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PURPOSE
With recent pharmaceutical trends in the application of biologics for therapeutic treatments, the necessity for cryogenic storage has become increasingly common. Cryo-storage is a preservation technique that involves freezing biological materials at extremely low temperatures, typically between -20 °C and -80 °C for cryopreservation purposes and -196 ℃ when using liquid nitrogen for preservation.1 In the pharmaceutical industry, cryogenic storage has emerged as a valuable tool for preserving and storing samples over extended periods of time, facilitating long-distance transportation of biological specimens and establishing sample banks.2 Maintaining the physical stability of semi-solid formulations stored in these extreme conditions can prove difficult. This research study aims to assess the impact of cryo-freezing on various topical formulations. The physical characteristics, including appearance and viscosity, of the resulting products were analyzed following weekly cycles of refrigeration or freezing for a cumulative duration of two weeks. This study will enable us to make informed decisions on formulation design when extreme temperature storage is needed.
METHODS
Thirty unique topical formulations, spanning three different dosage forms, were compounded (Table 1). Initially, a traditional non-ionic surfactant-based cream using 5% w/w Arlacel 165, was manufactured for testing. This cream was compared with two polymeric based creams, that utilized similar oil and water phases with the addition of either 4% w/w Sepineo P600 or 1% w/w Pemulen TR1. Similarly, two ointment formulas, a petrolatum-based ointment and a polyethylene glycol (PEG) ointment, were tested against each other. Lastly, aqueous (water) based gels containing either 0.25% w/w, 0.50% w/w, or 1% w/w of Hydroxyethyl Cellulose (HEC), Hydroxypropyl Cellulose (HPC), Xanthan Gum, Carbopol® 974P, or Carbopol® 980 were made. Additionally, aqueous gels with either 1% w/w, 2.5% w/w, or 4% w/w Sepineo P600 were made, and a 20% w/w Poloxamer 407 were formulated. To further explore the potential for developing aqueous formulas that can resist freezing at low cryo-temperatures, aqueous gel formulations containing either 0.5% w/w Carbopol® 980 or 1% w/w HEC were compounded with varying ratios of water to Propylene Glycol (PG) (50:50, 25:75, and 10:90). Similarly, an anhydrous 1% w/w HPC gel containing PG, Ethanol, and Propylene Glycol Diacetate was formulated. All formulas were stored at different temperatures: CRT, 5 ℃, -20 ℃, and -60℃ for 2 weeks. The formulas were tested for viscosity and appearance at T=0 and following storage at different temperatures for 1 week and 2 weeks. The samples were analyzed for appearance both immediately following removal from the freezer and after thawing to CRT. Viscosity measurements were conducted in the thawed samples to monitor any viscosity alterations over time.
RESULTS
The results in the cream formulations were significant. The traditional non-ionic surfactant cream separated and was unusable immediately after low temperature storage; however, it stayed stable at CRT. By contrast, the creams made with polymeric agents withstood the significant temperature changes. While all formulations did freeze, the polymeric creams seemed unchanged after thawing from their initial appearance and viscosity. The two ointment products resulted in different observations with similar outcomes. The petrolatum ointment fully froze during the cryo-storage, while the PEG ointment did not. However, both formulations appeared unchanged after thawing from the initial appearance and viscosity. All aqueous gel formulations, except for those blended with PG, fully froze under cryo-storage. However, after thawing, the formulations were unchanged from the initial viscosity measurements at 1 week (Image 1) and 2 weeks. Conversely, the aqueous gels blended with 75% w/w and 90% w/w PG did not freeze, even when stored at the -60 °C condition. The 50% w/w PG products did freeze at -60 ℃ and became visually less fluid when stored at -20 ℃. Additionally, the anhydrous HPC gel containing 49% w/w PG, 40% w/w Ethanol, and 10% w/w Propylene Glycol Diacetate did not freeze, even when stored at the -60 ℃ condition.
CONCLUSIONS
If working with a biological material, it may be necessary to determine whether the biological material in question would exhibit improved performance when preserved in fully frozen state or if preserving its semi-solid structure would be more advantageous for maintaining the formulation’s stability and functionality. The general conclusion is to never use traditional creams for products that necessitate cryostorage, as these are likely to separate. If a cream dosage form is needed, use a polymeric based cream formulation. If freezing is preferred, then a petrolatum ointment or aqueous based gel should be investigated. Selecting a gelling agent may be performed based on compatibility with the intended biologic since almost any gelling agent would be physically appropriate for cryo-storage. Additionally, if a biological material retains improved stability characteristics when the product is stored at -60 °C, but should remain unfrozen, aqueous and anhydrous gels blended with high concentrations of PG, or a PEG based ointment should be considered (Image 2).