WO2023138668A1 - Collagène recombinant de type i macromoléculaire stable et son utilisation - Google Patents

Collagène recombinant de type i macromoléculaire stable et son utilisation Download PDF

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Publication number
WO2023138668A1
WO2023138668A1 PCT/CN2023/073217 CN2023073217W WO2023138668A1 WO 2023138668 A1 WO2023138668 A1 WO 2023138668A1 CN 2023073217 W CN2023073217 W CN 2023073217W WO 2023138668 A1 WO2023138668 A1 WO 2023138668A1
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WIPO (PCT)
Prior art keywords
collagen
type
recombinant
recombinant collagen
months
Prior art date
Application number
PCT/CN2023/073217
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English (en)
Chinese (zh)
Inventor
范代娣
宇文伟刚
贺婧
段志广
徐茹
严建亚
刘琳
Original Assignee
陕西巨子生物技术有限公司
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Publication of WO2023138668A1 publication Critical patent/WO2023138668A1/fr

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Classifications

    • 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, 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
    • 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
    • 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
    • A61L27/60—Materials for use in artificial skin
    • 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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/04—Macromolecular materials
    • A61L31/043—Proteins; Polypeptides; Degradation products thereof
    • A61L31/044—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
    • A61L31/00—Materials for other surgical articles, e.g. stents, stent-grafts, shunts, surgical drapes, guide wires, materials for adhesion prevention, occluding devices, surgical gloves, tissue fixation devices
    • A61L31/14—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention belongs to the field of biotechnology, and relates to a novel genetic engineering recombinant collagen and its application.
  • 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. It is one of the most critical raw materials in the biotechnology industry and is widely used in medical materials, cosmetics, and food industries.
  • 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.
  • the amino acid composition and distribution of the recombinant collagen constructed by repeating the short amino acid sequence from natural human collagen is relatively monotonous. Theoretically speaking, this will cause a large charge load on its surface, and it is difficult to reach a stable equilibrium state as a whole. Therefore, it is easy to hydrolyze and denature in aqueous solution, and there is a tendency that the shorter the short amino acid sequence repeat unit and the more repetitions, the more unstable the recombinant collagen molecule is in aqueous solution.
  • 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 human 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 higher the molecular weight of collagen the better its mechanical strength and the lower its stability in aqueous solution, especially for recombinant collagen constructed by repeating short amino acid sequences from natural human collagen.
  • the molecular weight of each chain of natural human collagen is about 110-130kD. From a practical point of view, technical personnel generally believe that the molecular weight of collagen suitable for replacing natural human collagen as a tissue engineering material 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.
  • Recombinant collagen constructed by repeating acid sequences is unstable in aqueous solution, and recombinant collagen obtained through mutation engineering has immunogenicity problems. Therefore, how to obtain collagen suitable for replacing natural human collagen as tissue engineering material has become a limiting problem in this technical field.
  • the inventors conducted in-depth research.
  • the inventors first conducted technical literature research on recombinant collagens constructed repeatedly from short amino acid sequences from natural human collagen, selected some short amino acid sequences from natural human type I collagen (the most widely used tissue engineering material) in the prior art, and then used these short amino acid sequences as repeating units to construct recombinant collagens with different molecular weights, and investigated the long-term storage stability of these recombinant collagens in aqueous solution, in order to obtain large molecular weight (above 100kD) recombinant collagens that can be stored in aqueous solution for a long time and can meet the mechanical strength requirements of tissue engineering materials .
  • the inventor unexpectedly discovered that the recombinant type I collagen obtained by repeating a section of pentadecadecanoid (G A P G A P G S Q G A P GL Q) from natural human type I collagen for 75 to 110 times has exceptionally excellent stability. It is specifically reflected in: (1) Although the length of its repeating unit is the shortest among all the recombinant collagens tested by the inventor, its stability in aqueous solution is the best; and it is generally believed that the shorter the repeating unit, the more monotonous the composition and distribution of amino acids, 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 shown in SEQ ID No.: 1 (G A P G A P G S Q G A P GL Q), and 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 the group consisting of dermal fillers, artificial bones, artificial skin, oral cavity absorbable biofilms, bone implants, vascular scaffolds, intercellular matrix scaffolds and collagen sponges.
  • An aqueous collagen solution comprising the macromolecular type I recombinant collagen described in any one of items 1-5.
  • the collagen aqueous solution according to item 9 which has been stored at room temperature for more than 3 months, preferably for more than 6 months, more preferably for more than 12 months; or has been stored at 4°C for more than 12 months, preferably for more than 24 months, more preferably for more than 36 months.
  • the stability of recombinant collagen that repeats with a certain amino acid sequence as a repeat 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 reaching a certain number of repetitions, a certain spatial structure is just formed, making the surface load 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.
  • 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.
  • Yeast expression strains expressing No. 1-18 type I recombinant collagen shown in Table 1 were constructed. The specific operation is: after optimizing the codon preference of Pichia pastoris, synthesize the corresponding target gene by whole gene synthesis, add SnaB I and Not I restriction sites at both ends of the gene respectively, perform double digestion with SnaBI and Not I enzymes on the target gene, and connect it with pPIC9k, which has also been digested by SnaB I and Not I enzymes, under the action of T4 ligase.
  • Transformants linearized with SacI after plasmid extraction Afterwards, electroporation was transferred into Pichia pastoris GS115 competent cells, and multi-copy transformants were screened with G418 resistance plate, which was the expression strain of type I recombinant collagen.
  • the CM ion exchange column is used for column separation, eluted with 35% NaCl solution, the eluate is collected, desalted and concentrated, and then freeze-dried to prepare type I 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 exhibits exceptionally excellent stability in aqueous solution, and its purity can still be as high as 97% or more after being placed in aqueous solution for 12 months (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
  • Recombinant collagens No.1 and No.6 in Table 1 were formulated into solutions, and Raman spectra were measured.
  • ThermoFisher Omnic9.2 was used to smooth the baseline of the spectral data, and the spectral data in the 1700-1600 cm- 1 band was intercepted, and the second-order derivative peak fitting was performed with peakfit v4.12.
  • the processed data was plotted with orgin to obtain the secondary structure distribution, and the relative content of the secondary structure was calculated.
  • the comparison results of the secondary structures of the two proteins are shown in Table 4 and Figure 4.

Abstract

La présente invention concerne un collagène recombinant de type I macromoléculaire stable et son utilisation. Le collagène recombinant de type I macromoléculaire de la présente invention est formé en effectuant de multiples répétitions en utilisant une séquence d'acides aminés courte dérivée du collagène de type I humain naturel en tant qu'unité de répétition, la séquence d'acides aminés courte étant GAPGAPGSQGAPGLQ, et le nombre de répétitions étant de 75 à 110 fois. Le collagène recombinant de type I macromoléculaire de la présente invention présente une stabilité extrêmement bonne dans une solution aqueuse et peut être stocké dans une solution aqueuse pendant une longue période de temps.
PCT/CN2023/073217 2022-01-24 2023-01-19 Collagène recombinant de type i macromoléculaire stable et son utilisation WO2023138668A1 (fr)

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CN202210080354.6 2022-01-24
CN202210080354.6A CN114409807B (zh) 2022-01-24 2022-01-24 一种稳定的大分子i型重组胶原蛋白及其用途

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Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114409807B (zh) * 2022-01-24 2023-04-25 陕西巨子生物技术有限公司 一种稳定的大分子i型重组胶原蛋白及其用途
CN114369156B (zh) * 2022-01-27 2023-04-25 陕西巨子生物技术有限公司 包含稳定的大分子i型重组胶原蛋白的注射液

Citations (6)

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WO2003099465A1 (fr) * 2002-05-20 2003-12-04 Dow Corning Corporation Synthese de structures inorganiques par structuration et catalyse par des polymeres de proteines de repetition auto-assemblees
CN102906107A (zh) * 2010-03-11 2013-01-30 韩国科学技术院 高分子量重组丝或类丝蛋白、以及使用它们制造的微米或纳米级蜘蛛丝或者类蜘蛛丝纤维
CN111067662A (zh) * 2019-11-05 2020-04-28 南京艾澜德生物科技有限公司 重组胶原蛋白及其双层人造血管支架
US20210079064A1 (en) * 2020-04-23 2021-03-18 Jiangnan University Preparation of Type I Collagen-Like Fiber and Method for Regulating and Controlling the D-periodic of Fiber Thereof
CN113735966A (zh) * 2021-09-29 2021-12-03 陕西巨子生物技术有限公司 一种抗肿瘤重组胶原蛋白及其制备方法和应用
CN114409807A (zh) * 2022-01-24 2022-04-29 陕西巨子生物技术有限公司 一种稳定的大分子i型重组胶原蛋白及其用途

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CN112316214B (zh) * 2020-11-03 2022-09-30 陕西巨子生物技术有限公司 一种重组胶原蛋白可注射水凝胶及其制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003099465A1 (fr) * 2002-05-20 2003-12-04 Dow Corning Corporation Synthese de structures inorganiques par structuration et catalyse par des polymeres de proteines de repetition auto-assemblees
CN102906107A (zh) * 2010-03-11 2013-01-30 韩国科学技术院 高分子量重组丝或类丝蛋白、以及使用它们制造的微米或纳米级蜘蛛丝或者类蜘蛛丝纤维
CN111067662A (zh) * 2019-11-05 2020-04-28 南京艾澜德生物科技有限公司 重组胶原蛋白及其双层人造血管支架
US20210079064A1 (en) * 2020-04-23 2021-03-18 Jiangnan University Preparation of Type I Collagen-Like Fiber and Method for Regulating and Controlling the D-periodic of Fiber Thereof
CN113735966A (zh) * 2021-09-29 2021-12-03 陕西巨子生物技术有限公司 一种抗肿瘤重组胶原蛋白及其制备方法和应用
CN114409807A (zh) * 2022-01-24 2022-04-29 陕西巨子生物技术有限公司 一种稳定的大分子i型重组胶原蛋白及其用途

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