Category: Formulation and Quality
Purpose: The development of parenteral submicron particles for clinical and commercial use often faces the challenges of successful process scaling in a sterile or aseptic environment complying with good manufacturing practices. The path from lab-scale submicron particle formulations to the final drug approval is often covered with pitfalls due to the complexity of manufacturing nanomedicines .
Often, specific downstream processing is necessary to recover materials in the desired form and purity. In early stage development, submicron particle purification is usually performed by lab-scale centrifugation and decantation. However, the reproducibility of the results obtained by this process may be challenging. Moreover, in many cases it requires manual handling. Up scaling to larger volumes makes it even more labor intensive and time consuming. In this study, tangential flow filtration (TFF) was used as an alternative purification method. Here, the fluid is circulated along the surface of a membrane; no additional manual steps are required during the process.
The physical instability of particles in aqueous suspension can in general cause aggregation, flocculation, hydrolysis of particle-forming materials, and extraction of drugs out of the particles. Submicron particles are commonly dried via lyophilization to achieve long-term stability. Besides optimizing the lyophilization cycle, the optimal cryoprotectant formulation is critical in keeping the particles intact.
The objective of this study was 1) to evaluate TFF’s effectiveness in removing impurities, and 2) to develop a suitable lyophilization formulation using trehalose as cryoprotectant.
Methods: A colloidal suspension of particles in the range of 150-200 nm was prepared by the emulsification solvent evaporation method . One gram of RESOMER® RG 502 H (Evonik Nutrition & Care GmbH) was dissolved in dichloromethane (DCM) and dimethyl sulfoxide (DMSO) and mixed with an aqueous phase containing 1% (w/w) polyvinylalcohol (PVA) (Merck KGaA) using ultrasound. Purified water was then added to the emulsion as an extraction phase. The suspension was stirred at 350 rpm over night to evaporate the DCM.
The suspension was washed via TFF technique employing a KR2i TFF System® (Spectrum Labs) to remove excess stabilizer (PVA) and organic solvent (DMSO).
The Nuclear Magnetic Resonance (NMR) method was chosen as the analytical method for DMSO quantification. The suspension samples, before and after TFF, were measured by using a Bruker 600 MHz spectrometer.
The residual PVA was analyzed with a colorimetric assay. The absorption was calculated by setting the absorbance of an Infinite® M200 PRO (Tecan) at a wavelength of 689 nm.
The purified suspension was lyophilized with different concentrations of trehalose dihydrate (Carl Roth GmbH + Co. KG). A 40% (w/w) trehalose dihydrate stock solution was prepared and added to the colloidal suspension to reach 1, 2, 3 and 4% (w/w) final content, respectively. A control sample was prepared without any cryoprotectant.
Results: High quantities of DMSO and PVA were efficiently removed after 3 diafiltration volumes. An additional slight reduction after 5 diafiltration volumes was observed (Fig. 1A and 1B). The particle size remained unaffected by the filtration process (Fig. 1C). The total process time of a 1 g batch (100 mL suspension) using 3 diafiltration volumes was 1 hour. In comparison, the manual process by centrifugation and decantation took double as long. More than 98% of DMSO and about 86% of total amount PVA were removed from the submicron particle suspension.
The presence of trehalose in different percentages in the suspension prevented the agglomeration of particles while, without it, the average particle size and the PDI increased during the lyophilization process (Fig. 1D).
Conclusion: Tangential flow filtration has proven to be an effective alternative purification method to reduce the presence of impurities such as organic solvent residues and excess stabilizers compared to centrifugation, which is time- and labor consuming. It acts in a continuous and gentle way, keeping submicron polymeric particles unaltered without affecting the formulation specifications. In addition, it can be scaled up providing great benefits in terms of time and efficiency. Lyophilization has been shown to be more effective in stabilizing the submicron particles in the presence of trehalose.
This work is a first step in establishing effective downstream processes of purification and stabilization of submicron particles. The knowledge gained will be used in scale-up of sterile and aseptic nanomedicine production.
Andrea Engel– Head of Drug Delivery, Evonik Nutrition & Care GmbH, Darmstadt, Hessen, Germany
Maria Camilla Operti– Darmstadt, Hessen, Germany
David Fecher– Singapore, Singapore
Silko Grimm– Darmstadt, Hessen, Germany
Alexander Bernhardt– Darmstadt, Hessen, Germany
Oya Tagit– Nijmegen, Gelderland, Netherlands
Carl Figdor– Nijmegen, Gelderland, Netherlands