Methods for providing purified viral particles of Semliki Forest Virus (SFV), preparations obtainable thereby, and uses thereof

Abstract

The invention relates to purified vaccine preparations and methods for providing them. Provided is a method for providing purified viral particles of SFV. comprising the steps of i) providing a preparation of SFV replicon particles: ii) subjecting said preparation to an endonuclease treatment under conditions allowing for degradation of exogenous/host cell DNA and RNA: iii) bringing said endonuclease-treated preparation with a zwitterionic buffer solution to a conductivity of up to about 5.5 mS/cm: iv) contacting the preparation obtained in step (iii) with a strong anion exchange resin: v) eluting the bound SFV replicon particles from said anion exchange resin: vi) bringing the eluted SFV particles to a conductivity in the range of 7.0 to 9.0 mS/cm: vii) contacting the preparation obtained in step (vi) with a strong cation exchange resin under conditions and for a time sufficient to bind to said resin: viii) eluting the bound SFV replicon particles from said cation exchange resin with a zwitterionic buffer solution and collecting at least one fraction containing purified SFV replicon particles: and ix) stabilizing the at least one purified fraction by adding human serum albumin (HSA) to a final concentration in the range of about 0.5-2 w/v %. preferably about 1 w/v %.

Claims

1. A method for providing purified viral replicon particles of Semliki Forest Virus (SFV), comprising the steps of i) providing a preparation comprising crude SFV replicon particles; ii) subjecting said preparation to an endonuclease treatment under conditions allowing for degradation of exogenous/host cell DNA and RNA; iii) bringing said endonuclease-treated preparation with a zwitterionic buffer solution to a conductivity of up to about 5.5 mS/cm, preferably to a conductivity in the range of 4.5-5.0 mS/cm; iv) contacting the preparation obtained in step (iii) with a strong anion exchange resin under conditions and for a time sufficient to bind to said resin, followed by washing the resin with a zwitterionic buffer solution to remove the portion of the preparation which does not bind to said anion exchange resin from said anion exchange resin; v) eluting the bound SFV replicon particles from said anion exchange resin; vi) bringing the eluted SFV replicon particles to a conductivity in the range of 7.0 to 9.0 mS/cm, preferably in the range of 7.5-8.2 mS/cm; vii) contacting the preparation obtained in step (vi) with a strong cation exchange resin under conditions and for a time sufficient to bind to said resin, followed by washing the resin to remove the portion of the preparation which does not bind to said cation exchange resin from said cation exchange resin; viii) eluting the bound SFV replicon particles from said cation exchange resin with a zwitterionic buffer solution and collecting at least one fraction containing purified SFV replicon particles ix) stabilizing the at least one purified fraction by adding human serum albumin (HSA) to a final concentration in the range of 0.5-2 w/v %, preferably about 1 w/v %.

2. Method according to claim 1, wherein step i) comprises providing a host cell that is modified to produce viral particles, culturing the modified host cell in a medium under conditions allowing expression of the structural proteins and replication of the SFV replicon nucleic acid, and then packaging of the SFV replicon nucleic acid to form SFV replicon particles.

3. Method according to claim 1, wherein step ii) comprises a benzonase treatment.

4. Method according to claim 1, wherein the zwitterionic buffer solution used in at least one, preferably all, of the steps is selected from the group consisting of HEPES (N-2-hydroxyethyl piperazine-N-2-ethanesulfonic acid), MOPSO (3-[N-morpholino]-2-hydroxy propanesulfonic acid), BES (N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid), and TAPSO (3-[N-tris(hydroxymethyl)methylamino]-2-hydroxy propanesulfonic acid).

5. Method according to claim 4, wherein the buffer is a HEPES buffer pH 7.00.5, preferably a 40-60 mM HEPES buffer7.00.3.

6. Method according to claim 1, wherein step iv) and/or step vii) comprise(s) the use of a monolithic chromatography column, preferably having an average pore diameter in the range of about 600-750 nm.

7. Method according to claim 1, wherein the strong anion exchange resin is of the quaternary amine (QA) type.

8. Method according to claim 1, wherein the strong cation exchange resin is of the sulfonyl/SO3 type.

9. Method according to claim 1, further comprising performing a filtration or tangential flow filtration step in between steps iii) and iv).

10. Method according to claim 1, wherein said SFV particles comprise a vector system comprising a nucleic acid molecule encoding a pathogen-derived antigen or a cancer-derived antigen.

11. Method according to claim 10, wherein the antigen is derived from a virus, bacterium, fungus or parasite, preferably wherein the virus is human papilloma virus (HPV).

12. Method according to claim 11, wherein the nucleic acid molecule encodes at least one antigenic polypeptide fragment of human papilloma virus (HPV).

13. Method according to claim 12, wherein said polypeptide fragment is of a protein E6 or a protein E7 origin, preferably wherein said polypeptide fragment comprises an antigenic polypeptide fragment of the protein E6 and an antigenic polypeptide fragment of the protein E7.

14. A stabilized preparation of purified SFV replicon particles formulated in a zwitterionic buffer system comprising 0.5 to 2 w/v % HSA, preferably showing less than 0.3 log10 change in viral titer upon 18 months storage at 60 C. or lower.:

15. Preparation according to claim 14, comprising SFV replicon particles which direct the expression of a HPV antigen, preferably wherein said SFV particles comprise a recombinant, attenuated, replication-incompetent form of the SFV vector encoding the viral oncoproteins E6 and E7 derived from HPV.

16. A pharmaceutical composition comprising a stabilized preparation of purified SFV replicon particles according to claim 14, and a pharmaceutically acceptable carrier, vehicle or diluent.

17. Pharmaceutical composition according to claim 16, formulated with a HEPES buffer system, more preferably 15-25 mM HEPES pH 7.0, 200-250 mM NaCl, and 1-2 w/v % HSA in water for injection (WFI).

18. A method for vaccinating a subject, comprising administering to the subject an effective amount of a pharmaceutical composition according to claim 16.

19. Method according to claim 18, wherein said composition is administered by injection in at least a left and/or right limb, preferably in at least the right and left (upper) leg, of said subject.

Description

LEGEND TO THE FIGURES

[0070] FIG. 1 is a schematic representation of the production of SFV particles in Vero cells using three RNA transcripts, one encoding the replicase and the protein of interest, one encoding the SFV capsid protein, and one encoding the SFV spike proteins.

[0071] FIG. 2 presents the maps of the three plasmids pSFVeE6,7 encoding the replicase and E6,7 fusion protein (A), pSFV-helper-C-S219A encoding the capsid protein (B) and pSFV-helper-S2 encoding the spike proteins (C).

[0072] FIG. 3 presents the chromatograms of the CIMR-QA-8cGMP column (A) and CIMR-SO3-8cGMP column (B), showing the elution of SFV replicons from these columns in the purification run of SFV replicon particle batch number 16-A26-12. Curves represent the absorbance (Left axis in mAU) at 280 nm and the conductivity (right axis in mS/cm) of each run

[0073] FIG. 4 provides the results of SFV replicon particle purification on single staged CIM QA discs and CIM SO3 discs. Recovery in terms of % virus recovery (infectious viral particles), total (host cell) protein and total host cell DNA compared to viral load before purification are plotted.

EXPERIMENTAL SECTION

Example 1

Preparation of Host Cells Containing SFV Replicon Particles, Virus Harvest and Nuclease Treatment

[0074] In order to produce SFV replicon particles, Vero cells (kidney cells from an African green monkey) are electroporated with three transcripts (FIG. 1). These transcripts are derived from three plasmids i.e. pSFVeE6,7 encoding the replicase and E6,7 fusion protein, pSFV-helper-C-S219A encoding the capsid protein and pSFV-helper-S2 encoding the spike proteins. DNA of each individual plasmid was linearized via a digestion reaction using the BcuI (=SpeI) restriction enzyme, which has one recognition site in each plasmid (FIG. 2). The DNA was incubated with BcuI at 36-38 C. for 2 hours (+/10 minutes). The restriction reaction was stopped by adding a mixture of EDTA, ammonium acetate and ethanol. DNA was precipitated by centrifugation at 20,000 g and the resulting pellet is dried at room temperature. The DNA pellet was dissolved in nuclease-free water before storage.

[0075] The DNA was analyzed for concentration, absence of circular DNA by gel electrophoresis and absence of proteins by determination of the ratio OD 260/280 nm. RNA was then synthesized via in vitro transcription using SP6 RNA polymerase, which recognizes the SP6 promoter in each of the linearized DNA constructs. Linearized DNA of the three constructs was incubated with SP6 polymerase and nucleotides at 37 C. for 2 hours10 minutes. Next, the RNA was incubated with TURBO DNase to remove template DNA. The purified RNA was recovered by lithium chloride precipitation and centrifugation (20 minutes, 20,000 g, 4 C.) and the resulting pellet dissolved in nuclease-free water. The purified RNA was tested for concentration, purity and endotoxins.

[0076] SFV viral replicon particles were produced in Vero cells after electroporation of the three RNA transcripts. Vero cells were cultured in culture medium (Medium 199 w/o phenol red) with 10% FBS and 2 mM L-glutamine (size 175 cm2) at 36-38 C. and 4-6% CO.sub.2 to a cell confluency of 80-90%. At every cell passage, a single cell suspension was generated by recombinant trypsin (Tryple Select) treatment to dislodge the cells from the tissue flasks. Cells were passaged based on cell confluency and a passaging frequency of twice a week. Vero cells from subconfluent flasks were detached with Tryple Select and resuspended in Biorad electroporation buffer in a concentration of 2010.sup.6/ml. Cells were transferred to Gene pulser cuvettes and electroporated with the RNA transcripts using a Gene pulser Xcell electroporation unit. Each electroporation was performed with 1610.sup.6 cells and 11.3 g helper C, 15.4 g helper S and 36.0 g RNA E6,7 RNA.

[0077] The following electroporation conditions were applied: 195V, 5.0 msec pulse length, 4 pulses, pulse interval 0.1 sec. The transfected Vero cells were seeded back at a cell density of 2010.sup.6 cells/10 mL into 75 cm.sup.2 tissue culture flasks and incubated for virus propagation for up to two days at 37 C. and 5% CO.sub.2 in culture medium 199 with Earle's BSS containing 5% FBS and 2 mM L-glutamine. Viral particles were harvested after 24 and 48 hours by collection of the supernatant and centrifugation to remove Vero cells/cell debris. Viral titer and bioburden were determined for each harvest. Individual harvests were stored at 60 C. until purification. Virus titers of individual harvests typically range between 210.sup.7-1.310.sup.9 infectious particles/mL.

[0078] Individual crude SFV harvests were pooled for purification. Virus purification starts with thawing of the virus harvest(s) at 37 C. The thawed harvests were pooled in a sterile glass container. As shown in Table 1 herein below, titers of pooled harvests are typically between 410.sup.7-210.sup.8 infectious particles/mL. The pooled harvest was treated with benzonase (50 U/mL) in 2 mM magnesium chloride for 30 minutes at 37 C., in order to degrade all forms of host cell nucleic acid (DNA and RNA). The benzonase reaction was terminated by 1:1 v/v dilution with 10 mM EDTA in 50 mM HEPES buffer pH 7.0. The benzonase-treated material was then prepared for the first purification step by diluting with 50 mM HEPES buffer pH 7.0-7.5 to a conductivity of 4.5-5.0 mS/cm.

Example 2

Two-Step Chromatographic Purification

[0079] For the purification of the benzonase-treated preparation of SFV particles, an KTA pilot chromatography system was used. In the first step, the diluted harvest pool was loaded on a solid state ceramic strong anion exchange column (CIM-QA-8cGMP tube monolithic column from Bia Separations), which was used in a binding mode. After loading, the column was washed with a 50 mM HEPES buffer pH 7.0-7.5. Subsequently the virus particles were eluted using a 50 mM HEPES buffer 7.0-7.5 containing 0.3 M NaCl. An example of a chromatogram showing elution of the virus from the CIM-QA-8CGMP column is shown in FIG. 3A.

[0080] In a second step, the anion exchange purified virus was subjected to cation exchange chromatography using a CIM-SO3-8cGMP tube monolithic column from Bia Separations, again in binding mode. Before loading the virus on the column, the eluate obtained from the first chromatographic step was diluted with a 50 mM HEPES buffer pH 7.0-7.5 to a conductivity of 7.5-8.2 mS/cm. After loading, the column was washed with 50 mM HEPES buffer. Additional impurities were removed by elution with a 50 mM HEPES buffer containing 0.3 M NaCl. Subsequently, the viral particles were eluted with a 50 mM HEPES buffer containing 0.6 M NaCl. The eluted virus was collected in sub-fractions. An example of a chromatogram showing the elution of the virus from the CIM-SO3-8cGMP column is shown in FIG. 3B.

[0081] Human Serum Albumin (HSA) was added to a final concentration of 1 w/v % to the purified virus fractions as a stabilizer, and the fractions were stored at 60 C. for purity and quantity assessment prior to processing to the formulated Drug Substance.

[0082] The flowchart of Scheme 1 summarizes the individual steps performed in Examples 1 and 2.

Example 3

Characterization of the Purified SFV Replicon Particles

[0083] This example describes the characterization of products of the intermediate steps and the final product using a set of different assays (A through H). It demonstrates an appropriate removal of impurities, such as host cell DNA and protein, and recovery of infectious SFV replicon particles.

A) Viral Titer Assay to Detect Infectious Viral (SFV) Particles and Confirm Identity. The viral titer assay is used to determine the amount of infectious particles per milliliter (titer), as well as the identity of the SFV particles. The assay is performed by titration of viral particles by serial dilution on monolayers of BHK cells. Infection by SFV particles is determined by immunofluorescence using an antibody against SFV-nsp3 (replicase). This antibody was chosen because replicase is present in all cells infected with recombinant SFV. Positive staining therefore confirms the identity of the viral particles. Titers are calculated by counting positive cells and correction for the dilution factor.

B) Agarose Gel Electrophoresis to Determine the Size of Residual Host Cell DNA Fragments

[0084] Agarose gel electrophoresis is performed using a 0.8% agarose gel and Tris/Borate/EDTA running buffer. This method is used to determine the size of the residual DNA fragments in the crude pooled harvest. Biomolecules are separated by applying an electric field to move the negatively charged molecules through an agarose matrix. The separated DNA is visualized by using ethidium bromide stain and detection under UV light.

C) Host Cell DNA by Picogreen Assay

[0085] The Picogreen assay allows to determine the level of host cell DNA in purified SFV virus samples. The assay makes use of the Quant-iT PicoGreen dsDNA reagent, which is a fluorescent nucleic acid stain for quantifying double-stranded DNA in solution. Fluorescence is detected using a fluorescence microplate reader with excitation wavelength 480 nm and emission wavelength 520 nm. DNA concentration is determined using a DNA standard curve.

D) Host Cell Protein by ELISA

[0086] The residual Vero host cell protein levels were quantified using a Cygnus Technologies ELISA kit in which an affinity purified antibody directed against Vero cell proteins is attached to the solid phase (microtiter plate wells) and captured Vero proteins in SFV virus samples are detected by interaction with a horseradish peroxidase (HRP)-labeled anti-Vero antibody.

E) Microbca Assay to Detect Total Protein Including HSA

[0087] The MicroBCA assay is used to determine the level of total protein (i.e. host cell, virus protein in crude samples and HSA in purified samples) in SFV virus samples. The assay makes use of bicinchoninic acid (BCA) as the detection reagent for Cu+1, which is formed when Cu+2 is reduced by protein in an alkaline environment. A purple-colored reaction product is formed by the chelation of two molecules of BCA with one cuprous ion (Cu+1). Absorbance is detected at 562 nm using a fluorescence microplate reader. Protein concentration is determined using a BSA (bovine serum albumin) standard curve.

F) Benzonase ELISA

[0088] The residual amount of benzonase was tested in an enzyme immunoassay, using the Benzonase ELISA II kit supplied by Merck (product number 1.01681). The kit uses specific polyclonal antibodies to capture benzonase, if present.

G) Transmission Electron Microscopy to Measure Total Virus Particles

[0089] Transmission electron microscopy was performed to detect the total number of SFV viral particles (both infectious and non-infectious). In addition, this method enables assessing the size of the viral particles. The assay is performed by mixing virus particles with a standard quantity of latex beads. The mixture is then fixed on a grid and visualized using a transmission electron microscope. Latex beads and virus particles are counted and the concentration of virus particles is calculated based on the known concentration of latex beads.

H) Heterogeneity (Calculation)

[0090] The ratio of total virus particles versus infectious particles is calculated based on the results of the viral titer assay and the transmission electron microscopy. The ratio is an indication of the biological activity of the virus particles, i.e. when the ratio is 1 or lower, this indicates that all virus particles are infectious.

Test Results:

[0091] The results of the purification of 13 pooled harvest runs are shown in Table 1. It demonstrates that purification process as described in Example 2 efficiently removes Vero cell proteins and DNA and other process-related impurities such as Benzonase (As further described in Example 4 and Table 2). The ratio of 0.27 of the total: infectious particles demonstrated proficient biological activity of the purified SFV preparation. Given the variation of both assays, a ratio of 0.27 reflects that all physical particles are infectious. Importantly, the final titer after purification was sufficient for preparation of the final sterile formulation for dosing in humans.

TABLE-US-00001 TABLE 1 Characterization of intermediate products and final preparation of SFV replicon particles. Table 1A: Characterization of pooled harvest and QA load of 13 batches. Table 1B: Characterization of the QA peak fraction, SO3 load, and SO3 peak fraction of 13 batches. 16A26- 16B01- 16B02- 16B08- 16B08- 16B15- 16B15- Test 012 005 008 025 027 012 014 Crude pooled harvest Viral titer 2.0*10.sup.8 1.9*10.sup.8 1.9*10.sup.8 2.0*10.sup.8 1.6*10.sup.8 1.8*10.sup.8 1.0*10.sup.8 (infectious particles/mL) Purity: size ND ND ND ND ND ND ND residual DNA fragments QA load Viral titer 6.9*10.sup.7 4.2*10.sup.7 4.2*10.sup.7 5.0*10.sup.7 3.5*10.sup.7 2.8*10.sup.7 1.9*10.sup.7 (infectious particles/mL) QA peak fraction Viral titer 1.5*10.sup.8 9.5*10.sup.7 1.1*10.sup.8 1.6*10.sup.8 1.1*10.sup.8 1.6*10.sup.8 8.2*10.sup.7 (infectious particles/mL).sup.1 Purity: total 172 213 188 220 211 206 242 proteins (g/mL) Purity: 5 6 6 7 8 7 8 host cell DNA (ng/mL) SO3 load Viral titer 2.1*10.sup.7 1.9*10.sup.7 1.7*10.sup.7 2.6*10.sup.7 2.0*10.sup.7 2.3*10.sup.7 1.4*10.sup.7 (infectious particles/mL) SO3 peak fraction Purity: total 19 20 16 27 23 8 27 proteins(g/mL) Purity: Vero 0.84 host cell protein (g/mL) Purity: 2 3 4 4 5 3 3 host cell DNA (ng/mL) Total virus 1.1*10.sup.8 particles (total virus particles/mL) Viral titer 4.2*10.sup.8 6.4*10.sup.8 4.4*10.sup.8 5.9*10.sup.8 4.1*10.sup.8 7.6*10.sup.7 4.3*10.sup.8 (infectious particles/mL) after HSA addition Purity: total 0.27 particles/infectious particles ratio 16B15- 16B29- 16B29- 16B29- 16B29- 16B29- Test 016 036 034 035 037 038 Crude pooled harvest Viral titer 6.5*10.sup.7 1.7*10.sup.8 2.3*10.sup.8 9.3*10.sup.7 1.3*10.sup.8 1.1*10.sup.8 (infectious particles/mL) Purity: size ND ND ND ND ND ND residual DNA fragments QA load Viral titer 1.6*10.sup.7 3.9*10.sup.7 4.9*10.sup.7 1.7*10.sup.7 2.6*10.sup.7 2.7*10.sup.7 (infectious particles/mL) QA peak fraction Viral titer 5.5*10.sup.7 1.7*10.sup.8 1.6*10.sup.8 8.6*10.sup.7 8.1*10.sup.7 1.1*10.sup.8 (infectious particles/mL).sup.1 Purity: total 254 211 210 196 171 216 proteins (g/mL) Purity: 7 14 13 9 9 11 host cell DNA (ng/mL) SO3 load Viral titer 9.6*10.sup.6 2.7*10.sup.7 2.9*10.sup.7 1.4*10.sup.7 1.2*10.sup.7 1.8*10.sup.7 (infectious particles/mL) SO3 peak fraction Purity: total 29 26 22 15 19 28 proteins(g/mL) Purity: Vero 0.84 host cell protein (g/mL) Purity: 3 7 5 4 4 6 host cell DNA (ng/mL) Total virus 1.1*10.sup.8 particles (total virus particles/mL) Viral titer 2.3*10.sup.8 5.8*10.sup.8 4.1*10.sup.8 2.8*10.sup.8 2.7*10.sup.8 4.8*10.sup.8 (infectious particles/mL) after HSA addition Purity: total 0.27 particles/infectious particles ratio ND: not detectable

TABLE-US-00002 TABLE 2 Intermediate yields and overall yield of infectious viral particles (in percentage compared to crude pooled harvest set at 100) of representative exemplary batches subjected to the two-step chromatographic purification method. Crude QA Batch pooled QA peak SO3 SO3 peak Number harvest load fraction load fraction 16A26-012 100 136 112 64 22 16B01-005 100 79 49 40 22 16B15-014 100 69 57 39 22 16B29-038 100 97 66 42 20

Example 4

Preparation of Sterile Composition Comprising SFV Particles

[0092] The fractions that were collected from the SO3 cation exchange column according to Example 2 and depicted in Table 1 were pooled, and the pool was split in two portions and each portion was separately processed. They were diluted 2.5-fold with water for injection (WFI) containing 1 w/v % HSA, to a target concentration of 1.5610.sup.8 infectious particles/mL with formulation buffer. This resulted in two formulated drug substances 16E04-021 and 16H25-017, with a target concentration of 1.5610.sup.8 infectious particles/mL in 240 mM NaCl, 20 mM HEPES pH 7.0,1 w/v % HSA. Both formulations were sterile filtered using a SLGP033RS Millex-GP Syringe Filter Unit, 0.22 m, polyethersulfone, 33 mm, resulting in two clinical final product batches referred to as 16E8-011 and 16H25-004, respectively. In addition to the assays described in Example 3, the following methods were used to analyze the formulated drug substance and drug products:

Biological Activity and Identity by Western Blot E6E7

[0093] The identity of the recombinant E6E7 SFV virus in the formulated product was determined using Western Blot analysis using standard procedures. Briefly, BHK21 cells were infected with the recombinant SFV virus at different MOI (multiplicity of infection). BHK21cells were lysed after 24 hours of incubation and the cell lysate was analyzed for E7 expression by Western Blot gel electrophoresis, using a mouse-anti-HPV16-E7 primary antibody and a goat-anti-mouse-AP (alkaline phosphatase) secondary antibody. Finally, E7 containing bands on the Western Blot are visualized by addition of an AP substrate, resulting in a chemiluminescent reaction.

Absence of Replication Competent Viruses

[0094] Replication competent viruses were detected using a cell based assay. Vero cells were seeded in roller bottles to allow detection of a single replication competent virus in the maximum human dose. Spiking studies demonstrated that replication competent viruses outgrow the replication incompetent virus rapidly causing cytopathic effects and destruction of the Vero cells. After sufficient Vero cells have been obtained the product is inoculated into the roller bottles and incubated for 14 days. Within the culture period, the cells are passaged once. Cells were evaluated for cytopathic effects at the end of the incubation period, which is indicative for the presence of replication competent viruses.

Bioburden, pH, Osmolality, Appearance, Endotoxin, Visible particles and Extractable Volume.

[0095] These are compendial methods that are described in the European Pharmacopeia (Ph. Eur.)

Test Results:

[0096] The characterization of these batches show that infectious titers before (Table 3) and after (Table 4) sterile filtration are similar (within the variation of the assay) demonstrating that no loss of virus had occurred during the filtration step. Furthermore, both batches met the release criteria for use in clinical trials.

TABLE-US-00003 TABLE 3 Characteristics of exemplary formulated drug substance SFV replicon particle batches 16E04-021 and 16H25-004. Test Specification 16E04-021 16H25-004 Virus identity Confirmed ID Confirmed ID Confirmed ID Viral titer 1.25 10.sup.8 0.3log 1.9 10.sup.8 1.9 10.sup.8 Human serum 10 mg/mL 20% 11 mg/mL 11 mg/mL albumin Bioburden 1 CFU/mL 0 CFU/mL 0 CFU/mL Biological Protein expressed Expressed Expressed activity/ identity E6E7 pH 7.0 0.3 7.0 6.9 Osmolality 390 40 395 347 mOsmol/kg mOsmol/kg mOsmol/kg Appearance Clear or slightly Conform conform opalescent solution Absence of Not present in Conform Conform *.sup.5 replication one human dose competent viruses Benzonase Not detected not detected not detected * LOD = 0.2 ng/ml * Tests on absence of replication competent viruses and on benzonase were performed on representative samples of formulated drug substance (lot # 16E04-021)

TABLE-US-00004 TABLE 4 Characterization of exemplary formulated drug product SFV replicon particle batches 16E8-011 and 16H25-004 in HEPES, generated by sterile filtration of drug substance batches 16E04-021 and 16H25-004 and filling into glass vials. Test Specification 16E18-011 16H25-004 Sterility no growth no growth no growth (Ph. Eur.) Endotoxin <5 EU/mL <5 EU/mL <5 EU/mL concentration (Ph. Eur.) Visible particles Essentially free Conforms Conforms (Ph. Eur.) of visible particles Extractable 1 mL/vial Conforms Conforms volume (Ph. Eur.) Viral titer 1.25 10.sup.8 0.3log 1.7 10.sup.8 1.6 10.sup.8 (infectious particles/mL) Virus identity Confirmed ID Confirmed ID Confirmed ID Appearance Clear or slightly Conforms Conforms (Ph. Eur visual opalescent inspection) solution

Example 5

Comparison HEPES Versus Tris buffer

[0097] To stabilize the virus particles during downstream processing, buffering components are used to maintain a relatively constant pH. In this example, the suitability of two buffer systems, i.e. HEPES and Tris, in downstream purification of SFV viral particles according to the invention was compared. To this end, small scale purification runs were performed; 2 parallel runs on CIM QA discs using HEPES buffer, and 2 parallel runs using Tris buffer were performed. In addition, 2 parallel runs on CIM SO3 discs using HEPES buffer, and 2 parallel runs using Tris buffer were performed.

[0098] First, SFV crude harvest was split in two halves, one halve was diluted with 50 mM HEPES pH 7.0 and the other halve with 50 mM Tris pH 7.0. Between 3.5-4.210.sup.8 infectious particles of the diluted crude harvest were loaded on each disc. The CIM QA discs were washed with either 50 mM HEPES pH 7.0, or 50 mM Tris pH 7.0. Thereafter, virus was eluted using either 50 mM HEPES pH 7.0containing 0.3 M NaCl, or 50 mM Tris pH 7.0 containing 0.3 M NaCl. The CIM SO3 discs were washed with either 50 mM HEPES pH 7.0 containing 0.3 M NaCl, or 50 mM Tris pH 7.0 containing 0.3 M NaCl. Thereafter, virus was eluted using either 50 mM HEPES pH 7.0 containing 0.6 M NaCl, or 50 mM Tris pH 7.0containing 0.6 M NaCl.

[0099] The average % virus yield of the two parallel runs, as well as the % of total protein and host cell DNA are shown in FIG. 4. These results surprisingly show that, whereas the removal of protein and DNA impurities is similar using HEPES and Tris buffers, the SFV particle yields using the Zwitterionic HEPES buffer on the first (QA; 81.6%) and second (SO3; (31.9%) column is substantially higher than those using the non-zwitterionic Tris buffer (51.1 and 28.1%, respectively).

Example 6

Impact of Conductivity on Recovery of Virus from the Anion Exchange Column.

[0100] To evaluate the impact of the conductivity of the crude SFV replicon particle preparation on the recovery from the anion exchange column, a small scale experiment was performed using CIM QA 0.34 ml disks. To this end 3 parallel runs were performed: [0101] RUN 140 ml of SFV crude harvest was diluted with 50 mM HEPES pH 7.0to final conductivity of 4 mS/cm and loaded onto CIM QA 0.34 ml disk. [0102] RUN 2as in RUN 1 but diluted to final conductivity of 5 mS/cm. [0103] RUN 3as in RUN 1 but diluted to a final conductivity of 9 mS/cm.

[0104] After loading, the column was washed with 50 mM HEPES pH 7.0, and SFV particles were eluted with 50 mM Tris pH 7.0 containing 0.3 M NaCl and collected in two fractions, E1 and E2, where E1 contains the main viral peak. The titer of the loaded virus, flow through/wash, E1 and E2 fractions were titrated for infectious viral particles (Table 5). The results demonstrate that the conductivity of 5 mS/cm provides the highest recovery in the E1 fraction. The recovery using a conductivity of 4 mS/cm was very similar, but the recovery dropped while using a conductivity of 9 mS/cm. Hence, for optimal recovery in the QA column the conductivity is preferably in a range between 4-5.5 mS/cm, more specifically 4.5-5.0 mS/cm.

TABLE-US-00005 TABLE 5 Results of SFV particle purification CIM QA column using buffers with different conductivity (Runs 1-3). The viral titer in terms of infectious particles and recovery of total infectious virus particles in the different samples during purification are shown. Sample Volume (ml) Titer/ml Total amount of virus particles Recovery (%) Run 1 4 mS/cm Original 38.8 6.6E+07 2.6E+09 Load 177.7 1.5E+07 2.6E+09 103 FT 177.7 2.0E+03 3.6E+05 0 Wash 11.1 0.0E+00 0.0E+00 0 E1 8.6 2.0E+08 1.7E+09 68 E2 4.0 8.7E+07 3.5E+08 14 Run 2 5 mS/cm Original 41.0 6.6E+07 2.7E+09 Load 135.0 2.2E+07 2.9E+09 107 FT 135.0 7.0E+04 9.5E+06 0 Wash 10.4 0.0E+00 0.0E+00 0 E1 8.7 2.4E+08 2.1E+09 77 E2 4.0 8.5E+07 3.4E+08 13 Run 3 9 mS/cm Original 41.0 6.6E+07 2.7E+09 Load 70.0 3.6E+07 2.5E+09 92 FT 70.0 1.7E+07 1.2E+09 43 Wash 10.6 5.8E+05 6.1E+06 0 E1 8.8 1.4E+08 1.3E+09 47 E2 3.9 5.4E+07 2.1E+08 8 FT = Flow through. E1 and E2 are elution fractions.

Example 6

Stability of SFV Viral Particles

[0105] The stability of purified SFV replicon particles formulated into a clinical product as described in Examples 1-4, was tested up to 36 months storage at 60 C. The product was tested at different time points for appearance, 5 pH, Osmolality, and concentration of infectious virus particles. The data, as presented in Table 6, show that the clinical product is stable for at least 36 months in a 20 mM HEPES pH 7.0 buffer containing 1 w/v % HSA.

TABLE-US-00006 TABLE 6 Stability results for SFV clinical drug product (batch 16E18-011) during storage at 60 C. End of Test (and Release shelf life Storage period (months) metho specification specification 0 3 6 12 18 24 36 Appearance Clear or Clear or Conforms Conforms Conforms Conforms Conforms Conforms Conforms (visual slightly slightly inspection) opalescent opalescent solution solution pH 7.0 0.3 7.0 0.3 7.0 n.a. 7.0 7.0 7.0 7.0 7.0 (Ph. Eur. 2.2.20) Osmolality 390 40 390 40 342 n.a. 335 341 342 342 344 (Ph. Eur. 2.2.35) In mOsmol/kg Viral titer 1.25 10.sup.8 1.25 10.sup.8 1.6 10.sup.8 1.7 10.sup.8 1.8 10.sup.8 2.0 10.sup.8 1.8 10.sup.8 1.7 10.sup.8 1.4 10.sup.8 (infectious 0.3log10 0.3log10 particles/mL) (viral titer assay) Biological Expression Expression Conforms n.a. Conforms Conforms Conforms Conforms Conforms activity/ of protein protein identity E6E7 confirmed confirmed (Western Blot) no extra bands