Stabilized solid formulation of antibodies

Abstract

The invention provides salt coated granules comprising single-domain antibodies having improved stability.

Description

[0001] STABILIZED SOLID FORMULATION OF ANTIBODIES

[0002] Reference to a sequence listing

[0003] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to salt coated granules comprising single-domain antibodies.

[0006] BACKGROUND

[0007] Synthetic antibodies are now widely used in pharmaceutical applications as they can mimic the immune system (immunotherapy) in combatting challenging diseases, like viral infections and cancer. They are capable of binding very selectively to a biological target causing health problems.

[0008] The specificity of antibodies is largely determined by the “variable domain”, which can be tailored to bind a desired target. While this has been explored in the therapeutic and diagnostic industry for many years, it is now also gaining interest in other industries. The ability to selectively bind a biological target can for example be utilized to reduce pathogens in animal feed, as described in PCT application WO 2020 / 144164.

[0009] Antibodies are sensitive biological molecules that need particular attention to preserve the activity during heat and humidity treatments. In animal feed manufacturing it is highly desirous to be able to add all ingredients before feed pelleting. The present invention describes how to protect antibodies and improve stability during such challenging treatments.

[0010] SUMMARY OF THE INVENTION

[0011] The present invention provides, in a first aspect, a granule, comprising

[0012] (a) a core comprising a single-domain antibody, and

[0013] (b) a salt coating surrounding the core, which makes up 5-75% w / w of the granule and comprises at least 60% w / w of salt.

[0014] Other aspects and embodiments of the invention are apparent from the description and examples.

[0015] Unless otherwise indicated, or if it is apparent from the context that something else is meant, all percentages are percentage by weight (% w / w).

[0016] As used herein, the term "consists essentially of" (and grammatical variants thereof), as applied to the compositions and methods of the invention, means that the compositions / methods may contain additional components so long as the additional components do not materially alter the composition / method. As used herein, the term essentially free of (and grammatical variants thereof), as applied to the compositions and methods of the invention, means that the compositions / methods may contain minor amounts of the specified component so long as the amount of the component does not materially alter, or provide any material effect on, the composition / method. In an embodiment, "essentially free of" means 0% w / w.

[0017] DETAILED DESCRIPTION

[0018] We have found that single-domain antibodies can be efficiently protected against the challenging temperature / humidity conditions used during preparation of animal feed pellets. This is achieved by applying a salt as a coating surrounding a core particle that comprises the single-domain antibody. We have found that commonly used coating materials like fats / oils do not provide the same excellent stability provided by a separate salt coating.

[0019] Antibodies are characterized by their ability to bind to an antigen. The antigen binding site must be exposed to the surroundings to bind a target antigen. This makes single-domain antibodies vulnerable to inactivation by only minor modifications of the exposed antigen binding site. Contrary to enzymes and other reactive proteins, the reactive part of a single-domain antibody cannot be hidden or in other ways protected by other protein structures of the molecule. This makes maintenance of reactivity even more challenging in a harsh environment.

[0020] The antibody granule of the invention is particularly well suited for steam pelleting and as part of a steam treated pelletized feed composition.

[0021] Antibody granules

[0022] A granule of the invention is a small particle comprising a core which comprises a singledomain antibody, and a salt coating surrounding the core which makes up 5-50% w / w of the total granule and comprises at least 60% w / w of salt.

[0023] The granule may comprise the single-domain antibody in an amount of 0.1-75% w / w, such as 0.1-60% w / w, 0.1-50% w / w, 0.1-40% w / w, 0.5-30% w / w, or 0.5-20% w / w.

[0024] Core

[0025] The core comprises a single-domain antibody, as described below, and may include additional materials, such as fillers, fibre materials (cellulose or synthetic fibres), stabilizing agents, solubilizing agents, suspension agents, viscosity regulating agents, light spheres, plasticizers, salts, lubricants, fragrances, and binders, such as synthetic polymer, wax, fat, or carbohydrate. The ingredients of the core may be mixed as a homogenous blend, e.g., by high shear granulation.

[0026] The core may comprise the single-domain antibody in an amount of 0.1-75% w / w, such as 0.1-60% w / w, 0.1-50% w / w, 0.1-40% w / w, 0.5-30% w / w, or 0.5-20% w / w. The core may consist of an inert particle, or an inert particle with a single-domain antibody applied (coated) onto the surface of the inert particle, e.g., via seeded mixer granulation or layered granulation in a fluid bed. Such inert particles can be an organic particulate compound e.g. a natural compound such as agglomerated carbohydrates, e.g. sugars, starch, dextrins, flour (e.g. vegetable flour), or nonpareils. Nonpareils are spherical particles made of a seed crystal that has been built onto and rounded into a spherical shape. Nonpareils are typically made from a combination of a sugar such as sucrose, and a powder such as cornstarch. The inert particle can also be a sodium chloride or sodium sulfate crystal (or agglomerated crystals), also referred to as a seed, or other inorganic salt crystal; or a sucrose crystal.

[0027] The core can also be prepared by granulating a blend of the ingredients by a method comprising granulation techniques such as crystallization, precipitation, pan-coating, fluid bed coating, fluid bed agglomeration, rotary atomization, extrusion, prilling, spheronization, size reduction methods, drum granulation, and / or high shear granulation. Wet granulation processes are preferred.

[0028] Methods for preparing the core can be found in Handbook of Powder Technology; Particle size enlargement by C. E. Capes; Volume 1; 1980; Elsevier.

[0029] Salt coating

[0030] The core is surrounded by a salt coating (an outer layer). The salt coating comprises at least 60% w / w of a salt, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% w / w of a salt.

[0031] The salt coating may be applied in an amount of at least 5% w / w of the granule, e.g., at least 10%, 12% or 15% w / w. The amount may be at most 75%, 60%, 50%, 40% or 30% w / w.

[0032] The coating is preferably at least 0.1 pm thick, particularly at least 0.5 pm, at least 1 pm or at least 5 pm. In a particular embodiment the thickness of the coating is below 100 pm. In a more particular embodiment, the thickness of the coating is below 60 pm. In an even more particular embodiment, the total thickness of the coating is below 40 pm.

[0033] The coating should surround / encapsulate the core by forming a substantially continuous layer. A substantially continuous layer is to be understood as a coating having few or no holes (substantially impermeable or non-porous), so that the core unit is surrounded / encapsulated with few or no uncoated areas. The layer or coating should in particular be homogeneous in thickness.

[0034] The salt may be added from a salt solution where the salt is completely dissolved or from a salt suspension wherein the fine particles is less than 50 pm, such as less than 10 pm or less than 5 pm.

[0035] The salt coating may comprise a single salt or a mixture of two or more salts. The salt may be water soluble, in particular having a solubility at least 0.1 grams in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, e.g., at least 1 g per 100 g water, e.g., at least 5 g per 100 g water.

[0036] The salt may be an inorganic salt, e.g., salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids (less than 10 carbon atoms, e.g., 6 or less carbon atoms) such as citrate, malonate or acetate. Examples of cations in these salts are alkali or earth alkali metal ions, the ammonium ion or metal ions of the first transition series, such as sodium, potassium, magnesium, calcium, zinc or aluminium. Examples of anions include chloride, bromide, iodide, sulfate, sulfite, bisulfite, thiosulfate, phosphate, monobasic phosphate, dibasic phosphate, hypophosphite, dihydrogen pyrophosphate, tetraborate, borate, carbonate, bicarbonate, metasilicate, citrate, malate, maleate, malonate, succinate, lactate, formate, acetate, butyrate, propionate, benzoate, tartrate, ascorbate or gluconate. In particular alkali- or earth alkali metal salts of sulfate, sulfite, phosphate, phosphonate, nitrate, chloride or carbonate or salts of simple organic acids such as citrate, malonate or acetate may be used.

[0037] The salt in the coating may have a constant relative humidity at 20°C (also referred to as ‘humidity fixed point’) above 60%, particularly above 70%, above 80% or above 85%, or it may be another hydrate form of such a salt (e.g., anhydrate). The salt coating may be as described in WO 00 / 01793 or WO 2006 / 034710.

[0038] Specific examples of suitable salts are NaCI (CH2o°c=76%), Na2CO3 (CH2o°c=92%), NaNO3(CH2O"C=73%), Na2HPO4(CH2o"c=95%), Na3PO4(CH25°c=92%), NH4CI (CH2o"c = 79.5%), (NH4)2HPO4(CH2O"C = 93.0%), NH4H2PO4(CH2o"c = 93.1 %), (NH4)2SO4(CH2o°c=81 .1%), KOI (CH2O"C=85%), K2HPO4(CH2O"C=92%), KH2PO4(CH2O°C=96.5%), KNO3(CH2O"C=93.5%), Na2SO4(CH2O"C=93%), K2S04(CH2O"C=98%), KHSO4(CH2O"C=86%), MgSO4(CH2o"c=9O%), ZnSO4(CH2O°C=9O%) and sodium citrate (CH25°c=86%). Other examples include NaH3PO4, (NH4)H3PO4, CUSO4, Mg(NO3)2 and magnesium acetate.

[0039] The salt may be in anhydrous form, or it may be a hydrated salt, i.e. a crystalline salt hydrate with bound water(s) of crystallization, such as described in WO 99 / 32595. Specific examples include anhydrous sodium sulfate (Na3SO4), anhydrous magnesium sulfate (MgSO4), magnesium sulfate heptahydrate (MgSO47H3O), zinc sulfate heptahydrate (ZnSO47H3O), sodium phosphate dibasic heptahydrate (Na3HPO47H3O), magnesium nitrate hexahydrate (Mg(NO3)2(6H2O)), sodium citrate dihydrate and magnesium acetate tetrahydrate.

[0040] Preferably the salt is applied as a solution of the salt, e.g., using a fluid bed.

[0041] The salt coating may be supplemented with further coating(s) (surrounding the salt coating) that comprises, for example, polyethylene glycol (PEG), methyl hydroxy-propyl cellulose (MHPC), or polyvinyl alcohol (PVA). Such further coating(s) may be added to reduce dusting. Single-domain antibodies

[0042] The term “single-domain antibodies” or "immunoglobulin single variable domain", interchangeably used with "single variable domain", defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins or their fragments, wherein two immunoglobulin domains, in particular two variable domains interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation.

[0043] In contrast, the binding site of an immunoglobulin single variable domain is formed by a single VH or VL domain. Hence, the antigen binding site of an immunoglobulin single variable domain is formed by no more than three CDRs.

[0044] The terms "immunoglobulin single variable domains", or "single variable domain" hence do not comprise conventional immunoglobulins or their fragments which require interaction of at least two variable domains for the formation of an antigen binding site. This is also the case for embodiments of the invention which "comprise" or "contain" an immunoglobulin single variable domain. In the context of the present invention, such embodiments exclude conventional immunoglobulins or their fragments. Thus, a construct or peptide that "comprises" or "contains" an immunoglobulin single variable domain may relate to e.g. constructs comprising more than one immunoglobulin single variable domain. Alternatively, there may be further constituents other than the immunoglobulin single variable domains, e.g. auxiliary agents of different kinds, protein tags, colorants, dyes, etc. However, the terms "immunoglobulin single variable domains" or "single variable domain" do comprise fragments of conventional immunoglobulins wherein the antigen binding site is formed by a single variable domain.

[0045] The amino acid sequence and structure of an immunoglobulin sequence such as an immunoglobulin single variable domain, in particular a Nanobody, can be considered - without however being limited thereto - to be comprised of four framework regions or "FR's", which are referred to in the art and herein as "Framework region 1" or "FR1"; as "Framework region 2" or "FR2"; as "Framework region 3" or "FR3"; and as "Framework region 4" or "FR4", respectively; which framework regions are interrupted by three complementary determining regions or "CD's", which are referred to in the art as "Complementarity Determining Region I" or "CDR1"; as "Complementarity Determining Region 2" or "CDR2"; and as "Complementarity Determining Region 3" or "CDR3", respectively.

[0046] Thus, generally, single variable domains will be amino acid sequences that consist of, or essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively). "Essentially consist" in this context means that additional elements such as e.g. tags used for purification or labelling may be present, but such additional elements are small as compared to the immunoglobulin single variable domain per se, and do not interfere with the antigen binding activity of the immunoglobulin single variable domain. The total number of amino acid residues in a VHH immunoglobulin single variable domain, a humanized VHH or camelized VH, or a Nanobody, respectively, can be in the region of 110-120, is preferably 112-115, and is most preferably 113. It should however be noted that parts, fragments, analogs or derivatives (as further described herein) are not particularly limited as to their length and / or size, as long as such parts, fragments, analogs or derivatives meet the further requirements outlined herein, in particular show antigen binding activity, and are also preferably suitable for the purposes described herein.

[0047] "Suitable fragments" of immunoglobulin single variable domains relate to polypeptides which contain fewer amino acids than a native immunoglobulin single variable domain, but still show antigen binding activity (which will then usually contain at least some of the amino acid residues that form at least one of the CDR's, as further described herein). Such single variable domains and fragments most preferably comprise an immunoglobulin fold or are capable for forming, under suitable conditions, an immunoglobulin fold. More specifically, immunoglobulin single variable domains and their fragments are such that they are capable of binding to the target antigen. As such, the single variable domain may for example comprise a light chain variable domain sequence (e.g. a VL-sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g. a VH-sequence or VHH-sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e. a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit, as is for example the case for the variable domains that are present in for example conventional antibodies and scFv fragments that need to interact with another variable domain - e.g. through a VH / VL interaction - to form a functional antigen binding domain).

[0048] For example, the immunoglobulin single variable domains may be a domain antibody or may be a single domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb", “sdAb” (or an amino acid sequence that is suitable for use as a dAb / sdAb) or a Nanobody® {as defined herein, and including but not limited to a VHH sequence); other single variable domains, or any suitable fragment of any one thereof. For a general description of (single) domain antibodies, reference is also made to the prior art cited herein, as well as to EP 0 368684. For the term "dAb's", reference is for example made to Ward et al. 1989 (Nature 341 (6242): 544-6), to Holt et al. 2003 (Trends Biotechnol. 21(11): 484-490), as well as to for example WO 04 / 068820, WO 06 / 030220, WO 06 / 003388 and other published patent applications of Domantis Ltd. It should also be noted that, although less preferred in the context of the present invention because they are not of mammalian origin, single variable domains can be derived from certain species of shark (for example, the so-called "igNAR domains", see for example WO 05 / 18629}. In particular, the amino acid sequence of the invention may be a Nanobody or a suitable fragment thereof. For a further description of VHH s and Nanobodies, reference is made to the review article by Muyldermans 2001 (in Reviews in Molecular Biotechnology 74: 277-302); as well as to the following patent applications, which are mentioned as general background art: WO 94 / 04678, WO 95 / 04079 and WO 96 / 34103 of the Vrije Universiteit Brussel; WO 94 / 25591, WO 99 / 37681, WO 00 / 40968, WO 00 / 43507, WO 00 / 65057, WO 01 / 40310, WO 01 / 44301, EP 1134231 and WO 02 / 48193 of Unilever; WO 97 / 49805, WO 01 / 21817, WO 03 / 035694, WO 03 / 054016 and WO 03 / 055527 of the Vlaams Instituut voor Biotechnoiogie (VIB); WO 03 / 050531 of Algonomics N.V. and Ablynx N.V.; WO 01 / 90190 by the National Research Council of Canada; WO 03 / 025020 by the Institute of Antibodies; as well as WO 04 / 041867, WO 04 / 041862, WO 04 / 041865, WO 04 / 041863, WO 04 / 062551 , WO 05 / 044858, WO 06 / 40153, WO 06 / 079372, WO 06 / 122786, WO 06 / 122787 and WO 06 / 122825, by Ablynx N.V. and the further published patent applications by Ablynx N.V, Reference is also made to the further prior art mentioned in these applications, and in particular to the list of references mentioned on pages 41-3 of the international application WO 06 / 040153, which list and references are incorporated herein by reference. As described in these references, Nanobodies (in particular VHH sequences and partially humanized Nanobodies) can in particular be characterized by the presence of one or more "Hallmark residues" in one or more of the framework sequences. A further description of the Nanobodies, including humanization and / or camelization of Nanobodies, as well as other modifications, parts or fragments, derivatives or "Nanobody fusions", multivalent constructs (including some nonlimiting examples of linker sequences) and different modifications to increase the half-life of the Nanobodies and their preparations can be found e.g. in WO 07 / 104529.

[0049] Thus, in the meaning of the present invention, the term "immunoglobulin single variable domain", or "single variable domain" comprises polypeptides which are derived from a nonhuman source, preferably a camelid, preferably a camelid heavy chain antibody. They may be humanized, as previously described. Moreover, the term comprises polypeptides derived from non-camelid sources, e.g. mouse or human, which have been "camelized", as previously described.

[0050] Thus, in preferred embodiments of the methods according to the invention the immunoglobulin single variable domain comprises one or more selected from a VHH immunoglobulin single variable domain, a humanized VHH immunoglobulin single variable domain or a camelized VH immunoglobulin single variable domain or any suitable fragment or combination thereof.

[0051] Unless indicated otherwise, the term "immunoglobulin" - whether used herein to refer to a heavy chain antibody or to a conventional 4-chain antibody - is used as a general term to include both the full-size antibody, the individual chains thereof, as well as all parts, domains or fragments thereof (including but not limited to antigen-binding domains or fragments such as VHH domains or VH / VL domains, respectively). The terms antigen-binding molecules or antigen-binding protein are used interchangeably with immunoglobulin sequence and include Nanobodies.

[0052] The immunoglobulin single variable domains provided by the invention are preferably in isolated form or essentially isolated form, or form part of a protein or polypeptide of the invention, which may comprise or essentially consist of one or more immunoglobulin single variable domains and which may optionally further comprise one or more further amino acid sequences (ail optionally linked via one or more suitable Sinkers). For example, and without limitation, the one or more immunoglobulin single variable domains may be used as a binding unit in such a protein or polypeptide, which may optionally contain one or more further amino acid sequences that can serve as a binding unit {e.g. against, one or more other antigens and / or targets}, so as to provide a monovalent, multivalent or multispecific polypeptide of the invention, respectively, ail as described herein. Such a protein or polypeptide may also be in isolated or essentially isolated form. Thus, according to the invention, immunoglobulin single variable domains comprise constructs comprising two or more antigen binding units in the form of single variable domains, as outlined above. For example, two (or more) immunoglobulin single variable domains with the same or different antigen specificity can be linked to form e.g. a bivalent, trivalent or multivalent construct. By combining immunoglobulin single variable domains of two or more specificities, bispecific, trispecific etc. constructs can be formed. For example, a polypeptide according to the invention may comprise two immunoglobulin single variable domains directed against target A, and one immunoglobulin single variable domain against target B. Such constructs and modifications thereof, which the skilled person can readily envisage, are all encompassed by the present invention.

[0053] Generally, polypeptides that comprise or essentially consist of a single immunoglobulin single variable domain (such as a single Nanobody) will be referred to herein as "monovalent" polypeptides or as "monovalent constructs". Polypeptides that comprise or essentially consist of two or more immunoglobulin single variable domain (such as at least two Nanobodies) will be referred to herein as "multivalent" proteins or polypeptides or as "multivalent constructs". Some non-limiting examples of such multivalent constructs will become clear from the further description herein.

[0054] According to one specific, but non-limiting aspect, a polypeptide of the invention is a bivalent construct and comprises or essentially consists of two immunoglobulin single variable domains, such as two Nanobodies. According to another specific, but non-limiting aspect, a polypeptide of the invention is a trivalent construct and comprises or essentially consists of three immunoglobulin single variable domains, such as three Nanobodies.

[0055] In the above constructs, the one or more immunoglobulin single variable domains and / or Nanobodies may be directly linked to each other and / or suitably linked to each other via one or more linker sequences. The invention includes immunoglobulin sequences of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The invention also includes fully human, humanized or chimeric immunoglobulin sequences. For example, the invention comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized dAb as described by Ward et al (see for example WO 94 / 04678 and Davies and Riechmann (1994; Febs Letters 339: 285-290} and (1996; Prot. Engineering 9: 531-537)). Moreover, the invention comprises fused immunoglobulin sequences, e.g. forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Con rath et al. 2001 (J. Biol, Chem. 276: 7346-7350), as well as to for example WO 96 / 34103 and WO 99 / 23221), and immunoglobulin sequences comprising tags or other functional moieties, e.g. toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present invention.

[0056] In an embodiment, the single-domain antibody has an amino acid sequence identity to SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3, of at least 60%, e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%. In one aspect, the single-domain antibody amino acid sequence differs by up to 10 amino acids, e.g., 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, from the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

[0057] Amino acid alterations of single-domain antibodies, as described above, may be of a minor nature, that is conservative amino acid substitutions or insertions that do not significantly affect the folding and / or activity of the protein; small deletions, typically of 1-30 amino acids; small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue; a small linker peptide of up to 20-25 residues; or a small extension that facilitates purification by changing net charge or another function, such as a poly-histidine tract, an antigenic epitope or a binding module.

[0058] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are introduced at every residue in the molecule, and the resultant molecules are tested for enzyme activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271: 4699-4708. The biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899-904; Wlodaver et a / ., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved regions with a related polypeptide or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three- dimensional structures, functions, and significant sequence similarity.

[0059] Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, CRISPR gene editing, phage display (e.g., Lowman et al., 1991, Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et a / ., 1988, DNA 7: 127).

[0060] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle program to report the longest identity, the -nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0061] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0062] Animal feed

[0063] The invention is also directed to methods for the preparation of animal feed and feed additives comprising the granules of the invention.

[0064] The invention is further directed to a method for manufacturing a feed composition comprising mixing feed components with the granules of the invention, steam treating and pelleting said composition.

[0065] The granules of the invention may be used in feed for (i) non-ruminant animals; preferably (ii) mono-gastric animals; more preferably (iii) pigs, poultry, fish, and crustaceans; or, most preferably, (iv) pigs and poultry. The granules can be fed to the animal before, after, or simultaneously with the diet. The latter is preferred. The granules can be added to a pre-mix or to an animal feed.

[0066] The term feed, feed composition, or diet means any compound, preparation, mixture, or composition suitable for, or intended for intake by an animal. Ingredients used in animal feed compositions are well-known to the skilled artisan.

[0067] Further embodiments of the invention include: Embodiment 1. A granule, comprising

[0068] (a) a core comprising a single-domain antibody, and

[0069] (b) a salt coating surrounding the core, which makes up 5-75% w / w of the granule and comprises at least 60% w / w of salt.

[0070] Embodiment 2. The granule of the preceding embodiment, wherein the core comprises the single-domain antibody in an amount of 0.1-75% w / w.

[0071] Embodiment 3. The granule of the preceding embodiment, wherein the core comprises the single-domain antibody in an amount of 0.1-60% w / w.

[0072] Embodiment 4. The granule of the preceding embodiment, wherein the core comprises the single-domain antibody in an amount of 0.1-50% w / w.

[0073] Embodiment 5. The granule of the preceding embodiment, wherein the core comprises the single-domain antibody in an amount of 0.1-40% w / w.

[0074] Embodiment 6. The granule of the preceding embodiment, wherein the core comprises the single-domain antibody in an amount of 0.5-30% w / w.

[0075] Embodiment 7. The granule of the preceding embodiment, wherein the core comprises the single-domain antibody in an amount of 0.5-20% w / w.

[0076] Embodiment 8. The granule of the preceding embodiment, wherein the ingredients of the core are mixed as a homogenous blend.

[0077] Embodiment 9. The granule of any of the preceding embodiments, which further comprises an outermost polymer coating surrounding the salt coating.

[0078] Embodiment 10. The granule of any of the preceding embodiments, which further comprises an outermost polymer coating surrounding the salt coating, comprising polyethylene glycol, methyl hydroxy-propyl cellulose, or polyvinyl alcohol.

[0079] Embodiment 11. The granule of any of the preceding embodiments, wherein the salt coating comprises at least 70% w / w of salt.

[0080] Embodiment 12. The granule of any of the preceding embodiments, wherein the salt coating comprises at least 80% w / w of salt.

[0081] Embodiment 13. The granule of any of the preceding embodiments, wherein the salt coating comprises at least 90% w / w of salt.

[0082] Embodiment 14. The granule of any of the preceding embodiments, wherein the salt coating comprises at least 95% w / w of salt.

[0083] Embodiment 15. The granule of any of the preceding embodiments, wherein the salt of the salt coating has a constant relative humidity at 20°C of at least 60%.

[0084] Embodiment 16. The granule of any of the preceding embodiments, wherein the salt of the salt coating has a constant relative humidity at 20°C of at least 70%.

[0085] Embodiment 17. The granule of any of the preceding embodiments, wherein the salt of the salt coating has a constant relative humidity at 20°C of at least 80%. Embodiment 18. The granule of any of the preceding embodiments, wherein the salt of the salt coating has a constant relative humidity at 20°C of at least 85%.

[0086] Embodiment 19. The granule of any of the preceding embodiments, wherein the salt coating comprises sodium sulfate.

[0087] Embodiment 20. The granule of any of the preceding embodiments, wherein the salt coating makes up 5-60% w / w of the granule.

[0088] Embodiment 21 . The granule of any of the preceding embodiments, wherein the salt coating makes up 5-50% w / w of the granule.

[0089] Embodiment 22. The granule of any of the preceding embodiments, wherein the salt coating makes up 5-40% w / w of the granule.

[0090] Embodiment 23. The granule of any of the preceding embodiments, wherein the salt coating makes up 5-30% w / w of the granule.

[0091] Embodiment 24. The granule of any of the preceding embodiments, wherein the thickness of the salt coating is 1-100 pm.

[0092] Embodiment 25. The granule of any of the preceding embodiments, wherein the thickness of the salt coating is 1-60 pm.

[0093] Embodiment 26. The granule of any of the preceding embodiments, wherein the thickness of the salt coating is 5-60 pm.

[0094] Embodiment 27. The granule of any of the preceding embodiments, wherein the volume based average particle size of the granule is 20-2000 pm.

[0095] Embodiment 28. The granule of any of the preceding embodiments, wherein the volume based average particle size of the granule is 50-1500 pm.

[0096] Embodiment 29. The granule of any of the preceding embodiments, wherein the volume based average particle size of the granule is 100-1500 pm.

[0097] Embodiment 30. The granule of any of the preceding embodiments, wherein the volume based average particle size of the granule is 250-1200 pm.

[0098] Embodiment 31 . The granule of any of the preceding embodiments, which is water soluble or water dispersible.

[0099] Embodiment 32. The granule of any of the preceding embodiments, wherein the singledomain antibody has at least 60% amino acid sequence identity to any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3.

[0100] Embodiment 33. The granule of any of the preceding embodiments, wherein the singledomain antibody has at least 70% amino acid sequence identity to any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3.

[0101] Embodiment 34. The granule of any of the preceding embodiments, wherein the singledomain antibody has at least 80% amino acid sequence identity to any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3. Embodiment 35. The granule of any of the preceding embodiments, wherein the singledomain antibody has at least 90% amino acid sequence identity to any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3.

[0102] Embodiment 36. The granule of any of the preceding embodiments, wherein the singledomain antibody has at least 95% amino acid sequence identity to any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3.

[0103] Embodiment 37. The granule of any of the preceding embodiments, wherein the singledomain antibody has up to 30 alterations (substitutions, deletions, and / or insertions), preferably up to 25, up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 alteration(s), as compared to the amino acid sequence of any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3.

[0104] Embodiment 38. The granule of any of the preceding embodiments, wherein the singledomain antibody has up to 30 substitutions, preferably up to 25, up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 alteration(s), as compared to the amino acid sequence of any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3

[0105] Embodiment 39. A method for preparing an animal feed, comprising mixing the granule of any of the preceding embodiments with other animal feed components, steam treating, and pelleting the mixture.

[0106] Embodiment 40. An animal feed composition comprising the granule of any of the preceding embodiments.

[0107] Embodiment 41. Use of the granule according to any of the preceding embodiments as an additive or ingredient in animal feed.

[0108] EXAMPLES

[0109] Chemicals were commercial products of at least reagent grade.

[0110] The antigens targeted by the single domain antibodies (binding proteins) used in the examples are subunits of E. coli toxins, as shown in Table 1 (see also WO 2020 / 144164).

[0111] The amino acid sequences of the single domain antibodies used in the examples are shown in the sequence listing as SEQ ID NO: 1-3.

[0112] Table 1. Antibodies and their corresponding antigens. K88 fimbrial protein AB Fimbriae / pili 31.5 kDa

[0113] Analytical Assay

[0114] Residual activity of the samples after exposure was determined by analysis on LIPLC using a protein A column, phosphate buffer pH 6.8 as mobile phase, phosphate buffer pH 2.5 as eluent, and UV detection at 280 nm.

[0115] The binding proteins have affinity towards Protein A and are captured on the protein A resin while impurities are removed with mobile phase in the flow through. The binding of the binding proteins to the protein A column is impaired at low pH thus the binding proteins are eluted when the pH is lowered from 6.8 to 2.5.

[0116] EXAMPLE 1

[0117] Stability of sdAb granules

[0118] 1.1 Uncoated granules of single domain antibodies (sdAb)

[0119] Uncoated granules (cores) containing sdAb A, sdAb B, or sdAb C were produced by high shear granulation in a Lbdiger Mixer FM50 using water as granulation liguid. The ingredients used to prepare the wet granules (before drying) are shown in Table 2.

[0120] Table 2. Composition of wet granules in % w / w.

[0121] About 40% w / w of the spray dried sdAb powder is active antibody protein. The moist mixture was exposed to compaction and granulation as described in Example 1 of US 4,106,991. Subseguently, the granules were dried in a fluid bed and sieved to a particle size in the range of 250 to 850 microns.

[0122] 1.2 Salt coated granules

[0123] The uncoated granules produced in 1.1 above were salt coated by spraying a solution of

[0124] 1.2 kg sodium sulphate in 3 kg water onto 4 kg granules in a fluidized bed spray coater (Glatt Procell Labsystem).

[0125] The stability of the salt coated granules was measured by taking 3 g of each sdAb sample and exposing them to 85°C or 90°C at 85% relative humidity (see Table 3). These conditions were chosen to mimic a steam and extruder pelleting process, as used in animal feed production.

[0126] The stability results are shown as percent residual active sdAb compared to an unexposed reference of the salt coated granules.

[0127] Table 3. Stability of sdAb using a salt coating

[0128] 1.3 Oil coated granules

[0129] The uncoated granules produced in 1.1 above were oil coated as described in WO 97 / 39116, Example 17 (step 2) with 10% fully hydrogenated palm oil and 30% kaolin.

[0130] The stability of the oil coated granules was measured at the same conditions described above in 1.2, and the percentage of residual active sdAb, as compared to an unexposed reference of the oil coated granules, is shown in Table 4. Table 4. Stability of sdAb using an oil coating

[0131] Examples 1.2 and 1.3 show that the salt coatings provide a surprisingly high stability of the antibodies, as compared to a commonly used oil coating. Antibodies A, B, and C are directed to different antigens, so the improved stability is not limited to a specific antibody.

Claims

CLAIMS1. A granule, comprising(a) a core comprising a single-domain antibody, and(b) a salt coating surrounding the core, which makes up 5-75% w / w of the granule and comprises at least 60% w / w of salt.

2. The granule of any of the preceding claims, wherein the core comprises the single-domain antibody in an amount of 0.1-75% w / w, preferably in an amount of 0.1-60% w / w, 0.1-50% w / w, 0.1-40% w / w, 0.5-30% w / w, or 0.5-20% w / w.

3. The granule of any of the preceding claims, wherein the ingredients of the core are mixed as a homogenous blend.

4. The granule of any of the preceding claims, wherein the salt coating makes up 5-60% w / w of the granule, preferably 5-50% w / w of the granule.

5. The granule of any of the preceding claims, wherein the salt coating comprises at least 80% w / w of salt, preferably at least 90% w / w of salt.

6. The granule of any of the preceding claims, wherein the salt has a constant relative humidity at 20°C of at least 60%, preferably at least 80%.

7. The granule of any of the preceding claims, wherein the salt coating comprises sodium sulfate.

8. The granule of any of the preceding claims, wherein the salt has a solubility at least 0.1 grams in 100 g of water at 20°C, preferably at least 0.5 g per 100 g water, at least 1 g per 100 g water, or at least 5 g per 100 g water.

9. The granule of any of the preceding claims, wherein the volume based average particle size is 100-1500 pm, preferably 250-1200 pm.

10. The granule of any of the preceding claims, wherein the single-domain antibody has at least 80% amino acid sequence identity to any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3; preferably at least 90% or 95% amino acid sequence identity to any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO: 3.

11. The granule of any of the preceding claims, wherein the single-domain antibody has up to 30 alterations, preferably substitutions, preferably up to 25, up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 alteration(s), as compared to the amino acid sequence of any of SEQ ID NO: 1 , SEQ ID NO: 2, and SEQ ID NO:

312. A method for preparing an animal feed, comprising mixing the granule of any of the preceding claims with other animal feed components, steam treating, and pelleting the mixture.

13. An animal feed composition comprising the granule of any of the preceding claims.

14. Use of the granule according to any of the preceding claims as an additive in animal feed.

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