Thermostable Biopharmaceuticals For Delivery Without Reconstitution V. Bronshtein Supported by NIH Grants U01AI070350, 5R44AI080035, 5R21AI094508, and CDC Contract Number 200-2012-53276
Bottlenecks for Effective Applications of Vaccines and Other Fragile Biopharmaceuticals A need for maintaining a cold chain for vaccine storage and distribution. A need for reconstitution of vaccines with sterile water or aqueous solutions. A need for a conventional needle-based parental vaccine delivery. A need for using trained medical personal and special facilities for parental vaccination.
Could Biopharmaceuticals Be Delivered Without Reconstitution Using Methods Developed for Conventional Pharmaceuticals? Many conventional pharmaceuticals could be stored at ambient temperatures (AT) and delivered via oral (intestinal, sublingual, and buccal), transdermal, respiratory, vaginal, and anal delivery routes without reconstitution with water before the delivery, avoiding painful parenteral delivery and help of medical personal. Pharmaceutical industry had developed sophisticated methods and tools for production, tablets, dissolvable films, patches, suppositories, ointment, creams, and capsules (including enteric coated capsules for intestinal delivery) that are used for needle free delivery of the pharmaceuticals. The question is what is stopping us from doing the same for biopharmaceuticals?
Industry Problem: Most of Bio-pharmaceuticals (i.e. vaccines) currently produced using freeze-drying (FD) or spray-drying (SD) are not thermostable. Still, despite major limitations and shortcomings, freeze-drying and spraydrying remain the primary methods for stabilization of vaccines and other biopharmaceuticals in the dry state. Procedures that are currently used for preparation tablets, dissolvable films, patches, suppositories, ointment, etc. often include usage of materials and substances that are harmful for dry preserved biopharmaceuticals. In addition, they may include short term application of high temperatures, shear stresses, and other harmful conditions, destroying biologicals.
UST Solution for Production of Micronized Thermostable Biopharmaceuticals: First: Thermostabilization using PBV technology. Second: Micronization of the thermostable product using jet milling.
History and Development of Foam Drying and Preservation by Vaporization (PBV) 1956-1970. Annear preserved bacteria by foaming a syrup under vacuum. The syrup was obtained by evaporation. No freezing of the material was involved. Drawbacks: The process is applicable only to small volumes (several ml or less) of material. Syrup often does not foam. 1996. Roser and Gibbon proposed to use the same (Annear) process for other biologics. 1996. Bronshtein proposed using Preservation by Foam Formation (PFF) as an alternative to freeze-drying (Pharmaceutical Technology 28, 86-92, 2004). He suggested obtaining the syrup by boiling in order to make the process scalable. Drawbacks: Splashing, difficult to control and reproduce PFF process. 2004. Bronshtein proposed Preservation by Vaporization (PBV), during which a partially frozen material sublimates, boils and evaporates simultaneously (PCT Patent Application WO05117962A). It is scalable, easy to control and reproduce, and has minimum splashing...
Outside Research Supports the Superior Thermostability Offered by Foam Drying Monoclonal antibodies: Abdul-Fattah AM et al. 2007. J Pharm Sci. 96 (8): 1983-2008. Parainfluenza strain vaccine: Abdul-Fattah AM et al. 2007. Pharm Res. 24 (4): 715-727. Human serum albumin: Hajare AA et al. 2011. Curr Drug Deliv. Francisella tularensis live vaccine: Ohtake S et al. 2011. J Pharm Sci. 8 (6): 678-690. Salmonella typhi live vaccine: Ohtake S et al. 2011. Vaccine. 29 (15): 2761-2771. LaSota virus: Pisal S et al. 2006. AAPS PharmSciTech. 7 (3): 60.
Benefits of PBV Technology: Higher activity titer after drying and thermostability during subsequent storage (increased shelf-life). Eliminates the need of using a cold chain. Allows subsequent particle size reduction (micronization). Allows drying of vaccines encapsulated in gel microparticles for better intestinal delivery avoiding the need of reconstitution with water. Allows short-term (several hours) stability at 60 C to 90 C that could be used for encapsulation of dry powders in dissolvable polymeric films for buccal and transdermal delivery avoiding a need of reconstitution with water. Allows scalable continuous load barrier drying. Eliminate drawbacks of PFF.
Formulation of Patches and Quick-Dissolve Films for Delivery of Biopharmaceuticals 1. To make patches for transdermal delivery and quick-dissolve films for buccal, sublingual, and vaginal delivery, we cast liquid mixtures comprising water-soluble polymer, plasticizer, and sugar glass particles with biopharmaceuticals preserved inside the sugar glass into a mold. 2. The liquid mixtures can be prepared by melting polymer at elevated temperatures or by dissolving polymers in anhydrous solvents that do not dissolve the sugar glass. Sugar glass particles should be phase separated from the polymer in both solid and liquid state before casting. In addition, incorporating sugar glass particles inside a water-soluble polymer matrix should be performed in low humidity environment to avoid decrease of the sugar glass transition temperature. 3. Casting can be achieved by solidifying the melted polymers or polymer solutions in a mold by cooling, by evaporation of the solvent, fusion of polymeric powders by application of mechanical stresses, or by polymerizing monomers using irradiation or application of other physical and chemical factors. 4. To ensure that biopharmaceuticals can be placed inside melted polymer matrixes, we make preserved biopharmaceuticals thermostable for the short-term (i.e. an hour at 70 C to 90 C). For casting processes that include solvent evaporation we select solvents that are phase separated from sugar glass and do not harm biopharmaceuticals preserved inside the glass.
Quick-Dissolve Film Produced by Solvent Casting Method (In Collaboration with Dr. Lisa Rohan s Lab at MWRI) Magee Womens Research Institute
Examples of PBV Formulations for Proteins:
hbche Activity (%) Stability of Thermostabilized Butyrylcholiesterase (hbche) at High Temperatures 100 80 60 40 65 C 37 C Room Temperature 20 0 0 5 10 15 20 25 Time of Storage (month)
PA83 PBV Formulations: Immunogenicity
Immunogenicity of PA83 Foam-Dried Formulations P4 and P5 Experimental Design: Primary Immunization Secondary immunization Bleed 3 weeks 3 weeks Pre-bleed Day 21 Day 42 Day 70 Groups Balb/c 6wks Vaccine candidate Formulation Form Route Alhydrogel Dose at days 0 and 21 (µg/dose) 1 6 PA83 P4A Foam IM 0.3% 7.5 2 6 PA83 P4B Foam IM 0.3% 7.5 3 6 PA83 P5A Foam IM 0.3% 7.5 4 6 PA83 P5B Foam IM 0.3% 7.5 8 6 Saline Saline N/A IM 0.3% 7.5
Anthrax Lethal Toxin Neutralizing Antibody (TNA) Titers 7.5 (5) µg 0.3% Alhydrogel Formulation 4A 4B 5A 5B PA83 Saline Saline only GMT ED 50 ± SE Day 20 15±3 13±1 17±4 20±4 21±3 19±2 Day 42 249 ± 1112 89 ± 182 1083 ± 197 155 ± 396 113 ± 406 10 ± 0 Day 70 278 ± 362 172 ± 189 838 ± 167 138 ± 408 380 ± 404 10 ± 1 % Responders Day 42 67% 50% 100% 50% 50% 0% % Responders Day 70 67% 50% 100% 67% 67% 0% Data are represented at geometric mean effective dose 50 (ED 50 ) titers (GMT) % responders are determined when the GMT of a samples is to the GMT of a PA mab Results show that formulation P5A generated the highest TNA titers with 100% of mice responding after the booster dose. The response decreased slightly by study day 70, but remained significantly higher than the rest of the groups. The other foam dried formulations behaved similarly to the PA83 is saline.
PA83 Foam Dried Formulations: Stability at 37ºC
Foam dried Formulations Stability at 37 C: SDS PAGE and Western Blot Data
Summary and Conclusions SDS PAGE and Western blot data suggest that foam dried PA83 5A formulation is stable for 6 months at 37 C. Reconstituted PA83 after 6 month storage at 37 C does not lose its potential to bind to Alhydrogel adjuvant, as determined by the absence of the target molecule from the supernatant after Alhydrogel removal by centrifugation.
Examples of PBV Formulations for Viral Vaccines:
Thermostability and Activity of Live ERA Rabies Vaccine after PBV Drying and Encapsulation in Polyethylene Glycol (PEG) MW 35,000 10 Rabies vaccine activity titer (log FFU/ml) 8 6 4 2 Activity be fore drying Activity afte r drying Activity after 3 hours at 80 C Activity after encapsulation in PEG at 80 C Activity Activity ater 23 ater months 23 m at onths RT at RT Activity after 1 m onth at 37 C 0
Stability of PBV Preserved YF-VAX Vaccine17D at 37 C. Activity of YF-17D vaccine (log 10 PFU/0.5ml) 6 5 4 3 2 1 0 Form. that do not contain MAG (Metyl Glycoside) Control stored Control at -80 C Stored at -80 C Form. containing MAG Activity of Freeze-Dried YF-VAX 17D Activity Manufactured of freeze-dried by Sanofi YF-VAX Pasteur, 17D vaccine Inc. manufactured by Sanofi Pasteur Inc. Titer After 15 Months at RT: 4.55±0.23 (log 10 PFU/0.5ml) Formulations Including MAG 0 50 100 150 200 Days of storage at 37 C Formulations Not Including MAG
Particle Size Distribution After Jet Milling of PBV Preserved YF-VAX 17D 14 Statistics Graph (65 measurements) 10µm Volume (%) 12 10 8 6 4 2 0 0.01 2.01 4.01 6.01 8.01 10.01 12.01 14.01 16.01 18.01 Mean with +/- 1 Standard deviation error bar Particle Size (µm)
Stability of PBV Preserved MVA at 37 C 8 Activity Titer (Log 10 PFU/ml) 7 6 Formulation 1 Formulation 2 (Includes MAG) Formulation 3 (Includes MAG) Formulation 4 Stability of freeze-dried MVA vaccine (Baxter) 5 0 10 20 30 40 50 60 Time of Storage at 37 C (Weeks)
PBV Preserved YF-VAX 17D Can be Micronized Using Jet Milling with Minimum Activity Loss 7 6 Titer (log 10 PFU/0.5ml) 5 4 3 2 1 Titer of Preservation Mixture Before Drying Titer After Drying Titer Aftert Jet Milling (Micronization) 0
Stability of PBV Preserved Measles Vaccine at 37 C (Evaluated in Dr. Rota s Laboratory at CDC) % of Vaccine Activity Before Drying Measured in TCID50/ml 100 80 60 40 20 0 0 2 4 6 8 10 12 Time of storage at 37 C (Months)
Examples of PBV Formulations for Bacteria:
Survival of PBV bacteria (10 8 CFU/ml) at 37 C and 70 C Treatment Activity of L. rhamnosus Activity of L. jensenii Activity of L. crispatus Before drying Form. 1 Form. 2 Form. 3 139±17 --- 150±15 118±12 137±12 119±14 95±28 94±9 93±14 After drying Form. 1 Form. 2 Form. 3 After 1 hour at 70ºC Form. 1 Form. 2 Form. 3 After 3 months at 37ºC Form. 1 Form. 2 Form. 3 After 6 months at 37ºC Form. 1 Form. 2 Form. 3 After 11 months at 37ºC Form. 1 Form. 2 Form. 3 After 11 months at RT Form. 1 Form. 2 Form. 3 93±1.5 77±8 103±14 81±6 56±21 109±3 78±6 69±3 15±6 49±7 31±8 3.4±0.6 76±7 2.4±1.7 1±0.4 113±10 100±16 68±6 110±15 106±12 126±22 101±8 85±11 104±19 116±20 49±29 54±9 31±7 47±3 23±4 42±3 31±3 4.5±1 86±15 86±10 94±16 70±12 65±13 67±8 67±8 50±3 56±9 52±9 37±6 53±15 52±12 25±3 40±7 33±5 0 0 55±19 36±11 2.5±2.5
L. Rhamnosus PBV Formulation Micronized Using Jet Milling 200 μm 100 μm
Quick-Dissolve Film Produced by Solvent Casting Method (In Collaboration with Dr. Lisa Rohan s Lab at MWRI) Magee Womens Research Institute
Acknowledgements We would like to thank our collaborators from CDC and University of Pittsburg who allowed to share with you some results of our work.