Stabilized biopolymer composition, their manufacture and use

Abstract

The present invention relates to the field of stabilized biopolymer compositions, particularly to stabilized biopolymer compositions comprising a liquid mixture, in particular 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 / propylene oxide block copolymer capable to confer to said biopolymer composition superior properties in comparison to the presently known formulations, particularly with regard to aggregation tendency, hemolytic activity and / or solubility. The invention further relates to said block copolymers, to methods for preparing said stabilized compositions and to the use of said block copolymers for stabilizing an aqueous composition of said biopolymer The invention further relates said stabilized composition for use in medicine, in particular for diagnostic and / or therapeutic applications. The invention further relates to an essentially dry biopolymer composition, comprising said biopolymer and said block copolymers and to methods for preparing said essentially dry compositions. The invention further relates to said essentially dry biopolymer composition for use in medicine, in particular for diagnostic and / or therapeutic applications. Said compositions may be formulated as pharmaceutical compositions which may be delivered via suitable routes of administration such as via oral, rectal, transmucosal, topical, ophthalmic, otologic, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, as the case may be.

Description

[0001] Stabilized Biopolymer Composition, their Manufacture and Use

[0002] Field of the Invention

[0003] The present invention relates to the field of stabilized biopolymer compositions, particularly to stabilized biopolymer compositions comprising a liquid mixture, in particular aqueous solution, of at least one biopolymer component and at least one stabilizing surfactant.

[0004] More specifically, the present invention relates to stabilized biopolymer compositions comprising an ethylene oxide / propylene oxide block copolymer capable to confer to said biopolymer composition superior properties in comparison to the presently known formulations, particularly with regard to aggregation tendency, hemolytic activity and / or solubility.

[0005] The invention further relates to said block copolymers, to methods for preparing said stabilized compositions and to the use of said block copolymers for stabilizing an aqueous composition of said biopolymer

[0006] The invention further relates said stabilized composition for use in medicine, in particular for diagnostic and / or therapeutic applications.

[0007] The invention further relates to an essentially dry biopolymer composition, comprising said biopolymer and said block copolymers and to methods for preparing said essentially dry compositions.

[0008] The invention further relates to said essentially dry biopolymer composition for use in medicine, in particular for diagnostic and / or therapeutic applications.

[0009] Said compositions may be formulated as pharmaceutical compositions which may be delivered via suitable routes of administration such as via oral, rectal, transmucosal, topical, ophthalmic, otologic, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, as the case may be.

[0010] Background of the Invention

[0011] Due to favorable specificity and efficiency protein and cell- based therapies are finding widespread application in the treatment of a broad range of different diseases, for example cancer and autoimmune disorders. Antibodies have gained particular importance. However, due to their complex three-dimensional structure and the presence of many functional groups, their use is hampered by their susceptibility to aggregate during storage.

[0012] Antibody, and generally speaking protein, aggregates pose a safety risk because they can potentially be immunogenic and cause severe side effects.

[0013] Prevention of aggregation and surface adsorption, as well as ensuring long-term stability of the antibody formulations, while preserving antibody tertiary structure and function is still challenging.

[0014] Currently, polysorbate 20 (POE sorbitan monolaurate, PS20) and polysorbate 80 (POE sorbitan monooleate, PS80) and Poloxamer 188 are the most frequently used surfactants in marketed biopharmaceutical formulations.

[0015] Polysorbates are highly efficient, but difficult to manage due to their complex composition. Their multi-component nature makes them difficult to describe, control and it presents a particular challenge to monitor stability and degradation products. In general, oxidative or hydrolytic degradation is common and leads to the formation of reactive impurities. The stability of drug formulations is reduced because the polysorbate concentration decreases and also because reactive degradation products with aldehyde or peroxide structures trigger chemical change or degradation of biomolecules. Free fatty acids and altered protein structures can trigger the formation of proteinaceous particles, which are known to cause immunogenic reactions and are therefore a concern to patient safety. [ Dubey S, Giovannini R. Stability of Biologies and the Quest for Polysorbate Alternatives. Trends Biotechnol. 2021 Jun; 39(6): 546-549. doi:

[0016] 10.1016 / j.tibtech.2020.10.007. Epub 2020 Oct 30. PMID: 33139073.]

[0017] Furthermore, due to their similarity with the cell membrane lipids these surfactants may interact and disturb cellular equilibrium in erythrocytes, leading to membrane component reorganization and disturbance of the cell homeostasis which may eventually result in haemolysis. Polysorbates have also been described to cause immunogenic reactions, so a safer but equally effective stabilizer for parenteral formulations is 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).]

[0018] The chemical stability of the surfactants used in the formulation is therefore a key aspect to be considered in the preparation of the specific excipient formulation of choice.

[0019] Poloxamer 188 a non-ionic surfactant with a more defined chemical structure and a better chemical stability (no ester-bonds) has been used as an alternative to polysorbates. However, Poloxamer 188 is in general not as effective as a stabilizer and therefore typically requires higher concentrations to achieve a comparable performance. In many cases, the performance of Poloxamer 188 cannot match that of polysorbates, for example in the presence of residual silicon oil traces in pre-filled syringes [Grapentin, C. et a!.: Protein-Polydimethylsiloxane Particles in Liquid Vial Monoclonal Antibody Formulations Containing Poloxamer 188. Journal of Pharmaceutical Sciences, 2020).].

[0020] The stabilization challenge increases when the antibody is further engineered, coupled with other proteins or conjugated with small molecules presenting different physicochemical properties, such as small hydrophobic molecules and oligonucleotides.

[0021] An example thereof are antibody-fluorochrome conjugates as well as so-called antibody-drugs conjugates (ADCs), which are most commonly used in therapy to treat a broad range of different diseases and as detection reagents in diagnostic applications.

[0022] In that regard, the small molecule used for the labelling does influence the hydrophobicity and aggregation tendency of the antibody, which may possibly lead to cluster formation during storage and non-specific binding in imaging application.

[0023] Block-copolymers characterized by the presence of short hydrophilic blocks relative to the length of the hydrophobic block are known for their capacity to form wormlike micelles with high solubilisation capacity for poorly soluble aromatic drugs (Colloid Stability and Application in Pharmacy, edited by Tharwat F. Tadros, Weinheim 2007).

[0024] KR 20200077438 A describes Chitosan coated nanocapsules containing an active ingredient, in particular one which is non-soluble in water, and a pluronic. Chitosan, a polyaminosaccharide, is coated on nanoparticles prepared from the active ingredient and a pluronic in an organic solvent. Preferred pluronics are selected from P188, P407, L35, P123 and L81.

[0025] WO 2021 / 242908 provides a method of determining the concentration of a polysorbate in a composition comprising a protein and a polysorbate. Said method comprised the addition of a second surfactant, which may be a poloxamer, with a molecular weight in the range of 1.600 to 25.000 Da. The particular poloxamer as used in the examples is not further specified.

[0026] EP 1 598 074 A1 generically refers to a protein formulation containing a Poloxamer as surfactant. In the experimental section (Examples 1 , 2 and 3) the effect of Poloxamer 188 on the biological activity of an anti-human tissue factor antibody was examined. It was observed that Poloxamer 188 alone had the same effect on activity as Polysorbate 80 plus antioxidant L-methionine. Evidently, the teaching of said document is restricted to the use of Poloxamer 188 to obtain oxidation stability of certain antibody formulations in the absence of added antioxidants, like methionine. The granted claims relate to lgG4-type antibody compositions which do not contain added antioxidant and comprise Poloxamers 124, 237, 338, 407, 182, 184 or 331 or, preferably, Poloxamer 188, as surfactant. Poloxamers P334 and P335 (corresponding to Pluronic P104 and P105) while generically mentioned in the description within a long list of 22 different Pluronic surfactants have not been examined at all in EP 1 598 074 A1 , nor were they incorporated into the granted claims.

[0027] Consequently, a first problem to be solved by the invention relates to the identification of a highly water soluble polymer that demonstrates a water solubility and lack of haemolytic activity as observed for Poloxamer 188, but that in contrast to the Poloxamer 188 provides an improved aggregation stability to biopolymers, in particular antibody molecules, and therefore would enable the provision of a liquid biopolymer composition, in particular, liquid antibody composition of improved storage stability, in particular in combination with a low to absent hemolytic activity.

[0028] Summary of the Invention

[0029] The above mentioned problem could, surprisingly, be solved by the provision of Poloxamers (EO-PO-EO block-copolymers) with a specific range of molecular weight and ethylene oxide content (EO%).

[0030] More particularly, the above mentioned problem could be solved by the provision of Poloxamers presenting a molecular weight in the range of about 5 to 7 kDa and an EO% content of at 35 to 55 wt.-%.

[0031] These Poloxamers were found to be concomitantly characterized by at least two, more particularly all the following properties:

[0032] • high water solubility;

[0033] • high chemical stability;

[0034] • improved stabilization properties for antibody formulations regarding their capacity to inhibit antibody-surface interaction and / or aggregation; and

[0035] • low haemolytic activity.

[0036] Figure 1 : Illustration of selection criteria of particular exemplified Poloxamers as regards low haemolytic activity and their performance, in particular good water solubility, and long term stability. Detailed of the Invention

[0037] A. Abbreviations

[0038] ADC antibody drug conjugates

[0039] APC antibody payload conjugates

[0040] API active pharmaceutical ingredient

[0041] PO propylene oxide

[0042] CDCh deuterated chloroform

[0043] ECD equivalent circular diameter

[0044] EDTA Ethylenediaminetetraacetic acid

[0045] EO ethylene oxide

[0046] HPLC high-performance liquid chromatography

[0047] 1H-NHR proton nuclear magnetic resonance

[0048] IgG: immunoglobulin class G

[0049] PVDF polyvinylidene fluoride

[0050] MFI micro-flow imaging

[0051] PLA poly lactic acid

[0052] PLGA poly(lactic-co-glycolic acid)

[0053] PS20 Polysorbate 20

[0054] P188 Poloxamer 188

[0055] PS80 Polysorbate 80

[0056] PBS phosphate-buffered saline

[0057] RBC red blood cell

[0058] SFT Surface tension

[0059] B. Definitions

[0060] 1. General

[0061] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0062] In the context of the descriptions provided herein and of the appended claims, the use of “or” means “and / or” unless stated otherwise. Similarly, “comprise,” “comprises”, “comprising”, “include”, “includes,” and “including” are interchangeable and not intended to be limiting.

[0063] It is to be further understood that where descriptions of various embodiments use the term "comprising," those skilled in the art would understand that in some specific instances, an embodiment can be alternatively described using language "consisting essentially of' or "consisting of.”

[0064] The term “about” indicates a potential variation of ± 25% of the stated value, in particular ± 15% or ±10 %, more particularly ± 5%, ± 2% or ± 1%.

[0065] The term "substantially" describes a range of values of from about 80 to 100%, such as, for example, 85-99.9%, in particular 90 to 99.9%, more particularly 95 to 99.9%, or 98 to 99.9% and especially 99 to 99.9%.

[0066] “Predominantly” refers to a proportion in the range of above 50%, as for example in the range of 51 to 100%, particularly in the range of 75 to 99,9%; more particularly 85 to 98,5%, like 95 to 99%.

[0067] The term "essentially" describes a range of values of from about 90 to 100%, such as, for example, 90 to 99.9%, particularly 95 to 99.9%, more particularly 98 to 99.9% and especially 99 to 99.9%.

[0068] If the present disclosure refers to features, parameters and ranges thereof of different degree of preference (including general, not explicitly preferred features, parameters and ranges thereof) then, unless otherwise stated, any combination of two or more of such features, parameters and ranges thereof, irrespective of their respective degree of preference, is encompassed by the disclosure of the present description.

[0069] The terms “improved aggregation stability” and “improved inhibition of aggregation” in the context of the invention have to be regarded as synonyms. Both features are derived from the experimental observation that block copolymer surfactants of the invention reduce the aggregation tendency of a biopolymer, like a protein, in particular enzymes or immunoglobulin molecules, each optionally glycosylated, or adducts or conjugates of an optionally glycosylated immunoglobulin molecule and a payload molecule; or antibody payload conjugates (APC), particularly an antibody drug conjugates (ADC), as herein defined, in a liquid, optionally buffered mixture containing biopolymer and a block copolymer of the invention, relative to an otherwise identical liquid mixture which is not supplemented with said copolymer.

[0070] Such “improved aggregation stability” or “improved inhibition of aggregation” is in particular determined under “standard conditions” described in more detail in the experimental section, para.A.4. Typical standard conditions are: Protein concentration: in the range of about 1 to 50 mg / mL, particularly about 5 to 30 mg / mL or more particularly about 8 to 15 mg / mL; most particularly about 10 mg / mL;

[0071] Surfactant concentration: about 0,001 to 0,01% (w / v), particularly about 0,001 to 0,007% (w / v), more particularly 0,0015 to 0,005% (w / v); most particularly about 0,002% (w / v)

[0072] Solvent: deionized water or aqueous buffer pH about 5.5 to 7.5, particularly about 5.5 to 6.8, more particularly about 5,9 to 6,2, most particularly about 6; in a concentration of about 5 to 50 mM, particularly 10 to 40 mM, more particularly 15 to 30 mM, most particularly about 20 mM;

[0073] Particle size of aggregates determined in the range of 1 pm to 300 pm or 2pm to 50 pm;

[0074] Test temperature: ambient temperature (20 to 25 °C)

[0075] Blank: Protein aggregation in deionized water or buffer as defined above (without surfactant set to 100%)

[0076] Inhibition of aggregation is expressed the reduction of the number of aggregate particles in %, and is determined in a range between about less than 100%, like 99,9% to 0%; a “complete” inhibition is defined as a value in the range of about 5% to 0%.

[0077] A “reduced biopolymer-surface interaction” in the context of the invention is derived from the experimental observation that block copolymer surfactants of the invention reduce the static or dynamic surface tension of in a liquid, optionally buffered medium, containing a mixture of the copolymer surfactant of the present invention and a biopolymer, like a protein, in particular enzymes or immunoglobulin molecules, each optionally glycosylated, or adducts or conjugates of an optionally glycosylated immunoglobulin molecule and a payload molecule; or antibody payload conjugates (APC), particularly an antibody drug conjugates (ADC), as herein defined, relative to an otherwise identical liquid mixture which is not supplemented with said copolymer.

[0078] Such “reduced biopolymer-surface interaction” is in particular determined under “standard conditions” described in more detail in the experimental section, para. A.6 for measurements of the static or dynamic surface tension Typical standard conditions are:

[0079] Protein concentration: in the range of about 0.1 to 10 g / L, particularly about 0.5 to 3 m / L or more particularly about 0,75 to 2 g / L; most particularly about 1 g / L;

[0080] Surfactant concentration: about 0,01 to 1 g / L, particularly about 0,05 to 0,75 g / L, more particularly 0,075 to 0,75 g / L; most particularly about 0,1 g / L Solvent: Deionized water or aqueous buffer pH about 5.5 to 7.5, particularly about 5.5 to 6.8, more particularly about 5,9 to 6,2, most particularly about 6; in a concentration of about 5 to 50 mM, particularly 10 to 40 mM, more particularly 15 to 30 mM, most particularly about 20 mM;

[0081] Test temperature : ambient temperature ( 20 to 25 °C)

[0082] Blank: Protein dissolved in deionized water or in buffer as defined above (without surfactant)

[0083] Reduction of biopolymer-surface interaction is determined either statically by means of a drop shape analyzer at a fixed point of time of about 3 to 10 minutes, particularly about 5 minutes; or dynamically in a bubble pressure tensiometer over a suitable period of time, as for example 0,01 to 120 seconds, or more particularly 0,03 to 60 seconds; a reduction of the dynamic or static surface tension, expressed in [mN / m], of more than 5 to 10 mN / m relative to Blank is considered as significantly improved reduction of surface tension.

[0084] 2. Chemical Terms

[0085] The term “halogen” denotes in each case a fluorine, bromine, chlorine or iodine radical, in particular a fluorine radical.

[0086] “Alkyl” relates to a straight-chain or branched alkyl group having from 1 to 10, in particular 1 to 8, more particularly 1 to 4, 1 to 2 carbon atoms. Examples are methyl, ethyl, n-propyl, n-butyl, n-hexyl, n-heptyl or n-octyl.

[0087] “Alkylene” relates to a straight-chain or branched hydrocarbon bridging group having from 1 to 22, or 2 to 22, 1 to 6, 3 to 6 , 2 or 4, carbon atoms. Non limiting examples are -CH2-, -(CH2)2-, -(CH2)3-, -(CH2)4-, -(CH2) -, -(CH2)I5-, -(CH2)20-, -(CH2)22- and the respective branched analogues thereof.

[0088] Optionally said alkylene groups may be interrupted by one or more heteroatoms, such as oxygen.

[0089] “Alkyleneoxy” relates to a radical of the formula -R-O-, wherein R is a straightchain or branched alkylene group having from 1 to 22, or 2 to 22, 1 to 6, 3 to 6 ,2 or 4, carbon atoms as defined herein.

[0090] A “polyalkylene oxide” relates to a group in which at least two, identical or different repeating units of alkyleneoxy groups as defined above are covalently linked.

[0091] “Block copolymer” defines a macromolecular entity characterized by at least two alternating structurally different polymer blocks; wherein each block consists essentially of structurally analogous, in particular identical repeating monomeric units. Within the structure of said block copolymer may optionally be present chemical moieties linking two or more alternating blocks, such as polyvalent, as for example di- or trivalent organic or inorganic moieties.

[0092] The term “Poloxamer” refers to essentially linear block copolymers essentially consisting of alternating propylene oxide (PO) bocks and ethylene oxide (EO) blocks with a central PO block and one EO block at each of the two ends of the PO block.

[0093] “Static surface tension” within the meaning of the invention is to be preferably interpreted as the amount of energy per unit of surface area required to cause a deformation, such as a local increase, of the surface of a liquid sample presenting a given concentration of solute at the thermodynamic equilibrium at a predetermined temperature. The “static surface tension” is measured in millinewton per meter mN / m and is determined by means of the pendant drop technique.

[0094] “Ethylene oxide content” (EO%) and “propylene oxide content” (PO%) within the meaning of the invention are stated herein as either mole % (mol%) or as weight percentage (wt%) of ethylene oxide and propylene oxide monomer units within a given block copolymer as herein defined. If not otherwise stated “%” refers to wt%.

[0095] “Hemolysis” within the meaning of the invention relates to the tendency of a given excipient to cause breakdown of cells, particularly of red blood cells with consequent release of intracellular components.

[0096] “Equivalent circular diameter” (ECD) of a given, non-spherical particle, is defined as the diameter of a spherical particle which will give identical geometric, optical, electrical or aerodynamic behaviour to that of said non-spherical being examined.

[0097] Micro-flow imaging (MFI), is an analytical method, wherein microscopic images are automatically collected from a sample that passes the optics through a flow cell at a rate that is 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 count, shape and transparency can be obtained from the analysis software.

[0098] Unless otherwise stated the term “molecular weight” relates to the number average molecular weight “Mn”. Said molecular weight may be determined in a manner known per se. Typically, the OH value may be determined, as for example in line with DIN 53240-1 , or as described in European Pharmacopoeia (Ph. Eur.) or United States Pharmacopoeia (USP / NF).

[0099] 3. Biochemical Terms

[0100] The term “biopolymer” as used herein encompasses molecules, selected from oligopeptides, polypeptides, proteins, any type of antibody molecule or fragment or derivative thereof as defined below, glycosylated proteins, proteoglycans, oligo- and polynucleotides, DNA and RNA molecules, oligosaccharides, polysaccharides, as well as adducts or conjugates of such biopolymers, in particular of antibodies, with a further constituent selected from payload molecules as further defined below.

[0101] The term "antibody”, as used herein, broadly refers to any immunoglobulin (Ig) molecule comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such functional fragment, mutant, variant, or derivative antibody formats are known in the art. Nonlimiting embodiments of which 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 usually linked to one another via disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (also referred to herein as “variable heavy chain”, or abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region (also referred to herein as “variable light chain”, or abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass.

[0102] The terms "antigen-binding portion" of an antibody (or simply "antibody portion"), “antigen-binding moiety” of an antibody (or simply “antibody moiety”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (i.e. the immunogenic product of the invention), i.e. are 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. Such antibody embodiments may also be bispecific, dual specific, or multi-specific, specifically binding to two or more different antigens. Examples of binding fragments encompassed within 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 bridge 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 a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341 : 544-546, 1989; Winter et al., WO 90 / 05144 A1 , herein incorporated by reference), which comprises a single variable domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as 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 within 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 VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating 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 portions are known in the art (Kontermann and Dubel eds., Antibody Engineering, Springer-Verlag. New York. 790 pp_, 2001 , ISBN 3-540-41354-5).

[0103] The term "antibody”, as used herein, also comprises antibody constructs. The term “antibody construct” as used herein refers to a polypeptide comprising one or more of the antigen-binding portions of the invention linked to a linker polypeptide or an immunoglobulin constant domain. Linker polypeptides comprise two or more amino acid residues joined by peptide bonds and are used to link one or more antigen binding portions. 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).

[0104] An immunoglobulin constant domain refers to a heavy or light chain constant domain. Human IgG heavy chain and light chain constant domain amino acid sequences are known in the art.

[0105] Still further, a binding protein of the present invention (e.g. an antibody) may be part of a larger immunoadhesion molecule, formed by covalent or noncovalent association of the binding protein of the invention with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of the streptavidin core region to make a tetrameric scFv molecule (Kipriyanov et al., Human Antibodies and Hybridomas 6: 93-101 , 1995) and use of a cysteine residue, a marker peptide and a C-terminal polyhistidine tag to make 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, respectively, of whole antibodies. Moreover, antibodies, antibody portions and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

[0106] An "isolated antibody", as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds the immunogenic product of the invention may, however, have cross-reactivity to other antigens, such as Ap globulomers, e.g. A (20-42) globulomer or other Ap forms. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals and / or any other targeted AB form.

[0107] 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 include amino acid residues 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), for example in the CDRs and in particular in CDR3. 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.

[0108] The term "recombinant human antibody", as used herein, is intended to include all human antibodies that are prepared, expressed, created 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 J.V., and Larrick J.W. (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 (see e.g. Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21 :364- 370) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0109] The term “chimeric antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species and constant region sequences from another species, such as antibodies having murine heavy and light chain variable regions linked to human constant regions.

[0110] The term “CDR-grafted antibody” refers to antibodies which comprise heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of VH and / or VL are replaced with CDR sequences of another species, such as antibodies having murine CDRs (e.g., CDR3) in which one or more of the murine variable heavy and light chain regions has been replaced with human variable heavy and light chain sequences.

[0111] The terms "Kabat numbering", "Kabat definitions and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and , Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region ranges from amino acid positions 31 to 35 for CDR1 , amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region ranges from amino acid positions 24 to 34 for CDR1 , amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[0112] As used herein, the terms "acceptor" and "acceptor antibody" refer to the antibody or nucleic acid sequence providing or encoding at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% of the amino acid sequences of one or more of the framework regions. In some embodiments, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding the constant region(s). In yet another embodiment, the term "acceptor" refers to the antibody amino acid or nucleic acid sequence providing or encoding one or more of the framework regions and the constant region(s). In a specific embodiment, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, for example at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequences of one or more of the framework regions. In accordance with this embodiment, an acceptor may contain at least 1 , at least 2, at least 3, least 4, at least 5, or at least 10 amino acid residues that does (do) not occur at one or more specific positions of a human antibody. An acceptor framework region and / or acceptor constant region(s) may be, e.g., derived or obtained from a germline antibody gene, a mature antibody gene, a functional antibody (e.g., antibodies well-known in the art, antibodies in development, or antibodies commercially available).

[0113] As used herein, the term "CDR" refers to the complementarity determining region within antibody variable sequences. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term “CDR set” as used herein refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs have been defined differently according to 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 variable region of an antibody, 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)) found that certain sub- portions within Kabat CDRs adopt nearly identical peptide backbone conformations, despite having great diversity at the level of amino acid sequence. These sub-portions were designated as L1 , L2 and L3 or H1 , H2 and H3 where the "L" and the "H" designates the light chain and the heavy chains regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs overlapping with the Kabat CDRs have been 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 follow one of the above systems, but will nonetheless overlap with the Kabat CDRs, although they may be shortened or lengthened in light of prediction or experimental findings that particular residues or groups of residues or even entire CDRs do not significantly impact antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, particular embodiments use Kabat or Chothia defined CDRs.

[0114] As used herein, the term "canonical" residue refers to a residue in a CDR or framework that defines a particular canonical CDR structure as defined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia et al., J. Mol. Biol. 227:799 (1992), both are incorporated herein by reference). According to Chothia et al., critical portions of the CDRs of many antibodies have nearly identical peptide backbone confirmations despite great diversity at the level of amino acid sequence. Each canonical structure specifies primarily a set of peptide backbone torsion angles for a contiguous segment of amino acid residues forming a loop.

[0115] 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 species different from the antibody from which the framework regions are obtained or derived. In the context of a humanized antibody, the term "donor antibody" refers to a non-human antibody providing one or more CDRs.

[0116] As used herein, the term "framework" or "framework sequence" refers to the remaining sequences of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1 , -L2, and -L3 of light chain and CDR-H1 , -H2, and -H3 of heavy chain) also divide the framework regions on the light chain and the heavy chain into four sub-regions (FR1 , FR2, FR3 and FR4) on each chain, in which CDR1 is positioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. Without specifying the particular sub-regions as FR1 , FR2, FR3 or FR4, a framework region, as referred by others, represents the combined FR's within the variable region of a single, naturally occurring immunoglobulin chain. As used herein, a FR represents one of the four subregions, and FRs represents two or more of the four sub- regions constituting a framework region.

[0117] Human heavy chain and light chain acceptor sequences are known in the art.

[0118] As used herein, the term "germline antibody gene" or "gene fragment" refers to an immunoglobulin sequence encoded by non-lymphoid cells that have not undergone the maturation process that leads to genetic rearrangement and mutation for expression of a particular 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 of the advantages provided by various embodiments of the present invention stems from the recognition that germline antibody genes are more likely than mature antibody genes to conserve essential amino acid sequence structures characteristic of individuals in the species, hence less likely to be recognized as from a foreign source when used therapeutically in that species. As used herein, the term "key residues” refer to certain residues within the variable region that have more impact on the binding specificity and / or affinity of an antibody, in particular a humanized antibody. A key residue includes, but is not limited to, one or more of the following: a residue that is adjacent to a CDR, a potential glycosylation site (can be either N- or O-glycosylation site), a rare residue, a residue capable of interacting with the antigen, a residue capable of interacting with a CDR, a canonical residue, a contact residue between heavy chain variable region and light chain variable region, a residue within the Vernier zone, and a residue in the region that overlaps between the Chothia definition of a variable heavy chain CDR1 and the Kabat definition of the first heavy chain framework.

[0119] As used herein, the term "humanized antibody" is an antibody or a variant, derivative, analog or portion thereof which immunospecifically binds to an antigen of interest and which comprises a framework (FR) region having substantially the amino acid sequence of a human antibody and a complementary 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 at least 90%, at least 95%, at least 98% or at least 99% identical to the amino acid sequence of a 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 those of a human immunoglobulin consensus sequence. According to one aspect, a 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 the light chain as well as at least the variable domain of a heavy chain. The antibody also may include the CH1 , hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, a humanized antibody only contains a humanized light chain. In some embodiments, a humanized antibody only contains a humanized heavy chain. In specific embodiments, a humanized antibody only contains a humanized variable domain of a light chain and / or of a heavy chain.

[0120] The humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including without limitation IgG 1 , lgG2, lgG3 and lgG4. The humanized antibody may comprise sequences from more than one class or isotype, and particular constant domains may be selected to optimize desired effector functions using techniques well-known in the art. The framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g., the donor antibody CDR or the consensus framework may be mutagenized 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. In one embodiment, such mutations, however, will not be extensive. Usually, at least 90%, at least 95%, at least 98%, or at least 99% of the humanized antibody residues will correspond to those of the parental FR and CDR sequences. As used herein, the term "consensus framework" refers to the framework region in the consensus immunoglobulin sequence. As used herein, the term "consensus immunoglobulin sequence" refers to the sequence formed from the most frequently occurring amino acids (or nucleotides) 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 occurring most frequently at that position in the family. If two amino acids occur equally frequently, either can be included in the consensus sequence.

[0121] As used herein, "Vernier" zone refers to a subset of framework residues that may adjust CDR structure and fine-tune the fit to antigen as described by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which is incorporated herein by reference). Vernier zone residues form a layer underlying the CDRs and may impact on the structure of CDRs and the affinity of the antibody.

[0122] The term “antibody”, as used herein, also comprises multivalent binding proteins. The term "multivalent binding protein" is used in this specification to denote a binding protein comprising two or more antigen binding sites. The multivalent binding protein is engineered to have the three or more antigen binding sites, and is generally not a naturally occurring antibody. The term “multispecific binding protein” refers to a binding protein capable of binding two or more related or unrelated targets. Dual variable domain (DVD) binding proteins as used herein, are binding proteins that comprise two or more antigen binding sites and are tetravalent or multivalent binding proteins. Such DVDs may be monospecific, i.e. capable of binding one antigen or multispecific, i.e. capable of binding two or more antigens. DVD binding proteins comprising two heavy chain DVD polypeptides and two light chain DVD polypeptides are refered to a DVD Ig. Each half of a DVD Ig comprises a heavy chain DVD polypeptide, and a light chain DVD polypeptide, and two antigen binding sites. Each binding site comprises a heavy chain variable domain and a light chain variable domain with a total of 6 CDRs involved in antigen binding per antigen binding site. DVD binding proteins and methods of making DVD binding proteins are disclosed in US. Patent Application No. 11 / 507,050 and incorporated herein by reference.

[0123] The term “labeled binding protein”, as used herein, refers to a binding protein with a label incorporated that provides for the identification of the binding protein. Likewise, the term “labeled antibody” as used herein, refers to an antibody with a label incorporated that provides for the identification of the antibody. In one aspect, the label is a detectable marker, e.g., incorporation of a radiolabeled amino acid or attachment to a polypeptide of biotinyl moieties that can be detected by marked avidin (e.g., streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods). Examples of labels for polypeptides include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 35S, 90Y, 99Tc, 1111n, 1251, 1311, 177Lu, 166Ho, or 153Sm); fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic labels (e.g., horseradish peroxidase, luciferase, alkaline phosphatase); chemiluminescent markers; biotinyl groups; predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags); and magnetic agents, such as gadolinium chelates.

[0124] The term "antibody”, as used herein, also comprises antibody conjugates. The term “antibody conjugate” refers to a binding protein, such as an antibody, chemically linked to a second chemical moiety, such as a therapeutic agent.

[0125] The term "KD" (also "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 by dividing the dissociation rate constant (koff) by the association rate constant (kon). The association rate constant (kon), the dissociation rate constant (koff), and the equilibrium dissociation constant (KD) are used to represent 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. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used (e.g., instrument available from BIAcore International AB, a GE Healthcare company, Uppsala, Sweden). Additionally, a KinExA® (Kinetic Exclusion Assay) assay, available from Sapidyne Instruments (Boise, Idaho) can also be used.

[0126] “Internalize” or “internalization” of an immunoglobulin molecule relates to the ability of an immunoglobulin or ADC or APC as described herein binding to a cell surface receptor to induce a receptor-mediated endocytosis upon binding. “De-glycosylated” or “de-glycosylation” relates to the , partial and in particular complete, removal of one or more glycosyl-residues from a glycosylated species of a biomolecule, as for example a glycosylated immunoglobulin molecule. “Antibody formulation” is to be interpreted broadly and generally refers to a product in which said antibody is admixed in liquid or solid form with a pharmaceutically acceptable liquid or solid carrier comprising organic or inorganic excipients having the capacity to influence the physico-chemical properties of said antibodies.

[0127] “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 a payload

[0128] C. Particular aspects and embodiments of the invention

[0129] The present invention relates to the following aspects and particular embodiments thereof:

[0130] A first aspect of the invention relates to a stabilized biopolymer composition, comprising a liquid mixture, particularly a solution, and even more particularly an aqueous solution of at least one biopolymer component and at least one stabilizing block copolymer, especially of a mixture essentially consisting of at least one biopolymer component and at least one stabilizing block copolymer, wherein said block copolymer is in particular an essentially linear and optionally essentially symmetrical triblock-polymer, comprising a central PO block, and two flanking EO blocks, in particular of essentially similar length, which block copolymer shows a combination of the following features: a) a molecular weight Mn of 5.000 to 7.000 g / mol, particularly 5.300 to 6.800 g / mol, more particularly 5.500 to 6.700 g / mol, and especially 5.800 to 6.600 g / mol, as for example 5.800, 5.900, 6.000, 6.100, 6.200, 6,300, 6.400, or 6.500 g / mol, as calculated from a determination of the OH number and b) an EO content of 33 to 60 wt.-%, particularly 35 to 55 wt.-%, more particularly 38 to 53 wt.-%, especially 40 to 50 wt.-%, as for example 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 wt.-%, each based on the dry weight of said block copolymer.

[0131] In a particular embodiment of said composition said block copolymer is of the following general formula 1 : H-(-EO-)n-(-PO)m-(-EO)n-)-H

[0132] (1) wherein m represents an integer in the range of 50 to 60, in particular 52 to 58, like 52, 53, 54, 55, 56, 57 or 58; and n independently of each other represents an integer in the range of 23 to 45, in particular 28 to 40, as for example 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40;

[0133] Said ethylene oxide / propylene oxide block copolymer formally may also be designated as “triblock polymer”, or “EPE-block copolymer” as they contain a central PO block essentially consisting of PO monomers, flanked by two EO blocks each on one side of the PO block.

[0134] As particular combinations of features a) and b) there may be mentioned a Mn of 5.000 to 7.000g / mol and an EO content of 33 to 60 wt.-%, a calculated molecular weight of 5.300 to 6.800 g / mol and an EO content of 35 to 55 wt.-%, a calculated molecular weight of 5.500 to 6.700 g / mol and an EO content of 38 to 53 wt.-% a calculated molecular weight of 5.800 to 6.600 g / mol and an EO content of 40 to 50 wt.-%,

[0135] Preferably, said block copolymer shows at least one of the following additional features: c) a water solubility of at least 5 wt.-%, or more particularly at least 10% based on the total weight of the aqueous solution of the block copolymer ; d) an aqueous solution of said block copolymer (concentration 0.1 g / l) has a surface tension (SFT) in particular static SFT, of 53 to 30 mN / m, like, for example, of about 35, about 40, about 45 or about 50 mN / m, as for example 39 to 43 mN / m, particularly measured under the conditions of Section A.6 a), Sample preparation method 1 ; and e) lack of hemolytic activity, in particular less than 10% or more particularly less than 5%, or less than 1 , 2, 3 or 4% hemolysis, like 0 % or 0.1 to 0.9% hemolysis caused by a solution of 10Og / l block copolymer. Each of the above features b) to e) may be determined as described below by applying the respective analytical method and measurement conditions.

[0136] According to a particular embodiment, a preferred subgroup of block-copolymers shows a combination of anyone of the above features a), b) and e).

[0137] According to another particular embodiment, a preferred subgroup of blockcopolymers shows a combination of anyone of the above features a), b), c) and e).

[0138] According to still another particular embodiment, a preferred subgroup of blockcopolymers shows a combination of anyone of the above features a), b), c), d) and e).

[0139] In a particular embodiment of said first aspect, a stabilized biopolymer composition is provided, wherein said PO oxide block is composed of monomer units derived from propylene oxide monomers as a central moiety resulting from an appropriate bivalent starter alkanol, as for example 1 ,2-propylene glycol or dipropylene glycol

[0140] According to another particular embodiment of said first aspect, the invention relates to a stabilized biopolymer composition, wherein said block copolymer of general formula 1 shows a combination of the following features a) a calculated molecular weight 5.000 to 7.000 g / mole, and b) an EO content of 35 to 55 wt.-%, based on the dry weight of said blockcopolymer;

[0141] Said block copolymer may be selected from the following compounds having a) an Mn of about 5.900 and an EO content of about 40 wt.-%, based on the dry weight of the block copolymer; or b) an Mn of about 6.500 and an EO content of about 50 wt.-%, based on the dry weight of the block copolymer

[0142] According to another particular embodiment of said first aspect, the invention relates to a stabilized biopolymer composition, wherein the biopolymer is selected from oligopeptides, polypeptides, proteins, glycosylated proteins, proteoglycans, adducts or conjugates of such biopolymers with a further constituent selected from payload molecules, in particular a) pharmaceutically active compounds, b) labeling agents, c) biological small molecules such as lipids, phospholipids, glycolipids, sterols, vitamins, hormones, neurotransmitters, amino acids, nucleotides, monosaccharides; d) biological macromolecules such as peptides, oligopeptides, polypeptides, proteins, nucleic acids, such as any forms of DNA and RNA, oligosaccharides, and polysaccharides.

[0143] In the latter embodiment, said biopolymer may preferably be a diagnostically applicable or a therapeutically active biopolymer.

[0144] In the latter two embodiments, said biopolymer may preferably be selected from proteins, in particular enzymes and immunoglobulin molecules, each optionally glycosylated. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG-4, lgA1 and lgA2) or subclass, in particular IgG and any of the classes lgG1 , lgG2, lgG3 and lgG4.

[0145] In the latter three embodiments said biopolymer may preferably be selected from adducts or conjugates of an immunoglobulin molecule and a payload molecule.

[0146] In the latter embodiment, said biopolymer may preferably be an antibody payload conjugate (APC), particularly antibody drug conjugate (ADC).

[0147] In a particular embodiment of said first aspect, a stabilized biopolymer composition is provided, wherein said block copolymer of the general formula (I) is contained in a proportion of 0,001 to 30% or 0,001 to 10%, like 0,01 to 8%, 0,1 to 5% or 1 to 3%, each based on the total weight of the liquid composition.

[0148] In a further embodiment of said first aspect, a stabilized biopolymer composition is provided, in which said biopolymer is contained in a proportion of 0,01 to 30%, like 0,1 to 25%, 1 to 20%, 3 to 15% or 5 to 10%, each based on the total weight of the liquid composition.

[0149] In a particular embodiment of said first aspect, a stabilized biopolymer composition is provided, which is optionally in buffered form, having a pH in the range of 5 to 9, particularly 6, 7 or 8.

[0150] According to a second aspect, the 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 stabilizing ethylene oxide / propylene oxide block copolymer as defined in the above-identified first aspect.

[0151] In preferred embodiments said essentially dry biopolymer composition has a liquid content of 0% to 5% wt%, as for example 0,1 to 4,5 wt%, like 1 , 2, 3 or 4 wt%, based on the total weight of said composition. Optionally in said essentially dry biopolymer composition said at least one block copolymer (A) and said at least one biopolymer (B) are contained in a weight ratio (A) : (B) in the range of 1: 20.000 to 10:1, or 1 : 5.000 to 2:1 or of 1 : 100 to 1 ,2 : 1 , particularly 1 : 50 to 5 : 1 , or 1 : 20 to 2: 1 , and more particularly 1 : 10 to 1 , 1 : 1 .

[0152] In preferred embodiments said essentially dry biopolymer composition is characterized in that said block copolymer and said at least one biopolymer together are contained in a proportion of 1 to less than 100 wt%, in particular 5 to 60 wt%, more particular 10 to 50 wt%, or 20 to 40 wt.% or even 20 to 25% based on the total weight of said essentially dry composition.

[0153] Optionally said essentially dry biopolymer composition comprises at least one further excipient in a proportion of 0,1 to 99%, 40 to 95% and 50 to 90% wt.-% based on the total dry weight of said essentially dry composition.

[0154] According to another particular embodiment a liquid or essentially dry biopolymer composition is provided, comprising a nanoconstruct, in particular nanoparticles, composed of at least one biopolymer component as defined above and at least one stabilizing ethylene oxide / propylene oxide block copolymer as defined above.

[0155] More particularly, said nanoconstruct, or nanoparticle is composed of at least one biodegradable polymer (e.g. PLA, PLGA) or at least one lipid in addition to the at least one stabilizing ethylene oxide / propylene oxide block copolymer as defined above.

[0156] Even more particularly, said at least one biopolymer component is DNA, RNA or peptides.

[0157] A third aspect the invention relates to the use of a block copolymer defined in the above-identified first aspect for stabilizing an aqueous composition, in particular an aqueous solution, of a least one biopolymer as defined in the above-identified first aspect.

[0158] Particularly said aspect of the invention relates to the use of a block copolymer which is a triblock-polymer, comprising a central PO block, and two flanking EO blocks, which block copolymer shows a combination of the following features: a) a molecular weight Mn of 5.000 to 7.000 g / mol, particularly 5.300 to 6.800 g / mol, more particularly 5.500 to 6.700 g / mol, and especially 5.800 to 6.600. g / mol, as for example 5.800, 5.900, 6.000, 6.100, 6.200, 6,300, 6.400, or 6.500g / mol, as calculated from a determination of the OH number and b) an EO content of 33 to 60 wt.-%, particularly 35 to 55 wt.-%, more particularly 38 to 53 wt.-%, especially 40 to 50 wt.-%, as for example 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49 or 50 wt.-%, each based on the dry weight of said block copolymer; for improving aggregation stability of an aqueous composition, in particular aqueous solution, of a least one biopolymer, wherein said biopolymer is selected from i) proteins, in particular enzymes and immunoglobulin molecules, each optionally glycosylated; ii) adducts or conjugates of an optionally glycosylated immunoglobulin molecule and a payload molecule; and iii) antibody payload conjugates (APC), particularly an antibody drug conjugates (ADC).

[0159] More particularly, said block-copolymer improves aggregation stability of the biopolymer in said aqueous composition and / or further reduces biopolymer-surface interactions. Even more particularly said biopolymer comprises an immunoglobulin and wherein said block-copolymer improves aggregation stability of the immunoglobulin and / or reduces immunoglobulin-surface interactions.

[0160] Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) or subclass, in particular IgG and any of the classes lgG1 , lgG2, lgG3 and lgG4.

[0161] In another particular embodiment of said use said block copolymer is of the following general formula 1 :

[0162] H-(-EO-)n-(-PO)m-(-EO)n-)-H

[0163] (1) wherein m represents an integer in the range of 50 to 60, in particular 52 to 58 like 52, 53, 54, 55, 56, 57 or 58; and n independently of each other represents an integer in the range of 23 to 45, in particular 28 to 40, as for example 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40;

[0164] Said used ethylene oxide / propylene oxide block copolymer formally may also be designated as “triblock polymer”, or “EPE-block copolymer” as they contain a central PO block essentially consisting of PO monomers, flanked by two EO blocks each on one side of the PO block. As particular combinations of features a) and b) of said used copolymer there may be mentioned a Mn of 5.000 to 7.000 g / mol and an EO content of 33 to 60 wt.-%, a calculated molecular weight of 5.300 to 6.800 g / mol and an EO content of 35 to 55 wt.-%, a calculated molecular weight of 5.500 to 6.700 g / mol and an EO content of 38 to 53 wt.-% a calculated molecular weight of 5.800 to 6.600 g / mol and an EO content of 40 to 50 wt.-%,

[0165] More particularly, said block copolymer shows at least one of the following additional features c) a water solubility of at least 5 wt.-%, particularly at least 7 wt.-%, or more, particularly at least 10 wt.-% based on the total weight of the aqueous solution of the block copolymer; d) a surface tension (SFT) in particular static SFT, of 53 to 30mN / m as determined, like for example, of about 35, about 40, about 45 or about 50 mN / m, as for example 39 to 43 mN / m, based on a 0.1 g / l solution of said block copolymer, particularly measured under the conditions of Section A.6 a), Sample preparation method 1 ; and e) a lack of hemolytic activity, in particular less than 10% hemolysis or more particularly less than 5%, or less than 1 , 2, 3 or 4% hemolysis, like 0 % or 0.1 to 0.9% hemolysis, caused by a solution of 10Og / l.

[0166] According to a particular embodiment of said use, a preferred subgroup of blockcopolymers shows a combination of anyone of the above features a), b) and e).

[0167] According to another particular embodiment of said use, a preferred subgroup of block-copolymers shows a combination of anyone of the above features a), b), c) and e).

[0168] According to still another particular embodiment of said use, a preferred subgroup of block-copolymers shows a combination of anyone of the above features a), b), c), d) and e).

[0169] In a particular embodiment of said use, a stabilized biopolymer composition is applied, wherein said PO block is composed of monomer units derived from propylene oxide monomers an a central moiety resulting from an appropriate bivalent starter alkanol, as for example 1 ,2-propylene glycol or dipropylene glycol According to anther more particular embodiment of said use said block copolymer of general formula 1 shows a combination of the following features a) a molecular weight 5.000 to 7.000 g / mole, and b) an EO content of 35 to 55 wt.-%, more particularly 38 to 53 wt.-%, especially 40 to 50 wt.-%, based on the dry weight of said block copolymer;

[0170] The applied block copolymer may be selected from the following compounds having an Mn of about 5.900 and an EO content of about 40 wt.-%, based on the dry weight of the block copolymer; or an Mn of about 6.500 and an EO content of about 50 wt.-%, based on the dry weight of the block copolymer

[0171] Very particular examples of such block copolymers are selected from the following commercial products: Poloxamer P334 and Poloxamer 335.

[0172] According to another particular embodiment of said use the payload molecules are selected from a) pharmaceutically active compounds, b) labeling agents, c) biological small molecules such as lipids, phospholipids, glycolipids, sterols, vitamins, hormones, neurotransmitters, amino acids, nucleotides, monosaccharides; and d) biological macromolecules, such as peptides, oligopeptides, polypeptides, proteins, nucleic acids, such as any forms of DNA and RNA, oligosaccharides, and polysaccharides.

[0173] More particularly, said biopolymer is a diagnostically applicable or a therapeutically active biopolymer.

[0174] According to another particular embodiment of said use, the copolymer is contained in a proportion of 0,001 to 10 wt.-%, based on the total weight of the liquid composition; and / or said biopolymer is contained in a proportion of 0,01 to 30 wt.-%, based on the total weight of the liquid composition; and / or wherein said composition is in optionally buffered form, having a pH in the range of 5 to 9, particularly 6 to 8. A fourth aspect the invention relates to the composition according to the aboveidentified first or second aspect for use in medicine, in particular for diagnostic and / or therapeutic applications.

[0175] According to a further embodiment the invention relates to a composition according to the above-identified first or second aspect, which is a pharmaceutical composition optionally further supplemented by at least one pharmaceutically acceptable excipient.

[0176] A fifth aspect the invention relates to a method of preparing a stabilized composition according to the above-identified first aspect, which method comprises a) preparing in any order an aqueous, optionally buffered solution of the biopolymer; and an aqueous, optionally buffered solution of the block copolymer as defined above and b) preparing a mixture of both aqueous solutions as obtained in step a).

[0177] A sixth aspect the invention relates to a method of preparing the essentially dry stabilized according to the second aspect, which method comprises a) preparing in any order an aqueous, optionally buffered solution of said biopolymer; and an aqueous, optionally buffered solution of said block copolymer as defined above and b) preparing a mixture of both aqueous, optionally buffered, solutions as obtained in step a); c) optionally supplementing the aqueous, optionally buffered solutions prepared in step a) and / or the mixture of both aqueous, optionally buffered, solutions prepared in step b), with at least one pharmaceutically acceptable excipient; d) drying the mixture obtained in step b) or c).

[0178] Said drying step d) is performed by conventional, well-known methods. For example the drying step d) is performed by spray-drying or freeze-drying the mixture obtained in step b) or c).

[0179] D. Further embodiments

[0180] D.1. Synthesis of block copolymers of formula (1)

[0181] Methods for the preparation of stabilizing ethylene oxide / propylene oxide block copolymers of the general formula 1 are generally known in the art. Suitable methods for their preparation are reported, for example, in US 2,674,619, or EP-A-0 362 688 or Satacesaria et al., Chapter 15. Production of Ethylene oxide / propylene oxide block copolymers; in Handbook of Detergents, Part F, 253 - 269

[0182] Briefly, said block copolymers may, for example be prepared by a multistep protocol which foresees a first step in which an organic molecule comprising two active hydrogen atoms, like propylene glycol as starter, and propylene oxide are condensed to form a polyoxypropylene block. Thereafter, ethylene oxide is added and the reaction is let to proceed until the desired oxyethylene content is reached.

[0183] The reaction is preferably carried out under moisture-free conditions at elevated temperature and in presence of a suitable catalyst such as an alkali metal hydroxide or alkoxide, like alkali metal tert, butoxide.

[0184] The amount of catalyst employed should be from 0.05 to 1 percent by weight based on the total reactants. Reaction temperatures are in the range of from 80° to 200° C., with a temperature of about 110°C or 170°C being preferred during most of the reaction.

[0185] Superatmospheric pressures in the range of from 0.5 to 15 bar are ordinarily employed, very good results being obtained at pressures of from about 1 to 5 bar. The alkylene oxides employed are preferably substantially anhydrous, e.g. the moisture content of the oxides ordinarily should not exceed about 0.1 percent by weight. The alkylene oxides are also preferably as free as practical from contaminants, such as aldehydes, which give rise to side reactions and by-product formation.

[0186] The reaction may be conducted either batch-wise or continuously as desired. In batchwise operation, the commercially anhydrous organic molecule comprising two active hydrogen atoms, like propylene glycol is charged into a suitable dry reaction vessel, such as an autoclave, and mixed with an effective amount of catalyst, usually about 0.2 wt.-% of potassium hydroxide in terms of the total amount of reactants.

[0187] Prior to the introduction of propyleneoxide, the reaction vessel is advantageously flushed with a stream of dry inert gas, such as nitrogen, to remove any air or oxygen therefrom. The elimination of molecular oxygen from the reaction vessel is an important factor in obtaining colorless products and may, if desired, be carried out after adding the propylene glycol and catalyst to the reaction vessel.

[0188] After these preliminaries, the mixture of potassium hydroxide and said organic molecule comprising two active hydrogen atoms, like propylene glycol, is heated to a reaction temperature of about 140°C and propylene oxide is added at a fairly rapid rate. Usually, the rate of addition of propylene oxide is such as to maintain a pressure of about 3 bar in the reactor. Vigorous agitation is desirable to maintain a good dispersion of catalyst and uniform reaction rates throughout the mass.

[0189] The reaction of propylene oxide with propylene glycol is exothermic and it is therefore necessary to provide adequate cooling means.

[0190] By controlling the rate of addition of propylene oxide to maintain the pressure fairly constant, the reaction temperature may also be maintained constant.

[0191] The addition of propylene oxide is stopped upon obtaining the desired molecular weight of the polyoxypropylene glycol condensation product as determined by, for example, hydroxyl analysis or1H-NMR reckoning two free hydroxyl groups per molecule. Thereafter, ethylene oxide is condensed with the polyoxypropylene glycol condensation product to give a product in accordance with the invention. The addition of ethylene oxide is carried out in the same manner as the addition of propylene oxide already described.

[0192] Purification may be conducted by heating it at a reduced pressure under reflux or by stripping with inert gas to distill off any low boiling material.

[0193] D.2. Antibody-payload conjugates and their preparation

[0194] As stated above a biopolymer as part of a stabilized formulation of the invention may, in preferred embodiments, be an antibody payload conjugate (APC), and more particularly an antibody drug conjugate (ADC).

[0195] Antibody drug conjugates (ADC) combine two major therapies applied nowadays, namely chemotherapy and antibody therapy. Antibodies are important biologies, which bind to their specific antigen, e.g. to a receptor on a cell, which is overexpressed on a diseased cell, like a cancer cell compared to a healthy cell. The antibody activates the competent system and consequently, the cancer cell will be destroyed by killer cells. On the other hand, chemotherapy is the treatment with cytotoxic moieties, which can be absorbed by the cell and kill the cell by different pathways. Active cells, like cancer cells, can take up more of the cytotoxic drug than healthy cells. Nonetheless, this therapy shows huge side-effects. By combining antibodies with the killing effect of cytotoxic drugs, a directed and efficient cancer therapy is possible. Thereby, the choice of conjugation method, how the antibody is labeled with the drug, is very important.

[0196] The first ADCs on market were conjugated randomly, by utilizing cysteines or lysines of the antibody sequence to attach the toxic payload. This leads to a heterologous species with different kinds of drug-to-antibody ratios (DAR), which negatively influences pharmacokinetic and safety profile of the ADC (Senter, P. D. & Sievers, E. L. 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, J. R. et al. Site-specific conjugation of a cytotoxic drug to an antibody improves the therapeutic index. Nat. Biotechnol. 26, 925-932 (2008)).

[0197] Site-specific conjugation methods followed, utilizing the glycosylation of the antibody 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 restricted to specific sites and cannot be transferred to other positions in the antibody sequence.

[0198] One conjugation method, which is site-specific and unlimited in the choice of the position, is the use of the genetic code expansion technology. Therefore, a non-canonical amino acid (ncAA) is introduced at the translational level in the antibody sequence in response to a stop codon (e.g. amber stop codon, TAG), which is placed beforehand into the gene of the antibody. An orthogonal aminoacyl-tRNA-synthetase (aaRS) / tRNA pair has to be introduced into the antibody expression host, which is able to bind and introduce the ncAA 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 positioned freely in the antibody sequence and can used for the conjugation with the toxic payload depending on its chemical properties.

[0199] There are different ncAAs existing, based on various endogenous amino acids, like lysine or tryptophan. They can have different headgroups, which influences their chemical properties and give rise to which chemical reaction they can undergo. Tian et al. showed the incorporation of a ncAA containing a ketone headgroup into several antibodies expressed in CHO cells followed by coupling to a cytotoxic payload via copper-free click reaction. The reaction between an alkoxyamine functional group and the ketone could only be done at pH4, otherwise requiring additives (Tian, F. et al. A general approach to site-specific antibody drug conjugates. Proc. Natl. Acad. Sci. U. S. A. 111 , 1766-1771 (2014)).

[0200] The fastest bio-orthogonal chemical reaction nowadays, which can be even done at neutral pH, is the strain-promoted inverse electron demand Diels-Alder cycloaddition (SPIEDAC) between strained alkene or alkyne and a tetrazine group (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)). One special case of a SPIEDAC reaction is the conjugation of a cyclooctene- lysine (SCO) and a 1 ,2,4,5-tetrazine, which might not be an inverse electron demand reaction and does not show the same reaction speed as other strained alkenes or alkynes.. Therefore, SCO as well as the resulting reaction product, shows highest stability in the cellular environment compared to other strained alkene / 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)).

[0201] The toxic payload can be divided into a linker and a cytotoxic drug. There are many linker technologies existing nowadays, ranging from non-cleavable, to enzymatic, acidic and glutathione cleavable linkers. The linker is directly influencing the pharmacokinetics and pharmacodynamics of the ADC (Hafeez, U., Parakh, S., Gan, H. K. & Scott, A. M. Antibody-drug conjugates for cancer therapy. Molecules 25, 4764 (2020); Khongorzul, P., Ling, C. J., Khan, F. U., Ihsan, A. U. & Zhang, J. Antibody-Drug Conjugates: A Comprehensive Review. Mol. Cancer Res. 18, 3-19 (2020)).

[0202] A handful of different cytotoxic drug families are used nowadays as chemical warhead for an ADC, like auristatins, maytansinoids, calicheamicins and duocarmycins. They are either damaging DNA or microtubuli (Chau, C. H., Steeg, P. S. & Figg, W. D. Antibody-drug conjugates for cancer. Lancet 394, 793-804 (2019); Sievers, E. L. & Senter, P. D. Antibody-drug conjugates in cancer therapy. Annu. Rev. Med. 64, 15-29 (2013)).

[0203] Non-limiting examples of suitable antibodies and payload molecules within the meanings of the present invention are reported in the sections below.

[0204] D.3. Antibodies

[0205] The term “antibody” within the meaning of the present invention designates equally antibodies, antibody derivatives, antibody fragments, antibody (fragment) fusions (e.g. bi-specific and tri-specific mAb fragments or derivatives), polyclonal or monoclonal antibodies, such as human, humanized, mouse or chimeric antibodies (see also general definition provided above).

[0206] Typical non-limiting examples are selected form biologically, in particular pharmacologically active antibody molecules.

[0207] Non-limiting examples are selected form the following group: trastuzumab, bevacizumab, cetuximab, panitumumab, ipilimumab, rituximab, alemtuzumab, ofatumumab, gemtuzumab, brentuximab, ibritumomab, tositumomab, pertuzumab, adecatumumab, IGN101 , INA01 labetuzumab, hua33, pemtumomab, oregovomab, minretumomab (CC49), cG250, J591 , MOv-18, farletuzumab (MGRAb-003), 3F8, ch14,18, KW-2871 , hu3S193, lgN31 1 , IM- 2C6, CDP-791 , etaracizumab, volociximab, 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, lifastuzumab, glembatumumab, coltuximab, lorvotuzumab, indatuximab, anti-PSMA, MLN-0264, ABT-414, milatuzumab, ramucirumab, abagovomab, abituzumab, adecatumumab, afutuzumab, altumomab pentetate, amatuximab, anatumomab, anetumab, apolizumab, arcitumomab, ascrinvacumab, atezolizumab, bavituximab, bectumomab, belimumab, bivatuzumab, brontictuzumab, cantuzumab, capromab, catumaxomab, citatuzumab, cixutumumab, clivatuzumab, codrituzumab, conatumumab, dacetuzumab, dallotuzumab, daratumumab, demcizumab, denintuzumab, depatuxizumab, derlotuximab, detumomab, dinutuximab, drozitumab, duligotumab, durvalumab, dusigitumab, ecromeximab, edrecolomab, elgemtumab, emactuzumab, enavatuzumab emibetuzumab, enfortumab, enoblituzumab, ensituximab, epratuzumab, ertumaxomab, etaracizumab, farletuzumab, ficlatuzumab, figitumumab, flanvotumab, futuximab, galiximab, ganitumab, icrucumab, igovomab, imalumab, imgatuzumab, indusatumab, inebilizumab, intetumumab, iratumumab, isatuximab, lexatuzumab, lilotomab, lintuzumab, lirilumab, lucatumumab, lumretuzumab, margetuximab, matuzumab, mirvetuximab, mitumomab, mogamulizumab, moxetumomab, nacolomab, naptumomab, narnatumab, necitumumab, nesvacumab, nimotuzumab, nivolumab, nofetumomab, obinutuzumab, ocaratuzumab, ofatumumab, olaratumab, onartuzumab, ontuxizumab, oportuzumab, oregovomab, otlertuzumab, pankomab, parsatuzumab, pasotuxizumab, patritumab, pembrolizumab, pemtumomab, pidilizumab, pintumomab, polatuzumab, pritumumab, quilizumab, racotumomab, ramucirumab, rilotumumab, robatumumab, sacituzumab, samalizumab, satumomab, seribantumab, siltuximab, sofituzumab, tacatuzumab, taplitumomab, tarextumab, tenatumomab, teprotumumab, tetulomab, ticilimumab, tigatuzumab, tositumomab, tovetumab, tremelimumab, tucotuzumab, ublituximab, ulocuplumab, urelumab, utomilumab, vadastuximab, vandortuzumab, vantictumab, vanucizumab, varlilumab, veltuzumab, vesencumab, volociximab, vorsetuzumab votumumab, zalutumumab, zatuxima, combination and derivatives thereof, as well as other monoclonal antibodies targeting CAI 25, CAI 5-3, CAI 9-9, L6, Lewis Y, Lewis X, alpha fetoprotein, CA 242, 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, MllCI, CEA, gplOO, MARTI, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21 , MPG, and Neu oncogene product.

[0208] D.4. Payload molecules

[0209] Payload molecules typically used may be selected from bioactive compounds, in particular drugs, labeling agents, and chelators. Non-limiting examples thereof are given in the following sections.

[0210] D.4.1. Bioactive compounds

[0211] Bioactive compounds include, but are not limited to, the following:

[0212] Bioactive compounds applicable according to the present invention include but are not limited to: small organic molecule drugs, steroids, lipids, proteins, aptamers, oligopeptides, oligonucleotides, oligosaccharides, as well as peptides, peptoids, amino acids, nucleotides, oligo- or polynucleotides, nucleosides, DNA, RNA, toxins, glycans and immunoglobulins.

[0213] Exemplary classes of bioactive compounds that can be used in the practice of the present invention include but are not limited to hormones, cytotoxins, antiproliferative / antitumor agents, antiviral agents, antibiotics, cytokines, antiinflammatory agents, antihypertensive agents, chemosensitizing, photosensitizing and radiosensitizing agents, anti-AIDS substances, anti-viral agents, immunosuppressants, immunostimulants, enzyme inhibitors, anti-Parkinson agents, neurotoxins, channel blockers, modulators of cell-extracellular matrix interactions including cell growth inhibitors and anti-adhesion molecules, inhibitors of DNA, RNA or protein synthesis, steroidal and non-steriodal anti-inflammatory agents, anti-angiogenic factors, antiAlzheimer agents.

[0214] In some embodiments, the bioactive compound is a low to medium molecular weight compound (e.g. about 200 to 5000 Da, about 200 to about 1500 Da, preferably about 300 to about 1000 Da).

[0215] Exemplary cytotoxic drugs are particularly those which are used for cancer therapy. Such drugs include, in general, DNA damaging agents, anti-metabolites, natural products and their analogs, enzyme inhibitors such as dihydro folate reductase inhibitors and thymidylate synthase inhibitors, DNA binders, DNA alkylators, radiation sensitizers, DNA intercalators, DNA cleavers, microtubule stabilizing and destabilizing agents, topoisomerases inhibitors. Examples include but are not limited to platinum-based drugs, the anthracycline family of drugs, the vinca drugs, the mitomycins, the bleomycins, the cytotoxic nucleosides, taxanes, lexitropsins, the pteridine family of drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids, differentiation inducers, and taxols. Particularly useful members of those classes include, for example, auristatins, maytansines, maytansinoids, calicheamicins, dactinomycines, 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) (cf. US20210206763A1), methotrexate, methopterin, di ch loro methotrexate, 5-fluorouracil, DNA minor groove binders, 6- mercaptopurine, cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin, anthracyclines (doxorubicin, epirubicin, idarubicin, daunorubicin, PNU-159682 (cf. US 10,288,745 B2.) and its analogs, mitomycin C, mitomycin A, caminomycin, aminopterin, tallysomycin, podophyllotoxin and ;podophyllotoxin derivatives such as etoposide or etoposide phosphate, vinblastine, vincristine, vindesine, taxol, taxotere retinoic acid, butyric acid, N8-acetyl spermidine, staurosporin, colchicine, camptothecin, esperamicin, ene-diynes, and their analogues, hemiasterlin and its analogues.

[0216] Other exemplary drug classes are angiogenesis inhibitors, cell cycle progression inhibitors, P13K / m-TOR / AKT pathway inhibitors, MAPK signaling pathway inhibitors, kinase inhibitors, protein chaperones inhibitors, HDAC inhibitors, PARP inhibitors, Wnt / Hedgehog signaling pathway inhibitors, RNA polymerase inhibitors, and protein degraders (cf. https: / / pubs.acs.org / doi / 10.1021 / acschembio.0c00285).

[0217] Examples of auristatins include dolastatin 10, monomethyl auristatin E (MMAE), auristatin F, monomethyl auristatin F (MMAF), auristatin F hydroxypropylamide (AF HPA), auristatin F phenylene diamine (AFP), monomethyl auristatin D (MMAD), auristatin PE, auristatin EB, auristatin EFP, auristatin TP and auristatin AQ. Suitable auristatins are also described in U.S. ;Publication Nos. 2003 / 0083263, 2011 / 0020343, and 2011 / 0070248; PCT Application ;Publication Nos. WO09 / 117531 , W02005 / 081711 , W004 / 010957; W002 / 088172 and WO01 / 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; 5,767,237;

[0218] 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; ;5, 530, 097; 5,521 ,284;

[0219] 5,504,191 ; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; ;4, 879,278;

[0220] 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety. Exemplary drugs include the dolastatins and analogues thereof including: dolastatin A ( U.S. Pat No. 4,486,414), dolastatin B (U.S. Pat No. 4,486,414), dolastatin 10 (U.S. Pat No. 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, 5,780,588, 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 patent incorporated herein by reference in their entirety.

[0221] Exemplary maytansines, maytansinoids, such as DM-1 and DM-4, or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in U.S. Patent 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; 4,362,663; 4,364,866; 4,450,254; 4,322,348; 4,371 ,533;

[0222] 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 6,441 ,163;

[0223] 6,716,821 and 7,276,497.

[0224] Other examples include mertansine and ansamitocin; Pyrrolobenzodiazepines (PBDs), which expressly include 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],

[0225] Calicheamicins include, e.g. enediynes, esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116.

[0226] Examples of duocarmycins and analogs include CC1065, duocarmycin SA, duocarmycin A, duocarmycin B I, duocarmycin B2, duocarmycin Cl, duocarmycin C2, duocarmycin D, DU- 86, KW-2189, adozelesin, bizelesin, carzelesin, seco- adozelesin. Other examples include those described in, for example, US Patent No. 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; 6,756,397; 7,049,316; 7,553,816; 8,815,226; US20150104407; 61 / 988,011 filed May 2, 2014 and 62 / 010,972 filed June 11 , 2014; the disclosure of each of which is incorporated herein in its entirety.

[0227] Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine, and those disclosed in U.S. Publication Nos. 2002 / 0103136 and 2010 / 0305149, and in U.S. Patent No. 7,303,749, the disclosures of which are incorporated herein by reference in their entirety.

[0228] 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 WO97 / 19086; WO98 / 08849; W098 / 22461 ; W098 / 25929; W098 / 38192; WO99 / 01124; WO99 / 02514; WO99 / 03848; WO99 / 07692; WO99 / 27890; and W099 / 28324; the disclosures of which are incorporated herein by reference in their entirety.

[0229] Exemplary cryptophycin compounds are described in U.S. Patent Nos. 6,680,311 ; and 6,747,021 ; the disclosures of which are incorporated herein by reference in their entirety.

[0230] Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin.

[0231] Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracyclines, calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, and the like.

[0232] Exemplary microtubule stabilizing and destabilizing agents include taxane compounds, such as paclitaxel, docetaxel, tesetaxel, and carbazitaxel; maytansinoids, auristatins and analogs thereof, vinca alkaloid derivatives, epothilones and cryptophycins.

[0233] Exemplary topoisomerase inhibitors include camptothecin and camptothecin derivatives, camptothecin analogs and non-natural camptothecins, such as, for example, CPT-11 , SN-38,topotecan, 9-aminocamptothecin, rubitecan, gimatecan, karenitecin, silatecan, lurtotecan, exatecan, DXd, diflometotecan, belotecan, lurtotecan and S39625. Other camptothecin compounds that can be used include those described in, for example, J. Med. Chem., 29:2358-2363 (1986); J. Med. Chem., 23:554 (1980); J. Med Chem., 30: 1774 (1987).

[0234] Angiogenesis inhibitors include, but are not limited to, MetAP2 inhibitors, VEGF inhibitors, PIGF inhibitors, VGFR inhibitors, PDGFR inhibitors, MetAP2 inhibitors. Exemplary VGFR and PDGFR inhibitors include sorafenib, sunitinib and vatalanib. Exemplary MetAP2 inhibitors include fumagillol analogs, meaning compounds that include the fumagillin core structure.

[0235] Exemplary cell cycle progression inhibitors include CDK inhibitors such as, for example, BMS-387032 and PD0332991 ; Rho-kinase inhibitors such as, for example, AZD7762; aurora kinase inhibitors such as, for example, AZD1152, MLN8054 and MLN8237; PLK inhibitors such as, for example, Bl 2536, BI6727, GSK461364, ON- 01910; and KSP inhibitors such as, for example, SB 743921 , SB 715992, MK-0731 , AZD8477, AZ3146 and ARRY-520. Exemplary P13K / m-TOR / AKT signalling pathway inhibitors include phosphoinositide 3- kinase (P13K) inhibitors, GSK-3 inhibitors, ATM inhibitors, DNA-PK inhibitors and PDK-1 inhibitors.

[0236] Exemplary P13 kinases are disclosed in U.S. Patent No. 6,608,053, and include BEZ235, BGT226, BKM120, CAL263, demethoxyviridin, GDC-0941 , GSK615, IC87114, LY294002, Palomid 529, perifosine, PF-04691502, PX-866, SAR245408, SAR245409, SF1126, Wortmannin, XL147 and XL765.

[0237] Exemplary AKT inhibitors include, but are not limited to AT7867.

[0238] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.

[0239] Exemplary MEK inhibitors are disclosed in U.S. Patent No. 7,517,944 and include GDC- ;0973, GSKI 120212, MSC1936369B, AS703026, R05126766 and R04987655, PD0325901 , AZD6244, AZD8330 and GDC-0973.

[0240] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0241] Exemplary B p38 MAPK inhibitors include BIRB 796, LY2228820 and SB 202190. Exemplary receptor tyrosine kinases 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.

[0242] Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021 , BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.

[0243] Exemplary HDAC inhibitors include Belinostat (PXD101), CUDC-101 , Droxinostat, ITF2357 (Givinostat, Gavinostat), JNJ-26481585, LAQ824 (NVP-LAQ824, Dacinostat), LBH-589 (Panobinostat), MC1568, MGCD0103 (Mocetinostat), 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.

[0244] Exemplary Wnt / Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV-939.

[0245] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and proamanullin.

[0246] Exemplary cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 , TNF. As non-limiting examples of particular drugs there may be mentioned Auristatins, Maytansinoids, PBDs, topoisomerase inhibitors, anthracyclines

[0247] In another embodiment, a combination of two or more different drugs as described above are used.

[0248] According to another embodiment, the bioactive compound may be selected from any synthetic or naturally occurring compounds comprising one or more natural and / or non-natural, proteinogenic and / or non-proteinogenic amino acid residues, such as in particular oligo- or polypeptides or proteins.

[0249] Other suitable example of bioactive compound are immunoglobulin such as antibodies, antibodies derivatives and active fragments thereof. Suitable examples in that regard are reported in the section D.3 above.

[0250] D.4.2. Labelling Agents and Radionuclides

[0251] Labeling agents which may be used as biopolymer within the meanings of the present invention can comprise any type of label known in the art.

[0252] Examples are dyes (e.g. fluorescent, luminescent, or phosphorescent dyes (e.g. fluorescent, luminescent, or phosphorescent dyes), such as dansyl, coumarin, fluorescein, acridine, rhodamine, silicon-rhodamine, BODIPY, or cyanine dyes), molecules able to emit fluorescence upon contact with a reagent, chromophores (e.g., phytochrome, phycobilin, bilirubin, etc.), radiolabels (e.g. radioactive forms of hydrogen, fluorine, carbon, phosphorous, sulphur, or iodine, such as tritium, fluorine-18, carbon-11 , carbon-14, phosphorous-32, phosphorous-33, sulphur-33, sulphur-35, indium-111 , iodine-123, or iodine-125), MRI-sensitive spin labels, affinity tags (e.g. biotin, His-tag, Flag-tag, strep-tag, sugars, lipids, sterols, PEG-linkers, benzylguanines, benzylcytosines, or co-factors), polyethylene glycol groups (e.g., a branched PEG, a linear PEG, PEGs of different molecular weights, etc.), photocrosslinkers (such as p- azidoiodoacetanilide), NMR probes, X-ray probes, pH probes, IR probes, resins, solid supports and bioactive compounds as defied above.

[0253] In some embodiments, exemplary dyes can include an NIR contrast agent that fluoresces in the near infrared region of the spectrum. Exemplary near-infrared fluorophores can include dyes and other fluorophores with emission wavelengths (e.g., peak emission wavelengths) between about 630 and 1000 nm, e.g., between about 630 and 800 nm, between about 800 and 900 nm, between about 900 and 1000 nm, between about 680 and 750 nm, between about 750 and 800 nm, between about 800 and 850 nm, between about 850 and 900 nm, between about 900 and 950 nm, or between about 950 and 1000 nm. Fluorophores with emission wavelengths (e.g., peak emission wavelengths) greater than 1000 nm can also be used in the methods described herein.

[0254] 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, and CASCADE™ blue acetylazide (Molecular Probes, Inc., Eugene, Oreg.) and ATTO dyes.

[0255] Further labelling agents are 111-lndium, 64-Copper, 67-Copper, 124-lodine, 227- Thorium, 188-Rhenium, 177-Lutetium, 89-Zirkonium, 131-lod, 68-Gallium, 99m- Technecium, 225-Actinium, 213-Bismut, 90-Ytrium and 212-Plumbum.

[0256] D.4.3. Chelators

[0257] Lists of typically applicable chelators and their short names are given below; Corresponding salts thereof are also applicable.:

[0258] Acetyl acetone (ACAC), ethylene diamine (EN), 2-(2-aminoethylamino)ethanol (AEEA), diethylene triamine (DIEN), iminodiacetate (IDA), triethylene tetramine (TRIEN), triaminotriethylamine, nitrilotriacetate (NTA) and its saltslike Na3NTA or FeNTA, ethylenediaminotriacetate (TED), ethylenediamine tetraacetate (EDTA) and its salts like Na2EDTA and CaNa2EDTA, diethylene triaminpentaacetate (DTPA), 1 ,4,7,10- ztetraazacyclododecane-1 ,4,7,10-tetraacetate (DOT A), 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), dimercapto succinic acid (DMSA), 1 ,2-bis(diphenylphosphino)ethane (DPPE), sodium salicylate, methoxy salicylates, British anti-Lewisite or 2,3-dimercaprol (BAL), meso-2,3- dimercaptosuccinic acid (DMSA); Siderophores secreted by microorganisms, as for example desferrioxamine or deferoxamine B, also known as Deferral (Novartis), produced by Streptomyces spp.; deferoxamine (DFO) , a trihydroxamic acid secreted by Streptomyces pilosus; phytochemicals like curcuminoids and derivatives of mugineic acid, like 3-hydroxy-mugineic acid and 2 -deoxy-mugineic acid; synthetically produced chelators, like Ibuprofen; derivatives of catechol, hydroxamate and hydroxypyridinone, like hydroxamate desferal and hydroxypyridinone deferiprone; deferiprone (L1 or 1 ,2- dimethyl-3-hydroxypyrid-4-one); D-penicillamine (DPA or D-PEN) whoich is p-p- dimethylcysteine or 3-mercapto-D-valine; tetraethylenetetraamine (TETA) or trientine and its two major metabolites N1 -acetyltriethylenetetramine (MAT) and N1 ,N10 - diacetyltriethylenetetramine (DAT); hydroxyquinolines; clioquinol, which is a halogenated derivative of 8-hydroxyquinoline; and 5,7-dichloro-2-[(dimethylamino)methyl]quinolin-8- ol (PBT2).

[0259] D.5. Pharmaceutical composition

[0260] The stabilized composition (i.e. the stabilized active ingredients, in particular biopolymers) of this invention are generally given as “pharmaceutical compositions” comprised of a “therapeutically” and / or "prophylactically effective amount" or a “diagnostically” effective amount of at least one such active ingredient or its pharmaceutically acceptable salt and optionally at least one pharmaceutically acceptable excipient.

[0261] Thus the term "pharmaceutical composition" according to a particular embodiment of the present invention designates a stabilized liquid composition comprising or essentially consisting of at least one pharmaceutically active biopolymer compound (i.e. the active ingredient) and at least one stabilizing EO / PO block copolymer as described herein in a liquid, pharmaceutically acceptable medium. A dried powder of such liquid preparation can be obtained by lyophilization or any other suitable drying method that is typically applied.

[0262] Said pharmaceutical compositions may be delivered via suitable routes of administration such as via oral, rectal, transmucosal, topical, ophthalmic, otologic, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, as the case may be.

[0263] Depending on the nature or the mode of administration and dosage form said composition said at least one additional pharmaceutical excipient may be different

[0264] An “excipient” is a substance formulated alongside the active ingredient and is included for different purpose, as for example for long-term stabilization, bulking up solid formulations that contain potent active ingredients in small amounts (thus often referred to as "bulking agents", "fillers", or "diluents"), or to confer a therapeutic enhancement on the active ingredient in the final dosage form, such as for example facilitating drug absorption, reducing viscosity, or enhancing solubility. Excipients can also be useful in the manufacturing process of the pharmaceutical composition, to aid in the handling of the active substance concerns such as by facilitating powder flowability or non-stick properties, in addition to aiding in vitro stability such as prevention of denaturation or aggregation over the expected shelf life. The selection of appropriate excipients not only depends upon the route of administration and the dosage form, but also on the particular active ingredient and other factors.

[0265] Excipients may be selected from the following classes: immunological adjuvants, antiadherents, binders, coatings, colours, disintegrant, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles

[0266] Non limiting examples of excipients comprise diluents, preserving agents, stabilizers, emulsifying agents, like emulsifying polymers, such as polysorbates or poloxamers, antioxidants; anti-irritants, chelating agents and stabililizing salts, such as chlorides, sulfates, phosphates, diphosphates, hydrobromides and nitrates, suspending agents, antibacterial agents or antifungal agents. Further, buffering agents such as buffering systems of low molecular weight organic acids together with the respective salts, or inorganic buffering substances, such as phosphate buffers, can be used, Further suitable ingredients are also known from relevant pharmacological standard literature. Also the proportion of the various components will vary depending on the nature of the specific component used and is generally known to the person skilled in the art (Remington's Pharmaceutical science ("Handbook of Pharmaceutical Excipients", 2nd Edition, (1994), Edited by A Wade and PJ Weller or in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R Gennaro edit. 1985).

[0267] A pharmaceutical composition as used herein may be presented in the form of a “dosage form” or “unit dose” and may comprise one or more stabilized liquid composition, or essentially dry biopolymer composition comprising at least one pharmaceutically active biopolymer compound and at least one stabilizing EO / BPO block copolymer as described herein. Thus, a pharmaceutical composition as used herein could, for example, provide two active agents admixed together in a unit dose or provide two active agents combined in a dosage form wherein the active agents are physically separated.

[0268] Furthermore, one may administer said pharmaceutical composition in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.

[0269] The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, emulsifying, encapsulating, entrapping or combinations thereof. Proper formulation is dependent upon the route of administration chosen.

[0270] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of patients without excessive toxicity, irritation, allergic response, or other problem or complication commensurate with a reasonable risk / benefit ratio.

[0271] The invention includes all “pharmaceutically acceptable salt forms” of the active ingredient. Pharmaceutically acceptable salts are those in which the counter ions do not contribute significantly to the physiological activity or toxicity of the compounds and as such function as pharmacological equivalents. These salts can be made according to common organic techniques employing commercially available reagents. Some anionic salt forms include acetate, acistrate, besylate, bromide, chloride, citrate, fumarate, glucouronate, 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.

[0272] A "therapeutically effective amount" and / or "prophylactically effective amount" means an amount effective, when administered to a human or non-human patient, to provide any therapeutic and / or prophylactic benefit. More particularly, a “therapeutically effective amount” is an amount of an active ingredient disclosed herein or a combination of two or more such active ingredients, which inhibits, totally or partially, the progression of the condition or alleviates, at least partially, one or more symptoms of the condition.

[0273] A "diagnostically effective amount" means an amount effective to allow obtaining from the patient a diagnostically valuable information on status or progression of a disease state.

[0274] A therapeutic benefit may be an amelioration of symptoms of a diseased patient, e.g., an amount effective to decrease the symptoms of a diseased patient. In certain circumstances a patient may not present symptoms of a 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 indicia of a disease, disorder or condition e.g. an amount sufficient to significantly reduce the frequency and severity of disease symptoms to occur.

[0275] A therapeutically effective amount can also be an amount, which is prophylactically effective.

[0276] A “patient” as used herein means human or non-human, in particular human, animals.

[0277] A "dosage form" is any unit of administration (“unit dose”) of one or more active agents as described herein.

[0278] The term "treating" or “treatment” refers to: (i) preventing a disease, disorder or condition from occurring in a patient which may be predisposed to the disease, disorder and / or condition but has not yet been diagnosed as having it; (ii) inhibiting the disease, disorder or condition, i.e., arresting its development; and (iii) relieving the disease, disorder or condition, i.e., causing regression of the disease, disorder and / or condition. In particular it encompasses a prophylactic or therapeutic treatment or combinations thereof.

[0279] “Frequency” of dosage may vary depending on the compound used and the particular type of infection treated. A dosage regimen of once per day is possible. Dosage regimens in which the active agent is administered for several times daily, as for example 2 to 10 times, like 2, 3, 4, 5, 6, 7, 8, 9 or 10 times may occasionally be more helpful.

[0280] It will be understood, however, that the specific dose level and frequency for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, 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 undergoing therapy. Patients may generally be monitored for therapeutic or prophylactic effectiveness using assays suitable for the condition being treated or prevented, which will be familiar to those of ordinary skill in the art.

[0281] Particular examples of pharmaceutical composition according to the present invention are liquid form preparations such as solutions, suspensions, and emulsions and comprise, beside the block copolymer according to the present invention, a therapeutically effective amount of biopolymer component as defined above, optionally together with at least one further pharmaceutically acceptable excipient as defined above and may be administered through any suitable route.

[0282] Further examples of pharmaceutical composition according to the present invention are solid form preparations such as powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.

[0283] The following example serve for a better understanding of the present invention without limiting its scope.

[0284] Experimental Part

[0285] A. Material and Methods A.1 Chemicals and Buffers

[0286] Unless stated otherwise all chemicals as applied were of analytical grade and obtained from commercial sources.

[0287] APIs were obtained from commercial sources.

[0288] Bovine immunoglobulin (IgG) captured from pooled bovine plasma using a chromatographic method (MPBio, Cat-No: 08641402, Lot 21040)

[0289] A.2 Analytical methods

[0290] The HPLC measurement was done by gradient elusion using a RP-C18 column (Chromolith®, HighResolution, RP-18 endcapped, 100-4.6 mm, Merck KGaA) which was kept at 25°C. The mobile phase composed of solvent A (water, 0.1 % phosphoric acid) and solvent B (acetonitrile, 0.1% phosphoric acid) was at a flow rate of 1.5 ml / min. The samples were detected and quantified using a DAD detector. The herein used HPLC device was the 1260 Infinity II from Agilent Technologies.

[0291] 1H-NMR spectra were measured in CDCh with a Bruker AVANCE III 500 MHz spectrometer.

[0292] A.3 General protocol for determining of ethylene oxide content

[0293] Weight percent of ethylene oxide (wt% EG) in the claimed polymers was determined by1H-NMR spectroscopy. In1H-NMR spectroscopy, the integral of each peak is proportional to the molar concentration of the protons being analysed.

[0294] For propylene oxide and ethylene oxide block copolymers, the CH3 groups from polymerized proylene oxide give a multiplett signal at 5 = 1.15 ppm.

[0295] The ethylene oxide repeating group -O-CH2-CH2-O- and the propylene oxide repeating group -O-CH2-CH(Me)-O- give a broad multiplett from 5 = 3.0 to 4.0 ppm, whereas the contribution of each ethylene oxide repeating group is 4 protons (two CH2 groups), and each propylene oxide group contributes with 3 protons (one CH and one CH2 group) to the signal.

[0296] To determine the mol% of ethylene oxide and the wt% of ethylene oxide following calculations are done:

[0297] Integral at 8= 1.15 ppm is set as 3 protons is [Area PO]

[0298] Integral at 8 = 3.0 to 4.0 ppm is [Area EO+PO] [Area EO] = [Area EO+PO] - [Area PO] mol% EO = ([Area EOJ / 4) I (([area EO] / 4)+([Area POJ / 3)) *100 mol% BuO = ([Area POJ / 3) / (([area EO] / 4)+([Area POJ / 3)) *100

[0299] From mol% EO and mol% PO the weight percent of ethylene oxide wt% EO is calculated with molecular mass of ethylene oxide (44.05 g / mol) and molecular mass of propylene oxide (58.08 g / mol): wt% EO = mol% EO*44.05 / ((mol% EO*44.05)+(mol% P0*58.08))*100

[0300] A.4 General protocol for IgG aggregation assay

[0301] The test is performed with commercial bovine immunoglobulin (IgG) captured from pooled bovine plasma using a chromatographic method. For every test three references are tested in addition to the samples: IgG without surfactant (blank), IgG with PS20 (positive control), and IgG with P188 (negative control).

[0302] 1g IgG is dissolved in 50 mL 20 mM histidine buffer pH 6 by careful manual shaking. The IgG solution is stored overnight in the refrigerator at 4 °C. 40 mg of each surfactant is dissolved in 10 mL 20 mM histidine buffer pH 6 and stored overnight in the refrigerator at 4 °C.

[0303] To avoid particle contamination, all following steps are carried out under the clean bench. The surfactant solutions are diluted 1 :100 with 20 mM histidine buffer pH 6 to gain a concentration of 0.004% (w / v).

[0304] The IgG solution and the surfactant solutions are filtered through a PVDF syringe filter with 0.22 pm pore size. For each surfactant and for the blank 0.75 mL of the surfactant (histidine buffer for blank) and 0.75 mL of the IgG solution are added to five glass vials per sample. This leads to an IgG concentration of 10 mg / mL and a surfactant concentration of 0.002% (w / v). Four glass vials per sample are shaken at 200 rpm for 15 hours (T15) on an I KA HS 501 horizontal shaker.

[0305] The remaining glass vial (TO) is inverted 3 times, diluted 1 :10 in a deep-well plate with histidine buffer pH 6 and the particle count is quantified by micro-flow imaging (MFI) in the size range from 1 pm to 300 pm.

[0306] For all samples (TO and T15) the particle count is quantified by micro-flow imaging (MFI5000 by ProteinSimple). The samples are prepared for the measurement by pipetting 0.1 mL of each sample into a 96 deep-well plate, followed by 0.1 mL 200mM histidine buffer and 0.8 mL HPLC grade water.

[0307] All samples are measured in duplicates. The tests were run at ambient temperature (20 to 25 °C).

[0308] For the analysis 0.6 mL of the sample are used and the equivalent circular diameter (ECD) of the particles is determined in a range from 1 pm to 300 pm after removal of edge, stuck and slow particles.

[0309] The particle count for PS20 ranges from ca. 150 to 7,800 particles and for P188 from ca. 10,200 to 92,000 particles in 0.6 mL diluted sample, depending on the IgG batch that is used. For a good differentiation between different surfactants, an IgG batch should be used that, with the described protocol and in absence of surfactant (blank), results in at least 20,000 particles in 0.6mL diluted sample. While the particle count changes for different IgG batches, the trend between the different surfactants is consistent.

[0310] For analysis, the particle count of the blank is set in relation to the aggregation of the samples with surfactant (the particle count of the blank is set as 100% aggregation).

[0311] A.5 General protocol for water solubility assay

[0312] T o determine a polymer solubility of a 10 wt% solution in a 100 ml glass flask 7 g polymer (100%) and 63 g distilled water are placed at room temperature. The mixture is stirred with a magnetic stirrer until polymer is completely dissolved. To determine solubility at other concentrations, polymer solutions with various polymer content are prepared in a similar way.

[0313] A.6 General protocol for surface tension assay a) Static surface tension (Pendant Drop Method)

[0314] Sample preparation method 1

[0315] For the characterization of the static surface activity of block copolymers, samples were dissolved in deionized water at a concentration of 1 g / L and subsequently diluted to 0.1 g / L.

[0316] Sample preparation method 2

[0317] For the characterization of the static surface activity of block copolymers in the presence or absence of immunoglobulins, samples were dissolved 20 mM histidine buffer pH6 Surfactant was provided in a final concentration of 0.1 g / L.

[0318] Immunoglobulin was provided in a final concentration of 1 g / L

[0319] After stirring for 2 h and complete dissolution, the test solutions were filled into a disposable syringe, which was then mounted on a Drop Shape Analyser (DSA) 100 drop shape tensiometer from Kruss (Hamburg, Germany).

[0320] Static surface tension was measured at the indicated concentration by the pendant drop technique, where a free-hanging droplet of the test solution (typical volumes: 10-15 pL depending on surface tension) is generated at the outlet of the syringe. Then, a two-dimensional projection of the hanging droplet is acquired by an integrated camera system, from which the drop contour is determined via image analysis utilizing the instrument software Advance 1.9.2.

[0321] Fitting of the drop contour based on the Young-Laplace equation (C. Samuel et al., Polymer Testing, 2019, 78, 105995) yields the desired values for the surface tension.

[0322] The density of the solutions required for evaluation was assumed to be that of pure water.

[0323] Surface tension was monitored over a period of 5 minutes, and the measured values were averaged. All measurements were performed at 23 °C. b) Dynamic surface tension (Bubble Pressure Method)

[0324] Dynamic surface tension was measured using a bubble pressure tensiometer (SITA online t60) at 23 °C and surface ages ranging from 0.03 to 60 s. Solutions of 0.1 g / L (w / v) surfactant in histidine buffer (20 mM, pH 6.0) with and without added antibodies or antibodies alone (1 g / L (w / v) IgG) were studied in direct comparison.

[0325] A.7 General protocol for haemolysis assay

[0326] The principle of an RBC-test is described by Hoover (D.M: Hoover et al., Fundamental and Applied Toxicology 1990, 14, 589-597.) and Pape (W. J. W. 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 agitation of a cell suspension at different test compound concentrations. In case of RBC membrane damage due to the test substance, hemoglobin is released via the disrupted cell membrane into the test solution. The free hemoglobin concentration in the test solution is measured as correlate for the RBC membrane damage caused by the test substance.

[0327] Preparation of red blood cell (RBC) suspension: RBCs from EDTA blood of human blood donors were isolated by centrifugation and were washed three times with phosphate buffered saline plus glucose (PBS / glucose) to remove traces of plasma and the bulk of white blood cells. The washed RBCs were diluted with (PBS / glucose) and were adjusted to an approximate 2 % RBC suspension.

[0328] Test procedure: A test solution of the polymer of 1.33 % (=1 ,33g / 10 ml, i.e. final concentration in test 100 mg / ml) was prepared in PBS / glucose and adjusted to pH 7.4. Further test solutions (final concentration of polymer of 10 and 1 mg / ml) were made by dilution with PBS / glucose. One volume of the RBC suspension was added to three volumes of test solutions, resulting in final test compound concentrations of 100, 10, and 1 mg / ml. The assay mixtures were incubated at room temperature with shaking by an Eppendorf mixer (Model 5432; at 1000 rpm) for 60 minutes.

[0329] After incubation the samples were centrifuged to sediment remaining intact RBCs and membrane fragments.

[0330] Released free haemoglobin in the supernatant as a degree of hemolysis was determined spectrophotometrically at 540 nm.

[0331] Results were compared to totally lysed RBCs in distilled water (100% hemolysis) and to a fragility control with PBS / glucose (spontaneous, no substance related hemolysis). All samples were evaluated in triplicates.

[0332] Tested polymers were classified according to following scheme: low: < 10 % hemolysis at 100 mg / ml medium: < 10 % hemolysis at 10 mg / ml high: > hemolysis at 10 mg / ml

[0333] B. Examples

[0334] Example 1 : Pluronic PE 10400 (Poloxamer P334)

[0335] Commercially available product of BASF SE.

[0336] Example 2: Pluronic PE 10500 (Poloxamer P335)

[0337] Commercially available product of BASF SE.

[0338] C. Experimental results

[0339] C.1 Exemplary block copolymers

[0340] Table 1 : Exemplary Poloxamers according to the invention (Test samples No. 1 and 2) comparative examples CE1 to CE5 - Polysorbate and different Poloxamers; the Table also summarizes the experimental results on hemolytic activity, water solubility, surface tension and aggregation

[0341] 1)Determined in deionized water; Sample preparation method 1

[0342] Table 2: Static and dynamic surface tension measurements

[0343] Each sample determined in 20mM histidine buffer pH 6;

[0344] Static SFT: samples prepared according to Sample preparation method 2 C.2 Summary

[0345] Our attempts to provide polymeric surfactants as superior alternative to Poloxamer 188 allow the following conclusions.

[0346] The data of Table 1 show, that all tested samples proved to be good stabilizing agents for protein formulations. In that regard Examples 1 and 2 (corresponding to Poloxamers P334 and 335) prevented protein aggregation to a significantly higher extent than other Poloxamers. Said samples are also characterized by suitable surface tension (-within the range about 40 mN / m (0.1 g / l)) and a medium molecular weight with Mn 5.900 - 6.500 g / mol.

[0347] Comparative Poloxamers CE2 to CE4 showed a high degree of hemolysis.

[0348] The data of Table 2 show, that IgG and the herein tested surfactants are surface active and occupy the air / water interface in a time-dependent manner. By comparing the values of the dynamic surface tension and the static surface tension it can be determined that IgG is slower in reaching the air / water interface compared to P188, Pluronic PE 10400 or Pluronic PE 10500.

[0349] The values for the dynamic surface tension for each surfactant with or without IgG are similar (based on a pairwise comparison). Furthermore, the values for the static surface tension after 300 s are also very similar for each surfactant with or without IgG (based on a pairwise comparison). This indicates that the herein tested surfactants dominate the air / water interface and prevent the antibody from reaching the air / water interface.

[0350] Equal amounts of copolymer surfactant of the present invention (Ex. 1 and 2) and Poloxamer 188 (CE 1) differ significantly as regards the observed dynamic and static SFT. It is shown, that copolymer surfactants of the invention reduce both dynamic and static SFT surprisingly more efficiently. Thus, copolymer surfactants of the invention dominate the air / water interface so that the aggregation of antibody molecules caused by adsorption to the air / water interface is reduced to a surprisingly higher extent in comparison to Poloxamer 188.

[0351] Accordingly, copolymer surfactants of Examples 1 and 2 do fulfil all criteria necessary for their employment as surfactant for antibody formulations with superior properties in comparison to Polysorbates 20 and Poloxamer 188.

[0352] The surprising advantages associated with particular examples of Poloxamers of the present invention are summarized in attached Figure 1.

[0353] The content of documents referred to herein above is incorporated by reference. The content of any document cited herein is incorporated by reference.

Claims

Claims1. Use of a block copolymer which is a triblock-polymer, comprising a central PO block, and two flanking EO blocks, which block copolymer shows a combination of the following features: a) a molecular weight Mn of 5.000 to 7.000 g / mol, particularly 5.300 to 6.800 g / mol, more particularly 5.500 to 6.700 g / mol, and especially 5.800 to 6.

600. g / mol, as calculated from a determination of the OH number and b) an EO content of 33 to 60 wt.-%, particularly 35 to 55 wt.-%, more particularly 38 to 53 wt.-%, especially 40 to 50 wt.-%, each based on the dry weight of said block copolymer; for improving aggregation stability of an aqueous composition, in particular aqueous solution, of a least one biopolymer, wherein said biopolymer is selected from i) proteins, in particular enzymes and immunoglobulin molecules, each optionally glycosylated; ii) adducts or conjugates of an optionally glycosylated immunoglobulin molecule and a payload molecule; and iii) antibody payload conjugates (APC), particularly an antibody drug conjugates (ADC).

2. The use of claim 1 , wherein said block-copolymer improves aggregation stability of the biopolymer in said aqueous composition and further reduces biopolymersurface interactions.

3. The use of claim 1 or 2, wherein the biopolymer comprises an immunoglobulin and wherein said block-copolymer improves aggregation stability of the immunoglobulin and reduces immunoglobulin-surface interactions.

4. The use of anyone of the preceding claims, wherein said block copolymer is of the following general formula 1 :H-(-EO-)n-(-PO)m-(-EO)n-)-H(1) wherein m represents an integer in the range of 50 to 60, in particular 52 to 58 and n independently of each other represents an integer in the range of 23 to 45, in particular 28 to 40 ;5. The use of anyone of the preceding claims, wherein said block copolymer shows at least one of the following additional features c) a water solubility of at least 5 wt.-% based on the total weight of the aqueous solution of the block copolymer; d) a surface tension SFT of 53 to 30m N / m based on a 0.1 g / l solution of said block copolymer; and e) a lack of hemolytic activity, in particular less than 10% hemolysis caused by a solution of 10Og / l.

6. The use of anyone of the preceding claims, wherein said block copolymer of general formula 1 shows a combination of the following features a) a molecular weight 5.000 to 7.000 g / mole, and b) an EO content of 35 to 55 wt.-%, more particularly 38 to 53 wt.-%, especially 40 to 50 wt.-%, based on the dry weight of said block copolymer; and7. The use of anyone of the preceding claims, wherein the block copolymer is selected from the following commercial products: Poloxamer P334 and Poloxamer 335.

8. The use of anyone of the preceding claims, wherein the payload molecules are selected from pharmaceutically active compounds, labeling agents, biological small molecules such as lipids, phospholipids, glycolipids, sterols, vitamins, hormones, neurotransmitters, amino acids, nucleotides, monosaccharides; andbiological macromolecules, such as peptides, oligopeptides, polypeptides, proteins, nucleic acids, such as any forms of DNA and RNA, oligosaccharides, and polysaccharides.

9. The use of claim 8, wherein said biopolymer is a diagnostically applicable or a therapeutically active biopolymer.

10. The use of anyone of the preceding claims, wherein said block copolymer as defined in anyone of the claims 1 to 7 is contained in a proportion of 0,001 to 10 wt.-%, based on the total weight of the liquid composition; and / or said biopolymer is contained in a proportion of 0,01 to 30 wt.-%, based on the total weight of the liquid composition; and / or wherein said composition is in optionally buffered form, having a pH in the range of 5 to 9, particularly 6 to 8.

11. A biopolymer composition, comprising a liquid, in particular aqueous mixture, in particular aqueous solution, of at least one biopolymer component and at least one block copolymer wherein said block copolymer is a triblock-polymer, comprising a central PO block, and two flanking EO blocks, which block copolymer shows a combination of the following features: a) a molecular weight Mn of 5.000 to 7.000 g / mol, particularly 5.300 to 6.800 g / mol, more particularly 5.500 to 6.700 g / mol, and especially 5.800 to 6.

600. g / mol, as calculated from a determination of the OH number and b) an EO content of 33 to 60 wt.-%, particularly 35 to 55 wt.-%, more particularly 38 to 53 wt.-%, especially 40 to 50 wt.-%, each based on the dry weight of said block copolymer; wherein said biopolymer is selected from i) proteins, in particular enzymes and immunoglobulin molecules, each optionally glycosylated; ii) adducts or conjugates of an optionally glycosylated immunoglobulin molecule and a payload molecule; and iii) antibody payload conjugates (APC), particularly an antibody drug conjugates (ADC);with the proviso that the biopolymer composition is essentially free of any polysorbate surfactant; and with the further proviso that the biopolymer composition is essentially free of any polyaminosaccharide;12. The composition of claim 11 , wherein the block-copolymer is as further defined in anyone of the claims 4 to 7.

13. The composition of claim 11 or 12, wherein the biopolymer is selected from proteins, glycosylated proteins, proteoglycans, adducts or conjugates of such biopolymers with a further constituent selected from payload molecules, in particular pharmaceutically active compounds, labeling agents, biological small molecules such as lipids, phospholipids, glycolipids, sterols, vitamins, hormones, neurotransmitters, amino acids, nucleotides, monosaccharides; and biological macromolecules, such as peptides, oligopeptides, polypeptides, proteins, nucleic acids, such as any forms of DNA and RNA, oligosaccharides, and polysaccharides.

14. The composition of anyone of the claims 11 to 13, wherein said block copolymer as defined in claim 11 or 12 is contained in a proportion of 0,001 to 10%, based on the total weight of the liquid composition; and / or said biopolymer is contained in a proportion of 0,01 to 30 wt.-%, based on the total weight of the liquid composition; and / or wherein said composition is in buffered or non-buffered form, having a pH in the range of 5 to 9, particularly 6 to 8.

15. An essentially dry biopolymer composition, comprising at least one biopolymer component as defined in anyone of the claims 13 and at least one stabilizing ethylene oxide / propylene oxide block copolymer as defined in anyone of claims 1 to 7; which, in particular, has a liquid content of 0 to 5 wt.-% based on the total weight of said composition.

16. The composition of anyone of the claims 11 to 15 for use in medicine, in particular for diagnostic and / or therapeutic applications.

17. The composition of anyone of the claims 11 to 15, which is a pharmaceutical composition, optionally further supplemented by at least one pharmaceutically acceptable excipient.

18. A method of preparing a stabilized composition of anyone of the claims 11 to 14, which method comprises(1) preparing in any order an aqueous, optionally buffered solution of the biopolymer; and an aqueous, buffered or non-buffered solution of the block copolymer as defined in anyone of the claims 11 and 12; and(2) preparing a mixture of both aqueous solutions as obtained in step (1).

19. A method of preparing the essentially dry stabilized composition of claim 15, which method comprises(1) preparing in any order an aqueous, optionally buffered solution of said biopolymer; and an aqueous, buffered or non-buffered solution of said block copolymer as defined in anyone of the claims 11 and 12; and(2) preparing a mixture of both aqueous, buffered or non-buffered, solutions as obtained in step a);(3) optionally supplementing the aqueous, buffered or non-buffered solutions prepared in step a) and / or the mixture of both aqueous, buffered or nonbuffered, solutions prepared in step (2), with at least one pharmaceutically acceptable excipient; and(4) drying the mixture obtained in step (2) or (3).

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