Methods for the prevention of aggregation of viral components

Abstract

The present invention relates to a method for prevention and/or reduction of aggregation of viral components.

Claims

1. A method for producing a composition comprising Enteroviral particles, the method comprising, sequentially: (a) purifying Enteroviral particles from a medium containing Enteroviral particles, whereby during at least a part of the purification a basic amino acid or a derivative thereof is present at a concentration between 0.5-1000 mM to prevent or reduce aggregation of the Enteroviral particles; and, optionally, at least one of: (b) inactivating the Enteroviral particles; and, (c) formulating the Enteroviral particles, wherein the basic amino acid or derivative thereof is selected from the group consisting of arginine, lysine, histidine, arginine-HCl , lysine-HCl , histidine-HCl , agmatine, L-arginine ethyl ester dihydrochloride, tranexamic acid, DL-5-hydroxylysine hydrochloride, L-lysine methyl ester dihydrochloride, 3-methyl-L-histidine, salts thereof and combinations thereof.

2. The method according to claim 1, wherein the basic amino acid or derivative thereof is present at a concentration sufficient to prevent or reduce aggregation of the Enteroviral particles during at least a part of step (b), and wherein, optionally, also during step (c) the basic amino acid or derivative thereof is present at a concentration sufficient to prevent or reduce aggregation of the Enteroviral particles to formulate a pharmaceutical composition comprising the Enteroviral particles and optionally a concentration of the basic amino acid or derivative thereof sufficient to prevent or reduce aggregation of the Enteroviral particles.

3. The method according to claim 1, wherein the concentration of the basic amino acid or derivative thereof that is sufficient to prevent or reduce aggregation of the Enteroviral particles is maintained throughout the entire duration of at least one of steps (a), (b) and (c).

4. The method according to claim 1, wherein the concentration of the basic amino acid or derivative thereof is a concentration of at least 0.81 mM.

5. The method according to claim 4, wherein the concentration of the basic amino acid or derivative thereof is a concentration between 0.81-500 mM.

6. The method according to claim 1, wherein the Enteroviral particles are virus-like particles of an Enterovirus.

7. The method according to claim 1, wherein the Enteroviral particles are of an Enterovirus selected from the group consisting of polioviruses, Coxsackie A viruses, Coxsackie B viruses, Echoviruses, Rhinoviruses and Enteroviruses 68, 69, 70, 71 and 73.

8. The method according to claim 7, wherein the Enteroviral particles comprise polioviruses of the serotypes 1, 2 and 3.

9. The method according to claim 8, wherein the composition comprising Enteroviral particles is a vaccine.

10. The method according to claim 9, wherein the vaccine is an Inactivated Polio Vaccine (IPV).

11. A method of preventing or reducing aggregation of Enteroviral particles during purification of the Enteroviral particles from a medium, the method comprising adding to a composition comprising Enteroviral particles a basic amino acid or derivative thereof selected from the group consisting of arginine, lysine, histidine, arginine-HCl , lysine-HCl , histidine-HCl , agmatine, L-arginine ethyl ester dihydrochloride, tranexamic acid, DL-5-hydroxylysine hydrochloride, L-lysine methyl ester dihydrochloride, 3-methyl-L-histidine, salts thereof and combinations thereof, wherein during at least a part of the purification the basic amino acid or a derivative thereof is present at a concentration between 0.5-1000 mM to prevent or reduce aggregation of the viral component.

12. The method according to claim 11, wherein the Enteroviral particles are of an Enterovirus selected from the group consisting of polioviruses, Coxsackie A viruses, Coxsackie B viruses, Echoviruses, Rhinoviruses and Enteroviruses 68, 69, 70, 71 and 73.

13. The method according to claim 11, wherein the Enteroviral particles comprise polioviruses of the serotypes 1, 2 and 3.

14. The method according to claim 11, wherein the Enteroviral particles are an Inactivated Polio Vaccine (IPV).

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Schematics of the inactivated polio vaccine production process. During upstream processing cells are expanded using two pre-culture steps prior to cell culture and virus culture. The downstream processing consists of clarification, concentration, size exclusion chromatography and ion exchange chromatography followed by inactivation. To obtain trivalent polio vaccine this procedure is followed for each polio virus type separately prior to mixing for end product formulation (Bakker, 2011).

(2) FIGS. 2A, 2B, 2C, 2D, 2E, 2F, 2G, and 2H: Effect of the addition of different basic amino acids and derivatives to solutions containing aggregated virus on breaking up the existing aggregation. No additions (0 mM) was assumed to result in the maximum aggregation and has been set at 100% aggregated virus. Reduction is shown when different additions of basic amino acids and derivatives thereof in different concentrations are used. Detection of aggregation was performed using absorbance measurements at 590 nm. Other wavelengths may be used as well, leading to different spectra. Experiments carried out in FIG. 2A have been carried out using non-enveloped polio virus, FIG. 2B with enveloped influenza virus. FIGS. 2C, 2D and 2E depict the effect of respectively three different basic amino acids (L-arginine, L-lysine and L-histidine) on aggregation of three different influenza strains (Influenza A/Uruguay H3N2 (NIBSC) Influenza B/Florida/4/2006 (NIBSC) and Influenza A/PR/8/34 (NIBSC)). In FIGS. 2F, 2G and 2H depict the effect of respectively three different basic amino acids (L-arginine, L-lysine and L-histidine) on aggregation of three different poliovirus (Sabin) subtypes 1, 2 and 3.

(3) FIGS. 3A, 3B, 3C and 3D: Sabin-IPV rat potency (Albrecht P et al. 1984) using the regular IPV production process as e.g. described by Thomassen, 2013b, versus the optimized process according to the invention. Salk-IPV is used as reference standard (set at 1). Sabin-IPV prepared using the optimized process, i.e. using methods to prevent aggregation, yielded vaccines with comparable or better immunogenicity in rats compared to Sabin-IPV prepared as described by Thomassen 2013b. Salk-IPV is used as internal reference standard (set at 1).

(4) FIG. 4: Removal of L-arginine from a poliovirus (Sabin type 2) SEC product by diafiltration. Removal of L-arginine from a poliovirus (Sabin type 2) SEC product by diafiltration triggers aggregation of virus. An amount of L-arginine (squares) is removed by diafiltration, after ten volumes all L-arginine was removed. Aggregated virus (diamonds) was measured using absorbance measurements. After complete removal of the L-Arginine, poliovirus aggregates are formed in a time dependent manner. Thus removal of L-arginine, as followed by measurements and conductivity change, results in increased light scattering (UV increase) caused by formation of viral aggregates.

(5) FIG. 5: Effects of different basic amino acid and derivatives thereof on the reduction of poliovirus aggregates. The different compounds clearly show an effect, dependent on their type and concentration. For instance agmatine, L-lysine and the mixture of 3 basic amino acids the reduction most profound in the lower concentration range upto 50 mM. In all cases a reduction of aggregates from the starting material, as measured by UV, is clearly visible and dependent on their concentration.

EXAMPLES

Example 1

Virus Production

(6) Cell culture at lab-scale. Use of different systems. Production of inactivated attenuated poliovirus strains at lab-scale.

(7) The process starts with the cultivation of Vero Cells. The VERO cell line originates from African green monkey kidney cells (MKC) (ATCC CCL-81). Cultures are started with an ampoule from a well characterized frozen cell bank. To acquire the proper amount of cells to inoculate a bioreactor, a seed train with monolayer cultures in TC-flasks is started with M199 medium containing 5% Foetal bovine serum. M199 medium is as described in Morgan J. F. et al (1955) or Morgan J. F. et al (1950). It comprises mM amounts of basic amino acids: L-Arginine 0.33 mM, L-Histidine 0.1 mM, L-Lysine 0.38 mM (http://www.invitrogen.com/site/us/en/home/support/Product-Technical-Resources/media_formulation.86.html).

(8) The seed train is continued with sub cultivations in four T-flasks, three Hyper flasks and three Cell factories. The Cell factories have a total surface area of 3*6320 cm.sup.2. Cells are detached by trypsinisation. The entire seed cultivation takes about 2 weeks.

(9) VERO cell (earlier primary MKC was used) cultivation in bioreactors is performed on micro carriers (Cytodex 1 GE Healthcare, product number 17-0448-**). This technique was developed at RIVM in the late 1960's by van Wezel (1978). The microcarriers provide a large surface area for the attachment of Vero cells. The cultivation on micro carriers starts in batch mode in a 5 L (liter) bioreactor (3 L working volume). The 5 L bioreactor is operated with 3 L E-MEM cultivation medium supplemented with bovine serum (BS) (Minimum Essential Medium Eagle, Sigma Aldrich, M4642). The bioreactor is prepared with 4 g/L Cytodex 1 micro carriers and E-MEM cultivation medium. When cultivation conditions are stable, the bioreactor is inoculated with cells from the seed train to an initial cell concentration of 0.8*10.sup.6 cells/ml. The cultivation starts in batch mode for 1 day and is continued in recirculation mode for 3 days with 12 L E-MEM medium in the recirculation bottle. In recirculation mode cells start growing in multi layers. This way cell concentration of 4.0-4.5*10.sup.6 cells/ml can be reached in the 5 L bioreactor.

(10) Cells are detached from the micro carriers by trypsinisation. The released cells are used to start a cell culture in a 20 L. The 20 L bioreactor is prepared with 3 g/L micro carriers and E-MEM cultivation medium supplemented with BS. The final working volume after inoculation is 16 L. The inoculation level is 0.2*10.sup.6 cells/ml. The 20 L bioreactor is operated in batch mode. Cultivation takes 3-4 days depending on the lag phase after transfer of cells from the 5 L bioreactor to the 20 L bioreactor. Metabolites like glucose and glutamine are monitored to check if feeding of glucose or glutamine is necessary to maintain optimal growth conditions.

(11) Virus propagation is performed in the 20 L bioreactor. A medium switch from E-MEM to M199 is performed to create favorable conditions for virus propagation. The temperature is lowered from 37 C. to 32.5 C. Dissolved Oxygen percentage (DO %) is lowered from 50% to 25%. The multiplicity of infection level (MOI) is 0.01. Virus propagation and lysis take 3-5 days dependent on the virus subtype. Virus propagation and lysis are monitored by microscopic inspection of the Cytopathological effect (CPE). The virus culture is finished when the CPE is 90% and/or when oxygen consumption has ceased and at that time can be harvested for purification (down-stream processing, DSP).

(12) The bioreactor contains a steel 75 m mesh filter to retain microcarriers in the reactor. The virus harvest, containing cell debris, is clarified by two disposable filters in series. The depth filter cassette has incorporated of diatomaceous earth (HC Pod Filter grade COHC Millipore # MCOHC054H1). The final filter is a dual layer 0.5/0.2 m filter (Millipore Express SHC opticap XL #KHGES015FF3).

(13) The concentration step in the polio production process is performed by Tangential Flow Filtration (TFF) (also known as Cross Flow Filtration (CFF) or Ultrafiltration (UF)). The total concentration factor is 700-800. To avoid high losses of product in the dead volume of the TFF system, two systems are used in succession. Both systems use 100 kD flat screen filter cassettes (0.2 m.sup.2 and 50 cm.sup.2 resp.) Virus particles are retained and small molecules end up in the filtrate and are removed.

(14) Size Exclusion Chromatography (SEC) is a purification technique that separates particles and molecules by size. Large molecules elute faster than smaller ones. The column is packed with CL6B from GE healthcare. The first peak might contain aggregates and high molecular weight molecules like Host Cell Proteins (HCP's). The second peak is the product peak with most of the poliovirus. During SEC, the poliovirus particles eluted with a phosphate buffer with low ionic strength (20 mM) pH 7.00.2.

(15) Ion Exchange Chromatography (IEX) is preferably performed with a DEAE-ligand based matrix, Sephadex A50 from GE Healthcare. This particular resin is supplied as a dry powder from the supplier and, needs to be prepared by the user. This involves swelling and rinsing of the resin, column packing and column equilibration. The aim of this unit operation is to bind negatively charged components to the matrix. RNA/DNA/Host Cell Proteins are the main components that are bound to the column matrix. The poliovirus has limited interaction with the matrix.

(16) The purified virus obtained from the ion exchange chromatography is stabilized and diluted to a specific strength (when applicable). Stabilization and dilution is performed with M199 medium. The required concentration depends on the virus type

(17) Finally Glycine is added to a final concentration of 5 g/L in preparation of the inactivation

(18) The stabilized, diluted and prepared purified virus is inactivated using formaldehyde in a final concentration of 2-3 mM (or 1:4000). Inactivation takes 13 days and is performed at a temperature of 37 C. After 6-8 days of inactivation an intermediate filtration is performed to remove possible aggregates.

(19) After 13 days of inactivation the sterile monovalent pool is ready for storage at 2-8 C. At this point the obtained inactivated poliovirus is called monovalent bulk

(20) Mixing/Formulation

(21) A bulk is prepared by mixing the required serotypes together in the pre-determined concentrations. This can be either a single (type 1, type 2 or type 3), bi/divalent (type 1 and type 2 or type 1 and type 3 or type 2 and type 3) or trivalent mixture (type 1 and type 2 and type 3) formulation. Furthermore, the formulation can be combined with other vaccines like, Diphteria, pertussis, tetanus, Hepatitis, Haemophilus, meningitis, pneumococcal and/or others.

(22) Optimized Process

(23) The optimized process follows the same procedure as stated above with the following deviations: Clarification step is flushed with medium, after product filtration is finished The 2.sup.nd filter in the concentration step is not a 50 cm.sup.2 100 kD cassette filter, but a 115 cm.sup.2 hollow fiber is used as 2.sup.nd 100 kD filter (Spectrum labs # D02-E100-05-N). The buffer during the SEC is changed to a phosphate buffer supplemented with a basic amino acid like L-arginine at pH 7.00.2. During Ion exchange chromatography the buffer used for preparing the matrix and eluting the product is changed to a phosphate buffer supplemented with a basic amino acid like L-Arginine at pH 7.00.2.

(24) The improved process gives a higher D-antigen yield (DU/ml) (with comparable or better immunogenicity as can be assayed by a poliovirus neutralizing cell culture assay on sera from immunized rats (FIGS. 3A, 3B, 3C and 3D).

(25) In Table 1 the recoveries (percentages based on D-antigen yield) for the current and the optimized process (modification by addition of a basic amino acid) are given, as well as the total process yield for poliovirus types 1 and 2. The antigenicity of the polio virus or IPV product was tested using a polio D-antigen ELISA (ten Have et al. 2012).

(26) TABLE-US-00002 TABLE 1 Comparison of poliovirus yields as observed for the current and the improved purification process: Process step yield (%) Current with added L-Arginine Poliovirus Poliovirus Poliovirus Poliovirus Process step subtype 1 subtype 2 subtype 1 subtype 2 SEC 69 2.1 51 0.8 77 6.0 70 3.8 IEX (DEAE) 94 19.2 32 4.5 93 8.3 82 7.1 Overall 40 1.5 4 0.9 54 27

Example 2

Reduction of Aggregate Level

(27) Reduction of virus aggregation can be achieved by addition of a (concentrated) solution of a basic amino acid or derivative. Any skilled person can fine-tune this method to their specific need. See FIGS. 2A and 2B.

(28) In a further experiment, stock solutions of basic amino acids were prepared and added to batches containing different viruses in an aggregated form. Solutions were added in a 1:1 ratio. Aggregation was measured as the absorbance at 590 nm (Biowave DNA, WPA). Aggregated virus as measured prior to the addition of basic amino acids was set at 100%. The effects of different concentrations of amino acid addition on the percentage of aggregated virus are shown in FIGS. 2C, 2D and 2E for the effects of three different basic amino acids L-Arginine, L-Lysine and L-Histidine respectively) on three different influenza strains (Influenza A/Uruguay H3N2 (NIBSC), Influenza B/Florida/4/2006 (NIBSC) and Influenza A/PR/8/34 (NIBSC)). In FIGS. 2F, 2G and 2H results are depicted for the effect of respectively three different basic amino acids (L-arginine, L-lysine and L-histidine) on three different polio (Sabin) subtypes 1, 2 and 3.

Example 3

Offline Additions (Dissolving Existing Aggregation)

(29) Aggregated virus was produced according to the slightly modified Salk-IPV production protocol with a 20 mM phosphate buffer (Sabin batches types 1, 2 and 3; intermediate product fractions after SEC and IEX). The obtained virus fractions were frozen in 80 C. until use. The freezing positively facilitated and/or enhanced the aggregation effect.

(30) Influenza (with aggregates) was produced using a laboratory egg based process consisting of inoculating embryonated eggs (strain: A/Uruguay/H3N2 (NIBSC)), incubating, cooling and subsequent harvesting by disk-stack centrifugation followed by depth filtration (GE Healthcare). Virus was purified by sucrose density centrifugation, followed by a -propiolactone inactivation with filtration. Finally a diafiltration/concentration was performed.

(31) pH is measured using a calibrated pH meter (Orion scientific), with a gel-electrode (Mettler Toledo) and corrected using either sulfuric acid or sodium hydroxide.

(32) Optical density, as measurement of aggregation, was determined using a Biowave DNA spectrophotometer (WPA) at wavelengths 450 & 590 nm in either plastic disposable cuvettes (Greiner) or quartz cuvettes with a diameter of 10 mm.

(33) ELISA-assay; antigenicity of the polio virus or IPV product was tested using an ELISA (ten Have et al. 2012).

(34) The virus products, were used in offline testing with a range of concentrations of the different additives according to Table 2. The additive was weighed in a test tube and to this 1 or 2 ml of the product was added, the solution and additive were mixed. When the additives were dissolved the pH change was measured and corrected (when needed) to the initial product pH. After 1 hour the optical density was measured.

(35) TABLE-US-00003 TABLE 2 Additives tested for their ability to prevent or break up aggregation of virus Additive Remarks L-arginine D-arginine Chiral form of L-arginine L-lysine N--acetyl L-arginine Derivative of arginine Agmatine Sulfate salt Derivative of arginine Same concentration as Mixture composing of: used in medium Glycine M199 Alanine l-valine l-leucine l-isoleucine l-proline hydroxy-l-proline l-serine l-threonine l-glutamic acid l-glutamine l-aspartic acid l-aspargine M199 Cell culture medium (mix of >50 components)

(36) The offline additions of different additives perform differently in clearing the aggregates at different molarities and with different products (FIGS. 2A and 2B). The initial aggregated virus product was set at 100% as measured with no additions, a blank buffer solution was used as negative control e.g. no aggregation to be reached 0%.

(37) Addition of the M199 to virus was able to clear the formed aggregates from all virus products either stored at 80 C. stored material or freshly prepared solutions. For poliovirus, this resulted in a 22% +/12 increase of available D-antigen epitope as measured in an ELISA-assay (ten Have et al, 2012).

Example 4

Fast Purification of Influenza (Proof of Concept)

(38) Influenza was purified as a proof-of-concept using Arginine according to the following protocol. Virus feed was prepared from harvest of 10.000 SPF (Specific Pathogen Free) embryonated hen's eggs inoculated, at day 11 with influenza A/Uruguay H3N2 virus (EID50/ml of 9.17) using a semi-automatic inoculation machine located in a down flow booth with HVAC filters. Prior to inoculation, the eggs and the needles were disinfected using 70% ethanol. The eggs were incubated during 72 hr for 3 days at 35 C. and then cooled overnight to circa 4 C., with circa 12 hr temperature effectively below 8 C. Harvest of the allantoic fluid from the decapped eggs took place with a semi-automatic harvest machine in a down flow booth. Clarification of the crude harvested allantoic solution was performed with a Westfalia disk stack separator continuous centrifuge at 65 L/h, 1 bar backpressure, 10.000 rpm, followed by filtration using two parallel 10 inch 2.0 m GE ULTA prime capsule depth filters (GE Healthcare).

(39) From the obtained batch approximately 3 L was separated for small scale testing.

(40) The 3 L was again divided in 2 portions and each portion was concentrated approximately 15 times using a hollow fiber (GE Healthcare #UFP-750-E-3X2MA) and diafiltered, with 10 volumes, against either PBS (GIBCO) or PBS (GIBCO) supplemented with 0.35M L-Arginine (Sigma-Aldrich).

(41) From the thus acquired concentrated and diafiltered material, 10 ml was tested on a size exclusion column of 90 cm bedheight (column vl11/100 Millipore) containing Sepharose 6 fast flow matrix (GE Healthcare) using either PBS or PBS supplemented with 0.35M L-Arginine using an Akta explorer chromatographic system (GE Healthcare)

(42) All samples were inactivated using formaldehyde prior to analysis.

(43) Analytical Assays

(44) SRID (single radial immunodiffusion) assay is based on the reaction between the antibodies present in a flat agarose gel and the antigen that diffuses from an application spot in the gel. Once the concentration of antibody and antigen are equal a precipitation in the shape of a ring occurs. The precipitate was stained using Coomassie Brilliant Blue and the size of the ring was used as a measure for the concentration.

(45) Ovalbumin concentration was measured using a direct sandwich ELISA (Enzyme Linked Immuno Sorbent Assay). The assay was performed according to the instructions of the supplier of the ELISA kit (Serazym Ovalbumin ELISA: Cat. No. E041C, Seramun Diagnostica GmbH Wolzig). Independent duplicates of two different dilutions were uses as samples. Samples containing the antigen were pipetted in ELISA plate wells coated with polyclonal anti-ovalbumin antibodies. Anti-ovalbumine-linked to Horse Radish Peroxidase was added, followed by washing away unbound substances. The addition of a substrate initiated the development of a blue color. This process was stopped by adding sulphuric acid; the color changed from blue to yellow. The absorption at 450 nm was a measure for the quantity. A 630 nm filter was used as reference (Biotek reader with KC-jr and KC4 software).

(46) Results

(47) Material collected from the chromatographic run was tested in a SRID assay for the haemagglutinin antigen content. Analysis showed an 8 percent higher haemagglutinin antigen content in the material supplemented with 0.35 m L-Arginine.

(48) Furthermore the concentrated and diafiltrated (UF/DF) material from the hollow fiber which contained the supplemented L-Arginine mono hydrochloride contained significantly less (upto 76%) ovalbumine present in the sample, indicating that the diafiltration was far more efficient in removing impurities in presence of L-Arginine mono hydrochloride.

Example 5

Effect of Additions to Poliovirus Chromatography

(49) Breaking up aggregation after it has occurred is one way of solving the problem. However, during manufacturing it is more important to prevent the aggregation from the beginning. During the processing of (polio) virus, formed aggregates are removed during SEC and bind as impurities to the IEX (DEAF) column leading to high product losses (up to 70%).

(50) The chromatographic separations were performed in different solutions according to Table 3. The starting material is the concentrated poliovirus either from the 80 C. freezer or freshly prepared. Only the L-Arginine HCL addition to the 20 mM phosphate buffer was tested during chromatographic separations next to the control.

(51) The yield (in terms D-antigen recovery) was determined, the results are given in Tables 4 and 5 (nd=not done).

(52) TABLE-US-00004 TABLE 3 Chromatography additions Control Alternative 1 Alternative 2 SEC 20 mM phosphate M199 20 mM phosphate + pH 7.0 150 mM L-Arginine pH 7.0 IEX 20 mM phosphate M199 20 mM phosphate + pH 7.0 150 mM L-Arginine pH 7.0

(53) TABLE-US-00005 TABLE 4 SEC (size-exclusion column chromatography) recovery (percentage (%) based on D-antigen per milliliter) result with different elution buffers Poliovirus Recovery (%) Control: Poliovirus Phosphate Alternative 1: Alternative 2: Batch type buffer M199 L-Arginine 1 1 30 16 73 2 1 67 nd 81 3 2 72 59 66 4 2 66 nd 69 5 3 71 64 58 6 3 75 nd 73 7 3 83 nd 68

(54) TABLE-US-00006 TABLE 5 Ion-exchange column chromatography) recovery (percentage (%) based on D-antigen per milliliter) results with different elution buffers Poliovirus recovery (%) Control: Poliovirus Phosphate Alternative 1: Alternative 2: Batch type buffer M199 L-Arginine 1 1 37 102 99 2 1 100 nd 87 3 2 nd 21 90 4 2 28 nd 83 5 3 58 76 93 6 3 87 nd 92 7 3 100 nd 105

(55) The addition of L-Arginine to the elution buffer can have a positive effect, which is mainly observed during the SEC for types 1 and 2, while for the IEX it is especially clear for type 2. The addition of alternative 1 (M199) seems to be beneficial as well, however during purification this compound showed to reduce capacity on the IEC, limiting it's usage considerably when compared to alternative 2.

(56) In a further experiment, a high cell density product batch (semi-batch methodology, Thomassen et al., 2014) was produced, infected with Sabin subtype 2 and purified according to Thomassen et al, 2013a and example 1 optimized process, until a 400 times concentrated batch was made (by harvest, filtration and ultrafiltration). This material was aliquoted and stored at 80 C. This material was thawed and purified using size exclusion chromatography with a C16B-resin on an Akta explorer system (GE Healthcare) using different basic amino acid additions at different concentrations to the control 20 mM phosphate buffer. The variations and results are depicted in Table 6 below. The product concentration (D-antigen) is an average of a triplicate [ten Have et al, 2012], the product increase has been calculated and given in a percentage difference.

(57) TABLE-US-00007 TABLE 6 Size exclusion chromatography of Sabin subtype 2 using different basic amino acid additions at different concentrations to the control 20 mM phosphate buffer total additive concentration D-antigen % product (mM) (product/ml) increase Control 0 2330 L-Arginine 0.81 3065 32 2.5 3273 40 5 3285 41 25 3134 35 50 3200 37 100 2993 28 150 2993 28 D-Arginine 2.5 3394 46 25 3101 33 L-Lysine 2.5 3226 38 25 2735 17 150 2420 4 L-Histidine 2.5 2929 26 150 2386 2 D-Histidine 2.5 3577 54 25 3649 57 Agmatine 75 3318 42 2.5 mM D-His + 5 3418 47 2.5 mM L-Lys 25 mM D-his + 50 2735 17 25 mM L-Lys 50 mM L-Arg + 150 3220 38 50 mM L-His + 50 mM L-Lys

(58) From Table 6 it is obvious that different additions and different concentrations have a effect on the recovery of product from the size exclusion chromatography. In all cases the product yield increased from moderate (2%) to high (57%), clearly showing the improvement from the added basic amino acid.

Example 6

Removal of L-Arginine from a Poliovirus Intermediate Purified Product (SEC) Using Diafiltration

(59) The addition of a basic amino acid to a virus particle containing solution dissolves and/or prevents aggregate formation. By removing the basic amino acid again, aggregation is expected to return. This was illustrated by producing a poliovirus (Sabin type 2) batch using size exclusion chromatography (Cl6B in a xk26/80 column both GE Healthcare) using an elution buffer containing L-Arginine. The obtained virus product solution was subsequently washed/diafiltrated, as known in the art, using a 100 kD hollow fiber, against a similar buffer solution without the additive at the same rate in which filtrate is removed, thus maintaining a constant retentate volume. The absorbance, as measure for the amount of aggregates, was measured offline using a spectrophotometer (Biowave DNA, WPA), L-Arginine concentration was measured using a NMR. Pressure and conductivity were followed inline with Pendotech disposable sensors.

(60) One diafiltration volume corresponds to the total virus product volume (75 ml) present in the system. The system was left to run for 10 diafiltration volumes with constant flux and TMP (total time 2.5 hours) and samples were taken (and corrected for volume) after each diafiltration volume exchange for analysis.

(61) Results are depicted in FIG. 4 and show that the L-Arginine concentration quickly drops below the detection limit (1 mM) of the used NMR after 3-4 diafiltration volumes. Corresponding with this decreased concentration of L-Arginine the absorbance increased, indicating aggregation of virus particles. The L-Arginine can be indirectly followed by the conductivity as well. Conductivity drops quickly in parallel with the removal of L-Arginine to reach a final conductivity of 2.9 mS/cm corresponding to the control buffer, indicating that the additive has been removed. The absorbance has more than doubled after 10 diafiltration volumes compared to the starting value when this conductivity value has been reached. This clearly shows that the addition of the basic amino acid was the cause for the reduction in aggregation (as visualized by UV) and that the process is reversible, ie the additive may be removed, albeit aggregation will return.

Example 7

Purification of Wild Type Poliovirus in the Presence or Absence of Basic Amino Acids

(62) Wild type polio virus type 2 was produced and purified up to the chromatographic steps in a procedure as described by Thomassen et al (2013a). The material was purified using two different SEC columns, in 40 mM phosphate (pH7.0+/0.2) buffer. One buffer contained the additive (150 mM L-Arginine) and one buffer was without additive. Both obtained products were subsequently purified on an IEX (DEAF Sepharose Fast Flow, GE Healthcare), again using the two afore mentioned buffers. Table 7 shows the results for the IEX. It is clear that the presence of the additive resulted in a higher product yield by nearly doubling the product yield as measured by ELISA (Ten Have R et al, 2012) and peak area. In both cases, the purification resulted in good purity virus products (based on UV ratio's as determined by Koch and Koch 1985, data not shown).

(63) TABLE-US-00008 TABLE 7 SEC and IEX purification of wild type poliovirus in the presence or absence of 150 mM L-Arginine. The peak area corresponds with poliovirus amounts. The measured D-antigen corresponds with immunogenic virus. Without With 150 mM 150 mM L-arginine L-arginine Peak area (mAU*ml) 3621 6006 Poliovirus D-antigen ( DU/ml) 994 1845

(64) In a further experiment wild type polio virus type 3 was again produced and purified up to the chromatographic steps in a procedure as described by Thomassen et al (2013a). The material was purified using one SEC column in the regular 40 mM phosphate buffer pH 7.0+/0.2). The obtained product was split in 2 equal amount portions. To one portion, a highly concentrated buffer containing L-Arginine was added to make a final concentration of 149 mM, while to the other portion the same amount of regular buffer was added to compensate for the diluting effect.

(65) Both products were subsequently purified on a IEX (DEAE Sepharose Fast Flow, GE Healthcare), using the 40 mM phosphate buffer, either with or without additive (150 mM L-Arginine), dependent on the portion to be purified. In Table 8 shows the results for the IEX. It is clear again that the additive resulted in a product yield (based on peak area). In both cases, the purification resulted in good purity virus products (based on UV ratio's as determined by Koch and Koch 1985, data not shown).

(66) TABLE-US-00009 TABLE 8 IEX purification of wild typepoliovirus type 3 in the presence or absence of 150 mM L-arginine Without 150 mM With 150 mM L-Arginine L-Arginine Product Peak area 3887 4789 (mAU*ml)

(67) From Example 7 it is clear that the addition can be made in different ways to be effective, either directly and co-eluting or afterwards as a highly concentrated stock compound did not matter. The product containing the additive, in all cases, showed a higher yield. Different forms of additions, such as a solid, through diafiltration or as highly concentrated stock will all result in higher yields as well.

Example 8

Purification of a Chimeric Poliovirus in the Presence or Absence of Basic Amino Acids

(68) An experimental poliovirus was obtained, representing a combination/chimera/hybrid of both the wild type virus (bases of Salk-developed IPV) and attenuated virus (Sabin strains). This virus was furthermore crippled by genetic modification to make it less biologically active in mammals to further prevent any severe illness/reversals.

(69) This experimental virus was produced using the existing production process (Bakker et al, 2011 and Thomassen et al, 2013a) on laboratory scale to evaluate its potential. For the chromatographic separation part, both plain 20 mM phosphate buffer (control) and a 20 mM phosphate buffer containing 150 mM L-Arginine were used. The results are depicted in Table 9, which shows the yield of individual unit operations (%) and the combined chromatographic unit operation. Table 9 clearly shows the beneficial effects of the L-Arginine additive as higher overall recoveries are reached.

(70) TABLE-US-00010 TABLE 9 Chimeric poliovirus yield (%) for individual unit operations (SEC and IEX) and combined unit operations Yield (%) type 1 type 2 Control Additive Control additive SEC 67 90 60 100 IEX 75 88 59 70 Combined SEC & IEX 50 79 35 70

Example 9

Mixed-Mode/Multimodal Chromatography in the Presence of Basic Amino Acids

(71) The use of an additive in the elution buffer, opens the way for new purification options, such as the use of mixed-mode/multimodal chromatographic separations, instead of regular ion exchange. Hereby both electrostatic and hydrophobic effects are in play to allow bi-dimensional separation of particles, even allowing closely related particles, with regard to isoelectric point, to be separated.

(72) An example is given in Table 10. An HEA Hypercell aliphatic chromatographic resin (Pall Corporation) is used to purify Sabin type 2. Purity of the product is determined on the basis of UV 260/280 ratio in accordance with Koch and Koch (1985), whereby the target ratio for pure poliovirus is in the range of 1.60-1.80. Without L-Arginine present the HEA-purified virus has an UV 260/280 ratio of 0.98, whereas virus purified on the HEA column in the presence of 150 mM L-Arginine suddenly reaches a high purity as indicated by an UV 260/280 ratio of 1.76, i.e. within the target range.

(73) TABLE-US-00011 TABLE 10 Purity of viral polio product on a multimodal chromatographic resin target control additive UV260/280 1.60-1.80 0.98 1.76 ratio

(74) The use of new and more modern resins creates new options for viral purification.

Example 10

Effects of Different Basic Amino Acid and Derivatives Thereof on Reducing of Aggregation of Poliovirus

(75) A black 96-wells plate, chimney shaped with a clear bottom, was used for screening different compounds and concentrations against an aggregated intermediately purified batch of polio (Sabin, type 1, derived from a size exclusion chromatography step in a plain phosphate buffer).

(76) Stock solutions of the various compounds were prepared in a 96-deep wells plate by mixing concentrated stock solutions with buffer (both pH 7.0+/0.2) to a pre-determined molarity, after which these were added to the black 96 well plate in equal amounts with the virus preparation to be tested (1:1).

(77) The resulting plate was mixed on a shaker platform and absorbance (590 nm) was measured after 5 minutes. The results are depicted in FIG. 5.

REFERENCES

(78) Albrecht P., Van Steenis G., Van Wezel A L., Salk J. Standardization of poliovirus neutralizing antibody tests. Rev Infect Dis, 6 (May-June (Suppl. 2)) (1984), pp. S540-S544.

(79) Aylward B, Tangermann R. The global polio eradication initiative: Lessons learned and prospects for success. Vaccine 29(S4), D80-D85 (2011).

(80) Arakawa, T., John S Philo, Kouhei Tsumoto, Ryosuke Yumioka, Daisuke Ejima. Elution of antibodies from a Protein-A column by aqueous arginine solutions. Protein Expression and Purification. 2004 volume 36 (issue 2) Pages 244-248.

(81) Arakawa, T.; Kita, Y.; Koyama, A. H. Synergistic virus inactivation effects of arginine. Biotechnol. J. 2009, 4, 174-178.

(82) Bakker W A, Thomassen Y E, vant Oever A G, Westdijk J, van Oijen M G, Sundermann L C, vant Veld P, Sleeman E, van Nimwegen F W, Hamidi A, Kersten G F, van den Heuvel N, Hendriks J T, van der Pol L A. Vaccine. 2011 Sep. 22; 29(41):7188-96.

(83) Baynes B M, Trout B L. Rational design of solution additives for the prevention of protein aggregation. Biophys J. 2004 September; 87(3):1631-9.

(84) Baynes B M, Wang D I, Trout B L. Role of arginine in the stabilization of proteins against aggregation. Biochemistry. 2005 Mar. 29; 44(12):4919-25.

(85) Chumakov K, Ehrenfeld E, Wimmer E, Vadim I. Vaccination against polio should not be stopped. Nature Rev. Microbiol. 5, 952-958 (2007).

(86) Chumakov K, Ehrenfeld E, Plotkin S. New generation of inactivated poliovirus vaccines for universal immunization after eradication of poliomyelitis. Clin. Infect. Dis. 47(12), 1587-1592 (2008).

(87) Cromwell M E M, Hilario E, Jacobson F. Protein aggregation and bioprocessing. The American Association of Pharmaceutical Scientists. 2006 September; 8(3): E572-E579.

(88) Das T K. Protein particulate detection issues in biotherapeutics developmentcurrent status. AAPS PharmSciTech. 2012 June; 13(2):732-46.

(89) Desnues, C., and D. Raoult. 2010. Inside the lifestyle of the virophage. Intervirology 53:293-303.

(90) Duchene M, Peetermans, d'Hondt E, Harford N, Fabry L, Stephenne J. Production of poliovirus vaccines: past, present, and future. Viral Immunology 3(4), 243-272 (1990).

(91) Nathanson N, Kew O M. From Emergence to Eradication: The Epidemiology of Poliomyelitis Deconstructed. Am. J. Epidemiol. (2010) 172(11): 1213-1229.

(92) Fields Virology, 5.sup.th edition, 2007; Bernard N. Fields, David Mahan Knipe, Peter M. Howley, Wolters Kluwer.

(93) Gu Z et al. Inhibition of aggregation by media selection, sample loading and elution in size exclusion chromatographic refolding of denatured bovine carbonic anhydrase B.J. Biochem Biophys Methods. 2003. 56(1-3):165-75).

(94) Hamidi A, Bakker W A M. Innovative IPV from attenuated Sabin poliovirus or newly designed alternative seed strains. Pharmaceutical Patent Analyst (2012) 5(1):589-599.

(95) Heinsbroek E, Ruitenberg E J. The global introduction of inactivated polio vaccine can circumvent the oral polio vaccine paradox. Vaccine 28(22), 3778-3783 (2010).

(96) Heymann D L, Sutter R W, Aylward R B, A vision of a world without polio: The OPV cessation strategy. Biologicals 34(2), 75-79 (2006).

(97) Heymann D L, Sutter R W, Aylward R B. A global call for new polio vaccines. Nature 434, 699-700 (2005).

(98) Holmes E C. Viral evolution in the genomic age. PLoS Biol. 2007; 5(10):e278.

(99) Jonges M, Liu W M, van der Vries E, Jacobi R, Pronk I, Boog C, Koopmans M, Meijer A, Soethout E. Influenza virus inactivation for studies of antigenicity and phenotypic neuraminidase inhibitor resistance profiling. 2010, J. Clin. Microbiol. 48:928-940.

(100) Koonin E V, Senkevich T G, Dolja V V. The ancient Virus World and evolution of cells. Biol Direct. 2006 Sep. 19; 1:29.

(101) Kew O M, Sutter R W, de Gourville E M, Dowdle W R, Pallansch M A. Vaccine-derived polioviruses and the endgame strategy for global polio eradication. Ann. Rev. Microbiol. 59, 587-635 (2005).

(102) Koch and Koch. The molecular biology of poliovirus. 1985 Springer-Verlag Wien-New York ISBN 3-211-81763-8.

(103) Li Y, Weiss W F, Roberts C J. Characterization of high-molecular-weight non native aggregates and aggregation kinetics by size exclusion chromatography with inline multi-angle laser light scattering. Journal of pharmaceutical sciences 2009 vol 98 issue 11 p 3997-4016.

(104) Lee Sang-Won, Philip F. Markham, Mauricio J. C. Coppo, Alistair R. Legione, John F. Markham, Amir H. Noormohammadi, Glenn F. Browning, Nino Ficorilli, Carol A. Hartley, Joanne M. Devlin. Attenuated Vaccines Can Recombine to Form Virulent Field Viruses. Science 13 Jul. 2012: 188.

(105) Montagnon B J, Fanget B, Vincent-Falquet J C. Industrial-scale production of inactivated poliovirus vaccine prepared by culture of Vero cells on microcarrier. Rev Infect Dis. 1984 May-June; 6 Suppl 2:S341-4.

(106) Montagnon B, Vincent-Falquet J C, Fanget B. Thousand liter scale microcarrier culture of Vero cells for killed polio virus vaccine. Promising results. Dev Biol Stand. 1983; 55:37-42.

(107) Morgan, J. F. and Campbell, M. E. (1955) J. Natl. Cancer Inst., 16:557.

(108) Morgan, J. F., Morton, H. J. and Parker R. C. (1950) Proc. Soc. Exp. Biol. Med., 73:1.

(109) Nathanson & Kew. From emergence to eradication: the epidemiology of poliomyelitis deconstructed. Am J Epidemiol. 2010 Dec. 1; 172(11):1213-29.

(110) Pearson, H. 2008. Virophage suggests viruses are alive. Nature 454:677.

(111) Robinson H L. Viral attenuation by design. Nature Biotechnology. 2008 September; 26(9):1000-1.

(112) Sanders P, Edo-Matas D, Custers J H H V, Koldijk M R, Klaren V, Turk M, Luitjens A, Bakker W A M, Uytdehaag F, Goudsmit J, Lewis J A, Schuitemaker H, PER.C6 cells as a serum-free suspension cell platform for the production of high titer poliovirus: A potential low cost of goods option for world supply of inactivated poliovirus vaccine. Vaccine. 2013 Jan. 21; 31(5):850-6.

(113) Sun L, Young L N, Zhang X, Boudko S P, Fokine A, Zbornik E, Roznowski A P, Molineux I J, Rossmann M G, Fane B A. Icosahedral bacteriophage X174 forms a tail for DNA transport during infection. Nature. 2014 Jan. 16; 505(7483):432-5.

(114) Taylor, J. P.; Hardy, J.; Fischbeck; K. H. Toxic proteins in neurodegenerative disease. Science. 2002 Jun. 14; 296(5575):1991-5.

(115) Taylor D J, Ballinger M J, Bowman S M, Bruenn J. Virus-host co-evolution under a modified nuclear genetic code. Peed 2013 Mar. 5; 1e50.

(116) Ten Have R, Thomassen Y E, Hamzink M R, Bakker W A, Nijst O E, Kersten G, Zomer G. Development of a fast ELISA for quantifying polio D-antigen in in-process samples. Biologicals. 2012 January; 40(1):84-7.

(117) Thomassen Y E, van Sprang E N, van der Pol L A, Bakker W A. Multivariate data analysis on historical IPV production data for better process understanding and future improvements. Biotechnology & Bioengineering 2010 Sep. 1; 107(1):96-104.

(118) Thomassen Y E, Rubingh O, Wijffels R H, van der Pol L A, Bakker W A, Vaccine. 2014 May 19; 32(24):2782-8. doi: 10.1016/j.vaccine.2014.02.022. Epub 2014 Feb. 26.

(119) Thomassen Y E, van't Oever A G, Vinke M, Spiekstra A, Wijffels R H, van der Pol L A, Bakker W A. Scale-down of the inactivated polio vaccine production process. Biotechnol Bioeng. 2013a May; 110(5):1354-65.

(120) Thomassen, Y E, van't Oever, A G, van Oijen M G C T, Wijffels, R. H., van der Pol, L A, Bakker, W A M. Next generation inactivated polio vaccine manufacturing to support post polio-eradication biosafety goals. PLOS One. 2013b Dec. 12; 8(12):e83374Thompson K M, Tebbens R J. Current polio global eradication and control policy options: perspectives from modeling and prerequisites for oral poliovirus vaccine cessation. Expert Review of Vaccines 11(4), 449-459 (2012).

(121) Utsunimoya, H.; Ichinose, M.; Tsujimoto, K.; Katsuyama, Y.; Yamasaki, H.; Koyama, A. H.; Ejima, D.; Arakawa, T. Co-operative thermal inactivation of herpes simplex virus and influenza virus by arginine ans NaCl. Int. J. Pharm. 2009, 366, 99-102.

(122) Van Wezel A L, van Steenis G, Hannik C A, Cohen H. 1978. New approach to the production of concentrated and purified inactivated polio and rabies tissue culture vaccines. Develop, biol. Standard. 41: 159-168.

(123) Verdijk P, Rots N Y, Bakker W A M. Clinical development of a novel inactivated poliomyelitis vaccine based on attenuated Sabin poliovirus strains. Expert Review of Vaccines (2011) 10(5):635-644.

(124) Wei Wang. Protein aggregation and its inhibition in biopharmaceuticals. International journal of pharmaceuticles 2005; 289: 1-30.

(125) Westdijk, J., et al., Characterization and standardization of Sabin based inactivated polio vaccine: proposal for a new antigen unit for inactivated polio vaccines. Vaccine, 2011. 29(18): p. 3390-7.

(126) Widjojoatmodjo M N, Boes J, van Bers M, van Remmerden Y, Roholl P J, Luytjes W. A highly attenuated recombinant human respiratory syncytial virus lacking the G protein induces long-lasting protection in cotton rats. Virol J. 2010 Jun. 2; 7:114.

(127) Yamasaki, H.; Tsujimoto, K.; Koyama, A. H.; Ejima, D.; Arakawa, T. Arginine facilitates inactivation of enveloped viruses. J. Pharm. Sci. 2008, 97, 3063-3073.

(128) Zor and Selinger, Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem. 1996 May 1; 236(2):302-8.

ABBREVIATIONS

(129) AUAbsorbance units CCID5050% cell culture infectious dose CFFCross Flow Filtration cmcentimeter CPECytopathological Effect DEAEDiethylaminoethanol DFDiafiltration DLSDynamic Light Scattering DNADeoxyribonucleic acid DODissolved Oxygen DSPDown Stream Processing DUD-antigen Unit EID5050% egg infectious dose ELISAEnzyme-linked immuno sorbent assay E-MEMEagle's minimal essential medium FTUFormazin Turbidity Unit HCPHost Cell Protein HEAhexylamine HPVHuman papillomavirus HVACHeating, Ventilation, and Air Conditioning IEC/IEXIon Exchange Chromatography IPVinactivated polio Vaccine kDkiloDalton M199Medium 199 MALSMulti Angle Light Scattering MKCMonkey kidney cell mMmilliMolar MOImultiplicity of infection mSmilli-Siemens NIBSCNational Institute for Biological Standards and Control nmnanometer NMRnuclear magnetic resonance NTUNephelometric Turbidity Unit ODOptical Density OPVOral Polio Vaccine PBSPhosphate Buffered Saline RIVMNational Institute for Public Health and Environment RNARibonucleic Acid RSVRespiratory Syncytial Virus SECSize exclusion Chromatography sIPVSabin-based inactivated Polio Vaccine SPFSpecific pathogen Free SRIDSingle Radial Immunodiffusion TCID5050% Tissue Culture Infective Dose TFFTangential Flow Filtration UFUltra Filtration USPUpstream processing UVUltra Violet VAPPVaccine Associated Paralytic Poliomyelitis VDPVVaccine Derived Poliovirus VLPVirus Like Particle