Particles comprising biotherapeutic encapsulated with biodegradable polymer

Abstract

Disclosed herein are particles, methods of forming particles, and formulations comprising particles. The particles are encapsulated particles comprising a biodegradable polymer encapsulating a biotherapeutic. The biodegradable polymer is a polyesteramide (PEA) of defined structure. The particles are highly loaded with biotherapeutic and may exhibit desirable release properties in vivo.

Description

[0001] PARTICLES COMPRISING BIOTHERAPEUTIC ENCAPSULATED WITHBIODEGRADABLE POLYMER FIELD OF THE INVENTION

[0001] Aspects of the invention relate to particles of polyesteramide polymers encapsulating water-soluble biotherapeutic and more particularly those exhibiting advantageous in vivoperformance characteristics and other properties that render the particles suitable, according tospecific embodiments, for use in administering biotherapeutics with desirable release profiles.BACKGROUND

[0002] Biotherapeutics are medicinal products that are peptides, polypeptides, proteins, nucleic acidsor nucleic acid based, monoclonal antibodies, vaccines, or formulations thereof. Biotherapeutics are manufactured in, extracted from, or semi-synthesized from biologicalsources.

[0003] Biotherapeutics are administered to patients for myriad medical reasons, often by injection orinfusion. Due to limited plasma lifetime, administration of biotherapeutics may be required atfrequent intervals. Certain biotherapeutics may have their properties modified to allow administration at weekly intervals, but longer intervals between injections is still desired.

[0004] Sustained release or pulsatile release of the biotherapeutic after a single injection orimplantation would reduce the number of injections required to achieve the desired therapeutic effect. Sustained release systems may release the biotherapeutic over the course of a few daysto several months. Typically, limited burst release is desired so as to maintain efficient therapeutic concentration of the biotherapeutic.

[0005] A pulsatile drug delivery system may involve the simultaneous administration of an immediaterelease component and a modified release component. The immediate release component may act as a first dose or provide an immediately felt impact on the patient, while the modifiedrelease component may act as a second dose and / or provide a longer term benefit to the patient.

[0006] Drug delivery systems that are able to achieve the therapeutic effect of a biotherapeutic with areduced number of injections would be desirable.SUMMARY

[0007] One desired aspect of a drug delivery system is often that an initial “burst” of biotherapeuticupon injection should be maintained in a safe range. For sustained release, it is desired to ratherrelease the biotherapeutic slowly over time and over the course of a few weeks to severalmonths. For a pulsatile system, it is desired to release the dose, whether as a burst or gradually, sometime after initial injection. For example, after 25-30 days the majority of the delayed dose is released over ten days or less.

[0008] Achieving sustained release profiles in an injectable formulation while ensuring sufficientlyhigh loading of biotherapeutic is challenging. The administered formulation must be injectable,typically requiring particles of sufficiently small size. Increased biotherapeutic payload maynegatively affect particle formation generally as there is naturally less of the other componentsof the formulation needed to form particles as the amount of biotherapeutic increases. Achieving particles that are sufficiently stable once formed is also challenging, as is achievinga desired release profile once sufficient particles are formed.

[0009] Aspects of the invention are associated with the discovery that a class of biodegradablepolymers may inhibit the burst release of an array of water-soluble biotherapeutics from particles formed via a certain process. These polyesteramide (PEA) polymers exhibitsurprisingly beneficial properties for this purpose. This has been demonstrated, for example,by testing burst release in vitro of particles formed from water-soluble biotherapeutics andPEAs. Particularly, particles formed by an antisolvent method exhibit sustained release without significant burst release after subjecting the particles to a washing procedure as describedherein. Benefits of the disclosed inventions may include, alone or in combination, an improved release duration, limited burst release, reduced injection frequency, an improved dosingregimen, higher drug loading, patient comfort, patient compliance, reduced side effects, reduced gastrointestinal effect or discomfort, manufacturing efficiency or repeatability, and improved product stability.

[0010] The described process converts a micronized powder of biotherapeutics into injectablesustained release formulations. It is believed that the process allows formation of microparticleshighly-loaded with biotherapeutic wherein the contact of the biotherapeutic with the biodegradable polymer is minimized. Further, the process may be tunable in order to achieve a variety of release profiles of the encapsulated biotherapeutic.

[0011] These and other embodiments, aspects, and advantages relating to the present invention areapparent from the following Detailed Description. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] A more complete understanding of exemplary embodiments of the invention and theadvantages thereof may be acquired by referring to the following description in consideration of the accompanying figures, which serve to illustrate various features and certain principlesinvolved.

[0013] Fig. 1A is an SEM image of PEA X-50 encapsulating BSA particles formed by spray-drying.

[0014] Fig. 1B is an SEM image of PEA X-50 encapsulating BSA particles formed by spray-drying with1 w / v% Tween 60.

[0015] Fig. 1C is an SEM image of PEA X-50 encapsulating BSA particles formed by spray-drying with1 w / v % stearic acid.

[0016] Fig. 2 is a composite of SEM images of PEA X-50 encapsulating BSA particles formed byemulsification. Row A is 1:10 protein:polymer ratio, Row B is 1:5, Row C is 1:3, and Row D is6:4. The left column is before chloroform evaporation. The right column is after 3 days in PBS.

[0017] Fig. 3 is a graph of % released protein vs. time for PEA X-50 encapsulating BSA particles formedby emulsification at four different protein:polymer ratios.

[0018] Fig. 4 is a graph of % released protein vs. time for PEA X-50 encapsulating BSA particles formedby the antisolvent at two different protein:polymer ratios.

[0019] Fig. 5 is a graph of encapsulation efficiency vs. the acetone fraction in a water-acetone mixtureused as the antisolvent.

[0020] Fig. 6 is a graph of percentage of released BSA from encapsulated using the antisolvent methodat different water-acetone mixtures.

[0021] Fig. 7 is a graph of percentage of released BSA as a function time from particles encapsulated inPEA X-50 polymer by the antisolvent method with ethanol as the antisolvent and after washing.

[0022] Fig. 8 is a graph of (i) cumulative release as a percentage of total BSA load and (ii) daily dose asa function of time from particles encapsulated in PEA X-50 polymer by the antisolvent method with ethanol as the antisolvent and after washing. DETAILED DESCRIPTION

[0023] The disclosures herein relate to particles comprising a water-soluble biotherapeuticsencapsulated by certain biodegradable polymers. The biodegradable polymers are polyesteramides (PEAs) that are random copolymers comprising certain “units” as hereinafterdescribed.

[0024] The particles encapsulate a biotherapeutic that is water soluble. The biotherapeutic is presentwithin the encapsulated particles at high loading relative to the total weight of the particles. In an embodiment, the water-soluble biotherapeutic is present in an amount of from 60 wt% to 90 wt%, based on the total weight of the particles. In an embodiment, the water-solublebiotherapeutic is present at an amount of at least 65 wt%, more preferably at least 70 wt%, more preferably at least 75 wt%, more preferably at least 80 wt%, based on the total weight of the particles. In an embodiment, the water-soluble biotherapeutic is present at an amount of at most 88 wt%, based on the total weight of the particles. In an embodiment, the water-soluble biotherapeutic is present at an amount of at most 85 wt%, based on the total weight of the particles. In an embodiment, the average particle size (d50) of the particles is from 1 to 500 µm. In an embodiment, the average particle size (d50) of the particles is from 50 to 500 µm. Inan embodiment, the average particle size (d50) of the particles is from 1 to 50 µm.

[0025] The biotherapeutic is water-soluble. In an embodiment, the biotherapeutic is a peptide,polypeptide, protein, nucleic acid, monoclonal antibody, or a mixture thereof. In anembodiment, the biotherapeutic is a GLP-1 receptor agonist. In an embodiment, the peptide is aglucose-dependent insulinotropic polypeptide (GIP) agonist. In an embodiment, thebiotherapeutic is semaglutide or tirzepatide.

[0026] Some of the embodiments herein are associated with the surprising discovery that high loadingof the water-soluble biotherapeutic is obtained utilizing a washing procedure. The washing procedure generally involves contacting the encapsulated particles comprising such PEAs encapsulating a water-soluble biotherapeutic with a washing solvent. The washing solvent is capable of dissolving the biodegradable polymer. Such washing may involve agitation, such as by vortexing. The washing step may be repeated, such as by discarding a supernatant formedin a first washing step and starting with fresh washing solvent. In an embodiment, the washing step is repeated at least three times.

[0027] In an embodiment, the washing is carried out to the point that a certain amount of biodegradablepolymer is removed from the encapsulated particles. In an embodiment, that amount is until the encapsulated particles appear translucent. In an embodiment, that amount is until no or very little biodegradable polymer is detected in a supernatant. In an embodiment, the step of washing is repeated or carried out until the concentration of biodegradable polymer in the supernatant is below 5 w / v %, more preferably below 4 w / v %, more preferably below 3 w / v %, more preferably below 2 w / v %, most preferably below 1 w / v %. In an embodiment, that amount is until the encapsulated particles comprise from 60 wt% to 90 wt% of the biotherapeutic.

[0028] In an embodiment, the encapsulated particles are prepared by a so-called antisolvent method.Generally, the antisolvent method may be carried out as follows.

[0029] First, a first composition may be formed by dispersing particles of water-soluble biotherapeuticin a first solvent and dissolving the biodegradable polymer in the first solvent. The first solvent does not dissolve the water-soluble biotherapeutic but does dissolve the biodegradable polymer.

[0030] The particles of water-soluble biotherapeutic have an average particle size (d50) of from 1-200µm, preferably from 10 to 100 µm, more preferably from 10 to 60 µm. In another embodiment,the particles of water-soluble biotherapeutic have an average particle size (d50) of from 30-50 µm, from 10 to 20 µm, or from 1-5 µm.

[0031] In an embodiment, the weight ratio of water-soluble biotherapeutic to biodegradable polymerat this stage is from 6:4 to 1:15. In an embodiment, the weight ratio of water-solublebiotherapeutic to biodegradable polymer is from 1:5 to 1:15.

[0032] In a second step, the first composition is mixed with an antisolvent. The antisolvent does notdissolve the biodegradable polymer and is miscible with the first solvent. The encapsulated particles are thus obtained and are next separated and optionally dried before washing.

[0033] It may be desirable that the first solvent and / or the washing solvent are water miscible. In anembodiment, the first solvent is selected from the group consisting of ethanol, methanol, butanol, tert-butanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, and mixtures thereof. In an embodiment, the washing solvent is selected from the group consisting of ethanol, methanol, butanol, tert-butanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, and mixtures thereof. In an embodiment, the first solvent is ethanol. Inan embodiment, the washing solvent is ethanol. In an embodiment, the biotherapeutic is notsoluble in the washing solvent.

[0034] In an embodiment, the antisolvent is selected from the group consisting of water, acetone, ethylacetate, and mixtures thereof. In an embodiment, the antisolvent is water.

[0035] The particles are preferably delivered to the patient in an injectable formulation. Theformulation may comprise the particles along with a suitable pharmaceutically acceptable carrier, such as saline.

[0036] In an embodiment, the formulation may further comprise second particles. The second particlesmay comprise essentially the same components as the (first) particles but may differ in a desiredattribute. For example, the second particles may differ from the first particles in particle size, biotherapeutic loading, biodegradable polymer formulation, comprise additional biodegradable polymers, shell thickness, or comprise a further shell.

[0037] In an embodiment, the formulation further comprises unencapsulated water-solublebiotherapeutic. In this way, an immediate dose of biotherapeutic may be delivered with adelayed dose being delivered via the first particles. In an embodiment, the unencapsulated biotherapeutic is present as particles. In an embodiment, such unencapsulated biotherapeutic isthe same as the water-soluble biotherapeutic in the first particles.

[0038] In an embodiment, less than 20% of the water-soluble biotherapeutic is released from theparticles after twenty-four hours in phosphate buffer at pH 7.4 containing 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 0.1 % NaN3 at 37 °C. In an embodiment, less than 25% ofthe water-soluble biotherapeutic is released from the first particles after 7 days in phosphate buffer at pH 7.4 containing 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 0.1 % NaN3 at 37 °C.

[0039] An injectable formulation comprising the particles may be administered to a patient byinjecting the formulation. In an embodiment, less than 20% of the water-soluble biotherapeutic is released from the first particles after twenty-four hours. In an embodiment, less than 25% of the water-soluble biotherapeutic is released from the first particles after 7 days.

[0040] In addition to use of the encapsulated particles in an injected formulation, the encapsulatedparticles may be processed with another degradable or non-degradable biomaterial. In anembodiment, the particles are dispersed within a degradable or non-degradable polymer to become a (component of) a drug-eluting implant. In such an implant, the particles may be dispersed into another biomaterial to control the initial burst and drug release kinetics. In an embodiment, the particles may form the interior of an implant comprising an externaldegradable polymer layer or coating. In another embodiment, a melt is formed comprising adegradable or non-degradable polymer and the encapsulated particles, and a (component of) amedical implant is formed. In an embodiment the degradable or non-degradable polymer is a degradable polyester, such as PLA, PLLA, PLGA, or a combination thereof. In an embodiment the degradable or non-degradable polymer is ethylene-vinyl acetate (EVA) or a thermoplasticpolyurethane (TPU). In the case of a degradable polymer, upon degradation of the external layer or body of the component, the encapsulated particles may be released. The implant maybe formed by a hot-melt process, injection molding, or extrusion. Such an implant is typically a solid or a semi-solid.

[0041] In a further embodiment, a plurality of encapsulated particles is further encapsulated via a solid-oil-in-water emulsification process to form an injectable microparticles formulation. In thisembodiment the primary particles are suspended into an encapsulating polymer-containing oil phase which is added to an aqueous phase under the conditions of an emulsification process to yield injectable microparticles of average particle size (d50) of from 40 to 250 µm. In such anembodiment, each particle comprises a shell comprising a degradable encapsulating polymerencapsulating a plurality of the encapsulated particles.

[0042] The PEA has a structure that includes, or that may consist of, n units, each unit having residues,for example 2, 3, or 4 different types of residues, as defined below, which are present in eachunit at respective molar equivalents that are designated by m, p, q, and x, in which m+p+q+x=1. The n units may be characterized as “repeating” units to the extent that the defined values orranges of m, p, q, and x are consistent from one unit to the next, although it is not required foreach unit to be identical in structure, as long as the defined values or ranges are met. As is apparent from the structures of residues given below, they are linked by amide bonds [(- C=O)NH-)], whereas certain of these residues include ester bonds [(-C=O)O-)] that help confer water permeability. Given the teachings herein, those skilled in the art will be able to appreciate the structural modifications and corresponding adjustments in synthesis procedures that influence certain properties described herein.

[0043] For ease of understanding, dashed lines are used in structures of the individual residues, and inother structures, to emphasize that they represent points of attachment (bonding) and not methyl groups. To the extent that structures may include chiral carbon atoms, the lack of an express illustration of stereochemistry is meant to convey that all stereoisomers are intended. However, those skilled in organic chemistry, given the teachings herein including the specific synthesis procedures, will be able to ascertain particular stereochemical configurations that are representative of PEA polymers in preferred embodiments.

[0044] More specifically, the n units in the in the structure of the PEA may have:m molar equivalents of a first residue having the formula , , .

[0045] The values m, p, q, and x represent molar equivalents of the corresponding first, second, third,and fourth residues in the units of the random copolymer, which molar equivalents are scaledsuch that m+p+q+x=1, thereby indicating relative molar equivalents among the residues.

[0046] According to a first embodiment, m is from 0 to 0.8, p is from 0 to 0.95, m+p is from is from0.5 to 1, q is from 0 to 0.35, and x is from 0 to 0.25; n is from 5 to 300; R1 is (C2-C20)alkylene;R3and R4are selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl, -CH2SH, -(CH2)2S(CH3), -CH2OH, -CH(OH)CH3, -(CH2)4NH3+, -(CH2)3NHC(=NH2+)NH2, -CH2COOH, -CH2-CO-NH2, -CH2CH2-CO-NH2, -CH2CH2COOH,CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, H2N-(CH2)4-, Ph-CH2-, CH=C-CH2-, (CH3)2CH-,Ph-NH-, NH-(CH2)3-C-, or NH-CH=N-CH=C-CH2-, wherein R3and R4are the same or different; R5 is (C2-C20)alkylene, optionally having one or more alkylene carbon atoms (-C-)replaced by an oxygen atom (-O-), and / or optionally having 5 or 6 consecutive alkylene carbon atoms together form a respective 5- or 6-membered carbocyclic ring;R6is ; In such PEA polymers, highervalues of p, i.e., increased proportions of the second residue, can lead to a higher glass transition temperature, and more particularly a higher glass transition temperature of the polymer in its hydrated state (wet Tg).

[0047] As used herein, the term “alkyl” means a monovalent straight or branched chain hydrocarbongroup including methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-hexyl, and the like, with more specific alkyl groups being limited according to ranges of carbon atom numbers, as indicated in parentheses, preceding “alkyl.”

[0048] As used herein, the term “alkylene” means a divalent straight or branched chain hydrocarbongroup such as -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2)5-, and the like, with more specificalkyl groups being limited according to ranges of carbon atom numbers, as indicated in parentheses, preceding “alkylene.”

[0049] As used herein, the term “alkenyl” means a monovalent straight or branched chain hydrocarbongroup having at least one carbon-carbon double bond in the main chain or in a side chain, withmore specific alkenyl groups being limited according to ranges of carbon atom numbers, as indicated in parentheses, preceding “alkenyl.”

[0050] As used herein, the term “alkenylene” means a divalent straight or branched chain hydrocarbongroup having at least one carbon-carbon double bond in the main chain or in a side chain, withmore specific alkenylene groups being limited according to ranges of carbon atom numbers, as indicated in parentheses, preceding “alkenylene.”

[0051] As used herein, “alkynyl” means a monovalent straight or branched chain hydrocarbon grouphaving at least one carbon-carbon triple bond in the main chain or in a side chain, with morespecific alkynyl groups being limited according to ranges of carbon atom numbers, as indicated in parentheses, preceding “alkynyl.”

[0052] As used herein, “aryl” means an unsubstituted or optionally substituted phenyl radical or anunsubstituted or optionally substituted ortho-fused bicyclic carbocyclic radical having nine orten ring atoms, in which at least one ring is aromatic. Examples of aryl include, but are notlimited to, phenyl, naphthyl, and nitrophenyl.

[0053] As used herein, “biodegradable" means a material which is capable of being completely orsubstantially degraded or eroded when exposed to an in vivo environment. A polymer iscapable of being degraded or eroded when it can be gradually broken down, resorbed, absorbed,and / or eliminated by, for example, hydrolysis, enzymolysis, oxidation, metabolic processes, bulk or surface erosion, and the like. As noted above, in preferred embodiments, PEA polymersmay be characterized as being biodegradable. This leads to advantages in the use of such polymers in delivery of biotherapeutics.

[0054] According to the PEA structure as defined above and including n units, each having residues,for example 2, 3, or 4 different types of residues, in the case of a “random copolymer,” the residues, present in each unit at respective molar equivalents that are designated by m, p, q,and x, are distributed randomly throughout the units and throughout the copolymer.

[0055] Given the structures of the monomers above, it can be appreciated that, in any of theembodiments as defined herein, the PEA may have the following structure: above.

[0056] In more specific embodiments, the PEA is defined as according to the first embodiment,wherein m may be from 0, 0.10, 0.15, 0.20, or 0.25 to 0.75, 0.70, 0.65, 0.60, 0.55, 0.50, 0.45,0.40, or 0.35. In other more specific embodiments, the PEA is defined as according to the first embodiment, wherein p may be from 0.10, 0.20, 0.30, 0.35, or 0.40 to 0.95, 0.90, 0.85, 0.75,0.70, 0.65, 0.60, 0.55, 0.50, or 0.45. In other more specific embodiments, the PEA is defined as according to the first embodiment, wherein p may be greater than or equal to m. In othermore specific embodiments, the PEA is defined as according to the first embodiment, wherein mand p may be both greater than zero. In other more specific embodiments, the PEA is definedas according to the first embodiment, wherein m:p may be from 2:1, 1:1, or 2:3 to 1:5, 1:4, 1:3,or 1:2. In other more specific embodiments, the PEA is defined as according to the first embodiment, wherein q is from 0.05, 0.10, 0.12, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, or 0.2 to0.30, 0.25, 0.23, 0.21, 0.20, 0.19, 0.18, 0.17, 0.16, or 0.15. In other more specific embodiments, the PEA is defined as according to the first embodiment, wherein x is from 0, 0.02, 0.05, 0.06,0.07, 0.08, 0.09, or 0.10, or 0.125 to 0.25, 0.20, 0.15, 0.14, 0.13, 0.125, 0.12, 0.11, or 0.10. Inother more specific embodiments, the PEA is defined as according to the first embodiment, wherein the ratio q:x is from 9:1, 8:1, 7:1, 6:1, 5:1, 4:1 or 3:1 to 1:4, 1:3, 1:2, 1:1, 2:1, or 3:1.In other more specific embodiments, the PEA is defined as according to the first embodiment, wherein m is about 0.3, p is about 0.45, q is about 0.19, and x is about 0.06. n other more specific embodiments, the PEA is defined as according to the first embodiment, wherein m is about 0.3, p is about 0.45, q is about 0.125, and x is about 0.125. In other more specific embodiments, the PEA is defined as according to the first embodiment, wherein n is from 5,10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, or 75 to 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, or 150. In other more specific embodiments, the PEA is defined as according to the first embodiment and has a number average molecular weight (Mn) of at least 15,000 g / mol, at least 20,000 g / mol, at least 25,000 g / mol, at least 30,000 g / mol, or at least 35,000 g / mol. In other more specific embodiments, the PEA is defined as according to the first embodiment and has an Mn of at most 250,000 g / mol, at most 225,000g / mol, at most 200,000 g / mol, at most 175,000 g / mol, at most 150,000 g / mol, at most 125,000 g / mol, at most 100,000 g / mol, or at most 75,000 g / mol. Mn is measured via gel permeationchromatography (GPC) in tetrahydrofuran (THF) with polystyrene as standard.

[0057] In yet further specific embodiments, the PEA is defined as according to the first embodiment,and may have any one or more of: ranges of m as defined above; ranges of p as defined above;p being greater than m; m and p being both greater than zero; ranges of m:p as defined above; ranges of q as defined above; ranges of x as defined above; ranges of q:x as defined above; values of m, p, q, and x as defined above; ranges of n as defined above; lower limits of Mn as defined above; and / or upper limits of Mn as defined above.

[0058] In yet further specific embodiments, the PEA is defined as according to the first embodiment,and may be more particularly characterized by any one or more of: R3 is selected from thegroup consisting of hydrogen, (C1-C6)alkyl, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, Ph-CH2-,and (CH3)2CH-; R4 is selected from the group consisting of hydrogen, (C1-C6)alkyl, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, Ph-CH2-, and (CH3)2CH-; R3 and R4 are the same; R5 is (C2-C20)alkylene; R7 is (C6)aryl-CH2- (i.e. benzyl or phenylmethyl); and / or R8 is -(CH2)4-.

[0059] In yet further specific embodiments, the PEA is defined as according to the first embodiment,and may be more particularly characterized by any one or more of: p is from 0 to 0.8 and m+p is from 0.5 to 0.9; q is from 0.05 to 0.25; x is from 0.05 to 0.25; q:x is from 9:1 to 1:4, or more particularly from 4:1 to 1:4 or from 4:1 to 1:2; q is 0; x is 0; and / or m is 0.

[0060] In yet further specific embodiments, the PEA is defined as according to the first embodiment,and may be more particularly characterized by any one or more of: m is from 0.1 to 0.5, p isfrom 0.1 to 0.75, m+p is from 0.5 to 0.8, q is from 0.1 to 0.35, and x is from 0 to 0.1; R1 is (C2-C10)alkylene; R3 and R4 are (C1-C6)alkyl; R5 is (C2-C10)alkylene; R6is alkylene; and R8 is (C3-C6)alkylene. For example, themore of these definitions of m,1 3 4 p, m+p, q, x, R , R , R , R5, R6, R7, and R8, such as in the case of the PEA being characterized by all of these definitions.

[0061] In yet further specific embodiments, the PEA is defined as according to the first embodiment,and may be more particularly characterized by any one or more of: m is 0.3, p is 0.45, q is 0.25, and x is 0; R1 is octylene [(C8)alkylene]; R3 and R4 are isobutyl; R5 is hexylene [(C6)alkylene];R6is ; alkylene, or phenylmethyl]; and R8 is butylene [(C4)alkylene]. Forexample, the PEA may be characterized by one or more of these definitions of m, p, q, x, R1, R3, R4, R5, R6, R7, and R8, such as in the case of the PEA being characterized by all of these definitions.

[0062] Polyesteramide random copolymers may be synthesized by adapting a procedure known in theart from Katsarava et al. (WON J POLYM SCI A: POLYM CHEM 1999:37: 391–407). Briefly, thepolymers are prepared via solution polycondensation of di-p-toluenesulfonic or hydrochloricacid salts of bis-(α-amino acid) α,ω-diol diesters, lysine benzyl ester, lysine, and / or di-N-hydroxysuccinimide ester of sebacic acid in anhydrous DMSO. Typically, the salts areconverted to free amines by addition of triethylamine and these amines are further reacted with the di-acid derivative. The usage of pre-activated acid in the reaction allows polymerization at relatively low temperature, such as 65°C, affording side-product free polycondensates andpredictable degradation products. Subsequently, the obtained reaction mixture is purified viaa water precipitation followed by an organic precipitation and filtration. Drying under reduced pressure yields the polyesteramide random copolymer.

[0063] For example, such polymers may be prepared by reacting lysine, lysine benzyl ester, andhexahydrofuro[3,2-b]furan-3,6-diyl bis(2-amino-4-methylpentanoate) with di-N-hydroxysuccinimide ester activated sebacic acid in DMSO for 24 hours. The polymer is thenisolated from the reaction mixture in two precipitation steps and characterized by means ofproton NMR and THF-based GPC relative to polystyrene standards.EXAMPLES

[0064] The following examples are set forth as representative of the present invention. Theseexamples are not to be construed as limiting the scope of the invention as other equivalent embodiments will be apparent in view of the present disclosure and appended claims. Preparation of Copolymers

[0065] PEA-X50 is a random copolymer within the scope of PEA polymers as described herein andhas the structure:

[0066] which m is 0.30, p is 0.45, q is 0.125, and x is 0.125; R1is octylene [(C8)alkylene]; R3and R4are isobutyl; R5is hexylene [(C6)alkylene]; R6is

[0002] ; R7 lkylene, or phenylmethyl]; and R8 is butylene [(C4)alkylene]. This PEAcopolymer was prepared according to the following description.

[0067] Triethylamine (31 ml, 0.222 mole) and DMSO (54 ml, 0.76 mole) were added to a mixture ofdi-N-hydroxysuccinimide ester of sebacic acid (Di-NHS-sebacic acid) (39.805 g, 0.100 mole),L-leucine-(DAS)-2TosOH (32.390 g, 0.045 mole), L-leucine(6)-2TosOH (20.753 g, 0.030mole), L-lysine∙2HCl (2.750 g, 0.013 mole) and L-lysine(Bz)-2TosOH (7.289 g, 0.013 mole)in a nitrogen flushed 500 ml round bottomed flask equipped with an overhead stirrer at roomtemperature. The subsequent mixture was heated to 60°C to allow the reaction to proceed, withmonitoring by GPC analysis in THF. After 36 hours, a stable molecular weight was obtained.The reaction mixture was diluted with 250 ml DMSO and was allowed to cool to roomtemperature. At room temperature, acetic anhydride (1.89 ml, 0.0199 mole) was added toacylate the amino functional end groups of the polymer. Next, the mixture was stirred at roomtemperature for 24 hours.

[0068] The obtained crude polymer mixture was precipitated in water at a 10:1 ratio (water: reactionmixture). The polymer was collected and dissolved in ethanol (500 ml, 8.57 mole) and thenprecipitated a second time. The polymer was again dissolved in ethanol (500 ml, 8.57 mole)and precipitated in ethylacetate (5000 ml, 50.91 mole) by dropwise addition to a stirring solution. The precipitated polymer was washed with ethylacetate (100 ml, 1.00 mole), thesupernatant was removed, and the precipitate was washed again with ethylacetate (100 ml, 1.00mole). After the removal of the supernatant, the precipitate was dried and dissolved in ethanol(500 ml, 8.57 mole), and filtered over a 0.2 µm PTFE membrane filter. The filtered polymersolution was dried under reduced pressure at 65°C. From this synthesis procedure, a typicalyield is 75%, and the number average molecular weight (Mn) is normally in the range of 40-70 kDa, measured via gel permeation chromatography (GPC) using tetrahydrofuran (THF) asthe mobile phase on dried samples, and determined relative to polystyrene standards.

[0069] PEA III AcBz is a random copolymer within the scope of PEA polymers as described hereinand having the structure:

[0070] 0.3, p is 0.45, q is 0.25, and x is 0; R1is octylene [(C8)alkylene]; R3and R4are isobutyl; R5is hexylene [(C6)alkylene]; R6is ;

[0071] or phenylmethyl]; and R8 is butylene [(C4)alkylene]. This PEAcopolymer was prepared according to the following description.

[0072] Triethylamine (30.9 ml, 0.222 mole, 2.2 eq) and N,N-dimethylformamide (53.07 ml, 0.689mole) were added to a mixture of di-N-hydroxysuccinimide ester of sebacic acid (Di-NHS-sebacic acid) (39.940 g, 0.1008 mole, 1.0 eq), L-leucine(6)-2TosOH (20.823 g, 0.0302 mole, 0.30 eq), L-leucine-(DAS)-2TosOH (32.503 g, 0.0453 mole, 0.45 eq) and L-lysine(Bz)- 2TosOH (14.628 g, 0.0252 mole, 0.25 eq) in a nitrogen flushed 500 ml round bottomed flaskequipped with an overhead stirrer at room temperature. The subsequent mixture was heated to60°C to allow the reaction to proceed, with monitoring by GPC analysis in THF. After 36hours, a stable molecular weight was obtained. Subsequently, a portion of L-leucine(6)-2TosOH (4.338 g, 0.0063 mole) along with triethylamine (1.76 ml, 0.0126 mole) and N,N- dimethylformamide (4.54 ml, 0.0590 mole) were added to terminate the polymerizationreaction. The mixture was heated additionally for 24 hours, after which the viscous solutionwas further diluted with N,N-dimethylformamide (407.85 g, 5.301 mole) and allowed to coolto room temperature. At room temperature, acetic anhydride (1.89 ml, 0.0199 mole) was addedto acylate the amino functional end groups of the polymer. The mixture was stirred at roomtemperature for 24 hours.

[0073] The obtained crude polymer mixture was precipitated in water at a 10:1 ratio (water: reactionmixture). The polymer was collected and dissolved in ethanol (500 ml, 8.57 mole) and thenprecipitated a second time. The polymer was again dissolved in ethanol (500 ml, 8.57 mole)and precipitated in ethylacetate (5000 ml, 50.91 mole) by dropwise addition to a stirring solution. The precipitated polymer was washed with ethylacetate (100 ml, 1.00 mole), theethylacetate removed, and then the polymer was washed in ethylacetate again (100 ml, 1.00mole). The polymer was then dried and dissolved in ethanol (500 ml, 8.57mole) and filteredover a 0.2 µm PTFE membrane filter. The filtered polymer solution was dried under reducedpressure at 65°C. The yield was 75%, and the number average molecular weight (Mn) was43.3 kDa, measured via gel permeation chromatography (GPC) using tetrahydrofuran (THF)as the mobile phase on dried samples, and determined relative to polystyrene standards. Protein analysis by UPLC-PDA

[0074] BSA was analyzed via UPLC on a Shimadzu Nexera apparatus, equipped with LC-40DX3 deliverymodule and diode array UV-VIS detector (SPD-M40) operating at 220 nm. The chromatographic column was X-bridge C18 column, (150 mm x 4.6 mm, 3.5 µm particle size with column guardVanGard (Waters), 3.9 mm x 5 mm - C18, 3.5 µm). Binary gradient was used, with mobile phaseA = 0.1 % TFA in water and mobile phase B = ACN + 0.1 % TFA. The following gradient was used: A-80%, B-20% isocratic for 3 min, increase B to 60 % at 12 min, hold B = 60 % until 15 min, decrease B to 20 % until 15.01 min, hold B = 20 % until 20 min. Flow rate: 1 mL / min, Temperature: 30 °C, Injection volume: 35 µL. Protein Dissolution Assay

[0075] The protein dissolution assay was performed in a phosphate buffer at pH 7.4 containing: 138 mMNaCl, 2.7 mM KCl, 10 mM Na2HPO4and 0.1 % NaN3. Several droplets of pure phosphoric acid were used to adjust the pH of a 250 mL buffer solution to pH = 7.4, which introduced negligible dilution (< 1 %). The obtained particles were measured on an analytical balance and placed in 10 mL glass bottle. Phosphate buffer at 37 °C was then added to the glass bottle in a volume of 5 to 10 mL. The bottles were placed on a vortex (Vortex Genie 2 mixer) and vortexed at speed 3, which was sufficient to disperse the particles from the emulsion method as a uniform suspension. Particles from the antisolvent method remained as large aggregates in the solution. Aliquots of 1 mL were taken for analysis as a function of time. Each sample was filtered using 450 nm cellulose acetate (CA) syringe filters to remove undissolved material and then analyzed by UPLC to determine the released protein concentration (no fresh buffer had been added to the vials to compensate for thetaken samples). Encapsulation Efficiency Calculation Encapsulation efficiency was calculated for the emulsification and antisolvent methods based onmass balance considerations, which included the measurement of the concentration of BSA that had dissolved in the aqueous phase (viz. the concentration of “lost” protein) during the particle preparation protocol. For example, if the initial concentration of BSA in the organic solvent suspension was 1.5 w / v % in 2 mL, this equals 30 mg protein. Then, if the measured concentration of BSA in the 50 mL aqueous phase (e.g. used during emulsification) is 0.48 mg / mL, this equals24 mg BSA. Finally, by calculating [1-(24 / 30)]*100 we obtain the encapsulation efficiency of 20 %. See the following equation: where EE is the the concentration of protein in the organic solvent suspension, Vorg is the volume of the organic solvent suspension, Cw is the concentration of protein in the aqueous phase used for emulsification or antisolvent techniques, and Vwis the volume of the aqueous phase. For spray-drying, the mass of collected particles was used to calculate the yield (which was very low, around 1 %) and it was used in place of the encapsulation efficiency.

[0076] The protein particles used in all examples are spray-dried bovine serum albumin (BSA)particles having an average particle size of about 5 µm. Protein release from particles formed by spray-drying

[0077] BSA particle aggregation was first tested in ethanol, acetonitrile, acetone, dichloromethane,and methanol. BSA particle aggregation was found to be lowest in ethanol at 2.5 w / v % BSA,though still not completely suppressed. The addition of 1 w / v % PEA X-50 polymer significantly reduces BSA particle aggregation in ethanol. Addition of 1 w / v % surfactants (stearic acid, palmitic acid and Tween 60) to the protein dispersion containing PEA X-50 further improved the dispersion stability, yielding single, non-aggregating BSA particles in ethanol. However, as spray-drying of ethanol dispersions has the risk of explosion if inert atmosphere is not used, BSA particle aggregation in dichloromethane:ethanol 8:2 mixtures containing PEA X-50 and / or 1 w / v % surfactants (stearic acid, palmitic acid and Tween 60) was studied. Even in this solvent mixture, the presence of polymer and surfactants increased strongly the dispersion stability and single BSA particles were obtained.

[0078] The BSA particles were dispersed in methanol at a concentration of 2.5 w / v %. A sonotrode wasused for 1 min at 500 W to obtain a homogeneous suspension. Then PEA X50 polymer, 2.5 w / v % was added to obtain 5 w / v % solid material dispersion (2.5 w / v % BSA + 2.5 w / v % polymer). The suspension was then spray dried using Buchi mini spray drier B-290 at the following conditions: inlet temperature = 50°C, Q-flow = 30, Aspirator = 70 %, pump flow rate = 10 mL / min. Due tosticking of particles to the metal elements of the spray drying machinery, the particles were injectedmanually into the spray drier using a syringe.

[0079] Sufficient amounts of BSA particles encapsulated by the PEA X-50 polymer were recovered.Particles further comprising surfactants (tween 60, stearic acid) at 1 w / v % are can be characterized by SEM. The results are shown in Fig. 1A for particles without surfactant, Fig. 1B for particles with 1 w / v% Tween 60, and Fig.1C for particles with 1 w / v % stearic acid. The images show that some of the BSA particles have retained their size (around 5 μm diameter) and their surfaces appears rather homogenous, in contrast to other particles which appear to be enveloped in a polymer shell. Hence, the images hint that bare protein surface may be present on some of the particles, which could be due to unfavorable interactions between the protein surface groups and the precipitating polymer, leading to segregation of the polymer from the protein in some cases.

[0080] A sufficient amount of BSA particles encapsulated by the PEA polymer were recovered andprotein release experiments were performed with three systems: (1) PEA X-50, (2) PEA X-50+ tween 60 and (3) PEA X-50 + stearic acid. In all cases, the spray dried particles showed burst release with almost 100% released protein after 10 minutes, similarly to pure (non-encapsulated) BSA particles. See Table 1. Table 1: Dissolution of spray-dried BSA + PEA X-50 Particles System % Dissolved after 10 min BSA-PEA X-50 89 BSA-PEA X-501 w / v % Tween 60 100 BSA-PEA X-501 w / v % Stearic acid 80

[0081] Therefore, the obtained results showed that spray-drying is not recommended for proteinencapsulation by the PEA X-50 polymer. Analysis of the morphology of the particles (as visualized by the SEM images in the previous subsection) indicated that the fast dissolution of the BSA is most likely due to the inhomogeneous coating of the protein particles by the polymer, which leaves bare protein surface exposed and leads to surface dissolution, water channel formation, and quickprotein release. Protein release from particles formed by emulsification

[0082] Encapsulated BSA particles were prepared using emulsification method at different protein topolymer ratios (1:3, 1:5, 1:10, 6:4). To obtain ratios 1:3, 1:5 and 1:10, 1.5 w / v % BSA particles were dispersed in chloroform. A sonotrode was used for 1 min at 500 W to obtain homogeneous suspension. Then the polymer was dissolved to obtain 15 w / v % polymer solution. To obtain the 6:4 protein to polymer ratio, the polymer concentration was reduced to 10 w / v % and the protein concentration was increased to 15 w / v % (a sonotrode was again used to homogenize thesuspension).

[0083] After preparing the suspension of BSA particles in solution of the polymer in chloroform, 2 mL ofthe BSA-polymer mixture was emulsified in 50 mL 0.4 w / v % sodium oleate aqueous solution using an ULTRA-TURRAX® mixer at 13500 RPM for 5 minutes. Samples were taken formicroscope observations to study the particle size and morphology and for UPLC analysis to determine the encapsulation efficiency. The emulsion was then transferred to a flask and the chloroform was evaporated by using a rotary evaporator at 40 °C, 60 RPM for 1 hour. The particles were then separated from the aqueous phase by filtration using a paper filter (1:10 and 6:4 ratios) or by centrifugation at 4500 RPM (1:3 and 1:5 ratios). There were no significant losses of particles on the filter due to sticking on the filter surface. The obtained particles were dried under vacuum for 24 h. Samples were taken for microscope observation to study the morphology of the final particles.

[0084] The size and the morphology of the particles varied with the different protein to polymer ratios.See Fig. 2. At ratio of 1:10 protein:polymer, the obtained particles were irregular in shape withvariable size ranging from 30-40 µm to 100-200 µm. The irregular shape was at least partlydue to the high viscosity of the organic phase, which hinders droplet break-up during emulsification. The morphology of the particles remained unchanged after the emulsification process as well as during the dissolution experiments.

[0085] The 1:5 particles were smaller in size (20-100 µm) compared to the 1:10 particles, withspherical shape and very distinct tentacle-like polymer filaments protruding from the surface. The morphology of the particles remained unchanged after the emulsification process as well as during the dissolution experiments. The 1:3 ratio yielded the smallest particles, which were spherical with size around 50 µm in diameter. When working in excess of protein (the 6:4ratio), larger spherical particles (300-400 µm) were produced, which also kept their size and morphology during the dissolution process.

[0086] The encapsulation efficiency of the emulsification method was found to depend significantlyon the protein:polymer ratio (Table 2), with the general trend of increased encapsulation at higher polymer ratios when working in excess of polymer. When working in excess of protein (ratio of 6:4), an encapsulation efficiency of only 20 % was obtained: the majority of the protein had dissolved in the aqueous phase during the emulsification of the organic solvent protein suspension in water. Hence, the final protein:polymer ratio in the obtained particles was around 1:3.

[0087] In the case when 1:3 was selected as the starting protein-to-polymer ratio in the organic solventsuspension, the encapsulation efficiency was increased considerably to 74, yielding a final protein:polymer ratio of 1:4 in the encapsulated particles. Further increase of the initial protein:polymer ratio to 1:5 improved only slightly the encapsulation, whereas excellent encapsulation efficiency (around 100 %) was measured when working in a significant excess of polymer (1:10). Table 2: Encapsulation efficiency of BSA in PEA X-50 by emulsification Initial BSA:polymer ratio Encapsulation efficiency, % Final BSA:polymer ratio 6:4 20 1:31:3 74 1:41:5 79 1:61:10 100 1:10

[0088] The particles obtained by the emulsification method were shown to be effective in slowing downthe protein dissolution and achieving sustained release (Fig. 3). All prepared systems showedsustained protein release. After 7 days of dissolution, the protein found in the dissolution media was under 2% of the encapsulated protein for protein:polymer ratios of 1:3, 1:5 and 1:10 and slightly higher (around 10 %) for the 6:4 ratio after 3 days of dissolution.

[0089] Therefore, the obtained results clearly showed that BSA protein particles can be successfullyencapsulated via the PEA X-50 polymer and the obtained particles can yield protein release of less than 2 % for one week. The encapsulation efficiency, particle size and particle morphology was found to depend significantly on the protein-to-polymer ratio in the initial organic solvent suspension. Protein release from PEA X-50 particles formed by antisolvent method

[0090] Antisolvent particles were prepared at different protein to polymer ratios (1:5, 1:10, 6:4). To obtainthe 1:5 and 1:10 ratios, the BSA particles were dispersed in absolute ethanol to obtain 1.5 w / v % suspension. Sonotrode was used for 1 min at 500 W to obtain homogeneous suspension. Polymer was then added to obtain 15 w / v % polymer solution and the mixture was stirred on a magnetic stirrer until the polymer was completely dissolved. For the 6:4 protein to polymer ratio, the polymer concentration was reduced to 10 w / v % and the protein concentration was increased to 15 w / v %. Afterwards, the same protocol as the 1:5 and 1:10 ratios was followed.

[0091] After preparing the BSA suspension in solution of the polymer in ethanol, 2 mL of the suspensionwas added to 50 mL antisolvent while stirring using overhead stirrer at 600 RPM for 3 minutes. Particles were prepared using various antisolvent media: water, acetone, or water-acetone mixtures, as specified. Samples from the aqueous phase were taken for UPLC analysis to determine thequantity of non-encapsulated protein. The obtained particles were dried under vacuum for 24 h.

[0092] Effect of the protein-to-polymer ratio on encapsulation efficiency was studied similar to theemulsification method. It was found that the percentage of encapsulated protein increased significantly with increasing the polymer in the mixture (Table 3). When working at an excess of protein (6:4 protein:polymer ratio), most of the protein dissolved in the aqueous antisolvent phase during the mixing of the organic solvent BSA + polymer suspension in the water, yielding an encapsulation efficiency of 20 % and final protein-to-polymer ratio of 1:3.Table 3: Encapsulation efficiency of BSA in PEA X-50 by antisolvent method Initial BSA-polymer ratio Encapsulation efficiency, % Final BSA-polymer ratio6:4 20 1:31:5 71 1:71:10 78 1:13

[0093] Increasing the polymer fraction in the initial organic solvent suspension to 1:5 significantlyincreased the encapsulation efficiency to 71 %, resulting in final particle composition of 1:7. Further increase of the initial polymer fraction to 1:10 increased only slightly the encapsulation efficiency to 78 % and final particle composition of 1:13.

[0094] The morphology of the obtained particles depended on the polymer content. A large gum-likelump was obtained from the 1:10 ratio, whereas 1:5 ratio yielded thread like particles which formed a foil-like structure after drying.

[0095] The protein dissolution from the 1:5 and 1:10 particles is presented in Fig. 4. The particlesobtained from the 6:4 protein-to-polymer ratio were not studied because of the low encapsulation efficiency. No burst release was observed from the encapsulated particles, as demonstrated by the very low percentage of released BSA after 10 min for both formulations (1-2 %). The released protein increased significantly to 5-6 % after 7 days and remained constant at 7 % for the 1:10 formulation up to 21 days, whereas it increased gradually to ~7.5% for the 1:5 formulation after 21 days of dissolution.

[0096] The presented results showed that protein particles can be successfully encapsulated by the PEAX-50 polymer when using the antisolvent method and emulsification method, yieldingencapsulated particles with slow protein release and no burst-release events. Similarly to the emulsification method, the encapsulation efficiency depended significantly on the protein-to- polymer ratio. Use of acetone as anti-solvent

[0097] However, good encapsulation efficiency (e.g. above 80 %) was obtained only when working athigher polymer fractions of 1:5 or 1:10 protein-to-polymer. In the context of practical application, working at lower polymer fraction (higher drug loading) would be desirable. Since it is hypothesized that the major factor causing the decreased encapsulation efficiency is the dissolution of the protein in the aqueous phase during the preparation protocol, in the current section we explored the possibility to encapsulate the BSA particles by using acetone as an antisolvent for the polymer, instead of water. As the protein is insoluble in acetone, theoretically, this should result in significantly increased encapsulation efficiency while keeping the desired slow protein release.

[0098] The protein-to-polymer ratio was held constant at 6:4 (excess of protein), whereas the acetone-to-water ratio of the antisolvent media was varied. Excellent encapsulation efficiency (≥ 94 %) was obtained when at least 50 % of the antisolvent was composed of acetone. See Fig. 5. Decreasing the acetone fraction to 0.3 (3:7 acetone-to-water ratio) decreased strongly the encapsulation efficiency to 41 % which, however, was still higher compared to the encapsulation efficiency in 100 % water (20 %).

[0099] The protein dissolution from the particles was studied in dissolution experiments. The obtainedresults showed relatively quick release of the protein from the particles obtained at acetone fractions ≥ 0.5; more than 80 % of the protein was dissolved after 10 days. Encapsulation that yields slow release of the protein was observed only at an acetone fraction of 0.3 (3:7 acetone- to-water ratio), where less than 5 % of BSA was dissolved after 26 days of dissolution. Thefinal composition of the particles obtained by this method using a 3:7 acetone:water ratio (after accounting for the protein dissolved in the antisolvent media) was 1.0:1.7 protein:polymer. Washing of PEA X-50 particles

[0100] BSA particles encapsulated by PEA X-50 polymer at a protein-to-polymer ratio of 1:10 that wereformed by the antisolvent method with water as the antisolvent were washed from the excess polymer by following the following protocol.400 mg of particles are placed in a 2 mL centrifuge tube and 2 mL of absolute ethanol are added. The samples are vortexed until the polymer is dissolved, which could be visually confirmed by the surface of the particles becoming translucent after the addition of ethanol. The samples were then centrifuged for 5 min at 20000 x g. The supernatant containing dissolved polymer in ethanol was discarded, and 2 mL of fresh absolute ethanol was added to the sediment. The washing procedure was then repeated (total of three times) to remove most of the polymer. The remaining particles were then separated and dried under vacuum for 24 h.

[0101] The presumption was that the wash would remove all of the polymer, allowing quantification ofthe encapsulated BSA. It was attempted to dissolve the particles in water after washing, assuming that the particles should be composed of mainly BSA. Surprisingly, the particles did not dissolvein water and measurements by UPLC showed very low BSA concentration in the aqueous phase. The experiment was repeated three separate times and the same result was obtained.

[0102] The release of BSA from the washed particles was measured as a function of time. See Fig. 7. Thereleased BSA started from less than 1 % after 10 min dissolution and reached 22 % protein release after 26 days. Based on these finding, it was hypothesized that part of the polymer is bound stronglyto the surface of the BSA particles and was not removed during the washing protocol.

[0103] A modified protein dissolution assay was performed according to the following procedure and theresults averaged (n=2). Approximately 10 mg of particles were weighed into Eppendorf® LoBindtubes. 1 mL PBS buffer containing 0.05% sodium azide was added to give a concentration of approximately 10 mg / mL. Samples were incubated at 37 °C with shaking at 100 rpm for theduration of the study. At sampling points of day 1, 2, 3, 4, 7, 9, 11, 14, 17, 21, then weekly (or biweekly), 900 µL buffer was transferred to an HPLC vial and 900 µL fresh buffer was added tomaintain 1 mL volume. From the 900 µL sample two 200 µL portions were transferred to separate wells of a UV Star 96 well plate for BSA quantification through absorption measurement at 280 nm using a Thermo Fischer GO plate reader. BSA standards were prepared to produce a calibration line from 1 to 500 µg / mL. If samples were not measured immediately then they are stored at -20°C and thawed prior to measurement. The results are shown in Fig.8. Low initial burst release is observed, with less than 20% of the total protein load released in the first 24 hours and less than25% release in the first seven days. Steady release between day twenty and day 120 is also observed.

[0104] The presented results were very encouraging, since such particles formed by the antisolvent methodfollowed by washing were found to have the following advantages: (1) high protein-to-polymer ratio, (2) slow protein release, and (3) possibility to recycle the excess polymer for encapsulationof additional protein particles. The initial burst of the BSA from the particles is surprisingly low(<10%) considering the very high load of highly water-soluble BSA. The observed release profilewould be desirable for the delivery of various water-soluble biotherapeutics by injection.PEA III AcBz particles formed by antisolvent method with washing – small scale procedure

[0105] A protein solution of ovalbumin is first prepared as follows. 10% w / v dispersion of protein inwater (WPI, SMP, ovalbumin) was prepared by adding 10 mL deionized water to 1 g protein. The dispersion was stirred on magnetic stirrer for 30 minutes at ambient temperature. Then, the dispersion was split into five 2 mL centrifuge tubes and centrifuged at 20000 x g for 5 min to remove undissolved protein. The serum was then aspirated with a syringe and filteredthrough a syringe filter 450 nm CA. Clear supernatant should be obtained. The ovalbumin wasdifficult to filter through 450 nm CA and a considerable number of filters had to be used toobtain enough quantity clear solution.

[0106] 100 µL of the protein solution is then added to 10 mL octanol or dodecanol via micropipette.The emulsion is homogenized for 1 min with a pulse sonicator (SKL-650W, Syclon) at 1 s long pulses with 1 s power off at 500 W. Afterwards, the obtained dispersion was imaged via optical microscopy to check for formation of solid particles.

[0107] In order to accelerate water diffusion from the protein solution droplets to the dispersionmedium for some of the samples, the octanol or dodecanol in the initial emulsion was replaced by a water-miscible solvent as follows: wait for the particles to sediment on the bottom of the bottle; aspirate 8 mL octanol and replace it with 8 mL absolute ethanol (or other polymer; THF or THF-isopropanol 2:8).

[0108] Particles comprising ovalbumin encapsulated in PEA III AcBz were formed as follows. Afterwaiting for the protein particles to settle on the bottom (between 24 and 48 hours), 8 mL of the organic solvents was aspirated. 300 mg of PEA III AcBz was added to the correspondingvolume of solvent (see the solvents and targeted concentrations in Table 4) and stirred on amagnetic stirrer to dissolve the polymer (1 to 2 hours). 2 mL of this protein dispersion wasadded to 50 mL water while stirring on overhead stirrer with 4-bladed propeller stirrer (OS- 20S DLAB) at 600 RPM for 1 min. The resulting particles are then collected and dried overnight. Bradford assay of the antisolvent media (water) indicated negligible protein lossduring the particle formation process.

[0109] The collected particles are washed as follows. The particles were transferred to a centrifugetube and 1.5 mL ethanol was added. The sample was vortexed until the polymer dissolved(approximately 1 hour). The samples were then centrifuged for 5 min at 20000 x g. Thesupernatant containing dissolved polymer in ethanol was discarded and 1.5 mL of fresh ethanolwas added to the sediment. The washing procedure was then repeated (total of three times) to remove most of the polymer. The remaining particles were then separated and dried at 40 ºC. The resulting particles appeared similar to the PEA X-50 particles obtained above. Mostparticles were spherical and around 10-20 µm in diameter. In the PEA X-50 sample, somedented and broken particles were observed , while the PEA III AcBz sample showed a higher occurrence of aggregated particles.

[0110] The effect of varying the polymer solvent was investigated by varying the polymerconcentration and the polymer solvent. Encapsulation efficiency was calculated based on mass balance considerations, which included the measurement of the concentration of protein that had dissolved in the aqueous phase and how much of this protein is “lost” during the encapsulation of the particles. Samples from the antisolvent media and from the emulsion were taken and filtered through 450 nm CA syringe filters. The UV spectrum of the sample from400 to 200 nm was taken and the encapsulation efficiency is calculated through the specific absorption at 280 nm. For the systems with encapsulation efficiency > 90 %, no peaks wereobserved in the UV spectrum of the water antisolvent media and the limit of detection of the method was used to calculate encapsulation efficiency. The results are shown in Table 4.Table 4. Encapsulation efficiency of Ovalbumin- PEA AcBz antisolvent particles when varyingthe polymer solvent and polymer concentration Polymer Ovalbumin in Ovalbumin in Ovalbumin in THF: concentration, % EtOH, % THF, % isopropanol (8:2), % 5% PEA 66 51 Not measured10 % PEA >90 >90 >9015 % PEA >90 >90 Not measured

[0111] The final yield was determined by measuring the mass of particles after the washing step. First,the protein concentration in the initial stock solution was measured spectrophotometrically. By knowing the initial protein content in the emulsion and the amount of protein remaining after the washing process (which involved 10 washing cycles for the current particles), the percentage of the final yield was calculated (Table 5). Table 5. Particle yield of encapsulated protein particles Stock solution Encapsulated ovalbumin %, recovered Polymer Solvent (mg / mL)particles (mg)Ovalbumin EtOH2.4 28THF864.8 56THF: 2-propanol (8:2) 3.4 40Dissolution of PEA III AcBz particles formed by antisolvent method with washing in smallscale procedure

[0112] Particles were formed as described above at a polymer concentration of 15%, with ethanol asthe solvent, and washing three times to remove excess polymer. The obtained particles were transferred to a 10 mL jar and dissolution studies were performed.

[0113] The dissolution assay was performed in a phosphate buffer at pH 7.4 containing: 138 mMNaCl, 2.7 mM KCl, 10 mM Na2HPO4 and 0.1 % NaN3. Several droplets of pure phosphoric acid were used to adjust the pH of a 250 mL buffer solution to pH = 7.4, which introduced negligible dilution (< 1 %). The particles were measured on an analytical balance and placed in 10 mL glass bottle. Phosphate buffer at 37 °C was then added to the glass bottle in a volume of 10 mL. The bottles were placed on a vortex (Vortex Genie 2 mixer) and vortexed at speed 3.Aliquots of 1 mL were taken for analysis as a function of time. Each sample was filtered using 450 nm cellulose acetate (CA) syringe filters to remove undissolved material.

[0114] Samples for analysis were taken 2 weeks after the beginning of the experiment. The proteincontent in the sample was determined by the Bradford assay, described in the following paragraph. The protein amount found in the sample after 2 weeks was under the LOQ of thecalibration, which was equivalent to less than 5% released protein.

[0115] Bradford assay: 1 mL sample is mixed with 5 mL Bradford reagent. The sample was shakenand left for 2 minutes. The absorption is measure between 400 and 700 nm. When the dye is bound to a protein, its spectral properties in the visible range change. There is an increase in absorption at 595 nm and a decrease at 450 nm. The calibration curve is generated based onthe absorption ratio between these two wavelengths.

[0116] Bradford Reagent Recipe: 50 mg Coomassie blue was dissolved in 25 mL 95% ethanol. Next50 mL of 85% phosphoric acid were added. Next water was added to volume 500 mL. The reagent was filter through paper filter in a dark room and used within 3 days.

[0117] Microscopic observations of particles were performed throughout the assay. Substantialparticle aggregation was seen in each sample.

[0118] In an additional series of experiments, particles were prepared with ten washing steps. Thepolymer concentration and organic solvent used during the antisolvent encapsulation were varied to study their effect on protein particle dissolution. The results are summarized in Table 6. Only one of the prepared systems: ovalbumin-PEA AcBz, prepared in THF at 15% polymer concentration did show measurable ovalbumin concentration 9 days after the beginning of the experiment. For all the other systems, no peak was observed in the UV spectrum of the dissolution media, hence the LOQ of the analytical method was used to estimate the quantity of dissolved protein. Note that while LOQ was the same for all systems (as it originates from the ovalbumin calibration curve), the different initial mass of particles used in the dissolution assay leads to differences in the estimated fraction of dissolved protein.

[0119] The particles prepared in EtOH and THF were observed by optical microscopy at the 10th dayof the dissolution experiment. All samples looked very similar with large aggregates formed by the protein particles and no single particles. Table 6. Results from dissolution experiments with different Ovalbumin-PEA AcBz particles after 9 days dissolution. Ovalbumin in Ovalbumin in Ovalbumin in THF: 2- EtOH, % THF, % propanol (8:2), % 5% PEA AcBz < 4 < 6 -10% PEA AcBz < 3 < 3 < 615% PEA AcBz - 8.5 -PEA III AcBz particles formed by antisolvent method with washing – large scale procedure

[0120] Ovalbumin protein solution was prepared by sonication as follows. 1 mL of protein solutionwas added drop by drop to 100 mL n-octanol while homogenizing the emulsion for 30 sec with pulse sonicator (SKL-1500 II DN) at 1 s long pulses with 1 s power off at 20% power. The obtained emulsions were centrifuged for 12 min at 1500 x g. 80 mL octanol was replaced with 80 mL absolute ethanol or polymer solvent (THF, THF-isopropanol 2:8).

[0121] Ovalbumin protein solution was prepared by Ultra-Turrax® as follows. 1 mL of this proteinsolution was added to 100 mL n-octanol and homogenized on an Ultra-Turrax® for 1 min at13500 rpm. The obtained emulsions were centrifuged for 12 min at 1500 x g. 80 mL octanol was replaced with 80 mL absolute ethanol or other polymer solvent (THF, THF-isopropanol2:8).

[0122] Particles comprising ovalbumin encapsulated in PEA III AcBz were formed as follows usingprotein solution from either the sonication or Ultra-Turrax® method. The protein particles obtained as above were allowed to settle on the bottom (24 to 48 hours) and 80 mL of the organic solvents were aspirated. 2 g of PEA III AcBz was added and stirred on a magneticstirrer until the polymer dissolves (1 to 2 hours).20 mL of this protein dispersion was added to 500 mL water while stirring on an overhead stirrer at 600 RPM for 1 min. The resulting particles are then collected and dried overnight.

[0123] The particles are washed as follows. Half of the particles were transferred to a centrifuge tubeand 15 mL ethanol or THF is added. The sample was vortexed until the polymer dissolves. Thesamples are centrifuged at 5024 x g. The supernatant containing dissolved polymer in ethanolwas discarded and 15 ml of fresh ethanol or THF is added (this step is repeated ten times). Theorganic phase was then aspirated with a syringe and the particles were left to dry using compressed air while they are still in the centrifuge tubes.

[0124] Encapsulation efficiency was determined via UV-spectrophotometry and the final yield wasdetermined directly by measuring the mass of the dry encapsulated particles. The encapsulation efficiency with Ultra-Turrax® was estimated to be > 90%, as no protein peaks were found in the UV spectrum of the water antisolvent (Figure 17). As described in the previous section, the limit of quantification of the UV-Vis method was used to estimate the encapsulation efficiency in this case. However, protein was found in the sample prepared by Sonication, and the encapsulation efficiency was determined to be 80%. The emulsion prepared with Ultra-Turrax® was also superior to sonication in that more spherical and non-aggregated dropletswere obtained.

[0125] The final yield was determined by evaporating the organic solvent at the end of the ten cyclesof polymer washing and measuring the mass of the particles on an analytical scale. The Ultra- Turrax® was shown to be superior once again, this time in terms of yield, as 88% protein recovery was achieved compared to 69% for the sonication sample. All results, both pre- andpost-encapsulation, indicated that the Ultra-Turrax® is a superior emulsification technique compared to sonication, particularly for larger-scale applications. Dissolution of PEA III AcBz particles formed by antisolvent method with washing in large scaleUltra-Turrax® procedure

[0126] THF was chosen as a polymer solvent and the polymer concentration was set to 10%. Particleswere washed 10 times. Once obtained, dissolution studies were performed following the protocol described in the small scale procedure. The concentrations of the protein were measured with UV-spectrophotometer as also described in the small scale procedure. The protein was measured 4 and 7 days after the beginning of the dissolution. The release after 4 days was 12% and after 7 days, 13%, as shown in Table 7. Table 7 - Results from dissolution experiments in large scale procedureTime (days) Released proteinmg / mL %4 0.076 127 0.084 13

[0127] In both the small and large scale procedure, protein release from encapsulated particles wasvery low, regardless of the preparation protocol or solvent type, with a maximum of just 13% over a 7-day period. Therefore, it was concluded that the encapsulation process with washing successfully restricted rapid release. Further studies will be directed to reducing particleaggregation.

[0128] Those skilled in the art having knowledge of the present disclosure, will recognize that variouschanges can be made to these polymers and their applications, to attain these and otheradvantages, without departing from the scope of the present disclosure. As such, it should be understood that the features of the disclosure are susceptible to modifications and / or substitutions without departing from the scope of the invention. The specific embodimentsillustrated and described herein are for illustrative purposes only, and not limiting of the invention as set forth in the appended claims. EXEMPLARY DESCRIPTION OF CERTAIN EMBODIMENTS 1. A formulation for injection comprising:first particles comprising a water-soluble biotherapeutic encapsulated in a biodegradable polymer, wherein the water-soluble biotherapeutic is present at an amount of from 60 wt% to 90 wt%, based on the total weight of the first particles; wherein the biodegradable polymer comprises a random copolymer having a structure including n units of: m molar equivalents of a first residue having the formula , , , mis from 0 to 0.5, p is from 0.1 to 0.95, m+p is from 0.5 to 0.95, q is from 0.02 to 0.25,and x is from 0 to 0.25; nis from 5 to 300;if x > 0, the ratio q:x is from 9:1 to 1:9; R1is (C2-C20)alkylene; R3and R4are selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl, -CH2SH, -(CH2)2S(CH3), -CH2OH, -CH(OH)CH3, -(CH2)4NH3+, -(CH2)3NHC(=NH2+)NH2, -CH2COOH, -CH2-CO-NH2, -CH2CH2-CO-NH2, -CH2CH2COOH, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, H2N-(CH2)4-, Ph-CH2-, CH=C-CH2-, (CH3)2CH-, Ph-NH-, NH-(CH2)3-C-, or NH-CH=N-CH=C-CH2-, wherein R3 one or more by an oxygen atom (-O-), and / or optionally having 5 or 6 consecutive alkylene carbon atoms together form a respective 5- or 6-membered carbocyclic ring;R6is ; R8is (C3-C8)alkylene. The formulation of the previous exemplary embodiment, wherein, in the structure of thebiodegradable polymer, p is from 0.3 to 0.8 and m+p is from is from 0.6 to 0.9.The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, q is from 0.05 to 0.25. The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, x is 0. The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, x is from 0.02 to 0.25.The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, x is from 0.05 to 0.25. The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, q:x is from 5:1 to 1:9. The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, q:x is from 4:1 to 1:4. The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, m is 0. The formulation of any one of the previous exemplary embodiments, wherein the structureof the biodegradable polymer is further characterized by one or more of the following: mis from 0.1 to 0.5, p is from 0.1 to 0.75, m+p is from is from 0.5 to 0.8, q is from 0.2 to0.02, and x is from 0.02 to 0.2;R1is (C2-C10)alkylene; R3 and R4 are (C1-C6)alkyl or (CH3)2CH-CH2-;R5is (C2-C10)alkylene; R6is ; andR8is (C3-C6)alkylene. The formulation of any one of the previous exemplary embodiments, wherein the structureof the biodegradable polymer is further characterized by: m is 0.2-0.4, p is 0.3-0.6, q is 0.05-0.2, and x is 0.05-0.2;The formulation of any one of the previous exemplary embodiments, wherein the structureof the biodegradable polymer is further characterized by one or more of the following: m is 0.3, p is 0.45, q is 0.05-0.2, and x is 0.05-0.2; R1is octylene; R3and R4are isobutyl; R5is hexylene; R6is ; R8 is butylene.The formulation of any one of the previous exemplary embodiments, wherein thebiodegradable polymer has a glass transition temperature in the hydrated state of less than about 37 °C. The formulation of any one of the previous exemplary embodiments, wherein thebiodegradable polymer is amorphous. The formulation of any one of the previous exemplary embodiments, wherein thebiodegradable polymer has a water uptake of less than about 30 wt-% after two weeks inphosphate buffered saline (PBS) at 37 °C.The formulation of any one of the previous exemplary embodiments, wherein thebiodegradable polymer has a water uptake that remains within about + / -20%, over a period from 4 days to two weeks in phosphate buffered saline (PBS) at 37 °C.The formulation of any one of the previous exemplary embodiments, wherein the firstparticles further comprise a second biodegradable polymer.The formulation of any one of the previous exemplary embodiments, further comprisingunencapsulated water-soluble biotherapeutic.The formulation of any one of the previous exemplary embodiments, further comprisingunencapsulated biotherapeutic, wherein the unencapsulated biotherapeutic is present as particles.The formulation of any one of the previous exemplary embodiments, further comprisingunencapsulated water-soluble biotherapeutic, wherein the unencapsulated biotherapeutic isthe same composition as the water-soluble biotherapeutic in the first particles.The formulation of any one of the previous exemplary embodiments, wherein the water-soluble biotherapeutic is present at an amount of at least 65 wt%, more preferably at least 70 wt%, more preferably at least 75 wt%, more preferably at least 80 wt%, based on the total weight of the first particles.The formulation of any one of the previous exemplary embodiments, wherein the water-soluble biotherapeutic is present at an amount of at most 88 wt%, based on the total weightof the first particles.The formulation of any one of the previous exemplary embodiments, wherein the water-soluble biotherapeutic is present at an amount of at most 85 wt%, based on the total weight of the first particles. The formulation of any one of the previous exemplary embodiments, wherein the water-soluble biotherapeutic is a peptide, polypeptide, protein, nucleic acid, monoclonal antibody, or a mixture thereof.The formulation of any one of the previous exemplary embodiments, wherein the water-soluble biotherapeutic is a GLP-1 receptor agonist.The formulation of any one of the previous exemplary embodiments, wherein the water-soluble biotherapeutic is semaglutide or tirzepatide.The formulation of any one of the previous exemplary embodiments, wherein the averageparticle size (d50) of the first particles is from 50 to 500 µm.The formulation of any one of the previous exemplary embodiments, wherein less than20% of the water-soluble biotherapeutic is released from the first particles after twenty- four hours in phosphate buffer at pH 7.4 containing 138 mM NaCl, 2.7 mM KCl, 10 mMNa2HPO4 and 0.1 % NaN3 at 37 °C.The formulation of any one of the previous exemplary embodiments, wherein less than25% of the water-soluble biotherapeutic is released from the first particles after 7 days in phosphate buffer at pH 7.4 containing 138 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4and 0.1% NaN3 at 37 °C.A method for administering a biotherapeutic to a patient comprising the step of injectingthe formulation according to any one of the previous exemplary embodiments, wherein less than 20% of the water-soluble biotherapeutic is released from the first particles after twenty-four hours.A method for administering a biotherapeutic to a patient comprising the step of injectingthe formulation according to any one of the previous exemplary embodiments, wherein less than 25% of the water-soluble biotherapeutic is released from the first particles after 7 days.A method of forming particles comprising the steps of:a. providing encapsulated particles comprising a biodegradable polymerencapsulating a water-soluble biotherapeutic at a weight ratio of water-soluble biotherapeutic to biodegradable polymer of from 6:4 to 1:15; b. washing the encapsulated particles in a washing solvent, the washing solvent beingcapable of dissolving the biodegradable polymer, thereby forming first particles.A method of forming particles comprising the steps of:a. forming a first composition by dispersing particles of water-soluble biotherapeuticin a first solvent and dissolving a biodegradable polymer in the first solvent at aweight ratio of water-soluble biotherapeutic to biodegradable polymer of from 6:4 to 1:15, wherein the first solvent does not dissolve the water-soluble biotherapeutic;b. mixing the first composition with an antisolvent, wherein the antisolvent does notdissolve the biodegradable polymer and wherein the antisolvent is miscible with thefirst solvent, thereby obtaining encapsulated particles; c. separating the encapsulated particles from the antisolvent; andd. washing the encapsulated particles from step c in a washing solvent, the washingsolvent being the same as or different from the first solvent and being capable of dissolving the biodegradable polymer, thereby forming first particles.The method of forming particles of the previous exemplary embodiment, wherein the firstsolvent is water-miscible.The method of forming particles of the previous exemplary embodiment, wherein thewashing solvent is water-miscible.The method of forming particles of the previous exemplary embodiment, wherein thebiotherapeutic is not soluble in the washing solvent.The method of forming particles of the previous exemplary embodiment, wherein the firstsolvent and the washing solvent are selected from the group consisting of ethanol, methanol, butanol, tert-butanol, tetrahydrofuran, dimethyl sulfoxide, dimethylformamide, and mixtures thereof.The method of forming particles of any one of the previous exemplary embodiments,wherein the first solvent is ethanol.The method of forming particles of any one of the previous exemplary embodiments,wherein the washing solvent is ethanol.The method of forming particles of any one of the previous exemplary embodiments,wherein the antisolvent is selected from the group consisting of water, acetone, ethylacetate, and mixtures thereof.The method of forming particles of any one of the previous exemplary embodiments,wherein the antisolvent is water.The method of forming particles of any one of the previous exemplary embodiments,wherein the particles of water-soluble biotherapeutic have an average particle size (d50) offrom 1-200 µm, preferably from 10 to 100 µm, more preferably from 10 to 60 µmThe method of forming particles of any one of the previous exemplary embodiments,wherein the particles of water-soluble biotherapeutic have an average particle size (d50) of from 30-50 µm, from 10 to 20 µm, or from 1-5 µm.The method of forming particles of any one of the previous exemplary embodiments,wherein the weight ratio of water-soluble biotherapeutic to biodegradable polymer in step a is from 1:5 to 1:15.The method of forming particles of any one of the previous exemplary embodiments,wherein the step of washing further comprises washing the encapsulated particles until the surface of the encapsulated particles becomes translucent.The method of forming particles of any one of the previous exemplary embodiments,wherein the step of washing comprises contacting the washing solvent with the encapsulated particles from step c, optionally with agitation, to thereby form a supernatantcomprising washing solvent and biodegradable polymer, discarding the supernatant, andcollecting the encapsulated particles.The method of forming particles of any one of the previous exemplary embodiments,wherein the step of washing further comprises vortexing the encapsulated particles in thewashing solvent to form a first mixture, centrifuging the first mixture, discarding asupernatant formed thereby, and collecting the encapsulated particles.The method of forming particles of any one of the previous exemplary embodiments,wherein the step of washing is repeated or carried out until the concentration ofbiodegradable polymer in the supernatant is below 5 w / v %, more preferably below 4 w / v%, more preferably below 3 w / v %, more preferably below 2 w / v %, most preferably below 1 w / v %.The method of forming particles of the previous exemplary embodiment, wherein the stepof washing is repeated at least three times. The method of forming particles of any one of the previous exemplary embodiments,wherein the biodegradable polymer comprises a random copolymer having a structure including n units of: m molar equivalents of a first residue having the formula , , , m is from 0 to 0.5, p is from 0.1 to 0.95, m+p is from 0.5 to 0.95, q is from 0.02 to 0.25, and x is from 0 to 0.25; nis from 5 to 300;if x > 0, the ratio q:x is from 9:1 to 1:9; R1is (C2-C20)alkylene; R3and R4are selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl, -CH2SH, -(CH2)2S(CH3), -CH2OH, -CH(OH)CH3, -(CH2)4NH3+, -(CH2)3NHC(=NH2+)NH2, -CH2COOH, -CH2-CO-NH2, -CH2CH2-CO-NH2, -CH2CH2COOH, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, H2N-(CH2)4-, Ph-CH2-, CH=C-CH2-, (CH3)2CH-, Ph-NH-, NH-(CH2)3-C-, or NH-CH=N-CH=C-CH2-, wherein R3and R4are the same or different; R5is (C2-C20)alkylene, optionally having one or more alkylene carbon atoms (-C-) replaced by an oxygen atom (-O-), and / or optionally having 5 or 6 consecutive alkylene carbon atoms together form a respective 5- or 6-membered carbocyclic ring;R6is ; R8is (C3-C8)alkylene. The method of forming particles of any one of the previous exemplary embodiments,wherein in the structure of the biodegradable polymer, p is from 0.3 to 0.8 and m+p is from is from 0.6 to 0.9. The method of forming particles of any one of the previous exemplary embodiments,wherein in the structure of the biodegradable polymer, q is from 0.05 to 0.25.The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, x is 0.The formulation of any one of the previous exemplary embodiments, wherein, in thestructure of the biodegradable polymer, x is from 0.02 to 0.25.The method of forming particles of any one of the previous exemplary embodiments,wherein in the structure of the biodegradable polymer, x is from 0.05 to 0.25.The method of forming particles of any one of the previous exemplary embodiments,wherein in the structure of the biodegradable polymer, q:x is from 5:1 to 1:9.The method of forming particles of any one of the previous exemplary embodiments,wherein in the structure of the biodegradable polymer, q:x is from 4:1 to 1:4.The method of forming particles of any one of the previous exemplary embodiments,wherein in the structure of the biodegradable polymer, m is 0.The method of forming particles of any one of the previous exemplary embodiments,wherein the structure of the biodegradable polymer is further characterized by one or more of the following: m is from 0.1 to 0.5, p is from 0.1 to 0.75, m+p is from is from 0.5 to 0.8, q is from 0.2 to 0.02, and x is from 0.02 to 0.2; R1is (C2-C10)alkylene;R3 and R4 are (C1-C6)alkyl or (CH3)2CH-CH2-;R5is (C2-C10)alkylene; R6is

[0003] R7 is Ph-(C1-C6)alkylene; andR8is (C3-C6)alkylene. The method of forming particles of any one of the previous exemplary embodiments,wherein the structure of the biodegradable polymer is further characterized by: m is 0.2- 0.4, p is 0.3-0.6, q is 0.05-0.2, and x is 0.05-0.2; The method of forming particles of any one of the previous exemplary embodiments,wherein the structure of the biodegradable polymer is further characterized by one or more of the following: m is 0.3, p is 0.45, q is 0.05-0.2, and x is 0.05-0.2; R1is octylene; R3and R4are isobutyl; R5is hexylene; R6is ; R8 is butylene.The method of forming particles of any one of the previous exemplary embodiments,wherein the biodegradable polymer has a glass transition temperature in the hydrated state of less than about 37 °C. The method of forming particles of any one of the previous exemplary embodiments,wherein the biodegradable polymer is amorphous.The method of forming particles of any one of the previous exemplary embodiments,wherein the biodegradable polymer has a water uptake of less than about 30 wt-% after twoweeks in phosphate buffered saline (PBS) at 37 °C.The method of forming particles of any one of the previous exemplary embodiments,wherein the biodegradable polymer has a water uptake that remains within about + / -20%,over a period from 4 days to two weeks in phosphate buffered saline (PBS) at 37 °C.The method of forming particles of any one of the previous exemplary embodiments,wherein the first particles further comprise a second biodegradable polymer.The method of forming particles of any one of the previous exemplary embodiments,wherein the water-soluble biotherapeutic is present at an amount of at least 65 wt%, more preferably at least 70 wt%, more preferably at least 75 wt%, more preferably at least 80 wt%, based on the total weight of the first particles.The method of forming particles of any one of the previous exemplary embodiments,wherein the water-soluble biotherapeutic is present at an amount of at most 88 wt%, based on the total weight of the first particles.The method of forming particles of any one of the previous exemplary embodiments,wherein the water-soluble biotherapeutic is present at an amount of at most 85 wt%, based on the total weight of the first particles.The method of forming particles of any one of the previous exemplary embodiments,wherein the water-soluble biotherapeutic is a GLP-1 receptor agonist.The method of forming particles of any one of the previous exemplary embodiments,wherein the water-soluble biotherapeutic is semaglutide or tirzepatide.The method of forming particles of any one of the previous exemplary embodiments,wherein the average particle size (d50) of the first particles is from 50 to 500 µm.The method of forming particles of any one of the previous exemplary embodiments,wherein less than 20% of the water-soluble biotherapeutic is released from the first particlesafter twenty-four hours in phosphate buffer at pH 7.4 containing 138 mM NaCl, 2.7 mMKCl, 10 mM Na2HPO4 and 0.1 % NaN3 at 37 °C.The method of forming particles of any one of the previous exemplary embodiments,wherein less than 25% of the water-soluble biotherapeutic is released from the first particles after 7 days in phosphate buffer at pH 7.4 containing 138 mM NaCl, 2.7 mM KCl, 10 mMNa2HPO4 and 0.1 % NaN3 at 37 °C.Particles comprising a biodegradable polymer encapsulating a water-solublebiotherapeutic, wherein the particles are formed by the method of forming particles of any one of the previous exemplary embodiments.The formulation according to any one of the previous exemplary embodiments, whereinthe first particles are formed by the method of forming particles of any one of the previous exemplary embodiments.An implant comprising first particles dispersed within a degradable or non-degradablepolymer, wherein the first particles comprise the first particles present in any one of theprevious exemplary embodiments or formed by the method of forming particles of any oneof the previous exemplary embodiments, and wherein the implant is solid or semi-solid.Microparticles each comprising a shell comprising a degradable encapsulating polymer, theshell encapsulating a plurality of first particles.The microparticles of the previous exemplary embodiment, wherein the microparticles areformed from a solid-oil-in-water emulsion comprising the step of suspending the first particles in an encapsulating polymer-containing oil phase.The microparticles of any one of the previous exemplary embodiments, wherein themicroparticles have an average particles size (d50) of from 40 to 250 µmA process of forming microparticles comprising the step of suspending first particles in anencapsulating polymer-containing oil phase.An injectable formulation comprising the microparticles of any one of the previousexemplary embodiments and the first particles of any one of the previous exemplary embodiments.The method, microparticles, process, or injectable formulation of any one of the previousexemplary embodiments, wherein the resulting particles have any of the characteristics described in any one of the previous exemplary embodiments.

Claims

CLAIMS:

1. A formulation for injection comprising:first particles comprising a water-soluble biotherapeutic encapsulated in a biodegradable polymer, wherein the water-soluble biotherapeutic is present at an amount of from 60 wt% to 90 wt%, based on the total weight of the first particles; wherein the biodegradable polymer comprises a random copolymer having a structure including n units of: m molar equivalents of a first residue having the formula ,,,m is from 0 to 0.5, p is from 0.1 to 0.95, m+p is from 0.5 to 0.95, q is from 0.02 to 0.25, and x is from 0 to 0.25; nis from 5 to 300;if x > 0, the ratio q:x is from 9:1 to 1:9;R1is (C2-C20)alkylene; R3and R4are selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl, -CH2SH, -(CH2)2S(CH3), -CH2OH, -CH(OH)CH3, -(CH2)4NH3+, -(CH2)3NHC(=NH2+)NH2, -CH2COOH, -CH2-CO-NH2, -CH2CH2-CO-NH2, -CH2CH2COOH, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, H2N-(CH2)4-, Ph-CH2-, CH=C-CH2-, (CH3)2CH-, Ph-NH-, NH-(CH2)3-C-, or NH-CH=N-CH=C-CH2-, wherein R3and R4are the same or different; R5is (C2-C20)alkylene, optionally having one or more alkylene carbon atoms (-C-) replaced by an oxygen atom (-O-), and / or optionally having 5 or 6 consecutive alkylene carbon atoms together form a respective 5- or 6-membered carbocyclic ring;R6is ;R8is (C3-C8)alkylene.

2. The formulation of the previous claim, wherein, in the structure of the biodegradablepolymer, p is from 0.3 to 0.8 and m+p is from is from 0.6 to 0.9.

3. The formulation of any one of the previous claims, wherein, in the structure of thebiodegradable polymer, q is from 0.05 to 0.25.

4. The formulation of any one of the previous claims, wherein, in the structure of thebiodegradable polymer, x is from 0.02 to 0.25.

5. The formulation of any one of the previous claims, wherein, in the structure of thebiodegradable polymer, q:x is from 4:1 to 1:4.

6. The formulation of any one of the previous claims, wherein the structure of thebiodegradable polymer is further characterized by one or more of the following: m is from 0.1 to 0.5, p is from 0.1 to 0.75, m+p is from is from 0.5 to 0.8, q is from 0.2 to 0.02, and x is from 0.02 to 0.2; R1is (C2-C10)alkylene; R3 and R4 are (C1-C6)alkyl or (CH3)2CH-CH2-;R5is (C2-C10)alkylene; R6is ;andR8is (C3-C6)alkylene.

7. The formulation of any one of the previous claims, further comprising unencapsulatedwater-soluble biotherapeutic.

8. The formulation of any one of the previous claims, wherein the water-solublebiotherapeutic is present at an amount of at least 65 wt%, more preferably at least 70 wt%, more preferably at least 75 wt%, more preferably at least 80 wt%, based on the total weight of the first particles, and wherein the water-soluble biotherapeutic is present at an amount of at most 88 wt%, based on the total weight of the first particles.

9. The formulation of any one of the previous claims, wherein the water-solublebiotherapeutic is a GLP-1 receptor agonist.

10. The formulation of any one of the previous claims, wherein the average particle size (d50)of the first particles is from 50 to 500 µm.

11. A method of forming particles comprising the steps of:a. providing encapsulated particles comprising a biodegradable polymerencapsulating a water-soluble biotherapeutic at a weight ratio of water-soluble biotherapeutic to biodegradable polymer of from 6:4 to 1:15; b. washing the encapsulated particles in a washing solvent, the washing solvent beingcapable of dissolving the biodegradable polymer, thereby forming first particles.

12. A method of forming particles comprising the steps of:a. forming a first composition by dispersing particles of water-soluble biotherapeuticin a first solvent and dissolving a biodegradable polymer in the first solvent at a weight ratio of water-soluble biotherapeutic to biodegradable polymer of from 6:4 to 1:15, wherein the first solvent does not dissolve the water-soluble biotherapeutic; b. mixing the first composition with an antisolvent, wherein the antisolvent does notdissolve the biodegradable polymer and wherein the antisolvent is miscible with the first solvent, thereby obtaining encapsulated particles; c. separating the encapsulated particles from the antisolvent; andd. washing the encapsulated particles from step c in a washing solvent, the washingsolvent being the same as or different from the first solvent and being capable of dissolving the biodegradable polymer, thereby forming first particles.

13. The method of forming particles of the previous claim, wherein the first solvent is water-miscible and wherein the washing solvent is water-miscible.

14. The method of forming particles of any one of the previous claims, wherein thebiodegradable polymer comprises a random copolymer having a structure including n units of: m molar equivalents of a first residue having the formula, p q g ,,m is from 0 to 0.5, p is from 0.1 to 0.95, m+p is from 0.5 to 0.95, q is from 0.02 to 0.25, and x is from 0 to 0.25; nis from 5 to 300;if x > 0, the ratio q:x is from 9:1 to 1:9; R1is (C2-C20)alkylene; R3and R4are selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl, -CH2SH, -(CH2)2S(CH3), -CH2OH, -CH(OH)CH3,-(CH2)4NH3+, -(CH2)3NHC(=NH2+)NH2, -CH2COOH, -CH2-CO-NH2, -CH2CH2-CO-NH2, -CH2CH2COOH, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, H2N-(CH2)4-, Ph-CH2-, CH=C-CH2-, (CH3)2CH-, Ph-NH-, NH-(CH2)3-C-, or NH-CH=N-CH=C-CH2-, wherein R3by an oxygen atom (-O-), and / or optionally having 5 or 6 consecutive alkylene carbon atoms together form a respective 5- or 6-membered carbocyclic ring;R6is ;R8is (C3-C8)alkylene.

15. An implant comprising first particles dispersed within a degradable or non-degradablepolymer, the first particles comprising a water-soluble biotherapeutic encapsulated in a biodegradable polymer, wherein the water-soluble biotherapeutic is present at an amount of from 60 wt% to 90 wt%, based on the total weight of the first particles; wherein the biodegradable polymer comprises a random copolymer having a structure including n units of: m molar equivalents of a first residue having the formula ,, q q g,m is from 0 to 0.5, p is from 0.1 to 0.95, m+p is from 0.5 to 0.95, q is from 0.02 to 0.25, and x is from 0 to 0.25; nis from 5 to 300;if x > 0, the ratio q:x is from 9:1 to 1:9; R1is (C2-C20)alkylene; R3and R4are selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl, -CH2SH, -(CH2)2S(CH3), -CH2OH, -CH(OH)CH3, - (CH2)4NH3+, -(CH2)3NHC(=NH2+)NH2, -CH2COOH, -CH2-CO-NH2, -CH2CH2-CO-NH2, -CH2CH2COOH, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, H2N-(CH2)4-, Ph-CH2-, CH=C-CH2-, (CH3)2CH-, Ph-NH-, NH-(CH2)3-C-, or NH-CH=N-CH=C-CH2-, wherein R3and R4are theR5is (C2-C20)alkylene, optionally having one or more alkylene carbon atoms (-C-) replaced by an oxygen atom (-O-), and / or optionally having 5 or 6 consecutive alkylene carbon atoms together form a respective 5- or 6-membered carbocyclic ring;R6is ;R8is (C3-C8)alkylene, wherein the implant is solid or semi-solid.

16. Microparticles each comprising a shell comprising a degradable encapsulating polymer, theshell encapsulating a plurality of first particles, and the first particles comprising a water-soluble biotherapeutic encapsulated in a biodegradable polymer, wherein the water-soluble biotherapeutic is present at an amount of from 60 wt% to 90 wt%, based on the total weight of the first particles; wherein the biodegradable polymer comprises a random copolymer having a structure including n units of: m molar equivalents of a first residue having the formula ,,d mula ,m is from 0 to 0.5, p is from 0.1 to 0.95, m+p is from 0.5 to 0.95, q is from 0.02 to 0.25, and x is from 0 to 0.25; nis from 5 to 300;if x > 0, the ratio q:x is from 9:1 to 1:9; R1is (C2-C20)alkylene; R3and R4are selected from the group consisting of hydrogen, (C1-C6)alkyl, (C2-C6)alkenyl, (C2-C6)alkynyl, (C6-C10)aryl, -CH2SH, -(CH2)2S(CH3), -CH2OH, -CH(OH)CH3, -(CH2)4NH3+, -(CH2)3NHC(=NH2+)NH2, -CH2COOH, -CH2-CO-NH2, -CH2CH2-CO-NH2, -CH2CH2COOH, CH3-CH2-CH(CH3)-, (CH3)2CH-CH2-, H2N-(CH2)4-, Ph-CH2-, CH=C-CH2-, (CH3)2CH-, Ph-NH-, NH-(CH2)3-C-, or NH-CH=N-CH=C-CH2-, wherein R3one or more by an oxygen atom (-O-), and / or optionally having 5 or 6 consecutive alkylene carbon atoms together form a respective 5- or 6-membered carbocyclic ring;R6is; R8is (C3-C8)alkylene.

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