慢病毒载体冻干制剂的制备与稳定性研究

doi:10.3969/j.issn.1000-5641.2021.03.012

摘要/Abstract

摘要：

制备了一种新型慢病毒载体冻干制剂. 通过冻干保护剂处方的筛选与优化, 从外观、赋形性、色泽度、溶解性方面评价冻干制剂的理化性质, 确定最优处方为海藻糖0.30 g/mL、L-组氨酸0.31 mg/mL、L-丙氨酸0.178 mg/mL、CaCl₂ 0.020 mg/mL、MgSO₄ 0.015 mg/mL. 所制备的慢病毒载体冻干制剂外观良好, 残余水分含量低, 结构保持完整, 再分散性较好; 慢病毒载体生物滴度可高达9.37 × 10⁷ IU/mL, 滴度回收率为50.15%. 影响因素稳定性实验、高温加速实验和反复冻融稳定性实验等研究显示, 真空冷冻干燥技术可用于慢病毒载体固体制剂的制备, 并且能够有效改善慢病毒载体不同温度条件下的储存效果、反复冻融稳定性以及对高温等不良环境的耐受能力.

关键词: 慢病毒载体, 冻干制剂, 稳定性

Abstract:

In this paper, we studied a new preparation technique for lyophilized lentiviral vectors. We determined the optimal formulation for a freeze-drying protective agent by screening and optimizing potential candidates. The candidates were evaluated on the basis of physical and chemical properties of the freeze-drying process, including appearance, excipient, color, and solubility. The optimal formulation was determined to be trehalose 0.30 g/mL, L-histidine 0.31 mg/mL, L- alanine 0.178 mg/mL, CaCl₂ 0.020 mg/mL, and MgSO₄ 0.015 mg/mL. With this technique, the prepared lyophilized lentiviral vector had good appearance, low residual water content, intact structure, and good re-dispersibility. The biological titer of the lentiviral vector reached 9.37 × 10⁷ IU/mL, and the recovery rate of the titer was 50.15%. We also conducted research on potential influencing factors, including a high temperature accelerated experiment and repeated freeze-thaw stability experiments. These experiments showed that the lyophilizing technology can be used for the preparation of lentiviral vector solids and can be effectively used to improve the storage of lentiviral vectors under different temperature conditions, exposure to repeated freeze-thaw cycles, and tolerance to adverse environments (e.g., high temperatures).

Key words: lentiviral vector, lyophilized preparation, stability

中图分类号:

R944.2

沈鸿伟, 李明昊, 徐南, 邵佳琪, 王镜, 俞磊. 慢病毒载体冻干制剂的制备与稳定性研究[J]. 华东师范大学学报（自然科学版）, 2021, 2021(3): 114-127.

Hongwei SHEN, Minghao LI, Nan XU, Jiaqi SHAO, Jing WANG, Lei YU. Preparation and stability study of lyophilized lentiviral vector[J]. Journal of East China Normal University(Natural Science), 2021, 2021(3): 114-127.

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分组	冻干保护剂处方
Ⅰ	HBSS
Ⅱ	15% 海藻糖、0.010 mol/L L-组氨酸、0.010 mol/L L-丙氨酸、0.10 g/L CaCl₂、0.076 g/L MgSO₄
Ⅲ	3.0% 海藻糖、3.0% 甘露醇、2.0% 右旋糖酐、1.5% 肌醇
Ⅳ	0.30% 人血白蛋白、2.0% 海藻糖、1.0% 甘露醇、2.0% 右旋糖酐、2.0% 蔗糖
Ⅴ	1.0% 甘油、20 mg/mL 蔗糖、25 mg/mL 甘露醇、1.0 mg/mL L-精氨酸、 2.0 mg/mL 甘氨酸、2.0% PEG6000、1.5% 明胶

水平	因素
水平	海藻糖/g	L-丙氨酸/mg	L-组氨酸/mg	CaCl₂/mg	MgSO₄/mg
1	3.750	8.900	15.50	1.000	0.7600
2	15.00	222.5	387.5	25.00	19.00

实验号	海藻糖/g	L-丙氨酸/mg	L-组氨酸/mg	CaCl₂/mg	MgSO₄/mg
1	3.750	8.900	15.50	25.00	0.7600
2	15.00	222.5	15.50	25.00	0.7600
3	15.00	8.900	387.5	25.00	0.7600
4	9.375	115.7	201.5	13.00	9.880
5	15.00	222.5	15.50	1.000	19.00
6	3.750	222.5	15.50	25.00	19.00
7	15.00	8.900	387.5	1.000	19.00
8	9.375	115.7	201.5	13.00	9.880
9	3.750	8.900	387.5	25.00	19.00
10	3.750	8.900	15.50	1.000	19.00
11	3.750	222.5	387.5	1.000	19.00
12	15.00	222.5	387.5	1.000	0.7600
13	15.00	8.900	15.50	25.00	19.00
14	15.00	8.900	15.50	1.000	0.7600
15	15.00	222.5	387.5	25.00	19.00
16	9.375	115.7	201.5	13.00	9.880
17	3.750	222.5	15.50	1.000	0.7600
18	3.750	8.900	387.5	1.000	0.7600
19	3.750	222.5	387.5	25.00	0.7600

冻干保护剂处方	外观	色泽	复溶性
预处方	疏松, 粉末状	白色	良好, 30 s以内

冻干保护剂	外观	色泽	复溶性
Ⅰ组	疏松, 呈蜂窝状	橙黄	良好, 30 s以内
Ⅱ组	疏松, 呈蜂窝状	橙红	良好, 30 s以内
Ⅲ组	较差, 起泡皱缩	橙黄	一般, 60 s以内
Ⅳ组	疏松, 呈蜂窝状	橙黄	良好, 30 s以内
Ⅴ组	疏松, 呈蜂窝状	橙红	良好, 30 s以内