LENTIVIRAL VECTOR MANUFACTURING PROCESS IN PACKED BED BIOREACTOR

Abstract

The present invention relates to the development of a method for the manufacturing of LVV vectors in a packed bed bioreactor by multi-plasmid transient DNA transfection. More particularly, the present invention discloses and claims a process in which steps applied in perfusion or in batch mode are combined to obtain an effective process. Moreover, the invention identifies an effective range of total DNA to be successfully used in the step of transient transfection in a packed bed bioreactor for the manufacturing of lentiviral vectors.

Claims

1. A method for the manufacturing of lentiviral vectors in a packed bed bioreactor comprising the following steps: a. inoculating the cells; b. culturing cells for expansion for at least one day; c. transfecting the cells using multi-plasmid DNA in a total amount between 45 ng/cm.sup.2 and 100 ng/cm.sup.2 of the surface area of the packed bed, and the PEI transfection reagent; and d. harvesting the cell culture supernatant containing the recombinant lentiviral vectors.

2. A method according to claim 1, further comprising the change of the medium at the end of transfection wherein such change of medium is performed in perfusion mode or in batch mode.

3. A method according to claim 2, wherein the change of medium in batch mode is performed by draining from the bioreactor the cell culture medium containing the DNA transfecting particles and adding fresh medium to the cell culture.

4. A method according to claim 1, wherein harvesting of the cell culture supernatant is performed in perfusion mode.

5. A method according to claim 3, comprising starting, immediately after the addition of the fresh medium, collecting the recombinant lentiviral vectors by harvesting in perfusion mode the cell culture medium from the bioreactor.

6. A method according to claim 1, wherein the cells are transfected for a period of up to 18 hours.

7. A method according to claim 4, wherein harvesting in perfusion mode is performed for at least 39 hours after the post transfection change of medium.

8. A method according to claim 4, wherein harvesting in perfusion mode is performed for 39 hours after the post transfection change of medium.

9. A method according to claim 1, further comprising a total change of medium between the step of culturing for expansion (b) and the step of transfection (c).

10. A method according to claim 1, wherein the cells are cultivated for expansion in perfusion mode by supplying in the bioreactor up to the end of the expansion phase, fresh medium in a total amount of at least 0.1 ml/cm.sup.2 of the surface area of the packed bed.

11. A method according to claim 1, wherein the cells are cultivated for expansion in recirculation mode, using as reservoir, fresh medium in a total amount of at least 0.1 ml/cm.sup.2 of the packed bed.

12. A method according to claim 1, wherein the transfection step (c) is performed in absence of perfusion or recirculation of fresh medium.

13. A method according to claim 1, wherein the transfection is performed using a ratio between DNA amount and PEI amount corresponding to 1:1.

14. A method according to claim 1, wherein the cells are transfected under step (c) for a period of 8 hours.

15. A method according to claim 1, wherein the cells are selected from HEK293, HEK293 T, HEK 293E, HEK 293FT, 293 Vec, TE671, HT1080 or HeLa cell line.

16. A method according to claim 1, wherein the multi-plasmid DNA is composed by one plasmid carrying the lentiviral gag/pol genes, one plasmid carrying lentiviral rev gene, one plasmid carrying the gene encoding the envelope protein, and one plasmid carrying the transfer vector including the required portion of the lentiviral genome and the foreign gene of interest.

Description

DESCRIPTION OF THE DRAWINGS

[0152] FIG. 1: flow chart representing the processes performed in the experiments disclosed in Example 1.

[0153] FIG. 2: flow charts representing the processes performed in the experiments disclosed in Example 2: (*) Recirculation was applied from day 1 to day 3 for run PDE_B18172 and from day 0 to day 3 from run PDE_B18187 on; (**) Condition applied only for run PDE_B18172. (***) Conditions applied from run PDE_B19013 on, except for bio2 of run PDE_B19087 (media change 6 h after transfection).

EXAMPLES

Example 1—Description of Lentiviral Vector Production in iCellis Nano 0.53 m.SUP.2

Example 1.1—Materials and Methods Packaging Cells and Cell Culture Medium

[0154] Lentiviral vector is produced by HEK 293T cultured in IMDM without phenol red, 10% FBS and 2% L-Glutamine or 2% Glutamax).

[0155] Bioreactor

[0156] During feasibility study, all the experiments described in this Example 1 have been done in iCELLis nano with the characteristics reported in Table 1.

TABLE-US-00001 TABLE 1 iCellis nano characteristics Bioreactor FB Carrier Size height Comp- Density No. of (m.sup.2) (cm) action (g/L) carriers 0.53 2 1 96 472

[0157] Plasmids

[0158] The multi plasmid DNA system used is a third generation LVVs packaging system including 4 separate plasmids: one plasmid carrying gag-pol genes (pGag/Pol), one plasmid encoding rev (pREV), one plasmid carrying VSV-g gene (pENV-VSV-G)), and one transfer vector plasmid carrying the gene encoding GFP protein (pTransfer-GFP).

[0159] Analytical Methods

[0160] The following analytical methods have been applied to evaluate process performance: [0161] Physical viral titer: this analytical method is an ELISA to quantify p24 HIV protein, which can be used to evaluate the concentration of physical particles. [0162] Infectious Viral Titer: this analytical method is based on transduction of reference CEM A3.01 cell line. The expression of the GFP protein is evaluated by FACS analysis. [0163] Residual Host Cell Proteins: this analytical method is an ELISA to quantify residual proteins from HEK-293T packaging cell line in LVV samples. [0164] Residual Total DNA: this analytical method is based on a fluorescent dye that stains and allows quantification of residual DNA.

Summary of the Manufacturing Process

[0165] A flow chart of the processes applied in experiments disclosed in Example 1 is reported in FIG. 1. Briefly, for each experiment cells were expanded in flasks in IMDM w/o phenol red and containing 10% FBS and 2% L-glutamine (or glutamax). After split, cells were inoculated in the bioreactor iCELLis nano (5000 cells/cm.sup.2). After expansion phase in which pH, DO %, glucose and lactate concentration were monitored, the culture medium was changed with fresh complete medium and transfection step was performed at day 3 or 4. After 18 hours, a new media change was performed in order to remove every residuals of plasmid DNA and PEI Pro. Two harvests have been collected at 48 h and 72 h after transfection step. The lentiviral vector from each harvest was filtered 0.45 μm and stored at <−65° C. in samples for testing. From run PDE_B17054 B in the experiments shown in this example 1, harvest was performed in perfusion mode and the LV vectors were collected after 48 h of perfusion.

[0166] Transfection Step

[0167] In all the experiments disclosed in this Example 1, production of LV vectors was performed using Polyethylenimine (PEI)-mediated transfection method, that is easy-to-use, with a high degree of reproducibility and consistency. In order to identify the appropriate amount of plasmid DNA to be used in the PEI transfection protocol, the feasibility study was performed starting from the amount resulting functional in other cell culture systems (such as flasks) and consistent with amount disclosed in prior art (i.e.: from 300 ng/cm.sup.2 to 400 ng/cm.sup.2 of surface area as disclosed in Valkama et al 2018 or from 200 to 300 ng/cm.sup.2 as disclosed in Leinonen et al 2019). The amount of PEI used has been set to maintain a ratio of 1:1 between PEI and DNA.

[0168] Protocol of transfection: [0169] Perform medium change with pre-warmed complete medium 1 hour before transfection. [0170] Prepare a DNA mix, diluting the correct amount of the plasmid DNA in IMDM free. [0171] Dilute the correct amount of PEI in IMDM free [0172] Transfer the diluted PEI in the tube containing the diluted DNA and mix. [0173] Incubate the final mix (100 mL for 1 bioreactor) according to the manufacturer instructions. [0174] Transfer the mix into into the bioreactor.

Example 1.2—Amount of Total DNA

[0175] In this first group of experiments the following conditions were applied for the performance of manufacturing runs in iCellis Nano Bioreactor having a surface area of 0.53 m.sup.2: [0176] Cell seeding density: 5000 c/cm.sup.2; [0177] Media change post transfection in batch at 18 h; [0178] 2 harvests performed at 48 h and 72 h after transfection.

[0179] The optimal amount of DNA for transfection have been investigated. The transfection has been performed at day 3 after the inoculum for every run. In run PDE_M16142, PDE_M16155 Bio1 and PDE_M16177 the transfection was performed using DNA in a total amount of 181 ng/cm.sup.2. In order to investigate different ratio, the double amount of DNA (2×) has been used in run PDE_M16121 Bio1 i.e.: 363 ng/cm.sup.2 (consistent with DNA quantity disclosed in prior art) and the half amount (½) in run PDE_M16155 Bio2 i.e: 91 ng/cm.sup.2. The different conditions used are summarizes in Table 2.

TABLE-US-00002 TABLE 2 Experimental conditions DNA Total amount of (ng)/surface plasmid DNA Process ID area (cm.sup.2) (μg) PDE_M16121 Bio1 363 1925 PDE_M16142 181 962 PDE_M16155 Bio1 181 962 PDE_M16155 Bio2 91 481 PDE_M16177 Bio2 181 962

[0180] Briefly, in all the experiments, 5000 cells/cm.sup.2 were inoculated in iCELLis nano. Sampling have been performed from day 3 to day 6 in order to monitor cell growth, measuring pH, lactate and glucose concentration. During all the experiments, at day 3 before transfection, three microcarriers were taken from each bioreactor and lysated; a nuclei count was performed in order to estimate the cell density for cm.sup.2; the exhausted culture medium was removed and changed with fresh medium. PEI-mediated transfection was performed according to the protocol described previously (Example 1.1). 18 hours after transfection the exhausted culture medium was removed (drained from the bioreactor) and changed with fresh medium for production. Two harvests in batch were performed 24 h, 48 h after media change.

[0181] The number of cells for cm.sup.2 at the transfection day and lactate produced (mg/m L) from day 3 to day 6 are reported in Table 3.

TABLE-US-00003 TABLE 3 Cell density (cells/cm.sup.2) and lactate (mg/mL) data Cell density Lactate (mg/mL) Process ID n. cells/cm.sup.2 Day 3 Day 4 Day 5 Day 6 PDE_M16121 Bio1 1.22E+05 0.745 0.756 0.737 0.865 PDE_M16142 1.37E+05 0.756 1.11 1.02 1.22 PDE_M16155 Bio1 0.98E+05 0.832 1.17 1.04 1.25 PDE_M16155 Bio2 1.15E+05 0.847 1.25 1.2 1.48 PDE_M16177 Bio2 1.02E+05 0.746 1.1 1.0 1.31

[0182] The results are summarized in Table 4:

TABLE-US-00004 TABLE 4 Productivity data Physical viral titer ngP24/ Infectious viral titer Infectivity Process ID ngP24/mL tot ngP24 cm.sup.2 TU/mL Total TU TU/cm2 TU/ngP24 PDE_M16121 134 2.41E+05 45.5  8.0E+06 1.44E+10 2.72E+06 5.97E+04 Bio1 PDE_M16142 345 6.21E+05 117.2  1.5E+07 2.70E+10 5.09E+06 4.35E+04 PDE_M16155 289 5.20E+05 98.2  9.0E+06 1.62E+10 3.06E+06 3.11E+04 Bio1 PDE_M16155 163 2.93E+05 55.4  6.0E+06 1.08E+10 2.04E+06 3.68E+04 Bio2 PDE_M16177 262 4.72E+05 89.0 1.00E+07 1.80E+10 3.40E+06 3.82E+04 Bio2

[0183] As the results show, bulk productions obtained using in transfection total DNA in an amount of 181 ng/cm.sup.2 and 91 ng/cm.sup.2 result to have higher productivity compared to runs obtained with 363 ng/cm.sup.2 of total DNA. For this reason, in the following runs, 962 μg have been used for transfection step.

Example 1.3—Incubation of the Transfection Mix

[0184] In the following group of experiments, the transfection was performed at day 3 in iCellis Nano Bioreactor having a surface area of 0.53 m.sup.2 using multi-plasmid DNA in total amount of 962 μg per bioreactor (i.e.: 181 ng/cm.sup.2 of surface area) and PEI PRO (1 μg/ul) in total amount of 962 μg. In run PDE_B17028 A, media change was performed 6 h after the starting of the transfection phase, in order to investigate if an earlier medium change was associated to enhanced LVV productivity. In order to reduce the amount of FBS used in the process, in run PDE_M17028B a reduction of percentage of serum (2.5%) during the production phase was investigated.

[0185] The different tested conditions are indicated in Table 5.

TABLE-US-00005 TABLE 5 Experimental schedule Media change post Process ID % of FBS transfection PDE_M16203 A 10 18 h PDE_M17028 A 10  6 h PDE_M17028 B  2.5 18 h

[0186] 5000 cells/cm.sup.2 were inoculated in iCELLis nano at day 0. Sampling have been done from day 3 to day 6 in order to monitor cell growth; at day 3 before transfection, three carriers were taken from each bioreactor and lysates; a nuclei count was performed in order to estimate the cell density for cm.sup.2. The exhausted culture medium was removed, changed with new growing medium and Pei-mediated transfection was performed; 18 h after transfection step (6 hours for run PDE_M17028 A), exhausted medium change in batch mode was performed. In run PDE_M17028 B the exhausted media was replaced with IMDM 2.5% FBS and 2% L-glutamine. As in the previous runs, two harvests in batch were performed 24 h and 48 h after media change.

[0187] The results of physical and infectious viral titer and infectivity of pool harvest are summarized in Table 6.

TABLE-US-00006 TABLE 6 Productivity data Physical viral titer Infectious viral titer Infectivity Process ID ngP24/mL tot ngP24 ngP24/cm.sup.2 TU/mL Total TU TU/cm2 TU/ngP24 PDE_M16203 A 271 4.88E+05 92.0 1.0E+07 1.80E+10 3.40E+06 3.69E+04 PDE_M17028 A 155 2.79E+05 52.6 3.0E+06 5.40E+09 1.02E+06 1.94E+04 PDE_M17028 B 293 5.27E+05 99.5 8.0E+06 1.44E+10 2.72E+06 2.73E+04

[0188] The results of the run PDE_M17028 A showed that an earlier medium change after transfection (6 hours of incubation of cells with the transfection mix) decreased the production of the vector; reduction of percentage of FBS during the harvest phase (PDE_M17028 B) had no impact on production, but reduced viral titer and infectivity of the LV vector.

Example 1.4 Collection of LVVS by Harvesting in Perfusion

[0189] In order to increase the infectivity of viral vector produced and avoid the emptying/filling time between harvests of iCELLis 500, in run PDE_B17133, harvest in perfusion was evaluated and compared with harvest in batch. In detail, PDE_B17133 A was performed with two harvests in batch, while in run PDE_B17133B LV vector was harvested in perfusion mode. Both bioreactors followed the same process until transfection phase. Briefly, two bioreactors were inoculated with 5000 Cells/cm.sup.2 at day 0. After three days, medium change pre-transfection with complete medium was performed; followed by transfection with the same protocol followed in the previous runs. After 18 hours, media change post-transfection in batch was performed with new complete medium. Sampling have been done from day 3 to day 6 in order to monitor cell growth; before transfection, three carriers were taken from each bioreactor and lysate to perform nuclei count.

[0190] In PDE_B17133 A, the supernatant was collected in perfusion with at fix rate (13.5 mL/hour rate): fresh medium was transferred into an IN bottle, connected with the bioreactor and perfused in 48 hours. During harvest in perfusion, supernatant was collected in an OUT bottle. Samples from the OUT bottle were collected at 24 h and 48 h after media change, filtered 0.45 μm and stored at <−65° C. At 48 h, supernatant inside bioreactor and in OUT bottle were pooled, filtered and stored at <−65° C. Samples from pool were collected, filtered 0.45 μm and stored at <−65° C.

[0191] In PDE_B17133 B, two harvests were collected in batch at 24 h and 48 h after media change post transfection. Samples from each harvest were collected and stored at <−65° C. At the end of the process, the two harvest were pooled, filtered and stored at <−65° C.

[0192] In Table 7 are summarizes the conditions used for the two bioreactors.

TABLE-US-00007 TABLE 7 Experimental schedule of processes PDE_B17133A and PDE_B17133B Day PDE_B17133A PDE_B17133B 0 Seeding at 0.5 × 10.sup.4/cm.sup.2 1 Expansion phase 2 3 Media change pre-transfection PEI-mediated transfection step (as disclosed in example 1.3) 4 Media change post-transfection Start perfusion: 13.5 mL/h n.a. 5 Harvest in perfusion in 48 hours 1′ harvest 6 2′ harvest

[0193] Results are reported in the Table 8.

TABLE-US-00008 TABLE 8 Productivity data Physical viral titer Infectious viral titer Infectivity Process ID ngP24/mL tot ngP24 ngP24/cm.sup.2 TU/mL Total TU TU/cm2 TU/ngP24 PDE_B17133 A 168 2.18E+05 41.00 8.0E+06 1.04E+10 1.96E+06 4.76E+04 PDE_B17133 B 179 2.33E+05 43.00 8.0E+06 1.04E+10 1.96E+06 4.47E+04

[0194] The results demonstrated that the physical and infectious viral titer and infectivity obtained from the two bioreactors were comparable. The condition that has been introduced (supernatant harvested in perfusion started after post transfection change of medium in batch) allows to maintain the same level of productivity of the harvest in batch.

Example 1.5 Culturing Conditions Pre-Transfection

[0195] In order to optimize the scale up in iCellis 500 and avoid the emptying/filling time during all phases of the process, an alternative to media change pre-transfection in batch was evaluated. In particular, in runs PDE_B18034 and PDE_B18068, media change in perfusion and in recirculation were introduced. The Table 9 shows the investigated conditions in each run.

TABLE-US-00009 TABLE 9 Experimental schedule for Group #9 Medium change Volume of Process ID pre transfection medium change PDE_B18034 A Perfusion 1300 mL PDE_B18034 B* Batch  650 mL PDE B18068 A Recirculation  650 mL PDE_ B18068 B Perfusion 1300 mL PDE_B18068 C Perfusion  650 mL

[0196] Briefly, each bioreactor iCellis nano was inoculated at day 3 with 5000 Cells/cm.sup.2 and sampling have been done from day 3 to day 6 to monitor pH, lactate and glucose.

[0197] At day 3, medium change pre-transfection was performed as explained below: [0198] PDE_B18034 A and PDE_B18068 B: exhausted medium has been changed with new growing medium in perfusion mode. Perfused volume was 1300 mL. [0199] PDE_B18034 B: medium change was performed in batch; exhausted medium was removed and bioreactor was filled with 650 mL of new growing medium. [0200] PDE_B18068 C: exhausted medium has been changed with new growing medium in perfusion mode. Perfused volume was 650 mL.

[0201] PDE_B18068A: in this run media change was not performed, but at day 1 after inoculum, recirculation started with 650 mL of fresh medium, until day 3.

[0202] PEI-mediated transfection was performed as disclosed in example 1.3. From transfection phase to the end of the process, the harvest in perfusion for 48 hours has been performed in the three bioreactors During perfusion phase, samples have been collected at 24 h and 48 h post media change from OUT bottle in order to measure physical and infectious viral title.

[0203] Total pool from PDE_B18034 and PDE_B18068 were tested for physical and infectious viral title.

[0204] The results of physical and infectious viral title and infectivity of the experiments are reported in Table 10:

TABLE-US-00010 TABLE 10 Productivity data Physical viral titer Infectious viral titer Infectivity Process ID ngP24/mL tot ngP24 ngP24/cm.sup.2 TU/mL Total TU TU/cm2 TU/ngP24 PDE_B18034 239 3.11E+05 58.00 8.0E+06 1.04E+10 1.96E+06 3.35E+04 A PDE_B18034 170 2.21E+05 41.00 4.0E+06 5.20E+09 9.81E+05 2.35E+04 B PDE_B18068 211 2.74E+05 51.00 8.0E+06 1.04E+10 1.96E+06 3.79E+04 A PDE_B18068 259 3.37E+05 63.00 9.0E+06 1.17E+10 2.21E+06 3.47E+04 B PDE_B18068 212 2.76E+05 52.00 9.0E+06 1.17E+10 2.21E+06 4.25E+04 C

[0205] The results demonstrated that the productivity of LV vector is higher in runs in which cell culture for expansion is performed with media change in perfusion or recirculation mode, if compared with standard condition (run PDE_B18034B, medium change performed in batch).

Example 1.5 Post Transfection Change of Medium

[0206] The goal of this group of experiments was the study of the best technical solution to perform the medium change post transfection.

[0207] In detail, in run PDE_B18080 A, medium change post transfection was performed in perfusion in order to avoid the emptying and filling of the vessel (laborious and difficult in iCellis 500 and discouraged in prior art such as WO 2018/007873). 18 h after transfection, 1300 mL of complete fresh IMDM were perfused with a ratio of 9 mL/min in order to exchange 90% of medium.

[0208] In run PDE_B18080 B, holding time of 50′ was introduced during medium change post transfection in batch in order to mimic the emptying/filling timing of iCELLis 500. 18 hours after transfection phase, exhausted medium was removed and bioreactor was left empty for 50′ leaving only 16 mL inside it; this amount of medium correspond to the volume that cannot be recovered in iCellis 500 during the empting due to technical limitation of the system. After 50′ bioreactor was filled with new fresh medium.

[0209] In PDE_B18080C, medium change post transfection was performed with standard condition (batch, without holding time) in order to compare it with the new conditions introduced.

[0210] Samples have been done from the three bioreactors before and after medium change post transfection in order to evaluate plasmid residual DNA. In bioreactor A, samples have been done from the vessel after the half volume perfusion.

[0211] Different medium change conditions are summarized in Table 11.

TABLE-US-00011 TABLE 11 Medium change conditions tested Medium change post Holding Process ID transfection time PDE B18080 A Perfusion n.a. PDE B18080 B Batch 50 PDE B18080 C Batch n.a.

[0212] During the other phases of the process, all three bioreactors followed the standard conditions set in the previous run and reported in Table 12.

TABLE-US-00012 TABLE 12 Experimental schedule Day Step description 1 Inoculum at day 1, 5000 cells/cm.sup.2. 3 Sample from bioreactor to monitor pH and glu/lat. Medium change pre- transfection in perfusion (650 mL). Pei -mediated transfection according to protocol described in Example 1.3 4 Sample from bioreactor to monitor pH and glu/lat, and to evaluate contaminants. Medium change post transfection. Sample from bioreactor to evaluate contaminants. Start harvest in perfusion 5 Sample from bioreactor to monitor pH and glu/lat. Sample from out bottle to test p24. 6 Sample from bioreactor to monitor pH and glu/lat. Sample from out bottle to test p24. Harvest of total volume of supernatant-Sample from total pool to test physical and infectious viral titer.

[0213] Total pool of three bioreactors were tested for physical and infectious viral titer, HCP, plasmid and total residual DNA.

[0214] The results are reported in Table 13 and Table 14:

TABLE-US-00013 TABLE 13 Productivity data Physical viral titer Infectious viral titer Infectivity Process ID ngP24/mL tot ngP24 ngP24/cm.sup.2 TU/mL Total TU TU/cm2 TU/ngP24 PDE_B18080 A 158 2.05E+05 38.00 1.6E+07 2.08E+10 3.92E+06 1.01E+05 PDE_B18080 B 282 3.67E+05 69.00 2.0E+07 2.60E+10 4.91E+06 7.09E+04 PDE_B18080 C 217 2.82E+05 53.00 2.0E+07 2.60E+10 4.91E+06 9.22E+04

TABLE-US-00014 TABLE 14 Residuals data Process ID HCP (ng/mL) Total DNA (ng/ml) PDE_B18080 A 1882  997 PDE_B18080 B 2885 1597 PDE_B18080 C 2494 1262

[0215] As results show, it was surprisingly found that the productivity of LV vectors was lower in the bioreactor A, performed with medium change post transfection in perfusion mode (PDE_B18080 A), than the run performed with medium change post transfection performed in batch (PDE_B18080 C). The productivity of run PDE_B18080B, with medium change post transfection performed in batch mode with holding time, was also higher than that obtained in the run performed with medium change post transfection performed in perfusion (PDE_B18080 A), and even higher than the run PDE_B18080 C, while the infectious viral titer of run PDE_B18080 B is the same of the run PDE_B18080 C. The amount of contaminants, HCP, plasmid and total DNA is equivalent between the two runs PDE_B18080 B and PDE_B18080 C. In view of the scaling up to large scale in iCellis 500, medium change in batch with holding time in iCellis nano is more consistent with run performed in large scale iCellis 500 (since 50′ is the time needed for empty and filling the bioreactor). Therefore, it is more representative of the performance of the run in large scale.

Example 2—Lentiviral Vector Production in iCELLis Nano 1.06 m.SUP.2

Example 2.1—Materials and Methods Packaging Cells and Cell Culture Medium

[0216] The production of the LVV vectors described in this Example 2.1 has been performed with the HEK 293T cell line. HEK 293T cells were thawed and expanded in IMDM 10% FBS, 2% Glutamax.

[0217] Bioreactor

[0218] Except for the first experiment of example 2.2, in which one run has been performed in iCellis Nano having a surface area of 0.53 m.sup.2 bioreactor, all the other runs described in examples 2.2 and 2.3 have been performed with iCellis Nano having a surface area of 1.06 m.sup.2 bioreactor. Characteristics of the two kind of support are reported in Table 15.

TABLE-US-00015 TABLE 15 iCELLis nano characteristics Carrier Bioreactor FB height Density No. of Size (m.sup.2) (cm) Compaction (g/L) carriers 0.53 2 1 96 473 1.06 4 1 96 946

[0219] Plasmids

[0220] The production of the LVV vectors in iCELLis nano in this example 2 a multi-plasmid DNA system used is a third generation LVVs packaging system including 4 separate plasmids: one plasmid carrying gag-pol genes (pGag/Pol), one plasmid encoding rev (pREV), one plasmid carrying VSV-g gene (pENV-VSV-G)), and one transfer vector plasmid carrying the gene encoding GFP protein (pTransfer-GFP).

[0221] Analytical Methods

[0222] The following analytical methods has been applied to evaluate process performance: [0223] Physical viral titer: this analytical method is an ELISA to quantify p24 HIV protein, which can be used to evaluate the concentration of physical particles. [0224] Infectious Viral Titer: this analytical method is based on transduction of reference CEM A3.01 cell line. The expression of the GFP protein is evaluated by FACS analysis. [0225] Residual Host Cell Proteins: this analytical method is an ELISA to quantify residual proteins from HEK-293T packaging cell line in LVV samples. [0226] Residual Total DNA: this analytical method is based on a fluorescent dye that stains and allows quantification of residual DNA.

Summary of the Manufacturing Process

[0227] A flow chart of the processes applied in experiments disclosed in Example 2 is reported in FIG. 2. Briefly, for each experiment cells have been expanded in T-flasks in IMDM w/o phenol red and containing 10% FBS and 2% Glutamax. After split, cells have been inoculated in the bioreactor iCELLis nano (5000 cells/cm.sup.2). After expansion phase in which pH, DO %, glucose and lactate concentration were monitored, the culture medium was changed (in perfusion or through recirculation) with fresh complete medium and transfection step was performed at day 3. After 21 hours a media change in batch was performed in order to remove all residuals of plasmid DNA and PEI Pro. Harvest was performed in perfusion mode. Lentiviral vector supernatant has been collected 48 hours after media change post transfection. From run PDE_B18030 on, media change post transfection and harvest were anticipate at day 3 and day 5 respectively, to collect a vector supernatant with higher infectivity. Lentiviral vector's samples during harvest in perfusion have been filtered 0.45 μm and stored at <−65° C. for testing.

[0228] Transfection Step

[0229] In all the experiments reported in this example 2, production of LVV vector was performed using Polyethylenimine (PEI)-mediated transfection method. The optimization study was performed using the amount of μg of plasmid/mL previously tested in Example 1. Till the run PDE_B18187 DNA/cell ratio was maintained, doubling the volume of transfection mix and the quantity of DNA and PEI in 1.06 m2 bioreactor (1924 μg total DNA). Then the amount of DNA/cell was halved, using the same volume of transfection mix and DNA/mL ratio of 0.53 m2 iCELLis nano (962 μg total DNA). The amount of PEI used has been set to maintain a ratio of 1:1 between PEI and DNA. Protocol for transfection at day 3: [0230] 1. Perform medium change in perfusion (only if provided) with pre-warmed complete medium 1 hour before transfection. [0231] 2. Prepare a total DNA mix for the bioreactors, diluting the correct amount of DNA in IMDM free [0232] 3. Dilute the correct amount of PEI in IMDM free [0233] 4. Transfer the diluted PEI in the tube containing the diluted DNA and mix. [0234] 5. Incubate the final mix for according to the manufacturer instructions. [0235] 6. Remove the correct volume from bioreactor, transfer the mix into the bioreactor.

Example 2.2—Manufacturing in iCELLis Nano 1.06 m.SUP.2 .Vs iCELLis Nano 0.53 m.SUP.2

[0236] The goal of this first group of experiments was to test: [0237] Bioreactor with 1.06 m.sup.2 area; [0238] Recirculation during cell culture; [0239] Reduction of harvest volume;

[0240] In all these experiments, 5000 cells/cm.sup.2 were inoculated in iCELLis nano at day 0. After an expansion phase in flask T225, 26.5×10.sup.6 cells (in 0.53 m.sup.2 bioreactor) and 53×10.sup.6 cells (in 1.06 m.sup.2 bioreactor) were inoculated in the bioreactor. Control Petri dishes have been seeded in parallel at 5.50×10.sup.5 cells/petri in order to monitor the transfection phase.

[0241] Sampling have been done from bioreactor during the process in order to measuring pH, lactate and glucose concentration. In the first two runs, at day 3, three carriers were taken from each bioreactor and lysate; a nuclei count was performed in order to estimate the cell density for cm.sup.2.

[0242] Recirculation mode during cell culture for expansion has been evaluated. For all experiments, PEI-mediated transfection was performed according to the protocol described in Example 2.1. The transfection mix for the 0.53 m.sup.2 iCELLis nano was prepared using 962 μg of total DNA (corresponding to 181 ng/cm.sup.2 of surface area) and 962 μg PEI in a total volume of 100 ml. In 1.06 m.sup.2 iCELLis nano, volume of transfection mix and quantity of DNA and PEI have been doubled. 21 hours after transfection, the exhausted culture medium was removed and, after a holding time of 50 minutes, replaced with fresh medium for production. Harvests in perfusion was performed till 48 h after media change post transfection

TABLE-US-00016 TABLE 16 Experimental schedule Media Transfection Area change mix Process ID (m.sup.2) pre transf. Recirculation DNA (μg) PDE_B18172 Bio1 0.53 Yes no  962 PDE_B18172 Bio2 1.06 Yes (1300 mL) 1924 PDE_B18172 Bio3 1.06 No (1950 mL) 1924 PDE_B18187 Bio1 1.06 No (1300 mL) 1924 PDE_B18187 Bio2 1.06 No (1300 mL) 1924 PDE_B18187 Bio3 1.06 No (1300 mL) 1924 PDE_B19002 Bio1 1.06 No (1300 mL) 1924 PDE_B19002 Bio2 1.06 No (1300 mL) 1924 PDE_B19002 Bio3 1.06 No (1300 mL) 1924

[0243] The number of cells/cm.sup.2 and the amount of lactate produced (mg/mL) have been evaluated to monitor growth rate and are reported in Table 17.

TABLE-US-00017 TABLE 17 Cell densisty (cells/cm.sup.2) and lactate produced (mg/mL) Day 1 Day 3 Day 4 Day 5 Day 6 Process ID Lac in Lac in cells/cm.sup.2 Lac in Lac in Lac out Lac in Lac out cells/cm.sup.2 PDE_B18172 0.265/ 0.850/ 0.88E+05 1.52/ 1.27/ n.a. 1.48/ 1.03/ 3.1E+05 Bio1 0.261 0.842 1.52 1.23 1.53 1.07 PDE_B18172 0.409/ 0.964/ 1.39E+05 1.95/ 0.952/ n.a. 1.18/ 0.919/ 3.1E+05 Bio2 0.411 0.945 2.18 0.934 1.13 0.899 PDE_B18172 0.362/ 0.684/ 1.06E+05 2.00/ 0.797/ n.a. 0.937/ 0.788/ 2.3E+05 Bio3 0.368 0.663 2.10 0.815 0.917 0.767 PDE_B18187 0.422/ 0.914/ 1.40E+05 2.36/ 1.13/ n.a. 1.20/ 1.02/ n.a. Bio1 0.417 0.901 2.35 1.11 1.21 1.00 PDE_B18187 0.261/ 0.853/ 1.20E+05 2.28/ 1.12/ n.a. 1.25/ 1.01/ n.a. Bio2 0.264 0.871 2.29 1.13 1.25 1.00 PDE_B18187 0.429/ 0.833/ 0.93E+05 2.25/ 1.13/ n.a. 1.19/ 0.973/ n.a. Bio3 0.435 0.848 2.34 1.14 1.20 1.01 PDE_B19002 n.a. 0.838 n.a. 1.28 0.880 0.695 1.04 0.808 7.3E+05 Bio1 PDE_B19002 n.a. 0.821 n.a. 1.19 1.24 0.892 1.46 1.10 4.8 E+05 Bio2 PDE_B19002 n.a. 0.853 n.a. 1.21 1.03 0.780 1.14 0.942 4.2E+05 Bio3

[0244] The results of viral titer in terms of P24 and TU are summarized in Table 18.

TABLE-US-00018 TABLE 18 Productivity data Physical viral titer Infectious viral titer Infectivity Process ID ngP24/mL tot ngP24 ngP24/cm.sup.2 TU/mL Total TU TU/cm2 TU/ngP24 PDE_B18172 440 n.a. n.a. 1.6E+07 n.a. n.a. 3.6E+04 petri PDE_B18172 151 1.96E+05 37 7.0E+06 9.1E+09 1.7E+06 4.6E+04 Bio1 PDE_B18172 253 6.70E+05 62 1.2E+07 3.1E+10 2.9E+06 4.7E+04 Bio2 PDE_B18172 281 7.45E+05 69 1.2E+07 3.1E+10 2.9E+06 4.3E+04 Bio3 PDE_B18187 182 4.82E+05 45 1.0E+07 2.6E+10 2.5E+06 5.5E+04 Bio1 PDE_B18187 227 6.02E+05 56 1.5E+07 3.9E+10 3.7E+06 6.6E+04 Bio2 PDE_B18187 221 2.41E+05 45 1.2E+07 2.6E+10 2.4E+06 5.4E+04 Bio3 PDE_B19002 226 5.88E+05 55 9.4E+06 2.4E+10 2.3E+06 4.2E+04 Bio1 PDE_B19002 347 6.21E+05 70 1.5E+07 3.2E+10 3.0E+06 4.3E+04 Bio2 PDE_B19002 347 8.11E+05 70 1.5E+07 3.2E+10 2.3E+06 4.3E+04 Bio3

[0245] The amounts of HCP and total DNA of the pool harvest are reported in Table 19.

TABLE-US-00019 TABLE 19 HCP and total DNA (ng/mL) amounts HCP Tot DNA Pool Pool Process ID harvest harvest PDE_B18172 Bio1 1028 514 PDE_B18172 Bio2 2760 1766 PDE_B18172 Bio3 2831 1532 PDE_B18187 Bio1 1757 1144 PDE_B18187 Bio2 1981 1319 PDE_B18187 Bio3 2026 1337 PDE_B19002 Bio1 2151 1801 PDE_B19002 Bio2 2263 1479 PDE_B19002 Bio3 1840 1176

[0246] The results demonstrate that the infectious viral titer of vector produced in 1.06 m.sup.2 iCELLis bioreactor is higher than that produced in 0.53 m.sup.2 iCELLis nano. Recirculation mode doesn't cause any worsening to cell productivity of LVV vectors and replaces media change.

Example 2.3—Manufacturing in iCELLis Nano 1.06 m.SUP.2

[0247] In this example 2.3 the conditions set in the previous runs (example 2.2) were maintained: [0248] 1.06 m.sup.2 iCELLis bioreactor; [0249] No media change pre transfection; [0250] Recirculation during cell culture; [0251] Harvesting in perfusion.

[0252] Transfection step in a bioreactor having 1.06 m2 surface area and media change post transfection were investigated. In run PDE_B18197 different ratio of DNA/n° of cells and DNA/mL have been tested. In Bio 1 same transfection condition used in Example 2.2 were applied (i.e.: 1924 μg total DNA, i.e: 181 ng/cm.sup.2 of surface area, 1924 μg PEI, in transfection mix volume of 200 ml). In Bio2, halved volume of transfection mix (100 ml) has been used compared to the previous experiments with 1.06 m.sup.2 bioreactor. So the DNA/n° of cells ratio has been maintained, but the concentration of DNA and PEI was twice as much as the total volume. In Bio3 the same volume (100 ml) and amount of DNA (962 μg, i.e: 91 ng/cm2 of surface area) and PEI (962 μg) used for 0.53 m.sup.2 iCELLis nano has been tested. In this way DNA/mL ratio has been maintained, but the amount of DNA and PEI was half compared to the number of cells.

[0253] The different conditions used are summarizes in Table 20.

TABLE-US-00020 TABLE 20 Amount of plasmid DNA and PEI μg/Bioreactor Plasmid Bio1* Bio2** Bio3** Total DNA 1924 1924 962 Total PEI-PRO 1924 1924 962 (1μg/μl) *In 200 mL. **In 100 mL.

[0254] In run PDE_B19013 conditions of PDE_B18197 Bio3 have been reproduced, but different timing for transfection step and following media change has been tested. In Bio1 previous conditions were maintained (transfection at 11.30 on Monday and media change post transfection on Tuesday morning); in Bio2 both transfection step and media change have been performed on Monday, respectively at 9.30 and 17.30. Bio3 has been carried out as Bio2, but with a delay of 2 hours from Monday on. In all 3 bioreactors harvest in perfusion has started after media change post transfection.

[0255] Briefly, 5000 cells/cm.sup.2 were inoculated in iCELLis nano at day 0. Media recirculation has been done during cell culture for transfection. Then, transfection step and media change post transfection in batch have been performed as previously described. Harvest in perfusion has started after media change post transfection.

[0256] Sampling have been done from day 3 to day 6 (morning and afternoon) in order to monitor cell growth.

[0257] Experimental conditions are summarized in Table 21.

TABLE-US-00021 TABLE 21 Experimental schedule Media Harvest in Process ID Transfection change perfusion PDE_B18197 Bio1 21 h Tuesday 48 h PDE_B18197 Bio2 21 h Tuesday 48 h PDE_B18197 Bio3 21 h Tuesday 48 h PDE_B19013 Bio1 21 h Tuesday 48 h PDE_B19013 Bio2  8 h Monday 63 h PDE_B19013 Bio3  8 h Monday 61 h

[0258] The number of cells/cm.sup.2 and the amount of lactate produced (mg/mL) have been evaluated to monitor growth rate and are reported in Table 22.

TABLE-US-00022 TABLE 22 Cell densisty (cells/cm.sup.2) and lactate produced (mg/ml) Day 3 Day 4 Day 5 Day 6 Lac Lac Lac cells/ Process ID Lac in cells/cm.sup.2 Lac in out Lac in out Lac in out cm.sup.2 PDE_B18197 Bio1 0.928/ 1.44E+05 2.21/ n.a. 0.811/ 0.674/ 0.962/ 0.786/ 5.12E+05 0.916 2.33 0.821 0.694 0.983 0.800 PDE_B18197 Bio2 0.944/ 1.95E+05 2.47/ n.a. 1.09/ 0.833/ 1.28/ 1.03/ 2.26E+05 0.942 2.48 1.13 0.872 1.29 1.04 PDE_B18197 Bio3 0.901/ 1.64E+05 2.20/ n.a. 0.943/ 0.702/ 1.08/ 0.858/ 0.50E+05 0.900 2.19 0.951 0.708 1.09 0.866 PDE_B19013 Morning 0.687 n.a. 2.01 n.a. 0.964 0.722 1.22 0.843 n.a. Bio1 Afternoon n.a. n.a. 0.609 0.464 1.03 0.739 n.a. n.a. n.a. PDE_B19013 Morning 0.717 n.a. 0.835 0.648 1.27 0.928 1.54 1.08 n.a. Bio2 Afternoon 1.25 n.a. 1.04 0.737 1.31 0.936 n.a. n.a. n.a. PDE_B19013 Morning 0.660 n.a. 0.716 0.544 1.02 0.757 1.25 0.869 n.a. Bio3 Afternoon 1.24 n.a. 0.842 0.608 1.03 0.767 n.a. n.a. n.a.

[0259] The results of physical and infectious viral titer and infectivity of pool harvest are summarized in Table 23.

TABLE-US-00023 TABLE 23 Productivity data Physical viral titer ngP24/ ngP24/ Infectious viral titer Infectivity Process ID mL tot ngP24 cm.sup.2 TU/mL Total TU TU/cm2 TU/ngP24 PDE_B18197 Bio1 231 6.01E+05 58 9.0E+06 2.3E+10 2.2E+06 3.9E+04 PDE B18197 Bio2 198 5.15E+05 50 7.0E+06 1.8E+10 1.7E+06 3.5E+04 PDE_B18197 Bio3 231 6.01E+05 58 1.1E+07 2.9E+10 2.7E+06 4.8E+04 PDE_B19013 * 229 3.78E+05 36 7.8E+06 1.3E+10 1.2E+06 3.4E+04 Bio1 ** 267 5.74E+05 65 1.0E+07 2.2E+10 2.0E+06 3.7E+04 PDE_B19013 * 186 3.63E+05 34 1.4E+07 2.7E+10 2.6E+06 7.5E+04 Bio2 ** 194 5.02E+05 47 9.9E+06 2.6E+10 2.4E+06 5.1E+04 PDE_B19013 * 172 3.35E+05 32 1.1E+07 2.2E+10 2.0E+06 6.5E+04 Bio3 ** 198 5.00E+05 47 9.4E+06 2.4E+10 2.2E+06 4.7E+04 *: Wednesday **: Thursday

[0260] The amounts of HCP and total DNA of the pool harvest are reported in Table 24.

TABLE-US-00024 TABLE 24 HCP and total DNA (ng/mL) amounts Process ID HCP Tot DNA PDE_B18197 Bio1 2116 1192 PDE_B18197 Bio2 1794 864 PDE_B18197 Bio3 1870 1058 PDE_B19013 Bio1 * <866 1727 ** 1285 1370 PDE_B19013 Bio2 * <LLOQ (624 ng/ml) neg. ** <LLOQ (624 ng/ml) neg. PDE_B19013 Bio3 * <LLOQ (624 ng/ml) 545 ** <LLOQ (624 ng/ml) 959

[0261] The results show that the production of LVV vector in terms of P24 in experiment PDE_B18197 Bio3 is the same of Bio1, and higher than Bio 2 demonstrating that, surprisingly, it was possible to obtain the same or even higher productivity using half of the total amount of DNA. Moreover, this condition improves the infectious viral titer, bringing to an increase of infectivity.

[0262] Reduction of time of transfection from 21 h to 8 h in run PDE_B19013 does not reduce much the physical viral titer, but increases the infectivity. In particular, the results show that the infectivity of Bio2 and Bio3 on Wednesday is higher than in the other run, in which the harvest had been performed till Thursday. The results of HCP and Total DNA of run PDE_B19013 Bio2 and Bio3 are lower than in the previous run, demonstrating that supernatant has a lower presence of impurities.

Example 2.4—Transfection and Harvesting in Three Days

[0263] In order to confirm the results obtained, the conditions previously tested in example 2.3 and chosen as the best have been performed again in run described in the Table 25.

TABLE-US-00025 TABLE 25 Experimental schedule Transfection Harvest mix Trans- Media in Process ID DNA (μg) fection change perfusion PDE_B19030 Bio1 962  8 h Monday 39 h PDE_B19030 Bio2 962 21 h Tuesday 48 h PDE_B19030 Bio3 962  8 h Monday 39 h PDE_B19041 Bio2 962  8 h Monday 39 h PDE_B19041 Bio3 962  8 h Monday 39 h PDE_B19048 Bio1 962  8 h Monday 39 h PDE_B19048 Bio2 962  8 h Monday 39 h PDE_B19048 Bio3 962  8 h Monday 39 h PDE_B19054 Bio1 962  8 h Monday 39 h PDE_B19054 Bio2 962  8 h Monday 39 h PDE_B19054 Bio3 962  8 h Monday 39 h

[0264] As in the previous run, 5000 cells/cm.sup.2 were inoculated in iCELLis nano at day 0. Media recirculation has been performed up to day 3 with 1300 mL of fresh medium. PEI-mediated transfection has been performed at 10.00 am and media change post transfection in batch have been done 8 hours later, both on day 3. Harvest in perfusion has started after media change post transfection. Sampling have been done from day 3 to day 6 in order to monitor cell growth. The amount of lactate produced (mg/mL) have been evaluated to monitor growth rate and are reported in Table 26.

TABLE-US-00026 TABLE 26 Lactate produced (mg/mL) Day 3 Lac in Day 4 Day 5 Day 6 Process ID Morning Afternoon Lac in Lac out Lac in Lac out Lac in Lac out PDE_B19030 Bio1 0.809 1.37 0.716 0.544 1.04 0.736 n.a. n.a. PDE_B19030 Bio2 0.855 n.a. 2.30 n.a. 1.13 0.795 1.49 1.03 PDE_B19030 Bio3 0.834 1.49 0.832 0.600 1.13 0.808 n.a. n.a. PDE_B19041 Bio2 0.559 1.24 0.874 0.647 1.02 0.799 PDE_B19041 Bio3 0.613 1.31 0.857 0.637 1.03 0.769 PDE_B19048 Bio1 0.722 1.17 0.656 0.522 0.900 0.665 PDE_B19048 Bio2 0.787 1.30 0.891 0.657 1.22 0.915 PDE_B19048 Bio3 0.720 1.23 0.676 0.542 0.860 0.658 PDE_B19054 Bio1 0.671 1.25 0.713 0.576 0.880 0.702 PDE_B19054 Bio2 0.790 1.34 0.866 0.660 1.10 0.887 PDE_B19054 Bio3 0.677 1.15 0.782 0.583 1.02 0.860

[0265] The results of physical and infectious viral titer and infectivity of final pool harvest are reported in Table 27.

TABLE-US-00027 TABLE 27 Productivity data Physical viral titer ngP24/ ngP24/ Infectious viral titer Infectivity Process ID mL tot ngP24 cm.sup.2 TU/mL Total TU TU/cm.sup.2 TU/ngP24 PDE_B19030 302 0.59E+06 56 1.7E+07 3.2E+10 3.1E+06 5.5E+04 Bio1 PDE_B19030  329* 0.60E+06*  68* 1.1E+07* 2.5E+10* 2.4E+06* 3.5E+04* Bio2* PDE_B19030 248 0.48E+06 46 1.7E+07 3.4E+10 3.2E+06 7.0E+04 Bio3 PDE_B19041 347 0.68E+06 64 3.6E+07 4.7E+10 8.8E+06 6.0E+04 Bio2 PDE_B19041 317 0.62E+06 58 2.1E+07 4.1E+10 3.9E+06 5.4E+04 Bio3 PDE_B19048 257 0.50E+06 47 1.7E+07 3.4E+10 3.2E+06 5.1E+04 Bio1 PDE_B19048 389 0.76E+06 72 1.3E+07 2.6E+10 2.4E+06 5.2E+04 Bio2 PDE_B19048 391 0.76E+06 72 2.0E+07 4.0E+10 3.7E+06 4.1E+04 Bio3 PDE_B19054 402 0.78E+06 74 1.6E+07 3.1E+10 2.9E+06 6.7E+04 Bio1 PDE_B19054 422 0.82E+06 78 2.7E+07 5.3E+10 5.0E+06 7.5E+04 Bio2 PDE_B19054 283 0.55E+06 52 3.3E+07 6.4E+10 6.1E+06 5.2E+04 Bio3 *Physical titer, viral titer and infectivity on day 6 instead of day 5. The amounts of HCP and total DNA of the pool harvest are reported in Table 28.

TABLE-US-00028 TABLE 28 HCP and total DNA (ng/mL) amounts Process ID HCP Tot DNA PDE_B19030 Bio1 989 <LLOQ PDE_B19030 Bio2 1543 517 PDE_B19030 Bio3 769 <LLOQ (<0.6 μg/mL) PDE_B19041 Bio2 578 neg. PDE_B19041 Bio3 719 neg. PDE_B19048 Bio1 696 <LOQ (624 ng/ml) PDE_B19048 Bio2 1159 <LOQ (624 ng/ml) PDE_B19048 Bio3 379 <LOQ (624 ng/ml) PDE_B19054 Bio1 726 <LLOQ(<624 ng/mL) PDE_B19054 Bio2 784 <LLOQ(<624 ng/mL) PDE_B19054 Bio3 <LLOQ (<480) <LLOQ(<624 ng/mL)

[0266] As the results show, infectious viral titer and infectivity are higher when media change post transfection and final harvest are anticipate by one day i.e.: when transfection and harvesting are performed in three days. Moreover, pool harvests obtained performing transfection and harvesting in a total of three days result to have less contaminants with total DNA being undetectable or below the analytical method limit.

Example 3—Manufacturing in iCellis 500

Example 3.1—Manufacturing in iCellis 500 Having Surface Area 66 m.SUP.2

[0267] Two parallel vector productions in iCellis500 having a surface are of 66 m.sup.2 and iCellis nano having a surface area of 0.53 m.sup.2 have been performed as summarized in Table 29. The processes were performed using 293-T cell line and the third generation multi plasmid DNA system used in examples 1 and 2.

TABLE-US-00029 TABLE 29 Experimental schedule of the production runs Process Step for iCELLis500 vector Process Step for iCELLis nano vector Days production (PDE_B18116) production (PDE_B18117) Day 0 Inoculum seed cells in the bioreactor at 0.5 × 10.sup.4/cm.sup.2 0.5 × 10.sup.4/cm.sup.2 × 66 m.sup.2 = 3.3 × 10.sup.9 0.5 × 10.sup.4/cm.sup.2 × 0.053 m.sup.2 = 26.5 × 10.sup.6 Day 3 pre-transfection medium pre-transfection medium change in change in perfusion perfusion Addition of transfection mix Addition of transfection mix containing containing multi-plasmid DNA multi-plasmid DNA (181 ng/cm.sup.2) and PEI (181 ng/cm.sup.2) and PEI (ratio 1:1) (ratio 1:1) Day 4 Post-transfection medium Post-transfection medium change in batch change in batch (50′ of holding time) start harvest in perfusion start harvest in perfusion Day 6 Stop perfusion and empty the Stop perfusion and empty the vector vector inside the bioreactor inside the bioreactor:

[0268] The comparability between iCellis500 and iCellis nano of post clarification pre benzonase sample are reported in Table 30.

TABLE-US-00030 TABLE 30 Comparability between iCellis500 and iCellis nano iCellis500 iCellisnano Physical Particles (ng/cm.sup.2)  33  39 Viral Titer (TU/cm.sup.2) 3.2E+06 2.6E+06 Infectivity (TU/ng p24) 9.1E+04 6.6E+04 Host Cell Protein content 516 357 (HCP) (ng/cm.sup.2) Total DNA content (ng/cm.sup.2) 302 230 BSA (μg/cm.sup.2) 414 388

[0269] The results show that by upscaling the process to iCellis 500 productivity is maintained and infectious viral titer and infectivity result to be improved in respect to data obtained with iCellis nano. The impurity profile of the 2 runs is comparable and acceptable.

Example 3.2—Manufacturing in iCellis 500 Having Surface Area 133 m.SUP.2

[0270] Two parallel vector productions in iCellis500 having a surface are of 133 m.sup.2 and iCellis nano having a surface area of 1.06 m.sup.2 have been performed as summarized in Table 31. The processes were performed using 293-T cell line and the third generation multi plasmid DNA system used in examples 1 and 2.

TABLE-US-00031 TABLE 31 Experimental schedule of the production runs Process Step for iCELLis500 vector Process Step for iCELLis nano vector Days production (PDE_B19130) production (PDE_B19129) Day 0 Inoculum: seed cells in the bioreactor 133 m.sup.2 at 0.5 × 10.sup.4/cm.sup.2 0.5 × 10.sup.4/cm.sup.2 × 133 m.sup.2 = 6.65 × 10.sup.9 0.5 × 10.sup.4/cm.sup.2 × 0.106 m.sup.2 = 53 × 10.sup.6 Start recirculation with 176.1 L of Start recirculation 1.3 L of complete complete medium medium Day 3 Addition of transfection mix containing Addition of transfection mix multi-plasmid DNA (91 ng/cm.sup.2) and PEI containing multi-plasmid DNA (ratio 1:1) (91 ng/cm.sup.2) and PEI (ratio 1:1) Post-transfection medium change in batch Post-transfection medium change in of complete medium batch (50′ of holding time) Start harvest in perfusion Start harvest in perfusion Day 5 Stop perfusion and empty the vector inside Stop perfusion and empty the vector the bioreactor inside the bioreactor

[0271] The comparability between iCellis500 and iCellis nano of pre clarification sample are reported in Table 32.

TABLE-US-00032 TABLE 32 Comparability between iCellis500 and iCellis nano iCellis500 iCellisnano* Physical Particles (ng/cm.sup.2)  66  61 Viral Titer (TU/cm.sup.2) 7.7E+06 3.7E+06 Infectivity (TU/ng p24) 1.2E+05 6.0E+04 Host Cell Protein content 140 223 (HCP) (ng/cm.sup.2) Total DNA content (ng/cm.sup.2) <LLOQ <LLOQ

[0272] The results show that by upscaling the process to iCellis 500 productivity is maintained and infectious viral titer and infectivity result to be improved (doubled infectious viral titer and infectivity) in respect to data obtained with iCellis nano. The impurity profile of the bulk productions obtained in the 2 runs is comparable and the total DNA content of the production at large scale as well as at small scale results to be below the detection limit of the analytical method used, such results are obtained on samples collected before the treatment with endonuclease.

Example 4—Minimum Total DNA Quantity

[0273] The goal of this experiment is the evaluation of the productivity of the process obtained by performing transfection with DNA at total quantity of 45 ng/cm.sup.2 and 60 ng/cm.sup.2.

[0274] Packaging Cells and Cell Culture Medium

[0275] The production of the LVV vectors described in this Example 4 has been performed with the HEK 293T cell line. HEK 293T cells were thawed and expanded in IMDM 10% FBS, 2% Glutamax.

[0276] Bioreactor

[0277] The experiment was performed with iCellis Nano having a surface area of 1.06 m.sup.2 bioreactor.

TABLE-US-00033 TABLE 33 iCELLis nano characteristics Carrier Bioreactor FB height Density No. of Size (m.sup.2) (cm) Compaction (g/L) carriers 1.06 4 1 96 946

[0278] Plasmids

[0279] For the production of the LVV vectors in iCELLis nano in this example 4 the multi-plasmid DNA system used is a third generation LVVs packaging system including 4 separate plasmids: one plasmid carrying gag-pol genes (pGag/Pol), one plasmid encoding rev (pREV), one plasmid carrying VSV-g gene (pENV-VSV-G)), and one transfer vector plasmid carrying the gene encoding GFP protein (pTransfer-GFP).

[0280] Analytical Methods

[0281] The following analytical methods has been applied to evaluate process performance: Physical viral titer: this analytical method is an ELISA to quantify p24 HIV protein, which can be used to evaluate the concentration of physical particles.

[0282] Infectious Viral Titer: this analytical method is based on transduction of reference CEM A3.01 cell line. The expression of the GFP protein is evaluated by FACS analysis.

Summary of the Manufacturing Process

[0283] The process performed in this example 4 is summarized in table 34.

TABLE-US-00034 TABLE 34 process steps Days Process Steps Day 0 Inoculum: seed cells in the bioreactor at 0.5 × 10.sup.4/cm.sup.2: i.e. total cells 0.5 × 10.sup.4/cm.sup.2 × 0.106 m.sup.2 = 53 × 10.sup.6 Start recirculation 1.3 L of complete medium Day 3 Addition of transfection mix containing multi-plasmid DNA and PEI (ratio 1:1) Post-transfection medium change in batch (50′ of holding time) Start harvest in perfusion Day 5 Stop perfusion after 39 hours and empty the vector inside the bioreactor

[0284] Transfection Step

[0285] Production of LVV vectors was performed using Polyethylenimine (PEI)-mediated transfection method. In Bio 1 the transfection was performed using multi-plasmid DNA in a total quantity of 45 ng/cm.sup.2 of the surface area, in Bio2, the transfection was performed using multi-plasmid DNA in a total quantity of 60 ng/cm.sup.2 of the surface area. In both Bio1 and Bio 2 the amount of PEI used has been set to maintain a ratio of 1:1 between PEI and DNA.

[0286] Protocol for transfection at day 3: [0287] 1. Prepare a total DNA mix for the bioreactors, diluting the correct amount of DNA in IMDM free [0288] 2. Dilute the correct amount of PEI in IMDM free [0289] 3. Transfer the diluted PEI in the tube containing the diluted DNA and mix. [0290] 4. Incubate the final mix for according to the manufacturer instructions. [0291] 5. Remove the correct volume from bioreactor, transfer the mix into the bioreactor.

[0292] The results obtained are shown in table 35, which shows physical particles and viral titer obtained in Bio1 and Bio2 and, in the third column, a medium the values of the same parameters obtained in the indicated number of runs (“n”), performed by using in transfection multi-plasmid DNA in a total quantity of 90 ng/cm.sup.2 of the surface area.

TABLE-US-00035 TABLE 35 results of the process using minimum total DNA quantity in transfection Medium BIO 1 BIO 2 of n runs (45 ng/cm.sup.2) (60 ng/cm.sup.2) 90 ng/cm.sup.2 Physical Particles 51 56 55 (n* = 42) P24 (ng/cm.sup.2) Viral Titer 1.7E+06 2.0E+06 3.0E+06 (n* = 36) TU/cm.sup.2 *n is the number of runs performed

[0293] The results show that by reducing the total quantity of multi-plasmid DNA in transfection to 60 ng/cm.sup.2 and 45 ng/cm.sup.2, productivity in terms of physical particles is substantially maintained. The viral titer observes a slight decline using 60 ng/cm.sup.2 or 45 ng/cm.sup.2 of total multi-plasmid DNA, but data obtained still reveal a good level of infective viral particles that, surprisingly, is comparable to that observed in example 2.3 for the runs PDE_B18197 Bio1 and PDE_B18197 Bio2, in which transfection was performed using 181 ng/cm.sup.2 of the surface area and the TU/cm.sup.2 observed was 2.2E+06 and 1.7E+06 respectively (as shown in table 23).