Preparation and stability study of lyophilized lentiviral vector

SHEN Hongwei; LI Minghao; XU Nan; SHAO Jiaqi; WANG Jing; YU Lei

doi:10.3969/j.issn.1000-5641.2021.03.012

Issue 3

May 2021

Turn off MathJax

Article Contents

Article Navigation > Journal of East China Normal University (Natural Sciences) > 2021 > (3): 114-127

SHEN Hongwei, LI Minghao, XU Nan, SHAO Jiaqi, WANG Jing, YU Lei. Preparation and stability study of lyophilized lentiviral vector[J]. Journal of East China Normal University (Natural Sciences), 2021, (3): 114-127. doi: 10.3969/j.issn.1000-5641.2021.03.012

Citation:

SHEN Hongwei, LI Minghao, XU Nan, SHAO Jiaqi, WANG Jing, YU Lei. Preparation and stability study of lyophilized lentiviral vector[J]. Journal of East China Normal University (Natural Sciences), 2021, (3): 114-127. doi: 10.3969/j.issn.1000-5641.2021.03.012

Citation:

PDF( 1003 KB)

Preparation and stability study of lyophilized lentiviral vector

doi: 10.3969/j.issn.1000-5641.2021.03.012

School of Chemistry and Molecular Engineering, East China Normal University, Shanghai　200062, China

Received Date: 2019-12-25
Publish Date: 2021-05-01

Abstract

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).
- lentiviral vector,
- lyophilized preparation,
- stability

FullText(HTML)

References(29)

References

[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