A stabilizer for clinical-grade lentivirus and its application method
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A lentivirus and stabilizer technology, which is applied in the field of clinical-grade lentivirus stabilizers, can solve the problems of easy inactivation, increase the volume of lentiviral vector, and large impact on titer, so as to ensure the ability to infect cells and benefit the quality Control and use the effect with simple steps
Active Publication Date: 2020-09-11
上海埃秀马生物科技有限公司
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Problems solved by technology
[0003] The low stability of lentiviral vectors seriously affects production, making the clinical production of lentiviral vectors an elusive goal
Using cell culture medium to store lentiviral vectors is an effective way to improve stability, but cell culture medium has many components, which is unfavorable for the quality control of clinical applications, and the volume of lentiviral vectors has to be doubled during use. Thereby reducing the titer of the finished product and affecting the subsequent clinical application
[0004] The purified high-purity clinical-grade lentiviral vector is very easy to inactivate at -80°C due to multi-step purification, with a loss of up to 20%-70% within a week
In addition, the thawing process has a large and unstable effect on the titer
At present, there is no report of a stabilizer that can increase the stability of the lentiviral vector and meet the requirements of clinical application
Method used
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Embodiment 1
[0033] 1. Prepare or use a commercially available 0.01mol / L PBS buffer solution (pH7.4);
[0034] 2. Dissolve 10 grams of sucrose in 100 ml of the above buffer solution;
[0035] 3. Add 10g of recombinant albumin into the above buffer solution and fully dissolve;
[0036] 4. Add 4 mg of tocopherol to the above solution and mix well;
[0037] 5. Filter the above buffer solution into a sterile bottle with a 0.22 μm filter.
Embodiment 2
[0039] 1. Prepare or use a commercially available 0.01mol / L PBS buffer solution (pH7.4);
[0040] 2. Dissolve 20 grams of sucrose in 100 ml of the above buffer solution;
[0041] 3. Add 20g of recombinant albumin into the above buffer solution and fully dissolve;
[0042] 4. Add 10 mg of tocopherol to the above buffer solution and mix well;
[0043] 5. Filter the above solution with a 0.22 μm filter into a sterile bottle.
Embodiment 3
[0045] Step 1. Pack the lentivirus rLV-zsGreen correctly according to the conventional method.
[0046] Step 2. Collect the cell supernatant, that is, the virus stock solution.
[0047] Step 3. Purify according to the method for large-scale preparation of clinical-grade lentivirus to obtain clinical-grade lentivirus rLV-zsGreen.
[0048] Step 4. Take 1ml of the stabilizer for clinical lentivirus obtained in Example 1, add it to 9ml of clinical-grade lentivirus rLV-zsGreen, mix gently, divide into packages, and store at -80°C. Step 5. The effect of repeated freezing and thawing (1 time, 2 times, 3 times) and storage time (1 week, January, March, June, December) on the stability of the lentivirus was detected by titer determination.
[0049] 5.1 Take a 96-well plate one day before titer determination, add 1×10 4 cells / well of HEK293 cells.
[0050] 5.2 Add polybrene (polybrene) to the complete DMEM medium with a final concentration of 8 μg / ml.
[0051] 5.3 Dilute the lentivi...
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Abstract
The invention relates to the field of biochemical medicines, in particular to a stabilizer for clinical lentivirus and a use method of the stabilizer. The stabilizer for clinical lentivirus comprises10-20 g / ml of sucrose, 40-100 mg / L of tocopherol, 10-20g / ml of human albumin, and the balance of 0.01 mol / L of a PBS buffer with the pH value being 7.4, and is obtained by filtering with a 0.22-micronfilter. The use method of the stabilizer for clinical lentivirus comprises the following steps: gently and uniformly mixing a clinical lentivirus suspension with the stabilizer for clinical lentivirus at a volume ratio of 9:1; sub-packaging; and storing the sub-packaged clinical lentivirus suspension in an environment at -80 DEG C. After verification, the clinical lentivirus using the stabilizerprovided by the invention has no drop in titer after being stored at -80 DEG C for 6 months, has no significant drop in titer after one freeze-thaw cycle, and has a titer loss of only 10% after threefreeze-thaw cycles, thereby satisfying storage requirements in production, transportation and use processes of lentivirus. The stabilizer provided by the invention has simple components and few use steps, does not need to use additional instrument and equipment, is easy to control quality and meets requirements of clinical application.
Description
technical field [0001] The invention relates to the field of biochemical medicine, in particular to a stabilizer for clinical-grade lentiviruses and a method of use thereof. The invention can be applied to cell therapy and other fields that require long-term storage of lentiviruses. Background technique [0002] Lentivirus is modified from human immunodeficiency virus (HIV) by removing env, vif, vpr, vpu, nef and other toxic genes. Replication-deficient HIV vectors are usually packaged with vesicular stomatitis virus G glycoprotein (VSV-G) instead of the HIV-1 envelope, which is safer, has a wider host range, and can increase viral titer Spend. Lentiviral vectors can infect not only dividing cells, but also quiescent cells, and can be stably integrated into host cells for long-term expression. In the application of hematopoietic stem cell transplantation, lentivirus has become an effective tool for infecting HSCs and performing gene therapy because it can infect most of th...
Claims
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Application Information
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