Scalable microfluidic method for tunable liposomal production by a design of experiment approach.

Liposomes constitute a widespread drug delivery platform, gaining more and more attention from the pharmaceutical industry and process development scientists. Their large-scale production as medicinal products for human use is all but trivial, especially when parenteral administration is required. In this study an off-the-shelf microfluidic system and a methodological approach are presented for the optimization, validation and scale-up of highly monodisperse liposomes manufacturing. Starting from a Doxil®-like formulation (HSPC, MPEG-DSPE and cholesterol), a rational approach (Design of Experiments, DoE) was applied for the screening of the process parameters affecting the quality attributes of the product (mainly size and polydispersity). Additional DoEs were conducted to determine the effect of critical process parameters "CPPs" (cholesterol concentration, total flow rate "TFR" and flow rate ratio "FRR"), thus assessing the formulation and process robustness. A scale-up was then successfully accomplished. The procedure was applied to a Marqibo®-like formulation as well (sphingomyelin and cholesterol) to show the generality of the proposed formulation, process development and scale-up approach. The application of the system and method herein presented enables the large-scale manufacturing of liposomes, in compliance with the internationally recognized regulatory standards for pharmaceutical development (Quality by Design).

Vacuum-Induced Surface Freezing for the Freeze-Drying of the Human Growth Hormone: How Does Nucleation Control Affect Protein Stability?

In the present work, the effect of controlled nucleation on the stability of human growth hormone (hGH) during freeze-drying has been investigated. More specifically, the vacuum-induced surface freezing technique has been compared to conventional freezing, both with and without an annealing step. Size exclusion chromatography and cell-based potency assays have been used to characterize the formation of soluble aggregates and the biological activity of hGH, respectively. The results obtained indicate that controlled nucleation has a positive effect on both cycle performance and product homogeneity because of the formation of bigger ice crystals, and characterized by a narrower dimensional distribution. From the point of view of hGH stability, we observed that vacuum-induced surface freezing is not detrimental to the biological activity of the protein, or aggregate formation. In addition, the effect of 2 different formulations, including trehalose or cellobiose, on protein preservation was also considered for this study.

Vacuum Induced Surface Freezing as an effective method for improved inter- and intra-vial product homogeneity.

The stochastic nature of nucleation makes it difficult to control batch homogeneity in conventional freezing, and this lack of control is in contrast with the current emphasis on Quality by Design. Among the techniques which have been developed to overcome this problem, Vacuum Induced Surface Freezing is probably the most promising for application in manufacturing, because it does not require additional equipment and can be scaled-up more easily than other proposed approaches. In this work, we summarize the impact of Vacuum Induced Surface Freezing on product morphology, and the efficiency of the subsequent drying steps as well. We will show that this controlled freezing approach is extremely beneficial for both the efficiency of the freeze-drying process, and the quality and homogeneity of the final product. The hope is that this work could contribute to the commercial implementation of controlled nucleation technology, overcoming the final resistance to its widespread use. It is our opinion that this would be a substantial improvement, beneficial for both the pharmaceutical industry and the end users.

Influence of controlled ice nucleation on the freeze-drying of pharmaceutical products: the secondary drying step.

Vacuum Induced Nucleation is often discussed in the context of primary drying performances and its tunability, with the potential to tailor the nucleation temperature to the desired porous structure. Instead, here we investigate its influence on secondary drying dynamics and, in particular, on rate of desorption and vial-to-vial inhomogeneity. So as to track the evolution of residual moisture during secondary drying, vials were regularly collected through a vacuum-tight sampling device; the residual moisture and the morphology of the porous cake was then determined by Karl Fischer titration and Scanning Electron Microscopy, respectively. The control of freezing promotes the formation of larger ice crystals and, as a result, accelerates the sublimation of ice and slows down the desorption process. Overall, we found that it reduces the total (primary and secondary) drying time and produces much more uniform batches than those obtained by the conventional freezing, and this positive effect was observed since the end of primary drying. In conclusion, the control of freezing was beneficial to the total drying time reduction, vial-to-vial homogeneity and allowed a better control of product inhomogeneity.

Impact of vacuum-induced surface freezing on inter- and intra-vial heterogeneity.

This paper aimed to study the impact of freezing on both within-batch (inter-vial) and within-product (intra-vial) heterogeneity. This analysis has been carried out using two freezing protocols, the conventional shelf-ramped method and the Vacuum Induced Surface Freezing, and placebo formulations containing both crystallizing (mannitol) and amorphous (lactose and sucrose) excipients. The freezing conditions (i.e., the temperature of freezing, the temperature and time of the equilibration phase, and the filling volume) were found to have a dramatic impact on both the within-batch and the within-product homogeneity. Overall, we observed that the control of freezing can effectively minimize the variability in product characteristics, and moisture content, within the same batch. In addition to more uniform production, the control of freezing was found to be fundamental to achieve a more uniform product than that produced by the shelf-ramped freezing method. The influence of the freezing protocol on the crystallization process of mannitol was also investigated, showing that the temperature of freezing plays a key role in the formation of the mannitol polymorphs.