环孢菌素A柱层析硅胶再生工艺

doi:10.16085/j.issn.1000-6613.2022-1317

摘要/Abstract

摘要：

环孢菌素A工业化生产涉及真菌发酵、萃取、浓缩、结晶、硅胶柱层析、脱色、二次结晶等步骤，目前硅胶柱层析步骤的硅胶只能一次使用，现实生产过程产生了大量的废硅胶，环保处理压力大、企业成本高。据此本文开展了环孢菌素A柱层析硅胶再生工艺研究，针对硅胶上吸附的杂质性质，研究了硅胶再生洗脱溶剂系统，通过响应面法优化再生条件，结果表明：选用含有0.26%聚山梨醇的乙醇反向冲洗4个柱体积，再使用双氧水浓度为0.15%的去离子水正向冲洗2.1个柱体积，冲洗流速为5mL/min；使用压缩空气将再生后的硅胶压出，烘干至水分含量为6%。按照本方法得到的再生硅胶按原工艺使用时，环孢菌素A回收率完全达标，并且可以三次重复使用达标。本研究建立了一套切实可行的环孢素纯化硅胶再生工艺，可有效节约工厂生产成本和减轻环境压力。

关键词: 环孢菌素A, 柱层析, 硅胶再生, 纯化

Abstract:

The industrial production of cyclosporine A involves fungal fermentation, extraction, concentration, crystallization, silica gel column chromatography, decolorization, secondary crystallization and other steps, among which silica gel column chromatography produces a large amount of waste silica gel, which is under great pressure of environmental protection treatment and high cost for enterprises. Based on this, the silica gel regeneration process of cyclosporine A column chromatography was studied in this paper, and the regeneration conditions were optimized by response surface methodology: four column volumes were backwashed with ethanol containing 0.26% polysorbitol, and 2.1 column volumes were forward washed with deionized water containing 0.15% hydrogen peroxide at a flow rate of 5mL/min. The recycled silica gel was pressed out with compressed air and dried until the moisture content was 6%. The yield of the regenerated silica gel can reach more than 75% when used according to the original process, and the yield of the third regenerated silica gel can still reach 75%. This study established a set of feasible cyclosporine purified silica gel regeneration process, which can effectively save factory production cost and reduce environmental pressure.

Key words: cyclosporine A, column chromatography, silica gel regeneration, purification

中图分类号:

Q819

黄晔, 言行, 吴巧薇, 柴小涛, 潘恭赢, 张金峰, 李相前. 环孢菌素A柱层析硅胶再生工艺[J]. 化工进展, 2022, 41(S1): 461-468.

HUANG Ye, YAN Xing, WU Qiaowei, CHAI Xiaotao, PAN Gongying, ZHANG Jinfeng, LI Xiangqian. Study of silica gel regeneration applied on cyclosporine A column chromatography[J]. Chemical Industry and Engineering Progress, 2022, 41(S1): 461-468.

图/表 9

参考文献 18

1	ARSLANSOYU CAMLAR S, SOYLU A, KAVUKÇU S. Cyclosporine in pediatric nephrology[J]. Iranian Journal of Kidney Diseases, 2018, 12(6): 319-330.
2	WU Qinghua, WANG Xu, NEPOVIMOVA Eugenie, et al. Mechanism of cyclosporine A nephrotoxicity: oxidative stress, autophagy, and signalings[J]. Food and Chemical Toxicology, 2018, 118: 889-907.
3	WU Qinghua, KUCA Kamil. Metabolic pathway of cyclosporine A and its correlation with nephrotoxicity[J]. Current Drug Metabolism, 2019, 20(2): 84-90.
4	PATOCKA Jiri, NEPOVIMOVA Eugenie, KUCA Kamil, et al. Cyclosporine A: chemistry and toxicity — A review[J]. Current Medicinal Chemistry, 2021, 28(20): 3925-3934.
5	BAGRI Narendra Kumar. Cyclosporine for systemic onset juvenile idiopathic arthritis: current stand and future directions[J]. Indian Journal of Pediatrics, 2019, 86(7): 576-577.
6	DE PAIVA Cintia S, PFLUGFELDER Stephen C, Sueko M NG, et al. Topical cyclosporine A therapy for dry eye syndrome[J]. The Cochrane Database of Systematic Reviews, 2019, 9(9): CD010051.
7	LIDDICOAT Alex M, LAVELLE Ed C. Modulation of innate immunity by cyclosporine A[J]. Biochemical Pharmacology, 2019, 163: 472-480.
8	PATEL Dhrumi, WAIRKAR Sarika. Recent advances in cyclosporine drug delivery: challenges and opportunities[J]. Drug Delivery and Translational Research, 2019, 9(6): 1067-1081.
9	王博雅. 环孢素通过上调NR4A1表达促进人绒毛膜滋养细胞增殖与侵袭能力的研究[D]. 合肥: 安徽医科大学, 2022.
	WANG Boya. Cyclosporine A promotes human trophoblast cells proliferation and invasion by up-regulates NR4A1 expression[D]. Hefei: Anhui Medical University, 2022.
10	夏训明, 编译. 美国FDA批准首款环孢菌素 0.05%眼用乳剂仿制药用于治疗干眼病[J]. 广东药科大学学报, 2022, 38(2): 145.
	XIA Xunming, trans. FDA approved the first cyclosporin 0.05% ophthalmic emulsion as a generic drug for the treatment of dry eye disease [J]. Journal of Guangdong Pharmaceutical University, 2022, 38(2): 145.
11	王翔桂, 莫小兰, 江华, 等. 对儿童异基因造血干细胞移植患者环孢素浓度的影响因素分析[J]. 中国临床药理学杂志, 2022, 38(13): 1435-1439.
	WANG Xianggui, MO Xiaolan, JIANG Hua, et al. Analysis of factors influencing cyclosporine concentration in pediatric allogeneic hematopoietic stem cell transplant recipients[J]. The Chinese Journal of Clinical Pharmacology, 2022, 38(13): 1435-1439.
12	柳志文, 凌天牖, 贺建泽, 等. 复方环孢菌素A含漱液治疗口腔扁平苔藓的研究[J]. 湖南医科大学学报, 2000, 25(2): 183-184.
	LIU Zhiwen, LING Tianyou, HE Jianze, et al. Research of compound cyclosporin A mouthwash in the treatment of oral lichen planus[J]. Bulletin of Hunan Medical University, 2000, 25(2): 183-184.
13	方东升, 谢阳, 陈勇, 等. 高速逆流色谱法分离纯化环孢菌素[J]. 中国抗生素杂志, 2005, 30(1): 48-51.
	FANG Dongsheng, XIE Yang, CHEN Yong, et al. Separation of cyclosporins by high speed counter current chromatography[J]. Chinese Journal of Antibiotics, 2005, 30(1): 48-51.
14	陈秀明, 方剑英, 陈振伟, 等. 大孔吸附树脂柱层析结合高速逆流色谱分离纯化环孢菌素小组分[J]. 中国抗生素杂志, 2008, 33(5): 276-279, 292.
	CHEN Xiuming, FANG Jianying, CHEN Zhenwei, et al. Separation and purification of cyclosporin minor components by macroporous adsorption resin and high speed countercurrent chromatography[J]. Chinese Journal of Antibiotics, 2008, 33(5): 276-279, 292.
15	刘雨, 王会会, 赵建辉, 等. 雪白白僵菌高产环孢菌素A发酵条件优化[J]. 发酵科技通讯, 2021, 50(4): 198-201.
	LIU Yu, WANG Huihui, ZHAO Jianhui, et al. Optimization of fermentation conditions for high production of cyclosporin A from Beauvcria nivea[J]. Bulletin of Fermentation Science and Technology, 2021, 50(4): 198-201.
16	吴中豪, 龚子珊, 姜小梅, 等. 液质联用法同时测定干血斑中环孢菌素A、西罗莫司、他克莫司和依维莫司药物浓度[J]. 分析仪器, 2021(1): 11-18.
	WU Zhonghao, GONG Zishan, JIANG Xiaomei, et al. LC-MS/MS method for simultaneous determination of cyclosporin A, sirolimus, tacrolimus and everolimus in dried blood spots[J]. Analytical Instrumentation, 2021(1): 11-18.
17	王欣荣, 张雪霞, 褚以文, 等. 环孢菌素A菌渣无害化减量化技术研究[J]. 中国抗生素杂志, 2019, 44(2): 214-217.
	WANG Xinrong, ZHANG Xuexia, CHU Yiwen, et al. The reduction and harmlessness of cyclosporin A residue by waste minimization[J]. Chinese Journal of Antibiotics, 2019, 44(2): 214-217.
18	张常雄, 成颖, 周四元. 环孢菌素A制剂的研究进展[J]. 西北药学杂志, 2018, 33(1): 136-139.
	ZHANG Changxiong, CHENG Ying, ZHOU Siyuan. Advances in cyclosporine A preparation[J]. Northwest Pharmaceutical Journal, 2018, 33(1): 136-139.

因素	水平
因素	-1	0	+1
A：乙醇用量/BV	2	3	4
B：聚山梨醇浓度/%	0.1	0.2	0.3
C：双氧水浓度/%	0.1	0.2	0.3
D：去离子水用量/BV	2	3	4

因素	水平
因素	-1	0	+1
A：乙醇用量/BV	2	3	4
B：聚山梨醇浓度/%	0.1	0.2	0.3
C：双氧水浓度/%	0.1	0.2	0.3
D：去离子水用量/BV	2	3	4

来源	平方和	自由度	均方	F值	p值	显著性
合计	1553.21	28	—	—	—
模型	1521.66	14	108.69	48.22	< 0.0001	**
A：乙醇的使用量	286.65	1	286.65	127.18	< 0.0001	**
B：聚山梨醇浓度	93.30	1	93.30	41.39	< 0.0001	**
C：双氧水浓度	113.47	1	113.47	50.34	< 0.0001	**
D：去离子水使用量	18.03	1	18.03	8.00	0.0134	*
AB	37.76	1	37.76	16.75	0.0011	**
AC	0.65	1	0.6480	0.28	0.6002
AD	25.65	1	25.65	11.38	0.0045	**
BC	9.95	1	9.95	4.42	0.0542
BD	221.12	1	221.12	98.11	0.0020	**
CD	164.10	1	164.10	72.81	< 0.0001	**
A²	85.85	1	85.85	38.09	< 0.0001	**
B²	135.43	1	135.43	60.09	< 0.0001	**
C²	393.05	1	393.05	174.39	< 0.0001	**
D²	192.00	1	192.00	85.19	< 0.0001	**
残差	31.55	14	2.25	—	—
失拟项	24.60	10	2.46	1.41	0.3948
纯误差	6.96	4	1.74	—	—

来源	平方和	自由度	均方	F值	p值	显著性
合计	1553.21	28	—	—	—
模型	1521.66	14	108.69	48.22	< 0.0001	**
A：乙醇的使用量	286.65	1	286.65	127.18	< 0.0001	**
B：聚山梨醇浓度	93.30	1	93.30	41.39	< 0.0001	**
C：双氧水浓度	113.47	1	113.47	50.34	< 0.0001	**
D：去离子水使用量	18.03	1	18.03	8.00	0.0134	*
AB	37.76	1	37.76	16.75	0.0011	**
AC	0.65	1	0.6480	0.28	0.6002
AD	25.65	1	25.65	11.38	0.0045	**
BC	9.95	1	9.95	4.42	0.0542
BD	221.12	1	221.12	98.11	0.0020	**
CD	164.10	1	164.10	72.81	< 0.0001	**
A²	85.85	1	85.85	38.09	< 0.0001	**
B²	135.43	1	135.43	60.09	< 0.0001	**
C²	393.05	1	393.05	174.39	< 0.0001	**
D²	192.00	1	192.00	85.19	< 0.0001	**
残差	31.55	14	2.25	—	—
失拟项	24.60	10	2.46	1.41	0.3948
纯误差	6.96	4	1.74	—	—

[1]	孙鲁芹, 卢会霞, 王建友. 电渗析/超滤内在耦合过程分离蛋清中溶菌酶[J]. 化工进展, 2023, 42(5): 2262-2271.
[2]	王哲, 余颖, 石永杰, 杨顺, 陈久洲, 崔希利, 邢华斌. 尿素包合法分离费托轻质油中的正构烃[J]. 化工进展, 2023, 42(2): 677-683.
[3]	毛停停, 李双福, 黄李茗铭, 周川玲, 韩凯. 面向水处理与有机溶剂回收的太阳能界面蒸发系统与材料[J]. 化工进展, 2023, 42(1): 178-193.
[4]	齐亚兵, 贾宏磊. 熔融结晶技术分离纯化有机化合物的研究进展[J]. 化工进展, 2023, 42(1): 373-385.
[5]	马镓莉, 卢会霞, 苗晓雪. 基于膜技术分离纯化乳清蛋白的研究进展[J]. 化工进展, 2022, 41(6): 2826-2838.
[6]	史程风, 贾冉冉, 阎振丽, 惠丰立. 一株产低温脂肪酶酵母菌的鉴定及酶学性质[J]. 化工进展, 2022, 41(10): 5541-5548.
[7]	孔庆强, 黄显虹, 王振兵, 郭晓倩, 谢莉婧, 苏方远, 孙国华, 陈成猛. 超级电容器用活性炭国产化关键化学与化工问题[J]. 化工进展, 2021, 40(9): 5088-5096.
[8]	张春伟, 张学军, 赵阳. 应用于空分纯化系统的相变储热器建模及分析[J]. 化工进展, 2021, 40(6): 3099-3106.
[9]	李阳, 朱晨辉, 范代娣. 重组胶原蛋白的绿色生物制造及其应用[J]. 化工进展, 2021, 40(3): 1262-1275.
[10]	仲文雅, 俞汶佳, 谷艺明, 郭静, 樊博, 蔡志强. 生物合成安丝菌素的研究进展[J]. 化工进展, 2021, 40(2): 990-997.
[11]	陈亮, 田飞豹, 辛秀兰, 兰蓉, 于然, 吴志明, 梁浩. 核黄素超声结晶工艺优化[J]. 化工进展, 2021, 40(2): 1018-1024.
[12]	邹俊康, 鲍宗必, 杨启炜, 张治国, 任其龙, 杨亦文. 甘油磷酰胆碱的分析、制备及纯化研究进展[J]. 化工进展, 2021, 40(11): 6295-6304.
[13]	梁高峰, 陈学青, 王德武, 张少峰, 刘燕, 张继军. 淘洗六水氯化铝过程中晶体沉降速度与纯化规律分析[J]. 化工进展, 2021, 40(1): 386-393.
[14]	饶轶晟, 杨晓健, 张红, 胡国涛. 湿法磷酸净化脱砷的研究现状[J]. 化工进展, 2020, 39(S1): 219-224.
[15]	吴兴可,祁荃,卞婷婷,刘苏煜,谷艺明,郭静,蔡志强. 珍贵橙色束丝放线菌发酵产安丝菌素P-3的分离纯化工艺优化[J]. 化工进展, 2020, 39(3): 1122-1128.