METHOD FOR LARGE SCALE PRODUCTION AND PURIFICATION OF PARVOVIRUS
20180187166 · 2018-07-05
Assignee
Inventors
- Barbara Leuchs (Heidelberg, DE)
- Mandy Roscher (Schriesheim, DE)
- Marcus MÜLLER (Bad Rappenau, DE)
- Jean Rommelaere (Heidelberg, DE)
Cpc classification
C12N2750/14351
CHEMISTRY; METALLURGY
C12N7/00
CHEMISTRY; METALLURGY
International classification
Abstract
Described is a reproducible, effective and scalable process for parvovirus production including characterization strategies, preferably production of H-1PV.
Claims
1. A method for producing empty inactive or full active parvovirus particles, said method comprising: (a) providing the producer cell line NB-324K; (b) growing the cell line under suitable conditions and infecting the cells at a cell density from 2.0 to 5.0104 cells/cm2 with the parvovirus at a MOI of 0,5 to 210-2 PFU/cells; (c) harvesting the cells 2 to 6 days post-infection and obtaining a cell pellet by centrifugation; (d) subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method for obtaining a parvovirus containing cell lysate; (e) sonicating the cell lysate and subjecting it to DNAse treatment; (f) clarifying the DNAse-treated parvovirus harvest by filtration; and (g1) purifying the parvovirus by two successive density gradient ultracentrifugations, wherein the first gradient is a Iodixanol/PBS step gradient and the second gradient is a Iodixanol/Ringer step gradient or a Iodixanol/Ringer continuous gradient for obtaining full active parvovirus particles in one fraction and empty parvovirus particles in another fraction.
2. The method of claim 1, wherein the cell density of step (b) is from 3.0 to 4.0104 cells/cm2.
3. The method of claim 1, wherein for step (f) a 0.2-m filter with prefilter is used.
4. The method of claim 1, wherein the producer cell line NB-324K is characterized by (a) a viability of at least 95%; (b) a passage number below 20; and/or (c) lack of mycoplasma contamination.
5. The method of any one of claim 1 4, wherein virus production is performed in a collection system.
6. The method of claim 5, wherein the collection system is a 10-layer cell culture chamber.
7. The method of claim 1, wherein the parvovirus is H1-PV.
8. The method of claim 1 further comprising determining the ratio of native parvovirus capsids to non-assembled capsid proteins or denatured capsids.
9. The method of claim 8, wherein the ratio is determined by using a monoclonal antibody.
10. The method of claim 9, wherein the monoclonal antibody is the antibody BL-H1 (DSM ACC 3030).
11. (canceled)
12. (canceled)
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0021]
[0022] Protein extracts prepared from 293T HEK cells transfected with an infectious H-1PV molecular clone were analyzed by sucrose gradient centrifugation. Individual fractions were analyzed for the presence of GP, IU, HAU, and VP proteins. VPs were detected, respectively, by western dot blotting with BL-H1 or aVP antibodies. The data show that BL-H1 mAb specifically recognizes assembled capsids while VP recognizes VP proteins.
[0023]
[0024] The absorbance at 450 nm is plotted versus the number of H-1PV particles added (as determined by ELMI). The indicated values represent means with standard deviation bars of 3 independent measurements. A linear dose-response was derived from the regression analysis of data. The samples indicated as QC-L and QC-M contained, respectively, 4 and 810.sup.8 particles and were used subsequently as low- and medium-concentration quality control standards.
[0025]
[0026] Capsids were detected by either hemagglutination assay (HAU) or Capsid-ELISA (PP) while genome containing viral particles (GP) were quantified by Q-PCR.
[0027]
[0028] After production in a 10-layer CS, virus harvests were prepared and analyzed. PFU, GP, and PP titers were determined and are expressed relative to the number of cells in the CS at the time of infection. Values for 5 independent productions are shown.
[0029]
[0030] The PFU, GP, and PP titers of the indicated fractions are shown (means with standard deviations from 5 independent experiments).
[0031]
[0032] Virus harvest was clarified and then purified by IOD-PBS and VIS-Ringer density gradient centrifugation or by CsCl density gradient centrifugation. Samples of clarified harvest, virus-containing IOD-PBS gradient fraction, full- and empty-particle VIS-Ringer gradient fractions, and full- and empty-particle CsCl gradient fractions were analyzed. (a) Specific activity (PFU/mg), PFU recovery (%), and PP/PFU ratio in the intermediate and full-particle fractions. (b) Specific capsid titer (PP/mg), PP recovery (%), and PP/PFU ratio in the intermediate and empty-particle fractions.
[0033]
[0034] An H-1PV harvest was clarified and then purified by density gradient centrifugation, first in an IOD-PBS step gradient and then in a VIS-Ringer continuous gradient. GP and IU titers were determined in the clarified virus harvest, after the IOD-PBS step gradient, and in fractions 1 -10 of the continuous gradient. The IU-rich fractions 4 -6 were further analyzed, showing infectious H-1PV concentrations up to 3.010.sup.11 PFU/ml.
[0035]
[0036] Panels a,b: Protein extracts from virus samples (110.sup.10 PP) were analyzed by SDS-PAGE and revealed by (a) silver staining or (b) immunoblotting with VP antibodies.
[0037] Lane 1: Virus harvest, lane 2: clarified virus harvest, lanes 3-7: CsC1 gradient fractions corresponding to full (3), intermediate full/empty (4), empty (5), UV-irradiated intermediate full/empty (6), and UV-irradiated empty (7) particles, lane 8: virus particles from the IOD-PBS gradient, lanes 9-11: VIS-Ringer gradient fractions corresponding to full (9), intermediate full/empty (10), and empty (11) particles, M: size markers.
[0038] Panels c, d: Electron micrographs showing empty-particle fractions before (c) and after (d) UV-irradiation.
[0039]
[0040] Thus, the present invention relates to a method for producing full active parvovirus particles and empty inactive parvovirus particles, said method comprising:
[0041] (a) providing the producer cell line NB-324K;
[0042] (b) growing the cell line under suitable conditions and infecting the cells at a cell density from 2.0 to 5.010.sup.4 cells/cm.sup.2 with the parvovirus at a MOI of 0,5 to 210.sup.2 PFU/cells;
[0043] (c) harvesting the cells 2 to 6 days post-infection and obtaining a cell pellet by centrifugation;
[0044] (d) subjecting the resuspended cell pellet to a mechanical, physical or chemical cell lysis method for obtaining a parvovirus containing cell lysate;
[0045] (e) sonicating the cell lysate and subjecting it to DNAse treatment;
[0046] (f) clarifying the DNAse-treated parvovirus harvest by filtration; and
[0047] (g1) purifying the parvovirus by two successive density gradient ultracentrifugations, wherein the first gradient is a Iodixanol/PBS step gradient and the second gradient is a Iodixanol/Ringer step gradient or a Iodixanol/Ringer continuous gradient for obtaining full active parvovirus particles in one fraction and empty parvovirus particles in another fraction; or
[0048] (g2) purifying the parvovirus by a continuous CsCl gradient ultracentrifugation for obtaining empty parvovirus particles.
[0049] For optimum results, the producer cell line NB-324K is characterized by
[0050] (a) a viability of at least 95%, (b) a passage number below 20 (c) lack of mycoplasma contamination, and (d)lack of SV 40 production.
[0051] Preferably, the method of the present invention is used for production/purification of the parvovirus H1-PV.
[0052] The person skilled in the art knows common conditions for growing the producer cell line and for infecting the cells with the parvovirus. Usually, the cells are cultured at 37 C., e.g., in minimal essential medium with heat-inactivated fetal bovine serum (e.g. FBS 5%) in a 5% CO.sub.2 atmosphere. Preferably, the medium should be supplemented with penicillin, streptomycin and L-glutamine.
[0053] In a preferred embodiment of the present invention, the cell density of step (b) is from 3.0 to 4.010.sup.4 cells/cm.sup.2.
[0054] In a further preferred embodiment of the method of the present invention, virus production is performed in a single use cell culture system, preferably a 10-layer cell culture chamber, e.g. CellSTACK (CS) chamber. Further upscaling may be achieved with a 40-layer CS chamber or a carrier system.
[0055] Preferably, for harvesting, the culture medium is aspirated and infected cells are treated with a suitable buffer and/or enzyme e.g. PBS-EDTA or Trypsin. The medium supernatant and detached cells are centrifuged for obtaining a cell pellet, preferably at 5,000g, preferably for about 5 min. The person skilled in the art knows suitable mechanical, chemical or physical methods for releasing the parvovirus from the producer cells. Preferably, this can be done by freeze/thaw cycles, ultrasound treatment and/or Triton S100 treatment. The person skilled in the art also knows suitable methods for sonicating the cells and subsequent DNAse treatment. E.g., the cells can be sonicated at 30 to 70 W for a sufficient time and DNAse-treatment is carried out with 20-80 U/ml DNAse, usually at 37 C. for 10 to 50 min.
[0056] For the further purification and for the enrichment of full active parvoviral particles Iodixanol/PBS (IOD-PBS) and Visipaque/Ringer (Vis-Ringer) density gradients are carried out. These steps are described in further detail below.
[0057] The above mentioned Iodixanol is a synonym for Visipaque (for human injection use) or Iodixanolum(research grade). The chemical structure is
##STR00001##
[0058] The IUPAC name is 5-[acetyl-[3-[N-acetyl-3,5-bis(2,3-dihydroxypropylcarbamoyl)2,4,6,-triiodoanilino]2-hydroxypropyl]amino]-1-N,3,N-bis(2,3-dihydroxypropyl)-2,4,6-triiodobenzene-1,3-dicarboxamide. The CAS number is 92339-11-2. Visipaque is also a well known contrast agent for CT imaging.
[0059] (i) IOD-PBS and Vis-Ringer Density Gradients
[0060] Thus, to eliminate proteins, a step density gradient centrifugation was done as described by Zolotukhin (Zolotukhin et al., 1999). In a preferred embodiment, Quickseal tubes (Beckman, Germany, e.g. 2589 mm) were filled with 10-50 ml, preferably 20 ml, virus suspension. This suspension was underlaid with two to five, e.g. four, layers, of Iodixanol (Alexis Shield, Norway) in buffer, e.g. PBS. Preferred Iodixanol concentrations: 15, 25, 40, and 60%. Ultracentrifugation was performed for a suitable time and velocity in an ultracentrifuge, preferably 2 h at 4 C. in a 50.2 Ti rotor at 50,000 rpm (Beckman, L870M, Germany) that corresponds to 227,220 relative centrifugal force (RCF). Usually, 3.5 ml virus suspension was collected from the 40% Iodixanol layer. Afterwards, a second density gradient centrifugation was performed with Visipaque (GE Healthcare, Norway) diluted in buffer, preferably Ringer solution (B. Braun, Germany) for further protein elimination and separation of full from empty particles. In a preferred embodiment, Quickseal tubes (e.g. 2589 mm) were filled with virus suspension from the IOD-PBS density gradient, diluted at least 1:2.5 in buffer, e.g. Ringer solution. Then 1-10 ml (e.g. 5 ml) of 25%, 1-10 ml (e.g. 4 ml) of 40%, and 1-10 ml (e.g. 4 ml) of 55% Visipaque in Ringer solution were underlaid. For detection of the 40% layer, a reference gradient was made, where the 25% and 55% Visipaque/Ringer phases were colored with phenol red. Additionally, the 40% phase was labeled outside on the sample tube. Ultracentrifugation was performed under suitable conditions, e.g. for 2 h at 4 C. in a 50.2 Ti rotor at 50,000 rpm. Two fractions of the 40% phase were then collected with a syringe and hollow needle: preferably 1-5 ml, e.g. 2.5 ml, of the full-particle fraction (lower band in the 40% layer) and preferably 0.1-1.5 ml, e.g. 800 l, of the empty-particle fraction (upper band in the 40% layer). The refraction index of a 5-l sample was measured with a refractometer (AR200, Reichert Analytical Instruments, Germany) and the densities of the regions from which the fractions were taken were calculated with a reference table for Iodixanol (AXIS-SHIELD, Norway).
[0061] (ii) Continuous Vis-Ringer Gradient
[0062] For the continuous Vis-Ringer gradient, Quickseal tubes were filled with virus suspension diluted in Ringer solution to a refraction index of about 1.3 to 1.4, e.g. 1.3815, (corresponding to 30% Visipaque). The virus suspension was underlaid with 0.1-1 ml, preferably 0.5 ml, of 60-70%, preferably 65.2%, Visipaque cushion, and the tube was completely filled with preferably 30% Visipaque/Ringer solution. Ultracentrifugation was performed under suitable conditions, preferably for 10 h at 4 C. in a 70.1 Ti rotor at 63,000 rpm. Fractions of about 500 l were collected from the bottom under controlled dripping.
[0063] As mentioned above, according to the present invention, to obtain empty inactive particles a CsCl density gradient is carried out. A CsCl density gradient was established as described previously (Paradiso, 1981). In a preferred embodiment, polyallomere centrifuge tubes (Beckmann, Germany; 1495 mm) were filled with 1-10 ml, preferably 5 ml, CsCl at about 1.4 g/cm.sup.3 density and overlaid with 0.1-2 ml, preferably 1 ml, 1 M saccharose followed by 5 ml virus suspension. Ultracentrifugation was performed at suitable conditions, preferably 15 C. for at least 20 h at 39,000 rpm in an SW41 rotor. Different fractions were collected from the bottom (e.g. fr# 1: 500 l, fr# 2: 300 l, fr# 3-20: 200 l) and the capsid (physical particle, PP) content was measured by means of the new ELISA (C.sub.apsid-ELISA) described below or by hemagglutination assay. For detection of hemagglutination units (Kongsvik and Toolan, 1972), the fractions were diluted 1:10 to 1:50, e.g. 1:25, in buffer (e.g. PBS) and further diluted serially (e.g. 1:2) in a round-bottom 96-well plate (Greiner Bio-One, Germany). Next, a suitable amount, preferably 25 l, of a 2% suspension of guinea pig red cells (Charles River Laboratories, Germany) in PBS was added. The plate was incubated, e.g. for 1 h at 4 C., and the titer read as the highest dilution at which hemagglutination was complete. The refraction index was measured and the density calculated according to a reference table for CsCl (Griffith, 2006). Fractions containing full or empty capsids were pooled and directly dialyzed, e.g. against 1,000 volumes of VTE buffer for approximately 30 min at room temperature. This was followed by several, preferably three, dialysis cycles at 4 C. to eliminate the toxic CsCl.
[0064] In another preferred embodiment empty particles may also be obtained after the Vis-Ringer gradient centrifugation. As mentioned above the empty-particle fraction is located as the upper band in the 40% layer.
[0065] For inactivation of residual infectious particles in the empty-particle pool, the empty-particle fraction is subjected to a deactivation step. Suitable deactivation methods are UV inactivation (Tuynder et al, 2004), chemical, physical and/or thermical methods. The UV inactivation is particularly preferred.
[0066] The present invention also provides a method for determining the ratio of native parvovirus capsids to non-assembled capsid proteins or denatured capsids. In a preferred embodiment, the ratio is determined by using a monoclonal antibody. A particularly preferred monoclonal antibody is the monoclonal antibody BL-H1 (DSM ACC 3030). The person skilled in the art knows suitable formats for use of this antibody, e.g., ELISA.
[0067] The following examples are intended to illustrate, but not to limit the invention. While such examples are typical of those that might be used, other methods known to those skilled in the art may alternatively be utilized.
EXAMPLE 1
Material and Methods
[0068] (A) Producer Cell Line, H-1PV Virus Stock
[0069] NB-324K human newborn kidney cells transformed with simian virus 40 (SV40) (Tattersall and Bratton, 1983) were cultured at 37 C. in minimum essential medium (MEM, Sigma, Germany) with 5% heat-inactivated fetal bovine serum (FBS, Biowest, France) in a 5% CO.sub.2 atmosphere. The medium was supplemented with 100 U/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine (Life Technologies, Germany). For production, NB-324K cells propagated in 175-cm.sup.2 Y-flasks (Nunc, Denmark) were seeded into a 10-layer CeliSTACK culture chamber (Corning, Germany) with a 6,360cm.sup.2 growth area. Cell density and viability were measured by staining living cells with 0.4% trypan blue (Invitrogen, Germany). Cells were counted with a Countess Cell counter (Life Technologies, Germany). An in-house purified H-1PV virus stock was used to infect the cells.
[0070] (B) H-1PV Production
[0071] A 10-layer CellSTACK (CS) was chosen as a convenient single-use production system. For simultaneous cell seeding and infection, NB-324K cells were seeded at 3.610.sup.4 cells/cm.sup.2 into the 10-layer CS and infected immediately with H-1PV at a multiplicity of infection (MOI) of 0.01 plaque forming units (PFU) per cell. The pH during infection was 7.00.1. The infected cells were incubated for 4 days at 37 C. under 5% CO.sub.2 until the cytopathic effect (CPE), measured as the percentage of dead and detached cells observed under a microscope, reached at least 30%. For non-simultaneous seeding and infection, NB-324K cells were seeded at 7.910.sup.3 cells/cm.sup.2 into a 10-layer CS and allowed to grow for three days, by which time they had reached a density of approximately 3.6 x 10.sup.4 cells/cm.sup.2, as measured on a control-flask culture. These anchored cells were then infected at a MOI of 0.01 PFU/cell and incubated for 4 days as described above. For harvesting, the medium was aspirated and infected cells were treated with PBS/1 mM EDTA. The medium supernatant and detached cells were centrifuged for 5 min at 5,000g. The pellet was washed with PBS, resuspended in Virus Tris/EDTA buffer, pH 8.7 (VTE) containing 0.05 M Tris HCl, 0.5 mM EDTA, and subjected to three freeze/thaw cycles. After centrifugation for 5 min at 5,000g, cell debris were discarded. The cell lysate was then sonicated at 48 W for 1 min in a Sonorex Super 10 P ultrasonic homogenizer (Bandelin, Germany) and treated with DNAse (50 U/ml, Sigma, Germany) for 30 min at 37 C.
[0072] (C) H-1PV Purification
[0073] The DNase-treated virus harvest was clarified by filtration through a 0.2-m Sartolab P20 Plus filter (Sartorius, Germany). Two different methods were used to purify the virus, either two consecutive step gradientsone with Iodixanol-PBS (IOD-PBS) and one with Visipaque-Ringer (VIS-Ringer)or a cesium chloride density gradient followed by dialysis against VTE buffer.
[0074] (i) IOD-PBS and VIS-Ringer Density Gradients
[0075] To eliminate proteins, a step density gradient centrifugation was done as described by Zolotukhin (Zolotukhin et al., 1999). For this, 2589 mm Quickseal tubes (Beckmann, Germany) were filled with 20 ml virus suspension. This suspension was underlaid with four layers of Iodixanol (Alexis Shield, Norway) in PBS (Iodixanol concentrations: 15, 25, 40, and 60%). Ultracentrifugation was performed for 2 h at 4 C. in a 50.2 Ti rotor at 50,000 rpm (Beckmann, L870M, Germany) that corresponds to 227,220 relative centrifugal force (RCF). Usually, 3.5 ml virus suspension was collected from the 40% Iodixanol layer. Afterwards, a second density gradient centrifugation was performed with Visipaque (GE Healthcare, Norway) diluted in Ringer solution (B. Braun, Germany) for further protein elimination and separation of full from empty particles. For this, 2589 mm Quickseal tubes were filled with virus suspension from the IOD-PBS density gradient, diluted at least 1:2.5 in Ringer solution. Then 5 ml of 25%, 4 ml of 40%, and 4 ml of 55% Visipaque in Ringer solution were underlaid. For detection of the 40% layer, a reference gradient was made, where the 25% and 55% Visipaque/Ringer phases were colored with phenol red. Additionally, the 40% phase was labeled outside on the sample tube. Ultracentrifugation was performed for 2 h at 4 C. in a 50.2 Ti rotor at 50,000 rpm. Two fractions of the 40% phase were then collected with a syringe and hollow needle: 2.5 ml of the full-particle fraction (lower band in the 40% layer) and 800 pl of the empty-particle fraction (upper band in the 40% layer). The refraction index of a 5-l sample was measured with a refractometer (AR200, Reichert Analytical Instruments, Germany) and the densities of the regions from which the fractions were taken were calculated with a reference table for Iodixanol (AXIS-SHIELD, Norway).
[0076] (ii) Continuous Vis-Ringer Gradient
[0077] For the continuous Vis-Ringer gradient, Quickseal tubes were filled with virus suspension diluted in Ringer solution to a refraction index of 1.3815 (corresponding to 30% Visipaque). The virus suspension was underlaid with 0.5 ml of 65.2% Visipaque cushion, and the tube was completely filled with 30% Visipaque/Ringer solution. Ultracentrifugation was performed for 10 h at 4 C. in a 70.1 Ti rotor at 63,000 rpm. Fractions of about 500 l were collected from the bottom under controlled dripping.
[0078] (iii) Cesium Chloride Density Gradient and Hemagglutination Assay
[0079] A CsCl density gradient was established as described previously (Paradiso, 1981). For this, 1495 mm polyallomere centrifuge tubes (Beckmann, Germany) were filled with 5 ml CsC1 at 1.4 g/cm.sup.3 density and overlaid with 1 ml 1 M saccharose followed by 5 ml virus suspension. Ultracentrifugation was performed at 15 C. for at least 20 h at 39,000 rpm in an SW41 rotor. Different fractions were collected from the bottom (fr# 1: 500 l, fr# 2: 300 l, fr# 3-20: 200 l) and the capsid (physical particle, PP) content was measured by means of the new ELISA (Capsid-ELISA) described in this paper or by hemagglutination assay. For detection of hemagglutination units (Kongsvik and Toolan, 1972), the fractions were diluted 1:25 in PBS and further diluted serially 1:2 in a round-bottom 96-well plate (Greiner Bio-One, Germany). Next, 25 l of a 2% suspension of guinea pig red cells (Charles River Laboratories, Germany) in PBS was added. The plate was incubated for 1 h at 4 C. and the titer read as the highest dilution at which hemagglutination was complete. The refraction index was measured and the density calculated according to a reference table for CsCl (Griffith, 2006). Fractions containing full or empty capsids were pooled and directly dialyzed against 1,000 volumes of VTE buffer for approximately 30 min at room temperature. This was followed by three dialysis cycles at 4 C. to eliminate the toxic CsCl.
[0080] (D) UV Inactivation of the Empty-Particle Pool
[0081] For inactivation of residual infectious particles in the empty-particle pool, 500 l of empty-particle fraction was placed at the center of a 6-cm dish (Greiner Bio-One, Germany) under a sterile laminar flow hood. A UV lamp (Type NU-4, Herolab, Germany) emitting at 254 nm was used to irradiate the sample at 0.5 mW/cm.sup.2, as measured with a radiometer (VLX-3W, Benda, Germany). The sample was irradiated 4 times for 2 min with intervals of 5 min without UV.
[0082] (E) Virus Quantification and Characterization
[0083] (i) Plaque Formation Assay
[0084] Plaque assays were done essentially as described by Tattersall and Bratton, 1983. NB-324K cells were grown in monolayer cultures in MEM medium containing 5% FBS, 100 g/ml penicillin, 100 g/ml streptomycin, and 2 mM L-glutamine. They were infected at 60% confluence with serial dilutions of H-1PV and incubated for 1 h at 37 C. Then the inoculum was replaced with a bacto-agar overlay (1.7% in MEM containing 5% FBS). On day four post-infection, living cells were stained for 18-24 h by addition of 0.02% toluylene red staining solution (Sigma, Germany) containing bacto-agar (Becton Dickinson, Germany). The dishes were incubated at 37 C. under 5% CO.sub.2. Plaque-forming units were counted 5 days post-infection on a light box and their concentration expressed in PFU/ml.
[0085] (ii) DNA Hybridization Assay for Infectious H-1PV
[0086] NB-324K cells (7.610.sup.3 cells/well) were seeded into a 96-well plate 24 h prior to infection with H-1PV. They were infected with 10-fold serial dilutions of H-1PV and incubated for 72 h at 37 C. under 5% CO.sub.2. After freezing at 80 C. and alkaline lysis (1.5 M NaOH), their DNA was transferred to a nylon membrane, cross-linked with a CL-1000 Ultraviolet Crosslinker (UVP, USA) and hybridized with an NS1-specific .sup.32P-radiolabeled probe prior to autoradiography. Virus titration was performed in duplicate and the titer expressed in infectious units (IU) per ml (Lacroix et al., 2010).
[0087] (iii) Determination of Genome-Containing Viral Particles
[0088] The number of genome-containing viral particles (GP) was determined by Q-PCR, essentially as described previously (Lacroix et al., 2010). Each well received 20 l reaction mix containing 1 Premix Ex Taq (TaKaRa, France), 0.3 M labeled NS1-TaqManm probe, each primer at 0.3 M, and 3 l template. Q-PCR was run in an Abi Prism 7900 HT Sequence Detection System and results were processed with the SDS 2.1 software (Applied Biosystems, Germany).
[0089] (iv) H-1PV Capsid-ELISA
[0090] An ELISA was developed for detection of capsids (full or empty, henceforth called physical particles or PP). A flexible 96-well, U-bottomed plate (BD Falcon, N.J.) was coated overnight at 4 C. with 100 l monoclonal antibody BL-H1 at 2 ng/l in PBS. The development of this antibody (Leuchs et al., 2010) is described below. The coating solution was removed, 200 l blocking buffer (2 mg/ml casein (Sigma, Germany) and 0.05% Tween 20 (Sigma, Germany) in PBS) was added to each well, and the mixture incubated for 1 h at 37 C. Blocking buffer was aspirated and the wells were washed with PBS, 0.05% Tween 20, after which the positive control, the negative control, the sample, and serial dilutions of H-1PV standard (all of them in PBS buffer) were plated in duplicate at 100 l/well. The plate was incubated for 1 h at 37 C. The reaction mixture was aspirated and the wells were washed. Next, 100 l mAb BL-H1 labeled with horseradish peroxidase (0.1 g/ml) was added to each well and incubated for 1 h at 37 C. Samples were aspirated and the wells were washed with PBS, 0.05% Tween 20. After addition of 100 l 3,3,5,5-tetramethylbenzidine (Sigma, Germany) to each well, the plate was incubated for 15 min at room temperature in the dark. The reaction was stopped by adding 100 l of 1 M H.sub.2SO.sub.4 per well. Sample absorbance was measured at 450 nm with an Ascent Multiscan Plate Reader (Thermo Fisher Scientific, Germany).
[0091] (v) Bicinchoninic Acid (BCA) and Bradford Assays
[0092] Protein concentration was determined with the Pierce BCA Protein Assay Kit 23227 (Pierce, USA) in the 5-250 g/ml working range. The protein concentration was determined with a bovine serum albumin (BSA) standard and measured with Nanodrop 2000. The Bradford Assay was performed in a 96-well plate in the 50-2000 g/ml working range, with BSA as standard (Sigma, Germany).
[0093] (vi) SDS-PAGE, Silver Staining, and Western Blotting
[0094] To assess the purity of virus preparations, a 10% SDS-PAGE was performed (SERVA Electrophoresis, Germany), followed by silver staining (Invitrogen, CA) and western blotting. For viral capsid protein (VP) detection, polyclonal rabbit anti-VP (VP) (provided by C. Dinsart, DKFZ, Heidelberg) and a horseradish-peroxidase-conjugated anti-rabbit IgG (AmershamTM ECL Western Blotting Analysis) were used.
[0095] (vii) DNA Measurement
[0096] DNA was quantified by measuring the absorbance at 260 nm with a Nanodrop spectrophotometer. Human genomic DNA in the virus preparations was quantified by Q-PCR with the Quantifiler Human DNA Quantification Kit (Applied Biosystems, Germany) for detection of h-TERT (human telomerase reverse transcriptase), according to the manufacturer's instructions. The detection limit was 26 ng/ml and the amplicon size was 62 bp. As positive control, NB-324K cell genomic DNA was used.
[0097] (viii) Endotoxins and Sterility
[0098] Contamination by endotoxins was tested with the Endosafe Gel-Clot Limulus Amebocyte Lysate Assay (Charles River Laboratories, Germany). The sensitivity of the assay was 0.25 endotoxin unit (EU) per milliliter. Each H-1PV preparation was checked for the absence of bacterial or fungal contamination by incubating 2.5 l of the preparation on soy/peptone-agar for 5 days at 37 C.
[0099] (ix) Electron Microscopy
[0100] For qualitative analysis of virus preparations, electron microscopy pictures were taken. For this, 5 l virus suspension was added to a ready-to-use carbon-coated copper grid and incubated for 2 min. The grid was then washed with 5 l bidest water and coated with 2% uranyl acetate for 30 s. The drops were absorbed from the grid with Whatman 50 filter paper and the grid was dried for approximately 1 min. Photos were taken with a Zeiss transmission electron microscope at 20,100 magnification.
[0101] (F) Development and Characterization of the Monoclonal Antibody BL-H-1
[0102] To generate a monoclonal antibody against H-1PV capsids (PP), Balb/c mice (Charles River, Germany) were immunized intraperitoneally, 3 times over a 3-month period, with 1.210.sup.8 PFU each time. One week after the last H-1PV injection, their spleens were removed and spleen cells were fused with X63/Ag8 lymphoma cells (Kuck et al., 2007; Wobus et al., 2000). The hybridoma cells were propagated and the supernatants from single-clone were screened by western dot blotting against H-1PV. Positive wells were selected by single-colony analysis, and after three selection rounds, the selected hybridoma cells were used to produce BL-H1 antibodies in a CELLine 1000 bioreactor (Integra Biosciences AG, Switzerland). RPMI 1640 medium supplemented with 10% FBS, 100 g/ml penicillin, 100 g/ml streptomycin, 2 mM L-glutamine, 20 mM Hepes pH 7.2 was used to culture the hybridoma cells. The subclass of the BL-H1 antibody was determined with the Amersham mouse monoclonal antibody isotyping kit (Braunschweig, Germany). Purification was done with the HiTrap Protein A HP affinity column kit (GE Healthcare, Sweden) and the Akta prime system (GE Healthcare, Germany). The IgG.sub.2a concentration was determined with the Mouse IgG.sub.2a ELISA Set (BD Biosciences, Germany).
[0103] (i) H-1PV Analysis by Western Dot Blotting
[0104] Purified H-1PV (110.sup.8 PFU/dot) or sucrose density gradient fractions (diluted 1:10 in 100 l PBS) were transferred to a nitrocellulose membrane (AppliChem, Germany) with a vacuum blotter. Washing steps were carried out with PBS, 0.05% Tween 20. The membrane was blocked for 1 h with PBS containing 5% skimmed milk powder. For hybridoma screening, 40 l undiluted hybridoma supernatant was incubated for at least 3 h. For sucrose gradient analysis, either purified mAb BL-Hl (1:1,000) or VP antibody (1:500 diluted) was used. The membrane was then washed for 30 min at RT and incubated with secondary peroxidase-coupled goat anti-mouse (GE Healthcare, Germany) or goat anti-rabbit antibody (GE Healthcare, Germany) in PBS. Detection was done with ECL Plus and Hyperfilm ECL (Amersham Biosciences GmbH, Germany).
[0105] (ii) Sucrose Density Gradient Fractionation of Assembled and Non-Assembled Viral Capsid Proteins
[0106] Extract was prepared from 293T cells (CRL-11268, American Tissue Culture Collection) transfected with an infectious H-1PV molecular clone (Kestler et al., 1999) and harvested 72 h post-transfection. One ml of extract was transferred to a 10-50% linear sucrose gradient. Centrifugation was performed at 4 C. for 3.3 h at 28,000 rpm in a TST 41.14 (Kontron) rotor. Fractions (400 l) were collected and analyzed by western blotting and hemagglutination assay. The fractions were also assayed for infectivity and genome-containing particles.
EXAMPLE 2
Characterization of H1-PV
[0107] One of the aims of the present invention was to standardize the H-1PV manufacturing procedure. This involved characterizing each upstream and downstream process step, ensuring reproducibility, and fully characterizing the identity, purity, and safety of the final product in order to establish standard operating procedures.
[0108] Two alternative methods of cell preparation and infection were tested upstream from the core production process. Two different purification tracks downstream from core production were also compared. Table 1 summarizes the assays used to characterize intermediate steps in the production procedure and the final product as described below. To quantify physical particles more conveniently, it was necessary to establish a new assay, described below.
TABLE-US-00001 TABLE 1 Assays used for the quantitative and qualitative characterization of H-1PV stocks Type of Assay characterization Unit Plaque formation Biological assay for PFU/ml assay virus multiplication and spread. Virus titers expressed in plaque forming units DNA hybridization In situ assay for IU/ml assay for infectivity virus infection and DNA replication. Virus titers expressed in infectious units Viral genome quantification Physical GP/ml by real-time PCR (Q-PCR) characterization for presence of genome containing viral particles Capsid-ELISA Physical PP/ml determination of assembled viral particles, expressed in physical particles Hemagglutination Physical HAU/ml assay characterization for virus mediated guinea pig erythrocytes agglutination. Virus titer expressed in hemagglutination units Refraction index Physical determination mg/cm.sup.3 measurement of density from the refractive index (RI) Bicinchoninic and Colorimetric protein g protein/ml Bradford assays quantification SDS-PAGE and Determination of purity Western blot and identity of viral proteins Q-PCR with Quantification of cell ng DNA/ml h-TERT Quantifiler genomic DNA A.sub.260 Spectrometric ng DNA/ml measurement determination of DNA Electron microscopy Ultrastructural visualization of virus stocks LAL assay Determination of EU/ml endotoxin units Sterility assay Determination of CFU/ml bacteria and fungi colony-forming units on soy/peptone-agar plates
[0109] Two goals of this study were to separate full from empty particles and to determine the particle-to-infectivity ratio of the final product. Determining this ratio, which should influence both the therapeutic efficacy of parvoviral treatment and the expected immune response, requires determining the total number of physical particles (Rajendran et al.). To develop a convenient method for quantifying PP, we generated a monoclonal antibody against H-1PV, which recognizes native capsids but fails to recognize non-assembled capsid proteins or denatured capsids. This antibody, called BL-H1, is of type IgG.sub.2a. Its specificity is illustrated in
EXAMPLE 3
ELISA for H-1PV Capsid Quantification
[0110] To quantify physical particles throughout H-1PV production, we developed a Capsid-ELISA using mAb BL-H1. To standardize the ELISA, H-1PV was mixed with appropriate dilutions of a stock of adenovirus type 5 (Ad5, American Type Culture Collection) of known titer and the mixtures were visualized by electron microscopy. On 8 randomly chosen pictures, H-1PV and Ad5 particles were counted and the number of H-1PV particles was determined in relation to the known Ad5 titer. The ELISA showed a reproducible linear relationship between the number of PP (in the range 2.510.sup.9 to 3.910.sup.7 PP) and absorbance at 450 nm (
EXAMPLE 4
Large-Scale Production of H-1PV
[0111] (A) Optimization of Cell Seeding and Infection
[0112] We first optimized the cell density at the time of infection, the MOI, and the time of harvest. The highest virus yields were obtained with a cell density of 3.610.sup.4 cells/cm.sup.2, a MOI of 110.sup.2 PFU/cell, and harvesting 4 days post-infection, with approximately 30% CPE (data not shown). These conditions were used subsequently in all production experiments, the only difference being that some cells were grown in 175-cm.sup.2 Y-flasks, harvested, transferred to a CS, and infected immediately, while others were allowed to grow in the CS for three days prior to infection.
[0113] Table 2a shows that cells infected and seeded simultaneously and cells allowed to grow for three days in the CS prior to infection did not differ significantly as regards either virus production per infected cell or virus release into the supernatant per infected cell. In both cases, approximately 110.sup.3 PFU were produced per infected cell, corresponding to an average yield of 210.sup.11 PFU per 10-layer CellSTACK. Protein concentrations in the virus harvests obtained were also similar for the two procedures (210.sup.3 g/ml), indicating that the two cultures reached the same density at harvest time. As infection three days post-seeding saved working time and material as compared to simultaneous seeding and infection, the former approach was adopted. The culture medium supernatant contained only 10% of the total PFUs, at relatively low concentration (around 10.sup.7 PFU/ml). As concentrating the supernatant was cumbersome, time consuming, and inefficient, this fraction was discarded in routine productions.
TABLE-US-00002 TABLE 2a Infectious-particle recoveries after two different seeding/infection procedures Seeding/infection procedure PFU/infected cell g/ml protein Cell extract 3 days in CS prior 1.1 1.8E+03 2.0 0.9E+03 to infection simultaneous seeding 0.8 0.4E+03 1.7 0.4E+03 and infection Medium 3 days in CS prior 0.1 0.2E+03 0.8 0.4E+03 supernatant to infection simultaneous seeding 0.5 0.4E+03 0.9 0.4E+03 and infection Means with standard deviations of infectious particle and protein concentrations in virus harvests. Concentrations in cell extracts (20 ml/CS) and medium supernatants (1000 ml/CS) were calculated from 5 or more independent productions.
[0114] (B) Reproducibility of the Standardized Upstream Process and H-1PV Yields
[0115] As shown in
EXAMPLE 5
Purification of H-1PV
[0116] (A) DNase Digestion and Clarification of H-1PV Harvests
[0117] Unprocessed virus harvests were treated with DNase to digest non-encapsidated viral and host-cell DNA and then clarified by filtration through a Sartolab P20 Plus filter. Results obtained for five individual batches showed the significance of these steps. As measured with the human DNA Quantification Kit, 99.8% of the host-cell DNA was removed by DNase treatment. Yet only 37% of the total DNA was eliminated, as determined by A.sub.260nm measurement. The residual DNA might be protected viral genomes and/or cellular DNA fragments smaller than the h-TERT amplicons (62 bp) used for DNA detection. As illustrated in
TABLE-US-00003 TABLE 2b Virus harvest yields (cell extracts) and recoveries after clarification Virus harvest After clarification Recovery % PFU/ml 7.2 6.4E+09 5.8 6.1E+09 81 GP/ml 4.8 2.7E+12 6.1 3.4E+12 127 PP/ml 1.7 0.8E+13 1.9 1.3E+13 111 protein 2.1 0.9E+03 1.6 0.4E+03 76 g/ml Virus recovered from one 10-layer CS was dissolved in 20 ml VTE and the PFU, GP and PP were determined. Protein concentration was quantified by colorimetric assay. Means with standard deviations for 5 independent productions are shown. Recovery was calculated after clarification of the virus harvest by 0.2 m filtration.
[0118] (B) Separation of Full from Empty Particles
[0119] To separate full from empty H-1PV particles, two gradient centrifugation procedures were compared. Similar results were obtained with five individual H-1PV harvests from two 10-layer CS cultures. After clarification, each harvest was split into two equal parts, one of which was purified by two consecutive step gradients: IOD-PBS and VIS-Ringer, the Ringer and Visipaque formulations of the latter being well suited for injection into humans. As described under Materials and Methods, two fractions were collected, the full-particle fraction (lower band in the 40% phase) with an approximate density of 1.25 g/ml, and the empty-particle fraction (upper band in the 40% phase) with an approximate density of 1.23 g/ml. As shown in
TABLE-US-00004 TABLE 2c Empty particle fraction after Visipaque/Ringer gradient centrifugation Titer.sup.a empty particles fraction.sup.b PP/ml 3.9E+13 2.9E+13 GP/ml 4.6E+12 3.1E+12 PFU/ml 6.3E+09 5.8E+09 PP/PFU 6.1E+03 6.5E+03 .sup.aPP, physical particles; GP, genome-containing particles; PFU, plaque-forming units .sup.bAfter Visipaque/Ringer gradient centrifugation
[0120] As an alternative to obtain empty particles the second half of the virus harvest was fractionated on a continuous CsCl density gradient followed by dialysis against VTE. As shown in
[0121] (C) Recovery of and Enrichment in Infectious H-1PV Particles
[0122] As shown in
[0123] (D) Recovery of Empty Particles
[0124] As shown in
[0125] (E) H-1PV Concentration by Continuous VIS-Ringer Gradient Centrifugation
[0126] The titer of infectious particles in virus batches obtained by means of the above-mentioned IOD-PBS/VIS-Ringer and CsCl purification methods was about 110.sup.10 PFU/ml. Since a higher titer is required for some applications, we tried replacing the VIS-Ringer step gradient with a continuous gradient. This enabled us to achieve a titer of 310.sup.11 PFU/ml (
EXAMPLE 6
Inactivated Empty Particles
[0127] In the empty-particle fraction obtained after CsCl fractionation, infectious virus could still be detected (PFU-to-PP ratio: 1:10.sup.5). To allow the use of empty particles as a non-infectious control, it was necessary to develop an inactivation method that eliminates virus infectivity without altering capsid structure. This was achieved by UV irradiation (Table 3). While UV irradiation did not change the PP titer, it reduced the measured GP titer by 90%. This is likely due to interference of the DNA damage induced in the viral genomes with their titration by Q-PCR. Importantly, UV irradiation reduced virus infectivity by more than 7 logs, with a residual infectivity as low as 1 PFU per 710.sup.11 PP. This made it possible to use purified preparations of UV-irradiated empty particles as non-infectious controls in preclinical studies of the oncolytic effects of H-1PV (Kiprianova et al., 2011).
TABLE-US-00005 TABLE 3 Effect of UV irradiation on contamination of the empty-particle fraction by infectious virions Empty-particle UV-inactivated Titer.sup.a fraction.sup.b empty-particle fraction.sup.c PP/ml 1.1 0.7E+14 1.1 0.7E+14 GP/ml 1.2 1.0E+12 9.0 5.1E+10 PFU/ml 1.8 3.0E+09 1.6 0.8E+02 PP/PFU 4.6 4.3E+05 6.9 4.9E+11 .sup.aPP, physical particles; GP, genome-containing particles; PFU, plaque-forming units .sup.bAfter CsCl gradient centrifugation (see FIG. 6b) .sup.c254 nm, 0.5 mW/cm.sup.2, samples irradiated 4 times for 2 min at 5-min intervals
EXAMPLE 7
Purity of the H-1PV Preparations
[0128] The H-1PV preparations were analyzed for protein and DNA contamination, the presence of endotoxins, and sterility. Proteins from different steps and gradient fractions in the purification process were analyzed by SDS-PAGE and revealed by silver staining. As illustrated in
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