CN117414432A - 一种稳定腺病毒载体冻干制剂及其应用 - Google Patents
一种稳定腺病毒载体冻干制剂及其应用 Download PDFInfo
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Abstract
本发明涉及一种稳定腺病毒载体冻干制剂及其应用,所述冻干制剂,用于稳定具有生物活性的预防类疫苗或和治疗类的生物医药制品,同时还能够降低冻干过程对生物制品活性的损伤,该制剂由有效成分,盐类缓冲液,糖类冻干保护剂,蛋白质类保护剂,表面活性剂,赋形剂制备而成,pH为6.0‑7.0。
Description
技术领域
本发明涉及生物医药技术领域,尤其涉及一种解决生物制品活性成分长期稳定性的冻干制剂处方。
背景技术
时至今日,仍有数百万人死于疫苗及治疗性生物制品可预防和治愈的疾病,虽然这与在偏远农村地区、不发达国家和冲突地区生物医药的分发有直接关系,但也与生物制品的热稳定性较弱密切相关。换言之,生物制品的不稳定性一定程度上限制了其更广泛的使用。例如:新型冠状病毒的大流行凸显出疫苗在运输、储存及分发等环节的重要性,而疫苗的不稳定性往往造成较短的货架期以及对低温的依赖。提高疫苗的稳定性以及延长其货架期一直是几十年来的前沿问题,尤其对于具有活性的生物制品而言,研究如何在相对较长的时间内保证其良好的稳定性具有重要的意义。很多优秀的制剂科学家通过不断地研发,开发了大量用于维持生物医药活性的配方并优化了工艺参数,其研发目标是提供在储存期间和意外暴露于异常条件后仍能保持足够质量(效价、活性和免疫原性)的产品,一直到药物被使用。众所周知,病毒、蛋白等生物药品在8℃以上水介质中的稳定性是有限的,其热稳定性一直受到挑战,再加上冷链运输和储存条件的不确定性和不均衡性,液体制剂虽然制备方便,但也局限了药物长期使用的潜力。冻干方法在生物医药及食品工业领域有着广泛的应用,通过冻干的手段去除游离水(使最终水含量为产品总重量的1%~3%)可以通过充分抑制或减缓水介质中可能发生的降解等不稳定途径来显著增加病毒或者蛋白等的稳定性并延长其货架期。
本发明根据现有相关技术知识,通过一系列试验筛选了冻干配方中的关键组分。
发明内容
本发明旨在开发并提供一种冻干制剂配方,该制剂应用冻干手段,一方面能够改善生物制品在冻干完成后效价、活性和免疫原性降低的问题,另一方面能够保护并维持其生物质量长期稳定。
本发明同时利用该制剂配方,开发了重组腺病毒制剂的冻干工艺,用于重组腺病毒类制剂的冻干。
本发明的具体实施方案
本发明提供一种冻干制剂,由有效成分,盐类缓冲液,糖类冻干保护剂,蛋白质类保护剂,表面活性剂,赋形剂制备而成,pH为6.0~7.0。
所述有效成分为重组腺病毒疫苗。
所述的盐类缓冲液选自:柠檬酸、PB、Tris-HCl、生理盐水、DPBS及KPBS缓冲液;
所述糖类冻干保护剂选自:蔗糖、海藻糖、棉子糖或和环糊精;
所述蛋白质类保护剂选自:人血清白蛋白,牛血清白蛋白、明胶,水解乳清蛋白、水解酪蛋白;
所述表面活性剂选自:吐温20、吐温80、普洛沙姆F12、普洛沙姆F68、司盘20;
所述赋形剂选自:甘氨酸,右旋糖酐,聚乙烯吡咯烷酮,甘露醇,山梨醇,海藻糖。
所述液体制剂中,所述有效成分选自:呼吸道合胞病毒重组腺病毒疫苗,水痘-带状疱疹重组腺病毒疫苗,新型冠状病毒重组腺病毒疫苗,偏肺病毒重组腺病毒疫苗,水泡性口炎病毒重组腺病毒疫苗,单纯疱疹病毒I型重组腺病毒疫苗,单纯疱疹病毒II型重组腺病毒疫苗,幽门螺杆菌重组腺病毒疫苗,治疗性乙肝重组腺病毒疫苗,其中,优选的,所述有效成分为水痘-带状疱疹重组腺病毒疫苗。最优选的,所述有效成分呼吸道合胞病毒重组腺病毒疫苗包含的病毒颗粒数(virus particle,vp)以1.0×109~5.0×1013vp/ml的量存在。
其中,所述呼吸道合胞病毒重组腺病毒疫苗腺病毒疫苗,制备方法如下:
(1)构建并合成含有保护性抗原核苷酸的穿梭质粒;
(2)利用重组试剂盒将步骤(1)所述穿梭质粒与骨架质粒进行重组,得到含有保护性抗原蛋白基因的腺病毒重组子;
(3)将步骤(2)所述的腺病毒重组子转染入HEK-293或HEK-293.2sus;
(4)培养步骤(3)所述包装细胞为HEK-293或HEK-293.2sus;
(5)收获从步骤(4)所述包装细胞释放的复制缺陷型重组腺病毒;
(6)收获从步骤(5)所述复制缺陷型重组腺病毒进行噬斑纯化;
(7)对步骤(6)中的复制缺陷型重组腺病毒进行扩大培养;
(8)对步骤(7)中的培养的复制缺陷型重组腺病毒进行反复冻融,离心后去上清;
(9)对步骤(8)中的冻融产物进行物理除杂后超滤浓缩;
(10)对步骤(9)中的培养产物进行柱层析纯化。
优选的,所述盐类缓冲液选自KPBS或和PB缓冲液,其物质的量浓度为5mmol/L~100mmol/L。优选的,所述糖类冻干保护剂选自蔗糖、海藻糖或和棉子糖,其浓度为5%~30%。
优选的,所述蛋白质类保护剂为人血清白蛋白,其浓度为0.1~5%。
优选的,所述表面活性剂为吐温80,其浓度为0.001%~0.8%。
优选的,所述赋形剂选自右旋糖酐、甘露醇或和PVP,浓度为2%~12%。
本发明所述盐类缓冲液选自KPBS或和PB缓冲液,其物质的量浓度为10mmol/L~50mmol/L;
所述糖类冻干保护剂选自蔗糖、海藻糖或和棉子糖,其浓度为5%~20%;
所述蛋白质类保护剂为人血清白蛋白,其浓度为0.2%~3%;
所述表面活性剂为吐温80,其浓度为0.01%~0.6%或0.015%~0.4%;
所述赋形剂选自右旋糖酐、甘露醇或和PVP,浓度为3%~8%。
本发明进一步提供所述冻干制剂的制备方法,所述方法,步骤如下:按照2×的质量分数或摩尔物质的量称取各组分,使用注射用水将其溶解后定容,每次配制均为现配现用,并调节至指定pH值,使用0.22μM滤器进行过滤除菌,取生物制品原液进行适当稀释与冻干配方按照1:1体积比进行充分混匀,放入冻干机样品仓后,放置好温度电极,关紧仓门,设置冻干工艺流程及参数,进行冻干,冻干完成后,充入氮气破除真空,进行压塞和轧盖;所述的冻干工艺流程主要包括以下步骤:搁板预冷、冷冻、退火、一次干燥和二次干燥;所述的冻干工艺参数主要为:搁板预冷温度为-10~20℃,持续时间至少30min;冷冻温度为-60~-40℃,持续时间3~8h;退火温度为-30~-10℃,持续时间0.5~5h;一次干燥第一阶段为-60~-45℃持续时间4~6h,第二阶段为-40~-25℃持续时间至少12h,此阶段真空压力为0.08~0.5mbar;二次干燥温度为5~20℃,持续时间为6~12h,此阶段真空压力至为0.001~0.3mbar;其中,温度变化时的升温及降温速率为0.01~1.5℃/min,降温速率为0.5~5℃/min。优选的,其中,升温速率为0.1~1℃/min,降温速率为0.6~2℃/min。
本发明所述的最终冻干制剂中配方组分的确定,是通过冻干前后滴度的变化来筛选关键辅料,并综合冻干饼的评分来进行综合评估,确定可用的配方。
所述病毒感染力是通过测定病毒滴度,采用半数细胞感染量(CCID50)终点稀释法测定重组腺病毒在HEK-293T细胞中的感染效力,具体步骤如下:
(1)取病毒50μl,加入维持液450μl,混匀后,标记稀释度为10-1;取稀释度为10-1的病毒稀释液50μl,加维持液450μl,混匀后,标记稀释度为10-2;以此类推,将病毒稀释至10-10。
(2)将HEK-293T细胞消化后制成稀释度为2.0×105个/mL细胞悬液。将细胞悬液与梯度病毒稀释液等体积混合,备用。
(3)在96孔细胞培养板中接种细胞,每孔104个细胞,1A-1H列加入稀释度为10-6的病毒稀释液与细胞的混合液100μl/孔,2A-2H列加入稀释度为10-7的病毒稀释液与细胞的混合液各100μl/孔,以此类推,5A-5H列加入稀释度为10-10的病毒稀释液与细胞的混合液100μl/孔,6A-6H列加入维持液100μl/孔做为阴性对照。在7H-12H列中设置重复
(4)按Reed-Muench两氏法计算或Karer法计算CCID50。
所述冻干后对冻干饼的赋形效果评估由三个与此实验无关的人员对冻干饼进行评估打分,满分为3分,计算总分的平均值,分值越高赋形效果越好。
本发明试验中,所用病毒为重组黑猩猩63型腺病毒,也可以采用ChAd3、ChAd83、ChAd155、ChAd157、ChAdOx1和ChAdOx2等其他黑猩猩腺病毒以及血清1型~血清57型的人腺病毒中的一种或多种,比如为血清5型、7型、26型、35型或48型,并不影响本发明试验的结果。
本发明和现有技术相比,本发明的优点如下:
(1)通过不同种类、不同浓度的赋形剂单独或者组合搭配,保证冻干饼形状稳定。
(2)糖类和蛋白质冻干保护剂组合使用能更加有效地够维持生物制品的稳定性,同时降低冻干过程种的损伤。
(3)通过优化冻干工艺,保证冻干饼形态优质,同时能够降低冻干过程对生物制品质量(效价、活性和免疫原性)的损失。
具体实施方式
下面结合具体实施例对本发明所述的产品及其制备方法作进一步阐述,但本发明并不限于以下实施例。所述方法如无特别说明均为常规方法。所述原材料如无特别说明均能从公开商业途径获得。
实施例一:赋形剂的筛选
所述各种赋形剂,是单独存在或者与其他成分组合而成,以下所述不同赋形剂配方均含有20mmol/L的PB缓冲液且不含有生物制品。所述1号赋形剂为3%甘露醇;所述2号赋形剂为3%PVP;所述3号赋形剂为3%右旋糖酐;所述4号赋形剂3%甘氨酸;所述5号赋形剂3%山梨醇;所述6号赋形剂为成分为2%PVP和2%甘氨酸;所述7号赋形剂成分为2%PVP和2%甘露醇;所述8号赋形剂为成分为2%PVP和2%右旋糖酐;所述9号赋形剂成分为2%PVP和2%山梨醇;所述10号赋形剂成分为2%甘露醇和2%右旋糖酐;所述11号赋形剂成分为2%甘露醇和2%山梨醇;所述12号赋形剂成分为2%右旋糖酐和2%山梨醇;所述13号赋形剂成分为2%海藻糖和2%蔗糖。冻干结束后,对冻干饼进行赋形效果评分。以下为不同配方的评分。
编号 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | 13 |
冻干饼评分 | 2.98 | 2.80 | 2.78 | 2.33 | 0.8 | 2.06 | 3.00 | 2.20 | 1.50 | 2.00 | 2.78 | 2.5 | 2.00 |
根据以上结果可以看出:在只添加一种赋形剂时,山梨醇的赋形效果最差,甘露醇最好,PVP和右旋糖酐次之;在赋形剂的组合中,2%PVP和2%甘露醇以及2%甘露醇和2%山梨醇的赋形效果较好。
实施例二:冻干赋型效果及其保护效果实验
所述1号配方含有20mmol/L PB缓冲液,3%的甘露醇,0.1mol/L的蔗糖;所述2号配方含有20mmol/L PB缓冲液,3%的甘露醇,0.2mol/L的蔗糖;所述3号配方含有20mmol/L PB缓冲液,3%的甘露醇,0.3mol/L的蔗糖;所述4号配方含有20mmol/L PB缓冲液,3%的甘露醇,0.4mol/L的蔗糖;所述5号配方含有20mmol/L PB缓冲液,4%的甘露醇,0.1mol/L的蔗糖;所述6号配方含有20mmol/L PB缓冲液,4%的甘露醇,0.2mol/L的蔗糖;所述7号配方含有20mmol/L PB缓冲液,4%的甘露醇,0.3mol/L的蔗糖;所述8号配方含有20mmol/L PB缓冲液,4%的甘露醇,0.4mol/L的蔗糖;所述9号配方含有20mmol/L PB缓冲液,5%的甘露醇,0.1mol/L的蔗糖;所述10号配方含有20mmol/L PB缓冲液,5%的甘露醇,0.2mol/L的蔗糖;所述11号配方含有20mmol/L PB缓冲液,5%的甘露醇,0.3mol/L的蔗糖。冻干结束后,对冻干饼进行赋形效果评分,以下为不同配方的评分及冻干前后滴度。
根据以上结果可知:随着蔗糖浓度的增加,处方对病毒的保护效果更好,但是冻干饼的赋形效果变差。因此蔗糖浓度提高,为保证好的赋形效果,所需的赋形剂浓度也需要适当提高。
实施例三:不同缓冲体系对冻干保护效果的影响
所述1号配方含20mmol/L KPBS,0.02%的吐温80,0.1mol/L海藻糖,5%甘露醇;所述2号配方含生理盐水,0.02%的吐温80,0.1mol/L海藻糖,5%甘露醇;所述3号配方含20mmol/L Tris-HCl,0.02%的吐温80,0.1mol/L海藻糖,5%甘露醇;所述4号配方含20mmol/LDPBS,0.02%的吐温80,0.1mol/L海藻糖,5%甘露醇;所述5号配方含20mmol/LPB,0.02%的吐温80,0.1mol/L海藻糖,5%甘露醇;所述6号配方含20mmol/L柠檬酸,0.02%的吐温80,0.1mol/L海藻糖,5%甘露醇;以下为不同配方的评分及冻干前后滴度。
根据以上结果可知:不同的缓冲体系对冻干赋形效果没有影响,DPBS及PB缓冲体系在冻干制剂中是较好的缓冲液。
实施例四:不同无定形糖搭配
所述1号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L蔗糖,5%环糊精,5%甘露醇;所述2号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L蔗糖,0.1mol/L海藻糖,5%甘露醇;所述3号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L蔗糖,0.1mol/L棉子糖,5%甘露醇;所述4号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L海藻糖,5%环糊精,5%甘露醇;所述5号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L海藻糖,0.1mol/L棉子糖,5%甘露醇。所述6号配方含10mmol/L Tris-HCl,0.04%的吐温80,5%环糊精,0.1mol/L棉子糖,5%甘露醇;所述7号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L蔗糖,5%甘露醇;所述8号配方含10mmol/L Tris-HCl,0.04%的吐温80,5%环糊精,5%甘露醇;所述9号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L棉子糖,5%甘露醇;所述10号配方含10mmol/L Tris-HCl,0.04%的吐温80,0.1mol/L海藻糖,5%甘露醇。以下为不同配方的评分及冻干前后滴度。
为了提高处方对病毒的冻干保护效果,使用不同的糖类保护剂进行搭配组合。根据以上结果可知:不同糖类组合使用确实提高了一定的保护效果,但是赋形效果依然不佳,糖类单独使用赋形效果较好。
实施例五:不同蛋白类保护剂的比较
所述1号配方含20mmol/L KPBS,0.025%的吐温80,0.1mol/L蔗糖,4.5%甘露醇,0.5%水解乳清蛋白;所述2号配方含20mmol/L KPBS,0.025%的吐温80,0.1mol/L蔗糖,4.5%甘露醇,0.5%水解酪蛋白;所述3号配方含20mmol/L KPBS,0.025%的吐温80,0.1mol/L蔗糖,4.5%甘露醇,0.5%BSA;所述4号配方含20mmol/L KPBS,0.025%的吐温80,0.1mol/L蔗糖,0.5%HSA,4.5%甘露醇;所述5号配方含20mmol/L KPBS,0.025%的吐温80,0.1mol/L蔗糖,4.5%甘露醇,0.5%明胶。以下为不同配方的评分及冻干前后滴度。
根据以上结果可知:添加蛋白类保护剂后,处方对病毒的保护效果更好,滴度损失值较低,BSA和HSA的保护效果较好,明胶保护效果最差且赋形效果不佳。
实施例六:不同浓度表面活性剂及蛋白类保护剂的保护效果
所述1号配方含5mmol/L PB,0.02%的吐温80,0.1mol/L蔗糖,5%甘露醇;所述2号配方含5mmol/L PB,0.04%的吐温80,0.1mol/L蔗糖,5%甘露醇;所述3号配方含5mmol/LPB,0.06%的吐温80,0.1mol/L蔗糖,5%甘露醇;所述4号配方含5mmol/LPB,0.02%的吐温80,0.1mol/L蔗糖,0.25%HSA,5%甘露醇;所述5号配方含5mmol/LPB,0.02%的吐温80,0.1mol/L蔗糖,0.5%HSA,5%甘露醇;所述6号配方含5mmol/L PB,0.02%的吐温80,0.1mol/L蔗糖,1%HSA,5%甘露醇。以下为不同配方的评分及冻干前后滴度。
根据以上结果可知:表面活性剂的具体用量对保护病毒没有明显影响,HSA的用量在1%时,保护效果最佳。
实施例七:不同糖类与不同蛋白质类保护剂筛选
所述1号配方含5mmol/L PB,0.02%的吐温80,0.15mol/L蔗糖,0.5%HSA,5%甘露醇;所述2号配方含5mmol/L PB,0.02%的吐温80,0.15mol/L棉子糖,0.5%HSA,5%甘露醇;所述3号配方含5mmol/LPB,0.02%的吐温80,5%环糊精,0.5%HSA,5%甘露醇;所述4号配方含5mmol/L PB,0.02%的吐温80,0.15mol/L海藻糖,0.5%HSA,5%甘露醇。以下为不同配方的评分及冻干前后滴度。
根据以上结果可知:糖类及蛋白质类保护剂联合使用时,对病毒的保护效果明显更好。
实施例八:长期稳定性实验数据
按照所述权利要求的内容,制备了4种冻干制剂,并进行了长期稳定性测定。以下为4种不同处方冻干制剂的滴度监测结果。
其中,
1号处方如下:5mmol/PB、0.02%的吐温80,0.15mol/L蔗糖,5%甘露醇。
2号处方如下:5mmol/PB、0.02%的吐温80,0.15mol/L蔗糖,0.25%HSA,5%甘露醇。
3号处方如下:5mmol/PB、0.02%的吐温80,0.15mol/L蔗糖,0.5%HSA,5%甘露醇。
4号处方如下:5mmol/PB、0.02%的吐温80,0.15mol/L蔗糖,1%HSA,5%甘露醇。
结果显示,所述4种制剂处方,1号为无保护剂对照处方、在2~8℃条件下保存360d,3号处方保护效果最好,病毒滴度仅仅下降0.0375个lg值。2和4号处方滴度损失值小于0.5个lg值。
应当指出,对于熟悉本技术领域的技术人员,可轻易的实现优化改进,都应在本发明的保护范围之内,本申请所述的技术方案的各个技术特征均可根据需要进行适当的组合。
以上所述仅为本发明的优选实施方式,应当指出的是,本发明的实施方式并不受所述实施例的限制。在不脱离本发明原理的前提下,还可以做出若干的改变、修饰、替代、组合、简化,均应为等效的置换方式,这些也应视为本发明发保护范围。
Claims (10)
1.一种冻干制剂,其特征在于:由有效成分,盐类缓冲液,糖类冻干保护剂,蛋白质类保护剂,表面活性剂,赋形剂制备而成,pH为6.0-7.0。
2.根据权利要求1所述的冻干制剂,其特征在于:所述有效成分为重组腺病毒疫苗。
所述的盐类缓冲液选自:柠檬酸、PB、Tris-HCl、生理盐水、DPBS及KPBS缓冲液;
所述糖类冻干保护剂选自:蔗糖、海藻糖、棉子糖或和环糊精;
所述蛋白质类保护剂选自:人血清白蛋白,牛血清白蛋白、明胶,水解乳清蛋白、水解酪蛋白;
所述表面活性剂选自:吐温20、吐温80、普洛沙姆F12、普洛沙姆F68、司盘20;
所述赋形剂选自:甘氨酸,右旋糖酐,聚乙烯吡咯烷酮,甘露醇,山梨醇,海藻糖。
3.根据权利要求1所述的冻干制剂,其特征在于:所述盐类缓冲液选自为KPBS或和PB缓冲液,其物质的量浓度为5mmol/L-100mmol/L。
4.根据权利要求1所述的冻干制剂,其特征在于:所述糖类冻干保护剂选自蔗糖、海藻糖或和棉子糖,其浓度为5%-30%。
5.根据权利要求1所述的冻干制剂,其特征在于:所述蛋白质类保护剂为人血清白蛋白,其浓度为0.1-5%。
6.根据权利要求1所述的冻干制剂,其特征在于:所述表面活性剂为吐温80,其浓度为0.001%-0.8%。
7.根据权利要求1所述的冻干制剂,其特征在于:所述赋形剂选自右旋糖酐、甘露醇或和PVP,浓度为2%-12%。
8.根据权利要求2-7所述的冻干制剂,其特征在于:
所述重组腺病毒疫苗为:呼吸道合胞病毒重组腺病毒疫苗,水痘-带状疱疹重组腺病毒疫苗,新型冠状病毒重组腺病毒疫苗,偏肺病毒重组腺病毒疫苗,水泡性口炎病毒重组腺病毒疫苗,单纯疱疹病毒I型重组腺病毒疫苗,单纯疱疹病毒II型重组腺病毒疫苗,幽门螺杆菌重组腺病毒疫苗,治疗性乙肝重组腺病毒疫苗;
所述盐类缓冲液选自为KPBS或和PB缓冲液,其物质的量浓度为10mmol/L-50mmol/L;
所述糖类冻干保护剂选自蔗糖、海藻糖或和棉子糖,其浓度为5%-20%;
所述蛋白质类保护剂为人血清白蛋白,其浓度为0.2%-3%;
所述表面活性剂为吐温80,其浓度为0.01%-0.6%或0.015%-0.4%;
所述赋形剂选自右旋糖酐、甘露醇或和PVP,浓度为3%-8%。
9.权利要求1所述冻干制剂的制备方法,其特征在于,所述方法,步骤如下:按照2×的质量分数或摩尔物质的量称取各组分,使用注射用水将其溶解后定容,每次配制均为现配现用,并调节至指定pH值,使用0.22μM滤器进行过滤除菌,取生物制品原液进行适当稀释与冻干配方按照1:1体积比进行充分混匀,放入冻干机样品仓后,放置好温度电极,关紧仓门,设置冻干工艺流程及参数,进行冻干,冻干完成后,充入氮气破除真空,进行压塞和轧盖;所述的冻干工艺流程主要包括以下步骤:搁板预冷、冷冻、退火、一次干燥和二次干燥;所述的冻干工艺参数主要为:搁板预冷温度为-10~20℃,持续时间至少30min;冷冻温度为-60~-40℃,持续时间3~8h;退火温度为-30~-10℃,持续时间0.5~5h;一次干燥第一阶段为-60~-45℃持续时间4~6h,第二阶段为-40~-25℃持续时间至少12h,此阶段真空压力为0.08~0.5mbar;二次干燥温度为5~20℃,持续时间为6~12h,此阶段真空压力至为0.001~0.3mbar;其中,温度变化时的升温及降温速率为0.01~1.5℃/min,降温速率为0.5-5℃/min。
10.权利要求9所述的制备方法,其特征在于,其中,升温速率为0.1~1℃/min,降温速率为0.6~2℃/min。
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