CN114990077A - Lentiviral packaging and purification method - Google Patents

Description

A kind of lentivirus packaging and purification method

technical field

The invention relates to cell culture technology, in particular to a lentivirus packaging and purification method.

Background technique

With the development of the immunotherapy industry, more and more research institutions and pharmaceutical companies have devoted themselves to the research and development, clinical trials and industrialized preparation of CAR-T. The full name of CAR-T is Chimeric Antigen Receptor T-Cell Immunotherapy, CAR-T. It is a kind of CAR gene designed and synthesized by genetic engineering, and transferred into T cells by lentiviral vector, so as to obtain CAR-T. CAR-T cells are infused back into the patient. At present, CAR-T products have been launched in foreign and domestic markets, and many clinical trials are underway. It can be said that CAR-T therapy is likely to develop a tumor-curing cell therapy program for the benefit of mankind.

Lentivirus has the advantages of high transduction efficiency, integration of T cell genome and continuous expression of target protein, etc. It is an incomparable advantage of transfection, electroporation, retrovirus, adenovirus, etc. Nowadays, most of the clinical research of CAR-T cells and the production of marketed products are based on lentiviral vector-mediated CAR gene transfer. Therefore, lentiviruses must meet GMP standards.

At present, the preparation and production of lentiviruses are divided into two types, one for adherent cell packaging and the other for suspension cell packaging. Most of the processes for the preparation of lentivirus by adherent cell packaging are based on the adherent cell process relying on fetal bovine serum (FBS), which has the advantages of high lentiviral expression titer and low hardware investment; Products must be free of animal-derived components, which rely on fetal bovine serum, which is incompatible with current regulations. If fetal bovine serum is used for lentivirus preparation, the cost of downstream purification equipment, consumables and reagents will be greatly increased. In the process of preparing lentivirus by suspension cell packaging, 293T cells are first trained into cells that do not depend on fetal bovine serum, but the training process is cumbersome, and the trained cells are in poor condition during the lentivirus packaging process. It needs to invest a lot of equipment costs, and requires greater human, material, and financial costs, which increases an excessive burden on the development of the enterprise.

SUMMARY OF THE INVENTION

Based on the above problems, the problem to be solved by the present invention is to provide a lentivirus packaging and purification method with low purification difficulty, low cost, and no animal-derived components.

The technical scheme of the present invention is as follows:

A lentivirus packaging and purification method, comprising the following steps:

The 293T cells in logarithmic growth phase were resuspended in complete medium and transferred to the cell factory for static culture;

After standing for 24 hours, remove the complete medium, add serum-free medium, and continue to incubate;

The four plasmids, PEI and OPTI-MEM were mixed evenly and incubated, and then supplemented with serum-free medium to obtain a plasmid transfection mixture;

After incubating the 293T cells in the cell factory, remove the serum-free medium, and then add the plasmid transfection mixture to the cell factory for transfection;

After the transfection treatment, the plasmid transfection mixture was removed, and a new serum-free medium was added for static culture; during the static culture, the lentivirus supernatant was collected;

The lentivirus supernatant was sequentially subjected to Q membrane enrichment, nuclease enzymatic hydrolysis and ion exchange treatment to obtain a lentivirus purified solution;

The lentivirus purified solution is successively concentrated and filtered to obtain a purified chronic disease concentrate, which is then packaged, frozen, and used for later use.

In one example, in the lentivirus packaging and purification method, when the 293T cells are transferred into a cell factory: the transfer amount of the 293T cells is 5.0×10 ⁸ to 10.0×10 ⁸ ; the cell factory is a ten-layer cell factory and the complete medium is a complete medium containing 10% (v/v) FBS; preferably, when the 293T cells are transferred into the cell factory: the transfer amount of the 293T cells is 8.0×10 ⁸ .

In one embodiment, in the lentivirus packaging and purification method, after the stationary culture of the 293T cells is stopped, the serum-free medium is added in an amount of 500-700 ml, and the incubation time is 1 hour; preferably, the 293T After the stationary culture of cells was stopped, the added amount of the serum-free medium was 625 ml.

In one embodiment, in the lentivirus packaging and purification method, in the preparation step of the plasmid transfection mixture, the total number of PEI and the four plasmids is 2:1 (v/m); the incubation time is 10-30 minutes; Preferably, the incubation time is 20 minutes.

In one embodiment, in the lentivirus packaging and purification method, in the plasmid transfection treatment step, the transfection treatment time is 5-7 hours; preferably, the transfection treatment time is 6 hours.

In one embodiment, in the lentivirus packaging and purification method, after the plasmid transfection treatment is completed, the amount of the new serum-free medium added is 1000-1400 ml; preferably, the amount of the serum-free medium added is 1250ml.

In one embodiment, in the lentiviral packaging and purification method, the lentiviral supernatant is collected at the 24th hour and the 48th hour of static culture, respectively.

In one embodiment, in the lentivirus packaging and purification method, in the lentivirus supernatant processing step, the Q membrane is Mustang Q XT5; the ion exchange chromatography column filler used in the ion exchange treatment is Sepharose4FF.

In one embodiment, in the lentivirus packaging and purification method, the filtration treatment for the concentration of the lentivirus purified solution further comprises: adding the concentrated lentivirus with a final concentration of 10% (v/v) human albumin and A sucrose aqueous solution with a final concentration of 5% (v/v) was mixed uniformly, and then sterile filtered through a 0.22 membrane filter.

Compared with the prior art, the present invention has the following advantages:

1), in the present invention, before lentivirus packaging, 293T is cultured with 10% FBS complete medium, and the 293T cells in log phase are in the best state, ensuring the cell state during the lentivirus packaging process, and at the same time ensuring the lentivirus. high titer;

2), in the process of lentivirus packaging, the present invention completely uses serum-free medium, which makes downstream purification simpler and easier to obtain lentiviruses that meet GMP standards;

3) In the process of lentivirus packaging, the present invention controls the number of 293T cells to keep the cells in the best state during the packaging process;

4) In the lentivirus purification of the present invention, Q membrane enrichment, nuclease enzymatic hydrolysis, and ion exchange are used to obtain lentiviruses that meet GMP requirements after concentration, the process is simple, and the cost is low;

In the present invention, serum-free medium is used in the lentivirus packaging process, so that the 293T cells are kept in the state during the packaging process, and animal-derived components are not introduced, so that the downstream lentivirus purification is simpler; that is, the lentivirus is guaranteed. titer, reduce the cost of installation, and obtain GMP-compliant lentivirus more efficiently.

Description of drawings

Fig. 1 is the lentivirus packaging and purification process flow chart provided by the present invention;

Figures 2, 3, 4, and 5 are the effects of FBS on the purification peaks during the corresponding lentivirus packaging process of Comparative Example 1 and Examples 1 to 3, respectively;

Figure 6 shows the effect of 293T cell packaging number on lentivirus titers.

Detailed ways

The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

As shown in Figure 1, a kind of lentivirus packaging and purification method provided by the present invention comprises the following process steps:

S1. 5.0×10 ⁸ ~10.0×10 ⁸ 293T cells in log phase were resuspended in complete medium and transferred to the cell factory for static culture;

S2. After 24 hours of static culture, discard the complete medium in the cell factory, add 500-700ml of serum-free medium, and incubate for 1 hour; go to step S4;

S3. Mix the four plasmids, PEI and OPTI-MEM evenly, incubate for 10-30 minutes, and then add serum-free medium to 625ml to obtain a plasmid transfection mixture; transfer to step S4;

S4. After the incubation of the ten-layer cell factory in step S2 is completed, the serum-free medium is discarded, and the plasmid transfection mixture in step S3 is added, and the transfection is processed for 5-7 hours;

S5. After the transfection treatment, discard the plasmid transfection mixture and add 1000~1400ml of serum-free medium;

S6, collect lentiviral supernatant at 24h and 48h respectively;

S7, performing Q membrane enrichment, nuclease enzymatic hydrolysis, and ion exchange treatment on the collected lentivirus supernatant in sequence to obtain a lentivirus purified solution;

S8, the lentivirus purified solution is subjected to ultrafiltration concentration and sterile filtration to obtain a lentivirus concentrate;

S9, the lentivirus concentrate is divided into packs and frozen to obtain a purified lentivirus preparation.

In the above steps, steps S1-S4 are lentivirus packaging processes; and steps S5-S9 are lentivirus collection and purification processes.

A further scheme is that in the step S1, the number of 293T cells is 8.0×10 ⁸ , and the 293T cells are first resuspended in a complete medium containing 10% FBS (fetal bovine serum), and then transferred to a ten-layer cell factory. .

A further scheme is that, in the step S1, the complete medium contains 10v/v% FBS (fetal bovine serum), which can ensure that 293T is in the best state before lentivirus packaging; the cell factory adopts a ten-layer cell factory.

A further solution is that, in the step S2, discarding the complete medium in the cell factory is to completely remove the complete medium in the cell factory. The purpose of adding serum-free medium in step S2 is to minimize the interference of FBS on the four plasmids.

A further scheme is that in the step S3, PEIpro method is used to transfect 293T cells, and PEI:DNA (the amount of DNA in the total number of four plasmids) is transfected at 2:1 (v/m).

A further scheme is that in the step S3, ten layers of cell factory packaging are used, and a total of 158 ml of OPTI-MEM is used in the plasmid mixing process. After the incubation is completed, it needs to be supplemented to 625 ml of serum-free medium.

In a further scheme, in the step S4, the transfection time is 6 hours.

A further scheme is that in the steps S5 and S6, the ten-layer cell factory needs to be kept stable during the operation to prevent the medium from shaking strongly to make the 293T cells float; wherein, in step S5, the amount of serum-free medium added is 1250ml. .

A further scheme is that in the step S7, the Q membrane adopts Mustang Q XT5, and the filler of the ion exchange chromatography column used in the ion exchange treatment is Sepharose 4FF; wherein, the Q membrane enrichment and purification adopted in the ion exchange treatment are Sepharose 4FF. The instrument is AKTA pure25 from Cytiva.

A further scheme is that in the step S8, the concentrated lentivirus is added with human serum albumin (HSA) and sucrose aqueous solution by volume, and after mixing evenly, it is sterile filtered with a 0.22 filter; The final concentrations of human serum albumin (HSA) and sucrose in the concentrated filtration system were 10% v/v and 5% v/v, respectively, to ensure that the lentivirus remained in vitro and in vivo during cryopreservation and resuscitation. Osmolarity balance to prevent lentivirus damage.

In a further scheme, in the step S8, after lentivirus purification, concentration and filtration, a lentivirus preparation of about 16-25 ml is obtained.

A further scheme is that in the step S9, the lentiviruses are packaged according to the needs of the experiment, and can be stored at -20°C for short-term storage, and at -80°C for long-term storage.

The following is further described in detail through some embodiments.

Comparative Example 1

S01. 8.0×10 ⁸ 293T cells in logarithmic phase were resuspended in complete medium containing 10% FBS and transferred to a ten-layer cell factory for static culture;

S02. After standing for 24 hours, discard the complete medium, add 625ml of serum-free medium, and incubate for 1 hour;

S03. Mix the four plasmids, PEI and OPTI-MEM evenly in proportion, incubate for 20 min, and then add serum-free medium to 625 ml to obtain a plasmid transfection mixture; among them, the total number of PEI and four plasmids is 2:1 (v/m) ;

S04, discard the serum-free medium after the incubation of the ten-layer cell factory in step S02 is completed, add the plasmid transfection mixture in step S03, and conduct the transfection for 6 hours;

S05. After the transfection treatment, discard the plasmid transfection mixture and add 1250ml of complete medium;

S06, collect lentiviral supernatant at 24h and 48h respectively;

S07. Perform Q membrane enrichment, nuclease enzymatic hydrolysis, and ion exchange treatment on the collected lentivirus supernatant in turn to obtain a lentivirus purified solution; wherein, the Q membrane adopts Mustang Q XT5, and the ion exchange used in the ion exchange treatment The packing of the chromatography column is Sepharose 4FF; among them, the purification instrument used for Q membrane enrichment and ion exchange treatment is AKTA pure25;

S08, the lentivirus purified solution is subjected to ultrafiltration concentration and sterile filtration to obtain a lentivirus concentrate;

S09, the lentivirus concentrate is subpackaged and frozen to obtain a purified lentivirus preparation.

Example 1

S11. 5.5×10 ⁸ 293T cells in log phase were resuspended in complete medium containing 10% FBS and transferred to the ten-layer cell factory for static culture;

S12. After static culture for 24 hours, discard the complete medium, add 500 ml of serum-free medium, and incubate for 1 hour;

S13. Mix the four plasmids, PEI and OPTI-MEM evenly in proportion, incubate for 10 min, and then add serum-free medium to 625 ml to obtain a plasmid transfection mixture; among them, the total number of PEI and four plasmids is 2:1 (v/m) ;

S14. After the ten-layer cell factory incubation in step S12 is completed, the serum-free medium is discarded, and then the plasmid transfection mixture in step S13 is added, and the transfection is processed for 5 hours;

S15. After the transfection treatment, discard the plasmid transfection mixture and add 1000 ml of serum-free medium;

S16, collect lentiviral supernatant at 24h and 48h respectively;

1S7. Perform Q membrane enrichment, nuclease enzymatic hydrolysis, and ion exchange treatment on the collected lentiviral supernatant in sequence to obtain a lentivirus purified solution; wherein, the Q membrane adopts Mustang Q XT5, and the ion exchange used in the ion exchange treatment The packing of the chromatography column is Sepharose 4FF; among them, the purification instrument used for Q membrane enrichment and ion exchange treatment is AKTA pure25;

S18, the lentivirus purified solution is subjected to ultrafiltration concentration and sterile filtration to obtain a lentivirus concentrate;

S19, the lentivirus concentrate is divided and frozen to obtain a purified lentivirus preparation.

Example 2

S21. 8.0×10 ⁸ 293T cells in log phase were resuspended in complete medium containing 10% FBS and transferred to ten-layer cell factory for transfection treatment;

S22. After standing for 24 hours of culture, discard the complete medium, add 625 ml of serum-free medium, and incubate for 1 hour;

S23. Mix the four plasmids, PEI and OPTI-MEM evenly in proportion, incubate for 30 minutes and then supplement the serum-free medium to 625 ml to obtain a plasmid transfection mixture; among them, the total number of PEI and four plasmids is 2:1 (v/m) ;

S24, discard the serum-free medium after the incubation of the ten-layer cell factory in step S22 is completed, then add the plasmid transfection mixture in step S23, and perform the transfection for 6 hours;

S25. After the transfection treatment, discard the plasmid transfection mixture, and then add 1400 ml of serum-free medium;

S26, collect lentiviral supernatant at 24h and 48h respectively;

S27. Perform Q membrane enrichment, nuclease enzymatic hydrolysis, and ion exchange treatment on the collected lentivirus supernatant in sequence to obtain a lentivirus purified solution; wherein, the Q membrane adopts Mustang Q XT5, and the ion exchange used in the ion exchange treatment The packing of the chromatography column is Sepharose 4FF; among them, the purification instrument used for Q membrane enrichment and ion exchange treatment is AKTA pure25;

S28, the lentivirus purified solution is subjected to ultrafiltration concentration and sterile filtration to obtain a lentivirus concentrate;

S29, the lentivirus concentrate is divided and frozen to obtain a purified lentivirus preparation.

Example 3

S31. 10.0×10 ⁸ 293T cells in log phase were resuspended in complete medium containing 10% FBS and transferred to ten-layer cell factory for transfection treatment;

S32. After standing for 24 hours of culture, discard the complete medium, add 700 ml of serum-free medium, and incubate for 1 hour;

S33. Mix the four plasmids, PEI and OPTI-MEM evenly in proportion, incubate for 20 min and then supplement the serum-free medium to 625 ml to obtain a plasmid transfection mixture; among which, the total number of PEI and four plasmids is 2:1 (v/m) ;

S34. After the ten-layer cell factory incubation in step S32 is completed, discard the incubation base, then add the plasmid transfection mixture in step S33, and perform transfection for 7 hours;

S35. After the transfection treatment, discard the plasmid transfection mixture and add 1250 ml of serum-free medium;

S36, collect lentiviral supernatant at 24h and 48h respectively;

S37. Perform Q membrane enrichment, nuclease enzymatic hydrolysis, and ion exchange treatment on the collected lentivirus supernatant in sequence to obtain a lentivirus purified solution; wherein, the Q membrane adopts Mustang Q XT5, and the ion exchange used in the ion exchange treatment The packing of the chromatography column is Sepharose 4FF; among them, the purification instrument used for Q membrane enrichment and ion exchange treatment is AKTA pure25;

S38, the lentivirus purified solution is subjected to ultrafiltration concentration and sterile filtration to obtain a lentivirus concentrate;

S39, the lentivirus concentrate is divided and frozen to obtain a purified lentivirus preparation.

2. Lentivirus detection and analysis

The structural analysis of lentivirus detection is shown in Figure 2, 3, 4, 5 and Table 1.

Table 1 Detection results of lentiviruses by four packaging methods

In Comparative Example 1, according to the existing lentivirus packaging process, a complete medium of 10% FBS was used in the packaging process, as shown in Figure 2 and Table 1. During the purification process, the residual components of FBS will affect the lentivirus. At the same time, due to the use of complete medium to incubate 293T cells, there is residual serum, and the peak graph is very mixed, which greatly interferes with purification, resulting in the left peak curve not smooth and smooth. These resulted in a total DNA residue of 38960ng/ml (far exceeding the standard value of 100ng/ml) and BSA residues reaching 46850ng/5.0*10 ⁷ TULV (far exceeding the standard value of 500ng/5.0*10 ⁷ TULV).

In Example 1, the number of 293T cells less than that of Comparative Example 1 was used for packaging, and blood-free medium was used in the packaging process. As shown in Figure 3 and Table 1, during the purification process, the lentivirus was very clean and slow. The purity of the virus is high, and the residual detection is up to the standard, but the titer of the lentivirus is slightly lower; at the same time, since the 293T cells are incubated with serum-free medium, there is no serum residue, which has little interference with purification, making the left peak curve smooth and smooth, and the total DNA The residue reached 29.8ng/ml (100ng/ml lower than the standard value) and the BSA residue was 100ng/5.0*10 ⁷ TU LV (500ng/5.0*10 ⁷ TU LV lower than the standard value).

In Example 2, in the packaging process, the same number of 293T cells as in Comparative Example 1 was used, and blood-free medium was used in the packaging process, as shown in Figure 4 and Table 1, in the process of purification, the peak of lentivirus was clean. , the purity of the lentivirus is high, and the residual detection is up to the standard, and the titer of the lentivirus is much higher than that of Example 2; at the same time, since the 293T cells are incubated with serum-free medium, there is no serum residue, which has little interference with purification, making the left peak curve smooth. Fluency, the total DNA residue reached 50.9ng/ml (lower than the standard value of 100ng/ml) and the BSA residue was 98.6ng/5.0*10 ⁷ TU LV (lower than the standard value of 500ng/5.0*10 ⁷ TU LV).

In Example 3, compared with Comparative Example 1, the packaging process increased the number of lentivirus packaging 293T cells, and blood-free medium was used in the packaging process, as shown in Figure 5 and Table 1, during the purification process, the lentivirus The peak of 293T has spurious peaks, the purity of lentivirus decreases, the total DNA in residual detection exceeds the standard, and the titer of lentivirus decreases; at the same time, the amount of 293T cells increases, and due to the absence of serum, the cells have rapid apoptosis, at this time the cells The DNA in the tester will be released. During detection, the DNA will exceed the standard, and the total DNA residue will reach 348ng/ml (100ng/ml higher than the standard value) and the BSA residue will be 125.9ng/5.0*10 ⁷ TU LV (500ng/ml lower than the standard value). 5.0* ^107TULV ).

Therefore, it can be known from the scheme of the present invention that:

1. In the above comparative example 1 and examples 1-3, it is found by comparison that FBS can increase the titer of lentivirus, but it has higher requirements for downstream purification; the number of cells and the state of cells also affect the titer of lentivirus. certain influence;

2. FBS is added during lentivirus packaging, and the peak shape during purification is asymmetric, which increases the difficulty of purification; in the embodiment without FBS, when the number of packaging 293T cells is low, the state of the cells is very good, and the purified peak The shape of 293T cells is very clean. As the number of packaging 293T cells increases, there will be spurious peaks in the peak shape during purification, so the number of 293T cells of 8.0×10 ⁸ is the optimal ten-layer cell factory packaging number;

3. As shown in Figure 6, in Comparative Example 1, the complete medium containing FBS was added in the purification process to obtain the highest lentivirus titer; in Examples 1-3, the serum-free medium without FBS was added in the purification process, The titer of lentivirus will first increase and then decrease with the number of packaging cells 293T;

4. As shown in Table 1, the BSA of the lentivirus obtained in the example of adding FBS exceeds the standard, and at the same time, due to the influence of FBS, its total DNA also exceeds the standard, that is, due to the addition of FBS, other residual separation and purification are added; In the example of adding FBS, when the number of 293T cells is low, the index is the best, and as the number of cells increases, the state of 293T becomes worse, so that the total DNA residue in Example 3 exceeds the standard; therefore, 8.0 × 10 was selected. The number of 293T cells of ⁸ is the optimal ten-layer cell factory packaging number.

In conclusion, after the packaging and transfection of lentiviral cells 293T, 293T cells were purified by using serum-free medium for incubation. Compared with the complete medium (containing FBS) culture in Comparative Example 1, there were less DNA residues and BSA residues, and FBS has little effect on the purified peaks of 293T cells, which is beneficial to obtain lentiviruses that meet GMP standards.

It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed, and therefore should not be considered as a limitation on the scope of the patent protection of the present invention, and the patent protection scope of the present invention should be subject to the appended claims.

Claims (10)

1. a lentivirus packaging and purification method, is characterized in that, comprises the steps: The 293T cells in logarithmic growth phase were resuspended in complete medium and transferred to the cell factory for static culture; After 24 hours of static culture, remove the complete medium, add serum-free medium, and continue to incubate; The four plasmids, PEI and OPTI-MEM were mixed evenly and incubated, and then supplemented with serum-free medium to obtain a plasmid transfection mixture; After incubating the 293T cells in the cell factory, remove the serum-free medium, and then add the plasmid transfection mixture to the cell factory for transfection treatment; After the transfection treatment, the plasmid transfection mixture was removed, and a new serum-free medium was added for static culture; during the static culture, the lentivirus supernatant was collected; The lentivirus supernatant was sequentially subjected to Q membrane enrichment, nuclease enzymatic hydrolysis, and ion exchange treatment to obtain a lentivirus purified solution; After the lentivirus purified solution is subjected to ultrafiltration concentration and sterile filtration treatment in sequence, a purified chronic disease concentration solution is obtained, which is then packaged, frozen, and used for later use. 2. The lentivirus packaging and purification method according to claim 1, wherein when the 293T cells are transferred into a cell factory: the transferred amount of the 293T cells is 5.0×10 ⁸ to 10.0×10 ⁸ ; The factory was a ten-layer cell factory; and the complete medium was complete medium with 10% (v/v) FBS. 3. The lentivirus packaging and purification method according to claim 1, characterized in that, after the 293T cells are statically cultured for 24 hours, the added amount of the serum-free medium is 500-700 ml, and the incubation time is 1 Hour. 4 . The lentivirus packaging and purification method according to claim 1 , wherein, in the preparation step of the plasmid transfection mixture, the total number of PEI and four plasmids is 2:1 (v/m); the incubation time 10 to 30 minutes. 5 . The lentivirus packaging and purification method according to claim 1 , wherein 158 ml of OPTI-MEM is used in the plasmid mixing process, and after incubation, it needs to be supplemented to 625 ml of serum-free medium. 6 . 6 . The lentivirus packaging and purification method according to claim 1 , wherein, in the plasmid transfection treatment step, the transfection treatment time is 5-7 hours. 7 . 7 . The lentivirus packaging and purification method according to claim 1 , wherein, after the plasmid transfection treatment is completed, the amount of the new serum-free medium added is 1000-1400 ml. 8 . 8 . The lentivirus packaging and purification method according to claim 1 , wherein the collection time of the lentivirus supernatant is the 24th hour and the 48th hour of static culture, respectively. 9 . 9. The lentivirus packaging and purification method according to claim 1, wherein in the lentivirus supernatant treatment step, the Q membrane adopts Mustang Q XT5; the ion exchange used in the ion exchange treatment The packing of the chromatography column is Sepharose 4FF. 10. The lentivirus packaging and purification method according to claim 1, wherein the concentrated filtration treatment of the lentivirus purified solution further comprises: adding the concentrated lentivirus with a final concentration of 10% v/v human Serum albumin and sucrose aqueous solution with a final concentration of 5 (v/v) were mixed uniformly, and then sterile filtered through a 0.22 filter membrane.

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