Lyophilization Technology for Improving Stability of Small and Large Molecules

Introduction

The instability of drugs constitutes a major barrier to the commercial development and manufacture of pharmaceutical products. Lyophilization, or freeze-drying technology, remains an ideal method that leads to the immobilization of drugs and other components in a solid or powder state for an extended period without any undesired degradation.¹ Lyophilization eliminates the need for cold storage or sub-zero temperatures, making storage and transportation much more efficient. Lyophilization has also been applied to improve the solubility of poorly soluble molecules by amorphous solid dispersions (ASD).²

In this article, we will shed light on lyophilization to improve the stability of drug products. Over the years, many drugs have been approved as lyophilized products to improve shelf life, ease handling, and enhance stability. Those include trastuzumab (Herceptin), pembrolizumab (Keytruda), infliximab (Remicade), and Abatacept (Orencia) among others.³

Lyophilization Process

Lyophilization is a process that involves an initial freezing step that leads to solidification, followed by primary drying under cold or freezing conditions that lead to ice sublimation. A third drying step that leads to the removal of unfrozen water/moisture, as illustrated in Figure 1, is also done. Freezing and drying times, temperature,e and chamber pressure all should be adjusted, as necessary, depending upon product composition, structure, and volume.

Thermophysical properties, such as eutectic temperature (T_eu), glass transition temperature of cryo-concentrated solute (T_g’), collapse temperature (T_c ) of lyophilized produce,t and glass transition temperature of dried product (T_g ), are critical for lyophilization cycles and to achieve the quality product.

Sublimation is the primary driving force for drying a sample in a frozen state. It facilitates the removal of solvent from the sample. Primary drying remains the longest process for the removal of solvent by sublimation, while secondary drying takes 3-10 hours, depending upon the sample properties.⁴ Sublimation of water, for example, takes place at 4.5 mmHg and 0.009 °C as a eutectic phase interfacing solid, liquid, and vapor, the so-called triple point of the phase diagram.^5,6

Table 1 guides the selection of processing parameters for lyophilization cycles to achieve robust powders.¹

With the development of a freeze-dried product of nanoparticles, for instance, lyophilization leads to stabilization and maintaining the particle size distributions in the lyophilization. Other attributes, such as short reconstitution time, low residual moisture, and drug encapsulation efficiency, are important for an optimized drug product. For instance, freezing is carried out at -30°, -20° and -10 °C for freeze drying of fish oil in polycaprolactone (PCL) and primary drying takes place at -50 °C at 0.05 mbar pressure.⁷

For vitamin E in PCL, freezing is carried out at -45 °C for 120 minutes. Primary drying takes place at -20 °C for 8 hours and secondary drying takes about 6 hours at +20 °C.⁸ In encapsulation of docetaxel in hyaluronic acid, the freezing takes place at liquid nitrogen, the primary drying requires -35 °C for 60 h at 50 m Torr, while the secondary drying requires 0 oC for 24 h.⁹

Lyophilized products of a dispersed system are typically characterized for particle size, size distribution, zeta potential, polydispersity index for nanoparticles, drug contents, moisture contents, thermal analysis, reconstitution time, and microscopic analyses (SEM, TEM) among others.

Lyophilization of mRNA in LNPs

Meulewaeter et al. investigated the freeze drying of Covid-19 vaccines in lipid nanoparticles (LNPs) comprised of ionizable lipid C12-200, DSPC, cholesterol, Dimyristoyl-rac-glycerol-3-methoxypropylethylene glycol-2000 (DMG-PEG 2000) in composition of 50:10:38.5:1.5 mol%, respectively.¹⁰ Cytiva’s NxGen microfluidic system was used to prepare the LNP suspensions. Upon dialysis with the membrane (MWCO 12-14 kD), ethanol was removed, and the LNPs were stored in Tris, PB, S and at pH 7.4.

The freeze-drying of an aqueous suspension of LNPs was carried out with 25% sucrose or trehalose in Tris or PBS buffer comprised of 15.6 μg/ ml of mRNA and 12.5 m/v% protectant, which is subjected to freeze drying process in each 400 ul of protectant and mRNA LNP suspension. The encapsulation efficiency of mRNA/LNPs was determined by Quanti-iT RiboGreen assay. Ai et al. have demonstrated the stability of mRNA/LNPs after lyophilization at 4 °C and 25 oC but at 42 °C, the integrity of mRNA was compromised.¹¹

Conclusion and Future Perspectives

With the challenges stemming from the stability and solubility of small and large molecules, lyophilization remains one of the preferred methods for enhancing the shelf life and enhancing the bioavailability of life-saving medicines, respectively. Ascendia^® Pharmaceuticals’ AmorSol^®, EmulSol^®, NanoSol^® and LipidSol™ are enabling technologies that improve bioavailability of drug molecules. For vaccines and large molecules, requiring the lyophilization process, LipidSol™ can be used for the development of those drugs and improve the stability over an extended period. Our state-of-the-art cGMP clean room lyophilizer (Figure 2) can be used for the development of sterile drug products.

References

1. G. Degobert and D. Aydin, Lyophilization of nano-capsules: Instability sources, formulation and process parameters, Pharmaceutics, 2011, 13, 1112; (b) N. Kumar and U. Nautiyal, A review article on lyophilization techniques used in pharmaceutical manufacturing, Int. J. Pharm. Med. Res., 2017, 5, 478-484.
2. E. Valkama, O. Haluska, V-P. Lehto, O. Korhonen and K. Pajula, Production, and stability of amorphous solid dispersions produced by a Freeze-drying method from DMSO, Int. J. Pharm. 2021, 606, 120902.
3. Lyophilized marketed drugs: https://particlesciences.com/blog/lyophilization-of-pharmaceuticals-an-overview/
4. A. Molnar, T. Lakat, A. Hosszu, B. Szebeni, A. Balogh, L. Orfi, A. J. Sabo, A. Fekete and J. Hordea, Lyophilization and homogenization of biological samples improves reproducibility and reduces standard deviation in molecular biology techniques, Amino Acis, 2021, 53, 917-928.
5. K. A. Gaidhani, M. Harwalkar, D. Bhambre, and P. S. Nirgude, Lyophilization/freeze drying: A review, World J. Pharm. Res., 2015, 4, 516-543.
6. A. Baheti, L. Kumar, and A. K. Bansal, Excipients used in lyophilization of small molecules, Excipient, J. Excipients, and Food Chem. 2010, 1 (1), 41-53.
7. S. Bozdag, K. Dillen, J. Vandervoort, and A. Ludwig, The Effect of Freeze-Drying with Different Cryoprotectants and Gamma Irradiation Sterilization on the Characteristics of Ciprofloxacin HCl-Loaded Poly- (D, L-Lactide-Glycolide) Nanoparticles. J. Pharm. Pharmacol. 2010, 57, 699–707.
8. J. Crecente-Campo, S. Lorenzo-Abalde, A. Mora, J. Marzoa, N. Csaba, J. Blanco, A. González-Fernández, and M. J. Alonso, Bilayer Polymeric Nanocapsules: A Formulation Approach for a Thermostable and Adjuvanted E. Coli Antigen Vaccine. J. Control. Release 2018, 286, 20–32.
9. N. M. Bexiga, A. C. Bloise, A. M. Alencar, and M. A. Stephano, Freeze-Drying of Ovalbumin-Loaded Carboxymethyl Chitosan Nanocapsules: Impact of Freezing and Annealing Procedures on Physicochemical Properties of the Formulation during Dried Storage. Dry. Technol. 2018, 36, 400–417.
10.  S. Meulewaeter, G. Nuytten, M. H.Y. Cheng, S. C. De Smedt, P. R. Cullis, T. De Beer, I. Lentacker and R. Verbeke, Continuous freeze-drying of messenger RNA lipid nanoparticles enables storage at higher temperatures, J. Contr. Rel., 2023, 357, 149-160.
11.  L. Ai, Y. Li, W. Yao, H. Zhang, Z. Hu et al., Lyophilized mRNA lipid nanoparticle vaccines with long-term stability and high antigenicity against SARS-CoV-2, Cell Discovery, 2023, 9:9

Author Details 

Shaukat Ali, PhD and Jim Huang, PhD- Ascendia Pharma, Inc.

Publication Details 

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