Compositions containing ambient-temperature stable, inactivated but therapeutically active biopharmaceuticals and methods for formulation thereof

Abstract

The disclosure concerns compositions containing inactivated but therapeutically active biopharmaceuticals, and methods for formulation thereof. Biopharmaceuticals are encapsulated and immobilized in dry amorphous carbohydrate-glass and irradiated for inactivation while in the dry state. The resulting compositions provide ambient-temperature stable, therapeutically active but inactivated biopharmaceuticals for use in vaccines and other applications.

Claims

1. A composition, comprising: an amount of inactivated but therapeutically active (IBTA) ERAg333 virus immobilized in a dry amorphous carbohydrate-glass, said IBTA ERAg333 virus characterized as being inactivated by irradiation while in the dry state and having a loss of potency less than 0.5 logs after one year of storage at a storage temperature between 20 C. and 37 C., wherein said composition is ambient-temperature stable.

2. The composition of claim 1, wherein the dry amorphous carbohydrate-glass comprises two parts sucrose and one part methylglucoside.

3. The composition of claim 1, wherein the composition is stable for twenty-three months at 22 C.

4. The composition of claim 1, wherein the composition is stable for two months at 37 C.

5. A composition, comprising: an amount of inactivated but therapeutically active (IBTA) ERAg333 virus immobilized in a dry amorphous carbohydrate-glass, the carbohydrate-glass comprising two-parts sucrose and one-part methylglucoside, said IBTA ERAg333 virus characterized as being inactivated by irradiation while in the dry state and having a loss of potency less than 0.5 logs after one year of storage at a storage temperature between 20 C. and 37 C.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 is a plot illustrating survival of vegetative form of anthrax sterne strain vaccine after PBV drying and subsequent storage at 37 C.

(2) FIG. 2 is a plot illustrating metabolic activity of irradiated with 12.5 kGy anthrax vaccine in accordance with an MTT assay.

(3) FIG. 3 is a plot illustrating stability of L. Acidophilus after drying and subsequent storing at 37 C.

(4) FIG. 4 is a plot illustrating metabolic activity of PBV-preserved L. Acidophilus irradiated with 12.5 kGy.

(5) FIG. 5 illustrates a method for producing thermostable inactivated but therapeutically active biopharmaceuticals.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(6) For purposes of this invention, the terms foam drying and vacuum foam drying are herein to describe various drying techniques for obtaining preserved materials, including preservation by foam formulation (PFF) as described in U.S. Pat. No. 5,766,520; and preservation by vaporization (PBV) as described in WO 2005/117962; the contents of each of which are hereby incorporated by reference.

(7) The term preservation by vaporization (PBV) describes a current state of the art foam drying technique for preserving sensitive biological material in an amorphous sugar-glass by simultaneous sublimation, evaporation and boiling of water from within a partially frozen slush-state, as is further described in WO 2005/117962.

(8) The terms carbohydrate-glass or sugar-glass are used herein to describe an amorphous solid carbohydrate matrix including one or more carbohydrates, generally sugars. The matrix may further include amino acids, salts, surfactants and polymers that were dissolved in preservation solutions prior to drying.

(9) In accordance with aspects of the invention, it is a primary objective to provide vaccines and other biopharmaceutical products with improved therapeutic activity, and to enhance methods for formulating such products.

(10) In a general embodiment, a method for formulation of a therapeutic composition containing inactivated but therapeutically active (IBTA) biopharmaceuticals comprises: (i) preserving biopharmaceuticals in a dry state; (ii) irradiating the biopharmaceuticals in the dry state to yield IBTA biopharmaceuticals; and (iii) using the IBTA biopharmaceuticals to form the therapeutic composition.

(11) Preserving the biopharmaceuticals in the dry state can comprise any foam drying technique, including preservation by foam formulation, or preservation by vaporization as referenced above. In each of these techniques, the result is an amount of biopharmaceuticals being stabilized within a dry amorphous carbohydrate-glass matrix.

(12) Although not preferred, it is conceivable that a similar result can be achieved using lyophilization of a biopharmaceuticals suspension; however, at the risk of harming sensitive biomaterial structures of the biopharmaceuticals and thereby reducing therapeutic effectiveness. Other techniques can be similarly implemented for preserving the biopharmaceuticals.

(13) Once stabilized in the dry amorphous carbohydrate-glass matrix, the encapsulated and immobilized biopharmaceuticals are subjected to irradiation. The irradiation ideally comprises electron beam (EB) irradiation, but may alternatively include: Gamma, X-ray, proton, neutron, UV irradiation, or a combination thereof. Although radiation dose can be varied, the radiation dose should be at least 12 kGy in certain embodiments.

(14) Once preserved, and subsequently irradiated, the resulting inactivated but therapeutically active (IBTA) biopharmaceuticals can be further formed into a composition by any of: (i) reconstituting the encapsulated and immobilized IBTA biopharmaceuticals in an aqueous solution, (ii) combining the encapsulated and immobilized IBTA biopharmaceuticals with a dry compound to form a therapeutic mixture, or (iii) micronizing the encapsulated and immobilized IBTA biopharmaceuticals to form a therapeutic powder.

(15) In an embodiment, an amount of first therapeutically active biopharmaceuticals is preserved using a foam drying technique, the first preserved biopharmaceuticals are further inactivated by irradiation in the dry foam state, and subsequently micronized to yield a first preserved biopharmaceutical powder. An amount of second therapeutically active biopharmaceuticals is preserved using a foam drying technique, the second preserved biopharmaceuticals are inactivated by irradiation in the dry foam state, and subsequently micronized to yield a second preserved biopharmaceutical powder. Each of the first and second biopharmaceutical powders are then combined to yield a therapeutic composition, in this case a mixture of distinct preserved and inactivated biopharmaceuticals. Note that the foam drying technique can be any foam drying technique, and is independent for producing each of the first and second powders.

(16) In another embodiment, an amount of first therapeutically active biopharmaceuticals is mixed with an amount of second therapeutically active biopharmaceuticals. The first and second biopharmaceuticals are collectively preserved using a foam drying technique, and further irradiated when in the dry state. The resulting foam is micronized to yield a powder containing inactivated but therapeutically active (IBTA) biopharmaceuticals for biopharmaceutical applications.

(17) Experiments have shown that with biopharmaceuticals immobilized in carbohydrate-glass, electron beam irradiation can be used to reduce the viability of dry preserved bacteria more than one million times with minimal loss of metabolic activity, indicating that the internal bacterial proteins and enzymes are structurally and functionally intact after EB irradiation. In contrast, UV irradiation of cells in the liquid state can result in the formation of highly reactive radicals that can degrade proteins. An important benefit of using EB irradiation to produce vaccines is that radiation in the dry state will damage nucleic acids and not protein antigens on the bacterial surface. EB irradiation of dried biologicals is a simple and inexpensive procedure that does not require addition of psoralen.

(18) In the embodiments herein, it is preferred to first make the biopharmaceuticals ambient-temperature stable by encapsulating in a carbohydrate-glass matrix, and then attenuate them by EB irradiation. This approach enables one to evaluate survival of dry preserved bacteria prior to irradiation, and assess protein integrity after irradiation by measuring metabolic activity instead of performing expensive animal immunogenicity studies.

(19) Compositions containing ambient-temperature stable, inactivated but therapeutically active (IBTA) biopharmaceuticals present a unique opportunity for safe and efficacious treatment options.

(20) It is important to note that primary irradiation in the liquid state produces harmful free radicals that unpredictably damage the biological components. Accordingly, the methods described herein incorporate irradiation in the dry state, after stabilization, which effectively limits production of free radicals, and targets only the nucleic acids required for replication while maintaining the metabolism-associated structures intact.

(21) In contrast, methods which irradiate first and preserve second will fail to retain cellular metabolism. Because they are liquid-irradiated first, all the cells that are subsequently stabilized are preserved in the damaged state with much of their metabolic activity already lost.

(22) Thus, when inactivated properly, appropriate organisms will retain therapeutic potency and be a safer alternative to their live counterparts.

(23) In one embodiment, the step of encapsulating the therapeutically active biopharmaceuticals in a dry amorphous carbohydrate-glass matrix is achieved using the technique known as Preservation by Vaporization (PBV). PBV generally includes: (i) providing the biopharmaceuticals in one of an aqueous solution or a hydrogel to form a first composition; (ii) partially freezing the first composition to form a two-phase state thereof, wherein the first composition comprises an amount of ice and an amount of liquid water in the two-phase state; (iii) vaporizing the first composition, the vaporizing comprising simultaneously applying vacuum and heat, wherein water is removed from the first composition through simultaneous boiling of the liquid, sublimation of ice, and evaporation of water molecules from a surface of the liquid; and continuing the vaporization to transform the first composition into a dry foam, wherein the dry foam forms the carbohydrate-glass matrix that encapsulates the biopharmaceuticals.

(24) Where a hydrogel is incorporated into the first composition, the hydrogel can comprise calcium alginate.

(25) PBV is a current state of the art because the process yields high thermostability without significant disturbance of activity. For this reason, PBV is preferred, however, other foam drying techniques can be similarly implemented to encapsulate the biopharmaceuticals in a dry amorphous carbohydrate-glass matrix, for example, by using the technique known as preservation by foam formulation (PFF) as referenced above.

(26) FIG. 5 illustrates a method for producing thermostable inactivated but therapeutically active biopharmaceuticals.

EXAMPLES

Example 1: Preparation of Inactivated but Therapeutically Active (IBTA) Viral Lave Attenuated Vaccine

(27) Fixed rabies virus (RV), Evelyn-Rokitnicki-Abelseth (ERA) strain, was attenuated using a reverse genetics system. The recovered virus was sequenced and had only the desired change (R333E). The resulting virus, referred to as ERAg333, was grown.

(28) Supernatant was mixed (1:2) with 30% sucrose and 15% methylglucoside in phosphate buffer (pH=7.0). 0.5 ml of mixture was distributed into crimp vials and dried using Genesis and Virtis Ultra freeze-dryers that were modified to allow better vacuum pressure control. After two hours of processing, the solid material formed a stable dry foam. Secondary drying was performed under vacuum at 35 C. and 45 C. for 20-24 hours. RV preservation by vaporization (PBV) in crimp vials at 22 C. with desiccant was electron beam-irradiated at various doses. Virus titers were measured as described below except in 96-well plates on four consecutive days post infection.

(29) Commercially available RV vaccine RabAvert was purchased and reconstituted according to the manufacturer's instructions.

(30) RV PBV in crimp vials was placed at 22 C. with desiccant, in a dry incubator at 37 C., in mineral oil bath at 80 C. and 90 C. for viability, or in a water bath at 80 C. for electrochemiluminescent (ECL) assays. Vials were removed at different time points and reconstituted with 0.4 ml phosphate buffered saline (PBS) (0.01M, pH 7.4). Virus titers were measured by serially diluting vaccine with BHK-21 cells in an 8-well chamber slide as described previously. The mean focus forming units (ffu)/ml and standard deviation were calculated from at least three statistical replicates.

(31) The Meso Scale Discovery platform was used to perform RV antigen capture ECL assays as described previously. RV glycoprotein (G) monoclonal antibody (MAb) 62-80-6 was used at 1 g/ml for capture and 0.5 g/ml for detection.

(32) Approved animal use protocols were established with CDC IACUC. Blood was collected as described previously from female, 4-week-old, CD-1 mice assigned to groups of 10, and the geometric mean titer (GMT) of RV neutralizing antibodies (rVNA) in international units (IU)/ml was determined using a rapid fluorescent focus inhibition test (RFFIT) or a modified RV neutralization test for small volumes. Live attenuated RV PBV vaccine, placebo, and inactivated RV PBV, stored for 36 days at 22 C. in the dark with desiccant, were reconstituted with 0.4 ml of sterile PBS (0.01M, pH 7.4) without calcium or magnesium. Reconstituted vaccine and RV ERAg333 from frozen stock was subsequently diluted using the same PBS. On day 0, mice were vaccinated intramuscularly (IM) in the right leg as described previously. Back titrations of dilutions used to vaccinate mice were completed as described above. For inactivated vaccines, the BCA Protein Assay was used according to manufacturer's instructions to determine total protein concentration. Blood was collected again from all mice on day 14 and 30, and rVNA GMT was determined. On day 30 all mice were challenged IM in the left leg with 50 l of canine RV 3374R. Animals were monitored and euthanized when showing signs of rabies as described previously. The brain stem was collected from euthanized animals and subjected to the direct fluorescent antibody (DFA) test for rabies. The experiment was terminated 30 days after the last death in the placebo group. Probability values were calculated using chi-square test with a 95% confidence interval.

(33) Results

(34) The starting titer of RV ERAg333 before PBV was 8.3 log 10 ffu/ml. After PBV, about 0.2 log 10 of viable virus was lost resulting in 8.110.12 log 10 ffu/ml. For inactivated vaccines, electron beam-irradiation at all tested doses damaged RV and resulted in lower virus titers; no viable virus was recovered in samples treated with the highest dose of 12 kGy (Table 1). The complete inactivation of rabies virus after treatment with 12 kGy was confirmed in three blind cell passages.

(35) TABLE-US-00001 TABLE 1 Inactivation of RV PBV by electron beam Dose Rabies virus titer (log.sub.10 ffu/ml) (kGy) 24 hrs 48 hrs 72 hrs 96 hrs 0 8.16 0.1 TNC.sup.a TNC TNC 2.9 6.03 0.04 TNC TNC TNC 4.2 4.82 0.24 TNC TNC TNC 6 4.32 0.25 TNC TNC TNC 12 BLD.sup.b BLD BLD BLD .sup.aToo numerous to count (TNC) .sup.bBelow level of detection (BLD); no virus detected

(36) RV PBV was stored at 22 C. with desiccant for 1, 2, 3, 15, or 23 months. After a 0.5 log 10 drop in the first two months, the vaccine was stable up to the end of the experiment, when viability only decreased approximately 0.6 log 10 (Table 2). RV PBV was incubated at 37 C. for 1, 2, 15, or 23 months. After 2 months, viability dropped <1 log 10 and after 15 months dropped 1.5 log 10. RV PBV was placed at 80 C. or 90 C. After 3 hours at 80 C., viability was essentially the same, and only 1 log 10 of viable virus was lost after 16 hours. Incubation at 90 C. was significantly more damaging, and RV PBV lost >1 log 10 of activity after 1 hour at 90 C.

(37) TABLE-US-00002 TABLE 2 Viability of RV after PBV and storage at different temperatures Rabies virus titer (log.sub.10 ffu/ml) Temp. Initial 1 hrs 2 hrs 3 hrs 16 hrs 1 M 2 M 3 M 15 M 23 M 22 C. 7.91 .sup.ND.sup.a ND ND ND 7.51 7.40 7.39 7.42 7.33 0.05 0.07 0.09 0.12 0.04 0.05 37 C. 7.56 ND ND ND ND 7.13 6.99 ND 6.1 5.58 0.14 0.17 0.01 0.09 0.22 80 C. 7.51 7.56 7.48 7.46 6.53 ND ND ND ND ND 0.07 0.09 0.15 0.05 0.05 90 C. 7.51 6.07 ND ND ND ND ND ND ND ND 0.07 0.09 .sup.aNot determined (ND)

(38) MAb 62-80-6 was used for capture and detection of RV G in an antigen capture assay and counts g-1 ml-1 were estimated from the best fit linear regression. In agreement with the measured virus titers, live attenuated RV PBV had the same counts g-1 ml-1 as the original ERAg333 virus (Table 3).

(39) TABLE-US-00003 TABLE 3 Antigenic G content of different RV vaccines measured by antigen capture assay using the 62-80-6 RV G MAb Storage Conditions ECL Antigen Time Temperature Counts g.sup.1 ml.sup.1a ERAg333.sup.b 20 M 80 C. 2200 Live attenuated RV PBV 20 M 22 C. 2200 Commercial vaccine 25 M 4 C. 1400 3 hrs 80 C. 980 Inactivated RV PBV 20 M 22 C. 1300 3 hrs 80 C. 680 Native ERA G.sup.c 18 M 80 C. 9100 Denatured ERA G.sup.c 10 mins 98 C. 7 Placebo 20 M 22 C. 3 .sup.aEstimated from the best fit linear regression of means of at least four statistical replicates from at least two biological replicates of eight 5-fold serial dilutions .sup.bParent strain for both live attenuated and inactivated vaccines; generated by reverse genetics .sup.cPurified RV ERA glycoprotein

(40) Inactivation of RV PBV by electron beam irradiation resulted in a decrease in antigen content but was similar to a commercial inactivated vaccine. When inactivated RV PBV was placed at 80 C. with high humidity for 3 hours, antigen decreased 48% while decreasing 30% in a commercial vaccine incubated under the same conditions.

(41) Live attenuated and inactivated RV PBV was used to vaccinate mice IM. Both live and inactivated RV PBV effectively induced rVNA titers by day 14 (Table 4). Live vaccine induced rVNA titers similar to ERAg333 and commercial vaccine. On day 30 rVNA titers increased in groups that received live RV PBV surpassing ERAg333 and commercial vaccine. Inactivated RV PBV induced rVNA titers on day 30 similar to commercial vaccine on day 14.

(42) The different dilutions of live attenuated RV PBV induced similar rVNA titers on day 14 and 30. Only the undiluted and 10-1 dilution of inactivated RV PBV vaccine induced rVNA titers by day 30. The decreased immunogenicity of the inactivated RV PBV is consistent with the in vitro antigen capture results.

(43) On day 30 all mice were challenged with canine street RV IM in the hind leg. All animals that received commercial vaccine survived (Table 4, p<0.01 compared to placebo). All animals also survived in groups that received ERAg333 or live RV PBV, consistent with the observed rVNA responses. In groups that received inactivated RV PBV all animals survived except in the 10-2 group. In this group, 80% survived despite only 3 individuals having a measurable rVNA response. Survivorship in this group was significantly different compared to the placebo (p<0.05) but not compared to the commercial vaccine or other inactivated RV PBV groups. At the experimental endpoint, animals from each group were randomly selected for rabies diagnosis, and all were rabies DFA negative.

(44) TABLE-US-00004 TABLE 4 Immunogenicity and efficacy of rabies vaccine preserved by vaporization in mice Commercial Live Attenuated Vaccine ERAg333.sup.a Placebo Vaccine Inactivated Vaccine Group 1 2 3 4 5.sup. 6 7 8 9 Dilution 10.sup.1 10.sup.2 10.sup.3 10.sup.2 None None None 10.sup.1 10.sup.2 Titer 6.8.sup.b 5.7 4.4 7.9 NA.sup.c NA NA NA NA Load ND.sup.d ND ND ND 300.sup.e.sup. 620 350 34 2.3 GMT day 14.sup.f 0.26.sup.g 0.11 0.2 0.37 <0.05 0.23 0.07 <0.05 <0.05 SD day 14.sup.h 0.50.sup.g 0.70 0.95 0.98 .sup.0.0097 0.56 0.11 0.24 0.022 GMT day 30.sup.f 1.60.sup.g 0.96 1.7 0.84 <0.05 0.58 0.27 0.13 0.05 SD day 30.sup.h 2.4.sup.g 15 2.9 1.9 .sup.0.015 1.9 1.4 0.58 0.14 Survival.sup.i 100%.sup.j 100% 100% 100% 22% 100% 100% 100% 80%.sup.k .sup.aParent strain for both live attenuated and inactivated vaccines; generated by reverse genetics .sup.bLog.sub.10 ffu in 0.1 ml dose .sup.cNot applicable (NA); cannot be determined for inactivated vaccines .sup.dNot determined (ND) .sup.eg of total protein in 0.1 ml dose .sup.fGeometric mean titer (GMT) of rabies virus neutralizing antibodies .sup.gIU/ml .sup.hStandard deviation (SD) of rabies virus neutralizing antibody titers .sup.iGroup size = 10 except placebo n = 9 .sup.jp < 0.01 compared to placebo .sup.kp < 0.05 compared to placebo

(45) Discussion

(46) RV ERAg333 was successfully formulated into stable, dry foam using PBV technology. Live attenuated RV PBV was stable for 23 months at 22 C. and 2 months at 37 C. Stability decreased as temperature increased, yet RV PBV remained stable for at least 3 hours at 80 C.

(47) An antigen capture assay was used to compare the antigen content of different vaccines. Since the ERAg333 virus was used for RV PBV preparation, MAb 62-80-6 which binds a linear epitope in the G was used for both antigen capture and detection. By using the same antibody for capture and detection, only trimeric G is detected. This was confirmed by low ECL counts for heat denatured purified RV G antigen. While the antigen capture assay is not a substitute for potency testing, it can be used to project if vaccines are immunogenic. Live attenuated and inactivated RV PBVs were both antigenic and immunogenic.

(48) A single dose of live attenuated or inactivated RV PBV effectively induced rVNA and protected all mice from IM challenge with a canine RV. By day 30 the antibody response to live attenuated RV PBV surpassed commercial vaccine. Previous challenge experiments using the same RV, dose, and route found 100% mortality in unvaccinated mice. However, the IM challenge, while more closely modeling natural infection, introduces greater variability.

(49) The advantages of PBV are that live attenuated RV can be stabilized and formulated into an oral vaccine suitable for use in domestic or wild animals. These results also support the use of PBV technology for other vaccines, e.g. RV-vectored ebola vaccine. Inactivated RV PBV if formulated into a potent vaccine and paired with a needle-less delivery system could be considered for human use in the future. Access to safe, potent vaccines is paramount for canine rabies elimination and prevention of rabies in humans.

Example 2: Preparation of Ambient-Temperature Stable, Inactivated but Therapeutically Active (IBTA) Probiotics and Other Bacteria

(50) FIGS. 1-4 describe an example including formulation of ambient-temperature stable, inactivated and metabolically active vegetative form of Bacillus anthracis and Lactobacillus acidophilus bacteria.

(51) B. anthracis Sterne strain bacteria were dry preserved using PBV (FIG. 1) and subsequently exposed to electron beam (EB) irradiation. Bacterial survival (CFU) and metabolic (reducing) activity of bacteria were measured using an MTT assay for two hours after the bacteria were reconstituted. A 12.5 kGy dose decreased survival of B. anthracis bacteria measured by colony forming units on agar more than one million times with only 0.5 log decrease in metabolic activity during the first hour after reconstitution (FIG. 2). In studies with other PBV-preserved L. acidophilus (see FIG. 3) we demonstrated that a 12.5 kGy EB irradiation dose decreased bacterial survival more than a million times with no detectable loss in metabolic activity during the first 1.5 hours after reconstitution (see FIG. 4). These results suggest that EB processing did not damage integrity of bacterial membranes, integrity of the bacterial intermembrane proteins and integrity of intracellular reducing enzymes. Thus EB radiation damages mostly nucleic acids.