Stephen P. Cape

Successful respiratory immunization with dry powder live-attenuated measles virus vaccine in rhesus macaques

Measles remains an important cause of childhood mortality worldwide. Sustained high vaccination coverage is the key to preventing measles deaths. Because measles vaccine is delivered by injection, hurdles to high coverage include the need for trained medical personnel and a cold chain, waste of vaccine in multidose vials and risks associated with needle use and disposal. Respiratory vaccine delivery could lower these barriers and facilitate sustained high coverage. We developed a novel single unit dose, dry powder live-attenuated measles vaccine (MVDP) for respiratory delivery without reconstitution. We tested the immunogenicity and protective efficacy in rhesus macaques of one dose of MVDP delivered either with a mask or directly intranasal with two dry powder inhalers, PuffHaler and BD Solovent. MVDP induced robust measles virus (MeV)-specific humoral and T-cell responses, without adverse effects, which completely protected the macaques from infection with wild-type MeV more than one year later. Respiratory delivery of MVDP was safe and effective and could aid in measles control.

Stabilizing Formulations for Inhalable Powders of Live-Attenuated Measles Virus Vaccine

Carbon dioxide Assisted Nebulization with a Bubble Dryer((R)) (CAN-BD) processing allows particles to be made in the 3-5 mum size range, which is desirable for lung delivery, without destroying biological activity. In response to the Grand Challenge in Global Health Initiative #3, we have been developing an inhalable needle-free live-attenuated measles virus vaccine for use in developing countries. Measles was chosen because it is the number one vaccine preventable killer of children worldwide. Powders were processed by CAN-BD, where a solution containing excipients and live-attenuated measles virus in water was mixed intimately with supercritical or near superctitical carbon dioxide to form an emulsion. The emulsion was expanded to atmospheric pressure through a flow restrictor. The resulting plume was dried by heated nitrogen and the powders collected on a filter at the bottom of the drying chamber. Powders were analyzed using varying techniques including X-ray diffraction, scanning electron microscopy, Andersen cascade impaction, differential scanning calorimetery, Karl Fischer titration, and viral plaque assay. CAN-BD has been used to produce powders of live-attenuated measles virus vaccine with characteristics desirable for lung delivery. The powders retain viral activity through forming and drying the microparticles by CAN-BD, and have passed the WHO stability test for 1 week at 37 degrees C. The powders have an amorphous character and a glass transition temperature of around 60 degrees C. Lyophilization, the present standard commercial method of processing measles vaccine makes solids with a water content of less than 1%. By substituting myo-inositol for sorbitol and using the CAN-BD drying technique the water content can be lowered to 0.5%. The most successful formulations to date have been based conceptually on the current lyophilized formulation, but with modifications to the type and amounts of sugar. Of current interest are formulations containing myo-inositol, as they retain high viral activity and have low initial water content.

Preparation of Active Proteins, Vaccines and Pharmaceuticals as Fine Powders using Supercritical or Near-Critical Fluids

Supercritical or near-critical fluid processes for generating microparticles have enjoyed considerable attention in the past decade or so, with good success for substances soluble in supercritical fluids or organic solvents. In this review, we survey their application to the production of protein particles. A recently developed process known as CO2-assisted nebulization with a Bubble Dryer® (CAN-BD) has been demonstrated to have broad applicability to small-molecule as well as macromolecule substances (including therapeutic proteins). The principles of CAN-BD are discussed as well as the stabilization, micronization and drying of a wide variety of materials. More detailed case studies are presented for three proteins, two of which are of therapeutic interest: anti-CD4 antibody (rheumatoid arthritis), α1-antitrypsin (cystic fibrosis and emphysema), and trypsinogen (a model enzyme). Dry powders were formed in which stability and activity are maintained and which are fine enough to be inhaled and reach the deep lung. Enhancement of apparent activity after CAN-BD processing was also observed in some formulation and processing conditions.

Stabilization of measles virus for vaccine formulation

An attenuated live measles virus (MV) was characterized by several biophysical methods as a function of temperature and pH. Following a method developed previously, the resultant light scattering and spectroscopic data were synthesized into an empirical phase diagram that visually and simultaneously represents the entire data set. Using this empirically-based phase diagram, screening assays were developed to identify potential vaccine stabilizers. Various compounds are shown by these assays to inhibit the temperature-induced aggregation of viral particles, and also to protect the integrity of the viral envelope. Accelerated stability assays show that, upon thermal challenge, MV formulated with these excipients retains its infectivity to a significant extent. Thus, the enhanced physical stability produced by this method is shown to protect the biological activity of this important but labile vaccine.

Dry powder measles vaccine: Particle deposition, virus replication, and immune response in cotton rats following inhalation

A stable and high potency dry powder measles vaccine with a particle size distribution suitable for inhalation was manufactured by CO(2)-Assisted Nebulization with a Bubble Dryer(®) (CAN-BD) process from bulk liquid Edmonston-Zagreb live attenuated measles virus vaccine supplied by the Serum Institute of India. A novel dry powder inhaler, the PuffHaler(®) was adapted for use in evaluating the utility of cotton rats to study the vaccine deposition, vaccine virus replication, and immune response following inhalation of the dry powder measles vaccine. Vaccine deposition in the lungs of cotton rats and subsequent viral replication was detected by measles-specific RT-PCR, and viral replication was confined to the lungs. Inhalation delivery resulted in an immune response comparable to that following injection. The cotton rat model is useful for evaluating new measles vaccine formulations and delivery devices.

Inhalable Antibiotics Manufactured Through Use of Near-Critical or Supercritical Fluids

The development of unit-dose, inhalable, antibiotic microparticles for use in primary and combined therapy approaches to treating tuberculosis (TB), multi-drug-resistant (MDR-TB), and extensively drug-resistant TB is explored using the gentle drying process of Carbon-dioxide Assisted Nebulization with a Bubble Dryer (CAN-BD). The microparticles produced using this method contain capreomycin, kanamycin, and isoniazid, respectively, imbedded in L-leucine. Antibiotics were developed into inhalable antibiotic formulations for their utility in both first line and second line antibiotic treatment regimens. Capreomycin and kanamycin are typically administered by injection making them desirable candidates for the development of a needle-free delivery system that addresses the Grand Challenges in Global Health Initiative #3. In response to this challenge, unit-dose packaging that preserves powder properties by protecting them from moisture, oxidants, and UV exposure, and a low cost “active” dry powder inhaler, the PuffHaler, were developed and used as a prototype device, in addition to the Aerolizer, to disperse the microparticle antibiotic formulations. Antibiotic formulations show yields above 50% in small-scale powder production by CAN-BD. Capreomycin and kanamycin show improved powder yields in scale up experiments. The particle properties were characterized using scanning electron microscopy, Karl Fischer moisture analysis, Anderson Cascade Impaction studies, and X-ray diffraction. The inhalable antibiotic formulations are within a respirable size range (1–5 μm), and have less than 3% residual moisture. Unit-dose dry powder antibiotics have the potential to provide easy-to-use, stable products with improved safety profiles.

Synthesis of composite microparticles with a mixing cross

Fine particles of pharmaceuticals and other materials can be rapidly made by a patented drying process known as CO 2 -Assisted Nebulization with a Bubble-Dryer (CAN-BD), by aerosolization from a low-volume mixing device (e.g., a tee or cross). The compounds of interest are dissolved in water or an appropriate organic solvent and mixed intimately with supercritical or near-critical CO 2 by pumping the fluids through a mixing device at room temperature and about 83 bar. The mixture is then decompressed through a capillary tube flow restrictor into a drying chamber maintained at near atmospheric pressure. The aerosol plume is mixed with preheated nitrogen in the drying chamber and is dried in less than 5 s at temperatures between 0 and 60° C. The dry powders (hollow, porous, or solid) collected from the chamber can usually be in the particle size range (1-5 μm) optimum for pulmonary drug delivery. The use of a cross as a mixing device permits solutes or suspensions in two liquid streams and a nebulizing fluid (e.g., supercritical CO 2 ) to be mixed and desolvated to form heterogeneous microparticles. If one solid in the heterogeneous particles is a slowly dissolving coating material, the heterogeneous particles can release the second solid at a slower rate. If one solid is leached, a large porous particle with a high surface area can be obtained, yet its mean aerodynamic diameter is in the particle size optimum for pulmonary delivery. The primary advantages of this process are that (1) solids soluble in water and solids soluble in organic solvents can be micronized in a single-step process to generate heterogeneous particles, and (2) it facilitates drying of microbubbles and microdroplets at lower temperatures than those used in traditional spray drying processes.

Characterization and Application of Osmotic Dewatering to the Crystallization of Oligonucleotides

The recent increase in research and applications of oligonucleotides in new drug design has stimulated a need to determine the three‐dimensional structures of oligonucleotides and their complexes with various ligands. Three‐dimensional structure determination relies on X‐ray diffraction data from high‐quality crystals. However, only a very limited number of RNA molecules have been crystallized, and a broad record of experience in oligonucleotide crystallization is lacking. Osmotic dewatering is a crystallization method in which the oligonucleotide and its precipitant are concentrated at a controlled rate by the removal of water through a reverse‐osmosis membrane using a specified concentration gradient. Therefore, rates of nucleation and crystal growth can be controlled. In this study, a transport model was developed to predict the water removal rates at various concentration gradients. Experimental dewatering rate data agreed very well with theoretical calculations. Model predictions were applied to different osmotic dewatering crystallization devices to crystalize a model RNA, “U‐U dodecamer”. High‐quality dodecamer crystals were successfully grown and yielded a maximum X‐ray diffraction resolution of 2.46 Å and usable resolution of 3.0 Å. The results of this research will facilitate the applications of osmotic dewatering to crystallographic studies and industrial purification of biopharmaceuticals.