Lipids are gaining enhanced momentum due to their vital role in the field of ribonucleic acid (RNA) therapeutics and vaccine development for diseases such as cancer and COVID-19.
Currently, there are 18 liposomal drugs approved by the U.S. Food and Drug Administration (FDA) and hundreds of lipid-based drug candidates in the clinic for a range of ailments. Lipid-based formulations and lipid nanoparticles have shown promise in drug development and delivery due to their ability to:
- Enhance active pharmaceutical ingredient (API) stability by protecting the API from immune response, proteases and other factors (Yingchoncharoen, 2016)
- Boost the solubility and bioavailability of drugs with poor water solubility (Yingchoncharoen, 2016)
- Passively target inflamed or tumor tissues due to their leaky vasculature (Danhier, 2010)
- Improve the toxicity profile of the entrapped API; targeted drug delivery could improve the toxicity profile of the API further as the APIs are delivered directly to the site of action (Yingchoncharoen, 2016)
- Ability to deliver difficult APIs such as RNA, which are prone to instability, nucleasemediated lysis, strong immune responses, and inability to reach the site of action.
The newest advancements in lipid-based drug delivery research and drug development is in the field of nucleic acid delivery, for APIs such as short RNAs for gene silencing or activation (for e.g. siRNA, miRNA, saRNA) and long RNA (mRNA) for applications in cancer therapy, enzyme replacement therapy, vaccines, and more. The first vaccine to enter clinical trials for COVID-19 was a mRNA vaccine, where the mRNA of a viral antigen was encapsulated in a lipid nanoparticle.
Methods of Liposome Manufacturing
There are numerous methods for liposome manufacturing (Wagner and Vorauer-Uhl, 2011). The challenge for liposome drug producers lies in ensuring liposome manufacturing in a scalable, robust and efficient process.
The rehydration manufacturing process involves the dissolution of lipid molecules in a solvent, a drying step and a hydration step under agitation followed by energy input such as sonification or extrusion to downsize the vesicles to unilamellar vesicles of a homogeneous distribution, followed by purification. Depending on whether the API is hydrophobic or hydrophilic, the API is added either with the organic solvent during the initial dissolution of the lipid or with the aqueous solution during the hydration step.
Another manufacturing method of note is the solvent injection method, where the lipids are dissolved in a solvent-like ethanol and rapidly mixed with an aqueous medium containing the API as shown in Figure 1.
The manufacturing method is often based on the final application. For example, the ethanol injection method is suitable for the production of small unilamellar liposomes and stable nucleic acid lipid particles which are used in intravenous applications. However, this technology is not suitable to create large liposomes as multilamellar liposomes and multivesicular vesicles, which are used for vaccines administered by subcutaneous injection or intramuscular injection. In this case, the rehydration method is used.
Critical Aspects to Consider While Choosing Lipids
Lipid source and quality have a direct impact on the impurity profile and properties such as the particle characteristics, stability and release profile of the final formulation. To achieve reproducible results with the final formulation, consistent quality of lipids is required, which is dependent on the quality of the raw materials used to synthesize the lipids, and good material characteristics of the lipid itself.
Lipid Purity
Lipid purity is critical because it influences the lipid’s stability, the bilayer structure in the formulation, the formulation stability and release profile. Lipid purity can be optimized by the quality of the starting materials, and modifying the manufacturing and purification techniques.
Lipid purity starts with high and consistent quality raw materials that offer the following attributes:
- Low level of byproducts
- Defined stereochemistry (D/L) and isomeric purity (cis/trans)
- Low bioburden and endotoxin levels
- Plant-derived raw materials with bovine spongiform encephalopathy (BSE)/transmissible spongiform encephalopathy (TSE) and non-genetically modified organism (GMO) certificates
- Use of Class II and III solvents; Class I solvents should be avoided based on guidelines from the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) in ICH Q3C.
- The final good manufacturing practice (GMP) process needs to be scalable and reproducible regarding yield and product quality. Manufacturing costs are impacted by product concentration, yield and reaction/work-up time. Reaction conditions that could lead to isomerization should be avoided. Scalability should be considered from the very beginning to ensure economy of scale with increasing batch size. The manufacturing process should aim to reduce the number of chemical synthesis steps and clearly define the GMP steps.
The purification process steps must be scalable as well. If possible, crystallization or liquid/liquid extraction methods should be used. Similarly, chromatography should be avoided because the process is complex, expensive and not scalable. Filtration over silica gel is a good alternative.
Consistent Quality
Synthetic lipids need to have consistent quality in every step of the drug development process. This means avoiding variability in the formulation development process as well as avoiding bridging toxicity studies, which also saves time and reduces costs. Working with a specialized life science supplier offering a consistently high product quality is one strategy for achieving consistent quality.
Good Material Characteristics
The material’s characteristics - solubility, crystallinity, stability and flowability - play an important role in the drug product GMP manufacturing process. Lipids are waxy by nature, which can result in slow dissolution rates and lead to challenges when handling large amounts.
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Optimizing the lipid’s surface characteristics for a fast and complete dissolution is a prerequisite for a reproducible liposome manufacturing process. Cryo-milling, spray drying, crystallization and lyophilization are the four processes available to enhance a material’s surface. These processes provide solubility improvements, higher purity, enhanced stability and easier handling characteristics, all of which will enable an easier formulation process and is relevant to GMP requirements. The use of spray drying and lyophilization processes results in a material of very high surface area and good handling characteristics. Homogeneity in lipid mixtures can be ensured with spray drying and lyophilization.
Crystallization is one of the most commonly used methods for surface enhancement. Lipids such as DOPC and R,S-DOTAP Cl are typically available in the market as an amorphous material, which are difficult to weigh precisely due to their lumpiness. Additionally, these compounds have poor dissolution characteristics.
In contrast, crystalline DOPC and DOTAP Cl as offered by MilliporeSigma offer key advantages over the amorphous analogues/products including:
- Enhanced stability: confirmed by stability studies of more than seven years at 25 °C/60% rH
- Fast dissolution rate
- Free-flowing powder, allowing for easy weighing and portioning
- Another example of how process improvements can dramatically change the physical properties of lipids is DOPE as shown in Figure 3. Traditionally, DOPE is available as lumps, gel or foam format, and has limited dissolution even after lyophilization. Now, suppliers have improved the DOPE manufacturing process to make it available as a free-flowing powder for fast and complete dissolution. Powder DOPE also offers easier handling compared to the conventional wax-like material.
New Lipid Technologies
Due to innovations in the field of new lipid structures, it is possible to change the properties of the formulation completely. For example, the gene therapy field has seen several generations of cationic and ionizable lipids with novel headgroups, linkers containing hydrolyzable ester bonds and disulfide bridges, and lipidoids.
An unmet need in lipid-based delivery is targetability, to the site of action. Peptides or mAbs could be potential targeting agents when effectively displayed on the surface. However, conjugating peptides directly to lipids is not as straightforward as the functional groups of the side chains of the constituent amino acids can also react. The side reactions lead to unwanted by-products that are difficult to purify, and lead to low process yields, which results in a final, expensive product that is not scalable and unsuitable for GMP production.
A solution to this challenge is the in-solution lipidation of the peptide of interest utilizing a solid phase synthesis process. It starts with a lipidated amino acid attached to a resin and subsequent amino acids are conjugated one by one. Fast work-up can be accomplished by simply washing the resin. This solid phase synthesis results in a lower-priced product, and in a scalable process that is suitable for GMP production.
Product Development of Lipids
Product development should ideally follow the drug development stages as outlined in Figure 4. As drug development is a long and costly process, choosing the wrong material will lead to negative financial implications and delays.
Regulatory Aspects for Lipid-Based Drug Formulations
There is no clear path to regulatory approval of liposome drug products due to the absence of global harmonized regulatory requirements for lipid excipients. Since the purity and quality of the lipid components can affect the quality of the lipid-based formulation, detailed information on chemistry, manufacturing and controls is requested by regulatory authorities.
Due to the challenging regulatory environments, it is recommended that the drug manufacturer works closely with a supplier that provides regulatory expertise and counsel through all phases of clinical development and commercialization, covering all aspects of quality assurance and documentation.
Conclusion
For successful drug development with synthetic lipids, the GMP manufacturing process needs to be scalable and reproducible in quality and yield. This is a prerequisite for consistent quality of the final product. The quality of lipids used has a major impact on the performance of the liposomal formulation. Lipids are available in different formats, physical states, and purities from different suppliers, and it is essential to choose the right lipids with the best characteristics depending on the application. When working with any novel lipids, feasibility studies to find the most optimal synthesis route and purification steps, that can be scaled up for GMP production are key.
While the regulatory process for liposome drug products is complicated with many different guidelines from different global authorities, they all agree lipid quality is critical.
To avoid high costs and surprises later in the drug development process, it is important to plan the product development beforehand and work with the same quality of excipients throughout drug development. This highlights the importance of working with the right supplier that offers consistent high-quality products, understands all steps of the drug development process and the regulatory environment and provides a high-level customer support.
References
- Charcosset, C, A. Juban, et al. Preparation of liposomes at large scale using the ethanol injection method: Effect of scale-up and injection devices. Chemical Engineering Research and Design. 2015;94:508–515.
- Danhier F, Feron O, Préat V. To exploit the tumor microenvironment: passive and active tumor targeting of nanocarriers for anti-cancer drug delivery. J Control Release. 2010;148(2):135–146. doi:10.1016/j.jconrel.2010.08.02.
- Yingchoncharoen P, Kalinowski D, Richardson D. Lipid-based drug delivery systems in cancer therapy: what is available and what is yet to come. Pharmacological Reviews. July 1, 2016;68(3)701–787.
- Wagner A, Vorauer-Uhl K. Liposome technology for industrial purposes. J Drug Deliv. 2011;2011:591325.
- Shiksha Mantri has a DPhil in Chemical Biology from the University of Oxford. She is currently the global Technical Product Manager, Synthetic Lipids and Advanced Drug Delivery at MilliporeSigma.
Author Biography
Shiksha Mantri is the Global Technical Product Manager for synthetic lipids and responsible for the entire GMP lipids business (portfolio and custom manufacturing) at MilliporeSigma. In her current role, she supports the top industry players and young start-ups in the RNA delivery and vaccines fields. She holds a PhD in Chemical Biology from the University of Oxford, U.K. and did her postdoc at ETH Zurich, Switzerland.