Formulations for the extended release of antibodies or large proteins

Abstract

The present disclosure relates to a dosage form for the extended release of an antibody or large protein, the extended release dosage form comprising a biodegradable multiblock copolymer matrix and providing extended release of the antibody or large protein over a desired period of time.

Description

[Background technology]

[0001] I. Background Antibodies and antigen-binding fragments, such as monoclonal antibodies, bispecific antibodies, trispecific antibodies, antigen-binding fragments (Fabs), antibody drug conjugates (ADCs), and other large molecular weight biologics, such as Fc fusion proteins, enzymes, growth factors, and clotting factors, are becoming increasingly important in the development of more specific drugs for more effective drug therapy with fewer side effects. Rapid advances in this field, along with large-scale production of these compounds by recombinant DNA technology, among other techniques, have led to a significant increase in the number of antibody, antigen-binding fragment, and other large protein-derived drugs in development and already on the market. Unfortunately, the development of antibodies, antigen-binding fragments, and other large therapeutic proteins has far outpaced the ability to deliver these compounds systemically or locally using simple and effective delivery systems.

[0002] Biodegradable polymers have attracted increasing attention over the past decade for use in long-acting parenteral controlled-release systems for either systemic or site-specific drug delivery. Biodegradable controlled-release formulations can significantly improve the pharmacokinetics of therapeutic compounds. This is particularly relevant in the treatment of chronic diseases and for compounds with narrow therapeutic windows, as systemic plasma concentrations can be reduced while simultaneously reducing undesirable side effects. Furthermore, many new bioactive compounds have short half-lives and require frequent injections to achieve therapeutically effective plasma levels. Patient compliance and high costs associated with frequent dosing regimens for parenterally administered bioactive compounds have fueled interest in biodegradable parenteral sustained-release dosage forms.

[0003] Copolymers of lactic acid and glycolic acid, also known as poly(D,L-lactic acid) (PLA) and PLGA copolymers, are the most widely applied biodegradable polymers for use in parenteral sustained-release depot formulations. PLGA and PLA (collectively referred to as (PL(G)A) copolymers) have been successfully used for the development of sustained-release depot formulations for small molecules such as risperidone, and therapeutic peptides such as leuprolide, goserelin, or octreotide.

[0004] However, PL(G)A polymers have several drawbacks that limit their use and make them unsuitable for the delivery of protein therapeutics. First, PL(G)A copolymers are relatively hydrophobic polymers, which do not provide an optimal environment for encapsulated proteins. Proteins can adsorb to the polymer, resulting in slow and incomplete release, protein unfolding, and / or aggregation. Second, the ability to engineer the release of large protein molecules is limited because diffusion of such compounds through the relatively rigid and non-swelling PL(G)A matrix is ​​negligible. Therefore, the release of proteins from PL(G)A copolymers relies on diffusion through pores present in the matrix and on the degradation of the matrix. Typically, the encapsulated protein remains entrapped in the polymer matrix until the polymer matrix is ​​degraded to such an extent that it loses its integrity or dissolves, resulting in the biphasic or triphasic degradation-dependent release profile typically obtained with PL(G)A-based depot formulations. Finally, during degradation of PL(G)A copolymers, acidic moieties are formed and accumulate in the rigid and non-swelling PL(G)A matrix, resulting in the formation of an acidic microenvironment within the polymer matrix with an in situ pH that can be as low as pH 1-2. Under such acidic conditions, encapsulated proteins may form aggregates, resulting in incomplete protein release. Furthermore, the low pH can have adverse effects on the structural integrity and biological activity of encapsulated peptides or proteins, resulting in reduced therapeutic efficacy and increased immunogenicity. Chemical modifications of proteins and peptides, such as acylation and adduct formation, have been reported for PL(G)A-based sustained-release formulations.

[0005] Biodegradable phase-separated segmented multiblock copolymers (SynBiosys™, InnoCore Technologies BV, Groningen, The Netherlands), disclosed in WO 2012 / 005594 and WO 2013 / 015685, have been developed for the delivery of structurally intact and bioactive peptides and proteins for extended periods of up to 3-6 months (Stankovic et al., Eur. J. Pharm. Sci. 2013, 49(4), 578-587; Teekamp et al., Int. J. Pharm. 2017, 534(1-2), 229-236; Teekamp et al., J. Controlled Release 2018, 269(10), 258-265; Scheiner et al., ACS Omega 2019, 4(7), 11481-11492). SynBiosys multiblock copolymers are typically composed of two distinct blocks in which commonly used monomers, including D,L-lactide, L-lactide, glycolide, ε-caprolactone, p-dioxanone, and / or poly(ethylene glycol) (PEG), are copolymerized into a low-molecular-weight polymer (pre-polymer), which is then linked with a diisocyanate, typically 1,4-butane diisocyanate. The use of two chemically and physically distinct prepolymer blocks, such as a hydrophilic amorphous block and a compositionally crystalline block, results in phase-separated segmented multiblock copolymers that provide a mechanism for the extended release of drugs, including peptides and proteins. The hydrophilic amorphous block typically contains a high content of poly(ethylene glycol) (PEG), which causes the multiblock copolymer to swell under aqueous conditions. The hydrophobic crystalline block acts as a physical crosslink.Examples of peptides and proteins that have been successfully encapsulated in SynBiosys-based formulations and released structurally intact therefrom include goserelin (1269 g / mol), exenatide (4187 g / mol), recombinant insulin (5.8 kDa), insulin-like growth factor (7.6 kDa), lysozyme (14.7 kDa), carbonic anhydrase (29 kDa), bovine serum albumin (66.5 kDa), and hepatocyte growth factor (69 kDa), among others.

[0006] However, antibodies and antigen-binding fragments, monoclonal antibodies, bispecific antibodies, trispecific antibodies, antigen-binding fragments (Fabs), antibody drug conjugates (ADCs) and other high molecular weight biologics, such as Fc-fusion proteins, have molecular weights of up to or even above 200 kDa and can have complex three-dimensional structures.

[0007] Unfortunately, the delivery of monoclonal antibodies is burdensome. Monoclonal antibodies are typically administered via low-concentration intravenous (IV) infusion, which can take hours to deliver the full dose, causing patient discomfort and increasing the risk of infection. Furthermore, the patient must make frequent hospital visits to receive the infusion. For example, bevacizumab (Avastin™, Genentech, Inc.), a humanized anti-VEGF monoclonal IgG1 antibody with a molecular weight of 149 kDa used in the treatment of various cancers, is administered as a 25 mg / ml solution via infusion over a maximum of 90 minutes every two weeks.

[0008] There is a long-felt need to replace this inconvenient and burdensome administration procedure with more patient-friendly subcutaneous administration. The use of highly concentrated (and viscous) antibody solutions has enabled subcutaneous delivery of high doses of antibodies and antigen-binding fragments, but has not resolved the need for frequent administration. There remains a need in the art for extended release dosage forms that can be administered subcutaneously and that provide extended release of structurally intact and biologically active antibodies and antigen-binding fragments. The present invention fulfills this need. Summary of the Invention [Problem to be solved by the invention]

[0009] II. Overview The present disclosure addresses a long felt need in the art for parenteral extended release formulations of large proteins and antibodies. [Means for solving the problem]

[0010] Thus, in one aspect, the invention provides a formulation for extended release of an antibody (or antigen-binding fragment thereof), comprising: (a) an antibody (or an antigen-binding fragment thereof); (b) Biodegradable multiblock copolymer matrix Including, wherein the antibody (or the antigen-binding fragment thereof) is present in the multi-block copolymer matrix; wherein the biodegradable multi-block copolymer comprises one or more biodegradable, phase-separated, thermoplastic multi-block copolymers comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; where: The multi-block copolymer has a T of about 37° C. or less under physiological conditions. gand T of about 50°C to about 110°C m having; the segments are linked by a multifunctional chain extender; the segments are randomly distributed along the polymer chain; and the prepolymer (B) segment comprises an XYX triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by about 7 or more p-dioxanone monomer units; The present invention is directed to the above dosage form.

[0011] In a further aspect, the present invention provides a dosage form for the extended release of a protein of about 70 kDa or greater, comprising: (a) proteins of approximately 70 kDa or larger; (b) Biodegradable multiblock copolymer matrix Including, wherein the protein is present in the multi-block copolymer matrix; wherein the biodegradable multi-block copolymer comprises one or more biodegradable, phase-separated, thermoplastic multi-block copolymers comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; where: The multi-block copolymer has a T of about 37° C. or less under physiological conditions. g and T of about 50°C to about 110°C m having; the segments are linked by a multifunctional chain extender; the segments are randomly distributed along the polymer chain; and the prepolymer (B) segment comprises an XYX triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by about 7 or more p-dioxanone monomer units; The present invention is directed to the above dosage form.

[0012] In some embodiments, the dosage forms exhibit a release profile of about 1 week to about 2 months. In other embodiments, the dosage forms described herein can have an extended release profile of about 1 week to about 6 months.

[0013] The multi-block copolymer may comprise, or alternatively consist of, poly(ethylene glycol) (PEG) (or a polymer block containing PEG) and one or more other polymer blocks. The polymer matrix may be in the form of a microsphere or microparticle, but may also be included in other application forms, such as nanospheres or nanoparticles, rods, implants, coatings, films, sheets, tubes, membranes, meshes, fibers, plugs, scaffolds, or (in situ forming) gels.

[0014] The extended-release antibody and large protein formulations described herein may be useful in a variety of treatments, including, but not limited to, the treatment of cancer, neurodegenerative diseases, autoimmune diseases, cardiovascular diseases, transplant rejection, inflammatory diseases, and viral infections. Accordingly, a further aspect of the present disclosure relates to methods of administering the extended-release antibody formulations described herein and methods of treatment using the extended-release antibody formulations described herein. Such methods include a variety of treatments, including, but not limited to, the treatment of cancer, neurodegenerative diseases, autoimmune diseases, neurodegenerative diseases, transplant rejection, inflammatory diseases, and viral infections.

[0015] In another embodiment, methods are included that administer the described extended-release antibody formulations according to a regimen that achieves or approaches in vivo plasma levels of the antibody within the therapeutic range.

[0016] The invention described and claimed herein has many attributes and embodiments, including but not limited to those shown, described, or referred to in this Summary. Not intended to be comprehensive, the invention described and claimed herein is not limited to or by the features or embodiments specified in this Summary, which are included by way of example only and not by way of limitation. Additional embodiments may be disclosed in the following drawing descriptions and detailed description. [Brief explanation of the drawings]

[0017] III. BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1A] Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / O-based microencapsulation process. [Figure 1B] Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / O-based microencapsulation process. [Figure 1C] Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / O-based microencapsulation process. [Figure 1D]Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / O-based microencapsulation process. [Figure 2A] Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / W-based microencapsulation process. [Figure 2B] Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / W-based microencapsulation process. [Figure 2C] Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / W-based microencapsulation process. [Figure 2D] Scanning electron microscope images of mAbX-loaded microspheres with a 5% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / W-based microencapsulation process. [Figure 3]Cumulative release of mAbX from microspheres loaded with mAbX with a target loading of 5% mAbX. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / O-based microencapsulation process. [Figure 4] Cumulative release of mAbX from microspheres loaded with mAbX with a target loading of 5% mAbX. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 50:50, 25:75, and 0:100 w / w) and prepared by a W / O / W-based microencapsulation process. [Figure 5] Cumulative release of mAbX from microspheres loaded with mAbX with a 10% mAbX target loading. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 90:10, and 80:20 w / w) and prepared by a W / O / W-based microencapsulation process. [Figure 6] Cumulative release of mAbX from microspheres loaded with mAbX with a target loading of approximately 15% mAbX. The microspheres were composed of 80:20 (AMD17042) and 90:10 w / w (AMD17038) blends of 50CP30C40-LL40 and 50CP10C20-LL40, prepared by a W / O / W-based microencapsulation process using a polymer solution concentration of 15 wt%. [Figure 7A] Scanning electron microscope images of mAbX-loaded microspheres with 9.3% (AMD17022) and 19% (AMD17023) mAbX loading. The microspheres were composed of a 90:10 w / w blend of 50CP30C40-LL40 and 50CP10C20-LL40 and were prepared by a W / O / O-based microencapsulation process. [Figure 7B]Scanning electron microscope images of mAbX-loaded microspheres with 9.3% (AMD17022) and 19% (AMD17023) mAbX loading. The microspheres were composed of a 90:10 w / w blend of 50CP30C40-LL40 and 50CP10C20-LL40 and were prepared by a W / O / O-based microencapsulation process. [Figure 8] Cumulative release of mAbX from mAbX-loaded microspheres with 9.3% (AMD17022) and 19% (AMD17023) mAbX loading. The microspheres were composed of a 90:10 w / w blend of 50CP30C40-LL40 and 50CP10C20-LL40 and were prepared by a W / O / O-based microencapsulation process. [Figure 9A] Fluorescence spectroscopy (FLS) analysis of in vitro released mAbX showing the emission spectra of in vitro released mAbX samples taken at various time points (A). [Figure 9B] Emission maxima of in vitro released mAbX samples taken at various time points compared to native mAbX (B). [Figure 9C] Equation (C) used to calculate the percentage of correctly folded mAbX. [Figure 10A] Circular dichroism (CD) analysis of in vitro released mAbX showing molar ellipticity as a function of wavelength for in vitro released mAbX samples taken at various time points (A). [Figure 10B] Molar ellipticity at the 218 nm signal peak of in vitro released mAbX samples taken at various time points compared to native mAbX and fully unfolded mAbX (B). [Figure 10C] Equation (C) used to calculate the percentage of correctly folded mAbX. [Figure 11]In vitro release kinetics of mAbX microspheres showing the concentrations of total mAbX and intact mAbX in in vitro release samples taken at various time points as measured by SEC-UPLC and ELISA, and the percentage of folded mAbX as measured by fluorescence spectroscopy and circular dichroism. [Figure 12] In vivo pharmacokinetics of mAbX (PK study I) after subcutaneous administration of mAbX MSP (1, 4, and 8 mg of mAbX) in female NMRI mice. Blood samples were collected at the indicated time points, and serum concentrations of mAbX were measured by ELISA. [Figure 13] In vivo pharmacokinetics of mAbX (PK study II) after subcutaneous administration of mAbX-loaded microspheres (1, 2, 4, and 8 mg of mAbX) in female A431 xenograft-bearing NMRI nude mice. Blood samples were collected at the indicated time points, and serum concentrations of mAbX were measured by ELISA. [Figure 14A] Subcutaneous tumor growth as a function of time after subcutaneous administration of mAbX-loaded microspheres (1, 2, 4, 8 mg of mAbX) in NMRI nude mice bearing female A431 xenografts ((a) and (b) zoom). Tumor volume was determined by caliper measurement twice a week. [Figure 14B] Subcutaneous tumor growth as a function of time after subcutaneous administration of mAbX-loaded microspheres (1, 2, 4, 8 mg of mAbX) in NMRI nude mice bearing female A431 xenografts ((a) and (b) zoom). Tumor volume was determined by caliper measurement twice a week. [Figure 15] Cumulative release of mAb02 from microspheres loaded with mAb02 with a target loading of 19%. The microspheres were composed of blends of 50CP30C40-LL40 and 50CP10C20-LL40 (100:0, 90:10, 80:20, 70:30, 50:50, 25:75, and 100:0 w / w) and prepared by a W / O / O-based microencapsulation process. [Figure 16] Percentage of correctly folded mAb2 during in vitro release of mAb2 from mAb2-loaded microspheres as measured by fluorescence spectroscopy. A-F represent mAb2-loaded microspheres composed of different polymer compositions. [Figure 17] In vitro erosion of representative polymer-only microspheres composed of [poly(ε-caprolactone)-PEG-poly(ε-caprolactone)]-b-[poly(L-lactide)] (50CP10C20-LL40) and [poly(ε-caprolactone)-PEG-poly(ε-caprolactone)]-b-[poly(p-dioxanone)] (50CP10C20-D25, 50CP15C20-D25, and 20CP30C40-D23), expressed as percent mass remaining as a function of time. [Figure 18] Cumulative release of mAb02 from microspheres loaded with mAb02 at 20% mAb02 target loading. The microspheres were composed of blends of 50CP30C40-D25 and 50CP10C20-D25 with blend ratios of 100:0, 90:10, 80:20, 70:30, 50:50, 25:75, and 0:100 w / w and were prepared by a W / O / O-based microencapsulation process. [Figure 19-1] In vitro release of mAb02 from mAb02 microspheres composed of a blend of 50CP30C40-D25 and 50CP10C20-D25, showing the total and intact mAb02 concentrations (as measured by SEC-UPLC) and the percentage of correctly folded mAb02 (as measured by fluorescence spectroscopy) of in vitro release samples taken at various time points. A-G represent mAb02-loaded microspheres composed of different polymer compositions. [Figure 19-2]In vitro release of mAb02 from mAb02 microspheres composed of a blend of 50CP30C40-D25 and 50CP10C20-D25, showing the total and intact mAb02 concentrations (as measured by SEC-UPLC) and the percentage of correctly folded mAb02 (as measured by fluorescence spectroscopy) of in vitro release samples taken at various time points. A-G represent mAb02-loaded microspheres composed of different polymer compositions. [Figure 19-3] Figure 1. In vitro release of mAb02 from mAb02 microspheres composed of a blend of 50CP30C40-D25 and 50CP10C20-D25, showing the total and intact mAb02 concentrations (as measured by SEC-UPLC) and the percentage of correctly folded mAb02 (as measured by fluorescence spectroscopy) of in vitro release samples taken at various time points. A-G represent mAb02-loaded microspheres composed of different polymer compositions. DETAILED DESCRIPTION OF THE INVENTION

[0018] IV. Detailed Description The present invention is directed to an extended-release dosage form of a large therapeutic protein or antibody. There is a long-felt need in the art for such a dosage form, as current dosage forms of large therapeutic proteins or antibodies are intravenous infusions or solutions for intramuscular or subcutaneous injection. Furthermore, currently available antibodies and therapeutic proteins are typically administered as aqueous solutions. Depending on the half-life of the therapeutic protein or antibody, serum levels decline relatively rapidly after administration, requiring patients to undergo frequent infusions or injections to maintain desired plasma levels of the therapeutic protein or antibody throughout the treatment period. Frequent infusions or injections can be inconvenient, thereby resulting in poor patient compliance and leading patients and providers to choose less effective treatments.

[0019] The extended-release formulation comprises an antibody (e.g., a monoclonal antibody (mAb), a bispecific antibody, or a trispecific antibody), an antigen-binding fragment thereof (Fab), an antibody-drug conjugate (ADC), or a high-molecular-weight protein, such as an Fc-fusion protein, an enzyme, a growth factor, or a coagulation factor, encapsulated in a multiblock copolymer matrix. The multiblock copolymer may comprise, or alternatively consist of, poly(ethylene glycol) (PEG) (or a polymer block containing PEG) and one or more other polymers.

[0020] Embodiments according to the present disclosure will be described more fully below. However, aspects of the present disclosure may be embodied in different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and will fully convey the scope of the invention to those skilled in the art. The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0021] The term "phase-separated," as used herein, is meant to refer to a system, particularly a copolymer, constructed from two or more different prepolymers, at least two of which are (partially) incompatible with one another at or below body temperature (under physiological conditions, e.g., in the human body). Thus, the prepolymers do not form a homogeneous mixture when combined, i.e., as a physical mixture of the prepolymers, nor when the prepolymers are combined in a single chemical species as a "chemical mixture," i.e., a copolymer.

[0022] The term "prepolymer," as used herein, is meant to refer to polymer segments that are randomly linked by polyfunctional chain extenders and together constitute the multi-block copolymer. Each prepolymer can be obtained by polymerization of suitable monomers, which are thus the chemical units of each prepolymer. The desired properties of the prepolymer, and consequently of the multi-block copolymer, depend on the required T m or T g The composition and molecular weight (particularly, the number average molecular weight M n ) can be controlled by selecting the prepolymer.

[0023] The term "multiblock," as used herein, is meant to refer to the presence of at least two distinct prepolymer segments in a polymer chain. Additional prepolymer segments may optionally be present.

[0024] The terms "block" and "segment," as used herein, are meant to refer to distinct regions in a multi-block copolymer. These terms are used interchangeably herein.

[0025] The term "thermoplastic" as used herein is meant to refer to the non-crosslinked nature of the multi-block copolymer. When heated, a thermoplastic polymer becomes fluid, while when (re)cooled, it becomes solid. Thermoplastic polymers are soluble in suitable solvents.

[0026] The term "hydrolyzable," as used herein, is meant to refer to the ability of a chemical bond to be broken upon reaction with water. Hydrolyzable groups include ester, carbonate, phosphazene, amide, and urethane groups. Under physiological conditions, only ester, carbonate, and phosphazene groups react with water on a reasonable time scale.

[0027] The term "multifunctional chain extender," as used herein, is meant to refer to the presence of at least two reactive groups on the chain extender that chemically link reactive prepolymers, thereby forming a multi-block copolymer.

[0028] The term "random multi-block copolymer," as used herein, is meant to refer to a multi-block copolymer in which the distinct segments are randomly distributed along the polymer chain.

[0029] The term "water-soluble polymer," as used herein, is meant to refer to a polymer that has good solubility in aqueous media, preferably water, under physiological conditions. When this polymer is copolymerized with a more hydrophobic moiety, the resulting copolymer becomes swellable in water. The water-soluble polymer can be derived from a diol, diamine, or diacid. The diol or diacid is preferably used to initiate the ring-opening polymerization of the cyclic monomer.

[0030] The term "swellability," as used herein, is meant to refer to the uptake of water by a polymer. The swell ratio can be calculated by dividing the mass of the copolymer swollen with water by the mass of the dry copolymer.

[0031] The term "semi-crystalline," as used herein, is meant to refer to a morphology of the multi-block copolymer that includes two distinct phases: an amorphous phase and a crystalline phase. Preferably, the multi-block copolymer is composed of an amorphous phase and a crystalline phase.

[0032] A1. Antibody The term "antibody," as used herein, refers to an immunoglobulin molecule typically composed of two identical pairs of polypeptide chains, each pair consisting of one "heavy" and one "light" chain. Human light chains are classified as kappa and lambda. The heavy chains include various classes: mu, delta, gamma, alpha, or epsilon. These classes define the antibody's isotype, e.g., IgM, IgD, IgG, IgA, and IgE, respectively. These classes are important for the function of the antibody and help regulate the immune response. Both the heavy and light chains consist of a variable region and a constant region. The constant region of the heavy chain is significantly larger than the constant region of the light chain, explaining the nomenclature of the heavy and light chains. Each heavy chain variable region (VH) and light chain variable region (VL) contains framework regions (FR) interspersed with complementary determining regions (CDRs). The variable regions are composed of four FRs and three CDRs, arranged from amino to carboxyl terminus as follows: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The light and heavy chain variable regions together form the antibody binding site, which defines epitope specificity.

[0033] The term "antibody," as used herein, includes murine antibodies, humanized antibodies, deimmunized human antibodies, and chimeric antibodies, as well as antibodies in multimeric form, e.g., dimers, trimers, or higher multimers of monomeric antibodies. Antibodies also include monospecific, bispecific, or multispecific antibodies, and any other modified configuration of the immunoglobulin molecule that contains an antigen recognition site of the required specificity. The term antibody also includes antibodies linked or conjugated to non-antibody moieties. Furthermore, the term "antibody" is not limited by any particular method of producing the antibody. For example, it includes monoclonal antibodies (mAbs), recombinant antibodies, and polyclonal antibodies.

[0034] As used herein, antigen-binding fragments include Fab, F(ab'), F(ab')2, single-chain antibodies (scFv), and bivalent single-chain antibodies. In some instances, the term "antibody" as used herein is meant to also include antigen-binding fragments thereof. Thus, whenever the term "antibody" is used herein, it may be replaced by "antigen-binding fragment of an antibody." In particular, antigen-binding fragments encompass those fragments that contain at least two paired domains.

[0035] The antibodies disclosed herein may further comprise a moiety to increase the in vivo half-life of a molecule, such as, but not limited to, poly(ethylene glycol) (PEG), human serum albumin, glycosylation groups, fatty acids, and dextran. Such additional moieties may be conjugated to or otherwise combined with the antibody using methods well known in the art. In some embodiments, the antibodies disclosed herein may be coupled to an active compound, such as a toxin. Furthermore, the disclosed antibodies or antigen-binding fragments may be coupled to a label, such as a fluorescent protein, a chemical label, an organic dye, a colored particle, or an enzyme. The antibodies disclosed herein may be coupled to a drug to form an antibody-drug conjugate (ADC).

[0036] Preferably, the antibodies disclosed herein are humanized antibodies. The term "humanized antibody" refers to an antibody containing some or all of the CDRs from a non-human animal antibody, but the framework and constant regions of the antibody contain amino acid residues derived from human antibody sequences. Humanized antibodies are typically produced by grafting CDRs from a mouse antibody into human framework sequences, followed by back-substitution of certain human framework residues for the corresponding mouse residues from the source antibody. The term "deimmunized antibody" also typically refers to an antibody of non-human origin from which one or more epitopes have been removed in one or more variable regions, typically to reduce immunogenicity, that have a strong tendency to constitute human T-cell and / or B-cell epitopes. The amino acid sequences of the epitopes can be completely or partially removed. Typically, however, the amino acid sequence is altered by substituting one or more other amino acids with one or more of the amino acids that make up the epitope, thereby changing the amino acid sequence to one that does not make up a human T-cell and / or B-cell epitope, optionally with an amino acid that is present at one or more corresponding positions in the corresponding human variable heavy or variable light chain.

[0037] In some embodiments, the antibodies disclosed herein are human antibodies. The term "human antibody" refers to an antibody that consists entirely of amino acid sequences of human immunoglobulin sequences. Human antibodies can be prepared by various methods known in the art.

[0038] In some embodiments, the antibody is an isolated antibody. The term "isolated," as used herein, refers to material that is substantially or essentially free from components that normally accompany it in nature.

[0039] The antibodies disclosed herein can be produced by any method known to those of skill in the art. In some embodiments, antibodies can be prepared by immunizing animals and collecting polyclonal antibodies or using standard hybridoma techniques (Kohler et al., Nature 1975, 256(5517), 495-497). Antibodies can also be prepared using recombinant techniques, for example, by transfecting host cells with nucleic acids expressing the respective heavy and light chains. Suitable cell lines are known to those of skill in the art and include Chinese hamster ovary cells, NS0 cells, or PER-C6 cells. The transfected cells are cultured and the antibody is recovered from the culture medium. The antibody can be purified from the medium, but preferably, the antibody is affinity purified. Alternatively, the antibody can be synthetically produced. Methods for preparing bispecific antibodies are also known in the art (see, for example, WO 2013 / 157954).

[0040] The antibody preferably has a molecular weight of about 70 kDa or more, e.g., about 75 kDa or more, about 80 kDa or more, about 85 kDa or more, about 90 kDa or more, or about 100 kDa or more. Typically, the antibody or antigen-binding fragment thereof can have a molecular weight of about 200 kDa or less, e.g., about 190 kDa or less, about 180 kDa or less, about 170 kDa or less, about 160 kDa or less, or about 150 kDa or less.

[0041] A2. Giant proteins As used herein, a large protein refers to a protein having a molecular weight of about 70 kDa or more, e.g., about 75 kDa or more, about 80 kDa or more, about 85 kDa or more, about 90 kDa or more, or about 100 kDa or more. Typically, the large protein can have a molecular weight of about 200 kDa or less, e.g., about 190 kDa or less, about 180 kDa or less, about 170 kDa or less, about 160 kDa or less, or about 150 kDa or less. Preferably, the large protein is therapeutically active.

[0042] Some examples of large proteins that can be used in the dosage forms of the present invention include Fc-fusion proteins, antibody drug conjugates (ADCs), full-length immunoglobulins, clotting factors, growth factors, hormones, cytokines, enzymes, and the like.

[0043] B. Copolymer microspheres As used herein, the term "microsphere" refers to a spherical or spheroidal particle having a diameter of about 999 μm or less, which can be loaded with one or more therapeutic agents for drug delivery. In the context of this disclosure, a microsphere typically has a diameter of 1 μm or more. A microsphere can typically have a diameter of about 100 μm or less. Nevertheless, in some cases, the microsphere can have a diameter of about 100 μm or more, e.g., about 200 μm or more, about 300 μm or more, or even up to about 500 μm (e.g., microparticles for tissue engineering applications to construct scaffolds).

[0044] Aspects of the present disclosure relate to microspheres formed by copolymers. In some embodiments, these copolymers can be selected based on the copolymer or portion thereof having one or more of the following characteristics: (i) the polymer forms a matrix into which the antibody or protein can be incorporated, (ii) the polymer protects the antibody or protein from the environment in which it is stored and / or to which the microsphere is administered (e.g., temperature stability), (iii) the polymer is hydrophilic, (iv) the polymer allows diffusion of the antibody or protein, (v) the polymer will accommodate the hydrodynamic radius of the antibody or protein, (vi) the polymer is biodegradable, and / or (vii) the polymer degrades without affecting the purity of the antibody or protein.

[0045] The microspheres may release from less than about 3% to about 40% of the antibody or protein contained therein, based on the total weight of the microsphere, within about 24 hours.

[0046] It is surprising that stable extended-release formulations can be made utilizing the polymers described herein because large therapeutic proteins, particularly antibodies, are known to degrade (i) as a result of the harsh conditions (e.g., shear forces, oil-water interfaces, pH changes, etc.) used during the production of extended-release formulations for antibodies or large therapeutic proteins, (ii) at elevated temperatures, such as those used during hot-melt extrusion of extended-release implants or at body temperature, or (iii) in response to pH changes, such as those caused by acidic degradation products formed as a result of hydrolysis of the polymer. Thus, prior to the present invention, it was thought impossible to achieve long-term, extended in vivo release of antibodies or large proteins using biodegradable extended-release formulations comprising the polymers described herein. The present invention details the surprising discovery that the biodegradable polymers described herein can be used to form stable extended-release formulations of antibodies or large therapeutic proteins, wherein the structural integrity and biological activity of the antibodies or proteins is maintained during manufacture and storage of the extended-release formulations, and wherein the antibodies or proteins are released from the extended-release formulations after administration to the body while remaining structurally intact, properly folded, and biologically active in vivo.

[0047] Microspheres containing antibodies or large therapeutic proteins can be prepared by techniques known to those skilled in the art, including, but not limited to, solvent distillation and spray drying techniques. In some embodiments, the microspheres are formed with a water to polymer ratio of about 0.1 to about 1.0, for example, but not limited to, about 0.5 to about 1.0, about 0.55 to about 1.0, about 0.6 to about 1.0, about 0.65 to about 1.0, about 0.7 to about 1.0, about 0.75 to about 1.0, about 0.8 to about 1.0, about 0.85 to about 1.0, about 0.9 to about 1.0, or about 0.95 to about 1.0. In some embodiments, the water to polymer ratio of the microspheres is about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, or about 1.0. These water to polymer ratios can be adjusted to achieve a particular release profile based on the concentration of the antibody or protein and / or the polymer used to form the microspheres (Bos et al., Pharmaceutical Technology, October 2001, 110-120).

[0048] In some embodiments, the microspheres have a diameter of at least about 1 μm, at least about 2 μm, at least about 5 μm, at least about 10 μm, at least about 20 μm, at least about 30 μm, at least about 40 μm, at least about 50 μm, up to about 50 μm, up to about 60 μm, up to about 70 μm, up to about 80 μm, up to about 90 μm, up to about 100 μm, up to about 150 μm, up to about 200 μm, up to about 300 μm, or up to about 500 μm. In some embodiments, the microspheres have a diameter of about 15 μm to about 200 μm, about 20 μm to about 150 μm, about 25 μm to about 100 μm, about 30 μm to about 80 μm, or about 40 μm to about 100 μm.

[0049] In some embodiments, the concentration of the aqueous solution of antibody or protein used to prepare loaded microspheres is about 100 mg / ml or more, about 125 mg / ml or more, about 150 mg / ml or more, about 175 mg / ml or more, about 200 mg / ml or more, about 225 mg / ml or more, or about 250 mg / ml or more. The concentration of the aqueous solution of antibody or protein used to prepare loaded microspheres can be about 500 mg / ml or less, about 450 mg / ml or less, about 400 mg / ml or less, about 350 mg / ml or less, or about 300 mg / ml or less.

[0050] The microspheres can be homogeneous or monolithic, in which the antibody or protein is dispersed throughout the polymer matrix. The microspheres can also be reservoir-type, in which the antibody or protein is mononuclear or polynuclear and surrounded by polymer.

[0051] The microspheres can be prepared by techniques known to those skilled in the art, including but not limited to coacervation, solvent extraction / distillation, spray drying and spray freeze drying techniques. For example, the microspheres can be prepared by solvent extraction / distillation, which comprises dissolving the multiblock copolymer in an organic solvent such as dichloromethane and emulsifying the multiblock copolymer solution in an aqueous phase containing an emulsifier such as polyvinyl alcohol (as described, inter alia, in Okada, Adv. Drug Del. Rev. 1997, 28(1), 43-70).

[0052] The characteristics of the microspheres thus formed, such as particle size, porosity, and loading, vary depending on parameters such as the viscosity or concentration of the aqueous polyvinyl alcohol phase, the concentration of the multi-block copolymer solution, the ratio of the primary emulsion to the polyvinyl alcohol phase, and the stirring speed.

[0053] When the microspheres are formed by a spray-drying process, a low concentration of the multi-block copolymer is used in the organic solvent, such as dichloromethane, at about 0.5% to about 5%, in one embodiment about 2%, by weight of the total solution. Spray-drying generally results in the formation of porous, irregularly shaped particles.

[0054] Once the microspheres are formed, antibodies or large therapeutic proteins are encapsulated in the microspheres or microparticles. Generally, when the solvent extraction / distillation technique is used, the antibody or protein is first dissolved in a solution of the multiblock copolymer in an organic solvent, such as dichloromethane or ethyl acetate. The organic solution is then emulsified in an aqueous polyvinyl alcohol solution to produce an oil-in-water (O / W) emulsion. The organic solvent is then extracted into the aqueous phase and distilled to solidify the microspheres.

[0055] Generally, when the solvent distillation technique is used, an aqueous solution of the antibody or protein is first emulsified in a solution of the multiblock copolymer in an organic solvent, such as dichloromethane. This primary emulsion is then subsequently emulsified in an aqueous polyvinyl alcohol solution to produce a water-in-oil-in-water (W / O / W) emulsion. The organic solvent, such as dichloromethane or ethyl acetate, is then extracted in a manner similar to the O / W process route, solidifying the microspheres. Alternatively, a water-soluble agent can be directly dispersed in a solution of the multiblock copolymer in an organic solvent. The resulting dispersion is then subsequently emulsified in an aqueous solution containing a surfactant, such as polyvinyl alcohol, to produce a solid-in-oil-in-water (S / O / W) emulsion. The organic solvent is then extracted in a manner similar to the O / W process route, solidifying the microspheres.

[0056] The water-in-oil-in-oil (W / O / O) or solid-in-oil-in-oil (S / O / O) emulsification routes offer interesting alternatives for obtaining microspheres with sufficient encapsulation efficiency. In the W / O / O process, the antibody or protein is dissolved in an aqueous solution and emulsified with a solution of the polymer in an organic solvent, such as dichloromethane or ethyl acetate, similar to the W / O / W process. Next, a polymer precipitant, such as silicon oil, is slowly added under stirring to form embryonic microparticles, which are then poured into heptane or hexane to extract the silicone oil and organic solvent and solidify the microparticles. The microparticles can be collected by vacuum filtration, rinsed with additional solvent, and dried under vacuum. In the S / O / O emulsification route, the antibody or protein is dispersed as a solid powder in a solution of the polymer in an organic solvent, such as dichloromethane or ethyl acetate, similar to the S / O / W process. Next, a polymeric precipitant, such as silicon oil, is then slowly added under stirring to form embryonic microparticles, which are then poured into heptane or hexane to extract the silicone oil and dichloromethane and solidify the microparticles.

[0057] Stabilizers may be added to the aqueous solution of the antibody or antigen-binding fragment thereof to prevent loss of activity during processing into microspheres. Examples of such stabilizers are polyvinyl alcohol, Tween™ / polysorbate, human serum albumin, gelatin, and carbohydrates such as trehalose, inulin, and sucrose.

[0058] When the spray drying technique is used, an aqueous solution of the antibody or antigen-binding fragment thereof is emulsified in a solution of the copolymer in an organic solvent, such as dichloromethane, as described above, and the water-in-oil emulsion is then spray dried using a spray dryer.

[0059] C. Multiblock Copolymers The copolymer may be a block copolymer and may optionally comprise, or alternatively consist of, poly(ethylene glycol) (PEG) and one or more other polymers.

[0060] Exemplary polymers that can be utilized in the extended-release dosage forms of the present invention include SynBiosys™ polymers manufactured by Innocore Pharmaceuticals. InnoCore's SynBiosys™ technology provides a platform of bioabsorbable polymers specifically designed to function as drug delivery systems. These polymers are composed of D,L-lactide, L-lactide, glycolide, ε-caprolactone, p-dioxanone, and / or poly(ethylene glycol), and these monomers are used in several products, including bioimplants, drug delivery products, and combination products approved for human use. SynBiosys™ polymers are described in WO 2013 / 015685, the entire contents of which are incorporated herein by reference. Further exemplary polymers that can be utilized in the extended release dosage forms of the present invention include the multi-block copolymers described in non-prepublished European Patent Application Publication No. 19200879.5, which is also incorporated herein by reference in its entirety.

[0061] The form and properties under physiological conditions (i.e., in the body) may differ from those under ambient conditions (dry, room temperature). g and T m As used herein, refers to the corresponding value of a material when applied in vivo; i.e., when in equilibrium with an atmosphere saturated with water vapor and at body temperature. This can be simulated in vitro by equilibrating the material with an atmosphere saturated with water and then performing DSC measurements.

[0062] The characteristics of the polymer matrix, such as the rate of controlled release, degradation, swelling and strength, can be precisely controlled by the appropriate combination of the two copolymer segments.

[0063] The multi-block copolymers described herein are generally linear. However, it is also possible to prepare the copolymers in branched form. These non-linear copolymers can be obtained by using multifunctional chain extenders with three or more functional groups, such as trifunctional chain extenders, e.g., triisocyanates. Branched copolymers can exhibit improved creep properties.

[0064] The multi-block copolymers used in the present invention have a (soft) prepolymer (A) block and a (hard) prepolymer (B) block. The prepolymer (B) block can be based on poly(lactide) or poly(p-dioxanone), which are discussed separately below.

[0065] The morphology of the multi-block copolymer depends on the environmental conditions: DSC (differential scanning calorimetry) measurements can be performed under inert (dry) conditions, and the results can be used to determine the thermal properties of the dry material. However, the morphology and properties under physiological conditions (i.e., in the body) can differ from those under ambient conditions (dry, room temperature). The transition temperature, T g and T m It should be understood that, as used herein, refers to the corresponding value of a material when applied in vivo; i.e., when in equilibrium with an atmosphere saturated with water vapor and at body temperature. This can be simulated in vitro by equilibrating the material with an atmosphere saturated with water and then performing the DSC measurement.

[0066] The physicochemical properties (e.g., degradation, swelling, and thermal properties) of the multi-block copolymer can be easily adjusted by changing the types of monomers in the soft prepolymer (A) segment and the hard prepolymer (B) segment, their chain lengths, and chain ratios, as well as by selecting the type and amount of chain extender.

[0067] The biodegradable multi-block copolymer comprises a biodegradable, phase-separated, thermoplastic multi-block copolymer comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment, wherein: Under physiological conditions, the multi-block copolymer has a T of about 37°C or less. g and T of about 50°C to about 110°C m having; the segments are linked by a multifunctional chain extender; the segments are randomly distributed along the polymer chain; and The prepolymer (B) segment comprises an XYX triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by about 7 or more p-dioxanone monomer units.

[0068] The inventors have surprisingly found that these multiblock copolymers having crystalline poly(p-dioxanone) building blocks result in dosage forms that exhibit a more rapid in vitro degradation profile when compared with the multiblock copolymers having crystalline poly(lactide) building blocks. Multiblock copolymers having crystalline poly(lactide) building blocks have a degradation time of approximately 3 to 4 years. For the majority of sustained-release drug delivery formulations, this degradation time is undesirably long because it can lead to polymer accumulation upon repeated injections, potentially causing long-term tolerance issues. On the other hand, the multiblock copolymers having crystalline poly(p-dioxanone) building blocks exhibit a reduced in vitro degradation time of approximately 0.5 to 1.5 years, depending on the duration of release. At the same time, the degradation products of the multiblock copolymers result in little or no degradation of the antibody or protein. Thus, these bioactive compounds and their functionality remain intact (or remain largely intact).

[0069] The multi-block copolymer has a T of about 37° C. or less under physiological conditions. g This means that under physiological conditions, the T g This can be achieved by using a prepolymer (A) having a T of about 30° C. or less under physiological conditions. g For example, a T of about 25°C or less, about 15°C or less, or about 5°C or less g , can have.

[0070] The prepolymer (A) segment is derived from a prepolymer (A), which is typically completely amorphous under physiological (body) conditions.

[0071] Prepolymer (A) can be prepared, for example, by ring-opening polymerization. Thus, prepolymer (A) can be a hydrolyzable copolymer prepared by ring-opening polymerization initiated by a diol or diacid compound. The diol compound can be an aliphatic diol or a low molecular weight polyether, such as poly(ethylene glycol). The polyether can be part of prepolymer (A) by using it as an initiator, and can be further mixed with prepolymer (A) to form additional hydrophilic segments. Prepolymer (A) can comprise the reaction product of ester-forming monomers selected from diols, dicarboxylic acids, and hydroxycarboxylic acids. Prepolymer (A) can comprise the reaction product of cyclic and / or acyclic monomers. Exemplary cyclic monomers include glycolide, L-lactide, D-lactide, D,L-lactide, ε-caprolactone, δ-valerolactone, trimethylene carbonate, tetramethylene carbonate, 1,5-dioxepan-2-one, 1,4-dioxan-2-one (p-dioxanone), and / or cyclic anhydrides such as oxepane-2,7-dione. In one embodiment, ε-caprolactone is used.

[0072] When the prepolymer (A) contains poly(D,L-lactide), the L / D ratio of the lactide can be away from 1 (can be other than 50 / 50). For example, an L / D ratio of 85 / 15 to 15 / 85 will give a completely amorphous homopolymer. Furthermore, if one isomer (L or D) is in excess of the other, the T of poly(D,L-lactide) can be increased. g It is known to increase

[0073] Furthermore, the prepolymer (A) can be based on (or be a mixture of) condensed (acyclic) monomers, such as hydroxy acids (e.g., lactic acid, glycolic acid, hydroxybutyric acid), diacids (e.g., glutaric acid, adipic acid, succinic acid or sebacic acid) and diols, such as ethylene glycol, diethylene glycol, 1,4-butanediol or 1,6-hexanediol, which form ester and / or anhydride hydrolyzable moieties.

[0074] At least a portion of the prepolymer (A) segments, for example, about 30% or more, about 40% to about 95%, about 50% to about 90%, or about 60% to about 85% of the total weight of the prepolymer (A), can be derived from a water-soluble polymer.

[0075] The water-soluble polymer can include, for example, one or more selected from the group consisting of polyethers, such as poly(ethylene glycol) (PEG), poly(tetramethylene oxide) (PTMO), poly(propylene glycol) (PPG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(vinyl caprolactam), poly(hydroxyethyl methacrylate) (poly-(HEMA)), poly(phosphazenes), poly(orthoesters), poly(orthoester amides), or copolymers of any of these polymers. Preferably, the water-soluble polymer includes one or more selected from the group consisting of poly(ethylene glycol), poly(tetramethylene oxide), poly(propylene glycol), poly(vinyl alcohol), poly(vinyl pyrrolidone), and poly(vinyl caprolactam). More preferably, the water-soluble polymer includes or is poly(ethylene glycol).

[0076] Some non-limiting examples of suitable prepolymer (A) segments include poly(ε-caprolactone)-co-PEG-co-poly(ε-caprolactone), poly(D,L-lactide)-co-PEG-co-poly(D,L-lactide), poly(glycolide)-co-PEG-co-poly(glycolide), and poly(p-dioxanone)-co-PEG-co-poly(p-dioxanone).

[0077] Further, the prepolymer (A) segment comprises a copolymer of any of the above-mentioned monomers on both sides of the water-soluble polymer. Some non-limiting examples of such prepolymer (A) segments include [poly(ε-caprolactone-co-D,L-lactide)]-co-PEG-co-[poly(ε-caprolactone-co-D,L-lactide)], [poly(ε-caprolactone-co-glycolide)]-co-PEG-co-[poly(ε-caprolactone-co-glycolide)], [poly(ε-caprolactone-co-p-dioxanone)]-co-PEG-co-[poly(ε-caprolactone-co-p-dioxanone)], [poly(D,L-lactide-co-glycolide)]-co-PEG-co-[poly(D,L-lactide-co-glycolide)], [poly [D,L-lactide-co-p-dioxanone)]-co-PEG-co-[poly(D,L-lactide-co-p-dioxanone)], and [poly(glycolide-co-p-dioxanone)]-co-PEG-co-[poly(glycolide-co-p-dioxanone)].

[0078] Prepolymer (A) can further contain p-dioxanone. The introduction of p-dioxanone monomer into the prepolymer (A) segment can introduce additional crystallinity into the multiblock copolymer. The content of such p-dioxanone monomer in prepolymer (A) can be about 80% or less, for example, about 60% or less, about 50% or less, about 40% or less, about 30% or less, about 20% or less, about 10% or less, or about 5% or less, of the total weight of prepolymer (A). The content of p-dioxanone monomer in prepolymer (A) can be about 0.1% or more, for example, about 1% or more, or about 2% or more, of the total weight of prepolymer (A).

[0079] The prepolymer (A) segment has a number average molecular weight M of about 500 g / mole or more, e.g., about 1000 g / mole or more, about 1500 g / mole or more, or about 2000 g / mole or more. n Typically, the prepolymer (A) segment has a number average molecular weight M of about 10,000 g / mole or less, for example, about 9,000 g / mole or less, or about 8,000 g / mole or less. n It has.

[0080] The content of the prepolymer (A) segment in the copolymer can be about 5% to about 95%, for example, about 10% to about 90%, about 30% to about 75%, or about 50% to about 70%, based on the total weight of the multi-block copolymer.

[0081] The multi-block copolymer has a T of about 50°C to about 110°C, for example, about 60°C to about 110°C, about 60°C to about 100°C, about 70°C to about 100°C, or about 70°C to about 90°C under physiological conditions. m This is due to the prepolymer (B) segment. The prepolymer (B) has a T m The prepolymer (B) may have a T of about 0° C. or less, for example, about −5° C. or less, about −10° C. or less, about −15° C. or less, or about −20° C. or less. g may have.

[0082] The prepolymer (B) segment contains about 70% or more of poly(p-dioxanone) based on the total weight of the prepolymer (B) segment. The prepolymer (B) segment may contain about 80% or more, for example, about 85% or more, about 90% or more, or about 95% or more of poly(p-dioxanone) based on the total weight of the prepolymer (B) segment. The prepolymer (B) segment is based on a prepolymer consisting of poly(p-dioxanone). The phase-separated amorphous phase of the multiblock copolymer is mainly composed of soft prepolymer (A) segments.

[0083] Apart from poly(p-dioxanone), the prepolymer (B) segment may contain further monomer units, such as ε-caprolactone and / or δ-valerolactone.

[0084] The prepolymer (B) segment comprises an XYX triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment. The block length of the poly(p-dioxanone) segment X, expressed in terms of p-dioxanone monomer units, is about 7 or greater. Suitably, the block length of the poly(p-dioxanone) segment X can be about 7 to about 35, e.g., about 8 to about 30, about 9 to about 25, about 10 to about 20, or about 12 to about 15.

[0085] If the block length of the prepolymer (B) segment is too short, then the enthalpy of fusion is relatively low, and the crystallization of the polymer matrix during extraction with dichloromethane is slow and incomplete. This, in turn, leads to slow and incomplete hardening of the microspheres, which makes them sticky and causes the microspheres to aggregate and smear during production, resulting in a microsphere dry powder with a very wide particle size distribution and / or poor powder flowability. Furthermore, the slow and incomplete crystallization due to the small block length of the prepolymer (B) segment results in a significant loss of protein and / or antibody or antigen-binding fragment thereof from the microspheres during the extraction process, resulting in poor encapsulation and low content of protein and / or antibody or antigen-binding fragment thereof in the resulting microspheres. Furthermore, slow and incomplete crystallization due to the small block length of the prepolymer (B) segment results in an unstable product since further crystallization can occur during storage, thereby altering the critical properties (e.g., release rate) of the product.

[0086] Therefore, the minimum length of the crystalline prepolymer (B) segment plays an important role in obtaining a multiblock copolymer that combines good product stability and good processability. Suitable microspheres cannot be made using multiblock copolymers in which the prepolymer (B) segment comprises an XYX triblock copolymer, where X is composed of a short poly(p-dioxanone) block, because the short poly(p-dioxanone) block does not crystallize well and / or crystallizes very slowly. Such incompletely crystallized polymers are unstable during storage because further crystallization may occur. This, in turn, changes the critical properties of the polymer. Furthermore, short prepolymer (B) segments result in viscous polymers, which can cause difficulties during processing, such as aggregation and fusion of microspheres with each other during extraction / evaporation processes.

[0087] The polymerization initiator Y in the XYX triblock copolymer can be suitably a diol, such as an aliphatic diol having from about 2 to about 8 carbon atoms. Examples of suitable aliphatic diols for use as the polymerization initiator Y include ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 1,3-butanediol, 2,3-butanediol, diethylene glycol, dipropylene glycol, triethylene glycol, poly(ethylene glycol), 1,5-pentanediol, 1,6-hexanediol, neopentyl glycol, hydrogenated bisphenol A, and glycerol. Preferred polymerization initiators include ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 1,3-butanediol, 2,3-butanediol, 1,5-pentanediol, and 1,6-hexanediol. More preferred polymerization initiators include ethylene glycol, 1,4-butanediol, and 1,6-hexanediol.

[0088] The prepolymer (B) segment has a number average molecular weight M of about 1300 g / mol or greater, e.g., about 1500 g / mol or greater, about 2000 g / mol or greater, about 2200 g / mol or greater, or about 2500 g / mol or greater. n The number average molecular weight M of the prepolymer (B) segment may be n can be about 7200 g / mol or less, e.g., about 5000 g / mol or less, about 4500 g / mol or less, about 4000 g / mol or less, or about 3200 g / mol or less.

[0089] The prepolymer (B) segment has a weight average molecular weight M of about 1800 g / mol or greater, e.g., about 2100 g / mol or greater, about 2600 g / mol or greater, or about 3000 g / mol or greater. w The weight average molecular weight M of the prepolymer (B) segment may be w can be about 10080 g / mol or less, e.g., about 7000 g / mol or less, about 6300 g / mol or less, about 5600 g / mol or less, or about 4200 g / mol or less.

[0090] The prepolymer (B) segment has a molecular weight distribution M of about 1.0 or greater, for example, about 1.1 or greater, about 1.2 or greater, about 1.3 or greater, or about 1.4 or greater. w / M n The molecular weight distribution of the prepolymer (B) segment is typically about 3.0 or less, for example, about 2.0 or less, about 1.8 or less, about 1.6 or less, about 1.5 or less, or about 1.4 or less.

[0091] Prepolymer (B) is approximately 1.1 g / cm 3 or more, for example, about 1.15 g / cm 3 or more, or about 1.2 g / cm 3 The density of the prepolymer (B) may be about 1.5 g / cm or more (measured according to ASTM D1505). 3 For example, about 1.45 g / cm 3 or less, or about 1.4 g / cm 3 It can be the following:

[0092] The content of the prepolymer (B) segment in the copolymer can be about 5% to about 95% of the total weight of the multi-block copolymer, for example, about 10% to about 90%, about 25% to about 70%, or about 30% to about 50%. Such a content generally results in a desired material with good physical properties (e.g., swelling) and degradation characteristics at the temperature of application (i.e., about 37°C for medical applications).

[0093] The multifunctional chain extender can be a difunctional aliphatic chain extender, preferably a diisocyanate, such as 1,4-butane diisocyanate or 1,6-hexane diisocyanate. Selecting the type and amount of chain extender is a way to customize the polymer properties. For example, the chain extender can act as a softener or it can affect the degree of phase separation.

[0094] In an embodiment, the biodegradable multi-block copolymer is a [poly(ε-caprolactone)-co-poly(ethylene glycol)-co-poly(ε-caprolactone)]-b-[poly(p-dioxanone)] multi-block copolymer.

[0095] The multi-block copolymers having crystalline poly(p-dioxanone) building blocks also have the structure [(R 1 R 2 n R 3 ) q ] r [(R 4 p R 5 R 6 p )] s where R 1 and R 3 is independent,

[0096] [ka] and any combination thereof; R 2 teeth,

[0097] [ka] and R 4 and R 6 are respectively,

[0098] [ka] and Repeat R 2 The number of moieties, n, is from about 4 to about 120, preferably from about 13 to about 70, and more preferably from about 20 to about 46; Repeat R 4 and R 6The number of moieties, p, is about 7 or more, preferably about 7 to about 35, more preferably about 10 to about 20, and even more preferably about 11 to about 14; (R 1 R 2 n R 3 ) the number average molecular weight of the block, q, is from about 400 g / mol to about 10,000 g / mol, preferably from about 1,000 g / mol to about 6,000 g / mol, more preferably from about 1,400 g / mol to about 4,000 g / mol, even more preferably from about 1,600 g / mol to about 3,000 g / mol, and most preferably from about 1,800 g / mol to about 2,200 g / mol; The ratio of prepolymer (A) segments to prepolymer (B) segments, r / s, is from about 0.1 to about 2.5.

[0099] In a particular respect, n is about 20 to about 115, preferably about 35 to about 100, more preferably about 45 to about 85; p is about 7 or more, preferably about 7 to about 35, more preferably about 10 to about 20, and even more preferably about 10 to about 14; q is from about 1000 g / mol to about 7000 g / mol, preferably from about 3000 g / mol to about 5000 g / mol, more preferably from about 3800 g / mol to about 4200 g / mol; and r / s is about 0.10 to about 1.0, for example, about 0.15 to about 0.50, or about 0.20 to about 0.30.

[0100] When PEG polymers are present in the multi-block copolymers with crystalline poly(p-dioxanone) building blocks, the length of the PEG can vary from about 1000 g / mol to about 5000 g / mol. Non-limiting examples include PEG lengths of at least about 1000 g / mol, at least about 1200 g / mol, at least about 1400 g / mol, at least about 1600 g / mol, at least about 1800 g / mol, at least about 2000 g / mol, at least about 2200 g / mol, at least about 2400 g / mol, at least about 2600 g / mol, at least about 2800 g / mol, at least about 3000 g / mol, at least about 3200 g / mol, at least about 3400 g / mol, at least about 3600 g / mol, at least about 3800 g / mol, up to about 4000 g / mol, up to about 4200 g / mol, up to about 4400 g / mol, up to about 4600 g / mol, up to about 4800 g / mol, or up to about 5000 g / mol.

[0101] D. Formulation Aspects of the present disclosure relate to formulations comprising a plurality of microspheres. Such formulations may be comprised of a homogeneous or heterogeneous mixture of microspheres according to any one of the parameters disclosed herein. Furthermore, such formulations may optionally further comprise pharmaceutically acceptable excipients and / or other components relevant to the particular indication being treated.

[0102] E. Administration Methods and Release Profiles A feature of the microspheres disclosed herein is a release profile that allows for extended release of the antibody or protein. In some aspects, the release of the antibody or protein in the microsphere or microsphere formulation is less than or about 1 / 7, e.g., less than or about 1 / 14, less than or about 1 / 21, less than or about 1 / 28, less than or about 1 / 29, less than or about 1 / 30, less than or about 1 / 31, less than or about 1 / 33, less than or about 1 / 34, or ...31, less than or about 1 / 33, less than or about 1 / 34, or less than or about 1 / 7, e.g., less than or about 1 / 14, less than or about 1 / 21, less than or about 1 / 28, less than or about 1 / 29, less than or about 1 / 30, less than or about 1 / 31, less than or about 1 / 33, less than or about 1 / 34, or less than or about 1 / 3 Less than or about 1 / 34, 1 / 35, 1 / 42, 1 / 49, 1 / 56, 1 / 57, 1 / 58, 1 / 59, 1 / 60, 1 / 61, or 1 / 62 is released in the first 24 hours after administration. In some aspects, the antibody or protein in the microsphere or microsphere formulation is about 2% to about 40%, e.g., about 3%, about 3.5%, about 4%, about 4.5%, about 5%, about 5.5%, about 6%, about 6.5%, about 7%, about 7.5%, about 8%, about 8.5%, about 9%, about 9.5%, about 10%, about 10.5%, about 11%, about 11.5%, about 12%, about 12.5%, about 13%, about 13.5%, about 14%, about 14.5%, about 15%, about 15.5%, about 16%, About 16.5%, about 17%, about 17.5%, about 18%, about 18.5%, about 19%, about 19.5%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, or about 40%, or any range including two of these values, e.g., about 18.5% to about 26%, is released in the first 24 hours.

[0103] Without wishing to be bound by any theory, it is expected that the above release profiles will allow for extended release of the antibody or protein for a specified period of time, for example, but not limited to, about 1 week or more, about 2 weeks or more, about 3 weeks or more, about 4 weeks or more, about 5 weeks or more, about 6 weeks or more, about 7 weeks or more, about 8 weeks or more, about 12 weeks or more, about 16 weeks or more, about 20 weeks or more, about 24 weeks or more, about 28 weeks or more, about 1 month or more, about 2 months or more, about 3 months or more, about 4 months or more, about 5 months or more, about 6 months or more.

[0104] It is anticipated that the microspheres and formulations containing these microspheres may be administered according to any mode of administration known in the art, including, but not limited to, topical, parenteral, oral, sublingual, by inhalation, nasal, by injection, intradermal, transdermal, intramuscular, subcutaneous, intravitreal, intraarticular, intraarterial, intratumor, bolus administration, infusion, and / or any other suitable method.

[0105] For topical administration, the microspheres can be contained within a gel, cream, or ointment, and can be covered by a barrier, if desired, for example, the microspheres can be contained within a gel, such as a hyaluronic acid gel or a polymeric polysaccharide gel.

[0106] When administered by injection, the microspheres may be contained in a pharmaceutical carrier, such as water, saline solution (e.g., 0.9%), or a solution containing a surfactant in an amount of about 0.1% to about 0.5% w / v. An example of a surfactant that can be used is Tween 80 surfactant. The pharmaceutical carrier may further contain a viscosity enhancer, such as sodium carboxymethylcellulose.

[0107] In certain aspects, the present disclosure relates to methods of administration that achieve or approach a sustained release profile with minimal or burst release.

[0108] Aspects of the present disclosure relate to dosing regimens that allow for the maintenance of therapeutically appropriate plasma levels, including administration of an extended-release antibody or protein formulation about every week, about every 2 weeks, about every 3 weeks, about every 4 weeks, about every 5 weeks, about every 6 weeks, about every 7 weeks, about every 8 weeks, about every 12 weeks, about every 16 weeks, about every 20 weeks, about every 24 weeks, about every 28 weeks, or about every month, about every 2 months, about every 3 months, about every 4 months, about every 5 months, or about every 6 months.

[0109] F. Treatment Method The dosage forms disclosed herein can be used to treat a variety of diseases and can be administered according to appropriate frequency and dosage based on efficacy, which can be in humans or veterinary medicine. Exemplary indications include various cancers, acute myeloid leukemia, non-Hodgkin's lymphoma, rheumatoid arthritis, multiple sclerosis, cardiovascular disease, systemic lupus erythematosus, Crohn's disease, ulcerative colitis, psoriasis, atopic dermatitis, transplant rejection, Alzheimer's disease, inflammatory diseases, viral infections, hepatitis C-like infections, Ebola or HIV, ankylosing spondylitis, macular degeneration, allergic asthma, migraines, hemophilia A, hypercholesterolemia, as well as several more conditions associated with antibodies or therapeutic proteins and / or any other disease or condition indicated for oncology, hematology, cardiology / vascular disease, dermatology, endocrinology, gastroenterology, genetic diseases, immunology, infectious diseases, musculoskeletal disease, nephrology, ophthalmology, pulmonary / respiratory disease, and rheumatology. These examples are intended only to provide a list of diseases for which humans may receive antibodies or therapeutic proteins. This list is not exhaustive. Furthermore, as disease mechanisms are elucidated and antigens for which new drugs are being developed are identified, it is expected that new indications will be developed and discovered for existing and new antibodies and therapeutic proteins directed against additional target antigens. Thus, these antibodies or therapeutic proteins not listed here can be formulated in the same manner as described herein.

[0110] Furthermore, unlike methods disclosed in the art for administering antibodies or proteins, the antibody- or protein-containing microspheres and formulations disclosed herein require fewer administrations, e.g., less than about 10, less than about 9, less than about 8, less than about 7, less than about 6, less than about 5, less than about 4, or less than about 3 administrations, or about 1 or about 2 administrations, over a 3 or 6 month period, as opposed to 45 administrations of conventional antibodies. In some embodiments, the described formulations are in the form of a single injection, providing a therapeutic effect for more than 1 month.

[0111] G. General Definitions As used in the description of this invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly dictates otherwise.

[0112] The term "about," as used herein, when referring to a measurable value, such as an amount or concentration, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%, or even 0.1% of the specified amount.

[0113] When used to describe the selection of any component, range, dosage form, etc. disclosed herein, the words "acceptable," "effective," or "sufficient" mean that the component, range, dosage form, etc. is suitable for the disclosed purpose.

[0114] Also, as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and the lack of combinations when interpreted as alternatives ("or").

[0115] As used herein, the word "comprising" is intended to mean that the formulations and methods include the recited elements, but do not exclude others. As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) should be interpreted to include the recited substances or steps "and those that do not materially affect one or more of the basic and novel characteristics of the recited embodiment." Thus, the word "consisting essentially of," as used herein, should not be interpreted as equivalent to "comprising." "Consisting of" would mean excluding other ingredients, other than trace elements, and substantial method steps for administering the formulations disclosed herein. Perspectives defined by each of these transitional terms are within the scope of this disclosure.

[0116] A "formulation" is intended to mean a combination of an active agent with another compound or composition, inert (eg, a detectable agent or label) or active, such as an adjuvant.

[0117] A "pharmaceutical formulation" is intended to include the combination of an active agent with a carrier, inert or active, making the formulation suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0118] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the formulations of the present invention. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, poly(ethylene glycol), sodium carboxymethylcellulose, polyacrylates, waxes, polyoxyethylene-polyoxypropylene-block polymers, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a reference text in this field. These are preferably selected with regard to the intended form of administration, ie, oral tablet, capsule, elixir, syrup, etc., and consistent with conventional pharmaceutical practice.

[0119] The term "extended release" is used herein to refer to the ability to release an ingredient over a specified period of time. The term "burst release" refers to a rapid release period of an ingredient into the environment such that continuous release over an extended period of time cannot be sustained.

[0120] As used herein, the terms "subject" and "patient" are used interchangeably to mean any animal. In some embodiments, the subject can be a mammal; in further embodiments, the subject can be a human, a mouse, or a rat.

[0121] As used herein, "treating" or "treatment" of a disease in a subject refers to (1) preventing symptoms or a disease from occurring in a subject who is predisposed to the disease or who does not yet exhibit symptoms of the disease; (2) inhibiting or halting the development of the disease; or (3) alleviating or causing regression of the disease or symptoms of the disease. As understood in the art, "treatment" is an approach for obtaining beneficial or desired results, including clinical results. For purposes of the present technology, beneficial or desired results include, but are not limited to, one or more of the following: alleviation or relief of one or more symptoms, whether detectable or undetectable; attenuation of the severity of a condition (including a disease); a stabilized (i.e., non-worsening) status of a condition (including a disease); a delay or slowing of a condition (including a disease); progression, alleviation, or reduction of a condition (including a disease), condition, and remission (whether partial or total).

[0122] Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as those commonly understood by those skilled in the art to which this invention belongs. It will be further understood that words, such as those defined in commonly used dictionaries, should be interpreted as meanings consistent with their meanings in the context of this application and the relevant technical field, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. For example, descriptors can be used to refer to biological materials (e.g., tissues, organoids, samples) that exhibit the characteristics of a particular organ, such as the use of "hepatic" to describe liver-derived tissue or liver-like organoids. Unless expressly defined below, such terms should be interpreted according to their ordinary meaning.

[0123] The terminology used in the description herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0124] The practice of the techniques of the present invention will employ conventional techniques, within the skill of the art, unless otherwise indicated.

[0125] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein can be used in any combination. Furthermore, the present disclosure also contemplates that in some embodiments, any feature or combination of features described herein can be excluded or omitted. For illustration, if the specification describes a composite comprising components A, B, and C, it is specifically intended that any of A, B, or C, or combinations thereof, singly or in any combination, can be omitted and waived.

[0126] All numerical designations, e.g., pH, temperature, time, concentration, and molecular weight, are approximations, including ranges, and are varied by increments of 1.0 or 0.1, or alternatively, by a variation of + / - 15%, or alternatively, 10%, or alternatively, 5%, or alternatively, 2%, (+) or (-), as appropriate. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the word "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely exemplary and that equivalents of such are known in the art.

[0127] V. Working Examples The following examples are non-limiting and illustrative of procedures that can be used in various instances in carrying out the present disclosure. Additionally, all references disclosed herein are incorporated by reference in their entirety.

[0128] Example 1 – Synthesis of SynBiosys Multi-Block Copolymers This example describes the synthesis and characterization of [poly(ε-caprolactone)-co-poly(ethylene glycol)-co-poly(ε-caprolactone)]-b-[poly(L-lactide)] and [poly(ε-caprolactone)-co-poly(ethylene glycol)-co-poly(ε-caprolactone)]-b-[poly(p-dioxanone)] multiblock copolymers used in the preparation of mAb-loaded microspheres.

[0129] Molecular weight (M) of approximately 4000 g / mol n ) and a M of approximately 2000 g / mol. n Poly(ε-caprolactone)-co-PEG1000-co-(ε-caprolactone) prepolymers (abbreviated as PCL-PEG1000-PCL or CP10C20) with a molecular weight of approximately 4000 g / mol were prepared by ring-opening polymerization of ε-caprolactone using poly(ethylene glycol) with a molecular weight of 3000 g / mol (PEG3000) or 1000 g / mol (PEG1000) as the initiator and stannous octoate as the catalyst. n Poly(L-lactide) prepolymer (abbreviated as PLLA or LL40) having the molecular weight of 1 It was analyzed by H-NMR. M of approximately 2500 g / mol nPoly(p-dioxanone) prepolymer (abbreviated as PDO or D25) having the formula (I) was synthesized by ring-opening polymerization of p-dioxanone using 1,4-butanediol as an initiator and stannous octoate as a catalyst.

[0130] [PCL-PEG3000-PCL]-b-[PDO], [PCL-PEG1000-PCL]-b-[PDO], [PCL-PEG3000-PCL]-b-[PLLA], and [PCL-PEG1000-PCL]-b-[PLLA] multiblock copolymers with a block ratio of 50 / 50 w / w were prepared by chain extending PCL-PEG3000-PCL or PCL-PEG1000-PCL prepolymer with PDO or PLLA prepolymer in p-dioxane using 1,4-butanediisocyanate as a chain extender, followed by precipitation to remove the p-dioxane.

[0131] The polymers, abbreviated as 50CP30C40-LL40, 50CP10C20-LL40, 50CP30C40-D25 and 50CP10C20-D25, were analyzed for their chemical composition, intrinsic viscosity, residual p-dioxane content, and thermal properties.

[0132] The chemical composition (monomer ratio) and molecular weight (M n ), and the block ratio of the multi-block copolymer is 1 The NMR spectra were determined by H-NMR. For this determination, a Bruker Avance DRX 500 MHz NMR spectrometer (BAV-500) equipped with a Bruker Automatic Sample Changer (BACS 60) (Varian) operating at 500 MHz was used. The d1 delay time was set to 20 seconds, and the number of scans was 16. The spectra were recorded from 0 to 14 ppm. Approximately 1.3 g of deuterated chloroform was added to approximately 25 mg of polymer. 1 H-NMR samples were prepared.

[0133] Intrinsic viscosity was measured using an Ubbelohde Viscosimeter (DIN), Type 0C, Si Analytics, supplied with the Si Analytics Viscosimeter, including a water bath. Measurements were performed in chloroform at 25°C. The polymer concentration in chloroform was such that the relative viscosity was in the range of 1.2 to 2.0.

[0134] Residual p-dioxane content was determined using a GC-FID headspace method. Measurements were performed on a GC-FID Combi Sampler equipped with an Agilent column, DB-624 / 30 m / 0.53 mm. Samples were prepared in DMSO (dimethyl sulfoxide). Residual solvent content was determined using p-dioxanone calibration standards.

[0135] Modulated differential scanning calorimetry (MDSC) was used to determine the thermal behavior of the multiblock copolymers using a Q2000 MDSC (TA instruments, Ghent, Belgium). Approximately 5-10 mg of dry material was accurately weighed and heated from -85°C to 120°C under a nitrogen atmosphere at a heating rate of 2°C / min and a modulation amplitude of + / - 0.42°C every 80 seconds. The glass transition temperature (T g , midpoint), melting temperature (maximum of endothermic peak, T m ) and the enthalpy of fusion (ΔH m ) was determined from the reversing heat flow. Temperature and enthalpy were calibrated using indium standards.

[0136] Table 1 lists the characteristics of various multi-block copolymers.

[0137] [Table 1]

[0138] Example 2 – Generation of SynBiosys mAbX-loaded microspheres A monoclonal antibody (mAbX) with a molecular weight of 145.8 kDa was formulated in SynBiosys microspheres. mAbX-loaded microspheres (mAbX MSPs) with a target loading of 5 wt% were prepared from a blend of 50CP10C20-LL40 (RCP-1440) and 50CP30C40-LL40 (RCP1667) by water-in-oil-in-oil (W / O / O) and water-in-oil-in-water (W / O / W)-based microencapsulation processes.

[0139] The mAbX solution was concentrated to a target concentration of 100 mg / ml. Visual inspection of the concentrated protein solution showed the absence of insoluble particles. The integrity of mAbX was maintained during concentration. SEC-UPLC analysis confirmed the absence of aggregates and there was no sign of degradation. The mAbX concentration of the concentrated solution was 105.7 mg / ml, as determined by SEC-UPLC.

[0140] 50CP10C20-LL40 and 50CP30C40-LL40 were dissolved in dichloromethane at different weight ratios (100:0, 50:50, 25:75, and 0:100) to a final polymer concentration of 10 wt% and filtered through a 0.2 μm PTFE filter. 5.0 g of the polymer solution (O1) was then emulsified with 0.25 ml of concentrated mAbX solution (W1) at an O1 / W1 ratio of 20 wt / v using an Ultra Turrax (22,000 rpm for 40 s) to obtain a primary emulsion (dispersed phase, DP).

[0141] For the preparation of mAbX MSP via the W / O / O microencapsulation route, 4.5 g of silicone oil, which served as a coacervation agent, was then added to the DP under vortexing with a syringe pump to initiate dichloromethane extraction and form the initial MSP. Under constant stirring, the initial MSP was then transferred to a vessel containing 330 g of heptane to extract the dichloromethane and silicone from the initial MSP. After the extraction procedure was completed, the microspheres were collected by filtration using a 5 μm filter, washed with heptane (2 × 250 ml), and finally dried overnight at 40 °C under reduced pressure.

[0142] To prepare mAbX MSP via the W / O / W microencapsulation route, 5.0 ml of DP was then emulsified with 250 ml of 0.4% aqueous PVA solution (W2 or CP) containing 5 wt% NaCl by membrane emulsification using a membrane with 30 μm pores, thereby forming a water-in-oil-in-water (W / O / W) double emulsion. The W / O / W emulsion was stirred at room temperature for 1.5 hours to allow extraction and distillation of dichloromethane. After completion of solvent distillation, the mAbX MSP was collected by filtration, washed with 0.05 wt% aqueous Tween-80 solution and water for injection, and finally lyophilized.

[0143] The resulting microspheres were analyzed for their particle size distribution using a Horiba LA-960 Laser Particle Size Analyser (Table 2).

[0144] [Table 2]

[0145] The mean particle size of the mAbX MSP varied from 48 to 57 μm. The mAbX MSP prepared via the W / O / O route had a relatively broad particle size distribution with a coefficient of variation (CV) of 30 to 40% (FIG. 1), whereas the mAbX MSP prepared via the W / O / W process route had a very narrow particle size distribution (CV 11 to 12%).

[0146] Scanning electron microscopy (SEM) using a JEOL JCM-5000 Neoscope SEM confirmed the results obtained with laser diffraction. mAbX MSP prepared by W / O / O typically had a smooth surface with an irregular, raisin-like shape and a wide particle size distribution (Figure 1). The W / O / W microencapsulation process resulted in uniformly sized spherical microspheres with smooth surfaces (Figures 2B-D), except for mAbX-MSP (Figure 2A), which was composed entirely of 50CP30C40-LL40 and exhibited a rough surface with a fibrous structure present on the surface.

[0147] All mAbX MSPs were analyzed for their mAbX content. mAbX MSPs (approximately 10 mg) were completely dissolved in 1.0 ml of DMSO (80°C, 30 min), followed by the addition of 5.0 ml of 0.1 N aqueous NaOH 0.5% SDS solution to completely hydrolyze the polymer (room temperature, overnight stirring). The total protein content of the clear solution was then determined using a bicinchoninic acid (BCA) assay. The results, presented in Table 3, show that the coacervation-based W / O / O process, like the W / O / W double emulsion process, allows for the microencapsulation of mAbX with high encapsulation efficiencies (EEs) typically exceeding 90%, resulting in mAbX MSPs with mAbX loadings of 4.6–4.9 wt% (except for mAbX-MSP, which was composed entirely of 50CP30C40-LL40).

[0148] [Table 3]

[0149] The in vitro release kinetics of mAbX was determined by incubating 20 mg of mAbX MSP in a 2 ml polypropylene vial containing 1.8 ml of in vitro release buffer (100 mM phosphate buffer, pH 7.4, containing 0.025% Tween 20 and 0.02% NaN3) on an orbital shaker in a climate chamber thermostat at 37°C. At predetermined time points, after centrifugation of the vial, a 1.6 ml aliquot of buffer was removed and replaced with fresh buffer. The concentration of mAbX in the buffer was determined by SEC-UPLC with a Waters 2690 HPLC system using a TSKgel Size Exclusion HPLC column, 300 x 4.6 mm; 4 μm, isocratic analysis at 25°C (50 mM phosphate, 0.4 M perchlorate buffer pH 6.3:acetonitrile (90:10, v / v) and UV detection at 214 nm).

[0150] The release kinetics of mAbX was largely unaffected by the type of microencapsulation process (W / O / O or W / O / W), but was primarily influenced by the composition of the polymer matrix. mAbX MSPs prepared from 100% 50CP30C40-LL40 gradually and completely released mAbX over a period of one month. By (partially) replacing 50CP30C40-LL40 with 50CP10C20-LL40 (and thereby reducing the degree of swelling of the polymer matrix), the release rate of mAbX was effectively reduced. Based on the extrapolation of the release curves, the mAbX MSP prepared from a 50 / 50 blend of 50CP30C40-LL40 and 50CP10C20-LL40 releases mAbX gradually with an estimated total release period of approximately 3 months, while the mAbX MSP prepared from a 25 / 75 blend of 50CP30C40-LL40 and 50CP10C20-LL40 releases mAbX gradually with an estimated total release period of 4 (W / O / W) to 5 (W / O / O) months.

[0151] Example 3 - Production of SynBiosys mAbX MSP with high mAbX loading via W / O / W microencapsulation mAbX MSPs with target loadings of 10-20 wt% were produced on a 1 g scale by the W / O / W-based membrane emulsification process described in Example 2. The resulting mAbX microspheres were characterized using the method described in Example 2, except that their particle size distribution was measured using a Coulter Counter Multisizer III. The volume-average particle size (D50) and coefficient of variation (CV) were determined in the ranges of 4-120 μm or 8-240 μm.

[0152] mAbX MSPs with a 10% (wt) mAbX target loading were initially prepared from 100% 50CP30C40-LL40 in an attempt to produce mAbX MSPs with a 1-month sustained release of mAbX. However, the combination of a higher mAbX loading and a relatively high degree of swelling of 50CP30C40-LL40 resulted in a poor encapsulation efficiency of only 58% (AMD16172). By blending 50CP30C40-LL40 with 10-20% (wt) 50CP10C20-LL40, the encapsulation efficiency could be significantly improved (70-80%), yielding mAbX MSPs with 7.0% (AMD17013) and 8.2% (AMD17005) mAbX contents (Table 4).

[0153] [Table 4]

[0154] Figure 5 shows that the cumulative release kinetics of mAbX MSP prepared from a 90:10 polymer ratio was similar to that of mAbX MSP composed entirely of 50CP30C40-LL40, exhibiting sigmoidal release kinetics and complete release in 2-3 weeks. By increasing the weight fraction of 50CP10C20-LL40 to 20 wt%, the release of mAbX could be slowed.

[0155] To enable the preparation of mAbX MSP with a target loading of 19 wt%, the mAbX solution was further concentrated to approximately 230 mg / ml. Subsequently, mAbX MSP was prepared from a blend of 50CP30C40-LL40 and 50CP10C20-LL40 using the W / O / W process as described above, using the settings listed in Table 5. Unfortunately, the majority of mAbX formulations prepared in this manner had very poor encapsulation efficiencies, regardless of the polymer ratio, polymer concentration, and primary emulsification conditions. Significantly higher encapsulation efficiencies (70-75%) were obtained for mAbX MSP with a target loading of 14-15% prepared using a more concentrated polymer solution (15 wt%), resulting in mAbX MSP with approximately 10.6 wt% mAbX (AMD17042 and AMD17038) (Table 5).

[0156] [Table 5] a the weight ratio of 50CP30C40-LL40 to 50CP10C20-LL40; b DP prepared by 2 x 40 sec emulsification, Ultraturrax, 21600 rpm c The mAbX solution contained 50 or 150 mM L-arginine, 0.025% (w / v) Tween 80.

[0157] AMD17042 and AMD17038 were analyzed for their in vitro release kinetics (Figure 6). mAbX release from mAbX MSP (AMD17038) prepared from a 90:10 w / w blend of 50CP30C40-LL40 and 50CP10C20-LL40 was consistent with release kinetics previously generated for mAbX MSPs (e.g., AMD17013) prepared from the same polymer matrix but with lower mAbX content. Surprisingly, mAbX release from mAbX MSP prepared from an 80:20 w / w blend of 50CP30C40-LL40 and 50CP10C20-LL40 was significantly lower, demonstrating near-linear release of mAbX over the first 4 weeks for an estimated release period of approximately 2 months (based on extrapolation of the data). Both AMD17042 and AMD17038 released highly intact mAbX (greater than 96% integrity as determined by SEC-UPLC).

[0158] Example 4 - Generation of SynBiosys mAbX microspheres with high mAbX loading by W / O / O microencapsulation To enable the preparation of mAbX MSP with a mAbX loading of approximately 20% by weight, the W / O / W microencapsulation process was replaced by the water-in-oil-in-oil (W / O / O) based microencapsulation process used in Example 2. In the W / O / O-based microencapsulation process, dichloromethane extraction is performed using an anhydrous extraction process, which prevents loss of water-soluble API and hardening of the microspheres during dichloromethane extraction, thereby allowing for a high encapsulation efficiency of approximately 100%.

[0159] mAbX MSPs with target loadings of 10 and 19 wt% were prepared from a 90 / 10 blend of 50CP10C40-LL40 and 50CP10C20-LL40 by the W / O / O microencapsulation process described in Example 2 using the settings listed in Table 6.

[0160] [Table 6]

[0161] The mean particle sizes of the mAbX MSP (as measured by laser diffraction) were relatively high, with D50s of 78 and 109 μm, respectively. Both formulations had very broad particle size distributions with CVs ranging from 66 to 81%. SEM confirmed the results obtained by laser diffraction ( FIG. 7 ). As expected, the encapsulation efficiency was very high (>90%), yielding mAbX MSP with mAbX loadings of 9.3 and 19.3 wt %.

[0162] AMD17022 and AMD17023 were analyzed for their in vitro release kinetics (Figure 8). mAbX release from mAbX MSP (AMD17022) with a 9.3 wt% mAbX loading showed typical sigmoidal release kinetics, but had a slightly slower overall release when compared with mAbX MSP (e.g., AMD17038) with a similar mAbX loading prepared by a water-in-oil (W / O / W) microencapsulation process. mAbX MSP (AMD17023) with a 19 wt% mAbX loading showed significantly faster release with less burst release, followed by linear release for approximately 14 days. Both AMD17022 and AMD17023 released highly intact mAbX (greater than 96% integrity as determined by SEC-UPLC).

[0163] Example 5 - Generation of SynBiosys mAbX microspheres with high mAbX loading for in vitro and in vivo characterization To allow for more extensive in vitro and in vivo characterization of the lead formulation, mAbX MSP with a target loading of 19 wt% was prepared from a 90 / 10 wt% blend of 50CP10C40-LL40 and 50CP10C20-LL40 at a 10 g scale by a water-in-oil / oil microencapsulation process using the settings used for AMD17023 (see Table 6 in Example 4). Briefly, 9.0 g of 50CP30C40-LL40 (RCP-1667) and 1.0 g of 50CP10C20-LL40 (RCP-1440) were dissolved in dichloromethane to a concentration of 10 wt% and filter-sterilized through a 0.2 μm PTFE filter. The polymer solution (O1) was emulsified with a 220.0 mg / ml aqueous mAbX solution (220 mg / ml, pH 5.0) (W1) at an O1 / W1 ratio of 9.5 using an in-line Ultra Turrax (12,000 rpm) to obtain a primary emulsion (DP). The DP was directly homogenized with silicone oil using an in-line Ultra Turrax (12,000 rpm) at a silicone oil to dichloromethane ratio of 0.75 w / w. The nascent microspheres were continuously transferred to a vessel containing 6.7 L of heptane to extract the dichloromethane and silicone oil from the nascent microspheres (final heptane to dichloromethane ratio of 13.4 (w / w)). After solvent extraction was complete, the microspheres were collected by filtration, washed with heptane, and finally dried overnight at 40 °C under reduced pressure using a Nutsche filter setting. The dried mAbX MSP was sieved through a 200 μm sieve to remove oversized particles and stored at −18°C until further use.

[0164] mAbX MSP (AMD18012) was characterized using the method described in Example 2. The mAbX-MSP was spherical and had a smooth surface without any visible pores. The microspheres had a broad particle size distribution (CV 65%) with a mean particle size (D50) of 83 μm and a mAbX loading of 14.9 wt%, representing an encapsulation efficiency of 80%. Residual dichloromethane and heptane contents determined by GC-headspace (Agilent 6850 gas chromatograph equipped with a Combi-Pal headspace sampler) using DMSO to dissolve samples (50-500 mg), octane as an internal standard, and flame ionization were less than 18 ppm and 357 ppm, respectively.

[0165] The in vitro release kinetics of the mAbX-MSP was nearly identical to that of AMD17023 (Figure 8), with linear release kinetics up to 10 days without any burst and a recovery (percentage of encapsulated mAbX released) of 86% after 2 weeks of in vitro release.

[0166] Example 6 – In vitro characterization of mAbX formulations In this example, samples taken during in vitro release testing of mAbX MSP prepared in Example 5 were further characterized for (1) mAbX aggregation and fragmentation by size exclusion chromatography (SEC), (2) tertiary structure integrity by fluorescence spectroscopy (FLS), (3) secondary structure integrity by circular dichroism spectroscopy (CD), (4) protein concentration by spectrophotometric determination, and (5) ability to bind to its target by ELISA for quantification of functional mAbX.

[0167] Analysis method Aggregation and fragmentation of mAbX in in vitro release samples were analyzed by size-exclusion chromatography (SEC). Furthermore, the proportion of mAbX monomer was quantified. Samples were examined using a TSKgel Super SW3000 size-exclusion column (TOSOH) with a mobile phase (0.05 M sodium phosphate, 0.4 M sodium perchlorate, pH 6.3) and a Gel Filtration Standard (Bio-Rad). The different migration times of protein aggregates, monomers, and fragments through the column allowed for detection of peptide bonds and confirmation of molecular weights of the separated eluted fractions by UV at 214 nm. The area under the curve was used to calculate the percentage of eluted fractions.

[0168] The integrity of the tertiary structure of mAbX was analyzed by fluorescence spectroscopy (FLS) by excitation of tryptophan at a wavelength of 280 nm. The resulting emission was recorded from 290 nm to 450 nm. The emission wavelength of tryptophan shifts in the event of a change in the molecular environment, providing information about the conformational state of the protein. The proportion of correctly folded protein in a sample was determined by calculating the relationship between the emission maxima of the sample and a control sample of intact and denatured protein.

[0169] The integrity of the secondary structure of mAbX was analyzed by circular dichroism spectroscopy (CD). The optically active peptide bonds of intact antibodies absorb left- and right-polarized light to a certain extent, resulting in polarized light with a specific ellipticity. The secondary structure of mAbX was analyzed at a wavelength of 218 nm, revealing the signal of β-sheet, the predominant secondary structural element in antibodies. The proportion of correctly folded antibody in a sample was determined by the relationship between the CD signals of the sample and a control sample of intact and denatured mAbX in a suitable buffer system.

[0170] The protein concentration of the samples was determined spectrophotometrically. The absorbance of aromatic amino acids and disulfide bonds in proteins was detected at a wavelength of 280 nm and used to determine the protein concentration. After entering the reciprocal of the absorption coefficient for a particular protein, the concentration in mg / ml output format was automatically calculated by the Eppendorf Biophotometer Plus.

[0171] In the ELISA assay, 96-well plates were coated with soluble mAbX receptor and used to capture mAbX from in vitro and in vivo samples. Thus, only functional mAbX capable of binding to its target was immobilized. Next, an anti-human detection antibody coupled to horseradish peroxidase (HRP) was bound to the captured mAbX molecules. The resulting antigen-antibody-antibody-HRP complex was quantified by HRP-driven conversion of the chromogenic substrate, tetramethylbenzidine (TMB). The color intensity of the converted product was proportional to the amount of bound, functional mAbX from the sample.

[0172] result In Figure 9A, fluorescence spectroscopy (FLS) data are shown for a series of seven mAbX samples taken from an in vitro release study of mAbX MSP. No major changes could be demonstrated in the folding characteristics of the released mAbX over a period of up to 14 days, while only a minor red shift in the emission maximum at 2 nm was detected on day 17 compared to the native control mAbX sample (Figure 9B). The ratio of wavelengths at emission maxima (Figure 9C) was used to assess the folded versus unfolded fraction and to compare the FLS data with circular dichroism data (see also Figure 11).

[0173] In Figure 10A, circular dichroism data are shown for a series of seven mAbX samples after in vitro release experiments (Figure 10A). No major changes in the secondary structure of mAbX occurred over a period of up to 14 days (Figure 10B). Because the protein concentrations of samples taken after day 14 were below the detection limit of the CD method, CD analysis could not be performed at these later time points. The ratio of the signal peak at 218 nm divided by the signal of fully unfolded mAbX was used to compare the CD data with the FLS data (see also Figure 11).

[0174] Finally, the data obtained from FLS and CD (folded fraction) were plotted against the total protein concentration obtained by SEC-UPLC and the intact monomeric species of mAbX measured by SEC (Figure 11). Controlled release of intact, correctly folded, active mAbX over a 14-day period could be shown. After 14 days, only small amounts of mAbX were released by the polymer-based microsphere formulation.

[0175] Taken together, the data obtained from SEC-UPLC, fluorescence spectroscopy, circular dichroism, and ELISA demonstrate the suitability of the microencapsulation process with [PCL-PEG-PCL]- b -[PLLA]-based multiblock copolymers and the monoclonal antibody mAbX, a large protein of 145.8 kDA.

[0176] Example 7 – In vivo pharmacokinetics and antitumor efficacy of mAbX MSP mAbX MSPs prepared according to Example 5 were evaluated for their in vivo pharmacokinetics in healthy NMRI mice.

[0177] Female NMRI mice were used at 4–6 weeks of age. Mice were randomly divided into five groups of six animals and received either a single intravenous (No risk, but the delay in obtaining the grant publication will be postponed (meaning the examination procedure is still ongoing until such publication of the grant decision is made, for example, opened to third-party observations).) or subcutaneous (sc) injection of 1 mg of mAbX control solution or a single sc injection of a suspension of mAbX MSP in aqueous reconstitution medium (water for injection, 0.6 wt% carboxymethylcellulose (CMC)) (1, 4, or 8 mg of mAbX per mouse). Blood samples were collected at the indicated time points, and serum concentrations of mAbX were measured using a validated ELISA.

[0178] Figure 12 shows the plasma levels of mAbX after administration. In the iv control group, peak plasma levels were achieved after 3 hours, whereas in the sc control group, peak plasma levels were achieved after 1 day. Injection of the mAbX MSP formulation resulted in a shift in plasma peak levels to days 3-4, with a dose-dependent release of mAbX over a period of up to 23 days in the highest dose group. The data clearly demonstrate that mAbX is released from the microsphere formulation in vivo over an extended period. After in vivo release, mAbX could be detected in plasma by binding ELISA, indicating that the released mAbX was still able to bind to its receptor, indicating that the protein structure was functionally intact.

[0179] In a second pharmacokinetic study, mAbX plasma levels were monitored in A431 xenograft-bearing NMRI nude mice (Figure 13) and mAbX MSPs were evaluated for their antitumor efficacy. Subcutaneous tumors were 1x107 Tumor volumes of 50–250 mm were induced by inoculation of A431 cells into the flanks of NMRI nude mice. 3 Upon reaching 100 mg / mouse, mice were randomly assigned to each treatment group (n=7). The mice received a single injection of either mAbX solution (1 mg / mouse) or mAbX-loaded microspheres (0-8 mg / mouse). Blood samples were collected at the indicated time points, and serum concentrations of mAbX were measured using a validated ELISA. Tumor volumes were determined by caliper measurements twice weekly.

[0180] An intravenous control injection of 1 mg of mAbX resulted in a plasma peak after 3 hours, whereas a plasma peak could be observed after 1 day after a sc control injection of 1 mg of mAbX solution. The mAbX MSP formulations were subcutaneously injected at final mAbX doses of 1, 2, 4, or 8 mg, respectively. The plasma peak shifted from 2 to 4 days, whereas a sustained-release effect was observed for up to 27 days at the highest dose. This second pharmacokinetic study also confirmed that mAbX was successfully released from the microsphere formulation in vivo. mAbX could be detected in plasma by functional ELISA, demonstrating that the protein structure was still functionally intact.

[0181] In Figure 14, tumor growth in A431 tumor-bearing NMRI nude mice was monitored. Subcutaneous tumors were 1 x 10 7 Tumor volumes of 50–250 mm were induced by inoculation of A431 cells into the flanks of NMRI nude mice. 3Upon reaching 10 days, mice were randomly assigned to each treatment group (n=7). Tumor volumes were determined by caliper measurements twice weekly. After injection of the polymer-only group (without any mAbX), tumors continued to grow over the 42-day study period. Significant antitumor effects (p≦0.002) could be demonstrated for iv injection of mAbX and 8 mg of the mAbX MSP formulation 7 days later, and for sc injection of mAbX and 1, 2, or 4 mg of the mAbX MSP formulation 10 days later. This data set allowed us to demonstrate that mAbX could be successfully released from the microsphere formulation in vivo and that the protein remained structurally intact and biologically active after release.

[0182] Example 8 – Production of sustained-release microspheres of mAb02 In a separate study, a second monoclonal antibody with a molecular weight of 144.9 kDa (mAb02) was formulated into SynBiosys microspheres at a target loading of 19% by weight by a W / O / O process using the same polymer composition used to generate the mAbX-MSP formulation in Example 4.

[0183] Briefly, a total of 1 g of polymer (representing various blend ratios of 50CP30C40-LL40 and 50CP10C20-LL40) was dissolved in 9 g of dichloromethane and emulsified (Ultraturrax, 21600 rpm, 40 s) with 221.4 mg / ml mAb02 solution at a W / O ratio of 9.5. The primary emulsion was then homogenized (Ultraturrax, 21600 rpm, 30 s) with 5.3 g of silicon oil (350 CST). The nascent microspheres were then transferred to a vessel containing 250 ml of n-heptane, and the dichloromethane and silicone oil were extracted for 1 h. mAb02 MSP was collected on a 5 μm filter, washed twice with 250 ml of n-heptane, and finally dried overnight at 40° C. under reduced pressure.

[0184] Microspheres were produced according to this method for a variety of polymer blend ratios, including those listed in Table 7.

[0185] [Table 7]

[0186] The resulting mAb02 MSPs were characterized for particle size distribution, mAb02 content, and in vitro release kinetics of mAb02 using the same methods used for mAbX and described in previous examples. The mean particle size varied from 57 to 89 μm. Results indicate that the coacervation process allows for the encapsulation of high mAb02 contents (17.7 to 18.4 wt%) in microspheres while maintaining high encapsulation efficiencies (over 91%). The in vitro release kinetics of mAb02 for various microsphere formulations are presented in Figure 15. All formulations exhibit a low burst release of less than 5% during the first 2 hours. The total mAb02 release duration is increased from approximately 1 week to approximately 4 weeks by decreasing the weight fraction of 50CP30C40-LL40 compared to 50CP10C20-LL40.

[0187] For mAb02 MSP (SR19-002.B) composed of 90% by weight 50CP30C40-LL40 and 10% by weight 50CP10C20-LL40, the integrity of the released mAb02 was determined by SEC-UPLC by calculating the ratio of the peak area of ​​intact mAb02 to the soluble aggregates associated with mAb02. No additional peaks were observed in the chromatogram. Table 8 shows the total mAb02 released and the purity of the released mAb02 as determined by SEC-UPLC.

[0188] [Table 8]

[0189] Typically, the purity of the released mAb02 was 86-89%. The lower purity (80%) obtained at 14 days is due to the low concentration of mAb02 at that time point. Overall, approximately 90% of the microencapsulated mAb02 is released in intact form from the mAb02 MSP (as determined by SEC-UPLC).

[0190] In Figure 16, fluorescence spectroscopy (FLS) data are shown after in vitro release from mAb02 microsphere formulations utilizing different polymer blends (Figures 16A-F). Within these different polymer blend ratios, the hydrophilicity of the polymer blends decreases from A to F. The FLS data indicate that the intrinsic fluorescent tryptophan spectrum of mAb02 is not affected by the different polymer compositions.

[0191] Example 9 – In vitro erosion kinetics of 50CP10C20-LL40 Unfortunately, polymer-only microspheres composed of poly(L-lactide)-based 50CP10C20-LL40 multiblock copolymers, prepared as described in Example 2 using an oil-in-water (O / W)-based solvent extraction / distillation-emulsification process, were found to degrade very slowly (pH 7.4, 37°C). Based on extrapolation of the experimentally determined residual mass of the microspheres up to 12 months, the in vitro erosion time for 50CP10C20-LL40 microspheres is estimated to be approximately 4 years (Figure 17). The slow erosion of poly(L-lactide)-based multiblock copolymers was confirmed for other poly(L-lactides), such as 30CP30C40-LL40, and was attributed to the delayed hydrolysis of the crystalline poly(L-lactide) blocks.

[0192] At different weight ratios with various [PCL-PEG-PCL] prepolymers, an M of approximately 2500 g / mol was obtained. nPoly(p-dioxanone) (PDO)-based multiblock copolymers, prepared by chain extending a PDO prepolymer block with

[0193] Polymer-only microspheres composed of 50CP10C20-D25 were found to degrade approximately twice as rapidly as 50CP10C20-LL40. By using higher molecular weight PEG, such as PEG1500 or PEG3000, the erosion rate increased even more, as shown for 50CP15C20-D25, a [PCL-PEG1500-PCL]-b-[PDO] multiblock copolymer with a 50 / 50 block ratio, and 20CP30C40-D23, a [PCL-PEG3000-PCL]-b-[PDO] multiblock copolymer with a 20 / 80 block ratio (Figure 17).

[0194] Example 10 - Sustained release microspheres of mAb02 prepared from [PCL-PEG-PCL]-b-[PDO] multiblock copolymers This example describes the generation of mAb02 MSP composed of [PCL-PEG-PCL]-b-[PDO] multiblock copolymers with improved polymer degradation kinetics.

[0195] mAb02 MSPs with 20% mAb02 target loading were produced from a blend of 50CP30C40-D25 and 50CP10C20-D25 (synthesized as described in Example 1) and a mAb02 solution at a concentration of 221.4 mg / ml at a 1 g scale, following the same procedure as described in Example 8. The mAb02 MSPs were characterized for particle size distribution, mAb02 content, in vitro release kinetics of mAb02, and integrity of released mAb02 using the same methods as described in Example 8.

[0196] The resulting mAb02 MSPs had significantly larger particle sizes (approximately 120-330 μm) than mAb02 microspheres produced from mAbX and blends of 50CP30C40-LL40 and 50CP10C20-LL40 (Table 9). Microscopy by SEM showed extensive aggregation, explaining the relatively large particle size as measured by laser diffraction.

[0197] [Table 9]

[0198] The total protein content determined using the BCA-based content analysis method represented a mAb02 content varying from 13.2 to 17.3 wt % and an encapsulation efficiency varying from 66% to 87%, which was significantly lower compared to the typical encapsulation efficiency of over 90% obtained for mAb02 MSPs prepared from blends of 50CP30C40-LL40 and 50CP10C20-LL40.

[0199] The in vitro release kinetics of mAb02 from various microsphere formulations are shown in Figure 18.

[0200] All formulations showed a moderate burst release of 10-15%, followed by a sustained release with nearly perfect linear release kinetics. mAb02 MSP prepared from 100% 50CP30C40-D25 released mAb02 over a 6-week period. By partially replacing 50CP30C40-D25 with 50CP10C20-D25 (blend ratios from 90:10 to 0:100), the mAb02 release rate was effectively slowed, yielding mAb02 MSP formulations with release periods ranging from 2 months (blend ratios of 80:20 and 70:30) to 3-4 months (100% 50CP10C20-D25). The integrity of the released mAb02 was determined by SEC-UPLC by calculating the ratio of the peak areas of intact mAb02 and soluble aggregates associated with mAb02. No additional peaks were observed in the chromatogram. Table 10 shows the total released mAb02 and purity of released mAb02 as determined by SEC-UPLC for mAb02 MSP composed of a 90:10 blend of 50CP30C40-D25 and 50CP10C20-D25. After 6 weeks, approximately 85% of the total released mAb02 was still released in its intact form, confirming the compatibility of mAb02 with the polymer and manufacturing procedure used.

[0201] [Table 10]

[0202] Characterization of released mAb02 in vitro by FLS, according to the method described in Example 6, showed that the intrinsic fluorescent tryptophan spectrum of mAb02 was unaffected after release from microsphere formulations composed of various 50CP30C40-D25 / 50CP10C20-D25 polymer blends.

[0203] Figure 19 shows the concentration of total and intact mAb02 (measured by SEC-UPLC) and the percentage of correctly folded mAb02 (determined by fluorescence spectroscopy (FLS) according to the method shown in Figure 9C) in in vitro release samples taken at various time points during in vitro release testing of mAb02-loaded microsphere formulations composed of various 50CP30C40-D25 / 50CP10C20-D25 polymer blends (Figures 19A-G). Within these different polymer blend ratios, the hydrophilicity of the polymer blends decreases from A to G.

[0204] The percentage of intact mAb02 determined by SEC-UPLC was typically 80-90%. The percentage of correctly folded mAb02 determined by FLS varied from 65-100%, independent of the release period, and was not affected by the composition of the polymer blend.

[0205] Taken together, the data obtained from SEC-UPLC and fluorescence spectroscopy indicate that the release of intact and correctly folded mAb02 for periods of up to 3 months is feasible, demonstrating the compatibility of the faster degrading [PCL-PEG-PCL]-b-[PDO]-based multiblock copolymer and microencapsulation process with the monoclonal antibody mAb02, a large protein of 144.9 kDa. The present disclosure may be configured as follows. [Section 1] 1. A dosage form for extended release of an antibody or antigen-binding fragment thereof, comprising: (a) an antibody or an antigen-binding fragment thereof; (b) Biodegradable multiblock copolymer matrix Including, wherein the antibody or antigen-binding fragment thereof is present in the multi-block copolymer matrix; wherein the biodegradable multi-block copolymer comprises one or more biodegradable, phase-separated, thermoplastic multi-block copolymers comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; The multi-block copolymer has a T of about 37° C. or less under physiological conditions. g and T of about 50°C to about 110°C m having; the segments are linked by a multifunctional chain extender; the segments are randomly distributed along the polymer chain; and the prepolymer (B) segment comprises an XYX triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by about 7 or more p-dioxanone monomer units; The dosage form. [Section 2] Item 1. The dosage form according to Item 1, wherein the multiblock copolymer matrix releases less than about 3% to about 40% of the protein or antibody or antigen-binding fragment thereof, based on the total weight of the protein or antibody or antigen-binding fragment thereof present in the multiblock copolymer matrix, within about 24 hours. [Section 3] Item 3. The dosage form according to Item 1 or 2, wherein the antibody or antigen-binding fragment thereof comprises one or more selected from the group consisting of a monoclonal antibody, a bispecific antibody, a trispecific antibody, an antibody-drug conjugate, an antigen-binding fragment including Fab, F(ab'), F(ab'), a single-chain antibody (scFv), and a bivalent single-chain antibody. [Section 4] Item 4. The dosage form according to any one of Items 1 to 3, wherein the antigen-binding fragment comprises at least two paired domains. [Section 5] Item 5. The dosage form according to any one of Items 1 to 4, wherein the antibody or antigen-binding fragment thereof has a molecular weight of about 70 kDa or more, for example, about 75 kDa or more, or about 80 kDa or more. [Section 6] A dosage form for the extended release of a protein of about 70 kDa or greater, comprising: (a) proteins of approximately 70 kDa or larger; (b) Biodegradable multiblock copolymer matrix Including, wherein the protein is present in the multi-block copolymer matrix; wherein the biodegradable multi-block copolymer comprises one or more biodegradable, phase-separated, thermoplastic multi-block copolymers comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; where: The multi-block copolymer has a T of about 37° C. or less under physiological conditions. g and T of about 50°C to about 110°C m having; the segments are linked by a multifunctional chain extender; the segments are randomly distributed along the polymer chain; and the prepolymer (B) segment comprises an XYX triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by about 7 or more p-dioxanone monomer units; The dosage form. [Section 7] Item 7. The dosage form according to Item 6, wherein the protein comprises one or more selected from the group consisting of an Fc-fusion protein, an antibody-drug conjugate (ADC), a full-length immunoglobulin, a coagulation factor, a growth factor, a hormone, a cytokine, and an enzyme. [Section 8] Item 8. The dosage form according to any one of Items 1 to 7, wherein the prepolymer (B) segment comprises poly(p-dioxanone) in an amount of about 70% or more, for example, about 80% or more, about 85% or more, about 90% or more, or about 95% or more of the total weight of the prepolymer (B) segment. [Section 9] Item 9. The dosage form according to any one of Items 1 to 8, wherein the block length of the poly(p-dioxanone) segment X, expressed in terms of p-dioxanone monomer units, is about 7 to about 35, for example, about 8 to about 30, about 9 to about 25, about 10 to about 20, or about 12 to about 15. [Section 10] Item 10. The dosage form according to any one of Items 1 to 9, wherein the polymerization initiator Y is a polymerization initiator selected from the group consisting of an aliphatic diol having about 2 to about 8 carbon atoms, such as ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 1,3-butanediol, 2,3-butanediol, diethylene glycol, dipropylene glycol, triethylene glycol, poly(ethylene glycol), 1,5-pentanediol, 1,6-hexanediol, neopentyl glycol, hydrogenated bisphenol A, and glycerol. [Section 11] The prepolymer (B) segment has a number average molecular weight M of about 1300 g / mol or more to about 7200 g / mol, for example, about 1500 g / mol or more to about 5000 g / mol, about 2000 g / mol or more to about 4500 g / mol, about 2200 g / mol or more to about 4000 g / mol, or about 2500 g / mol or more to about 3200 g / mol. n Item 11. The dosage form according to any one of items 1 to 10, comprising: [Section 12] The prepolymer (B) segment has a weight average molecular weight M of about 1800 g / mol to about 10080 g / mol, for example, about 2100 g / mol to about 7000 g / mol, about 2600 g / mol to about 6300 g / mol, or about 3000 g / mol to about 5600 g / mol. w Item 12. The dosage form according to any one of items 1 to 11, comprising: [Section 13] Item 13. The dosage form according to any one of Items 1 to 12, wherein the content of the prepolymer (B) segment in the copolymer is about 5% to about 95% of the total weight of the multi-block copolymer, for example, about 10% to about 90%, about 25% to about 70%, or about 30% to about 50%. [Section 14] Item 14. The dosage form according to any one of Items 1 to 13, wherein the prepolymer (A) segment comprises a reaction product of glycolide, lactide (D and / or L), ε-caprolactone, δ-valerolactone, trimethylene carbonate, tetramethylene carbonate, 1,5-dioxepan-2-one, 1,4-dioxan-2-one (p-dioxanone), and / or a cyclic anhydride, such as oxepane-2,7-dione. [Section 15] Item 15. The dosage form according to any one of Items 1 to 14, wherein the prepolymer (A) comprises a reaction product of glycolide, lactide (D and / or L), and / or ε-caprolactone. [Section 16] Item 16. The dosage form according to any one of Items 1 to 15, wherein about 30% or more, for example, about 40% to about 95%, about 50% to about 90%, or about 60% to about 85% of the total weight of the prepolymer (A) is derived from a water-soluble polymer. [Section 17] Item 17. The dosage form according to any one of items 1 to 16, wherein the water-soluble polymer comprises one or more selected from the group consisting of polyethers, such as poly(ethylene glycol) (PEG), poly(tetramethylene oxide) (PTMO), poly(propylene glycol) (PPG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(vinylcaprolactam), poly(hydroxyethyl methacrylate) (poly-(HEMA)), poly(phosphazene), poly(orthoester), poly(orthoester amide), or copolymers of any of these polymers. [Section 18] Item 18. The dosage form according to any one of items 1 to 17, wherein the water-soluble polymer comprises one or more selected from the group consisting of poly(ethylene glycol), poly(tetramethylene oxide), poly(propylene glycol), poly(vinyl alcohol), poly(vinyl pyrrolidone), and poly(vinyl caprolactam). [Section 19] Item 19. The dosage form according to any one of Items 1 to 18, wherein the water-soluble polymer comprises or is poly(ethylene glycol). [Section 20] Item 20. The dosage form according to any one of items 1 to 19, wherein the prepolymer (A) segment comprises poly(ε-caprolactone)-co-PEG-co-poly(ε-caprolactone). [Section 21] The prepolymer (A) segment has a number average molecular weight M of about 500 g / mol to about 10,000 g / mol, for example, about 700 g / mol to about 9,000 g / mol, about 1,000 g / mol to about 8,000 g / mol, about 2,000 g / mol to about 8,000 g / mol, about 3,000 g / mol to about 8,000 g / mol, or about 4,000 g / mol to about 8,000 g / mol. n Item 21. The dosage form according to any one of items 1 to 20, comprising: [Section 22] Item 22. The dosage form according to any one of Items 1 to 21, wherein the content of the prepolymer (A) in the multi-block copolymer is about 5% to about 95%, for example, about 10% to about 90%, about 30% to about 75%, or about 50% to about 70%, based on the total weight of the multi-block copolymer. [Section 23] Item 23. The dosage form according to any one of items 1 to 22, wherein the polyfunctional chain extender is a difunctional aliphatic chain extender. [Section 24] Item 24. The dosage form according to any one of items 1 to 23, wherein the polyfunctional chain extender is a diisocyanate, such as 1,4-butane diisocyanate or 1,6-hexane diisocyanate. [Section 25] Item 25. The dosage form according to any one of items 1 to 24, wherein the dosage form is in the form of a microsphere, microparticle, nanosphere, nanoparticle, rod, implant, film, sheet, tube, membrane, mesh, fiber, plug, coating, or gel. [Section 26] Item 26. The dosage form according to any one of items 1 to 25, wherein the dosage form is in the form of a microsphere. [Section 27] Item 27. The dosage form according to any one of Items 1 to 26, wherein the microspheres are in the form of microspheres having an average diameter of about 1 μm to about 200 μm, for example, about 10 μm to about 150 μm, or about 20 μm to about 100 μm. [Section 28] Item 28. A method for administering the dosage form according to any one of Items 1 to 27, wherein the multiblock copolymer matrix releases less than about 20%, preferably less than about 10%, more preferably less than 5% of the protein or antibody or antigen-binding fragment thereof, relative to the total weight of the protein or antibody or antigen-binding fragment thereof present in the multiblock copolymer matrix, within about 24 hours. [Section 29] 29. The method of paragraph 28, wherein the administration is via intradermal, transdermal, intramuscular, subcutaneous, intravitreal, intraarticular, or intratumoral injection. [Section 30] A method for treating a subject in need of a protein or antibody, or an antigen-binding fragment thereof, comprising administering the dosage form according to any one of Items 1 to 27. [Section 31] Item 28. The dosage form according to any one of items 1 to 27, for use in treating a subject in need of the protein or antibody, or an antigen-binding fragment thereof, the method comprising administering the dosage form to the subject. [Section 32] 28. A method of using biodegradable polymer microspheres, preferably biodegradable polymer microspheres as defined in any one of items 1 to 27, for the sustained release of proteins or antibodies or antigen-binding fragments thereof.

Claims

1. 1. A dosage form for extended release of an antibody or antigen-binding fragment thereof, comprising: (a) an antibody or antigen-binding fragment thereof; (b) Biodegradable multi-block copolymer matrix Including, wherein the antibody or antigen-binding fragment thereof is present in the multi-block copolymer matrix; wherein the biodegradable multi-block copolymer comprises one or more biodegradable, phase-separated, thermoplastic multi-block copolymers comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; The multi-block copolymer has a T of 37° C. or less under physiological conditions. ｇ and T of 50°C to 110°C ｍ having the segments are linked by a multifunctional chain extender; the segments are randomly distributed along the polymer chain; and the prepolymer (B) segment comprises an X-Y-X triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by 7 or more p-dioxanone monomer units; The dosage form is in the form of a microsphere, microparticle, or nanoparticle. The dosage form.

2. The dosage form of claim 1, wherein the multi-block copolymer matrix releases less than 1 / 7 of the antibody or antigen-binding fragment within 24 hours.

3. 3. The dosage form of claim 1 or 2, wherein the multi-block copolymer matrix releases less than 3% of the antibody or antigen-binding fragment thereof relative to the total weight of the antibody or antigen-binding fragment thereof present in the multi-block copolymer matrix within 24 hours.

4. The antibody or antigen-binding fragment thereof may be a monoclonal antibody, a bispecific antibody, a trispecific antibody, an antibody-drug conjugate, Fab, F(ab'), F(ab') ２ The dosage form according to any one of claims 1 to 3, comprising one or more selected from the group consisting of an antigen-binding fragment comprising: a single-chain antibody (scFv), and a bivalent single-chain antibody.

5. The dosage form of any one of claims 1 to 4, wherein the antigen-binding fragment comprises at least two paired domains.

6. The dosage form of any one of claims 1 to 5, wherein the antibody or antigen-binding fragment thereof has a molecular weight of 70 kDa or greater.

7. A dosage form for the extended release of a protein of 70 kDa or greater, comprising: (a) a protein of 70 kDa or greater; (b) Biodegradable multi-block copolymer matrix Including, wherein the protein is present in the multi-block copolymer matrix; wherein the biodegradable multi-block copolymer comprises one or more biodegradable, phase-separated, thermoplastic multi-block copolymers comprising at least one amorphous hydrolyzable prepolymer (A) segment and at least one semi-crystalline hydrolyzable prepolymer (B) segment; where: The multi-block copolymer has a T of 37° C. or less under physiological conditions. ｇ and T of 50°C to 110°C ｍ having the segments are linked by a multifunctional chain extender; the segments are randomly distributed along the polymer chain; and the prepolymer (B) segment comprises an X-Y-X triblock copolymer, where Y is a polymerization initiator and X is a poly(p-dioxanone) segment having a block length represented by 7 or more p-dioxanone monomer units; The dosage form is in the form of a microsphere, microparticle, or nanoparticle. The dosage form.

8. 8. The dosage form of claim 7, wherein the protein comprises one or more selected from the group consisting of an Fc-fusion protein, an antibody drug conjugate (ADC), a full-length immunoglobulin, a coagulation factor, a growth factor, a hormone, a cytokine, and an enzyme.

9. The dosage form according to any one of claims 1 to 8, wherein the prepolymer (B) segment contains poly(p-dioxanone) in an amount of 70% or more of the total weight of the prepolymer (B) segment.

10. 10. The dosage form of any one of claims 1 to 9, wherein the block length of the poly(p-dioxanone) segment X, expressed in terms of p-dioxanone monomer units, is 7 to 35.

11. 11. The dosage form according to any one of claims 1 to 10, wherein the polymerization initiator Y is an aliphatic diol having 2 to 8 carbon atoms.

12. The dosage form according to any one of claims 1 to 11, wherein the polymerization initiator Y is selected from the group consisting of ethylene glycol, 1,2-propanediol, 1,3-propanediol, 1,4-butanediol, 1,3-butanediol, 2,3-butanediol, diethylene glycol, dipropylene glycol, triethylene glycol, poly(ethylene glycol), 1,5-pentanediol, 1,6-hexanediol, neopentyl glycol, hydrogenated bisphenol A, and glycerol.

13. The prepolymer (B) segment has a number average molecular weight M of 1300 g / mol or more to 7200 g / mol. ｎ The dosage form according to any one of claims 1 to 12, having

14. The prepolymer (B) segment has a weight average molecular weight M of 1800 g / mol to 10080 g / mol. ｗ The dosage form according to any one of claims 1 to 13, having

15. The dosage form according to any one of claims 1 to 14, wherein the content of the prepolymer (B) segment in the copolymer is 5% to 95% of the total weight of the multi-block copolymer.

16. 16. The dosage form of any one of claims 1 to 15, wherein the prepolymer (A) segment comprises a reaction product of glycolide, lactide (D and / or L), ε-caprolactone, δ-valerolactone, trimethylene carbonate, tetramethylene carbonate, 1,5-dioxepan-2-one, 1,4-dioxan-2-one (p-dioxanone), and / or a cyclic anhydride.

17. 17. The dosage form of claim 16, wherein the cyclic anhydride is oxepane-2,7-dione.

18. The dosage form of any one of claims 1 to 17, wherein the prepolymer (A) comprises a reaction product of glycolide, lactide (D and / or L), and / or ε-caprolactone.

19. The dosage form according to any one of claims 1 to 18, wherein 30% or more of the total weight of the prepolymer (A) is derived from a water-soluble polymer.

20. 20. The dosage form of claim 19, wherein the water-soluble polymer comprises one or more selected from the group consisting of polyethers.

21. 20. The dosage form of claim 19, wherein the water-soluble polymer comprises one or more selected from the group consisting of poly(ethylene glycol) (PEG), poly(tetramethylene oxide) (PTMO), poly(propylene glycol) (PPG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(vinylcaprolactam), poly(hydroxyethyl methacrylate) (poly-(HEMA)), poly(phosphazene), poly(orthoester), poly(orthoester amide), and copolymers of any of these polymers.

22. 20. The dosage form of claim 19, wherein the water-soluble polymer comprises one or more selected from the group consisting of poly(ethylene glycol), poly(tetramethylene oxide), poly(propylene glycol), poly(vinyl alcohol), poly(vinylpyrrolidone), and poly(vinylcaprolactam).

23. 20. The dosage form of claim 19, wherein the water-soluble polymer comprises or is poly(ethylene glycol).

24. The dosage form of any one of claims 1 to 23, wherein the prepolymer (A) segment comprises poly(ε-caprolactone)-co-PEG-co-poly(ε-caprolactone).

25. The prepolymer (A) segment has a number average molecular weight M of 500 g / mol to 10,000 g / mol. ｎ The dosage form according to any one of claims 1 to 24, having

26. The dosage form according to any one of claims 1 to 25, wherein the content of the prepolymer (A) in the multi-block copolymer is 5% to 95% based on the total weight of the multi-block copolymer.

27. 27. The dosage form of any one of claims 1 to 26, wherein the multifunctional chain extender is a difunctional aliphatic chain extender.

28. 28. The dosage form of any one of claims 1 to 27, wherein the multifunctional chain extender is a diisocyanate.

29. The dosage form of any one of claims 1 to 28, wherein the multifunctional chain extender is 1,4-butane diisocyanate or 1,6-hexane diisocyanate.

30. 30. The dosage form of any one of claims 1 to 29, wherein the dosage form is in the form of a microsphere.

31. 31. The dosage form of any one of claims 1 to 30, wherein the dosage form is in the form of microspheres having an average diameter of 1 μm to 200 μm.

32. 32. The dosage form of any one of claims 1 to 31, wherein the dosage form is administered to a subject and the multi-block copolymer matrix releases less than 20% of the protein or antibody or antigen-binding fragment thereof relative to the total weight of the protein or antibody or antigen-binding fragment thereof present in the multi-block copolymer matrix within 24 hours.

33. 33. The dosage form of claim 32, wherein the administration is via intradermal, transdermal, intramuscular, subcutaneous, intravitreal, intraarticular, or intratumoral injection.

34. 32. The dosage form of any one of claims 1 to 31 for use in treating a subject in need of the protein or antibody or antigen-binding fragment thereof.

35. 32. The dosage form of any one of claims 1 to 31 for use in treating a subject in need of a protein or antibody or antigen-binding fragment thereof, comprising administering the dosage form to the subject.

36. A dosage form according to any one of claims 1 to 31 for sustained release of a protein or antibody or antigen-binding fragment thereof.

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