Controlled Ice Nucleation in the Pharmaceutical Industry

In order to keep a drug product’s structural integrity, potency, and homogeneity, which are all vital to the success of a pharmaceutical product, stabilization by lyophilization is ideal.

Lyophilization starts by lowering the product’s temperature to less than its freezing point in order to form ice crystals (nucleation). Yet, this process is random and often results in poor product reconstitution and heterogeneity.

This article outlines a number of methods that are employed to control ice nucleation which has been widely utilized in research and development environments and illustrate its use in product manufacturing.

It will focus on one of these techniques, ControLyo® which employs a pressurization and depressurization method to supply simultaneous ice nucleation. The benefits of implementing ControLyo will be looked at, including its role in creating a smooth transition between lab- and commercial- scale lyophilization.


Key objectives of the manufacturing process for all drugs are controlling reproducibility and inter-/intra- batch variability, but especially in the case of parenteral drugs. Lyophilization is part of this process that stabilizes a drug for transport and storage but requires retention of its structural integrity, potency, and homogenous quality.

In order to fully control the freeze-drying process, it is vital that every stage of lyophilization and the individual products are understood thoroughly and optimized while at the same time considerate of future upscaling of product manufacturing from the R&D laboratory to commercial production.

Ice nucleation

Successful freeze-drying begins with the initiation of the freezing phase (ice nucleation). Ice nucleation is a stochastic (uncontrolled and random) process that makes it difficult to create a uniform batch of lyophilized products of consistent quality.

The contents of individual vials usually nucleate over a wide scope of temperatures which can range between 10–15 °C less than the formulation’s thermodynamic freezing point in a laboratory freeze dryer and even more in a manufacturing cleanroom environment.

Vials that nucleate at separate temperatures that will greatly influence the resulting ice crystal size, will dry at different rates and have varying cake morphology, pore structure, and specific surface area.

A high degree of supercooling (colder nucleation temperature) results in small ice crystals, which leads to lower sublimation rates and small pores of high resistance in the product, as seen in Figure 1.

Using 1 °C/min shelf cooling rate (“uncontrolled”).

Figure 1. Using a 1 °C/min shelf cooling rate (“uncontrolled”). Images reprinted with kind permission of Dr. Margit Gieseler at GILYOS GmbH

A number of techniques and technologies have been investigated to initiate nucleation and optimize the drying process in order to overcome limitations. The most common of these include the utilization of a controlled nucleation technique, such as pressurization and depressurization technique or the addition of an annealing cycle.

For many years, adding an annealing cycle to maintain the formulation above its sub-ambient glass transition temperature (Tg’), and below the ice melting temperature for a specific length of time, has been the standard technique used to produce bigger ice crystals and minimize the fluctuation in drying rates, as seen in Figure 21.

Using 1 °C/min shelf cooling rate plus annealing (“anneal”).

Figure 2. Using 1 °C/min shelf cooling rate plus annealing (“anneal”). Images reprinted with kind permission of Dr. Margit Gieseler at GILYOS GmbH

Yet, this method can be detrimental to protein stability and does not standardize the nucleation temperature which can still lead to batch variability. In order to create nucleation, there are less common alternative methods, but a number of these options are only feasible at the laboratory-scale production of lyophilized samples and are not possible for larger-scale commercial production.

Although one of these techniques, the ice-fog method, controls nucleation, the vials typically nucleate within a minute or two from each other instead of simultaneously. This is because the distribution of the ice-fog in the convoluted freeze dryer chamber can be challenging.

The pressurization/depressurization method of controlled ice nucleation involves cooling the entire batch of vials to a temperature that is less than the thermodynamic freezing point but higher than the temperature at which stochastic nucleation starts, followed by pressurization of the freeze-dryer chamber with an inert gas like argon or nitrogen.

The excess pressure is quickly released (depressurization) when thermal equilibrium has been reached, causing ice crystals to form at the top of the solution and propagate throughout the vial within seconds. Using this technique, ice formation is induced at virtually the same time for all vials in the batch, as seen in Figure 3.

Using controlled nucleation at -3 °C (“CL@-3 °C”.

Figure 3. Using controlled nucleation at -3 °C (“[email protected] °C”). Images reprinted with kind permission of Dr. Margit Gieseler at GILYOS GmbH

The advantages of controlled ice nucleation (CIN) technology

ControLyo from SP Scientific is one pressurization/depressurization method that creates homogeneity of vials and between batches during the lyophilization process. It uses a series of pressurization and rapid depressurization steps and inert gas to generate instantaneous ice nucleation in all vials in the product chamber at a higher temperature.

This minimizes supercooling and produces the largest ice crystals possible. As the ice sublimes, large crystals produce bigger cavities, resulting in less resistance for subsequent drying of internal areas and the shortest possible drying time.

Some of the studies utilizing CIN have seen a number of benefits to the lyophilization process:

Reduced vial breakage

In the absence of annealing, some products are able to transition randomly through crystalline phases during the drying process, which can lead to vial cracking. Vial breakage is disruptive and costly but by using ControLyo technology which induces nucleation at warmer temperatures, it can be substantially mitigated.

Working with a large pharmaceutical company, it has been shown that lyophilization of a small molecule drug was enhanced with controlled nucleation happening at a higher temperature of -4 to -6 °C  with no cracked vials compared to uncontrolled nucleation at -16 to -19 °C with 14% cracked vials. The cake appearance was also consistent and considerably improved.

Decreased cycle time

The structural integrity of a biologic drug product relies on a gradual, slow drying process which can take a number of days to complete. ControLyo allows controlled ice nucleation to happen at warmer temperatures which produce bigger ice crystals with less resistance to mass transfer and also shorten drying time.

Previous studies have demonstrated that the primary drying time is reduced by 3% for every 1 °C increase in nucleation temperature2 In some cases, ControLyo has been shown to decrease the cycle time from 7.5 to 5.5 days which heightens productivity and supplies economic benefits (unpublished data).

Maintained drug potency

The success of the freeze-drying process can be measured by the end-product, which would be a drug product in many instances. Maintenance of drug activity is particularly challenging in the case of biologic drugs.

Increased repeatability, reproducibility, and homogeneity are good indicators of optimal lyophilization and are all enhanced with controlled ice nucleation, like utilizing ControLyo technology. A research study examined protein degradation with respect to freeze-drying heterogeneity in order to show the influence of controlled nucleation on drug activity3.

The study compared CIN using ControLyo and annealing processes for their effect on sodium phosphate buffer crystallization and degradation of β-galactosidase. Annealed lyophilized formulations exhibited a 12–40% loss of activity. In contrast, CIN formulations exhibited a 9–14% loss of activity.

Reduced reconstitution time

More time for reconstitution could be detrimental to the patients, especially in emergency situations. So, knowledge of extrinsic and intrinsic parameters that affect reconstitution time and reproducibility are vital performance factors for lyophilization.

Reconstitution time is shorter for products lyophilized with CIN, such as ControLyo, for the majority of formulations. This is thought to be because of the larger pores produced with higher ice nucleation temperatures.

Product quality

Every part of the freeze-drying process has a big effect on product appearance, quality, and uniformity. Consistency between and within batches is a key quality parameter. As all vials freeze at the same product temperature, variation among vials with ControLyo technology is minimal.

Suboptimal freezing causes defects in cake appearance. In order to optimize cake elegance, controlled nucleation with ControLyo can adjust freezing behavior. Using controlled nucleation the yield of the product can also be enhanced.

Protein aggregation, which can decrease product yield, tends to happen on the surface of ice crystals. Yet, the larger crystals associated with ControLyo have a decreased surface area and ControLyo users have seen a reduction in protein aggregation with better product yield.

Implementing ControLyo technology in a commercial environment

As previously discussed, there are a number of different approaches to control ice nucleation in a laboratory-scale freezer, yet a lot of these are not appropriate for commercial purposes. For instance, patient safety could be compromised by toxic issues and biological contaminants with the addition of nucleating agents.

Yet, it is fairly simple to implement ControLyo technology into a commercial dryer which is capable of steam-in-place (SIP) and has available ports to evacuate the chamber in the required time. Only minimal alterations must be made to the system and so, there is a nominal impact on existing protocols and validation.

A new control interface is one of the advantages of ControLyo implementation, which is easy to utilize whilst still complying to FDA’s 21 CFR Part 11 regulations on electronic record keeping.

Its software also allows any type of cycle (even those that do not need CIN) to be performed without any alterations to the external and internal equipment, making this method an ideal solution to maximizing usage and enhancing production output.

One of the first commercially available freeze dryers with the capability to control nucleation was SP Scientific’s LyoStar 3 freeze dryer with ControLyo technology. The success of ControLyo over the years means it is now possible to retrofit freeze dryers with this technology regardless of manufacturer.

Although other cycles that do not require ControLyo can still be run on the existing equipment, the retrofit can repurpose existing production equipment and expand the capabilities of the freeze dryer. The retrofit is time efficient as it is typically shorter than the time taken for the manufacturing cycle for a new freeze dryer.

SP Scientific also has a freeze dryer refurbishing service to extend the life of existing equipment by refurbishing, updating, repairing, and retrofitting previously owned lyophilization systems including retrofitting and upgrading controls systems.

This LyoRenewal capability allows each installation to be customized to the company’s standard control and operating procedures and other site requirements or corporate policies.

S20 LyoConstellation Freeze Dryer.

S20 LyoConstellation Freeze Dryer. Image Credit: SP Scientific

Scaling up lyophilization from R&D to commercial product manufacturing

Whether it is in early-stage development, clinical stages, pilot batches, or commercial manufacturing, the critical parameters which lead to the efficient production of a quality product are the same.

Even though these factors are the same at every lyophilization step, transferring from one stage to another frequently requires repeated optimization of these parameters because of the differences between equipment at each stage.

Understanding the limitations of the commercial freeze dryer before starting to scale up the drug development process can decrease the optimization time for this process. ControLyo technology is based on pressure and so, each vial, regardless of shelf location or tray position experiences extremely similar conditions at the same time.

The ControLyo nucleation process remains the same when scaling up from lab to commercial production; pressurizing to 28.5 PSIG and depressurizing to 2 PSIG in under 2 seconds. Successful nucleation has also been seen with ControLyo at pressures as low as 17.5 PISG to 2 PSIG in a 96 ft2 chamber.

The only requirement of the commercial freeze dryer for ControLyo technology is that there is an appropriate port or combination of ports to attain the speed necessary to achieve ice nucleation.

ControLyo is part of a range of tools (equipment and technologies) created by SP Scientific called Line of Sight® (LoS) which can be used at each stage of development and production for better efficiency, control, quality, and consistency in the lyophilization process.

This suite of technologies and PAT tools (LyoFlux® TDLAS, SMART™, Tempris®, and ControLyo®) are built into small-scale freeze dryers (LyoStar™ 3, LyoCapsule™) through to large scale commercial dryers (LyoConstellation™) which ensures the utilization of methodology and technology from one equipment can be reproduced and directly compared to another.

This technique enables higher confidence in the methodology and meaningful comparison of the results at every stage.

Process changes and meeting regulatory compliance

The US Federal Drugs Administration (FDA) strongly recommends a Quality by Design (QbD) method in order to assure quality product performance in drug manufacturing. This includes better product knowledge and so more control of the lyophilization process.

It must be established that product quality (e.g. purity, strength, and potency) and long-term stability using ControLyo is comparable or better than the current process, as with any modifications to a process.

The key difference between ControLyo and other freeze-drying methods is the utilization of pressurization and depressurization, as described earlier, which leads to simultaneously controlled ice nucleation.

So, it is not thought that there would be a need for concern. Even so, the FDA requires a series of evaluation experiments to establish if there is any effect of ControLyo on product attributes.

Yet, if there is an alteration which falls outside the determined design space, implementing ControLyo into the lyophilization process will need the end-user to submit a supplement (21 314.70 (e) or 21 601.12 (for a biological drug)) to the approved New Drug Application (NDA) or Biological License Application (BLA).


The ability to control ice nucleation so that every vial freezes simultaneously and generates a high-quality product with greater efficiency and less variability is one of the keys to successful lyophilization.

Although there are a number of techniques that can be used to achieve controlled nucleation outlined in this article, SP Scientific’s ControLyo technology utilizes a series of pressurization and depressurization steps and has been proven to achieve all of these goals.

ControLyo technology heightens the homogeneity of vials and improves cake morphology, decreases cycle time, and reduces vial breakage between batches. Implementation of ControLyo is quick and efficient, needing minimal downtime and having little effect on existing validation which also minimizes the impact of re-submitting supplement applications for FDA approval.

It is also viable to retrofit ControLyo onto existing equipment regardless of manufacturer, making ControLyo an extremely versatile technology. ControLyo’s benefits are not only within a small-scale freeze dryer, but ControLyo can be reproduced and compared directly to other larger freeze dryers during product scale-up with minimal effort through SP Scientific’s LoS suite of technologies.

This permits a product to move through different stages of development to commercialization more efficiently. Overall, ControLyo streamlines the development process and establishes consistent product quality for lower long-term economic burden and support of best patient outcomes.

References and Further Reading

  1. Wang B.S. and Pikal M.J. (2012) Stabilization of Lyophilized Pharmaceuticals by Process Optimization: Challenges and Opportunities. Am. Pharma. Review
  2. Searles J.A., Carpenter J.F. and Randolph T.W. (2001) The ice nucleation temperature determines the primary drying rate of lyophilization for samples frozen on a temperature-controlled shelf. J. Pharm. Sci.
  3. Mudhivarthi V.K. (2015) Controlled ice nucleation in freeze-drying: freezing heterogeneity and protein stability. SP Scientific Lyolearn Webinar.

About SP Scientific Products

SP Scientific is the synergistic collection of well-known, well-established, and highly regarded scientific equipment brands — VirTis, FTS Systems, Hotpack, Hull, Genevac, PennTech, and most recently i-Dositecno — joined to create one of the largest and most experienced companies in freeze-drying/lyophilization, production fill-finish lines, centrifugal evaporation and concentration, temperature control/thermal management, glassware washers and controlled environments.

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Last updated: Sep 4, 2020 at 8:43 AM


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