Methods and compositions for stabilizing dried biological materials

Abstract

The present invention relates to methods for producing dried formulations of biopharmaceutical agents that aim to minimize the loss of activity of the agents upon drying and to provide dried formulations with an extended shelf life. The method comprises the step of drying an aqueous solution comprising, in addition to the biopharmaceutical agent, at least an amino acid, a polyol and a metal salt. Preferably the amino acid is glutamate, the polyol is sorbitol and optionally also mannitol and the metal salt is a magnesium salt. The solution is dried by vacuum drying or by lyophilization. The methods are particularly useful for preparing dried formulations of viruses such as poliovirus or respiratory syncytial virus to be used for vaccination. The invention also relates to dried formulations prepared in accordance with the methods of the invention and to their use as medicaments, e.g. as vaccines.

Claims

1. A method for producing a formulation of a biopharmaceutical agent, comprising drying a solution comprising: (a) a biopharmaceutical agent comprising poliovirus, (b) an amino acid selected from the group consisting of glutamate, arginine, histidine, glycine and mixtures thereof, (c) a polyol comprising sorbitol and/or mannitol, and (d) at least 0.2% (w/v) of a metal salt and water, wherein the metal salt is Mg.sup.2+, Ca.sup.2+, Li.sup.+ or a mixture thereof.

2. The method according to claim 1, wherein the solution consists essentially of 1 pg-10 g per ml of the biopharmaceutical agent, 0.01-20% (w/v) of the amino acid, 0.5-20% (w/v) of the polyol, 0.2-10% (w/v) of the metal salt and water.

3. The method according to claim 1, wherein the glutamate is dissolved in the solution in the form of monosodium glutamate, and/or wherein the arginine is in the form of poly-L-arginine.

4. The method according to claim 3, wherein the solution consists essentially of 1 pg-10 g per ml of the biopharmaceutical agent, 5-20% (w/v) sorbitol, 5-20% (w/v) monosodium glutamate, 2-10% (w/v) of a magnesium salt, and optionally 5-20% (w/v) mannitol.

5. The method according to claim 1, wherein the solution comprises a pharmaceutically acceptable buffer and is buffered at a neutral pH.

6. The method according to claim 1, wherein the drying is by air drying, vacuum drying, spray drying or by lyophilization.

7. The method according to claim 1, wherein the poliovirus is one or more of poliovirus serotypes 1, 2 or 3.

8. The method according to claim 1, wherein the poliovirus is inactivated.

9. The method according to claim 1, wherein the formulation, upon reconstitution in a liquid, retains at least 50% of the activity of the biopharmaceutical agent present in the solution prior to drying.

10. The method according to claim 9, wherein the formulation comprises at least two different poliovirus serotypes, and wherein the difference in loss of activities for the different agents is less than 50%, whereby the retained activity of the agent with the most loss in activity is expressed as percent of the retained activity of the agent with the least loss, which is set at 100%.

11. The method according to claim 1, wherein the formulation upon reconstitution after storage for at least one week at 45° C., retains at least 50% of the activity of the biopharmaceutical agent present in the solution prior to drying.

12. The method according to claim 11, wherein the formulation comprises at least two different poliovirus serotypes, and wherein the difference in loss of activities for the different agents is less than 50%, whereby the retained activity of the agent with the most loss in activity is expressed as percent of the retained activity of the agent with the least loss, which is set at 100%.

13. The method according to claim 4, wherein the magnesium salt is MgCl.sub.2 and/or MgSO.sub.4.

14. A method for producing a formulation of a biopharmaceutical agent, comprising drying a solution comprising poliovirus, glutamate, sorbitol, and at least 0.2% (w/v) of Mg.sup.2+ metal salt and water.

15. The method according to claim 14, wherein the drying is by lyophilisation.

16. The method according to claim 15, wherein the drying is by vacuum drying.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1 D-Antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations (experiment A). The stabilizing effect of sucrose (SUC), trehalose (TREH), mannitol (MAN), dextran (DEX) and sodium chloride (NaCl) was tested.

(2) FIG. 2 Response surface plots representing D-antigen recovery percentages of the three serotypes directly after lyophilization of IPV-formulations containing 10% of mannitol and 0% dextran.

(3) FIG. 3 D-Antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations (experiment B). The stabilizing effect of sucrose (SUC), trehalose (TREH), mono-sodium glutamate (MSG), hydroxyethyl starch (HES) and sodium chloride was tested.

(4) FIG. 4 D-Antigen recovery directly after vacuum drying (V), lyophilization with a slow freezing rate (L) and lyophilization with a fast freezing rate (S). The stabilizing effect of sucrose and trehalose, compared to IPV vaccine without addition of stabilizers (negative control) was investigated.

(5) FIG. 5 D-antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations as indicated (experiment C).

(6) FIG. 6 D-antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations (experiment D). All formulations contain 5% sorbitol and 125 mM NaCl and, excluding D11-D16, 5% peptone and are further as indicated.

(7) FIG. 7 Accelerated stability testing of lyophilized serotype 1, 2 and 3 IPV-formulations as indicated (experiment D) as determined by D-antigen recovery after one week incubation at 45° C.

(8) FIG. 8 D-antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations as indicated (experiment E). The stabilizing effects of sorbitol (SOR), sucrose (SUC), mannitol (MAN), mono-sodium glutamate (MSG), peptone (PEP) and MgCl.sub.2 were tested by using fast (panel A) and slow (panel B) freezing rates.

(9) FIG. 9 Stability of lyophilized serotype 1, 2 and 3 IPV-formulations as indicated (experiment E) after one week incubation at 45° C. and using fast (panel A) and slow (panel B) freezing rates. Only the most promising formulations directly after lyophilization are shown.

(10) FIG. 10 D-antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations (experiment F, n=3). The stabilizing effect of peptone was investigated in an IPV-formulation containing 10% sorbitol, 10% MSG and 5% MgCl.sub.2 with or without 10% mannitol. All formulations were fast frozen prior to the drying phase of the lyophilization process.

(11) FIG. 11 Stability of lyophilized serotype 1, 2 and 3 IPV-formulations (experiment F, n=3) after one week incubation at 45° C. This experiment investigates whether it is possible to improve the IPV-formulation containing 10% sorbitol, 10% MSG and 5% MgCl.sub.2 with or without 10% mannitol during accelerated stability with the addition of peptone or single amino acids.

(12) FIG. 12 D-antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations with different buffer components as indicated (experiment G). Four formulations as indicated in panels A, B, C and D, all containing 10% sorbitol, 10% MSG and were tested with 10 mM McIlvaine, 10 mM citrate, 10 mM histidine, 10 mM HEPES and 10 mM phosphate buffer. The control formulation was not dialyzed.

(13) FIG. 13 Stability of lyophilized serotype 1, 2 and 3 IPV-formulations with different buffer components as indicated (experiment G) after lyophilization and subsequent storage at 45° C. for one week. Four formulations, containing 10% sorbitol, 10% MSG and MgCl.sub.2 without or with mannitol and without or with peptone were tested in combination with a 10 mM McIlvaine, 10 mM citrate, 10 mM histidine, 10 mM HEPES and 10 mM phosphate buffers. The control formulations were not dialyzed and 1:1 diluted with McIlvaine buffer containing the excipients as indicated.

(14) FIG. 14 D-Antigen recovery directly after vacuum drying (V), lyophilization with a slow freezing rate (L) and lyophilization with a fast freezing rate (S). The stabilizing effect of different formulations compared to IPV vaccine without addition of stabilizers (negative control) and sucrose or trehalose formulated IPV.

(15) FIG. 15 D-antigen recovery directly after lyophilization of serotype 1, 2 and 3 IPV-formulations as indicated.

(16) FIG. 16 Stability of serotype 1, 2 and 3 IPV-formulations as indicated after lyophilization and subsequent storage at 45° C. for one week, as determined by D-Antigen recovery.

EXAMPLES

(17) 1. Materials and Methods

(18) 1.1 Materials

(19) The trivalent inactivated polio vaccine (Salk-IPV), containing the inactivated Mahoney strain for type 1, MEF for type 2 and Saukett for type 3, was obtained from the process development department of the RIVM-Vaccinology (Bilthoven, The Netherlands). The Salk-IPV trivalent bulk (10×) was formulated as a ten times concentrated 40-8-32 DU/single human dose (1 ml). The concentration of the IPV 05-126B bulk that was used in this study was determined at 411-90-314 DU/ml with the QC-ELISA as described by Westdijk et al. [6].

(20) The excipients sucrose, D-sorbitol, D-trehalose dihydrate, D-glucose monohydrate, mannitol, L-glutamic monosodium salt monohydrate (referred to as glutamate, sodium glutamate, monosodium glutamate or MSG herein), myo-inositol, D-raffinose, hydroxy ethyl starch, glycine, L-proline, L-leucine, calciumchloride dihydrate, maltitol, magnesiumchloride hexahydrate, lithium chloride, and ovalbumin were all purchased from Sigma (St. Louis, Mo.). Peptone (vegetable), dextran (6 kDa, from Leuconostoc ssp), L-histidine, L-alanine, zinc chloride, calcium lactobionate monohydrate were from Fluka (Buchs, Switzerland). Lactitol (Lacty®-M) was from Purac Biochem (Gorinchem, The Netherlands), L-arginine (EP, non-animal origin) and Tween80 were from Merck (Darmstadt, Germany), polyvinylpyrrolidone 25 (PVP, 29 kDa) was from Serva Feinbiochemica GmbH (Heidelberg, Germany), Sol-U-Pro, a hydrolyzed porcine gelatin, was from Dynagel Inc. (Calumet City, Ill.) and Ficoll was from Pharmacia (Uppsala, Sweden). As buffer components sodium dihydrogen phosphate dihydrate (NaH.sub.2PO.sub.4), sodium chloride (NaCl), potassium dihydrogen phosphate (KH.sub.2PO.sub.4) and EDTA from Merck were used. Trisodium citrate dihydrate, citric acid and HEPES were from Sigma-Aldrich (St. Louis, Mo.) and disodium hydrogen phosphate dihydrate (Na.sub.2HPO.sub.4) was from Fluka (Buchs, Switzerland). All excipients used were of reagent quality or higher grade.

(21) To prepare 10 mM McIlvaine buffer, 10 mM citric acid was added to 10 mM Na.sub.2HPO.sub.4 in a ratio of 1:6 and a pH-value of 7.0. For the 10 mM citrate buffer the components trisodiumcitrate dihydrate (10 mM) and citric acid (10 mM) were mixed together till pH 7.0 was reached. The 10 mM phosphate buffer of pH 7.0 consisted of 10 mM KH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4. The 10 mM HEPES and 10 mM histidine buffers were prepared by weighing and dissolving the buffer components followed by adjustment of the pH-value at 7.0 using HCl and/or NaOH.

(22) 1.2 Methods

(23) 1.2.1 Dialysis

(24) Unless otherwise indicated, the trivalent IPV bulk material was dialyzed against 10 mM McIlvaine buffer (pH 7.0) using a 10 kDa molecular weight cut-off, low-binding regenerated cellulose membrane dialysis cassette (Slide-A-Lyzer®, Pierce, Thermo Scientific, Rockford, Ill.) to replace the buffer components of the IPV bulk (M199 medium).

(25) 1.2.2 Solutions to be Dried

(26) All excipients were dissolved in McIlvaine buffer at a double concentration of the indicated end concentration. The dialyzed IPV was equally mixed with the formulation to be tested. Subsequently 2 ml glass injection vials (Müller+Müller, Holzminden, Germany) were filled with 0.2 ml of the IPV-excipient mixtures and provided with 13 mm pre-dried (overnight at 90° C.) rubber stoppers (type V9250 from Helvoet Pharma, Alken, Belgium).

(27) 1.2.3 Lyophilization and Vacuum-Drying Process

(28) For lyophilization, filled and half-stopped vials were loaded into a Leybold GT4 freeze-dryer or Zirbus pilot/laboratory freeze-drying unit sublimator 2-3-3 at a shelf temperature of −50° C., or at a shelf of 4° C. and then frozen to −50° C. by reducing the temperature at a rate of 1° C./min, which will be denoted as fast and slow freezing, respectively. The vials were kept at a temperature of −50° C. for 2 h. For the primary drying phase the shelf temperature was increased at 0.1° C./min to −45° C., then at 0.02° C./min to −40° C., followed by incubation for 42 h. The secondary drying phase was performed by further increase of the shelf temperature at 0.02° C./min to 10° C., followed by an 8 h during incubation at 10° C. Thereafter, the shelf temperature was increased at 0.02° C./min to 25° C.

(29) For vacuum drying, filled and half-stopped vials were loaded into a Zirbus freeze-drying unit sublimator 2-3-3 at a shelve temperature of 15° C. and kept at that temperature for 10 minutes. The chamber pressure was reduced till 1 mbar in ramping steps of 15 minutes with different rates (1 mbar/min, 0.3 mbar/min, 0.1 mbar/min) and starting at a 25 mbar chamber pressure. The temperature was decreased till −10° C. for 1 h at 0.05 mbar and for 1 h at 0.03 mbar, resulting in no freezing of the formulations (product temperature above eutectic temperature of the formulations). Subsequently, shelf temperature was increased at 0.05° C./min to 30° C. At the end of the cycle, the vials were closed under vacuum, sealed with alu-caps and kept at 4° C. until analysis. An example of the shelf temperatures and chamber pressures during the course of vacuum drying process is shown in Table 1.

(30) TABLE-US-00001 TABLE 1 T.sub.shelf Period Pressure (° C.) (min) (mbar) FT01 15 10 — D01 15 15 25 D02 15 15 10 D03 15 15 5 D04 15 15 3 D05 15 15 1 D06 −10 60 0.05 D07 −10 60 0.03 D08 −5 120 0.03 D09 5 120 0.03 D10 10 120 0.03 P01 20 240 — P02 30 240 — P03 4 60 —
1.2.4 D-Antigen ELISA

(31) Polystyrene 96-well microtiter plates were coated overnight at room temperature with serotype-specific bovine anti-polio serum (RIVM, Bilthoven, The Netherlands). After washing with 0.1% Tween20 in PBS (wash buffer), twofold dilutions of an IPV reference standard and a single dilution of IPV-formulations diluted in assay buffer (PBS with 0.5% Protifar and 0.1% Tween20) were added (100 μl/well, in duplicate). The plates were incubated at 37° C. for 30 minutes under gentle shaking, extensively washed and a mixture of serotype-specific monoclonal mouse antibodies (mab 3-4-E4 (type 1), 3-14-4 (type 2), 1-12-9 (type 3), all from RIVM, Bilthoven, The Netherlands) and HRP-labeled anti-mouse IgG (GE Healthcare, Buckinghamshire, UK) was added. Subsequently, plates were incubated at 37° C. for 30 minutes under gentle shaking. Plates were washed extensively and ELISA HighLight signal reagent from (Zomerbloemen BV, Zeist, The Netherlands) was added and chemiluminescence was measured during 10-15 minutes by using a luminometer (Berthold Centro LB960).

(32) 1.2.5 Moisture-Content Analysis

(33) The water content was determined using a Karl Fischer coulometric titrimeter (Model CA-06 Moisture meter, Mitsubishi). The principle of the water residue determination by Karl Fischer method is based on the fact that iodine and sulphurdioxide only react in the presence of water. The samples were weighted and subsequently reconstituted in the Karl-Fischer reagent, Hydranal Coulomat A (Fluka, Buchs, Switzerland). The reconstituted sample was withdrawn into a syringe and injected into the titration vessel. Each vial was measured in triplicate. The empty vials were weighted and the water content was calculated based on the water content measured by the titrimeter, the weight of the lyophilized product in the vial, the reconstitution volume of the reagent, titration volume and the water content of the blank titration.

(34) 1.2.6 Differential Scanning Calorimetry (DSC)

(35) The thermodynamic behaviour of the formulations was determined by differential Scanning calorimetry (DSC), a method which measures the temperatures and heat flow, associated with phase transitions in materials, as a function of time and temperature. The freeze-dried formulations were filled in an aluminium DSC pan and subjected to a controlled temperature program in a differential scanning calorimeter (DSC Q100, TA Instruments). The sample was heated from 0° C. to 150° C. at a heating rate of 20° C./min and the sample chamber was purged with nitrogen gas (50 ml/min). The glass transition temperatures (Tg) were determined as the midpoint of the discontinuities in the heat flow curves using software (Universal Analysis 2000, TA Instruments).

(36) 2. Results

(37) 2.1 Stabilizing Different IPV Subtypes During Lyophilization

(38) In the first experiment (Experiment A) four well known stabilizing sugars/polyols (sucrose, trehalose, mannitol and dextran), as well as sodium chloride were evaluated for their stabilizing potential (FIG. 1). The study was set up with a design of experiments approach in order to obtain an optimal formulation for lyophilization of IPV.

(39) Different IPV-formulations were lyophilized as described above (section 1.2.3). Lyophilized cakes were reconstituted by adding an equal amount of water as the starting volume and the D-antigen recovery was determined by an ELISA (section 1.2.4). Recoveries were shown as the percentages of the D-antigen content in the liquid formulations, which were measured before lyophilization.

(40) The trivalent IPV formulation, without any additives, IPV 1:1 diluted with McIlvaine buffer, showed recoveries <10% for all serotypes after lyophilization (FIG. 1; A1). Type 2 IPV showed to be the most stable serotype in all formulations with maximum D-antigen recoveries of ±80%. Dextran seemed to have a negative effect on D-antigen recovery of the lyophilized IPV formulations, especially for type 1 and 3. Best results, with maximum recoveries of ±55%, ±85% and ±50% for serotype 1, 2 and 3 respectively, were obtained with formulations containing sucrose and/or trehalose in combination with mannitol (FIG. 1; formulations A3, A4, A12 and A16). Addition of NaCl had no positive effect on the recovery of IPV after lyophilization.

(41) This first pilot experiment clearly shows the complexity of lyophilizing a trivalent polio vaccine in which each IPV serotype prefers its own stabilizing agents. In a formulation with 10% mannitol type 1 and type 3 preferred the presence of high concentrations sucrose without trehalose, whereas type 2 preferred a high concentration of trehalose without sucrose (FIG. 2).

(42) In the next experiment (Experiment B) the stabilizing potential of a mixture of glutamate, a saccharide, and a polymer was investigated. Different combinations of the excipients sucrose, trehalose, monosodium glutamate (MSG), hydroxyethyl starch (HES) and NaCl were investigated. Lyophilization of trivalent IPV with formulations based on MSG together with disaccharide, sucrose and/or trehalose, showed D-antigen recoveries of 50-60%, 70-95% and 50-65% for the three serotypes respectively (FIG. 3; formulations B4, B7, B9, B12, B13). Formulations based on only 8% HES or 63 mM NaCl did not protect IPV during lyophilization (FIG. 3; B5 and B10). The addition of NaCl to a formulation with sucrose showed an 5-10% increase in D-antigen recovery after lyophilization (FIG. 3; B2 and B16).

(43) 2.2 Impact of the Drying Process and Formulation

(44) In the next test results are shown of typical formulations used for drying of biopharmaceuticals in relation to the drying process. Formulations containing trivalent IPV were dried by vacuum drying (a drying method without freezing), freeze drying using a fast freezing step (direct placement of the product on pre-cooled shelves of −50° C.) and freeze drying using a slow freezing step (placement of product on shelves of 4° C. and freezing towards −50° C.). As shown in FIG. 4 standard formulations, e.g. based on sucrose or trehalose, partially protect IPV upon vacuum drying. However, these formulations do not give protection upon freeze drying.

(45) 2.3 Screening of Excipients

(46) In the next experiment (Experiment C) different formulations containing sorbitol, mannitol, sucrose and/or MSG combined with some amino acids, proteins/peptides or other stabilizing agents were tested (Table 2). In order to investigate the effect of salt in the lyophilized IPV-formulation, the C-formulations were also tested with addition of 125 mM NaCl. No clear effect of the NaCl on the D-antigen recoveries was observed (data not shown). Having a first look on the antigenicity results directly after lyophilization, it was clear that formulation C13, containing 5% sorbitol, 5% peptone and 1% lithium chloride (LiCl), showed the highest recoveries for all serotypes; ±85%, ±100% and ±85% for type 1, 2 and 3 respectively (FIG. 5). Substitution of the LiCl-compound in 1.8% MgCl.sub.2 resulted in a decrease of ±5%±20% and 15% in D-antigen recovery for the three serotypes. However, these formulations showed relative high residual moisture contents, 2.1% for the MgCl.sub.2-containing formulation and 6.2% for the formulation with LiCl. The addition of a very small amount of surfactant Tween 80 to a formulation containing sucrose, trehalose and the amino acids glycine and lysine increased the D-antigen recoveries with 5-15% (FIG. 5; C8, C9). The formulations based on sorbitol, mannitol and MSG (C2, C3, C4 and C7) showed recoveries of more than 65%, 80% and 70% for type 1, 2 and 3 respectively.

(47) In this study, the combination of sorbitol, peptone and the salts LiCl or MgCl.sub.2 seemed to have a positive effect on the D-antigen recovery directly after lyophilization of IPV. Another notable formulation is the mixture of sorbitol, mannitol and MSG, which showed that the presence of polyols in combination with MSG stabilizes the IPV during lyophilization.

(48) The glass transition temperature of the lyophilized formulations was measured (Table 2), but showed no clear relation with the D-antigen recoveries. Formulations containing ovalbumine and peptone showed the highest glass transition temperatures.

(49) TABLE-US-00002 TABLE 2 Composition of the lyophilized IPV-formulations (Experiment C). Residual moisture content (RMC) was determined by Karl Fischer and the T.sub.g of the dried cake by DSC. Amino RMC T.sub.g SOR MAN SUC MSG Sugars/polyols acids Proteins Other (%) (° C.) C1 — — — — — — — — 3.6 n.d. C2 7% 7% — 2% — 2% 7% — 0.3 37.2 Glycine Ovalbumin C3 7% 7% — 2% — 2% 7% 1 mM n.d. 38.3 Glycine Ovalbumin EDTA C4 7% 7% — 2% — 2% — — 1.9 53.5 Glycine C5 — — 3% — 3% Dextran — 3% — 1.1 54.9 3% Myo- Ovalbumin Inositol C6 5% 5% 5% — — 2% — — 1.0 37.1 Glycine 3% Lysine 3% L- Arg C7 5% 5% 5% 3% — 2% — — 3.3 37.2 Glycine 3% Lysine C8 — — 5% — 5% Trehalose 3% — — 0.5 32.3 Lysine 3% Alanine C9 — — 5% — 5% Trehalose 3% — 0.01% 0.5 34.1 Lysine Tween80 3% Alanine C10 — — 5% — — 3% — 3% Ca- 1.0 31.2 Lysine Lactobionate 3% Alanine C11 — — 5% — — 3% 3% Rec. — 0.3 35.7 Lysine Gelatin 3% Alanine C12 5% — — — — — 5% 1.8% MgCl.sub.2 2.1 44.7 Peptone C13 5% — — — — — 5% 1% LiCl 6.2 n.d. Peptone C14 — — 5% — 5% Trehalose — 5% — 0.5 35.6 Peptone

(50) Based on these findings a new screening experiment (Experiment D) was designed. Since the most promising recoveries were obtained with formulations based on sorbitol, peptone and Mg or Li-chloride, we designed an experiment based on 10% sorbitol, 5% peptone and 125 mM NaCl. Ovalbumine was discarded since it is from animal origin, thus an undesirable excipient in a vaccine for human use. In order to get more insight in the IPV stabilizing mechanism of several excipients, formulations containing 10% sorbitol, 5% peptone and 125 mM NaCl were combined with either a sugar/polyol, an amino acid (instead of 5% peptone), a salt or other stabilizing agents, like surfactants or proteins. The formulation with sorbitol, NaCl and 1% histidine showed recoveries of 90-100% directly after the freeze-drying process (FIG. 6; D11). As observed earlier, the MgCl.sub.2-containing formulation showed auspicious stabilizing capacity during lyophilization, which was similar for the calciumchloride (CaCl.sub.2)- and lithium chloride (LiCl)-containing IPV formulations (FIG. 6; D17-19). During the screening phase accelerated stability has been evaluated to select the excipients on their ability to provide a stable lyophilized product as well, even after subsequent storage at high temperature.

(51) 2.4 Accelerated Stability Testing

(52) Although some formulations showed acceptable D-antigen recoveries directly after lyophilization, after one week incubation at 45° C. the D-antigen recoveries of these four formulations were dropped till percentages below 30%, 60% and 10% for respectively serotype 1, 2 and 3 (FIG. 7). The formulation with 5% myo-inositol represented the highest recoveries after incubation at 45° C., but still a decrease of ±40%, 10% and 30% was observed for the three serotypes, which showed again IPV type 2 to be the most stable serotype (FIG. 7; D5). Upon one week storage at 45° C., the formulation containing 1% lactitol showed less than 10% decrease in D-antigen recovery for serotype 2, unfortunately serotype 1 and 3 showed a decrease of >30% (FIG. 7; D7).

(53) In order to further investigate the combination with sorbitol, mannitol, MSG and the stabilizing potential of peptone and MgCl.sub.2, a new design of experimental set up was performed to determine the relationship between the different excipients and D-antigen recovery after lyophilization. The following variables were included in Experiment E: 0/10% sorbitol, 0/10% sucrose, 0/10% mannitol, 0/10% MSG, 0/5% peptone and/or 0/5% MgCl.sub.2 and freezing speed was investigated in this experiment. Slow freezing means that the vials were placed on shelves at 4° C. and subsequently cooled till −50° C. at a rate of 0.1° C./min, where fast freezing means that the vials were directly placed at shelves pre-cooled at −50° C. The results are shown in FIG. 8A for the fast freezing rate and in FIG. 8B for the slow freezing rate.

(54) Having a first look on the D-antigen recoveries after lyophilization, the fast frozen formulations containing MSG and MgCl.sub.2 in combination with a sugar/polyol showed the highest recoveries of ±80-90% for all serotypes (FIG. 8A; E22-24, E29-32). For the slow frozen samples the MgCl.sub.2-containing formulations showed recoveries of 75-90% for type 1 and 3 (FIG. 8B; E54-57, E63-66). However, for serotype 2 only formulations of the sugar(s) in combination with peptone showed recoveries of ±70% (FIG. 8B, E43-48). To have an indication of the reproducibility of the experiment formulation H33 and H66 were tested in triplicate and showed standard deviations <10% for all serotypes. From the comparison of the D-antigen recoveries of these two formulations, directly after lyophilization (FIG. 8) or after one week incubation at 45° C. (FIG. 9), it is clear that freezing rate did not influence the recovery significantly for these IPV formulations.

(55) Experiment E was set up on the basis of ‘Design of Experiment’ using the “Modde” software from Umetrics. Besides recovery of D-antigen after lyophilization, also recoveries after lyophilization and subsequent storage at 37° C. or 45° C. were used as output in the design. The output as function of the formulations was modulated and put in a model using Modde. This revealed which formulation parameters affected the recovery after lyophilization and storage (data not shown).

(56) The most important formulation parameters for each of the viral subtypes are summarized in Tables 3-5.

(57) TABLE-US-00003 TABLE 3 Recovery of D-antigen of type 1 formulation after after storage after storage parameter lyophilization % at 37° C. % at 45° C. % MSG 9 10 7 Sorbitol 8 6 4 MgCl.sub.2 7 6 3 Peptone 6 4 Mannitol 4 6 MSG* MgCl.sub.2 4

(58) TABLE-US-00004 TABLE 4 Recovery of D-antigen of type 2 formulation after after storage after storage parameter lyophilization % at 37° C. % at 45° C. % MSG 7 9 9 Sorbitol 7 7 5 MgCl.sub.2 Peptone 10 10 Mannitol 3 4 4 Sucrose 2

(59) TABLE-US-00005 TABLE 5 Recovery of D-antigen of type 3 formulation after after storage after storage parameter lyophilization % at 37° C. % at 45° C. % MSG 14 10 10 Sorbitol 8 7 7 MgCl.sub.2 4 4 4 Peptone 4 12 12 Mannitol 6 5 5 Sucrose 4 1 1 MSG* MgCl.sub.2 5 5
2.5 Substitution of Peptone

(60) Since peptone seemed to stabilize the lyophilized IPV during the subsequent storage at a temperature of 45° C., we performed an experiment to investigate the role of peptone in a formulation with 10% sorbitol, 5% MSG and 5% MgCl.sub.2 and the same formulation combined with 10% mannitol. No significant differences were found with the addition of 10% mannitol to the formulation containing sorbitol, MSG and MgCl.sub.2. Adding 5% peptone did not affected the antigenicity of both formulations (FIG. 10).

(61) To find out whether the addition of single amino acids could take over the stabilizing role of peptone during subsequent storage of the lyophilized IPV, amino acids were added to the formulation containing sorbitol, MSG and MgCl.sub.2 with or without mannitol. After a week incubation at 45° C. neither peptone or one of the added amino acids showed improved stability of the D-antigen recovery when compared to the control formulation, which contain 10% sorbitol, 10% MSG and 5% MgCl.sub.2 (FIG. 11, F1.0-F1.5). In the presence of mannitol the stabilizing capacity of peptone was only shown for type 3, whereas the D-antigen recovery was improved with ±15% when compared to the control sample. However, a significant decreased recovery (p<0.001) was found for type 1 and, even though not significant, type 2 showed also a ±10% lower recovery (FIG. 11; F2.0, F2.1). For all formulations containing sorbitol, MSG and MgCl.sub.2 a decrease of ±10% of D-antigen content type 2 was shown after one week 45° C., where the addition of mannitol was found to be stable for type 2 during accelerated stability. Arginine seemed to have stabilizing potential in a formulation (FIG. 11, F2.3) with sorbitol, MSG, MgCl.sub.2 and mannitol and showed only ±15% and ±30% decrease in D-antigen recovery for type 1 and 3 respectively after incubation at 45° C. This was comparable to the stabilization by almost the same formulation with peptone instead of arginine (FIG. 11, F2.1).

(62) Due to the fact that an undefined excipient, such as peptone, is not preferred in a human vaccine, a possible substitute for peptone, which could stabilize the IPV during storage, was investigated. Analysis by mass spectrometry and HPLC showed the most abundant amino acids present in peptone (data not shown). The addition of several single amino acids to the formulation containing sorbitol, MSG and MgCl.sub.2 did not improve the stability at 45° C. when compared to the control formulation. Where peptone seemed to stabilize serotype 3 in the formulation containing sorbitol, MSG, MgCl.sub.2 and mannitol, arginine is able to improve the stability of both serotype 1 and 3. Serotype 2 showed already in the control formulation full maintenance of D-antigen recovery during accelerated stability. Although the exact composition of peptone is hard to determine, the amino acid quantification by reverse-phase HPLC with non-hydrolyzed versus chemical hydrolyzed peptone showed that peptone consists of both single amino acids and peptides, however >90% w/w of the peptone remains undefined. Since peptone seemed to increase glass transition temperature of the studied IPV-formulations, it might be possible to replace the peptone by an excipient with high T.sub.g, like sucrose or trehalose.

(63) TABLE-US-00006 TABLE 7 Glass transition temperatures (Tg′ and Tg) of IPV- formulations containing sorbitol, MSG and MgCl.sub.2 with/without mannitol were determined by DSC. The effect of peptone on the glass transition of these formulations was investigated. Single measurements were shown. 10% sorbitol + 10% MSG + 10% sorbitol + 10% MSG + 5% MgCl.sub.2 5% MgCl.sub.2+ 10% mannitol T.sub.g′ (° C.) T.sub.g (° C.) T.sub.g′ (° C.) T.sub.g (° C.) Control −47.9 35.2 −44.4 38.8 +5% −44.1 39.8 −42.8 48.7 peptone

(64) The previous experiment did not yielded a worthy substitute for peptone and showed that the formulations with sorbitol, MSG, MgCl.sub.2 with or without mannitol gave the best results, even after accelerated stability tests. During this study all formulations were prepared with McIlvaine buffer, which is known to be a suitable buffer for lyophilization of IPV [52]. In order to further optimize the formulation a buffer screening was performed with buffers that are frequently used for lyophilization of biopharmaceuticals [18]. IPV batches in each buffer were prepared by dialysis and non-dialyzed IPV acted as control.

(65) The formulation with sorbitol, MSG and MgCl.sub.2 showed recoveries of ±95%, ±85% and ±90% for the three serotypes directly after lyophilization (FIG. 12A; G1.0-G1.5). After addition of peptone the type 1 and 3 D-antigen recoveries were dropped with 10-15%, where type 2 showed similar recoveries when compared to the formulation without peptone (FIG. 12B). McIlvaine buffer showed 10-15% lower recoveries for serotype 2 in comparison with the other buffer components. Histidine buffer seemed to increase the D antigen yield of IPV after lyophilization the IPV-formulation with sorbitol, MSG and MgCl.sub.2 (recoveries of >95% for type 1 and 3; FIG. 12A, G1.3). After lyophilization, a D-antigen recovery of 88% for type 2 was reached with the phosphate buffer, where the McIlvaine buffer showed 73% recovery for type 2 (FIG. 12A, G1.5).

(66) The addition of mannitol to the formulations of sorbitol, MSG, MgCl.sub.2. without peptone revealed that serotype 2 prefers the presence of mannitol in the formulation during lyophilization, since recoveries of 85-100% for type 2 were found (FIG. 12C). For type 1 recoveries of 80-90% and for type 3±80% were found for the sorbitol, MSG, MgCl.sub.2 and mannitol containing IPV-formulations. The addition of peptone showed ±10% reduction in recovery of type 1 and 3 and similar D-antigen contents for type 2 after lyophilization (FIG. 12D).

(67) 2.6 The Effect of Buffer Components

(68) An accelerated stability experiment showed formulation-dependent differences between the used buffers. Whereas the control formulations showed high recoveries directly after lyophilization, recoveries of ±40%, ±50% and <15% for serotype 1, 2 and 3, respectively, were found for the peptone-lacking control formulations after one week incubation at 45° C. (FIG. 13; G1.0 and G3.0). For the control formulations, peptone showed clear improvement of stability of type 1 and 2 when where type 3 showed a decrease of ±25% and ±15% in D-antigen recovery, instead of >90% and 80%, respectively for the formulations without and with mannitol after storage at 45° C. (FIG. 13; G2.0 and G4.0). For the formulation containing sorbitol, MSG and MgCl.sub.2 the highest recoveries were observed with HEPES buffer, D-antigen contents of ±80%, ±85% and ±60% for the three serotypes after lyophilization and storage. This means that only 10%, 0% and ±30% reduction in D-antigen recovery occurred during accelerated stability testing for type 1, 2 and 3 respectively. Only for type 3 a better result was observed with citrate buffer; a reduction of ±20% and remained recovery of 79% (FIG. 13A; G1.2). Addition of peptone showed overall 15-25% lower recoveries for all serotype when compared to the same formulation and buffer without peptone (FIG. 13A). Obvious improved stability was shown with the addition of peptone to the formulation with sorbitol, MSG, MgCl.sub.2 and mannitol. Where the different buffer formulations without peptone showed maximum recoveries of ±55%, ±75% and ±45%, the same buffers with peptone showed recoveries of ±50%, ±80% and ±55% (FIG. 13B). The formulation based on phosphate buffer with the excipients sorbitol, MSG, MgCl.sub.2, mannitol and peptone showed to be the most stable formulation with reduced recoveries of ±30% for type 1 and only ±10% for type 2 and 3 after incubation at 45° C.

(69) 2.7 Formulations Suitable for Stabilization Independent of Drying Method

(70) FIG. 14 shows that only formulations containing sorbitol can resist stresses caused by the different drying methods. This could further be improved by inclusion of MSG and MgCl.sub.2 (especially type 3), resulting in formulations that gave comparable or even higher recoveries than vacuum drying with trehalose or sucrose. From earlier results (Experiment E) we concluded that the formulations based on sorbitol, MSG and MgCl.sub.2 possesses the best recovery after accelerated stability testing.

(71) 2.8 Formulations without Peptone

(72) One of the better formulation without peptone that we found in above experiments, is a formulation containing 10% sorbitol+10% MSG+5% MgCl.sub.2. In an additional experiment we evaluated what the impact was of the primary excipients in this formulation. The results were compared with 2 standard formulations based on sucrose and trehalose. The results direct after lyophilization are shown in the FIG. 15. From FIG. 15 we conclude that primarily sorbitol is responsible for recovery of IPV after lyophilization, especially for subtypes 1 and 2. The combination of MSG and MgCl.sub.2 shows significant stabilization of the three IPV subtypes. Compared to sorbitol only, the combination of sorbitol, MSG and MgCl.sub.2, showed the best recoveries after lyophilization, especially for type 3.

(73) Upon accelerated stability evaluation as shown in FIG. 16, most of the formulations showed an enormous decrease in recovery, also the formulation containing only sorbitol. However, the formulations containing 10% sorbitol+10% MSG+5% MgCl.sub.2 (with/without mannitol) showed high recoveries after storage at 45° C. for one week.

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ABBREVIATIONS

(75) BCG—Bacillus Calmette-Guérin

(76) DoE—Design of Experiments

(77) DSC—Differential Scanning calorimetry

(78) DU/D-Ag—D-Unit/D-antigenicity

(79) ELISA—Enzyme-Linked ImmunoSorbent Assay

(80) HES—Hydroxyethyl starch

(81) HRP—Horseradish peroxidase

(82) IgG—Immunoglobulin G

(83) IPV—Inactivated Polio Vaccine

(84) MAN—Mannitol

(85) MS—Mass Spectrometry

(86) MSG—Mono-Sodium Glutamate

(87) OPV—Oral Polio Vaccine

(88) PEP—Peptone

(89) QC—Quality Control (department at RIVM)

(90) RIVM—National Institute for Public Health and Environment

(91) RMC—Residual Moisture Content

(92) RP-HPLC—Reversed-Phase High-Performance Liquid Chromatography

(93) sIPV—Sabin Inactivated Polio Vaccine (based on Sabin strains)

(94) SOR—Sorbitol

(95) SUC—Sucrose

(96) T.sub.g—Glass transition temperature

(97) TREH—Trehalose

(98) VAPP—Vaccine Associated Paralytic Poliomyelitis

(99) VDPV—Vaccine Derived Poliovirus

(100) WHO—World Health Organisation