New Developments in Controlled Nucleation: VERISEQ Nucleation Technology

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The VERISEQ nucleation technology offers a commercially viable technique for cryogenically generating a uniform dispersion of microscopic ice crystals (or ice-fog).

Freeze drying, also known as lyophilization, plays an important role in drug manufacturing by stabilizing delicate pharmaceutical and biological products. Over the past several decades, significant progress has been made in the design of freeze-drying processes enabling the industry to take advantage of recently developed technologies, including those in the field of controlled nucleation.

Freeze drying was developed commercially in the 1940s to produce a dry product that can be readily reconstituted to its original form by adding solvent (usually, water) when required (1). Removal of moisture facilitates the slowing down of chemical, microbiological, and physical degradation processes, therefore, extending product shelf life.

The freeze-drying process is comprised of three main steps-freezing (solidification), primary drying (ice sublimation), and secondary drying (moisture desorption)-and can be time-consuming, taking up to several days. The overall efficiency and consistency of the entire process, as well as ensuring high-quality products, largely depends on the nucleation temperature. This temperature directly affects the size of ice crystals, which in turn, determines the pore size distribution and therefore, resistance of the porous freeze-dried matrix to vapour flow.

For nucleation to occur, two process conditions must be met-the product temperature has to be lower than the freezing point of the solution, and nucleation sites must be present to trigger the process. The temperature difference between the equilibrium freezing point and the ice nucleation point is known as super-cooling. A lower nucleation temperature, or a higher degree of super-cooling, results in more ice-nuclei and smaller ice crystals. On the other hand, higher nucleation temperature, or a lower degree of super-cooling, results in fewer ice-nuclei and larger ice crystals, which eventually form pores and pore networks. Larger pores enable higher sublimation rates, and hence shorter drying cycles, as well as reduced reconstitution times and improved finished product attributes. It is also important that all vials nucleate at the same temperature to ensure consistency of the product morphology, the resultant cake structure and appearance, as well as uniformity of the product from vial to vial. However, in absence of nucleation sites (i.e., uncontrolled conditions), which is common in smooth-wall sterilised glass vials, the spread in nucleation temperatures between different vials, and hence, non-uniformity of the final product, could be quite significant.  

The freezing step is one of the most important steps in the lyophilization process. Handling it in a “controlled” versus “uncontrolled” or “random” fashion results in a number of benefits to product manufacturers and end users.

Products that could benefit from implementation of controlled nucleation include biological products such as protein and peptide formulations, vaccines, liposome, and small-molecule drugs susceptible to physical and chemical degradation, as well as injectables that must remain effective from manufacture to patient administration (2).  Due to the rapid growth in biological therapies, the demand for freeze drying has never been higher, and this trend is expected to continue for the next decade (3).

The challenge of providing a solution to enable controlled nucleation of the liquid preparations in terms of nucleation temperature and time greatly depends on its scalability. A number of the proposed techniques that work well in laboratory conditions are, however, difficult or sometimes impossible to implement at a production level (4). Technologies utilizing ice crystals as nucleation sites are commonly referred to as “ice-fog” technologies.

VERISEQ nucleation technology: background, technical approach, and scale-up


The VERISEQ nucleation technology, developed by Linde Gases in cooperation with IMA Life North America, offers a commercially viable technique for cryogenically generating a uniform dispersion of microscopic ice crystals (or ice-fog). The ice-fog is formed by combining cold nitrogen (produced from liquefied sterile-filtered nitrogen gas) and hot steam in a mixing device outside the lyophilization chamber. Upon introduction into pre-cooled vials (or seeding the vials) containing the product to be freeze dried, these ice-fog crystals serve as nucleation sites. This causes a rapid and uniform nucleation of the product in a vial as well as between vials of the same batch at low degrees of super-cooling (5).

Hence, the ice-fog introduction follows a two-step approach: the product-containing vials are first cooled to a selected suitable temperature at or below their freezing point, and then, the ice-fog is introduced to facilitate nucleation.

A key challenge for the commercial implementation of VERISEQ nucleation technology was to generate a sufficient amount of ice-fog to fill the chamber, and to ensure its penetration inside the vials with various lyophilizer volumes and vial/stopper geometries. This was achieved by implementing a proprietary mixing assembly, which provided an efficient mechanism of quickly forming the ice-fog and circulating it throughout the freeze chamber. The VERISEQ nucleation system has no moving parts or other complicated components that would be difficult to steam or otherwise sterilise. It requires no chamber pressurisation and can be retrofitted to any freeze dryer.

While there are many approaches to achieve controlled nucleation, only few techniques can do so on a commercial scale. The above-described ice-fog technology can be effectively utilized in  laboratory and/or production-scale lyophilizers to induce uniform ice nucleation at reduced levels of super-cooling and eliminate vial-to-vial variability, which in turn, can help mitigate a host of related issues and lead to improved process parameters as well as product quality.

1. A. Siew, Pharm. Technol. 37 (5) 36-40 (2013).
2. A. Gupta, Int. J. Drug Dev. Res. 4 (3) 35-40 (2012).
3. Market Overview Freeze-Drying for Biotechnology and Pharmaceuticals (July 24, 2009),, accessed Mar. 16, 2015.
4. P. Thomas, Controlled Ice Nucleation Moves into Manufacturing: Interview with Michael Pikal (2011),, accessed Mar. 16, 2015.
5. P. Chakravarty et al., Ice-fog as a means to induce uniform ice nucleation during lyophilization, BioPharm Int. 25 (1) 33-38 (2012).


Eugene Wexler, PhD, is senior project manager, Chemicals & Environment, Linde.

Joseph Brower is process services manager at IMA Life.