Sustained delivery of biologics and non-aqueous manufacture of same

Abstract

Sustained release biologics can be prepared by emulsifying a Solid-in-Hydrocarbon (S / H) mixture in a fluorocarbon solution to form an emulsion of the S / H in the fluorocarbon solution. Advantageously, the Solid-in-Hydrocarbon (S / H) mixture can be prepared by dispersing spray dried biologic particles in a solution of a sustained release polymer in a hydrocarbon solvent. Further, the spray dried biologic particles can be prepared by atomizing a feed solution including a biologic drug in an aqueous carrier to form an aerosol and exposing the aerosol to a stream of drying gas at a temperature of greater than about 80 °C, e.g., from about 80 °C to about 150 °C, to dry the aerosol to form the spray dried biologic particles. In some aspects, emulsifying the S / H mixture in the fluorocarbon solution can be carried out with a microporous membrane.

Description

SUSTAINED DELIVERY OF BIOLOGICS AND NON-AQUEOUS MANUFACTURE OF SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 704,929, filed 8 October 2024, titled SUSTAINED DELIVERY OF BIOLOGICS; U.S. Provisional Application No. 63 / 743,817, filed 10 January 2025, titled NOVEL NON-AQUEOUS EMULSIFICATION-BASED MICROSPHERE PLATFORM FOR SUSTAINED DELIVERY OF PROTEINS; and U.S. Provisional Application No. 63 / 794,892, filed 25 April 2025, titled SUSTAINED DELIVERY OF BIOLOGICS AND NON-AQUEOUS MANUFACTURE OF SAME; the entire disclosures of which are hereby incorporated by reference herein.TECHNICAL FIELD

[0002] The present disclosure relates to sustained release biologies as microparticles and non-aqueous based processes for their preparation.BACKGROUND

[0003] Over the past decade, the development and commercialization of therapeutic proteins, monoclonal antibodies, and other biologies have surged exponentially. These biologies are primarily administered through systemic injections, mainly via the intravenous route (Skalko-Basnet, 2014). However, alternative delivery methods, such as subcutaneous injections, are becoming more common (Bittner et al., 2018). Biologies, when delivered systemically, are particularly susceptible to physiological degradation (Mitragotri et al., 2014; Yadav et al., 2010), necessitating administration of higher doses to achieve therapeutic efficacy. This, in turn, may increase off-target effects and immune responses (Chauhan et al., 2020).

[0004] To circumvent higher doses, biologies are administered frequently to maintain the therapeutic threshold and efficacy. As such, there is a significant opportunity to enhance patient compliance by reducing the dosing frequency and minimizing off- target effects experienced after systemic injections. To enable this, diverse delivery platforms that protect biologies from physiological degradation via either encapsulation and / or a sustained therapeutic delivery may be utilized. Advances in delivery system innovation for biologies areoften inspired by the development journey of small molecule delivery systems, which have been prevalent for many years before the advent of biologies as therapeutic interv entions. Such delivery' platforms include Microspheres (MS), lipid nanoparticles, polymeric implants, etc., which have been successful in reducing off-target effects and improving therapeutic outcomes for small molecules / peptides / genetic materials. In particular, MS as sustained delivery systems, have yet to succeed in delivering biologies. This limited utility may be attributed to the complex set of formulation and process variables that affect biologic encapsulation, stability, and release.

[0005] There are currently less than 20 approved microsphere (MS) products in the USA, all of which are utilized to deliver small molecules and peptides (Wan et al., 2023). Traditionally, MS are prepared using an aqueous emulsification (AqE) process, which involves an organic phase comprising hydrocarbon solvents and an aqueous phase containing water and stabilizers (van der Kooij et al., 2022). This emulsification process results in MS prepared via oil-in-water (O / W) emulsions, water-in-oil (W / O) emulsions, or more complex emulsions such as water-in-oil-in-water (W / O / W). Historically, this platform has been effective for small molecules and peptides. However, biologies have encountered significant challenges in achieving high encapsulation efficiency (due to loss from solubility in aqueous vehicles) and stability (as porous MS leads to increased physiological exposure and leakage) when loaded onto MS using this aqueous platform (Vaishya et al., 2015). To address the limitation of encapsulation efficiency due to solubility of drugs in aqueous vehicles, researchers have explored eliminating water as the continuous phase, leading to the development of oil-in-oil (O / O) emulsification platforms for MS manufacturing, primarily’ for hydrophilic small molecules (Mana et al., 2007). An O / O or non- AqE typically involves two immiscible nonpolar solvents: one serving as the continuous phase and the other as a solubilizer for the polymer used. The continuous phase in O / O emulsions is often spiked with stabilizers or surfactants (to prevent phase separation and aide the emulsification process), while hydrocarbon solvents such as dichloromethane (DCM) or ethyl acetate (EAc) are used to solubilize the polymer and form an emulsion. However, this platform is not well established for biologies, primarily due to the poor compatibility of liquid-state biologies with commonly used non-polar solvents (van de Weert et al., 2000). However, there is an opportunity to formulate solid-state biologies using non-aqueous platforms, as solid biologies are potentially more stable and less prone to degradation and denaturation by non-polar solvents such as DCM.This approach could pave the way for more effective encapsulation and delivery' of biologies using MS systems. Non-aqueous emulsion methods involving protein powder / hydrocarbon / fluorocarbon to produce polymer-coated microparticles are disclosed in US Patent Nos. 11.730,793 and 12,239,687.

[0006] However, a continuing need exists to prepare biologic drug formulations that have high potency, are stable for adequate storage periods and that can be tuned for extended release.SUMMARY OF THE DISCLOSURE

[0007] Advantages of the present disclosure include spray dried biologic particles that can be used in preparing a sustained release biologic.

[0008] These and other advantages are satisfied, at least in part, by producing spray dried biologic particles by atomizing a feed solution, which includes a biologic drug in an aqueous carrier, to form an aerosol: and exposing the aerosol to a stream of drying gas at a temperature of 80 °C or greater (e.g., from about 80 °C to about 150 °C) to dry the aerosol to form spray dried particles of the biologic drug. Advantageously, the aqueous carrier can be a buffered aqueous carrier such as a physiological compatible phosphate buffered saline. The biologic drug can include a protein drug such as an antibody or antigen binding fragment thereof, a fusion protein, a recombinant protein, or a fragment or truncated version thereof, or any combination of the foregoing. In some aspects, the spray dried particles of the biologic drug can have an average particle diameter (Dv50) of at least about 0.5 pm, and no more than 15 pm, such as from about 1 pm to about 10 pm.

[0009] In an implementation, the spray dried particles can be formed into a sustained release biologic by emulsifying a Solid-in-Hydrocarbon (S / H) mixture in a fluorocarbon solution to form an emulsion of the S / H mixture in the fluorocarbon solution, wherein the S / H mixture comprises the spray-dried biologic particles dispersed in a solution of the sustained release polymer in a hydrocarbon solvent: and removing the hydrocarbon solvent to form the microparticles.

[0010] In an implementation, the spray dried particles can be formed into a sustained release biologic by passing a Solid-in-Hydrocarbon (S / H) mixture through pores and into a channel of a microporous membrane which channel includes a cross flow of a fluorocarbon solution to form an emulsion of the S / H mixture in the fluorocarbon solution, wherein the S / Hmixture comprises spray dried biologic particles in a solution of a sustained release polymer in a hydrocarbon solvent, and wherein the channel of the microporous membrane includes an insert which the cross flow of the fluorocarbon solution flows around, and which insert is positioned at a distance of at least 0.1 mm from the pores of the microporous membrane; removing the hydrocarbon solvent to form microparticles in the fluorocarbon solution; and separating the microparticles from the fluorocarbon solution, wherein the microparticles comprise the spray dried biologic particles and the sustained release polymer.

[0011] In some aspects, the microparticles have an average diameter (Dv50) of from about 20 pm to 200 pm, e.g., from about 30 pm to 80 pm. In other aspects, an average microparticle size to average spray dried biologic particle size ratio can be no less than about 5: 1, e.g., no less than about 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, etc. In further aspects, the microparticles can have a sustained release of the biologic drug in a physiological aqueous environment at about 37 °C over a period of at least about 7 days, at least about 14 days or at least about 28 days.

[0012] Additional advantages of the present disclosure will become readily apparent to those skilled in the art from the following detailed description, wherein only certain embodiments are shown and described, simply by way of illustration of carrying out certain subject matter. As will be realized, the invention is capable of other and different embodiments, and its several details are capable of modifications in various respects, all without departing from the invention. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Reference is made to the attached drawings, wherein elements having the same reference numeral designations represent similar elements throughout and wherein:

[0014] FIG. 1 A illustrates a schematic of functional features of a spray drying apparatus for producing spray dried biologic particles according to aspects of the present disclosure.

[0015] FIG. IB illustrates a schematic representation of a lab-scale non-aqueous emulsion process of preparing a sustained release biologic according to an implementation of the present disclosure. The acronyms in the figure include: DCM, dichloromethane; FC-40, fluorocarbon continuous phase; rFcP, recombinant fusion protein; SDP. spray dried protein; S / H / F, solid / hydrocarbon / fluorocarbon.

[0016] FIG. 1C illustrates a schematic representation of functional features of a cross flow non-aqueous emulsion process of preparing a sustained release biologic according to an implementation of the present disclosure.

[0017] FIG. 2A and FIG. 2B illustrate scanning electron micrograph (SEM) images of spray dried protein (SDP) obtained from non-aqueous emulsification process. In particular FIG. 2A is an SEM image of SDP obtained from process 1. A toroidal surface morphology can be seen with a wide particle size distribution. FIG. 2B is an SEM image of SDP obtained from process 2 with a distinct morphology- as compared to process 1. The feed solution in process 2 did not undergo dialysis and was spray dried using a smaller nozzle as compared to process 1 which resulted in a distinct morphology and tighter particle size distribution.

[0018] FIG. 2C illustrates a table providing particle size distribution for SDP obtained from both processes. The median particle size by volume at 50% (Dv50) for SDP obtained from process 1 was two-fold higher as compared to SDP obtained in process 2.

[0019] FIG. 3 A illustrates a plot of the mean particle size of microspheres prepared as formulations F 1-F6 in the Examples below.

[0020] FIG. 3B illustrates SEM images of certain microspheres prepared as formulations F13, F4, F5 and -F6 in the Examples below; Particle diameter was determined using ImageJ opensource software by measuring 15 particles across SEM images captured for all formulations (B)(i-iv) SEM images representing microspheres for formulation F3, F4, F5, and F6, respectively. As seen in the images, particles formed in F3 were non-spherical while formulations F4 and F5 boasted excess SDP on particle surface. Formulation F6 resulted in relatively lesser SDP on microsphere surface while maintaining the spherical shape of the particles. Data represents mean ± SD with n=15.

[0021] FIG. 4A illustrates an SEM image of microspheres obtained from aqueous emulsification. Particles prepared via aqueous process clearly depicts presence of pores (SEE magnified subset).

[0022] FIG. 4B illustrates an SEM image of microspheres prepared via non-aqueous emulsification according to an implementation of the present disclosure. Such particles prepared via the non-aqueous process demonstrate non-porous surface morphology-.

[0023] FIG. 4C and 4D illustrate plots. FIG. 4C is s a plot of % encapsulation efficiency of SDP loaded via aqueous and non-aqueous emulsification process; FIG. 4D is aplot of % burst release of SDP encapsulated in microspheres prepared via aqueous and nonaqueous emulsification. Data represents mean ± SD with n=l or 3.

[0024] FIG. 4E illustrates a plot of % SDP loading of the biologic (w / w) via aqueous and non-aqueous emulsification processes (p, 0.014).

[0025] FIG. 5A, FIG. 5B and FIG. 5C illustrate plots. In particular, FIG. 5A plot represents the particle size distribution (DvlO, Dv50, and Dv90) values for formulations F5 and F6 evaluated using Mastersizer 3000 provided in the Examples. Formulations F5 and F6 have similar size distribution across DvlO and Dv50 values, however, F6 has a wider size distribution, indicated by the relatively higher Dv90 value. FIG. 5B plot represents % total HMW and % purity for SDP prior to microsphere encapsulation followed by after encapsulation in formulation F5 and F6. It can be seen that formulation F6 maintains the pre manufacturing attributes of the SDP in terms of purity and HMW, while formulation F5 results in severe aggregation of SDP. FIG. 5C Plot represents a 2-hour burst release profile for formulations F5 and F6 with F6 leading to lesser amount of SDP being released after 2 hours.

[0026] FIG. 6A illustrates a plot of % cumulative release of SDP from formulation F6 at pH 6.5 (red curve, circles) and pH 7.4 (green curve, triangles). After the initial burst release, a clear sustained release of SDP, independent of pH was observed over 216 hours. At 216 hours, about > 90% of SDP was released from microspheres at a rate of ~8% released at every timepoint. Data represents mean ± SD with n=2 or 3.

[0027] FIG. 6B illustrates a Table of the R2 values calculated for five release models. These R2 values are calculated for the entire duration of release as well as from 24hrs to 216hrs to understand the mechanism of sustained release of SDP. Data represents mean ± SD with n=2 or 3.

[0028] FIG. 6C illustrates a plot of % aggregation and % purity of encapsulated SDP at the end of release (day 8), compared with the start of release and pre-encapsulated SDP.

[0029] FIG. 6D illustrates a plot of % relative potency for a control spry dried biologic protein, after encapsulated with a sustained release polymer, and after the end of release at end of release (day 8) and an assay acceptance criteria of 50-150% activity relative to control. Data represents mean ± SD with n= 2 (aggregation) or 3 (IVR and potency)

[0030] FIG. 7A, and FIG. 7B illustrate plots. In particular, FIG. 7A Plot represents Dv50 values for F6 at TO and Tim stored at accelerated and thermal stress conditions of 25 °C and 40°C / 75% RH. The mean particle size distribution at 25 °C was similar across bothtimepoints, however, slight aggregation was observed for samples stored at 40°C / 75% RH; FIG. 7B Plot represents the % loading efficiency across TO and Tim for samples stored at two different conditions. Storage at accelerated conditions does not affect the loading of SDP on microspheres.

[0031] FIG. 7C, FIG. 7D and FIG. 7E illustrate SEM images of the surface morphology of microspheres incubated in lx PBS at 37 °C for a period of T-0 days, T-4 days. And T-23 days, respectively. A clear surface erosion of microspheres can be seen over time, confirming diffusion via surface erosion as one of the mechanisms responsible for sustained release of SDP. Data represents mean ± SD with n= 6 or 1.

[0032] FIG. 8 illustrates a plot representing cell viability data for HEK-293 cells after exposure to a modified cell nutrient media. Blank MS were incubated with the nutrient media for 24 hours at 37°C to extract any leachable excipient that may contribute to cytotoxicity. Data represents mean ± SD with n= 1 or 3.DETAILED DESCRIPTION OF THE DISCLOSURE

[0033] The present disclosure relates to methods for producing a sustained release biologic, e.g., a protein drug, and to methods for producing spray dried biologic particles that are suitable for encapsulation with sustained release polymers.

[0034] Advantageously, processes of the present disclosure enable high encapsulation of biologies within microparticles, e.g., microspheres, good compatibility with manufacturing process, achieve a tunable release profile, and high stability and high potency of the encapsulated / released biologic, e.g., protein.

[0035] In some aspects, relatively small spray dried biologic particles (e.g., small spray dried protein particles) result in an increased loading in the microparticle and can also reduce encapsulation. Further it was also observed that relatively small spray dried biologic particles tend to adhere less to the surface of the microparticle thereby advantageously reducing burst release of the biologic upon administration to a subject. In certain aspects, the spray dried biologic particles have an average particle diameter (Dv50) of no more than about 15 pm. Further it was found that an average microparticle size to average spray dried biologic particle size ratio can significantly improve encapsulation, such as a ratio of no less than about 5: 1, e.g., no less than about 6: 1, 7: 1, 8: 1, 9: 1, 10: 1, etc.

[0036] Loading and conditions for spray drying can also facilitate potency of the biologic in the microparticle. In other aspects, the biologic comprising the microparticles can retain at least 50% potency, such as at least 70% potency, e.g., at least 80%, 90%, or higher potency.

[0037] In still other aspects, the microparticles are stable for at least one month at 25 °C as determined by a change in Dv50 value of no more than 40%, e.g., no more than 30%, 20%, 15%, such as a change in Dv50 value of no more than about 10%.

[0038] Biologies were formulated as solids using spray-drying process capable of producing stable protein particles of desired sizes. In addition, spray dried biologies (e.g. spray dried protein (SDP)) particles can be encapsulated with sustained release polymers to form polymeric microspheres (MS) using non-aqueous vehicles (hydrocarbons and fluorocarbons), thereby facilitating high encapsulation efficiency while maintaining stability of the biologic and making them less prone to degradation and denaturation. Moreover, process of the present disclosure can harden the encapsulating polymer into to form solid non-porous microparticles, such as microspheres (MS) thereby limiting leakage of the biologic.

[0039] Spray-Dried Biologic Drug

[0040] In some implementations, spray dried biologic particles can be prepared by atomizing a feed solution including a biologic, e g., a therapeutic protein, in an aqueous carrier, e.g., buffered aqueous carrier, to form an aerosol. FIG. 1 A illustrates a schematic of functional features of a spray drying apparatus (100) for producing spray dried biologic particles according to aspects of the present disclosure. As illustrated, atomization can be carried out using an atomizing spray nozzle (102) by supplying a liquid (104), e.g. a feed solution, into the nozzle together with a compressed spray gas (106), e.g. a compressed inert gas such as nitrogen. Atomizing the feed solution in such a manner results in an aerosol of the solution as very fine droplets (110) in the spray gas.

[0041] In some aspects it is advantageous to include a buffering agent with the aqueous carrier for the feed solution since such a buffered aqueous carrier can facilitate forming stable biologic drug particles with minimal degradation of the biologic. Examples of buffered aqueous carriers include a physiological compatible phosphate buffered saline having a pH from about 7.0 to about 7.8, e.g., having a pH of from about 7.2 to about 7.6. Such phosphate buffered saline includes sodium chloride, sodium phosphate, and optionally potassium chlorideand potassium phosphate. Lower pH buffers can also be used, and unbuffered water can be used as well.

[0042] In some aspects, a concentration of the biologic drug in the feed solution can be at least about 1 mg / ml such as a range from about 1 mg / ml to about 500 mg / ml. about 5 mg / ml to about 150 mg / ml, about 10 mg / ml to about 100 mg / ml, and any value or range thereof or therebetween.

[0043] The aerosol formed by atomizing a feed solution can be dried by exposing the aerosol to a stream of drying gas at elevated temperature to dry the aerosol to form spray dried particles of the biologic. Such elevated temperature can be about 80 °C or greater, such as from about 80 °C to about 150 °C, e.g., from about 95 °C to about 130 °C. The drying gas at elevated temperature can be air or an inert gas such as nitrogen. Such a process can be carried out by introducing the aerosol into a drying chamber (122) which includes an inlet for the drying gas (120). The drying gas can be preheated to an elevated temperature with a heater (126). The drying gas at elevated temperature (e.g., at least 80 °C) can be fed into the port (124) of the drying chamber (122) together with the aerosol dries the aerosol to form the spray dried biologic as particles. The formed spray dried particles can be collected using centrifugal forces, such as with a cyclone (130) and collected in a second chamber (140) with exhaust gas exiting the apparatus (128).

[0044] The nozzle orifice, feed rates of the feed solution, and pressure of the compressed gas into the nozzle and the temperature of the dry ing gas at the inlet of the chamber (i.e., drying gas inlet temperature) each influences the average size of the spray dried biologic particles. In an implementation of preparing spray dried biologic particles, the nozzle atomizer can have an orifice of no greater than about 0.7 mm (e.g., from about 0.5 mm to about 0.7 mm), for example. In addition, or alternatively, the compressed spray gas fed into the nozzle can be at a pressure of at least about 50 psi. The table below summarizes various process parameters that can be used in spray drying biologies according to aspects of the present disclosure.Spray Process parameters

[0045] In some aspects, the spray dry biologic particles can have an average particle diameter (Dv50) of no more than about 15 pm, such no more than about 12 pm, 10 pm, 9 pm, 8 pm, 7 pm and even in a range of from about 1 pm to about 10 pm, e.g., from about 2 pm to about 9 pm, from about 2 pm to about 8 pm, and from about 2 pm to about 7 pm. In some aspects, the spray dried biologic particles can have an average particle diameter (Dv50) of at least about 0.5 pm, and no more than 15 pm, such as from about 1 pm to about 10 pm.

[0046] In addition or alternatively, the spray dried biologic particles of the present disclosure can have no more than about 9% of high molecular weight (HMW) species such no more than about 8%, 7%, 6% of HMW species. High molecular weight species are aggregated forms of the biologic (e.g., protein) that are larger than the intended monomeric or functional biologic unit. In other aspects, the spray dried biologic particles retain at least 50% potency, e.g., at least 60%, 70%, 80% or more potency of the biologic drug relative to a control that has not undergone spray drying.Biologies

[0047] Advantageously a variety of biologies, i.e., biological drugs, can be prepared as spray dried particles by the methods of the present disclosure. For example, in some aspects the biologic that can be produced as spray dried particles by methods of the present disclosure include a protein drug. Exemplary protein drugs include but are not limited to proteins, fusion proteins and fragments thereof, antibodies and antigen binding fragments thereof. In one embodiment, the protein is VEGF Trap protein (e.g., Aflibercept, which contains the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 of the VEGF receptor Flkl fused to Fc ofhlgGl for example as described in US PatentNos. 7,087,411, 7.279,159, and 8144840 which are herein incorporated by reference in their entirety. In some embodiments, the VEGF Trap protein is a truncated form of VEGF Trap as described in US Patent No. 7,396, 664 which is incorporated by reference in its entirety.

[0048] In some embodiments, the biologic is an antibody, a human antibody, a humanized antibody, a chimeric antibody, a monoclonal antibody, a multispecific antibody, a bispecific antibody, an antigen binding antibody fragment, a single chain antibody, a diabody,triabody or tetrabody, a dual-specific, tetravalent immunoglobulin G-like molecule, termed dual variable domain immunoglobulin (DVD-IG), an IgD antibody, an IgE antibody, an IgM antibody, an IgG antibody, an IgGl antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In one embodiment, the antibody is an IgGl antibody. In one embodiment, the antibody is an IgG2 antibody. In one embodiment, the antibody is an IgG4 antibody. In another embodiment, the antibody comprises a chimeric hinge. In still other embodiments, the antibody comprises a chimeric Fc. In one embodiment, the antibody is a chimeric IgG2 / IgG4 antibody. In one embodiment, the antibody is a chimeric IgG2 / IgGl antibody. In one embodiment, the antibody is a chimeric IgG2 / IgGl / IgG4 antibody.

[0049] In some embodiments, the antibody is selected from the group consisting of an anti -Programmed Cell Death 1 antibody (e.g., an anti-PDl antibody as described in U.S. Pat. No. 9,987,500, an anti-Programmed Cell Death Ligand-1 (e.g., an anti-PD-Ll antibody as described in in U.S. Pat. No. 9,938,345), an anti-D114 antibody, an anti-Angiopoetin-2 antibody (e.g., an anti-ANG2 antibody as described in U.S. Pat. No. 9,402,898), an anti- Angiopoetin- Like 3 antibody (e.g., an anti-AngPtl3 antibody as described in U.S. Pat. No. 9,018,356), an anti -platelet derived growth factor receptor antibody (e.g., an anti-PDGFR antibody as described in U.S. Pat. No. 9.265,827). an anti-Erb3 antibody, an anti- Prolactin Receptor antibody (e.g.. anti-PRLR antibody as described in U.S. Pat. No. 9.302.015). an antiComplement 5 antibody (e.g., an anti-C5 antibody as described in U.S. Pat. No 9,795,121), an anti-TNF antibody, an anti-epidermal growth factor receptor antibody (e.g., an anti-EGFR antibody as described in U.S. Pat. No. 9,132,192 or an anti -EGFRv III antibody as described in U.S. Pat. No. 9,475,875), an anti-Proprotein Convertase Subtilisin Kexin-9 antibody (e.g., an anti-PCSK.9 antibody as described in U.S. Pat. No. 8,062,640 or U.S. Pat. No. 9,540,449), an Anti-Growth and Differentiation Factor-8 antibody (e.g. an anti-GDF8 antibody, also known as anti-myostatin antibody, as described in U.S. Pat Nos. 8,871,209 or 9,260,515), an anti- Glucagon Receptor (e.g. anti-GCGR antibody as described in U.S. Pat. Nos. 9.587,029 or 9,657,099), an anti-VEGF antibody, an anti-ILlR antibody, an interleukin 4 receptor antibody (e.g., an anti-IL4R antibody as described in U.S. Pat. Appln. Pub. No. US2014 / 0271681A1 (abandoned) or U.S. Pat Nos. 8,735,095 or 8,945,559), an anti-interleukin 6 receptor antibody (e.g., an anti-IL6R antibody as described in U.S. Pat. Nos. 7,582,298, 8,043,617 or 9,173.880), an anti-ILl antibody, an anti-IL2 antibody, an anti-IL3 antibody, an anti-IL4 antibody, an anti- IL5 antibody, an anti-IL6 antibody, an anti-IL7 antibody, an anti-interleukin 33 (e.g., anti- IL33antibody as described in U.S. Pat. Nos. 9,453,072 or 9,637,535), an anti-Respiratory syncytial virus antibody (e.g., anti-RSV antibody as described in U.S. Pat. Nos. 9,447,173 and 10,125.188, and U.S. Pat. Appl. Pub. No. US2019 / 0031741A1), an anti-Cluster of differentiation 3 (e.g., an anti-CD3 antibody, as described in U.S. Pat. No. 9,657,102), an antiCluster of differentiation 20 (e.g., an anti-CD20 antibody as described in U.S. Pat. Nos. 9,657,102 and US20150266966A1, and in U.S. Pat. No. 7,879,984), an anti-CD19 antibody, an anti-CD28 antibody, an anti- Cluster of Differentiation-48 (e.g., anti-CD48 antibody as described in U.S. Pat. No. 9,228,014). an anti-Fel dl antibody (e.g., as described in U.S. Pat. No. 9,079,948), an anti-Middle East Respiratory Syndrome virus (e.g. an anti-MERS antibody as described in U.S. Pat. No. 9,718,872), an anti-Ebola virus antibody (e.g., as described in U.S. Pat. No. 9,771,414), an anti-Zika virus antibody, an anti-Lymphocyte Activation Gene 3 antibody (e.g., an anti-LAG3 antibody, or an anti-CD223 antibody), an anti-Nerve Growth Factor antibody (e.g.. an anti-NGF antibody as described in U.S. Pat. Appln. Pub. No. US2016 / 0017029 (abandoned) and U.S. Pat. Nos. 8,309,088 and 9,353,176) and an antiProtein Y antibody. In some embodiments, the bispecific antibody is selected from the group consisting of an anti-CD3 x anti-CD20 bispecific antibody (as described in U.S. Pat. Nos. 9,657,102 and US20150266966A1), an anti-CD3 x anti-Mucin 16 bispecific antibody (e.g., an anti-CD3 x anti-Mucl6 bispecific antibody), and an anti-CD3 x anti- Prostate-specific membrane antigen bispecific antibody (e.g., an anti-CD3 x anti-PSMA bispecific antibody). In some embodiments, the protein of interest is selected from the group consisting of abciximab, , adalimumab, adalimumab-atto, ado-trastuzumab, alemtuzumab, alirocumab. atezolizumab, avelumab, basiliximab, belimumab, benralizumab, bevacizumab, bezlotoxumab, blinatumomab, brentuximab vedotin, brodalumab, brolucizumab, canakinumab, capromab pendetide, certolizumab pegol, cemiplimab, cetuximab, denosumab, dinutuximab, dupilumab, durvalumab, eculizumab, elotuzumab, emicizumab-kxwh, emtansinealirocumab, evinacumab, evolocumab, fasinumab, golimumab, guselkumab, ibritumomab tiuxetan, idarucizumab, infliximab, infliximab-abda, infliximab-dyyb, ipilimumab, ixekizumab, mepolizumab, necitumumab, nesvacumab, nivolumab, obiltoxaximab, obinutuzumab, ocrelizumab, ofatumumab, olaratumab, omalizumab, panitumumab, pembrolizumab, pertuzumab, ramucirumab, ranibizumab. raxibacumab, reslizumab, rinucumab, rituximab, sarilumab, secukinumab, siltuximab, tocilizumab, tocilizumab, trastuzumab, trevogrumab, ustekinumab, and vedolizumab.

[0050] In some embodiments, the protein included as spray dried particles is a recombinant protein that contains an Fc moiety and another domain, (e.g., an Fc-fusion protein). In some embodiments, an Fc-fusion protein is a receptor Fc-fusion protein, which contains one or more extracellular domain(s) of a receptor coupled to an Fc moiety. In some embodiments, the Fc moiety comprises a hinge region followed by a CH2 and CH3 domain of an IgG. In some embodiments, the receptor Fc-fusion protein contains two or more distinct receptor chains that bind to either a single ligand or multiple ligands. For example, an Fc-fusion protein is a TRAP protein, such as for example an IL-1 trap (e.g., rilonacept, which contains the IL-lRAcP ligand binding region fused to the I1-1R1 extracellular region fused to Fc of hlgGl ; see U.S. Pat. No. 6,927,004, which is herein incorporated by reference in its entirety), or a VEGF trap (e.g., aflibercept or ziv-aflibercept, which comprises the Ig domain 2 of the VEGF receptor Fltl fused to the Ig domain 3 oftheVEGF receptor Flkl fused to Fc of hlgGl 0). In other embodiments, an Fc-fusion protein is a ScFv-Fc-fusion protein, which contains one or more of one or more antigen-binding domain(s), such as a variable heavy chain fragment and a variable light chain fragment, of an antibody coupled to an Fc moiety.

[0051] Solid-in-Hydrocarbon-in-Fluorocarbon (S / H / F) Emulsions

[0052] In accordance with various implementations, the spray dried biologic particles of the present disclosure can be formed into microparticles with a sustained release polymer by a non-aqueous emulsion processes such as a solid-in-hydrocarbon-in-fluorocarbon emulsion. Two such non-limiting solid-in-hydrocarbon-in-fluorocarbon emulsions processes are provided in the present disclosure. In an implementation, a solid-in-hydrocarbon-in- fluorocarbon emulsion includes emulsifying a Solid-in-Hydrocarbon (S / H) mixture in a fluorocarbon solution to form an emulsion of the S / H mixture in the fluorocarbon solution. Advantageously, the S / H mixture comprises the spray -dried biologic particles dispersed in a solution of a sustained release polymer in a hydrocarbon solvent. Removing the hydrocarbon solvent in such an emulsion forms microparticles that includes the spray dried biologic particles and the sustained release polymer. In some aspects the spray dried biologic particles are coated, at least in part, with or encapsulated by the sustained release polymer.

[0053] Non-aqueous processes were previously reported in US Patent Nos. 11,730,793 and 12.239,687 by Regeneron Pharmaceuticals, Inc. (Zhao and Chen). A FluoriPack platform employs a Solid / Hydrocarbon / Fluorocarbon (S / H / F) system. Utilizing solid-state proteins in this technology offers multiple advantages over their liquid counterparts, including enhancedstability within the hydrocarbon phase and improved encapsulation efficiency within the polymer matrix. The hydrocarbon phase, which includes the solid protein and polymer, comprises an organic solvent capable of solubilizing the polymer. A fluorocarbon solution can form the fluorocarbon continuous phase as an inert, immiscible solution. The fluorocarbon solution can include a fluorocarbon liquid, e.g. a perfluoro amine, such as 3M™ Fluorinert™ Electronic Liquid FC-40 (hereafter referred to as Fluorinert or FC-40), which is inert and immiscible with most commercially available organic hydrocarbon solvents. Additionally, a fluorocarbon solution can include a fluorinated surfactant, e.g., a perfluoropolyether such as Pico-Surf™l. to stabilize MS. Together, FC-40 and Pico-Surf 1 constitute the continuous phase for emulsification. The emulsification of the hydrocarbon phase and the continuous phase followed by solvent evaporation results in the microprecipitation of the polymer, enabling efficient protein encapsulation. Such Solid / Hydrocarbon / Fluorocarbon (S / H / F) system can be used with the spray dried biologic particles of the present disclosure with improved loading, potency, storageability, and / or extended release profile.

[0054] For example, FIG. IB is a schematic representation of atype of FluoriPack (FP). This platform demonstrates excellent process robustness, reproducibility, and tunability for producing polymeric microspheres. As shown in FIG. 1A. spray dried biologic particles can be prepared with a spray drying apparatus (100). A non-aqueous mixture is prepared by combining spray dried powder of biologic particles (202), e.g., an spry-dried protein (SDP) of rFcP particles for this example, in a solution of a sustained release polymer (204) in a hydrocarbon solvent (e.g., dichloromethane (DCM)) to form a solid in hydrocarbon S / H mixture (Phase 1 )(210). The S / H mixture (210) can be dispersed in a fluorocarbon solution (Phase 2) (212). For this example, the fluorocarbon solution includes FC-40 (a fluorocarbon liquid) and Pico-Surf™l (a fluorosurfactant). The S / H mixture can be dispersed in the fluorocarbon solution (Phase 2) by adding the mixture to the fluorocarbon solution with agitation such as sonication to form an emulsion of the mixture as droplets in the fluorocarbon solution (S / H / F emulsion) (214). Removing the hydrocarbon solvent (e.g., DCM) from the droplets forms solid microparticles in the fluorocarbon solution (216). Removing the hydrocarbon solvent can be carried out by vacuum removal with or without stirring. The microparticles can be isolated by fdtration and further dried to remove residual fluorocarbon solution or hydrocarbon solvent (220). The microparticles (230) include one or more spray dried biologic particles (202) entrapped or coated by the sustained release polymer (204).

[0055] In certain aspects, methods for producing a sustained release biologic can include dispersing spray dried biologic particles in a solution of a sustained release polymer in a hydrocarbon solvent as a Solid-in-Hydrocarbon (S / H) mixture. In some aspects the spray- dried biologic particles comprises a therapeutic protein, which is also referred to here as a protein drug, such as an antibody or antigen binding fragment thereof, a fusion protein, a recombinant protein, recombinant fusion protein, or a fragment or truncated version of a fusion protein, a recombinant protein, recombinant fusion protein, or any combination of the foregoing. The S / H mixture can be emulsified in a fluorocarbon solution to form an emulsion of the S / H in the fluorocarbon solution (S / H / F). The methods further include removing the hydrocarbon solvent to form microparticles in the fluorocarbon solution. The microparticles can then be readily separated from the fluorocarbon solution. Advantageously, the microparticles comprise the spray dried biologic particles, e.g., spray dried particles of a therapeutic protein, and the sustained release polymer. In some aspects the microparticles comprise the spray dried biologic particles coated, at least in part, with or encapsulated by the sustained release polymer.

[0056] The amounts of the spray dried biologic and sustained release polymer in the mixture will influence the release profde of the microparticles formed thereof. In some aspects, the amount of the spray dried particles of the biologic drug in the mixture can be from about 2.5 % w / v to about 25 % w / v relative to the total volume of the mixture. In addition, the amount of the sustained release polymer in the mixture can be from about 10 % w / v to about 60 % w / v, such as from about 20 %w / v to about 60 % w / v relative to the total volume of the mixture.

[0057] Further, and as shown in the Examples below, the rate of agitating the S / H / F emulsion, such as the rate of stirring, can influence the size of the droplets of the mixture in the fluorocarbon solution and subsequently formed microparticles. In addition, the type of agitation can influence the shape of the microparticle. For example, stirring or vortexing the emulsion will generally lead to microparticles in the form of microspheres. In some aspects, the microparticles, e.g., microspheres, can have an average diameter (Dv50) of from about 20 pm to 200 pm, such as an average diameter (Dv50) of from about 20 pm to about 80 pm, e.g., from about 20 pm to about 60 pm.

[0058] In a particular aspect, the microparticles of the present disclosure can have an average size (Dv50) of from about 30 pm to about 50 pm and can include the spray driedbiologic particles with an average size of no more than Dv50 of 10 pm, e.g., from about 2 pm to about 6 pm, and in an amount of from about 3- 25% w / w of the sustained release polymer, e.g., a polyorthoester.

[0059] S / H / F Cross Flow emulsification

[0060] Another implementation of a solid-in-hydrocarbon-in-fluorocarbon emulsion includes emulsifying a Solid-in-Hydrocarbon (S / H) mixture in a fluorocarbon solution by passing the S / H mixture through pores and into a channel of a microporous membrane. The channel includes a cross flow of the fluorocarbon solution to form the emulsion of the S / H mixture in the fluorocarbon solution. For example, and as illustrated in FIG. 1C. a Solid-in- Hydrocarbon (S / H) mixture (Dispersed phase) (310) can be passed through the pores of a microporous membrane (320) using controlled pressure into a channel (322) of a microporous membrane. The microporous membrane (320) can be housed in a container (324). Advantageously, the S / H mixture (310) includes the spray-dried biologic particles as solids dispersed in a solution of a sustained release polymer in a hydrocarbon solvent. Simultaneously, the continuous phase, i.e., a fluorocarbon solution (312), flows tangentially (cross flow) across the membrane surface in the channel of a microporous membrane. As the S / H mixture exits the membrane pores, droplets (314) form at the pore openings. The shear force from the cross-flowing continuous fluorocarbon phase detaches the droplets, carrying them away into the bulk fluid as an emulsion (31 ) of the S / H mixture (310) in the fluorocarbon solution (312). This drop-by-drop mechanism allows for precise control over droplet size and distribution. Further, and as illustrated, the channel of the microporous membrane includes an insert (350) which the cross flow of the fluorocarbon solution flows around. The insert (350) modifies internal flow, potentially reducing backpressure and improving droplet formation. It was found that when the insert is positioned at a distance of at least 0.1 mm from the pores of the microporous membrane, appropriately droplets formed. Also, this configuration imparts a low shear on the biologic making it a gentler emulsification process than stirring.

[0061] Upon exit of the emulsion (316) from the channel (322), the hydrocarbon can be removed thereby forming microparticles that includes the spray dried biologic particles and the sustained release polymer. Further, the microparticles can be separated from the fluorocarbon solution. In some aspects the spray dried biologic particles are coated, at least in part, with or encapsulated by the sustained release polymer by this process.

[0062] In some aspects, the S / H mixture can be passed through the pores of the microporous membrane at a rate of at least 0. 1 rnl / min such as about 0.5 mL / min or greater. In other aspects, the pores of the microporous membrane can have an average size of at least 0.2 microns such as from about 10 microns to about 100 microns. In still further aspects, the cross flow of the continuous phase, i.e., a fluorocarbon solution, can be at least 1 ml / min, such as at least 5 ml / min, and higher.

[0063] Further, the flow rates of the S / H mixture through the membrane pores and continuous fluorocarbon solution in the channel as well as the insert and its position can influence the size of the droplets of the S / H mixture in the fluorocarbon solution emulsion and subsequently formed microparticles. In some aspects, the microparticles, e.g., microspheres, can have an average diameter (Dv50) of from about 20 pm to 200 pm, such as an average diameter (Dv50) of from about 20 pm to about 80 pm, e.g., from about 20 pm to about 60 pm.

[0064] In a particular aspect, the microparticles of the present disclosure can have an average size (Dv50) of from about 30 pm to about 50 pm and can include the spray dried biologic particles with an average size of no more than Dv50 of 10 pm, e.g., from about 2 pm to about 6 pm, and in an amount of from about 3-25% w / w of the sustained release polymer, e.g., a poly orthoester.

[0065] Advantageously, the sustained release biologic microparticles of the present disclosure prepared by any of the foregoing emulsion processes can release the biologic, e.g., therapeutic protein, in a physiological aqueous environment at about 37 °C at a relatively constant rate over an extended period of time. For example, sustained release biologic microparticles of the present disclosure can release the biologic can have a sustained release of the biologic drug in a physiological aqueous environment at about 37 °C over a period from about 1 to about 60 days, or any range therebetween or value thereof such as over a period of at least 1 day, at least about 7 days, at least about 14 days, at least about 21 days, or at least about 28 days. Advantageously, the sustained release of the biologic drug can be independent of physiological pH, e.g., the sustained release of the biologic drug is independent of pH in a range of from about 6.0 to about 8.0, such as a pH range of about 6.5 to about 7.4. Moreover, in an aspect, the sustained release biologic of the present disclosure can have a burst rate of no more than 10% such as no more than about 5%.

[0066] Hydrocarbon Solvents

[0067] A variety of hydrocarbon solvents (also referred to as hydrocarbon liquids) can be used to form the mixture with spray dried biologic particles and a sustained release polymer. Useful hydrocarbon solvents include those that dissolve the sustained release polymer, e.g., a biodegradable and / or bioerodible polymer. For example, hydrocarbon solvents can include dichloromethane, chloroform, toluene, ethyl acetate, tetrahydrofuran, or a combination thereof. In aspects, the hydrocarbon solvent can include other solvents such as acetonitrile, dimethylformamide, dimethylsulfoxide, acetone, ethanol, methanol, pentane, propanol, hexane, or a combination thereof.

[0068] Fluorocarbon Solution

[0069] As explained above, the fluorocarbon solution forms the continuous phase or bulk of the S / H / F emulsion. The fluorocarbon solution includes a fluorocarbon liquid and optionally a fluorosurfactant. A variety of fluorocarbon liquids can be used in the methods of the present disclosure and include for example a fluoro or perfluoro C5-C18 compound, a perfluoro C1-C18 ether, amine, etc. An exemplary fluorocarbon liquid includes a perfluoro amine having a formula of N(Rfl, Rf2, Rf3) in which each of Rfl, Rf2, and Rf3 are independently linear or branched fluoroalkyl groups having 1 -8 carbon atoms and mixtures of such perfluoro amines. Such a perfluoro amine can include Flourinert™ FC-40 (average MW = 650 g / mol) l,l,2,2,3,3,4,4.4-nonafluoro-N,N-bis(1.1.2,2,3,3,4,4,4-nonafluorobutyl)butan-l- amine (N(CF2CF2CF2CF3)3), Fluorinert™ FC-70 (average MW = 821 g / mol) or a combination thereof.

[0070] Fluorosurfactant

[0071] In some implementations, the fluorocarbon solution can include one or more fluorosurfactants. Such a fluorosurfactant can be a perfluorinated polyether (PFPE) or a block copolymer of the perfluorinated polyether with another polymer block such as a polyethylene glycol. For example, the fluorosurfactant (FS) can have the following structure:

[0072] in which n is about 37, x + z is about 6.0, y is about 12.5, or wherein n = 3.7, x+ z ~ 3.6, y ~ 9.0. (Lee, M. et al., Lab Chip., 7: 14(3): 509-13(2014)). An exemplary7FS is Perfluoropolyether-b-Polyethylene glycol-b- Perfluoropolyether (PFPE-PEG-PFPE) tri-block co-polymer which is commercially available as Pico-Surf1M1. In some aspects, the fluorocarbon solution includes the fluorosurfactant in an amount of from about 0.1 % w / v to about 5 % w / v, e.g., from about 0. 1 % w / v to about 1 % w / v.

[0073] Hydrofluoroether (HFE)

[0074] In some aspects, a fluorocarbon solution can include a hydrofluoroether (HFE). Such HFE can be represented as RfORh in which Rf is a fluoroalkyl group and Rh is an alkyl group. HFEs have molecular structures which can be linear, branched, or cyclic, or a combination thereof (such as alkylcycloaliphatic), and are preferably free of ethylenic unsaturation, having a total of about 4 to about 20 carbon atoms. Such HFEs are known and are readily available, either as essentially pure compounds or as mixtures. Due to the lipophilicity and fluorophilicity of HFEs, they are miscible with both fluorocarbon and hydrocarbon. When added to the hydrocarbon / fluorocarbon emulsion they can act as a cosolvent to extract hydrocarbon to the fluorocarbon phase and accelerate forming microparticles, e.g. accelerate the hardening process.

[0075] In one aspect, the fluorocarbon solution can include an HFE having the following chemical structure:CH3CH-O F E FCF3CF3CF F pF2-(Trifluoromethyl)-3-ethoxy dodecafluorohexane.

[0076] Other exemplary HFE includes but are not limited to NOVEC™ 7000 (1- methoxyheptafluoropropane), NOVEC™ 7100 (methoxy -nonafluorobutane), NOVEC™ 7200 (ethoxy -nonafluorobutane), NOVEC™ 7500 (2-(Trifluoromethyl)-3- ethoxy dodecafluorohexane. In still other aspects, the fluorocarbon solution includes FC-40, FC-70, Novec™ 7500, Novec™ 7100, Novec™ 7000, or combinations thereof.

[0077] Sustained release Polymers

[0078] A variety of sustained release polymers can be included in the mixture with spray dried biologies to form sustained release microparticles. Such sustained release polymers include erodible and / or biodegradable polymers. Such polymers include, for example, branched or linear polyethylene glycol (PEG), polylactic acid (PLA), polygly colic acid (PGA), polylactic-polyglycolic copolymer (PLGA), poly-D,L-lactide-co-glycolide (PLGA), PLGA- ethylene oxide fumarate, PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), polyanhydride poly[1.6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric acid-cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly (lactic acid) copolymer (PEG-PLA), poly- -caprolactone (PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate (PEC), polyisobutyl cyanoacrylate, poly-N-(2- hydroxypropyl)methacrylamide (poly(HPMA)). poly-|3-R-hydroxy butyrate (PHB), poly-|3-R- hydroxy alkanoate (PHA), poly-P-R-malic acid, phospholipid-cholesterol polymers, 2- dioleoyl-sn-glycero-3-phosphatidylcholine / polyethyleneglycol- distearoylphosphatidylehtanolamine (DOPC / PEG-DSPE) / Cholesterol, polysaccharides, cellulose, ethyl cellulose, methyl cellulose, alginates, dextran and dextran hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin, heparin, hyaluronic acid, cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes, polyaspartates, polyglutamates, polylucine, leucine-glutamate co-polymers, polybutylene succinate, gelatin, collagens, fibrins, fibroin, polyorthoesters, polyorthoester-polyamidine copolymer, polyorthoester-diamine copolymers, poly orthoesters incorporating latent acids, poly(ethylene glycol) / poly(butylene terephthalate) copolymer, and combinations and copolymers thereof. In one embodiment, the polymer is poly-e-caprolactone (PCL) or a derivative or copolymer thereof. In one embodiment, the sustained release polymer includes a PLGA or a derivative or copolymer thereof. In one embodiment, the sustained release polymer includes an ethyl cellulose or a derivative or copolymer thereof. In one embodiment, the sustained release polymer includes apoly orthoester or a derivative or copolymer thereof. In one embodiment, the sustained release polymer includes a polyesteramide.

[0079] As used herein, the term “polymer” refers to a macromolecule comprising repeating monomers connected by covalent chemical bonds. Polymers can be biocompatible, biodegradable, and bioerodible. A biocompatible and biodegradable polymer can be natural or synthetic. Natural polymers include polynucleotides, polypeptides, such as naturally occurring proteins, recombinant proteins, gelatin, collagens, fibrins, fibroin, polyaspartates, polyglutamates, polylysine, leucine-glutamate co-polymers; and polysaccharides, such as cellulose alginates, dextran and dextran hydrogel polymers, amylose, inulin, pectin and guar gum, chitosan, chitin, heparin, and hyaluronic acid. Synthetic biocompatible or biodegradable polymers include polylactic acid (PLA), polyglycolic acid (PGA), polylactic-polyglycolic copolymer (PLGA), poly-D.L-lactide-co-glycolide (PLGA), PLGA-ethylene oxide fumarate, PLGA-alpha-tocopheryl succinate esterified to polyethylene glycol 1000 (PLGA-TGPS), polyanhydride poly[l,6-bis(p-carboxyphenoxy)hexane] (pCPH), poly(hydroxbutyric acid- cohydroxyvaleric acid) (PHB-PVA), polyethylene glycol-poly (lactic acid) copolymer (PEG- PLA), poly- -caprolactone (PCL), poly-alkyl-cyano-acrylate (PAC), poly(ethyl)cyanoacrylate (PEC), polyisobutyl cyanoacrylate. poly-N-(2- hydroxypropyl)methacrylamide (poly(HPMA)), poly- -R-hydroxy butyrate (PHB), poly- - R-hydroxy alkanoate (PHA), poly- -R-malic acid, phospholipid-cholesterol polymers, 2- dioleoyl-sn-glycero-3-phosphatidylcholine / polyethyleneglycol- distearoylphosphatidylehtanolamine (DOPC / PEG-DSPE) / Cholesterol. ethyl cellulose, cyclodextrin (CD)-based polyrotaxanes and polypseudorotaxanes, polybutylene succinate (PBS), polyorthoesters, polyorthoester-polyamidine copolymers, polyorthoester-diamine copolymers, polyorthoesters incorporating latent acids tom control rates of degradation, and inter alia poly(ethylene glycol) / poly(butylene terephthalate) copolymers.

[0080] Ethyl cellulose (EC) is a well-known and readily available biomaterial used in the pharmaceutical and food sciences. It is a cellulose derivative in which some of the glucose hydroxyl groups are replaced with ethyl ether. See Martinac et al., J. Microencapsulation, 22(5): 549-561 (2005) and references therein, which describe methods of using ethyl cellulose as biocompatible polymers in the manufacture of microspheres. See also US 4,210.529 (1980) and references therein for a detailed description of ethyl cellulose and methods of making derivatives of ethyl cellulose.

[0081] Poly-D,L-lactide-co-glycolide (PLGA) is also a well-known Food and Drug Administration (FDA) approved biocompatible and biodegradable polymer used in tissue engineering and pharmaceutical delivery systems. PLGA is a polyester comprising glycolic acid and lactic acid monomers. For a description of the synthesis of PLGA and manufacture of PLGA nanoparticles, see Astete and Sabliov, Biomater. Sci. Polym. Ed., 17(3): 247-89 (2006) and references therein.

[0082] Poly-e-caprolactone (PCL) is another biocompatible and biodegradable polymer approved by the FDA for use in humans as a drug delivery device. PCL is a polyester of s-caprolactone, which hydrolyses rapidly in the body to form a non-toxic or low toxicity hydroxycarboxylic acid. For a description of the manufacture of PCL, see Labet and Thielemans, Chemical Society Reviews 38: 3484-3504 (2009) and references therein. For a description of the manufacture and use of PCL-based microspheres and nanospheres as delivery systems, see Sinha et al., Int. J. Pharm., 278(1): 1-23 (2004) and references therein.

[0083] Polyorthoester (POE) is a bioerodible polymer designed for drug delivery. It is generally a polymer of a ketene acetal, preferably a cyclic diketene acetal, such as e.g., 3,9- dimethylene-2,4,8,10-tetraoxa spiro [5.5] -undecane, which is polymerized via glycol condensation to form the orthoester linkages. A description of polyorthoester synthesis and various types can be found e.g. in US 4,304,767. Polyorthoesters can be modified to control their drug release profile and degradation rates by swapping in or out various hydrophobic diols and polyols, such as e.g., replacing a hexanetriol with a decanetriol.; as well as adding latent acids, such as e.g., glycolide. octanedioic acid or the like, to the backbone to increase pH sensitivity. Custom forms of POE can include glycolic acid in the POE backbone to tune mass loss and drug release. Other modifications to the polyorthoester include the integration of an amine to increase functionality7. The formation, description, and use of polyorthoesters are described in US 5,968,543; US 4,764,364; Heller and Barr, Biomacromolecules, 5(5): 1625- 32 (2004); and Heller, Adv. Drug. Deliv. Rev., 57: 2053-62 (2005).

[0084] The following examples further illustrate forming microparticles of spray dried biologic particles with a sustained release polymer by a solid-in-hydrocarbon-in-fluorocarbon emulsion and characterization of the microparticles.EXAMPLES

[0085] The following examples are intended to further illustrate certain aspects of the subject technology and are not limiting in nature. Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein.

[0086] 1. METHODS

[0087] To develop MS using FluoriPack system, a protein (97 kDa) was encapsulated in polyester (PE) based MS. The protein was first spray dried using an in-house developed process, which can yield stable solid biologies of desired particle sizes. SDP was characterized for particle morphology and size distribution. Following this. 5-25% w / w SDP was dispersed in a solution of 10-40% w / v PE in di chloromethane (DCM) or ethyl acetate (EtAc) as the Solid in Hydrocarbon phase (S / H). S / H phase was emulsified with the fluorocarbon solution phase (e.g., Fluorinert™ FC-40, NOVEC™ 7500, etc.) containing fluorosurfactants such as PicoSurf- 1™. Bulk emulsification was performed via homogenization of both phases for 60 seconds and followed by evaporation of DCM. Protein loaded MS were evaluated for physical characteristics (scanning electron microscopy), PSD (Malvern 3000), % SDP loading (SoloVPE), in-vitro release (SoloVPE), accelerated stability (PSD and % SDP loading), and toxicity of blank microspheres (HEK-293 cell-based assay). In addition, encapsulated protein released from MS was evaluated for integrity (SE-UPLC) and potency (bioassay).

[0088] 1.1 RESULTS

[0089] Preliminary analysis of SDP revealed minimal protein aggregation after spray drying, as analyzed using SE-UPLC. It was observed that SDP had purity of between 94-99% and total % HMW species between 0.5 - 6%. Moreover, the produced SDP retained >98% biological activity relative to the liquid-state protein formulation, indicating excellent quality of solid protein. These SDP particles were utilized as model solid protein for further studies. Multiple formulations were prototyped as part of a DoE study to understand the impact of MS formulation parameters such as polymer concentration, initial SDP % loading, SDP size, choice of hydrocarbon solvent, concentration of surfactant, and process vanables such as homogenization shear, time, and emulsification ratio. An optimized formulation comprised of 40% w / v of polymer stabilized with 1% PicoSurf-1™ with 5% w / w SDP loading.

[0090] Preliminary evaluation of SDP-loaded MS reveals superior performance for MS prepared via FluoriPack (MS-FP) as compared to aqueous emulsification (MS-AqE),evidenced by non-porous versus porous surface and bulk morphology (scanning electron microscopy), respectively, enhanced encapsulation efficiency (> 60 % for MS-FP versus < 10 % for MS-AqE), and reduced burst release (< 40% for MS-FP versus > 75 % for MS-AqE within 2 hours). In-depth characterization of MS-FP revealed a mean microsphere particle size of ~40 pm with minimal impact on protein aggregation (A -1.1%) and potency (-90% relative to the liquid-state biologic). In-vitro release evaluation revealed an initial burst release of < 40% followed by a sustained release over 9 days with -8% of encapsulated protein being released every 24 hours. MS-FP were found to be stable at accelerated conditions (25°C) tested over 1 month, as indicated by their mean particle size (A +0.8 pm) and % loading (A -0.21 % w / w). At thermal stress conditions (40°C / 75% RH), a slight increase in mean microsphere particle size (A +13 pm) w as observed at the 1-month timepoint for SDP-loaded MS, and there was no impact on measured SDP loading (A -0. 1 % w / w). Further, biocompatibility evaluation of blank MS-FP was performed using non-cancerous mammalian cells (HEK-293). All tested concentrations of blank MS-FP resulted in > 80% viability of HEK-293 cells after 24 hours of incubation with 200 mg / ml of particles being the highest concentration that was tested.

[0091] 1.3 CONCLUSIONS FOR METHOD 1

[0092] Our studies successfully demonstrate the development and optimization of SDP-loaded MS prepared using a novel non-aqueous emulsification platform to overcome limitations of sustained biologic delivery using conventional emulsification techniques. Studies are ongoing to fabricate MS using membrane emulsification technique for scalable and reproducible production. The data generated shows that MS prepared using this novel nonaqueous emulsification platform achieved the desired quality attributes, enabling FluoriPack to be a promising tool for sustained delivery of biologies.

[0093] 2. Materials

[0094] A model recombinant fusion protein (rFcP) (97 kDa) (85-115 kDa, -850 amino acid sequences) was provided by Regeneron (Tarrytown, NY, USA). Polyorthoester (POE) polymer used in these examples was procured from Chempilots (Farum, Denmark) and has a MW range of 12-19 kDa with low glycolic acid content with a co-monomer ratio of 1:0.9999:0.0001 (DETOSU:CDM+CM:CDM-GL). This material has a Tg of 91°C with a solution viscosity of 18 cP (n-PAc). DCM and FC-40 solvents were procured from Sigma- Aldrich (St. Louis, MO, USA). 5% Pico-Surf 1 in FC-40 was procured from Sphere Bio (Cambridge, United Kingdom). lOx phosphate buffered solution (PBS) pH 7.4 was procuredfrom Invitrogen™ Corporation (Waltham, MA, USA) and polyvinyl alcohol (99 to 100% hydrolyzed) was procured from Spectrum® Chemicals (New Brunswick, NJ, USA). The stock solution of rFcP was prepared to reach a final concentration of 10 mg / mL using lx PBS pH 7.4 ± 0.3 and filtered using a 0.22 pm syringe filter.L0095] 3. Methods

[0096] 3.1 Preparation of spray dried protein

[0097] A Buchi B-290 mini spray dryer (Biichi Labortechnik, Switzerland) with a nozzle atomizer and a nozzle orifice of 0.7 mm (process 1) or 0.5 mm (process 2) was used to produce spray dried protein (SDP). SDP indicates the active protein in the dried powder blend and any reference to SDP here after refers to the active protein alone and not the excipients involved. This study develops and optimizes a process to produce stable spray dried powder with tunable size range. The rFcP stock solution (10 mg / mL) was spray dried with the feed pump at 10% (3 mL / min) capacity with an atomizing airflow of compressed ultra-high purity grade nitrogen at 75 psi with the aspirator capacity set at 85% and an inlet temperature of 130°C (corresponding outlet temperature was ~65 to 75°C). SDP was collected using a high- performance cyclone (Biichi Labortechnik, Switzerland) and stored in clear glass vials. The spray-drying process was completed within 10 to 15 minutes. A summary of spray-drying parameters is shown in Table 1 below.Table 1 : Example Spray -drying process parameters

[0098] Spray dried protein particles were analyzed for particle size distribution (PSD) using Mastersizer 3000 (Malvern Panalytical, Malvern, United Kingdom), surface morphology using Thermo Scientific™ Phenom™ XL scanning electron microscopy (SEM) (Nanoscience Instruments, Phoenix, AZ, USA), and aggregation using size-exclusion chromatography (SEC) (Waters™ Corporation, Milford, MA, USA). Protein content in the spray dried product was analyzed using CTech™ SoloVPE® (Repligen Corporation, Waltham. MA, USA) ultraviolet (UV) method at 280 nm. Individual analytical methods have been elaborated in section 2.4.

[0099] 3.2 Fabrication of POE MS via non-AqE (example of a FP platform)

[0100] POE MS were manufactured via an in-house developed novel non-aqueous bulk emulsification process (FIG. 1). MS were prepared by first dispersing SDP (5% or 25% w / w) in 500 pL DCM containing 40% w / v POE to compnse phase 1. This phase was then bath sonicated for 3 minutes to ensure even distribution of SDP. Phase 2 comprised of FC-40 with 1% w / v of Pico-Surf 1. Phase 1 was added to phase 2 dropwise to reach a 1:20 ratio and homogenized at low shear (50% capacity, -17.000 RPM). DCM was then evaporated under vacuum stirring for three hours resulting in hardened POE MS. Formed MS particles were washed using FC-40 (3x with 1 mL FC-40) to remove polymeric debris and unencapsulated free SDP. Washed MS were dried overnight at room temperature and collected via vacuum filtration by using a 0.22 pm PES filter membrane. Blank MS were prepared using the same procedure, void of SDP.

[0101] Process parameters impacting quality attributes were identified and listed as part of Table 2 below.Table 2: Microsphere characteristicsDv50, volume median diameter; mAbs, monoclonal antibody; POE, polyorthoester; rFcP, recombinant fusion protein; SDP, spray dried protein.

[0102] 3.3 Fabrication of POE MS via AqE

[0103] POE MS were manufactured via a traditional bulk emulsification process using a homogenizer VWR® 200 (VWR International. Radnor. PA, USA). The process was optimized for SDP used in this study with respect to solvent evaporation rate and time (higher than FP), stabilizer / surfactant used (PVA versus PicoSurf), washing (water wash versus FC- 40) and drying of formed MS (longer drying times than FP). Multiple iterations of AqE were prepared to optimize microsphere size, morphology and encapsulation. The resulting process represents the best outcome for an O / W (single emulsion for direct comparison with FP) AqE for encapsulating SDP in MS.

[0104] Briefly, SDP (5% w / w) was dispersed in 500 pL DCM containing 40% w / v POE to comprise phase 1 which was bath sonicated (Branson 3510, Branson Ultrasonics Corp., Danbury, CT, USA) for 3 minutes at room temperature to ensure homogenous dispersion of SDP. Phase 2 (continuous phase) was comprised of 1% w / v polyvinyl alcohol (PVA) solution in water. Phase 1 was added to phase 2, dropwise, to reach a 1:20 ratio and homogenized for 1 minute at low intensity. DCM was then evaporated under vacuum stirring for three hours resulting in hardened POE MS. The MS were washed with 2 mL water three times (total of 100: 1 polymer to water ratio at each washing step) to remove polymeric debris and free SDP. The washed MS were collected and dried overnight by vacuum fdtration using 0.22 pm PES filter membrane.

[0105] 3.4 Physical characterization of SDP and MS

[0106] 3.4.1 Surface morphology evaluation

[0107] The surface morphology of the SDP encapsulated MS was imaged using a SEM.Briefly, dried MS were adhered on the SEM stubs (Ted Pella Inc., Redding, CA, USA) using double-sided conductive carbon tabs (Ted Pella Inc., Redding, CA, USA). The sample wasthen sputter coated with gold at 15 mA for 180 seconds followed by imaging on a Phenom XL desktop SEM (Nanoscience instruments, Phoenix, AZ, USA) at 10 kV. The average particle diameter was calculated using ImageJ open-source software by performing measurements for at least 15 separate MS for each formulation.

[0108] 3.4.2 Particle size distribution

[0109] Particle-size distribution was measured using Mastersizer 3000 (Malvern Panalytical, Malvern, PA, USA). Briefly, a 10 mg / mL suspension of SDP-loaded MS was prepared in degassed ethanol and bath sonicated for 5 minutes at room temperature to obtain a homogenous distribution. The sonicated sample was injected into 6 mL of degassed ethanol contained in an automated small volume wet-sample distribution cartridge (Hydro SV) under constant stirring (500 rpm) to achieve an obscuration between 10 to 20%. The intensity of scattered light was measured at different angles for calculating the Dv50 (volume median diameter), which is the diameter at 50% of the volume distribution.

[0110] 3.4.3 Percent loading and encapsulation efficiency

[0111] To measure the protein content loaded into the MS, 200 pL of DCM was added to ~10 mg of SDP-encapsulated MS and was bath sonicated for 5 minutes at room temperature. Encapsulated protein was extracted by partitioning into 1 mL of lx PBS. The dispersion was centrifuged (Eppendorf Centrifuge 5424, Eppendorf Group, Hamburg, Germany) for 5 minutes at 10,000x g to separate any undissolved polymer debris. The clear aqueous supernatant was collected, filtered using a 0.22rm syringe filter (Arodisc™, Supor™ membrane #4602, Cytiva, Amsterdam), and analyzed using SoloVPE UV at a wavelength of 280 nm to determine the encapsulated SDP concentration. Blank controls were evaluated prior to analyzing rFcP concentrations. Percent (%) loading and % encapsulation efficiency was calculated using the formulae described below.% Loading% Encapsulation Efficiency =

[0112] 3.4.4 Residual Moisture Content Analysis

[0113] Moisture content of SDP loaded FP MS was evaluated using Computrac® Vapor Pro XL moisture analyzer (AMETEK Brookfield, MA, USA). Briefly, ~50 mg of SDP loaded microspheres were sealed in 6R ready -to-use (RTU) borosilicate vials (Schott Pharma, Mainz, Germany) and loaded onto the Vapor Pro XL. The sample was heated to 75°C (below Tg of the polymer, 91 °C) and % moisture content was calculated after normalizing against the weight of MS sample. Moisture analysis was performed on three separate batches of SDP loaded MS.

[0114] 3.5 Characterization of Encapsulated SDP

[0115] 3.5.1 Protein aggregation via size-exclusion chromatography[001 16] Aggregation was evaluated at various intermediate manufacturing process steps using a size exclusion chromatography (SEC). To evaluate the impact of spray drying on protein aggregation, samples were collected both pre and post spray drying and were diluted to 10 mg / mL in lx PBS. To understand the impact of the FP manufacturing process, SDP was extracted from the MS via the method described in section 3.4.3. Briefly, MS were incubated in lx PBS for 2 hours at 37°C, and the released protein was analyzed. All samples were evaluated on SEC using 10 mM sodium phosphate containing 500 mM sodium chloride as the mobile phase at a flow rate of 0.3 mL / min and detected using a UV detector at 280 nm. Total % punty and % high molecular weight (HMW) species were determined.

[0117] 3.5.2 Evaluation of SDP structure post encapsulation

[0118] The impact of FP process on the secondary structure of SDP was evaluated using circular dichroism (CD) using far-UV (200 nm-260 nm) measurements. Briefly, SDP and post-encapsulation SDP were diluted to 0.30 mg / mL in lx PBS. CD measurements (10 replicates per sample, n=3) were conducted on a J-1500 Spectrophotometer (Jasco Inc., MD, USA) using a cuvette of 0.1 cm path length. Data analysis was performed on Spectra Analysis (v2.0) software designed by the manufacturer. Briefly, replicates were averaged into a single accumulated spectrum and normalized with signal from buffer control. Spectra were then converted to molar ellipticity by factoring in molar concentration, molecular weight, and number of amino acid sequences of the protein. Finally, structural details were estimated using CONTIN analysis to identify correlation between sample spectra and reference spectra of well- defined protein structures.

[0119] 3.5.3 Potency of encapsulated SDP

[0120] To determine the potency of the entrapped SDP, a cell-based bioassay was performed using 293 / D9 (NfkB-luciferase-IRES-GFP cells) / Flt (l-7)-IL18Ralpha / Flt (1-7)- IL18Rbeta clone. Briefly, cells were cultivated in Dulbecco's Modified Eagle Medium (DMEM) (with glutamine) spiked with 10% fetal bovine serum (FBS) and 1% penicillin- streptomycin-glutamine (pen / strep / glutamine). At 90% confluency, cells were seeded in white 96-well plates at a density of 10,000 cells / well and allowed to adhere overnight at 37°C / 5% CO2. The SDP dose-response curve was prepared by preparing SDP dilutions (3 nM to 51 fM) in Opti-MEM™ medium spiked with 0.5% FBS and 1% pen / strep / glutamine. Passively released SDP (2 hours at 37°C) was collected and stored in a L-histidine buffer matrix to evaluate the impact of process parameters on SDP potency. Cell viability was determined using ONE-Glo™ substrate and analyzing the luminescence produced post-treatment.

[0121] 3.6 In vitro release testing (IVRT)

[0122] To determine drug release from POE MS, an in vitro release study was designed using lx PBS, pH 6.5 and 7.4 as the release media. Briefly, 10 mg of SDP-loaded MS were collected in a low-binding 2 ml Eppendorf Tube® with 1.8 mL of release media, maintaining at least lOx sink condition throughout the IVRT. At each timepoint, 80% (1.4 mL) of the release media was collected and analyzed for SDP concentrations. Following this, the release tube was replenished with the same volume of fresh media to maintain sink conditions. Samples w ere collected and analyzed at 2 hrs, 24 hrs, 48 hrs, 72 hrs, 96 hrs, 168 hrs and 216 hrs. SDP concentrations were analyzed using SoloVPE UV detection at 280 nm. IVR data w as subjected to release kinetics analysis to determine the order of release (zero order, first order, Korsmeyer- Peppas, Higuchi, Hixson-Crowell). Aggregation and purity of the released protein was analyzed using SEC, as described in section 3.5.1. Bioactivity of the released protein was also analyzed via a cell-based bioassay, described in section 3.5.3.

[0123] 3.6 Stability testing

[0124] Stability’ of SDP-loaded MS was evaluated by storage under long-term, accelerated, and thermal stress conditions. Briefly, ~15 mg of MS was sealed in 2R ready -to- use (RTU) borosilicate vials (Schott Pharma, Mainz, Germany) and stored at 25°C and 40°Cfor 4 weeks. At each timepoint, MS were recovered and analyzed for particle size and loading efficiency, as described in sections 3.4.2 and 3.4.4, respectively.

[0125] 3.7 In vitro degradation of SDP-loaded POE MS

[0126] Qualitative in vitro degradation of SDP-loaded MS was determined based on the changes to the surface morphology of particles, as examined using SEM. Briefly, ~10 mg of MS were placed in 1.8 mL (sink condition) of lx PBS at 37°C for ~3 weeks. At each time interval (Day 0, Day 4 and Day 23), MS were collected, washed and evaluated for surface morphology, as described in section 3.4.1.

[0127] 3.8 In vitro toxicity of blank POE MS

[0128] 3.8.1 Cell Viability via MTT assay

[0129] Cytotoxicity of blank POE MS was evaluated to determine biocompatibility of the formulation composition, excipients, and any residual solvents from the manufacturing process. For this assay, human embryonic kidney 293 (HEK-293) cells were used, that were cultivated in high -glucose DMEM (with glutamine and sodium pyruvate) containing 10% FBS and 1% penicillin-streptomycin. According to International Standards Organization (ISO) 10993-5, the cell culture media elution method was followed where blank MS were incubated with cell nutrient media for 24 hrs. at 37°C to extract any potentially toxic substances. The resulting extracts were then serially diluted to achieve effective MS concentrations ranging from 200.0 mg / ml to 12.5 mg / ml. HEK-293 non-malignant mammalian cells were seeded in a 96-well, tissue culture (TC)-treated plate (30,000 cells / well) and were allowed to adhere overnight. The following day. cells were treated with varying concentration of the extracts in triplicate and further incubated for 24 hours at 37°C / 5% CO2. At each time interval, 5 mg / ml 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (ThermoFisher Scientific, Waltham, MA, USA) was added to each well and plates were incubated at 37°C / 5% CO2 for 3 hrs.. The MTT solution was aspirated, and dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. Absorbance values for each well were calculated at 560 nm using GloMax® Discover (Promega Corporation, Madison, WI, USA), and % cell viability was determined relative to a no -treatment control group (nutrient medium only - DMEM with 10% FBS and 1% pen / strep / glutamine) of cells.

[0130] 3.8.2 Live / Dead Cell Staining Assay

[0131] Blank microspheres were incubated with cell culture media for 24 hrs. at 37°C for extraction of any potentially residual solvents. The resulting extracts were then serially diluted to achieve blank microspheres concentrations ranging from 200 mg / ml to 12.5 mg / ml. HEK-293 cells were seeded at a density of 30.000 cells per well in a 96-well plate and allowed to incubate overnight at 37 °C / 5% CO2. Cells were treated with varying concentrations of blankmicrospheres extracts (12.5 mg / ml - 200 mg / ml). After 24hrs. of treatment, Live / dead cell viability assay was performed as follows, media was removed, and cells were washed with lx DPBS. Calcein AM and Ethidium homodimer-1 (EthD-1) dyes were prepared at a concentration of 2 pM and 4 pM. respectively, in DPBS. Dye solution was added at 100 pl volume per well and allowed to incubate at RT for 30 mins. Following that, fluorescence microscopy was performed using the EVOS M5000 Imaging System (Invitrogen) and cells were imaged in the green and red fluorescence channels along with non-fluorescent phase contrast channel. All channels were overlaid to assess the live / dead cell population.

[0132] 3.9 Statistical analysis

[0133] All data presented here are mean ± standard deviation (SD) or standard error of measurement (SEM) (n=3-6), unless otherwise stated. Cytotoxicity studies represent an average of 3 independent trials (n=3 for each trial). Unpaired Student’s t-test was used to compare two groups, whereas to compare more than two groups, one-way analysis of variance (ANOVA) followed by Tukey’s post hoc multiple comparison test was used. P<0.05 was considered statistically significant.

[0134] 4. Results

[0135] 4. 1 Production of spray dried rFcP

[0136] The spray-drying process described above was used to manufacture the dry powder rFcP used for encapsulation into POE MS. Based on the historical data across multiple modalities, an in-house spray-drying process has been developed to produce stable dry powder biologies with high yield, without affecting the biologic potency. The process further produced distinct SDP in terms of surface morphology and particle size. Process 1 used a 0.7 mm diameter nozzle tip while process 2 utilized a 0.5 mm diameter nozzle tip, both of which resulted in atomization of the drug substance (DS) feed solution. Furthermore, process 1 utilized a pure protein feed solution (i.e., protein in lx PBS pH 7.4) and process 2 feed solution comprised of excipients 10 mM L-histidine buffer, 5% w / v sucrose, and 0.001% polysorbate 20, pH 6.0). All other parameters remained constant across both processes, including the outlet temperature which was recorded to be 60 to 75 °C. As seen in FIG. 2A, SDP manufactured via process 1 exhibited toroidal and dimple shape morphology' with somewhat wrinkled / smooth surfaces while process 2 resulted in SDP particles with distinctly wrinkled surface morphology (FIG. 2B). This change in morphology may be attributed to the smaller nozzle tip and presence of excipients. The 0.5 mm diameter spray nozzle resulted in a smaller average particle size forthe manufactured SDP. As seen in FIG. 2C, process 1 and process 2 resulted in SDP particles with a volume median diameter (Dv50) value of 11.25 ± 1.63 pm and 4.05 ± 0.08 pm, respectively. Protein aggregation after manufacture of the SDP was analyzed using SE-UPLC to understand the impact of the spray drying process on protein purity and high molecular weight (HMW) species. It was observed that both processes resulted in SDP total % HMW < 3.0 and purity > 90%.

[0137] Process 1 resulted in 2.18 ± 0.64 % total HMW species and 97.83 ± 0.64 % purity as compared to 0.8% total HMW and 99.0% purity of the feed solution (i.e., prior to spray drying). Process 2 resulted in SDP with total % HMW 2.25 ± 0.96 % and 97.72 ± 1.01% purity. The manufactured SDP particles from both processes were utilized as a model dry powder biologic for encapsulation in POE MS.

[0138] Based on these results, the process of the present disclosure can produce particles of desired sizes to suit diverse applications. In some aspects, the spray dried biologic particles have no more than 9% of HMW species such no more than 8%, 7%, 6% of HMW species. In other aspects, the spray dried biologic particles retain at least 50% potency, e.g., at least 60%, 70%, 80% or more potency.

[0139] 4.2 Fabrication rFcP-loaded POE MS via FP, a novel non-AqE platform

[0140] Spray dried rFcP for both size ranges were utilized to prepare SDP-loaded POE MS formulations using our novel non-AqE technique, and the critical quality attributes were characterized. Table 3 details the composition and manufacturing process variables for MS formulations prepared during development as per design considerations (Table 2). As described earlier, the particle size values reported in this study were generated using ImageJ analysis software with 15 particle measurements performed for each formulation (F).Table 3: Formulation composition and manufacturing parametersF, formulation; No., number: SDP, spray dried protein *Bath sonication w as done for 3 minutes at room temperature

[0141] Fl was made using 10% w / v of POE and EAc as the hydrocarbon solvent with 25% w / w of SDP loading. Primary emulsion for SDP in the hydrocarbon solvent was fabricated via simply vortexing the mix. The secondary emulsification was homogenized at extremely high shear, and this resulted in an average MS particle size of 5.01 ± 1.02 pm (Fig. 3A), which was out of range per the desired MS considerations represented in Table 2. Homogenization intensity and time was adjusted to low shear and 1 minute, respectively, to manufacture F2. and the resulting average MS size for the F2 batch was 5.45 ± 1.13 pm (Fig. 3A). For batch F3, the homogenization shear intensity was kept the same as the F2 batch (i.e., low shear); however, the POE concentration w as increased from 10% w / v to 40% w / v. This resulted in an average MS particle size of 23.40 ± 9.34 pm (Fig. 3A), which indicates that the polymer concentration has a significant impact on MS size as compared to the process parameters. F3 yielded MS with non-smooth surfaces and non-spherical shape with SDP particles present on the surface (Fig. 3B-i). The impact of the hydrocarbon solvent was evaluated in F4 with EAc being replaced with DCM, while keeping all other process and formulation parameters the same as F3. This resulted in an average MS size of 34.13 ± 9.67 pm (Fig. 3A) with a marked improvement in MS shape (Fig. 3B-ii). However, SDP particles were present at a greater amount (qualitatively assessed from SEM image) on the MS surface. To resolve this, SDP loading was reduced from 25% w / w to 5% w / w in F5 along with an improved primary emulsification process (vortex±bath sonication), and that resulted in an average particle size of 34.87 ± 5.01 pm Fig. 3A. Reducing the SDP loading significantly reduced the amount of SDPpresent on the MS surface. The F6 batch was manufactured using smaller SDP particles (process 2; average size 4.05 ± 0.08 pm) which significantly reduced the amount of SDP adhered on particle surface, as seen qualitatively in Fig. 3B-iii. The average particle size of F6 MS was found to be 27.94 ± 8.62 pm (Fig. 3 A) and showed smooth surface morphology and spherical shape.

[0142] This formulation development effort successfully establishes the novel FP process to produce rFcP-loaded POE MS with smooth non-porous surface morphology' and an average particle size of ~30 pm. The study revealed that polymer concentration has the biggest impact on MS size, whereas the choice of hydrocarbon solvent is critical in producing MS with smooth spherical morphology. For this purpose, a POE concentration of 40% w / v was chosen and dissolved in DCM which was the preferred hydrocarbon solvent. Another critical observation from this developmental effort was the impact of SDP size on the encapsulation efficiency. It was revealed that the utility of a smaller SDP size resulted in a lower surface retention on MS. Based on MS particle size, SDP surface retention, and MS shape / morphology, F6 was selected for further analyses.

[0143] However, F5 was also characterized for aggregation to ascertain the impact of SDP size on aggregation. Imaging reveals high aggregation of SDP when fabricated as batch F5 (28.59 ± 2.86% HMW). The high aggregation may be attnbuted to the larger particle size of SDP used in F5 coupled with poor encapsulation within MS, leading to high surface retention and aggregation. Poor encapsulation within MS increases the probability' of SDP being exposed to processing solvents for extended duration during collection and washing. Moreover, any residual DCM present on MS surface may interact with the surface retained SDP, potentially leading to high aggregation.

[0144] 4.3 Fabrication rFcP-loaded POE MS via AqE

[0145] POE MS were prepared using conventional AqE technique to facilitate comparison between aqueous and non- AqE processes. Fig. 4A represents MS prepared via the aqueous process. SEM imaging shows numerous pores on the MS surface, which may be attributed to solvent evaporation during the MS hardening process and / or the dissolution of SDP particle in the aqueous vehicle. In comparison, Fig. 4B represents formulation F6, generated using non-AqE, which does not exhibit porous MS surface.

[0146] The porous nature of the manufactured MS and SDP dissolution in the aqueous vehicle resulted in negligible SDP being encapsulated, while MS prepared via non-AqEdemonstrated 63% encapsulation of the biologic (Fig. 4C). In addition, the porous nature of the manufactured MS and SDP dissolution in the aqueous vehicle resulted in negligible SDP loading (0.145 ± 0.001%), while MS prepared via non-AqE demonstrated 3.55 ± 0.7 % SDP loading of the biologic (Fig. 4E). Moreover, particles prepared via AqE led to increased particle aggregation which led to higher average particle size represented as a Dv50 value of 82.48 ± 12.20 pm as compared to 39.9 ± 5.2 pm for particles prepared via non-AqE (formulation F6). Next, the burst release from MS prepared using both the aqueous and non-aqueous systems was evaluated. Fig. 4D represents the release of SDP after 2 hours of incubation at 37°C. The release of SDP from AqE MS is about 80% as compared to the non-AqE (about 34%); and this high burst from AqE may be attributed to the porous morphology of the MS, providing a path for the SDP to release.

[0147] These results clearly portray the need for the novel non-AqE platform to encapsulate biologies. It was observed that an AqE process resulted in the formation of porous MS, which facilitates a faster release of rFcP from within the MS. This, coupled with low encapsulation efficiency and high particle size, results in a delivery system which may be incapable of delivering rFcP for a sustained application (beyond 1 week). This direct comparison between the traditional aqueous process and the novel FP process highlights the need for developing a non-AqE platform to fully utilize the potential of MS in delivering biologies for sustained delivery applications.

[0148] 4.4 Evaluation of critical quality attributes for rFcP-loaded POE MS

[0149] 4.4.1 % yield, encapsulation, and loading efficiency

[0150] Batch F6 resulted in a 75% yield for SDP-loaded POE MS prepared using non- AqE, and the collected MS have an encapsulation efficiency of 63% resulting in 3.5 ± 0.7% w / w protein loading (target loading was 5% w / w).

[0151] 4.4.2 Particle size distribution

[0152] F5 and F6 were evaluated for their particle size distribution using a Mastersizer 3000. Fig. 5 A represents DvlO, Dv50 and Dv90 values for formulations F5 and F6. The Dv50 values for F5 and F6 were found to be 28.85 ± 2.05 pm and 39.9 ± 5.2 pm, respectively. Formulations F5 and F6 have similar size distribution across DvlO and Dv50 values, however, F6 has a wider size distribution, indicated by the relatively higher Dv90 value.

[0153] 4.4.3 % yield, encapsulation, and loading efficiency

[0154] Batch F6 resulted in a -75% yield for SDP-loaded POE MS prepared using non- AqE, and the collected MS have an encapsulation efficiency of 61.57 ± 9.94 % resulting in 3.5 ± 0.7% w / w protein loading (target loading was 5% w / w).

[0155] 4.4.4 Moisture Content of FP-MS

[0156] Moisture content analysis of batch F6 revealed an average residual moisture of 1.47 ± 0.01 % (Table 4). Since FP processing utilizes non-aqueous solvents, the extent of moisture present in MS would be limited to residual moisture that may have been absorbed by the product upon storage or during processing.Table 4: Characterization of quality attributes for FP-MS

[0157] Based on extensive physical characterization of F5 and F6, it was revealed that F6 maintains higher purity of the rFcP while resulting in a lower burst release. See FIG. 5C. These observations can be attributed to the lower amount of rFcP adhered to the surface of the MS in F6, which can be correlated to the smaller particle size of the SDP utilized in F6 relative to F5. Based on characterization parameters such as particle shape, SDP presence on surface, average particle size, % yield and encapsulation efficiency, and burst release, F6 exhibited better performance than F5.

[0158] 4.5 Evaluation of encapsulated SDP

[0159] 4.5.1 Aggregation analysis via SE-UPLC

[0160] Encapsulated SDP was extracted from the MS formulations and analyzed using SE-UPLC. Fig. 5B represents the total % HMW and % urity of SDP extracted from MS. F5 and F6 resulted in distinct SDP aggregation profiles, as indicated by the total HMW of about 29% and about 5%, respectively. It was evident that higher SDP size resulted in higher aggregation (F5) and corresponding low protein purity levels. Encapsulated SDP in formulation F6 had purity level of >90% which was comparable to SDP purity' prior to MSmanufacturing (about 97%), indicating that the non-AqE process does not significantly affect the SDP quality in terms of purity.

[0161] 4.5.2 Evaluation of secondary' protein structure

[0162] Spray dried protein, both pre- & post- encapsulation were analyzed and evaluated for any changes in their secondary structure that may occur due to the FP process and MS encapsulation. Far-UV CD scan followed by secondary structure analysis on deconvoluted CD spectra revealed no significant difference between any of the prevalent secondary structures, a-helix for pre-encapsulated SDP was 3.5 ± 0.1 % while F6-SDP demonstrated 2.9 ± 0.4%. No major differences were observed among the prevalence of antiparallel and parallel P-sheets, % turn and random coil structures. The similarities between secondary structure presentations indicate that FP-processing did not cause any significant change in protein structure, and may be correlated to having no impact on protein structure conformation and related attributes, post-encapsulation.

[0163] 4.5.3 Bioactivity evaluation of rFcP via cell-based potency assay

[0164] Spray dried protein potency is a critical attribute that determines whether a formulation or process is compatible to encapsulate the biologic within a drug delivery' system. Multiple factors in the non-AqE process may contribute to compromising potency of the SDP, including spray-drying process, hydrocarbon interaction, homogenization, drying, and / or storage thereafter. In practicing aspects of the present disclosure, however, relative to the control, spray-drying process did not have any significant impact on potency of the biologic (98% potent). That is, it was observed that the spray dried biologic prepared according to process of the present disclosure exhibited 98% potency relative the biologic that did not undergo spray drying. Further, the potency of the SDP released from within the MS was 82% relative to the control and within an assay acceptance criteria (50%-150% acceptance criteria). This confirms that the non-AqE has negligible impact on potency of the encapsulated protein and is a viable process to fabricate SDP-loaded MS.

[0165] 4.6 In-vitro release testing

[0166] In vitro release of encapsulated protein from POE MS was evaluated at pH 6.5 and pH 7.4 using lx PBS buffer while maintaining sink conditions. The selected pH conditions represent the range of physiological pH within tissues, e g., subcutaneous, muscle, and solid tumors. As seen in Fig. 6A, protein release from POE MS is pH independent, indicating this sustained delivery platform would exhibit consistent performance within physiological pHrange. Burst release within the first two hours of 36.75 ± 0.71% (pH 6.5) and 34.60 ± 1.60% (pH 7.4), was followed with a sustained release at an average rate of ~8% encapsulated SDP per day. 80.38 ± 0.12% SDP was released at pH 6.5, and 85.8 ± 3.7% SDP was released at pH 7.4 by 168 hours. Almost all SDP was released by 216 hours (93.00 ± 0.72 at pH 6.5 and 93.90 ± 2.40% at pH 7.4). At all time points, no statistical significance was observed between the tested conditions of pH 6.5 and 7.0, indicating the similarity in MS performance across a w ide range of physiologically relevant pH conditions, e.g. a pH from about 6.5 to about 7.4.

[0167] 4.6.1 Release kinetics

[0168] Release kinetics of SDP from F6 was assessed using numerous mathematical models, including zero-order release, first-order release, Higuchi release model, Hixson- Crowell model, and Korsmeyer-Peppas release model, utilizing data obtained from the in vitro release study at both studied pH of 6.5 and 7.4. Order of release w as calculated with the initial burst release as well as for the sustained release of SDP after 24 hours of burst release. As seen in Fig. 6B, SDP release (0h-216h) at pH 6.5 follows a combination of first order and Hixson- Crowell release model with R2 value as 0.9880 for both, while it follows a first-order release at pH 7.4 (R2 = 0.9766). However, after the initial burst, the release at pH 6.5 follows the Korsmeyer-Peppas model of release (R2 = 0.9950), indicating a surface erosion and diffusion phenomenon for SDP release (24h-216h). At pH 7.4 after the initial burst. SDP release follows a Higuchi model of release, how ever, due to the closeness in R2 values for Higuchi (0.9864), Hixson-Crowell (0.9849), and Korsmeyer-Peppas (0.9859) models, the order of release remains inconclusive. However, surface erosion and diffusion are common factors across all these models, indicating that as the primary mechanism of sustained SDP release from POE MS.

[0169] Observations from IVRT shows that F6 can release rFcP in a sustained manner over 9 days across two tested pH conditions of pH 6.5 (slightly acidic pH to replicate tumor microenvironment) and pH 7.4 (physiological blood pH). At both tested pH. the release of rFcP was overlapping, indicating no impact of pH erosion of the prepared MS, and this may be attributed to the innate behavior of the custom-made POE used in this study. The release rate is dependent on the erosion of POE and diffusion of rFcP, and this w as confirmed via release kinetic calculations.

[0170] 4.6.2 Aggregation of released SDP

[0171] Encapsulated SDP was allowed to release over the course of 8 days followed by collection and analysis on SEC for the extent of aggregation and purity. Fig. 6C represents the. SDP from F6 demonstrated total % HMW of 5.03 ± 0.33 %, while total % HMW and % purity of SDP at the end of release from MS towards the end of release, as anticipated, an increase in aggregation was observed (12.59 ± 4.7 %). This is not unusual since the model biologic is prone to thermally enabled aggregation and in an in vitro setting, in the absence of thermal stabilizer as part of the MS delivery vehicle, aggregation of SDP is anticipated.

[0172] 4.6.3 Biological activity of released SDP

[0173] The potency of encapsulated SDP at the end of release period (day 8), relative to pre- and post-encapsulated SDP was evaluated. It can be seen in Fig. 6D that the biological activity of SDP is slightly reduced (61% relative to control) at the end of the release period. However, this trend is acceptable in an in vitro setting, in the absence of a formulation vehicle including a thermal stabilizer to prevent thermally enabled reduction in biological activity. Moreover, post FP process, SDP biological activity’ was advantageous at 82% relative to the control. Therefore, relative to post-encapsulated SDP (82%), about 74.4% retention of biological activity’ was observed at the end of release. It is also advantageous that the reported potency for end of release SDP samples are within the assay acceptance criterion of 50-150% relative to control, as described in section 4.5.3. This assay provides an early insight into the stability of SDP after 8 days of incubation in lx PBS at 37°C, in vitro.

[0174] 4.7 Accelerated and stressed stability’ assessment of rFcP-loaded POE MS

[0175] POE MS prepared via non-AqE were staged at 25°C and 40°C for 1 month in RTU borosilicate glass vials at storage conditions to evaluate stability of these particles. After 1 month, particles were evaluated for their size distribution and loading efficiency. Fig. 7A represents the Dv50 values for samples at T=0 and T=1 month. Samples stored at 25°C for 1 month did not show any significant change in mean particle size (39.9 ± 5.1 pm [TO] versus 40.70 ± 0.88 pm [Tim]) whereas storage at 40°C / 75% RH for 1 month resulted in significant increase in mean particle diameter from 39.9 ± 5. 1 pm to 52.9 ± 2. 1 pm (P<0.0001). In terms of % loading efficiency, there was no significant differences in SDP loading across all conditions and timepoints, indicating no impact on the encapsulated biologic upon storage (Fig. 7B) (25°C: 3.49 ± 0.00% [TO] vs 3.28 ± 0.15% [Tim], 40°C / 75%RH: 3.49 ± 0.00% [TO] vs 3.39 ± 0.04% [Tim]). This demonstrates that non-porous MS prepared using a non AqEplatform yields highly stable MS, capable of maintaining the average particle size and SDP load upon storage at accelerated conditions (25°C) for 1 month.

[0176] 4.7.1 Evaluation of rFcP-loaded POE MS degradation

[0177] SDP-loaded POE MS were suspended in lx PBS (pH 7.4) at 37°C for three weeks. Samples were retrieved at day 4 and day 23 and imaged using SEM to qualitatively assess morphology and understand degradation behavior. FIGS. 7C-7E reveal the behavior of POE MS under these conditions. Surface erosion and formation of surface pores is observed on day 4, which increases further by day 23. This qualitative analysis confirms the mechanism POE MS degradation via surface erosion.

[0178] 4.8 Toxicity evaluation of blank POE MS on HEK-293 cells

[0179] 4.8.1 Cell Viability assay

[0180] To evaluate the effect of residual fluorocarbons and POE on non-cancerous mammalian cells, blank POE MS were extracted in cell nutrient media for 24 hours at 37°C, and this extraction media was incubated with HEK-293 cells for further 24 hours. The dosing range (12.5 to 200.0 mg / ml) of these MS, which was equivalent to 0.6 to 10.0 mg target encapsulated SDP, was evaluated. Fig. 8 illustrates the impact of MS extraction media on viability of HEK-293 cells. Blank MS were incubated with the nutrient media for 24 hours at 37°C to extract any leachable excipient that may contribute to cytotoxicity. Data represents mean ± SD with n= 1 or 3. As shown by the data in FIG. 8, relative to the control, HEK-293 cells retained > 80% viability for all tested concentrations (200 mg / ml reported a viability of 78.7 ± 2.2%). This high viability after 24 hours of treatment indicates negligible in vitro toxicity of the fluorocarbons used in the non-AqE process. It has previously been demonstrated that if the cell viability is reduced to < 70% of the blank control, then the tested sample is considered potentially cytotoxic. However, results from the current study indicate a vi ability of > 80% of HEK-293 cells at all tested concentrations, providing an insight into potential toxicity of extracted residual solvents. Further in-depth tox studies are warranted to ascertain the toxicity profile of MS products fabricated using FP process.

[0181] 4.8.2 Live / Dead cell toxicity assay

[0182] A qualitative live / dead assay was performed to visualize the live and dead cells post treatment with blank microspheres. This visualization was a result of calcein AM (green fluorescence) and ethidium bromide (red fluorescence) staining of live and dead cells, respectively. The testing involved one control group of cells along with three representativesets of cells treated with microsphere extracted nutrient media (as described in section 3.8.2.), equivalent to microsphere concentrations of 50-, 100-, and 150 mg / mL. It was observed that all groups of cells exhibit high levels of green fluorescence while red fluorescence is not detectable, indicating, negligible dead cell population, post-treatment. Moreover, phase contrast images indicate that no evident change in cellular morphology was observed posttreatment, indicating negligible toxicity of blank MS.

[0183] 5. Discussion of Examples

[0184] These experiments describe a non-aqueous platform for encapsulating solid biologies in polymeric MS. The use of solid biologies has several advantages over their liquid counterparts, including higher stability in hydrocarbon solvents and improved encapsulation within the polymer matrix. Previous research by Sawasdee et al. demonstrated the efficiency of a solid-oil-water (S / O / W) emulsification system in encapsulating an enzyme into poly (lactic-co-gly colic) acid (PLGA) nanoparticles (Sawasdee et al.. 2018). Nanoparticles prepared using S / O / W showed enhanced enzyme activity compared to those prepared using O / W and W / O / W systems, attributed to reduced exposure of the enzy me to organic solvents (Sawasdee et al., 2018). Solid-state biologies experience organic solvent-mediated denaturation only at the particle interface and thus protecting the biologic activity’ more effectively than liquid biologies. An example of the successful implementation of this S / O / W manufacturing process is Zilretta®, a triamcinolone acetoamide microparticle suspension approved by the United States Food and Drug Administration (US FDA) for managing osteoarthritis. To achieve such solid-state biologies, historically, lyophilization has been the preferred method, known for its ability to improve shelf life and meet supply chain requirements (Tang and Pikal. 2004). However, lyophilization is a lengthy process, often taking several days to weeks, leading to increased capital costs and reduced efficiencies (Stratta et al., 2020). Moreover, for this application, lyophilization doesn’t readily produce micronized particles that are suitable for encapsulation in MS. This unsuitability’ may be attributed to the bulk cake produced after lyophilization which requires an additional micromzation step to make it fit for encapsulation. Alternatively, methods such as spray dry ing have been successful in achieving similar outcomes as lyophilization. Kanojia et al compared the attributes of infliximab powder produced using lyophilization and spray drying and concluded that both processes are well suited to achieve dry powder biologies with minor differences in biologic thermostability (Kanojia et al., 2016). For the FP platform, the spray-drying process was selected to produce asolid-state rFcP as the model SDP. This process is a fast and cost-effective way to achieve dry powder biologies that exhibit superior stability, relative to liquid biologies. SDP was formulated using an in-house developed spray-dry ing process capable of producing solid biologies of desired size while retaining biological activity, as seen by the aggregation and potency evaluations. Two processes have been reported in this study that demonstrate the distinct nature of SDP with respect to the particle size distribution and surface morphology7.

[0185] It was observed that the smaller SDP size resulted in an increased encapsulation and lower surface retention on MS. The reduced particle size provides for a much larger surface area for the polymer to coat and entangle around “smaller particles”, resulting in a desirable encapsulation & loading efficiency. Further it was found that a target MS size to SDP size ratio can significantly improve encapsulation as a size ratio of 4:1 (~40 pm MS with ~11 pm SDP) as recorded for F1-F5, and as evaluated, demonstrated lower encapsulation and high burst relative to a 10: 1 ratio (~40 pm MS with ~4 pm SDP), as recorded for F6. Hence a MS size to SDP size ratio of no less than about 5: 1 and upto and over about 10: 1 can achieve improved encapsulation and reduced burst release.

[0186] Conventionally, S / O / W or W / O / W have been used to fabricate biologic loaded MS but has been challenging due to poor encapsulation efficiency, low drug loading, and impact on properties such as protein purity, aggregation, potency, and post-translation modifications. Efficient encapsulation of hydrophilic molecules, such as biologies, has often been cumbersome due to the aqueous solubility of biologies. Furthermore, MS solidification process often produces pores on the surface of the MS which result in an increased probability of high burst release, as seen in section 4.3. Strategies to improve loading efficiency include replacing a solvent with a non-solvent, however, using organic solvents or oils as part of an O / O emulsion often compromises the biologic activity' and performance. In lieu of this, the FP process evaluates the applicability of fluorinated solvents such as perfluorocarbons since they are inert non-solvents that are neither hydrophilic nor hydrophobic and thus do not interact with biologies and hydrocarbons, making them suitable candidates to be used as a continuous phase in the emulsification process. This non-reactive nature facilitates formation of biologic loaded hydrocarbon droplets that harden and form polymeric MS without biologic leakage into the continuous phase. Moreover, perfluorocarbons have been demonstrated to be safe and relatively less toxic, and this may be attributed to their inert nature (Hoogendijk et al.. 2020). In vitro cytotoxicity assay results from this study (section 4.9) also indicate the safety of thesefluorocarbons used in FP process, where it was observed that > 80% cells were viable even at the highest tested concentration of 200 mg / mL following a 24-hour treatment using HEK-293 cells. Additionally, for MS stabilization, surfactants / stabilizers have been deployed to minimize particle aggregation. For the FP process, utility of fluorinated surfactant (fluorosurfactants) was evaluated. FP process utilizes PicoSurf- 1 as an amphiphilic fluorosurfactant which is a biocompatible fluorinated surfactant blended with a fluorocarbon carrier oil (Fc-40). Such commercially available amphiphilic fluorosurfactants that have been evaluated in the past and have successfully provided stabilization of droplet-based emulsions (Holtze et al., 2008; van de Wouw et al.. 2024) and these amphiphilic molecules exhibit solubility’ in organic solvents including fluorocarbons. A study conducted by Mana et al summarizes the need for a fluorocarbon-based emulsification platforms and reiterates the challenges with O / W and O / O emulsions to fabricate ibuprofen (IF) (hydrophobic) microemulsions (Mana et al., 2007). The study demonstrates that particles prepared using an O / O emulsion resulted in poor IF loading and this was attributed to the solubility of IF in the organic phase. O / W emulsions for IF resulted in porous microparticles which led to an increased burst release of IF, however, loading efficiency was improved since it was poorly soluble in the aqueous phase. This outlines the criticality of having no interactions between the continuous phase and the active molecule. The current study confirms the impact of interaction between the continuous phase and the active rFcP, with POE MS prepared using AqE resulting in porous particles with an extremely high burst release of SDP, whereas the non- AqE process which included inert fluorocarbon continuous phase resulted in SDP-loaded MS with smooth continuous surface, more efficient drug loading, and reduced burst release. In addition, possibly due to minimal interaction between SDP and hydrocarbon (DCM), rFcP demonstrated high retention of potency, post encapsulation.

[0187] A Design of Experiments (DoE) was performed to understand the impact of factors, such as polymer concentration, homogenization shear, hydrocarbon solvents, and initial SDP loading amounts on outcomes, such as MS size and burst release of SDP. Briefly, it was revealed that average MS particle size increases as the polymer concentration increases (2.7 pm with 10% w / v of POE versus 34.8 pm with 40% w / v of POE). A similar trend was observed while altering the % loading of SDP with 5% w / w SDP load resulting in an average size of 20.6 pm while 30% w / w SDP load resulted in an average size of 40.7 pm. A decrease in average MS size was correlated with increasing homogenization shear (16. 1 pm with lowshear as compared to 5.4 pm for high shear) and has been similarly reported by others (Mulia et al., 2019). For evaluating the impact of hydrocarbon solvents on MS size, DCM and EAc solvents were used to manufacture MS while keeping all other process and formulation parameters constant. Interestingly. MS prepared using DCM as a hydrocarbon solvent resulted in an average size of 30.7 pm while MS prepared using EAc solvent resulted in an average size of 21.2 pm. Moreover, it was revealed that MS prepared using EAc as the hydrocarbon solvent led to non-spherical surface morphology, whereas when DCM was used as the hydrocarbon solvent, MS exhibited a spherical morphology. The observations in MS attributes (surface and bulk morphology) coupled with the performance characterization in the current study are indicative that the novel non-AqE technology, exhibits superior MS attributes and in vitro performance (non-porous MS with 34% burst release and 63% encapsulation) in comparison to the conventional AqE process (porous surface and bulk morphology with high burst release and low encapsulation efficiency).

[0188] Current studies using the FP process demonstrate that it can reproducibly manufacture biologic-loaded MS, especially for large molecules (100-150kDa). Currently, there is an unmet need for sustained delivery' of biologies that would enable reduced systemic exposure, improved safety profile, and a tunable system that can provide delivery over a period of weeks to months for improved patient compliance and comfort. Currently approved polymeric MS-based products demonstrate the extent of tunability and are mainly long-acting weekly injections (e.g., Bydureon® exenatide 2 mg / week, Arestin® minocycline HC1 1 mg / 2 weeks, etc.) or monthly injections (e.g., Lupron Depot® leuprolide acetate 7.5 mg / month, Vivitrol® naltrexone 380 mg / month, etc.). Sustained release is contingent of polymer degradation and diffusion of the encapsulated therapeutic from within the MS into the physiological environment. One of the most common pathways for polymeric degradation is via hydrolysis. As studied by Tipnis et al, degradation of the PLGA MS product Zilretta, which is a triamcinolone acetoamide suspension resulted in the formation of porous channels (Tipnis et al., 2020). These porous channels were directly correlated with the sustained release of the active small molecule over multiple weeks. In the current study, sustained release of SDP follows a similar diffusion mechanism combined with the slow and sustained formation of porous channels post incubation in the release media, which was visualized using SEM. Clearly defined surface pores and porous channels were seen to form, as MS were incubated in the IVRT medium, resulting in a sustained release of SDP from the MS core over 9 days.Evaluation of the released SDP attributes (aggregation and potency) is a critical indicator of the effectiveness of the FP process to be utilized to produce MS for biologies deliver}7. It is common knowledge that proteins such as rFcP are prone to degradation under stress, and putting such molecules through an emulsification process to produce MS may lead to degradation and a loss in bioactivity. The release of degraded protein may lead to unwanted immunogenic reactions; and thus, a critical process development parameter to consider is the impact on biologic integrity7post encapsulation. The FP platform disclosed herein was carefully curated with the maintenance of rFcP integrity at the forefront of development. This study reports two such quality attributes for the released rFcP. aggregation and bioactivity7(potency). There yvas no aggregation observed as a result of the FP process at accelerated temperatures at 25°C condition for 1 month, as seen by the presence of HMW species pre and post manufacturing (section 4.4.3). Moreover, the released protein retained its biological activity7as seen by the potency evaluation in section 4.8. This clearly demonstrates negligible process impact on rFcP integrity7, making this platform suitable to manufacturing MS for delivering potent biologies.

[0189] 6. Conclusion

[0190] These experiments disclose a novel non-aqueous technology to fabricate polymeric MS suitable for sustained delivery of biologies. A direct comparison between aqueous and non-aqueous processes to encapsulate solid biologies, shows that MS prepared via AqE exhibited porous morphology- y hich potentially led to > 75% burst release of SDP within 2 hours. On the other hand, MS fabricated using the novel FP emulsification platform exhibited non-porous surface morphology, yvhich was integral to significantly enhance encapsulation efficiency of SDP with minimal aggregation and ensured potency of the released SDP. Moreover, the fabricated MS exhibited a sustained release profile for entrapped SDP yvith a release rate of ~8% SDP ever}724 hours, which is a function of diffusion and polymer degradation rate.

[0191] The FP process is dependent on appropriate selection of organic solvents and stabilizing surfactants to achieve a thermodynamically stabile emulsion until the microspheres are hardened. Furthermore, the use of an alternate polymer system and changes in SDP physiochemical properties may require extensive process optimization to achieve the desired target product profile. . Certain challenges such as tunability of the platform, pre-clinical / clinical tolerability, process scalability, and environmental considerations need to be addressed to enable large-scale adoption of this platform for localized and sustained delivery’ of biologies.

[0192] In summary, the examples demonstrate SDP-loaded MS prepared using the FP platform to overcome limitations of sustained biologic delivery. MS prepared using the FP platform achieved the desired quality' attributes, enabling it to be a promising tool for sustained delivery of biologies.

[0193] Only certain features and aspects of the subject technology’ and examples of its versatility are shown and described in the present disclosure. It is to be understood that the technology disclosed herein is capable of use in various other combinations and environments and is capable of changes or modifications. Thus, for example, those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances, procedures and arrangements described herein. Such equivalents are considered to be within the scope of the invention, and are covered by the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method of producing a sustained release biologic, comprising: producing spray dried biologic particles by atomizing a feed solution, which comprises a biologic drug in an aqueous carrier, to form an aerosol, and exposing the aerosol to a stream of drying gas at a temperature of from about 80 °C to about 150 °C to dry the aerosol to produce the spray dried biologic particles; and forming microparticles of the spray dried biologic particles with a sustained release polymer by a solid-in-hydrocarbon-in-fluorocarbon emulsion.

2. The method of claim 1, wherein atomizing the feed solution comprises supplying the feed solution and a compressed gas to a nozzle atomizer having an orifice of no greater than about 0.7 mm and wherein the compressed gas is at a pressure of at least about 50 psi.

3. The method of any one of claims 1-2, wherein the aqueous carrier comprises a phy siological compatible phosphate buffered saline.

4. The method of any one of claims 1-3, wherein a concentration of the biologic drug in the feed solution is from about 1 mg / ml to about 500 mg / ml.

5. The method of any one of claims 1-4, wherein the spray dried biologic particles have no more than about 7% of high molecular weight (HMW) species and retains at least 50% potency.

6. The method of any one of claims 1-5, wherein the microparticles have a sustained release of the biologic drug in a physiological aqueous environment at about 37 °C over a period of at least about 7 days.

7. The method of claim 6, wherein the sustained release of the biologic drug is independent of pH in a range of from about 6.0 to about 8.0.

8. The method of any one of claims 1-7, wherein the microparticles have an average diameter (Dv50) of from about 30 pm to 50 pm.

9. The method of any one of claims 1-8, wherein the spray dried biologic particles have an average particle diameter (Dv50) of at least about 0.5 pm.

10. The method of any one of claims 1-9, wherein a ratio of average microparticle size to average spray dried biologic particle size is no less than about 7: 11 1 . The method of any one of claims 1-10, wherein the biologic drug comprises an antibody or antigen binding fragment thereof, a fusion protein, a recombinant protein, recombinant fusion protein, or a fragment or truncated version of a fusion protein, a recombinant protein, recombinant fusion protein, or any combination of two or more thereof.

12. The method of any one of claims 1-11, wherein forming the microparticles comprises emulsifying a Solid-in-Hydrocarbon (S / H) mixture in a fluorocarbon solution to form an emulsion of the S / H mixture in the fluorocarbon solution, wherein the S / H mixture comprises the spray dried biologic particles dispersed in a solution of the sustained release polymer in a hydrocarbon solvent; and removing the hydrocarbon solvent to form the microparticles.

13. The method of claim 12, wherein the S / H mixture includes the spray dried biologic particles in an amount from about 2.5 % w / v to about 25 % w / v and the sustained release polymer in an amount from about 20 % w / v to about 60 % w / v.

14. The method of claim 12, wherein emulsifying the Solid-in-Hydrocarbon (S / H) mixture in the fluorocarbon solution to form the emulsion comprises passing the S / H mixture through pores and into a channel of a microporous membrane which channel includes a cross flow of the fluorocarbon solution to form the emulsion of the S / H mixture in the fluorocarbon solution.

15. The method of any one of claims 1-14, wherein the sustained release polymer comprises a polyorthoester (POE).

16. A method of producing a sustained release biologic, comprising: passing a Solid-in-Hydrocarbon (S / H) mixture through pores and into a channel of a microporous membrane which channel includes a cross flow of a fluorocarbon solution to form an emulsion of the S / H mixture in the fluorocarbon solution, wherein the S / H mixture comprises spray dried biologic particles in a solution of a sustained release polymer in a hydrocarbon solvent, and wherein the channel of the microporous membrane includes an insert which the cross flow of the fluorocarbon solution flows around, and which insert is positioned at a distance of at least 0. 1 mm from the pores of the microporous membrane; removing the hydrocarbon solvent to form microparticles in the fluorocarbon solution; and separating the microparticles from the fluorocarbon solution, wherein the microparticles comprise the spray dried biologic particles and the sustained release polymer.

17. The method of claim 1 , wherein the spray dried biologic particles are produced by atomizing a feed solution comprising a biologic drug in an aqueous carrier to form an aerosol; and exposing the aerosol to a stream of drying gas at a temperature of from about 80 °C to about 150 °C to dry the aerosol to form the spray dried biologic particles..

18. The method of any one of claims 16-17, wherein the S / H mixture is passed through the pores of the microporous membrane at a rate of at least 0.1 mL / min.

19. The method of any one of claims 16-18, wherein the pores of the microporous membrane have an average size of at least 0.2 microns.

20. The sustained release biologic obtained from any one of claims 1-19.

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