CN112341542B - 一种3-羟基丁酰化修饰蛋白质药物及其制备方法和应用 - Google Patents
一种3-羟基丁酰化修饰蛋白质药物及其制备方法和应用 Download PDFInfo
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Abstract
本发明涉及医药技术领域，公开了一种3‑羟基丁酰化修饰蛋白质药物（例如抗体）及其制备方法和应用，特别是一种3‑羟基丁酰化修饰抗体及其制备方法和应用。发明人经过大量实验发现，3‑羟基丁酸及其类似物修饰蛋白质药物（例如抗体）后，可以显著提高蛋白质药物的热稳定性、对蛋白酶水解的抗性，降低蛋白质药物的等电点，并显著延长其在受试者体内的半衰期，进而提高其药效。修饰后所得蛋白质药物在科研和临床方面具有广阔的应用前景和较高的商业价值。
Description
技术领域
本发明涉及医药技术领域，具体涉及一种3-羟基丁酰化修饰蛋白质药物（例如抗体）及其制备方法和应用。
背景技术
随着基因重组技术的发展，蛋白质药物的商品化生产已成为现实，例如单克隆抗体和基因工程抗体、重组疫苗，等等。与以往的小分子药物相比，蛋白质药物往往具有高活性、特异性强、低毒性、生物功能明确、有利于临床应用等特点，其已成为医药产品中的重要组成部分。
蛋白质药物分子大，通常分子量为几千或几万甚至十几万道尔顿，另外，蛋白质常常具有多种官能团，可在同一时相内发生多种反应，因此通常具有不稳定性的特点，且其不稳定因素有酶、温度、pH等。例如，人体可天然产生蛋白酶对蛋白质进行消化和重塑，治疗性蛋白也会受到消化和重塑。在非病原体引发的人血浆中存在着多种类型的蛋白酶，人蛋白酶、纤溶酶、基质金属蛋白酶和嗜中性粒细胞弹性蛋白酶在各蛋白酶特有的残基处切割抗体重链多肽。在一些炎症性疾病或者癌症发展过程中，蛋白酶的水平会相应的升高，促进抗体在血浆中的水解（Terness, P., et al., The natural human IgG anti-F(ab')2 antibody recognizes a conformational IgG1 hinge epitope. J Immunol, 1995. 154(12): p. 6446-52.）。另外，在一些微生物感染中，微生物分泌的酶类也会参与抗体的降解过程。如金黄色葡萄球菌球菌可以分泌谷氨酰胺内肽酶，直接对抗体进行切割。通常情况下抗体的铰链区容易被蛋白酶切割。铰链区的切割会导致Fc片段断裂，使得抗体与受体不能结合而失去抗体靶向细胞和杀灭病菌的功能（Sun, H., et al., Plasminogen is a critical host pathogenicity factor for group A streptococcal infection.Science, 2004. 305(5688): p. 1283-6.）。因此，蛋白质的不稳定性在很大程度上限制了其医学以及商业应用。
发明内容
为克服现有技术的不足，本发明提供一种如下所示化合物及其药学上可接受的盐、酯、立体异构体修饰的蛋白质药物，
（Ⅰ）
其中，R1选自：H、OH、烷基、环烷基、环烷基烷基、芳基、芳烷基、卤素、烷氧基、烷氧基烷基、-COR3、-C(O)OR3、-NR3R4、-C(O)NR3R4；
R2选自：H、烷基、环烷基、环烷基烷基、芳基、芳烷基；
n为1-20的整数（例如1、2、3、4、5、6、7、8、9、10、12、14、16、18、20）；
R3和R4独立地选自：H、烷基、环烷基、环烷基烷基、芳基、芳烷基。
在本发明的一个实施方式中，R1为烷基，例如C1-C6烷基，特别是C1-C4烷基，例如甲基、乙基、正丙基、异丙基、正丁基。
在本发明的一个实施方式中，R2为H。
在本发明另一实施方式中，R2为烷基，例如C1-C6烷基，特别是C1-C4烷基，例如甲基、乙基、正丙基、异丙基、正丁基。
具体地，n为1-10的整数，特别是1-6的整数。
在本发明的一个实施方式中，n=1。
在本发明的另一实施方式中，n=2-6。
具体地，上述化合物可以选自如下结构：
具体地，上述化合物可以选自如下结构中的一种或多种的组合：
具体地，上述化合物可以是D型或L型的，也可以是D型和L型的混合物。
具体地，上述化合物可通过各类聚羟基脂肪酸PHA的水解和醇解等多种方法得到，经过蒸馏纯化，通过GC分析确认纯度极高，没有双键等危害细胞生长的副产物（Chen GQ，WuQ.Microbial Production and Applications of Chiral Hydroxyalkanoates.ApplMicrobiol Biotechnol67(2005)592-599）。
具体地，上述蛋白质药物可以选自：抗体、抗体药物偶联物（ADC）、重组蛋白、融合蛋白等，特别是抗体。
具体地，上述修饰是式Ⅰ所示化合物中的羰基与蛋白质药物中的游离氨基形成酰胺键。
具体地，上述修饰的蛋白质药物中一个或多个（例如2、3、4、5、6、7、8、9、10个）氨基酸残基被修饰。
具体地，上述修饰的蛋白质药物中，被修饰的氨基酸残基选自：赖氨酸、精氨酸、组氨酸、丝氨酸、谷氨酰胺、天冬酰胺，特别是赖氨酸。
具体地，上述修饰的蛋白质药物中至少一个（例如1、2、3、4、5、6、7、8、9、10个）赖氨酸残基被修饰。
具体地，上述蛋白质药物为抗体，其可为现有产品，也可通过现有技术已知的任何合适的方法制备得到。
在本发明的一个实施例中，上述蛋白质药物为抗SARS-CoV-2或其结构蛋白（如S蛋白、M蛋白、E蛋白、N蛋白中的一种或多种）的抗体，例如，抗SARS-CoV-2 S蛋白的抗体，例如“Wu, Y., et al., A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science, 2020. 368(6496): p.1274-1278.”中所述抗体B38。
在本发明的一个实施例中，上述修饰的蛋白质药物为3-羟基丁酸修饰的抗SARS-CoV-2 S蛋白的抗体、3-羟基戊酸修饰的抗SARS-CoV-2 S蛋白的抗体、3-羟基己酸修饰的抗SARS-CoV-2 S蛋白的抗体（其中，抗SARS-CoV-2 S蛋白的抗体，例如“Wu, Y., et al., A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science, 2020. 368(6496): p. 1274-1278.”中所述抗体B38）。
在本发明的另一实施例中，上述修饰的蛋白质药物为一种或多种3-羟基丁酸寡聚物修饰的抗SARS-CoV-2 S蛋白的抗体，特别是3-羟基丁酸寡聚物的混合物修饰的抗SARS-CoV-2 S蛋白的抗体，更特别是
本发明还提供上述修饰的蛋白质药物的制备方法，其包括蛋白质药物与上述式Ⅰ所示化合物及其药学上可接受的盐、酯、立体异构体进行反应的步骤。
具体地，上述反应在缩合剂的存在下进行。
具体地，上述缩合剂可以为碳二亚胺类缩合剂、鎓盐类缩合剂、有机磷类缩合剂，以及其他任何合适的缩合剂，特别是碳二亚胺类缩合剂（例如，1-乙基-(3-二甲基氨基丙基)碳二亚胺，二环己基碳二亚胺，二异丙基碳二亚胺，及其合适的盐）；在本发明的一个实施例中，上述缩合剂为1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐。
具体地，上述反应温度可以为20-30℃，例如室温（约25℃）。
具体地，上述反应中，蛋白质药物与上述式Ⅰ所示化合物及其药学上可接受的盐、酯、立体异构体的比例为10-1000g：1mol（例如，20g：1mol、40g：1mol、50g：1mol、60g：1mol、80g：1mol、100g：1mol、150g：1mol、200g：1mol、300g：1mol、400g：1mol、500g：1mol、600g：1mol、800g：1mol）。
具体地，上述方法还包括纯化步骤（例如通过超滤离心除去未反应的化合物）。
本发明还提供一种药物组合物，其包含本发明上述修饰的蛋白质药物，以及一种或多种药学上可接受的辅料。
具体地，上述药学上可接受的辅料为药学领域常规的辅料，例如，稀释剂、赋形剂、填充剂、粘合剂、湿润剂、崩解剂、吸收促进剂、表面活性剂、吸附载体、润滑剂等。
具体地，上述药物组合物可以采用任意的剂型或给药形式，本领域技术人员可以根据情况选用，包括，但不限于，片剂（包括糖衣片剂、膜包衣片剂、舌下片剂、口腔崩解片、口腔片剂等等）、丸剂、粉剂、颗粒剂、胶囊剂（包括软胶囊、微胶囊）、锭剂、糖浆剂、液体、乳剂、混悬剂、控制释放制剂（例如，瞬时释放制剂、缓释制剂、缓释微囊）、气雾剂、膜剂（例如，口服崩解膜剂、口腔粘膜-粘附膜剂）、注射剂（例如，皮下注射、静脉注射、肌内注射、腹膜内注射）、静脉滴注剂、透皮吸收制剂、软膏剂、洗剂、粘附制剂、栓剂（例如，直肠栓剂、阴道栓剂）、小药丸、鼻制剂、肺制剂（吸入剂）、眼睛滴剂等等。特别是，可通过注射、喷射、滴鼻、滴眼、渗透、吸收、物理或化学介导的方法导入机体如肌肉、皮内、皮下、静脉、粘膜组织；或是被其他物质混合或包裹后导入机体。
本发明还提供上述修饰的蛋白质药物、药物组合物在制备预防、治疗或诊断疾病的药物中的应用。
具体地，上述疾病可以为，但不限于，肿瘤、糖尿病、心脑血管疾病、免疫系统介导疾病、皮肤病、神经系统疾病、烧伤或创伤引发的病症、由病原体感染引起或病原体感染相关的疾病。
具体地，上述肿瘤为恶性肿瘤，例如，但不限于：淋巴瘤、母细胞瘤、髓母细胞瘤、视网膜母细胞瘤、肉瘤、脂肪肉瘤、滑膜细胞肉瘤、神经内分泌肿瘤、类癌肿瘤、胃泌素瘤、胰岛细胞癌、间皮瘤、神经鞘瘤、听神经瘤、脑膜瘤、腺癌、黑素瘤、白血病或淋巴样恶性肿瘤、鳞状细胞癌、上皮鳞状细胞癌、肺癌、小细胞肺癌、非小细胞肺癌、腺癌肺癌、肺鳞癌、腹膜癌、肝细胞癌、胃癌、肠癌、胰腺癌、成胶质细胞瘤、子宫颈癌、卵巢癌、肝癌、膀胱癌、肝癌、乳腺癌、转移性乳腺癌、结肠癌、直肠癌、结肠直肠癌、子宫癌、唾液腺癌、肾癌、前列腺癌、外阴癌、甲状腺癌、肝癌、肛门癌、阴茎癌、梅克尔细胞癌、食管癌、胆道肿瘤、头颈部癌和血液恶性肿瘤。
具体地，上述心脑血管疾病是心脏血管和脑血管疾病的统称，例如，但不限于，冠状动脉疾病、高血压、血脂异常、先天性心脏病、瓣膜疾病、心律失常等。
具体地，上述神经系统疾病是指发生于中枢神经系统、周围神经系统、植物神经系统的以感觉、运动、意识、植物神经功能障碍为主要表现的疾病，例如，脑血管病，包括脑梗死、脑出血、脑供血不足、短暂性脑缺血发作等；脊髓病变，例如急性脊髓炎、脊髓灰质炎、脊髓空洞等；中枢神经系感染，比如脑炎、脑膜炎等；周围神经病变，例如坐骨神经损伤、面神经麻痹、格林-巴利综合征等；运动障碍性疾病，例如帕金森病、小舞蹈病、肝豆状核变性；癫痫；神经系统变性疾病，流入运动神经元病、阿尔茨海默病、痴呆；神经系统遗传性疾病，例如神经皮肤综合征、脊髓小脑性共济失调；神经系统发育异常性疾病，例如先天性脑积水、脑性瘫痪；自主神经系统疾病，例如自主神经功能不全；等。
具体地，上述免疫系统介导疾病是指机体对自身抗原发生免疫反应而导致自身组织损害所引起的疾病，例如，但不限于，牛皮癣、牛皮癣关节炎、胃溃疡、关节病炎、溃疡性结肠炎、葡萄膜炎、系统性红斑狼疮、强直性脊椎炎、系统性血管炎、过敏性和特应性疾病、哮喘和特应性哮喘、湿疹、过敏性鼻炎、过敏性接触性皮炎、器官移植排斥反应、移植物抗宿主疾病、多发性硬化症、重症肌无力、风湿热、类风湿性关节炎、结节病、硬皮病、斯耶格伦氏综合征、克罗恩氏病。
具体地，上述病原体可以为微生物、寄生虫（原虫、蠕虫等）或其他媒介。具体地，上述微生物可选自：病毒、衣原体、立克次体、支原体、细菌、螺旋体、真菌等中的一种或多种。
具体地，上述病毒可以为，但不限于，腺病毒科（如腺病毒）、疱疹病毒科（如HSV1（口腔疱疹）、HSV2（外生殖器疱疹）、VZV（水痘）、EBV（埃-巴二氏病毒）、CMV（巨细胞病毒））、痘病毒科（如天花病毒、牛痘病毒）、乳多泡病毒科（如乳头瘤病毒）、细小病毒科（如B19病毒）、嗜肝DNA病毒科（如乙型肝炎病毒）、多瘤病毒科（如多瘤病毒）、呼肠孤病毒科（如呼肠弧病毒、轮状病毒）、小核糖核酸病毒科（如肠道病毒、口蹄疫病毒）、嵌杯样病毒科（如诺沃克病毒、戊型肝炎病毒）、披膜病毒科（如风疹病毒）、沙粒病毒科（如淋巴细胞性脉络丛脑膜炎病毒）、逆转录病毒科（HIV-1、HIV-2、HTLV-1）、黄病毒科（如登革热病毒、寨卡病毒、乙型脑炎病毒、基孔肯亚病毒、黄热病病毒、丙型肝炎病毒、西尼罗病毒等）、正粘病毒科（如流感病毒（如甲型流感病毒、乙型流感病毒、丙型流感病毒等））、副粘病毒科（如1型人副流感病毒(HPV)、2型HPV、3 型HPV、4型HPV、仙台病毒、腮腺炎病毒、麻疹病毒、呼吸道合胞病毒、新城疫病毒等）、布尼亚病毒科（如加利福尼亚脑炎病毒、汉坦病毒）、弹状病毒科（如狂犬病毒）、丝状病毒科（如埃博拉病毒、马尔堡病毒）、冠状病毒科（如HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、SARS-CoV、MERS-CoV、SARS-CoV-2等）、星状病毒科（如星状病毒）、博尔纳病毒科（如博尔纳病毒）。
具体地，上述由病原体感染引起或病原体感染相关的疾病包括但不限于，流感、SARS、COVID-19、病毒性肝炎（如甲型肝炎、乙型肝炎、丙型肝炎、丁型肝炎等）、AIDS、狂犬病、登革热、埃博拉病毒病等。
本发明还提供上述式Ⅰ所示化合物及其药学上可接受的盐、酯、立体异构体在蛋白质药物的修饰的应用。
本发明还提供上述式Ⅰ所示化合物及其药学上可接受的盐、酯、立体异构体在提高蛋白质药物的稳定性的应用。
具体地，上述稳定性包括热稳定性、对蛋白酶（例如胰蛋白酶、纤维蛋白酶）水解的抗性。
本发明还提供上述式Ⅰ所示化合物及其药学上可接受的盐、酯、立体异构体在延长蛋白质药物的半衰期的应用。
具体地，上述半衰期为蛋白质药物在受试者体内的半衰期。
具体地，上述受试者为动物，特别是哺乳动物，例如，鼠、兔、猫、犬、猴、牛、马、羊、猪、人类，特别是人类。
本发明还提供上述式Ⅰ所示化合物及其药学上可接受的盐、酯、立体异构体在提高蛋白质药物的酰化修饰水平中的应用。
具体地，上述酰化包括丁酰化、戊酰化、己酰化，特别是3-羟基丁酰化、3-羟基戊酰化、3-羟基己酰化或其寡聚物酰化。
本发明还提供一种预防和/或治疗疾病的方法，其包括向有此需要的受试者施用预防有效量或治疗有效量的本发明上述修饰的蛋白质药物、药物组合物的步骤。
具体地，上述方法中的疾病、受试者、修饰蛋白质药物、药物组合物具有本发明上述相应定义。
本发明的发明人经过大量实验发现，3-羟基丁酸及其类似物修饰蛋白质药物（例如抗体）后，可以显著提高蛋白质药物的热稳定性、对蛋白酶水解的抗性，降低蛋白质药物的等电点，并显著延长其在受试者（例如哺乳动物，特别是人类）体内的半衰期，进而提高其药效。修饰后所得蛋白质药物在科研和临床方面具有广阔的应用前景和较高的商业价值。
附图说明
图1所示为纯化抗体的SDS-PAGE(reduced)分析结果，M：蛋白质分子质量标准；1：纯化抗体。
图2所示为质谱测定抗体分子量的结果，其中图2A为未修饰抗体，图2B为3-羟基丁酸修饰的抗体。
图3所示为3-羟基丁酰化修饰抗体的修饰位点检测结果，数据为修饰抗体的二级质谱数据，测定出的氨基酸序列是HK（3HB）VYACWVTHQGLSSPVTK，即其中第二位的赖氨酸发生了三羟基丁酰化修饰。
图4所示为3-羟基丁酰化修饰抗体的酶切实验结果，其中92表示无修饰抗体，92-3HB为3-羟基丁酸修饰的抗体。
图5所示为3-羟基丁酰化修饰抗体的热稳定性实验结果。
图6所示为3-羟基丁酰化修饰抗体等电点测定结果，A：未修饰抗体等电点；B：修饰抗体等电点。
图7所示为3-羟基丁酰化修饰抗体小鼠体内半衰期变化结果，其中92表示无修饰抗体，92-3HB为3-羟基丁酸修饰的抗体。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
本发明中，术语“抗体”指的是与抗原特异性结合的免疫球蛋白分子。抗体可为源于自然源或源于重组源的完整的免疫球蛋白，并可为完整免疫球蛋白的免疫反应部分。抗体通常为免疫球蛋白分子的四聚物。本发明中的抗体可以以多种形式存在，包括例如，多克隆抗体、单克隆抗体、Fv、Fab和F(ab)2，以及单链抗体、人源化抗体、嵌合抗体，等等。
术语“烷基”指的是直链或支链的且不含不饱和键的烃链自由基，且该烃链自由基以单键与分子其它部分连接。典型的烷基基团含有1至12个（例如1、2、3、4、5、6、7、8、9、10、11、12）碳原子，如甲基、乙基、正丙基、异丙基、正丁基、叔丁基、正戊基、异戊基、新戊基、叔戊基、正己基、异己基等。如果烷基被环烷基取代，其相应为“环烷基烷基”自由基，如环丙基甲基、环丙基乙基、环丁基甲基、环戊基甲基、环己基甲基等。如果烷基被芳基取代，那么其相应为“芳烷基”自由基，如苄基、二苯甲基或苯乙基。
术语“烷氧基”指的是羟基中的氢被烷基取代后形成的取代基，C1-C6的烷氧基指含有1-6个碳原子的烷氧基，如甲氧基、乙氧基、丙氧基、丁氧基等。
术语“环烷基”指的是脂环烃，如含1至4个单环和/或稠环、含3-18个碳原子，优选3-10（例如3、4、5、6、7、8、9、10）个碳原子，如环丙基、环丁基、环戊基、环己基或金刚烷基等。
术语“芳基”指的是单环或多环自由基，包括含单芳基基团和/或稠芳基基团的多环自由基，如包含1-3个单环或稠环及6-18（例如6、8、10、12、14、16、18）个碳环原子，如苯基、萘基、联苯基、茚基等。
术语“卤素”是指溴、氯、碘、氟。
术语“盐”应理解为本发明相应化合物的任意形式，其中该化合物为离子形式或者为带电荷的且与带相反电荷的离子（阳离子或阴离子）耦合或在溶液中。该定义特别地包括药学上可接受的盐。盐包括酸加成盐和碱加成盐。酸加成盐包括但不限于来自无机酸诸如盐酸、硝酸、磷酸、硫酸、氢溴酸、氢碘酸和膦酸的盐，以及来自有机酸如脂肪族单羧酸和二羧酸、苯基取代的链烷酸、羟基链烷酸、链烷二酸、芳香酸和脂肪族和芳香族磺酸的盐。因此，这些盐包括但不限于硫酸盐、焦硫酸盐、硫酸氢盐、亚硫酸盐、亚硫酸氢盐、硝酸盐、磷酸盐、磷酸一氢盐、磷酸二氢盐、偏磷酸盐、焦磷酸盐、盐酸盐、氢溴酸盐、碘酸盐、乙酸盐、丙酸盐、辛酸盐、异丁酸盐、乙二酸盐、丙二酸盐、琥珀酸盐、辛二酸盐、癸二酸盐、富马酸盐、马来酸盐、苦杏仁酸盐、苯甲酸盐、氯代苯甲酸盐、甲基苯甲酸盐、二硝基苯甲酸盐、酞酸盐、苯磺酸盐、甲苯磺酸盐、苯基乙酸盐、柠檬酸盐、乳酸盐、马来酸盐、酒石酸盐和甲磺酸盐，还包含氨基酸的盐如精氨酸盐、葡糖酸盐、半乳糖醛酸盐等。酸加成盐可以通过以常规方式使游离碱形式与足够量的所需酸接触形成盐的方式制备。可通过使盐形式与碱接触重新生成游离碱形式，并且以常规方式分离该游离碱。碱加成盐是指与金属或者胺形成的盐，诸如碱金属和碱土金属的氢氧化物，或者与有机胺形成。用作阳离子的金属的例子包括但不限于钠、钾、镁、钙。适当的胺的例子包括但不限于N,N′-二苄基乙二胺、氯普鲁卡因、胆碱、二乙醇胺、乙二胺（乙烷-1,2-二胺）、N-甲基葡糖胺和普鲁卡因。碱加成盐可通过以常规方式使游离酸形式与足够量的所需碱接触形成盐的方式制备。可通过使盐形式与酸接触重新生成游离酸形式，并且以常规方式分离游离酸。
术语“酯”应理解为酸与本发明相应化合物的羟基（如果存在的话）反应生成的化合物。该定义特别地包括药学上可接受的酯。
此外，本发明中所涉及的任何化合物可以互变异构体形式存在。特别地，术语互变异构体是指化合物的两个或多个结构异构体中的一个，这些异构体间存在平衡，可以相互转换。常见的互变异构体对为烯胺-亚胺、酰胺-亚胺酸、酮-烯醇、内酰胺-内酰亚胺等。
术语“药学上可接受的”，表示考虑到待治疗的疾病或病症以及相应的施用途径，所指出的材料不具有引起合理谨慎的医师避免将该材料施用于患者的性质，理论上无毒、刺激性及过敏反应的，并且能够实现或提供药物分子临床上可接受的药代动力学性质、吸收、分布及代谢性质，可达到预期目的材料。
本文所引用的各种出版物、专利和公开的专利说明书，其公开内容通过引用整体并入本文。
下面通过具体实施例对本发明进行说明，但本发明并不局限于此。
下述实施例中所述实验方法，如无特殊说明，均为常规方法；所述试剂和生物材料，如无特殊说明，均可从商业途径获得。
实施例1：抗体的重组表达（抗体制备参考“Wu, Y., et al., A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science, 2020. 368(6496): p. 1274-1278.”中的抗体B38制备）
1.1 质粒抽提
（1）取400mL过夜培养的大肠杆菌菌液，9000r/min离心10min，尽可能地倒干上清，收集菌体；
（2）用25mL溶液P1重悬菌体沉淀，涡旋振荡至彻底悬浮；
（3）加25mL的溶液P2，温和地上下翻转4-7次使菌体充分裂解，室温放置4min；
（4）加25mL溶液P3，立即温和地上下翻转4 -7次，充分混匀，此时会出现白色絮状沉淀；冰上静置3-5min，13000r/min离心10min，小心取上清至一个50mL的离心管，冰上预冷；
（5）加入0.1体积冰预冷的内毒素清除剂到上一步所得上清，颠倒旋转混匀7-10次，冰浴10min，中间偶尔颠倒混匀几次；
（6）42℃水浴，颠倒混匀后，42℃温育20-30min；
（7）室温1200 r/min离心10 min分相；
（8）溶液分为上下两相；上层水相含DNA，下层油状相含内毒素；将含DNA的上层水相转移到新管，弃油状层；
（9）向上一步所得上层水相中加入0.5体积结合液PB后，充分混匀，转入吸附柱中（吸附柱放入收集管中），13000r/min离心30-60s，倒掉收集管中的废液；
（10）加入漂洗液，13000r/min离心30-60s，弃掉废液；
（11）将吸附柱放回空收集管中，13000 r/min离心2min，除去漂洗液；
（12）取出吸附柱，放入离心管中，在吸附膜的中间部位加500吸附洗脱缓冲液，室温放置2min，13000r/min离心1min。
转染
（1）培养293F细胞使其处于对数生长期，活力大于95%，细胞处于1.8-2.2数生长期，活力大于附膜；
（2）需提前37℃预热，DNA提前室温预热10min，转染缓冲液37℃预热；
（3）转染体系；
表1 转染条件
| 转染体积 | 摇瓶规格 | 细胞体积 | 转染缓冲液 | DNA | 293(max) |
| 每30mL | 125mL | 27mL | 3mL | 30LL | 150LL |
（4）将所需质粒及MAX添加入转染缓冲液里面，将两者混合一起充分摇匀，37℃孵育30 min；
（5）将孵育好的质粒添加到细胞中，37℃悬浮培养；
（6）转染24h以后补充N源和生长因子；
（7）第4d计数细胞，观察细胞状态及死亡率，收获细胞；
（8）5000 r/min，10 min，收集细胞上清抗体纯化。
抗体纯化
（1）准备Protein A纯化柱，用5-10倍柱体积的PBS平衡柱子；
（2）将PBS平衡过的Protein A 柱加入到收集的上清中，在旋转孵育器上4℃孵育3-4h；
（3）用PBS缓冲液以0.5 mL/min的流速洗杂，至流出液OD280值到达基线；
（4）洗杂至核酸蛋白检测仪数值稳定不变时准备洗脱；
（5）用Elution-Buffer（0.1M NaCl，0.1M甘氨酸，pH3.0）以1mL/min流速洗脱目的抗体，收集流出液，并用1M Tris-HCl pH9.0使其pH值恢复至中性，将收集的抗体进行SDS-PAGE纯度分析后，将含目的蛋白的组分加入透析袋中，用PBS（pH7.4）透析过夜；
（6）透析后的样品BCA浓度检测，如图1所示。
实施例2：抗体的3-羟基丁酸修饰后分子量检测
将实施例1所得抗体浓度调整成1mg/mL，加入1M的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和1M 3-羟基丁酸至终浓度10mM，室温反应2h，之后用10kd的超滤柱离心4次，洗去未反应的化合物，进行质谱分析。
用0.1%甲酸溶液将经过脱盐的抗体配成0.2 mg/mL的溶液用于流动注射分析。用于LC-MS分析的抗体蛋白样品直接用超纯水配制每次进样2μL蛋白样品直接用超纯水配微量注射器吸取蛋白质样品，使用nanoACQUITY UPLC系统以1 µL/min的流速通过30分钟的梯度洗脱分离分析物。由流动注射或LC-MS分析得到的多电荷谱图，再经ProMass软件去卷积后得到样品相对分子质量信息，结果如图2所示。
实施例3：抗体的3-羟基丁酸修饰后修饰位点检测
将实施例2所得抗体进行LC-MS/MS检测。通过 SDS-PAGE分离蛋白质，从凝胶上切下目标凝胶带，用5mM二硫苏糖醇还原，并用11mM碘乙酰胺烷基化。然后，在凝胶中用测序级修饰的胰蛋白酶在50mM碳酸氢铵中于37°C过夜进行消化。将肽用在50％乙腈水溶液中的0.1％三氟乙酸萃取两次，每次30分钟。将胰蛋白酶解的肽重新溶解在20μL 0.1％TFA中，并通过LC-MS/MS分析。对于LC-MS/MS分析，使用Thermo-Dionex Ultimate 3000 HPLC系统以0.30μL/min的流速通过85分钟的梯度洗脱分离多肽，该系统直接与Thermo Scientific QExactive质谱仪连接。分析柱是填充有C-18树脂的自制熔融石英毛细管色谱柱（内径75µm，长度150mm， Upchurch）。流动相由0.1％甲酸组成，流动相B由80％乙腈和0.1％甲酸组成。QExactive质谱仪使用Xcalibur 2.1.2软件在与数据相关的采集模式下运行，并且在轨道阱中只有一个全扫描质谱图（300-1800 m/z，70,000分辨率），然后有20个与数据相关的MS/MS以27％的归一化碰撞能量（HCD）进行扫描。
使用内部蛋白质组发现程序（PD1.4版，美国赛默飞世尔科技公司），快速搜索每个LC-MS / MS运行中的MS/MS质谱图，以查找目的蛋白名称。搜索标准如下：需要完全的胰蛋白酶特异性；允许进行两次错位切割；碳酰氨基甲基（C）设置为固定修饰；氧化（M）被设定为可变修饰。对于在orbitrap质量分析仪中采集的所有MS，前驱体离子质量公差设置为20ppm；对于获得的所有MS2质谱图，碎片离子质量容差均设置为0.02Da。使用PD提供的Percolator计算肽错误发现率（FDR）。当q值小于1％时，认为肽谱匹配（PSM）是正确的。针对反向诱饵数据库搜索时，基于PSM确定FDR。仅分配给给定蛋白质组的肽被认为是独特的。对于蛋白质鉴定，错误发现率（FDR）也设置为0.01。修饰位点检测结果如图3所示。
图3测定出的氨基酸序列是HK（3HB）VYACEVTHQGLSSPVTK，即其中第二位的赖氨酸发生了三羟基丁酰化修饰。
实施例4：3-羟基丁酸修饰的抗体的蛋白酶敏感性检测
（1）利用修饰和未修饰的抗体（分别为实施例2所得修饰抗体和实施例1所得抗体），稀释到一定的浓度，取40μL蛋白，加入10μL的酶，37℃反应相应的时间，之后加入蛋白上样缓冲液，95℃加热5min，之后进行电泳检测；
（2）电泳结束后进行转膜，300mA转膜90min；
（3）转膜后进行脱脂奶封闭1小时；
（4）加入稀释好的一抗，室温孵育1h；
（4）TBST洗涤三次，每次5min，之后加入二抗孵育1h；
（4）TBST洗涤三次后进行发光检测，结果如图4所示。
由图4可知，抗体经3-羟基丁酸修饰后对蛋白酶（例如胰蛋白酶、纤维蛋白酶）水解的抗性大大提高。
实施例5：3-羟基丁酸修饰的抗体的热稳定性检测
对修饰抗体和未修饰的抗体（分别为实施例2所得修饰抗体和实施例1所得抗体）进行热稳定性评价，把抗体分装入PCR管，与PCR仪器上设置不同的温度进行加热反应，加热完成后进行拍照。结果如图5所示。
由图5可知，抗体经3-羟基丁酸修饰后热稳定性提高，在70和75℃下的热稳定性明显优于未修饰的抗体。
实施例6：3-羟基丁酸修饰的抗体的等电点检测
蛋白质是同时含有带正电荷和负电荷官能团的两性分子，其总电荷通常是由其周围环境的pH值所决定。当外部pH使蛋白质分子的酸性解离和碱性解离相等，即分子所带的正电荷和负电荷相互抵消，此时的pH值被称为该蛋白质的等电点（isoelectric point，pI）。当pH=pI时，蛋白质表面总电荷为零，蛋白质溶解度最小。特定蛋白的等电点是固定的，与该蛋白的组分与构象相关，因此精确测定等电点常用作蛋白鉴定的有效手段。此外，蛋白等电点也是许多常见生化分析、蛋白组学实验中的一项重要指标，例如双向凝胶电泳（2D-PAGE）、X射线结晶、液质联用等。全柱成像毛细管等电聚焦电泳（icIEF）是一种特殊的微分离技术，是表征蛋白质电荷不均一性的强有力工具。其原理是在毛细管两端加上直流电压，管内的载体两性电解质可以形成一定范围的pH梯度，蛋白质依据其所带电荷向阳极或阴极移动，直到净电荷为0的某个pH值（即等电点pI）时停止，最终蛋白质聚焦成很窄的区段，从而达到分离的目的。
将抗体（实施例2所得修饰抗体和实施例1所得抗体）与0.35%甲基纤维素、4.22和9.46的pI marker以及水，混合均匀后上样分析。预聚焦电压1.5kV，持续1min；聚焦电压3.0kV，持续7min。检测结果如图6所示。
结果表明，3-羟基丁酸修饰的抗体的等电点显著性被降低。
实施例7：3-羟基丁酸修饰抗体的小鼠体内半衰期评价
取正常的6-8周的C57BL/6小鼠随机分为两组，每只鼠尾静脉注射50μg的实施例2所得修饰抗体和实施例1所得抗体。后于在第 2、4、6、8、10、12和18天收集血样，在前18天是通过尾静脉采血的方式采取5μL的全血，用500μL生理盐水稀释，3000rpm的转速下离心15min收集上清，冻存于-80℃。在第18天时通过心脏穿刺从麻醉的小鼠中收集血清。
利用ELISA对血清中的抗S蛋白的抗体进行检测，具体步骤如下：利用碳酸盐缓冲液（0.05mol/L、pH9.6）对重组的新冠病毒S蛋白按照10倍新冠病毒浓度进行稀释，4℃包被过夜。第二日，用PBS-T洗涤3次，之后用1%的BSA进行封闭，37℃ 1h，弃掉上清，PBS-T洗涤3次。每孔加入100μL稀释好的血清，37℃孵育2h，弃掉上清，用PBS-T洗涤4次。随后加入辣根过氧化物酶标记多克隆山羊抗人IgG，37℃孵育1h，用PBS-T洗涤4次，加入显色底物，待颜色较清楚时加入2N的硫酸终止反应，于OD450测定吸光值。结果如图7所示。
通过对数据进行分析表明，3-羟基丁酸修饰的抗体在动物体内相较于未修饰组有较长的半衰期。
实施例8：抗体的3-羟基己酸修饰
将实施例1所得抗体浓度调整成1mg/mL，加入1M的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和1M 3-羟基己酸至终浓度10mM，室温反应2h，之后用10kd的超滤柱离心4次，洗去未反应的化合物，进行质谱分析。
用0.1%甲酸溶液将经过脱盐的抗体配成0.2mg/mL的溶液用于流动注射分析。用于LC-MS分析的抗体蛋白样品直接用超纯水配制每次进样2μL蛋白样品直接用超纯水配微量注射器吸取蛋白质样品，使用nanoACQUITY UPLC系统以1 µl / min的流速通过30分钟的梯度洗脱分离分析物。由流动注射或LC-MS分析得到的多电荷谱图，再经ProMass软件去卷积后得到样品相对分子质量信息。
实施例9：3-羟基己酸修饰的抗体的热稳定性检测
对修饰抗体和未修饰的抗体（分别为实施例8所得修饰抗体和实施例1所得抗体）进行热稳定性评价，把抗体分装入PCR管，与PCR仪器上设置不同的温度进行加热反应，加热完成后进行拍照。
实验结果表明，抗体的3-羟基己酸修饰可以提高热稳定性。
实施例10：抗体的3-羟基戊酸修饰
将实施例1所得抗体浓度调整成1mg/mL，加入1M的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和1M3-羟基戊酸至终浓度10mM，室温反应2h，之后用10kd的超滤柱离心4次，洗去未反应的化合物，进行质谱分析。
用0.1%甲酸溶液将经过脱盐的抗体配成0.2mg/mL的溶液用于流动注射分析。用于LC-MS分析的抗体蛋白样品直接用超纯水配制每次进样2μL蛋白样品直接用超纯水配微量注射器吸取蛋白质样品，使用nanoACQUITY UPLC系统以1 µl / min的流速通过30分钟的梯度洗脱分离分析物。由流动注射或LC-MS分析得到的多电荷谱图，再经ProMass软件去卷积后得到样品相对分子质量信息。
实施例11：3-羟基戊酸修饰的抗体的热稳定性检测
对修饰抗体和未修饰的抗体（分别为实施例10所得修饰抗体和实施例1所得抗体）进行热稳定性评价，把抗体分装入PCR管，与PCR仪器上设置不同的温度进行加热反应，加热完成后进行拍照。
实验结果表明，抗体的3-羟基戊酸修饰可以提高热稳定性。
实施例12：3-羟基丁酸寡聚物修饰
抗体浓度调整成1mg/mL，加入1M的1-乙基-(3-二甲基氨基丙基)碳二亚胺盐酸盐和1M 3-羟基丁酸寡聚物（其为混合物：6.4% v/v 二聚物，30.7% v/v 三聚物，39.9% v/v四聚物，16.6% v/v五聚物，6.2% v/v六聚物）至终浓度0.1mg/mL，室温反应2h，之后用10kd的超滤柱离心4次，洗去未反应的化合物。
实施例13：3-羟基丁酸寡聚物修饰的抗体的热稳定性检测
将修饰抗体和未修饰的抗体（分别为实施例12所得修饰抗体和实施例1所得抗体）进行热稳定性评价，把抗体分装入PCR管，与PCR仪器上设置不同的温度进行加热反应，加热完成后进行拍照。
实验结果表明，抗体的3-羟基丁酸寡聚物修饰可以提高热稳定性。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
本发明中描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。
Claims (8)
1.一种化合物修饰的蛋白质药物，其中，所述化合物具有如下结构：
；所述蛋白质药物为抗SARS-CoV-2 S蛋白B38，其中，所述抗SARS-CoV-2 S蛋白B38的氨基酸序列HKVYACEVTHQGLSSPVTK中第二位氨基酸发生3-羟基丁酰化修饰。
2.一种权利要求1所述的修饰的蛋白质药物的制备方法，其包括所述蛋白质药物与所述化合物进行反应的步骤。
3.如权利要求2所述的制备方法，其特征在于，所述反应在缩合剂的存在下进行。
4.如权利要求3所述的制备方法，其特征在于，所述缩合剂选自：1-乙基-(3-二甲基氨基丙基)碳二亚胺、二环己基碳二亚胺、二异丙基碳二亚胺，及其盐。
5.如权利要求2-4任一项所述的制备方法，其特征在于，所述蛋白质药物与所述化合物的比例为10-1000g：1mol。
6.一种药物组合物，其包含权利要求1所述的修饰的蛋白质药物，以及一种或多种药学上可接受的辅料。
7.如权利要求1所述的修饰的蛋白质药物、权利要求6所述的药物组合物在制备治疗或诊断疾病的药物中的应用，所述疾病为SARS-CoV-2感染引起的疾病。
8.一种化合物在提高蛋白质药物的稳定性、延长蛋白质药物的半衰期、提高蛋白质药物的酰化修饰水平中的应用，其中，所述化合物具有如下结构：
；所述蛋白质药物为抗SARS-CoV-2 S蛋白B38，其中，所述抗SARS-CoV-2 S蛋白B38的氨基酸序列HKVYACEVTHQGLSSPVTK中第二位氨基酸发生3-羟基丁酰化修饰。
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