WO2023143391A1 - 低内毒素胶原蛋白的制备方法 - Google Patents

低内毒素胶原蛋白的制备方法 Download PDF

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WO2023143391A1
WO2023143391A1 PCT/CN2023/073213 CN2023073213W WO2023143391A1 WO 2023143391 A1 WO2023143391 A1 WO 2023143391A1 CN 2023073213 W CN2023073213 W CN 2023073213W WO 2023143391 A1 WO2023143391 A1 WO 2023143391A1
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collagen
endotoxin
low
liquid
solution
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PCT/CN2023/073213
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French (fr)
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范代娣
史静静
古娟
段志广
徐茹
严建亚
刘琳
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陕西巨子生物技术有限公司
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Publication of WO2023143391A1 publication Critical patent/WO2023143391A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2400/00—Materials characterised by their function or physical properties
    • A61L2400/06—Flowable or injectable implant compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/34—Materials or treatment for tissue regeneration for soft tissue reconstruction

Definitions

  • the invention belongs to the field of biotechnology, and relates to a collagen, which has a low endotoxin content, and can be prepared into a collagen injection for facial filling through the aseptic processing technology of filtration and sterilization, which guarantees maximum protection Safe and effective for facial implants.
  • Collagen is a biological polymer protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. Collagen is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, etc. It is one of the most critical raw materials in the biotechnology industry. It is used in medical materials, cosmetics , Food industry are widely used.
  • the source of natural human collagen is limited.
  • the natural collagen currently used in industry is mainly extracted from animal skin or bone by acid, alkali or enzymatic methods, and its main source is animal connective tissue.
  • collagen extracted from animal tissues has risks such as animal-derived diseases, and at the same time, large-scale preparations have caused huge pressure on animal feeding on the supply side.
  • human collagen may be that its amino acid sequence contains more sites that are prone to hydrolysis. Therefore, technicians construct recombinant collagen by selecting and repeating short amino acid sequences from natural human collagen in order to avoid sites prone to hydrolysis, thereby improving the stability of collagen while maintaining the excellent properties of natural human collagen. performance.
  • the recombinant collagen constructed by repeating the short amino acid sequence from natural human collagen has a relatively monotonous amino acid composition and distribution. In theory, this will cause a large charge load on its surface, and it is difficult to achieve a stable equilibrium state as a whole.
  • the homology between the recombinant collagen obtained by such mutation and natural human collagen is reduced, and immunogenicity problems may arise, so it is not suitable for use in biomaterials that need to be in long-term contact with the human body.
  • tissue engineering materials such as dermal fillers are an important application direction of collagen.
  • collagen is required to have good mechanical strength and stability in aqueous solution (it can be stored in aqueous solution for a long time).
  • the greater the molecular weight of collagen the better its mechanical strength, and the poorer its stability in aqueous solution, especially for recombinant collagen constructed by repeating short amino acid sequences from natural human collagen. in this way.
  • the single-chain molecular weight of natural human collagen is about 110-130kD. From a practical point of view, technicians generally believe that the molecular weight of collagen suitable for replacing natural human collagen as tissue engineering materials needs to reach more than 100kD.
  • the source of natural human collagen is limited, animal-derived collagen has the risk of spreading diseases, and human collagen expressed by genetic engineering is prone to degradation during fermentation, purification and storage.
  • short amino acid Recombinant collagen constructed by repeating sequences is unstable in aqueous solution, and recombinant collagen obtained through mutational transformation has immunogenicity problems. Therefore, how to obtain collagen suitable for replacing natural human collagen as tissue engineering materials has become a technical challenge. field limitations.
  • the inventors conducted in-depth research.
  • the inventor first conducted a technical literature survey on the recombinant collagen constructed by repeating the short amino acid sequence from natural human collagen, and selected some of the prior art from natural human type I collagen (the most widely used tissue engineering material) short amino acid sequences, and then use these short amino acid sequences as repeating units to construct recombinant collagens with different molecular weights, and investigate the long-term storage stability of these recombinant collagens in aqueous solution, in order to obtain long-term stable storage in aqueous solution and meet the requirements of The large molecular weight (above 100kD) recombinant collagen required for the mechanical strength of tissue engineering materials.
  • the inventors unexpectedly discovered that the recombinant type I collagen obtained by repeating 75 to 110 times of a segment of pentapentadecantide (G A P G A P G S Q G A P G L Q) from natural human type I collagen has exceptionally excellent stability. It is embodied in: (1) although the length of its repeating unit is the shortest among all recombinant collagens tested by the inventor, its stability in aqueous solution is the best; The more monotonous the distribution, the greater the surface charge load of the recombinant collagen thus constructed, the less likely it is to reach a stable equilibrium state, and thus the easier it is to hydrolyze.
  • the present invention includes:
  • a macromolecular type I recombinant collagen which is composed of a short amino acid sequence from natural human type I collagen as a repeating unit, wherein the short amino acid sequence is as SEQ ID No.: 1( G A P G A P G S Q G A P G L Q), the number of repetitions is 75 to 110 times.
  • the macromolecular type I recombinant collagen according to item 1 which has a molecular weight of more than 120kD.
  • tissue engineering material is selected from dermal fillers, artificial skin, bone implants, intercellular scaffolds and collagen sponges.
  • the inventors are conducting more in-depth research on the reason why the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability.
  • Preliminary research results show that this may be because: the stability of recombinant collagen that repeats with a certain amino acid sequence as a repeating segment is closely related to the surface charge, and the surface charge is related to the composition of amino acids and the spatial structure of the protein. After a specific number of repetitions, a certain spatial structure is just formed, so that the surface load is in a balanced or near-balanced state, so it will show an abnormally stable state.
  • the inventor just found that the 15 amino acid repeat sequence collagen is in the range of load balance, so the macromolecular type I recombinant collagen of the present invention has exceptionally excellent stability.
  • the collagen liquid or collagen freeze-dried product prepared by this method has high purity (for example, more than 99%) and low endotoxin content (for example, less than 0.01 EU/mg).
  • the collagen liquid prepared by the method or the low-endotoxin collagen freeze-dried product can be used to prepare collagen injection, and the collagen injection can be used for Examples include facial fillers to correct dynamic forehead lines, including glabellar, forehead and crow's feet.
  • the preparation method of the low-endotoxin collagen liquid or the low-endotoxin collagen freeze-dried product of the present invention is particularly suitable for purifying the collagen liquid containing the macromolecular type I recombinant collagen of the present invention to obtain low-endotoxin collagen containing the present invention.
  • Collagen liquid or collagen freeze-dried product of macromolecular type I recombinant collagen is particularly suitable for purifying the collagen liquid containing the macromolecular type I recombinant collagen of the present invention to obtain low-endotoxin
  • the present invention also includes:
  • the low-endotoxin collagen solution can be prepared by, for example, the preparation method of the low-endotoxin collagen solution described below.
  • a preparation method of low endotoxin collagen liquid which comprises the following steps:
  • Step A Load the collagen sample solution on the cation exchange chromatography column, the collagen sample solution is composed of collagen, 1-3v/v% PEG-8 caprylic acid/capric glycerides and 2-5v/v% A solution of v% Tween 80, 0.1644w/v% sodium dihydrogen phosphate, 0.0894% w/v% disodium hydrogen phosphate and 0.585w/v% sodium chloride, with a pH of 6.5;
  • Step B Use buffers 1 to 3 to rebalance, wash and elute the cation exchange chromatography column respectively, collect the eluted peaks in the endotoxin-removing collection tube to obtain a low-endotoxin collagen solution,
  • the buffer solution 1 is used to rebalance the cation exchange chromatography column, which contains 1-3v/v% PEG-8 caprylic/capric glycerides, 2-5v/v% Tween 80, 0.1644w
  • the aqueous solution of the sodium dihydrogen phosphate of /v%, the disodium hydrogen phosphate of 0.0894%w/v% and the sodium chloride of 0.585w/v%, the pH is 6.5, and the volume is 0.5 ⁇ of the volume of described collagen sample liquid 2 times, such as about 1 times;
  • the buffer solution 2 acts as a wash, which is an aqueous solution comprising 0.1644w/v% sodium dihydrogen phosphate, 0.0894%w/v% disodium hydrogen phosphate and 1.17w/v% sodium chloride, with a pH of 6.5, the volume is 1 to 3 times the volume of the collagen sample solution, for example about 1.5 times;
  • the buffer 3 is used for elution, which is an aqueous solution comprising 0.1644w/v% sodium dihydrogen phosphate, 0.0894w/v% disodium hydrogen phosphate and 5.85w/v% sodium chloride, with a pH of 6.5 , the volume is 0.3-1 times, for example about 0.5 times, the volume of the collagen sample solution.
  • the elution peak can be confirmed by monitoring the A220 value of the eluate.
  • step A equilibrate the cation exchange chromatography column with the base solution until the baseline is stable .
  • the base solution comprises a buffer (pH 6.5) of 0.1644w/v% sodium dihydrogen phosphate, 0.0894w/v% disodium hydrogen phosphate and 0.585w/v% sodium chloride.
  • the cation exchange chromatography column is a Capto S cation exchange chromatography column, and is treated with 0.5M NaOH for 30min to remove Pyrogen.
  • the purity of collagen can be confirmed by SDS-PAGE detection.
  • a method for preparing a low-endotoxin collagen freeze-dried product which includes: preparing a low-endotoxin collagen solution by using the method for preparing a low-endotoxin collagen solution described in any one of items 2-5 to 2-10. collagen liquid, and freeze-drying the low-endotoxin collagen liquid to obtain a low-endotoxin collagen freeze-dried product.
  • PEG-8 caprylic/capric glycerides are polyethylene glycol-8 caprylic/capric glycerides, and Tween 80 is polysorbate 80.
  • FIG. 1 is an SDS-PAGE electrophoresis image of a portion of type I recombinant collagen prepared in Example 1. Take No.4, 6, 7, and 10 as examples for control proteins.
  • Fig. 2 is the HPLC picture of part of the test samples prepared in Example 2 after standing for 12 months. Take No.4, 7, 10, and 13 as examples for control proteins.
  • Fig. 3 is the infrared spectrogram of No.1 and No.6 type I recombinant collagen.
  • Fig. 4 is the Raman spectrum of No.1 and No.6 type I recombinant collagens.
  • Fig. 5 is the measurement result of the endotoxin content in the low-endotoxin collagen freeze-dried fiber after removing endotoxin according to the operation steps of Example 5 and freeze-drying.
  • Fig. 6 is the detection result of the purity of collagen in the low-endotoxin collagen freeze-dried fibers after removing endotoxin and freeze-drying according to the operation steps of Example 5.
  • the enzymes used in the specific embodiment are all purchased from TaKaRa Company, the plasmid DNA extraction kit and the DNA gel recovery kit are purchased from Beijing Suolaibao Company, and the gene recombination kits (Reorganization Kits) are purchased from For Tiangen Biology, the specific operation was carried out in accordance with the instructions of the kit.
  • Yeast expression strains expressing No. 1-18 type I recombinant collagen shown in Table 1 were constructed. The specific operation is: after optimization according to the codon preference of Pichia pastoris, synthesize the corresponding target gene by whole gene synthesis, and add SnaB I and Not I restriction sites at both ends of the gene respectively, and use SnaBI and NotI enzymes to The target gene was double-enzymatically digested, and then ligated with pPIC9k, which was also digested with SnaB I and Not I enzymes, under the action of T4 ligase. After connecting overnight at 16°C, it was transferred into Top10 competent cells and coated with ampicillin-resistant plates.
  • the supernatant prepared by fermentation is concentrated by 30kD ultrafiltration membrane, it is separated by CM ion exchange column, eluted with 35% NaCl solution, and the eluate is collected, desalted and concentrated, and then freeze-dried to obtain type I Preparation of recombinant collagen. Take 0.1g of lyophilized powder and dissolve it in 100ml of normal saline, fully dissolve it and perform SDS-PAGE gel electrophoresis to confirm the molecular weight and protein electrophoresis purity.
  • Embodiment 2 Stability experiment of various type I recombinant collagens in aqueous solution
  • the type I recombinant collagen (No.1 ⁇ 3) of the present invention shows exceptionally excellent stability in aqueous solution, and it is placed in aqueous solution for 12 months, and the purity can still be as high as more than 97% (see Figure 2A-G).
  • Example 3 Preliminary research on the reasons why type I recombinant collagen No. 1-3 has abnormal stability in aqueous solution
  • Embodiment 4 Low endotoxin collagen liquid and preparation of freeze-dried product
  • the 60L protein sample solution which contains No.1 type I recombinant collagen prepared in Example 1, 0.1644w/v% sodium dihydrogen phosphate, 0.0894% w/v% disodium hydrogen phosphate and 0.585w/v% sodium chloride, pH 6.5, measuring its endotoxin content is about 50EU/mg
  • PEG-8 caprylic acid/capric glycerides and Tween 80 so that their concentration is 0.5% (v /v) and 1% (v/v), and load it on the above equilibrated Capto S cation exchange column.
  • the column was rebalanced with 60L of buffer solution 1, the composition of the buffer solution 1 was 0.1644w/v% sodium dihydrogen phosphate, 0.0894% w/v% disodium hydrogen phosphate and 0.585 W/v% sodium chloride, pH 6.5+0.5% (v/v) PEG-8 caprylic/capric glycerides+1% (v/v) Tween 80; then wash with 90L buffer 2 Miscellaneous, the composition of described buffer solution 2 is the sodium dihydrogen phosphate of 0.1644w/v%, the disodium hydrogen phosphate of 0.0894%w/v% and the sodium chloride of 1.17w/v%, pH 6.5; Buffer 3 is used for elution, and the composition of the buffer 3 is sodium dihydrogen phosphate comprising 0.1644w/v%, disodium hydrogen phosphate 0.0894w/v%, and sodium chloride 5.85w/v%, pH 6.5 .
  • the low-endotoxin collagen liquid was freeze-dried after the endotoxin content was measured to obtain the low-endotoxin collagen freeze-dried product of Example 2.
  • Example 5 Operate as in Example 4, and prepare the low-endotoxin collagen liquid and lyophilized product of Examples 5-13, the difference is that PEG-8 caprylic acid/capric glycerin is added to the protein sample solution in Example 1 Esters and Tween 80, so that their concentrations are shown in Table 5;
  • the endotoxin content (EU/mg collagen) in the low-endotoxin collagen freeze-dried product (fibrous) prepared in each example was measured, and the measurement results are shown in Table 5.
  • Fig. 5 is the measurement result of the endotoxin content in the low-endotoxin collagen freeze-dried fiber after removing endotoxin and freeze-drying according to the operation steps of Example 5, and the collagen content in the test sample is prepared to be 4 mg/mL.
  • Determination of collagen content Determination according to the method stipulated in the first method of Kjeldahl method in the fourth general rule 0731 of "Pharmacopoeia of the People's Republic of China".
  • Collagen purity test the low-endotoxin collagen freeze-dried fiber was prepared into a 1mg/mL solution with water, and it was determined according to the "Size Exclusion Chromatography" in the fourth general rule 0514 "chromatography" of the Pharmacopoeia of the People's Republic of China. Purity was calculated by area normalization.
  • Fig. 6 is the test result of the purity of collagen in the low-endotoxin collagen freeze-dried fibers after removing endotoxin and freeze-drying according to the operation steps of Example 5.
  • Fig. 5 and Fig. 6 respectively show the detection results of endotoxin content and protein purity of 2211001 batches of recombinant collagen freeze-dried fibers prepared according to the operation of Example 5.
  • the pharmaceutical industry standard YY0954-2015 stipulates that the endotoxin content in the protein injection is lower than 0.5 EU/mL, and the endotoxin content detection results of the above-mentioned Examples 5, 6, 8-10 show that the low-endotoxin collagen liquid of the present invention ( The clinical use concentration is 4mg/mL) endotoxin is lower than 0.04EU/mL, which is less than 10 times of the standard requirement, indicating that the endotoxin removal process of the present invention has a remarkable effect and can fully meet the safety requirements of facial injection fillers.

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Abstract

提供一种包括低内毒素胶原蛋白液和低内毒素胶原蛋白冻干物的低内毒素胶原蛋白及其制备方法，步骤包括上样、再平衡、洗杂和洗脱；对胶原蛋白液进行冷冻干燥得到低内毒素胶原蛋白冻干物。该胶原蛋白纯度为99％以上，且内毒素含量低于0.01EU/mg，当以4mg/mL的浓度作为胶原蛋白注射液临床使用时，所述胶原蛋白注射液中的内毒素含量低于0.04EU/mL，远低于医药行业标准YY0954-2015规定的内毒素限值0.5EU/mL。

Description

低内毒素胶原蛋白的制备方法 技术领域
本发明属于生物技术领域，涉及一种胶原蛋白，所述胶原蛋白的内毒素含量低，通过过滤除菌的无菌加工处理技术可制备成用于面部填充的胶原蛋白注射液，最大程度保证了用于面部植入剂的安全及有效。
背景技术
胶原蛋白是一种生物高分子蛋白，是动物结缔组织中的主要成分，也是哺乳动物体内含量最多、分布最广的功能性蛋白，占蛋白质总量的25％～30％。胶原蛋白与组织的形成、成熟、细胞间信息的传递以及关节润滑、伤口愈合、钙化作用、血液凝固和衰老等有着密切的关系，是生物科技产业最关键的原材料之一，在医学材料、化妆品、食品工业中均有广泛应用。
天然的人胶原蛋白来源受限，目前工业上使用的天然胶原蛋白主要是通过酸、碱或者酶法提取动物的皮肤或骨骼中的胶原蛋白，其主要来源为动物结缔组织。但从动物组织中提取的胶原蛋白存在动物源疾病等风险，同时大规模的制备对供给侧的动物饲养造成巨大的压力。
随着基因工程技术的广泛应用，通过采用合适的工程菌株(大肠杆菌、毕赤酵母等)外源性表达人胶原蛋白，成功地解决了人胶原蛋白大规模制备的瓶颈问题。但是，利用工程菌株例如毕赤酵母基因工程菌株表达人胶原蛋白时，人胶原蛋白会在发酵、纯化及保存过程中发生降解，这增加了生产成本，并且影响了这种方法生产的人胶原蛋白的性能。
推测人胶原蛋白发生降解的原因可能是其氨基酸序列中含有较多的易发生水解的位点。因此，技术人员通过选择来自天然人胶原蛋白的短氨基酸序列进行重复来构建重组胶原蛋白，以期回避易发生水解的位点，从而提高胶原蛋白的稳定性，同时还能保持天然人胶原蛋白的优良性能。但是，通过来自天然人胶原蛋白的短氨基酸序列重复构建的重组胶原蛋白，其氨基酸组成及分布都相对单调，理论上讲，这会造成其表面的电荷负载大，整体不容易达到稳定的平衡状态，因而在水溶液中容易水解、变性，且存在短氨基酸序列重复单元越短、重复次数越多，重组胶原蛋白分子在水溶液中越不稳定的倾向。
可以尝试通过对来自天然胶原蛋白的短氨基酸序列进行突变并以此作为重复单元，从而获得更耐降解的重组胶原蛋白。但是，这样通过突变改造得到的重组胶原蛋白与天然人胶原蛋白的同源性降低，可能出现免疫原性问题，因而不适合应用于需与人体长期接触的生物材料。
另一方面，组织工程材料例如皮下填充剂等是胶原蛋白的一个重要应用方向，作为组织工程材料，要求胶原蛋白具有良好的力学强度和在水溶液中的稳定性(可在水溶液中长期保存)。一般而言，胶原蛋白的分子量越大，其力学强度就越好，而其在水溶液中的稳定性越差，对于通过来自天然人胶原蛋白的短氨基酸序列重复构建的重组胶原蛋白而言更是如此。
天然人胶原蛋白的单链分子量约为110-130kD，从实用性的观点出发，技术人员普遍认为适合替代天然人胶原蛋白用作组织工程材料的胶原蛋白的分子量需要达到100kD以上。然而，正如前述，天然的人胶原蛋白来源受限，动物源胶原蛋白存在传播疾病的风险，基因工程表达的人胶原蛋白容易在发酵、纯化及保存过程中发生降解，通过来自天然人胶原蛋白的短氨基酸 序列重复构建的重组胶原蛋白在水溶液中不稳定，而通过突变改造得到的重组胶原蛋白又存在免疫原性问题，因此，如何获得适合替代天然人胶原蛋白用作组织工程材料的胶原蛋白成为本技术领域的限制性问题。
另一个技术问题是，重组胶原蛋白是通过工程菌株表达的，其中容易包含内毒素。而按照医药行业标准，用于组织工程医疗产品的胶原蛋白对内毒素含量的限值有着严格的要求，因此需要尽可能地去除其中的内毒素。然而，特别是在工业规模的生产中，内毒素去除工艺的效果难以令人满意。
发明内容
为了解决现有技术中存在的上述技术问题，发明人进行了深入研究。发明人首先对通过来自天然人胶原蛋白的短氨基酸序列重复构建的重组胶原蛋白进行了技术文献调研，选取了现有技术中的一些来自天然人I型胶原蛋白(应用最广泛的组织工程材料)的短氨基酸序列，然后分别将这些短氨基酸序列作为重复单元，构建不同分子量的重组胶原蛋白，考察这些重组胶原蛋白在水溶液中长期保存的稳定性，以期获得能够在水溶液中长期稳定保存且能够满足作为组织工程材料的力学强度要求的大分子量(100kD以上)重组胶原蛋白。
上述研究的结果是，发明人意外发现，通过将来自天然的人I型胶原蛋白的一段十五肽(G A P G A P G S Q G A P G L Q)进行75～110次重复而得到的重组I型胶原蛋白具有异常优异的稳定性。具体体现在：(1)尽管其重复单元的长度是发明人测试的所有重组胶原蛋白中最短的，但其在水溶液中的稳定性是最好的；而通常认为，重复单元越短，氨基酸组成及分布就越单调，由此构建的重组胶原蛋白的表面电荷负载越大，越不容易达到稳定的平衡状态，因而更易水解。(2)其甚至比将该十五肽进行52次或62次或72次重复而 得到的重组I型胶原蛋白更加稳定，而通常认为，重复次数越多，分子量越大，重组胶原蛋白的表面电荷负载越大，越不容易达到稳定的平衡状态，因而更易水解。
基于上述发现，发明人完成了本发明。即本发明包括：
1.一种大分子I型重组胶原蛋白，其由来自天然人I型胶原蛋白的短氨基酸序列作为重复单元进行多次重复而构成，其中，所述短氨基酸序列如SEQ ID No.:1(G A P G A P G S Q G A P G L Q)所示，重复次数为75～110次。
2.根据项1所述的大分子I型重组胶原蛋白，其中，所述重复次数为80～105次，优选82～102次。
3.根据项1所述的大分子I型重组胶原蛋白，其分子量为120kD以上。
4.根据项1所述的大分子I型重组胶原蛋白，其还带有使其易于纯化的标签。
5.根据项4所述的大分子I型重组胶原蛋白，其中，所述标签为His标签、Flag标签或c-Myc标签。
6.根据项1～5中任一项所述的大分子I型重组胶原蛋白作为组织工程材料的用途。
7.根据项6所述的用途，其中，所述组织工程材料选自皮下填充剂、人工皮肤、骨植入剂、细胞间质支架和胶原蛋白海绵。
8.根据项1～5中任一项所述的大分子I型重组胶原蛋白作为皮下填充剂、人工皮肤、骨植入剂、细胞间质支架或胶原蛋白海绵的用途。
关于本发明的大分子I型重组胶原蛋白具有异常优异的稳定性的原因，发明人正在进行更为深入的研究。初步的研究结果表明，这可能是因为：以某一氨基酸序列作为重复片段进行重复的重组胶原，其稳定性与表面电荷密切相关，而表面电荷与氨基酸组成及蛋白的空间结构相关联，达到某一特定的重复次数后刚好形成了某一空间结构，使得表面荷载处于平衡或近平衡的状态，因此会表现出异常稳定的状态。发明人刚好找到了该15个氨基酸重复序列胶原蛋白处于荷载平衡的范围，因而本发明的大分子I型重组胶原蛋白具有异常优异的稳定性。
通过该方法制备的胶原蛋白液或胶原蛋白冻干物的纯度高(例如99％以上)且内毒素含量低(例如0.01EU/mg以下)。该方法制备的胶原蛋白液或低内毒素胶原蛋白冻干物可用于制备胶原蛋白注射液，该胶原蛋白注射液可用于 例如面部填充，以纠正额部动力性皱纹，包括眉间纹、额头纹和鱼尾纹。本发明的低内毒素胶原蛋白液或低内毒素胶原蛋白冻干物的制备方法特别适合于对包含本发明大分子I型重组胶原蛋白的胶原蛋白液进行纯化，得到内毒素低的包含本发明大分子I型重组胶原蛋白的胶原蛋白液或胶原蛋白冻干物。
因此，本发明还包括：
2-1.一种低内毒素胶原蛋白液或低内毒素胶原蛋白冻干物，其中，胶原蛋白的纯度为99％以上，且内毒素含量低于0.01EU/mg。所述低内毒素胶原蛋白液可以采用例如下述的低内毒素胶原蛋白液的制备方法进行制备。
2-2.根据项2-1所述的低内毒素胶原蛋白液或低内毒素胶原蛋白冻干物，其中，所述胶原蛋白是项1所述的大分子I型重组胶原蛋白。
2-3.根据项2-1或2-2中所述的低内毒素胶原蛋白液或低内毒素胶原蛋白冻干物在制备胶原蛋白注射液中的用途。
2-4.根据项2-3所述的用途，其中，所述胶原蛋白注射液用于面部填充，以纠正额部动力性皱纹，所述额部动力性皱纹包括眉间纹、额头纹和鱼尾纹。
2-5.一种低内毒素胶原蛋白液的制备方法，其包括下述步骤：
步骤A：将胶原蛋白样本液上样于阳离子交换层析柱，所述胶原蛋白样本液是包含胶原蛋白、1～3v/v％的PEG-8辛酸/癸酸甘油酯类和2～5v/v％的吐温80、0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和0.585w/v％的氯化钠的溶液，pH为6.5；
步骤B：依次用缓冲液1～3分别进行阳离子交换层析柱再平衡、洗杂、洗脱，收集洗脱峰于去内毒素的收集管中，得到低内毒素胶原蛋白液，
所述缓冲液1作用为阳离子交换层析柱的再平衡，其为包含1～3v/v％的PEG-8辛酸/癸酸甘油酯类、2～5v/v％的吐温80、0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和0.585w/v％的氯化钠的水溶液，pH为6.5，体积是所述胶原蛋白样本液的体积的0.5～2倍，例如约1倍；
所述缓冲液2作用为洗杂，其为包含0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和1.17w/v％的氯化钠的水溶液，pH为6.5，体积是所述胶原蛋白样本液的体积的1～3倍，例如约1.5倍；
所述缓冲液3作用为洗脱，其为包含0.1644w/v％的磷酸二氢钠、0.0894w/v％的磷酸氢二钠和5.85w/v％的氯化钠的水溶液，pH为6.5，体积是所述胶原蛋白样本液的体积的0.3～1倍，例如约0.5倍。
所述洗脱峰可以通过监测洗脱液的A220值来确认。
2-6.根据项2-5所述的低内毒素胶原蛋白液的制备方法，其中，所述步骤A之前还进行下述步骤：用基础溶液平衡阳离子交换层析柱，直至基线稳定不变。
所述基础溶液包含0.1644w/v％的磷酸二氢钠、0.0894w/v％的磷酸氢二钠和0.585w/v％的氯化钠的缓冲液(pH 6.5)。
2-7.根据项2-5所述的低内毒素胶原蛋白液的制备方法，其中，所述阳离子交换层析柱为Capto S阳离子交换层析柱，使用前采用0.5M NaOH处理30min以去除热原。
2-8.根据项2-5所述的低内毒素胶原蛋白液的制备方法，其中，所述步骤A中，所述胶原蛋白样本液的量为10L以上。
2-9.根据项2-5所述的低内毒素胶原蛋白液的制备方法，其中，所述步骤B中，将经检测确认胶原蛋白纯度为99％以上的收集于去内毒素的收集管中的洗脱峰进行超滤浓缩，得到超滤浓缩液，将该超滤浓缩液作为低内毒素胶原蛋白液。
可以通过SDS-PAGE检测确认胶原蛋白的纯度。
2-10.根据项2-5所述的低内毒素胶原蛋白液的制备方法，其中，所述胶原蛋白是项1～5中任一项所述的大分子I型重组胶原蛋白。
2-11.一种低内毒素胶原蛋白冻干物的制备方法，其包括：采用项2-5～2-10中任一项所述的低内毒素胶原蛋白液的制备方法制备低内毒素胶原蛋白液，并将所述低内毒素胶原蛋白液冷冻干燥，从而得到低内毒素胶原蛋白冻干物。
需要说明的是，PEG-8辛酸/癸酸甘油酯类即聚乙二醇-8辛酸/癸酸甘油酯类，吐温80即聚山梨脂80。
附图说明
图1为实施例1制备的部分I型重组胶原蛋白的SDS-PAGE电泳图。对照蛋白以No.4、6、7、10为例。
图2为实施例2制备的部分测试样本放置12个月后的HPLC图。对照蛋白以No.4、7、10、13为例。
图3为No.1和No.6的I型重组胶原蛋白的红外光谱图。
图4为No.1和No.6的I型重组胶原蛋白的拉曼光谱图。
图5为按照实施例5的操作步骤去除内毒素并冻干后的低内毒素胶原蛋白冻干纤维中内毒素含量的测定结果。
图6为按照实施例5的操作步骤去除内毒素并冻干后的低内毒素胶原蛋白冻干纤维中胶原蛋白纯度的检测结果。
具体实施方式
以下将通过具体的实施例对本发明进行详细地描述。需要特别指出的是，这些描述仅仅是示例性的描述，并不构成对本发明范围的限制。
一般性说明：具体实施方式中所用到的酶全部从TaKaRa公司购买，质粒DNA抽提试剂盒和DNA凝胶回收试剂盒均购自北京索莱宝公司，基因重组试剂盒(Reorganization Kits)购自天根生物，具体操作完全按照试剂盒的说明进行。
实施例1.利用酵母表达系统制备各种I型重组胶原蛋白
1、酵母表达菌株的构建
构建了分别表达表1所示的No.1～18的I型重组胶原蛋白的酵母表达菌株。具体操作是：根据毕赤酵母密码子偏好优化后，通过全基因合成的方式合成对应的目标基因，并在基因的两端分别添加SnaB I和Not I酶切位点，以SnaBI和NotI酶对目标基因进行双酶切，与同样经SnaB I和Not I酶酶切的pPIC9k在T4连接酶的作用下进行连接，16℃连接过夜后，转入Top10感受态细胞，涂布氨苄抗性平板，挑取阳性转化子，提取质粒后用SacI进行线性化后，电击转入毕赤酵母GS115感受态细胞中，以G418抗性平板筛选多拷贝转化子，即 为I型重组胶原蛋白的表达菌株。
表1各酵母表达菌株表达的I型重组胶原蛋白
2、目标蛋白的诱导表达
(1)挑取酵母表达菌株的单菌落加入到5ml YPD液体培养基中(1％酵母提取物，2％蛋白胨和2％葡萄糖)，30℃，200rpm培养过夜进行活化；
(2)以1％的接种量接种于100ml的BMGY液体培养基，30℃，200rpm培养至OD600＝6.0～9.0；
(3)在1500g离心力作用下，25℃离心6min收集菌体，并将其悬浮于200ml BMMY液体培养基中，使其起始浓度为OD600＝1.0，在30℃，200rpm条件下培养；
(4)每隔24h加甲醇，终浓度为0.5～1.0％,进行诱导表达；
(5)诱导72h，取培养液在12000rpm条件下离心2min，取上清液。即为重组I型胶原蛋白粗表达液。
3、I型重组胶原蛋白制备
将发酵制备的上清液经过30kD超滤膜超滤浓缩后，采用CM离子交换柱进行柱分离，以35％的NaCl溶液进行洗脱，收集洗脱液，脱盐浓缩后冻干即为I型重组胶原蛋白制备。取0.1g冻干粉融入100ml生理盐水中，充分溶解后上SDS-PAGE凝胶电泳，进行分子量大小及蛋白电泳纯度的确认。
结果显示构建的18株表达菌均能成功的表达目标蛋白，经分离纯化后制备的蛋白电泳纯度均在99％以上，电泳结果如图1所示。
实施例2：各种I型重组胶原蛋白在水溶液中的稳定性实验
A实验材料
实验所用材料为实施例1中制备的I型重组胶原蛋白No.1～-18。
B实验方法
将A中的实验材料用ddH2O配置成蛋白浓度为1mg/mL的蛋白溶液，在超净工作台中用0.22μm的无菌滤器过滤后分装到无菌离心管中密封，置于25℃±2℃的条件下，分别于0个月、6个月、12个月取样，每次取样3管，检测蛋白纯度(高效液相色谱法测定蛋白纯度)，根据纯度变化判定蛋白的稳定性。
C实验结果
测试结果如下表：
表2重组胶原蛋白溶液12个月稳定性测试结果(纯度，％)
通常认为，(1)分子量相同或相近的重组胶原蛋白，重复单元越短，氨基酸组成及分布就越单调，表面电荷负载越大，越不容易达到稳定的平衡状态，因而更易水解；(2)相同重复单元的重组胶原蛋白，重复次数越多，分子量越大，重组胶原蛋白的表面电荷负载越大，越不容易达到稳定的平衡状 态，因而更易水解。
但是，由表2可知，本发明的I型重组胶原蛋白(No.1～3)表现出异常优异的水溶液中稳定性，其在水溶液中放置12个月，纯度仍可高达97％以上(参见图2A～G)。
实施例3：I型重组胶原蛋白No.1～3具有反常的水溶液中稳定性的原因初探
1)红外光谱测定
将表1中No.1和No.6的重组胶原蛋白分别配制成溶液后，进行红外光谱测定，用Bruker OPUS7.2对光谱数据进行傅里叶退卷积，截取1700～1600cm-1波段光谱数据，用peakfit v4.12进行二阶导分峰拟合处理，将处理后的数据用orgin作图得到二级结构分布，并计算二级结构的相对含量。两种蛋白的二级结构比较结果如表3及图3所示。
表3红外光谱测定的No.1和No.6的重组胶原蛋白的二级结构
2)拉曼光谱测定
将表1中No.1和No.6的重组胶原蛋白分别配制成溶液后，进行拉曼光谱测定，用ThermoFisher Omnic9.2对光谱数据进行平滑基线校正处理，截取1700～1600cm-1波段光谱数据，用peakfit v4.12进行二阶导分峰拟合处理，将处理后的数据用orgin作图得到二级结构分布，并计算二级结构的相对含量。两种蛋白的二级结构比较结果如表4及图4所示。
表4拉曼光谱测定的No.1和No.6的重组胶原蛋白的二级结构
从实施例3的测定结果可以看出，重复单元相同但重复次数不同的重组胶原蛋白在二级结构上存在较大差异，这也暗示两者的三级结构存在差异。可以推测No.1～3的I型重组胶原蛋白的空间结构使其表面电荷更为平衡，从而表现出良好的水溶液中的稳定性。
实施例4低内毒素胶原蛋白液及冻干物的制备
首先，用基础溶液(其成分为0.1644w/v％的磷酸二氢钠、0.0894w/v％的磷酸氢二钠和0.585w/v％的氯化钠的缓冲液pH为6.5)平衡柱子(Capto S阳离子交换柱)，直至基线稳定不变。
然后，向60L蛋白上样液(其中包含实施例1中制备的No.1的I型重组胶原蛋白、0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和0.585w/v％的氯化钠，pH 6.5，测定其内毒素含量约为50EU/mg)中加入PEG-8辛酸/癸酸甘油酯类和吐温80，使得它们浓度分别为0.5％(v/v)和1％(v/v)，并将其上样于上述经过平衡的Capto S阳离子交换柱。
上样完毕后，先用60L的缓冲液1进行柱子的再平衡，所述缓冲液1的成分为0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和0.585w/v％的氯化钠，pH 6.5+0.5％(v/v)PEG-8辛酸/癸酸甘油酯类+1％(v/v)吐温80；再用90L的缓冲液2进行洗杂，所述缓冲液2的成分为0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和1.17w/v％的氯化钠，pH 6.5；最后用30L的缓冲液3进行洗脱，所述缓冲液3的成分为包含0.1644w/v％的磷酸二氢钠、0.0894w/v％的磷酸氢二钠和5.85w/v％的氯化钠，pH 6.5。
洗脱过程中监测流出的洗脱液的A220值，当出现洗脱峰时，将其收集至去内毒素的收集管中。对于收集至各收集管中的洗脱液，采用SDS-PAGE检测其中胶原蛋白的纯度，将胶原蛋白纯度在99％以上的洗脱液进行超滤浓缩得到超滤浓缩液，以此液作为实施例2的低内毒素胶原蛋白液。
进一步，对于所述低内毒素胶原蛋白液，在进行了内毒素含量测定后，将其进行冷冻干燥，得到实施例2的低内毒素胶原蛋白冻干物。
实施例5～13低内毒素胶原蛋白液及冻干物的制备
像实施例4那样进行操作，制备了实施例5～13的低内毒素胶原蛋白液及冻干物，不同在于：向实施例1中的蛋白上样液中加入PEG-8辛酸/癸酸甘油酯类和吐温80，使得它们浓度分别如表5所示；以及，使所述缓冲液1中的PEG-8辛酸/癸酸甘油酯类和吐温80的浓度分别如表5所示。
实施例14低内毒素胶原蛋白的内毒素含量的测定
对于各实施例制备的低内毒素胶原蛋白冻干物(纤维状)，测定其中的内毒素含量(EU/mg胶原蛋白)，测定结果示于表5。
内毒素含量测定方法：依据《中华人民共和国药典》(2020版)四部通则1143细菌内毒素检查法的光度测定法进行检测。图5为按照实施例5的操作步骤去除内毒素并冻干后的低内毒素胶原蛋白冻干纤维中内毒素含量的测定结果，测试样本中的胶原蛋白含量配制成4mg/mL。
胶原蛋白含量测定：按《中华人民共和国药典》第四部通则0731蛋白质含量测定法第一法凯氏定氮法规定的方法测定。
胶原蛋白纯度检测：用水将低内毒素胶原蛋白冻干纤维配制成1mg/mL的溶液，按《中华人民共和国药典》第四部通则0514“色谱法”的“分子排阻色谱法”测定，按面积归一化法计算纯度。图6为按照实施例5的操作步骤去除内毒素并冻干后的低内毒素胶原蛋白冻干纤维中胶原蛋白纯度检测结果。
表5

需要说明的是，图5、图6分别为按照实施例5的操作制备的2211001批次重组胶原蛋白冻干纤维的内毒素含量及蛋白纯度检测结果。
由实施例5、6、8～10的内毒素含量检测结果可知，当使用的PEG-8辛酸/癸酸甘油酯类的量在1～3％、且吐温80的量在2～5％时，相比于PEG-8辛酸/癸酸甘油酯类和/或吐温80的量不在上述范围的实施例，去除内毒素的效果更加显著，同时实施例12、13也说明了PEG-8辛酸/癸酸甘油酯类与吐温80联用的效果优于单个使用的效果。特别是，由实施例5的内毒素含量检测结果可知，当使用的PEG-8辛酸/癸酸甘油酯类的量为1％、且吐温80的量为2％时，去除内毒素的效果最为显著。需要说明的是，图5和图6显示了2211001批次产品的内毒素含量及胶原蛋白纯度测定结果，可知该批次产品的内毒素含量为0.004075EU/mg，且胶原蛋白纯度在99％以上。
而且，医药行业标准YY0954-2015规定蛋白注射剂中内毒素含量低于0.5EU/mL，上述实施例5、6、8～10的内毒素含量检测结果表明，本发明的低内毒素胶原蛋白液(临床使用浓度为4mg/mL)内毒素低于0.04EU/mL,是标准要求的10倍以下，说明本发明的内毒素去除工艺效果显著，可完全满足面部注射填充剂的安全性要求。

Claims (11)

  1. 一种低内毒素胶原蛋白液的制备方法，其包括下述步骤：
    步骤A：将胶原蛋白样本液上样于阳离子交换层析柱，所述胶原蛋白样本液是包含胶原蛋白、1～3v/v％的PEG-8辛酸/癸酸甘油酯类和2～5v/v％的吐温80、0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和0.585w/v％的氯化钠的水溶液，pH为6.5；
    步骤B：依次用缓冲液1～3分别进行阳离子交换层析柱再平衡、洗杂、洗脱，收集洗脱峰于去内毒素的收集管中，得到低内毒素胶原蛋白液，
    所述缓冲液1作用为阳离子交换层析柱的再平衡，其为包含1～3v/v％的PEG-8辛酸/癸酸甘油酯类、2～5v/v％的吐温80、0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和0.585w/v％的氯化钠的水溶液，pH为6.5，体积是所述胶原蛋白样本液的体积的0.5～2倍，例如约1倍；
    所述缓冲液2作用为洗杂，其为包含0.1644w/v％的磷酸二氢钠、0.0894％w/v％的磷酸氢二钠和1.17w/v％的氯化钠的水溶液，pH为6.5，体积是所述胶原蛋白样本液的体积的1～3倍，例如约1.5倍；
    所述缓冲液3作用为洗脱，其为包含0.1644w/v％的磷酸二氢钠、0.0894w/v％的磷酸氢二钠和5.85w/v％的氯化钠的水溶液，pH为6.5，体积是所述胶原蛋白样本液的体积的0.3～1倍，例如约0.5倍。
  2. 根据权利要求1所述的低内毒素胶原蛋白液的制备方法，其中，所述步骤A之前还进行下述步骤：用基础溶液平衡阳离子交换层析柱，直到基线稳定不变。
  3. 根据权利要求1所述的低内毒素胶原蛋白液的制备方法，其中，所述阳离子交换层析柱为Capto S阳离子交换层析柱，使用前采用0.5M NaOH处理30min以去除热原。
  4. 根据权利要求1所述的低内毒素胶原蛋白液的制备方法，其中，所述步骤A中，所述胶原蛋白样本液的量为10L以上。
  5. 根据权利要求1所述的低内毒素胶原蛋白液的制备方法，其中，所述步骤B中，将经检测确认胶原蛋白纯度为99％以上的收集于去内毒素的收集管中的洗脱峰进行超滤浓缩，得到超滤浓缩液，将该超滤浓缩液作为低内毒素胶原蛋白液。
  6. 根据权利要求1所述的低内毒素胶原蛋白液的制备方法，其中，所述胶原蛋白是大分子I型重组胶原蛋白，所述大分子I型重组胶原蛋白，其由来自天然人I型胶原蛋白的短氨基酸序列作为重复单元进行多次重复而构成，其中，所述短氨基酸序列如SEQ ID No.:1(G A P G A P G S Q G A P G L Q)所示，重复次数为75～110次，优选80～105次，更优选82～102次。
  7. 一种低内毒素胶原蛋白冻干物的制备方法，其包括：采用权利要求1～6中任一项所述的低内毒素胶原蛋白液的制备方法制备低内毒素胶原蛋白液，并将所述低内毒素胶原蛋白液冷冻干燥，从而得到低内毒素胶原蛋白冻干物。
  8. 一种低内毒素胶原蛋白液或低内毒素胶原蛋白冻干物，其中，胶原蛋白的纯度为99％以上，且内毒素含量低于0.01EU/mg。
  9. 根据权利要求8所述的低内毒素胶原蛋白液或低内毒素胶原蛋白冻干物，其中，所述胶原蛋白是大分子I型重组胶原蛋白，所述大分子I型重组胶原蛋白，其由来自天然人I型胶原蛋白的短氨基酸序列作为重复单元进行多次重复而构成，其中，所述短氨基酸序列如SEQ ID No.:1(G A P G A P G S Q G A P G L Q)所示，重复次数为75～110次，优选80～105次，更优选82～102次。
  10. 根据权利要求8所述的低内毒素胶原蛋白液或低内毒素胶原蛋白冻干物在制备胶原蛋白注射液中的用途。
  11. 根据权利要求10所述的用途，其中，所述胶原蛋白注射液用于面部填充，以纠正额部动力性皱纹，所述额部动力性皱纹包括眉间纹、额头纹和鱼尾纹。
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