METHOD FOR OBTAINING EFFICIENT COMPOSITIONS COMPRISING VIRAL VECTORS FOR VACCINATION OR GENE THERAPY

Abstract

The present invention relates to a method for preparing a composition comprising a viral vector, the method comprising the steps of a) providing viral vectors, (b) providing a solution comprising at least one sugar and at least three different excipients selected from hydrophilic and amphiphilic excipients, wherein the excipients are characterized by polar, aliphatic, aromatic, negatively charged, and/or positively charged functional groups, and wherein the solution is free or substantially free of Mg2+ or of any divalent cations and/or salts thereof; and (c) mixing the replication deficient viral vectors of step (a) with the solution of step (b). Furthermore, the invention relates to a composition obtained or obtainable by the method of the invention, and to a composition comprising a viral vector and the solution of step (b).

Claims

1. A method for preparing a composition comprising a viral vector, the method comprising the steps: (a) providing viral vectors; (b) providing a solution comprising at least one sugar and at least three different excipients selected from hydrophilic and amphiphilic excipients, wherein the excipients are characterized by polar, aliphatic, aromatic, negatively charged, and/or positively charged functional groups, and wherein preferably the at least three different excipients comprise or are amino acids; and wherein the solution is free or substantially free of Mg.sup.2+ and/or salts thereof; (c) mixing the viral vectors of step (a) with the solution of step (b).

2. The method of claim 1, wherein the solution is free or substantially free of Ca.sup.2+, Mn.sup.2+, Cu.sup.2+, Zn.sup.2+, and or Ni.sup.2+ and/or salts thereof, or wherein the solution is free or substantially free of any divalent cations.

3. The method of claim 1 or 2, wherein the at least three amino acids, at least provide one anti-oxidative functional group and at least one osmolytic function and at least one buffering function and at least one charged functional group.

4. The method of any of the preceding claims further comprising the step (d) of storing the composition obtained by mixing the viral vectors of step (a) with the solution of step (b) in liquid state.

5. The method of claim 4, wherein the composition is stored in liquid state in step (d) for at least 30 days, more preferably at least 6 months, even more preferably 9 months and most preferably at least 12 months.

6. The method of claims 4 and 5, wherein the composition is stored in liquid state in step (d) at a temperature between 4° and 30°, preferably between 20° C. and 27° C.

7. The method of claims 4 to 6, wherein the composition storage in liquid state in step (d) for 6 months at 25° C. leads to a loss of infectivity titer of no more than between 1.5 and no more than 2 log levels.

8. The method of claims 4 to 6, wherein the composition storage in liquid state in step (d) for over 12 months at 25° C. in step (d) of the method of the invention, the loss in infectivity titer of the viral vector comprised in the composition may be no more than 3 log levels.

9. The method of any of the preceding claims, wherein the viral vector is selected from the group consisting of adenovirus, Adenovirus-associated virus (AAV), lentivirus, vesicular stomatitis virus (VSV), MVA, or herpesviruses.

10. The method of any of the preceding claims, wherein the viral vector is a virus like particle.

11. The method of any of the preceding claims, wherein the viral vectors of (a) are viral vectors that have been reconstituted immediately after harvesting from cell cultures and purification.

12. The method of any of the preceding claims, wherein the viral vector-based particles present in the composition have a particle size distribution with a polydispersity index (PDI) of less than 0.5.

13. A composition obtained or obtainable by the method according to any of the preceding claims.

14. A composition comprising a viral vector and a solution according to claim 1.

15. The composition according to claim 13 or 14, wherein the composition is characterized by a loss of infectivity titer of no more than between 1.5 and 2 log levels upon storage for 6 months at 25° C. and/or by a loss of infectivity titer of no more than 3 log levels upon storage of the composition over 12 months at 25° C.

Description

[0164] The invention is illustrated with the following figures which show:

[0165] FIG. 1: (A) shows the infectivity of Ad5 in liquid formulations (round 1) after accelerated aging at 37° C.; (B) to (D) depict the linear influence on Ad5 stability (negative or positive) of single amino acids (AA) after short and long term storage.

[0166] FIG. 2: (A) shows the infectivity of Ad5 in the best performing formulations after storage for up to 35 days at 37° C., (B) shows the infectivity of Ad5 in the worst performing after storage for up to 35 days at 37° C., (C) Best performing formulations are shown as time kinetics for up to 6 days at 25° C., (D) shows the infectivity of Ad5 in the best performing formulations after storage for up to 24 months at 5° C. The values show the mean±SD generated of at least 5 measurements per infected well.

[0167] FIG. 3: (A) shows the infectivity of Ad5 in liquid formulations (round 2) after different storage for up to 28 days at 37° C., (B) shows the infectivity of Ad5 in liquid formulations (round 2) after different storage for up to 12 days at 25° C., (C) shows the infectivity of Ad5 in liquid formulations (round 2) after different storage for up to 24 days at 5° C. The values show the mean±SD generated of at least 15 measurements (5 measurements from 3 biological replicates) per infected well.

[0168] The examples illustrate the invention:

EXAMPLE 1: INITIAL EXCIPIENT SELECTION AND LIQUID STORAGE

1.1 Materials and Methods

1.1.1 Pre-Selected Formulations

[0169] For initial excipient selection, the formulation matrix, tailored for the Ad5 viral vectors, was composed of 40 formulations containing eight different amino acid combinations selected from Arg, Ala, Gly, Lys, His, Glu and Met in a concentration between 0.75 g/l and 31,235 g/l was used all formulation comprised 40 mg/ml saccharose, 2 mM MgCl.sub.2 at a pH of 7.4. For comparison, original suppliers formulation (OF) 1,552 g/l Histidin, 50 g/l saccharose, 1 mM MgCl.sub.2 at a pH of 7.4 and a positive control formulation comprising 2 mM MgCl.sub.2 60 g/l trehalose*2H2O, 0.05 g/l polysorbat 80 at a pH of 8 was used. An adenoviral stock solution (Ad5-CMV-EGFP: E1/E3-deleted human adenovirus, Serotype 5) stored at −80° C. with a concentration of 7.5×1010 IFU/ml in the original supplier formulation (OF), which was designed for frozen storage (Sirion Biotech GmbH; Germany) was employed.

1.1.2 Sample Preparation and Storage

[0170] Ad5 was diluted to 1×10.sup.8 IU/ml in original formulation (Sirion Biotech; Germany), or different amino acid based comprising different combinations and amounts of the eight different pre-selected amino acids in combination with sucrose, MgCl.sub.2 according to 1.1.1.

[0171] The resulting Ad5 formulations were initially stored under short-term stress conditions at 37° C. for up to 35 days. In order to evaluate the predictive capability of the applied approach using a short-term storage model at 37° C. for liquid storage under real time conditions these samples were additionally stored for up to 6 months at 25° C. and for up to 24 months at 5° C. Infectivity of the Ad5 viruses was analyzed at day 0 and at different time point as indicated in FIGS. 1 and 2.

1.1.3 Infectivity Assay

[0172] In order to analyze the infective titer of the adenoviral vector formulations, antibody-based virus titration assays in adherent HEK 293 cell cultures were conducted. Antibody-mediated immunostaining of the adenoviral hexon protein was applied after successful amplification of the adenovirus in the infected cells. Therefore, 2.5×10.sup.5 HEK 293 cells in 500 μl per well were seeded in a 24-well plate and further used when cells started attaching to the surface (after 2-3 hours). Serial dilutions of the adenoviral samples were prepared and 50 μl of the resulting dilutions per well were used for infection of the cells. As for positive controls, aliquots of Ad5 in the original supplier formulation (Sirion Biotech, Germany) stored at −80° C. with a concentration of 1×10.sup.8 IU/ml were used. Cells were inoculated for 42±2 hours at 37° C., 5% CO.sub.2 and subsequently fixed with methanol (Carl Roth GmbH & Co. KG; Germany). Immunostaining was done stepwise by incubation with the primary anti-Hexon protein antibody (Santa Cruz Biotechnology, Inc.; USA), the secondary horse radish peroxidase (HRP)-conjugated anti-mouse antibody (Cell Signaling Technology; USA) and an HRP enzymatic reaction with diaminobenzidine (Carl Roth GmbH & Co. KG; Germany). The number of infected cells was quantified by counting the stained (brown colored) cells under the light microscope. Each stained cell was considered as one infectious viral particle in order to calculate the Infective Units per milliliter (IU/ml) according to the standardized calculation procedure. The scope of detection allows titer determination between 9.87×10.sup.4 IU/ml and 2.04×10.sup.8 IU/ml.

1.1.4 Data Analysis

[0173] Data obtained for each time point during storage as provided in the Figures were analysed by DoE-based linear regression (R-Software; F-Statistics). For each amino acid F-values between −1, 0 and +1 indicate the linear influence of single amino acids on Ad5 stability and functionality calculated as Infective Unit per mL [IFU/ml] from hexon immunostaining. All experiments were done at least in triplicates and data are depicted as Mean±SD, except when indicated otherwise. Effects were considered statistically significant at p<0.1 (.), p<0.05 (*), p<0.01 (**), p<0.001 (***), respectively.

1.2 Results

[0174] After formulation of the original Ad5 viral vector preparation in these 40 formulations and in the original supplier formulation as a control, liquid storage under accelerated aging conditions at 37° C. was performed. Based on the first order regression of the infectivity results (FIG. 1A), the linear influences of each single amino acid used in the DoE calculation of the 40 formulations on Ad5 infectivity during liquid storage at 37° C. were evaluated (FIG. 1B). Either a positive, neutral or negative linear influence was determined for each single amino acid. The anti-oxidative effective amino acid methionine (AA8) demonstrated a significant (p<0.01) positive influence on the Ad5 stability after 14 days as well as 21 days short term liquid storage at 37° C. (FIG. 1B). The osmolytic amino acids alanine (AA2) and glutamine (AA7) also revealed a minor positive effect on Ad5 stability during liquid storage up to 21 days at 37° C. In contrast, the radical scavenging amino acid tryptophan (AA6) elicited a significant (p<0.01) negative influence up to 21 days short term liquid storage at 37° C. (FIG. 1B).

[0175] The identified linear effects of single amino acids were in line with formulations comprising these amino acids as determined in liquid storage experiments (37° C.) up to 21 days. The most effective stabilizing formulations (F1_3, F1_4, F1_10, F1_13, F1_16, F1_29, F1_39) partially retained Ad5 infectivity upon liquid storage at 37° C. for up to 21 days (see FIG. 2A) with a titer loss of approx. 1 to 2 log levels. In contrast, when the original supplier formulation was used, Ad5 infectivity was already lost after 14 days storage at 37° C. The composition of the

TABLE-US-00002 TABLE 2 Composition of most effective stabilizing formulations L-Lysin MgCl2 mono- *6H20 Formulation Ala Gly HCl His Glu Met Saccharose (2 mM ) pH No. g/l F1_3 3 10 0.750 40 0.407 7.4 F1_4 24.988 3 1.500 40 0.407 7.4 F1_10 10 10 3 2 1.500 40 0.407 7.4 F1_13 20 3 4 40 0.407 7.4 F1_16 20 3 1.500 40 0.407 7.4 F1_29 10 31.235 3 1.500 40 0.407 7.4 F1_39 15 3 0.750 40 0.407 7.4

[0176] After liquid storage for more than one month (5 weeks) at 37° C., only Ad5 in formulation F1_29 remained active with a titer loss of approx. 3 log levels while no infectivity (limit of detection (LOD) reached) could be found with all other formulations. All stabilizing formulations contained either 2 or 3 selected amino acids with positive linear influence (FIG. 1) and tryptophan (AA6) which was shown earlier to elicit significant negative influence on Ad5 stability.

[0177] In contrast to the best stabilizing formulations such as F1_29, the weakest stabilizing formulations only maintained Ad5 infectivity up to 14 days storage at 37° C. (see FIG. 2B). In line with the results of the linear regression analysis of the DoE-based infectivity data these weakly stabilizing formulations contained amino acid tryptophan (AA6). The best performing formulations did not comprise tryptophan (AA6).

[0178] Based on these results, the most effective stabilizing excipients methionine (AA8), alanine (AA2) and glutamine (AA7) as well as the elimination of tryptophan (AA6) were considered for long-term storage experiments and further iterative optimization of the formulations.

[0179] The linear regression of the DoE based Ad5 infectivity results analyzed at indicated time points, according to guideline ICH Q1, during liquid storage at 25° C. (up to 12 months) and 5° C. (up to 24 months) revealed similar influences of single amino acids on the Ad5 stability during storage at both temperatures compared to liquid storage at 37° C.). For example, similar to the results regarding the linear influences of the single amino acids during short-term storage of Ad5 at 37° C. (FIG. 1B), the amino acid methionine (AA8) also significantly stabilized Ad5 during liquid storage at 25° C. (FIG. 10) and at 5° C. (FIG. 1D). In contrast, amino acid tryptophan (AA6) elicited significant negative effects on Ad5 stability during liquid storage at all temperatures (FIGS. 1B-D).

[0180] Results of the seven best-of stabilizing at 37 C liquid formulations (F1_3, F1_4, F1_10, F1_13, F1_16, F1_29, F1_39) are shown in FIG. 2A. FIG. 2C depicts the results obtained at indicated time points during liquid storage for up to 6 months at 25° C. These formulations are similar to the best-of formulations during liquid storage under accelerated aging conditions at 37° C. The loss of only maximal 1 log level of the virus infectivity was observed after 3 months and 1-2 log after 6 months storage at 25° C. (FIG. 2C). In contrast, during storage for 3 months the Ad5 virus formulated in the original supplier formulation (OF) completely lost infectivity.

[0181] As shown in FIG. 2D, the stabilizing effects of the seven best-of stabilizing formulations (F1_3, F1_4, F1_10, F1_13, F1_16, F1_29, F1_39) and their impact on Ad5 infectivity during liquid storage at 5° C. are shown over time up to 24 months. A dramatic loss of infectivity was found for Ad5 in OF already after three months storage at 5° C. (FIG. 2D). Accordingly, after 24 months storage at 5 C a complete loss of the Ad5 infectivity in the OF was observed. All stabilizing formulations almost completely maintained the Ad5 viral infectivity during long-term storage at 5° C. for up to 24 months. These results are in line with the calculation of the linear influences of single amino acids on the Ad5 stability. For example, the formulations F1_3, F1_4, F1_10, F1_13, F1_16, F1_29, F1_ 39 lack acid tryptophan (AA6) that was shown to have negative linear effects at 37° C. (see FIG. 1). Formulations without methionine (AA8) (e.g. F1_14, F1_15, F1_25, F1_26, F1_30, F1_31, F1_33) resulted in a total loss after 24 months at 5° C. or to a loss of infectivity up to 1 to 2 log levels during liquid storage for 24 months at 5° C. (FIG. 2D). During long-term liquid storage for up to 6 months at 25° C. the same formulations showed only minor stabilizing effects on functional integrity of the viral vector.

[0182] Interestingly, the most effective stabilizing formulations (F1_20, F1_22, F1_34, F1_36) after 24 months liquid storage at 5° C. all contained amino acid tryptophan, except formulation 34, but also contained methionine (AA8), suggesting a partially masking of the negative influence of acid tryptophan (AA6) by the stabilizing effect of methionine (AA8) during long term liquid storage. In contrast, during liquid storage under accelerated aging conditions the negative influence of acid tryptophan (AA6) is more pronounced also in combination with methionine (AA8).

[0183] The DoE-based overall evaluated linear influences of single amino acids on the Ad5 stability during liquid storage were found likely to be superimposed by the negative or positive effects of specific excipients in the particular formulations during liquid storage at different temperatures. For example, poor stabilizing formulations (F1_5, F1_7, F1_17, F1_22, F1_25, F1_28, F1_31, F1_34; see FIG. 1A) containing high amounts of AA1 were associated with negative effects on Ad5 stability during short-term storage at 37° C. However, as shown in FIG. 1, the evaluation of the linear influence of single amino acids revealed a neutral influence of AA1. The rather poor stabilizing effects of formulations 22 and 34 during short term storage at 37° C. associated with AA1 may be overcome by the stabilizing effects of methionine (AA8) during long-term storage. Moreover, the previously evaluated significant negative influence of the aromatic amino acid tryptophan (AA6) reflected in the poor stabilizing effects of formulations F1_14, F1_15, F1_25, F1_30, F1_31, F1_33 was completely abolished during long-term storage in formulations F1_20, F1_22 and F1_36 that all contained AA6 in combination with tryptophan (AA8).

EXAMPLE 2: OPTIMIZATION OF THE STABILIZING AD5 FORMULATIONS

2.1 Materials and Methods

[0184] Based on formulations F1-16 and F1-29 the formulations were modified as shown in Table 3.

TABLE-US-00003 TABLE 3 Modified formulations L- MgCl2 Lysin *6H20 mono- (2 mM Formulation Arg Ala Gly HCl His Glu Met Saccharose Mannitol fix) pH No g/l F1_29 F2_1 10 31.235 3 0 1.500 40 0.407 7.4 F2_2 8.426 31.235 3 0 1.500 40 0.407 7.4 F2_3 29.804 10 3 0 1.500 40 0.407 7.4 F2_4 10 31.235 3 0 1.500 40 7.4 F2_5 10 31.235 3 0 1.500 21.290 0.407 7.4 F2_6 10 31.235 3 4 1.500 40 0.407 7.4 F1_13 F2_7 20 3 4 40 0.407 7.4 F2_8 20 3 4 40 7.4 F2_9 20 3 4 1.500 40 0.407 7.4

[0185] The modified formulations were stored for 37° C. for up to 28 days, 25° C. for up to 12 months and 5° C. for up to 24 months. Samples were analysed as described for Example 1 at the time points indicated in FIG. 3.

2.2 Results

[0186] The identified most effective stabilizing amino acids methionine (AA8), alanine (AA2) and glutamine (AA7) and two of the most effective stabilizing amino acid compositions (FIG. 3A; formulations F1_13 and F1_29) of the first round were the basis for the second stabilization round for the Ad5 vector. Although formulation F1_13 did not contain methionine (AA8), this formulation was one of the most effective Ad5 stabilizing formulations in the DoE round (FIG. 3A). It was assumed that formulations containing high amounts of the osmolytic amino acid alanine (AA2) and intermediate amounts of the osmolytic acidic amino acid glutamine (AA7) as well as sucrose and MgCl.sub.2 may overcome the lack of the anti-oxidative amino acid methionine (AA8). Formulation F1_13 and F1_29 were modified accordingly. Modified formulations are shown in Table 3.

[0187] Surprisingly, in both of the modified initial formulations, omission of MgCl.sub.2 resulted in an increased stability under long term storing conditions at 25° C. as shown in FIG. 3B (F2_1 vs F2_4 and F2_7 vs F2_8.

[0188] In comparison to long term storing conditions at 25° C. (FIG. 38) the effect of the omission of MgCl.sub.2 was less pronounced at short term storage at 37° C. (FIG. 3A) or storage at 5° C. (FIG. 3A).

[0189] Liquid storage for 6 months at 25° C. led to a loss of titer only between 1.5 and 2 log levels of the Ad5 infectivity for most formulations. Ad5 viral vector formulated in the formulations F2_1; F2_2; F2_4; F2_5; F2_6; F2_8 and F2_9 (see; see Table 3) retained the infective titer even after 9 months storage at 25° C. The best stabilizing effect were observed with the formulation F2_4 w/o MgCl.sub.2, which performed best with a titer of 1.23×10.sup.5 IFU/ml after 12 months at 25° C. (FIG. 38).

[0190] These results suggest that in contrast to the teachings of the prior art, which rely on the presence of MgCl.sub.2 for stabilizing viral vectors, stabilization of viral vectors may be improved in solutions which are free or substantially free of Mg.sup.2+ and/or salts thereof, and preferably free or substantially free of divalent cations.