DOI: 10.15129/91299007-146d-4b44-b1ad-cec8d309612c
title: Data for "Using microfluidics for scalable manufacturing of nanomedicines from bench to GMP: A case study using protein-loaded liposomes."

description: Nanomedicines are well recognised for their ability to improve therapeutic outcomes. Yet, due to their complexity, nanomedicines are challenging and costly to produce using traditional manufacturing methods. For nanomedicines to be widely exploited, new manufacturing technologies must be adopted to reduce development costs and provide a consistent product. Within this study, we investigate microfluidic manufacture of nanomedicines. Using protein-loaded liposomes as a case study, we manufacture liposomes with tightly defined physico-chemical attributes (size, PDI, protein loading and release) from small-scale (1 mL) through to GMP volume production (200 mL/min). To achieve this, we investigate two different laminar flow microfluidic cartridge designs (based on a staggered herringbone design and a novel toroidal mixer design); for the first time we demonstrate the use of a new microfluidic cartridge design which delivers seamless scale-up production from bench-scale (12 mL/min) through GMP production requirements of over 20 L/h using the same standardised normal operating parameters. We also outline the application of tangential flow filtration for down-stream processing and high product yield. This work confirms that defined liposome products can be manufactured rapidly and reproducibly using a scale-independent production process, thereby de-risking the journey from bench to approved product. Figure 1: Micromixer cartridge designs used within these studies. Schematics illustrate the staggered herringbone micromixer (SHM) with embossed chevrons allowing consistent fluid mixing and the toroidal mixer (TrM) with planar geometry employing centrifugal forces to encourage uniform mixing allowing for greater fluid stream velocities. The flow rate capacities for each of the microfluidic mixers and the microfluidic platforms that are used are listed. Figure 2: Comparison of micromixer design on the physio-chemical attributes of liposomes. Liposomes were prepared using either a staggered herringbone (SHM) in the NanoAssemblr® Benchtop or a toroidal mixer (TrM) in the IgniteTM and their physico-chemical attributes compared. Anionic liposomes (DSPC:Chol:DOPS 10:5:4 w/w) and neutral liposomes (DSPC:Chol 2:1 w/w) were prepared at a 3:1 flow rate ratio and a total flow rate of 15 mL/min and purified by tangential flow filtration. Cationic liposomes DOPE:DOTAP (1:1 w/w) were produced at a flow rate ratio of 1:1 and a total flow rate of 10 mL/min and purified using a 1/10 dilution with Tris to reduce the solvent concentrations. All formulations were prepared at an initial lipid concentration of 4 mg/mL dissolved in ethanol (DOPE:DOTAP) and methanol (DSPC:Chol:DOPS and DSPC:Chol). The liposome z-average diameter (columns) and PDI (open circles) (A) and zeta potential (B) were measured. Results represent mean ± SD of three independent batches. Figure 3: Production of drug loaded liposomes using different microfluidic mixers. Liposomes were prepared using either a staggered herringbone (SHM) in the NanoAssemblr® Benchtop or a toroidal mixer (TrM) in the IgniteTM. Anionic liposomes (DSPC:Chol:DOPS 10:5:4 w/w) and neutral liposomes (DSPC:Chol 2:1 w/w) were prepared at a 3:1 flow rate ratio and a total flow rate of 15 mL/min. All formulations were prepared at an initial lipid concentration of 4 mg/mL dissolved in methanol and loaded with 0.25 mg/mL initial OVA concentration dissolved in PBS pH 7.4. Non-entrapped protein was removed by tangential flow filtration and protein loading was quantified via RP-HPLC. Cationic liposomes (DOPE-DOTAP (1:1 w/w) were prepared at a flow rate ratio of 1:1 and a total flow rate of 10 mL/min. An initial lipid concentration of 4 mg/mL (dissolved in ethanol) and an initial PolyA concentration of 166 µg/mL (dissolved in Tris buffer pH 7.4, 10 mM) was used. Cationic liposomes were purified by dilution and drug loading quantified by using a Ribogreen assay. The liposome z-average diameter (columns) and PDI (open circles) (A), drug loading (B) and zeta potential (C) were measured. Results represent mean ± SD of three independent batches. Figure 4: Production of liposomes entrapping protein are room temperature irrespective of phospholipid transition temperature. Liposomes were prepared from DMPC, DSPC or HSPC in combination with cholesterol at a 2:1 w/w ratio using either a staggered herringbone (SHM) in the NanoAssemblr® Benchtop or a toroidal mixer (TrM) in the IgniteTM. Liposomes were manufactured at a 3:1 flow rate ratio, 15 mL/min total flow rate, an initial lipid concentration of 4 mg/mL (dissolved in methanol) and an initial ovalbumin protein concentration of 0.25 mg/mL (dissolved in PBS pH 7.4) which was quantified by RP-HPLC after purification. All liposomes were prepared at room temperature. Liposomes were purified by tangential flow filtration. The liposome z-average diameter (columns) and PDI (open circles) (A), loading (B), and zeta potential (C) was compared. Results represent mean ± SD of three independent batches. Figure 5: Small-scale production of liposomes using different process parameters. Liposomes (DSPC:Chol 2:1 w/w) were produced by either a staggered herringbone (SHM) in the NanoAssemblr® Benchtop or a toroidal mixer (TrM) in the IgniteTM and the effect of flow rate ratio (A and C) and total flow rate (B and D) investigated on liposome z-average diameter (columns) and PDI (open circles) (A and B) and protein loading (C and D). Liposomes entrapping OVA were manufactured at flow rate ratios of 3:1 or 5:1 and a total flow rates of 12, 15 or 20 mL/min. An initial lipid concentration of 4 mg/mL (dissolved in methanol) and an initial OVA concentration of 0.25 mg/mL (dissolved in PBS) was used. Liposomes were purified by tangential flow filtration and protein loading quantified by RP-HPLC. Results represent mean ± SD of three independent batches. Figure 6: Investigating the impact of solvent choice and lipid concentration when using different microfluidic mixers. Liposomes (DSPC:Chol 2:1 w/w) entrapping OVA were produced using either methanol or ethanol to dissolve the lipid and either a staggered herringbone (SHM) in the NanoAssemblr® Benchtop or a toroidal mixer (TrM) in the IgniteTM. The liposome z-average diameter (columns) and PDI (open circles) (A), protein loading (B; quantified by RP-HPLC) and zeta potential (C) was measured. DSPC:Chol liposomes were manufactured using either methanol or ethanol at a flow rate ratio of 3:1, a total flow rate of 15 mL/min, an initial lipid concentration of 4 mg/mL and OVA concentration of 0.25 mg/mL. Liposomes (DSPC:Chol 2:1 w/w) entrapping OVA were also produced at final lipid concentrations from 0.5 to 10 mg/mL and their particle size (D) and protein loading (E) was measured by a combination of RP-HPLC and micro-BCA. Liposomes were purified by tangential flow filtration and protein loading quantified by HPLC. Results represent mean ± SD of three independent batches. Figure 7: Liposome morphology, lipid recovery and protein release profiles of liposomes produced using different microfluidic mixers. Liposomes (DSPC:Chol 2:1 w/w) entrapping OVA were produced using either a staggered herringbone (SHM) in the NanoAssemblr® Benchtop or a toroidal mixer (TrM) in the IgniteTM. (A) and (B) show the morphology of liposomes produced using the staggered herring bone mixer. (C) and (D) show the morphology of formulations produced using the toroidal mixer. (E) Phospholipid recovery in liposomes produced by each mixer before and after purification via tangential flow filtration. (F) Protein release from liposomes produced by the two different micromixer designs and incubated at 37 °C for 120 h under agitation. DSPC:Chol liposomes were produced at a flow rate ratio of 3:1, and a total flow rate of 15 mL/min. An initial lipid concentration of 4 mg/mL (dissolved in methanol) and an initial OVA concentration of 0.25 mg/mL (dissolved in PBS) was used for (A) to (E). For protein release studies (F), liposome formulations were produced at a 4-folds higher concentration (initial lipid concentration of 16 mg/mL and initial OVA of 1 mg/mL). Protein loading and release was quantified by RP-HPLC. Results represent mean ± SD of three independent batches (E, F). Figure 8: Scale-independent production of liposomes entrapping protein - from bench to GMP. Liposomes (DSPC:Chol 2:1 w/w) entrapping OVA were produced using a toroidal mixer in the IgniteTM, NxGen BlazeTM or a GMP microfluidic manufacturing system. All three systems were run at the same flow rate ratio (3:1). Given the system selected to total flow rate was increased to demonstrate scale-independent production from 12 mL/min (IgniteTM) to 60 mL/min (NxGen BlazeTM) or 200 mL/min (GMP system). The liposome z-average diameter and PDI (A), size intensity (B) and protein loading quantified by micro-BCA (C) are shown. Results represent mean ± SD of three independent batches for the Ignite and NxGen Blaze systems and 1 large-scale batch on the GMP system.