PID control of the aqueous flowrate resulted in the rapid convergence of particle size to the target size of 85 nm (<10 minutes, Figure 4C). Liposome size was then maintained at the desired target by continuous PID control. This led to improved size stability in time as compared to continuous manufacturing without PID control. A supervised system led to a Z-average standard deviation of 3 nm, as compared to 4 nm for an unsupervised system, with a standard error of the mean of 0.1 nm for the supervised system compared to 0.2 nm for an unsupervised system. Using the NanoFlowSizer as PAT tool for PID control of the DIANT Pharma LiFT system not only allowed for the automation of flowrates to achieve a desired liposome size, but thus also improved the quality and consistency of the product by continuously fine-tuning the process.
Figure 4. Turbulent Injection automated control via liposome size measurement. A. Set-up of an NFS integrated in liposome continuous manufacturing LiFT system (DIANT Pharma Inc.) in an in-line loop for real-time particle size measurement and automated feedback control of the internal aqueous flow rate. B. Schematic of the LiFT-NFS set-up. The aqueous (i) and the ethanolic (ii) phases were pumped (iii and iv) to the turbulent jet mixer (viii). Before entering the NanoFlowSIzer (ix), the suspension was diluted - required from product perspective, not for NFS measurement- from a secondary aqueous deposit (vi and vii). The PID feedback (blue arrows) allows NFS-control (ix) of the aqueous flow rate during turbulent jet injection (viii). The flow then continued for further processing (x) and final collection. C. Continuous, real-time measurement of liposome particle size (Z-Average) during automated aqueous flowrate regulation via PID control by a NanoFlowSizer. The line at 85 nm indicates the input target size in the PID controller of the LiFT system, updated continuously with the measured Z-average data from the NFS. The target size was achieved after 10 minutes after the start of the process and maintained at precisely 85 nm during continuous manufacturing. Aqueous flow rate as regulated by the NanoFlowSizer from the real-time particle size measurements. Flowrate was automatically tuned (from ~130 mL/min to ~100 mL/min) until the target liposome size was achieved, and it was continuously adapted to maintain the continuous liposome production at the target size. Image Credit: InProcess-LSP
Continuous process control via real-time particle size measurement was achieved for High-Pressure Homogenization (HPH) of emulsions and Turbulent Injection continuous manufacturing of liposomes. The NanoFlowSizer was integrated into the processes in an in-line configuration, and PID control was achieved via the dedicated PID module of the XsperGo software. Communication between the NanoFlowSizer and other equipment was established via PLC with a Profinet protocol.
For HPH (GEA XStream Lab Homogenizer 1000), the NanoFlowSizer allowed for the automated control of the internal pressure at the High-Pressure Valve of the system, thus controlling the resulting droplet size of a 5% oil-in-water emulsion. Furthermore, the process was automatically stopped at the desired size to avoid overprocessing. For the Turbulent jet Injection method for continuous liposome manufacturing, the NanoFlowSizer allowed for the automated adjustment of the aqueous flow rate in a LiFT skid system (DIANT Pharma Inc.), resulting in the rapid convergence and stability of liposome size to a target size value. This value was then sustained in time, leading to improved stability and product uniformity during continuous liposome manufacturing.
In conclusion, the NanoFlowSizer can be easily integrated into processes in which particle size is a CQA and can be used to tune CPPs such as pressure or flow rate. The dedicated XsperGo software allows for the easy deployment of PID control with customizable features to adapt to a multitude of needs, such as achieving a target size, avoidance of overprocessing or establishment of precise continuous manufacturing for real-time quality control.
MATERIALS AND METHODS
Sunflower oil (Sigma-Aldrich), Tween 20 (Sigma-Aldrich), water.
Standard NanoFlowSizer set-up for HPH
A FIDES I type NanoFlowSizer equipped with a 1.5-inch flow cell was connected in an in-line configuration (see Figure 3B). The connection between the system of interest and the flow cells was done via standard tri-clamps. The HPH was connected to a Recirculation Tank, from which the sample was pumped through the NanoFlowSizer by a micro gear pump (LongerPump, WT3000-1JA) and back to the hopper of the HPH. Flow rates for the micro gear pump were manually changed to match the process speed over time.
Emulsion preparation for HPH
A 5% mixture of sunflower oil in water was prepared with 1% Tween 20 as surfactant. The emulsion was pre-homogenized to 600 nm (at 200 bar) prior to the NanoFlowSizer homogenization run to set a standard known starting point.
NanoFlowSizer integration and control of HPH
An Ethernet LAN cable was used to connect the HPH PLC to the NanoFlowSizer PC, and a Profinet (TCP/IPv4) connection was established. The controller module of XsperGo software for HPH applications was then initiated. Proportional-only control was established with proportionality constants P (Kp): -0.13, I: 0 and D: 0, with a particle target size of 200 nm. Pressure changes were applied by the NanoFlowSizer every 2 minutes after size measurements. The initial pressure was set to 200 bar and the maximum possible change in pressure per step was set to 20 bar.
NanoFlowSizer for liposome production
A FIDES I type NanoFlowSizer was connected to a LiFT (DIANT Pharma Inc.) liposome manufacturing skid (see Figure 4B). The parameters regarding the PID control of the LiFT system and liposome formulation are property of DIANT Pharma Inc. and are available upon reasonable request.
The authors would like to acknowledge the contributions of Eric Ardeche for his help integrating the NanoFlowSizer with the GEA Xstream Lab Homogenizer 1000 HPH and of Dr. Costa for providing the data on liposome production by a LiFT skid (DIANT Pharma Inc.) with an integrated NanoFlowSizer.
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