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Microfluidic Extractive Crystallisation for Spherical Drug/Drug-Excipient Microparticle Production Eunice W.Q. Yeap, Andrew J. Acevedo, and Saif A. Khan Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.8b00432 • Publication Date (Web): 01 Feb 2019 Downloaded from http://pubs.acs.org on February 1, 2019

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Organic Process Research & Development

Microfluidic Extractive Crystallisation for Spherical Drug/DrugExcipient Microparticle Production Eunice. W. Q. Yeap†, Andrew J. Acevedo§, and Saif A. Khan* † BioSystems and Micromechanics (BioSym), Singapore-MIT Alliance for Research and Technology, 1 CREATE Way, #04-13/14, Enterprise Wing, Singapore 138602, Singapore § Department of Biomedical Engineering, Boston University, 44 Cummington Street, Boston, MA 02215, United States  Department of Chemical and Biomolecular Engineering, National University of Singapore, 4 Engineering Drive 4, Singapore 117585, Singapore

* Corresponding Author. Email: [email protected]

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FOR TABLE OF CONTENTS ONLY


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ABSTRACT Granulation is a common manufacturing step for pharmaceutical drug products, which improves powder flowability, compactibility and ensures tablet content uniformity. Granules of uniform content can conventionally be challenging to obtain due to powder segregation and mixing issues prior to granulation. Spherical crystallisation – a method where drug crystals are directly formed into spherical granules, is a promising way to overcome issues with mixing and form granules with uniform content. However, a common challenge of existing quasi emulsion solvent diffusion or solvent extraction methods for spherical crystallisation involving miscible solvents in stirred batch vessels is the coarse control over particles sizes, as they are sensitive to multiple scale-up factors (mixing efficiency, impeller and vessel geometry, inlet configuration). This limits the method in terms of content uniformity, which in turn limits the extent to which granules with tunable dissolution profiles can be created. Here, we propose a method for the formation of monodisperse drug-excipient microparticles with tunable release profiles via microfluidic spherical extractive crystallisation using drug and excipient-loaded ethyl acetate-in-water emulsions. Monodisperse droplets are generated using microfluidics and droplet saturation via solvent extraction leads to eventual and direct monodisperse spherical particle formation within minutes. We demonstrate this method using ethyl acetate droplets loaded with naproxen or naproxen and ethyl cellulose, as a hydrophobic drug and drug-excipient model system respectively, and obtained monodisperse spherical microparticles in both cases. Lastly, preliminary investigations of in vitro drug release from a range of microparticles made from droplets containing different naproxenethyl cellulose ratios displayed clear differences in the release profiles. When coupled with microfluidic droplet generators that operate at high volumetric throughputs, this 3 ACS Paragon Plus Environment


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method has the potential to be applied in continuous manufacturing platforms for the production of monodisperse spherical drug particles or drug-excipient composites with excellent content uniformity and tunable release profiles at a kg/day scale throughput. (300 words) KEYWORDS: Pharmaceutical Formulations, Advanced Continuous Manufacturing, Antisolvent Crystallisation, Particle Engineering


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INTRODUCTION Granulation is an important manufacturing step used in the pharmaceutical industry for the conversion of crystalline drug material into solid dosage forms, particularly tablets and capsules. This step transforms loose drug crystals or active pharmaceutical ingredients (API) into granules that exhibit better flowability, compressibility and content uniformity for subsequent portioning and tableting1, 2. Prior to granulation, drug crystals are often either blended with various excipients (functional inactive substances added to the formulation) to protect the active drug from moisture, modify drug dissolution, or act as a binder for the final product3. Industrially, content uniformity at the individual granule level can be challenging due to the difficulty of obtaining homogeneous drug powder blend for granulation4, 5; powder blend uniformity is high dependant on drug crystal and excipient powder size and shape6, bulk density7, and adhesive or cohesive forces between different compounds within the mixture8. Kawashima et al. introduced the concept of spherical crystallisation nearly three decades ago; this is a particularly facile process of granulation, in which spherical crystal agglomerates are directly formed during crystallisation via the introduction of a bridging liquid9 or by conducting antisolvent crystallisation within transient droplets (quasi emulsions) formed from miscible solvent pairs using high shear and agitation10,

11.

The main challenge of the aforementioned methods when

conducted in agitated vessels is the coarse control over particle sizes as operation is sensitive to multiple scale-up factors such as mixing efficiency, impeller and vessel geometry, inlet configuration. Some recent studies that have addressed this challenge couple microfluidics with drug crystallisation, allowing for facile production of neat drug and also drug-excipient microparticles with tunable sizes and structures using microfluidic evaporative crystallisation12-14. In these demonstrations, monodisperse 5 ACS Paragon Plus Environment


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drug or drug-excipient mixture loaded droplets are generated in immiscible continuous fluids and subjected to solvent removal via evaporation to induce drug supersaturation for direct particle formation. Besides evaporative crystallisation, recent demonstrations by Watanabe et al.15 and Kalny et al.16 also showcased monodisperse droplet generation from partially miscible solvent systems using microfluidics prior to saturation by solvent extraction which enabled good size control over sodium poly-(styrenesulfonate) or potassium-chloride particle formation respectively. In this work, we have developed a novel method of emulsion-based processing for drug microparticle production with tunable release profiles via microfluidic extractive crystallisation from drug-loaded ethyl acetate droplets formed in a continuous aqueous phase. Monodisperse drug-loaded ethyl acetate-in-water droplets are serially produced using microfluidics and transferred to a separate collection unit for the extraction of ethyl acetate which generates drug supersaturation in the individual droplets for crystallisation. Here, the usage of microfluidics enables monodisperse drug-loaded (or drug-excipient mixture-loaded) ethyl acetate droplets of tunable sizes but with precise and uniform content to be formed in water at controlled volume (flow) ratios of the dispersed and continuous phases. The droplets are directly transferred into a static collection bath with excess water for droplet saturation and crystallisation, where solvent extraction of ethyl acetate from the droplets into the surrounding aqueous medium leads to droplet shrinkage, drug supersaturation, and the formation of spherical microparticles containing drug crystals. The usage of ethyl acetate, a Class 3 solvent, as the dispersed phase in this method is favourable as a greener and less toxic alternative to chlorinated solvents17. To the best of our knowledge, this is the first demonstration of work that produces monodisperse crystalline drug and drug6 ACS Paragon Plus Environment

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composite microparticles with miscible solvents that exhibit tunable release profiles and have excellent content uniformity without the usage of bridging liquids18 or binding polymers19 for spherical crystallisation. We showcase the generality of our method by formulating two types of hydrophobic drug and drug-polymeric excipient microparticles, using naproxen and naproxen-ethyl cellulose respectively. Preliminary investigations of in vitro drug release from the naproxen and four different naproxenethyl cellulose composite microparticles with varying drug-polymer ratios highlight the range of release profiles achievable, which are a function of internal microparticle structure.

EXPERIMENTAL Materials Naproxen (N8280), poly(vinyl) alcohol (PVA) (M.W. 67,000), and ethyl cellulose (viscosity 10 cP) were purchased from Sigma-aldrich.

Ethyl acetate (HiPerSolv

CHROMANORM® for HPLC, ≥ 99.8%) was purchased from VWR International, LLC and used as per received. Ultrapure water (18.3 MΩ) obtained using a Millipore MilliQ purification system was used to prepare the aqueous continuous phase. The ultrapure water was also used directly for the collection of generated drug-loaded ethyl acetate droplets. Polytetrafluoroethylene (PTFE) tubes of 0.02” and 0.04” I.D. were purchased from Cole Parmer, and Polyether Ethyl Ketone (PEEK) MicroTees (P-727, 0.020” thru hole, and P-890, 0.006” thru hole) were purchased from IDEX Health & Science LLC. Potassum-Buffer Saline Tablets (5g Gibco PBS tablet) were purchased from Thermo Fisher Scientific Inc.



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Methods Particle Formation – droplet generation, supersaturation and collection: the drug loaded dispersed phase (DP) was prepared by dissolving either (i) 50 mg of naproxen, (ii) 50 mg of naproxen and 20 mg of ethyl cellulose, (iii) 50 mg of naproxen and 40 mg of ethyl cellulose, (iv) 50 mg of naproxen and 80 mg of ethyl cellulose, or (v) 30 mg of naproxen and 40 mg of ethyl cellulose, per mL of ethyl acetate. The continuous phase (CP) was a 3 w/w% aqueous PVA solution using ultrapure water. The two phases were introduced into a PEEK MicroTee (P-727, 0.020” thru hole or P-890, 0.006” thru hole) using Harvard Pumps (Harvard PHD 22/2000 series), where the continuous and dispersed flow rates were adjusted to generate droplets of different diameters; for naproxen droplet generation, continuous flow rates of 200 - 800 µL/min and dispersed flow rates of 8 – 170 µL/min, were used to generate monodisperse droplets with mean diameters in the 140 – 600 µm range. For naproxen-ethyl cellulose droplets, the continuous flow rates of 50 – 120 µL/min and dispersed flow rates of 200 – 500 µL/min, were used to generate droplets with mean diameters in the 400 – 500 µm range using the P-727 MicroTee. The droplets were then directly transferred into a collection cylinder vessel (50 cm in height) containing an excess of ultrapure water (> 1L), which is the extracting medium for ethyl acetate, using a 30 cm, 0.04” I.D. PTFE tube connection from the outlet of the MicroTee. The outlet of the tube was placed at 10 cm from the base of the collection vessel, and the residence time of the droplets in this 30 cm tube was ≤ 10 s. A high-speed digital camera (Basler pI640) was mounted on a stereomicroscope (Leica MZ16) to capture images of the droplets entering the collection vessel from the connection tube, at 100 fps. A schematic and optical image of the experimental set up may be found in Figure 1 in the Results and Discussion section and Figure S1 of the Supporting Information


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respectively. All collected particles were collected on filter paper, rinsed thrice with ultrapure water to remove traces of surfactant and vacuum dried at room temperature for 8 hours prior to structural and polymorphic characterisation. Drug release studies from neat naproxen and naproxen-ethyl cellulose particle: drug release studies from neat naproxen and naproxen-ethyl cellulose particles made using the protocol described above were conducted using the small-volume method described by Acevedo et al.20. A Phosphate-Buffered Saline (PBS) stock solution was prepared by dissolving a 5g Gibco PBS tablet into 500 mL of ultrapure water., and the test media used for all samples was 50 mL of the PBS solution. The mass of each particle type used per test was set such that the expected amount of naproxen was approximately 2.5 mg. A 60 mL round-bottom, plastic test tube (BRAND®, SigmaAldrich, St. Louis, MO, USA) was used as the test vessel. To start the release test, particle samples were pre-wetted with a 400 µL aliquot of test media and transferred to the test vessel using a micropipette. A 40 kHz ultrasonic generator with a 0.125 in diameter microprobe horn (Vibracell VCX134FSJ; Sonics, Newtown, CT, USA) was used to apply ultrasonic agitation to the test media for 7200 sec after the particles were added at an amplitude setting of 45% and a probe height of 55 mm. A continuous, closed-loop sampling system was set up to monitor the amount of drug release over time – a peristaltic pump (BT50S/YZ15, Lead Fluid, Baoding, Heibei, China) transported dissolved drug solution from the test vessel through a 1 µm pore size cannula filter (QLA, Telford, PA, USA) and 0.2 µm in-line syringe filter (0.25 mm diameter, FLL/MLS CA; GVS Life Sciences, Sanford, Maine, USA) to a flow cell (20 mm path length, SMA-Z-20-PEEK, FIA Labs, Seattle, WA, USA) and back into the test vessel. A modular spectrophotometer (FLAME-T, Ocean Optics, Largo, FL, USA) connected to a UV-light source (DH-2000-BAL, Ocean Optics) measured 9 ACS Paragon Plus Environment


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absorbance of the sampled solution at 270 nm. OceanView software was used to record and export absorbance data for analysis. At the end of each test, the system was cleaned by passing 10 mL of test media through the sampling system. Each sample was tested in triplicate. Post-release drug microparticles were also collected and vacuum dried at room temperature for further structural characterization.

Characterisation A field-emission scanning electron microscope (JEOL JSM-6700F) at 5 kV accelerating voltage was used to image the microparticles. All samples were prepared on conventional SEM stubs with carbon tape and were coated with ~10 nm of platinum by sputter coating. To reveal the microparticle cross sections, a 21G needle was used to manually shear the microparticles under an optical microscope prior to SEM observation. Polymorphic characterization of the neat naproxen and naproxenethyl cellulose composite microparticles were analysed using powder X-ray diffraction (PXRD) to examine their crystallinity. An X-ray diffractometer (Bruker, D8 Advance) was operated at 40 kV, 30 mA, and at a scanning rate of 1.06 °/min over a range of 2θ from 2.5−30°, using a Cu radiation wavelength of 1.54 Å. Droplet size measurements were made for the initial and final droplets using the open source software Image J (NIH). Optical microscopy images of dried particles were captured using a Qimaging MicroPublisher 5.0 RTV camera mounted on an Olympus SZX7 microscope, with illumination using a Leica CLS 150 XE light source prior to particle


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size measurements using Image J. Average droplet diameters and standard deviations are based on measurements of at least 100 droplets or particles per sample.

RESULTS AND DISCUSSION Formation of monodisperse crystalline drug-containing microparticles Using naproxen as a model hydrophobic drug, monodisperse naproxen-loaded ethyl acetate droplets of different sizes were generated at a microfluidic T-junction and immediately routed (in

Drug

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