Method For Purifying An Enveloped Virus

Abstract

The present invention relates to a method for purifying an enveloped virus. The present invention further relates to an enveloped virus or a plurality of enveloped viruses obtainable by said method.

Claims

1. A method for purifying an enveloped virus comprising the steps of: (i) binding an enveloped virus comprised in a preparation to a mixed mode chromatography carrier, and (ii) eluting the enveloped virus from the mixed mode chromatography carrier, wherein the mixed mode chromatography carrier is a hydrophobic ion exchange chromatography carrier.

2. The method of claim 1, wherein the preparation comprising an enveloped virus in step (i) is subjected to one or more of the following steps selected from the group consisting of: (a) cell lysis, (b) virus clarification, and (c) nuclease treatment prior to binding the enveloped virus to the mixed mode chromatography carrier.

3. The method of claim 1, wherein the mixed mode chromatography carrier is equilibrated with an equilibration buffer.

4. The method of claim 1, wherein the method further comprises the step of: (iii) washing the mixed mode chromatography carrier with a washing buffer, wherein the enveloped virus remains bound to the mixed mode chromatography carrier.

5. The method of claim 1, wherein the enveloped virus is eluted from the mixed mode chromatography carrier with an elution buffer.

6. The method of claim 3, wherein the eluting in step (ii) is achieved using an elution buffer having a higher salt concentration than the equilibration buffer and washing buffer, an elution buffer having a higher pH than the equilibration buffer and washing buffer, or an elution buffer having a higher salt concentration and a higher pH than the equilibration buffer and washing buffer.

7. The method of claim 5, wherein the elution buffer comprises arginine.

8. The method of claim 1, wherein by eluting the enveloped virus from the mixed mode chromatography carrier in step (ii), a mixed mode eluate is formed.

9. The method of claim 8, wherein the eluate is further subjected to one or more of the following steps selected from the group consisting of: (a) filtration, (b) chromatography, and (c) nuclease treatment.

10. (canceled)

11. (canceled)

12. An elution buffer comprising arginine.

13. (canceled)

14. A kit for purifying an enveloped virus comprising: (i) a mixed mode chromatography carrier, wherein said mixed mode chromatography carrier is a hydrophobic ion exchange chromatography carrier, (ii) one or more of the following buffers: equilibration buffer, washing buffer, and elution buffer, and (iii) optionally a nuclease.

15. The kit of claim 14, wherein said elution buffer comprises arginine.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0250] The following Figures and examples are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

[0251] FIG. 1: Schematic presentation of an exemplarily method of enveloped virus purification according to the present invention.

[0252] FIG. 2: Effect of Arginine during elution from a mixed mode medium. (a) Representative chromatogram of virus capture and recovery under indicated conditions. Virus lysate was loaded to the mixed mode resin, washed and finally eluted by applying either buffer supplied with 2 M NaCl only (grey) or elution buffer containing 2 M NaCl and 0.25 M Arginine (black). b) Typical recoveries of infectious particles (IVP) and Peak areas during the individual chromatography steps shown in a) in absence or presence of arginine (+/−Arg) in the elution buffer.

[0253] FIG. 3: Reproducibility of virus capture using a mixed mode medium. Overlay of successive chromatography runs (n=10) performed on the same mixed mode resin under optimised conditions demonstrates the robustness of virus capture through the described method. In each step, optimised amounts of virus lysates from the primary clarification step were loaded, washed and finally recovered using elution buffer supplied with 2 M NaCl and 0.25 M arginine.

[0254] FIG. 4: High arginine concentrations can substitute high salt requirements during elution and allow further improved virus recoveries from a mixed mode carrier. a) Representative chromatogram of virus capture and recovery under indicated conditions. Virus lysate was loaded to the mixed mode resin, washed and finally eluted by applying buffer supplied either with 2 M NaCl and 0.25 M Arginine (grey) or elution buffer containing 0.5 M NaCl and 0.75 M Arginine (black). b) Typical recoveries of infectious particles (IVP) and Peak areas during the individual chromatography steps shown in a) in absence or presence of arginine (+/−Arg) in the elution buffer.

EXAMPLES

[0255] The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

Example 1

Modified Vaccinia Ankara (MVA) Virus Production

[0256] MVA-CR19.gfp, a fluorescent version of the parental MVA-CR19 clone (U.S. Pat. No. 9,732,325 B2), was produced in AGE1.CR.pIX cells (Jordan I, Vos A, Beilfuss S, Neubert A, Breul S, Sandig V. An avian cell line designed for production of highly attenuated viruses. Vaccine. 2009; 7:748-756) cultivated in growth medium until a viable cell density of 2×10.sup.6 cells/ml. For virus production, 50% of the growth medium CD-U4 (GE Healthcare, USA) was replaced by CD-VP4 medium (Gibco, USA) to induce cellular aggregates supporting virus spread. Cells were infected with a multiplicity of infection (MOI) of 0.005 and cultured for up to three days before harvest. Cells were not separated from culture supernatant but lysed directly using ultrasound. Titers of 10{circumflex over ( )}9/ml or above were regularly obtained in this process.

Example 2

MVA Virus Titration (TCID50, qTCID50)

[0257] Virus titration of MVA was performed on adherent AGE1.CR.pIX for both methods TCID50 and qTCID50. For the TCID50 (Tissue Culture Infection Dose 50) assay, 96-well plates were infected 24 h after seeding 2.5×10.sup.5 cells/ml in DMEM/F12 (Gibco, USA) containing 5% FCS by serial virus dilutions. The evaluation was 48 h after the incubation at 37° C. and calculated according to Reed and Muench (Reed L J, Muench H. A simple method of estimating fifty per cent endpoints. Am J Hyg 1938; 27: 493-497). Furthermore qTCID50, the titer determination by quantitative polymerase chain reaction (qPCR), for MVA was developed and established. Similarly to TCID50 the cell-plates were infected by the defined standard diluted 10.sup.−2-10.sup.−6 and the samples diluted 10.sup.−3. After the incubation for 6 h the medium was removed and the wells were washed with PBS. For cell lysis 50 μl per well QuickExtract DNA Extraction Solution 1.0 (Epicentre, USA) was used and the plate heated to 65° C. for 15 min, following by an incubation step at 95° C. for 5 min. After adding 100 μl WFI, the samples were used for qPCR. To 15 μl of the final mastermix which contains the primer pair MVA128L 5′-CGTTTTGCATCATACCTCCATCTT-3′ (SEQ ID NO: 1) and 5′-GCGGGTGCTGGAGTGCTT-3′ (SEQ ID NO: 2), TIB MolBiol, Germany), Power SYBR Green PCR Master Mix (Thermo Fisher Scientific, USA) and WFI 5 μl probe was added each. For the non-template control (NTC) WFI was used. The qPCR was performed in a StepOnePlus RealTime PCR System (Applied Biosystems, USA) programmed to 95° C. for 10 min followed by 40 cycles of denaturation at 95° C. for 15 s and annealing and DNA amplification at 60° C. for 1 min. The evaluation was based on the ct-values and the titer of the MVA standard.

Example 3

MVA Virus Purification via Mixed Mode Chromatography

[0258] At harvest, part of the virus can be found in the supernatant, however, the majority of the virus remains inside cells. Although the virus fraction in the supernatant is higher for MVA-CR19 (U.S. Pat. No. 9,732,325 B2), the intracellular fraction is still important and cell lysis is preferably included into the process. For release of intracellular MVA virus, cells in production medium were incubated with chaotropic salts (250 mM NaBr, 250 mM NaCl, and/or 150 mM KCl, see U.S. Pat. No. 9,273,289 B2) to detach host cell DNA from virus particles and lysed using ultrasound (amplitude 10%, 45 s).

[0259] Primary clearance of the lysate was performed with prefilters (Sartopure, Sartorius, Germany) to remove cellular debris and larger particles that might cause challenges in the following purification steps (FIG. 1).

[0260] Already after this stage, nuclease treatment can be applied to reduce host cell DNA. However, precipitates were observed in lysates treated with nuclease, requiring an additional filtration step as a prerequisite to chromatography. This step removed more than 50%, sometimes even 80% of the virus. Moreover, at the high volume of the cleared lysate nuclease treatment is not economic. Therefore, although nuclease treatment can be performed at this stage which would ease removal of nuclease in the following steps, it is preferred to omit nuclease treatment at this stage but to perform it after chromatography instead.

[0261] The central component for virus purification is an efficient capture step allowing in parallel reduction of harvest volumes, concentration of virus and removal of major contaminants early during downstream purification. Due to their various modes of binding interactions, mixed mode carriers were anticipated to be particularly suitable for enveloped viruses with their inhomogeneous structure, lipid and protein composition as well as their heterogeneous surface charges. Moreover, mixed mode carriers were considered applicable to various viruses as a platform step. However, the typical conditions binding to and eluting from mixed mode carriers may cause inactivation of fragile enveloped viruses. Mixed mode carrier compatible for large scale production have recently become commercially available through various vendors but have not been used for enveloped viruses. By screening different mixed mode ligands, capture of MVA for a number of ligands under conditions compatible with viable virus was indeed observed. Most efficient capture was achieved efficient with mixed mode cation exchangers Capto MMC™ (available from GE Healthcare) and Nuvia™ cPrime™ (available from Bio-Rad) under the applied conditions (pH 7.2±0.2, 55 mS/cm) as judged by substantial reduction of infectious units in the flow through fraction (FIG. 2).

[0262] For a preferred process, 5×10.sup.9 infectious virus particles were loaded per ml of resin (Nuvia cPrime, Biorad, Germany) equilibrated with 50 mM Tris, 0.5 M NaCl, pH 7.2. Contaminants were washed out with 25 CV of Wash buffer (50 mM Tris, 0.5 M NaCl, pH 7.2) as evident by the increase in UV absorption (FIG. 2a).

[0263] To release viral particles from the mixed mode carrier, increasing the salt concentration in the elution buffer was tested. Elution with 50 mM Tris. HCL and 2 M NaCl allowed to recover about 24% of infectious virus, whereas the residual bound material was released during CIP (FIG. 2b). Interestingly, virus recoveries were significantly improved from 24% to about 40% when arginine was added to the elution buffer and the CIP peak area diminished accordingly (FIG. 2a, b)

[0264] It is, therefore, preferred to elute virus with an elution buffer containing 50 mM Tris.HCL, 2 M NaCl, and 250 mM arginine, pH 7.2. Considering the negative impact of arginine on viruses, this finding is surprising. Notably, the eluted material was relieved from about 97.5% of contaminating cellular DNA (Table 1) as well as about 98% of protein contaminants and, thus, can be designated as substantially purified when compared to the initial material applied to the carrier.

[0265] Consistency of virus capture using the mixed mode ligand was examined by successive runs on the same mixed mode carrier using an elution buffer containing arginine. As shown in FIG. 3, virus capture and elution could be reproducibly performed with the method described here, even at extended use of the carrier (n=10). Besides demonstration of process robustness, this finding has major impact on the column size required for scale-up and suggests that the process described here is not restricted by such issues.

Example 4

Reduction of DNA by Nuclease Treatment and Removal of Nuclease

[0266] To further reduce cellular DNA burden and meet the requirement for human vaccines/therapeutic products, another chromatography might be considered but nuclease treatment might be the most appropriate step. However, the high salt concentration in the eluate might be not compatible with the activity of many nucleases. To digest, DNA buffer can be exchanged by TFF to reduce the salt concentration, an additional step that will reduce overall yield. Alternatively, a nuclease active in higher salt concentration such as SAN (Artic Enzymes, Norway) may be used. The recommended salt concentration for this nuclease is 500 mM with, some activity preserved at 1M. Surprisingly, an 150 fold reduction of DNA per virus dose was found when the partially purified material/eluate was treated with 50 U/ml of SAN overnight at room temperature.

[0267] After digestion, nuclease can be removed either by tangential flow filtration or by using a gel filtration carrier separating the enzyme from the virus particles due to size differences. Diafiltration against elution buffer using hollow fibers (750 kDa mPES, SpectrumLabs, USA) and mild shear rates (500 1/s) removed nuclease with a step yield of 98%. The same step is applied to change buffer to a suitable formulation for application.

TABLE-US-00001 TABLE 1 Representative virus yields and DNA levels during purification of recombinant MVA using an elution buffer containing 2M NaCl and 250 mM Arginine (examples 3 and 4). Cell lysis and primary filtration were performed as described in example 3. Nuvia cPrime was loaded with 5 × 10.sup.9 IVP/ml resin. Contaminants were washed out with 25 CV of Wash buffer (50 mM Tris, 0.5M NaCl, pH 7.2) and bound virus eluted with 5 CV of Elution buffer (50 mM Tris.HCL, 2M NaCl, 250 mM Arginine, pH 7.2). The partially purified material/eluate was further treated with a nuclease as described in example 4. DNA DNA DNA/ Purification IVP Yield IVP Yield Reduction Reduction Dose* step (Step) (Overall) (Step) (Overall) [ng] Cell Lysis 100%  100%   0%  0.00% 1200 Primary 95% 95% 20% 20.00% 960 Filtration Capture 40% 38% 97%  97.5% 30 Nuclease 90% 34% 99% 99.98% 0.2 *Based on calculations considering 1 × 10.sup.8 IVP/dose

Example 5

Mixed Mode Chromatography at High Arginine Concentration

[0268] It is desired to lower the salt concentration in the elution step to facilitate nuclease digestion without prior buffer exchange. Since it was observed that the addition of arginine improves virus recovery, it was reasoned that a very high arginine concentration could substitute for NaCl.

[0269] Lysate was prepared, pretreated and applied to the chromatography resin (Nuvia cPrime, Biorad, Germany) as described in example 3. Contaminants were washed out with 25 CV of Wash buffer (50 mM Tris, 0.5 M NaCl, pH 7.2). For the elution buffer, arginine concentration was raised threefold (0.75 M) and the salt concentration was decreased fourfold (0.5 M). Due to this approach, virus recoveries were further improved to about 83% (FIG. 4a, b).

[0270] The eluate was subjected to nuclease treatment without prior buffer exchange. DNA content was reduced more than 500× by treatment with 50 U/ml of SAN overnight at room temperature.

[0271] Consequently, SAN nuclease has higher activity at 500 mM NaCl as expected but was also insensitive to high concentrations 750 mM of arginine. The overall process yield and purity is described in Table 2.

TABLE-US-00002 TABLE 2 Representative virus yields and DNA levels during purification of recombinant MVA using an elution buffer containing 0.5M NaCl and 750 mM Arginine (example 5). Cells lysis and primary filtration were performed as described in example 3. Nuvia cPrime was loaded with 5 × 10.sup.9 IVP/ml resin. Contaminants were washed out with 25 CV of Wash buffer (50 mM Tris, 0.5M NaCl, pH 7.2) and bound virus eluted with 5 CV of Elution buffer (50 mM Tris.HCL, 0.5M NaCl, 750 mM Arginine, pH 7.2). The partially purified material was further treated with a nuclease as described in example 5. DNA DNA DNA/ Purification IVP Yield IVP Yield Reduction Reduction Dose* step (Step) (Overall) (Step) (Overall) [ng] Cell Lysis 100% 100%   0%  0.00% 1200 Primary  95% 95% 20% 20.00% 960 Filtration Capture  83% 79% 97% 97.3% 33 Nuclease 100% 79% 100%  100.00%   0.06 *Based on calculations considering 1 × 10.sup.8 IVP/dose

Example 6

Quantitation of Host Cell DNA

[0272] The measurement of purified samples with a low amount of DNA was performed by qPCR. Before DNA extraction the samples with high salt levels were diluted 10.sup.−1 and the defined standard 10.sup.−1-10.sup.−6. To 5 μl QuickExtract DNA Extraction Solution 1.0 (Epicentre, USA) 20 μl sample was added and heated to 65° C. and incubate for 15 min, following by an incubation step at 95° C. for 5 min using a C1000 Touch Thermal Cycler (Bio Rad, USA). Afterwards 50 μl of WFI were added. The qPCR method and the composition of the mastermix were identical to the one used for the qTCID50 except for the primer pair duPseudo2 (5′-CAGGCAGGTTTCTTTAGGAAGG-3′ (SEQ ID NO: 3) and 5′-GTAGGTAGCAAGGAGGTTTAGC-3′ (SEQ ID NO: 4), (TIB MolBiol, Germany).

Example 7

Newcastle Disease Virus (NDV) Production

[0273] Newcastle disease virus (NDV) was produced in AGE1.CR.pIX cells. Suspension cultures were maintained in a shaking incubator (HT Multitron Cell, Infors AG, Bottmingen, Switzerland) on a rotating platform with amplitude of 5 cm and rotation speed of 180 min.sup.−1. CO.sub.2 atmosphere was set to 8% and temperature to 37° C. All culture vessels, shake tubes (Tubespin 50, TPP Techno Plastic Products AG, Switzerland) or baffled shake flasks (Corning, N.Y., USA), were equipped with 0.2 μm filtered lids to allow gas exchange. Culture volumes were maintained at 20-50% of the vessel size.

[0274] The DASBox (DASGip, Eppendorf, Hamburg, Germany) bioreactor units were equipped with a Marine impeller with three blades and 60-250 ml working-volume vessels. Gas mixing was performed with N.sub.2, air, CO.sub.2 and O.sub.2, pH was adjusted with CO.sub.2 and 1 M Na.sub.2CO.sub.3. Inoculation was usually performed to 1×10.sup.6 cells/ml in CD-U3 medium and the culture was allowed to proliferate for 3 days to approximately 4×10.sup.6 cells/ml. The parameters for the cell proliferation phase were 37° C. culture temperature, 60% DO (dissolved oxygen) saturation in the medium, 180 rpm for the impeller, and a pH gradient that decreased from 7.25 to 7.00 units in the cell culture during cell proliferation. The pH was usually kept at 7.1 units during infection.

[0275] Propagation of NDV virus was furthermore supported by feeding recombinant trypsin (rTrypsin, Novozym 6395020) into the infected culture from a solution kept at 4° C. with an activity adjusted such that a feeding rate of 0.17 ml/h (4 ml per day) resulted in a final concentration of 8 U/ml of culture volume each day. The incubation temperature was set to 35° C.

Example 8

Newcastle Disease Virus Purification via Mixed Mode Chromatography

[0276] The cell suspension was subjected to three freeze-thaw cycles and cell debris was removed by filtration (Sartopure, Sartorius, Germany) as described for the MVA process. The obtained material was subjected to a chromatography step using a mixed mode cation exchanger (Nuvia cPrime, Biorad, Germany) Contaminants were washed out with 25 CV of Wash buffer and bound virus eluted with 5 CV of Elution buffer and live virus was recovered.

[0277] The partially purified material/eluate was further treated with 50 U/ml of nuclease (SAN, Artic Enzymes, Norway) overnight at room temperature.

[0278] The enzyme as well as DNA fragments and smaller impurities were removed by diafiltration against Elution buffer using hollow fibers.

Example 9

Determination of Infectious Units

[0279] Infectious titres of Newcastle disease virus (NDV) were determined on Vero cells. 1.5×10.sup.6 cells in DMEM:F12 medium containing 2 mM GlutaMAX I (both Gibco) and 5% foetal calf serum (Biochrom) were seeded into CellBIND 96-well plates (Corning) at 100 μl of cell suspension. The medium was replaced on the following day against DMEM:F12 containing 2 mM GlutaMAX I and 1.5 μg/ml trypsin (type IX-S, Sigma T0303), but no foetal calf serum. Serial dilutions in steps of 10 of NDV samples were prepared in DMEM:F12 medium free of serum, and 10 μl each of the dilutions were added to the Vero cultures. Virus replication was allowed at 37° C. for 72 h.

[0280] Detection of NDV replication was facilitated by immunostaining: the cells were fixed in methanol for 10 min, allowed to dry to completion, and rehydrated with PBS containing 0.05% Tween-20. NDV antiserum (GD Animal Health Deventer, the Netherlands) was added to a dilution of 1:2000 in PBS containing 1% foetal calf serum and incubated for 1 h at room temperature. After two washes with PBS, secondary antibody (anti-chicken, Alexa Fluor 488 labelled, host rabbit, Dianova, 303-545-003 at 1 μg/μl) was added at a dilution of 1:2000 for 2 h at ambient temperature or overnight at 4° C. Infected wells were identified by fluorescence after two washes with PBS. Calculation of TCID50 values was performed according to Reed et al. (Reed U, Muench H. A simple method of estimating fifty per cent endpoints. Am J Hyg 1938; 27: 493-497).