CN116240241A - Purification and preparation method of recombinant lentiviral vector - Google Patents

Abstract

The invention discloses a purification preparation method of a recombinant lentiviral vector; comprises the steps of crude virus liquid harvesting, first nuclease digestion, deep filtration, TFF concentration liquid exchange, second nuclease digestion and chromatographic purification; in the TFF concentration liquid exchange step, a hollow fiber membrane is adopted to concentrate the crude virus liquid after deep filtration, buffer solution is used to wash and filter the concentrated crude virus liquid to 1/10-1/20 of the original crude virus liquid, the virus liquid is used to carry out reverse circulation cleaning on a pipeline and a hollow fiber bundle membrane column, and the virus liquid after ultrafiltration concentration liquid exchange is obtained. The invention updates the TFF concentration liquid changing process, and the key steps of the new TFF process are that the liquid flowing direction in the system pipeline is changed after the concentration is finished, the original forward circulation cleaning is changed into reverse circulation cleaning, and the reverse circulation cleaning is required to meet the requirement of more than 30 minutes, so that the current lentivirus purification recovery rate is improved to more than 40 percent.

Description

Purification and preparation method of recombinant lentiviral vector

Technical Field

The present invention belongs to the technical field of lentiviral vector purification, and relates to a method for purifying and preparing a recombinant lentiviral vector.

Background Art

Currently, in terms of lentivirus purification, the virus liquid is usually ultrafiltered, concentrated and replaced with a 300kDa-500kDa cutoff pore size in a 20mM Tris buffer system. The concentrated virus liquid is digested with 50U/ml nuclease at 8°C overnight. The digested lentiviral vector crude liquid is purified by chromatography, and the chromatography includes two steps. The first step is composite mode resin chromatography purification (Capto Core 300), followed by a one-step anion exchange chromatography purification (Capto DEAE). The purified and recovered lentiviral vector is ultrafiltered, concentrated and replaced with a second 300kDa-500kDa solution, and finally filtered and sterilized under a 0.2μm filter membrane to obtain the final product. This scheme has the problems of low lentivirus recovery rate and poor lentivirus activity after recovery.

The purification process of lentivirus includes deep filtration, TFF concentration, chromatography, and secondary TFF concentration. Each process is a major test for the final purification recovery rate. Each step of the process needs to minimize the loss of virus in a single step, so as to ensure a higher efficiency of lentivirus recovery.

There is currently no more efficient purification method for lentiviruses, so now more optimization and modification are made on the process details of each process to make the recovery efficiency of each step in the purification process higher, and then gradually increase the final recovery rate of purification. The current mature lentivirus recovery process has basically fixed the recovery efficiency at around 35%, and every 1% increase in recovery rate in the future will be an important step in lentivirus purification research.

Lentivirus is smaller and more fragile than adenovirus. During the purification process, pH, conductivity, temperature and exposure time are all reasons for the relative poor activity of lentivirus. Without affecting the activity of lentivirus, the selected pH makes the lentivirus and other impurity proteins have similar charges and similar anion binding conditions, making it difficult to separate impurities and seriously affecting the recovery rate. After changing the pH, the salt concentration used for elution of lentivirus is often higher, which also has a serious impact on the activity of lentivirus.

Summary of the invention

The purpose of the present invention is to provide a method for purifying and preparing a recombinant lentiviral vector in view of the deficiencies in the above-mentioned prior art.

The objective of the present invention is achieved through the following technical solutions:

The present invention relates to a method for purifying and preparing a recombinant lentiviral vector, the method comprising the following steps:

Harvesting of crude virus fluid: The crude virus fluid was centrifuged;

First nuclease digestion: The crude virus solution after centrifugation is incubated with the first nuclease;

Deep filtration;

TFF concentration and liquid exchange: Use hollow fiber membrane to concentrate the crude virus solution after deep filtration, use buffer to wash and filter the concentrated crude virus solution to 1/10 to 1/20 of the original crude virus solution, and obtain the concentrated virus solution after liquid exchange and dilution; Use the concentrated virus solution after liquid exchange and dilution to reversely cycle and clean the pipeline and hollow fiber bundle membrane column, collect, and obtain the virus solution after ultrafiltration concentration and liquid exchange;

Second nuclease digestion: The virus solution after ultrafiltration, concentration and replacement is incubated with nuclease for the second time;

Purification by chromatography.

As an embodiment, the centrifugal treatment adopts low-speed centrifugation, the centrifugal speed is 2000-3000g, the centrifugal temperature is 4-8°C, and the centrifugal time is 5-10min. The purpose of the centrifugal treatment is to remove some impurities with larger particles. In some implementation examples, the centrifugal speed is 2500g, the centrifugal temperature is 4°C, and the centrifugal time is 5-3min.

As an embodiment, the first nuclease incubation is performed with 20-30 U/ml of nuclease and 2-5 mM DTT solution. The first nuclease incubation breaks down the larger nucleic acids into small fragments, which are removed in the subsequent TFF& chromatography. In some implementation examples, the first nuclease incubation is performed with 25 U/ml of nuclease and 2 mM DTT solution.

Furthermore, the nuclease is selected from ArcticZymes or Cimage, the incubation temperature is 30-33°C, and the incubation time is 0.5-1h. In some implementation examples, the incubation temperature is 33°C, and the incubation time is 1h.

As an embodiment, the deep filtration is performed using a filter membrane with a pore size of 0.45 μm. Although centrifuged, there are still large particles of impurities in the crude virus solution, such as a small amount of cell debris, etc. In order to prevent these large particles from having a negative impact on the purification process, the present invention performs a deep filtration treatment on the crude virus solution before performing TFF.

During deep filtration operation, a peristaltic pump is used to slowly pump the virus solution through the filter membrane and collect it. The speed of the peristaltic pump is 30-60rpm/min.

As an embodiment, the buffer contains Hapes, NaCl and histidine-AC buffer, and the buffer pH = 3.0-3.4. The reverse cycle cleaning time is 10-30min. TFF concentration and liquid exchange are used to remove some small molecule impurities in the crude venom solution, such as nucleic acid fragments, BSA, etc. Precool the buffer at 4°C in advance to ensure that the buffer temperature can remain at a low temperature when changing the liquid. The histidine-AC buffer contains 10mM Hapes and 150mM NaCl. In some implementation examples, the purification buffer system used in the TFF concentration and liquid exchange process of the present invention is a 20mM histidine buffer system (20mM histidine-AC buffer, pH = 3.0, 10mM Hapes)

As an embodiment, in the TFF concentration and liquid replacement step, after reverse circulation cleaning, the pipeline and hollow fiber bundle membrane column are reversely circulated and cleaned with a dilution buffer, and the mixture is collected together; the volume of the buffer is 1/4 to 1/3 of the volume of the collected virus liquid.

As an embodiment, the second nuclease incubation is carried out with 50-100U/ml nuclease and 2-5mM DTT solution. The purpose of the second nuclease incubation is to completely remove the nucleic acid remaining in the virus solution. In some implementation examples, the second nuclease incubation is carried out with 100U/ml nuclease and 2mM DTT solution.

Furthermore, the nuclease is acid-resistant and alkali-resistant, and the incubation temperature is 30-33° C. for 0.5-1 h. In some implementation examples, the incubation temperature is 33° C. for 1 h.

As an embodiment, the chromatography purification adopts a composite mode chromatography, using Capto Core 300 or Viral L filler; the single loading volume is 3-4CV. The composite mode chromatography is used to separate the virus particles from other impurities, and finally achieve the purpose of purification.

As an embodiment, in the composite mode chromatography, the loading/chromatography flow rate is 480 cm/h, the initial collection UV280 is 15 mAU, and the final collection UV280 is 15 mAU.

Compared with the prior art, the present invention has the following beneficial effects:

1) The present invention updates the TFF concentration and liquid replacement process, so that the current lentivirus purification recovery rate is increased to more than 40%; the key step of the new TFF process is that after the concentration is completed, the liquid flow direction in the system pipeline needs to be changed, and the original forward circulation cleaning is changed to reverse circulation cleaning, and the reverse circulation cleaning must be satisfied for more than 30 minutes, so as to ensure the final lentivirus recovery efficiency.

2) In the present invention, auxiliary reagents for the work of nuclease are additionally added in both nuclease digestion steps to improve the working efficiency of the enzyme itself.

BRIEF DESCRIPTION OF THE DRAWINGS

Other features, objects and advantages of the present invention will become more apparent from the detailed description of non-limiting embodiments made with reference to the following drawings:

FIG1 is a schematic diagram of the purification and preparation process of a recombinant lentiviral vector.

DETAILED DESCRIPTION

The present invention is described in detail below in conjunction with embodiments. The following embodiments will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that, for those of ordinary skill in the art, some adjustments and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

The recombinant lentiviral vector in the present invention refers to a lentiviral vector produced by introducing a specific plasmid into a specific packaging cell. The multi-mode composite chromatography resin refers to a composite mode filler Capto Core 300, Viral L with multiple chromatography properties.

Example 1

This embodiment provides a method for purifying and preparing a recombinant lentiviral vector, as shown in FIG1 , which includes the following steps: harvesting a crude virus solution, a first nuclease digestion, deep filtration, TFF concentration and liquid replacement, a second nuclease digestion, and chromatography purification.

The specific process details of each process are as follows:

1) Harvest of crude virus solution:

Although the cells used in the virus packaging system are adherent systems, cell death is inevitable during the culture process, so there are some impurities such as large cell fragments in the suspended virus liquid. Although there is a deep filtration step in the subsequent process, in order to avoid the fragments and impurities clogging the filter membrane and causing pressure to increase, thereby damaging the activity of the virus, the present invention chooses to centrifuge the crude virus liquid at a low speed to remove some of the large impurities.

Centrifugation parameters:

Centrifuge speed: 2500g

Centrifugation temperature: 4°C

Centrifugation time: 5-3min

2) Deep filtration

Although centrifuged, there are still large particle impurities in the crude virus liquid, such as a small amount of cell fragments. In order to prevent these large particle impurities from having a negative impact on the purification process, the present invention performs deep filtration on the crude virus liquid before TFF.

Filter parameters:

Filter membrane manufacturers: Cobetter, Pall

Membrane pore size: 0.45 μm

Filter material: hydrophilic polyethersulfone

Filter operation parameters:

Use a peristaltic pump to slowly pump the virus solution through the filter membrane and collect it. The speed of the peristaltic pump is 30-60 rpm/min.

3) First nuclease digestion:

The crude venom contains a large amount of host DNA and exogenously added plasmids. The intact nucleic acid has a high viscosity, which will have an adverse effect on the TFF & chromatography purification of the virus. Therefore, before the subsequent steps, the present invention requires advanced nuclease treatment of the crude venom to break down the larger nucleic acids into small fragments, and remove them in the subsequent TFF & chromatography.

Nuclease digestion parameters:

Nuclease brands: ArcticZymes, Cimetrix

Nuclease concentration: 25U/ml

Incubation temperature: 33°C

Incubation time: 1h

Additional auxiliary reagents: 2mM DTT solution

4) TFF concentration and liquid replacement:

The environment in which the crude venom is located is not suitable for chromatography purification. Therefore, the present invention changes the environment in which the virus is located to conditions suitable for chromatography purification, and at the same time can remove some small molecule impurities in the crude venom, such as nucleic acid fragments, BSA, etc.

Hollow fiber parameters:

Hollow fiber membrane column brand: Acefab/Cobetter

Membrane pore size: 500kDa

Membrane material: mPES

Buffer environment: Histidine-AC buffer containing 10mM Hapes & 150mM NaCl pH = 3.0-3.4

Process flow:

The forward cycle concentrates the crude virus solution after deep filtration to 1/20 of the original volume.

Add buffer to dilute to six times the concentrated volume (six-fold dilution), and reduce the system flow rate to half of the concentration to carry out the concentration after the liquid change and dilution. The concentrated volume is 1/20 of the original crude venom solution (the virus solution after deep filtration). Then, after blocking the outlet, use the virus solution (the concentrated virus solution after the liquid change and dilution) to reversely cycle and clean the pipeline and hollow fiber bundle membrane column for 30 minutes, and collect the virus solution. Then use a dilution buffer with a volume of one-third of the collected virus solution to reversely clean the pipeline and column, and combine the cleaning solution with the concentrated virus solution to collect, and finally obtain the virus solution after ultrafiltration concentration and liquid change.

Process parameters:

System shear force control: 2000s ^-1

Pf: 10.3-10.8psi

Pr: 8.9-9.1psi

Pp: -0.1psi

Δp: 1.9-2.0psi

TMP: 9.5-10.4psi

Temperature Control:

Buffer: Precool the buffer to 4°C in advance to ensure that the buffer temperature can remain low when changing the solution.

Virus liquid: Currently, the device does not support temperature control of the liquid storage barrel. Therefore, during the TFF process, ice packs are used to cool the liquid storage barrel to ensure that the virus is kept at a low temperature during the TFF process.

5) Second nuclease digestion:

The purpose of this nuclease digestion is to completely remove the nucleic acid remaining in the virus solution.

Nuclease digestion parameters:

Nuclease brand: ArcticZymes or Cimex

Nuclease concentration: 100U/ml

Incubation temperature: 33°C

Incubation time: 1h

Additional auxiliary reagents: 2mM DTT solution

6) Chromatographic purification:

By utilizing the characteristics of the composite filler, the virus particles can be separated from other impurities, ultimately achieving the purpose of purification.

Packing parameters:

Filler manufacturer: Cytiva

Medium type: Capto Core 300

Chromatographic purification parameters:

Single loading volume: 3-4CV

Sample loading & chromatography flow rate: 480cm/h

Initial collection UV280: 1mAU

Stop collecting UV280: 5mAU (if the UV absorbance value decreases gradually above 5mAU, you can stop collecting).

The results of nuclease digestion in this example are shown in Tables 1-3 below:

Table 1 Comparison of digestion results with and without ArcticZymes auxiliary reagents

Table 2 Results of digestion with Cemig nuclease

Table 3 Repeated comparison of the effects of auxiliary reagents on nucleic acid digestion

The p24 titer detection and recovery rate calculation of this embodiment are shown in Table 4:

Table 4p24 physical titer test results and recovery calculation

In this example, the new TFF process was used to compare the p24 titer detection and recovery rate calculation with the old process as shown in Table 5:

Table 5 New and old process p24 physical titer test results and recovery calculation

The overall process flow p24 titer detection and recovery rate calculation after using the new TFF process in this embodiment are shown in Table 6:

Table 6p24 physical titer test results and recovery calculation

In this example, the titer detection and recovery rate of lentivirus p24 after secondary TFF concentration are shown in Table 3:

Table 3p24 physical titer test results and recovery calculation

The infection titer detection and recovery rate calculation of this example are shown in Table 8:

Table 8 Activity titer detection and recovery calculation

In summary, the present invention uses a new nuclease and compares the digestion conditions of the old nuclease and the new nuclease. The new nuclease is superior in digestion efficiency. Under the same conditions, the digestion of nucleic acids is more thorough. The initial nuclease digestion has reached the final acceptance standard. In addition, the present invention has changed the new TFF process, and the recovery rate has reached more than 30%, while the single-step recovery rate of chromatography can be controlled at more than 60%, and the final recovery rate can basically reach more than 40%.

The above describes the specific embodiments of the present invention. It should be understood that the present invention is not limited to the above specific embodiments, and those skilled in the art may make various modifications or variations within the scope of the claims, which do not affect the essence of the present invention.

Claims (10)

1. A method for purifying and preparing a recombinant lentiviral vector, characterized in that the method comprises the steps: Crude virus fluid harvest: Centrifuge the crude virus fluid; The first nuclease digestion: carry out the first nuclease incubation on the centrifuged crude virus liquid; deep filter; TFF Concentrated Liquid Replacement: Using a hollow fiber membrane to diafilter and concentrate the deep-filtered crude virus liquid to 1/10-1/20 of the original crude virus liquid with a buffer solution, to obtain a concentrated virus liquid after liquid exchange and dilution; use liquid exchange The diluted concentrated virus liquid is cleaned and collected in reverse circulation on the pipeline and the hollow fiber bundle membrane column to obtain the virus liquid after ultrafiltration and concentrated liquid exchange; The second nuclease digestion: carry out the second nuclease incubation on the virus liquid after the ultrafiltration concentration and replacement; Chromatographic purification. 2. The purification preparation method of the recombinant lentiviral vector according to claim 1, characterized in that, the centrifugation process adopts low-speed centrifugation, the centrifugation speed is 2000-3000g, the centrifugation temperature is 4-8°C, and the centrifugation time is 5- 10min. 3. The purification preparation method of the recombinant lentiviral vector according to claim 1, characterized in that, the first nuclease incubation adopts 20-30U/ml nuclease and 2-5mM DTT solution to incubate. 4. The method for purifying and preparing the recombinant lentiviral vector according to claim 3, wherein the nuclease is ArcticZymes or Cimege, the incubation temperature is 30-37°C, and the incubation time is 0.5-1h. 5. The method for purifying and preparing recombinant lentiviral vector according to claim 1, characterized in that, the deep layer filtration adopts a filter membrane with a pore size of 0.45 μm for filtration. 6. The purification preparation method of recombinant lentiviral vector according to claim 1, characterized in that, the buffer contains Hapes, NaCl histidine-AC buffer, buffer pH=7.0-7.4; reverse cycle The cleaning time is 10-30min. 7. The purification preparation method of the recombinant lentiviral vector according to claim 1, characterized in that, in the step of concentrating and changing the liquid of the TFF, after reverse circulation cleaning, the pipeline and the hollow fiber membrane column are carried out with a dilution buffer solution. Reverse cycle washing, combined collection; the volume of the buffer is 1/4-1/3 of the volume of the collected virus liquid. 8. The method for purifying and preparing the recombinant lentiviral vector according to claim 1, wherein the second nuclease incubation is incubated with 50-100 U/ml of nuclease and 2-5mM DTT solution. 9 . The method for purifying and preparing the recombinant lentiviral vector according to claim 8 , wherein the nuclease is an acid-resistant and alkali-resistant nuclease, the incubation temperature is 30-37° C., and the incubation time is 0.5-1 h. 10. The purification preparation method of the recombinant lentiviral vector according to claim 1, characterized in that, the chromatographic purification adopts multi-mode chromatography, using Capto Core 700 or Viral L filler; a single load is 3- 4CV.

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