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Advantages of Enavail's Rapid Freezing (RF) Technologies
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In biopharmaceutical applications, chemical and physical instability of proteins and peptides is problematic in both formulation and storage. Proteins and peptides are more often stable in the solid state than in solution. The most commonly used method for preparing solid protein powder is lyophilization, in which particle growth during slow cooling (∼0.01 K/s) limits the particle diameter to a minimum of a few microns and very low surface areas (approximately 1 m2/g or less). This process also generates a variety of freezing and drying stresses due to solute concentration, formation of ice crystals, pH changes, and freeze-induced phase separation. All of these stresses can denature proteins to various degrees. Thus, stabilizers are often required in a protein formulation to protect protein stability during freezing and the drying processes. The same limitation is true for plunge freezing ultra-thin walled tubes and vials filled with protein solution into liquid nitrogen. |
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Submicron protein particles may be precipitated from aqueous solution by a variety of processes including spray drying and spray freeze drying (SFD). In spray drying, the feed solution is atomized into droplets that dry rapidly because of their high surface area and intimate contact with the drying gas. SFD involves atomizing a protein solution into a cold vapor phase over a cryogenic liquid to form droplets, which then rapidly freezes upon contact with the cryogenic liquid. Though smaller particle sizes and larger surface area can be produced, biologically active proteins can be inactivated due to denaturation at the air–water interface and unfold upon interfacial adsorption as well as by exposure to heat during solvent evaporation for spray drying. |
Enavail's RF technologies are novel particle engineering processes that can generate superior dry powder forms of nanostructured proteins and peptides.
In the RF processes, protein solution droplets come into direct contact with a cryogenic substrate, resulting in rapid freezing (~ 100K/s) of the solution. The resulting frozen particles are then lyophilized to obtain a more stable dry powder form of the protein or peptide with very high surface area (~100 m2/g). The RF-processed dry nanostructured microparticle aggregates can also be easily redispersed to produce a homogenous nanoparticle distribution in various dosage forms for uniform delivery of the protein/peptides. The rapid cooling of aqueous solutions without the need for atomization minimizes protein and peptide denaturation. In addition, no organic solvents or elevated temperatures are utilized in the RF processes when applied to water soluble peptides and proteins. Because of these unique advantages,
Enavail's RF technologies allow for the production of high surface area stable submicron and micron-sized protein and peptide particles without the loss of biological activity seen in other technologies and can open the door to new opportunities for depot, pulmonary, and transdermal protein and peptide delivery applications. |
Enavail's Elimination of Protein Agglomeration and "Burst Release"
Typically, protein and peptide drugs that require prolonged systemic delivery but have short biological half-life or poor oral bioavailability are most effectively delivered by incorporation into a biodegradable depot delivery system. However, a common problem associated with depot drug delivery systems is burst release, in which periodic releases of large quantities of encapsulated drug (accounting anywhere from 10-80% of the total drug loading) occur randomly through the degradation of depot delivery system. Burst release is caused by aggregation and the non-homogenous distribution and loading of drug materials within biodegradable delivery systems. As the degradable systems begin to erode, large particles of aggregated drug material are exposed and release rapidly resulting in unwanted bursts of systemically available drug material. These bursts usually occur in the early stages of degradation due to agglomeration of drug materials at the surface of biodegradable particle delivery systems, leading to inefficient delivery and significant toxicity hazards, particularly for hormones, growth factors, and anticancer therapeutics.
In the images below on the left a fluorescently labeled micronized protein was incorporated within a biodegradable microparticle system to illustrate the aggregation and non-homogenous distribution pattern that is seen in depot systems when traditional micronized protein materials are utilized. In the particles displayed in the image on the right, Enavail's RF technologies were utilized to create a nanostructured version of the same fluorescently labeled protein which was then incorporated into the same biodegradable microparticle delivery system as shown on the left.
From the image on the right it is clearly evident that Enavail's creation of its nanostructured protein material allows for reduced agglomeration and more homogenous loading of drug within the biodegradable system, allowing for more uniform and controlled release of drug material from the degradable depot system and elimination of burst release.
Advantages of Enavail’s Rapid Freezing Compared to Existing Methods
| Existing Methods |
| 1. Standard Lyophilization/Plunge Freezing |
2. Spray Drying/Spray Freeze Drying/Atomization |
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Advantages
- No agglomeration or denaturation
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Advantages
- Small particle sizes(submicron)
- High surface area
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Disadvantages
- Large particles(>micron)
- Low surface area
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Disadvantages
- Loss of protein(denaturation due to high interfacial surface area)
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