Production of Nanoemulsion Adjuvants using High Shear Fluid Processing
Posted on April 29, 2016
BY: DR. YANG SU AND STEVEN MESITE
Microfluidics International Corporation, An IDEX Material Processing Technologies Unit
90 Glacier Dr. Suite 1000, Westwood, MA 02459 USA
Since their first introduction more than two centuries ago, vaccines have demonstrated to be the most cost effective and efficient medical intervention to prevent pandemics and save people’s lives. Vaccines are prepared to improve immunity to particular diseases and their efficacy can be greatly improved through the combination of adjuvants. Traditional aluminum salts adjuvants, which are still the most commonly used adjuvants, have limitations mainly due to their humoral (Th2)-biased immune response. Stable nanoemulsion adjuvants have evolved as advanced novel adjuvants due to their ability to enhance immune response without the addition of immune stimulator along with other advantages:
• Effective: Enhance both cellular (Th1), humoral (Th2) and major histocompatibility complexes (MHC) responses.1, 2
• Well tolerable.
Schematic of Immune Responses Induced by Different Adjuvants
Key Production Requirements & Challenges
One of the key challenges during nanoemulsion adjuvants production is to precisely control the droplet size and the size distribution since both will not only determine the stability of the nanoemulsions, but also greatly affect sterile filtration, an essential post processing operation. The existence of even a small population of micron-size droplets may destabilize the emulsion through mechanisms such as Ostwald ripening. In addition, droplets well over 220 nm in diameter may plug the sterilization filters and lead to substantial losses, and therefore their presents should be limited in the emulsion.Other key requirements for the production of nanoemulsion adjuvants include:
• Process efficiency.
• Sterile production.
The schematic of Microfluidizer® processor and the fixed geometry interaction chamber are shown in Figure 2 and Figure 3, respectively. Microfluidizer® technology utilizes high pressure to pump multi-phase fluids through the microchannels of an interaction chamber, exposing the fluids to high shear. The system can operate either in batch processing mode or continuous processing mode and can accommodate materials with high solid content, high viscosities, as well as a wide range of temperatures. The product temperature can be regulated by a heat exchanger.
Schematic of Microfluidizer® Processor
Schematic of Y Type Interaction Chamber
“Y” Single-Slotted & “Y” Multi-Slotted
Microfluidics offer two different types of interaction chamber: Z type and Y type. Figure 3 shows the Y type interaction chamber. The Z chambers are typically used for solid dispersions including cell disruption. The Y chambers are typically used for processing liquid-liquid types of dispersions such as emulsions, liposomes, etc. When using a Y chamber, inlet fluid is split into two jets which subsequently impinge on each other inside the high impact zone. Besides high shear rate, intense turbulent energy dissipation is also achieved to help break the droplets into nanometer range. A multi-slotted scale-up design is also shown in Figure 3. By using parallel array of identical microchannels, scale-up results are ensured since fluids inside each microchannel will be subjected to exact same shear rate and generate the same impact force/energy dissipation.
The major advantage of combining intensifier pump and fixed geometry interaction chamber is the delivery of constant process pressure profile. As shown in Figure 4, the process pressure of the Microfluidizer processor stays constantly at the target pressure for majority of the compression strokes. Therefore, virtually the entire product is passed through the Microfluidizer processor at the target pressure and smaller particle size with narrow size distribution can be achieved. Note that the zero pressure portion represents the suction strokes where no materials are pushed and processed through the interaction chamber, hence does not affect the final product quality. The pressure profile of the high pressure homogenizer, on the other hand, is continuously varying throughout the entire process due to the movement of the homogenizing valve. This is because the high pressure homogenizers are designed to deliver constant volume instead of pressure. Figure 4 indicates that the process pressure barely stays at the target pressure and can drop as much as 50 percent from target pressure for the high pressure homogenizer. As a result of such large pressure fluctuations, the product obtained with the high pressure homogenizer usually carries large particles and broad size distribution.
Pressure Profile Comparison between Microfluidizer® Processor and High Pressure Homogenizer
In summary, Microfluidizer technology offers a number of advantages:
• Velocities of over 400 m/s in microchannels results in shear rates of up to 108 s-1, which are orders of magnitude higher than conventional technologies.
• Constant pressure pumping system combined with fixed geometry interaction chamber deliver true constant process pressure.
• Variable microchannel shapes and sizes provide tunable shear rate.
• Parallel arrays of identical microchannels ensure linear scalability to tens of liters per minute.
The advantages of producing nanoemulsion adjuvants using Microfluidics’ high pressure, high shear fluid processors are presented here. In this study, the production of an oil-in-water nanoemulsion adjuvant was investigated by comparing Microfluidizer technology with traditional high pressure homogenization technology.
MATERIALS & METHODS
The model oil-in-water nanoemulsions were formulated, prepared and analyzed as follows:
• The oil phase consists 5 wt.% squalane oil and 0.75 wt.% Span 85; the aqueous phase consists 0.75 wt.% Tween 80 and deionized water.
• Pre-emulsion was prepared using a low energy rotor-stator mixer (Quadro HV0) for both emulsification methods.
• Final nanoemulsions were created by a Microfluidizer® (MF) processor (M110-P) and a high pressure homogenizer (HPH).
• Parameters varied during processing: pressure and number of passes through each processor.
• Particle size analyzed using a dynamic light scattering instrument (Malvern Zetasizer Nano-S) and an optical microscope (Olympus BH2).
RESULTS & DICUSSION
Results demonstrated that the Microfluidizer processor is more efficient in producing nanoemulsions with repeatable results.
Average particle size of nanoemulsions obtained with Microfluidizer and high pressure homogenizer
Particle size increase of homogenized nanoemulsion comparing to Microfluidized nanoemulsions
Figure 5 shows the fast particle size reduction when using Microfluidizer. At three different test pressures of 10, 20, and 30 kpsi, the particle size of Microfluidized nanoemulsions were all smaller than that of the homogenized nanoemulsions. The average particle size of the homogenized nanoemulsion could not be reduced to smaller than 180 nm even after 5 passes at the highest process pressure setting. Figure 6 shows the relative size comparison between the two processing technologies under identical process conditions. For a given energy input, e.g., same pressure, and number of passes, the average particle size of Microfluidized nanoemulsions was 18-55% smaller than that of the homogenized nanoemulsions.
Polydispersity index of nanoemulsions obtained with Microfluidizer and high pressure homogenizer
Figure 7 shows the polydispersity index (PDI) of the nanoemulsions produced with both methods. For majority of the test conditions, Microfluidized nanoemulsions showed uniform size distribution with PDI smaller than 0.1. All homogenized nanoemulsions showed broad size distribution with much larger PDI.
Power consumption comparison between Microfluidizer and high pressure homogenizer
The power consumption comparison between Microfluidizer and high pressure homogenizer is summarized in Table 1. To be able to achieve droplet size of ~180 nm, the nanoemulsion needed to be processed with high pressure homogenizer method for 5 passes at pressure of 30,000 psi. On the other hand, Microfluidizer method only required 2 passes at pressure of 10,000 psi to achieve similar droplet size. Power consumption calculation indicated that the high pressure homogenizing method would consume as much as 7.5 times more power than the Microfluidizer processor.
Repeatability in terms of particle size of homogenized nanoemulsion comparing to Microfluidized nanoemulsions
Finally, the repeatability results are shown in Figure 8 with the average particle size of nanoemulsions obtained from 3 separate runs by each method under 30 kpsi. Microfluidized nanoemulsions showed very repeatable results with much lower standard deviations between different runs compared to homogenized nanoemulsions: 0.1-2.6 vs. 3.8-14.8.
A nanoemulsion adjuvant was created using both of the Microfluidizer method and high pressure homogenizer method. Results of particle size and size distributions, power consumption and process repeatability demonstrated that MF technology is better than traditional HPH technology when producing such nanoemulsion. This would lead to potential benefits such as improved stability and post process sterile filtration.
• Microfluidizer® processors are well suitable for manufacturing nanoemulsion vaccine adjuvants.
• Microfluidizer® processor consumed 7.5 times less power than the high pressure homogenizer.
• Microfluidized emulsion were 18-55% smaller than the homogenized emulsions with the same energy input.
• Emulsions created by Microfluidizer® were 17-91% less polydisperse than that created by high pressure homogenizer.
• The standard deviations of Microfluidized emulsions (0.1-2.6) were much lower than that of homogenized emulsions (3.8-14.8).
1. Vaccine adjuvants review, www. Innivogen.com, 2011
2. Subunit vaccine delivery, Springer, 2015
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Related Topics and Keywords
Biodegradable, cellular, enhance immune response, High Shear Fluid Processing, humoral, major histocompatibility complexes, MHC, microfluidics, nanoemulsion, Nanoemulsion Adjuvants, Steven Mesite, Th1, Th2), vaccine, Well tolerable, YANG SU
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