CN115960962A

CN115960962A - Protective agent for enhancing stability of lentivirus vector and application thereof

Info

Publication number: CN115960962A
Application number: CN202111177141.7A
Authority: CN
Inventors: 王宇涛; 宣春玲; 孙伟娟; 刘虎; 宋扬胜
Original assignee: Jiangsu Jinsirui Biotech Co ltd
Current assignee: Jiangsu Jinsirui Biotech Co ltd
Priority date: 2021-10-09
Filing date: 2021-10-09
Publication date: 2023-04-14
Also published as: WO2023056968A1

Abstract

The present specification provides a protective agent for enhancing the stability of a lentiviral vector, the protective agent comprising: the buffer solution is histidine buffer solution or 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution; a stabilizer comprising human serum albumin; and an inorganic salt; wherein the protective agent does not comprise a saccharide. The protective agent can ensure that the lentivirus vector stored in the protective agent keeps higher activity titer and transduction efficiency after long-time low-temperature storage and repeated freeze-thaw and under the condition of long-time working temperature, and enhances the stability of the lentivirus vector.

Description

Protective agent for enhancing stability of lentivirus vector and application thereof

Technical Field

The specification relates to the technical field of biological agent preservation, in particular to a protective agent for enhancing the stability of a lentiviral vector and application thereof.

Background

The lentivirus vector is a virus vector derived from human immunodeficiency virus-1 (HIV-1), contains genetic information required by packaging, transfection and stable integration, and is a main component of a lentivirus vector system. The lentivirus vector can effectively integrate the exogenous gene onto the host chromosome, thereby realizing the persistent expression, and in addition, the lentivirus vector has stronger infection capacity to dividing cells and non-dividing cells, the transferred gene has large capacity, and the immune response of the host is not easy to be initiated. In recent years, lentiviral vectors have played an increasingly important role in research of RNAi, gene therapy, transgenic animals, and the like. Thus, lentiviruses have become the primary vectors for gene therapy and in vitro gene modification.

Despite the great progress in the study of lentiviral vectors, the stability of recombinant lentiviral formulations has yet to be improved, making it difficult to meet the needs of in vivo applications. For example, if the cryopreservation is carried out for too long or the freezing and thawing are repeated during the use process, the activity titer (generally expressed as the virus titer) of the recombinant lentiviral vector tends to rapidly and greatly decrease, thereby limiting the normal use of the recombinant lentiviral vector. Therefore, there is a need for a cryopreservation protection reagent that is safe and simple in composition, can preserve lentiviral vectors at a lower temperature for a long period of time, and does not affect their activity.

Disclosure of Invention

One embodiment of the present disclosure provides a protective agent for enhancing the stability of a lentiviral vector, the protective agent comprising: a buffer solution, wherein the buffer solution is Histidine (Histidine) buffer solution or 4-hydroxyethyl piperazine ethanesulfonic acid (HEPES) buffer solution; a stabilizer comprising human serum albumin; and an inorganic salt; wherein the protective agent does not comprise a saccharide.

In some embodiments, the stabilizing agent further comprises a cholesterol lipid.

In some embodiments, the cholesterol lipid is present in the protectant at a concentration of 0.1-1% by volume.

In some embodiments, the cholesterol lipid is present in the protectant at a concentration of 0.4% by volume.

In some embodiments, the human serum albumin is recombinant human serum albumin expressed in an exogenous expression system; the mass volume percentage concentration of the human serum albumin in the protective agent is 1-5%.

In some embodiments, the inorganic salt comprises sodium chloride; the molar concentration of the sodium chloride in the protective agent is 75-100mmol/L.

In some embodiments, the inorganic salt comprises magnesium chloride; the molar concentration of the magnesium chloride in the protective agent is 1.5-3mmol/L.

In some embodiments, the molar concentration of the magnesium chloride in the protecting agent is 2mmol/L.

In some embodiments, the buffer solution in the protective agent is a histidine buffer at 10-20mmol/L.

In some embodiments, the buffer solution in the protectant is 20-50mmol/L of 4-hydroxyethylpiperazine ethanesulfonic acid buffer.

In some embodiments, the pH of the protectant is from 6.8 to 7.4.

In some embodiments, the pH of the protectant is 7.2.

In some embodiments, the activity titer of a lentiviral vector stored in the protective agent decreases by 0-10% with 8 freeze-thaw cycles compared to 0 freeze-thaw cycles; the activity titer of the lentiviral vector stored in the protective agent decreases by 0-25% in the case of 7 days at 4 ℃ compared to 0 days at 4 ℃; the activity titer of lentiviral vectors stored in the protective agent decreased by 0-50% when stored at 37 ℃ for 48 hours, compared to 0 hours at 37 ℃.

One of the embodiments of the present specification further provides the use of the above-described protective agent for enhancing the stability of a lentiviral vector in the purification and/or storage of a lentiviral vector.

One of the embodiments of the present specification also provides a lentiviral vector formulation comprising a lentiviral vector and the protective agent described above for enhancing the stability of the lentiviral vector.

Drawings

The present description will be further explained by way of exemplary embodiments, which will be described in detail by way of the accompanying drawings. These embodiments are not intended to be limiting, and in these embodiments like numerals are used to indicate like structures, wherein:

FIG. 1 is a graph comparing pH as a function of temperature for virus solutions formulated with different protective agents, according to some examples presented herein.

FIG. 2 is a graph showing the effect on specific lentiviral activity of virus solutions formulated with different protective agents at 37 ℃ for different time periods, according to some examples of the present disclosure.

FIG. 3 is a graph showing the effect of virus fluids formulated with different protectants on specific activity of lentiviruses after different number of freeze-thaw cycles, according to some embodiments of the present disclosure.

FIG. 4 is a graph showing the effect of virus fluids formulated with different protective agents on specific activity of lentiviruses after standing at 4 ℃ for different periods of time, according to some examples of the present disclosure.

FIG. 5 is a graph showing the effect of virus fluids formulated with

protectants

3, 6, and 7 on lentivirus activity titer at 37 ℃ for various periods of time, according to some examples of the present disclosure.

FIG. 6 is a graph showing the effect of virus fluids formulated with

protectants

3, 6, and 7 on lentivirus activity titer after standing at 4 ℃ for various periods of time, according to some examples of the present disclosure.

FIG. 7 is a graph showing the effect of virus fluids formulated with

protectants

3, 6, 7 on lentivirus activity titer after various times of freeze-thawing according to some examples of the present disclosure.

Figure 8 is a flow scatter plot of different volumes of

virus fluids

1 and 2 after infection of T cells according to some embodiments presented herein. Among them, FIGS. 8A to 8C are flow scattergrams after infection of T cells with 6.6. Mu.L, 1.1. Mu.L and 0.18. Mu.L of virus fluid 2, respectively; FIGS. 8D to 8F are flow scattergrams of 6.6. Mu.L, 1.1. Mu.L and 0.18. Mu.L of virus fluid 1 after infection of T cells, respectively. In the flow scattergram, the ordinate represents the number of cell particles, and the numerical unit M represents millions; the abscissa represents the fluorescent signal intensity of the FL1 channel.

Detailed Description

In order to more clearly illustrate the technical solutions of the embodiments of the present specification, the drawings used in the description of the embodiments will be briefly described below. It is obvious that the drawings in the following description are only examples or embodiments of the present description, and that for a person skilled in the art, without inventive effort, the present description can also be applied to other similar contexts on the basis of these drawings. Unless otherwise apparent from the context, or stated otherwise, like reference numbers in the figures refer to the same structure or operation.

As used in this specification and the appended claims, the terms "a," "an," "the," and/or "the" are not intended to be inclusive in the singular, but rather are intended to be inclusive in the plural, unless the context clearly dictates otherwise. In general, the terms "comprises" and "comprising" merely indicate that steps and elements are included which are explicitly identified, that the steps and elements do not form an exclusive list, and that a method or apparatus may include other steps or elements.

The stability of lentiviral vectors is an important issue that limits their use. The envelope of lentiviral vectors is susceptible to inactivation by a variety of physical and chemical factors, including temperature, osmotic pressure, pH, shear forces, etc., thereby reducing the stability of the vector. In terms of production, the production process of lentiviral vector includes transfection, purification, storage and transduction of target cells to complete the gene delivery process, and the change of the influencing factors such as temperature, osmotic pressure, pH and the like in the process makes the lentiviral vector easy to inactivate, which may lead to the reduction of the final quality and effectiveness of the vector. In particular, during the purification of lentiviral vectors, viral vectors become increasingly unstable due to the removal of components that maintain the stability of the lentivirus. Thus, there is also a need for lentiviral formulation components that maintain vector stability throughout the purification process. In terms of storage, lentiviral vectors need to be stored at temperatures of-70 ℃ or lower to maintain their infectivity (Harper, virology ed. Bios Scientific Publishers Limited, oxford, UK, 1993), whereas proteins are easily denatured at low temperatures. In terms of use, the lentiviral vector was operated at 37 ℃. In general, the half-life of lentiviruses in solution at 37 ℃ is no more than 12 hours, even about 6 hours as reported in the literature. Thus, suitable lentiviral formulation components are required to ensure that the lentivirus is both stable during manufacturing and storage at low temperatures for extended periods of time, and infectious over a period of time after multiple freeze/thaw cycles, and at operating temperatures.

According to one aspect of the present application, there is provided a protective agent for enhancing the stability of a lentiviral vector, comprising a buffer solution, a stabilizer, and an inorganic salt, and the protective agent does not comprise a saccharide. The protective agent selects proper components, and the lentivirus vector stored in the protective agent can keep higher activity titer and transduction efficiency after long-time low-temperature storage, repeated freeze thawing and long-time working temperature condition through the cooperation of the components, so that the stability of the lentivirus vector is enhanced.

In some embodiments, the stabilizing agent may include human serum albumin. Specifically, human serum albumin can be compatible with envelope glycoprotein of a lentiviral vector, and the stability of the lentivirus is enhanced. The human serum albumin can include blood-derived human serum albumin (HAS) and recombinant human serum albumin (rHSA) expressed by an exogenous expression system. The recombinant human serum albumin and the blood source human serum albumin have the same amino acid composition and structural characteristics, and the functions and functions are completely consistent. Compared with blood source human serum albumin, the recombinant human serum albumin is easier to obtain, and the possibility that blood can carry virus pollution is avoided; compared with animal source serum albumin, the safety is higher, and the requirement of clinical use can be met. In some embodiments, the human serum albumin can be recombinant human serum albumin expressed in an exogenous expression system. In some embodiments, preferably, the human serum albumin can be recombinant human serum albumin expressed by a transgenic yeast.

In some embodiments, the human serum albumin concentration in the protectant may be 1-5% by mass volume. In some embodiments, the human serum albumin concentration in the protective agent can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by mass volume. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary. In some embodiments, preferably, the human serum albumin concentration in the protectant may be 2% by weight.

In some embodiments, the stabilizing agent further comprises a cholesterol lipid. Specifically, the cholesterol lipid and the human serum albumin can act on the membrane structure of the lentiviral vector in a synergistic way, and can protect the membrane structure so as to reduce the influence of factors such as temperature, osmotic pressure and the like on the activity of the lentiviral vector, thereby enhancing the stability of the lentiviral vector. The protective agent containing human serum albumin and cholesterol lipid can effectively improve the transduction efficiency of the lentiviral vector. In some embodiments, the cholesterol lipid may be present in the protectant at a concentration of 0.1-2% by volume. For example, the volume percent concentration of the cholesterol lipid in the protectant may be 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1.1%, 1.3%, 1.5%, 1.7%, 1.9%, or 2%. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary. In some embodiments, preferably, the volume percent concentration of the cholesterol lipid in the protectant may be 0.4%.

Lentiviral vectors are extremely pH sensitive, such as the commonly used VSV-G enveloped lentiviral vectors, and the infection potential gradually decreases below pH 7.0. Therefore, the selection of an appropriate buffer solution to maintain the protective agent in an appropriate pH range is very important to enhance the stability of the lentiviral vector.

In some embodiments, the pH of the protectant may be 6.8-7.4. For example, the pH of the protectant may be 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4. Any range characterized by a combination of the preceding endpoints is also encompassed and is not further described herein. In some embodiments, in order to make the preservation environment of the lentivirus constructed by the protective agent closer to the environment of human body fluid, the pH value of the protective agent can be 7.2-7.4. In some embodiments, more preferably, the pH of the protectant may be 7.2.

The buffer solution can limit the change in pH of the protectant. The extent of ionization of the buffer solution will vary with temperature, meaning that the pH of the buffer will vary as the protectant is cooled or heated. Therefore, the selection of an appropriate buffer is very important to stabilize the biological activity of the lentivirus. In some embodiments, the buffer solution may be a histidine buffer or a 4-hydroxyethylpiperazine ethanesulfonic acid buffer. Herein, the histidine buffer refers to a buffer containing histidine. Non-limiting examples of histidine buffers include, but are not limited to, histidine-hydrochloric acid buffer, histidine-acetic acid buffer, histidine-phosphoric acid buffer, histidine-sulfuric acid buffer. In some embodiments, preferably, the histidine buffer may be a histidine-hydrochloric acid buffer.

In some embodiments, the buffer solution can be a histidine buffer, and the final concentration of the histidine buffer in the protective agent can be 10-20mmol/L. For example, the final concentration of histidine buffer in the protecting agent may be 10mmol/L, 12mmol/L, 14mmol/L, 16mmol/L, 18mmol/L or 20mmol/L. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary. In some embodiments, preferably, the buffer solution may be a histidine buffer, and the final concentration of the histidine buffer in the protective agent may be 10mmol/L.

In some embodiments, the buffer solution is 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and the final concentration of 4-hydroxyethylpiperazine ethanesulfonic acid buffer in the protecting agent can be 20-50 mmol/L. For example, the final concentration of 4-hydroxyethylpiperazine ethanesulfonic acid buffer in the protecting agent can be 20mmol/L, 22mmol/L, 24mmol/L, 26mmol/L, 28mmol/L, 30mmol/L, 32mmol/L, 34mmol/L, 36mmol/L, 38mmol/L, 40mmol/L, 42mmol/L, 44mmol/L, 46mmol/L, 48mmol/L, or 50mmol/L. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary. In some embodiments, preferably, the buffer solution is 4-hydroxyethylpiperazine ethanesulfonic acid buffer, and the final concentration of the 4-hydroxyethylpiperazine ethanesulfonic acid buffer in the protective agent can be 20mmol/L of HEPES buffer.

The inorganic salt in the protective agent can play a role in adjusting the osmotic pressure of the protective agent, preventing the aggregation of the lentiviral vector and the like. In some embodiments, the inorganic salt may include sodium chloride. In some embodiments, the molar concentration of the sodium chloride in the protectant may be 75-100mmol/L. For example, the molar concentration of the sodium chloride in the protecting agent may be 75mmol/L, 77.5mmol/L, 80mmol/L, 82.5mmol/L, 85mmol/L, 87.5mmol/L, 90mmol/L, 92.5mmol/L, 95mmol/L, 97.5mmol/L, 100mmol/L. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary. In some embodiments, preferably, the molar concentration of the sodium chloride in the protecting agent can be 75mmol/L.

In some embodiments, the inorganic salt may include magnesium chloride. In some embodiments, the molar concentration of the magnesium chloride in the protecting agent may be 1.5 to 3mmol/L. For example, the molar concentration of the magnesium chloride in the protecting agent may be 1.5mmol/L, 1.75mmol/L, 2mmol/L, 2.25mmol/L, 2.5mmol/L, 2.75mmol/L, 3mmol/L. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary. In some embodiments, preferably, the molar concentration of the magnesium chloride in the protective agent may be 2mmol/L.

Specifically, the protective agent containing magnesium chloride and/or sodium chloride can maintain the osmotic pressure of the protective agent in a range close to that of human body fluid, thereby being beneficial to keeping the virus preparation for a long time and avoiding the disintegration of the virus vector caused by low osmotic pressure; on the other hand, the osmotic pressure is maintained in a range close to the osmotic pressure of human body fluid, which is beneficial to directly applying the protective agent to clinical treatment. The protective agent comprising magnesium chloride and/or sodium chloride may prevent aggregation of the lentiviral vector, which may inhibit the activity of the lentiviral vector.

In some embodiments, the protective agent does not comprise a saccharide selected from one or more of a monosaccharide, a disaccharide, an oligosaccharide, a polysaccharide. Non-limiting examples of excluded sugars include, but are not limited to, glucose, sucrose, fructose, xylose, lactose, galactose, maltose, isomaltose, mannose, sorbose, trehalose, raffinose, and the like. In some embodiments, the excluded sugars may be selected from one or more of glucose, sucrose, fructose, xylose, trehalose, raffinose.

In some embodiments, the activity titer of a lentiviral vector stored in the protective agent can be reduced by 0-10% in the case of 8 freeze-thaw cycles compared to 0 freeze-thaw cycles. For example, an FCAS method can be used to assay the activity titer of a lentiviral vector stored in a protective agent, which is decreased by 0%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% after 8 times of freeze-thawing compared to the activity titer determined in the absence of freeze-thawing (i.e., 0 times of freeze-thawing). Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary.

In some embodiments, the lentiviral vector stored in the protective agent has a 0-25% decrease in titer of activity when placed at 4 ℃ for 7 days as compared to 0 days at 4 ℃. For example, the activity titer of a lentiviral vector stored in a protective agent, as determined by the FCAS method, can be reduced by 0%, 1%, 3%, 5%, 7%, 9%, 11%, 13%, 15%, 17%, 19%, 21%, 23%, or 25% after 7 days at 4 ℃ compared to the activity titer determined at 0 days at 4 ℃. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary.

In some embodiments, the titer of activity of a lentiviral vector stored in the protective agent decreases by 0-50% when stored at 37 ℃ for 48 hours compared to 0 hours at 37 ℃. For example, an FCAS method can be used to assay the activity titer of a lentiviral vector stored in a protective agent, which can be reduced by 0%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% after 7 days at 37 ℃ compared to the activity titer determined at 0 days at 37 ℃. Any range characterized by a combination of the preceding endpoints is also encompassed, and no further description is deemed necessary.

According to another aspect of the present application there is provided the use of a protective agent for enhancing the stability of a lentiviral vector in the purification and/or storage of the lentiviral vector. The addition of the above-mentioned protective agent for enhancing the stability of a lentiviral vector enables maintenance of the stability of the lentiviral vector at one or more steps in the production process of the lentivirus, such as transfection, purification, storage and transduction of target cells to accomplish gene delivery. The lentiviral vector needs to be stored at a temperature of-70 ℃ or below to maintain its infectivity, while the protein is easily denatured at a low temperature, and the protective agent mentioned above is added to the lentiviral vector to enable the lentiviral vector to be antifreeze and stored at a low temperature of-70 ℃ for a long period of time.

According to another aspect of the present application, there is provided a lentiviral vector formulation comprising a lentiviral vector and a protective agent for enhancing the stability of the lentiviral vector. The buffer solution and other auxiliary materials in the lentiviral vector preparation are in medicinal grade and have definite chemical components, so that the lentiviral vector preparation can be used as a gene therapy medicament to be injected into a human body, and can meet the requirements of clinical treatment modes such as intradermal injection, subcutaneous injection, intramuscular injection, vascular administration and the like.

In some embodiments, the lentiviral vector referred to herein can be a lentiviral vector selected from HIV-1, HIV-2, SIV, FIN, EIAV and VISNA. In some embodiments, the lentiviral vector may be an HIV-1 vector. The lentiviral vector may be a vector expression system. In some embodiments, a lentiviral vector expression system can comprise a nucleic acid vector comprising the gag-pol gene, a lentiviral structural protein, and a nucleic acid vector comprising the env gene or a functional substitute thereof. The env gene or functional substitute thereof may encode a vesicular stomatitis virus glycoprotein (VSV-G) or a derivative protein thereof. In some embodiments, the lentiviral vector expression system may further comprise a nucleic acid vector comprising the auxiliary gene rev or a gene analogous thereto. In some embodiments, the lentiviral vector expression system may also include a nucleic acid vector comprising a heterologous nucleic acid.

For example, a lentiviral vector expression system may include a nucleic acid vector comprising the gag-pol gene, a nucleic acid vector comprising the env gene or a functional substitute thereof, a nucleic acid vector comprising the rev gene or a similar gene thereof, and a transfer nucleic acid vector comprising a heterologous nucleic acid. As another example, a lentiviral vector expression system can comprise a nucleic acid vector comprising a gag-pol-rev gene, a nucleic acid vector comprising an env gene or a functional replacement thereof, and a transfer nucleic acid vector comprising a heterologous nucleic acid.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Example 1 Lentiviral preparation

And (3) transiently transfecting the HEK293T cells subjected to serum-free suspension domestication by adopting a four-plasmid packaging system to prepare the lentivirus. In a four plasmid packaging system, the viral envelope protein VSVG is provided by the pMD2.G plasmid (Addgene, cat # 12259); the pMDLg/pRRE plasmid (Addgene, cat # 12251) provides the viral structural protein gag-pol; the pRSV Rev plasmid (Addgene, cat # 12253) provides the viral regulatory protein Rev; the pLJM1-EGFP (Addgene, cat. 19319) plasmid is a transfer plasmid transfer containing a marker gene (green fluorescent protein, eGFP).

1.1 cell inoculation. HEK293T cells (from ECACC) were used to prepare cell suspensions in serum-free medium with cell density adjusted to 4X 10 ⁶ Per mL; inoculating 27mL of the cell suspension in a cell culture flask, 37 ℃,5% CO ₂ Shaking culture is carried out in an incubator at 125 rpm.

1.2 preparation of transfection mixture. The plasmid was as follows: gag-pol: rev: transfer =1:2:2:3.75, preparing mixed transfection plasmids according to the mass ratio; and (3) respectively dissolving the mixed transfection plasmid and the PEI transfection reagent (1 mg/mL) in a serum-free culture medium to obtain a mixed transfection plasmid diluent and a PEI transfection reagent diluent. Dropwise adding the PEI transfection reagent diluent into the mixed transfection plasmid diluent, wherein the mass ratio of the transfection plasmid to the PEI is 1.

1.3 transfection. 3mL of transfection mixture (10% production volume) was added dropwise to the cell culture shake flask of step 1.1 for transfection. After 48h of incubation, virus supernatants were collected and used for virus purification.

1.4 purifying. The virus supernatant was collected in a 50mL ultracentrifuge tube, centrifuged at 19400rpm,4 ℃ for 2h, and the supernatant was discarded to obtain a lentivirus pellet.

Example 2 protectant preparation and stability testing

2.1 preparation of protective Agents

1 XDPBS buffer (Gibco, cat 14040133) was used as protectant 1, and protectant 1 was used as a control. The protectants 2-7 were prepared according to the formulations shown in table 1. Wherein, in the stabilizer aspect, the

protective agents

2, 3 and 6 contain recombinant human serum albumin and do not contain saccharide, the protective agent 7 contains recombinant human serum albumin and cholesterol lipid and does not contain saccharide, and the

protective agents

4 and 5 contain saccharide and do not contain recombinant human serum albumin and cholesterol lipid.

Table 1 formula of protective agent

Note: cholesterol lipids (250 ×, cholesterol lipid) were purchased from Thermo Fisher, cat # 12531018.

2.2 preparation of lentivirus resuspension

Taking equal amount of lentivirus precipitate, and respectively suspending in equal amount of protective agents 1-7 to obtain 7 groups of virus solutions, wherein the numbers of the virus solutions are 1-7 in sequence; each group contained 16 tubes of virus solution, numbered A-P in sequence, 2mL per tube.

2.3 determination of the pH stability of the Virus solutions under various conditions

For group 7 virus solutions: the A tube virus liquid in each group is placed at room temperature for 3 hours, and then the pH value is tested; placing the virus liquid of the No. B tube in each group in a refrigerator at 4 ℃ for 3h, and then testing the pH value; the C number tube virus liquid in each group is heated in water bath at 37 ℃ for 3h, then the pH value is tested, and the D number tube virus liquid in each group is frozen and thawed (placed in a refrigerator at-80 ℃ for 90min, and then placed at room temperature for melting for 40 min) and then the pH value is tested at room temperature.

The results of the pH test are shown in FIG. 1. As shown in FIG. 1, the virus solution of group 1 was a control group, and the pH values of the virus solutions of the other groups except the virus solution of group 5 were small in the range of pH change with temperature within 6.8-7.4. Under different temperature conditions and at room temperature after freeze thawing, the virus solution prepared by using the

protective agents

2, 3, 4, 6 and 7 shows better pH stability.

2.4 determination of lentivirus stability at 37 ℃ for different standing times

2.4.1 standing the virus solution at 37 DEG C

For group 7 virus solutions: placing the virus liquid of the tube E in each group at 37 ℃ for 0h, and detecting the titer; placing the F tube virus liquid in each group at 37 ℃ for 24h, and detecting the titer; placing the virus liquid of the G tube in each group at 37 ℃ for 36h, and detecting the titer; the H tube virus solution in each group was left at 37 ℃ for 48H, and the titer was determined. The titer test was performed on the same day and same batch for each group from tube E to tube H.

2.4.2 determination of the physical titer of lentiviruses by ELISA

The ELISA method only measures the particle count of the lentivirus and does not measure the actual infectious capacity of the lentivirus to target cells, so the ELISA method measures the physical titer of the lentivirus rather than the actual (biological) titer. Since there are about 2000 molecules of P24 protein in one Lentiviral Particle (LP), the Particle number (LP) of the Lentiviral vector can be calculated using the following formula: 1ng P24=1.25X 10 ⁷ LPs. P24 ELISA kit (TAKARA, cat # 632200) was used for determination of lentivirus titer by following the protocol and calculating the physical titer from the assay results.

2.4.3 determination of active Titers of lentiviruses Using FACS method

The FACS method can accurately determine the actual infection capacity of the recombinant lentivirus, and the measured titer can accurately reflect the actual value of the virus functional titer. The method mainly comprises the following steps: target cells HEK293T diluted to 3X 10 ⁵ one/mL, mixed with polybrene at working concentration 30. Mu.g/mL. After diluting the virus solution in a cell culture medium gradient, adding lentivirus into target cells, continuously culturing for 72h at 37 ℃, and then determining the proportion of successfully infected cells in the target cells by a flow cytometer. Wells with fluorescence intensity (positive rate) between 5% and 10% were selected and the dilution factor of the wells was recorded. Calculating the activity titer of the virus sample to be tested according to the following formula:

activity titer (TU/mL) = [ (sample positivity-background pore positivity) × total number of cells upon transduction × dilution ]/volume of inoculated virus (mL) of the test virus sample.

2.4.4 Lentiviral stability assay

Since the present example pre-dispenses the virus solutions to be tested, and then takes out the corresponding virus solutions according to different processing conditions for detection, in order to eliminate the concentration difference between dispensed samples, the present study evaluates the lentivirus stability by the change of specific lentivirus activity (i.e., by the obtained value of physical titer/functional titer). Theoretically, when the stability of lentivirus is reduced, the functional titer is correspondingly reduced, while the physical titer is maintained, so that the specific activity of lentivirus is correspondingly increased.

In this example, the specific activity changes of lentiviruses after the virus solution is left at 37 ℃ for 0h, 24h, 36h and 48h are shown in FIG. 2. As shown in FIG. 2, the change in specific activity was greater in the

group

4 and 5 virus solutions than in the group 1 virus solution as a control group, the change in specific activity was substantially equal to that in the group 1 virus solution in the group 2 virus solution, and the change in specific activity was smaller in the

group

3, 6 and 7 virus solutions; after standing at 37 ℃ for 48h, the specific activities of the virus solutions of

groups

3, 6 and 7 were lower than those of the other virus solutions. The

protective agents

3, 6 and 7 have better effect of enhancing the stability of the lentivirus when being placed for a long time at the working temperature of 37 ℃.

2.5 determination of lentivirus stability under repeated Freeze-thaw conditions

2.5.1 freezing and thawing of Virus fluid

For group 7 virus solutions: freezing and thawing No. I tube virus liquid in each group for 0 times, and detecting titer; freezing and thawing the virus liquid of the No. J tube in each group for 2 times, and detecting the titer; freezing and thawing the K tuba virus liquid in each group for 5 times, and detecting the titer; the L number tube virus liquid in each group is frozen and thawed 8 times, and the titer is detected. The titer test was performed on the same day for each group of tubes I to L. The virus liquid is placed in a refrigerator at minus 80 ℃ for 90min, then placed at room temperature for melting for 40min, and recorded as 1 freeze thawing.

2.5.2 Lentiviral stability assay

Physical and activity titers were determined for group 7 virus fluids of step 2.5.1 in tubes I to L with reference to step 2.4, and specific activity of lentiviruses was calculated.

In this example, the changes in specific activity after 0, 2, 5 and 8 times of freezing and thawing of the virus solution are shown in FIG. 3. As shown in FIG. 3, the specific activity of the virus solutions of

groups

2, 3, 6 and 7 varied in a small amount; in particular, the virus solutions of

groups

3, 6 and 7 had lower specific activities than those of group 2 and group 1 as a control group after 8 times of freeze thawing. For the situation of repeated freeze thawing of the virus liquid, the

protective agents

3, 6 and 7 have better function of enhancing the stability of the lentivirus.

It should be noted that no data was collected from tubes I to L of the virus solutions in

groups

4 and 5 due to systematic errors.

2.6 determination of lentivirus stability at 4 ℃ for different standing times

2.6.1 standing the virus solution at 4 DEG C

For group 7 virus solutions: placing the M number tube virus liquid in each group at 4 ℃ for 0 day, and detecting the titer; placing the N-type tube virus liquid in each group at 4 ℃ for 1 day, and detecting the titer; placing the O-tube virus liquid in each group at 4 ℃ for 3 days, and detecting the titer; the virus solution of the P-tube in each group was left at 4 ℃ for 7 days, and the titer was measured. The titer of each group was determined on the same day for tubes M to P in the same batch.

2.6.2 Lentiviral stability assay

Physical and activity titers of tubes M to P of group 7 virus solutions of step 2.6.1 were determined with reference to step 2.4, and specific activities of lentiviruses were calculated.

In this example, the change in specific activity after the virus solution was left at 4 ℃ for 0 day, 1 day, 3 days, and 7 days is shown in FIG. 4. As shown in FIG. 4, the change in specific activity of the virus fluid of the 4 th group was substantially equal to that of the virus fluid of the 1 st group, the change in specific activity of the virus fluid of the 5 th group was larger than that of the virus fluid of the 1 st group, and the change in specific activity of the virus fluids of the 2 nd, 3 rd, 6 th and 7 th groups was smaller than that of the virus fluid of the 1 st group as a control group. And for the condition that the virus liquid is placed for a long time at 4 ℃, the

protective agents

2, 3, 6 and 7 have better effects of enhancing the stability of the lentiviruses.

2.7 Integrated analysis

In conclusion, it can be seen from the detection and analysis in steps 2.3 to 2.7 that the virus solutions in

groups

3, 6 and 7 exhibited better pH stability at different temperatures and under freeze-thaw conditions, and showed better lentivirus stability at 4 ℃ for long time, 37 ℃ for long time and under repeated freeze-thaw. The overall effect of the virus solutions of

groups

3, 6 and 7 in enhancing lentivirus stability was significantly better than that of the virus solution of group 1 as a control group, and also better than that of the virus solutions of

groups

2, 4 and 5.

Aiming at the virus solutions of the

groups

3, 6 and 7, the percentage ratio of the activity titer of the F number tube virus solution placed for 24 hours at 37 ℃, the activity titer of the G number tube virus solution placed for 36 hours, the activity titer of the H number tube virus solution placed for 48 hours and the activity titer of the E number tube virus solution placed for 0 hour in each group of virus solution is respectively calculated so as to analyze the change condition of the activity titer of the lentivirus under different placing time at 37 ℃, and the result is shown in figure 5.

For the virus solutions of

groups

3, 6 and 7, the percentage ratio of the activity titer of the virus solution of number N in each group at 4 ℃ for 1 day, the activity titer of the virus solution of number O at 4 ℃ for 3 days, the activity titer of the virus solution of number P at 4 ℃ for 7 days and the activity titer of the virus solution of number M at 4 ℃ for 0h was calculated respectively, so as to analyze the change of the activity titer of lentiviruses under different standing times at 4 ℃, and the result is shown in FIG. 6.

For the 3 rd, 6 th and 7 th groups of virus solutions, the percentage ratio of the activity titer of the virus solution in the J-tube, the K-tube and the L-tube in the L-tube to the activity titer of the virus solution in the I-tube is respectively calculated for 2 times of freezing and thawing, 5 times of freezing and thawing, 8 times of freezing and thawing, so as to analyze the change condition of the activity titer of the lentivirus under the repeated freezing and thawing condition, and the result is shown in FIG. 7.

FIG. 5 is a graph showing the effect of virus solutions formulated with

protectants

3, 6, and 7 on lentivirus activity titer at 37 ℃ for various periods of time. As shown in FIG. 5, the virus solutions of

groups

3, 6 and 7 had an activity titer of 50% or more of that of 0h at 37 ℃ in 24h, 36h and 48h at 37 ℃. In particular, the virus solutions of

groups

6 and 7 did not show a significant decrease in activity titer after prolonged storage at 37 ℃. For the group 6 virus fluid resuspended and prepared with protectant 6, the activity titer at 37 ℃ for 24h and 36h is more than 80% of the activity titer at 37 ℃ for 0 h; the activity titer after being placed at 37 ℃ for 48h is 62.97 percent of the activity titer after being placed at 37 ℃ for 0h, and the activity titer is reduced by 37.03 percent. For group 7 virus fluid resuspended and prepared with protectant 7, the activity titer at 37 ℃ for 24h and 36h is more than 80% of the activity titer at 37 ℃ for 0 h; the activity titer after being placed at 37 ℃ for 48h is 60.31 percent of the activity titer after being placed at 37 ℃ for 0h, and the activity titer is reduced by 39.69 percent.

FIG. 6 is a graph of the effect of virus fluids formulated with

protectants

3, 6, and 7 on lentivirus activity titer after standing at 4 ℃ for various periods of time. As shown in FIG. 6, the virus solutions of

groups

3, 6 and 7 had activity titers of 75% or more of those of the virus solutions stored at 4 ℃ for 1 day, 3 days and 7 days, respectively, and those of the virus solutions stored at 4 ℃ for 0 day. In particular, the virus solutions of

groups

6 and 7 did not show a significant decrease in activity titer after prolonged storage at 4 ℃. For group 6 virus solutions formulated with protectant 6, the activity titers at 4 ℃ for 1 day and 3 days were greater than 90% of the activity titer at 4 ℃ for 0 days; the activity titer of the cells after being placed at 4 ℃ for 7 days is 87.86 percent of the activity titer of the cells after being placed at 4 ℃ for 0 days, and the activity titer is reduced by 12.14 percent. For group 7 virus solutions formulated with protectant 7, the activity titers at 4 ℃ for 1 day and 3 days were 95% or more of the activity titer at 4 ℃ for 0 days; the activity titer after being placed at 4 ℃ for 7 days is 79.44 percent of the activity titer after being placed at 4 ℃ for 0 days, and the activity titer is reduced by 20.56 percent.

FIG. 7 is a graph of the effect of virus fluids formulated with

protectants

3, 6, 7 on lentivirus activity titer after various times of freeze-thawing. As shown in FIG. 7, compared with the activity titer obtained by freezing and thawing 0 times, the activity titer of the virus fluid of the

groups

3, 6 and 7 is reduced by 0-7.5% in the freezing and thawing 2 times, 5 times and 8 times, and the activity titer is not significantly reduced.

Based on the above analysis, the virus solution prepared by using the

protective agents

3, 6 and 7 can keep high activity titer of lentivirus, especially the protective agent 6 and the protective agent 7 under the conditions of long-time standing at 4 ℃, long-time standing at 37 ℃ and repeated freeze thawing.

Example 3 Effect of

protective agents

6 and 7 on lentivirus transduced T cells

CART cells are T cells that deliver foreign genes via lentiviruses, enabling T cells to specifically recognize and kill tumor cells. To assess the effect of lentiviral protective agents on the efficiency of lentiviral transduction of T cells, human primary T cells were infected with lentiviruses (containing the marker gene eGFP, encoding green fluorescent protein) stored in protective agent 6 and protective agent 7, respectively, to detect the positive rate of CART expression.

The transduction efficiency test method based on the PBMC T cell infection lentivirus is as follows:

3.1 preparing virus solutions by using an equal amount of the lentivirus precipitate of example 1 and equal amounts of the protective agent 6 and the protective agent 7 of step 2.1 of example 2; wherein, the virus liquid 1 contains lentivirus and a protective agent 6, and the virus liquid 2 contains lentivirus and a protective agent 7.

3.2 in cell culture plates, at 1X 10 ⁶ Density of individual cells/mL human primary T cells were plated.

3.3 for each virus fluid: the virus fluids were added to the sample wells containing the cell suspension of step 3.2 in different volumes (6.6. Mu.L, 1.1. Mu.L and 0.18. Mu.L), lentiviruses were co-cultured with human primary T cells for 72 hours, and lentivirus-infected cells were harvested by centrifugation, discarding the supernatant.

3.4 lentivirus-infected cells were resuspended in 200. Mu.L DPBS and analyzed on a flow cytometer using uninfected lentivirus cells as negative controls.

The results are shown in FIG. 8, in which FIGS. 8A to 8C are flow scattergrams after infection of T cells with 6.6. Mu.L, 1.1. Mu.L and 0.18. Mu.L of virus fluid 2, respectively; FIGS. 8D to 8F are flow scattergrams of 6.6. Mu.L, 1.1. Mu.L and 0.18. Mu.L of virus fluid 1 after infection of T cells, respectively. For lentivirus stored in protectant 7, the positive rates after T cell infection with different volumes (6.6 μ L, 1.1 μ L and 0.18 μ L) of virus fluid were 61.1%, 35.8% and 22.7%, respectively, higher than for protectant 6; and as the volume of the virus liquid increases, that is, as the multiplicity of infection increases, the advantage of the lentivirus preserved in the protective agent 7 in transduction efficiency becomes more significant. It was found that the protective agent 7 was superior to the protective agent 6 in terms of maintaining the efficiency of lentiviral transduction.

EXAMPLE 4 osmolarity testing of protectant

Protectant

3, 6 and 7 were formulated as in example 2, step 2.1. The

protective agents

3, 6 and 7 were each subjected to an osmotic pressure test. The osmolalities of

protectants

3, 6 and 7 were 504mOsm/L, 183mOsm/L and 425mOsm/L, respectively. The osmotic pressure of the

protective agents

3, 6 and 7 is lower than 600mOsm/L. Can meet the requirements of clinical treatment modes such as intradermal injection, subcutaneous injection, intramuscular injection, vascular administration and the like.

The protective agents disclosed herein for enhancing the stability of lentiviral vectors may have beneficial effects including, but not limited to: (1) The lentivirus vector can keep higher titer and transduction efficiency under the conditions of long-time storage at 4 ℃, long-time storage at 37 ℃ and repeated freeze thawing, maintain the activity of the lentivirus vector and enhance the stability of the lentivirus vector; (2) the components are safe and simple, and are suitable for clinical treatment; (3) Can be used for direct cryopreservation of the lentivirus vector without freeze-drying, and can reduce the production cost of the recombinant lentivirus preparation. It is to be noted that different embodiments may produce different advantages, and in different embodiments, any one or combination of the above advantages may be produced, or any other advantages may be obtained.

It should be understood by those skilled in the art that the above examples are only illustrative and not limiting of the present invention. Any modification, equivalent replacement or change made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A protective agent for enhancing the stability of a lentiviral vector, wherein the protective agent comprises:

the buffer solution is histidine buffer solution or 4-hydroxyethyl piperazine ethanesulfonic acid buffer solution;

a stabilizer comprising human serum albumin; and

an inorganic salt;

wherein the protective agent does not comprise a saccharide.

2. The protective agent for enhancing lentiviral vector stability according to claim 1, wherein the stabilizer further comprises a cholesterol lipid.

3. The protective agent for enhancing the stability of a lentiviral vector of claim 2, wherein the cholesterol lipid is present in the protective agent at a concentration of 0.1 to 1% by volume.

4. A protective agent for use in enhancing the stability of a lentiviral vector according to claim 3, wherein the concentration of cholesterol lipid in the protective agent is 0.4% by volume.

5. The protective agent for enhancing lentiviral vector stability according to any one of claims 1 to 4, wherein the human serum albumin is recombinant human serum albumin expressed by an exogenous expression system; the mass volume percentage concentration of the human serum albumin in the protective agent is 1-5%.

6. The protective agent for enhancing lentiviral vector stability according to any one of claims 1-4, wherein the inorganic salt comprises sodium chloride; the molar concentration of the sodium chloride in the protective agent is 75-100mmol/L.

7. The protective agent for enhancing lentiviral vector stability according to any one of claims 1 to 4, wherein the inorganic salt comprises magnesium chloride; the molar concentration of the magnesium chloride in the protective agent is 1.5-3mmol/L.

8. The protective agent for enhancing the stability of a lentiviral vector according to claim 7, wherein the molar concentration of the magnesium chloride in the protective agent is 2mmol/L.

9. The protective agent for enhancing the stability of a lentiviral vector according to any one of claims 1 to 4, wherein the buffer solution in the protective agent is a histidine buffer of 10 to 20mmol/L.

10. The protective agent for enhancing lentiviral vector stability according to any one of claims 1 to 4, wherein the buffer solution in the protective agent is 20 to 50 mmol/L4-hydroxyethylpiperazine ethanesulfonic acid buffer.

11. The protective agent for enhancing the stability of a lentiviral vector of any one of claims 1 to 4, wherein the protective agent has a pH of 6.8 to 7.4.

12. The protective agent for enhancing the stability of a lentiviral vector of claim 1, wherein the protective agent has a pH of 7.2.

13. The protective agent for enhancing lentiviral vector stability according to any one of claims 1 to 4, wherein the activity titer of lentiviral vectors stored in the protective agent is reduced by 0 to 10% in the case of 8 times of freeze-thawing compared to 0 times of freeze-thawing; the activity titer of the lentiviral vector stored in the protective agent decreases by 0-25% in the case of 7 days at 4 ℃ compared to 0 days at 4 ℃; the activity titer of lentiviral vectors stored in the protective agent decreases by 0-50% when stored at 37 ℃ for 48 hours, compared to 0 hours when stored at 37 ℃.

14. Use of a protective agent according to any one of claims 1 to 13 for enhancing the stability of a lentiviral vector in the purification and/or storage of a lentiviral vector.

15. A lentiviral vector formulation comprising a lentiviral vector and the protective agent of any one of claims 1 to 13 for enhancing the stability of the lentiviral vector.