Stabilized biopolymer compositions, their preparation and uses

Abstract

The present invention relates to the field of stabilized biopolymer compositions, and in particular to stabilized biopolymer compositions comprising a liquid mixture, particularly an aqueous solution, of at least one biopolymer component and at least one stabilizing surfactant. More specifically, the present invention relates to stabilized biopolymer compositions comprising an ethylene oxide / butylene oxide block copolymer, which can impart superior properties to the biopolymer composition compared to currently known formulations, particularly with respect to aggregation tendency, hemolytic activity, and solubility. The present invention further relates to the block copolymer, a method for preparing the stabilized composition, and the use of the block copolymer to stabilize an aqueous composition of the biopolymer. The present invention further relates to the stabilized composition for use in medicine, particularly for diagnostic and / or therapeutic applications. The present invention further relates to an essentially dry biopolymer composition comprising the biopolymer and the block copolymer, and a method for preparing the essentially dry composition. The present invention further relates to the essentially dry biopolymer composition for use in medicine, particularly for diagnostic and / or therapeutic applications. The compositions may be formulated as pharmaceutical compositions that can be delivered via a suitable route of administration, such as oral, rectal, transmucosal, topical, ophthalmic, otorhinological, or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and, in some cases, epidural, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

Description

[Technical Field]

[0001] The present invention relates to the field of stabilized biopolymer compositions, and in particular to stabilized biopolymer compositions comprising a liquid mixture, in particular an aqueous solution, of at least one biopolymer component and at least one stabilizing surfactant.

[0002] More specifically, the present invention relates to stabilized biopolymer compositions comprising ethylene oxide / butylene oxide block copolymers that can impart superior properties to the biopolymer compositions compared to currently known formulations, particularly with respect to aggregation tendency, hemolytic activity and solubility.

[0003] The present invention further relates to said block copolymers, to methods for preparing said stabilized compositions and to the use of said block copolymers for stabilizing aqueous compositions of said biopolymers.

[0004] The present invention further relates to said stabilized compositions for use in medicine, in particular for diagnostic and / or therapeutic applications.

[0005] The present invention further relates to an essentially dry biopolymer composition comprising said biopolymer and said block copolymer, and a method for preparing said essentially dry composition.

[0006] The present invention further relates to said essentially dry biopolymer composition for use in medicine, in particular for diagnostic and / or therapeutic applications.

[0007] The compositions may be formulated as pharmaceutical compositions that can be delivered via a suitable route of administration, such as oral, rectal, transmucosal, topical, ophthalmic, otorhinological, or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and, in some cases, epidural, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection. [Background technology]

[0008] Due to their favorable specificity and efficiency, protein and cell-based therapeutics have been widely applied in the treatment of a wide range of different diseases, such as cancer and autoimmune disorders, with antibodies being of particular importance.

[0009] However, due to its complex three-dimensional structure and the presence of many functional groups, its use is hindered by its tendency to aggregate during storage.

[0010] Antibodies, and proteins in general, carry safety risks as they are potentially immunogenic and can cause serious side effects.

[0011] Maintaining antibody tertiary structure and function while preventing aggregation and surface adsorption and ensuring the long-term stability of antibody formulations remains challenging.

[0012] Currently, polysorbate 20 (POE sorbitan monolaurate, PS20) and polysorbate 80 (POE sorbitan monooleate, PS80) and poloxamer 188 are the most frequently used surfactants in commercial biopharmaceutical formulations.

[0013] Polysorbates are highly effective but difficult to manage due to their complex composition. Their multicomponent nature makes them difficult to describe and control, and monitoring stability and degradation products is particularly challenging. In general, oxidative or hydrolytic degradation is common, resulting in the formation of reactive impurities. Drug formulation stability is reduced as polysorbate concentration decreases and reactive degradation products with aldehyde or peroxide structures induce chemical changes or degradation of biomolecules. Free fatty acids and altered protein structures can induce the formation of proteinaceous particles, which are known to cause immunogenic reactions and therefore pose a concern for patient safety. (Dubey S, Giovannini R.Stability of Biologics and the Quest for Polysorbate Alternatives.Trends Biotechnol.2021 Jun; 39(6):546-549.doi: 10.1016 / j.tibtech.2020.10.007.Epub 2020 Oct 30.PMID: 33139073.)

[0014] Furthermore, these surfactants, due to their similarity to cell membrane lipids, can interact and disrupt cellular equilibrium in red blood cells, causing reorganization of membrane components and disruption of cellular homeostasis, ultimately resulting in hemolysis. Polysorbates have also been described to induce immunogenic responses, so safer yet equally effective stabilizers for parenteral formulations are highly desirable for the industry. (Maggio, Edward. 2017. "Reducing or Eliminating Polysorbate Induced Anaphylaxis and Unwanted Immunogenicity in Biotherapeutics—Review Article." Journal of Excipients and Food Chemicals 8(3).)

[0015] Therefore, the chemical stability of the surfactants used in the formulation is an important aspect to be considered in the preparation of the particular excipient formulation selected.

[0016] Poloxamer 188, a nonionic surfactant with a more defined chemical structure and better chemical stability (no ester bonds), has been used as an alternative to polysorbates. However, poloxamer 188 is generally not as effective as a stabilizer and therefore typically requires a higher concentration to achieve comparable performance. In many cases, the performance of poloxamer 188 cannot match that of polysorbates, for example, in the presence of residual silicone oil traces in prefilled syringes (Grapentin, C. et al.: Protein-Polydimethylsiloxane Particles in Liquid Vial Monoclonal Antibody Formulations Containing Poloxamer 188. Journal of Pharmaceutical Sciences, 2020).

[0017] The challenge of stabilization increases when antibodies are further engineered, coupled to other proteins, or conjugated to small molecules exhibiting different physicochemical properties, such as small hydrophobic molecules and oligonucleotides.

[0018] One example is antibody-fluorochrome conjugates and so-called antibody-drug conjugates (ADCs), which are most commonly used in therapies to treat a wide range of different diseases and as detection reagents in diagnostic applications.

[0019] In this regard, small molecules used for labeling can affect the hydrophobicity and aggregation tendency of antibodies, possibly leading to cluster formation during storage and non-specific binding in imaging applications.

[0020] Block copolymers, characterized by the presence of a short hydrophilic block relative to the length of the hydrophobic block, are known for their ability to form worm-like micelles with high solubilizing capacity for poorly soluble aromatic drugs (Colloid Stability and Application in Pharmacy, edited by Tharwat F. Tadros, Weinheim 2007).

[0021] U.S. Pat. No. 5,300,295 describes the use of polyoxyalkylenes, particularly polyethylene oxide and polybutylene oxide block copolymers exhibiting sol-gel properties, for use in drug delivery systems or for use in ophthalmic applications as surgical adjuncts.

[0022] WO 1998 / 029127 A1 describes the use of polyethylene oxide and polybutylene oxide block copolymers in the prevention of post-operative adhesion formation / reformation in mammals following injury to organs of a body cavity.

[0023] WO 2003 / 024425 A1 teaches the use of surfactants as possible surface stabilizers adsorbed onto the surface of nanoparticulate insulin compositions intended to enhance insulin delivery in high-dose dosage forms. A long list of ionic and nonionic surfactants is described. Possible surface stabilizers include polyoxyalkylene block copolymers (e.g., polyethylene- or polybutylene oxide triblock copolymers), without any preference for such compounds. Indeed, in the experimental section, it was reported that preferred stabilizers selected from PVP / sodium deoxycholate, Pluronic® F68 / sodium deoxycholate, and cationic surfactants designated S1001 and S1004 were adsorbed onto the surface of ball-milled nanoparticulate insulin, and that "in some instances" a reduction in insulin particle size was observed one week after milling. The surface stabilizer acts as a steric barrier to other solid insulin particles, thereby preventing aggregation and particle size growth and protecting the insulin from degradation. This document does not describe the preparation of insulin solutions, and does not investigate the hemolytic activity or water solubility of such copolymers. Stabilized biopolymer solutions are not mentioned therein.

[0024] U.S. Patent No. 5,587,143 describes nanoparticles of poorly soluble diagnostic or therapeutic agents, the surface of which is adsorbed a triblock polymer of the PEO-PBuO-PEO type, with a molecular weight of 3,000-5,000 and an EO content of 60% by weight. Butronics B20-3000 and B20-5000 are mentioned. There is no mention of biopolymer solutions, nor of stabilized biopolymer solutions. This document does not investigate such copolymers with regard to further important characteristics, such as their hemolytic activity, water solubility, or surface tension.

[0025] European Patent Application Publication No. A-0179583 discloses anhydrous compositions of poorly water-soluble small molecule drugs mixed with a surfactant. The choice of surfactant is considered "uncritical." Examples include, among others, polysorbate 20 and 80, Pluronic 25R4, and a surfactant designated Butronic L-1, of unknown composition and molecular weight. This document does not investigate such copolymers with respect to further important characteristics, such as their hemolytic activity, water solubility, or surface tension. International Publication No. WO 2017 / 112828 A1 teaches the use of block copolymers, such as di- or triblock copolymers containing polyethylene oxide units, as stabilizers for encapsulating water-soluble biomolecules. Polyethylene or polybutylene oxide copolymers are presented as possible candidates suitable for forming encapsulation shells. Summary of the Invention [Problem to be solved by the invention]

[0026] The first problem that the present invention aims to solve therefore relates to identifying highly water-soluble polymers that exhibit surface activity in the range of polysorbates, but that, in contrast to polysorbates, exhibit higher chemical stability (no ester bonds) and no hemolytic activity, thus making it possible to provide liquid biopolymer compositions, in particular liquid antibody compositions, with improved long-term stability, in particular in combination with low or no hemolytic activity.

[0027] Surprisingly, the above-mentioned problems can be solved by providing Butronics (EO-BuO-EO block copolymers) having a specific range of molecular weight and ethylene oxide content (EO%). [Means for solving the problem]

[0028] More specifically, the above mentioned problems could be solved by providing Butronics which exhibit a molecular weight of more than 2 KDa and an EO% content of at least 60%.

[0029] These Butronics are at least Two, or more specifically all, of the following characteristics: High water solubility; High chemical stability; improving the stabilizing properties of antibody formulations with respect to their ability to inhibit antibody surface inhibition and / or aggregation; and / or Low hemolytic activity It was found that the above characteristics were simultaneously

[0030] Butronics, which have approximately 60% by weight EO based on the total weight of ethylene and butylene oxide monomer units and a calculated molecular weight of 5,900 Da or greater, are also particularly well able to dissolve small molecules compared to Butronics of different structures and therefore may be particularly useful as stabilizers for ADCs and antibody-based probes for imaging applications involving such small molecules conjugated to antibody molecules.

[0031] Butronics, which contain about 50-85% by weight EO based on the total weight of their ethylene and butylene oxide monomer units and have a calculated molecular weight of 3,000 Da to about 8,500 Da, are particularly suitable for stabilizing antibody-based formulations. [Brief explanation of the drawings]

[0032] [Figure 1] 1 is a description of the selection criteria for certain exemplified Butronics with respect to good water solubility, low hemolytic activity, and / or long-term stability. DETAILED DESCRIPTION OF THE INVENTION

[0033] A. Abbreviation ADC Antibody Drug Conjugate APC antibody payload conjugates API Active Pharmaceutical Ingredient BuO or OBu butylene oxide CDCl3 deuterated chloroform ECD Equivalent Circle Diameter EDTA Ethylenediaminetetraacetic acid EO or OE Ethylene Oxide HPLC High Performance Liquid Chromatography 1 H-NHR proton nuclear magnetic resonance IgG: immunoglobulin class G PVDF Polyvinylidene Fluoride MFI Microflow Imaging PLA Polylactic Acid PLGA Poly(lactic-co-glycolic acid) PS20 Polysorbate 20 P188 Poloxamer 188 PS80 Polysorbate 80 PBS Phosphate-buffered saline RBC red blood cells

[0034] B. Definition 1. Overview Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. The meaning and scope of the terms should be clear, but in the event of any potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless the context requires a different interpretation, singular terms shall include the plural and plural terms shall include the singular.

[0035] In connection with the description provided herein and the appended claims, the use of "or" means "and / or" unless specifically stated otherwise. Similarly, "comprise," "comprises," "comprising," "include," and "including" are interchangeable and are not intended to be limiting.

[0036] It is further understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific instances, the embodiments may alternatively be described using "consisting essentially of" or "consisting of."

[0037] The term "about" or "approximately" indicates a potential variation of ±25%, particularly ±15% or ±10%, more particularly ±5%, ±2% or ±1% of the stated value.

[0038] The term "substantially" refers to a range of values ​​such as approximately 80 to 100%, for example 85 to 99.9%, specifically 90 to 99.9%, more specifically 95 to 99.9%, or 98 to 99.9%, especially 99 to 99.9%.

[0039] "Mainly" refers to a proportion in the range of more than 50%, for example, in the range of 51 to 100%, particularly in the range of 75 to 99.9%, more specifically, 85 to 98.5%, 95 to 99%, etc.

[0040] Where the present disclosure refers to features, parameters and ranges thereof of different priority (including general, not explicitly preferred, features, parameters and ranges thereof), unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof is encompassed by the disclosure herein, regardless of their respective preferences.

[0041] 2.Chemical terms The term "halogen" denotes in each case a fluorine, bromine, chlorine or iodine radical, in particular a fluorine, chlorine or bromine radical.

[0042] "Alkyl" refers to a straight-chain or branched alkyl group having 1 to 10, in particular 1 to 8, more particularly 1 to 4, or 1 to 2 carbon atoms. Examples are methyl, ethyl, n-propyl, n-butyl, n-hexyl, n-heptyl or n-octyl.

[0043] "Alkylene" refers to a straight or branched chain hydrocarbon bridging group having 1 to 22, or 2 to 22, 1 to 6, 3 to 6, 2, or 4 carbon atoms. Non-limiting examples include -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2) 10 -, -(CH2) 15 -, -(CH2) 20 -, -(CH2) 22 - and their respective branched analogs.

[0044] Optionally, the alkylene group may be interrupted by one or more heteroatoms such as oxygen.

[0045] "Alkyleneoxy" refers to a radical of the formula -RO- where R is a straight or branched alkylene group having 1 to 22, or 2 to 22, 1 to 6, 3 to 6, 2 or 4 carbon atoms, as defined herein.

[0046] "Polyalkylene oxide" refers to a group in which at least two identical or different repeating units of an alkyleneoxy group as defined above are covalently linked.

[0047] A "block copolymer" defines a macromolecular entity characterized by at least two alternating, structurally distinct polymer blocks; each block consists essentially of structurally similar, in particular identical, repeating monomer units. Optionally, within the structure of said block copolymer, there may be polyvalent, e.g., divalent or trivalent, organic or inorganic moieties, chemical moieties connecting two or more alternating blocks.

[0048] "Butronics" should be broadly construed and generally refers to block copolymers of alternating butylene oxide and ethylene oxide blocks, with an ethylene oxide molar content (EO%) of about 10 to less than 100 mole %, and a molecular weight of 1,000 to 15,000 g / mol. Specific Butronics within the meaning of the present invention are exemplified in the general and experimental sections below.

[0049] "Static surface tension" within the meaning of the present invention should preferably be interpreted as the amount of energy per unit surface area required to cause a deformation, such as a local increase in the surface of a liquid sample exhibiting a given concentration of solute in thermodynamic equilibrium at a given temperature. "Static surface tension" is measured in millinewtons per meter (mN / m) and is determined by the pendant drop method.

[0050] "Ethylene oxide content" (EO%) and "butylene oxide content" (BuO%) within the meaning of the present invention are described herein as either the mole percent (mol%) or weight percent (wt%) of ethylene oxide and butylene oxide monomer units within a given block copolymer defined herein. Unless otherwise specified, "%" refers to weight percent.

[0051] The term "EO%" refers to a weight percent value calculated from the atomic masses of all atoms of the copolymer molecule of Formula 1 according to the following formula: EO%=[(ΣEO mass) / (ΣEO mass + ΣBuO mass + starting material X mass)]*100

[0052] The term "calculated molecular weight of x to y g / mol" includes integers x, y, and any integer between x and y. For example, a molecular weight of "2.500 to 12.500 g / mol" includes at least the following integers: 2,500, 2,600, 2,700, 2,800, 2,900; 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900; 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900; 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900; 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900; 7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900; 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600, 8,700, 8,800, 8,900; 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900; 10,000, 10,100, 10,200, 10,300, 10,400, 10,500, 10,600, 10,700, 10,800, 10,900; 11,000, 11,100, 11,200, 11,300, 11,400, 11,500, 11,600, 11,700, 11,800, 11,900; 12,000, 12,100, 12,200, 12,300, 12,400, and 12,500.

[0053] "Hemolysis" within the meaning of the present invention relates to the tendency of a given excipient to cause the breakdown of cells, in particular red blood cells, with the consequent release of intracellular components.

[0054] The "equivalent circular diameter" (ECD) of a given non-spherical particle is defined as the diameter of a spherical particle that gives the same geometric, optical, electrical, or aerodynamic behavior as the non-spherical particle under investigation.

[0055] Microflow imaging (MFI) is an analytical method that automatically collects microscopic images of a sample passing through a flow cell, fast enough to analyze thousands of particles in a few minutes. Particle size is reported as the equivalent circular diameter (ECD), which is the diameter of a circle with the same projected area as the particle. In addition, information on particle number, shape, and transparency can be obtained from the analysis software.

[0056] 3.Biochemistry terminology The term "biopolymer" as used herein encompasses oligopeptides, polypeptides, proteins, as defined below, any type of antibody molecule as defined below or fragments or derivatives thereof, glycosylated proteins, proteoglycans, oligo- and polynucleotides, DNA and RNA molecules, oligosaccharides, polysaccharides, and adducts or conjugates of such biopolymers, particularly antibodies, together with further components selected from payload molecules as further defined below.

[0057] The term "antibody," as used herein, broadly refers to any immunoglobulin (Ig) molecule composed of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivative thereof, that retains the essential epitope-binding properties of an Ig molecule. Such functional fragment, mutant, variant, or derivative antibody formats are known in the art. Non-limiting embodiments thereof are discussed below. A "full-length antibody," as used herein, refers to an Ig molecule comprising four polypeptide chains, two heavy chains and two light chains. The chains are typically linked to each other via disulfide bonds. Each heavy chain is composed of a heavy chain variable region (also referred to herein as a "variable heavy chain," or abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (also referred to herein as a "variable light chain," or abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions are further subdivided into regions of hypervariability called complementarity-determining regions (CDRs) and more conserved, interspersed regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0058] The term "antigen-binding portion of an antibody" (or simply "antibody portion"), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (i.e., an immunogenic product of the invention), i.e., functional fragments of an antibody. It has been shown that the antigen-binding function of an antibody can be performed by one or more fragments of a full-length antibody. Embodiments of such antibodies may also be bispecific, dual-specific, or multispecific, specifically binding to two or more different antigens. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bond at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of one arm of an antibody; (v) a dAb fragment comprising a single variable domain (Ward et al., Nature 341:544-546, 1989; Winter et al., WO 90 / 05144 A1, incorporated herein by reference); and (vi) an isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant techniques with a synthetic linker to produce a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988). Such single-chain antibodies are also encompassed by the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also encompassed.Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains on the same chain, so that the domains pair with the complementary domains on another chain and form two antigen-binding sites (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure 2:1121-1123, 1994). Such antibody-binding moieties are known in the art (Kontermann and Dubel eds., Antibody Engineering, Springer-Verlag, New York, 790 pp., 2001, ISBN 3-540-41354-5).

[0059] The term "antibody," as used herein, also includes antibody constructs. As used herein, the term "antibody construct" refers to a polypeptide comprising one or more antigen-binding moieties of the present invention linked to a linker polypeptide or immunoglobulin constant domain. A linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen-binding moieties. Such linker polypeptides are well known in the art (see, e.g., Holliger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448, 1993; Poljak et al., Structure 2:1121-1123, 1994).

[0060] Immunoglobulin constant domain refers to either a heavy or light chain constant domain. Human IgG heavy and light chain constant domain amino acid sequences are known in the art.

[0061] Furthermore, the binding proteins (e.g., antibodies) of the present invention may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of the binding protein of the present invention with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of a streptavidin core region to generate tetrameric scFv molecules (Kipriyanov et al., Human Antibodies and Hybridomas 6:93-101, 1995) and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov et al., Mol. Immunol. 31:1047-1058, 1994). Antibody portions, such as Fab and F(ab')2 fragments, can be prepared from whole antibodies using conventional techniques, such as papain or pepsin digestion of whole antibodies, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0062] As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities. However, an isolated antibody that specifically binds to an immunogenic product of the present invention may have cross-reactivity to other antigens, such as Aβ globulomers (e.g., Aβ(20-42) globulomers) or other Aβ forms. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals and / or any other targeted Aβ forms.

[0063] The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may contain amino acid residues, for example, in the CDRs, particularly CDR3, not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0064] As used herein, the term "recombinant human antibody" is intended to include all human antibodies that are prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described further in Section B below), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom, TIB Tech. 15:62-70, 1997; Azzazy and Highsmith, Clin. Biochem. 35:425-445, 2002; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (e.g., Taylor, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al. (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies have been subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when transgenic animals of human Ig sequences are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but may not naturally occur within the human antibody germline repertoire in vivo.

[0065] The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0066] The term "CDR-grafted antibody" refers to an antibody that comprises heavy and light chain variable region sequences from one species, but in which the sequences of one or more of the CDR regions of the VH and / or VL have been replaced with CDR sequences from another species, e.g., an antibody having a murine CDR (e.g., CDR3) in which one or more of the murine variable heavy and light chain regions have been replaced with human variable heavy and light chain sequences.

[0067] The terms "Kabat numbering," "Kabat definition," and "Kabat label" are used interchangeably herein. These art-recognized terms refer to a numbering system for amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable regions range from amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 102 for CDR2, and amino acid positions 95 to 65 for CDR3. In the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 97 for CDR2, and amino acid positions 89 to 56 for CDR3.

[0068] As used herein, the terms "acceptor" and "acceptor antibody" refer to an antibody or nucleic acid sequence that provides or encodes at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. In some embodiments, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes the constant regions. In yet other embodiments, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence that provides or encodes one or more of the framework and constant regions. In certain embodiments, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, e.g., at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. In accordance with this embodiment, the acceptor comprises at least one, at least two, at least three, at least four, at least five, or at least 10 amino acid residues that do not occur at one or more specific positions in a human antibody. The acceptor framework regions and / or acceptor constant regions can be derived or obtained from, for example, germline antibody genes, mature antibody genes, functional antibodies (e.g., antibodies known in the art, antibodies in development, or commercially available antibodies).

[0069] As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. Each heavy and light chain variable region has three CDRs, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs present in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently in different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, but also provides precise residue boundaries defining the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) have used the Kabat CDRs to We have found that certain subportions within CDRs adopt nearly identical peptide backbone conformations despite significant diversity at the amino acid sequence level. These subportions are designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" refer to the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs and have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly adhere to any of the above systems and still overlap with the Kabat CDRs, but specific residues or groups of residues, or entire CDRs, may be shortened or extended in light of predictions or experimental results that do not significantly affect antigen binding.The methods used herein can utilize CDRs defined according to any of these systems, with particular embodiments using Kabat- or Chothia-defined CDRs.

[0070] As used herein, the term "canonical" residues refers to residues within a CDR or framework that define a particular canonical CDR structure as defined by Chothia et al. (See J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both of which are incorporated herein by reference). According to Chothia et al., significant portions of the CDRs of many antibodies identify nearly identical peptide backbones, despite great diversity at the amino acid sequence level. Each canonical structure primarily specifies a set of peptide backbone torsion angles for a contiguous segment of amino acid residues that form a loop.

[0071] As used herein, the terms "donor" and "donor antibody" refer to an antibody providing one or more CDRs. In one embodiment, the donor antibody is an antibody from a different species than the antibody from which the framework regions are obtained or derived. In the context of humanized antibodies, the term "donor antibody" refers to a non-human antibody providing one or more CDRs.

[0072] As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of a variable region, excluding the CDRs. The exact definition of a CDR sequence can be determined by different systems, and the meaning of a framework sequence is subject to different interpretations accordingly. The six CDRs (CDRs-L1, -L2, -L3 in the light chain and CDRs-H1, -H2, -H3 in the heavy chain) also divide the framework regions of the light and heavy chains into four subregions (FR1, FR2, FR3, FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Unless a specific subregion is designated as FR1, FR2, FR3, or FR4, the framework region, as otherwise referred to, represents the composite FR within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of the four subregions, and FR represents two or more of the four subregions that make up the framework region.

[0073] Human heavy and light chain acceptor sequences are known in the art.

[0074] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by a non-lymphoid cell that has not undergone the maturation process that results in gene rearrangement and mutation for expression of a specific immunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med Biol. 484:13-30 (2001)). One advantage provided by various embodiments of the present invention arises from the recognition that germline antibody genes are more likely than mature antibody genes to preserve essential amino acid sequence structures characteristic of individuals of a species and, therefore, are less likely to be recognized as of foreign origin when used therapeutically in that species.

[0075] As used herein, the term "critical residues" refers to specific residues in the variable region that have a greater impact on the binding specificity and / or affinity of antibodies, particularly humanized antibodies. Critical residues include, but are not limited to, one or more of the following: residues adjacent to the CDRs, potential glycosylation sites (which may be either N- or O-glycosylation sites), rare residues, residues that can interact with antigens, residues that can interact with the CDRs, canonical residues, contact residues between the heavy and light chain variable regions, residues within the Vernier zone, and residues within the overlap region between the Chothia definition of the variable heavy chain CDR1 and the Kabat definition of the original heavy chain framework.

[0076] As used herein, the term "humanized antibody" refers to an antibody or variant, derivative, analog, or portion thereof that immunospecifically binds to an antigen of interest and comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. As used herein, the term "substantially" in the context of a CDR refers to a CDR having an amino acid sequence that is at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. A humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, FabC, Fv), in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are human immunoglobulin consensus sequences. According to one embodiment, the humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. In some embodiments, a humanized antibody contains both a light chain and at least the variable domain of a heavy chain. The antibody may also include the CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody contains only a humanized light chain. In some embodiments, a humanized antibody contains only a humanized heavy chain. In certain embodiments, a humanized antibody contains only humanized variable domains of the light and / or heavy chain.

[0077] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA, and IgE, and any isotype, including but not limited to IgG1, IgG2, IgG3, and IgG4. The humanized antibody can comprise sequences from multiple classes or isotypes, and particular constant domains can be selected to optimize desired effector functions using techniques well known in the art.

[0078] The framework and CDR regions of a humanized antibody need not correspond exactly to the parental sequences; for example, the donor antibody or consensus framework may be altered by substitution, insertion, and / or deletion of at least one amino acid residue so that the CDR or framework residue at that site does not correspond to either the donor antibody or the consensus framework. However, in one embodiment, such mutations are not extensive. Typically, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in a consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to a sequence formed from the amino acids (or nucleotides) that occur most frequently in a family of related immunoglobulin sequences (see, e.g., Winnaker, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany 1987)). In a family of immunoglobulins, each position in the consensus sequence is occupied by the amino acid that occurs most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0079] As used herein, "Vernier" zone refers to a subset of framework residues that can adjust CDR structure and fine-tune fit to the antigen, as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and can influence the structure of the CDR and the affinity of the antibody.

[0080] The term "antibody" as used herein also includes multivalent binding proteins. The term "multivalent binding protein" is used herein to refer to a binding protein containing two or more antigen-binding sites. Multivalent binding proteins are engineered to have three or more antigen-binding sites and are generally not naturally occurring antibodies. The term "multispecific binding protein" refers to a binding protein capable of binding to two or more related or unrelated targets. As used herein, a dual variable domain (DVD) binding protein is a binding protein containing two or more antigen-binding sites and is a tetravalent or multivalent binding protein. Such DVDs can be monospecific, i.e., capable of binding to one antigen, or multispecific, i.e., capable of binding to two or more antigens. A DVD binding protein containing two heavy chain DVD polypeptides and two light chain DVD polypeptides is referred to as a DVD Ig. Each half of a DVD Ig contains a heavy chain DVD polypeptide and a light chain DVD polypeptide and two antigen-binding sites. Each binding site contains a heavy chain variable domain and a light chain variable domain, with a total of six CDRs per antigen-binding site involved in antigen binding. DVD-binding proteins and methods for making DVD-binding proteins are described in US Patent Application No. 11 / 507,050, which is incorporated herein by reference.

[0081] The term "labeled binding protein," as used herein, refers to a binding protein that incorporates a label that provides for the identification of the binding protein. Similarly, the term "labeled antibody," as used herein, refers to an antibody that incorporates a label that provides for the identification of the antibody. In one aspect, the label is the incorporation of a detectable marker, e.g., a radiolabeled amino acid that is detectable by labeled avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that is detectable by optical or colorimetric methods), or the attachment of a biotin moiety to the polypeptide. Examples of labels for polypeptides include, but are not limited to, radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 111In, 125I, 131I, 177Lu, 166Ho, or 153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), enzyme labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents such as gadolinium chelates.

[0082] The term "antibody," as used herein, also includes antibody conjugates, which refer to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic agent.

[0083] The term "KD" (also referred to as "Kd" or "KD"), as used herein, is intended to refer to the "equilibrium dissociation constant" and refers to the value obtained in a titration measurement at equilibrium or the value obtained by dividing the dissociation rate constant (koff) by the association rate constant (k). The association rate constant (koff), dissociation rate constant (koff), and equilibrium dissociation constant (KD) are used to express the binding affinity of a binding protein (e.g., an antibody) to an antigen. Methods for determining association and dissociation rate constants are well known in the art. The use of fluorescence-based techniques offers high sensitivity and the ability to test samples in physiological buffer at equilibrium. Other experimental approaches and instruments, such as the BIAcore® (Biomolecular Interaction Analysis) assay can be used (e.g., instruments available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, the KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho), can also be used.

[0084] "Internalize" or "internalization" of an immunoglobulin molecule refers to the ability of an immunoglobulin or ADC or APC described herein to bind to a cell surface receptor and, upon binding, induce receptor-mediated endocytosis.

[0085] "Deglycosylated" or "deglycosylation" refers to the partial, especially complete, removal of one or more glycosyl residues from a glycosylated species of a biological molecule, such as a glycosylated immunoglobulin molecule. "Antibody formulation" should be interpreted broadly and generally refers to a product in which the antibody, in liquid or solid form, is mixed with a pharmaceutically acceptable liquid or solid carrier containing organic or inorganic excipients capable of affecting the physicochemical properties of the antibody.

[0086] An "antibody conjugate" refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic agent (i.e., a drug) or payload.

[0087] C. Specific Aspects and Embodiments of the Invention The present invention relates to the following aspects and specific embodiments thereof.

[0088] A first aspect of the present invention relates to a polymerizable composition comprising at least one biopolymer component and a polymerizable compound of general formula 1: H-(-OE) n -(OBu) m -X-(BuO) m -(EO) n -H (1) (In the formula, X is -O-; or represents a divalent organic moiety, in particular a moiety derived from an organic molecule containing two active hydrogen atoms, such as -O-(C2-C4-alkylene)-O-; m's independently represent an integer in the range of 4 to 25, such as 10 to 22, 12 to 18, or an integer selected from 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25; n's each independently represent an integer in the range of 15 to 100, such as 20 to 80, 25 to 70, 30 to 60, or 35 to 65, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30; 31, 32, 33, 34, 35, 36, 37, 38, 39, 40; 41, 42, 43, 44, 45, 46, 47, 48, 49, 50; 51, 52, 53, 54, 55, 56, 57, 58, 59, 60; 61, 62, 63, 64, 65, 66, 67, 68, 69, or 70. with at least one stabilized ethylene oxide / butylene oxide block copolymer; The block copolymers exhibit the following combination of characteristics: a) a calculated molecular weight of 2,500 to 12,500 g / mol, particularly 2,800 to 9,000 g / mol, more particularly 3,000 to 8,000 g / mol, most particularly 3,500 to 8,000 g / mol, or 3,500 to 7,000 g / mol, particularly calculable from the sum of the atomic masses of all atoms in the copolymer molecule of Formula 1; and b) an EO content of more than 50% to 85% by weight, in particular 53 to 85% by weight, or 55 to 80% by weight, more particularly 57 to 75% by weight or 57 to 80% by weight, for example 60 to 70% by weight, each based on the dry weight of the block copolymer, in particular as can be calculated from the atomic masses of all atoms of the copolymer molecule of formula 1.

[0089] The ethylene oxide / butylene oxide block copolymers may also be formally referred to as "triblock polymers" because they contain a central BuO block consisting essentially of BuO monomers flanked by two EO blocks.

[0090] As a particular combination of features a) and b), A calculated molecular weight of 2,500 to 12,500 g / mol and an EO content of more than 50% to 85% by weight, · Calculated molecular weight of 2,800-9,000 g / mol and EO content of 55-85 wt.%; · Calculated molecular weight of 2,800-9,000 g / mol and EO content of 55-80 wt.%; · Calculated molecular weight of 3,000-8,000 g / mol and EO content of 55-80 wt.%; · Calculated molecular weight of 3,500-8,000 g / mol and EO content of 57-80 wt%; A calculated molecular weight of 3,000 to 8,000 g / mol and an EO content of 57 to 75% by weight, or Calculated molecular weight of 3,500-7,000 g / mol and EO content of 60-70 wt% can be mentioned, where in particular the molecular weight in each case can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1, and the EO content in each case can be calculated from the atomic masses of all atoms in the copolymer molecule of formula 1.

[0091] Preferably, the block copolymer exhibits at least one of the following additional characteristics: c) at least 5% by weight, more specifically at least 10% water solubility, based on the total weight of the aqueous solution of the block copolymer; d) an aqueous solution of the block copolymer (measured at a concentration of 0.1 g / l) has a surface tension SFT of less than 60 mN / m to more than 25 mN / m, in particular 53 to 30 mN / m, for example about 35, about 40, about 45, or about 50 mN / m; and e) Absence of hemolytic activity, in particular less than 10%, more particularly less than 5%, or less than 1, 2, 3, or 4% hemolytic activity, for example 0% or 0.1-0.9% hemolysis caused by a solution of 100 g / l of the block copolymer.

[0092] Each of the above properties a) to e) can be determined by applying the respective analytical methods and measurement conditions as described in the experimental section below.

[0093] According to a particular embodiment, a preferred subgroup of block copolymers exhibits a combination of any one of the above characteristics a), b) and e).

[0094] According to another particular embodiment, a preferred subgroup of block copolymers exhibits a combination of any one of the above characteristics a), b), c) and e).

[0095] According to yet another particular embodiment, a preferred subgroup of block copolymers exhibits a combination of any one of the above characteristics a), b), c), d) and e).

[0096] According to yet another embodiment, at least one biopolymer component and at least one polymer of general formula 1 H-(-OE) n -(OBu) m -X-(BuO) m -(EO) n -H (1) (In the formula, X represents -O-; or a divalent organic moiety; m's each independently represent an integer ranging from 4 to 25; n's each independently represent an integer ranging from 15 to 100. and an ethylene oxide / butylene oxide block copolymer of the formula (I) in an aqueous solution; where: the biopolymer is selected from an adduct or conjugate of such a biopolymer with a further moiety selected from an oligopeptide, a polypeptide, a protein, an antibody molecule or a fragment or derivative thereof, a glycosylated protein, a proteoglycan, an oligonucleotide and a polynucleotide, a DNA and an RNA molecule, an oligosaccharide, a polysaccharide, and a payload molecule; The block copolymers exhibit the following combination of characteristics: a) a molecular weight of 2,500 to 12,500 g / mol, particularly 2,800 to 9,000 g / mol, more particularly 3,000 to 8,000 g / mol, and most particularly 3,500 to 8,000 g / mol, each of which can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1; b) an EO content of greater than 50% to 85% by weight, particularly 53 to 85% by weight, more particularly 55 to 80% by weight, and most particularly 57 to 80% by weight, each based on the dry weight of the block copolymer and calculable from the atomic masses of all atoms in the copolymer molecule of Formula 1; c) at least 5% by weight water solubility, based on the total weight of the aqueous solution of the block copolymer; and d) less than 10% hemolytic activity caused by a 100 g / l solution, or more specifically less than 5%, or less than 1, 2, 3, or 4% hemolysis, e.g., 0% or 0.1-0.9% hemolysis caused by a 100 g / l solution of the block copolymer.

[0097] According to more particular embodiments thereof, said biopolymer is selected from an adduct or conjugate of such a biopolymer with a further moiety selected from an oligopeptide, a polypeptide, a protein, an antibody molecule or a fragment or derivative thereof, a glycosylated protein, and a payload molecule.

[0098] According to yet another more specific embodiment thereof, said biopolymer is selected from a polypeptide, a protein, or a glycosylated protein, such as an enzyme.

[0099] According to yet another more specific embodiment thereof, said biopolymer is selected from an adduct or conjugate of such a biopolymer with a further moiety selected from an antibody molecule or a fragment or derivative thereof, and a payload molecule.

[0100] According to yet another more specific embodiment thereof, said biopolymer is selected from an adduct or conjugate of such a biopolymer with a further moiety selected from a monoclonal or polyclonal antibody molecule or a fragment or derivative thereof, and a payload molecule.

[0101] According to yet another particular embodiment thereof, said biopolymer is selected from a polyclonal antibody, a fragment or derivative thereof.

[0102] According to yet another particular embodiment thereof, said biopolymer is selected from a monoclonal antibody, a fragment or derivative thereof.

[0103] According to yet another particular embodiment thereof, said biopolymer is selected from a monoclonal antibody or a fragment thereof conjugated to a further moiety selected from a payload molecule.

[0104] According to yet another particular embodiment thereof, said biopolymer is selected from adducts or conjugates of such biopolymers with further moieties selected from proteoglycans, oligo- and polynucleotides, DNA and RNA molecules, oligosaccharides, and polysaccharides, and payload molecules.

[0105] In another particular embodiment of the first aspect, a stabilized biopolymer composition is provided, wherein the butylene oxide block is composed of monomer units derived from 1,2-butylene oxide, 2,3-butylene oxide, isobutylene oxide, or a mixture thereof, particularly essentially forming 1,2-butylene oxide, and more particularly consisting of 1,2-butylene oxide.

[0106] In another particular embodiment of the first aspect, a stabilized biopolymer composition is provided, wherein X is —O—, —O-alkylene-O—, particularly —O—(C-C 22 -alkylene)-O-, more particularly -O-(C3-C6-alkylene)-O-, where the alkylene chain is linear or branched and optionally interrupted by one or more heteroatoms, particularly oxygen atoms; more particularly -O-, -O-(n-butylene)-O-, -O-(1,2-butylene)-O-, a group of formula 2 below, particularly -O-(n-butylene)-O-; or X is a group of formula 2. [ka]

[0107] According to another particular embodiment of said first aspect, the present invention relates to a stabilized biopolymer composition, wherein said block copolymer of general formula 1 exhibits the following combination of characteristics: a) a calculated molecular weight of 3,500 to 7,000 g / mol, which can be calculated in particular from the sum of the atomic masses of all atoms of the copolymer molecule of formula 1; and b) an EO content of 60 to 70% by weight, based on the dry weight of the block copolymer, particularly as calculated from the atomic masses of all atoms in the copolymer molecule of formula 1; and c) X = -On-butylene-O-.

[0108] According to another particular embodiment of said first aspect, said block copolymer may be chosen from compounds of general formula 1 below, in which X, m and n have the following meanings: X = -On-butylene-O-; m = 10, n = 17 X = -On-butylene-O-; m = 10, n = 27 X = -On-butylene-O-; m = 5, n = 35 X = -On-butylene-O-; m = 16, n = 40 X = -On-butylene-O-; m = 12, n = 48 X = -On-butylene-O-; m = 21, n = 54 X = -On-butylene-O-; m = 10, n = 70 X = part of Equation 2, m = 10; and n = 17; X = part of Equation 2, m = 10, and n = 27; X = part of Equation 2, m = 5, and n = 35; X = part of Equation 2, m = 16, and n = 40; X = part of Equation 2, m = 12, and n = 48; X = part of Equation 2, m = 21, and n = 54; X = a moiety of Formula 2, m = 11, and n = 68; or X = part of Equation 2, m = 10, and n = 70.

[0109] According to yet another more specific embodiment, there is provided a biopolymer composition wherein the block copolymer is selected from compounds of the following general formula 1, wherein X, m, and n have the following meanings: X=-On-butylene-O-, m=12, and n=48, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 6,050; or X=-On-butylene-O-, m=10, and n=70, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,658; X=-On-butylene-O-, m=16, and n=40, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 5,922; or X=the moiety of formula 2, m=11, and n=68, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,724.

[0110] According to yet another more specific embodiment, X is a group of formula 2 [ka] Biopolymer compositions are provided, wherein: The block copolymers exhibit the following combination of characteristics: a) a molecular weight of 3,000 to 8,000 g / mol, 5,500 to 8,000 g / mol, in particular 5,800 to 7,900 g / mol, in particular calculable from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1; b) an EO content of 55 to 85 wt. % based on the dry weight of said block copolymer; particularly, as calculable from the atomic masses of all atoms in the copolymer molecule of Formula 1; c) at least 5% by weight water solubility, based on the total weight of the aqueous solution of the block copolymer; and d) Less than 10% hemolytic activity caused by a 100 g / l solution.

[0111] According to yet another particular embodiment, said biopolymer is an immunoglobulin or protein molecule or an antibody payload conjugate (APC), in particular an antibody drug conjugate (ADC), each optionally glycosylated, in which said at least one ethylene oxide / butylene oxide block copolymer is of general formula 1 above, During the ceremony, X represents -O-; or a divalent organic moiety; m's are each independently an integer ranging from 10 to 20; n's are each independently an integer ranging from 25 to 75; The block copolymers exhibit the following combination of characteristics: a) a molecular weight of 3,500 to 8,000 g / mol, which can be calculated in particular from the sum of the atomic masses of all atoms of the copolymer molecule of general formula 1; and b) an EO content of, in particular, 55 to 85% by weight, each based on the dry weight of said block copolymer, in particular calculable from the atomic masses of all atoms of the copolymer molecule of formula 1; c) at least 5% by weight water solubility, based on the total weight of the aqueous solution of the block copolymer; and d) Less than 10% hemolytic activity caused by a 100 g / l solution.

[0112] According to another particular embodiment of said first aspect, the present invention relates to a stabilized biopolymer composition, wherein the biopolymer is selected from the group consisting of an oligopeptide, a polypeptide, a protein, a glycosylated protein, a proteoglycan, an antibody molecule or a fragment or derivative thereof, a payload molecule (in particular a) a pharmaceutically active compound; b) labeling agents; c) small biological molecules such as lipids, phospholipids, glycolipids, sterols, vitamins, hormones, neurotransmitters, amino acids, nucleotides, and monosaccharides; or d) Biopolymers such as peptides, oligopeptides, polypeptides, proteins, nucleic acids such as DNA and RNA in any form, oligosaccharides, and polysaccharides. and adducts or conjugates of such biopolymers with additional moieties selected from:

[0113] In the latter embodiment, said biopolymer may preferably be a diagnostically applicable or therapeutically active biopolymer.

[0114] In the latter two embodiments, said biopolymers may preferably be selected from optionally glycosylated proteins, in particular enzymes and immunoglobulin molecules, respectively.

[0115] In the latter three embodiments, the biopolymer may preferably be selected from an adduct or conjugate of an immunoglobulin molecule and a payload molecule.

[0116] In the latter embodiment, said biopolymer may preferably be an antibody payload conjugate (APC), in particular an antibody drug conjugate (ADC).

[0117] In certain embodiments of the first aspect, a stabilized biopolymer composition is provided, wherein the block copolymer of general formula (I) is present in an amount of 0.001 to 30% or 0.001 to 10%, for example, 0.01 to 8%, 0.1 to 5%, or 1 to 3%, based on the total weight of the liquid composition.

[0118] In a further embodiment of the first aspect, there is provided a stabilized biopolymer composition, wherein the biopolymer is present in an amount of 0.01 to 30%, such as 0.1 to 25%, 1 to 20%, 3 to 15%, or 5 to 10%, based on the total weight of the liquid composition.

[0119] In particular embodiments of the first aspect, a stabilized biopolymer composition is provided, optionally in buffered form, having a pH in the range of 5-9, particularly 6, 7 or 8.

[0120] According to a second aspect, the present invention relates to an essentially dry biopolymer composition comprising at least one biopolymer component as defined in the above-identified first aspect and at least one stabilized ethylene oxide / butylene oxide block copolymer of general formula (1) as defined in the above-identified first aspect.

[0121] In a preferred embodiment, the essentially dry biopolymer composition has a liquid content of 0% to 5% by weight, such as 0.1 to 4.5% by weight, for example 1, 2, 3 or 4% by weight, based on the total weight of the composition.

[0122] Optionally, in the essentially dry biopolymer composition, the at least one block copolymer (=A) of general formula (1) and the at least one biopolymer (=B) are contained in a weight ratio (A):(B) ranging from 1:20,000 to 10:1, or from 1:5,000 to 2:1, or from 1:100 to 1.2:1, in particular from 1:50 to 5:1, or from 1:20 to 2:1, more particularly from 1:10 to 1.1:1.

[0123] In a preferred embodiment, the essentially dry biopolymer composition is characterized in that the block copolymer and the at least one biopolymer together comprise from 1 to less than 100% by weight, in particular from 5 to 60% by weight, more particularly from 10 to 50% by weight, or from 20 to 40% by weight, or even from 20 to 25% by weight, based on the total weight of the essentially dry composition.

[0124] Optionally, the essentially dry biopolymer composition comprises at least one additional excipient in an amount of 0.1-99%, 40-95%, and 50-90% by weight, based on the total dry weight of the essentially dry composition.

[0125] According to another particular embodiment, there is provided a liquid or essentially dry biopolymer composition, comprising nanostructures, in particular nanoparticles, composed of at least one biopolymer component as defined above and at least one stabilized ethylene oxide / butylene oxide block copolymer of general formula (1) as defined above.

[0126] More particularly, said nanostructures or nanoparticles are composed of at least one stabilized ethylene oxide / butylene oxide block copolymer of general formula (1) as defined above, as well as at least one biodegradable polymer (e.g., PLA, PLGA) or at least one lipid.

[0127] Even more specifically, said at least one biopolymer component is DNA, RNA or a peptide.

[0128] A third aspect of the present invention relates to the use of a block copolymer as defined in the first aspect identified above for stabilising an aqueous composition, in particular an aqueous solution, of at least one biopolymer as defined in the first aspect identified above.

[0129] A fourth aspect of the invention relates to a composition according to the first or second aspect identified above for use in medicine, in particular for diagnostic and / or therapeutic applications.

[0130] According to a further embodiment, the present invention relates to a composition according to the above-identified first or second aspect, which is a pharmaceutical composition optionally further supplemented with at least one pharmaceutically acceptable excipient.

[0131] A fifth aspect of the present invention relates to a method for preparing a stabilised composition according to the above-identified first aspect, the method comprising: a) preparing, in any order, an optionally buffered aqueous solution of a biopolymer; and an optionally buffered aqueous solution of a block copolymer of general formula (I); and b) preparing a mixture of both aqueous solutions obtained in step a);

[0132] A sixth aspect of the invention relates to a method for preparing an essentially dry stabilised composition according to the second aspect, the method comprising: a) preparing, in any order, an optionally buffered aqueous solution of the biopolymer; and an optionally buffered aqueous solution of the block copolymer of general formula (1); and b) preparing a mixture of both optionally buffered aqueous solutions obtained in step a); c) optionally supplementing the optionally buffered aqueous solution prepared in step a) and / or the mixture of both optionally buffered aqueous solutions prepared in step b) with at least one pharmaceutically acceptable excipient; d) drying the mixture obtained in step b) or c).

[0133] The drying step d) is carried out by a method known in the art, for example by spray drying or freeze drying the mixture obtained in step b) or c).

[0134] A seventh aspect of the present invention relates to a block copolymer selected from compounds of the following general formula (I), in which X, m and n have the following meanings: X = -On-butylene-O-, m = 10; and n = 17; X = -On-butylene-O-, m = 10, and n = 27; X = -On-butylene-O-, m = 5, and n = 35; X = -On-butylene-O-, m = 16, and n = 40; X = -On-butylene-O-, m = 12, and n = 48; X = -On-butylene-O-, m = 21, and n = 54; X = -On-butylene-O-, m = 10, and n = 70; X = part of Equation 2, m = 10; and n = 17; X = part of Equation 2, m = 10, and n = 27; X = part of Equation 2, m = 5, and n = 35; X = part of Equation 2, m = 16, and n = 40; X = part of Equation 2, m = 12, and n = 48; X = part of Equation 2, m = 21, and n = 54; X = a moiety of Formula 2, m = 11, and n = 68; or X = part of Equation 2, m = 10, and n = 70.

[0135] In particular, the present invention relates to block copolymers selected from compounds of the following general formula (I), in which X, m and n have the following meanings: X=-On-butylene-O-, m=12, and n=48, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 6,050; X=-On-butylene-O-, m=10, and n=70, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,658; X=-On-butylene-O-, m=16, and n=40, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 5,922; or X=the moiety of formula 2, m=11, and n=68, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,724.

[0136] D. Further Embodiments D.1. Synthesis of block copolymer of formula (1) Methods for preparing stabilizing ethylene oxide / butylene oxide block copolymers of general formula 1 are generally known in the art.

[0137] Suitable methods for their preparation are reported, for example, in US Pat. No. 2,828,345.

[0138] Briefly, the block copolymers can be prepared by a multi-step protocol that involves the first step of condensing an organic molecule containing two active hydrogen atoms, such as 1,4-butanediol or 1,3-butanediol, with butylene oxide to form polyoxybutylene, followed by the addition of ethylene oxide and allowing the reaction to proceed until the desired oxyethylene content is reached.

[0139] Examples of suitable butylene oxides are 1,2-butylene oxide and 2,3-butylene oxide.

[0140] The reaction is preferably carried out under elevated temperature, moisture-free conditions, and in the presence of a suitable catalyst such as an alkali metal hydroxide or alkoxide (such as an alkali metal tert-butoxide).

[0141] The reaction can be carried out in the presence of water, but reacting butylene oxide with water (particularly water from the aqueous catalyst solution or water contained in the starting materials or in EO or BuO added to the reaction (discussed further in the experimental section below)) results in the in situ formation of an organic molecule with two active hydrogen groups, such as 1,2-butanediol (similar to the disclosure of CA 698,568).

[0142] The amount of catalyst used should be 0.05 to 1 weight percent based on the total reactants. The reaction temperature ranges from 80 to 200°C, with temperatures of about 110°C or 170°C being preferred during the majority of the reaction.

[0143] Typically, superatmospheric pressures in the range of 0.5 to 15 bar are used, with very good results being obtained at pressures of about 1 to 5 bar. The alkylene oxide used is preferably substantially anhydrous; for example, the water content of the oxide should typically not exceed about 0.1% by weight. The alkylene oxide is also preferably as free as practical from contaminants such as aldehydes, which cause side reactions and by-product formation.

[0144] The reaction can be carried out either batchwise or continuously, as desired. In a batchwise operation, a commercially available anhydrous organic molecule containing two active hydrogen atoms, such as 1,4-butanediol or butylene glycol, is charged to a suitable dry reaction vessel, such as an autoclave, and mixed with an effective amount of catalyst (typically about 0.2% by weight of potassium hydroxide based on the total amount of reactants).

[0145] Prior to introducing butylene oxide, the reaction vessel is advantageously flushed with a stream of dry, inert gas, such as nitrogen, to remove air or oxygen therefrom. Removal of molecular oxygen from the reaction vessel is an important factor in obtaining a colorless product and can be carried out, if desired, after the butylene glycol and catalyst are added to the reaction vessel.

[0146] After these preparations, a mixture of potassium hydroxide and an organic molecule containing two active hydrogen atoms, such as 1,4-butanediol or butylene glycol, is heated to a reaction temperature of about 140°C, and butylene oxide is added at a fairly rapid rate.

[0147] Typically, the rate of addition of butylene oxide is such as to maintain a pressure in the reactor of about 3 bar. Vigorous stirring is desirable to maintain good dispersion of the catalyst and a uniform reaction rate throughout the mass.

[0148] The reaction between butylene oxide and butylene glycol is exothermic and therefore it is necessary to provide adequate cooling means.

[0149] By controlling the rate of addition of butylene oxide to maintain a nearly constant pressure, the reaction temperature can also be maintained constant.

[0150] The addition of butylene oxide, for example, calculates the hydroxyl analysis or two free hydroxyl groups per molecule. 1 The reaction is stopped when the desired molecular weight of the polyoxybutylene glycol condensation product is obtained, as measured by H-NMR. Ethylene oxide is then condensed with the polyoxybutylene glycol condensation product to obtain the product of the present invention. The addition of ethylene oxide is carried out in the same manner as the addition of butylene oxide described above.

[0151] Purification can be done by heating it under reflux at reduced pressure or by stripping with an inert gas to distill off low boiling materials.

[0152] D.2. Antibody-Payload Conjugates and Their Preparation As noted above, the biopolymer as part of the stabilized formulation of the present invention may in a preferred embodiment be an antibody payload conjugate (APC), more particularly an antibody drug conjugate (ADC).

[0153] Antibody-drug conjugates (ADCs) combine the two major therapies currently used: chemotherapy and antibody therapy. Antibodies are important biologics that bind to their specific antigens, e.g., receptors on cells that are overexpressed on diseased cells, such as cancer cells, compared to healthy cells. Antibodies activate the competency system, resulting in the destruction of cancer cells by killer cells. Chemotherapy, on the other hand, is a treatment using cytotoxic moieties that can be absorbed by cells and kill them through a different pathway. Activated cells, such as cancer cells, can take up more cytotoxic drugs than healthy cells. Nevertheless, this treatment has significant side effects. Combining antibodies with the killing effect of cytotoxic drugs allows for targeted and efficient cancer therapy. Therefore, the choice of conjugation method, i.e., how the antibody is labeled with the drug, is very important.

[0154] The first commercially available ADCs were randomly conjugated by utilizing cysteines or lysines in the antibody sequence to attach toxic payloads, resulting in heterogeneous species with different drug-to-antibody ratios (DARs), which adversely affected the pharmacokinetics and safety profile of the ADCs (Senter, PD & Sievers, EL. The discovery and development of brentuximab vedotin for use in relapsed Hodgkin lymphoma and systemic anaplastic large cell lymphoma. Nat. Biotechnol. 30, 631-637 (2012); Junutula, JR et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925-932 (2008)).

[0155] Subsequently, site-specific conjugation methods were carried out using antibody glycosylation or enzymatic coupling (Van Geel, R. et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody-Drug Conjugates. Bioconjug. Chem. 26, 2233-2242 (2015); Dennler, P. et al. Transglutaminase-based chemo-enzymatic conjugation approach yields homogeneous antibody-drug conjugates. Bioconjug. Chem. 25, 569-578 (2014)). These methods are limited to specific sites and cannot be transferred to other positions in the antibody sequence.

[0156] One site-specific, open-ended conjugation method is the use of genetic code extension technology. Thus, non-standard amino acids (NCAAs) are introduced at the translational level into the antibody sequence in response to a stop codon (e.g., amber stop codon, TAG) pre-positioned in the antibody gene. An orthogonal aminoacyl-tRNA-synthetase (aaRS) / tRNA pair must be introduced into the antibody expression host, which can bind to the NCAA and incorporate it into the growing antibody protein sequence (Lemke, E.A. The exploding genetic code. ChemBioChem 15, 1691-1694 (2014); de la Torre, D. & Chin, J.W. Reprogramming the genetic code. Nat. Rev. Genet. 22, 169-184 (2021)). The NCAA can be freely positioned within the antibody sequence and used for conjugation with toxic payloads depending on its chemical properties.

[0157] There are various ncAAs based on various endogenous amino acids, such as lysine or tryptophan. These can have different head groups, which affect their chemical properties and determine the types of chemical reactions they can undergo. Tian et al. demonstrated the incorporation of ncaAs containing a ketone head group into several antibodies expressed in CHO cells, followed by coupling to cytotoxic payloads via a copper-free click reaction. The reaction between the alkoxyamine functional group and the ketone can only be carried out at pH 4; otherwise, additives are required (Tian, ​​F. et al., A General Approach to Site-Specific Antibody-Drug Conjugation. Proc. Natl. Acad. Sci. USA 111, 1766-1771 (2014)).

[0158] Today, the fastest bioorthogonal chemical reaction, which can be performed even at neutral pH, is the strain-promoted reverse electrophilic Diels-Alder cycloaddition (SPIEDAC) between strained alkenes or alkynes and tetrazine groups (Nikic, I. & Lemke, E. A. Genetic code expansion enabled site-specific dual-color protein labeling: superresolution microscopy and beyond. Curr. Opin. Chem. Biol. 28, 164-173 (2015)).

[0159] One special case of the SPIEDAC reaction is the conjugation of cyclooctene-lysine (SCO) with 1,2,4,5-tetrazine, which may not be a reverse electrophilic reaction and does not exhibit the same reaction rate as other strained alkenes or alkynes. Therefore, SCO and the resulting reaction products exhibit the highest stability in the cellular environment compared to other strained alkenes / alkynes tested (Wagner, J.A., Mercadante, D., Nikic, I., Lemke, E.A. & Grater, F. Origin of Orthogonality of Strain-Promoted Click Reactions. Chem.-A Eur. J. 21, 12431-12435 (2015); Reinkemeier, C.D. et al. Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells. Chem.-A Eur. J. 27, chem. 202100322 (2021)).

[0160] Toxic payloads can be divided into linkers and cytotoxic drugs. Today, many linker technologies exist, ranging from non-cleavable linkers to enzymatic, acidic, and glutathione-cleavable linkers. The linker directly influences the pharmacokinetics and pharmacodynamics of ADCs (Hafeez, U., Parakh, S., Gan, HK, & Scott, AM Antibody-drug conjugates for cancer therapy. Molecules 25, 4764 (2020); Khongorzul, P., Ling, CJ, Khan, FU, Ihsan, AU, & Zhang, J. Antibody-Drug Conjugates: A Comprehensive Review. Mol. Cancer Res. 18, 3-19 (2020)).

[0161] Currently, several different cytotoxic drug families are used as chemical warheads in ADCs, including auristatins, maytansinoids, calicheamicins, and duocarmycins, which damage DNA or microtubules (Chau, CH, Steeg, PS & Figg, WD. Antibody-drug conjugates for cancer. Lancet 394, 793-804 (2019); Sievers, EL & Senter, PD. Antibody-drug conjugates in cancer therapy. Annu. Rev. Med. 64, 15-29 (2013)).

[0162] Non-limiting examples of suitable antibodies and payload molecules within the meaning of the present invention are reported in the following sections.

[0163] D.3. Antibodies The term "antibody" within the meaning of the present invention refers equally to antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g., bispecific and trispecific mAb fragments or derivatives), polyclonal or monoclonal antibodies, such as human antibodies, humanized antibodies, murine antibodies or chimeric antibodies (see also the general definition provided above).

[0164] Typical, non-limiting examples are selected from biologically, in particular pharmacologically active antibody molecules.

[0165] Non-limiting examples are selected from the group consisting of: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumab, tositumomab, pertuzumab, adecatumumab, IGN101, INA01 labetuzumab, hua33 , pemtumomab, oregovomab, minletumomab (CC49), cG250, J591, MOv-18, farletuzumab (MORAb-003), 3F8, ch14,18, KW-2871, hu3S193, lgN311, IM-2C6, CDP-791, etaracizumab, volocicimab, nimotuzumab, MM-121, AMG 102, METMAB, SCH 900105, AVE1642, IMC-A12, MK-0646, R1507, CP 751871, KB004, III A4, mapatumumab, HGS-ETR2, CS-1008, denosumab, sibrotuzumab, F19, 81 C6, pinatuzumab, rifatuzumab, glenbatumumab, coltuximab, lorvotuzumab, indatuximab, anti-PSMA, MLN-0264, ABT-414, milatuzumab, ramucirumab, abagovomab, abituzumab, adecatumumab, afutuzumab, altumomab pentetate, amatuximab, anatumomab, anetumab, apolizumab, arcitumumab, asclinvacumab, atezolizumab, bavituximab, bectumumab, belimumab, vivax tuzumab, brontuzumab, cantuzumab, capromab, catumaxomab, sitatuzumab, cixutumumab, clivatuzumab, codrituzumab, conatumumab, dacetuzumab, darotuzumab, daratumumab, demcizumab, denintuzumab, depatukizumab, delrotuximab, detumomab, dinutuximab, drozitumab, durigotumab, durvalumab, dusigitumab, ecloneximab, edrecolomab, elgemtuzumab, emactuzumab, enabatuzumabEmibetuzumab, enfortumab, enoblitutuzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, framvotumab, futuximab, galiximab, ganitumab, icrucumab, igovomab, imalumab, imgatuzumab, indusatumab, inebilizumab, intetumumab, iratumumab, isaximab, lexatuzumab, rilotomab , lintuzumab, lirilumab, lucatumumab, lumletuzumab, margetuximab, matuzumab, mirvetuximab, mitumomab, mogamulizumab, moxetumomab, nacolomab, naptumomab, narunatumumab, necitumumab, nesbacumab, nimotuzumab, nivolumab, nofetumomab, obinutuzumab, ocaratuzumab, ofatumumab, olaratuzumab, onartuzumab, ontuxizumab, oportuzumab, and oregovomab , otlertuzumab, pancomab, palsatuzumab, pasotuximab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, kiruzumab, racotumab, ramucirumab, rilotumumab, lobatumumab, sacituzumab, samalizumab, satumomab, seribantumab, siltuximab, sofituzumab, tacatuzumab, taplituzumab, talexuzumab, tenatumab, tep Rotumumab, tetulomab, ticilimumab, tigatuzumab, tositumomab, tobetumab, tremelimumab, tucotuzumab, ublituximab, urocupulumab, urelumab, utomilumab, vadatuximab, bundletuzumab, vanticizumab, vanucizumab, varlilumab, veltuzumab, besencumab, volociximab, borsetuzumab, votumumab, zalutumumab, zalutumumab, combinations and derivatives thereof, and CAI 25, CAI 5-3, CAI 9-9, L6, Lewis Y, Lewis X, alpha-fetoprotein, CA242, other monoclonal antibodies targeting placental alkaline phosphatase, prostate-specific antigen, prostate-specific membrane antigen, prostatic acid phosphatase, epidermal growth factor, MAGE-1, MAGE-2, MAGE-3, MAGE-4, transferrin receptor, p97, MUCI, CEA, gplOO, MARTI, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21, MPG, and Neu oncogene product.

[0166] D.4. Payload Molecules Typically used payload molecules may be selected from biologically active compounds, in particular drugs, labeling agents, and chelating agents, non-limiting examples of which are given in the following sections.

[0167] D.4.1. Biologically active compounds Bioactive compounds include, but are not limited to:

[0168] Bioactive compounds applicable according to the present invention include, but are not limited to, organic small molecule drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, peptides, peptoids, amino acids, nucleotides, oligo- or polynucleotides, nucleosides, DNA, RNA, toxins, glycans, immunoglobulins.

[0169] Exemplary classes of biologically active compounds that can be used in the practice of the present invention include, but are not limited to, hormones, cytotoxins, antiproliferative / antitumor agents, antivirals, antibiotics, cytokines, anti-inflammatory agents, antihypertensive agents, chemosensitizers, photosensitizers and radiosensitizers, anti-AIDS agents, antivirals, immunosuppressants, immunostimulators, enzyme inhibitors, anti-Parkinson's agents, neurotoxins, channel blockers, modulators of cell-extracellular matrix interactions including cell proliferation inhibitors and anti-adhesion molecules, inhibitors of DNA, RNA or protein synthesis, steroidal and non-steroidal anti-inflammatory agents, anti-angiogenic factors, anti-Alzheimer's agents.

[0170] In some embodiments, the biologically active compound is a compound of low to medium molecular weight (eg, about 200 to 5000 Da, about 200 to about 1500 Da, preferably about 300 to about 1000 Da).

[0171] Exemplary cytotoxic drugs are those used in cancer treatment in particular.Such drugs generally include DNA damaging agents, antimetabolites, natural products and their analogs, enzyme inhibitors such as dihydrofolate reductase inhibitors and thymidylate synthase inhibitors, DNA binders, DNA alkylating agents, radiosensitizers, DNA intercalators, DNA cleavage agents, microtubule stabilizers and destabilizers, and topoisomerase inhibitors.Examples include, but are not limited to, platinum-based drugs, anthracyclines, vinca drugs, mitomycin, bleomycin, cytotoxic nucleosides, taxanes, lexitropsins, pteridine drugs, diynenes, podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxol. Particularly useful members of these classes include, for example, auristatins, maytansines, maytansinoids, calicheamicins, dactinomycins, duocarmycins, CC1065 and its analogs, camptothecin and its analogs, SN-38 and its analogs; DXd, tubulysin M, cryptophycins, pyrrolobenzodiazepines and pyrrolobenzodiazepine dimers (PBDs), pyridinobenzodiazepines (PDDs) and indolinobenzodiazepines (IBDs) (see U.S. Patent Application Publication No. 20210206763A1), methotrexate, and the like. Examples of suitable anti-inflammatory drugs include methadone, methopterin, dichloromethotrexate, 5-fluorouracil, DNA minor groove binders, 6-mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin), PNU-159682 (see U.S. Pat. No. 10,288,745 B2) and its analogs, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, and podophyllotoxin. Podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere, retinoic acid, butyric acid, N8-acetylspermidine, staurosporine, colchicine, camptothecin, esperamicin, enediyne and its analogs, hemiasterin and its analogs.

[0172] Other exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperone inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / hedgehog signaling pathway inhibitors, RNA polymerase inhibitors, and protein degraders (see https: / / pubs.acs.org / doi / 10.1021 / acschembio.0c00285).

[0173] Examples of auristatins include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylenediamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP, and auristatin AQ. Suitable auristatins are also described in U.S. Patent Application Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248; WO 09 / 117531, WO 2005 / 081711, WO 04 / 010957; WO 02 / 088172 Fret and brochure 01 / 24763, and U.S. Patent Nos. 7,498,298; 6,884,869; 6,323,315; 6,239,104; 6,124,431; 6,034,065; 5,780,588; and 5,767,237. ; Specification No. 5,665,860; Specification No. 5,663,149; Specification No. 5,635,483; Specification No. 5,599,902; Specification No. 5,554,725 Specification; Specification No. 5,530,097; Specification No. 5,521,284; Specification No. 5,504,191; Specification No. 5,410,024; Specification No. 5,138,036 Nos. 5,076,973; 4,986,988; 4,978,744; 4,879,278; 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entireties.

[0174] Exemplary drugs include dolastatins and their analogs, including dolastatin A (U.S. Pat. No. 4,486,414), dolastatin B (U.S. Pat. No. 4,486,414), dolastatin 10 (U.S. Pat. Nos. 4,486,444, 5,410,024, 5,504,191, 5,521,284, 5,530,097, 5,599,902, 5,635,483, 5,663,149, 5,665,860, 5,666,861, 5,666,862, 5,666,863, 5,666,864, 5,666,865 ... Nos. 5,780,588, 6,034,065, 6,323,315), dolastatin 13 (U.S. Pat. No. 4,986,988), dolastatin 14 (U.S. Pat. No. 5,138,036), dolastatin 15 (U.S. Pat. No. 4,879,278), dolastatin 16 (U.S. Pat. No. 6,239,104), dolastatin 17 (U.S. Pat. No. 6,239,104), and dolastatin 18 (U.S. Pat. No. 6,239,104) (each of which is incorporated herein by reference in its entirety).

[0175] Exemplary maytansinoids, such as maytansine, DM-1 and DM-4, ​​or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Pat. Nos. 4,424,219; 4,256,746; 4,294,757; 4,307,016; 4,313,946; 4,315,929; 4,331,598; 4,361,650; and 4,362,663. ;4,364,866;4,450,254;4,322,348;4,371,533;5,208,020;5,416,064;5,475,092;5,585,499;5,846,545;6,333,410;6,441,163;6,716,821 and7,276,497.

[0176] Other examples include mertansine and ansamitocin; pyrrolobenzodiazepines (PBDs), explicitly including dimers and analogs, include, but are not limited to, those described in (Denny, Exp. Opin. Ther. Patents, 10(4):459-474(2000)), (Hartley et al., Expert Opin Investig Drugs. 2011, 20(6):733-44), Antonow et al., Chem Rev. 2011, 111(4), 2815-64).

[0177] Calicheamicins include, for example, enediynes, esperamicins, and those described in US Pat. Nos. 5,714,586 and 5,739,116.

[0178] Examples of duocarmycins and analogs include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, DU-86, KW-2189, adozelesin, bizelesin, carzelesin, and secoadzelesin. Other examples include, for example, U.S. Patent Nos. 5,070,092; 5,101,092; 5,187,186; 5,475,092; 5,595,499; 5,846,545; 6,534,660; 6,548,530; 6,586,618; 6,660,742; and 6,756,397. Nos. 7,049,316; 7,553,816; 8,815,226; U.S. Patent Application Publication No. 20150104407; U.S. Patent Application No. 61 / 988,011, filed May 2, 2014, and U.S. Patent Application No. 62 / 010,972, filed June 11, 2014, the disclosures of each of which are incorporated herein in their entirety.

[0179] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U.S. Patent Application Publication Nos. 2002 / 0103136 and 2010 / 0305149, and U.S. Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entireties.

[0180] Exemplary epothilone compounds include epothilone A, B, C, D, E, and F, and derivatives thereof. Suitable epothilone compounds and derivatives thereof are described, for example, in U.S. Patent Nos. 6,956,036; 6,989,450; 6,121,029; 6,117,659; 6,096,757; 6,043,372; 5,969,145; and 5,886,026; and WO 97 / 19086; WO 98 / 08849; Nos. 98 / 22461; 98 / 25929; 98 / 38192; 99 / 01124; 99 / 02514; 99 / 03848; 99 / 07692; 99 / 27890; and 99 / 28324, the disclosures of which are incorporated herein by reference in their entireties.

[0181] Exemplary cryptophycin compounds are described in US Pat. Nos. 6,680,311 and 6,747,021, the disclosures of which are incorporated herein by reference in their entireties.

[0182] Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, and tetraplatin.

[0183] Exemplary DNA binding or alkylating agents include CC-1065 and its analogs, anthracyclines, calicheamicin, dactinomycin, mithromycin, pyrrolobenzodiazepines, and the like.

[0184] Exemplary microtubule-stabilizing and destabilizing agents include taxane compounds such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel; maytansinoids, auristatins and their analogs, vinca alkaloid derivatives, epothilones, and cryptophycins.

[0185] Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs, and unnatural camptothecins, such as CPT-11, SN-38, topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, ciratecan, lurtotecan, exatecan, DXd, diflometotecan, belotecan, lurtotecan, and S39625. Other camptothecin compounds that can be used include, for example, those described in J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med. Chem., 30:1774 (1987).

[0186] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, and MetAP2 inhibitors.Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib, and vatalanib.Exemplary MetAP2 inhibitors include fumadillol analogs, which refer to compounds that contain the fumagillin core structure.

[0187] Exemplary cell cycle progression inhibitors include CDK inhibitors, such as BMS-387032 and PD0332991; Rho kinase inhibitors, such as AZD7762; Aurora kinase inhibitors, such as AZD1152, MLN8054 and MLN8237; PLK inhibitors, such as BI 2536, BI6727, GSK461364, ON-01910; and KSP inhibitors, such as SB 743921, SB 715992, MK-0731, AZD8477, AZ3146 and ARRY-520.

[0188] Exemplary P13K TOR / AKT signaling pathway inhibitors include phosphoinositide 3 kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors, and PDK-1 inhibitors.

[0189] Exemplary P13 kinases are disclosed in U.S. Pat. No. 6,608,053 and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridine, GDC-0941, GSK615, IC87114, LY294002, Palomid 529, Perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, wortmannin, XL147, and XL765.

[0190] Exemplary AKT inhibitors include, but are not limited to, AT7867.

[0191] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.

[0192] Exemplary MEK inhibitors are disclosed in U.S. Pat. No. 7,517,944 and include GDC-0973, GSK1 120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901, AZD6244, AZD8330 and GDC-0973.

[0193] Exemplary B-RAF inhibitors include CDC-0879, PLX-4032, and SB590885.

[0194] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820, and 202190. Exemplary receptor tyrosine kinase inhibitors include, but are not limited to, AEE788 (NVP-AEE 788), BIBW2992 (afatinib), lapatinib, erlotinib (Tarceva), gefitinib (Iressa), AP24534 (ponatinib), ABT-869 (linifanib), AZD2171, CHR-258 (dovitinib), sunitinib (sutent), sorafenib (Nexavar), and vatalinib.

[0195] Exemplary protein chaperone inhibitors include HSP90 inhibitors, including 17AAG derivatives, BIIB021, BIIB028, SNX-5422, NVP-AUY-922, and KW-2478.

[0196] Exemplary HDAC inhibitors include belinostat (PXD101), CUDC-101, droxinostat, ITF2357 (divinostat, gabinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, dacinostat), LBH-589 (panobinostat), MC1568, MGCD0103 (mosetinostat), MS-275 (entinostat), PCI-24781, pyroxamide (NSC 696085), SB939, trichostatin A, and vorinostat (SAHA). Exemplary PARP inhibitors include iniparib (BSI 201), olaparib (AZD-2281), ABT-888 (veliparib), AG014699, CEP9722, MK 4827, KU-0059436 (AZD2281), LT-673, 3-aminobenzamide, A-966492, and AZD2461.

[0197] Exemplary Wnt / hedgehog signaling pathway inhibitors include vismodib, cyclopamine, and 939.

[0198] Exemplary RNA polymerase inhibitors include amatoxins, including α-amanitin, β-amanitin, γ-amanitin, η-amanitin, amanulin, amanuric acid, amanisamide, amanone, and proamanulin.

[0199] Exemplary cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21, TNF.

[0200] Non-limiting examples of specific drugs include auristatins, maytansinoids, PBDs, topoisomerase inhibitors, and anthracyclines.

[0201] In another embodiment, a combination of two or more different drugs as described above is used.

[0202] According to another embodiment, the biologically active compound may be selected from any synthetic or naturally occurring compound comprising one or more natural and / or non-natural, proteinogenic and / or non-proteinogenic amino acid residues, such as, in particular, oligopolypeptides or proteins.

[0203] Other suitable examples of biologically active compounds are immunoglobulins, such as antibodies, antibody derivatives, and active fragments thereof. Suitable examples in this regard are reported in section D.3 above.

[0204] D.4.2. Labelling agents and radionuclides Labeling agents that may be used as biopolymers within the meaning of the present invention may include any type of label known in the art.

[0205] Examples include dyes (e.g., fluorescent, luminescent, or phosphorescent dyes, e.g., dansyl, coumarin, fluorescein, acridine, rhodamine, silicon rhodamine, BODIPY, or cyanine dyes), molecules that fluoresce upon contact with a reagent, chromophores (e.g., phytochromes, phycobilins, bilirubin, etc.), radiolabels (e.g., tritium, fluorine-18, carbon-11, carbon-14, phosphorus-32, phosphorus-33, sulfur-33, sulfur-35, indium-111, iodine-123, iodine-125, etc.), and chromophores (e.g., hydrogen, fluorine, carbon, etc.). radioactive forms of iodine, phosphorus, sulfur, iodine), MRI-sensitive spin labels, affinity tags (e.g., biotin, His-tag, Flag-tag, Strep-tag, sugars, lipids, sterols, PEG linkers, benzylguanine, benzylcytosine, or cofactors), polyethylene glycol groups (e.g., branched PEG, linear PEG, PEG of different molecular weights, etc.), photocrosslinkers (e.g., p-azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, solid supports, and biologically active compounds as defined above.

[0206] In some embodiments, exemplary dyes can include NIR contrast agents that fluoresce in the near-infrared region of the spectrum. Exemplary near-infrared fluorophores can include dyes and other fluorophores having emission wavelengths (e.g., peak emission wavelengths) of about 630-1000 nm, e.g., about 630-800 nm, about 800-900 nm, about 900-1000 nm, about 680-750 nm, about 750-800 nm, about 800-850 nm, about 850-900 nm, about 900-950 nm, or about 950-1000 nm. Fluorescent materials with emission wavelengths (e.g., peak emission wavelengths) greater than 1000 nm can also be used in the methods described herein.

[0207] In some embodiments, exemplary fluorophores include 7-amino-4-methylcoumarin-3-acetic acid (AMCA), TEXAS RED™ (Molecular Probes, Inc., Eugene, Oreg.), 5-(and-6)-carboxy-X-rhodamine, Lissamine rhodamine B, 5-(and-6)-carboxyfluorescein, fluorescein-5-isothiocyanate (FITC), 7-diethylaminocoumarin-3-carboxylic acid, tetramethylrhodamine-5-(and-6)-isothiocyanate, 5-(and-6)-carboxytetramethylrhodamine, 7-hydroxycoumarin-3-carboxylic acid, 6-[fluorescein-5-(and-6)-carboxamido]hexanoic acid, N-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-3-indacenepropionic acid), eosin-5-isothiocyanate, erythrosin-5-isothiocyanate, CASCADE™ Blue acetyl azide (Molecular Probes, Inc., Eugene, Oreg.) and ATTO dyes.

[0208] Further labeling agents are 111-indium, 64-copper, 67-copper, 124-iodine, 227-thorium, 188-rhenium, 177-lutetium, 89-zirconium, 131-iodine, 68-gallium, 99m-technetium, 225-actinium, 213-bismut, 90-yttrium, 212-plumbum.

[0209] D.4.3. Chelating Agents Below is a list of typically applicable chelating agents and their short names, the corresponding salts of which are also applicable:

[0210] Acetylacetone (ACAC), ethylenediamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylenetriamine (DIEN), iminodiacetic acid (IDA), triethylenetetramine (TRIEN), triaminotriethylamine, nitrilotriacetic acid (NTA) and its salts (Na3NTA or FeNTA, etc.), ethylenediaminotriacetic acid (TED), ethylenediaminetetraacetic acid (EDTA) and its salts (Na2EDTA, CaNa2EDTA, etc.), diethylenetriaminepentaacetic acid (D TPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetate (DOTA), 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA), oxalate (OX), tartrate (TART), citrate (CIT), dimethylglyoxime (DMG), 8-hydroxyquinoline, 2,2'-bipyridine (BPY), 1,10-phenanthroline (PHEN), dimercaptosuccinic acid (DMSA), 1,2-bis(diphenyl) dimercaptosuccinic acid (DMSA); siderophores secreted by microorganisms, such as deferrioxamine or deferoxamine B (also known as deferal (Novartis)) produced by Streptomyces sp.; deferoxamine (DFO), a trihydroxamic acid secreted by Streptomyces pilosus. Phytochemicals such as curcuminoids and derivatives of mugineic acid (3-hydroxymugineic acid, 2'-deoxymugineic acid, etc.); synthetic chelating agents (such as ibuprofen); derivatives of catechol, hydroxamate, and hydroxypyridinone (hydroxamate desferal, hydroxypyridinone deferiprone, etc.); deferiprone (L1 or 1,2-dimethyl-3-hydroxypyrid-4-one); D-penicillamine (DPA or D-PEN), which is β-β-dimethylcysteine ​​or 3-mercapto-D-valine;Tetraethylenetetraamine (TETA) or trientine and its two major metabolites, N1-acetyltriethylenetetramine (MAT) and N1,N10-diacetyltriethylenetetramine (DAT); hydroxyquinoline; clioquinol, a halogenated derivative of 8-hydroxyquinoline; and 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8-ol (PBT2).

[0211] D.5. Pharmaceutical Compositions The stabilized compositions (i.e., stabilized active ingredients, particularly biopolymers) of the present invention are generally provided as "pharmaceutical compositions" comprised of a "therapeutically" and / or "prophylactically" or "diagnostically" effective amount of at least one such active ingredient or a pharmaceutically acceptable salt thereof, and optionally at least one pharmaceutically acceptable excipient.

[0212] Thus, the term "pharmaceutical composition" according to certain embodiments of the present invention refers to a stabilized liquid composition comprising or consisting essentially of at least one pharmaceutically active biopolymer compound (i.e., active ingredient) and at least one stabilized EO / BuO block copolymer described herein in a liquid pharmaceutically acceptable medium. Dry powders of such liquid preparations can be obtained by lyophilization or any other suitable drying method typically applied.

[0213] The pharmaceutical compositions may be delivered via any suitable route of administration, such as oral, rectal, transmucosal, topical, ophthalmic, otorhinological, or enteral administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injection, and, in some cases, epidural, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injection.

[0214] Depending on the nature or mode of administration and dosage form, the at least one additional pharmaceutical excipient of the composition may vary.

[0215] "Excipients" are substances formulated with an active ingredient and are included for different purposes, such as long-term stabilization, bulking up a solid dosage form containing a small amount of potent active ingredient (thus often referred to as a "bulking agent," "filler," or "diluent"), or to impart therapeutic enhancements to the active ingredient in the final dosage form, such as facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients can also be useful in the manufacturing process of pharmaceutical compositions to help address active agent concerns, such as by promoting powder flow or non-stickiness, in addition to aiding in in vitro stability, such as preventing denaturation or aggregation over the expected shelf life. The selection of appropriate excipients depends not only on the route of administration and dosage form, but also on the particular active ingredient and other factors.

[0216] Excipients may be selected from the following classes: immunoadjuvants, antiadhesives, binders, coating agents, colorants, disintegrants, flavoring agents, glidants, lubricants, preservatives, adsorbents, sweeteners, and vehicles.

[0217] Non-limiting examples of excipients include diluents, preservatives, stabilizers, emulsifiers (such as emulsifying polymers such as polysorbates or poloxamers), antioxidants (e.g., epigallocatechin-3-O-gallate, lycopene, ellagic acid, coenzyme Q, 10, indole-3-carbinol, genistein, quercetin, ascorbic acid, glutathione, melatonin, catechin, taurine, captopril, gallic acid, N-acetylcysteine, α-lipoic acid, BHT, tocopherols, tocotrienols, and other compounds), or enzymes (such as superoxide dismutase and catalase); anti-irritants, chelating agents, stabilizing salts (such as chlorides, sulfates, phosphates, diphosphates, hydrobromides, and nitrates), suspending agents, antibacterial agents, and antifungal agents. Furthermore, buffers, such as buffer systems of low-molecular-weight organic acids and their respective salts, or inorganic buffer substances such as phosphate buffers, can also be used. Further suitable ingredients are also known from the relevant pharmacological standards. The proportions of the various ingredients will also vary depending on the nature of the specific ingredients used and are generally known to those skilled in the art (see Remington's Pharmaceutical Sciences ("Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), Edited by A. Wade and P. J. Weller or in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro, edited. 1985)).

[0218] As used herein, a pharmaceutical composition may be provided in the form of a "dosage form" or "unit dose" and may comprise one or more stabilized liquid or essentially dry biopolymer compositions comprising at least one pharmaceutically active biopolymer compound and at least one stabilized EO / BuO block copolymer as described herein. Thus, as used herein, a pharmaceutical composition may provide, for example, two active agents mixed together in a unit dose, or may provide two active agents combined in a dosage form in which the active agents are physically separated.

[0219] Furthermore, the pharmaceutical composition may be administered in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.

[0220] The pharmaceutical compositions of the present invention may be prepared in a manner known per se, for example by conventional mixing, dissolving, emulsifying, encapsulating, entrapping or combinations thereof. Proper formulation depends upon the chosen route of administration.

[0221] The phrase "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a patient without undue toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk / benefit ratio.

[0222] The present invention includes all "pharmaceutically acceptable salt forms" of the active ingredient. Pharmaceutically acceptable salts are those in which the counterion does not significantly contribute to the physiological activity or toxicity of the compound and therefore function as pharmacological equivalents. These salts can be prepared according to common organic techniques using commercially available reagents. Some anionic salt forms include acetate, besylate, bromide, chloride, citrate, fumarate, glucuronate, hydrobromide, hydrochloride, hydroiodide, iodide, lactate, maleate, mesylate, nitrate, pamoate, phosphate, succinate, sulfate, tartrate, tosylate, and xinofoate. Some cationic salt forms include ammonium, aluminum, benzathine, bismuth, calcium, choline, diethylamine, diethanolamine, lithium, magnesium, meglumine, 4-phenylcyclohexylamine, piperazine, potassium, sodium, tromethamine, and zinc.

[0223] A "therapeutically effective amount" and / or a "prophylactically effective amount" means an amount effective to provide any therapeutic and / or prophylactic benefit when administered to a human or non-human patient. More specifically, a "therapeutically effective amount" is an amount of an active ingredient disclosed herein, or a combination of two or more such active ingredients, that completely or partially inhibits the progression of a condition or at least partially alleviates one or more symptoms of a condition.

[0224] "Diagnosically effective amount" means an amount effective to enable diagnostically useful information regarding the status or progression of a disease state to be obtained from a patient.

[0225] The therapeutic benefit can be an improvement in the symptoms of a disease patient, for example, an amount effective to alleviate the symptoms of a disease patient. In certain circumstances, the patient may not exhibit the symptoms of the condition for which the patient is being treated. Thus, a prophylactically effective amount of a compound is also an amount sufficient to provide a significant positive effect on any manifestation of a disease, disorder, or condition, for example, an amount sufficient to significantly reduce the frequency and severity of occurring disease symptoms.

[0226] A therapeutically effective amount can also be a prophylactically effective amount.

[0227] As used herein, a "patient" refers to a human or non-human, particularly a human, animal.

[0228] A "dosage form" is a unit of administration ("unit dose") of one or more active agents described herein.

[0229] The term "treating" or "treatment" refers to (i) preventing a disease, disorder, or condition from occurring in a patient who may be predisposed to, but has not yet been diagnosed as having, the disease, disorder, or condition; (ii) inhibiting the disease, disorder, or condition, i.e., arresting its development; and (iii) alleviating the disease, disorder, or condition, i.e., causing regression of the disease, disorder, and / or condition. In particular, this includes prophylactic or therapeutic treatment, or a combination thereof.

[0230] The "frequency" of administration can vary depending on the compound used and the particular type of infection being treated. A once-daily administration regimen is possible. In some cases, a dosing regimen in which the active agent is administered several times daily, for example, 2-10 times, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times, may be more useful.

[0231] It will be understood, however, that the specific dose level and frequency for any particular patient will depend on a variety of factors, including the activity of the specific compound used, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination, and the severity of the particular disease in the patient being treated. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays appropriate for the condition being treated or prevented, which will be well known to those skilled in the art.

[0232] Particular examples of pharmaceutical compositions according to the present invention include liquid formulations such as solutions, suspensions, emulsions, etc., which contain, in addition to the block copolymer according to the present invention, a therapeutically effective amount of a biopolymer component as defined above, optionally together with at least one further pharmaceutically acceptable excipient as defined above, and which may be administered by any suitable route.

[0233] Further examples of pharmaceutical compositions according to the present invention include solid dosage forms such as powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.

[0234] The following examples serve to better understand the invention without limiting its scope.

[0235] Experimental part A. Materials and Methods A.1 Chemicals and buffers Unless otherwise stated, all applied chemicals were of analytical grade and obtained from commercial sources.

[0236] The API was obtained from commercial sources.

[0237] Commercially available bovine immunoglobulin (IgG) captured from pooled bovine plasma using a chromatographic method (MPBio, catalog number 08641402, lot 21040)

[0238] A.2 Analysis method HPLC measurements were performed using a gradient elution method on an RP-C18 column (Chromolith®, High Resolution, RP-18 endcap, 100-4.6 mm, Merck KGaA), and the column temperature was maintained at 25°C. The mobile phase consisted of solvent A (water, 0.1% phosphoric acid) and solvent B (acetonitrile, 0.1% phosphoric acid), and the flow rate was 1.5 ml / min. Samples were detected and quantified using a DAD detector. The HPLC device used here was an Agilent Technologies 1260 Infinity II.

[0239] 1 H-NMR spectra were measured in CDCl3 on a Bruker Avance III 500 MHz spectrometer.

[0240] A.3 1 General Protocol for Determination of Ethylene Oxide Content by H-NMR The weight percent of ethylene oxide (EO wt%) in the claimed polymer is: 1 Determined by H-NMR spectroscopy. 1 In H-NMR spectroscopy, the integral of each peak is proportional to the molar concentration of the proton being analyzed.

[0241] For butylene oxide and ethylene oxide block copolymers (Butronics), the CH3 groups from polymerized butylene oxide give a triplet signal at δ=0.95 ppm.

[0242] The ethylene oxide repeat group -O-CH2-CH2-O- and the butylene oxide repeat group -O-CH2-CH(Et)-O- give a broad multiplet at δ = 3.0-4.2 ppm, but each ethylene oxide repeat group contributes four protons (two CH2 groups) and each butylene oxide group contributes three protons (one CH and one CH2 group) to the signal.

[0243] To determine the mol % ethylene oxide and wt % ethylene oxide, perform the following calculations: The integral at δ=0.95 ppm is set to three protons [area BuO]. The integral at δ = 3.0 to 4.2 ppm is [area EO + BuO] [EO area]=[EO+BuO area]-[BuO area] EO mol%=([EO area] / 4) / (([EO area] / 4)+([BuO area] / 3))*100 BuO mol%=([BuO area] / 3) / (([EO area] / 4)+([BuO area] / 3))*100

[0244] From EO mol % and BuO mol %, the weight percentage of ethylene oxide, EO wt %, is calculated using the molecular weight of ethylene oxide (44.05 g / mol) and the molecular weight of butylene oxide (72.11 g / mol). EO wt%=EO mol%*44.05 / ((EO mol%*44.05)+(BuO mol%*72.11))*100

[0245] A.4 General protocol for IgG agglutination assay Tests are performed using commercially available bovine immunoglobulin (IgG) captured from pooled bovine plasma using a chromatographic method. For each test, three reference substances are tested in addition to the sample: IgG without detergent (blank), IgG with PS20 (positive control), and IgG with P188 (negative control).

[0246] Dissolve 1 g of IgG in 50 mL of 20 mM histidine buffer (pH 6) by careful manual shaking. Store the IgG solution in a refrigerator at 4° C. overnight. Dissolve 40 mg of each surfactant in 10 mL of 20 mM histidine buffer (pH 6) and store in a refrigerator at 4° C. overnight.

[0247] To avoid particle contamination, all the following procedures are performed under a clean bench. Dilute the surfactant solution 1:100 with 20 mM histidine buffer (pH 6) to a concentration of 0.004%.

[0248] The IgG and surfactant solutions were filtered through a 0.22 μm PVDF syringe filter. For each surfactant and blank, 0.75 mL of surfactant (histidine buffer for the blank) and 0.75 mL of IgG solution were added to five glass vials per sample. This resulted in an IgG concentration of 10 mg / mL and a surfactant concentration of 0.002%. Four glass vials per sample were shaken at 200 rpm on an IKA HS 501 horizontal shaker for 15 hours (T15).

[0249] Invert the remaining glass vial (T0) three times and dilute 1:10 with histidine buffer pH 6 in a deep-well plate. Quantify particle numbers by microflow imaging (MFI) in the size range of 1 µm to 300 µm.

[0250] For all samples (T0 and T15), particle counts are quantified by microflow imaging (Protein Simple MFI5200). Samples are prepared for measurement by pipetting 0.1 mL of each sample into a 96-deep-well plate, followed by 0.1 mL of 200 mM histidine buffer and 0.8 mL of HPLC-grade water. All samples are measured in duplicate.

[0251] For analysis, use 0.6 mL of sample and determine the particle's equivalent circle diameter (ECD) in the range of 1 µm to 300 µm after removing edge particles, adhered particles, and slow particles.

[0252] The particle count for PS20 ranges from approximately 150 to 7,800 particles in a 0.6 mL diluted sample, depending on the IgG batch used, and the particle count for P188 ranges from approximately 10,200 to 92,000 particles. To effectively distinguish between different surfactants, it is necessary to use an IgG batch that, using the described protocol, yields at least 20,000 particles in a 0.6 mL diluted sample in the absence of surfactant (blank). While particle counts vary for different IgG batches, the trends between different surfactants are consistent.

[0253] For the analysis, a blank particle count is set relative to the aggregation of the sample containing surfactant (blank particle count is set as 100% aggregation).

[0254] A.5 General protocol for aqueous solubility assays To determine the polymer solubility of a 10 wt% solution in a 100 ml glass flask, 7 g of polymer (100%) and 63 g of distilled water were placed at room temperature. The mixture was stirred with a magnetic stirrer until the polymer was completely dissolved. To determine the solubility at other concentrations, polymer solutions with various polymer contents were prepared in a similar manner.

[0255] A.6 General protocol for surface tension assays For characterization of the surface activity of the synthetic polymers, samples were dissolved in deionized water at a concentration of 1 g / L and subsequently diluted to 0.1 g / L.

[0256] After stirring for 2 hours to ensure complete dissolution, the surfactant solution was loaded into a disposable syringe, which was then attached to a Drop Shape Analyser (DSA) 100 drop shape tensiometer from Kruess (Hamburg, Germany).

[0257] Static surface tension was measured at 0.1 g / L by the pendant drop method. Here, a freely hanging droplet of surfactant solution (typical volume: 1–10 μL depending on surface tension) was generated at the syringe outlet. A two-dimensional projection of the hanging droplet was then acquired by the integrated camera system, and the droplet contour was determined by image analysis using the instrument software Advance 1.9.2.

[0258] Fitting the droplet contour based on the Young-Laplace equation (C. Samuel et al., Polymer Testing, 2019, 78, 105995) yields the desired value for the surface tension.

[0259] The density of the solution required for evaluation was assumed to be that of pure water.

[0260] Surface tension was monitored over a 5 minute period and measurements were averaged. All measurements were performed at 23°C.

[0261] A.7 General protocol for hemolysis assay The principle of the RBC assay has been described by Hoover (DM: Hoover et al., Fundamental and Applied Toxicology 1990, 14, 589-597) and Pape (WJW Pape et al., Molecular Toxicology 1987, 1:525-536). The test is based on the integrity of the red blood cell (RBC) membrane and determines the degree of hemolysis after stirring a cell suspension with different test compound concentrations. In the event of RBC membrane damage by the test substance, hemoglobin is released into the test solution through the disrupted cell membrane. The concentration of free hemoglobin in the test solution is measured as a correlation with the RBC membrane damage caused by the test substance.

[0262] Preparation of red blood cell (RBC) suspension: RBCs were isolated from EDTA blood of human blood donors by centrifugation and washed three times with phosphate-buffered saline plus glucose (PBS / glucose) to remove traces of plasma and most of the white blood cells. The washed RBCs were diluted with PBS / glucose to an approximate 2% RBC suspension.

[0263] Test procedure: A 1.33% test solution of polymer (=1.33 g / 10 ml, i.e., final test concentration 100 mg / ml) was prepared in PBS / glucose and adjusted to pH 7.4. Further test solutions (final polymer concentrations of 10 and 1 mg / ml) were made by dilution with PBS / glucose. One volume of RBC suspension was added to three volumes of test solution to obtain final test compound concentrations of 100, 10, and 1 mg / ml. The assay mixture was incubated at room temperature for 60 minutes while shaking in an Eppendorf mixer (model 5432, 1000 rpm).

[0264] After incubation, the samples were centrifuged to pellet any remaining intact RBCs and membrane fragments.

[0265] The degree of hemolysis was measured by spectrophotometrically measuring the amount of free hemoglobin released into the supernatant at 540 nm.

[0266] Results were compared to RBCs completely lysed in distilled water (100% hemolysis) and a fragile control with PBS / glucose (spontaneous, no substance-related hemolysis). All samples were evaluated in triplicate.

[0267] The polymers tested were classified according to the following scheme: Low: Less than 10% hemolysis at 100 mg / ml Medium: Less than 10% hemolysis at 10 mg / ml High: 10mg / ml or more of hemolysis

[0268] A.8 General Protocol for Measuring Small Molecule Solubilization Capacity with Butronics A 10% (m / m) aqueous solution of the test substances (Butronics and the reference substances Kolliphor EL and HS15) is prepared in phosphate buffer pH 7.4. For each test substance and reference substance, three 5 ml amber glass ampoules with screw caps are filled with 5 g of the prepared aqueous solution.

[0269] Next, add each API to be analyzed (e.g., fenofibrate / itraconazole / nilotinib, etc.) to obtain a supersaturated solution (approximately 30 mg to 500 mg per 5 g of 10% test substance solution, to the extent that the added powder does not dissolve any more).

[0270] After stirring for 72 hours at room temperature, each sample is filtered through a 0.22 μm PVDF filter and the concentration of each API in the filtrate is quantified by HPLC.

[0271] The average of three replicates for each API was calculated and the resulting concentrations are shown relative to Kolliphor EL, which was set at 100%.

[0272] Table 7 in section C.6 shows the mean results for fenofibrate, itraconazole and nilotinib.

[0273] A.9 General protocol for cloud point determination The cloud point is determined in accordance with DIN EN 1890, method A.

[0274] In a 100 ml glass flask, 0.4 g of polymer (100) and 39.6 g of distilled water are placed at room temperature. The mixture is stirred with a magnetic stirrer until the polymer is completely dissolved. Approximately 30 ml of this solution is filled into a test glass. A thermometer is placed inside the test glass to monitor the temperature. The test glass is slowly heated in a hot air oven until a persistent turbidity is observed.

[0275] The cloud point is determined as the temperature at which a solution changes from clear to cloudy. A value of 20°C indicates that the solution is cloudy at room temperature. A cloud point above 95°C means that no cloudiness is observed up to 95°C.

[0276] A.10 General Protocol for the Preparation of PLGA Nanoparticles PLGA nanoparticles were prepared by nanoprecipitation. A 50 mg / mL solution of PLGA (Resomer® RG502H) in acetone was prepared. 0.5 mL of this solution was slowly injected into 9.5 mL of HPLC-grade water while vigorously stirring the water. For injection, the tip of an Eppendorf tip was immersed in the vigorously stirred water, and the PLGA solution was slowly released from the tip. The final acetone concentration in the resulting slightly turbid suspension was 5%, and the PLGA concentration was 2.5 mg / mL. The particle size of the resulting PLGA particles (measured by DLS using a Malvern Zetasizer Nano S) was approximately 230 nm, with a PDI of approximately 0.1 nm.

[0277] For each surfactant (Poloxamer P188 and Butronics), prepare a 5% (w / w) aqueous solution by adding 9.5 mL of HPLC-grade water to 0.5 g of surfactant and shaking at 1000 rpm in a thermoshaker for at least 20 minutes until all surfactant is completely dissolved. The solution is then filtered through a 0.2 μm PVDF syringe filter.

[0278] For each surfactant, 0.2 mL of 5% surfactant solution was added to 1.8 mL of PLGA suspension. The resulting suspension was mixed briefly and then incubated for approximately 5 minutes. The particle size and dispersity of the undiluted suspension were measured by DLS (Malvern Zetasizer Nano S) according to the manufacturer's instructions. The solution was then transferred to a 2 mL Eppendorf tube and centrifuged at 14,800 rpm for 20 minutes. A white pellet formed at the bottom of the tube. The supernatant was removed, and each pellet was resuspended in 2 mL of HPLC-grade water. All samples were vortexed briefly (for a few seconds), and the particle size and dispersity of the undiluted suspension were then measured again by DLS (Malvern Zetasizer Nano S) according to the manufacturer's instructions.

[0279] A.11 General protocol for further biopolymer aggregation assays A.11.1 β-Casein Protocol The test is carried out using commercially available β-casein extracted from cow's milk and is carried out according to the protocol given in section A.4. The final protein concentration is 3.125 mg / mL.

[0280] A.11.2 Lysozyme Protocol The test is carried out using commercially available lysozyme and is carried out according to the protocol given in section A.4.

[0281] Dissolve 0.1 g of lysozyme in 50 mL of phosphate-buffered saline (PBS) pH 7.4 by careful manual shaking. Store the lysozyme solution overnight in a refrigerator at 4°C. The detergent solution is prepared as described in Section A.4, whereby PBS is used instead of the histidine buffer.

[0282] For each surfactant and blank, 0.4 mL of surfactant (PBS for the blank) and 0.4 mL of lysozyme solution were added to four glass vials per sample. This resulted in a lysozyme concentration of 1 mg / mL and a surfactant concentration of 0.002%. The glass vials were shaken at 500 rpm in a Thermo Fisher Scientific MaxQ™ 6000 incubation shaker at room temperature for 24 hours.

[0283] For all samples, particle numbers are quantified by microflow imaging (Protein Simple MFI5200). Sample preparation and measurements are carried out as described in section A.4.

[0284] For the analysis, a blank particle count is set relative to the aggregation of the sample containing surfactant (blank particle count is set as 100% aggregation).

[0285] A.11.3 Thrombin Protocol The test is carried out using commercially available thrombin and is carried out according to the protocol given in section A.4. The final protein concentration is 1 mg / mL and the final volume in the vial is 0.8 mL.

[0286] A.11.4 Carbonic Anhydrase Protocol The test is carried out using commercially available carbonic anhydrase and is carried out according to the protocol given in section A.4. The final protein concentration is 1 mg / mL and the final volume in the vial is 0.8 mL. The vials are shaken at 300 rpm on an IKA HS 501 horizontal shaker for 4 hours.

[0287] A.11.5 Protocol for IgG Extracted from Human Serum The test is carried out using commercially available IgG extracted from human serum and is carried out according to the protocol given in section A.4. The final protein concentration is 1 mg / mL and the final volume in the vial is 0.8 mL. The vials are shaken for 4.5 hours at 300 rpm on an IKA HS 501 shaker plate.

[0288] A.11.6 Monoclonal Antibody 4 (mAb4) Protocol The tests are carried out using a recombinant humanized IgG1 monoclonal antibody (mAb4). For each test, four reference substances are tested in addition to the sample: mAb4 stored at room temperature without detergent and shaking (unstressed mAb4), mAb4 stressed by shaking without detergent (blank), mAb4 with PS20 (positive control), and mAb4 with P188 (negative control).

[0289] The antibody is already dissolved in 20 mM histidine buffer (pH 6) and stored in a -80°C freezer. After thawing in a 4°C refrigerator, the antibody solution is further diluted to a concentration of 2 mg / mL. The detergent solution is prepared as described in Section 4.

[0290] For each surfactant, blank, and unstressed sample, 0.4 mL of surfactant (histidine for blank and unstressed samples) and 0.4 mL of diluted mAb4 solution were added to four glass vials per sample. This resulted in a mAb4 concentration of 1 mg / mL and a surfactant concentration of 0.002%. The glass vials were shaken at 300 rpm on an IKA HS 501 shaker plate for 15 hours at room temperature. The vials containing unstressed mAb4 were stored at room temperature without shaking.

[0291] Because the blank samples are very turbid, they are not further analyzed by microflow imaging, while the remaining samples are analyzed as described in section A.4.

[0292] For analysis, the particle count of the sample is set relative to the aggregation of unstressed mAb4 (the particle count of unstressed mAb4 is set as 100% aggregation).

[0293] A.11.7 Monoclonal Antibody 3 (mAb3) Protocol The tests are carried out using a recombinant humanized IgG2 monoclonal antibody (mAb3). For each test, four reference substances are tested in addition to the sample: mAb3 stored at room temperature without detergent and shaking (unstressed mAb3), mAb3 stressed by shaking without detergent (blank), mAb3 with PS20 (positive control), and mAb4 with P188 (negative control).

[0294] The antibodies were previously dissolved in 20 mM histidine buffer (pH 6) and stored in a -80°C freezer. After thawing in a 4°C refrigerator, the antibody solution was further diluted to a concentration of 2 mg / mL. 40 mg of each surfactant was dissolved in 10 mL of 20 mM histidine buffer (pH 6) containing 600 mM NaCl and stored in a refrigerator at 4°C overnight.

[0295] To avoid particle contamination, all the following procedures are performed under a clean bench. The surfactant solution is diluted 1:100 with 20 mM histidine buffer (pH 6) containing 600 mM NaCl to a concentration of 0.004%.

[0296] For each surfactant, blank, and unstressed sample, 0.4 mL of surfactant (histidine with 600 mM NaCl for blank and unstressed samples) and 0.4 mL of diluted mAb3 solution were added to four glass vials per sample. This resulted in a mAb3 concentration of 1 mg / mL, a surfactant concentration of 0.002%, and a NaCl concentration of 300 mM. The glass vials were shaken at 400 rpm in a Thermo Fisher Scientific MaxQ™ 6000 incubation shaker for 4.5–5 h at room temperature, while the vials containing unstressed mAb3 were stored at room temperature.

[0297] All further analyses will be performed as described in Section A.4.

[0298] B. Synthesis example Exemplary Butronics according to the present invention (Test Sample Nos. 1-10) and comparative Butronics (CE1-CE5) are characterized by the following general formula 1a: [ka]

[0299] All polymers were synthesized by applying a synthetic protocol similar to that described in Synthesis Example 1 below (X is derived from 1,4-butanediol).

[0300] The given molecular weights are calculated from the molar ratios of the starting materials used. The ethylene oxide content (EO wt%) was determined according to the protocol described above (see item 3).

[0301] Synthesis Example 1: 1,4-Butanediol Butoxylated with 20 Moles of 1,2-Butylene Oxide and Ethoxylated with 34 Moles of Ethylene Oxide (Synthesis Method A) Step a: Synthesis of butoxylated 1,4-butanediol with 20 moles of 1,2-butylene oxide A 2-L autoclave was charged with 90.12 g of 1,4-butanediol and 3.1 g of potassium tert-butoxide, and the reactor was purged with nitrogen three times. The mixture was heated to 140°C. 1,440.0 g of 1,2-butylene oxide was added within 20 hours. To complete the reaction, the mixture was post-reacted at 140°C for an additional 10 hours. The reaction mixture was stripped with nitrogen, and volatile compounds were removed under vacuum at 90°C for 2 hours. 1,528.0 g of a pale orange oil was obtained.

[0302] in CDCl 1 H-NMR confirmed complete conversion to the expected polymer.

[0303] Step b: Synthesis of 1,4-butanediol butoxylated with 20 moles of 1,2-butylene oxide and ethoxylated with 34 moles of ethylene oxide A 2 L autoclave was charged with 404.5 g of 1,4-butanediol butoxylated with 20 moles of 1,2-butylene oxide (from step a) and 1.6 g of potassium tert-butoxide, and the reactor was purged with nitrogen three times. The mixture was heated to 110°C. 395.5 g of ethylene oxide was added within 20 hours. To complete the reaction, the mixture was post-reacted at 140°C for an additional 10 hours. The reaction mixture was stripped with nitrogen, and 1.3 g of acetic acid was added for neutralization. The collected droplets were dried in an oven at 90°C for 2 hours. 800.0 g of a beige solid was obtained.

[0304] in CDCl 1 H-NMR confirmed complete conversion to the expected polymer. The hydroxyl number was measured to be 37.2 mg KOH / g and the water content was 0.1%.

[0305] Synthesis Example 2: 1,4-Butanediol butoxylated with 24 moles of 1,2-butylene oxide and ethoxylated with 96 moles of ethylene oxide - Synthesis Step 1 in the presence of water (Synthesis Method B) Step a: Synthesis of 1,4-butanediol butoxylated with 10 moles of 1,2-butylene oxide (in the presence of water) In a 2 L autoclave, 85.6 g of 1,4-butanediol and 3.4 g of potassium hydroxide (50% in water) were placed, and the reactor was heated to 100° C. The reactor was purged with nitrogen three times. The mixture was heated to 140° C. 771.0 g of 1,2-butylene oxide was added within 6 hours. To complete the reaction, the mixture was post-reacted at 140° C. for an additional 3 hours. The reaction mixture was stripped with nitrogen, and volatile compounds were removed under vacuum at 90° C. for 2 hours. 852.0 g of a pale orange oil was obtained.

[0306] in CDCl 1 H-NMR confirmed complete conversion to the expected polymer. The hydroxyl number was measured to be 130.0 mg KOH / g and the water content was 0.13 wt%.

[0307] Step b: Synthesis of 1,4-butanediol butoxylated with 24 moles of 1,2-butylene oxide and ethoxylated with 96 moles of ethylene oxide A 2 L autoclave was charged with 145.8 g of 1,4-butanediol butoxylated with 10 moles of 1,2-butylene oxide (from step a) and 1.6 g of potassium hydroxide (50% in water). A vacuum (<25 mbar) was applied, and the mixture was heated to 125°C. The mixture was stirred at 125°C under a vacuum <25 mbar for 2.5 hours. Nitrogen was introduced, and the vacuum was released. The mixture was heated to 140°C. 181.4 g of butylene oxide was added within 2 hours. The mixture was post-reacted for 6 hours to complete the reaction. 761.0 g of ethylene oxide was added within 6 hours. The mixture was post-reacted at 140°C for an additional 2 hours to complete the reaction. The reaction mixture was stripped with nitrogen, and 2.5 g of phosphoric acid (75% in water) was added for neutralization. Volatile compounds were removed under vacuum at 90°C for 2 hours. 0.10 g of α-tocopherol was added and the mixture was stirred for 0.25 h. After cooling, 1081.0 g of a beige solid was obtained.

[0308] in CDCl 1 H-NMR confirmed complete conversion to the expected polymer. The hydroxyl number was measured to be 24.8 mg KOH / g and the water content was 0.1%.

[0309] Synthesis Example 3: Synthesis of isosorbide butoxylated with 24 moles of 1,2-butylene oxide and ethoxylated with 96 moles of ethylene oxide (Synthesis Method C) Step a: Synthesis of isosorbide butoxylated with 10 moles of 1,2-butylene oxide in the presence of water A 2 L autoclave was charged with 146.1 g of isosorbide and 15.6 g of potassium hydroxide (50% in water), and the reactor was heated to 90°C. A vacuum (less than 25 mbar) was applied, and the mixture was heated to 120°C. The mixture was stirred at 125°C and a vacuum of less than 25 mbar for 2.5 hours. The reactor was purged with nitrogen three times. The mixture was heated to 140°C. 721.1 g of 1,2-butylene oxide was added within 8 hours. To complete the reaction, the mixture was post-reacted at 140°C for an additional 3 hours. The reaction mixture was stripped with nitrogen, and volatile compounds were removed under vacuum at 90°C for 2 hours. 865.0 g of a pale orange oil was obtained.

[0310] in CDCl 1 H-NMR confirmed complete conversion to the expected polymer. The water content was 0.12 wt%.

[0311] Step b: Synthesis of isosorbide butoxylated with 24 moles of 1,2-butylene oxide and ethoxylated with 96 moles of ethylene oxide A 2 L autoclave was charged with 104.7 g of isosorbide butoxylated with 10 moles of 1,2-butylene oxide (from step a). The reactor was purged with nitrogen three times. The mixture was heated to 160°C. 120.4 g of butylene oxide was added within 2 hours. The mixture was post-reacted for 6 hours to complete the reaction. The reactor was cooled to 150°C, and 504.4 g of ethylene oxide was added within 6 hours. The mixture was post-reacted at 150°C for an additional 2 hours to complete the reaction. The reaction mixture was stripped with nitrogen, and 1.5 g of phosphoric acid (75% in water) was added for neutralization. Volatile compounds were removed under vacuum at 90°C for 2 hours. 0.10 g of α-tocopherol was added, and the mixture was stirred for 0.25 hours. After cooling, 719.0 g of a beige solid was obtained.

[0312] in CDCl 1 H-NMR confirmed complete conversion to the expected polymer. The hydroxyl number was measured to be 24.8 mg KOH / g.

[0313] C. Experimental Results C.1 Exemplary Block Copolymers (Butronics)

[0314] [Table 1]

[0315] [Table 2]

[0316] [Table 3]

[0317] C.2 IgG agglutination assay results Aggregation propensity assays on selected sample sets were performed as described above (see section A.4).

[0318] For each study, three reference substances are tested in addition to the samples: IgG without surfactant (blank), polysorbate 20 (positive control), and P188 (negative control).

[0319] The results are summarized in Table 3.

[0320] [Table 4]

[0321] As can be seen, all samples, including comparative Butronics CE1-CE5, prevent significant protein aggregation. In this regard, samples 1, 2, 4, 5, 7, 12-15, and 17 were shown to prevent protein aggregation to a greater extent than the commonly used polysorbate 20.

[0322] C.3 Water Solubility Assay Results Water solubility assays on selected sample sets were performed as described above (see section A.5).

[0323] The results are summarized in Table 4.

[0324] [Table 5]

[0325] Kolliphor® EL is a registered trademark for polyethoxylated castor oil. It is prepared by reacting 35 moles of ethylene oxide with 1 mole of castor oil. Kolliphor® EL is a synthetic nonionic surfactant used to stabilize emulsions of nonpolar substances in water. Kolliphor® EL is an excipient or additive used in drugs.

[0326] Solutol® HS 15 is a registered trademark for polyoxyethylated 12-hydroxystearic acid, another excipient or additive used in drugs.

[0327] As can be seen, all test samples except for Test Sample 1 and CE5 exhibited water solubility greater than 10%. Therefore, CE5 is unsuitable as a formulation excipient due to its insufficient water solubility.

[0328] C.4 Surface tension assay results Surface tension assays on selected sample sets were performed as described above (see section A.6).

[0329] The results are summarized in Table 5.

[0330] [Table 6]

[0331] As can be observed, all test samples exhibited surface tension values ​​comprised between 53 and 30 mN / m (0.1 g / l).

[0332] C.5 Hemolysis Assay Results Hemolysis refers to the phenomenon that results in the rupture and lysis of red blood cells. Assays on selected sample sets were performed as described above (see section A.7).

[0333] The results are summarized in Table 6.

[0334] [Table 7]

[0335] While CE1-4 showed a high degree of hemolysis, test samples 1-7 and CE5 showed significantly lower hemolytic activity compared to polysorbate 80, indicating their lower toxicity and therefore suitability as possible formulation additives.

[0336] C.6 Small molecule solubilization ability The suitability of Butronics according to the invention for solubilizing model small molecules was tested as described above (see section A.8).

[0337] The results are summarized in Table 7.

[0338] [Table 8]

[0339] Test samples 4 and 6, and comparative copolymers CE1 and CE5, demonstrated superior small molecule solubilization efficacy to Kolliphor® EL (reference material).

[0340] C.7 Results of preparation of PLGA nanoparticles using Butronics PLGA particles were prepared using different surfactants as described above (see section A.10). The stabilizing properties of Butronics were compared to the stability of surfactant-free particles (water only) and poloxamer P188.

[0341] The results are summarized in Table 8.

[0342] [Table 9]

[0343] As can be seen, Test Samples 2 and 3 best stabilized the PLGA particles. CE5 was also able to stabilize the particles, but due to its low water solubility, CE5 is less suitable as a formulation excipient compared to Test Samples 2 and 3. In all other test samples, the pellets could not be resuspended after centrifugation, indicating insufficient stabilization of the PLGA particles. PDI=polydispersity index DLS = Dynamic Light Scattering PLGA = poly(lactic-co-glycolic acid)

[0344] C.8 Further biopolymer aggregation assay results C.8.1 β-Casein Results β-Casein aggregation assays on a selected set of samples were performed as described in section A.11.1.

[0345] For each test, three reference substances are tested in addition to the sample: surfactant-free β-casein (blank = 100%), polysorbate 20 (positive control), and P188 (negative control).

[0346] The results are summarized in Table 9.

[0347] [Table 10]

[0348] As can be seen, all samples prevent significant protein aggregation. In this regard, samples 1-5 were proven to prevent protein aggregation to the same extent as the commonly used polysorbate 20.

[0349] C.8.2 Lysozyme Results Lysozyme aggregation assays on a selected set of samples were performed as described in section A.11.2.

[0350] For each test, three reference substances are tested in addition to the sample: lysozyme without surfactant (blank = 100%), polysorbate 20 (positive control), and P188 (negative control).

[0351] The results are summarized in Table 10.

[0352] [Table 11]

[0353] As can be seen, all samples prevent significant protein aggregation.

[0354] C.8.3 Thrombin Results Thrombin aggregation assays on a selected set of samples were performed as described in section A.11.3.

[0355] For each test, three reference substances are tested in addition to the sample: thrombin without detergent (blank = 100%), polysorbate 20 (positive control), and P188 (negative control).

[0356] The results are summarized in Table 11.

[0357] [Table 12]

[0358] As can be seen, all samples prevent significant protein aggregation. In this regard, all Butronics samples 1-9 have been shown to prevent protein aggregation to a greater extent than the commonly used polysorbate 20.

[0359] C.8.4 Carbonic anhydrase results Carbonic anhydrase agglutination assays on a selected set of samples were performed as described in section A.11.4.

[0360] For each test, three reference substances are tested in addition to the sample: thrombin without detergent (blank = 100%), polysorbate 20 (positive control), and P188 (negative control).

[0361] The results are summarized in Table 12.

[0362] [Table 13]

[0363] Samples 1-9 prevent protein aggregation more efficiently than polysorbate 20. The high ethylene oxide content (approximately 80%) significantly prevents aggregation of carboanhydrase during shaking.

[0364] C.8.5 Results for IgG extracted from human serum IgG from human serum agglutination assays for a selected set of samples were performed as described in section A.11.5.

[0365] For each test, three reference substances are tested in addition to the sample: thrombin without detergent (blank = 100%), polysorbate 20 (positive control), and P188 (negative control).

[0366] The results are summarized in Table 13.

[0367] [Table 14]

[0368] As can be observed, Samples 1-9 prevent significant protein aggregation.

[0369] C.8.6 Monoclonal Antibody 4 (mAb4) Results The mAb4 assay on a selected set of samples was performed as described in section A.11.6.

[0370] For each test set, four reference substances are tested in addition to the samples: mAb4 stored at room temperature without detergent and shaking (unstressed mAb4), mAb4 stressed with shaking and without detergent (blank = 100%), mAb4 with PS20 (positive control) and mAb4 with P188 (negative control).

[0371] Because the blank samples were very turbid, they were not further analyzed by microflow imaging. For analysis, the particle counts of the samples were set relative to the aggregation of unstressed mAb4 (the particle count of unstressed mAb4 was set as 100% aggregation).

[0372] The results are summarized in Table 14.

[0373] [Table 15]

[0374] All samples significantly prevent mAb4 aggregation.

[0375] C.8.7 Monoclonal Antibody 3 (mAb3) Results The mAb3 assay on a selected set of samples was performed as described in section A.11.7.

[0376] For each test set, four reference substances are tested in addition to the samples: mAb3 stored at room temperature without detergent and shaking (unstressed mAb3), mAb3 stressed with shaking and without detergent (blank = 100%), mAb3 with PS20 (positive control) and mAb3 with P188 (negative control).

[0377] The results are summarized in Table 15.

[0378] [Table 16]

[0379] As can be seen, all samples prevent significant protein aggregation.

[0380] C.9 Summary Our attempt to provide polymeric surfactants as superior alternatives to polysorbate 20 / 80 and poloxamer 188 has led to the following conclusions:

[0381] For polyclonal IgG, all samples tested (samples 1-17 and 5a) proved to be good stabilizers against aggregation of protein formulations.

[0382] Looking at the stabilization performance of Butronics using biopolymers, samples 4, 5, 7, 8, and 9 in particular showed excellent performance, preventing protein aggregation to a greater extent than the commonly used polysorbate 20.

[0383] Samples 5, 7, 8, and 9 are the best performing samples in the assays tested here. They are also characterized by favorable surface tensions (within the range of approximately 53-30 mN / m (0.1 g / L)) and molecular weights (Mw of approximately 6,000-8,000 g / mol). Samples 1 and CE5 were found to be less water soluble, while the comparative Butronics CE1-CE4 exhibited a high degree of hemolysis.

[0384] Thus, samples 5, 7, 8, and 9 perform best with respect to all criteria required for use as surfactants in biopolymer formulations with superior properties compared to Polysorbate 20 / 80 and Poloxamer 188.

[0385] Furthermore, samples 2, 4, 5, 7, 8, and 9 perform best with respect to all criteria required for use as surfactants in antibody formulations with superior properties compared to polysorbate 20 / 80 and poloxamer 188.

[0386] Furthermore, Samples 4 and 6 were characterized by particularly excellent small molecule solubilizing effects, which indicated that they were particularly suitable surfactants for ADC formulations.

[0387] The surprising advantages associated with this particular example of Butronics of the present invention are summarized in accompanying FIG.

[0388] The contents of all documents mentioned above are incorporated herein by reference.

Claims

1. At least one biopolymer component and at least one compound represented by general formula 1 2015 ｎ -(Bu) ｍ -X-(BuO) ｍ -(59) ｎ -2 (1) (In the formula, X represents —O— or a divalent organic moiety; m's are each independently an integer ranging from 4 to 25; n's each independently represent an integer ranging from 15 to 100. and an ethylene oxide / butylene oxide block copolymer of said biopolymers are selected from adducts or conjugates of such biopolymers with further moieties selected from oligopeptides, polypeptides, proteins, antibody molecules or fragments or derivatives thereof, glycosylated proteins, proteoglycans, oligonucleotides and polynucleotides, DNA and RNA molecules, oligosaccharides, polysaccharides, and payload molecules; and The block copolymer has the following characteristics: a) a molecular weight of 2,500 to 12,500 g / mol, in particular 2,800 to 9,000 g / mol, more particularly 3,000 to 8,000 g / mol, and most particularly 3,500 to 8,000 g / mol, each of which can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1; b) an EO content of greater than 50% to 85% by weight, particularly 53 to 85% by weight, more particularly 55 to 80% by weight, and most particularly 57 to 80% by weight, each based on the dry weight of the block copolymer and each calculable from the atomic masses of all atoms in the copolymer molecule of Formula 1; c) at least 5% by weight water solubility, based on the total weight of the aqueous solution of the block copolymer; and d) a hemolytic activity of less than 10% caused by a solution of said block copolymer at 100 g / l , a biopolymer solution showing a combination of

2. The block copolymer may have the following additional characteristics: a) at least 10% by weight water solubility, based on the total weight of the aqueous solution of said block copolymer; b) a surface tension SFT of 53 to 30 mN / m, based on a 0.1 g / l solution of said block copolymer; and c) lack of hemolytic activity The biopolymer solution of claim 1 , wherein the biopolymer solution exhibits at least one of the following:

3. the butylene oxide block is composed of monomer units derived from 1,2-butylene oxide, 2,3-butylene oxide, isobutylene oxide, or mixtures thereof, in particular essentially forming 1,2-butylene oxide, more particularly consisting of 1,2-butylene oxide; and / or X is —O—, —O-alkylene-O—, particularly —O—(C ２ -C ２２ -alkylene)-O-, more specifically -O-(C ２ -C ６ -alkylene)-O-, or even more specifically -O-(C ２ -C ４ -alkylene)-O-, where the alkylene chain is linear or branched and is optionally interrupted by one or more heteroatoms, in particular oxygen or -O-(n-butylene)-O-; or X is a group of formula 2 【Chemistry 1】 The biopolymer solution according to claim 1 or 2, wherein the group is

4. The block copolymer of general formula 1 has the following characteristics: a) a molecular weight of 5,500 to 8,000 g / mol, which can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1; b) an EO content of 60 to 85 wt. % based on the dry weight of the block copolymer, calculable from the atomic masses of all atoms in the copolymer molecule of Formula 1; and c) X=-O-n-butylene-O- The biopolymer solution according to any one of claims 1 to 3, which exhibits a combination of:

5. The block copolymer is selected from compounds of the following general formula 1, wherein X, m and n have the following meanings: X=-O-n-butylene-O-, m=10; and n=17; X = -O-n-butylene-O-, m = 10, and n = 27; X = -O-n-butylene-O-, m = 5, and n = 35; X = -O-n-butylene-O-, m = 16, and n = 40; X = -O-n-butylene-O-, m = 12, and n = 48; X = -O-n-butylene-O-, m = 21, and n = 54; X = -O-n-butylene-O-, m = 10, and n = 70; X = the moiety of Formula 2, m = 10; and n = 17; X = moiety of Formula 2, m = 10, and n = 27; X = moiety of Formula 2, m = 5, and n = 35; X = moiety of Formula 2, m = 16, and n = 40; X = moiety of Formula 2, m = 12, and n = 48; X = moiety of Formula 2, m = 21, and n = 54; X = a moiety of Formula 2, m = 11, and n = 68; or X = the moiety of Formula 2, m = 10, and n = 70 The biopolymer solution of any one of claims 1 to 4, wherein

6. The block copolymer is selected from compounds of the following general formula 1, wherein X, m and n have the following meanings: X=—O-n-butylene-O—, m=12, and n=48, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 6,050; X=—O-n-butylene-O—, m=10, and n=70, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,658; X=—O-n-butylene-O—, m=16, and n=40, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 5,922; or X = the moiety of formula 2, m = 11, and n = 68. The molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,724.

6. The biopolymer solution of claim 5, having:

7. X is a compound of formula 2 【Chemistry 2】 is the basis of The block copolymer has the following characteristics: a) a molecular weight of 3,000 to 8,000 g / mol, 5,500 to 8,000 g / mol, particularly 5,800 to 7,900 g / mol, which can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1; b) an EO content of 55 to 85 wt. % based on the dry weight of the block copolymer, calculable from the atomic masses of all atoms in the copolymer molecule of general formula 1; c) at least 5% by weight water solubility, based on the total weight of the aqueous solution of the block copolymer; and d) less than 10% hemolytic activity caused by a 100 g / l solution 4. The biopolymer solution of claim 3, which exhibits a combination of:

8. the biopolymer is an oligopeptide, a polypeptide, a protein, a glycosylated protein, a proteoglycan, an antibody molecule or a fragment or derivative thereof; a) a pharmaceutically active compound; b) a labeling agent; c) small biological molecules such as lipids, phospholipids, glycolipids, sterols, vitamins, hormones, neurotransmitters, amino acids, nucleotides, and monosaccharides; and d) Biopolymers such as peptides, oligopeptides, polypeptides, proteins, nucleic acids such as DNA and RNA in any form, oligosaccharides, and polysaccharides.

8. The biopolymer solution of claim 1, wherein the biopolymer is selected from the group consisting of:

9. The biopolymer is a diagnostically applicable or therapeutically active biopolymer, in particular the biopolymer is selected from proteins, in particular enzymes and immunoglobulin molecules, each optionally glycosylated; or the biopolymer is selected from an adduct or conjugate of an immunoglobulin molecule and a payload molecule; or The biopolymer solution of claim 8 , wherein the biopolymer is an antibody payload conjugate (APC), in particular an antibody drug conjugate (ADC).

10. The biopolymer is an immunoglobulin or protein molecule or an antibody payload conjugate (APC), in particular an antibody drug conjugate (ADC), each optionally glycosylated, wherein the at least one ethylene oxide / butylene oxide block copolymer has the general formula 1 2015 ｎ -(Bu) ｍ -X-(BuO) ｍ -(59) ｎ -2 (1) (In the formula, X represents —O—; or a divalent organic moiety; m's are each independently an integer ranging from 10 to 20; n are, independently of each other, integers ranging from 25 to 75; The block copolymer has the following characteristics: a) a molecular weight of 3,500 to 8,000 g / mol, which can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1; b) an EO content of 55 to 85 wt. % based on the dry weight of the block copolymer, calculable from the atomic masses of all atoms in the copolymer molecule of general formula 1; c) at least 5% by weight water solubility, based on the total weight of the aqueous solution of the block copolymer; and d) less than 10% hemolytic activity caused by a 100 g / l solution 10. The biopolymer solution according to claim 1, which exhibits a combination of:

11. The block copolymer of general formula 1 is contained in an amount of 0.001 to 10% based on the total weight of the biopolymer solution; and / or the biopolymer is contained in an amount of 0.01 to 30% based on the total weight of the biopolymer solution; and / or the biopolymer solution is optionally in a buffered form and has a pH in the range of 5 to 9, in particular 6 to 8. The biopolymer solution according to any one of claims 1 to 10.

12. 11. A composition comprising at least one biopolymer component as defined above and at least one ethylene oxide / butylene oxide block copolymer of general formula 1 as defined in any one of claims 1 to 7 or 10; and having a liquid content of 0% to 5% by weight based on the total weight of the composition; Optionally, the following: The at least one block copolymer (A) and the at least one biopolymer (B) are contained in a weight ratio (A):(B) ranging from 1:20,000 to 10:1, or from 1:5,000 to 2:1, from 1:100 to 1.2:1, in particular from 1:10 to 1.1:1; and / or the at least one block copolymer and the at least one biopolymer together comprise from 1 to less than 100% by weight, in particular from 5 to 60% by weight, more particularly from 10 to 50% by weight, or from 20 to 40% by weight, or from 20 to 25% by weight, based on the total weight of the essentially dry composition; and / or and at least one additional excipient in an amount of 0.1 to 99 wt. %, 40 to 95 wt. %, and 50 to 90 wt. %, based on the total dry weight of the essentially dry composition. The essentially dry biopolymer composition, further characterized as:

13. Use of a block copolymer according to any one of claims 1 to 7 or 10 for stabilising an aqueous composition, in particular an aqueous solution, of at least one biopolymer as defined above.

14. A composition according to any one of claims 1 to 12 for use in medicine, in particular for diagnostic and / or therapeutic applications.

15. The composition according to any one of claims 1 to 12, which is a pharmaceutical composition optionally further supplemented with at least one pharmaceutically acceptable excipient.

16. A method for preparing a stabilized biopolymer solution according to any one of claims 1 to 11, comprising the steps of: a) preparing, in any order, an optionally buffered aqueous solution of the biopolymer; and an optionally buffered aqueous solution of a block copolymer of general formula 1; and b) preparing a mixture of both aqueous solutions obtained in step a); A method comprising:

17. 13. A method for preparing the essentially dry stabilized biopolymer solution of claim 12, comprising: a) preparing, in any order, an optionally buffered aqueous solution of said biopolymer; and an optionally buffered aqueous solution of said block copolymer of general formula 1; b) preparing a mixture of both optionally buffered aqueous solutions obtained in step a); c) optionally supplementing the optionally buffered aqueous solution prepared in step a) and / or the mixture of both optionally buffered aqueous solutions prepared in step b) with at least one pharmaceutically acceptable excipient; d) drying the mixture obtained in step b) or c). A method comprising:

18. The compound is selected from the compounds of the general formula 1 below, wherein X, m and n have the following meanings: X=-O-n-butylene-O-, m=10; and n=17; X = -O-n-butylene-O-, m = 10, and n = 27; X = -O-n-butylene-O-, m = 5, and n = 35; X = -O-n-butylene-O-, m = 16, and n = 40; X = -O-n-butylene-O-, m = 12, and n = 48; X = -O-n-butylene-O-, m = 21, and n = 54; X = -O-n-butylene-O-, m = 10, and n = 70; X = the moiety of Formula 2, m = 10, and n = 17; X = moiety of Formula 2, m = 10, and n = 27; X = moiety of Formula 2, m = 5, and n = 35; X = moiety of Formula 2, m = 16, and n = 40; X = moiety of Formula 2, m = 12, and n = 48; X = moiety of Formula 2, m = 21, and n = 54; X = a moiety of Formula 2, m = 11, and n = 68; or X = the moiety of Formula 2, m = 10, and n = 70 A block copolymer having the formula:

19. X, m and n have the following meanings: X=—O-n-butylene-O—, m=12, and n=48, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 6,050; X=—O-n-butylene-O—, m=10, and n=70, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,658; X=—O-n-butylene-O—, m=16, and n=40, and the molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 5,922; or X = the moiety of formula 2, m = 11, and n = 68. The molecular weight that can be calculated from the sum of the atomic masses of all atoms in the copolymer molecule of general formula 1 is approximately 7,724. The block copolymer of claim 18 having the formula:

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