Introduction
Liposomes are nano-scale spherical-shaped vesicles comprised of one or more phospholipid bilayers, that have the ability to encapsulate hydrophilic or lipophilic drugs for the purposes of targeted drug delivery (Akbarzadeh et al. 2013). A number of strategies have been demonstrated for liposome manufacture but have suffered from the lack of reliable methods with sufficient throughput to enable a commercial scale (Mozafari 2005, Maherani et al. 2011, Worsham et al. 2019). Strategies for liposome synthesis focus on addressing and optimizing one or several of the key driving forces of vesicle assembly including the component solubilities, concentrations, and process thermodynamic parameters (i.e., temperature, pressure, etc.) (Mozafari 2005, Maherani et al. 2011). Manufacturing methods can be designed to fine-tune liposomes with various properties and, in doing so, can lend both advantages and disadvantages amenable to large-scale processing. In addition, selection of the manufacturing method often depends on the end product requirements for clinically efficacy including liposome size and size distribution, lipid composition, and the drug release characteristics, which together dictate the pharmacokinetic demonstration of adsorption, distribution, metabolism, and elimination (ADME).
The most successful examples of scaled methods for liposome manufacture to date have followed the principles of alcohol injection (Figure 1A) or crossflow techniques (Figure 1B), wherein dissolved lipids are precipitated from an organic solvent into an aqueous solution (anti-solvent) by means of reciprocal diffusion of the organic and aqueous phases (Jaafar-Maalej et al. 2010, Wagner et al. 2002, Wagner et al. 2002, Wagner et al. 2011, Wagner et al. 2002). A change in the local solubility of the lipids during this process ultimately leads to the spontaneous formation of liposomes that encapsulate a small volume of the aqueous solution. Depending on the chemical nature of the API, it can be encapsulated in the aqueous core or embedded in the lipid layer. The critical parameters for the formation of liposomes by this method are residence time and geometry of the mixing/intersection of organic-solvated lipid and the antisolvent which are dictated by programmed flow conditions. After liposome formation, the mixture containing undesired organic solvent and unencapsulated API can then be refined to the desired formulation strength and composition using TFF or similar methods (Wagner et al. 2002, Kim et al. 2012, Li et al. 2011).
The aforementioned production methods were designed to operate as a batch process, but the crossflow method is based on a liposome formation step which is continuous in its inherent mechanism (Figure 1B). So long as each feed stream is continuously fed, liposomes will be continuously generated. With continuous formulation of the feed solutions, the liposome formation step can proceed indefinitely. By implementing a continuous version of TFF, which supports refinement of the drug product to the desired end formulation, continuous manufacturing of liposomal drug products is a feasible concept.
Continuous versions of TFF have been explored for similar applications in the biologics sector. For continuous perfusion cell culture, the industry has moved from internal spin-filters to external retention devices such as alternating tangential flow (ATF) or TFF systems for media exchange (Pollock et al. 2013, Castilho 2015, Whitford 2015). Single pass tangential flow filtration (SPTFF) has been evaluated for cell culture harvest concentration and for protein concentration allowing this process step to happen in a continuous fashion instead of the batch mode required by traditional TFF (Arunkumar et al. 2017, Casey et al. 2011, Brower et al. 2015, Jungbauer 2013, Dizon-Maspat et al. 2012, Subramanian 2015). TFF concentrates product through multiple passes of a recirculating loop while SPTFF concentrates in an inline fashion with a single pass through multiple TFF cassettes in series. SPTFF enables product to be continuously fed to the next unit operation or process step with the additional benefits of lower system hold-up volumes. Designs for multiple SPTFFs in series, such as the CadenceĀ® In-line Diafiltration Module (ILDF), are becoming available and have been explored (Gjoka et al. 2017). Applying the ILDF design as the TFF/formulation refinement step in a continuous process is shown in Figure 1C.
Previous work with continuous TTF designs has focused on biologics manufacture. To date, no examination of continuous TFF designs have been performed for liposomal drug product manufacture. This paper investigates the impact of the organic solvent used in liposome formation, namely ethanol, on the design of continuous inline diafiltration for a liposomal drug product. To explore the continuous TFF/formulation refinement for liposomal drug product, a pilot scale ILDF system mimicking Figure 1C was explored (Figure 1D). The set up in Figure 1D evaluated a simulated ILDF configuration by analysing each concentration pass and dilution step individually with the objective of determining the number of passes/stages and the buffer consumption needed to achieve target solvent removal. The results were compared to the batch process option.