Method for purifying enveloped viruses or viral vectors

Abstract

The invention relates to a process for purifying enveloped viruses. The process of the invention is useful for recovering at a large scale enveloped viruses under conditions complying with good manufacturing practices and allowing viruses of a clinical grade to be obtained.

Claims

1. A process for purifying a pseudotyped lentivirus comprising an anion exchange chromatography step, the buffers used during said chromatography being: of a pH below 6, or of a pH greater than or equal to 6 and further comprising a polyol.

2. The process according to claim 1, said anion exchange chromatography buffer(s) having a pH of less than 6 and also comprising a polyol.

3. The process according to claim 1, the pH of the buffers being between 5.5 and 6.

4. The process according to claim 1, the anion exchange chromatography step being preceded with an ultrafiltration/diafiltration step.

5. The process according to claim 4, the ultrafiltration/diafiltration step comprising the use of one or several buffers with a pH between 5.5 and 7.5, said buffer(s) optionally comprising a polyol.

6. The process according to claim 1, the process comprising: (a) clarification of a cell culture medium containing cells producing said pseudotyped lentivirus thereby obtaining a clarified lentivirus; (b) an ultrafiltration/diafiltration step for the clarified lentiviruses; (c) an anion exchange chromatography; and (d) an exclusion chromatography.

7. The process according to claim 6, step (a) being carried out by filtration of the culture medium on a retention filter for which the retention threshold is between 0.2 and 0.45 m.

8. The process according to claim 6, step (b) being carried out by means of a tangential flow filtration.

9. The process according to claim 6, step (d) comprising the use of an exclusion resin having an exclusion size between 300 and 1,000 kDa.

10. The process according to claim 6, the resin used for the exclusion chromatography being a multimode resin, having a dual functionality of exclusion and adsorption.

11. The process according to claim 1, the purified lentivirus being produced in a neutral medium or in a moderately acid medium.

12. The process according to claim 1, the polyol being selected from sucrose, mannitol, sorbitol and trehalose.

13. The process according to claim 5, the polyol being present in the buffer at a concentration between 1.5% and 15% by weight in the buffer.

14. The process according to claim 4, the polyol being present in the buffers used during the ultrafiltration/diafiltration step and an anion exchange chromatography.

15. The process according to claim 6, the polyol being present in the buffers in all the steps of the purification process.

16. The process according to claim 1, the buffers used during said process also comprising a magnesium salt at a concentration between 0.1 mM and 5 mM.

17. The process according to claim 1, wherein the anion exchange chromatography is a weak anion exchange chromatography and/or an anion exchange chromatography on a column.

18. A process for purifying a pseudotyped lentivirus, said process comprising an ultrafiltration/diafiltration step, said step being carried out using buffers containing a polyol.

19. The process according to claim 1, wherein the lentivirus is pseudotyped with the GaLV-TR, VSV-g or MV envelope glycoprotein.

20. The process according to claim 1, wherein the lentivirus is pseudotyped with the GaLV-TR envelope glycoprotein.

21. The process according to claim 1, wherein the lentivirus is pseudotyped with the VSV-g envelope glycoprotein.

22. The process according to claim 13, wherein the polyol in the buffer is sucrose at a concentration between 2% and 8% by weight.

23. The process according to claim 22, where the polyol in the buffer is sucrose at a concentration of 5% by weight.

Description

LEGEND OF THE FIGURES

(1) FIG. 1. Two processes for purifying LV-GaLV-TR lentiviral particles: process (A) is a simplified process of applying a single exclusion chromatography step (gel filtration) after the step of tangential flow filtration; and process (B) is a more elaborate process aimed at obtaining a higher purity than during the use of process (A), for example, with the aim of producing vectors for clinical use.

(2) FIG. 2. Comparison of the membranes with cutoffs of 500 kDa and of 750 kDa in the removal of contaminating proteins (SDS-PAGE (at the top) and Western blot anti-p24 (at the bottom)). The 24/25 kDa band (=p24) is well visible for all the samples on the Western blot: 1) size markers; 2) diafiltration (a) (test 1) at 750 kDa; 3) diafiltration (b) (test 2) at 750 kDa; 4) ultracentrifugation at 68,338 g for 3 h (resuspension in a culture medium X-vivo 20); 5) diafiltration (a) (test 1) at 500 kDa; 6) diafiltration (b) (test 2) at 500 kDa; 7) culture supernatant containing LV-GaLV-TR vectors.

(3) FIG. 3. Effect of NaCl on the stability of the LV-GaLV-TR vectors coding for a GFP, stored at room temperature. The vectors are incubated for 4 hours at a pH of 7.0 (PBS) at room temperature (RT). In order to optimize the anion exchange chromatography step, we tested the stability of the vectors in a saline NaCl medium. For this, the vectors were incubated after the UF/DF step in PBS buffers of pH 7.0 with different NaCl concentrations for 4 h at room temperature. Next, the vectors were titrated on HCT116 cells. 48 hours later, the cells are passed on FACS (flow cytometry) in order to measure the expression percentage of GFP.

(4) FIG. 4. Effect of the pH and of the salinity of the elution buffer on the purification yield of the infectious lentiviral vectors after an anion exchange chromatography step. The preparation of vectors was produced by transfection of HEK293T cells, clarified and concentrated/diafiltered by TFF with a view to being used for evaluating various substrates of anion exchange chromatography (low). Various substrates were evaluated: Toyopearl 650C DEAE, CIM D (DEAE) and Poros D. The yield of 100% is equivalent to the infectious titer after the preceding TFF step.

(5) FIG. 5. Purification of a preparation of GaLV-TR lentiviral vectors by exclusion chromatography (Capto Core 700). Three mL of a lentiviral preparation were concentrated/diafiltered and then passed over a column of Capto Core 700 (4.5 mL). A PBS buffer (pH 7.0), 5% sucrose, and 2 mM MgCl.sub.2 were used during this step for equilibration of the column and of the formulation. 1 mL fractions were harvested and analyzed for vector concentration (TU): a) Chromatogram showing the titer (TU) by fraction, the accumulation of the vector amount for the fractions 4-9 and accumulative recovery (%) for the fractions 4-9; b) Western blot of all the fractions; c) SDS-PAGE of all the fractions.

(6) FIG. 6. Transduction of the CD34+SC cells (blood from the umbilical cord) with an HIV-GaLV-TR vector from MOI 20.

(7) Crude: percentage of cells expressing the GFP determined by flow cytometry of the CD34+ cells transduced with the crude product HIV-GaLV-TR.

(8) DF/UF: percentage of cells expressing the GFP determined by flow cytometry of the CD34+ cells transduced with the preparation of HIV-GaLV-TR vectors obtained after purification and concentration by TFF of the crude product.

EXAMPLES

(9) The studies reported in the present application benefited from a subsidy via the 7.sup.th Framework Program of the European Community (FP7/2007-2013), under number 222878.

(10) Materials and Methods

(11) Cells:

(12) The HEK293T and HCT116 cell lines (colorectal cancer cells CCL-247, origin: ATCC) are cultivated at 37 C., with 5% CO.sub.2 in Dulbecco's modified Eagle's medium (Gibco) (DMEM+Glutamax) supplemented with 2 to 10% of fetal calf serum (FCS) (Life Technologies). Culture medium: DMEM/FCS buffered at pH 6.0 by adding hydrochloric acid (HCl 37%, Sigma-Aldrich), and then filtered by means of a Corning 1,000 mL filter (0.22 m PES (polyethersulfone)).

(13) Production of Viral Vectors:

(14) Viral vectors derived from HIV-1, pseudotyped with various glycoproteins are produced by transient quadri-transfection with calcium phosphate in 293T cells, by 4 plasmids as described by Merten et al. (2011). 210.sup.8 293T cells are sown in a 1,760-cm.sup.2 Hyperflask (Corning) in 550 mL of DMEM 10% FCS (Kutner et al. 2009). 24 hours later, the culture medium is replaced with the transfection medium, by combining therein the DNA/CaCl.sub.2/HBS complex. The 4 plasmids: gagpol (pKLgagpol) 136 g, rev (pKrev) 52.25 g, the transgenic plasmid (pCCL-eGFP) 206.8 g, the suitable envelope plasmid for each pseudotype: GaLV-TR: pBA.GALV-TR/Ampho-Kana (Gibbon ape Leukemia Virus) 223 g for generating LV-GaLV-TR; VSV-GpMDG (Vesicular stomatitis virus-g) 68.13 g for generating LV-VSV-g; pF30 and pHCMH2 (modified envelope proteins of the measles virus) 40 g and 14 g for generating the LV-MV; a sufficient amount for 18 mL of H.sub.2O and 8.9 mL of TE0.1, mixed with 3 mL of CaCl.sub.2 (2.5M) and then add 30 mL of HBS2, await the formation of the complex for 4 min and add the mixture to the culture medium. After 16 hours, the supernatant is replaced with fresh medium of 2% FCS 15U Benzonase (Merck) and 2 mM of MgCl.sub.2 (Sigma-Aldrich). The harvest is made after 48 hours post-transfection; the supernatant is filtered on a 0.45 m filter in cellulose acetate (CA) 1 L (Corning).

(15) The retroviral vectors MLV-GaLV are produced by PG13 cells. These are cells producing MLV-GaLV vectors (Miller et al. 1991). The cells are maintained in Dulbecco's modified Eagle's medium (Gibco) (DMEM+Glutamax) supplemented with 2 to 10% of FCS, at 37 C., 5% of CO.sub.2. The harvesting of the vectors is made after 24 hours of having changed the culture medium. Next, the production supernatant is clarified on a 0.45 m filter in cellulose acetate (Corning).

(16) Concentration of the Viral Vectors by Tangential Flow Filtration (TFF):

(17) This step consists of concentrating the production supernatant and then replacing the culture medium with an adequate buffer for the continuation of the process.

(18) Ultrafiltration (UF) is carried out after the preparation of the UF cassette and the determination of the permeability normalized to water NWP at 0.5 bars at 20 C. The membrane is then equilibrated with a Bis-Tris buffer at pH 6.0, 5% sucrose, and 2 mM MgCl.sub.2 or with other buffers with a view to carrying out concentration/diafiltration at other pHs (for example: PBS, pH 7.0, 5% sucrose and 2 mM MgCl.sub.2). The whole process is carried out at about 4 C. and the tank of the product to be concentrated is put in an ice box.

(19) Principle: A first concentration up to a volume of 20 mL followed by diafiltration with 10 volumes of buffer for loading ion exchange chromatography (in this case: 1020 mL) are carried out. These steps are followed by a second concentration down to the minimum possible volume (10 mL in the present case).

(20) Membrane 750 kDa, 410 cm.sup.2: Hollow fiber cartridge (GE Healthcare, Ref: UFP750-E3MA) by using the Kros-Flow Research II TFF system (Spectrum).

(21) After validation of the integrity of the membrane, the concentration of the vectors begins with an initial volume of 500 mL of crude supernatant and is concentrated by the membrane from 500 mL to 20 mL.

(22) The concentrated product is diafiltered against 200 mL of Buffer A (with a view to diafiltering 10 times 20 mL of concentrate). This represents a concentration factor of 25. The final volume of the diafiltrate is 10 mL. In this case, the concentration factor is 50.

(23) Anion Exchange Chromatography:

(24) In a first procedure, the anion exchange chromatography step is carried out downstream from the TFF. Several chromatography substrates are tested: monolithic column CIMD DEAE, CIMD Q (BIA Separations, Villach, Austria), volume of the column: 1 mL; Sartobind D 75MA, volume: 2.1 mL (Sartorius Stedim Biotech); Poros PI, volume of the column: 4 mL; Poros D 50, volume of the column: 4 mL; Poros HQ, volume of the column: 4 mL (LifeTechnologies); Toyopearl 650C DEAE, volume of the column: 2 mL (Tosoh).

(25) The column to be tested is connected to a Biologic-LP chromatograph (Bio-Rad) equipped with a 280 UV absorbance reader, with a conductivity-meter, a plotter (Chart recorder 1327, Bio-Rad), and a Fraction collector (Model 2110, Bio-Rad).

(26) The column is equilibrated with 5 column volumes (5 CV) of buffer A at 2 mL/min. After loading the sample on the column, the column is washed with 5 CV of a suitable equilibration buffer (depending on the desired pH, according to table A). An elution in two steps is then carried out: 0.3 M of NaCl, 20 mM of Bis-Tris, 5% sucrose, 2 mM of MgCl.sub.2 (pH 6.0) and then 650 mM of NaCl, 20 mM of Bis-Tris, 5% sucrose, 2 mM of MgCl.sub.2 (pH 6.0) for eluting the vectors. Three other pHs are tested, pH 5.5, 7.0 and 8.0, by using the suitable buffers (including buffers such as bis-propane, PBS, L-His), in the presence (5%) and in the absence of 5% sucrose and of MgCl.sub.2 (2 mM).

(27) Finally, the fraction is loaded straightaway on the gel filtration column (exclusion chromatography) for removing the contaminating salts and proteins eluted with the vectors at 650 mM of NaCl.

(28) In a second procedure, the clarified production supernatant is loaded on an anion exchange chromatography column Poros D, without any preliminary ultrafiltration/diafiltration step in order to evaluate the yield of the chromatography under these conditions. The equilibration buffer used has a pH of 5.5, and contains 5% sucrose and 2 mM of MgCl.sub.2.

(29) Exclusion Chromatography:

(30) This is the final step before the sterilizing filtration for processes A and B (FIG. 1). This step consists of removing the contaminants having a size of less than that of the gel used (for example, 750 kDa, or else 500 kDa). The Capto Core 700 column was used for this step. This is a gel with dual functionality: exclusion chromatography and adsorption gel chromatography.

(31) Before beginning the loading, the column is sanitized with NaOH 1M and equilibrated with the formulation buffer. The UF/DF product (process A), or the fractions corresponding to the chromatography peak of anion exchange chromatography (AXC) (process B) is loaded on the column. 8 mL (UF/DF or AXC fraction) are loaded with a flow rate of 0.5 mL/min. Next the formulation buffer is injected with 0.5 mL/min (5 CV of formulation buffer). The fraction corresponds to the UV peak, is collected (about 16 mL) and then filtered on a 0.22 m filter (sterilizing filtration). The samples are stored at 80 C.

(32) Titration of the Viral Vectors:

(33) The viral titer in transduction units (TU) of the vectors having the reporter gene eGFP, is analyzed by transduction of HCT116 cells. 72 hours after transduction the cells are passed to FACS for determining the titer in TU/mL as described earlier (Pfeifer et al. 2009). For physical analysis of the viral particles, the kit ELISA p24 (PerkinElmer) was used for quantifying the capsid protein of the lentiviruses p24 according to the instructions of the supplier.

(34) Transduction of CD34+ Blood Cells of the Umbilical Cord:

(35) The CD34+ cells are isolated from blood from the umbilical cord by immunomagnetic selection (Miltenyi Biotec). The cultivation and the transduction of the CD34+ cells is accomplished as described (Charrier et al. 2011): first the cells are pre-stimulated overnight in a medium X-Vivo 20 (Lonza) and supplemented with cytokines. The pre-activated cells are sown in a 48-well plate (510.sup.4 cells/100 l). The transduction is accomplished by adding 100 l of vectors (10.sup.6 TU) purified in the presence of 8 g/mL of vectofusine-1 (Fenard et al., 2013). After 6 hours of incubation, 1 mL of differentiation medium (X-VIVO-20 supplemented with 10% serum, and in the presence of cytokines (hSCF, h-Il-3 h-Flt3 h-Il-6) as described (Charrier et al. 2011)) is added into each well, and after 5 days, the transduction efficiency is evaluated by measuring the expression of GFP by FACS (FC500, BD Biosciences).

(36) SDS-PAGE Western Blot:

(37) The culture samples containing lentiviral vectors or purified samples are analyzed with SDS-PAGE and by Western blot in order to detect the presence of p24 capsid proteins. The revealing of the p24 proteins is carried out according to the method developed by LI-COR, with the Odyssey apparatus and the Odyssey 2.1 software package. The primary antibody used is an anti-p24 (Santa Cruz # SC-57823) for detecting p24 capsid proteins of HIV. The antibody is used with a dilution of 1/200 in 0.1% PBS1-Tween+blocker Odyssey (1:1). The secondary goat antibody used coupled with the fluorochrome Dye 800 of LiCOR is directed against the primary antibodies.

(38) Quantification of the Residual Proteins and of the Specific Residual DNA:

(39) The total proteins are quantified by Bradford's method (Bio-Rad) with serum albumin as a standard. The test is conducted according to the instructions of the supplier.

(40) Residual DNA: the quantification of residual DNA of plasmid origin and/or stemming from the host cell is accomplished by quantitative PCR. The samples are treated with proteinase K (Roche) and then the DNA is extracted by using the system: MagNA Pure DNA and viral NA small volume kit (MagNA Pure 96 Roche). Quantitative real time PCR is then carried out, with specific primers for the gene of kanamycin in order to detect residual DNA of plasmid origin. In order to detect the residual DNA from the host cell, primers are used which target the E1A gene. Absolute quantification is carried out relative to a reference plasmid containing the regions amplified by quantitative PCR and for which the number of copies is known.

(41) TABLE-US-00001 TABLE A buffers used during the process Sucrose MgCl.sub.2 (Sigma- (Sigma- Buffers pH Aldrich) Aldrich) buffer A L-Histidine 20 mM 5.0 5% W/V 2 mM (Sigma-Aldrich) buffer B Bis-Tris 20 mM 5.5 (Sigma-Aldrich) buffer C Bis-Tris 20 mM 6 (Sigma-Aldrich) buffer D PBS (GIBCO) 7.2 buffer E Bis-Propane 20 mM 8 (Sigma-Aldrich)
Results

(42) This invention relates to the development and the establishment of a novel purification protocol for lentiviral vectors derived from HIV-1 or from other retroviruses produced by transient transfection or with stable and pseudotyped producing cells with different envelope glycoproteins, such as GaLV-TR, VSV-G, measles virus and -retroviral vectors GaLV produced from stable cells, such as PG13, while ensuring a good yield and good quality of the purified viral particles. This development is essentially, but not exclusively, based on three purification techniques: TFF (tangential flow filtration), anion exchange chromatography and exclusion chromatography. The different combinations are illustrated in FIG. 1.

(43) Cultivation of Cells and Clarification:

(44) These steps consist of producing retroviral and lentiviral vectors by using stable cells such as PG13 characterized by a stable and continuous production of retroviral vectors in a continuous cultivation with a regular exchange of medium and with cells such as HEK293 or HEK293T which have to be transfected with 3 or 4 plasmids (providing the helper functions of the lentivirus and the sequence of the recombinant vector) with a view to inducing the production of lentiviral vectors. Transient production is limited in time and allows one or several harvests a few days after transfection. The titers generally depend on the construction (sequence) of the vector but also on the envelope protein. The following titers may be obtained with these production systems (Table 1).

(45) TABLE-US-00002 TABLE 1 Concentrations of vectors obtained with the different production systems: Production cell, Vector concentration pseudotype vector (TU/mL, gi/mL) References PG13, MLV-GaLV-TR 5 10.sup.6 TU/mL Miller et al. 1991 HEK293T, LV (HIV-1) - 5 10.sup.5 TU/mL Sakuma et al. 2010 GaLV-TR HEK293T, LV (HIV-1) - 1-5 10.sup.7 TU/mL Merten et al. 2011 VSVg

(46) Before any further treatment, it is possible to remove the cell debris and the aggregates present in the supernatant of the production. Conventionally, a 0.45 m filter (cellulose acetate) is used. The yield of this step is of 805%. However, one skilled in the art may use other membranes or cascades of membranes, characterized by a similar behavior and yield.

(47) Tangential Flow Filtration:

(48) Tangential flow filtration comprises two successive steps of ultrafiltration and diafiltration (UF/DF). Both of these steps give the possibility of removing a great portion of the contaminants of which the size is less than the exclusion size of the pores of the membrane used. This UF/DF step also gives the possibility of concentrating the viral particles and of reducing the volume of the product to be purified. A membrane of 110 cm.sup.2 with a pore exclusion size of 750 kDa (GE Healthcare) was used. In order to begin UF, different concentrations of sucrose (notably 5% of sucrose (weight/volume)) and various concentrations of MgCl.sub.2 (notably 2 mM of MgCl.sub.2 (final concentration)) are added to the clarified product. Next the UF concentration step is performed with a flow of 80 mL/mn, 7 psig. The TFF tank is placed in an ice box in order to ensure low temperature during UF/DF. The diafiltration step begins after having reduced the volume from 500 mL to 20 mL during UF. For DF, 200 mL (10 volumes of the concentrated product) of the diafiltration buffer (PBS, 5% sucrose, 2 mM MgCl.sub.2) are used. At the end of this step, 20 mL of UF/DF product are recovered in a 50 mL Corning tube. The selection of the buffer depends on the use of the preparation or on the optimum conditions of the step following concentration/diafiltration (e.g., in this case, other buffers may be used like Bis-Tris (pH 6.0), 5% sucrose, 2 mM MgCl.sub.2; see Table A). The samples are titrated on HCT116 cells as described by Fenard et al. (2013).

(49) Studies for Optimization of the Concentration/Diafiltration Conditions:

(50) 1. The lentiviral particles have a diameter ranging from 80 to 120 nm meaning that the pore size of membranes which may be used for the concentration/diafiltration may range at most up to about 50 nm (or 750 kDa). Within the scope of this invention, the cutoff sizes of 500 kDa and 750 kDa were evaluated. The yields (in TU) were the following: 64% for the 750 kDa membrane versus 34% yield in TU for the 500 kDa membrane.

(51) FIG. 2 shows electrophoresis gels (SDS-PAGE and Western blot) for the preparation of vectors after tangential filtration by using membranes with cutoffs of 500 kDa and 750 kDa. In addition to the higher yields obtained upon using 750 kDa membranes, it is clear that a cutoff of 750 kDa has a positive effect (FIG. 2, columns 2, 3) compared with the use of the 500 kDa membrane as regards the removal of contaminating proteins (FIG. 2, columns 5, 6). Further, the concentrate generated with the 750 kDa membrane contains bands of proteins much less intense than observed for the crude supernatant.

(52) 2. Given that the tangential filtration step is characterized by the generation of shearing fields leading to the inactivation of the retroviral/lentiviral particles, it was necessary to optimize this step with a view to maintaining the functionality of these vectors. The addition of a polyol at various concentrations was evaluated with a view to protecting the lentiviral vector from the adverse conditions of tangential filtration.

(53) TABLE-US-00003 TABLE 2 Concentration/diafiltration yield of LV-GaLV-TR by using various concentrations of sucrose. Yield % (TU) 0% sucrose 50.83 2% sucrose 80.31 5% sucrose 80.40 10% sucrose 52.37 15% sucrose 68.49 Note: 190 mL of crude supernatant were concentrated to 17 mL and diafiltered several times with PBS (pH 7) + various % of sucrose and 2 mM of MgCl.sub.2.

(54) These results clearly show the benefit of carrying out the concentration/diafiltration of supernatant containing LV-GaLV-TR vectors in the presence of sucrose and MgCl.sub.2. The best yields are obtained at concentrations from 2% to 5% of sucrose (Table 2).

(55) Further, the use of a moderate sucrose concentration has the advantage that the sample to be concentrated is less viscous since high sucrose concentrations (10%-15%) lead to an increase in viscosity.

(56) 3. Evaluation of the pH and its effect on tangential filtration and yield of functional vectors:

(57) In the application FR 13 58909 filed by the present applicant, it was shown that the production of enveloped vectors pseudotyped with different envelope proteins is increased upon the use of a pH of 6.0 (up to 2). It was decided to evaluate the impact of the selection of the pH of the supernatant containing the lentiviral vectors on the efficiency of the tangential filtration. In this context, two different pHs were evaluated (pH 6 and pH 7) during the concentration/diafiltration of the GaLV-TR pseudotyped lentiviral vectors (Table 3). The reduction of the pH from 7.0 to 6.0 led to a reduction of the yield by about 10% (from 73.6% to 64%). This yield however remains acceptable and it is therefore possible to envision concentration/diafiltration with an acid pH.

(58) TABLE-US-00004 TABLE 3 Impact of the pH of the supernatant to be concentrated/of the diafiltration buffer on the concentration/diafiltration yields of GaLV-TR and VSV-g pseudotyped lentiviral vectors. Tangential filtration condition LV vector Yield (%, TU) PBS, 5% sucrose 2 mM MgCl.sub.2, LV-GaLV-TR 73.64 pH 7.0 Bis-Tris 20 Mm, 5% sucrose, LV-GaLV-TR 63.99 2 mM MgCl.sub.2, pH 6.0

(59) 4. Identification of the best condition for concentration/diafiltration of GaLV-TR lentiviral vectors:

(60) As regards the GaLV-TR lentiviruses, the best concentration/diafiltration (tangential filtration) condition was the following: the LV-GaLV-TR vectors (1 L) are clarified through a 0.45 m cellulose acetate membrane, in the presence of 5% sucrose and 2 mM MgCl.sub.2, followed by the TFF step (cartridge 750 kDa, 410 cm.sup.2) with reduction of the volume to be reached of 20 mL (50). A diafiltration step is then carried out against a volume of 200 mL of suitable buffer (for example, Bis-Tris 20 mM, pH 6.0, 5% sucrose and 2 mM MgCl.sub.2 or PBS, pH 7.0, 5% sucrose and 2 mM MgCl.sub.2).

(61) The yield of this step for LV-GaLV-TR vectors is 86%5% for an initial volume of 550 mL of crude product. The volume of the concentrated product is 15 mL with a concentration factor of 36.6 and the removal of the contaminants attained more than 90%.

(62) 5. Evaluation of the established tangential filtration conditions for the concentration/diafiltration of other retroviral and lentiviral vectors pseudotyped with different envelope proteins:

(63) In the scientific literature, various envelope proteins were evaluated with a view to studying and improving tropism of the retroviral and lentiviral vectors. In this context, the conditions established for concentration/diafiltration of the GaLV-TR lentiviral vectors were evaluated for the concentration/diafiltration of retroviral and lentiviral vectors pseudotyped with various envelope proteins (Table 4). The results obtained with the GaLV-TR pseudotyped lentiviral vectors are indicated as a reference.

(64) TABLE-US-00005 TABLE 4 Concentration/diafiltration of retroviral vectors pseudotyped with various envelope proteins: Tangential filtration condition Retroviral vector Yield % (TU) Bis-Tris, 5% sucrose, 2 mM MgCl.sub.2, MLV-GaLV 94.2 pH 6.0 (PG13) PBS, 5% sucrose, 2 mM MgCl.sub.2, LV-GaLV-TR 73.64 pH 7.0 Bis-Tris, 5% sucrose, 2 mM MgCl.sub.2, LV-GaLV-TR 63.99 pH 6.0 Bis-Tris 5% sucrose 2 mM MgCl2 LV-MV- 61.22 pH 6.0 CMHII PBS, 5% sucrose, 2 mM MgCl.sub.2, LV-MV- 65.67 pH 7.0 CMHII PBS 5% sucrose, 2 mM MgCl.sub.2, LV-VSV-g 107 pH 7.0 Bis-Tris, 5% sucrose, 2 mM MgCl.sub.2, LV-VSV-g 104 pH 6.0 Note: MLV-GaLV: GaLV pseudotyped murine retrovirus; LV-GaLV-TR: GaLV-TR pseudotyped lentivirus; LV-MV: pseudotyped lentivirus with the env of the measles virus (CMHII modified); LV-VSV-g: VSV-g pseudotyped lentivirus.

(65) The results presented in Table 4 show that all the retroviral or lentiviral vectors pseudotyped with different envelope proteins may be concentrated in the presence of sucrose and MgCl.sub.2 at a pH of 7.0 leading to yields ranging from about 74% for LV-GaLV-TR to about 100% for VSVg. As regards the use of a pH of 6.0 no difference was observed for VSVg pseudotyped vectors.

(66) As regards the GaLV-TR pseudotyped lentiviral vectors, these vectors proved to be more stable at pH 7.0 during tangential filtration. The concentration/diafiltration yield was greater than 90%, while the yield was around 74% for GaLV-TR lentiviral vectors.

(67) Anion Exchange Chromatography:

(68) The concentration/diafiltration step by tangential flow filtration considerably reduced the load of proteins and DNA (see above) meaning that a significant portion of contaminants which may be competitors of the vectors to be purified for accessing the ligands of the chromatography is reduced. In principle, according to the subsequent use, it is possible to imagine two different ways for contemplating purification. They are shown in FIG. 1: a simplified process applying a single exclusion chromatography step (A in FIG. 1) and a more elaborate process applying an additional anion exchange chromatography step with a view to preparing lentiviral vectors for clinical use (B in FIG. 1).

(69) The different chromatography possibilities are developed subsequently:

(70) After the TFF UF/DF step and in order to reduce the contaminants and well separate the viral particles, an anion exchange chromatography step is added. This technique allows separation of the biomolecules according to their isoelectric points depending on the pH and on the salt concentration. Therefore, at a given pH value, a certain salt concentration (often NaCl) is required in order to detach the retained biomolecules and this concentration has to be selected according to the interaction force between the biomolecules and the ligands: the greater this interaction, the higher has to be the salt concentration (salinity). Further, the closer the pH of the chromatography buffer is to the isoelectric point of the species of biomolecules to be purified, the less salt is required for detaching the biomolecules from the chromatographic ligands. However, it is known that retroviral and lentiviral vectors rapidly lose their infectiosity depending on the salt concentration (review by Segura et al. 2006). Therefore, in a first phase, the stability of the lentiviral vectors towards different NaCl concentrations was evaluated.

(71) 1. Impact of Salinity on the Stability of the GaLV-TR Lentiviral Vectors:

(72) As indicated above, the elution of the biomolecules retained by a chromatography column is often accomplished with salt gradients (buffers containing NaCl) or a step for increasing the salt concentration (NaCl). Therefore, with a view to evaluating the effect of the NaCl concentration, incubation tests of post-TFF lentiviral vectors were conducted in different NaCl concentrations ranging from 50 mM to 1,500 mM, at room temperature for 4 h. FIG. 3 illustrates the infectivity of the lentiviral vectors at room temperature according to the NaCl concentration as compared with conditions without any added NaCl or the same preparation of vectors incubated at 4 C. without addition of NaCl. This test clearly shows that an NaCl concentration comprised between 50 mM and 1M has a moderately detrimental effect on the stability of the GaLV-TR lentiviral vectors with a loss of infectivity ranging from 29.52% (50 mM NaCl) to 43.86% (1M NaCl) (percentage relative to the preparation stored at 4 C.). On the other hand, the concentration of 1.5M of NaCl leads to a loss of 63.8% of infectivity when the preparation of vectors is stored at room temperature for 4 h. It should be noted that the storage at 20 C. (room temperature) for 4 h without addition of NaCl also leads to a certain loss of infectivity of the vectors by about 23% as compared with storage at 4 C.

(73) These results mean that it is indispensable to elute the GaLV-TR lentiviral vectors of the chromatographic substrates with the lowest possible salinity, therefore ideally below 1M of NaCl with a view to maintaining maximum infectivity. Further, it is also preferable to carry out the totality of the purification (all the steps) at a reduced temperature (ideally between 4 C. and 10 C.).

(74) 2. Evaluation of Different Anion Exchange Chromatography Substrates (AEX):

(75) We used low anion exchange chromatography substrates (DEAE (D)) in order to determine whether it was possible to limit the inactivation of the vectors with this type of substrate, in particular by attempting to reduce the required salt concentration for detaching said vectors from the chromatography column. In preliminary tests using a concentrated supernatant from cultures of PG13 cells (MLV-GaLV), it was possible to show that the use of a chromatographic substrate based on DEAE (Tosoh TSK gel DEAE 5PW) leads to an infectious vector yield of about 71% higher than during the use of a strong exchanger (Q Sepharose FF from GE Healthcare) for which the yield was only 16%, due to the too strong interaction leading to inactivation during elution. In this example, the required salt concentration for detaching the retroviral vectors was 655 mM and 915 mM, respectively.

(76) Based on these results, low anion exchangers were selected for the continuation of the development: several chromatography substrates were evaluated: Monolithe CIM D (DEAE), Poros D50 (Life Technologies), Sartobind D (Sartorius) (Bandeira et al. 2012) and Toyopearl 650C DEAE (Merten et al. 2011).

(77) As preliminary tests, three substrates were evaluated aiming to purification of GaLV-TR lentiviral vectors at a pH of 5.5 or 6.0 and 7.0. For all the substrates tested at a pH of 5.5 or 6.0 and 7.0, the low pH choice (5.5 or 6.0) was beneficial as regards infectious vector yield: as regards the CIM D DEAE substrate, the yield was increased from 23% (pH 7.0) to 64% (pH 6.0) during reduction of the pH of the buffers used for the chromatography from 7.0 to 6.0 (FIG. 4). Similar results were observed for the substrates Sartobind 75D (increase in the yield from 5.8% to 15.6%) and Poros D (increase in the yield from 32% (pH 7.0) to 80.2% (pH 6.0) and about 100% (pH 5.5)), and for the gel Toyopearl 650C (increase in the yield from 23% (pH 7.0) to 89% (pH 5.5)) (FIG. 4). Further, in order to detach the vectors from the substrates, the salinity of the elution buffer was able to be lower during chromatography at pH 6.0 (therefore, milder for the lentiviral vectors). As regards the substrate Poros D used at pH 6.0, the elution of the vectors is accomplished at 650 mM of NaCl (see below). In terms of general efficiency, the modern substrates (developed more recently, generating reduced shearing forces (essentially due to the larger porosity than the other substrates) during chromatography and characterized by the incompressibility of the substrate during the modification of the buffer flow rate, like the monolith CIM D DEAE or Poros D) have exhibited yields greater than the yields of substrates with the membrane (Sartobind 75D) or substrates based on a compressible gel (Toyopearl 650C).

(78) Finally, the selection was made on the recent substrates since their efficiency for separating and recovering vectors was greater as compared with more conventional substrates. Both of these supports were therefore more widely evaluated and their use was optimized with a view to purifying lentiviral vectors. Both supports, CIM D DEAE and Poros D, have an interesting yield of more than 60%. The elution occurs at 650 mM NaCl, Bis-Tris 5%, sucrose 2 mM and MgCl.sub.2 pH 6.0. An increase in the pH of the elution buffer to 7.0 (PBS) causes a drop in the yield to a value below 7%, but the addition of sucrose 5% to PBS causes a significant increase from about 7% to 40%. However in this case it is necessary to use an NaCl concentration of more than 1M. Indeed, it was observed that for pH 7.0 for eluting the vectors (buffer without additional sucrose) about 1.5 M of NaCl is necessary in PBS, which is probably the explanation of the low yield. The negative effect of the concentration of salts on the stability of the viral particles is known (Segura et al. 2005).

(79) 3. Evaluation of Different pH Values on the Chromatography Efficiency by Using the Substrate Poros D:

(80) The pH varied in a range from 5.5 to 8.0 in the presence and in the absence of 5% sucrose. The presence of 5% sucrose has a positive effect on the yield during the anion exchange chromatography step when the pH is greater than 5.5 (for example, Poros D) (Table 5). The positive effect of the presence of sucrose on the yield is no longer observed at pH 5.5. On the other hand, the presence of sucrose is indispensable at a pH of 8.0 with a view to recovering about 58% of infectious vectors. Whereas during the use of a pH ranging from 6.0 to 7.0, the yield is between 52 and 65%, the best yield (about 100%) is obtained at a pH of 5.5.

(81) Generally, the presence of 5% sucrose leads to a reduction in the salinity required for initiating elution of the lentiviral vector with a required decrease of the NaCl concentration by about 25 mM.

(82) TABLE-US-00006 TABLE 5 Comparison of the yields of LV-GaLV-TR by chromatography on Poros D by using buffers of various pH (5.5-8.0) in the presence or in the absence of sucrose. With/without sucrose Yield in TU % pH 5.5 5% sucrose 105.98 (Bis-Tris) 0% sucrose 101.33 pH 6.0 5% sucrose 52.32 (Bis-Tris) 0% sucrose 29.29 pH 7.0 5% sucrose 65.52 (PBS) 0% sucrose 10 pH 8.0 5% sucrose 57.76 (Bis-Tris- 0% sucrose 0 propane)

(83) 4. Evaluation of an Alternative Procedure Comprising Anion Exchange Chromatography as a First Step:

(84) We evaluated the yield obtained upon applying an anion exchange chromatography step immediately after the clarification step. Under these conditions, the observed yield is lower than when an ultrafiltration/diafiltration step is applied between the clarification and the anion exchange chromatography. The latter procedure was therefore selected for the subsequent purification.

(85) Exclusion Chromatography:

(86) Exclusion chromatography is a method of choice for separating biomolecules according to their molecular size thus allowing separation of the particles from the contaminants.

(87) The filtration gel Capto Core 700 (GE Healthcare) was used, but other substrates may be contemplated. This step allows us to replace the buffer of the preceding step with the desired formulation buffer, to remove the contaminant molecules of a size of less than 750 kDa and to avoid dilution of the sample to be loaded. This chromatography step may be directly used after tangential flow filtration (concentration/diafiltrationprocess A) or after an ion exchange chromatography step (process B) (FIG. 1). The sample from the tangential flow filtration or the sample from fractions of the anion exchange chromatography containing the lentiviral vectors is loaded on the exclusion chromatography column. In both cases, the yield of this step is 86%4, according to the fractions retained for subsequent use.

(88) FIG. 5 shows the purification of lentiviral vectors (concentrated and diafiltered by tangential flow filtration) by filtration on a gel (Capto Core 700). The elution peak of the vector is found at the passage front of the buffer and exit the column at fractions 4-9, covering about 70% of the amount of vectors initially loaded on the column (FIG. 5a). FIGS. 5B and 5C represent the analysis of each fraction by electrophoresis (Western blot, SDS-PAGE) clearly indicating the absence of contaminating bands (FIG. 5C) and the presence of the band at 24-25 kDa corresponding to the protein p24 of the capsid of the lentiviral vector.

(89) Yields and Purities:

(90) The most important parameters relate to the overall yield as well as to the purity of the preparation of lentiviral vectors at the load reduction in contaminating proteins and in contaminating DNA.

(91) As regards the procedure B (including a TFF, an anion exchange chromatography (AEX) and an exclusion chromatography (SEC)) (FIG. 1) intended for purifying lentiviral vectors for clinical use, the yield is of about 50% and this procedure allows 99.9% removal of contaminating proteins and 99.9% of contaminating DNA.

(92) The procedure A (including a TFF and an exclusion chromatography (SEC)) (FIG. 1) intended for the purification of lentiviral vectors for use in research is simpler, since it is without the ion exchange chromatography step. The overall yield is higher because of the reduction in the number of purification steps and attains 60.2%. The removal of the residual DNA contaminants of this simplified procedure is on the order of 96.17% and a reduction of the contaminating proteins of 99.63% is observed.

(93) Practical Examples of Transduction of Target Cells:

(94) Transduction of CD34+ Cells:

(95) In order to determine the quality of the purified vectors, CD34+ umbilical cord blood cells are transduced. The cells are thawed after 18 hours of pre-stimulation with cytokines. The transduction is accomplished for 6 hours. Next, the cells are put in a differentiation medium for 5 days. The cells are then passed to FACS FC500 (BD Biosciences) for measuring the percentage of expression of GFP. The following results are typically obtained (FIG. 6): the purification by concentration/diafiltration of the lentiviral vectors (GaLV-TR) leads to an increase in the transduction efficiency of the CD34+ cells (expressed as a percentage of cells expressing the GFP) ranging from 9% upon the use of a crude supernatant at 70% for use of a preparation of concentrated/diafiltered LV vectors.

(96) Purification of a Pseudotyped Lentiviral Vector by Means of a Modified Envelope of the Measles Virus

(97) A process for purifying a lentiviral vector pseudotyped by means of the modified glycoprotein of an envelope of the measles virus (MV pseudotyping) is described here. The LV-MV-CMHII (CMHII=anti-CMHII) lentiviral vectors produced according to the procedure indicated above are purified according to the following steps:

(98) 1) Concentration/Diafiltration by Means of a TFF Step

(99) membrane used: GE #UFP-750-E-3MA 110 cm.sup.2, for purification of one liter of product diafiltration buffer: PBS (pH 7.0, 2 mM MgCl.sub.2, 5% sucrose) volume reduction: from 500 mL/1,000 mL to 20 mL, the buffer being replaced with the diafiltration buffer yield of infectious vectors: 64-70%
2) Exclusion Chromatography (Gel Filtration): column used: Capto Core 700 4.7 mL formulation buffer: PBS, 5% sucrose, 2 mm MgCl.sub.2, or else X-vivo or HANKS, containing 5% sucrose and 2 mM MgCl.sub.2 equilibration of the column with 10 CV of a formulation buffer loading the concentrate from the TFF on the Capto Core 700 column at a rate of 0.5 mL/min washing the column with 20 CV of the formulation buffer harvesting the samples corresponding to the OD peak (volume of 21 mL at 50) yield of infectious vectors: >90% in TU

(100) The overall yield of this purification is from 60 to 63% of infectious vectors, which represents major progress for purification, and therefore the utilization of lentiviral vectors pseudotyped by means of the modified MV glycoprotein.

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