Currently, clinically approved medicines with liposomal formulations have a combined annual revenue of approximately $100 million USD, but their difficult and expensive production methods make more widespread use prohibitive. Microfluidic devices overcome the quality and scalability issues inherent in previous liposome production methods, but post-encapsulation purification is still a time-consuming process preventing the widespread use of liposomal drugs. A team of researchers from University College London, Aston University, and University of Strathclyde in the UK, investigated how the NanoAssemblr™ Benchtop™ can be combined with a tangential flow filtration (TFF) device to generate purified liposomal therapeutics in under 4 minutes. The combination of microfluidic liposomal drug encapsulation and purification by tangential flow filtration into a single continuous process allows the ideal lipid compositions, operating conditions, and scale-up parameters for liposomal drug formulation to be rapidly identified, which significantly reduces the time to develop and produce new liposomal drugs.

 

The authors first investigated how backpressure in the TFF device affected liposome size and purification. They discovered that there was no significant change in neutral liposome size or polydispersity index (PDI) at backpressures between 7 and 80 psi, but that neutral liposomes were being pushed across the filtration membrane into the permeate at backpressures above 75 psi. This suggests that 75 psi is the upper limit of backpressure for this system, which is within the 5 to 80 psi range commonly found in industrial filtration purification processes. The authors also investigated the production and purification of cationic and anionic liposomes at a backpressure of 49 psi and discovered that liposome size, PDI, zeta potential, and particle concentration were not notably changed by the TFF process and that no liposomes were detected in the permeate at this lower backpressure. These results demonstrate that TFF can retain a variety of liposomes produced on the NanoAssemblr™ Benchtop™ without affecting their physicochemical characteristics.

 

The authors also investigated the efficiency of TFF at removing unincorporated drugs, proteins, and residual ethanol from liposomal nanomedicine formulations by adding propofol or ovalbumin to liposomal formulations before performing TFF. For propofol, they found that 99% was removed after three diafiltration cycles and the residual ethanol concentration was 3%. For ovalbumin, they found that at least 70% was removed after three diafiltration cycles and the residual ethanol concentration was below 5%. These results show that TFF is a very effective method for purifying liposomal nanomedicines made on the NanoAssemblr™ Benchtop™.

 

Finally, the authors developed a continuous liposome manufacturing and purification process by feeding the TFF device with liposomes produced on the NanoAssemblr™ Benchtop™. An intermediate collection vial was used to hold the liposomes before TFF to independently control and optimize the process for each device. Using an intermediate collection vial also allowed purification or concentration of the liposomes depending on if the buffer was replenished after each diafiltration cycle or not. This system was then tested for the production and purification of liposomes incorporating propofol and cationic liposomes modified by the addition of ovalbumin within the intermediate collection vial. Both formulations were purified to a similar extent as those described above, and no unexpected changes in liposome size, PDI, or zeta potential were detected in the continuous process.

 

This paper demonstrates the feasibility of on-chip purification of liposomes during the production process, which reduces the sometimes hours-long process of downstream purification by dialysis to a continuous-flow production and purification process that can be completed in under 4 minutes. The authors anticipate that this process will allow the efficient investigation of liposomal drug candidates and provide an easy path to their industrial production, which will help overcome the quality and scalability issues preventing the widespread use of liposomal therapeutics and speed their transition from the lab to the clinic.

 

Liposomes are lipid based bilayer vesicles that can encapsulate, deliver and release low-soluble drugs and small molecules to a specific target site in the body. They are currently exploited in several nanomedicine formulations. However, their development and application is still limited by expensive and time-consuming process development and production methods. Therefore, to exploit these systems more effectively and support the rapid translation of new liposomal nanomedicines from bench to bedside, new cost-effective and scalable production methods are needed. We present a continuous process flow system for the preparation, modification and purification of liposomes which offers lab-on-chip scale production. The system was evaluated for a range of small vesicles (below 300 nm) varying in lipid composition, size and charge; it offers effective and rapid nanomedicine purification with high lipid recovery (> 98%) combined with effective removal of non-entrapped drug (propofol >95% reduction of non-entrapped drug present) or protein (ovalbumin >90% reduction of OVA present) and organic solvent (ethanol >95% reduction) in less than 4 minutes. The key advantages of using this bench-top, rapid, process development tool are the flexible operating conditions, interchangeable membranes and scalable high-throughput yields, thereby offering simultaneous manufacturing and purification of nanoparticles with tailored surface attributes.