TWI758285B - A method for renaturation and purification of recombinant human granulocyte colony stimulating factor - Google Patents
A method for renaturation and purification of recombinant human granulocyte colony stimulating factor Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/53—Colony-stimulating factor [CSF]
- C07K14/535—Granulocyte CSF; Granulocyte-macrophage CSF
Abstract
Description
本發明涉及的是生物醫藥及生物工程下游蛋白純化領域，具體的涉及一種重組人粒細胞刺激因子(recombinant human granulocyte colony stimulating factor，rhG-CSF)的復性及純化方法，本發明復性率高，操作簡單，適合於工業化放大生產。 The invention relates to the field of biomedicine and downstream protein purification of bioengineering, in particular to a method for renaturation and purification of recombinant human granulocyte colony stimulating factor (rhG-CSF). , the operation is simple, and it is suitable for industrial scale production.
人粒細胞集落刺激因子(human granulocyte colony stimulating factor，hG-CSF)屬於造血生長因子家族。hG-CSF是由單核巨噬細胞、血管內皮細胞及成纖維細胞合成的蛋白，由174個胺基酸組成，分子量為19KD，其序列是公知的，如序列1所示。hG-CSF的主要作用機理有：(1)特異地作用於骨髓粒細胞和巨噬細胞的前驅細胞，促進其向成熟粒細胞分化、增殖；(2)作用於骨髓成熟中性粒細胞，促進其從骨髓向外周血釋放；(3)啟動成熟粒細胞的功能，增強其遊走、吞噬及殺菌能力，延長其存活時間；(4)刺激骨髓造血幹細胞向外周血釋放。 Human granulocyte colony stimulating factor (human granulocyte colony stimulating factor, hG-CSF) belongs to the family of hematopoietic growth factors. hG-CSF is a protein synthesized by mononuclear macrophages, vascular endothelial cells and fibroblasts. It consists of 174 amino acids and has a molecular weight of 19KD. Its sequence is well known, as shown in SEQ ID NO: 1. The main mechanisms of action of hG-CSF are: (1) specifically acting on the precursor cells of bone marrow granulocytes and macrophages to promote their differentiation and proliferation into mature granulocytes; (2) acting on bone marrow mature neutrophils to promote It is released from bone marrow to peripheral blood; (3) activates the function of mature granulocytes, enhances their ability to migrate, phagocytose and kill bacteria, and prolongs their survival time; (4) stimulates the release of bone marrow hematopoietic stem cells to peripheral blood.
hG-CSF在臨床上有非常廣泛的用途，治療各種原因引起的中性粒細胞減少症，目前主要用於防治化療及放療後引起的骨髓抑制；此外還用於自身骨髓移植、某些類型的白血病、愛滋病的白血球減少和再生障礙性貧血等，均顯示具有顯著的療效。 hG-CSF has a very wide range of clinical uses in the treatment of neutropenia caused by various reasons. At present, it is mainly used for the prevention and treatment of myelosuppression caused by chemotherapy and radiotherapy; Leukemia, AIDS leukopenia and aplastic anemia, etc., all show significant curative effect.
hG-CSF的天然來源有限，產量甚微，不能滿足臨床應用的需要。同時聚乙二醇修飾的hG-CSF實現了長效增加外周血中性粒細胞數的目的，其規模化生產同樣需要大量的hG-CSF原液，因此hG-CSF原液的大規模生產有重大的經濟意義和社會意義。 The natural sources of hG-CSF are limited and the yield is very small, which cannot meet the needs of clinical application. At the same time, polyethylene glycol-modified hG-CSF achieves the purpose of increasing the number of neutrophils in peripheral blood in a long-term manner. Its large-scale production also requires a large amount of hG-CSF stock solution, so the large-scale production of hG-CSF stock solution has a significant impact. economic and social significance.
大腸桿菌是目前最為廣泛用來表達外源蛋白的表達系統，然而rhG-CSF在大腸桿菌系統中基本以包涵體形式表達，表達產物沒有生物活性，欲利用其生物活性，必須先經變性劑充分溶解，然後經復性過程恢復其天然構象，才能得到高活性的蛋白樣品，爾後藉由純化步驟得到高純度，有功能的產品。利用rhG-CSF不依賴於糖基化修飾即可發揮天然分子相應的生物學活性的特點，選擇在大腸桿菌中表達該蛋白是最為簡便可行而且產率較高的方法。 E. coli is the most widely used expression system for expressing foreign proteins. However, rhG-CSF is basically expressed in the form of inclusion bodies in the E. coli system, and the expression product has no biological activity. After dissolving and restoring its natural conformation through the renaturation process, a highly active protein sample can be obtained, and then a high-purity, functional product can be obtained through a purification step. Taking advantage of the fact that rhG-CSF can exert the corresponding biological activities of natural molecules independent of glycosylation modification, it is the most convenient and feasible method to express this protein in E. coli with high yield.
工藝研發過程中，影響得率的主要步驟在於復性，復性效果好，得率必然高。復性完全是自發和隨機的，因此復性速度慢，回收率低。目前國內外所用的蛋白質復性方法均是依據排除使蛋白變性的環境，具體方法有透析法、超濾復性、管柱復性、稀釋法等。 In the process of process research and development, the main step that affects the yield is renaturation. If the renaturation effect is good, the yield must be high. Refolding is completely spontaneous and random, resulting in slow renaturation and low recovery rates. At present, the protein renaturation methods used at home and abroad are all based on the exclusion of the environment that denatures the protein. Specific methods include dialysis, ultrafiltration renaturation, column renaturation, and dilution methods.
透析法需要時間長，且需要多次更換透析溶液，同時 透析袋內部溶液在透析過程中是不均一的，靠近透析膜的溶液變性劑濃度下降快而內部下降慢，只有部分蛋白處於適於復性的環境，其他蛋白很容易聚集、絮凝、沉澱，使復性回收率下降，更主要的是受透析袋規格的限制，透析法難以放大至生產規模。 The dialysis method takes a long time and requires multiple changes of the dialysis solution, while The solution inside the dialysis bag is not uniform during the dialysis process. The concentration of the denaturant in the solution near the dialysis membrane decreases rapidly while the inner decreases slowly. Only some proteins are in an environment suitable for renaturation, and other proteins are easily aggregated, flocculated, and precipitated. The recovery rate of renaturation decreases, and more importantly, it is difficult to scale up the dialysis method to the production scale due to the limitation of the size of the dialysis bag.
Amgen公司的美國專利5,849,883在1998年公開了一種G-CSF純化方法，其中rhG-CSF的復性採用了在攪拌條件下加入硫酸銅，藉由金屬離子氧化的方式進行蛋白復性。該方法缺點是有痕量金屬離子殘留的風險，進而影響藥品相關檢測及藥品安全性，目前在工業化生產中已很少採用。 US Patent No. 5,849,883 of Amgen Corporation in 1998 disclosed a G-CSF purification method, wherein the renaturation of rhG-CSF adopts the addition of copper sulfate under stirring conditions, and the protein renaturation is carried out by means of metal ion oxidation. The disadvantage of this method is that there is a risk of trace metal ions remaining, which in turn affects drug-related detection and drug safety, and is rarely used in industrial production at present.
中國專利CN1167150A報導了使用中空纖維超濾的方式進行rhG-CSF的復性，雖然可以用於大規模生產，但存在以下缺點：①受濃差極化現象影響，復性蛋白溶液在中空纖維柱內濃度並不均一，容易出現蛋白絮集、沉澱甚至堵塞中空纖維柱的情況；②大規模生產中往往需要長度超過90cm的中空纖維柱提供足夠的膜面積，研發階段的中空纖維柱長度都是在30-60cm範圍內，因此中空纖維超濾的操作無法線性放大，對於複雜的復性過程難以進行細緻的工藝研究來保障工藝的穩定性。 Chinese patent CN1167150A reported the use of hollow fiber ultrafiltration to renature rhG-CSF. Although it can be used for large-scale production, it has the following disadvantages: The internal concentration is not uniform, and it is easy to cause protein flocculation, precipitation or even blockage of the hollow fiber column; ② In large-scale production, a hollow fiber column with a length of more than 90 cm is often required to provide sufficient membrane area, and the length of the hollow fiber column in the research and development stage is all In the range of 30-60cm, the operation of hollow fiber ultrafiltration cannot be linearly scaled up, and it is difficult to carry out detailed process research for the complex renaturation process to ensure the stability of the process.
中國專利CN102344931A提供了一種鎳親和層析管柱復性的方法，該方法操作繁瑣，難以放大。中國專利CN104120159 A提供了一種Sephadex G-25管柱層析復性的方式，層析管柱直徑在5-20cm，管柱床高度60-100cm，層析 上樣蛋白濃度1.0-2.0mg/ml，上樣體積小於管柱床體積的20%，上樣和洗脫流速為5-10cm/h；按其報導的規模計算，批量約為10-20g，難以滿足商業化生產的需要。 Chinese patent CN102344931A provides a method for renaturation of nickel affinity chromatography column, which is complicated to operate and difficult to scale up. Chinese patent CN104120159 A provides a method for renaturing Sephadex G-25 column chromatography. The loading protein concentration is 1.0-2.0 mg/ml, the loading volume is less than 20% of the column bed volume, and the loading and elution flow rates are 5-10 cm/h; It is difficult to meet the needs of commercial production.
稀釋復性同其他方法相比而言具有設備要求簡單、操作方便、成本低、容易放大的優點，但是也存在大量錯誤的折疊和聚合，以至蛋白沉澱，使復性收率大大降低，平均只有20%左右。 Compared with other methods, dilution renaturation has the advantages of simple equipment requirements, convenient operation, low cost, and easy amplification. 20% or so.
中國專利CN1718739 A報導的rhG-CSF的復性方法是：第一次稀釋復性20小時以上→超濾濃縮→第二次稀釋復性96小時以上→超濾濃縮，即進行兩次稀釋復性操作和兩次超濾處理，該方法雖然結合使用了稀釋復性和超濾處理，復性率較高，但是存在復性步驟多、復性時間過長、效率低的缺點，這勢必影響整體收率並增加工藝放大的難度。 The renaturation method of rhG-CSF reported in Chinese patent CN1718739 A is: the first dilution and renaturation for more than 20 hours→ultrafiltration concentration→the second dilution and renaturation for more than 96 hours→ultrafiltration and concentration, that is, carry out two dilution and renaturation operation and two ultrafiltration treatments, although this method uses a combination of dilution renaturation and ultrafiltration treatment, the renaturation rate is high, but there are many renaturation steps, long renaturation time, and low efficiency, which will inevitably affect the overall yield and increase the difficulty of process scale-up.
針對上述問題，從包涵體變性溶解及復性的整個過程考慮，在包涵體蛋白變性溶解過程中需加入還原劑對其進行還原，通常使用二硫蘇糖醇(DL-Dithiotheitol,DTT)，然而，由於DTT容易被氧化，穩定性較差，而且容易揮發，作用易受環境影響，難以控制，因此在包涵體溶解後增加去除DTT的步驟再稀釋復性是穩妥的做法。 In view of the above problems, considering the whole process of inclusion body denaturation, dissolution and renaturation, a reducing agent needs to be added to reduce the inclusion body protein in the process of denaturation and dissolution, usually using dithiothreitol (DL-Dithiotheitol, DTT). However, , because DTT is easily oxidized, has poor stability, and is easy to volatilize, its effect is easily affected by the environment, and it is difficult to control. Therefore, it is a safe practice to add a step to remove DTT after the dissolution of inclusion bodies and then dilute and renature.
中國專利CN101045742A提供了一種重組人粒細胞刺激因子的復性純化方法，在復性前增加了將裂解液進行Sephadex G-25分離，去除多餘的還原劑DTT的步驟，復性品質收率高於50%，純化後rhG-CSF的RP-HPLC純度高於 97%，該方法藉由Sephadex G-25層析雖然可以達到除去裂解液中過量還原劑的目的，但是含有Urea的裂解液鹽濃度極高，進行Sephadex G-25層析時存在反壓大，存在層析流速慢，處理時間長，工業化放大難度大的缺點。 Chinese patent CN101045742A provides a method for renaturing and purifying recombinant human granulocyte stimulating factor. Before renaturation, the lysate is separated by Sephadex G-25 and the excess reducing agent DTT is removed. The renaturation quality yield is higher than 50%, the RP-HPLC purity of rhG-CSF after purification is higher than 97%, although this method can achieve the purpose of removing excess reducing agent in the lysate by Sephadex G-25 chromatography, the salt concentration of the lysate containing Urea is extremely high, and there is a large back pressure during Sephadex G-25 chromatography. There are disadvantages of slow flow rate of chromatography, long processing time, and difficulty in industrial scale-up.
同時，在復性過程中需要為蛋白分子內二硫鍵的正確形成提供適當的氧化還原環境，通常所用的氧化還原劑為GSH/GSSG，其中GSH和GSSG的比例也會影響蛋白的復性效率。藉由研究不同GSH和GSSG的添加比例對復性效果的影響，本發明創新性地開發了僅添加GSSG進行rhG-CSF稀釋復性的方法，開發了一種rhG-CSF更簡單、高效的復性和純化工藝。 At the same time, it is necessary to provide an appropriate redox environment for the correct formation of disulfide bonds in the protein molecule during the renaturation process. The redox agent usually used is GSH/GSSG, and the ratio of GSH and GSSG will also affect the renaturation efficiency of the protein. . By studying the effect of different addition ratios of GSH and GSSG on the renaturation effect, the present invention innovatively develops a method for rhG-CSF dilution and renaturation only by adding GSSG, and develops a simpler and more efficient renaturation method for rhG-CSF. and purification process.
本發明提供了一種重組人粒細胞刺激因子的製備方法，包括以下步驟：a)對rhG-CSF基因工程菌進行發酵培養，經分離和洗滌，獲得精製的包涵體；b)包涵體經變性液溶解得變性蛋白液；c)對變性蛋白液進行緩衝液置換，得置換後變性液；d)將置換後變性液進行稀釋復性；e)對復性後的rhG-CSF進行純化，獲得rhG-CSF原液。 The present invention provides a preparation method of recombinant human granulocyte stimulating factor, comprising the following steps: a) fermenting and culturing rhG-CSF genetically engineered bacteria, separating and washing to obtain refined inclusion bodies; b) denaturing the inclusion bodies by denaturing liquid Dissolving the denatured protein solution; c) performing buffer replacement on the denatured protein solution to obtain a denatured solution after replacement; d) diluting and renaturing the denatured solution after replacement; e) purifying the renatured rhG-CSF to obtain rhG - CSF stock solution.
其中，該步驟a中rhG-CSF基因工程菌係由帶有rhG-CSF基因的重組質粒轉化的大腸桿菌，如重組質粒pBV220/G-CSF轉化的大腸桿菌DH5α菌株。 Wherein, in step a, the rhG-CSF genetically engineered bacteria are Escherichia coli transformed by a recombinant plasmid with rhG-CSF gene, such as Escherichia coli DH5α strain transformed by recombinant plasmid pBV220/G-CSF.
包涵體洗滌緩衝液可以是：緩衝液A(5-100mmol/L Tris-HCl，2-20mmol/L EDTA，100-500mmol/L NaCl，2-4mol/L Urea，pH 7-9)，緩衝液B(5-100mmol/L Tris-HCl，2-20mmol/L EDTA，pH 8-10)；洗滌方式為緩衝液A→緩衝液B依次洗滌。 Inclusion body washing buffer can be: buffer A (5-100mmol/L Tris-HCl, 2-20mmol/L EDTA, 100-500mmol/L NaCl, 2-4mol/L Urea, pH 7-9), buffer B (5-100mmol/L Tris-HCl, 2-20mmol/L EDTA , pH 8-10); the washing mode is buffer A→buffer B to wash sequentially.
該步驟b的變性溶解條件可以是：包涵體按1：20至1：25(W/V)加入變性液(6-10mol/L Urea，2-20mmol/L DTT，2-20mmol/L EDTA，5-100mmol/L Tris-HCl，pH 7-9)中，攪拌裂解10-18h。 The denaturing and dissolving conditions of this step b may be: the inclusion bodies are added to a denaturing solution (6-10mol/L Urea, 2-20mmol/L DTT, 2-20mmol/L EDTA, 1:20 to 1:25 (W/V), 5-100mmol/L Tris-HCl, pH 7-9), stirring and cracking for 10-18h.
該步驟b的變性溶解條件較佳為：包涵體按1：20至1：25(W/V)加入變性液(8mol/L Urea，10mmol/L DTT，5mmol/L EDTA，20mmol/L Tris-HCl，pH 8.2)中，攪拌裂解10-18h。 The denaturing and dissolving conditions of this step b are preferably as follows: the inclusion bodies are added to a denaturing solution (8mol/L Urea, 10mmol/L DTT, 5mmol/L EDTA, 20mmol/L Tris- HCl, pH 8.2), stirring and lysing for 10-18 h.
該步驟c中置換用的緩衝液可包含6-10mol/L Urea，2-20mmol/L EDTA，5-100mmol/L Tris-HCl，pH 7-9，較佳為8mol/L Urea，5mmol/L EDTA，20mmol/L Tris-HCl，pH 8.2；置換用的緩衝液溫度控制為2至30℃，更佳為10至20℃；緩衝液置換的方式包括但不限於超濾、脫鹽管柱層析；其中超濾包括但不限於中空纖維超濾和膜包超濾；如若採用超濾進行緩衝液置換，超濾膜孔徑包括但不限於3KD，5KD和10KD，更佳為5KD；如若採用GE Healthcare公司的中空纖維柱進行超濾緩衝液置換，剪切速率可以控制在16000sec-1以下，更佳為約8000sec-1； 如若採用中空纖維超濾進行緩衝液置換，超濾條件可以是：①變性蛋白液在超濾系統中適當濃縮；②採用恒體 積置換，在超濾系統內保持樣品體積恒定，使流加的洗濾液速度與透出液速度相同；③跨膜壓力(TMP)不大於50PSI，更佳為10-18PSI；④共超濾置換樣品體積的3倍以上，考慮到合理的工藝用時，更佳為7倍體積，即共流加洗濾液體積為置換起始樣品體積的3倍以上，更佳為7倍體積。 The buffer used for replacement in this step c can comprise 6-10mol/L Urea, 2-20mmol/L EDTA, 5-100mmol/L Tris-HCl, pH 7-9, preferably 8mol/L Urea, 5mmol/L EDTA, 20mmol/L Tris-HCl, pH 8.2; the temperature of the buffer solution used for replacement is controlled to be 2 to 30°C, preferably 10 to 20°C; the methods of buffer replacement include but are not limited to ultrafiltration, desalting column chromatography ; Wherein ultrafiltration includes but is not limited to hollow fiber ultrafiltration and membrane package ultrafiltration; if ultrafiltration is used for buffer exchange, ultrafiltration membrane pore size includes but not limited to 3KD, 5KD and 10KD, preferably 5KD; if GE Healthcare is used The company's hollow fiber column is used for ultrafiltration buffer replacement, and the shear rate can be controlled below 16000sec -1 , more preferably about 8000sec -1 ; if the hollow fiber ultrafiltration is used for buffer replacement, the ultrafiltration conditions can be: ① Denaturation The protein solution is properly concentrated in the ultrafiltration system; ②Constant volume replacement is adopted, and the sample volume is kept constant in the ultrafiltration system, so that the flow rate of the filtrate is the same as that of the permeate; ③Transmembrane pressure (TMP) is not greater than 50PSI , more preferably 10-18PSI; ④ co-ultrafiltration replaces more than 3 times the volume of the sample, taking into account the reasonable process time, it is more preferably 7 times the volume, that is, the volume of the co-flow and washing filtrate is 3 times the volume of the initial sample to replace more than 7 times the volume.
該步驟d中的復性緩衝液可包含GSSG，所述GSSG的濃度可以是0.1-1mmol/L。較佳地，該復性緩衝液可以是0.5mmol/L GSSG，20mmol/L Tris-HCl，pH8.2，置換後變性液加入復性緩衝液前控制復性緩衝液溫度可為2至8℃； 該步驟d中稀釋復性的條件可以是：復性液蛋白終濃度0.1-0.4g/L，較佳地，該復性液蛋白終濃度0.3g/L，置換後變性液緩慢加入復性緩衝液，繼續攪拌30min後停止攪拌，2至8℃靜置復性10-18小時； 該步驟e中對rhG-CSF復性液可採用依次進行鹽析、疏水管柱層析、脫鹽管柱層析進行純化，其中鹽析可採用硫酸銨沉澱或氯化鈉沉澱，更較佳為硫酸銨沉澱；如若採用硫酸銨沉澱的方式，條件可為：攪拌條件下，緩慢加入(NH4)2SO4使其終濃度為0.9mol/L，並補加EDTA使其終濃度為5mmol/L，停止攪拌並靜置30min； 鹽析過程中的膜過濾可採用中空纖維切向流過濾、膜包過濾或死端過濾，更佳為中空纖維切向流過濾；如若採用中空纖維切向流過濾，優化後的條件為：膜孔徑0.2μm，跨膜壓(TMP)為3-8PSI，剪切速率約8000sec-1； 該步驟e中的管柱層析如若採用GE Healthcare公司的層析介質，優化後的層析純化策略是：先由苯基瓊脂糖凝膠Phenyl Sepharose FF管柱層析再到低吸附的葡聚糖凝膠Sephadex(如Sephadex G-25)管柱層析。 The renaturation buffer in this step d may contain GSSG, and the concentration of the GSSG may be 0.1-1 mmol/L. Preferably, the renaturation buffer can be 0.5mmol/L GSSG, 20mmol/L Tris-HCl, pH 8.2, and the temperature of the renaturation buffer can be controlled to be 2 to 8°C before the denaturation buffer is added to the renaturation buffer after replacement. ; The conditions for dilution and renaturation in this step d may be: the final protein concentration of the renaturing liquid is 0.1-0.4 g/L, preferably, the final protein concentration of the renaturing liquid is 0.3 g/L, and the denaturing liquid is slowly added to the renaturing liquid after replacement. Buffer solution, continue stirring for 30min, stop stirring, and stand at 2 to 8°C for renaturation for 10-18 hours; in this step e, the rhG-CSF renaturation solution can be subjected to salting out, hydrophobic column chromatography, and desalting column in sequence. Chromatography for purification, wherein salting out can be ammonium sulfate precipitation or sodium chloride precipitation, more preferably ammonium sulfate precipitation; if using ammonium sulfate precipitation, the conditions can be: under stirring conditions, slowly add (NH 4 ) 2 SO4 to make the final concentration 0.9mol/L, and add EDTA to make the final concentration 5mmol/L, stop stirring and let stand for 30min; Membrane filtration in the process of salting out can use hollow fiber tangential flow filtration, membrane bag Filtration or dead end filtration, preferably hollow fiber tangential flow filtration; if hollow fiber tangential flow filtration is used, the optimized conditions are: membrane pore size 0.2μm, transmembrane pressure (TMP) 3-8PSI, shear rate About 8000sec -1 ; If the column chromatography in this step e adopts the chromatographic medium of GE Healthcare company, the optimized chromatographic purification strategy is: firstly by the Phenyl Sepharose FF column chromatography of Phenyl Sepharose Sephadex (eg Sephadex G-25) column chromatography with low adsorption.
使用本發明所提供的重組人粒細胞刺激因子的製備方法得到的重組人粒細胞刺激因子原液可用於製備相應的製劑產品，也可用於製備聚乙二醇修飾的重組人粒細胞刺激因子，如WO9611953以及CN101172161A中描述的聚乙二醇修飾的重組人粒細胞刺激因子，其製備方法可包括本發明所述的重組人粒細胞刺激因子的製備方法製備重組人粒細胞刺激因子的步驟，以及將重組人粒細胞刺激因子與聚乙二醇偶聯的步驟。其中，聚乙二醇修飾的重組人粒細胞刺激因子的結構可以是如式I所示，
本發明的有益效果是： The beneficial effects of the present invention are:
(1)優化的包涵體洗滌工藝，為後續的純化步驟奠定了基礎。 (1) The optimized inclusion body washing process laid the foundation for the subsequent purification steps.
(2)稀釋復性前增加去除還原劑的步驟，提高了復性率。 (2) The step of removing the reducing agent is added before the dilution and renaturation, which improves the renaturation rate.
(3)藉由確定復性之前變性液中DTT的可接受殘留量，在保證復性率不受影響的前提下，顯著縮短了工藝操 作時間，且有利於蛋白活性的維持。 (3) By determining the acceptable residual amount of DTT in the denaturing solution before renaturation, the process operation is significantly shortened on the premise that the renaturation rate is not affected. time, and is conducive to the maintenance of protein activity.
(4)復性蛋白濃度高於一般的稀釋復性，降低了樣品處理體積。 (4) The concentration of renatured protein is higher than that of general dilution and renaturation, which reduces the sample processing volume.
(5)復性效率大幅提高至70%以上。 (5) The renaturation efficiency is greatly increased to more than 70%.
(6)本發明涉及的復性方法，優化了復性條件，大大降低了生產成本，且易於控制，提高了工藝操作的穩定性。 (6) The renaturation method involved in the present invention optimizes the renaturation conditions, greatly reduces the production cost, is easy to control, and improves the stability of the process operation.
(7)復性後蛋白純度即達到85%以上，降低了後續純化步驟的壓力。 (7) After renaturation, the protein purity reaches more than 85%, which reduces the pressure of subsequent purification steps.
(8)復性液保持澄清，未出現沉澱現象。 (8) The renaturation solution remained clear, and no precipitation occurred.
(9)工藝流程中沒有離心、透析等難以放大的方法，工藝放大的可行性好，成功放大至生產規模。 (9) There are no difficult-to-scale methods such as centrifugation and dialysis in the process flow, and the process scale is feasible and successfully scaled up to a production scale.
(10)整體工藝成本低，純化週期短。 (10) The overall process cost is low and the purification cycle is short.
(11)可獲得高純度，高活性的rhG-CSF蛋白原液，經檢測其SDS-PAGE電泳純度達到99%以上，RP-HPLC純度達到98%以上，比活性範圍在(10.0±4.0)×107IU/mg，高於《中華人民共和國藥典2015版》第三部“重組人粒細胞刺激因子注射液”項下原液檢定的要求。 (11) High-purity, high-activity rhG-CSF protein stock solution can be obtained. After testing, its SDS-PAGE electrophoresis purity reaches more than 99%, RP-HPLC purity reaches more than 98%, and the specific activity range is (10.0±4.0)×10 7 IU/mg, which is higher than the requirements of the original solution test under the item "Recombinant Human Granulocyte Stimulating Factor Injection" in Part III of "Pharmacopoeia of the People's Republic of China 2015 Edition".
第1圖為實施例一中洗滌後包涵體的SDS-PAGE電泳純度檢測圖譜，其中電泳道1：洗滌後的包涵體，電泳道2：rhG-CSF對照品(自製)。 Figure 1 is the SDS-PAGE electrophoresis purity detection map of the washed inclusion bodies in Example 1, wherein electrophoresis lane 1: washed inclusion bodies, electrophoresis lane 2: rhG-CSF reference substance (self-made).
第2圖為實施例一中rhG-CSF原液的SDS-PAGE電泳純度檢測圖譜，其中電泳道1：分子量標準蛋白Mark 12，電泳道2：rhG-CSF原液。 Figure 2 is the SDS-PAGE electrophoresis purity detection map of the rhG-CSF stock solution in Example 1, wherein electrophoresis lane 1: molecular weight standard protein Mark 12, and electrophoresis lane 2: rhG-CSF stock solution.
下面藉由具體實施例，對本發明的技術方案進行清晰、完整的描述，所描述的實施例僅僅是本發明的一部分實施例，而不是全部的實施例，基於本發明的實施例，本領域普通技術人員在沒有作出創造性勞動前提下所獲得的所有其他實施例，都屬於本發明保護的範圍。 The technical solutions of the present invention will be clearly and completely described below by means of specific embodiments. The described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, the common All other embodiments obtained by the skilled person without creative work fall within the protection scope of the present invention.
重組人粒細胞刺激因子包涵體的復性及純化方法，主要包括以下步驟： The method for renaturation and purification of recombinant human granulocyte-stimulating factor inclusion bodies mainly includes the following steps:
pBV220/G-CSF轉化的大腸桿菌DH5α菌株接入一級種子培養基(蛋白腖10g/L，酵母粉5g/L，NaCl 5g/L)，30℃ 220rpm培養7小時；一級種子接入二級種子培養基(蛋白腖10g/L，酵母粉5g/L，NaCl 5g/L，葡萄糖5g/L)，30℃ 220rpm培養17小時；二級種子接入發酵培養基(蛋白腖10g/L，酵母粉5g/L，NaCl 5g/L，葡萄糖5g/L，KH2PO4 2.7g/L，Na2HPO4 11g/L，MgSO4 0.3g/L)，30℃培養至pH、溶氧雙反彈(pH 7.0、溶氧
發酵後，取濕菌體1kg，用5倍體積菌體重量TE緩衝 液(20mmol/L Tris-HCl,5mmol/L EDTA,pH8.2)懸浮菌體，混合均勻，泵入高壓勻質機進行勻漿破碎。之後，離心分離獲得濕的包涵體粗品500g。 After fermentation, take 1kg of wet thalline and buffer with 5 times the volume of thalli weight TE liquid (20mmol/L Tris-HCl, 5mmol/L EDTA, pH8.2) to suspend the bacterial cells, mix well, and pump into a high-pressure homogenizer for homogenization and crushing. After that, 500 g of wet crude inclusion bodies were obtained by centrifugation.
將上述得到的粗包涵體用緩衝液A(20mmol/L Tris-HCl,5mmol/L EDTA,4mol/L Urea,0.25mol/L NaCl,pH8.2)和緩衝液B(20mmol/L Tris-HCl,5mmol/L EDTA,pH8.2)依次洗滌，獲得高純度的包涵體。 The crude inclusion bodies obtained above were treated with buffer solution A (20mmol/L Tris-HCl, 5mmol/L EDTA, 4mol/L Urea, 0.25mol/L NaCl, pH8.2) and buffer solution B (20mmol/L Tris-HCl). , 5mmol/L EDTA, pH8.2) washed sequentially to obtain high-purity inclusion bodies.
對包涵體進行SDS-PAGE電泳純度檢測，電泳檢測的條件和結果如下： The inclusion bodies were tested for purity by SDS-PAGE electrophoresis. The conditions and results of electrophoresis were as follows:
SDS聚丙烯醯胺凝膠：NuPAGE® 4-12% Bis-Tris Gel，Life technologies SDS Polyacrylamide Gel: NuPAGE® 4-12% Bis-Tris Gel, Life technologies
樣品緩衝液：NuPAGE® LDS Sample Buffer(4×)，Life technologies Sample buffer: NuPAGE® LDS Sample Buffer (4×), Life technologies
電泳緩衝液：NuPAGE® MOPS SDS Running Buffer(20×)，Life technologies Running buffer: NuPAGE® MOPS SDS Running Buffer (20×), Life technologies
染色液：GelcodeTM Blue safe protein Stain，Thermo scientific Staining solution: Gelcode TM Blue safe protein Stain, Thermo scientific
脫色液：純化水，自製 Decolorization solution: purified water, self-made
上樣後150V恒壓電泳至溴酚藍遷移膠底處，考馬斯亮藍快速染色法染色，凝膠成像儀進行純度分析。 After loading, the samples were electrophoresed at a constant voltage of 150V to the bromophenol blue migration gel bottom, stained by Coomassie brilliant blue fast staining, and the purity was analyzed by a gel imager.
從說明書第1圖電泳道1可以看出獲得的包涵體SDS-PAGE電泳純度為72.5%。
It can be seen from the
取20g初步純化的包涵體，以固液比1：20的比例用變性液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,10mmol/L DTT,pH8.2)變性溶解，室溫攪拌14至16小時，得變性蛋白液400ml。 Take 20g of preliminarily purified inclusion bodies, denature and dissolve them with a denaturing solution (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, 10mmol/L DTT, pH8.2) at a ratio of solid-liquid ratio of 1:20, Stir at room temperature for 14 to 16 hours to obtain 400 ml of denatured protein solution.
樣品澄清過濾：上述步驟2的變性蛋白液經10μm和0.45μm兩級過濾澄清；平衡中空纖維：選用5KD中空纖維柱，用平衡液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,pH8.2)平衡中空纖維及系統；濃縮：將澄清後樣品加入中空纖維系統，以8000sec-1的剪切速率運行系統，控制跨膜壓(TMP)為10-18PSI，濃縮至300ml；恒體積置換：向中空纖維系統中連續流加置換緩衝液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,pH 8.2)，控制流加速度與透出端流速相同，保持變性蛋白液體積恒定，超濾過程中共向變性蛋白液中連續補加2100ml置換緩衝液(20mmol/L Tris-HCl,5mmol/L EDTA,8mol/L Urea,pH 8.2)，期間控制跨膜壓(TMP)為10-18PSI，置換緩衝液溫度在10至20℃。 Sample clarification and filtration: the denatured protein solution in the above step 2 was clarified by 10 μm and 0.45 μm two-stage filtration; equilibrium hollow fiber: 5KD hollow fiber column was selected, and the equilibrium solution (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L) was used. Urea, pH8.2) to balance the hollow fiber and the system; concentration: add the clarified sample to the hollow fiber system, run the system at a shear rate of 8000sec -1 , control the transmembrane pressure (TMP) to 10-18PSI, and concentrate to 300ml; Constant volume displacement: Add displacement buffer (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, pH 8.2) to the hollow fiber system continuously, and control the flow acceleration to be the same as the flow rate at the permeate end to keep the denatured protein The liquid volume was constant. During the ultrafiltration process, 2100ml of replacement buffer (20mmol/L Tris-HCl, 5mmol/L EDTA, 8mol/L Urea, pH 8.2) was continuously added to the denatured protein solution, and the transmembrane pressure (TMP) was controlled during the process. For 10-18 PSI, replace the buffer temperature at 10 to 20 °C.
樣品收集：從中空纖維系統底閥處收集樣品，用100ml置換緩衝液沖洗中空纖維系統，沖洗液併入樣品中，得置換後變性蛋白液400ml。 Sample collection: collect the sample from the bottom valve of the hollow fiber system, rinse the hollow fiber system with 100ml of replacement buffer, and incorporate the rinse into the sample to obtain 400ml of denatured protein solution after replacement.
取置換後變性蛋白液20μl用RP-HPLC法檢測樣品中rhG-CSF蛋白濃度，RP-HPLC條件如下： Take 20 μl of the denatured protein solution after replacement and use RP-HPLC to detect the protein concentration of rhG-CSF in the sample. The RP-HPLC conditions are as follows:
rhG-CSF對照品：自製，蛋白濃度為0.85mg/ml rhG-CSF reference substance: homemade, protein concentration of 0.85mg/ml
色譜管柱：Symmetry ShieldTM RP18，3.5μm，100mm×4.6mm Chromatography column: Symmetry ShieldTM RP18, 3.5μm, 100mm×4.6mm
A相：三氟乙酸-水溶液(取1.0ml三氟乙酸加水至1000ml，充分混勻超聲脫氣20min) Phase A: trifluoroacetic acid-water solution (take 1.0 ml of trifluoroacetic acid and add water to 1000 ml, fully mix and ultrasonically degas for 20 minutes)
B相：三氟乙酸-乙腈溶液(取1.0ml三氟乙酸加入色譜純乙腈至1000ml，超聲脫氣20min) Phase B: trifluoroacetic acid-acetonitrile solution (take 1.0 ml of trifluoroacetic acid, add chromatographically pure acetonitrile to 1000 ml, and ultrasonically degas for 20 min)
室溫條件下，按下表進行梯度洗脫，檢測波長214nm。 At room temperature, gradient elution was performed according to the following table, and the detection wavelength was 214 nm.
根據檢測樣品及對照品中rhG-CSF的峰面積，用面積歸一化法計算得置換後變性蛋白液中rhG-CSF的濃度為7.5mg/ml。 According to the peak area of rhG-CSF in the test sample and the reference substance, the concentration of rhG-CSF in the denatured protein solution after replacement was calculated to be 7.5 mg/ml by the area normalization method.
配製緩衝液(20mmol/L Tris-HCl,pH8.2)9.6L，控制其溫度在2至8℃，將置換後變性蛋白液緩慢加入緩衝液中，即蛋白終濃度為0.3g/L，同時加入GSSG至終濃度為0.5mmol/L，攪拌30min混勻後停止攪拌，2至8℃靜置復性16至18小時。 Prepare 9.6L of buffer solution (20mmol/L Tris-HCl, pH8.2), control its temperature at 2 to 8°C, and slowly add the denatured protein solution after replacement into the buffer solution, that is, the final protein concentration is 0.3g/L, and at the same time Add GSSG to a final concentration of 0.5 mmol/L, stir for 30 minutes, stop stirring, and let stand at 2 to 8 °C for renaturation for 16 to 18 hours.
取復性液50μl用RP-HPLC法檢測樣品中rhG-CSF蛋白濃度(條件同步驟3中的RP-HPLC條件)，根據檢測樣品及對照品中rhG-CSF的峰面積，用面積歸一化法計算得復性液中rhG-CSF的濃度為0.23mg/ml。 Take 50 μl of the renaturation solution to detect the protein concentration of rhG-CSF in the sample by RP-HPLC (the conditions are the same as the RP-HPLC conditions in step 3), and normalize the area according to the peak area of rhG-CSF in the detected sample and reference substance. The concentration of rhG-CSF in the renaturation solution was calculated to be 0.23 mg/ml.
復性後的rhG-CSF溶液中加入硫酸銨至終濃度為0.9mol/L，加入EDTA至終濃度為5mmol/L，攪拌混勻後靜置30min，0.2μm中空纖維切向流澄清過濾後進行管柱層析純化。 Ammonium sulfate was added to the renatured rhG-CSF solution to a final concentration of 0.9 mol/L, and EDTA was added to a final concentration of 5 mmol/L. After stirring and mixing, let it stand for 30 min, and 0.2 μm hollow fiber tangential flow clarification and filtration were carried out. Column chromatography purification.
Phenyl Sepharose FF管柱層析：Phenyl Sepharose FF column chromatography:
(1)以含0.9mol/L(NH4)2SO4，pH8.2的20mmol/L Tris-HCl緩衝液平衡層析管柱，流速150cm/小時，平衡3倍柱體積；(2)澄清液樣品上樣，流速150cm/小時；(3)以含0.65mol/L(NH4)2SO4，pH8.2的20mmol/L Tris-HCl沖洗層析管柱，流速150cm/小時，沖洗5倍管柱體積；(4)用含0.1mol/L(NH4)2SO4，pH8.2的20mmol/L Tris-HCl 洗脫，流速150cm/小時，收集目標蛋白峰。 (1) Equilibrate the column with 20 mmol/L Tris-HCl buffer containing 0.9 mol/L (NH 4 ) 2 SO 4 , pH 8.2, flow rate 150 cm/hour, and equilibrate 3 times the column volume; (2) Clarify (3) Rinse the column with 20 mmol/L Tris-HCl containing 0.65mol/L (NH 4 ) 2 SO 4 , pH 8.2, at a flow rate of 150 cm/h, rinse 5 double the column volume; (4) Elute with 20 mmol/L Tris-HCl containing 0.1 mol/L (NH 4 ) 2 SO 4 , pH 8.2, flow rate 150 cm/hour, and collect the target protein peak.
Sephadex G-25管柱層析：Sephadex G-25 column chromatography:
(1)以pH 5.0的50mmol/L醋酸鹽緩衝液平衡層析管柱，流速150cm/小時，平衡2倍管柱體積。 (1) Equilibrate the column with 50 mmol/L acetate buffer at pH 5.0, the flow rate is 150 cm/hour, and equilibrate 2 times the column volume.
(2)Phenyl Sepharose FF管柱層析洗脫收集液上樣，單次上樣體積不超過管柱體積的1/4，流速150cm/小時。 (2) Phenyl Sepharose FF column chromatography elution collection solution was used for sample loading, and the single sample loading volume did not exceed 1/4 of the column volume, and the flow rate was 150 cm/hour.
(3)pH 5.0的50mmol/L醋酸鹽緩衝液洗脫，流速150cm/小時，收集目標蛋白峰。 (3) 50 mmol/L acetate buffer at pH 5.0 was eluted, and the flow rate was 150 cm/hour, and the target protein peak was collected.
蛋白收樣除菌過濾後即為重組人粒細胞刺激因子原液。 The recombinant human granulocyte-stimulating factor stock solution is obtained after sterilization and filtration of protein samples.
2)樣品檢測：2) Sample detection:
rhG-CSF原液用SDS-PAGE電泳、RP-HPLC方法進行純度分析。 The purity of rhG-CSF stock solution was analyzed by SDS-PAGE and RP-HPLC.
(1)SDS-PAGE法檢測rhG-CSF原液純度的條件同步驟1中的SDS-PAGE電泳分析條件
(1) The conditions for SDS-PAGE to detect the purity of rhG-CSF stock solution are the same as the conditions for SDS-PAGE electrophoresis analysis in
SDS-PAGE法進行rhG-CSF原液純度的檢測分析結果如第2圖所示，從分析結果可以看出，最終所得rhG-CSF原液的SDS-PAGE電泳純度為100%。 The SDS-PAGE method for the detection and analysis of the purity of the rhG-CSF stock solution is shown in Figure 2. It can be seen from the analysis results that the SDS-PAGE electrophoresis purity of the final rhG-CSF stock solution is 100%.
(2)RP-HPLC法檢測rhG-CSF原液純度的條件和結果如下： (2) The conditions and results of the RP-HPLC method to detect the purity of the rhG-CSF stock solution are as follows:
色譜管柱：Symmetry ShieldTM RP18，3.5μm，100mm×4.6mm Chromatography column: Symmetry ShieldTM RP18, 3.5μm, 100mm×4.6mm
A相：三氟乙酸-水溶液(取1.0ml三氟乙酸加水至1000ml，充分混勻超聲脫氣20min) Phase A: trifluoroacetic acid-water solution (take 1.0 ml of trifluoroacetic acid and add water to 1000 ml, fully mix and ultrasonically degas for 20 minutes)
B相：三氟乙酸-乙腈溶液(取1.0ml三氟乙酸加入色譜純乙腈至1000ml，超聲脫氣20min) Phase B: trifluoroacetic acid-acetonitrile solution (take 1.0 ml of trifluoroacetic acid, add chromatographically pure acetonitrile to 1000 ml, and ultrasonically degas for 20 min)
室溫條件下，按下表進行梯度洗脫，檢測波長280nm。 At room temperature, gradient elution was performed according to the following table, and the detection wavelength was 280 nm.
RP-HPLC法進行rhG-CSF原液純度的檢測，從結果分析可以看出，最終所得rhG-CSF原液的RP-HPLC純度為99.14%。 The RP-HPLC method was used to detect the purity of the rhG-CSF stock solution. From the analysis of the results, it can be seen that the RP-HPLC purity of the final rhG-CSF stock solution was 99.14%.
從說明書第2圖電泳道2可以看出獲得的rhG-CSF原液電泳純度為100%。 It can be seen from the electrophoresis lane 2 in Figure 2 of the manual that the electrophoretic purity of the obtained rhG-CSF stock solution is 100%.
以重組人粒細胞集落刺激因子活性測定國家標準品為活性標準品，用NFS-60細胞/MTT比色法測定rhG-CSF原液的生物學活性，並根據樣品蛋白濃度計算rhG-CSF原液比活性為1.35×108IU/mg。 Using the national standard for the determination of recombinant human granulocyte colony-stimulating factor activity as the activity standard, the biological activity of the rhG-CSF stock solution was determined by the NFS-60 cell/MTT colorimetric method, and the specific activity of the rhG-CSF stock solution was calculated according to the sample protein concentration. 1.35×10 8 IU/mg.
重組人粒細胞刺激因子包涵體的復性及純化方法，主要包括以下步驟： The method for renaturation and purification of recombinant human granulocyte-stimulating factor inclusion bodies mainly includes the following steps:
步驟1、2、3同實施例一的步驟1、2、3。
步驟4 rhG-CSF的稀釋復性Step 4 Dilution and renaturation of rhG-CSF
配製緩衝液(20mmol/L Tris-HCl,pH8.2)9.6L，控制其溫度在2至8℃，將置換後變性蛋白液緩慢加入緩衝液中，即蛋白終濃度為0.3g/L，同時加入GSSG至終濃度為0.3mmol/L，加入GSH至終濃度為0.1mmol/L，攪拌30min混勻後停止攪拌，2至8℃靜置復性16至18小時。 Prepare 9.6L buffer solution (20mmol/L Tris-HCl, pH8.2), control its temperature at 2 to 8°C, and slowly add the denatured protein solution after replacement into the buffer solution, that is, the final protein concentration is 0.3g/L, and at the same time Add GSSG to a final concentration of 0.3 mmol/L, add GSH to a final concentration of 0.1 mmol/L, stir for 30 minutes, stop stirring, and stand at 2 to 8 °C for renaturation for 16 to 18 hours.
取復性液50μl用RP-HPLC法檢測樣品中rhG-CSF蛋白濃度(條件同步驟3中的RP-HPLC條件)，根據檢測樣品及對照品中rhG-CSF的峰面積，用面積歸一化法計算得復性液中rhG-CSF的濃度為0.20mg/ml。 Take 50 μl of the renaturation solution to detect the protein concentration of rhG-CSF in the sample by RP-HPLC (the conditions are the same as the RP-HPLC conditions in step 3), and normalize the area with the peak area of rhG-CSF in the detected sample and reference substance The concentration of rhG-CSF in the renaturation solution was calculated to be 0.20 mg/ml.
同實施例一步驟5的工藝步驟。 The process steps are the same as those of step 5 in the first embodiment.
rhG-CSF原液用SDS-PAGE電泳、RP-HPLC方法進行純度分析，檢測方法同實施例一步驟5的樣品檢測方法，用NFS-60細胞/MTT比色法測定rhG-CSF的活性，所得rhG-CSF原液的SDS-PAGE電泳純度為100%，RP-HPLC純度為98.61%，比活性為1.26×108IU/mg。 The purity of the rhG-CSF stock solution was analyzed by SDS-PAGE electrophoresis and RP-HPLC. The detection method was the same as the sample detection method in step 5 of Example 1. The activity of rhG-CSF was measured by NFS-60 cell/MTT colorimetric method, and the obtained rhG - The SDS-PAGE electrophoresis purity of the CSF stock solution was 100%, the RP-HPLC purity was 98.61%, and the specific activity was 1.26×10 8 IU/mg.
採用實施例一、實施例二所述的方法進行重組人粒細胞刺激因子的復性及純化，製備rhG-CSF原液，同時採用現有技術CN101045742A公開的方法進行對比，對比結果如下：
可見，採用本發明所述的rhG-CSF復性和純化方法，簡化了操作步驟，工藝過程簡單，易於控制，復性蛋白濃度高於現有技術，降低了樣品處理體積，同時目的蛋白損失少，蛋白收率和品質明顯提高，適合於大規模工業生產。 It can be seen that the use of the rhG-CSF renaturation and purification method of the present invention simplifies the operation steps, the technological process is simple, easy to control, the renatured protein concentration is higher than the prior art, the sample processing volume is reduced, and the loss of the target protein is small at the same time, The protein yield and quality are significantly improved, and it is suitable for large-scale industrial production.
雖然本發明已將較佳實施例揭示如上，但是這並非用以限制本發明的內容，本發明的保護範圍以申請專利的實際申請專利範圍為准。 Although the preferred embodiments of the present invention have been disclosed above, this is not intended to limit the content of the present invention, and the protection scope of the present invention is subject to the actual scope of the patent application.
<110> 江蘇恆瑞醫藥股份有限公司(JIANGSU HENGRUI MEDICINE CO.,LTD.) <110> JIANGSU HENGRUI MEDICINE CO.,LTD.
<120> 一種重組人粒細胞刺激因子的復性及純化方法 <120> A kind of renaturation and purification method of recombinant human granulocyte stimulating factor
<130> 770026CPCT <130> 770026CPCT
<160> 1 <160> 1
<170> PatentIn version 3.3 <170> PatentIn version 3.3
<210> 1 <210> 1
<211> 175 <211> 175
<212> PRT <212> PRT
<213> 智人(Homo sapiens) <213> Homo sapiens
<400> 1
由於本案的圖為實驗數據，並非本案的代表圖。故本案無指定代表圖。 Since the graph of this case is experimental data, it is not a representative graph of this case. Therefore, there is no designated representative map in this case.
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| CN110041423A (en) * | 2018-01-16 | 2019-07-23 | 江苏奥赛康药业股份有限公司 | A kind of renaturation and purification process of recombinant human granulocyte colony stimulating factor |
| CN114224853B (en) * | 2022-01-04 | 2022-09-23 | 山东新时代药业有限公司 | Freeze-dried preparation for injection of polyethylene glycol recombinant human granulocyte stimulating factor |
| CN116874555B (en) * | 2023-09-08 | 2023-11-28 | 成都华任康生物科技有限公司 | Replacement liquid, kit and related application thereof |
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| CN101045742A (en) * | 2006-03-27 | 2007-10-03 | 华北制药集团新药研究开发有限责任公司 | Dipurification process of recombinant humangranulocyte colony stimulating factor |
| CN101616678A (en) * | 2006-11-17 | 2009-12-30 | 东亚制药株式会社 | Polyethylene glycol-G-CSF conjugate |
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| CA1339757C (en) * | 1987-04-16 | 1998-03-17 | Robert F. Halenbeck | Production of purified biologically active, bacterially produced recombinant human csf-1 |
| JP2517100B2 (en) * | 1989-03-08 | 1996-07-24 | 協和醗酵工業株式会社 | Purification method of protein having human granulocyte colony-stimulating factor activity |
| CN101220082B (en) * | 2008-01-28 | 2012-09-05 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN103014100B (en) * | 2012-12-25 | 2014-10-01 | 哈药集团生物工程有限公司 | Purifying method for recombinant human granulocyte stimulating factor |
| CN103233053B (en) * | 2013-04-03 | 2014-03-05 | 北京四环生物制药有限公司 | Production method for recombinant human granulocyte colony-stimulating factor |
| LT6161B (en) * | 2013-09-27 | 2015-06-25 | Uab Profarma | Fused proteins of granulocyte colony-stimulating factor with other partners of growh factor, preferably with stem cell factor, and method of preparation thereof |
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| CN101045742A (en) * | 2006-03-27 | 2007-10-03 | 华北制药集团新药研究开发有限责任公司 | Dipurification process of recombinant humangranulocyte colony stimulating factor |
| CN101616678A (en) * | 2006-11-17 | 2009-12-30 | 东亚制药株式会社 | Polyethylene glycol-G-CSF conjugate |
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