What is the role of organic co-solvent (t-Butanol) in freeze-dried and frozen formulations?

In the past year, over 40% of FDA-approved drugs have been lyophilized.1 The lyophilization process stabilizes the drug product, offering easier transportation and extending shelf life at room temperature.

Processing steps typically involved in the lyophilization process (freezing, primary drying and secondary drying) have the potential to expose proteins to harsh conditions and stresses, resulting in denaturation, aggregation and a loss of functionality in some circumstances.

Mitigation of stresses experienced by proteins during freezing, drying and storage is typically achieved through the use of cryo- or lyo-protectants in specific ratios. This remains a central theme in formulation development.

Jayesh Sonje, a Ph.D. candidate from Dr. Raj Suryanarayanan’s lab at the University of Minnesota, USA, recently delivered a webinar around the thermal characterization of an organic solvent - tert-Butyl alcohol (TBA) – and its use as an excipient in the freeze-drying process. This article summarizes that webinar.

Role of excipients in freeze-drying

A number of excipients are employed in freeze-drying; for example, buffers, surfactants, stabilizers, bulking agents and organic solvents. These are used either individually or in combination to ensure the protein is retained in its native state - the factor responsible for its biological activity.

The complicated interplay of excipients, protein composition, and processing conditions makes it challenging to optimize formulation composition and process parameters effectively.

It should be noted that an excipient’s physical form during freezing, drying, and storage will dictate its functionality once it enters the formulation. For example, stabilizers like sugars perform better in an amorphous state while bulking agents like mannitol are more suited to a crystalline state.

tert-Butyl alcohol (TBA)

Organic solvents such as TBA offer a number of benefits to freeze-dried formulations. These include improving the solubility of hydrophobic drugs, reducing drying time, enhancing product stability and improving reconstitution characteristics.2

TBA exhibits a high freezing point (24 °C) and is primarily crystalline at room temperature, subliming during freezing. TBA becomes entirely crystallized during the freezing stage, exhibiting long needle-shaped ice crystals. It also produces a lower cake resistance during the drying stage, with a larger specific surface area.

These beneficial properties can reduce drying times from 100 hours (with 5% w/v sucrose) to 10 hours (with 5% w/v sucrose plus 5% w/v TBA), substantially reducing the manufacturing time required for a drug product.3

TBA also enhances the stability of a drug – a benefit demonstrated for the approved Pfizer drug, Caverject® whereby the introduction of 20% v/v TBA solution increased stabilization of the active ingredient, alprostadil.2

Thermal behavior of TBA-water

Designing and optimizing ideal freezing and drying conditions frequently requires determination of eutectic temperature (Teu) - the temperature at which water and solute crystallize simultaneously. This be measured using X-ray diffractometry (XRD) and differential scanning calorimetry (DSC).

A binary phase diagram for the excipients utilized offers a wealth of useful information for developing and optimizing a freeze-drying cycle. Ascertaining the eutectic temperature also assists in determining the primary drying temperature – this should ideally be lower than the eutectic temperature in order to prevent melt back.

TBA is an organic co-solvent that has seen a great deal of research into its use freeze-drying. Despite this, some ambiguity remains around the phase behavior of TBA with respect to Teu, composition and solid phases.

Dr. Raj Suryanarayanan’s lab has been working on an approach to address some of this ambiguity, aiming to generate a refined phase diagram of TBA in water.

Eutectic composition in TBA-water system

An assessment was performed using TBA from 0-25% w/w under various conditions. This was analyzed using DSC and XRD techniques.4

Preliminary studies highlighted complex thermal events occurring during the heating of frozen TBA-water systems. A number of melting endo- and exotherms were produced on the heating curves at various concentrations, meaning it proved challenging to unambiguously attribute to phases.

The addition of an annealing step allowed the production of a more defined heating curve, but DSC experiments alone did not result in sufficient understanding of TBA phase behavior.

XRD experiments were conducted via the synchrotron located at the Argonne National Labs, Illinois, USA. This instrument enabled a rapid scan time of 1 second in transmission mode, allowing data to be acquired in almost real-time.

By combining DSC and XRD it was possible to identify different phases determined by TBA composition. Less than 20% w/w TBA resulted in the production of three endotherms that corresponded to TBA-dihydrate melting, TBA heptahydrate and ice melting (eutectic composition), and ice melting.

A TBA concentration higher than 20% w/w resulted in the creation of an exotherm event as TBA-heptahydrate crystallized and a further endotherm event as TBA heptahydrate and ice melted (eutectic composition).

DSC was used to confirm the eutectic composition, and this was then compared to binary phase diagrams which had been generated by other research groups.

Refined Phase Diagram. Eutectic Composition 22.5% w/w TBA, Eutectic Temperature - 8 °C, Phases TBA heptahydrate + Ice.

Figure 1. Refined Phase Diagram. Eutectic Composition 22.5% w/w TBA, Eutectic Temperature - 8 °C, Phases TBA heptahydrate + Ice. Image Credit: SP Scientific Products

This allowed the eutectic composition to be defined as 22.5% w/w TBA at a eutectic temperature of -8 °C with phases of TBA heptahydrate and ice.3

Dual functionality of mannitol in TBA solutions

Once the phase composition of TBA had been determined, mannitol was added to the mixture in order to evaluate its cryoprotective ability and characterize its phase behavior in frozen TBA-water systems (5-30% w/w TBA).5

Mannitol is a commonly used bulking agent in freeze-dried formulations. Aqueous mannitol has been well characterized as demonstrating two glass transition temperatures and the tendency for partial crystallization during freezing as a hemihydrate.

DSC heating curves of 5% w/w mannitol in 5-30% w/w TBA suggested that mannitol’s glass transition temperatures were not impacted by TBA. It was also noted that TBA at specific concentrations actually delayed mannitol crystallization during the freezing stage.

Increasing the concentration of TBA (at 30% TBA) led to overlapping endotherms attributable to crystallization of both mannitol and TBA. Further XRD experiments were useful in helping to resolve these complicated thermal events.

When these results were combined with the XRD data using 22.5% w/w TBA and 2% mannitol, it was clear that mannitol kept its amorphous properties during freezing and heating until -20 °C. At this temperature, mannitol crystallized into the anhydrous delta form in the presence of TBA during heating.

Amorphous mannitol’s capacity to function as a cryoprotectant was explored further by employing human serum albumin (HSA) as a model protein in the TBA-water system. Formulations were freeze-thawed or freeze-dried before being assessed using dynamic light scattering (DLS).

Individually, TBA and mannitol were found to be ineffective as stabilizers. When these were combined with TBA ≤ 22.5% w/w, however, mannitol remained in an amorphous state and there was minimal HSA aggregation. These results were comparable to those achieved using a common lyoprotectant, such as sucrose.

Results showing particulate count (subvisible particles) measured using dynamic light scattering in prelyophilization, freeze‑thawed and freeze-dried samples.

Figure 2. Results showing particulate count (subvisible particles) measured using dynamic light scattering in prelyophilization, freeze‑thawed and freeze-dried samples. Image Credit: SP Scientific Products

Without the addition of TBA, mannitol was observed to crystallize as a hydrate and HSA aggregation took place.4

Conclusions

Characterization of TBA as an excipient and in combination with mannitol was described in Jayesh Sonje’s webinar. The thermal events noted during warming, and their characterization by DSC and XRD, enabled the generation of well-defined phase boundaries as well as TBA’s eutectic temperature and composition.

It was also clear that when used in the presence of ≤ 22.5% w/w TBA, mannitol offered the potential to act as a cryoprotectant during freezing, as well as a bulking agent in the final lyophile during drying.

References

  1. BCC research, 2018 - 2026
  2. Teagarden, DL et al, Eur. J. Pharm. Sci. (2002) 15:115
  3. Kasraian K et al. Pharm Res. 1995; 12 (4): 491-5
  4. Bhatnagar, BS et al, Phys. Chem. Chem. Phys. (2020) 22(3):1583-90
  5. Sonje, J et al, Mol. Pharm. (2020) 17(8):3075–3086

Acknowledgments

Produced from materials originally authored by Jayesh Sonje from the Department of Pharmaceutics, University of Minnesota.

About SP Scientific Products

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

SP is part of SP Industries, Inc., a leading designer, and manufacturer of state-of-the-art laboratory equipment, pharmaceutical manufacturing solutions, laboratory supplies and instruments, and specialty glassware. SP's products support research and production across diverse end-user markets including pharmaceutical, scientific research, industrial, aeronautic, semiconductor, and healthcare. In December 2015, SP Industries was acquired by Harbour Group, a private investment firm founded in 1976. Harbour Group is a privately owned, operations focused company based in St. Louis, Missouri. Headquartered in Warminster, Pennsylvania, SP has production facilities in the USA and in Spain and the UK in Europe and offers a world-wide sales and service network with full product support including training and technical assistance.


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Last updated: Aug 19, 2021 at 2:45 AM

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