comprises cell disruption, and / or the filtration comprises membrane filtration.

10. The method of claim 2 or 3, wherein the cation exchange chromatography comprises cation exchange chromatography containing sulfopropyl or carboxymethyl as ligands, preferably using an SP Bestrarose FF column, preferably comprising equilibration with phosphate buffer, preferably 10 mM PB, pH 5.9-6.1 before and after sample loading, elution with phosphate buffer containing sodium chloride, preferably 10 mM PB, 0.2 M NaCl, pH 5.9-6.1, and collection of the eluent; The anion exchange chromatography described herein includes anion exchange chromatography containing quaternary ammonium or amino groups as ligands, preferably using a QBestrarose FF column, preferably including equilibration with sodium chloride-containing phosphate buffer, preferably 10 mM PB, 0.2 M NaCl, pH 5.9-6.1 before and after sample loading, and collection of flow-through; and / or wherein the exchange solution contains 10 mM sodium citrate-citric acid, 8.1-8.3 g / L NaCl, 0.16-0.20 g / L EDTA, pH 6.4-6.6; and / or concentrating the flow-through to a protein concentration of 95-105 mg / ml.

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