Process Development of a Drug Delivery Nanoemulsion and Post Process Sterile Filtration
Posted on April 25, 2016
By: Dr. Yang Su, Kyle Jandrasitz, and Steven Mesite
Microfluidics International Corporation, 90 Glacier Dr. Suite 1000, Westwood, MA 02459, USA
Ross Turmell, Dr. Martha Folmsbee, and Kevin Marion
Pall Life Sciences, 25 Harbor Park Dr., Port Washington, NY 11050, USA
Emulsions are mixtures of two or more immiscible liquids with one finely dispersed in the other. The many advantages of nanoemulsions, such as enhanced bioavailability with increased drug solubility, protection of encapsulated drugs from hydrolysis and enzymatic degradation, minimal side effects or toxicities due to increased drug retention time, and better controlled release capabilities, make them attractive drug delivery systems. Additionally, nanoemulsions have also evolved as promising novel vaccine adjuvants in the past two decades since the discovery of their ability to trigger improved immune response without the addition of immunostimulator1.
Most adjuvanted vaccines are administered through injection therefore sterilization is a crucial step. For nanoemulsion systems with heat-labile substances, terminal sterilization with heat is not always an option and sterile filtration is employed. It has been observed that some lipid-containing solutions such as nanoemulsions and liposomes are among fluids with high potential of bacteria penetration of sterilizing-grade filters. Furthermore, low filtration throughput, low or variable volumes through filters, and low recovery of oil phase have also been observed with these solutions.
Microfluidizer® technology utilizes intensifier pump to push multi-phase fluids through the microchannels of an interaction chamber at constant pressure. The fluids are exposed to extremely high shear and intense turbulent energy dissipation inside the fixed geometry interaction chamber. The results are smaller particles and a tighter particle size distribution. The system can either running continuously or under a batch processing mode and can accommodate materials with high solid content, high viscosity, and a wide range of temperatures. The product temperature can be regulated by a heat exchanger.
Figure 1. Schematic of Microfluidizer® Processor
Generally, particulate or microbial contamination can come either from external sources, within the process, or during maintenance. During filtration, the contaminants are removed by the filter matrix through three removal mechanisms and two retention mechanisms.
Removal mechanisms work to get the particles to interact with the filter matrix:
• Direct interception: removes the contaminant particles via physical sizes, particles are physically too large to pass through the filtration matrix are retained in or on the matrix.
• Diffusional interception: removes the contaminant particles due to random motion as particles pass through the filter matrix.
• Inertial impaction: contaminant particles repeatedly change moving directions inside the filter matrix to follow the tortuous pathway. Inertial forces cause those particles to impact on the filtration medium and are retained even though the particles are smaller than the ratings of the filtration membrane.
Retention mechanisms work to ensure that particles stay in place:
• Mechanical retention: occurs due to the relative particles size been greater than that of the filtration pore size.
• Adsorption: occurs due to electrochemical charge differentials between the filtration matrix and the contaminant particles in the fluid stream.
Figure 2 shows the surface and cross-section of a 0.2 μm rated filter membrane challenged with bacteria. The surface view indicates that even the membrane is rated as 0.2 μm, many pore openings at the membrane surface are actually greater than 0.2 μm. The edge view shows that the challenge bacteria can penetrate through membranes up to 10 microns, while the thickness of filter membranes is roughly between 40 to 150 microns. Therefore, the thickness of the membranes along with the use of multilayer approach is critical to bacterial retention, especially with the high risk fluids.
Figure 2. Surface View (Left) and Edge View (Right) of 0.2 μm Rated Membrane Challenged With Bacteria2
Filter Challenge Tests
Both sterilizing rating tests and process specific filter validation or qualification tests are conducted according to industry standards3,4. It’s required that sterilizing filters are challenged with the micro-organism Brevundimunas diminuta at a minimum concentration of 107 CFU/cm2 of effective filter area. For sterilizing grade filters, the filters much provide a complete sterile effluent which means the downstream count of micro-organisms must be zero.
Validation of sterile filtration and achieving high efficient filtration-based sterilization require a robust solution with combined process development and filtration technology. In this study, Microfluidics and Pall collaborate to develop a filtration-based alternative to terminal heat sterilization for a biopharma nanoemulsion.
- Model Drug Delivery System
The drug delivery system studied was an oil-in-water nanoemulsion. The dispersed phase or the oil phase consists of 5 wt% squalane oil and 0.75% Span 85 as surfactant. The aqueous phase was formulated with 0.75 wt% Tween 80 and deionized water.
- Nanoemulsion Generation
Nanoemulsions were generated using a high shear Microfluidizer® processor (M110-P) equipped with a 75 μm F12Y interaction chamber and a 200 μm H30Z auxiliary processing module. Each emulsion sample was processed at various pressures for a number of passes.
- Particle Size Analysis
Particle size and size distribution of the obtained nanoemulsions were determined by a laser diffraction instrument (Horiba LA-950).
- Sterile Filtration
All nanoemulsion samples were passed through Pall Supor® EKV sterilizing grade filter first as a pre-filtration step. The final sterile filtrations were performed with a Pall Fluorodyne® EX Grade EDF sterilizing grade filter.
- Bacterial Challenge Tests
The Fluorodyne® EX EDF filter was challenged with Brevundimonas diminuta using the selected squalane nanoemulsions as the test solution. The tests were conducted under three different test pressures of 10, 30, and 60 psi following the standards. Three individual filters were challenged at each test pressure. Particle sizes of the test nanoemulsions were also measured post filtration.
Particle Size & Size Distribution
The particle size and size distribution of Microfluidized nanoemulsions under different process conditions were given in Table 1 and Figure 1. In this study, the goal was to produce nanoemulsions with various sizes and determine their influences on the subsequent sterile filtration. As shown in both of Table 1 and Figure 1, the goal was successfully achieved by simply control the process pressure and the number of passes during the Microfluidizing processes. The droplet diameters of the generated nanoemulsions varied in the ranges of 113-171 nm (D50) and 182-303 nm (D95). All samples also have uniform particle size distributions.
Table 1. Particle Size of the Squalane Nanoemulsions
The bench scale throughput and average filtration flux of passing squalane nanoemulsions with various sizes through Supor EKV filter are plotted in Figure 4. As expected, Figure 4 indicates that both of the throughput and the flux are inversely proportional to the droplet size of the nanoemulsions. Very high throughput of 1315 L/m2 and flux of 2187 L/m2/hr were observed with squalane nanoemulsion that has a D50 size of 124 nm.
Figure 4. Filtration Throughput and Flux of Squalane Nanoemulsions Using Supor EKV Filters
Using this nanoemulsion sample, additional throughput and flux studies were performed with Supor EKV filter placed directly over Fluorodyne EX EDF filter. A throughput of greater than 1000 L/m2 at an average flux of more than 4300 L/m2/hr were achieved at a filtration pressure of 30 psi.
The results of bacteria challenge tests are summarized in Table 2. Table 2 confirmed that all challenge levels were higher than the minimum required level of 107 CFU/cm2. All challenged filters showed complete retention with zero count of bacteria in the effluent.
Table 2. Bacteria Challenge Test Results
The particle sizes (D50) of the squalane nanoemulsion pre- and post-filtration through the 0.2 μm rated Fluorodyne EX EDF filter membrane are shown in Figure 5. Only data for tests under filtration pressure of 30 and 60 psi are presented. It can be seen from Figure 5 that there were no changes in the nanoemulsion particle sizes after filtration. This suggests that the filter membrane did not affect the oil phase recovery of this test solution.
Figure 5. Particle Size of Squalane Nanoemulsions Pre- & Post-filtration
Process of producing a drug delivery nanoemulsion was developed using Microfluidizer® processor. High sterile filtration efficiency can be achieved by combining the nanoemulsion with optimum particle size distribution and selected sterile filtration membrane material. All filters were successfully validated with the bacterial challenge tests. Conducting bacterial retention tests as early screening is a critical step in the development of drug delivery nanoemulsion formulations and evaluation of proposed manufacturing processes prior to their finalization.
• A high shear Microfluidizer® processor was used to develop a drug delivery nanoemulsion.
• High filtration efficiency can be achieved by combining a nanoemulsion with optimum particle size distribution and appropriate sterile filtration membrane material.
• All challenged filters showed complete retention at 10, 30, and 60 psi test pressures during bacterial challenge tests.
• The particle sizes of test nanoemulsions did not change post filtration.
• This study demonstrated the benefits of early screening to provide a technical solution for complex applications.
1. Editors: C. Foged, T. Rades, Y. Perrie, and S. Hook, Subunit vaccine delivery, Springer, 2015
2. M. Osumi, N. Yamada, and M. Toya, PDA J. Pharma. Sci. & Technol., 1996 (50): 30-34.
3. ASTM, Standard Test Method for Determining Bacterial Retention of Membrane Filters Utilized for Liquid Filtration American Society for Testing and Materials (ASTM) 2005, ASTM Standard F838-05.
4. PDA, PDA Technical Report No 26, Sterilizing Filtration of Liquids. PDA J Pharm Sci Technol 2008, 62, (S-5), 1-60.
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Related Topics and Keywords
bioavailability, Drug Delivery Nanoemulsion, encapsulated drugs, enzymatic degradation, hydrolysis, immune response, immunostimulator, increased drug solubility, Kyle Jandrasitz, microfluidics, minimal side effects, Post Process Sterile Filtration, process development, protection, Steven Mesite, toxicities, vaccine adjuvants, YANG SU
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