Stabilization

 

Brief History 

 

The scientific community recognizes that the stabilization of biologicals requires arresting molecular mobility to stop the degradation processes of biological materials during long-term storage. This can be achieved only by vitrification, which is the transformation from a liquid into a supercooled or supersaturated, noncrystalline, amorphous solid state, known as the “glass state”. The basic premise is that the high viscosity of the glass state will arrest all diffusion-limited physical processes and chemical reactions, including the processes responsible for the degradation of biological materials. This premise is based on Einstein’s theory that establishes the inverse proportionality between viscosity and molecular mobility (or diffusion coefficients of molecules). In general terms, glasses are thermodynamically unstable, amorphous materials; however, they can maintain the same state for long periods of time because of their very high viscosity (10^12 - 10^14 Pa.s.); for example, a typical liquid has a flow rate of 10 m/s compared to 10^-14 m/s in the glass state.  

 

The presence of water in a sample has a strong plasticizing effect, which decreases the glass transition temperature (Tg) and thus limits stability at higher temperatures.  For example, for water, Tg is about –147°C , for 80% sucrose, Tg is about –40°C; Tg of 99% sucrose is about +52°C.  Therefore, if biopharmaceuticals are to be preserved without degradation in the glass state at an ambient temperature, they must be immobilized in strongly dehydrated “sugar” solutions.  In the immobilized (VitriLife™) state biopharmaceuticals are dormant, but can be returned to the active (or live) state after reconstitution with water.

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Dehydration Drying

 

Dehydration (drying) can be very damaging to vaccines and other biologicals if performed in the absence of protective molecules (i.e. sucrose, trehalose) that adsorb at the surface of biological membranes and macromolecules and replace water of hydration at the surfaces, and this way protects the biologicals from destruction associated with hydration forces that arise during dehydration.  Because of this proper selection of protective molecules are key to successful stabilization of biologicals at ambient temperatures without loss of their activity.

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Evaporative Drying 

 

A simple method way of drying is by evaporative drying (or desorbtion).  During evaporation water leaves a specimen from its surface into a dry air or vacuum.  However, before reaching the surface water should diffuse through the body of the specimen.  Thus evaporative drying is a diffusion limited process which is why desorption could be applied only for drying of small drops or very thin specimens with large surface to volume ratios.  After desorption a specimen should be cooled to achieve the glass state.

 

Evaporative drying (ED) was very successfully applied for producing ambient temperature (AT) stable formulations of many biopharmaceuticals including LAVs.  However, (ED) is very difficult to scale for most applications.  For this reason that freeze-drying (FD) and spray-drying (SD) technologies have conventionally been used as the primary methods for the stabilization of vaccines and fragile pharmaceuticals in the dry state.  However, there are fundamental reasons preventing FD and SD from delivering thermostable vaccines and many other biologicals. 

 

Freeze Drying & Spray Drying

 

Freeze-drying and spray-drying have failed to deliver thermostable vaccines and other biologicals. Despite its limitations and shortcomings, freeze-drying has remained, for more than 50 years, the primary method to stabilize fragile biopharmaceuticals and biologics (vaccines, therapeutic proteins, probiotics, etc.) in the dry state.  This is, in part, because of erroneous conventional belief that drying at low temperature would be less damaging, and in part because for many years no viable alternatives for scalable drying technologies were available.  Conventional freeze-drying takes too long, costs too much, and—in many cases—produces low yields because it is a very damaging process for many biopharmaceuticals.  Freeze-dried biopharmaceuticals, such as vaccines, require refrigeration and a cold chain to maintain stability and viability during transportation, storage, and delivery to the point of use.  Lyophilization-induced injury happens both during freezing and during subsequent ice sublimation from frozen specimens at intermediate low temperatures (between –50°C and –20°C).  It is at these temperatures that most damaging cryochemical reactions occur.  

 

PBV Vacuum Foam Drying

 

Vacuum foam drying was introduced to scale up evaporative drying.  In brief, the technique of foam drying is composed of boiling a very concentrated and viscous aqueous solution (syrup) under vacuum at ambient temperature (AT), such that it transforms into a foam.  During this process shear stresses that occur in the viscous liquid during the growth of vapor bubbles nucleate new bubbles that split thick films into thin films that can quickly dry under vacuum. The large surface area of the foam allows efficient desorption of the water from the bubbling syrup and solidification of the material in the foam; and at the end of the vacuum drying period the solution becomes a mechanically stable dehydrated foam.  

 

At least three independent groups have contributed to the development of foam drying:

 

• 1956-1970.  Annear preserved bacteria by foaming a concentrated bacterial syrup under vacuum.  The syrup was obtained by evaporation above the freezing point. 

• Drawbacks: The process is applicable only to small volumes (several ml or less) of material.  The syrup often did not foam.

• 1996. Roser and Gibbon proposed using a process similar to Annear’s for other biologics and they demonstrated the applicability of using modern freeze-drying equipment for executing the process.

•  1996. Bronshtein proposed preparing the syrup and subsequent foam by boiling a preparation above the freezing point in order to make the process scalable. (see: Preservation by Foam Formulation (PFF) as an alternative to freeze-drying. Pharmaceutical Technology 28, 86-92, 2004).  Drawbacks: Splashing during boiling before foaming begins is difficult to control resulting in a PFF process that is not reproducible.  Download the publication here.

• 2004. Bronshtein proposed Preservation by Vaporization (PBV) to eliminate the drawbacks of PFF.  During PBV, a partially frozen material sublimates, boils and evaporates simultaneously (see WO05117962A). This process has minimum splashing; thus it is scalable, easy to control and reproduce.

• PBV dries material by vaporization (simultaneous sublimation, boiling, and evaporation) from a slush state several degrees below 0°C. The PBV technology provides gentle, cost-effective and efficient industrial scale stabilization of proteins, viruses, bacteria, and other sensitive biologicals, thereby allowing production of products that are not possible by existing methods of manufacture. The PBV process comprises primary drying under vacuum from a partially frozen state (i.e. slush) at near subzero temperatures, followed by stability-drying at elevated temperatures (i.e., above 40 °C). 

• PBV is more efficient and less damaging than the freeze-drying process.  PBV primary drying is performed at near -0°C temperatures at which water vapor pressure is about 50 times higher than it is during primary freeze-drying.  PBV allows completely aseptic barrier drying as well as continuous load aseptic production.

• To learn more about VitriLife® formulations developed with PBV drying click here.