Preparation and stability study of lyophilized lentiviral vector
-
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.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
-
图 1 慢病毒载体冻干前后滴度值与回收率
注: Da表示对照组原液, 即加入HBSS溶解的病毒样品; Db表示实验组原液, 即加入冻干保护剂溶解的病毒样品; Dc表示对照组–80 ℃样品, 即加入HBSS –80 ℃保存的病毒样品; Dd表示对照组用于冻干样品, 即加入HBSS冻干之后的病毒样品; De表示实验组–80 ℃样品, 即加入冻干保护剂 –80 ℃保存的病毒样品; Df表示实验组冻干样品, 即加入冻干保护剂冻干之后的病毒样品; Dc/Da代表对照组–80 ℃样品滴度回收率; Dd/Da代表对照组冻干样品滴度回收率; De/Db代表实验组冻存样品滴度回收率; Df/Db代表实验组冻干样品滴度回收率.
Fig. 1 Titer value and recovery rate of lentiviral vector before and after freeze-drying
表 1 不同冻干保护剂的效果
Tab. 1 Effects of different lyophilized protectants
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% 明胶表 2 正交实验因素水平
Tab. 2 Factor level of orthogonal experiment
水平 因素 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 表 3 正交实验设计表
Tab. 3 Orthogonal experimental design table
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 表 4 慢病毒冻干制剂的理化性质评价
Tab. 4 Evaluation of physical and chemical properties of the freeze-drying preparation technique
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 表 5 不同冻干保护剂的效果
Tab. 5 Effects of different lyophilized protectants
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 表 6 正交实验结果表
Tab. 6 Results of orthogonal experiment
实验号 海藻糖/g L-丙氨酸/mg L-组氨酸/mg CaCl2/mg MgSO4/mg 生物滴度/(IU·mL–1) 1 3.750 8.900 15.50 25.00 0.7600 2.12 × 107 2 15.00 222.5 15.50 25.00 0.7600 2.32 × 107 3 15.00 8.900 387.5 25.00 0.7600 2.01 × 107 4 9.375 115.7 201.5 13.00 9.880 3.17 × 107 5 15.00 222.5 15.50 1.000 19.00 2.16 × 107 6 3.750 222.5 15.50 25.00 19.00 2.29 × 107 7 15.00 8.900 387.5 1.000 19.00 3.47 × 107 8 9.375 115.7 201.5 13.00 9.880 3.20 × 107 9 3.750 8.900 387.5 25.00 19.00 4.54 × 107 10 3.750 8.900 15.50 1.000 19.00 4.91 × 107 11 3.750 222.5 387.5 1.000 19.00 4.22 × 107 12 15.00 222.5 387.5 1.000 0.7600 3.89 × 107 13 15.00 8.900 15.50 25.00 19.00 2.60 × 107 14 15.00 8.900 15.50 1.000 0.7600 5.11 × 107 15 15.00 222.5 387.5 25.00 19.00 3.96 × 107 16 9.375 115.7 201.5 13.00 9.880 4.09 × 107 17 3.750 222.5 15.50 1.000 0.7600 2.15 × 107 18 3.750 8.900 387.5 1.000 0.7600 2.81 × 107 19 3.750 222.5 387.5 25.00 0.7600 1.46 × 107 表 7 正交实验方差分析表
Tab. 7 Analysis of variance of orthogonal experiment
来源 自由度 Adj SS Adj MS F 值 P 值 模型 12 2.035 45 × 1015 1.696 21 × 1014 189.23 0 线性 5 8.070 11 × 1014 1.614 02 × 1014 180.06 0 海藻糖 1 6.563 38 × 1012 6.563 38 × 1012 7.320 0 0.042 L-丙氨酸 1 1.645 09 × 1014 1.645 09 × 1014 183.53 0 L-组氨酸 1 4.521 50 × 1013 4.521 50 × 1013 50.440 0.001 CaCl2 1 3.439 20 × 1014 3.439 20 × 1014 383.67 0 MgSO4 1 2.468 04 × 1014 2.468 04 × 1014 275.33 0 2 因子交互作用 7 1.228 44 × 1015 1.754 92 × 1014 195.78 0 海藻糖 × L-丙氨酸 1 7.207 12 × 1013 7.207 12 × 1013 80.400 0 海藻糖 × MgSO4 1 4.591 37 × 1014 4.591 37 × 1014 512.21 0 L-丙氨酸 × L-组氨酸 1 2.660 03 × 1014 2.660 03 × 1014 296.75 0 L-丙氨酸 × CaCl2 1 4.330 05 × 1013 4.330 05 × 1013 48.310 0.001 L-组氨酸 × CaCl2 1 4.194 36 × 1013 4.194 36 × 1013 46.790 0.001 L-组氨酸 × MgSO4 1 2.089 88 × 1014 2.089 88 × 1014 233.15 0 CaCl2 × MgSO4 1 1.369 98 × 1014 1.369 98 × 1014 152.83 0 误差 5 4.481 91 × 1012 8.963 83 × 1011 失拟 4 4.438 46 × 1012 1.109 61 × 1012 25.530 0.147 纯误差 1 4.345 75 × 1010 4.345 75 × 1010 合计 17 2.039 93 × 1015 表 8 回归拟合模型汇总表
Tab. 8 Summary table of regression fitting model
S R-sq/% R-sq(调整)/% R-sq(预测)/% 946775 99.78 99.25 94.96 表 9 多响应预测结果
Tab. 9 Results of multiple response prediction
变量 海藻糖/g L-丙氨酸/mg L-组氨酸/mg CaCl2/mg MgSO4/mg 设置 15 8.9 15.5 1.0 0.76 响应 拟合值 拟合值标准误差 95% 置信区间 95% 预测区间 响应值 5.13287×107 8.49756×105 (4.91443×107, 5.35131×107) (4.80584×107, 5.45989×107) 表 10 高温加速实验(42 ℃)条件下慢病毒冻干制剂的保存效果
Tab. 10 Preservation effects of freeze-drying preparations at 42 ℃
时间/d 外观 色泽 复溶性 1 疏松, 呈蜂窝状 橙红 良好, 30 s以内 3 疏松, 呈蜂窝状 橙红 良好, 30 s以内 5 皱缩, 吸潮黏连 橙红 一般, 90 s以内 7 皱缩, 吸潮黏连 橙红 一般, 90 s以内 14 皱缩, 吸潮黏连 橙红 较差, 120 s以内 21 皱缩, 吸潮黏连 橙红 较差, 120 s以内 30 皱缩, 吸潮黏连 橙红 较差, 120 s以内 表 11 反复冻融对慢病毒冻干制剂保存效果的影响
Tab. 11 Effects of repeated freeze-thaw cycles on the preservation of freeze-drying preparations
反复冻融次数/次 外观 色泽 复溶性 1 疏松, 呈蜂窝状 橙红 良好, 30 s以内 2 疏松, 呈蜂窝状 橙红 良好, 30 s以内 3 疏松, 呈蜂窝状 橙红 良好, 30 s以内 4 疏松, 呈蜂窝状 橙红 良好, 30 s以内 5 疏松, 呈蜂窝状 橙红 良好, 30 s以内 6 疏松, 呈蜂窝状 橙红 良好, 30 s以内 -
[1] NALDINI L, TRONA D, VERMA I M. Lentiviral vectors, two decades later [J]. Science, 2016, 353(6304): 1101-1102. doi: 10.1126/science.aah6192 [2] MILONE M C, O DOHERTY U. Clinical use of lentiviral vectors [J]. Leukemia, 2018, 32(7): 1529-1541. doi: 10.1038/s41375-018-0106-0 [3] DUNBAR C E, HIGH K A, JOUNG J K, et al. Gene therapy comes of age [J]. Science, 2018, 359(6372): n4672. doi: 10.1126/science.aan4672 [4] CARMO M, ALVES A, RODRIGUES A F, et al. Stabilization of gammaretroviral and lentiviral vectors: from production to gene transfer [J]. The Journal of Gene Medicine, 2009, 11(8): 670-678. doi: 10.1002/jgm.1353 [5] YANNARELL D A, GOLDBERG K M, HJORTH R N. Stabilizing cold-adapted influenza virus vaccine under various storage conditions [J]. Journal of Virological Methods, 2002, 102(1): 15-25. [6] KUMRU O S, WANG Y, GOMBOTZ C W R, et al. Physical Characterization and Stabilization of a Lentiviral Vector Against Adsorption and Freeze-Thaw [J]. Journal of Pharmaceutical Sciences, 2018, 107(11): 2764-2774. doi: 10.1016/j.xphs.2018.07.010 [7] 李仲艺. 真空冷冻干燥技术在生物制药方面的应用分析 [J]. 中国新技术新产品, 2018(1): 76-77. doi: 10.3969/j.issn.1673-9957.2018.01.042 [8] 耿锟锟, 熊非, 朱家壁, 等. 用于药物制剂的冷冻干燥技术及相关影响因素 [J]. 药学进展, 2011, 35(3): 104-109. [9] 田烨, 吴明媛. 生物制品冻干保护方法研究进展 [J]. 中国医药生物技术, 2018, 13(1): 73-76. doi: 10.3969/j.issn.1673-713X.2018.01.015 [10] HANSEN L J J, DAOUSSI R, VERVAET C, et al. Freeze-drying of live virus vaccines: A review [J]. Vaccine, 2015, 33(42): 5507-5519. doi: 10.1016/j.vaccine.2015.08.085 [11] MORGAN C A, HERMAN N, WHITE P A, et al. Preservation of micro-organisms by drying: A review [J]. Journal of Microbiological Methods, 2006, 66(2): 183-193. doi: 10.1016/j.mimet.2006.02.017 [12] DE LAS MERCEDES SEGURA M, KAMEN A, GARNIER A. Downstream processing of oncoretroviral and lentiviral gene therapy vectors [J]. Biotechnology Advances, 2006, 24(3): 321-337. doi: 10.1016/j.biotechadv.2005.12.001 [13] KUTNER R H, ZHANG X, REISER J. Production, concentration and titration of pseudotyped HIV-1-based lentiviral vectors [J]. Nature Protocols, 2009, 4(4): 495-505. doi: 10.1038/nprot.2009.22 [14] TISCORNIA G, SINGER O, VERMA I M. Production and purification of lentiviral vectors [J]. Nature Protocols, 2006, 1(1): 241-245. doi: 10.1038/nprot.2006.37 [15] 上海比昂生物医药科技有限公司. 一种慢病毒冷冻干燥保护剂及慢病毒冻干粉: CN201810995003.1 [P]. 2018-10-09. [16] 张一折, 姜春来, 王忠诚, 等. 重组MVA病毒载体疫苗的冻干保护剂研究 [J]. 中国生物制品学杂志, 2006(2): 174-176. doi: 10.3969/j.issn.1004-5503.2006.02.021 [17] 张一折, 滕洪刚, 吕帅然, 等. 重组HIV-1腺病毒载体活疫苗的冻干保护剂研究 [J]. 中国生物制品学杂志, 2007(2): 104-106. doi: 10.3969/j.issn.1004-5503.2007.02.010 [18] ADEBAYO A A, SIM-BRANDENBURG J W, EMMEL H, et al. Stability of 17D yellow fever virus vaccine using different stabilizers [J]. Biologicals, 1998, 26(4): 309-316. doi: 10.1006/biol.1998.0157 [19] PRABHU M, BHANUPRAKASH V, VENKATESAN G, et al. Evaluation of stability of live attenuated camelpox vaccine stabilized with different stabilizers and reconstituted with various diluents [J]. Biologicals, 2014, 42(3): 169-175. doi: 10.1016/j.biologicals.2014.02.001 [20] SARKAR J, SREENIVASA B P, SINGH R P, et al. Comparative efficacy of various chemical stabilizers on the thermostability of a live-attenuated peste des petits ruminants (PPR) vaccine [J]. Vaccine, 2003, 21(32): 4728-4735. doi: 10.1016/S0264-410X(03)00512-7 [21] SIVA SANKAR M S, BHANUPRAKASH V, VENKATESAN G, et al. Comparative efficacy of chemical stabilizers on the thermostabilization of a novel live attenuated buffalopox vaccine [J]. Biologicals, 2017, 49: 39-45. doi: 10.1016/j.biologicals.2017.07.002 [22] 马海燕, 方彧聃, 张敬之. 应用荧光实时定量PCR方法检测重组慢病毒滴度及其感染效率 [J]. 生命科学研究, 2009, 13(5): 394-398. [23] 玛尔江·木坎, 卢琳. 卡尔费休氏试剂与水分测定 [J]. 畜禽业, 2016(5): 42-43. doi: 10.3969/j.issn.1008-0414.2016.05.032 [24] 傅晖, 顾菁. 腺病毒冻干条件的改进研究 [J]. 上海医药, 2017, 38(13): 67-69. doi: 10.3969/j.issn.1006-1533.2017.13.019 [25] HAN Y, JIN B S, Lee S B, et al. Effects of sugar additives on protein stability of recombinant human serum albumin during lyophilization and storage [J]. Arch Pharm Res, 2007, 30(9): 1124-1131. doi: 10.1007/BF02980247 [26] 张光磊, 张新创, 翟雷. 活菌制剂冻干保护剂的研究进展 [J]. 微生物学免疫学进展, 2015, 43(4): 80-85. [27] 薛菲, 王凤山. 蛋白质的冻干保护剂及其保护机制研究进展 [J]. 中国药学杂志, 2018, 53(10): 765-770. [28] OLSSON C, JANSSON H, SWENSON J. The Role of Trehalose for the Stabilization of Proteins [J]. The Journal of Physical Chemistry B, 2016, 120(20): 4723-4731. doi: 10.1021/acs.jpcb.6b02517 [29] ZHANG M, OLDENHOF H, SYDYKOV B, et al. Freeze-drying of mammalian cells using trehalose: preservation of DNA integrity [J]. Scientific Reports, 2017, 7(1): 6198. doi: 10.1038/s41598-017-06542-z