Discussion
The assessment of the continuous ILDF set up for liposomal drug product formulation refinement was successful and provided a range of intriguing results. The notion of the permeate being impacted by the ethanol concentration in the batch process was reinforced by the results from the continuous ILDF experiment(s).
The use of the simulated ILDF (Figure 1D) showed that reduction of the initial ethanol concentration produced an ethanol concentration dependent rate of reduction in the number of passes/stages necessary to achieve the target ethanol removal. This led to the derivation of an equation (Equation 2) for the simulated ILDF concentration/dilution arrangement (Figure 1D) including the derivation of a function for ethanol concentration dependency of the rejection coefficient (Equation 3).
While these equations prove applicable and predictable under the simulated ILDF set up from Figure 1D, these equations proved less applicable to a real-world set up such as that shown in Figure 1C. The simulated ILDF method showed that the permeability of the hollow fiber was limited by the initial exposure of ethanol. The permeability then only improved a limited amount as the ethanol concentration decreased with each pass. By using the same hollow fiber to simulate each pass of a ILDF system, the efficiency of each subsequent pass was limited and not representative of a true ILDF arrangement. By using the data from the initial passes of the separate runs, a true ILDF data set was extrapolated and assessed.
The continuous ILDF model showed an almost linear reduction as compared to the exponential reduction in the simulated ILDF model (Figure 4B vs. Figure 3B). This may be because the permeability of the hollow fibers in the continuous ILDF model were set independently and had no impact on the subsequent hollow fibers as the ethanol concentration decreased. Additionally, the linear reduction of the ethanol in the continuous ILDF model limited the overall buffer consumption to that of a batch process where the rejection coefficient is zero. This showed that continuous ILDF would be more efficient than a solvent-based batch TFF with respect to buffer required.
Based on these findings, the impact of ethanol on the continuous ILDF design is less driven by a traditional rejection coefficient concept and more on ethanol’s effect on hollow fiber permeability. By reducing the initial exposure of each individual hollow fiber in a continuous ILDF system, the performance and overall efficiency of the system is improved. The most optimal design for a liposomal continuous ILDF process would involve significant upfront dilution (i.e. 5% initial ethanol concentration) in order to start the ILDF with minimal ethanol concentration. This would minimize the amount of ILDF stages needed and buffer required. This is similar to the dilution strategy recommended for albumin diafiltration though optimization is not specifically correlated to the impact of ethanol concentration on permeability (Jaffrin et al. 1994, Paulen et al. 2011). Additionally, these finding may prove applicable and beneficial to mRNA-LNP vaccines, which often look to minimize mRNA exposure to organic solvents as well as having massive production demands (Hou et al. 2021, Schoenmaker et al. 2021, Verma et al. 2023), which a continuous process design could help to meet.