Stabilisation method for viruses or bacteria

Abstract

The present invention relates to a method for stabilising viruses or bacteria, the method comprising embedding the viruses or bacteria in an aqueous solution, wherein the solution comprises: (i) at least three different amino acids; or (ii) at least one dipeptide or tripeptide and wherein the solution is free or substantially free of sugar(s), silanes and protein(s).

Claims

1. A method for stabilizing viruses or bacteria, the method comprising: (a) embedding the viruses or bacteria in an aqueous solution, wherein the aqueous solution comprises: at least three different amino acids selected from at least three different groups of (i) amino acids with non-polar, aliphatic R groups; (ii) amino acids with polar, uncharged R groups; (iii) amino acids with positively charged R groups; (iv) amino acids with negatively charged R groups; and (v) amino acids with aromatic R groups; and wherein the aqueous solution is free or substantially free of sugar(s), silanes and protein(s), (b) storing the embedded viruses or bacteria in the aqueous solution for at least 7 days, wherein the viruses are whole viruses which maintain their infectivity and/or the ability to replicate after being embedded and stored in the aqueous solution for at least 7 days.

2. The method of claim 1, wherein the solution comprises at least one amino acid selected from each group of (1) an amino acid with non polar, aliphatic R groups; (2) an amino acid with polar, uncharged R groups; (3) an amino acid with positively charged R groups; (4) an amino acid with negatively charged R groups; and (5) an amino acid with aromatic R groups.

3. The method according to claim 1, wherein the solution comprises at least the amino acids selected from the groups of: (1) alanine, glutamate, lysine, threonine and tryptophan; (2) aspartate, arginine, phenylalanine, serine and valine; (3) proline, serine, asparagine, aspartate, threonine, and phenylalanine; (4) tyrosine, isoleucine, leucine, threonine, and valine; (5) arginine, glycine, histidine, alanine, glutamate, lysine, and tryptophan; and (6) alanine, arginine, glycine, glutamate, and lysine.

4. The method according to claim 1, wherein one or more of the amino acids are selected from the group consisting of natural non-proteinogenic amino acids and synthetic amino acids.

5. The method according to claim 1, wherein the solution further comprises an amphiphilic molecule.

6. The method according to claim 5, wherein the amphiphilic molecule is selected from the group consisting of a saponin or a fatty acid or derivatives thereof.

7. The method of claim 6, wherein the saponin is glycyrrhizic acid or a derivative thereof.

8. The method of claim 6, wherein the fatty acid is selected from the group consisting of short chain and medium chain fatty acids.

9. The method according to claim 1, wherein the w/w ratio between the excipients of the solution and the virus or bacteria is between 1:1 and 500:1.

10. The method of claim 1, further comprising the step of subsequently storing the stabilized viruses or bacteria at a temperature selected from about −90° C. to about 45° C.

11. The method of claim 1, wherein the stabilized viruses or bacteria are supplied for storage as a liquid preparation.

12. The method of claim 1, wherein the stabilized viruses or bacteria are supplied for storage as a dried preparation.

13. The method of claim 1, further comprising a subsequent inactivation step.

14. The method of claim 1, wherein the viruses are selected from the group consisting of influenza viruses, polio viruses, herpes simplex viruses-1, vaccinia viruses and adenoviruses.

15. The method of claim 1, wherein the bacteria are selected from the group consisting of pertussis, tetanus, diphtheria, meningococcus, pneumococcus, Haemophilus influenza, cholera, typhoid and anthrax.

16. The method of claim 1, wherein the aqueous solution further comprises at least one dipeptide of (ii) selected from the group consisting of carnosin, glycyltyrosine, glycylglycine and glycylglutamine.

17. The method of claim 12, further comprising irradiating the solution.

18. A method for stabilizing viruses or bacteria, the method comprising: (a) embedding the viruses or bacteria in an aqueous solution, wherein the aqueous solution comprises: at least three different non-peptidic independent amino acids selected from at least three different groups of (i) amino acids with non-polar, aliphatic R groups; (ii) amino acids with polar, uncharged R groups; (iii) amino acids with positively charged R groups; (iv) amino acids with negatively charged R groups; and (v) amino acids with aromatic R groups; and wherein the aqueous solution is free or substantially free of sugar(s), silanes and protein(s), (b) storing the embedded viruses or bacteria in the aqueous solution for at least 7 days, wherein the viruses are whole viruses which maintain their infectivity and/or the ability to replicate after being embedded and stored in the aqueous solution for at least 7 days.

Description

(1) The figures show:

(2) FIG. 1: HSV-1 was stored at different storage conditions (RT, 4° C., −20° C., 37° C.) in the presence of or absence of the protecting solution of the invention for different amounts of time (0, 7, 14, 21, 28 days). Subsequently, HSV-1 was used for inoculation in cell culture and the virus titer was titrated.

(3) FIG. 2: The activity of influenza A is higher in virus preparations with the protecting procedure according to the present invention as indicated by lattice formation (no visible red button) even at high dilutions of virus.

(4) FIG. 3: Adenovirus type 5 (Ad5) infectivity assay. (A/B) Ad5 samples were β-irradiated at 25 kGy (A) and 40 kGy (B) with and without nano-coating. The titers of infective virus particles before and after irradiation are shown. (C/D) In the same setting an IgM antibody was irradiated. The relative antigen binding capacity before and after irradiation is shown.

(5) FIG. 4: (A) SDS-PAGE; Lane 1: OVA control, Lane 2: OVA degraded, Lane 3: (OVA+ inventive solution) SD, Lane 3: Marker (PAGERULER™ (prestained protein ladder)); (B) CD spectra of OVA control, OVA degraded and (OVA+ inventive solution) SD (spectra of protein only).

(6) FIG. 5: Enhancement of fluorescence intensity of ANS on binding to OVA.

(7) FIG. 6: Fluorescence intensity of OVA control and (OVA+ inventive solution) SD (spectra of protein only) after binding with ANS.

(8) FIG. 7: Effects of different compositions on the functionality of hemagglutinine under stress conditions.

(9) Activity of an inactivated influenza A H1N1 split vaccine, respective hemagglutination activity, formulated with different compositions of the protecting solution against different types of stresses like freeze drying and β irradiation with 40 kGy. Influenza A H1N1 split vaccine was rebuffered in compositions according to the present invention, containing seven amino acids (amino acid mixture 1: Ala, Arg, Gly, Glu, Lys, His, Trp) or five amino acids (amino acid mixture 2: Ala, Arg, Gly, Glu, Lys and amino acid mixture 3: Ala, Gly, Glu, His, Trp, respectively), supplemented with the dipeptide carnosin, other dipeptides and glycyrrhizic acid, respectively.

(10) The examples illustrate the invention:

EXAMPLE 1

Testing of Infectivity after Liquid Storage

(11) HSV-1 was propagated in cell culture. After determination of the virus titer by titration the virus was split into different samples for different storage conditions (RT, 4° C., −20° C., 37° C.) in the presence of or absence of protecting solution with and without glycyrrhizic acid. As a control, PBS was used. At different times (0, 7, 14, 21, 28 days) after experimental set-up HSV-1 was used for inoculation in cell culture and titration of the titer, accordingly. The maximum observation period was four weeks.

EXAMPLE 2

Influenza a HA Assay Showing Maintenance of Antigenicity

(12) Inactivated influenza A virus was dialyzed at 2 to 8° C. versus the protecting solution according to the present invention. The w/w (virus/excipients of the protecting solution) ratio was between 1:10 and 1:100. Lyophilization was done in 100 μl volumes and lyophilisates were irradiated with 25 kGy β-irradiation. Additional data were obtained for a variety of different compositions of the protecting solution as shown in table 1. Furthermore, irradiation with 40 kGy β-irradiation was carried out as an alternative approach.

(13) After irradiation and reconstitution, the functionality of the samples was evaluated in the hemagglutination assay (HA). As shown in FIG. 2, in contrast to the control, which showed a complete loss of antigenicity of influenza A in storage buffer, the hemagglutination activity was almost fully maintained after reconstitution of influenza A lyophilized in the inventive solution.

(14) Table 1 provides an overview over the different compositions of the protecting solution of the present invention used in the example. Compositions with seven amino acids (amino acid mixture 1; Ala, Arg, Glu, Gly, Lys, His, Trp) or with five amino acids (amino acid mixture 2; Ala, Arg, Glu, Gly, Lys and amino acid mixture 3: Ala, Gly, Glu, His, Trp, respectively) were used. Compositions containing the dipeptide carnosine, glycyrrhicic acid, additional dipeptides, and combinations thereof, resulted in the best protection against lyophilisation-mediated and/or irradiation-mediated loss of functionality. The experiments revealed that the exclusion of amino acids that may have unappreciated side effects upon prolonged storage (e.g. oxidation sensitive; hygroscopic) resulted in reduced protection. When GA and/or carnosine, and/or dipeptides (Gly-Tyr; Gly-GLy; Gly-Gln) were supplemented to these compositions, the protecting effects of the composition could be increased. After the substitution of selected amino acids with dipeptides and/or GA the osmolarity of the composition is lower and may therefore be beneficial for therapeutical purposes.

(15) TABLE-US-00001 TABLE 1 Compositions of the applied formulations 1 to 18 Amino acids Supplements Formulation 1 Ala, Arg, Gly, Glu, Lys, His, Trp Formulation 2 Ala, Gly, Glu, His, Trp Formulation 3 Ala, Arg, Gly, Glu, Carnosin, N-acetyl-Trp Lys Formulation 4 Ala, Arg, Gly, Glu, Carnosin, glycyrrhizic acid Lys, Trp Formulation 5 Ala, Arg, Gly, Glu, N-acetyl-Trp, β-alanine Lys, His Formulation 6 Ala, Arg, Gly, Glu, Carnosin, Gly-Tyr, N-acetyl-Trp, Lys N-acetyl-His Formulation 7 Ala, Arg, Gly, Glu, Carnosin, Gly-Tyr, Gly-Gly, Gly- Lys Gln Formulation 8 Ala, Arg, Gly, Glu, Gly-Tyr, Gly-Gly, Gly-Gln Lys Formulation 9 Ala, Arg, Gly, Glu, N-acetyl-Trp, N-acetyl-His, Lys β-alanine, glycyrrhizic acid Formulation 10 Ala, Arg, Gly, Glu, Carnosin, Gly-Tyr, glycyrrhizic Lys acid Formulation 11 Ala, Arg, Gly, Glu, Gly-Tyr, Gly-Gly, glycyrrhizic Lys acid Formulation 12 Ala, Arg, Gly, Glu, Gly-Gly, Gly-Gln, glycyrrhizic Lys acid Formulation 13 Ala, Arg, Gly, Glu, Carnosin, Gly-Tyr, N-acetyl-Trp, Lys N-acetyl-His, glycyrrhizic acid Formulation 14 Ala, Arg, Gly, Glu, Carnosin, Gly-Gly, N-acetyl-Trp, Lys N-acetyl-His, glycyrrhizic acid Formulation 15 Ala, Arg, Gly, Glu, Carnosin, Gly-Gln, N-acetyl-Trp, Lys N-acetyl-His, glycyrrhizic acid Formulation 16 Ala, Arg, Gly, Glu, Carnosin, Gly-Tyr, Gly-Gly, Lys Gly-Gln, glycyrrhizic acid Formulation 17 Ala, Arg, Gly, Glu, Carnosin, N-acetyl-Trp, Gly-Tyr, Lys Gly-Gly, Gly-Gln, glycyrrhizic acid Formulation 18 Ala, Arg, Gly, Glu, Gly-Tyr, Gly-Gly, Gly-Gln, Lys glycyrrhizic acid

EXAMPLE 3

Virus Irradiation and Maintenance of Antigenicity

(16) Virus inactivation studies were performed using human adenovirus type 5 (Ad5). Specifically, 50 μL of virus suspension were dried at 37° C. on the bottom of sterile polystyrol tubes. The dried virus was then overlaid with 50 μL of the protecting solution and dried again at 37° C. After β-irradiation at 25 kGy or 40 kGy (controls were not irradiated), the virus/protective solution bilayer was resuspended in 1 mL of MEM, and the titer of infectious virus was determined by means of end-point titration (FIG. 3A/B). In parallel, the same experimental setup was employed with IgM (LO-MM-3; FIG. 3C/D).

(17) We show that β-irradiation led to quantitative inactivation of Ad5 (25 kGy,≥99.9% reduction, 40 kGy ≥99.999% reduction; FIG. 3 A/B) while the functionality of an IgM antibody was maintained. These results demonstrate that the inventive solution selectively stabilizes and protects protein structures outside the virus while at the same time allowing inactivation of the infectivity.

EXAMPLE 4

Failure of Protecting Solution to Stabilize Living Eukaryotic Cells During Freezing and Thawing

(18) Cultured fibroblasts were harvested after reaching confluency in culture plates. Cells were counted and reconstituted with a) DMSO as a standard procedure or b) in protecting solution or c) in different mixtures of DMSO and protecting solution (SPS®) before freezing and storing in −80° C. After thawing, the viability of cells was analyzed by trypan blue and the ability to form new cultures in vitro. The results are shown in table 2 below.

(19) The samples were reconstituted in (all in DMEM/20% FKS (v/v)): 1 10% DMSO (v/v)/0 (v/v) SPS (20 g/l)=negative control 2 7.5% DMSO (v/v)/2.5% (v/v) SPS (20 g/l) 3 5% DMSO (v/v)/5% (v/v) SPS (20 g/l) 4 2.5% DMSO (v/v)/7.5% (v/v) SPS (20 g/l) 5 0% DMSO (v/v)/10% (v/v) SPS (20 g/l) 6 0 DMSO (v/v)/0% (v/v) SPS (20 g/l)=positive control
Protecting solution with 20% FCS: 7 SPS 10 mg/ml+20% FKS/10% DMSO 8 SPS 10 mg/ml+20% FKS/0% DMSO

(20) TABLE-US-00002 TABLE 2 Lack of stabilization of eurkayotic cells in the inventive solution Total Recovery in % Cells/ml Cells/ml number of Vitality (relative to amount vital dead live cells in % of cells frozen) 1 1.6 × 10.sup.5 — 8.0 × 10.sup.5 100 85.6 2 2.2 × 10.sup.5 0.2 × 10.sup.5 11.0 × 10.sup.5  91.7 100 3 0.8 × 10.sup.5 0.2 × 10.sup.5 4.0 × 10.sup.5 80 42.3 4 0.4 × 10.sup.5 0.4 × 10.sup.5 2.0 × 10.sup.5 50 21.4 5 — 0.6 × 10.sup.5 — 0 — 6 — 1.2 × 10.sup.5 — 0 — 7 0.6 × 10.sup.5 — 3.0 × 10.sup.5 100 32.1 8 — 0.8 × 10.sup.5 — 0 —

(21) It was further tested how cells attached to the culture dish 24 hours after thawing. The level of attachment was determined microscopically. The results are summarized in table 3 below.

(22) TABLE-US-00003 TABLE 3 Attachment to cell culture dishes 24 hours after thawing Attachment 1 cells attach well, only a few individual loose cells 2 cells attach well, only a few individual loose cells 3 cells attach well, some individual loose cells or cell fragments 4 cells attach well, some individual loose cells or cell fragments 5 only loose cells or cell fragments 6 only loose cells or cell fragments 7 cells attach well but have a slightly round shape, only a few individual loose cells 8 only loose cells or cell fragments

EXAMPLE 5

Material and Methods

Preparation of the Protecting Solution

(23) The protecting solution employed in the examples above contains L-alanine, L-arginine, L-glutamic acid, glycine, L-histidine, L-lysine monohydrochloride and L-tryptophan.

(24) The protective solution was prepared by specifically combining the different amino acids according to the present invention and, optionally, glycosidic excipients (here, glycyrrhizic acid) to reach a stock concentration of about 100 g/L. The weight:weight (w/w) ratio of the solid content of final solution (1-25 g/L) to the agents to be protected was >2:1. All components were non-toxic. Amino acids are approved for intravenous infusion (Fong and Grimley), Glycyrrhizic acid has been approved for intravenous application in the treatment of chronic hepatitis, and its safety has been well documented in several clinical studies. In the inventive solution, glycyrrhizic acid can be used in a range between 1-10000 μg/mL.

(25) Virus Infectivity Assay

(26) For the infectivity/inactivation study, adenovirus type 5, strain Adenoid 75 (American Type culture collection, ATCC-VR-5) was propagated on human lung cancer cell line A549 (ATTC-CCL-185). For virus propagation, cells were grown at 37° C. and 5% CO2 in minimum essential medium (MEM) supplemented with 5% fetal calf serum (FCS). The virus titer was determined by means of end-point titration (eight wells per dilution in a 96-well microtiter plate), with 50 μL virus dilution and 50 μL A549 cells (10-15×10.sup.3 cells) per well. For the experiments, a titer of 1.1×10.sup.9 tissue culture infectious dose (TCID50)/mL was used. Specifically, a volume of 50 μL virus suspension was dried at 37° C. on the bottom of sterile polystyrol tubes. The dried virus was then overlaid by 50 μL of the protective solution (20 g/l) and dried again at 37° C. After β-irradiation at 25 kGy or 40 kGy (controls were not irradiated), the virus/protective solution bilayer was resuspended in 1 mL MEM. The virus titer was determined again as described above. The cultures were observed for cytopathic effects (CPE) after 7 days of inoculation.

(27) Virus controls were treated identically but without β-irradiation. The virus titers are expressed as TCID50/mL including standard deviation. Titer reduction is expressed as the difference between the virus titer after β-irradiation and control virus titer.

(28) Statistics

(29) All experiments were done at least five times. Where relevant, data are presented as mean+/− SEM. Statistical analyses were performed by Student's t-Test (GraphPad Prism Program, version 5, GraphPad Software, San Diego, Calif.). P values of less than 0.05 were considered statistically significant.

EXAMPLE 6

Stabilization of Ovalbumin During Spray Drying

(30) Ovalbumin, a model antigen commonly employed in immunization, was used as a proof-of-principle to confirm the usefulness of the inventive solution for the stabilization of antigens during spray drying. The dried powder was then characterized for integrity using Circular dichroism (CD), Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence spectroscopy.

(31) Materials and Methods

(32) Ovalbumin was purchased from Sigma (St. Louis, Mo., USA). The inventive solution was as described above (see embedding solution). 1-Anilino-8-naphthalene sulfonate (ANS) was also purchased from Sigma (St. Louis, Mo., USA). All other reagents were of analytical grades.

(33) Preparation of Spray-Dried Protein

(34) 3% (w/v) of Ovalbumin along with 6% (w/v) of the inventive solution were spray dried using a Büchi B-290 laboratory spray dryer (Büchi, Flawil, Switzerland). Inlet air temperature was set to 120° C., the air flow was 470 L/h, the pump rate was 7.5 ml/min and the outlet air temperature was between 50-55° C.

(35) SDS-PAGE

(36) SDS-PAGE was carried out using a Bio-Rad MINI-PROTEAN® 3 gel electrophoresis system (Bio-Rad Laboratories, Hercules, USA). 10 μl of protein samples (0.5 mg/ml) was loaded into each well after incubating (95° C., 5 min) with 10 μl of sample buffer and subjected to electrophoresis at voltage of 200 V for about 55 min. The proteins were visualised with Coomassie blue staining.

(37) Circular Dichroism (CD)

(38) Jasco J-715 spectropolarimeter (JASCO International Co. Ltd, Hachioji city, Japan) was used to assess the secondary structure of proteins after spray drying. CD spectra were recorded in far UV range (200 to 250 nm) region with a sampling interval of 1.0 nm in a 0.05 cm path length cuvette. The solution of the invention was separated from the protein using an ultra-filtration device (VIVASPIN® 6 from Sartorius Stedim biotech, Gottingen, Germany) since it has some signal in CD. All spectra are average of two scans and were background corrected and normalised. Spectra were then compared to that of pure/unprocessed protein.

(39) Fluorescence Spectroscopy

(40) The fluorescence spectra were recorded in a fluorescence spectrometer (LS55 from PerkinElmer, Waltham, USA) using a 1 cm path length cuvette. Intrinsic fluorescence of ANS was measured by exciting it at 360 nm and the emission spectrum was recorded in 390-550 nm range. Fluorescence spectra were corrected for the background spectrum of solvent.

(41) Results

(42) SDS-PAGE analysis of OVA control and spray dried OVA with the inventive solution is shown in FIG. 4(A). The same gel pattern is observed in both samples and no low molecular weight bands are present, indicating that no degradation of the OVA molecule has taken place upon spray drying.

(43) In addition, as shown in FIG. 4(B), the CD spectra of OVA after spray drying with the inventive solution resembles the OVA control indicating that no change in secondary structure can be observed after re-dispersion.

(44) 1-Anilino-8-naphthalene sulfonate (ANS) is practically non-fluorescent in water, but shows fluorescence upon binding to hydrophobic sites that exists on proteins. Accordingly, this fluorescence is greatly increased when the protein is denatured and thus serves as a measure of the degree of denaturation (FIG. 5). This phenomenon was used to characterise the integrity of OVA after spray drying. As seen in FIG. 6, the control and spray dried OVA with the inventive solution possess the same fluorescence intensity indicating that no denaturation event had taken place during spray drying.

(45) In conclusion, the inventive solution is suitable for preserving the secondary structure during spray drying of Ovalbumin.