Tocopherol alkoxylates for biopolymer stabilization

Abstract

The present invention relates to a buffered protein containing formulation comprising as stabilizing ingredient an amphiphilic surfactant. Said surfactant is formed by polyalkoxylating an a-tocopherol compound. The polyalkoxy residue is either a block copolymer or a statistical or random copolymer composed of a majority of ethylene oxide moieties and a smaller amount of propylene oxide and / or butylene oxide moieties. Said surfactant significantly improves stability, in particular aggregation stability of the protein formulation, while showing low hemolytic activity and good water solubility. Said surfactant is particularly suitable for preparing stabilized formulations of enzymes and immunoglobulin molecules as well as adducts and conjugates comprising such biomolecules. Said formulations may be provided in liquid or dry form and are suitable for the preparation of pharmaceutical compositions. According to another aspect of the invention the use of such formulations in medicine, in particular for diagnostic and / or therapeutic applications is provided. The present invention also relates to a method of preparing such formulations as well as a method for preparing such specific amphiphilic surfactants. According to a further aspect a method for preventing or suppressing protein aggregation of a liquid protein containing formulation in vitro by using an amphiphilic surfactant of the present invention as a stabilizer is provided. A prefilled syringe, comprising a reservoir prefilled with an amount of a formulation of the invention constitutes another aspect of the invention.

Description

[0001] BASF SE 240647W001

[0002] 1

[0003] Tocopherol Alkoxylates for Biopolymer Stabilization

[0004] Field of the Invention

[0005] The present invention relates to a buffered protein containing formulation comprising as stabilizing ingredient an amphiphilic surfactant. Said surfactant is formed by polyalkoxylating a tocopherol-type compound, in particular an a-tocopherol-type compound. The polyalkoxy residue is either a block copolymer or a statistical or random copolymer composed of a majority of ethylene oxide moieties and a smaller amount of propylene oxide and / or butylene oxide moieties. Said surfactant significantly improves stability, in particular aggregation stability of the protein formulation, while showing low hemolytic activity and good water solubility. Said surfactant is particularly suitable for preparing stabilized formulations of enzymes and immunoglobulin molecules as well as adducts and conjugates comprising such biomolecules.

[0006] Said formulations may be provided in liquid or dry form and are suitable for the preparation of pharmaceutical compositions.

[0007] According to another aspect of the invention the use of such formulations in medicine, in particular for diagnostic and / or therapeutic applications is provided.

[0008] The present invention also relates to a method of preparing such formulations as well as a method for preparing such specific amphiphilic surfactants.

[0009] According to a further aspect a method for preventing or suppressing protein aggregation of a liquid protein containing formulation in vitro by using an amphiphilic surfactant of the present invention as a stabilizer is provided.

[0010] Background of the Invention

[0011] Vitamin E Polyethylene Glycol Succinate (abbr. TPGS or Tocofersolan ®, Vitamin E TPGS NF, d-a-Tocopheryl Polyethylenglycol 1000 Succinat) is used in pharmaceutical formulations as emulsifier, solubilizer or as vitamin E supplement. It is synthesized by esterification of tocopherol succinate with PEG 1000. As a consequence, TPGS is a mixture composed of monoesterified polyethylene glycol 1000, diesterified polyethylene glycol 1000, free polyethylene glycol 1000, and free tocopherol.

[0012] Ethoxylates from tocopherols are described as surfactant, antioxidants, emulsifiers in Cosmetics & Toiletries 1993, 708, 63-78.

[0013] GB 2,254,080, DE 4210112 or US 5,235,073 are disclosing a-tocopherol ethoxylates with 2-100 mol ethylene oxide per OH. The ethoxylates are obtained from a- tocopherol by ethoxylation with NaOH, KOH or NaOMe as catalyst under standard conditions. The ethoxylates are used in cosmetic formulations.

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[0015] 2

[0016] WO99 / 62896 (EP1091951) describes tocopherol alkoxylates as surfactants, which are obtained by first ethoxylation and followed by propoxylation. They are used in cosmetic formulations.

[0017] EP1178044 (Kolb, 2000) describes tocopherol alkoxylates, a process to obtain tocopherol alkoxylates and their use in cosmetic or pharmaceutical formulations. Claimed tocopherol alkoxylates are alkoxylated with a first block of minimum 1 mole of propylene oxide or butylene oxide, followed by a second block consisting of up to 100 moles ethylene oxide. These tocopherol alkoxylates are used as emulsifiers, and the described examples are very likely not water soluble at concentrations of > 5 wt%.

[0018] WO2021 / 245125 (BASF) describes the use of a-tocopherol alkoxylates as emulsifiers for polymerization reactions or the use in cleaning compositions. Tocopherol is alkoxylated with ethylene oxide, propylene oxide and / or butylene oxide. The degree of alkoxylation is >1 to <100 per OH group.

[0019] US2016 / 0166516 describes pharmaceutical compositions with curcuminoids, and water soluble vitamin E derivative (preferably a polyalkylene glycol derivative of vitamin E and most preferably Vitamin E-TPGS).

[0020] Cucuzza et al. ( / nt. J. Pharm. 2024, 650, 123692) mention “TPG-light” as alternative to TPGS for stabilization of protein therapeutics. TPG-light is a tocopherol, ethoxylated with 16 moles ethylene oxide and end- capped with a methyl group. This shows a significant higher hemolytic activity than the benchmark TPGS, and no information is given about the protein stabilizing properties, in particular aggregation stability conferred by TPG-light.

[0021] Consequently, a problem to be solved by the invention relates to the identification of a highly water soluble surfactant that confers to biomolecules, like in particular proteins and more particularly enzymes and immunoglobulins, a degree of aggregation stability higher that Tocofersolan ®, but that in contrast to Tocofersolan ®, shows a higher chemical stability and concomitantly no hemolytic activity or a haemolytic activity comparable to Tocofersolan ®, and, therefore, would enable the provision of a liquid protein composition, in particular liquid antibody composition, of improved long term stability.

[0022] Summary of the Invention

[0023] The above mentioned problem could, surprisingly, be solved by the provision of tocopherol type surfactants of the general structure A-Toco, wherein “Toco” refers to a tocopherol-type moiety as defined herein below, polyalkoxylated with a polyalkoxylate moiety A as defined herein below; and more particularly by the provision of particular a-

[0024] M / 65044- PCT BASF SE 240647W001

[0025] 3 tocopherol-type polyalkoxylates as further defined in the attached claims.

[0026] These a-tocopherol polyalkoxylates were found to be concomitantly characterized

[0027] • high water solubility;

[0028] • high chemical stability;

[0029] • improved stabilization properties for antibody formulations regarding their capacity to inhibit antibody-surface interaction and / or aggregation; and / or

[0030] • low haemolytic activity.

[0031] Detailed Description of the Invention

[0032] A. Abbreviations

[0033] ADC antibody drug conjugates

[0034] APC antibody payload conjugates

[0035] API active pharmaceutical ingredient

[0036] BuO or OBu butylene oxide

[0037] CDCh deuterated chloroform

[0038] ECD equivalent circular diameter

[0039] EDTA Ethylenediaminetetraacetic acid

[0040] EO or OE ethylene oxide

[0041] HPLC high-performance liquid chromatography

[0042] 1H-NHR proton nuclear magnetic resonance

[0043] I FT interfacial tension

[0044] IgG: immunoglobulin class G

[0045] MFI micro-flow imaging

[0046] PVDF polyvinylidene fluoride

[0047] PS20 Polysorbate 20

[0048] P188 Poloxamer 188

[0049] PBS phosphate-buffered saline

[0050] RBC Red blood cell

[0051] RT Room temperature (20°C)

[0052] PO or OP propylene oxide

[0053] SFT Surface tension

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[0055] 4

[0056] B. Definitions

[0057] 1. General

[0058] 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.

[0059] 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.

[0060] 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.”

[0061] The terms “about” or “approximately” indicate a potential variation of ± 25% of the stated value, in particular ± 15% or ±10 %, more particularly ± 5%, ± 2% or ± 1%.

[0062] 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%.

[0063] “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%.

[0064] 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.

[0065] Unless otherwise specified the term “ambient temperature” or “room temperature” refers to a temperature in the range of 20 to 25°C.

[0066] 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 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

[0067] M / 65044- PCT BASF SE 240647W001

[0068] 5 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 copolymer surfactant of the invention, relative to an otherwise identical liquid mixture which is not supplemented with said copolymer.

[0069] 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.3. Typical standard conditions are:

[0070] Protein concentration: in the range of about 0,1 to 50 mg / mL, particularly about

[0071] 1 to 30 mg / mL or more particularly about 1 to 15 mg / mL; most particularly about

[0072] 1 to 10 mg / mL;

[0073] 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)

[0074] 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;

[0075] Particle size of aggregates determined in the range of 1 pm to 300 pm or 2pm to 50 pm;

[0076] Test temperature: ambient temperature (20 to 25°C);

[0077] Blank: Protein aggregation in deionized water or buffer as defined above (without surfactant set to 100%);

[0078] 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%.

[0079] A “reduced biopolymer-surface interaction” or “reduced protein-surface interaction” in the context of the invention is derived from the experimental observation that 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

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[0081] 6 defined, relative to an otherwise identical liquid mixture which is not supplemented with said copolymer.

[0082] Such “reduced biopolymer-surface interaction” or “reduced protein-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:

[0083] 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;

[0084] 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;

[0085] 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;

[0086] Test temperature: ambient temperature (20 to 25°C);

[0087] Blank: Protein dissolved in deionized water or in buffer as defined above (without surfactant);

[0088] 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.

[0089] 2. Chemical Terms

[0090] The term “tocopherol-type” in the context of the invention encompasses a group of compounds of the general formula wherein R1and R2are identical or different and selected from H and methyl, in stereoisomerically pure form or as a mixture of at least two stereoisomers, said group of compounds being also known as a-, -, y - or 5- tocopherol-type compound.

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[0092] 7

[0093] The term “a-tocopherol” denotes a compound of the chemical name (2R)-2,5,7,8-

[0094] Tetramethyl-2-[(4F?,8R)-4,8,12-trimethyltridecyl]-3,4-dihydro-2 / 7-chromen-6-ol (CAS

[0095] Number 10191-41-0) having the following structural formula and stereochemistry:

[0096] “Alkyl” relates to a straight-chain or branched alkyl group having from 1 to 4, in particular 1 , 2, 3 or 4 carbon atoms. Example are methyl, ethyl, n-propyl, n-butyl, i-butyl and tert.-butyl.

[0097] “Alkylene oxy unit” or “alkylene oxide unit” relates to a radical of the formula -X- O-, or -O-X- wherein X is a straight-chain or branched alkylene group having 1 , 2, 3 or 4 carbon atoms as defined herein.

[0098] 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.

[0099] “Ethylene oxide” (EO) as used in the context of the invention relates an 1-2-epoxy ethane.

[0100] “Ethylene oxy units” or “ethylene oxide units” (-EO- or-OE-) may be incorporated in a polymer or copolymer moiety of the invention in any orientation.

[0101] “Propylene oxide” (PO) as used in the context of the invention relates an 1-2- epoxy propane.

[0102] “Propylene oxy units” or “propylene oxide units” (-PO- or -OP-) as incorporated in a polymer or copolymer moiety of the invention are selected from -O-CH2-CH(CH3)- and -CH2-CH(CH3)-O- and may be incorporated in a polymer or copolymer moiety of the invention in any orientation.

[0103] “Butylene oxide” (BuO) as used according to the invention encompass 1,2-epoxy butane and any isomer thereof, like 2,3-epoxy butane.

[0104] “Butylene oxy units” or “butylene oxide units” (-BuO- or -OBu-) as incorporated in a polymer or copolymer moiety of the invention are selected from -O-CH2-CH(C2Hs)-, -CH2-CH(C2HS)-O- or -O-CH(CH3)-CH(CH3)- and may be incorporated in a polymer or copolymer moiety of the invention in any orientation.

[0105] “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.

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[0107] 8

[0108] “Random copolymer" or “statistical copolymer” define a macromolecular entity characterized by at least two structurally different monomer moieties, randomly or statistically distributed over the length of the copolymer chain.

[0109] “Static surface tension” (SFT) 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.

[0110] “Ethylene oxide content” (EO%), “Propylene oxide content” (PO%) and “butylene oxide content” (BuO%) within the meaning of the invention are stated herein as either mole % (mol%) or as weight percentage (wt.-%) of ethylene oxide, propylene oxide or butylene oxide monomer units within a given copolymer unit A as herein defined. If not otherwise stated “%” refers to wt.-%.

[0111] The term “EO% calculated from the atomic masses of all atoms of the copolymer moiety A in formula 1 refers to a wt.-% value obtained according to the following formula:

[0112] EO% = [( S masses EO) / (( £ masses EO + S masses PO and / or + S masses BuO)] * 100

[0113] The term “a calculated molecular weight of x to y g / mol” (also designated herein as “Mw calc.”) encompasses the integers x , y and any integer between x and y. For example, a molecular weight of ”700 to 2.500 g / mol” encompasses at least the integers:

[0114] 700, 750, 800, 850, 900, 950, 1.000, 1.100, 1.200, 1.300, 1.400, 1.500, 1.600, 1.700, 1.800, 1.900, 2.000, 2.100, 2.200, 2.300, 2.400, and 2.500,

[0115] “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.

[0116] “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.

[0117] 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.

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[0119] 9

[0120] 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).

[0121] 3. Biochemical Terms

[0122] The term “biopolymer” as used herein in principle encompasses molecules, selected from oligopeptides, polypeptides, and in particular proteins, any type of antibody molecule or fragment or derivative thereof as defined below, glycosylated proteins, proteoglycans, 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.

[0123] 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.

[0124] 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,

[0125] M / 65044- PCT BASF SE 240647W001

[0126] 10 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).

[0127] 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).

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[0129] 11

[0130] 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.

[0131] 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.

[0132] 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 A forms. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals and / or any other targeted AB form.

[0133] 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.

[0134] 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.,

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[0136] 12 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.

[0137] 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.

[0138] 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.

[0139] The terms "Kabat numbering", "Kabat definitions and "Kabat labelling" 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.

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[0141] 13

[0142] 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).

[0143] 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

[0144] M / 65044- PCT BASF SE 240647W001

[0145] 14

[0146] 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.

[0147] 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.

[0148] 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.

[0149] 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.

[0150] Human heavy chain and light chain acceptor sequences are known in the art.

[0151] 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

[0152] M / 65044- PCT BASF SE 240647W001

[0153] 15 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.

[0154] 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.

[0155] As used herein, the term "humanized antibody" is an antibody or a variant, derivative, analogue 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

[0156] M / 65044- PCT BASF SE 240647W001

[0157] 16 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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

[0162] M / 65044- PCT BASF SE 240647W001

[0163] 17 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 referred 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.

[0164] The term “labelled 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 “labelled 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 radiolabelled 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, 111ln, 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.

[0165] 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.

[0166] The term "KD" (also "Kd" or “KD”), as used herein, is intended to refer to the "equilibrium dissociation constant", and refers to the value obtained in a titration measurement at equilibrium, or 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

[0167] M / 65044- PCT BASF SE 240647W001 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.

[0168] “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.

[0169] “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.

[0170] “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.

[0171] C. Particular aspects and embodiments of the invention

[0172] The present invention relates to the following aspects and particular embodiments thereof:

[0173] A first aspect of the invention relates to a formulation comprising at least one protein, at least one buffer and at least one amphiphilic surfactant, wherein said amphiphilic surfactant is a polyalkoxylated a-tocopherol compound, in particular a polyalkoxylated a-tocopherol-type compound of the general formula 1 in stereoisomerically pure form or as a mixture of at least two stereoisomers, wherein

[0174] A represents

[0175] M / 65044- PCT BASF SE 240647W001

[0176] 19 i) a polyalkylene oxide block copolymer moiety of the general formula 2:

[0177] R1-(OE)n-(OR)m-O-

[0178] (2) wherein m represents an integer in the range of 1 to 10; particularly 1 to 8, more particularly 1 to 7 or 1 to 6, like 1 , 2, 3, 4, 5 or 6; n represents an integer in the range of 10 to 100; particularly 10 to 60, more particularly 10 to 40, like 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40; even more particularly 15 to 40, like 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40; and most particularly 18 to 35, like 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or 35;

[0179] OE represents an ethylene oxide unit

[0180] OR represents the same or different alkylene oxide unit selected from a propylene oxide (-OP-) or a butylene oxide (-OBu-) unit; and and

[0181] R1represents H or C1-C4 alkyl; like methyl, ethyl and n-propyl; particularly H or methyl, and most particularly H. or ii) a polyalkylene oxide random copolymer moiety wherein said random copolymer moiety is composed of m randomly distributed alkylene oxide units selected from propylene oxide (-OP-) units, butylene oxide (-OBu-) units or mixtures thereof wherein m represents an integer in the range of 1 to 10; particularly 1 to 8, more particularly 1 to 7 or 1 to 6, or 2 to 5, like 1 , 2, 3, 4, 5 or 6; and

[0182] M / 65044- PCT BASF SE 240647W001

[0183] 20 n randomly distributed ethylene oxide (-OE-) units wherein n represents an integer in the range of 10 to 100, particularly 10 to 60, more particularly 10 to 40, and even more particularly 2 to 27; like 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40; and the terminal outer alkylene oxide unit or outer ethylene oxide unit is capped with a residue R1selected from H or C1-C4 alkyl; particularly H or methyl, and most particularly H; or a polyalkylene oxide random copolymer moiety wherein said random copolymer moiety is composed of m randomly distributed propylene oxide (-OP-) units, wherein m represents an integer in the range of 2 to 10; particularly 2 to 8, more particularly 2 to 7, 2 to 6, and most particularly 2 to 5, like 2, 3, 4 or 5; and n randomly distributed ethylene oxide (-OE-) units wherein n represents an integer in the range of 10 to 100, particularly 10 to 60, more particularly 10 to 40, and even more particularly 23 to 27; like 23, 24, 25, 26 or 27; and most particularly 24 to 26, like in particular 25; and the terminal outer alkylene oxide unit or outer ethylene oxide unit is capped with a residue R1 selected from H or C1-C4 alkyl; particularly H or methyl, and most particularly H.

[0184] Particularly, said propylene oxide (-OP-) units are 1 , 2- propenyloxy units and said butylene oxide (-OBu-) units are 1 ,2- butenyloxy units.

[0185] M / 65044- PCT BASF SE 240647W001

[0186] 21

[0187] According to another particular embodiment of the first aspect, the said polyalkoxylated tocopherol-type compounds of the invention are block copolymers for the following general formula 5:

[0188] R represents methyl or ethyl; and

[0189] R1represents H or Ci- C4 alkyl; like particularly H or methyl, more particularly H; m represents an integer in the range of 1 to 10; particularly 1 to 8, more particularly 1 to 7 or 1 to 6, like 1 , 2, 3 or 4, n represents an integer in the range of 10 to 100; particularly 12 to 60, more particularly 15 to 40, like 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40; and wherein the (OE)nblock constitutes either the outer or the inner of said two polymer blocks; or,

[0190] R represents methyl and

[0191] R1represents H or Ci- C4 alkyl; like particularly H or methyl, more particularly H; m represents an integer in the range of 2 to 10; particularly 2 to 8, more particularly 2 to 7 or most particularly an integer in the range of 2 to 6, like, 2, 3 , , 4, 5 or 6; n represents an integer in the range of 15 to 40, like 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, and most particularly an integer in the range of 18 to 25, like 18, 19, 20, 21, 22, 23, 24 or 25; and wherein the (OE)nblock constitutes the outer of said two polymer blocks;

[0192] M / 65044- PCT BASF SE 240647W001

[0193] 22 or

[0194] R represents ethyl; and

[0195] R1represents H or Ci- C4 alkyl; like particularly H or methyl, more particularly H; m represents an integer in the range of 1 to 10; particularly 1 to 8, more particularly 1 to 7 or 1 to 6, like 1 , 2, 3 or 4; and most particularly 1 or 2; n represents an integer in the range of 10 to 100; particularly 12 to 60, more particularly 15 to 40, like 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39 or 40; and most particularly 25 to 35, like 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34 or 35; and wherein the (OE)nblock constitutes the outer of said two polymer blocks.

[0196] Particularly, said polyalkoxylated tocopherol type compound (1) is a polyalkoxylated derivative of (2R)-2,5,7,8-Tetramethyl-2-[(4R,8R)-4,8,12- trimethyltridecyl]-3,4-dihydro-2 / 7-chromen-6-ol (alpha-Tocopherol).

[0197] In particular, a formulation of is provided, wherein the polyalkoxy moiety A is either a statistical copolymer moiety of the ethylene oxide units and the same or different alkylene oxide units selected from propylene oxide units, butylene oxide units or mixtures thereof; or is composed of a first, inner propylene oxide block or inner butylene oxide block or mixed inner propylene oxide / butylene oxide block covalently bound to the tocopherol moiety and a second, outer ethylene oxide block covalently bound to the terminal end of to the first, inner propylene oxide block or inner butylene oxide block or mixed inner propylene oxide / butylene oxide block.

[0198] More particularly, a formulation is provided, wherein the polyalkoxy moiety is composed of a first, inner propylene oxide block or butylene oxide block covalently bound to the tocopherol moiety and a second, outer ethylene oxide block covalently bound to the terminal end of to the first inner propylene oxide block or inner butylene oxide block, and wherein in formula 1 m is an integer of 1 to 9, particularly 1 to 8, more particularly 1 to 7 or 1 to 6; like 1 , 2, 3, 4, 5 or 6; and n is an integer of 15 to 50, particularly 15 to 40, more particularly 20 to 40, or 25 to 35, 18 to 35 or 18 to 25; and

[0199] M / 65044- PCT BASF SE 240647W001

[0200] 23

[0201] R1is H or methyl, more particularly H.

[0202] According to another particular embodiment the polyalkoxy moiety is composed of a first, inner propylene oxide block covalently bound to the tocopherol moiety and a second, outer ethylene oxide block covalently bound to the terminal end of to the first inner propylene oxide block, wherein the polyalkoxy moiety shows molar proportion (mole-%) of the propylene oxide units in the range about 7 to 25, particularly about 8 to 24, more particularly 9 to 23 or even more particularly about 10 to 20 mole-% propylene oxide moieties based on the sum of moles of propylene oxide and moles of ethylene oxide of the polyalkoxy moiety.

[0203] According to another particular embodiment the polyalkoxy moiety is composed of a first, inner butylene block covalently bound to the tocopherol moiety and a second, outer ethylene oxide block covalently bound to the terminal end of to the first inner butylene oxide block, wherein the polyalkoxy moiety shows molar proportion (mole-%) of the butylene oxide units in the range about 2 to 10, particularly about 3 to 7, more particularly 3.5 to 6 or even more particularly about 4 to 5.5 mole-% butylene oxide moieties based on the sum of moles of butylene oxide and moles of ethylene oxide of the polyalkoxy moiety.

[0204] According to another particular embodiments the calculated molecular weight of polyoxyalkylene modified alpha-Tocopherols of the invention is in an approximate range of 1.200 - 2.500, particularly 1.300- 2.200 and more particularly 1.300 - 1.900 g / mole.

[0205] According to another particular embodiment of said formulation, the a-tocopherol- type compound of formula (1) or (5) shows at least one of the following additional features a) a water solubility of at least 10 wt.-% based on the total weight of the aqueous solution of the block copolymer, b) less than 10% hemolytic activity caused by a solution of said block copolymer of 100 mg / L c) an interfacial surface tension (IFT) in the range of less than 22,3 mN / m, particularly 10 to 22 mN / m, or more particularly 12 to 21 mN / m.

[0206] According to a more particular embodiment of said formulation, the a-tocopherol- type compound of formula (1) or (5) shows at least one of the following additional features a) a water solubility of at least 10 wt.-% based on the total weight of the aqueous solution of said compound of formula (1) or (5), b) less than 10% hemolytic activity caused by a solution of said compound of formula (1) or (5) at a concentration of 100 mg / L in PBS / glucose (0.18% w / v glucose), pH 7.4 at 20 °C (RT) and a RBC content of approximately

[0207] M / 65044- PCT BASF SE 240647W001

[0208] 24

[0209] 0.5% (v / v); and c) an interfacial surface tension (IFT) in the range of less than 22,3 mN / m, particularly 10 to 22 mN / m, or more particularly 12 to 21 mN / m; as determined by pendant drop tensiometry between a silicone reservoir and a droplet of a 1 g / l (w / v) solution of said compound of formula (1) or (5) at 23 °C.

[0210] Suitable methods for determining the above identified parameters water solubility, hemolytic activity and IFT, while being described in more detail in the experimental section, are considered to form part of the general disclosure.

[0211] According to another particular embodiment of said formulation, said block copolymer shows calculated molecular weight of about 1 .000 to 2.500 g / mol, particularly about 1.200 to about 2.000 g / mol, calculated on the basis of parameters n, m, R and R1of formula 1.

[0212] More particularly said formulation may be provided in dry or liquid form.

[0213] The protein as contained in a formulation of the invention is particularly selected from oligopeptides, polypeptides, proteins, glycosylated proteins, proteoglycans, antibody molecules or fragments or derivatives thereof, adducts or conjugates of such proteins with a further constituent selected from payload molecules, which 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.

[0214] More particularly, said protein is a diagnostically applicable or a therapeutically active protein, and even more particularly said protein is selected from enzymes and immunoglobulin molecules, each optionally glycosylated; or said protein is selected from adducts or conjugates of an optionally glycosylated immunoglobulin molecule and a payload molecule; or said protein is an optionally glycosylated antibody payload conjugate (APC), particularly an optionally glycosylated antibody drug conjugate (ADC).

[0215] M / 65044- PCT BASF SE 240647W001

[0216] 25

[0217] According to a further particular embodiment of said formulation, said protein is an antibody molecule or an antibody payload conjugate (APC), particularly an antibody drug conjugate (ADC), each optionally glycosylated.

[0218] Optionally glycosylated natural or recombinant 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.

[0219] According to still another particular embodiment of said formulation, the formulation is provided in liquid form, wherein said surfactant of the general formula 1 is contained in a proportion of 0,001 to 10%, based on the total weight of the liquid formulation; and / or said protein is contained in a proportion of 0,01 to 30%, based on the total weight of the liquid formulation; and / or wherein said liquid formulation is in buffered form, in particular having a pH in the range of pH 5 to 9, more particularly pH 6 to 8.

[0220] According to still another particular embodiments, said formulation is a pharmaceutical composition, optionally further supplemented by at least one pharmaceutically acceptable excipient.

[0221] A second aspect of the invention relates to an essentially dry formulation comprising at least one protein component as defined above, and at least surfactant as defined above, which has a liquid content of 0% to 5% wt.-% based on the total weight of said formulation; and is optionally further characterized as follows: wherein said at least one surfactant (Y) and said at least protein (Z) are contained in a weight ratio (Y) : (Z) in the range of 1 : 20.000 to 10:1 , or 1 : 5.000 to 2:1 , 1 : 100 to 1 ,2 : 1 , particularly 1 : 10 to 1 , 1 : 1 ; and / or wherein said at least one surfactant (Y) and said at least one protein (Z) together (i.e. (Y) plus (Z)) 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 20 to 25 wt.-% based on the total weight of said essentially dry formulation and / or further comprises at least one further excipient in a proportion of 0,1 to 99 wt.-%, 40 to 95 wt.-% and 50 to 90 wt.-% based on the total dry weight of said essentially dry formulation.

[0222] A third aspect of the invention relates to the use of a surfactant as defined above,

[0223] M / 65044- PCT BASF SE 240647W001

[0224] 26 for stabilizing an aqueous composition, in particular aqueous solution, of a least one optionally glycosylated protein as defined above. Particular examples of optionally glycosylated proteins are optionally glycosylated natural or recombinant immunoglobulin molecules, which 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.

[0225] Particularly, said use is for improving aggregation stability of an aqueous composition, in particular aqueous solution, of said least one protein.

[0226] A fourth aspect of the invention relates to a formulation as herein above defined for use in medicine, in particular for diagnostic and / or therapeutic applications. Particularly said formulation contains at least one optionally glycosylated, natural or recombinant immunoglobulin molecule, which 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.

[0227] A fifth aspect of the invention relates to a method of preparing a protein formulation as defined herein above, which method comprises a) preparing in any order an aqueous, optionally buffered solution of the protein; and an aqueous, optionally buffered solution of the surfactant as defined herein above, wherein at least one of said aqueous solutions is applied as buffered solution; and b) preparing a mixture of both aqueous solutions as obtained in step a).

[0228] A sixth aspect of the invention relates to a method of preparing the essentially dry protein formulation as defined above which method comprises a) preparing in any order an aqueous, optionally buffered solution of said protein; and an aqueous, optionally buffered solution of said surfactant as defined herein above, wherein at least one of said aqueous solutions is applied as buffered solution; 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;

[0229] M / 65044- PCT BASF SE 240647W001

[0230] 27 d) drying the mixture obtained in step b) or c).

[0231] A seventh aspect of the invention relates to a method for preventing or suppressing protein aggregation of a liquid protein-containing formulation in vitro by using an amphiphilic surfactant as a stabilizer in said formulation, wherein said amphiphilic surfactant is defined herein above, particularly wherein said amphiphilic surfactant is applied in a proportion in the range of 0,0001 to 10 wt.-%, particularly 0,005 to 5 wt.-%, most particularly 0,001 to 0,2 wt.-%, based on the total weight of said liquid formulation and wherein more particularly the protein is defined herein above.

[0232] As particular examples of such proteins there may be mentioned optionally glycosylated natural or recombinant immunoglobulin molecules, which 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 I gG 1 , I gG2, lgG3 and lgG4.

[0233] An eighth aspect of the invention relates to a prefilled syringe, comprising a reservoir prefilled with an amount of a formulation as defined above.

[0234] A ninth aspect of the invention relates to a method of preparing an amphiphilic surfactant of general formula 1 , in stereoisomerically pure form or as a mixture of at least two stereoisomers, wherein

[0235] A is as defined above for the first aspect of the invention which method comprises admixing an alpha-tocopherol-type compound of formula (4)

[0236] M / 65044- PCT BASF SE 240647W001

[0237] 28 in stereoisomerically pure form or as a mixture of at least two stereoisomers, with a base catalyst under inert gas in a reactor vessel, optionally remove water being present, and then a) either adding propylene oxide or butylene oxide or a mixture of propylene oxide and butylene oxide in a suitable amount in order to polymerize a first propylene oxide block or first butylene oxide block or first mixed propylene oxide and butylene oxide block to the terminal hydroxy group of said tocopherol, followed by the addition of ethylene oxide in a suitable amount in order to polymerize a second ethylene oxide block to the terminal end of the first propylene oxide block or first butylene oxide block or first mixed propylene oxide and butylene oxide block in order to obtain a polyalkylene oxide moiety A with a polymer block structure; b) or adding a mixture of ethylene oxide and propylene oxide or a mixture of ethylene oxide and butylene oxide or a mixture of ethylene oxide and propylene oxide and butylene oxide in a suitable amount and in a suitable ratio of the proportion of ethylene oxide and propylene oxide or ethylene oxide and butylene oxide ethylene oxide and propylene oxide and butylene oxide in order to generate a statistically polymerized polyalkylene oxide moiety A.

[0238] Particularly, said alpha-tocopherol type compound of formula (4) is (2R)-2,5,7,8- Tetramethyl-2-[(4R,8R)-4,8,12-trimethyltridecyl]-3,4-dihydro-2 / 7-chromen-6-ol.

[0239] A tenth aspect of the invention relates to particular compounds of general formula (1) or (5), selected from: a) Tocopherol derivatives of general formula (1) or (5), wherein A represents a block copolymer unit composed of an inner PO or BuO monomer block and an outer EO monomer block, terminated with R1= H

[0240] M / 65044- PCT BASF SE 240647W001

[0241] 29

[0242] Inner Outer block block alpha-Tocopherol + 2 PO / OH + 18 EO / OH alpha-Tocopherol + 2 PO / OH + 20 EO / OH alpha-Tocopherol + 2 PO / OH + 25 EO / OH alpha-Tocopherol + 3 PO / OH + 25 EO / OH alpha-Tocopherol + 3 PO / OH + 25 EO / OH alpha-Tocopherol + 4 PO / OH + 25 EO / OH alpha-Tocopherol + 5 PO / OH + 25 EO / OH alpha-Tocopherol + 6 PO / OH + 25 EO / OH alpha-Tocopherol + 1 BuO / OH + 25 EO / OH alpha-Tocopherol + 2 BuO / OH + 35 EO / OH and b) Tocopherol derivatives of general formula (1) or (5), wherein A represents a copolymer unit composed of randomly distributed EO and PO monomers, terminated with R1= H: alpha-Tocopherol + 25 EO / OH+ 2 PO / OH (random) alpha-Tocopherol + 25 EO / OH+ 5 PO / OH (random)

[0243] D. Further embodiments

[0244] D.1. Synthesis of copolymers of formula (1)

[0245] Methods for the preparation of stabilizing compounds of the general formula 1 are generally known in the art.

[0246] Suitable methods for their preparation are reported, for example, in EP1 178 044 or WO2021 / 245125.

[0247] Examples of suitable alkylene oxides are ethylene oxide, propylene oxide and the butylene oxides 1 ,2-butylene oxide and 2,3-butylene oxide.

[0248] The reaction is preferably carried out under moisture-free conditions at elevate temperature and in presence of a suitable catalyst such as an alkali metal tert-butoxide or an alkali metal hydroxide

[0249] 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.

[0250] M / 65044- PCT BASF SE 240647W001

[0251] 30

[0252] 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.

[0253] The reaction may be conducted either batch-wise or continuously as desired. In batchwise operation, the tocopherol starting molecule 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.

[0254] Prior to the introduction of the alkylene oxide or mixture of alkylene oxides, the reaction vessel is advantageously flushed with a stream of dry inert gas, such as nitrogen, to remove any traces of air or oxygen or traces of water therefrom. The elimination of molecular oxygen and water from the reaction vessel is an important factor in obtaining colorless products and may, if desired, be carried out after adding the alkylene oxide(s) and catalyst to the reaction vessel. Preferably, the content of residual water should be in the range of 0,1 wt.-% or less, more preferably in the range of 0,05 wt.-% or less, each based on the total weight of the added alkylene oxides.

[0255] The reaction mixture of tocopherol and catalyst is then heated to a reaction temperature of about 120 to 140°C and PO or BuO, or a mixture of EO and PO, or EP and BuO, or EO, PO and BuO is added at a fairly rapid rate.

[0256] Usually, the rate of addition of alkylene 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.

[0257] By controlling the rate of addition of alkylene oxide to maintain the pressure fairly constant, the reaction temperature may also be maintained constant.

[0258] The addition of alkylene oxide is stopped upon obtaining the desired molecular weight of the polyoxyalkylene condensation product as determined by, for example, hydroxyl analysis or1H-NMR reckoning one free hydroxyl groups per molecule.

[0259] In the case of preparing a block copolymer side chain (with a first inner block consisting of PO and / or BuO moieties), ethylene oxide is condensed with the said first block to give a second outer EO block., essentially consisting of EO moieties The addition of ethylene oxide is carried out in the same manner as the addition of PO and or BuO already described.

[0260] 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. For example, the reaction

[0261] M / 65044- PCT BASF SE 240647W001

[0262] 31 mixture was cooled to a temperature below 100°C, and stripped with nitrogen. Volatile compounds were removed in vacuo at said temperature. Phosphoric acid is added for neutralization.

[0263] 1H-NMR in CDCI3 is used to confirm the complete conversion to the expected polymer.

[0264] D.2. Antibody-payload conjugates and their preparation

[0265] 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).

[0266] 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.

[0267] 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)).

[0268] 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.

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[0270] 32

[0271] 25, 569-578 (2014)). These methods are restricted to specific sites and cannot be transferred to other positions in the antibody sequence.

[0272] 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.

[0273] 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)).

[0274] 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)).

[0275] 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)).

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[0277] 33

[0278] 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, II., 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. II., Ihsan, A. II. & Zhang, J. Antibody-Drug Conjugates: A Comprehensive Review. Mol. Cancer Res. 18, 3-19 (2020)).

[0279] 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)).

[0280] Non-limiting examples of suitable antibodies and payload molecules within the meanings of the present invention are reported in the sections below.

[0281] D.3. Antibodies

[0282] 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).

[0283] Typical non-limiting examples are selected form biologically, in particular pharmacologically active antibody molecules.

[0284] 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

[0285] M / 65044- PCT BASF SE 240647W001

[0286] 34 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, MUCI, CEA, gplOO, MARTI, IL-2 receptor, CD20, CD52, CD33, CD22, human chorionic gonadotropin, CD38, CD40, mucin, P21 , MPG, and Neu oncogene product.

[0287] D.4. Payload molecules

[0288] 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.

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[0290] 35

[0291] D.4.1. Bioactive compounds

[0292] Bioactive compounds include, but are not limited to, the following:

[0293] 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.

[0294] 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.

[0295] 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).

[0296] 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.

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[0298] 36

[0299] 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.

[0300] 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).

[0301] Examples of a uri statins 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;

[0302] 5,665,860; 5,663,149; 5,635,483; 5,599,902; 5,554,725; ;5, 530, 097; 5,521 ,284;

[0303] 5,504,191 ; 5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; ;4, 879, 278;

[0304] 4,879,278; 4,816,444; and 4,486,414, the disclosures of which are incorporated herein by reference in their entirety.

[0305] 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.

[0306] Exemplary maytansines, maytansinoids, such as DM-1 and DM-4, or maytansinoid analogs, including maytansinol and maytansinol analogs, are described in

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[0308] 37

[0309] 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;

[0310] 5,208,020; 5,416,064; 5,475,092; 5,585,499; 5,846,545; 6,333,410; 6,441 ,163;

[0311] 6,716,821 and 7,276,497.

[0312] 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],

[0313] Calicheamicins include, e.g. enediynes, esperamicin, and those described in U.S. Patent Nos. 5,714,586 and 5,739,116.

[0314] 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.

[0315] 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.

[0316] 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.

[0317] 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.

[0318] Exemplary platinum compounds include cisplatin, carboplatin, oxaliplatin, iproplatin, ormaplatin, tetraplatin.

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[0320] 38

[0321] Exemplary DNA binding or alkylating drugs include CC-1065 and its analogs, anthracyclines, calicheamicins, dactinomycines, mitromycines, pyrrolobenzodiazepines, and the like.

[0322] 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.

[0323] 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).

[0324] 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.

[0325] 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.

[0326] 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.

[0327] 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.

[0328] Exemplary AKT inhibitors include, but are not limited to AT7867.

[0329] Exemplary MAPK signaling pathway inhibitors include MEK, Ras, JNK, B-Raf and p38 MAPK inhibitors.

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[0331] 39

[0332] 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.

[0333] Exemplary B-raf inhibitors include CDC-0879, PLX-4032, and SB590885.

[0334] 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.

[0335] Exemplary protein chaperon inhibitors include HSP90 inhibitors. Exemplary inhibitors include 17AAG derivatives, BIIB021 , BIIB028, SNX-5422, NVP-AUY-922 and KW-2478.

[0336] 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.

[0337] Exemplary Wnt / Hedgehog signalling pathway inhibitors include vismodegib, cyclopamine and XAV-939.

[0338] Exemplary RNA polymerase inhibitors include amatoxins. Exemplary amatoxins include alpha-amanitins, beta amanitins, gamma amanitins, eta amanitins, amanullin, amanullic acid, amanisamide, amanon, and proamanullin.

[0339] Exemplary cytokines include IL-2, IL-7, IL-10, IL-12, IL-15, IL-21 , TNF.

[0340] As non-limiting examples of particular drugs there may be mentioned Auristatins, Maytansinoids, PBDs, topoisomerase inhibitors, anthracyclines

[0341] In another embodiment, a combination of two or more different drugs as described above are used.

[0342] 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.

[0343] Other suitable examples of bioactive compounds are immunoglobulins such as antibodies, antibodies derivatives and active fragments thereof. Suitable examples in that regard are reported in the section D.3 above.

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[0345] 40

[0346] D.4.2. Labelling Agents and Radionuclides

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] M / 65044- PCT BASF SE 240647W001

[0352] 41

[0353] 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.

[0354] D.4.3. Chelators

[0355] Lists of typically applicable chelators and their short names are given below. Corresponding salts thereof are also applicable:

[0356] 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 (DOTA), 1 ,4,7-triazacyclononane-1 ,4,7- triacetic acid (NOTA), Oxalate (OX), tartrate (TART), citrate (CIT), dimethylglyoxime (DMG), 8-hydroxyquinoline, 2,2'-bipyridine (BPY), 1 ,10-phenanthroline (PHEN), 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) which 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).

[0357] D.5. Pharmaceutical composition

[0358] The stabilized composition (i.e. the stabilized active ingredients, in particular biopolymers, more particularly proteins selected from oligopeptides, polypeptides,

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[0360] 42 proteins, glycosylated proteins, proteoglycans, antibody molecules or fragments or derivatives thereof, adducts or conjugates of such proteins with a further constituent as further defined herein above) 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.

[0361] 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 copolymer of the invention 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.

[0362] 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.

[0363] Depending on the nature or the mode of administration and dosage form said composition said at least one additional pharmaceutical excipient may be different

[0364] 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.

[0365] Excipients may be selected from the following classes: immunological adjuvants, antiadherents, binders, coatings, colours, disintegrant, flavours, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles

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[0367] 43

[0368] Non limiting examples of excipients comprise diluents, preserving agents, stabilizers, emulsifying agents, like emulsifying polymers, such as polysorbates or poloxamers, antioxidants, as for example chemical compounds, like epigallocatechin-3- O-gallate, lycopene, ellagic acid, coenzyme Q , indole-3-carbinol, genistein, quercetin, ascorbic acid, glutathione, melatonin, catechin, taurine, captopril, gallic acid, N-acetyl cysteine, a-lipoic acid, BHT, tocopherols and tocotrienols, or enzymes like superoxide dismutase and catalase; anti-irritants, chelating agents and stabilizing 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).

[0369] 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 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.

[0370] Furthermore, one may administer said pharmaceutical composition in a targeted drug delivery system, for example, in a liposome coated with endothelial cell-specific antibody.

[0371] 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.

[0372] 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.

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[0374] 44

[0375] 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.

[0376] 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.

[0377] 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.

[0378] 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.

[0379] A therapeutically effective amount can also be an amount, which is prophylactically effective.

[0380] A “patient” as used herein means human or non-human, in particular human, animals.

[0381] A "dosage form" is any unit of administration (“unit dose”) of one or more active agents as described herein.

[0382] 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,

[0383] M / 65044- PCT BASF SE 240647W001

[0384] 45 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.

[0385] “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.

[0386] 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.

[0387] Particular examples of pharmaceutical composition according to the present invention are liquid form preparations such as solutions, suspensions, and emulsions and comprise, beside the 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.

[0388] 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.

[0389] The following example serve for a better understanding of the present invention without limiting its scope.

[0390] Experimental Part

[0391] A. Material and Methods

[0392] A.1 Chemicals and Buffers

[0393] Unless stated otherwise all chemicals as applied were of analytical grade and

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[0395] 46 obtained from commercial sources.

[0396] Bovine immunoglobulin (IgG) captured from pooled bovine plasma using a chromatographic method (MPBio, Cat-No: 08641402)

[0397] A.2 Analytical methods

[0398] 1H-NMR spectra were measured in CDCh with a Bruker AVANCE III 500 MHz spectrometer.

[0399] A.3 General protocol for IgG aggregation assay

[0400] 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, IgG with P188 and IgG with Tocofersolan.

[0401] 1 g 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.

[0402] 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).

[0403] 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.

[0404] The remaining glass vial (TO) is inverted 3 times, diluted 1 :10 in a deep-well plate with 20 mM 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.

[0405] For all samples (TO and T15) the particle count is quantified by micro-flow imaging (MFI5200 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 0.9 mL 20mM histidine buffer pH 6.

[0406] All samples are measured in duplicates. The tests were run at ambient

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[0408] 47 temperature (20 to 25°C).

[0409] 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.

[0410] 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.

[0411] 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).

[0412] A.4 General protocol for water solubility assay

[0413] To 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.

[0414] A.5 General protocol for surface tension (SFT) measurements a) Static surface tension (pendant drop technique)

[0415] Sample preparation method 1

[0416] For the characterization of the static surface activity of surfactants of the invention and comparative compounds, samples were dissolved in deionized water (Milli-Q quality) at a concentration of 1 g / L and subsequently diluted with the same deionized water to 0.1 g / L.

[0417] Sample preparation method 2

[0418] For the characterization of the static surface activity of surfactants of the invention and comparative compounds in the presence or absence of immunoglobulins, samples were dissolved in 20 mM histidine buffer (pH 6). Surfactant was provided at a final concentration of 0.1 g / L (w / v), while immunoglobulin was provided at a final

[0419] M / 65044- PCT BASF SE 240647W001

[0420] 48 concentration of 1 g / L (w / v).

[0421] After stirring for 2 h and complete dissolution of surfactants and / or immunoglobulins, the test solutions were filled into a disposable syringe, which was then mounted on a DSA 100 drop shape tensiometer from Kruss (Hamburg, Germany).

[0422] 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. 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. The density of the solutions required for evaluation was assumed to be that of pure water. Surface tension was monitored over a period of 5 minutes, and the final values obtained from two independent determinations were averaged. All measurements were performed at 23°C. b) Dynamic surface tension (maximum bubble pressure method)

[0423] 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) surfactants of the invention and comparative compounds in histidine buffer (20 mM, pH 6.0) with and without added 1 g / L (w / v) of immunoglobulins or immunoglobulins alone were studied in direct comparison.

[0424] A.6 General protocol for interfacial surface tension (IFT) measurements

[0425] The interfacial tension between silicone oil and 1 g / L (w / v) solutions of surfactants of the invention and comparative compounds in 20 mM histidine buffer (pH 6) was determined by pendant drop tensiometry on a PAT 1 M instrument from Sinterface (Berlin, Germany). For this purpose, a droplet of surfactant solution (typical volume: 25 pL) was generated at the outlet of a vertical syringe into a reservoir filled with silicone oil (grade AK 5 from Wacker Chemie AG (Burghausen, Germany)) at 23 °C. 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 software of the manufacturer. 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. The density of the solutions required for evaluation was assumed

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[0427] 49 to be that of pure water, while the density of the silicone oil was taken to be 0.919 g / cm3. The interfacial tension between the aqueous phase and the oil was monitored over a period of 100 s and the final values obtained in two independent determinations were averaged.

[0428] A.7 General protocol for haemolysis assay

[0429] 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.

[0430] 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 (0.18 % (w / v)) 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 % (v / v) RBC suspension.

[0431] Test procedure: A test solution of the surfactant of the invention and comparative compounds 1 ,33g / 10 ml (i.e. final concentration in test 100mg / ml) was prepared in PBS / glucose and adjusted to pH 7.4. Further test solutions (final concentration of surfactant of the invention and comparative compounds of 10 and 1 mg / ml in RBC suspension) were made by serial 1 :10 dilutions with PBS / glucose. One volume of the RBC suspension was added to three volumes of test solutions, resulting in final test compound concentrations of 0,1 mg / ml. The assay mixtures were incubated at room temperature with shaking by an Eppendorf mixer (Model 5432; at 1000 rpm) for 60 minutes.

[0432] After incubation the samples were centrifuged to sediment remaining intact RBCs and membrane fragments.

[0433] Released free haemoglobin in the supernatant as a degree of hemolysis was determined spectrophotometrically at 540 nm.

[0434] 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.

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[0436] 50

[0437] B. Synthesis examples

[0438] Exemplary a-tocopherol copolymers according to the invention (Test samples No. 1 to 10) were synthetized by applying synthetic protocols similar to those described in synthesis examples 1 , 2 or 3 below.

[0439] Molecular weights given are calculated from the used molar ratio of starting materials.

[0440] Synthesis Example 1 : a-Tocopherol, propoxylated with 5 moles propylene oxide and ethoxylated with 25 moles ethylene oxide (Sample No.: 5)

[0441] In a 2 I autoclave 250.0 g a-tocopherol and 2.0 g potassium tert.-butoxide were placed and the reactor was purged three times with nitrogen. The mixture was heated to 130°C. 169.0 g propylene oxide was added within 2 hours. To complete the reaction, the mixture was allowed to post-react for additional 4 hours at 130°C. 639.2 g ethylene oxide was added within 6 hours, followed by 4 hours post-reaction time to complete conversion. The reaction mixture was cooled to 90°C and stripped with nitrogen.

[0442] Volatile compounds were removed in vacuo at 90°C for 2 hours. 2.4 g Phosphoric acid (75% in water) was added, and 1022.0 g of a light brown paste was obtained.1H- NMR in CDCI3 and hydroxyl number of 32.4 mg KOH / g confirmed the complete conversion to the expected polymer.

[0443] Synthesis Example 2: a-Tocopherol, propoxylated with 4 moles propylene oxide and ethoxylated with 25 moles ethylene oxide (Sample No 4)

[0444] In a 2 I autoclave 250.0 g alpha-tocopherol and 2.3 g potassiumhydroxide (45% aqueous solution) were placed and the reactor was purged three times with nitrogen. The mixture was heated to 125°C and vacuum of 25 mbar was applied. The mixture was dewatered for 2 hours at 125°C and 25 mbar. Vacuum was replaced with nitrogen, and 134.8 g propylene oxide was added within 2 hours. To complete the reaction, the mixture was allowed to post-react for additional 6 hours at 125°C. 639.2 g ethylene oxide was added within 11 hours, followed by 6 hours post-reaction time to complete conversion. The reaction mixture was cooled to 90°C, and was stripped with nitrogen. Volatile compounds were removed in vacuo at 90°C for 2 hours. 2.4 g Phosphoric acid (75% in water) was added, and 988.7 g of a light brown paste was obtained.1H-NMR in CDCI3

[0445] M / 65044- PCT BASF SE 240647W001

[0446] 51 and hydroxyl number of 32.2 mgKOH / g confirmed the complete conversion to the expected polymer.

[0447] Synthesis Example 3: a-Tocopherol, propoxylated with 3 moles propylene oxide and ethoxylated with 25 moles ethylene oxide (Sample NO. 3)

[0448] In a 2 1 autoclave 140.0 g alpha-tocopherol and 1.2 g potassium hydroxide (45% aqueous solution) were placed and the reactor was purged three times with nitrogen. The mixture was heated to 125°C and vacuum of 25 mbar was applied. The mixture was dewatered for 2 hours at 125°C and 25 mbar. Vacuum was replaced with nitrogen, and 56.6 g propylene oxide was added within 2 hours. To complete the reaction, the mixture was allowed to post-react for additional 6 hours at 125°C. 357.9 g ethylene oxide was added within 9 hours, followed by 6 hours post-reaction time to complete conversion. The reaction mixture was cooled to 90°C, and was stripped with nitrogen. Volatile compounds were removed in vacuo at 90°C for 2 hours. 1.0 g Phosphoric acid (75% in water) was added, and 550.0 g of a light brown paste was obtained.1H-NMR in CDCh and hydroxyl number of 33.2 mgKOH / g confirmed the complete conversion to the expected polymer.

[0449] C. Experimental results

[0450] Experimental results are summarized in the following Table 1 .

[0451] M / 65044- PCT BASF SE 240647W001

[0452] 52

[0453] Table 1: Summary of experimental results

[0454] 1)mole% PO = (m moles PO / m moles PO +n moles EO) * 100 mole% BuO = (m moles BuO / m moles BuO + n moles EO) * 100

[0455] M / 65044- PCT

[0456] BASF SE 240647W001

[0457] 53

[0458] C.2 Summary

[0459] The data further underline the surprising advantages of copolymers of the invention (samples 1 to 10) over prior art surfactants as used for biopolymer stabilization (Polysorbates, P188 and Tocofersolan (TPGS)).

[0460] Tocopherol alkoxylates of the invention do not contain ester bonds and consequently show improved stability against lipases (data not shown).

[0461] Tocofersolan is a mixture of mono-TPGS, di-TPGS and PEG (see: dx.doi.org / 10.1021 / ac403195f | Anal. Chem. 2014, 86, 1567-1574), whereas tocopherol alkoxylates show more defined chemical structures.

[0462] Tocopherol alkoxylate polymers of the invention show an improved effect on protein aggregation compared to Tocofersolan.

[0463] Tocopherol alkoxylate polymers of the invention show an interfacial tension against silicone oil of 13 < IFT < 21 ,5mN / m. This underlines the particular utility of the surfactants of the invention for preparing liquid biomolecule preparations to be formulated in pre-filled syringes usually being preconditioned with silicone oil.

[0464] The content of all documents referred to herein above is incorporated by reference.

[0465] M / 65044- PCT

Claims

BASF SE 240647W00154Claims1. A formulation comprising at least one protein, at least one buffer and at least one amphiphilic surfactant, wherein the amphiphilic surfactant is a polyalkoxylated alpha-tocopherol-type compound of the general formula 1in stereoisomerically pure form or as a mixture of at least two stereoisomers, whereinA represents i) a polyalkylene oxide block copolymer moiety of the general formula 2:R1-(OE)n-(OR)m-O-(2) wherein m represents an integer in the range of 1 to 10; n represents an integer in the range of 15 to 40;OE represents an ethylene oxide unit;OR represents the same or different alkylene oxide unit selected from a propylene oxide (-OP-) or a butylene oxide (-OBu-) unit; andR1represents H or C1-C4 alkyl; or ii) a polyalkylene oxide random copolymer moiety wherein said random copolymer moiety is composed of: m randomly distributed alkylene oxide units selected from propylene oxide (-OP-)units wherein m represents an integer in the range of 2 to 5;240647 WO01M / 65044- PCTBASF SE 240647W00155 and n randomly distributed ethylene oxide (-OE-) units wherein n represents an integer in the range of 23 to 27 and the terminal outer alkylene oxide unit or outer ethylene oxide unit is capped with a residue R1selected from H or C1-C4 alkyl.

2. The formulation of claim 1 , wherein(I) the polyalkoxy moiety is a block copolymer moiety is composed of a) a first, inner propylene oxide block covalently bound to the tocopherol moiety and a second, outer ethylene oxide block covalently bound to the terminal end of to the first inner propylene oxide block, and wherein in formula 1 m is an integer of 2 to 9, particularly 2 to 8, more particularly 2 to 7 or most particularly 2 to 6; and n is an integer of 15 to 35, particularly 15 to 30, more particularly 18 to 30 or most particularly 18 to 25. or b) a first, inner butylene oxide block covalently bound to the tocopherol moiety and a second, outer ethylene oxide block covalently bound to the terminal end of to the first inner butylene oxide block, and wherein in formula 1 m is an integer of 1 or 2, and n is an integer of 15 to 40, particularly 20 to 40, or more particularly 25 to 35. or(II) the polyalkoxy moiety a polyalkylene oxide random copolymer moiety wherein said random copolymer moiety is composed of m randomly distributed alkylene oxide units selected from propylene oxide (-OP-) units,M / 65044- PCTBASF SE 240647W00156 wherein m represents an integer in the range of 2 to 5, like 2, 3, 4 or 5; and n randomly distributed ethylene oxide (-OE-) units wherein n represents an integer in the range of 23 to 27; particularly 24 to 26 and most particularly 25.

3. The formulation of claim 1 or 2, wherein said alpha-tocopherol-type compound of formula (1) shows at least one of the following additional features a) a water solubility of at least 10 wt.-% based on the total weight of the aqueous solution of said compound of formula (1), b) less than 10% hemolytic activity caused by a solution of said compound of formula (1)at a concentration of 100 mg / L in PBS / glucose (0.18 % (w / v) glucose), pH 7.4 at 20 °C (RT) and a RBC content of approximately 0.5% (v / v); and c) an interfacial surface tension (I FT) in the range of less than 22,3 mN / m, particularly 10 to 22 mN / m, or more particularly 12 to 21 mN / m; as determined by pendant drop tensiometry between a silicone reservoir and a droplet of a1 g / l (w / v) solution of said compound of formula (1) at 23 °C.

4. The formulation of anyone of the preceding claims which is in dry or liquid form.

5. The formulation of anyone of the preceding claims, wherein the protein is selected from oligopeptides, polypeptides, proteins, glycosylated proteins, proteoglycans, antibody molecules or fragments thereof, adducts or conjugates of such proteins with a further constituent selected from payload molecules, which 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.M / 65044- PCTBASF SE 240647W001576. The formulation of claim 5, wherein said protein is a diagnostically applicable or a therapeutically active protein, in particular said protein is selected from enzymes and immunoglobulin molecules, each optionally glycosylated; or said protein is selected from adducts or conjugates of an optionally glycosylated immunoglobulin molecule and a payload molecule; or said protein is an optionally glycosylated antibody payload conjugate (APC), particularly an optionally glycosylated antibody drug conjugate (ADC).

7. The formulation of anyone of the preceding claims, wherein said protein is an antibody molecule or an antibody payload conjugate (APC), particularly an antibody drug conjugate (ADC), each optionally glycosylated.

8. The formulation of anyone of the preceding claims in liquid form, wherein said surfactant of the general formula 1 is contained in a proportion of 0,001 to 10%, based on the total weight of the liquid formulation; and / or said protein is contained in a proportion of 0,01 to 30%, based on the total weight of the liquid formulation; and / or wherein said liquid formulation is in buffered form, in particular having a pH in the range of pH 5 to 9, more particularly pH 6 to 8.

9. The formulation comprising at least one protein component as defined in claim 5 and at least surfactant as defined in anyone of claim 1 to 3, which has a liquid content of 0% to 5% wt.-% based on the total weight of said formulation; and is optionally further characterized as follows: wherein said at least one surfactant (Y) and said at least protein (Z) are contained in a weight ratio (Y) : (Z) in the range of 1 : 20.000 to 10:1 , or 1 : 5.000 to 2:1 , 1 : 100 to 1 ,2 : 1 , particularly 1 : 10 to 1 , 1 : 1 ; and / or wherein said at least one surfactant (Y) and said at least one protein (Z) together (i.e. (Y) plus (Z)) 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 20 to 25 wt.-% based on the total weight of said essentially dry formulation and / or further comprises at least one further excipient in a proportion of 0,1 to 99 wt.-%, 40 to 95 wt.-% and 50 to 90 wt.-% based on the total dry weight of saidM / 65044- PCTBASF SE 240647W00158 essentially dry formulation.

10. Use of a surfactant of anyone the claims 1 to 3, for stabilizing an aqueous composition, in particular aqueous solution, of a least one protein as defined in above claims 5 to 7, and in particular for improving aggregation stability of an aqueous composition, in particular aqueous solution, of said least one protein.

11. The formulation of anyone of the claims 1 to 9 for use in medicine, in particular for diagnostic and / or therapeutic applications.

12. The formulation of anyone of the claims 1 to 9, which is a pharmaceutical composition, optionally further supplemented by at least one pharmaceutically acceptable excipient.

13. A method of preparing a protein formulation of anyone of the claims 1 to 9 or 12 which method comprises a) preparing in any order an aqueous, optionally buffered solution of the protein; and an aqueous, optionally buffered solution of the surfactant of anyone of the claims 1 to 3, wherein at least one of said aqueous solutions is applied as buffered solution; and b) preparing a mixture of both aqueous solutions as obtained in step a).

14. A method of preparing a protein formulation of claim 9, which method comprises a) preparing in any order an aqueous, optionally buffered solution of said protein; and an aqueous, optionally buffered solution of said surfactant as defined in anyone of the claims 1 to 3, wherein at least one of said aqueous solutions is applied as buffered solution; 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).

15. A method for preventing or suppressing protein aggregation of a liquid proteincontaining formulation in vitro by using an amphiphilic surfactant as a stabilizer inM / 65044- PCTBASF SE 240647W001 said formulation, wherein said amphiphilic surfactant is defined according to any one of claims 1 to 3, particularly wherein said amphiphilic surfactant is applied in a proportion in the range of 0,0001 to 10 wt.-%, particularly 0,005 to 5 wt.-%, most particularly 0,001 to 0,2 wt.-%, based on the total weight of said liquid formulation; wherein in particular the protein is defined according to any one of claims 5 to 7.

16. A prefilled syringe, comprising a reservoir prefilled with an amount of a formulation as defined in anyone of the claims 1 to 9.

17. A method of preparing an amphiphilic surfactant of general formula 1 ,in stereoisomerically pure form or as a mixture of at least two stereoisomers, whereinA is as defined in claim 1 which method comprises admixing alpha-tocopherol of formula (4)in stereoisomerically pure form or as a mixture of at least two stereoisomers, with a base catalyst under inert gas in a reactor vessel, optionally remove water being present, and then a) either adding propylene oxide or butylene oxide or a mixture ofM / 65044- PCTBASF SE 240647W00160 propylene oxide and butylene oxide in a suitable amount in order to polymerize a first propylene oxide block or first butylene oxide block or first mixed propylene oxide and butylene oxide block to the terminal hydroxy group of said tocopherol, followed by the addition of ethylene oxide in a suitable amount in order to polymerize a second ethylene oxide block to the terminal end of the first propylene oxide block or first butylene oxide block or first mixed propylene oxide and butylene oxide block in order to obtain a polyalkylene oxide moiety A with a polymer block structure; b) or adding a mixture of ethylene oxide and propylene oxide in a suitable amount and in a suitable ratio of the proportions of ethylene oxide and propylene oxide in order to generate a statistically polymerized polyalkylene oxide moiety A.M / 65044- PCT

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