WO2018103601A1 - 一种腺病毒冷冻干燥添加剂及腺病毒冻干制剂 - Google Patents

一种腺病毒冷冻干燥添加剂及腺病毒冻干制剂 Download PDF

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WO2018103601A1
WO2018103601A1 PCT/CN2017/114401 CN2017114401W WO2018103601A1 WO 2018103601 A1 WO2018103601 A1 WO 2018103601A1 CN 2017114401 W CN2017114401 W CN 2017114401W WO 2018103601 A1 WO2018103601 A1 WO 2018103601A1
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adenovirus
freeze
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replication
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朱涛
侯丽华
吴诗坡
段磊
宇学峰
毛慧华
许丽锋
邵忠琦
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康希诺生物股份公司
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    • A—HUMAN NECESSITIES
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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  • the invention relates to the field of biological products, in particular to an adenovirus freeze-drying additive and an adenovirus lyophilized preparation.
  • the adenovirus production process is simple and low in cost, has little pathogenicity to humans and does not induce carcinogenesis, has high reproductive titer and large loading capacity, and can efficiently transfer foreign genes to target cells, allowing them to be expressed in cells, producing body fluids and cells. Immunization, therefore, recombinant adenoviral vectors are widely used in biopharmaceutical fields such as gene therapy and vaccine development.
  • Adenoviral vectors were originally used for gene therapy, and breakthroughs have been made. For example, recombinant adenoviral vectors expressing transgenes in gene therapy of human cancer show better therapeutic effects; such as expression of granulocyte-macrophage colony-stimulating factor (GM-CSF) Recombinant adenovirus for the treatment of melanoma.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • adenoviral vector vaccine As a novel vaccine, recombinant adenoviral vector vaccine has been extensively studied in various human and animal viral diseases, bacterial diseases and parasitic diseases, and cancer prevention.
  • the expressed diseases are human immunodeficiency virus (HIV), rabies virus (RV), hepatitis B virus (HBV), hepatitis C virus (HCV), dengue virus (DEN), herpes virus (EB), and Ebola virus (EBOV).
  • HCV human immunodeficiency virus
  • RV rabies virus
  • HBV hepatitis B virus
  • HCV hepatitis C virus
  • DEN dengue virus
  • EB herpes virus
  • Ebola virus Ebola virus
  • adenovirus is sensitive to repeated freeze-thawing, easy to inactivate, and has poor thermal stability.
  • the adenovirus is usually stored in a buffer containing 10% glycerol and stored and transported at -80 °C to ensure adenovirus.
  • Lyophilized preparations are commonly used in biotechnological medicines. However, it has been reported that the adenovirus stock solution and the lyophilized protective agent are lyophilized, and the adenovirus is inactivated to a large extent, and the lyophilized preparation cannot be stably stored at 2-8 °C. To date, there have been no freeze-dried adenovirus preparations that can be stored at 2-8 ° C for a long time.
  • the technical problem to be solved by the present invention is to provide an adenovirus freeze-dried additive.
  • Another technical problem to be solved by the present invention is to provide an adenovirus lyophilized preparation in combination with the above-described adenovirus freeze-dried additive.
  • the technical solution of the present invention is:
  • An adenovirus freeze-drying additive consisting of mannitol, sucrose, glycerin, magnesium chloride, phosphate buffer, and Tween 80, wherein
  • the above-mentioned adenovirus freeze-drying additive has a glycerin content of 0.1 to 1.8%.
  • the above-mentioned adenovirus freeze-drying additive is as follows:
  • Each 1 vial (0.5ml after reconstitution) contains:
  • the above-mentioned adenovirus lyophilized preparation has a pH of 7.0-8.0 after reconstitution.
  • An adenovirus lyophilized preparation consisting of an adenovirus and the above-described adenovirus freeze-dried additive.
  • the adenovirus lyophilized preparation is an recombinant replication-deficient human type 5 adenovirus, or an adenovirus of type 4, type 7, type 11, or type 55.
  • the above-described adenovirus lyophilized preparation is a replication-defective human type 5 adenovirus expressing a Zaire type (Guinea, 2014) Ebola virus envelope glycoprotein gene; or
  • the recombinant replication-deficient human type 5 adenovirus is a replication-defective human type 5 adenovirus expressing the Mycobacterium tuberculosis Ag85A gene.
  • the above-mentioned adenovirus lyophilized preparation contains, per 1 vial (0.5 ml after reconstitution):
  • the above-mentioned adenovirus lyophilized preparation has a pH of 7.0-8.0 after reconstitution.
  • the above-mentioned adenovirus lyophilized preparation is white and loose.
  • the above-described adenovirus lyophilized preparation can be used as a gene therapy drug or a vaccine for preventing infectious diseases, a multivalent vaccine, or a combination vaccine.
  • the adenovirus freeze-dried additive of the present invention can be obtained by rational screening and compounding of excipients, and the synergistic action of each component to optimize the ratio of each other, so that the obtained recombinant adenovirus Ebola virus vaccine can be frozen.
  • the dry preparation is stably stored at 2-8 °C for 1 year, stable at 37 °C for 2 weeks, and the adenovirus activity is good, minimizing the loss and inactivation of the adenovirus during the dispensing and lyophilization process, ensuring the adenovirus Activity and effectiveness of the formulation, animal experiments and humans Clinical trials have shown that the vaccine is safe and immunogenic.
  • the obtained adenovirus lyophilized preparation overcomes the shortcomings of the existing adenovirus liquid preparation-80 ° C cryopreservation and ultra-low temperature cold chain during transportation, is convenient for transportation and use, and avoids the addition of human serum albumin in the adenovirus vaccine.
  • recombinant adenoviral vector vaccine has a good application value, confirmed by clinical trials, the vaccine is safe, and immune protection can be achieved by inoculation two weeks in advance.
  • Non-human primate model tests have shown that the vaccine protection rate is 100%. Stability tests showed that the formulation was stable for 1 year at 2-8 °C and stable for 2 weeks at 37 °C.
  • Figure 1 is the appearance of a recombinant adenovirus Ebola vaccine lyophilized formulation.
  • the cells were lysed, nuclease-treated, centrifuged, and separated by column chromatography to obtain an adenovirus.
  • the recombinant adenovirus constructed by the Academy of Military Sciences was used to construct a recombinant adenovirus carrying the Ebola virus envelope glycoprotein (GP protein) expression gene, and the adenoviral shuttle plasmid of the foreign gene was cloned.
  • pDC316 was co-transfected into 293 cells with the packaging plasmid pBHGlox_E1, 3Cre carrying most of the adenovirus genome, and recombined by the action of Cre/loxP system to produce recombinant adenovirus.
  • the virus was cultured and proliferated to obtain a large number of viruses. For the vaccine stock solution.
  • Vaccine semi-finished product at a ratio of mannitol 50 mg/ml, sucrose 25 mg/ml, magnesium chloride 1 mM, phosphate buffer 10 mM, Tween 80 (mass) 0.1% (w/w), glycerol (mass) 0.3% (w/w) .
  • Control the semi-finished product pH 7.0-8.0.
  • the adenovirus freeze-dried additive is reconstituted with water for injection, sterile filtered, and the adenovirus is concentrated according to the amount of replication-deficient human type 5 adenovirus 4 ⁇ 10 10 VP per 1 vial (0.5 ml after reconstitution).
  • the liquid is added to the lyophilized solution to obtain a vaccine semi-finished product.
  • test results are as follows:
  • the loss rate of live virus before and after lyophilization of the formulation was about 25%.
  • the quality standards for recombinant Ebola virus vaccine products require a live virus concentration of ⁇ 8 ⁇ 10 8 IFU/ml per dose.
  • the above results show that at 37 °C, 8 ⁇ 10 8 IFU/ml or more is maintained at 2 weeks. , indicating that the lyophilized preparation under this formula can be stabilized at 37 ° C for 2 weeks.
  • the IFU concentration of the finished product has dropped below 8 ⁇ 10 8 IFU/ml, and the finished product has not met the quality standard.
  • the quality standard of recombinant Ebola virus vaccine products requires that the live virus concentration of each dose is ⁇ 8 ⁇ 10 8 IFU/ml.
  • the above results show that under the condition of 2-8 °C, 8 ⁇ 10 8 IFU/ml is maintained at 12M.
  • the lyophilized preparation under the formulation can be stable for 1 year at 2-8 °C.
  • adenovirus freeze-dried additive and the adenovirus lyophilized preparation according to the above reference examples is illustrative and not limiting, and several embodiments can be enumerated according to the limited scope, so Variations and modifications of the present general inventive concept are intended to be within the scope of the present invention.

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Abstract

一种腺病毒冷冻干燥添加剂及腺病毒冻干制剂，所述腺病毒冷冻干燥添加剂由甘露醇、蔗糖、甘油、氯化镁、磷酸盐缓冲剂和吐温80组成，所述腺病毒冻干制剂由腺病毒和该腺病毒冷冻干燥添加剂组成，腺病毒是重组腺病毒埃博拉病毒病疫苗。

Description

一种腺病毒冷冻干燥添加剂及腺病毒冻干制剂 技术领域
本发明涉及生物制品技术领域，尤其是一种腺病毒冷冻干燥添加剂及腺病毒冻干制剂。
背景技术
腺病毒生产工艺简单成本低，对人致病性小且不诱导癌变，繁殖滴度高，装载容量大，可以高效转移外源基因至靶细胞中，使其在细胞内表达，产生体液和细胞免疫，因此，重组腺病毒载体广泛应用于基因治疗、疫苗开发等生物制药领域中。
腺病毒载体最初用于基因治疗，且取得突破进展，比如人类癌症的基因治疗中表达转基因的重组腺病毒载体显示出比较好的治疗效果；如表达粒性巨噬细胞集落刺激因子(GM-CSF)的重组腺病毒治疗黑色素瘤。
重组腺病毒载体疫苗作为一种新型疫苗，在各种人和动物病毒病，细菌病和寄生虫病、癌症的免疫预防中进行了大量研究。表达的病原有人体免疫缺陷病毒(HIV)、狂犬病毒(RV)、乙肝病毒(HBV)、丙肝病毒(HCV)、登革热病毒(DEN)、疱疹病毒(EB)以及埃博拉病毒(EBOV)。但是，腺病毒对反复冻溶敏感，容易失活，且热稳定性差，通常将腺病毒保存于含有10％甘油的缓冲液中，并于-80℃条件下存储和运输，以保证腺病毒的活性。因此，即便是含有稳定剂的液体重组腺病毒制剂对温度也较为敏感，2014年埃博拉病毒疫情在西非爆发之后，美国国家过敏症和传染病研究所(NIAID)与英国制药企业葛兰素史克公司(GSK)合作开发了黑猩猩腺病毒载体-埃博拉疫苗(cAd3-ZEBOV  vaccine)，该疫苗是利用黑猩猩3型腺病毒(cAd3)为载体，表达Zaire型EBOV GP蛋白，临床试验表明疫苗的安全性和免疫原型良好，但该疫苗需要在-80℃条件下存储和运输，在地处热带且冷链不健全的西非使用极为不方便。
冻干制剂是生物技术药物普遍采用的剂型。但报道显示，腺病毒原液及冷冻干燥保护剂经分装冻干之后，腺病毒有较大程度的失活，且冻干制剂在2-8℃条件下不能稳定存储。迄今为止，尚未有能在2-8℃长期保存的冻干腺病毒制剂上市。
发明内容
本发明所要解决的技术问题在于提供一种腺病毒冷冻干燥添加剂。
本发明所要解决的另一技术问题在于提供配合使用上述腺病毒冷冻干燥添加剂的腺病毒冻干制剂。
为解决上述技术问题，本发明的技术方案是：
一种腺病毒冷冻干燥添加剂，由甘露醇、蔗糖、甘油、氯化镁、磷酸盐缓冲剂和吐温80组成，其中，
甘露醇  10-80mg/ml
蔗糖  10-80mg/ml
甘油  0.1-2％(w/w)
氯化镁  0.1-2mM
磷酸盐缓冲剂  5-30mM
吐温80  0.01％-10％(w/w)。
优选的，上述腺病毒冷冻干燥添加剂，所述甘油的含量为0.1-1.8％。
优选的，上述腺病毒冷冻干燥添加剂，组方如下：
每1西林瓶(复溶后0.5ml)含：
甘露醇  50mg/ml
蔗糖  25mg/ml
甘油  0.2％-0.3％(w/w)
氯化镁  1mM
磷酸盐缓冲剂  10mM
吐温80  0.1％(w/w)
优选的，上述腺病毒冻干制剂，复溶后PH值为7.0-8.0。
上述腺病毒冷冻干燥添加剂的制备方法，具体步骤为：
(1)按组方量称取甘露醇、蔗糖、甘油、氯化镁、磷酸盐缓冲剂和吐温80；
(2)将甘露醇、蔗糖、甘油、氯化镁和吐温80分别加入到磷酸盐缓冲剂中，充分溶解，用孔径0.22μm滤膜过滤除菌。
上述腺病毒冷冻干燥添加剂在制备腺病毒冻干制剂方面的应用。
一种腺病毒冻干制剂，由腺病毒和上述腺病毒冷冻干燥添加剂组成。
优选的，上述腺病毒冻干制剂，所述腺病毒是重组复制缺陷型人5型腺病毒，或者是4型、7型、11型、或55型腺病毒。
优选的，上述腺病毒冻干制剂，所述重组复制缺陷型人5型腺病毒是表达Zaire型(Guinea，2014)埃博拉病毒包膜糖蛋白基因的复制缺陷型人5型腺病毒；或
所述重组复制缺陷型人5型腺病毒是表达结核分支杆菌Ag85A基因的复制缺陷型人5型腺病毒。
优选的，上述腺病毒冻干制剂，每1西林瓶(复溶后0.5ml)含：
复制缺陷型人5型腺病毒4×1010-8×1011VP
甘露醇  10-80mg/ml
蔗糖  10-80mg/ml
甘油  0.1-2％(w/w)
氯化镁  0.1-2mM
磷酸盐缓冲剂  5-30mM
吐温80 0.01％-10％(w/w)。
优选的，上述腺病毒冻干制剂，复溶后PH值为7.0-8.0。
优选的，上述腺病毒冻干制剂，呈白色疏松状。
优选的，上述腺病毒冻干制剂，可以作为基因治疗药物或预防传染性疾病的疫苗、多价疫苗、或联合疫苗。
上述腺病毒冻干制剂的制备方法，具体步骤为：
(1)向腺病毒冷冻干燥添加剂中加入注射用水，充分溶解后过滤除菌；
(2)再将重组腺病毒浓缩液加入到上述溶液中进行无菌灌装，每只西林瓶分装0.5ml,经真空冷冻干燥、轧盖密封瓶口即得冷冻干燥剂型的重组腺病毒埃博拉病毒病疫苗。
本发明的有益效果：
冻干保护剂制备领域中，保护剂组分的选择和配比对疫苗的保存效果至关重要。本发明所述腺病毒冷冻干燥添加剂通过对赋形剂等的合理筛选和复配，各组分协同作用，优化彼此间的配比浓度，可使获得的重组腺病毒埃博拉病毒病疫苗冻干制剂稳定存贮于2-8℃1年，37℃条件下稳定2周，且腺病毒活性良好，最大限度地减少腺病毒在分装和冻干过程中的损失和失活，保证腺病毒的活性和制剂的有效性，动物实验和人 体临床试验表明，该疫苗安全性和免疫原性良好。
所得腺病毒冻干制剂克服了现有腺病毒液体制剂-80℃低温冻存及运输过程中的超低温冷链的缺点，便于运输和使用，并且避免了人血白蛋白的加入，在腺病毒疫苗及重组腺病毒载体疫苗方面有很好的应用价值，经临床试验证实，疫苗安全性良好，提前两周接种可实现免疫保护。非人灵长类动物模型试验表明，疫苗保护率100％。稳定性试验表明，该制剂配方可在2-8℃稳定存储1年，37℃稳定2周。
附图说明
图1是重组腺病毒埃博拉疫苗冻干制剂外观。
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。
实施例1重组腺病毒埃博拉疫苗冻干制剂的制备
(1)按照常规的腺病毒疫苗生产方法制备能够表达Zaire型(Guinea，2014)埃博拉病毒包膜糖蛋白(GP蛋白)基因的复制缺陷型人5型腺病毒疫苗原液：取冷冻保存的病毒种子进行复苏和种子培养，然后接种到50L培养基中继续培养，控制溶解氧、通气量、pH、搅拌转速等使细胞增殖，72小时候将腺病毒种子Ad5-EBOV感染细胞，感染后继续培养。培养结束后裂解细胞，核酸酶处理，离心，再经柱层析分离得到腺病毒。具体操作为委托军科院构建的重组腺病毒，采用AdMax包装系统构建带有埃博拉病毒包膜糖蛋白(GP蛋白)表达基因的重组腺病毒，将克隆了外源基因的腺病毒穿梭质粒pDC316与携带了腺病毒大部分基因组的包装质粒pBHGlox_E1,3Cre共转染293细胞，利用Cre/loxP系统的作用实现重组，产生重组腺病毒，该病毒经过培养增殖得到大量的病毒，经纯化后即为疫苗原液。
(2)配制腺病毒冷冻干燥添加剂。按照每1西林瓶(复溶后0.5ml)含
甘露醇50mg/ml、蔗糖25mg/ml、氯化镁1mM、磷酸盐缓冲剂10mM、吐温80(质量)0.1％(w/w)、甘油(质量)0.3％(w/w)的比例配制疫苗半成品。控制半成品的PH7.0-8.0。
(3)配制疫苗半成品。将腺病毒冷冻干燥添加剂用注射水复溶，经无菌过滤，再按照每1西林瓶(复溶后0.5ml)含复制缺陷型人5型腺病毒4×1010VP的量将腺病毒浓缩液加入冷冻干燥溶液中，即得到疫苗半成品。
(4)分装冻干得到疫苗成品。将疫苗半成品进行无菌灌装，每只西林瓶分装0.5ml,经真空冷冻干燥，经轧盖密封瓶口即制成冷冻干燥剂型的重组腺病毒埃博拉病毒病疫苗，外观如图1所示，外观良好，呈白色疏松状。
实施例2
重组腺病毒埃博拉疫苗冻干前后的腺病毒活性比较
按照实施列中1中所述的生产工艺和配方生产3个批次的重组腺病毒埃博拉疫苗冻干制剂成品，并对冻干前的半成品和冻干后的成品进行IFU(活病毒滴度)测定，检测结果如下：
表1 重组腺病毒埃博拉疫苗冻干前后的活病毒浓度的检测结果
批次 冻干前(半成品) 冻干后(成品)
1 2.1×109IFU/ml 1.5×109IFU/ml
2 2.4×109IFU/ml 1.8×109IFU/ml
3 2.0×109IFU/ml 1.4×109IFU/ml
根据以上检测结果发现，在该配方下冻干制剂前后活病毒的损失率在25％左右。
实施例3
重组腺病毒埃博拉疫苗冻干制剂加速稳定性研究(37℃)
按照实施列中1中所述的生产工艺和配方生产3个批次的冻干制剂成品，并对3批成品分别进行IFU(活病毒滴度)4周的观察，检测结果如下：
表2 重组腺病毒埃博拉疫苗冻干制剂37℃下4周的IFU检测结果
批次 0点 1周 2周 4周
1 1.5×109IFU/ml 1.2×109IFU/ml 1.1×109IFU/ml 7.5×108IFU/ml
2 1.8×109IFU/ml 1.3×109IFU/ml 1.2×109IFU/ml 7.8×108IFU/ml
3 1.4×109IFU/ml 1.2×109IFU/ml 1.0×109IFU/ml 7.6×108IFU/ml
重组埃博拉病毒病疫苗制品的质量标准要求每剂成品活病毒浓度在≥8×108IFU/ml，以上研究结果发现37℃条件下，2周时还维持8×108IFU/ml以上，说明该配方下冻干制剂可以在37℃条件下稳定2周。但4周时成品的IFU浓度已降至8×108IFU/ml以下，成品已经不符合质量标准。
实施例4
重组腺病毒埃博拉疫苗冻干制剂2-8℃的稳定性研究
表3 重组腺病毒埃博拉疫苗冻干制剂2-8℃下12M的IFU检测结果
Figure PCTCN2017114401-appb-000001
重组埃博拉病毒病疫苗制品的质量标准要求每剂成品活病毒浓度在≥8×108IFU/ml，以上研究结果发现2-8℃条件下，12M时还维持8×108IFU/ml以上，符合质量标准，说明该配方下冻干制剂可以在2-8℃条件下可以稳定1年。
实施例5
上述实施例1所得埃博拉疫苗临床试验由江苏省疾控中心研究员朱凤才 主持，在泰州中国医药城完成了对120名志愿者的剂量递增、随机双盲、安慰剂对照I期临床试验，结果表明，疫苗安全性好，主要不良反应为注射部位疼痛，未发生严重不良事件，接种后14天细胞免疫水平达到最高，28天抗体水平达到峰值，提前2周接种可以实现免疫保护。
上述参照实施例对该一种腺病毒冷冻干燥添加剂及腺病毒冻干制剂进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。

Claims (10)

  1. 一种腺病毒冷冻干燥添加剂，其特征在于：由甘露醇、蔗糖、甘油、氯化镁、磷酸盐缓冲剂和吐温80组成，其中，
    Figure PCTCN2017114401-appb-100001
  2. 根据权利要求1所述的腺病毒冷冻干燥添加剂，其特征在于：所述甘油的含量为0.1-1.8％。
  3. 根据权利要求1所述的腺病毒冷冻干燥添加剂，其特征在于：组方如下：
    每1西林瓶复溶后0.5ml含：
    Figure PCTCN2017114401-appb-100002
  4. 权利要求1所述腺病毒冷冻干燥添加剂的制备方法，其特征在于：具 体步骤为：
    (1)按组方量称取甘露醇、蔗糖、甘油、氯化镁、磷酸盐缓冲剂和吐温80；
    (2)将甘露醇、蔗糖、甘油、氯化镁和吐温80分别加入到磷酸盐缓冲剂中，充分溶解，用孔径0.22μm滤膜过滤除菌。
  5. 权利要求1所述腺病毒冷冻干燥添加剂在制备腺病毒冻干制剂方面的应用。
  6. 一种腺病毒冻干制剂，由腺病毒和权利要求1所述腺病毒冷冻干燥添加剂组成。
  7. 根据权利要求6所述腺病毒冻干制剂，其特征在于：所述腺病毒是重组复制缺陷型人5型腺病毒，或者是4型、7型、11型、或55型腺病毒。
  8. 根据权利要求7所述腺病毒冻干制剂，其特征在于：所述重组复制缺陷型人5型腺病毒是表达Zaire型(Guinea，2014)埃博拉病毒包膜糖蛋白基因的复制缺陷型人5型腺病毒；或
    所述重组复制缺陷型人5型腺病毒是表达结核分支杆菌Ag85A基因的复制缺陷型人5型腺病毒。
  9. 根据权利要求6所述腺病毒冻干制剂，其特征在于：每1西林瓶复溶后0.5ml含：
    复制缺陷型人5型腺病毒4×1010-8×1011VP
    Figure PCTCN2017114401-appb-100003
    Figure PCTCN2017114401-appb-100004
  10. 权利要求6所述腺病毒冻干制剂的制备方法，其特征在于：具体步骤为：
    (1)向腺病毒冷冻干燥添加剂中加入注射用水，充分溶解后过滤除菌；
    (2)再将重组腺病毒浓缩液加入到上述溶液中进行无菌灌装，每只西林瓶分装0.5ml,经真空冷冻干燥、轧盖密封瓶口即得冷冻干燥剂型的重组腺病毒埃博拉病毒病疫苗。
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