Optimised fc molecules

Abstract

The present invention relates to binding molecules comprising an optimised Fc domain, wherein the optimised Fc domain comprises one or more mutations which decrease protein A binding, increase protein A binding, or improve stability. The present invention also relates to methods of purifying heterodimeric proteins comprising an optimised Fc domain comprising one or more mutations which decrease protein A binding or increase protein A binding.

Description

[0001] x xptimised Fc Molecules Related Applications and Incorporation by Reference All documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference. This application claims the benefit of and priority from United Kingdom Patent Application No. 2311984.5, filed August 042023, the content of which is incorporated herein by reference in its entirety. SEQUENCE LISTING The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on 18 July 2024, is named P45177WO1.xml, and is 157,627 bytes in size. Field of Invention The present invention relates to companion animal antigen binding proteins and antibodies comprising immunoglobulin heavy chains which have optimised heavy chains and / or fragment crystallisable (Fc) regions. The optimised molecules comprise differential affinity for a binding affinity reagent and / or improved stability. The differential affinity of the immunoglobulin heavy chains allows for optimised isolation of said binding proteins or antibodies. The invention also relates to methods of purifying these molecules. Introduction Large scale and high-throughput purification of monospecific and bispecific antibodies typically relies on commercially available resins, most commonly those based on staphylococcal protein A (SpA). The affinity of an IgG to binding moieties, such as protein A, through binding of the Fc portion, determines how well the IgG can be bound and subsequently eluted from a purification resin such as protein A. Modulation of binding characteristics, such as binding to protein A, can therefore be a valuable tool not only to assist in the commercial purification of monospecific IgG isotypes with naturally poor binding affinities, but also as a means to help with the separation of bispecific antibodies. In addition to protein A binding, biophysical properties encoded within the Fc sequences of natural IgG isotypes may be suboptimal for purification. For example, it is known that human IgG4 has hinge disulphide bond instability and this results in IgG4 Fab arm exchange and the production of half antibody formation in non-reducing conditions (Angal et al 199330:105-108; Kolfschoten et al 2007 Science 307:1554-1557). Another unfavourable property of human IgG4 is the acid induce aggregation, which limits its manufacturability as low pH is a necessary step in antibody elution from protein A resin and subsequent incubation in acid condition is a necessary and important step for viral inactivation, as part of the manufacturing requirement (Namisaki et al., 2020 PLoS One 15(3): e0229027). Where similar instability is observed in companion animal antibodies, there is a need for Fc modifications to natural isotypes to improve their manufacturability. However, changes in Fc-mediated functions by editing amino acid sequences in the Fc region may result in changes in biophysical properties, which may lead to undesired outcomes such as reduced thermostability, increased aggregation propensity and compromised in vivo pharmacokinetics. In addition, modifications can also cause conformational changes which may impact Fc interactions such as Fc effector functions. These changes are unpredictable based on sequence alone. Therefore, when modifying Fc fragments to alter binding properties, it is important to consider and carefully evaluate the impact of any mutations on physicochemical properties, including stability and aggregation. This is critical in developing clinically useful molecules. There is a need to develop improved antibodies for veterinary medicine that have modified resin binding and other properties such as increased stability. There is also a need for methods for making such antibodies for veterinary medicine. The invention is aimed at addressing this need, in particular by providing modified immunoglobulin Fc regions. Summary of Invention The present invention relates to companion animal binding molecules comprising at least one variant IgG domain wherein the binding molecule comprises optimised characteristics. The optimised characteristics may be selected from decreased affinity for a binding affinity reagent, increase affinity for a binding affinity reagent, and / or improved stability. An aspect of the invention relates to a binding molecule comprising at least one variant IgG Fc domain comprising at least one amino acid substitution that reduces binding affinity to Protein A relative to the wild-type IgG Fc domain, wherein the binding molecule is a companion animal binding molecule. An aspect of the invention relates to a binding molecule comprising at least one variant IgG Fc domain comprising an amino acid substitution at position 315 according to EU numbering, wherein the binding molecule is a companion animal binding molecule and has increased binding affinity to Protein A relative to the wild-type IgG Fc domain. An aspect of the invention relates to a binding molecule comprising at least one variant IgG heavy chain comprising an amino acid substitution in the CH3 domain and / or in the hinge domain that increases stability relative to the wild-type IgG heavy chain, wherein the binding molecule is a companion animal binding molecule. An aspect of the invention relates to heterodimeric protein comprising at least one of the binding molecules described herein. An aspect of the invention relates to a pharmaceutical composition comprising one of the binding molecules described herein. An aspect of the invention relates to a nucleic acid encoding one of the binding molecules described herein. An aspect of the invention relates to a vector comprising one of the binding molecules described herein. An aspect of the invention relates to a host cell comprising a nucleic acid as described herein or a vector as described herein. An aspect of the invention relates to a kit comprising one of the binding molecules as described herein or a pharmaceutical composition as described herein. A method for purifying a heterodimeric protein, as disclosed herein, comprising a first polypeptide comprising a variant IgG Fc domain comprising a decreased affinity for Protein A as described herein, wherein the method comprises a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the first polypeptide comprises a variant IgG Fc domain comprising a decreased affinity for Protein A, and wherein the first polypeptide has lower affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide, and b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range. A method for purifying a heterodimeric protein, as disclosed herein, comprising a first polypeptide comprising a variant IgG Fc domain comprising an increased affinity for Protein A as described herein, wherein the method comprises a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the first polypeptide comprises a variant IgG Fc domain comprising an increased affinity for Protein A, and wherein the first polypeptide has a greater affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide, and b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range. Accordingly, it is an object of the invention not to encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product. It may be advantageous in the practice of the invention to be in compliance with Art.53(c) EPC and Rule 28(b) and (c) EPC. All rights to explicitly disclaim any embodiments that are the subject of any granted patent(s) of applicant in the lineage of this application or in any other lineage or in any prior filed application of any third party is explicitly reserved. Nothing herein is to be construed as a promise. It is noted that in this disclosure and particularly in the claims and / or paragraphs, terms such as "comprises", "comprised", "comprising" and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean "includes", "included", "including", and the like; and that terms such as "consisting essentially of" and "consists essentially of" have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description. Figures Figures 1-11, 24 relate to canine molecules, Figures 12-23, 25-30 relate to feline molecules. Figure 1A shows amino acid sequences of wild type canine IgG (cIgG) IgG-A (SEQ ID NO: 1), IgG-B (SEQ ID NO: 2), IgG-C (SEQ ID NO: 3) IgG-D (SEQ ID NO: 4) and wild type feline IgG (fIgG) IgG-1 (SEQ ID NO: 20), IgG-2 (SEQ ID NO: 21), and IgG-3 (SEQ ID NO: 22).The constant region begins at residue 118. Highlighting identifies the lower hinge, proline sandwich and SHED areas. Figure 1B shows a focussed image of the hinge region of the amino acid sequences highlighted in grey in Figure 1A. Figure 2 shows wild type canine IgGs have been analysed for Protein A binding using Biacore 8K. Sensogram showing raw data (grey dotted lines) and fitting obtained with 1:1 Langmuir fit (black solid lines) are shown. Figure 3A shows a model of canine IgG-Bwt (Fc only) in complex with Protein A which has been generated using homology modelling (based on human experimental structure available). Figure 3B: The model was used to calculate impact of alanine mutations on single residues. Delta values (DDG and DG) are indicative of energy changes in either interaction between Fc- Protein (DDG) or within Fc only (DG). Positive values indicate reduced strength / stability and vice versa. Figure 4 shows single point and multiple mutations on different Protein A interacting domain on the Fc of different canine IgGs are shown in bold / underlined characters. Figure 5: Mutant canine IgGs have been analysed for Protein A binding using Biacore 8K. Sensogram showing raw data (grey dotted lines) and fitting obtained with 1:1 Langmuir fit (black solid lines) are shown. Figure 6A shows a plot of Canine FcRn binding affinity (on y-axis Logarithmic scale) of wild type and protein A binding mutated canine IgGs. Figure 6Bshows a able for the same. Figures 7A-7B show column plots of % of monomer (H-SEC; Figure 7A) and % of main species (H-SCX; Figure 7B) for wild type and protein A binding mutated canine IgGs. Figures 7C and 7D show the same as tabulated values (H-SEC; Figure 7C) and (H-SCX; Figure 7D). Figure 8 shows acartoon representation of heterodimer Protein A+ / - design and its rationale for bispecific canine IgG purification using protein A affinity chromatography. Figures 9A-9B show an example chromatogram from AKTA MabSelect SuRe LX purification (overall (Figure 9A) and zoomed view (Figure 9B) on the elution profile) for mutant 428 and wild type Fc bispecific canine IgG. It is worth noting that 3 peaks can be resolved using this optimised method. Figure 10 shows LC-MS intact mass analyses of acetate peak from sample 428 showing all masses detected corresponds to heterodimers 428 mAb with different degrees of glycosylation. Figure 11A shows an HSCX chromatogram of acetate peak from sample 428. Figure 11B shows a column graph showing % of heterodimers content found in acetate peak. Figure 12A shows amino acid sequences of wild type feline IgG1, 2 and 3 as well as feline IgG1 SpA- deficient mutants (1-SpA-1 to 10) and IgG3 SpA+ proficient mutants (3-SpA+1 to 3) as described herein. Mutations are in two regions within the CH2 domain (amino acid residues 251- 256 and 307-317 respectively), and the CH3 domain (amino acid residues 427-436). In bold are highlighted amino acids that are mutated in SpA mutants compared to wild type feline IgG. Figure 12B shows wild-type feline chimeric fIgG1 (SEQ ID NO: 20) (a), fIgG2 (SEQ ID NO: 21) (b) and fIgG3 (SEQ ID NO: 22) with human ofatumumab variable region as visualised via non- reducing SDS-PAGE. Note that IgG2 has hinge instability resulting in a HL half antibody band migrating at a lower molecular weight around 75 kDa. Figure 13 shows SPR measurements of binding affinity of WT and SpA mutants with ofatumumab variable region to protein A. Wild type IgG1 has significantly higher binding affinity to protein A compared to wild type IgG3 (KD 4.74e-9 versus KD 2.01e-7 respectively). This difference in protein A affinity can be modulated by mutating two single amino acids at positions 309 and 315 within the CH2 domain of IgG1 to that of IgG3 and vice versa. Figure 14 showsSPR measurements of binding affinity of WT and SpA mutants with variable region B showing no statistical difference in binding affinity to feline FcRn complex. Figure 15 shows complement-dependent cytotoxicity (CDC) activity of IgG1-WT (SEQ ID NO: 20), IgG1-SpA -ve (Feline IgG 1 SpA-3, SEQ ID NO:25), IgG3-WT (SEQ ID NO: 22) and IgG3- SpA +ve (Feline IgG 3 SpA+3, SEQ ID NO: 35) antibodies containing the Ofatumumab variable region. Modulation of protein A does not alter IgG’s natural CDC activity. CDC assay showing the complement dependent killing of feline B cells expressing human CD20 (MS4 hCD20) by effector function proficient IgG1 WT and IgG1-SpA- deficient mutant when compared to the effector function deficient IgG3 WT and IgG3-SpA+ proficient mutants. All antibodies used in this assay have Ofatumumab variable regions. Data are plotted as percentage of killing where 100% means all cells are killed and 0% means signal was identical to what obtained in control cells (no antibody added). Experiments were conducted using three biological replicates (n=3 individual cat serum samples). Figure 16A shows IgG1-WT (SEQ ID NO: 20) IgG1-SpA- (Feline IgG 1 SpA-3, SEQ ID NO: 25), IgG3-WT (SEQ ID NO: 22) and IgG3-SpA+ (Feline IgG 3 SpA+3, SEQ ID NO: 35), with human variable region for antigen B as visualised via non-reducing SDS-PAGE. Note that IgG3 and IgG3-SpA+ exhibits signs of aggregation. Figure 16B shows a plot of IgG integrity. All protein A mutants showed no significant reduction in % as well as area of monomeric peak as determined by HPLC-SEC compared to their WT counterparts meaning that the overall protein stability is not affected by the introduced mutations in protein A binding regions. Note that IgG3 has an inherently lower percentage of monomeric species having a propensity to aggregate, which is also found in the SpA+ proficient mutant. Figure 17A shows amino acid sequences of wild type feline IgG1, 2 and 3 as well as feline IgG2 hinge stabilised mutants (MUT5 and MUT6) as described herein. Mutations are within the hinge region (amino acid residues 219-226). In bold are highlighted amino acids that are mutated in hinge stabilised mutants compared to wild type feline IgG. Figure 17B shows IgG1-WT (SEQ ID NO:20) IgG2-WT (SEQ ID NO: 21), IgG2 MUT 5 (SEQ ID NO: 39) and IgG2 MUT6 (SEQ ID NO: 40), with human ofatumumab variable region as visualised via non-reducing SDS-PAGE. IgG2 hinge stabilised mutants demonstrate lack of HL species compared to the WT IgG2 control. Figure 17C shows a reduction in % as well as area of monomeric peak as determined by HPLC- SEC was observed when comparing the IgG1 and IgG2 WT isoforms. Regardless of the lack of HL species in the mutant IgG2 hinge stabilised mutants no improvement in integrity was observed via HPLC-SEC, indicating that these mutants as well as the IgG2 WT isoform have a propensity to aggregate. Figure 18A shows amino acid sequences of wild type feline IgG1, 2 and 3 as well as IgG2 and IgG3 aggregation stabilised mutants (IgG3-SpA+ve + R409K) as described herein. Mutations are within the CH3 domain (amino acid residues 406 and 409). In bold are highlighted amino acids that are mutated in the aggregation stabilised mutants compared to wild type feline IgG. Figure 18B shows IgG3-SpA+ (SEQ ID NO: 35) and IgG3 SpA-ve + R409K (SEQ ID NO: 41), with human ofatumumab variable region as visualised via non-reducing SDS-PAGE. IgG3 aggregation stabilised mutant lacks the additional high molecular weight band (>250kDa) observed in the loading well of the IgG3 SpA+ control. C) A significant improvement in integrity was observed via HPLC-SEC when comparing the aggregation stabilisation mutant in the IgG3 Fc backbone compared to both the SpA+ and WT IgG3 controls. Figure 19 showspercentage aggregation as measured by HPLC-SEC of IgG3 WT (SEQ ID NO: 22), IgG3 SpA+ (SEQ ID NO: 35) and IgG3 SpA+ve + R409K (SEQ ID NO: 41) IgG following incubation at pH 3.5 for 10 and 60 minutes at 37°C. The R409K mutant was resistant to aggregation compared to both the IgG3 WT and protein A proficient mutant, being on par with the feline IgG1 WT molecule. Figure 20 shows SPR measurements of binding affinity of IgG3 WT (SEQ ID NO: 22), IgG3 SpA+ (SEQ ID NO: 35), and IgG3 SpA+ve + R409K (SEQ ID NO: 41) IgG to protein A. An additional mutation within the protein A proficient IgG3 backbone (R409K) does not significantly alter its binding affinity to protein A, with it still remaining over one log different from the WT control (IgG3 WT =2.01e-007M, IgG3 SpA+ =7.91e-009M, IgG3 R409K =1.21e-008M). Figure 21 shows CDC activity of SEQ ID NO:20), IgG3 WT (SEQ ID NO: 22), IgG3 SpA+ (SEQ ID NO: 35), and IgG3 SpA+ve + R409K (SEQ ID NO: 41), antibodies containing the Ofatumumab variable region. Mutation of position R409K does not alter the IgG’s natural CDC activity. CDC assay showing the complement dependent killing of feline B cells expressing human CD20 (MS4 hCD20) by effector function proficient IgG-1 WT deficient mutant when compared to the effector function deficient IgG3 WT, IgG3 SpA+ proficient and IgG3 SpA+ve + R409K aggregation stabilised mutants. All antibodies used in this assay have Ofatumumab variable regions. Data are plotted as percentage of killing where 100% means all cells are killed and 0% means signal was identical to what obtained in control cells (no antibody added). Experiments were conducted using three biological replicates (n=3 individual cat serum samples). Figures 22A-22B show SPR measurements of binding affinity of IgG1 WT, IgG3 WT (SEQ ID NO: 22), IgG3 SpA+ (SEQ ID NO: 35), and IgG3 SpA+ve + R409K (SEQ ID NO: 41), mutant showed no statistical difference in binding affinity to human Fc gamma receptor 1 (Figure 22A) or feline FcRN (Figure 22B). Figure 23 shows an experimental overview of in vivo FcRN binding validation of feline IgG aggregation stabilised mutants. Figure 24A shows a chromatogram from AKTA MAbSelect SuRe LX purification showing the overall washing and elution profiles for mutant 437 Fc bispecific canine IgG. Two peaks were resolved using this method for this variant. Figure 24B shows LC-MS intact mass analysis of acetate peak from sample 437 showing two main masses detected corresponding to heterodimer AB and homodimer BB with different degrees of glycosylation. Figure 24C shows LC-MS intact mass analysis of acetate peak from sample 428 showing all masses detected corresponding to heterodimer AB with different degrees of glycosylation. Figure 24D shows comparison of total protein yield and percentage species from all fractions analysed for variants 428 vs 437. Figures 25A-25B show SPR measurements of binding affinity of Protein A following mutations of amino acid positions 309 and 315 alone and in combination. Figure 25A shows 1:1 binding affinity result for IgG3 WT (SEQ ID NO: 22), IgG3 SpA+1 (SEQ ID NO: 33), IgG3 SpA+2 (SEQ ID NO: 34) and IgG3 SpA+3 to protein A (SEQ ID NO: 35). The single mutation T315K marginally improved the IgG3 Fc’s protein A binding capacity. The single mutation V309L shows a greater improvement in IgG3 Fc’s protein A binding capacity compared to T315K alone. The combination mutant shows the greatest protein A binding proficiency compared to the mutations alone, being two log different from the WT control (IgG3 WT =2.55e-007M, IgG3 SpA+1 =5.5e-008M, IgG3 SpA+2 =9.09e-009M, IgG3 SpA +3 =3.23e-009M). Figure 25B shows 1:1 binding affinity result for IgG1 WT (SEQ ID NO: 20), IgG1 SpA-1 (SEQ ID NO: 23), IgG1 SpA-2 (SEQ ID NO: 24) and IgG1 SpA-3 to protein A (SEQ ID NO: 25). The single mutation K315T marginally reduces the IgG1 Fc’s protein A binding capacity. The single mutation L309V shows a greater reduction in IgG1 Fc’s protein A binding capacity compared to K315T alone. The combination mutant shows the greatest protein A binding deficiency compared to the mutations alone, being one log different from the WT control (IgG1 WT =1.31e-009M, IgG1 SpA-1 =4.05e-009M, IgG1 SpA-2 =2.8e-008M, IgG1 SpA -3 =7.82e-008M). Figures 26A-26B show SPR measurements of binding affinity of WT and individual versus combination SpA mutants (IgG1 - Figure 26A; IgG3 - Figure 26B) in position 309 and 315. The kinetic rate constants were obtained using a simple first order (1:1) bimolecular interaction model (Langmuir) or a steady state affinity model supplied by the BIAevaluation software. Figure 27 shows PK comparison of wild type and mutant variants. Sprague Dawley rats (n=5 per group) were IV-injected with 5mg / kg doses. Blood concentrations over time are shown, with data points representing the mean antibody concentration over time (hrs) ± SD. PK parameters were determined in GraphPad Prism using the one-phase exponential decay fitting function. Figures 28A-28B show examples of chromatograms from AKTA purification comparing the elution profile following use of a MabSelect Protein A column (Figure 28A) vs Capture Sure CH1-XL column (Figure 28B) for mutant SpA-8. Figure 28C shows a comparison of the yield obtained following purification of 30ml of supernatant from CHO-expressing cells using both columns for the various IgG1 SpA deficiency mutants shows those mutants which could not be purified with Protein A can be efficiently purified with CH1 columns. Figure 29 shows sensograms of protein A binding affinity of IgG1 WT and protein A deficiency mutants SpA-1 to SpA-15. Sensograms were generated using Biacore 8K with light grey dotted lines showing the raw data and the dark grey lines being those obtained with 1:1 Langmuir fit. Figure 30 shows sensograms of FcRn binding affinity of IgG1 WT and protein A deficiency mutants SpA-1 to SpA-15. Sensograms generated using Biacore 8K with black lines showing the WT IgG1 control and the light grey lines those obtained for each mutant variant. The kinetic rate constants were obtained using a simple first order (1:1) bimolecular interaction model (Langmuir) or a steady state affinity model supplied by the BIAevaluation software. Detailed Description The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols. 1 and 2, Ontermann and Duebel, eds., Springer-Verlag, Heidelberg (2010). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Variant IgG Molecules The present invention relates to companion animal binding molecules comprising at least one immunoglobulin heavy chain wherein the binding molecule comprises optimised characteristics. The optimised characteristics may be selected from decreased affinity for a binding affinity reagent, increased affinity for a binding affinity reagent, and / or improved stability. More specifically, the inventors have identified specific mutations in canine and feline Fc backbones of which both rescue and induce protein A binding deficiency. This could enable these Fcs to be used in therapeutic mAbs or Fc fusions, as the capacity of protein A binding is an important consideration for manufacturability. Companion animals of the invention are suitably selected from dogs, cats, horses, birds, rabbits, goats, reptiles, fish and amphibians. A dog is a preferred companion animal of the invention. A cat is a preferred companion animal of the invention. A horse is a preferred companion animal of the invention. For the avoidance of doubt, a human is not a companion animal and the present invention does not extend to human molecules. In one aspect, the companion animal is a dog. In one aspect, the companion animal is a cat. In one aspect, the companion animal is a horse. The term "isolated" molecule, protein or polypeptide refers to a molecule, protein or polypeptide that is substantially free of other proteins or polypeptides, having different antigenic specificities. Moreover, protein or polypeptide may be substantially free of other cellular material and / or chemicals. Thus, the protein, nucleic acids and polypeptides described herein are preferably isolated. Thus, as used herein, an "isolated" protein, or polypeptide means protein or polypeptide that has been identified and separated and / or recovered from a component of its natural cell culture environment. Contaminant components of its natural environment are materials that would interfere with diagnostic or therapeutic uses for the protein or polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Peptides, oligopeptides, dimers, multimers, and the like, are also composed of linearly arranged amino acids linked by peptide bonds, and whether produced biologically, recombinantly, or synthetically and whether composed of naturally occurring or non-naturally occurring amino acids, are included within this definition. Both full-length proteins and fragments thereof are encompassed by the definition. The terms also include co-translational and post-translational modifications of the polypeptide, such as, for example, disulfide-bond formation, glycosylation, acetylation, phosphorylation, proteolytic cleavage (e.g., cleavage by furins or metalloproteases and prohormone convertases (PCs)), and the like. Furthermore, for purposes of the present invention, a "polypeptide" encompasses a protein that includes modifications, such as deletions, additions, substitutions and post-translational modifications (generally conservative in nature as would be known to a person in the art), to the native sequence, as long as the protein maintains the desired activity. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental, such as through mutations of hosts that produce the proteins, or errors due to PCR amplification or other recombinant DNA methods. Polypeptides or proteins are composed of linearly arranged amino acids linked by peptide bonds, but in contrast to peptides, have a well- defined conformation. Proteins, as opposed to peptides, generally consist of chains of 50 or more amino acids. For the purposes of the present invention, the term "peptide" as used herein typically refers to a sequence of amino acids of made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds. Generally, peptides contain at least two amino acid residues and are less than about 50 amino acids in length. The term “amino acid sequence” refers to a sequence of amino acids residues in a peptide or protein. The term "antibody" as used herein broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion thereof, 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 mutant, variant, or derivative antibody formats are known in the art. As used herein, the term "antibody" encompasses not only intact polyclonal or monoclonal antibodies. In a fuIl-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) 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 or domain (abbreviated herein as LCVR) and a light chain constant region. The light chain constant region is comprised of one domain, CL. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N- terminus, a variable domain (VH) followed by three constant domains (CH) for each of the α and γ chains and four CH domains for μ and ε isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CH1). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. The "variable region" or "variable domain" of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody. The variable domains of the heavy chain and light chain may be referred to as "VH" and "VL", respectively. These domains are generally the most variable parts of the antibody (relative to other antibodies of the same class) and contain the antigen binding sites. The term "variable" refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and defines the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the entire span of the variable domains. Instead, it is concentrated in three segments called hypervariable regions (HVRs) both in the light-chain and the heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta- sheet structure. The HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen binding site of antibodies. The constant domains are not involved directly in the binding of antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody- dependent cellular toxicity. The heavy chain and light chain variable 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 heavy chain and light chain variable region 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. A "fragment crystallizable (Fc) polypeptide" or “Fc domain” is the portion of an antibody molecule that interacts with effector molecules and cells. It comprises the C-terminal portions of the immunoglobulin heavy chains. As used herein, an Fc polypeptide or Fc domain includes fragments of the Fc domain having one or more biological activities of an entire Fc polypeptide. In some embodiments, a biological activity of an Fc polypeptide is the ability to bind FcRn. In some embodiments, a biological activity of an Fc polypeptide is the ability to bind protein A. An "effector function" of the Fc polypeptide is an action or activity performed in whole or in part by any antibody in response to a stimulus and may include complement fixation and / or ADCC (antibody-dependent cellular cytotoxicity) induction. In embodiments the Fc polypeptide or Fc domain comprises the CH2 domain and CH3 domain. In embodiments the Fc polypeptide or Fc domain comprises the hinge, CH2 domain and CH3 domain. In embodiments the Fc domain does not comprise CH1 or CL domain. Immunoglobulin molecules can be of any type, class or subclass. For example, for canine molecules that immunoglobulin may be IgG, IgE, IgM, IgD and IgA. Further, for canine molecules the immunoglobulin may be selected from one of the IgG subtypes for example, IgG-A, IgG-B, IgG-C, and IgG-D. For example, for feline molecules that immunoglobulin may be IgG, IgE, IgM, IgD, or IgA. Further, for feline molecules the immunoglobulin may be selected from one of the IgG subtypes for example, IgG-1a, IgG-1b, IgG-2, and IgG-3. The isolated companion animal protein of the present invention comprises IgG. In canine, there are four IgG heavy chains referred to as A, B, C, and D. These heavy chains represent four different subclasses of dog IgG, which are referred to as IgG-A, IgG-B, IgG-C and IgG-D. The DNA and amino acid sequences of these four heavy chains were first identified by Tang et al. (Vet. Immunol. Immunopathol.80: 259-270 (2001)). Exemplary amino acid and DNA sequences for these heavy chains are also available from the GenBank data bases (IgG-A: accession number AAL35301.1, IgG-B: accession number AAL35302.1, IgG-C: accession number AAL35303.1, IgG-D: accession number AAL35304.1). Canine antibodies also contain two types of light chains, kappa and lambda (GenBank accession number kappa light chain amino acid sequence ABY 57289.1, GenBank accession number ABY 55569.1). In feline, there are four IgG heavy chains referred to as 1a, 1b, 2 and 3. These heavy chains represent four different subclasses of cat IgG, which are referred to as IgG-1a, IgG-1b, IgG-2, and IgG-3 (WO 2023 / 012486 A1; Kanai et al Veterinary Immunology and Immunopathology 73 (2000) 53-62; Strietzel et al, Veterinary Immunology and Immunopathology 158(2014) 214-223; Lu et al Scientific reports 7(2017) 12713. Amino acid sequences for canine IgG-A, IgG-B, IgG-C and IgG-D as used by the inventors and according to the aspects and embodiments of the invention are shown in the below Sequences section. Amino acid sequence for feline IgG-1a, IgG-1b, IgG-2, and IgG-3 as used herein and according to aspect of the invention are shown in the below Sequences section. Thus, the invention specifically encompasses aspects that include a modification of the CH2 domain, CH3 domain and or hinge in canine IgG-A, IgG-B, IgG-C, or IgG-D. The invention specifically encompasses aspects that include a modification of the CH2 domain, CH3 domain and or hinge domain in feline IgG-1a, IgG-1b, IgG-2, or IgG-3. The term “IgG Fc” refers to an Fc region derived from an IgG isotype. The term “IgG heavy chain” refers to a heavy chain polypeptide derived from an IgG isotype. The term "hinge" refers to any portion of an Fc polypeptide or variant Fc polypeptide that is proline-rich and comprises at least one cysteine residue located between CH1 and CH2 of a full- length heavy chain constant region. As used herein the hinge is used to refer to amino acid residues at any of positions 215 to 230. The term “CH2” domain refers to a constant domain of the heavy chain, the CH2 domain is found between the hinge and the CH3 domain. As used herein the CH2 domain is used to refer to amino acid residues at any of positions 231 to 340. The term “CH3” domain refers to a constant domain of the heavy chain, the CH3 domain is found at the terminal end of the antibody. As used herein the CH2 domain is used to refer to amino acid residues at any of positions 341 to 447. 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" 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 can be defined differently according to different systems known in the art. The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol. 196:901 -917 (1987)). The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1 -113 of the heavy chain). Another system is the ImMunoGeneTics (IMGT) numbering scheme. The IMGT numbering scheme is described in Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005). 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. In the present invention, the residues in Fc region are numbered according to the EU indexes for the immunoglobulin heavy chain (Edelman, G.M. et al., Proc. Natl. Acad. USA, 63, 78-85 (1969)). The EU indexes of Kabat system refer to the EU residue numbering for human IgG1 antibody. The positions in the amino acid sequence of antibody Fc region are indicated with the EU indexes mentioned in Kabat, et al. Antigen binding fragments are also contemplated according to the aspects and embodiments of the invention. Antigen binding fragments include, for example, Fab, Fab', F(ab')2, Fd, Fv, single domain antibodies (sdAbs), e.g., VH single domain antibodies, fragments including complementarity determining regions (CDRs), single chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. An “Fv" is the minimum antibody fragment which contains a complete antigen- recognition and -binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. From the folding of these two domains emanate six hypervariable loops (3 loops each from the H and L chain) that contribute the amino acid residues for antigen binding and confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. “Single-chain Fv” also abbreviated as “sFv” or “scFv” are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. The term “antigen binding site” refers to the part of the antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen binding site may be provided by one or more antibody variable domains. Preferably, an antigen binding site is comprised within the associated VH and VL of an antibody or antibody fragment. A “chimeric antibody” is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, while the constant domains of the antibody molecule are derived from those of another species, e.g., a canine antibody. An exemplary chimeric antibody is a chimeric human – canine antibody. A “humanized antibody” is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences. As used herein, the term "caninized antibody" refers to forms of recombinant antibodies that contain sequences from both canine and non-canine (e.g., murine) antibodies. In general, the caninized antibody will comprise substantially all of at least one or more typically, two variable domains in which all or substantially all of the hypervariable loops correspond to those of a non- canine immunoglobulin, and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) are those of a canine immunoglobulin sequence. A caninized antibody may comprise both the three heavy chain CDRs and the three light chain CDRS from a murine or human antibody together with a canine frame or a modified canine frame. A modified canine frame comprises one or more amino acids changes that can further optimize the effectiveness of the caninized antibody, e.g., to increase its binding to its target. As used herein, the term "felinized antibody" refers to forms of recombinant antibodies that contain sequences from both feline and non-feline (e.g., murine) antibodies. In general, the felinized antibody will comprise substantially all of at least one or more typically, two variable domains in which all or substantially all of the hypervariable loops correspond to those of a non- canine immunoglobulin, and all or substantially all of the framework (FR) regions (and typically all or substantially all of the remaining frame) are those of a feline immunoglobulin sequence. A felinized antibody may comprise both the three heavy chain CDRs and the three light chain CDRS from a murine or human antibody together with a feline frame or a modified feline frame. A modified feline frame comprises one or more amino acids changes that can further optimize the effectiveness of the felinized antibody, e.g., to increase its binding to its target. In an embodiment the present invention relates to fully canine antibodies. In an embodiment the present invention relates to caninized antibodies. In an embodiment the present invention relates to fully feline antibodies. In an embodiment the present invention relates to felinized antibodies. A “canine antibody” refers to an antibody derived from canine or comprising sequences from a canine antibody. A “feline antibody” refers to an antibody derived from feline or comprising sequences from a feline antibody. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and / or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they are synthesized by the hybridoma culture, uncontaminated by other immunoglobulins. The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen (to which an immunoglobulin, antibody or antibody fragment, specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term specifically includes linear epitopes and conformational epitopes. Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non- contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antibody fragment (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antibody fragment. An antibody binds "essentially the same epitope" as a reference antibody, when the two antibodies recognize identical or sterically overlapping epitopes. The most widely used and rapid methods for determining whether two epitopes bind to identical or sterically overlapping epitopes are competition assays, which can be configured in different formats, using either labelled antigen or labelled antibody. The binding molecules of the present invention comprise variant molecules. The term “variant” refers to a polypeptide that differs from a reference polypeptide by single or multiple non-native amino acid substitutions, deletions, and / or additions. In some embodiments, a variant retains at least one biological activity of the reference polypeptide. In some embodiments, a variant (e.g., a variant canine IgG-A Fc, a variant canine IgG-B Fc a variant canine IgG-C Fc, a variant canine IgG-D Fc, a variant feline IgG-1a Fc, a variant feline IgG-1b Fc, a variant feline IgG-2 Fc, a variant feline IgG-3 Fc) has an activity that the reference polypeptide substantially lacks. For example, in some embodiments, a variant canine IgG-A Fc, a variant canine IgG-C Fc, a variant canine IgG-D Fc, variant feline IgG-1a Fc, a variant feline IgG-1b Fc, a variant feline IgG-2 Fc, a variant feline IgG-3 Fc has increased binding to Protein A compared to the wildtype sequence. For example, in some embodiments, a variant canine IgG-A Fc, a variant canine IgG-C Fc, a variant canine IgG-D Fc, variant feline IgG-1a Fc, a variant feline IgG-1b Fc, a variant feline IgG- 2 Fc, a variant feline IgG-3 Fc has decreased binding to Protein A compared to the wildtype sequence. For example, in some embodiments, a variant canine IgG-A heavy chain, a variant canine IgG-C heavy chain, a variant canine IgG-D heavy chain, variant feline IgG-1a heavy chain, a variant feline IgG-1b heavy chain, a variant feline IgG-2 heavy chain, a variant feline IgG-3 heavy chain has increased stability compared to the wildtype sequence. By "amino acid" herein is meant one of the 20 naturally occurring amino acids or any non- natural analogues that may be present at a specific, defined position. Amino acid encompasses both naturally occurring and synthetic amino acids. Although in most cases, when the protein is to be produced recombinantly, only naturally occurring amino acids are used. As used herein, a "substitution of an amino acid residue" with another amino acid residue in an amino acid sequence of heterodimeric protein or polypeptide as described herein (an antibody for example), is equivalent to "replacing an amino acid residue" with another amino acid residue and denotes that a particular amino acid residue at a specific position in the original (e.g. wild type / germline) amino acid sequence has been replaced by (or substituted for) by a different amino acid residue. This can be done using standard techniques available to the skilled person, e.g., using recombinant DNA technology. The amino acids are changed relative to the native (wild type / germline) sequence as found in nature in the wild type (WT), but may be made in IgG molecules that contain other changes relative to the native sequence. By "wild type" or "WT" or "native" herein is meant an amino acid sequence or a nucleotide sequence that is found in nature, including allelic variations. A WT protein, polypeptide, antibody, immunoglobulin, IgG, etc. has an amino acid sequence or a nucleotide sequence that has not been intentionally modified. Decreased Protein A Binding The present inventors have introduced amino acid substitutions at key positions to generate molecules with reduced binding affinity for Protein A. More importantly, ablation of protein A affinity was of particular interest to bispecific mAb purification. Making a heterodimer with a protein A proficient and a protein A deficient chain enables a one-step protein A purification method leading to almost 100% heterodimer purity which is extremely advantageous for large scale manufacturing processes. An aspect of the invention relates to a binding molecule comprising at least one variant IgG Fc domain comprising at least one amino acid substitution that reduces binding affinity to Protein A relative to the wild-type IgG domain, wherein the binding molecule is a companion animal binding molecule. The amino acid substitution that reduces binding affinity to Protein A of the variant IgG Fc domain, may comprise one or more of the following amino acid substitutions; a. an amino acid substitution at position 252, b. an amino acid substitution at position 253, c. an amino acid substitution at position 309, d. an arginine at position 311, i.e.311R, e. an amino acid substitution at position 315, f. an amino acid substitution at position 433, g. an amino acid substitution at position 435, and / or h. an amino acid substitution at 436, wherein the positions are numbered according to the EU numbering system. The amino acid substitutions listed above at one or more of positions 252, 253, 309, 311, 315, 433, 435 and / or 436 may be combined in any manner. The amino acid substitution at one or more of positions 252, 253, 309, 315, 433, 435 and / or 436 may comprise any amino acid substitution wherein the amino acid present in the wild type / native sequence is replaced with any other amino acid, amino acid derivative or non- natural analogues. The amino acid substitution at position 311 is 311R i.e., wherein the naturally occurring or wild-type residue is replaced with an arginine residue. In an embodiment the binding molecule comprises a canine variant IgG domain. In an embodiment the binding molecule comprises a variant canine IgG domain with reduced binding affinity to Protein A, wherein the variant canine IgG domain comprises an amino acid substitution at one or more of any of; position 252, 253, 311, 315 and / or 435, wherein the amino acid substitution at position 311 comprises arginine i.e., 311R and wherein the positions are numbered according to the EU numbering system. In an embodiment the binding molecule comprises a feline variant IgG domain. In an embodiment the binding molecule comprises a variant feline IgG domain with reduced binding affinity to Protein A, wherein the variant feline IgG domain comprises an amino acid substitution at one or more of any of; position 253, 309, 315, 435, 436, wherein the positions are numbered according to the EU numbering system. In some embodiments combinations of amino acid substitutions may be used to reduce binding affinity of Protein A. In an embodiment the variant IgG Fc domain of the binding molecule of the invention comprises: a. an amino acid substitution at one or more of position 252, 253 and / or 315 according to EU numbering; b. an amino acid substitution at one or more of position 252, 253, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; c. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; d. an amino acid substitution at one or more of position 252, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; e. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; f. an arginine residue at position 311 according to EU numbering; g. an amino acid substitution at one or more of position 253 and / or 435 according to EU numbering; h. an amino acid substitution at position 435 according to the EU numbering system; i. an amino acid substitution at position 436 according to EU numbering; or j. an amino acid substitution at one or more of position 309 and / or 315 according to EU numbering. In an embodiment the binding molecule comprises a variant canine IgG Fc domain which comprises a. an amino acid substitution at one or more of position 252, 253 and / or 315 according to EU numbering; b. an amino acid substitution at one or more of position 252, 253, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; c. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; d. an amino acid substitution at one or more of position 252, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; e. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; f. an amino acid substitution at one or more of position 253 and / or 435 according to EU numbering. As discussed above the amino acid substitution may comprise the replacement of the amino acid present in the native or wild-type sequence with any other amino acid, derivative or non- natural analogue. In some embodiments there are preferred amino acids which may replace the wild type or native amino acid in said sequence. In an embodiment the amino acid substitution at position 252 comprises 252V or 252R. Where, for example the term “252V” is used, this indicates that the original amino acid found in the native or wild type sequence is replaced with V (valine), this terminology is used herein throughout. In an embodiment the amino acid substitution at position 253 comprises 253T or 253D. In an embodiment the amino substitution at position 309 comprises 309E or 309V or 309Q. In an embodiment the amino acid substitution at position 311 comprises 311R. In an embodiment the amino acid substitution at position 315 comprises 315S or 315T. In an embodiment the amino acid substitution at position 433 comprises 433A. In an embodiment the amino acid substitution at position 435 comprises 435R or 435Q or 435A. In an embodiment the amino acid substitution at position 436 comprises 436F. As disclosed herein the variant IgG domain may be canine. Where the binding molecule of the invention has a decreased affinity for Protein A the variant IgG domain may be selected from a canine IgG-A Fc domain, a canine IgG-B Fc domain, or a canine IgG-D Fc domain. Any of the aforementioned amino acid substitutions to decrease affinity for Protein A may be present in a canine IgG-A Fc domain, a canine IgG-B Fc domain, or a canine IgG-D Fc domain In an embodiment the variant IgG Fc domain is a canine IgG-A domain and comprises one of the following; a. R252V, b. I253T or I253D c. E309V or E309Q, d. Q311R, e. T315S, f. Q433A, g. H435R or H435A, h. Y436F i. any combination of the above amino acid substitutions. As used herein the term “R252V” refers to an amino acid substitution wherein the R at position 252 is replaced with V, this terminology is used throughout. In an embodiment the variant IgG Fc domain is a canine IgG-A domain and comprises one or more of the following amino acid substitutions; R252V, I253T, I253D, Q311R, T315S, H435R or H435A. In an embodiment the variant IgG Fc domain is a canine IgG-A domain and comprises one or more of the following amino acid substitutions; R252V, I253T, Q311R, T315S, H435A. In an embodiment the variant IgG Fc domain is the variant IgG Fc domain is a canine IgG-B domain and comprises one of the following; a. L252V or L252R, b. I253T or I253D c. G309V or G309Q, d. Q311R e. K315S or K315T, f. H433A, g. H435R or H435A, h. Y436F i. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is the variant IgG Fc domain is a canine IgG-B domain and comprises one or more of the following amino acid substitutions; L252V, L252R, I253T, I253D, Q311R, K315T, K315S, H435R, H435A. In an embodiment the variant IgG Fc domain is the variant IgG Fc domain is a canine IgG-B domain and comprises one or more of the following amino acid substitutions; L252V, I253T, Q311R, K315S, H435A. In an embodiment the variant IgG Fc domain is a canine IgG-D domain and comprises and comprises one of the following; a. R252V, b. I253T or I253D c. E309V or E309Q, d. Q311R e. T315S, f. Q433A, g. H435R or H435A, h. Y436F i. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following amino acid substitutions; R252V, I253T, I253D, Q311R, T315S, H435R, H435A. In an embodiment the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following amino acid substitutions; R252V, I253T, Q311R, T315S, H435A. As disclosed herein the variant IgG domain may be feline. Where the binding molecule of the invention has a decreased affinity for Protein A the variant IgG domain may be selected from a feline IgG1a Fc domain, a feline IgG1b Fc domain, or a feline IgG2 Fc domain. Any of the aforementioned amino acid substitutions to decrease affinity for Protein A may be present in a feline IgG1a Fc domain, a feline IgG1b Fc domain, or a feline IgG2 Fc domain. In an embodiment the variant IgG Fc domain is a feline IgG1a and comprises one or more of the following; a. S252V or S252R, b. I253T, I253D, c. L309V, L309Q, d. Q311R, e. K315T, K315S, f. H433A, g. H435R or H435Q, h. H435A, i.H436F and / or j. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is a feline IgG1a and comprises one or more of the following amino acid substitutions; I253T, I253D, L309V, L309Q, K315T, K315S, H435R, H435Q, H435A, and or H436F. In an embodiment the variant IgG Fc domain is a feline IgG1a and comprises one or more of the following amino acid substitutions; I253T, I253D, L309V, L309Q, K315T, H435R, H435Q, H435A, and or H436F. In an embodiment the variant IgG Fc domain is a feline IgG1b and comprises one or more of the following; a. S252V or S252R, b. I253T, I253D, c. L309V, L309Q, d. Q311R, e. K315T, K315S, f. H433A, g. H435R or H435Q, h. H435A, i.H436F and / or j. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is a feline IgG1b and comprises one or more of the following amino acid substitutions; I253T, I253D, L309V, L309Q, K315T, K315S, H435R, H435Q, H435A, and or H436F. In an embodiment the variant IgG Fc domain is a feline IgG1b and comprises one or more of the following amino acid substitutions; I253T, I253D, L309V, L309Q, K315T, H435R, H435Q, H435A, and or H436F. In an embodiment the variant IgG Fc domain is a feline IgG2 and comprises one or more of the following; a. S252V or S252R, b. I253T, I253D, c. L309V, L309Q, d. Q311R, e. K315T, K315S, f. H433A, g. H435R or H435Q, h. H435A, i.H436F and / or j. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is a feline IgG2 and comprises one or more of the following amino acid substitutions; I253T, I253D, L309V, L309Q, K315T, K315S, H435R, H435Q, H435A, and or H436F. In an embodiment the variant IgG Fc domain is a feline IgG2 and comprises one or more of the following amino acid substitutions; I253T, I253D, L309V, L309Q, K315T, H435R, H435Q, H435A, and or H436F. In an embodiment the variant IgG Fc domain comprises the following amino acid substitutions a. I253T, L252V and K315S; b. I253T, L252V, K315S and Q311R; c. L252V and K315S d. I253T and H433A, or e. L309V and K315T In an embodiment the variant canine IgG-B Fc domain comprises the following amino acid substitutions a. I253T, L252V and K315S; b. I253T, L252V, K315S and Q311R; c. L252V and K315S, or d. I253T and H433A In an embodiment the variant feline IgG-1a Fc domain comprises L309V and K315T. In some embodiments the variant IgG Fc domain is a CH2 domain, wherein the amino acid substitution is present in the CH2 domain. In some embodiments the variant IgG Fc domain is a CH3 domain, wherein the amino acid substitution is present in the CH3 domain. In some embodiments the variant IgG Fc domain comprises both a CH2 and CH3 domain, wherein both the CH2 and CH3 domain comprises amino acid substitutions. In certain embodiments the binding molecule with decreased affinity to Protein A may comprise an IgG Fc domain comprising one of SEQ ID NO: 7 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 8 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 12 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 13 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 14 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 42 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 43 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 23 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 24 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 25 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 26 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 27 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 28 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 29 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 30, SEQ ID NO: 31 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 32 or a sequence with at least 90% or 95% sequence identity thereto,SEQ ID NO:SEQ ID NO:. In certain embodiments the canine binding molecule with decreased affinity to Protein A may comprise an IgG Fc domain comprising one of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:SEQ ID NO: 42, SEQ ID NO: 43, or a sequence with at least 90% or 95% sequence identity thereto. In certain embodiments the feline binding molecule with decreased affinity to Protein A may comprise an IgG Fc domain comprising one SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32. The binding molecules of the invention may comprise one of the sequences described herein or a sequence with or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The molecules of the invention may comprise one of the sequences described herein or a sequence with or a sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid differences. For example the binding molecules of the invention may comprise further modifications such as those described herein to modulate effector function, as such molecules of the invention may comprise one of the sequences described herein such as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO:SEQ ID NO: 42, SEQ ID NO: 43, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32 or a sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid differences. In some embodiments the binding molecules of the invention comprise a decreased affinity for Protein A, wherein the affinity is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% decreased compared to the wild-type sequence. In some embodiments the binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A which is lower than 1x10-10M, lower than 2x10-10M, lower than 3x10-10M, lower than 4x10-10M, lower than 5x10-10M, lower than 6x10-10M, lower than 7x10-10M, lower than 8x10-10M, lower than 9x10-10M, lower than 1x10-11M, lower than 2x10-11M, lower than 3x10-11M, lower than 4x10-11M, lower than 5x10-11M, lower than 6x10-11M, lower than 7x10-11M, lower than 8x10-11M, lower than 9x10-11M. The tern “lower than” when referring to a binding affinity indicates that the strength of the binding interaction is lower than the recited KD i.e., the binding interaction is weaker. In some embodiments the binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A in the range of 1x10-11M to 1x10-6M, 1x10-10M to 1x10-6M, 1x10-9M to 1x10-6M, 1x10-8M to 1x10-6M, 1x10-7M to 1x10-6M, 2x10-11M to 2x10-6M, 2x10-10M to 2x10-6M, 2x10-9M to 2x10-6M, 2x10-8M to 2x10-6M, 2x10-7M to 2x10-6M, 5x10-11M to 5x10-6M, 5x10-10M to 5x10-6M, 5x10-9M to 5x10-6M, 5x10-8M to 5x10-6M, or 5x10-7M to 5x10-6M. In some embodiments the canine binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A which is lower than 1x10-10M, lower than 2x10-10M, lower than 3x10-10M, lower than 4x10-10M, lower than 5x10-10M, lower than 6x10-10M, lower than 7x10-10M, lower than 8x10-10M, lower than 9x10-10M, lower than 1x10-11M, lower than 2x10-11M, lower than 3x10-11M, lower than 4x10-11M, lower than 5x10-11M, lower than 6x10-11M, lower than 7x10-11M, lower than 8x10-11M, lower than 9x10-11M. In some embodiments the canine binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A in the range of 1x10-11M to 1x10-6M, 1x10-10M to 1x10-6M, 1x10-9M to 1x10-6M, 1x10-8M to 1x10-6M, 1x10-7M to 1x10-6M, 2x10-11M to 2x10-6M, 2x10-10M to 2x10-6M, 2x10-9M to 2x10-6M, 2x10-8M to 2x10-6M, 2x10-7M to 2x10-6M, 5x10-11M to 5x10-6M, 5x10-10M to 5x10-6M, 5x10-9M to 5x10-6M, 5x10-8M to 5x10-6M, or 5x10-7M to 5x10-6M. In some embodiments the feline binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A which is lower than 1x10-10M, lower than 2x10-10M, lower than 3x10-10M, lower than 4x10-10M, lower than 5x10-10M, lower than 6x10-10M, lower than 7x10-10M, lower than 8x10-10M, lower than 9x10-10M, lower than 1x10-11M, lower than 2x10-11M, lower than 3x10-11M, lower than 4x10-11M, lower than 5x10-11M, lower than 6x10-11M, lower than 7x10-11M, lower than 8x10-11M, lower than 9x10-11M. In some embodiments the feline binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A in the range of 1x10-11M to 1x10-6M, 1x10-10M to 1x10-6M, 1x10-9M to 1x10-6M, 1x10-8M to 1x10-6M, 1x10-7M to 1x10-6M, 2x10-11M to 2x10-6M, 2x10-10M to 2x10-6M, 2x10-9M to 2x10-6M, 2x10-8M to 2x10-6M, 2x10-7M to 2x10-6M, 5x10-11M to 5x10-6M, 5x10-10M to 5x10-6M, 5x10-9M to 5x10-6M, 5x10-8M to 5x10-6M, or 5x10-7M to 5x10-6M. In an embodiment the binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A of approximately 1x10-7M for example 1x10-6M to 1x10-8M. In an embodiment the feline binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A of approximately 1x10-7M for example 1x10-6M to 1x10-8M. In an embodiment the canine binding molecules of the invention with decreased affinity for protein A, comprise a binding affinity for protein A of approximately 1x10-7M for example 1x10-6M to 1x10-8M. An aspect of the invention relates to a method of decreasing the affinity for Protein A of a binding molecule comprising an IgG Fc domain comprising introducing into said IgG Fc domain one or more of the amino acid substitutions described herein which decrease Protein A affinity. Increased Protein A Binding The present inventors have used a structure guided approach to identify amino acid residues which are important for Protein A binding. It has been possible to introduce amino acid substitutions at these key positions to generate molecules with increased binding affinity for Protein A. An aspect of the invention relates to a binding molecule comprising at least one variant IgG Fc domain comprising an amino acid substitution at position 315 according to EU numbering that increases binding affinity to Protein A relative to the wild-type IgG domain, wherein the binding molecule is a companion animal binding molecule. Position 315 has been identified as a key position to modulate increased binding to Protein A, however in some embodiments further amino acid substitutions may be present in the variant IgG Fc domain to increase binding affinity to Protein A. For example, further amino acid substitutions which increase binding affinity to Protein A may be present at one or more of any of; a. position 252, b. position 253, c. position 309, and / or d position 433, wherein the positions are numbered according to EU numbering Amino acid substitutions at one or more of positions 252, 253, 309, 315 and / or 433 may be combined in any manner. The amino acid substitution at one or more of positions 252, 253, 309, 315 and / or 433 may comprise any amino acid substitution wherein the amino acid present in the wild type / native sequence is replaced with any other amino acid, amino acid derivative or non- natural analogues. In an embodiment the binding molecule comprises a canine variant IgG domain. In an embodiment the binding molecule comprises a variant canine IgG domain with increased binding affinity to Protein A, wherein the variant canine IgG domain comprises an amino acid substitution at one or more of any of; position 252, 253, 309, 315 and / or 433, wherein the positions are numbered according to the EU numbering system. In an embodiment the binding molecule comprises a feline variant IgG domain. In an embodiment the binding molecule comprises a variant feline IgG domain with increased binding affinity to Protein A, wherein the variant feline IgG domain comprises an amino acid substitution at one or more of any of; position 252, 253, 309, 315 and / or 433, wherein the positions are numbered according to the EU numbering system. In some embodiments combinations of amino acid substitutions may be used to increase binding affinity of Protein A. In an embodiment the variant IgG Fc domain of the binding molecule of the invention comprises: a. an amino acid substitution at one or more of position 252, 253 and / or 315 according to EU numbering; or b. an amino acid substitution at one or more of position 252, 315, and / or 433 according to EU numbering. In an embodiment the variant IgG Fc domain of the binding molecule of the invention comprises: a. an amino acid substitution at position 252, 253 and 315 according to EU numbering; or b. an amino acid substitution at position 252, 315, and 433 according to EU numbering. As discussed above the amino acid substitution may comprise the replacement of the amino acid present in the native or wild-type sequence with any other amino acid, derivative or non- natural analogue. In some embodiments there are preferred amino acids which may replace the wild type or native amino acid in said sequence. In an embodiment the amino acid substitution at position 252 comprises 252L. In an embodiment the amino acid substation at position 253 comprises 253I. In an embodiment the amino acid substitution at position 309 comprises 309L. In an embodiment the amino acid substitution at position 315 comprises 315K. In an embodiment the amino acid substitution at position 433 comprises 433H. As disclosed herein the variant IgG domain may be canine. Where the binding molecule of the invention has an increased affinity for Protein A the variant IgG domain may be selected from a canine IgG-A Fc domain, a canine IgG-C Fc domain or a canine IgG-D Fc domain. In an embodiment the variant IgG Fc domain is a canine IgG-A Fc domain and comprises one or more of the following; a. R252L, b. E309L, c. T315K, d. Q433H and / or e. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is a canine IgG-A Fc domain and comprises one or more of the following R252L, T315K, and / or Q433H. In an embodiment the variant IgG Fc domain is a canine IgG-C domain and comprises one or more of the following; a. V252L, b. T253I, c. G309L, d. S315K. and / or e. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is a canine IgG-C domain and comprises one or more of the following V252L, T253I and / or S315K. In an embodiment the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following; a. R252L, b. E309L, c. T315K, d. Q433H, and / or e. any combination of the above amino acid substitutions. In an embodiment the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following R252L, T315K and / or Q433H. As disclosed herein the variant IgG domain may be feline. Where the binding molecule of the invention has an increased affinity for Protein A the variant IgG domain may be selected from a feline IgG1b Fc domain, a feline IgG2 Fc domain or IgG3 Fc domain. In an embodiment the variant IgG Fc domain is a feline IgG1b Fc domain and comprises the following amino acid substitution S252L. In an embodiment the variant IgG Fc domain is a feline IgG2 Fc domain and comprises the amino acid substitution S252L. In an embodiment the variant IgG Fc domain is a feline IgG3 and comprises one or more of the following amino acid substitutions S252L, V309L and / or T315K. In an embodiment the variant IgG Fc domain is a feline IgG3 Fc domain and comprises V309L and / or T315K. In an embodiment the binding molecule of the invention comprises a. a variant canine IgG-A Fc domain and comprises R252L, T315K and Q433H. b. a variant canine IgG-C Fc domain and comprises V252L, T253I and S315K. c. a variant canine IgG-D Fc domain and comprises R252L, T315K and Q433H. d. a variant feline IgG3 Fc domain and comprises V309L and / or T315K. In some embodiments the variant IgG Fc domain is a CH2 domain, wherein the amino acid substitution is present in the CH2 domain. In some embodiments the variant IgG Fc domain is a CH3 domain, wherein the amino acid substitution is present in the CH3 domain. In some embodiments the variant IgG Fc domain comprises both a CH2 and CH3 domain, wherein both the CH2 and CH3 domain comprises amino acid substitutions. In certain embodiments the binding molecule with increased affinity to Protein A may comprise an IgG Fc domain comprising one of SEQ ID NO: 5 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 6 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 15 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 16 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 17 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 18 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 33 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 34 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 35 or a sequence with at least 90% or 95% sequence identity thereto. In certain embodiments the canine binding molecule with increased affinity to Protein A may comprise an IgG Fc domain comprising one of SEQ ID NO: 5 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 6 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 15 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 16 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 17 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 18 or a sequence with at least 90% or 95% sequence identity thereto. In certain embodiments the feline binding molecule with increased affinity to Protein A may comprise an IgG Fc domain comprising one SEQ ID NO: 33 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 34 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 35 or a sequence with at least 90% or 95% sequence identity thereto. The binding molecules of the invention may comprise one of the sequences described herein or a sequence with or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The molecules of the invention may comprise one of the sequences described herein or a sequence with or a sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid differences. For example the binding molecules of the invention may comprise further modifications such as those described herein to modulate effector function, as such molecules of the invention may comprise one of the sequences described herein such as SEQ ID NO: 5, 6, 15, 16, 17, 18, 33, 34, or 35 or a sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid differences. In some embodiments the binding molecules of the invention comprise an increased affinity for Protein A, wherein the affinity is 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% increased compared to the wild-type sequence. In some embodiments the binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A in the range of which is more than 1x10-8M, more than 2x10-8M, more than 3x10-8M, more than 4x10-8M, more than 5x10-8M, more than 6x10-8M, more than 7x10-8M, more than 8x10-8M, more than 9x10-9M, more than 1x10-9M, more than 2x10-9M, more than 3x10-9M, more than 4x10-9M, more than 5x10-9M, more than 6x10-9M, more than 7x10-9M, more than 8x10-9M, more than 9x10-9M. The tern “more than” when referring to a binding affinity indicates that the strength of the binding interaction is more than the recited KD i.e., the binding interaction is stronger. In some embodiments the binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A in the range of 1x10-12M to 1x10-7M, 1x10-11M to 1x10-7M, 1x10-10M to 1x10-7M, 1x10-9M to 1x10-7M, 1x10-8M to 1x10-7M, 1x10-12M to 1x10-8M, 1x10-11M to 1x10-8M, 1x10-10M to 1x10-8M, 1x10-9M to 1x10-8M, 1x10-12M to 1x10-7M, 1x10-12M to 1x10-8M, 1x10-12M to 1x10-9M, 1x10-12M to 1x10-11M, 1x10-12M to 1x10-11M. In some embodiments the canine binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A which is more than 1x10-8M, more than 2x10-8M, more than 3x10-8M, more than 4x10-8M, more than 5x10-8M, more than 6x10-8M, more than 7x10-8M, more than 8x10-8M, more than 9x10-9M, more than 1x10-9M, more than 2x10-9M, more than 3x10-9M, more than 4x10-9M, more than 5x10-9M, more than 6x10-9M, more than 7x10-9M, more than 8x10-9M, more than 9x10-9M. In some embodiments the canine binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A in the range of 1x10-12M to 1x10-7M, 1x10-11M to 1x10-7M, 1x10-10M to 1x10-7M, 1x10-9M to 1x10-7M, 1x10-8M to 1x10-7M, 1x10-12M to 1x10-8M, 1x10-11M to 1x10-8M, 1x10-10M to 1x10-8M, 1x10-9M to 1x10-8M, 1x10-12M to 1x10-7M, 1x10-12M to 1x10-8M, 1x10-12M to 1x10-9M, 1x10-12M to 1x10-11M, 1x10-12M to 1x10-11M. In some embodiments the feline binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A which is more than 1x10-8M, more than 2x10-8M, more than 3x10-8M, more than 4x10-8M, more than 5x10-8M, more than 6x10-8M, more than 7x10-8M, more than 8x10-8M, more than 9x10-9M, more than 1x10-9M, more than 2x10-9M, more than 3x10-9M, more than 4x10-9M, more than 5x10-9M, more than 6x10-9M, more than 7x10-9M, more than 8x10-9M, more than 9x10-9M. In some embodiments the feline binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A in the range of 1x10-12M to 1x10-7M, 1x10-11M to 1x10-7M, 1x10-10M to 1x10-7M, 1x10-9M to 1x10-7M, 1x10-8M to 1x10-7M, 1x10-12M to 1x10-8M, 1x10-11M to 1x10-8M, 1x10-10M to 1x10-8M, 1x10-9M to 1x10-8M, 1x10-12M to 1x10-7M, 1x10-12M to 1x10-8M, 1x10-12M to 1x10-9M, 1x10-12M to 1x10-11M, 1x10-12M to 1x10-11M, 1x10-11M to 1x10-7M, 1x10-11M to 1x10-8M, 1x10-11M to 1x10-9M, 1x10-10M to 1x10-7M, 1x10-10M to 1x10-8M, 1x10-10M to 1x10-9M. In an embodiment the binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A of approximately 1x10-11M for example 1x10-12M to 1x10-10M. In an embodiment the canine binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A of approximately 1x10-11M for example 1x10-12M to 1x10-10M. In an embodiment the binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A of approximately 1x10-9M for example 1x10-10M to 1x10-8M. In an embodiment the feline binding molecules of the invention with increased affinity for protein A, comprise a binding affinity for protein A of approximately 1x10-9M for example 1x10-10M to 1x10-8M. An aspect of the invention relates to a method of increasing the affinity for Protein A of a binding molecule comprising an IgG Fc domain comprising introducing into said IgG Fc domain one or more of the amino acid substitutions described herein which increase Protein A affinity. Improved stability The present inventors have also identified strategies to improve the stability of certain IgG Fc domains. In particular advantageous amino acid substitutions have been identified within the CH3 domain and / or the hinge domain / region. In particular, the inventors have corrected naturally occurring deficiencies in the feline IgG2 and IgG3 isotypes. The feline IgG2 isotype has hinge instability resulting in a HL (heavy chain + light chain) half antibody. With the hinge region being the crucial linker of the Fab region to the Fc, instability within this region can have important impacts on an antibody drug's safety, efficacy, and production yield. Strengthening the heavy– heavy chain interaction within the IgG2 hinge would therefore be of benefit, making IgG2 more stable toward reduction during manufacturing and prevent Fab-arm exchange. As such an aspect of the present invention relates to a binding molecule comprising at least one variant IgG heavy chain comprising an amino acid substitution in the CH3 domain and / or in the hinge domain, wherein the binding molecule is a companion animal binding molecule and has increased stability relative to the wild-type IgG heavy chain. In an embodiment the variant IgG heavy chain comprises an amino acid substitution within the CH3 domain for example an amino acid substitution at any one of positions 341 to 447 according to EU numbering. In an embodiment the variant IgG heavy chain comprises an amino acid substitution at position 409 according to EU numbering. In an embodiment the amino acid at position 409 in the wild-type sequence is replaced with any amino acid which causes an increase in stability of the IgG Fc domain. The skilled person would be able to introduce various amino acids at position 409 and identify those which increase stability using standard techniques in the art such as those disclosed in the examples section herein. In an embodiment the amino acid substitution at position 409 comprises 409K, according to EU numbering. The variant IgG heavy chain may be canine. Where the variant IgG domain has increased stability, the variant IgG domain may be selected from a canine IgG-A heavy chain, a canine IgG-C heavy chain, a canine IgG-B heavy chain, or a canine IgG-D heavy chain. The variant IgG heavy chain may be feline. Where the variant IgG heavy chain has increased stability, the variant IgG heavy chain may be selected from a selected from a feline IgG-1a heavy chain, a feline IgG-1b heavy chain, a feline IgG-2 heavy chain or a feline IgG-3 heavy chain. In a preferred embodiment the variant IgG heavy chain may be selected from a selected from a feline IgG-1b heavy chain, a feline IgG-2 heavy chain or a feline IgG-3 heavy chain. In an embodiment the variant feline IgG-1b heavy chain comprises R409K according to EU numbering. In an embodiment the variant feline IgG-2 heavy chain comprises R409K according to EU numbering. In an embodiment the variant feline IgG-3 heavy chain comprises R409K. In an embodiment the variant IgG heavy chain comprising an amino acid substation at position 409 has an improved stability, as evaluated by SDS PAGE. The present inventors have found that certain amino acid substitutions within the hinge region can improve the stability of the binding molecule. In an embodiment the amino acid substitution within the hinge region may be at one or more of; a. position 219, b.220, c.221, d.222, e.223, f.224, g.225, h.226 and / or i. any combination of the above amino acid substitutions, wherein the positions are numbered according to EU numbering. The variant IgG heavy chain may be canine. Where the variant IgG heavy chain comprises an amino acid substitution in the hinge region which increases stability, the variant IgG domain may be selected from a canine IgG-A heavy chain, a canine IgG-B heavy chain, a canine IgG-C heavy chain, or a canine IgG-D heavy chain. The variant IgG heavy chain may be feline. Where the variant IgG heavy chain comprises an amino acid substitution in the hinge region which increases stability, the variant IgG heavy chain may be selected from a selected from a feline IgG-1a heavy chain, a feline IgG-1b heavy chain, a feline IgG-2 heavy chain or a feline IgG-3 heavy chain. In a preferred embodiment the variant IgG heavy chain may be a feline IgG-2 heavy chain. Where the variant IgG heavy chain is a feline IgG-2 heavy chain, the variant IgG heavy chain may comprise one or more of the following amino acid substitutions, a. A219D, b. S220H, c. T221P, d. I222P, e. E223G, f. S224P, g. T225aP, h. G225bC, i. E225cD, j. G226C and / or l. any combination of the above amino acid substitutions, wherein the positions are numbered according to EU numbering. As used herein position 225a, 225b, 225c refer to residues in the hinge region according to EU numbering and as set out in Figure 1B. The variant feline IgG-2 heavy chain may comprise: a. A219D, S220H, T221P, I222P, E223G, S224P, T225aP, G225bC, E225cD, and G226C, or b. G225bC, E225cD and G226C The amino acid substitutions, disclosed herein, within the CH3 domain and the hinge region which increase stability may be combined in any manner. In an embodiment the binding molecule with improved stability may comprise an IgG heavy chain comprising one of SEQ ID NO: 39 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO 40 or a sequence with at least 90% or 95% sequence identity thereto, SEQ ID NO: 41 or a sequence with at least 90% or 95% sequence identity thereto. The binding molecules of the invention may comprise one of the sequences described herein or a sequence with or a sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity thereto. The molecules of the invention may comprise one of the sequences described herein or a sequence with or a sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid differences. For example the binding molecules of the invention may comprise further modifications such as those described herein to modulate effector function, as such molecules of the invention may comprise one of the sequences described herein such as SEQ ID NO: 39, 40 or 41 or a sequence comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid differences. In an embodiment the variant IgG domain comprising an amino acid substitution at one or more of positions 219, 220, 221, 222, 223, 224, 225, 226 has an improved stability as evaluated by SDS PAGE and HPLC-SEC. An aspect of the invention relates to a method of improving the stability of a binding molecule comprising an IgG heavy chain comprising introducing into said IgG heavy chain one or more of the amino acid substitutions described herein which improve stability. Decreased aggregation Antibody aggregation can be a problem not only in terms of developability but can also influence the safety and efficacy of the therapeutic treatment resulting in for example immunogenicity and unwanted complement activation. The present inventors have observed, in particular, feline IgG3 isotypes display increased susceptibility to aggregation as compared with feline IgG1 antibodies. It is therefore advantageous to resolve this instability, in order to generate aggregation resistant effector function deficient isoforms. Additional engineering The binding molecules of the present invention may comprise further amino acid substitutions to modulate other characteristics of the binding molecules. For example, the binding molecule of the present invention may be used in a dimer format e.g., heterodimer or homodimer format. Where the binding molecules are in a heterodimer format it may be advantageous for the two molecules to comprise charge steering amino acid substitutions such as those described in WO2021214460 (incorporated herein by reference in its entirety). As described above, the binding molecules of the present invention may comprise one or more further amino acid substitutions. In one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within one or more the CDR region and / or one or more framework region the antibody or fragment of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (using the functional assays described herein or known in the art. Thus, these amino acid changes can typically be made without altering the biological activity, function, or other desired property of the polypeptide, such as its affinity or its specificity for antigen. In general, single amino acid substitutions in nonessential regions of a polypeptide do not substantially alter biological activity. Furthermore, substitutions of amino acids that are similar in structure or function are less likely to disrupt the polypeptides' biological activity. Abbreviations for the amino acid residues of the polypeptides and peptides described herein, and conservative substitutions for these amino acid residues are shown in Table A below. Table A. Amino Acid Residues and Examples of Conservative Amino Acid Substitutions Binding molecules described herein may comprise suitable Fc regions. In one embodiment, for example a canine Fc region may be present, for example a canine IgG-B Fc region. In one embodiment, the Fc portion of the antibody may be modified to improve certain properties, e.g. to provide reduced complement- and FcγR- mediated effector functions. Exemplary modified Fc regions for canine antibodies sequences comprise modifications compared to wild type Fc IgG-B regions. The modified Fc regions reduce or abolish canine IgG- B effector function when compared to the same polypeptide comprising a wild-type IgG-B Fc domain. The amino acid substitutions reside in the lower hinge, proline sandwich region and SHED region. Thus, an antibody of the invention may include a modified Fc region having the modifications. Thus, with reference to the wild type residue in canine IgG-B constant region (SEQ ID NO: 4) the antibodies may have the following amino acid substitutions at the following positions in the Fc domain: E233G; M234S or A; L235A; D267G; P268R; D270N; N325H; K326I; A327G; P329G and / or P331S. In one embodiment, the canine IgG-B Fc region comprises a) an amino acid substitution at position 234 according to EU numbering to S and b) an amino acid substitution at position 325 according to EU numbering to H, an amino acid substitution at position 326 according to EU numbering to I and an amino acid substitution at position 327 according to EU numbering to G. In one embodiment, the Fc region comprises a) an amino acid substitution at position 234 according to EU numbering to S; b) an amino acid substitution at position 267 according to EU numbering to G and an amino acid substitution at position 268 according to EU numbering to R and c) an amino acid substitution at position 325 according to EU numbering to H, an amino acid substitution at position 326 according to EU numbering to I and an amino acid substitution at position 327 according to EU numbering to G. In one embodiment, the Fc region comprises a) an amino acid substitution at position 234 according to EU numbering to S; b) an amino acid substitution at position 267 according to EU numbering to G and an amino acid substitution at position 268 according to EU numbering to R and c) an amino acid substitution at position 325 according to EU numbering to H, an amino acid substitution at position 326 of according to EU numbering to I and an amino acid substitution at position 327 according to EU numbering to G and an amino acid substitution at position 331 of according to EU numbering to S. In one embodiment, the Fc region comprises a) an amino acid substitution at position 234 according to EU numbering to S; b) an amino acid substitution at position 267 according to EU numbering to G and an amino acid substitution at position 268 according to EU numbering to R and c) an amino acid substitution at position 325 according to EU numbering to H, an amino acid substitution at position 326 according to EU numbering to I and an amino acid substitution at position 327 according to EU numbering to G and an amino acid substitution at position 329 of according to EU numbering to G. In one embodiment, the Fc region comprises an amino acid substitution at position 234 according to EU numbering to A and at position 235 to A. In one embodiment, the Fc region comprises a) an amino acid substitution at position 234 according to EU numbering to A and at position 235 to A and b) an amino acid substitution at position 331 according to EU numbering to S. In one embodiment, the Fc region comprises a) an amino acid substitution at position 234 according to EU numbering to A and at position 235 to A and b) an amino acid substitution at position 329 according to EU numbering to G. In one embodiment, the Fc region comprises a) an amino acid substitution at position 233 according to EU numbering to G and b) an amino acid substitution at position 270 according to EU numbering to N. In one embodiment, the Fc region comprises a) an amino acid substitution at position 234 according to EU numbering to A and at position 235 to A; b) an amino acid substitution at position 270 according to EU numbering to N and c) an amino acid substitution at position 331 according to EU numbering to S. Heterodimers In certain embodiments the binding molecules of the present invention may form homodimeric or heterodimeric polypeptides. The heterodimeric polypeptide may be a bispecific or multispecific binding molecule wherein the binding molecule can bind at least two antigen targets. A heterodimer or heterodimeric protein generally refers to a protein made up of two similar, but not identical subunits. An example of a heterodimeric protein is a bispecific antibody. The ability to separate homodimer contaminants from heterodimer in bispecific antibody purification is important to achieve product purity. Often this involves multi-step process with the first step being protein A affinity purification followed by ion exchange chromatography, which relies on distinctive pI separation between the homodimers and the heterodimer. Although methods such as knob-into-hole or charge pair-based mutations have been successfully used to enrich the heterodimer formation in canine bispecific antibody production, these methods cannot fully eliminate homodimer contaminants and downstream purification method development is important to resolve the heterodimer from homodimers. By reducing or abolishing the protein A binding capacity of a single Fc chain (Chain A) and maintaining full protein A binding capacity of the second Fc chain (Chain B), the resulting bispecific heterodimer (AB) will have an intermediate protein A binding strength, while the AA homodimer will not bind to the protein A column and BB homodimer will have strong protein A binding capacity, thus can be eluted with more stringent condition. Effectively, this could provide a mean of purifying canine bispecific antibodies with a single protein A affinity chromatography step. In certain embodiments the heterodimeric molecules comprise at least one binding molecule, wherein the binding molecule comprises a variant IgG Fc domain comprising an increased or decreased affinity for Protein A. In an embodiment the heterodimeric protein comprises; a) a first polypeptide comprising a variant IgG Fc domain comprising a decreased affinity for Protein A as described herein, b) a second polypeptide comprising an IgG Fc domain. In certain embodiments the first polypeptide comprises a canine variant IgG Fc domain with decreased affinity for Protein A. The variant IgG domain may be selected from a canine variant IgG-A Fc domain, a canine variant IgG-B Fc domain, a canine variant IgG-D Fc domain, wherein the variant canine Fc domain comprises one or more of the amino acid substitutions described herein as decreasing the affinity for Protein A. In certain embodiments the first polypeptide comprises a feline variant IgG Fc domain with decreased affinity for Protein A. The variant IgG domain may be selected from a feline variant IgG-1a Fc domain, a feline variant IgG-1b Fc domain, a feline variant IgG-2 Fc domain, wherein the variant feline Fc domain comprises one or more of the amino acid substitutions described herein as decreasing the affinity for Protein A. In certain embodiments the heterodimeric protein is an antigen binding protein comprising a first and a second polypeptide, the first polypeptide comprising, from N-terminal to C-terminal, a first antigen-binding region that selectively binds a first antigen, followed by a constant region that comprises a variant IgG Fc region comprising an amino acid substitution that decreases the affinity of the IgG Fc region to Protein A as described herein; and, a second polypeptide comprising, from N-terminal to C-terminal, a second antigen-binding region that selectively binds a second antigen, followed by a constant region that comprises an IgG Fc region. The IgG Fc region in the second polypeptide does not comprise an amino acids substitution that decreases the affinity of the IgG Fc region to Protein A. The IgG Fc region in the second polypeptide may be selected from a canine IgG-A, IgG-B, IgG-C, IgG-D, feline IgG-1a, IgG-1b, IgG-2, IgG-3 Fc. In an embodiment the heterodimeric protein comprises; a) a first polypeptide comprising a variant IgG Fc domain comprising an increased affinity for Protein A as described herein, b) a second polypeptide comprising an IgG Fc domain. In certain embodiments the first polypeptide comprises a canine variant IgG Fc domain with decreased affinity for Protein A. The variant IgG domain may be selected from a canine variant IgG-A Fc domain, a canine variant IgG-C Fc domain, a canine variant IgG-D Fc domain, wherein the variant canine Fc domain comprises one or more of the amino acid substitutions described herein as increasing the affinity for Protein A. In certain embodiments the first polypeptide comprises a feline variant IgG Fc domain with increased affinity for Protein A. The variant IgG domain may be selected from a feline variant IgG-1a Fc domain, a feline variant IgG-1b Fc domain, a feline variant IgG-3 Fc domain, wherein the variant feline Fc domain comprises one or more of the amino acid substitutions described herein as increasing the affinity for Protein A. In certain embodiments the heterodimeric protein is an antigen binding protein comprising a first and a second polypeptide, the first polypeptide comprising, from N-terminal to C-terminal, a first antigen-binding region that selectively binds a first antigen, followed by a constant region that comprises a variant IgG Fc region comprising an amino acid substitution that increases the affinity of the IgG Fc region to Protein A as described herein; and, a second polypeptide comprising, from N-terminal to C-terminal, a second antigen-binding region that selectively binds a second antigen, followed by a constant region that comprises an IgG Fc region. The IgG Fc region in the second polypeptide does not comprise an amino acids substitution that increases the affinity of the IgG Fc region to Protein A. The IgG Fc region in the second polypeptide may be selected from a canine IgG-A, IgG-B, IgG-C, IgG-D, feline IgG-1a, IgG-1b, IgG-2, IgG-3 Fc. In an embodiment the heterodimeric protein comprises; a) a first polypeptide comprising an IgG Fc domain, b) a second polypeptide comprising an IgG Fc domain. wherein the first and / or second polypeptide comprises a variant IgG Fc domain comprising increased stability as described herein. In certain embodiments the heterodimeric protein is an antigen binding protein comprising a first and a second polypeptide, the first polypeptide comprising, from N-terminal to C-terminal, a first antigen-binding region that selectively binds a first antigen, followed by a constant region that comprises an IgG heavy chain region; and, a second polypeptide comprising, from N-terminal to C-terminal, a second antigen-binding region that selectively binds a second antigen, followed by a constant region that comprises an IgG heavy chain region, wherein said first and / or second polypeptide comprises a variant IgG heavy chain region comprising an amino acid substitution that increases the stability of the IgG heavy chain region as described herein. In certain embodiments only one of the first or second polypeptide comprises a variant IgG heavy chain region with increased stability. The IgG heavy chain region in the first and / or second polypeptide may be selected from a canine IgG-A, IgG-B, IgG-C, IgG-D, feline IgG-1a, IgG-1b, IgG-2, IgG-3 Fc. In an embodiment the IgG heavy chain region in the first and / or second polypeptide may comprise a feline IgG-3 Fc. In certain embodiments the heterodimeric protein further comprises an immunoglobulin light chain. The immunoglobulin light chain may be a feline or canine immunoglobulin light chain. In certain embodiments, the heterodimeric protein comprises the specified amino acid substitutions and may have additional mutations in the Fc domain or in another location in the amino acid sequence. For example, the hetero dimeric protein may comprise further amino acid substitutions such as those disclosed herein which improve half-life, reduce effector function or allow charge steering. In other embodiments, the heterodimeric protein only has the specified amino acid substitutions in the Fc domain / heavy chain region and does not have any other amino acid substitutions and / or other mutations in the Fc domain / heavy chain region. In other embodiments, the heterodimeric protein only has the specified amino acid substitutions in the Fc domain / heavy chain region and does not have any other mutations in the protein sequence. Thus, in one embodiment, the amino acid substitutions provided consist of those recited. Nucleic Acids An aspect of the invention relates to an isolated nucleic acid encoding the isolated companion animal protein of the invention. In an aspect, the invention relates to a vector, plasmid, transcription, expression cassette or nucleic acid construct comprising a nucleic acid encoding the isolated companion animal protein of the invention. In an aspect, the invention relates to a host cell comprising a nucleic acid encoding the isolated companion animal protein of the invention or a vector, plasmid, vector, transcription, expression cassette or construct as described above. Expression vectors of use in the invention may be constructed from a starting vector such as a commercially available vector. After the vector has been constructed and the nucleic acid molecule has been inserted into the proper site of the vector, the completed vector may be inserted into a suitable host cell for amplification and / or polypeptide expression. The term “vector” means a construct, which is capable of delivering, and in some aspects expressing one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. The invention also relates to an isolated recombinant host cell comprising one or more nucleic acid molecule plasmid, vector, transcription or expression cassette as described above. The transformation of an expression vector into a selected host cell may be accomplished by well- known methods including transfection, infection, calcium phosphate co-precipitation, electroporation, microinjection, lipofection, DEAE-dextran mediated transfection, or other known techniques. The method selected will in part be a function of the type of host cell to be used. The host cell may be eukaryotic or prokaryotic, for example a bacterial, viral, plant, fungal, mammalian or other suitable host cell. In one embodiment, the cell is an E. coli cell. In another embodiment, the cell is a yeast cell. In another embodiment, the cell is a Chinese Hamster Ovary (CHO) cell, HeLa cell or other cell that would be apparent to the skilled person. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC) and any cell lines used in an expression system known in the art can be used to make the recombinant polypeptides of the invention. In general, host cells are transformed with a recombinant expression vector that comprises DNA encoding a protein. Among the host cells that may be employed are prokaryotes, yeast or higher eukaryotic cells. Prokaryotes include gram negative or gram-positive organisms, for example E. coli or bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the COS-7 cells, L cells, CI27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, or their derivatives and related cell lines which grow in serum free media, HeLa cells, BHK cell lines, the CVIIEBNA cell line, human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Optionally, mammalian cell lines such as HepG2 / 3B, KB, NIH 3T3 or S49, for example, can be used for expression of the polypeptide when it is desirable to use the polypeptide in various signal transduction or reporter assays. Other suitable host cells include insect cells, using expression systems such as baculovirus in insect cells, plant cells, transgenic plants and transgenic animals, and by viral and nucleic acid vectors. Alternatively, it is possible to produce the polypeptide in lower eukaryotes such as fungal cell lines and yeast or in prokaryotes such as bacteria. Suitable yeasts include S. cerevisiae, S. pombe, Kluyveromyces strains, Pichia pastoris, Candida, or any yeast strain capable of expressing heterologous polypeptides. Suitable bacterial strains include E. coli, B. subtilis, S. typhimurium, or any bacterial strain capable of expressing heterologous polypeptides. If the protein is made in yeast or bacteria, it may be desirable to modify the product produced therein, for example by phosphorylation or glycosylation of the appropriate sites, in order to obtain a functional product. Such covalent attachments can be accomplished using known chemical or enzymatic methods. A host cell, when cultured under appropriate conditions, can be used to express a protein that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted). The selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule. In another aspect, the invention also relates to the use of a binding molecule of the invention as in a fusion protein with another moiety, e.g., with a half-life extending moiety as described in more detail below. Therefore, the binding molecule of the invention can be provided covalently linked or couple to a half-life extending moiety. Alternatively, it may be provided incorporated in a liposome. In some embodiments the binding molecule is provided as in a fusion protein with another moiety e.g., a half-life extending moiety, in order to improve its pharmacokinetic (PK) properties. In a further aspect, the invention relates to a method for making a heterodimeric protein or a polypeptide comprising a binding molecule according to the invention comprising the steps of a) transforming a host cell with a nucleic acid or a vector described herein; b) culturing the host cell and expressing a first polypeptide comprising a variant IgG Fc domain as described herein second polypeptide comprising an IgG Fc domain and c) recovering the heterodimeric protein or polypeptide from the host cell culture. Pharmaceutical Compositions The binding molecules of the invention may have therapeutic uses. Therefore, in an aspect there is provided a pharmaceutical composition comprising the binding molecule or heterodimer as described herein or nucleic acid as described herein. The isolated companion animal protein provided in the pharmaceutical composition, comprises an IgG domain comprising an amino acid substitution that reduces or eliminates binding of the domain to Protein A. The isolated companion animal protein may have any one or more of the other features set out above. The pharmaceutical composition comprising the binding molecule as described herein or the heterodimer as described herein may be formulated for administration by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intratumoural, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation. In another embodiment, delivery is of the nucleic acid encoding the drug, e.g., a nucleic acid encoding the isolated companion animal protein of the invention is delivered. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical, intra-articular or subcutaneous administration. Preferably, the compositions are administered parenterally. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier or vehicle, The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term "carrier" refers to a diluent, adjuvant or excipient, with which a drug antibody conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and coloring agents can be used. In one embodiment, when administered to a subject, the polypeptide of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. The pharmaceutical composition can be in the form of a liquid, e.g., a solution, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneous. When intended for oral administration, the composition can be in solid or liquid form, where semi- solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye / colorant and flavour enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included. Compositions can take the form of one or more dosage units. In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion. The amount of the binding molecule, heterodimer or pharmaceutical composition described herein that is effective / active in the treatment of a particular disease or condition will depend on the nature of the disease or condition and can be determined by standard clinical techniques. In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disease, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Typically, the amount is at least about 0.01% of a polypeptide of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the polypeptide of the present invention by weight of the composition. Compositions can be prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the polypeptide of the present invention. For administration by injection, the composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the subject’s body weight, preferably, between about 0.1 mg / kg and about 20 mg / kg of the subject’s body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the subject’s body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks or more. Treatment can for example be once a month or bi-monthly. This is advantageous over daily administration as this improves compliance and minimises stress to the subject. As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disease. For example, treatment can include a postponement of development of the symptoms associated with a disease or disease, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the mammals, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment. The term "subject" or "patient" refers to a human which is the object of treatment, observation, or experiment. An aspect of the invention relates to the binding molecule or heterodimer of the invention for use in treatment or prevention of disease. An aspect of the invention relates to a method of treatment or prevention of disease comprising administering a therapeutically effective amount of the binding molecule or heterodimer of the invention or pharmaceutical composition to a subject in need thereof. Kits An aspect of the invention relates to a kit comprising one or more of the binding molecules according to the invention with an optimised Fc domain or heavy chain domain as described herein, and optionally instructions for use. The kit may further comprise suitable buffers, excipients and / or adjuvants etc. Methods of Purification of Molecules with Decreased Protein A Binding The binding molecules and heterodimers described herein allow for more efficient isolation and purification of said molecules. Differential binding to an affinity matrix can be manipulated by changing the pH and / or ionic strength of a solution passed over the affinity matrix. The binding molecules described herein have different affinities for Protein A and so by taking advantage of these modulated affinities and by utilising a gradient of pH / ionic strength improved separation of proteins comprising the binding molecules of the invention can be achieved. Accordingly, the present invention also relates to methods of purifying said molecules. A method for purifying a heterodimeric protein, as disclosed herein, comprising a first polypeptide comprising a variant IgG Fc domain comprising a decreased affinity for Protein A as described herein, wherein the method comprises a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the first polypeptide comprises a variant IgG Fc domain comprising a decreased affinity for Protein A, and wherein the first polypeptide has lower affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide, and b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range. In embodiments the first polypeptide comprises a variant IgG Fc domain comprising a one or more of the amino acid substitutions described herein as decreasing the affinity for Protein A. In an embodiment the mixture of multimeric proteins comprises a second homodimer comprising two copies of the first polypeptide, wherein the first polypeptide comprises a variant IgG Fc domain comprising a decreased affinity for Protein A. The second homodimer may elute at a third pH range. In one embodiment, the heterodimer is eluted from the affinity matrix in a buffer having a first pH range, and the first homodimer is eluted from the affinity matrix in a buffer having a second pH range and, in some embodiments, the second homodimer is eluted from the affinity matrix in a buffer having a third pH range. As shown in the Examples the first homodimer comprises two copies of the second polypeptide which does not comprise amino acid substitution to decrease affinity to Protein A, the heterodimer comprises one copy of a polypeptide comprising at least one amino acid substitution which decreases affinity for Protein A and the second homodimer comprises two copies of a polypeptide comprising at least one amino acid substitution which decreases affinity for Protein A. As such the affinity for Protein A of the first homodimer is greater than that of the heterodimer which is in turn greater than the affinity for Protein A of the second homodimer. The first pH range at which the heterodimer is eluted is a higher pH range (i.e. a more basic pH range) than the second pH range at which the first homodimer is eluted. Where a second homodimer is present, the third pH range at which the second homodimer is eluted is a higher pH range (i.e. a more basic pH range) than the first pH range at which the heterodimer is eluted. As shown in the examples the second homodimer may not bind to the affinity matrix and may be eluted by the wash buffer. In an embodiment the first pH range at which the heterodimer is eluted is between pH 5.5 to pH 4.0. The pH at which the heterodimer is eluted may be pH 5.5, 5.4, 5.3, 5.2, 5.1, 5.0, 4.9, 4.8, 4.7, 4.6, 4.4, 4.4, 4.3, 4.2, 4.1 or 4.0. In an embodiment the second pH range at which the first homodimer is eluted is between pH 4.0 to pH 2.7. The pH at which the first homodimer is eluted may be pH 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8 or 2.7. In an embodiment the third pH range at which the second homodimer is eluted is pH 5.5 and above, preferably pH 5.5 to pH 7.0. The pH at which the second homodimer is eluted may be pH 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.7, 6.8, 6.9, or 7.0. Prior to eluting the mixture of proteins, a wash buffer may be used to wash the affinity matrix. This wash buffer may have a pH above pH 5.5. In certain embodiments this buffer may elute one of the homodimers, wherein the homodimer comprises two copies of the polypeptide comprises one or more amino acid substitutions which decrease Protein A binding. In some embodiments of the methods of purification described herein, a first buffer is used to elute the homodimers and heterodimer. The first buffer used to elute the homodimer and heterodimers described above will have the pH range as described above. The first buffer may further comprise additional components for example the buffer may contain acetate and a chaotropic agent. The chaotropic agent can be a salt, having a cation selected from lithium, magnesium, calcium, and guanidinium, and an anion selected from chloride, nitrate, bromide, chlorate, iodide, perchlorate, and thiocyanate. In certain embodiments to chaotropic agent is selected from NaCl, CaCl2, or MgCl2. In an embodiment the concentration of the chaotropic agent is between 100 to 500 mM, 100 to 400 mM, 100 to 300 mM, 100 to 200 mM, 200 to 500 mM, 200 to 400 mM, 200 to 300 mM, 200 to 250 mM, 250 to 450 mM, 300 to 400mM. In one embodiment the chaotropic agent is NaCl and the concentration is between 200 to 500 mM, preferably approximately 200mM. In certain embodiments the buffer comprises acetate, for example sodium acetate. The concentration of sodium acetate may be between 10 to 300 mM, 10 to 250 mM, 10 to 200 mM, 10 to 150 mM, 10 to 100 mM, 10 to 50 mM, 50 to 300 mM, 5 to 250 mM, 50 to 200 mM, 50 to 150 mM, 50 to 100 mM. In an embodiment the sodium acetate is present at approximately 200 mM. The first buffer may have a pH between pH 4.0 and pH 5.5, the buffer may be applied with a pH gradient spanning the pH range of pH 4.0 to 5.5. In some embodiments methods of purification described herein, a second buffer is used to elute the homodimers and heterodimer. The second buffer may comprise glycine-HCl The concentration of the glycine-HCl may be between 10 to 300 mM, 10 to 250 mM, 10 to 200 mM, 10 to 150 mM, 10 to 100 mM, 10 to 50 mM, 50 to 300 mM, 5 to 250 mM, 50 to 200 mM, 50 to 150 mM, 50 to 100 mM. In an embodiment the glycine-HCl is present at approximately 100 mM. The glycine-HCl buffer may have a pH between pH 4.0 and 2.5. The glycine-HCl buffer may be applied with a pH gradient spanning pH 4.0 and 2.5, alternatively it may be applied at a set pH for example pH 2.7. Methods of Purification of Molecules with Increased Protein A Binding A method for purifying a heterodimeric protein, as disclosed herein, comprising a first polypeptide comprising a variant IgG Fc domain comprising an increased affinity for Protein A as described herein, wherein the method comprises a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the first polypeptide comprises a variant IgG Fc domain comprising an increased affinity for Protein A, and wherein the first polypeptide has a greater affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide, and b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range. In embodiments the first polypeptide comprises a variant IgG Fc domain comprising a one or more of the amino acid substitutions described herein as increasing the affinity for Protein A. In an embodiment the mixture of multimeric proteins comprises a second homodimer comprising two copies of the first polypeptide, wherein the first polypeptide comprises a variant IgG Fc domain comprising an increased affinity for Protein A. The second homodimer may elute at a third pH range. In one embodiment, the heterodimer is eluted from the affinity matrix in a buffer having a first pH range, and the first homodimer is eluted from the affinity matrix in a buffer having a second pH range and, in some embodiments, the second homodimer is eluted from the affinity matrix in a buffer having a third pH range. In the method of purifying mixture of multimeric proteins wherein the multimeric protein comprises a variant IgG engineered to have increase affinity to Protein A, the first homodimer comprises two copies of the second polypeptide which does not comprise amino acid substitution to increase affinity to Protein A, the heterodimer comprises one copy of a polypeptide comprising at least one amino acid substitution which increases affinity for Protein A and the second homodimer comprises two copies of a polypeptide comprising at least one amino acid substitution which increases affinity for Protein A. As such the affinity for Protein A of the second homodimer is greater than that of the heterodimer which is in turn greater than the affinity for Protein A of the first homodimer. The first pH range at which the heterodimer is eluted is a lower pH range (i.e. a more acidic pH range) than the second pH range at which the first homodimer is eluted. Where a second homodimer is present, the third pH range at which the second homodimer is eluted is a lower pH range (i.e. a more acidic pH range) than the first pH range at which the heterodimer is eluted. In an embodiment the first pH range at which the heterodimer is eluted is between pH 2.5 to pH 4.0. The pH at which the heterodimer is eluted may be pH 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9 or 4.0. The methods of purification described herein may comprise using a buffer comprising a chaotropic agent. In an embodiment of the methods of purification described herein the affinity matrix comprises Protein A affixed to a substrate, the substrate may be a solid support. The substrate or solid support may be agarose, poly(styrene divinylbenzene), polymethacrylate, controlled pore glass, spherical silica, cellulose. The methods of purification may comprise a step of loading the mixture of multimeric proteins onto the affinity matric. The mixture of proteins may be applied a concentration of 1 to 100 mg / mL, 10 to 100 mg / mL, 20 to 100 mg / mL, 30 to 100 mg / mL, 40 to 100 mg / mL, or 50 to 100 mg / mL. The pH gradients used, in the methods of purification described herein, to elute the heterodimer and homodimer may be applied in a step-wise manner or a continuous gradient. Further aspects and embodiments of the invention will be apparent to those skilled in the art given the present disclosure including the following experimental exemplification. Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety, including any references to gene accession numbers and references to patent publications. "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. The term “comprising” or “comprises” where used herein means including the component(s) specified but not to the exclusion of the presence of other components. The term “consisting essentially of” or “consists essentially of” means including the components specified but excluding other components except for materials present as impurities, unavoidable materials present as a result of processes used to provide the components and the like, further they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. The term “consisting of” or “consists of” means including the components specified but excluding other components. Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists essentially of” or “consisting essentially of”. Whenever appropriate, depending upon the context, the use of the term “comprises” or “comprising” may also be taken to include the meaning “consists of” or “consisting of”. The optional features set out herein may be used either individually or in combination with each other where appropriate and particularly in the combinations as set out in the accompanying claims. The optional features for each aspect or exemplary embodiment of the invention, as set out herein are also applicable to all other aspects or exemplary embodiments of the invention, where appropriate. In other words, the skilled person reading this specification should consider the optional features for each aspect or exemplary embodiment of the invention as interchangeable and combinable between different aspects and exemplary embodiments. The invention is further described in the following non-limiting numbered embodiments. Numbered Embodiments 1. A binding molecule comprising at least one variant IgG Fc domain comprising at least one amino acid substitution that reduces binding affinity to Protein A relative to the wild-type IgG domain, wherein the binding molecule is a companion animal binding molecule. 2. The binding molecule according to embodiment 1, wherein the variant IgG Fc domain comprises one or more of the following amino acid substitutions, a. an amino acid substitution at position 252, b. an amino acid substitution at position 253, c. an amino acid substitution at position 309, d. an arginine at position 311, i.e.311R, e. an amino acid substitution at position 315, f. an amino acid substitution at position 433, g. an amino acid substitution at position 435, and / or h. an amino acid substitution at 436, wherein the positions are numbered according to the EU numbering system. 3. The binding molecule according to embodiment 1 or 2, wherein said IgG domain comprises: a. an amino acid substitution at one or more of position 252, 253 and / or 315 according to EU numbering; b. an amino acid substitution at one or more of position 252, 253, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; c. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; d. an amino acid substitution at one or more of position 252, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; e. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; f. an arginine at position 311 according to EU numbering; g. an amino acid substitution at one or more of position 253 and / or 435 according to EU numbering; h. an amino acid substitution at position 435 according to the EU numbering system; i. an amino acid substitution at position 436 according to EU numbering; or j. an amino acid substitution at one or more of position 309 and / or 315 according to EU numbering. 4. The binding molecule according to any of embodiments 2 or 3, wherein the amino acid substitution at position 252 comprises 252V or 252R, the amino acid substitution at position 253 comprises 253T or 253D, the amino substitution at position 309 comprises 309E or 309V or 309Q, the amino acid substitution at position 311 comprises 311R , the amino acid substitution at position 315 comprises 315S or 315D or 315T, the amino acid substitution at position 433 comprises 433A, the amino acid substitution at position 435 comprises 435R or 435Q or 435A, the amino acid substitution at position 436 comprises 436F. 5. The binding molecule according to any preceding embodiment, wherein the IgG domain is canine and is selected from a canine IgG-A Fc domain, a canine IgG-B Fc domain, or a canine IgG-D Fc domain. 6. The binding molecule according to any of embodiments 1 to 4, wherein the IgG domain is feline and is selected from a feline IgG1a Fc domain, a feline IgG1b Fc domain, or a feline IgG2 Fc domain. 7. The binding molecule of any of the preceding embodiments, wherein the variant IgG Fc domain is a CH2 or CH3 domain. 8. The binding molecule according to embodiment 5, wherein the variant IgG Fc domain is a canine IgG-A domain and comprises one or more of the following amino acid substitutions R252V, I253T I253D, E309V, E309Q, Q311R, T315S, T315D Q433A, H435R, H435A, and / or Y436F. 9. The binding molecule according to embodiment 5, wherein the variant IgG Fc domain is a canine IgG-B domain and comprises one or more of the following amino acid substitutions L252V, L252R, I253T, I253D, G309E, G309V, G309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435A, and / or Y436F. 10. The binding molecule according to embodiment 5, wherein the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following amino acid substitutions R252V, I253T, I253D, E309V, E309Q, Q311R, T315S, T315D, Q433A, H435R, H435A, and / or Y436F. 11. The binding molecule of embodiment 6 wherein the variant IgG Fc domain is a feline IgG1a and comprises one or more of the following amino acid substitutions S252V, S252R, I253T, I253D, L309V, L309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435Q, H435A, and or H436F 12. The binding molecule of embodiment 6 wherein the variant IgG Fc domain is a feline IgG1b and comprises one or more of the following amino acid substitutions S252V, S252R, I253T, I253D, L309V, L309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435Q, H435A, and or H436F 13. The binding molecule of embodiment 6 wherein the variant IgG Fc domain is a feline IgG2 and comprises one or more of the following amino acid substitutions S252V, S252R, I253T, I253D, L309V, L309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435Q, H435A, and or H436F. 14. The binding molecule of embodiment 5 wherein the canine IgG-B domain comprises the following amino acid substitutions a. I253T, L252V and K315S; b. I253T, L252V, K315S and Q311R; c. L252V and K315S, or d. I253T and H433A 15. A binding molecule comprising at least one variant IgG Fc domain comprising an amino acid substitution at position 315 which increases binding affinity to Protein A relative to the wild- type IgG domain according to EU numbering, wherein the binding molecule is a companion animal binding molecule. 16. The binding molecule according to embodiment 15, wherein the variant IgG Fc domain further comprises an amino acid substitution at one or more of any of positions 252, 253, 309, 433 according to EU numbering 17. The binding molecule according to embodiment 15 or 16, wherein the variant IgG Fc domain comprises: a. an amino acid substitution at one or more of position 252, 253 and / or 315 according to EU numbering; or b. an amino acid substitution at one or more of position 252, 315, and / or 433 according to EU numbering. 18. The binding molecule according to any one of embodiments 16 or 17, wherein the amino acid substitution at position 252 comprises 252L, the amino acid substation at position 253 comprises 253I, the amino acid substitution at position 309 comprises 309L, the amino acid substitution at position 315 comprises 315K, the amino acid substitution at position 433 comprises 433H. 19. The binding molecule according to any one of embodiments 15 to 18, wherein the IgG Fc domain is canine and is selected from a canine IgG-A Fc domain, a canine IgG-C Fc domain or a canine IgG-D Fc domain. 20. The binding molecule according to any one of embodiments 15 to 18, wherein the IgG Fc domain is feline and is selected from a feline IgG1a Fc domain, a feline IgG1b Fc domain, or a feline IgG3 Fc domain. 21. The binding molecule according to embodiment 19, wherein the variant IgG Fc domain is a canine IgG-A domain and comprises one or more of the following amino acid substitutions R252L, E309L, T315K, Q433H. 22. The binding molecule according to embodiment 19, wherein the variant IgG Fc domain is a canine IgG-C domain and comprises one or more of the following amino acid substitutions V252L, T253I, G309L, S315K. 23. The binding molecule according to embodiment 19, wherein the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following amino acid substitutions R252L, E309L, T315K, Q433H. 24. The binding molecule of embodiment 20 wherein the variant IgG Fc domain is a feline IgG1b and comprises the following amino acid substitution S252L. 25. The binding molecule of claim 20 wherein the variant IgG Fc domain is a feline IgG2 and comprises the amino acid substitution S252L. 26. The binding molecule of embodiment 20 wherein the variant IgG Fc domain is a feline IgG3 and comprises one or more of the following amino acid substitutions S252L, V309L, T315K. 27. The binding molecule of embodiment 19 wherein the variant IgG Fc domain is a canine IgG-A Fc domain and comprises R252L, T315K and Q433H. 28. The binding molecule of embodiment 19 wherein the variant IgG Fc domain is a canine IgG-C Fc domain and comprises V252L, T253I and S315K. 29. The binding molecule of embodiment 19 wherein the variant IgG Fc domain is a canine IgG-D Fc domain and comprises R252L, T315K and Q433H. 30. The binding molecule of embodiment 20, wherein the variant IgG Fc domain in a feline IgG3 Fc and comprises V309L and / or T315K. 31. The binding molecule of any of the preceding embodiments, wherein the IgG domain is a CH2 or CH3 domain 32. A binding molecule comprising at least one variant IgG heavy chain comprising an amino acid substitution in the CH3 domain and / or in the hinge domain that increases stability relative to the wild-type IgG heavy chain, wherein the binding molecule is a companion animal binding molecule. 33. The binding molecule according to embodiment 32, wherein the variant IgG heavy chain comprises an amino acid substitution at position 409 according to EU numbering. 34. The binding molecule according to embodiment 32 or 33 wherein the amino acid substitution at position 409 comprises 409K. 35. The binding molecule according to any one of embodiments 32 to 34, wherein the variant IgG heavy chain is selected from a feline IgG-1b heavy chain, a feline IgG-2 heavy chain or a feline IgG-3 heavy chain. 36. The binding molecule according to embodiment 32, wherein the variant IgG heavy chain comprises amino acid substitution at one or more of positions 219, 220, 221, 222, 223, 224, 225, and / or 226 according to EU numbering. 37. The binding molecule according to embodiment 36, wherein the variant IgG heavy chain is a feline IgG2 heavy chain. 38. The binding molecule according to embodiment 36 or 37, wherein the variant IgG heavy chain comprises one or more of the following amino acid substitutions, A219D, S220H, T221P, I222P, E223G, S224P, T225aP, G225bC, E225cD, G226C. 39. The binding molecule according to any one of embodiments 36 to 38, wherein the IgG heavy chain comprises: a. A219D, S220H, T221P, I222P, E223G, S224P, T225aP, G225bC, E225cD, and G226C, or b. G225bC, E225cD and G226C 40. The binding molecule of any of the preceding embodiments, wherein the protein is a heterodimeric protein. 41. The binding molecule of any of the preceding embodiments, wherein the IgG domain comprises one of SEQ ID NO:SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 42, SEQ ID NO: 43 or a sequence with at least 90% or 95% sequence identity thereto. 42. A pharmaceutical composition comprising the binding molecule of any of the preceding embodiments. 43. A nucleic acid encoding a binding molecule of any of the preceding embodiments. 44. A vector comprising a nucleic acid according to embodiment 43. 45. A host cell comprising a nucleic acid according to embodiment 43 or a vector according to embodiment 44. 46. A kit comprising a protein of any of embodiments 1 to 39 or a pharmaceutical composition according to embodiment 42. 47. A binding molecule according to any of embodiments 1 to 39 wherein the variant IgG Fc domain or variant IgG heavy chain domain is non-immunogenic or substantially non- immunogenic in a companion animal. 48. A method for purifying a heterodimeric protein comprising a binding molecule according to any of embodiments 1 to 14, comprising a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the first polypeptide comprises a variant IgG Fc domain comprising a decreased affinity for Protein A, and wherein the first polypeptide has lower affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide; and b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range. 49. A method for purifying a heterodimeric protein comprising a binding molecule according to any one of embodiments 15 to 31, comprising c. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the first polypeptide comprises a variant IgG Fc domain comprising an increased affinity for Protein A, and wherein the first polypeptide has greater affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide; and d. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range. 50. The method according to embodiments 48 or 49, wherein the mixture of multimeric proteins comprises a second homodimer comprising two copies of the second polypeptide. 51. The method according to any one of embodiments 48 to 50, wherein the affinity matrix comprises a Protein A ligand affixed to a substrate. 52. The method according to any one of embodiments 48 to 51, comprising the step of applying a pH gradient to the loaded affinity matrix of step (a). The invention is further described in the non-limiting examples. EXAMPLES Example 1: Protein A binding capacity of wild type Canine IgG isotypes To understand wild type protein A binding capacity of different canine IgG isotypes, we generated chimeric antibodies with human ofatumumab variable region (ofa) (sequence available on Drugbank https: / / go.drugbank.com / drugs / DB06650) joined to canine IgG-A, IgG-B, IgG-C, and IgG-D (“dIgG”). The amino acid sequences for wild type canine IgG-A, IgG-B, IgG- C, and IgG-D and (SEQ ID NO: 1 to 4) are given in Table 1. Sequence alignments are shown in Figure 1A with human EU numbering scheme. To generate such antibody expressing constructs, the variable regions and Fc were PCR amplified using Q5 high fidelity DNA polymerase and assembled into standard mammalian expression vectors using NEBuilder HIFI DNA Assembly (New England Biolabs). In the expression vectors, the heavy chain and the antibiotic resistant gene expression units are flanked by DNA transposon piggyBac terminal inverted repeats to mediate stable integration into host cells in the presence of piggyBac transposase. The expression vectors encoding the heavy chain and light chain were co-transfected into a suitable mammalian cell line such as CHO cells together with PiggyBac transposase to obtain stable transgene integration. For antibody production, 3 × 106selected CHO cells were seeded in 3 ml culture media and incubated at 32 C, 5% CO2 with shaking at 200 rpm.4 % HyClone Cell Boost 7a supplement + 0.4 % HyClone Cell Boost 7b supplement + 1 % glucose was added to the media on days 1, 4, 7 and 10. Culture supernatants were collected on day 12 and the IgG concentration determined using surface plasmon resonance using protein A chip (Biacore 8K, Cytiva Life Sciences). Harvested supernatant from CHO (ofa-IgGA,B,C and D) expressing cells were loaded on 5mL MabSelect SuRe (Cytiva) column using PBS as a running buffer, washed with PBS and eluted with 0.1M Glycine pH2.7 and immediately neutralised with 10% 1M TRIS-HCl pH 8. Purified protein samples were diluted from 1uM to 4nM (6 concentrations with 1:3 dilutions) in Running Buffer and kinetics were assessed using a single cycle kinetics method (Biacore Assay Handbook, Cytiva). Purified proteins were concentrated using centrifugal concentrators (Sartorious - VS02H22) to 5 mg / mL. Protein concentration was assessed using UV absorbance at 280nm with NanoDrop™ One (Thermo Scientific™). Binding affinity to Protein A was assessed using Biacore 8K (Cytiva). Briefly, Sensor Chip Protein A (Cytiva) was docked into Biacore 8K, equilibrated for 30 minutes at RT and then Running Buffer (10mM HEPES pH7.4150mM NaCl 3mM EDTA and 0.005% Tween20) was applied to the SPR chip surface. Kinetics and / or Affinity quantification were performed using Biacore Insight following standard analyses methods. The protein A binding kinetics and affinity for wild type canine isotypes are shown in Figure 2 and Table 2 Kinetics values are based on 1:1 Langmuir fitting. IgG-B has the strongest affinity and slowest off-rate. This binding kinetics profile is very similar to human IgG1. The weaker protein A binding affinity of IgG-A are IgG-D are due to their fast off-rate. IgG-C has the weakest protein A binding capacity, mainly due to its slow on-rate and low Rmax. These results suggests that, apart from IgG-B, the other three main isotypes are not suitable for protein A based purification for manufacturing purposes. Enhancement of their protein A binding could fix this issue. Equally, generating protein A deficient variants of IgG-B could be attractive to applications in bispecific antibody purification. Example 2: Generation and characterisation of canine variants with enhancing and reducing A binding properties Based on the protein A binding domain knowledge in human and mouse Fcs, three regions of the Fc in CH2 and CH3 domains of canine Fc isotypes were identified as protein A interaction sites. Structural analysis was performed (see Figure 3A) and the model was used to calculate impact of alanine mutations on single residues. DeltaDeltaG (thermodynamic stability) energy values were used to predict the positions within these three protein A binding regions for the impact on protein A binding Figure 3B. Delta values (DDG and DG) are indicative of energy changes in either interaction between Fc-Protein (DDG) or within Fc only (DG). Positive values indicate reduced strength / stability and vice versa. Residues 252, 253, 311, 433, 434 and 435 of canine IgG-B were identified as those predicted to destabilise canine protein A binding capacity of canine IgG-B. Interestingly, by aligning sequences in the protein A binding regions for the four canine isotypes, we discovered the sequence variations between IgG-B and other three Fc isotypes were residues 252, 253, 315, 433 (Figure 4). Based on these results, we generated a series of protein A variants for IgG-A, IgG-C, and IgG-D with the aims to enhancing their protein A binding capacity. We also generated variants for IgG-B aiming at abolishing its protein A binding capacity. The variant sequences are shown in Figure 4. The canine IgG protein A mutant variants were generated by PCR based site directed mutagenesis. As before, chimeric antibodies of these Fc variants were generated with human ofatumumab as the variable region as described above. Construct cloning, antibody production, purification and protein A binding kinetics and affinity were as described in Example 1. Mutant variants were analysed for Protein A binding using Biacore 8K. Figure 5 shows a Sensogram showing raw data (grey dotted lines) and fitting obtained with 1:1 Langmuir fit (black solid lines). Single point mutation R252L in IgG-A (A1 variant) and IgG-D (D1 variant) and T253I in IgG-C (C1 variant) were able to significantly increase protein A affinity, highlighting the importance of the hydrophobic interaction particularly around the CH2 domain with protein A. T253I and R252L improved the association phase and the on rate while the relative fast off-rate could not be rescued. The addition of further mutations to Lysine (K) at position 315 and to Histidine (H) at position 433 in IgG-A, IgG-C and IgG-D(A3 variant for IgG-A and D3 variant for IgG-D) resulted in improved off rate. Conversely, a single point mutation I253T and H435A in IgG-B significantly reduced protein A binding in terms of off-rate and R-max. Taken together, this data suggests that different residues in different protein A binding regions have distinct influence on protein A binding kinetics, and additive effect of these positions constitute the full binding characteristics as observed in IgG-B. Further characterisations of canine protein A Fc variants for FcRn binding and biophysical properties Since the same Fc regions involved in protein A binding is also involved in FcRn binding, FcRn binding characteristics were examined for the wild type canine Fc isotypes and the mutated variants. FcRn binding affinity was measured using Biacore 8K (Cytiva). A CM5 Sensor Chip (Cytiva) was docked into Biacore 8K, equilibrated for 30’ at RT and then Running Buffer (10mM HEPES pH6150mM NaCl 3mM EDTA and 0.005% Tween20) was applied to the SPR chip surface. Canine FcRn-B2M recombinant protein (Immunitrack, ITF12 - ITF08) was diluted into 10mM acetate buffer pH4.5 at 22nM (1:1000 dilution from stock) and immobilised using a standard amine coupling reaction. Chimeric antibodies dilutions were prepared from 3uM to 37nM (5 concentrations with 1:3 dilutions) in Running Buffer and kinetics was assessed using multi-cycle kinetics method (30sec association – 300sec dissociation). Kinetics and / or Affinity quantifications were performed using Biacore Insight following standard analyses methods. The FcRn binding affinity is shown in Figures 6A and B. Wild type IgG-A, IgG-C and IgG-D had undetectable FcRn binding affinity, and only IgG-B wild type was measurable in this setting. Interestingly protein A enhancing mutant variants, A1 (R252L) for IgG-A and C1 T253I for IgG- C, also resulted in enhancement of FcRn binding. Conversely, single point mutation in I253T abolishes IgG-B FcRn binding capacity. This suggests that the hydrophobic region in CH2 domain 1 centred around position 252 and 253 are important for interacting with FcRn. Surprisingly, the inability of FcRn binding to IgG-D could not be rescued by reversion of IgG-B like R252L mutation (D1 variant) and in combination with T315K and Q433H. To examine whether these variants could also alter the chimeric antibodies biophysical properties, size exclusion chromatography (H-SEC) and cation exchange chromatography (H- SCX) were used to examine the oligomeric and charge variation, respectively. HPLC-SEC chromatography (column: XBridge Premier Protein SEC 250A 2.5um, 7.8 x 150mm Column) was performed using ACQUITY H-class Bio from WATERS using 0.5x PBS as mobile phase at flow rate of 1mL / min. HPLC-SCX chromatography (column: BioResolve SCX mAb Column, 3 µm, 4.6 mm x 100 mm) was performed using ACQUITY H-class Bio from WATERS using 50mM MES pH5 as mobile phase with salt gradient to 500mM NaCl used to separate charge variants at a flow rate of 0.9mL / min. Integration of each chromatography peak was performed using Empower 3 software, MW (for HSEC) or charge (for HSCX) standards have been included in each run. IgG-B and IgG-C isotypes have the best monomeric content and charge homogeneity shown in H-SEC and HSCX analysis respectively (Figure 7). Apart of IgG-C3 and IgG-D3 variants, monomeric content was not significantly changed for the engineered variants tested. In terms of charge homogeneity, IgG-A3 and IgG-D3 showed a reduction, while other tested mutant variants remain unchanged. Example 3: Utility of reduced protein A binding IgG-B variants in improving canine bispecific antibody purification A series of IgG-B mutants (B3, B6, 428, 429, 431, 432, and 436, Figure 4) were tested. These mutants aimed at abolishing protein A binding ability in a fully canine bispecific antibody format, where a mutant in the first chain is combined together with a wild type IgG-B protein A binding domain in the second chain. In addition, charge pair mutations (as described in WO 2021 / 214460 A1) were introduced to both chains to improve heterodimerisation in combination with 428, 429, 431, 432, and 436 mutation variants (Figure 4). A schematic is shown in Figure 8. Bispecific canine antibody production was as described before in Example 1. To selectively purify heterodimer in a single protein A step, a pH gradient protocol was applied. Briefly, harvested CHO supernatant was spiked with 5M NaCl to create a final concentration of 0.83M NaCl. Samples were loaded on a 5mL MabSelect SuRe (Cytiva) column using PBS as a running buffer. The column was washed with 200mM Sodium Acetate, 300mM NaCl at pH5.5, before initiating a 30CV gradient into 200mM Sodium Acetate, 300mM NaCl at pH4. The final elution step was performed with 100mM GlycineHCl at pH 2.7. All peaks from the gradient elution and final glycine elution were collected and concentrated and subjected to HSCX analysis and using charge variation as a readout for proportion of heterodimer and homodimer. Although single I253T (B3 variant) and H435A (B6 variant) protein A reducing mutation can completely eliminate AA homodimer, i.e., both chains containing the protein A binding mutation, heterodimer AB cannot be effectively separated from BB homodimer, i.e. both chains containing non-mutated IgG-B protein A binding domains (Table 3). This data suggests that single mutation I253T and H435A are not sufficient to reduce the protein A binding affinity sufficiently to allow the separation of heterodimer AB from homodimer BB with two wild type protein A binding domains. To further reduce the protein A binding capacity, mutants 428, 429, 431, 432, and 436 were tested. These mutations were designed based on the variants present in IgG-C (position L252V, I253T, K315S,) with the assumption that IgG-C is completely protein A binding deficient, and these positions contribute to this characteristic. In addition, Q311R was tested in different combinations with the IgG-C like mutations as this is a known mutation to reduce protein A binding capacity in the context of mouse Fc (Zwolak A et al 2017 Scientific reports 7:15521, 1- 11). The protein A pH gradient elution profiles for bispecific antibodies containing mutation variants 428, 429, 431, 432, and 436 together with charge pair mutations were overall very similar, with a protein peak eluting from the column at a pH of 5.1 during the acetate gradient. Consistently, a second peak was detected during the glycine elution in all separations. An additional peak at the beginning of the pH gradient around pH5.4 was detected in variant 431. An example trace for variant 428 is shown in Figure 9. All recovered peaks were collected, concentrated, and subjected to HPLC cation ion exchange analysis and intact mass analysis by LC-MS. Overall, all variants tested achieved around 90% or above heterodimer content in the peak at pH 5.1 in the acetate gradient (Figure 9A). A zoomed in version of the elution profile of molecule 428 during the gradient purification is shown in Figure 9B. The additional peak at pH 5.4 for variant 431 consisted of protein A binding mutated homodimer AA, suggesting that the specific mutation combination may not be completely protein A deficient, and still retains some column retention capacity at neutral pH. The glycine pH 2.7 peak for all variants consisted around 90% or more homodimer BB, i.e., the protein A proficient homodimer as expected. Total protein yield from all fractions were also analysed and the results are summarised in Table 4. overall % of heterodimer to total. LC-MS analysis was performed on each eluted peak for all variants using Waters BioAccord System with Acquity Premier using Acquity Premier BEH C4 300Å 1.7µ 2.1 x 50mm and ACQUITY UPLC® I-Class Plus from WATERS. 20ul of sample was treated with 500 units of PNGase F (New England Biolabs) and incubated at 37⁰C for a minimum of one hour to remove attached glycans. The samples were then separated using a gradient of water +0.1% formic acid and acetonitrile +0.1% formic acid. Intact mass analysis showed good molecular weight matches for heterodimers, i.e., the bispecific antibody, and homodimers, along with matches for the proteins with either 1 or 2 glycan groups not removed by the PNGase F. An example heterodimer peak from 428 variant containing homogenous fully canine bispecific antibody is demonstrated in Figure 10. Although the H435A (B6 variant) protein A reducing mutation was able to completely eliminate AA homodimer, i.e., both chains containing the protein A binding mutation, heterodimer AB could not be effectively separated from BB homodimer, i.e., both chains containing non-mutated IgG- B protein A binding domains (Table 3). A further mutation H435R / H436F (437 variant), described in the human literature to be utilised for bispecific purification, was assessed to determine if any improvements in the canine IgG-B bispecific molecule purification could be achieved. The protein A pH gradient elution profiles for this mutant variant (H435F / H436F – 437 SEQ ID NO: 156) and the 428 variant which could effectively separate homodimers BB from heterodimer AB, were compared. Overall, the elution peaks appeared very similar, with a protein peak eluting from the column at a pH of 5.1 during the acetate gradient (Figure 24A). Consistently, a second peak was detected during the glycine elution. No additional peaks at the beginning of the pH gradient around pH5.4 were detected for this variant (Figure 24A). All recovered peaks were collected, concentrated, and subjected to intact mass analysis by LC-MS (Figures 24B- 24C). Intact mass analysis showed good molecular weight matches for heterodimers, i.e., the bispecific antibody. In comparison to variant 428 which showed 100% heterodimeric protein following analysis by LC-MS, variant 437 had significant homodimer BB contaminant (21% homodimer BB (ProtA+ / +), 79% heterodimer AB (ProtA+ / -) de-glycosylated version). The total protein yield from all fractions were analysed and the percentage of heterodimer to total results are summarised in Figure 24D. Quantification of HSCX heterodimers / homodimers For heterodimer / homodimer quantification, the method of HSCX as described above was followed. Each different fraction from MabSelect purification showed peaks at different RT, corresponding to either homodimers AA (ProtA- / -), heterodimers AB (ProtA+ / -), and homodimers BB (ProtA+ / +). Particularly, early acetate elution showed two peaks the first at RT potentially corresponding to heterodimers (i.e. ~23min, based on protein charges prediction from aa seq) and at later RT corresponding to homodimers AA (ProtA- / -). The acetate peak showed mainly one peak in each variant tested corresponding to RT of heterodimers. Glycine fractions showed two peaks, one at RT close to 21-22min corresponding to homodimers BB (based on charge prediction) and another at 23-24min potentially corresponding to heterodimers. Example of acetate peak chromatogram from HSCX as well as % of heterodimers found in acetate peak for all samples have been shown in Figures 11A-11B. After the identity and composition of each peak was confirmed by intact mass and HSCX, analyses of titre, purity and enrichment of heterodimers was performed. In the first set of molecules (lacking charge pair mutations to favour heterodimerisation), a preferred expression of homodimers BB (IgG-Bwt) is quite evident, in contrast to heterodimers AB / homodimers AA (see Table 3). Interestingly, even if the expression of chain B is preferred over chain A, single protein A deficient mutations (B3 and B6) are still capable of increasing the heterodimer content around 2-fold compared to wt molecules. The total titre of individual species from the second set of molecules tested is reported in Table 4. Results from both AKTA and HSCX quantification showed that all protein A mutations together with charge pair mutations increased the total yield of heterodimers recovered compared to the charge pair mutation alone. Protein A and FcRn binding characteristics of fully canine bispecific antibodies containing a single chain protein A deficient mutation variants Since single Fc protein A binding capacity is important for manufacturability, protein A binding capacity was determined with bispecific antibodies when a protein A deficient single chain is used. In addition, since protein A and FcRn binding to IgG-Fc significantly overlap, determination of FcRn binding capacity is also important for such a format. To this end, protein A binding affinity determination for homodimer AA (both chain protein A deficient), heterodimer AB (single chain protein A deficient) and homodimer BB (both chain protein A proficient) were conducted using SPR method as described earlier in Example 2 and 3 respectively. The protein A binding and Canine FcRn binding affinity results were summarised in Table 4 and 5. As expected, protein A binding affinity of homodimers AA with different protein A mutation variants are not detectable, while heterodimers AB are 10-100 fold weaker than homodimers BB in affinity, yet could be recovered sufficiently using a standard protein A resin MabSelect SuRe LX. Canine FcRn binding affinity of heterodimers AB generated from different protein A mutation variants were measured. With the exception of 436, the affinity of all variants lies within approximately 3 fold difference to wild type IgG-B Fc. This is in sharp contrast to dual chain protein A deficient Fc shown in Example 3, even a single mutation I253T (B3) and H435A (B6) can abolish FcRn binding resulting in undetectable KD by SPR Figure 6A. This data suggests that a single wild type FcRn binding site in the heterodimer is sufficient for FcRn bispecific antibody interaction, which usually correlates with similar in-vivo plasma half-life as its corresponding IgG wt isotype. This has also been observed in the context of human and mouse bispecific heterodimers with protein A binding modulations (Zwolak et al Scientific Reports (2017) 7:15521; Smith et al Scientific reports (2016) 5, 17943). Example 4: Modulating Protein A Binding Capacity of Feline IgG Isotypes We have previously shown that feline IgG1 is capable of binding to protein A with nanomolar affinity (WO 2023 / 012486). Likewise, the protein A binding characteristics of feline IgG2 have been described in Strietzel et al (Strietzel et al Veterinary Immunology and Immunopathology (2014) 158;214-223) who showed that both feline IgG1 and IgG2 are capable of binding to protein A with similar nanomolar affinities (KD value from 13nM to 43nM). At present no published data is available with regard to feline IgG3. Production of feline protein A modulated antibody constructs To determine regions responsible for binding of the Fc to protein A, sequences for the three known natural feline IgG were aligned and differences in known regions involved in binding of protein A, as determined in other species including humans and dogs, were compared. Based on sequence conservation we predicted that feline IgG3 would have differing ability to bind protein A, compared to IgG1 and IgG2 (Figure 12A). The amino acid sequences for wild type feline IgG 1, 2 and 3 (SEQ ID NO: 20, 21 and 22) are given in Table 1. Figure 12A illustrates amino acid substitutions to generate protein A deficient IgG1 (IgG1 SpA-) and protein A proficient IgG3 (IgG3 SpA+) variants, corresponding amino acid sequences can be found in Table 1 (SEQ ID NO: 23 to 35). DNA constructs were generated to encode chimeric antibodies comprising selected feline IgG constant regions fused to human ofatumumab variable regions (as described above) and variable regions specific for a second antigen, designated “antigen B”. The cloning strategies, protein A binding variant generation, antibody production and purification procedures are as described for canine antibodies in Example 1 and Example 2. Chimeric antibodies containing all 3 wild type IgG isotypes were assessed by non-reducing SDS-PAGE (using 4-12% Bis-Tris NuPAGE gels, MES SDS running buffer and SeeBlue Plus 2 standards, ThermoFisher), Figure 12B. While IgG1 and IgG3 show molecular weight consistent with 4 chain intact antibody, IgG2 containing antibody contains a smaller species with molecular weight consistent with heavy and light chain, demonstrating hinge disulphide bond instability. Protein A binding affinity characterisation To confirm the protein A binding affinity, Biacore 8K (Cytiva) was used to determine the different mutant IgGs binding affinity compared to their wild type counterparts. Briefly, Sensor Chip Protein A (Cytiva) was docked into Biacore 8K, equilibrated for 30’ at RT and then Running Buffer (10mM HEPES pH7.4150mM NaCl 3mM EDTA and 0.005% Tween20) was applied to the SPR chip surface. Antibodies dilutions were prepared by diluting IgG1-WT (SEQ ID NO: 20) IgG1-SpA- (SEQ ID NO: 25), IgG3-WT (SEQ ID NO: 22) and IgG3-SpA+ (SEQ ID NO: 35) from 500nM to 31.25nM (5 concentrations with 1:2 dilutions) in Running Buffer (10mM HEPES pH7.4150mM NaCl 3mM EDTA and 0.005% Tween20) and kinetics was assessed using single cycle kinetics method (Biacore Assay Handbook, Cytiva). Kinetics and / or affinity quantification have been performed using Biacore Insight following standard analyses methods. The results show that feline IgG1 has high binding affinity to protein A (KD: 4.74e-9 M) whereas feline IgG3 has much lower affinity (KD: 2.01 e-7M) (Figure 13). Mutating two amino acids (L309V and K315T) in IgG1 to IgG3 like significantly diminishes its binding affinity (affinity of 4.74e-9M to 7.91e-7M). Conversely, mutating these two amino acids in IgG3 to IgG1 like (V309L and T315K) significantly increases its protein A binding affinity (affinity of 2.01e-7M to 7.91e- 9M). To confirm if both the position 309 and 315 mutations are required to alter the protein A binding capacity of feline IgG Fc’s, DNA constructs were generated containing each of the mutations alone and then compared to those mutations in combination. Protein A proficiency resulting from individually mutating T315K or V309L in IgG3 (IgG3 SpA+1; SEQ ID NO 33 and IgG3 SpA+2; SEQ ID NO 34 respectively) was compared to the IgG3 combination mutant (IgG3 SpA+3; SEQ ID NO 35) and IgG3 WT control (SEQ ID NO 22) via SPR as described previously. Protein A deficiency resulting from individually mutating K315T or L309V in IgG1 (IgG1 SpA-1; SEQ ID NO 23 and IgG SpA-2; SEQ ID NO 24 respectively) was similarly compared to the IgG1 combination mutant (IgG1 SpA-3; SEQ ID NO 25) and the IgG1 WT control (SEQ ID NO 20). The 1:1 binding affinity result demonstrated, although each individual mutation in position 309 and 315 influence the IgG Fc’s protein A binding capacity, the combination mutant has the greatest effect on binding (Figure 25). Mutations within position 315 have the weakest contribution to protein A modulation, demonstrating a one log fold change compared to the WT controls for both the IgG3 proficient variant (IgG3 WT =2.55e-007M, IgG3 SpA+1 =5.5e-008M) and IgG1 deficiency variant (IgG1 WT =1.31e-009M, IgG1 SpA-1 =4.05e-009M). Mutations within position 309 demonstrate a greater contribution to protein A modulation than position 315 alone, for both the IgG3 proficiency variant (control (IgG3 WT =2.55e-007M, IgG3 SpA+1 =5.5e- 008M, IgG3 SpA+2 =9.09e-009M) and the IgG1 deficiency variant (IgG1 WT =1.31e-009M, IgG1 SpA-1 =4.05e-009M, IgG1 SpA-2 =2.8e-008M). The combination mutants in both cases demonstrates an additive effect when both 309 and 315 are mutated. FcRN binding affinity characterisation As described earlier, protein A and FcRn share similar binding sites on the Fc and modulation of the protein A binding domain can have an impact on antibody serum half-life (DeLano WL et al. Science 2000; 287:1279-1283). Therefore, FcRn binding affinity was examined using Biacore 8K (Cytiva). Briefly, CAP Series S Sensor Chip (Cytiva) was docked into Biacore 8K, equilibrated overnight at RT and then Running Buffer (1X PBS and 0.05% Tween20) was applied to the SPR chip surface. Feline FcRn-B2M recombinant protein (Acro Biosystems) was diluted into 0.5ug / mL in running buffer and immobilised using the Biotin CAPture kit standard protocol. Antibodies dilutions were prepared by diluting IgG1-WT, IgG1-SpA -ve, IgG3-WT and IgG3-SpA +ve from 25nM to 0.781nM (6 concentrations with 1:2 dilutions) in Running Buffer and kinetics was assessed using multi-cycle kinetics method (60sec association – 120sec dissociation). Kinetics and / or affinity quantification have been performed using Biacore Insight following standard analyses methods. Affinity to FcRn of IgG1-SpA -ve, and IgG3-SpA +ve was comparable to that of WT IgG1 and IgG3 counterparts (Figure 14), suggesting that mutations L309V and K315T alter protein A binding only. To confirm that mutations in position 309 and 315 do not alter FcRn binding, binding affinity of both the individually mutated and combination mutants to FcRn were compared (Figure 26). Minimal disruption in FcRn binding capability is observed following mutations within position 309 and 315, with the combination mutants in both cases being less than 1-fold difference to the control. Complement Dependent Cytotoxicity (CDC) Activity in protein A binding modulated feline Fc’s To ensure modulation of the protein A binding domain does not affect the natural effector function of the IgG, CDC activity was assessed. To assess CDC activity, un-transfected (wild type) target lymphoid cells or equivalent human CD20-expressing cells were used in a cell killing assay. In this assay, 5,000 target cells per well of 96-well plate were incubated with anti-human CD20 feline Fc chimeric antibody and feline complement preserved serum (BioIVT) at a final dilution of 1:2, for 2 hours at 370C, 5% CO2. The assay was set up using media Advanced RPMI + 15% fetal bovine serum made using heat inactivated serum so that feline complement preserved serum would be the only source of complement. Live cells were then quantified using CellTitre-Glo® Luminescent Cell Viability Assay (Promega) following manufacturer’s protocol. This assay uses the ATP content of live cells as an indication of cell viability. Luminescence was measured on a CLARIOstar (BMG Labtech). Data was analyzed using MARS software (BMG Labtech) and the number of live cells remaining was used to calculate the percentage of killing in the presence of antibodies using Microsoft Excel. Background signal was obtained from a sample of cells treated with 1 % triton (where no cells were left alive) and subtracted from the signal obtained from each test sample. Max signal (0% killing) was obtained from a sample of cells treated identically but where antibodies were omitted. Graphs were plotted in Microsoft Excel or GraphPad Prism. CDC activity was assessed using hCD20 expressing cells and feline chimeric IgG-1 wt, IgG-1 SpA -ve, IgG3-WT and IgG-3 SpA +ve antibodies containing the Ofatumumab variable region. Antibodies were used at serial 1:3 dilutions ranging from 30µg / ml to 0.0003 µg / ml. As shown in Figure 15, protein A deficient and proficient mutations do not affect effector function. Wild type IgG1 and the IgG1 SpA-ve mutant variant mutant are still capable of eliciting CDC mediated killing in a dose dependent manner, whereas the wild type IgG3 and IgG3 SpA +ve mutant remain unable to elicit CDC activity even when exposed to up to 10ug / ml of antibody. This data suggests that protein A binding modulation at positions 309 and 315 do not impact effector function of feline IgGs. Biophysical properties of protein A modulated feline Fcs IgG1-WT, IgG1-SpA -ve, IgG3-WT and IgG3-SpA +ve mutants were assessed by SDS-Page using 4-12% Bis-Tris NuPAGE gels, MES SDS running buffer and SeeBlue Plus 2 standards (ThermoFisher) under non-reducing conditions. No clear differences were observed, however both IgG3 WT and the SpA+ proficient mutant IgGs show signs of aggregates as shown by the high molecular weight band (>~250kDa) observed in the loading well (Figure 16A). HPLC-SEC chromatography (column: BioResolve SEC mAb 200A, 2.5um column WATERS) was performed using ACQUITY H-class Bio from WATERS using PBS as mobile phase with isocratic 0.575mL / min flow rate. All the above-mentioned molecules were centrifuged 5’ at 20000 x g (using standard tabletop centrifuge) to remove any precipitates and then 10uL of each sample were injected in H-SEC using the above-mentioned protocol. Percentage of monomeric species and area (indicative of antibody concentration) was determined for each molecule. The results are summarised in Figure 16B and show that no significant reduction in percentage as well as area of monomeric peak was determined when comparing protein A modulated IgGs to their WT counterparts meaning that the overall protein stability is not affected by the introduced mutations in protein A binding regions. WT IgG3 was shown to have an inherently lower percentage of monomeric species when compared to IgG1 which remained following modulation of protein A. Utility of reduced protein A binding IgG-1 feline variants to allow for future feline bispecific antibody purification. Although the IgG1 deficiency variant IgG1 SpA-3 (SEQ ID NO: 25) could reduce the binding efficiency of IgG1 to protein A, its binding ability was not abolished as it was still purifiable using protein A resin. For utility of reduced protein A binding to allow for bispecific antibody purification, further mutants (IgG1 SpA-1 to SpA-15) were tested to determine if complete abolishment of protein A binding could be achieved (SEQ ID NO 23-32 and 157-161). The mutations tested were generated by PCR based site directed mutagenesis and as before, chimeric antibodies of these Fc variants were generated with human ofatumumab as the variable region as described previously. Construct cloning, antibody production and purification were as described in Example 1. As shown in Figure 28, several of the IgG-1 protein A deficiency mutants could not be purified using MabSelect Protein A columns. As such to demonstrate these mutants were purifiable, CH1 columns were utilised. Harvested supernatants from CHO expressing cells were loaded onto 1 mL CaptureSelect™ CH1-XL pre-packed column (Thermo Scientific™). Columns were then washed with 20mM Tris-HCl 150mM NaCl pH 7.5, the second wash with 20mM Tris pH7.5 and eluted with 100mM NaOAc pH 4.7 and then immediately neutralised with 10% 1M TRIS-HCl pH 8. Purified protein samples were assessed for protein A binding kinetics using single cycle kinetics method (Biacore Assay Handbook, Cytiva) and FcRn binding kinetics were assessed using a multi-cycle kinetics with capture method (Biacore Assay Handbook, Cytiva) as previously described. Protein concentrations were assessed using UV absorbance at 280nm with NanoDrop™ One (Thermo Scientific™). The CH1 purified mutant variants were then analysed for Protein A binding using Biacore 8K. Figure29 shows SPR sensogram raw data (light grey dotted lines) and fitting data obtained with 1:1 Langmuir fit (dark grey solid lines) of each IgG1 SpA mutant. As observed in canine IgG-B the single point mutation 253T in feline IgG-1 (SpA-5) significantly reduced protein A binding in terms of off-rate and R-max and the antibody could not be purified using a MAbSelect Protein A column (Figure 28C). Similarly, substitution 253D (SpA-6) resulted in significantly reduced protein A binding with minimal antibody being recovered following purification using a MAbSelect Protein A column. Mutating I253D in human IgG1 is known to disrupt FcRn interaction (Zwolak A et al 2017 Scientific reports 7:15521, 1-11.) FcRn binding affinity was similarly determined for the feline mutants (Figure 30). Both 253T and 253D mutants (SpA-5, SpA-6) had undetectable FcRn binding, suggesting that the hydrophobic region in CH2 domain 1 centred around position 253, like that in both human and canine IgGs, is important for interacting with FcRn. When combined with mutations within position 309 and 315, which do not affect FcRn binding, the addition of 253T lead to undetectable FcRn (SpA-8) further emphasising the contribution of position 253 in FcRn binding. The single point mutations H435A in canine IgG-B was capable of significantly reducing protein A binding to a similar extend to that of the IgG-B I253T mutant. In feline IgG-1, although there was a significant reduction in protein A binding following introduction of the 435A mutant (SpA- 10), it was not abolished and could still be purified using MabSelect Protein A columns albeit at low concentrations (Figures 28C- and Figure 29). In contrast when this position was mutated to a Glutamine (SpA-9) or Arginine (SpA-13) as opposed to an Alanine (SpA-10), protein A binding was completely abolished. All mutations generated at position 435 resulted in disruption of FcRn binding, highlighting the importance of this position in interacting with FcRn (Figure 30). Human IgG1 mAbs harbouring the mutation: H435R have similarly been shown to be important for FcRn interactions resulting in reduced serum half-life. The addition of Y436F has been shown to mediate recovery of FcRn interactions, for the H435F mutation and thus recovery of serum half- life in human IgG1 (Zwolak A et al 2017 Scientific reports 7:15521, 1-11). In the feline Fc, addition of a mutation in 436F does not elicit improvements in FcRn interaction, with it remaining undetectable. In addition, Q311R was tested alone and in combination with the IgG-3 like mutations within position 309 and 315 as this mutation is known to reduce protein A binding capacity in the context of the mouse Fc (Zwolak A et al 2017 Scientific reports 7:15521, 1-11). Interestingly, Q311R was able to further decrease the protein A binding capacity whist retaining its affinity for FcRn when used in combination with 309Q and 315T (Figure 28C and Figure 29). When Q113R was used in combination with 343R a 3-fold increase in FcRn binding was observed as well as a one- log fold decrease in protein A binding affinity. Whether FcRn binding affinity changes manifest in significant half-life effects in vivo compared to wild-type IgG1 is still to be determined. Example 5: Enhancing the Stability of Feline IgG2 As shown in Figure 17B the feline IgG2 isotype has hinge instability driven by Fab-arm exchange, resulting in a HL (heavy chain + light chain) half antibody produced even in the non- reducing SDS PAGE condition. With the hinge region being the crucial linker of the Fab region to the Fc, instability within this region can have important impacts on an antibody drugs efficacy and production manufacturability. Strengthening the heavy–heavy chain disulphide bond interaction within the IgG2 hinge would therefore be of benefit, making IgG2 more stable toward reduction during manufacturing and prevent Fab-arm exchange. As the hinge region, which is located between amino acid position 215-230 (Figure 17A), is not conserved between the three wild type feline IgG we hypothesised that this would account for the hinge instability observed in feline IgG2. Production of feline hinge stabilised mutants The sequences for the three known natural feline IgG were aligned and differences in the hinge region between the isoforms were compared. The amino acid sequences for wild type feline IgG 1, 2 and 3 (SEQ ID NO: 20, 21 and 22) are given in Table 1. Figure 17A shows amino acid substitutions to generate hinge stabilised IgG2 variants, corresponding amino acid sequences can be found in Table 1 (SEQ ID NO: 39 and 40). These mutants were generated by fully swapping the hinge regions of IgG1 and IgG2 (MUT5) or partially swapping the cystine rich hinge regions of IgG1 into IgG2 (MUT6). For antibody production, DNA constructs were generated to encode chimeric antibodies comprising selected feline IgG constant regions fused to human Ofatumumab variable regions. The feline IgG2 hinge stabilised mutant variants (IgG2 SEQ ID NO: 39 and 40) were generated by site directed mutagenesis as described in Example 1. Stability of hinge stabilised mutants To evaluate the effects of hinge mutations on stability, IgG2-WT, IgG2 MUT5, and IgG2 MUT6 mutants were assessed by SDS-PAGE using 4-12% Bis-Tris NuPAGE gels, MES SDS running buffer and SeeBlue Plus 2 standards (ThermoFisher) under non-reducing conditions (Figure 17B). The presence of the HL species observed at approximately 75 kDa in the WT IgG2. Such HL species no longer exist in MUT5 and MUT6, resembling that observed for wild type IgG1. To further evaluate the stability HPLC-SEC chromatography (column: BioResolve SEC mAb 200A, 2.5um column WATERS) was performed as described in Example 3. Percentage of monomeric species and Area (indicative of antibody concentration) was determined for each molecule. Comparing the percentage of monomeric species between the IgG1-WT and IgG2- WT and mutant molecules shows that although the presence of HL species is reduced there is still an inherent issue with the IgG2 isoform in forming aggregates (Figure 17C). Example 6: Reducing aggregation propensity for feline IgG The IgG subtype selected for a therapeutic target is typically selected according to the desired functional activity of that antibody. For example, for therapeutic applications in which the mechanism of action is target cell depletion, selecting an isotype which has effector functions such as ADCC and CDC is crucial. In contrast for therapeutic strategies which require antagonist or agonist function such effector functions are undesirable as they present as safety concerns. As feline IgG3 has been shown to be naturally effector function deficient it presents as a useful tool for this purpose, unlike feline IgG1 which is effector function proficient. As described in Example 5, feline IgG3 displays increased susceptibility to aggregation as compared with feline IgG1. This susceptibility to aggregation is not only observed in feline antibodies, human IgG4 is also known to undergo Fc-mediated aggregation (Chennamsetty et al., 2009 Journal of Molecular Biology 391(2): 404-413). This increased aggregation can be a problem not only in terms of developability but can also influence the safety and efficacy of the therapeutic treatment resulting in for example immunogenicity and unwanted complement activation (Namisaki et al., 2020 PLoS One 15(3): e0229027). It is therefore advantageous to resolve this instability, in order to generate aggregation resistant effector function deficient isoforms. Production of feline aggregation stabilised mutants It has previously been demonstrated that substituting Arginine 409 to a Lysine in human IgG4 increases the stability of human IgG4 (Namisaki et al., 2020 PLoS One 15(3): e0229027). The amino acid sequences for wild type feline IgG 1, 2 and 3 were aligned to that of the human IgG1 and IgG4 sequence. Like human IgG4, feline IgG3 has a lysine present at position 409. We therefore hypothesised that substituting the feline IgG3 Arginine 409 to a Lysine may improve its stability. The amino acid substitutions to generate aggregation stabilised IgG3 variants, corresponding amino acid sequences can be found in Figure 18 and Table 1. For antibody production, DNA constructs were generated to encode chimeric antibodies comprising selected feline IgG constant regions fused to human variable regions ofatumumab and human variable for a second antigen, designated “antigen B”. The feline aggregation stabilised mutant variant IgG3- SpA+ve + R409K was generated by site directed mutagenesis as described in Example 1. This aggregation mutation was introduced into the IgG3 SpA+ proficient Fc (SEQ ID NO: 25) identified in Example 3. This is exemplified by SEQ ID NO: 41. Stability of aggregation stabilised mutants To evaluate the effects of introduced mutation on stability, IgG3-SpA+ve and IgG3- SpA+ve + R409K mutants were assessed by SDS-PAGE using 4-12% Bis-Tris NuPAGE gels, MES SDS running buffer and SeeBlue Plus 2 standards (ThermoFisher) under non-reducing conditions (Figure 18B). Higher molecular weight protein species is observed with IgG3 isotype which is not present in R409K mutation. To further evaluate the antibody oligomerisation status, HPLC-SEC chromatography (column: BioResolve SEC mAb 200A, 2.5um column WATERS) was performed as described previously. Percentage of monomeric species and area (indicative of antibody concentration) was determined for each molecule. Comparing the percentage of monomeric species between the IgG3-WT, IgG3-SpA+ and the aggregation stabilised mutant molecule (R409K) shows a significant increase in percentage of intact mAb (~15%) following addition of the aggregation stabilised mutation R409K (Figure 18C). Aggregation at low pH One possibility for this decreased stability is that IgG3 aggregation could occur during the acidic elution from the protein A affinity chromatography stage as documented for human IgG4 isotype (Namisaki et al., 2020). To evaluate whether the R409K mutation could prevent acid induced aggregation of feline IgG3, IgG3-SpA+ve and IgG3- SpA+ve + R409K mutants were purified using AKTA Pure systems. Chromatographic purification steps included an affinity chromatography (Protein A: MabSelect Sure LX), and buffer exchange (G-25 Fine) into 20mM Sodium Acetate pH 5.5. Samples were buffer exchanged into 0.1M Glycine pH 3.5 before being left for 10 minutes and 60 minutes in a 37C incubator. Following incubation samples were neutralising to pH 5.5 using 1M Tris-HCl pH 7.5. Samples were then analysed by HPLC-SEC as described previously. For WT IgG1, minimal aggregation was observed following incubation at low pH at 3.5 for up to 60 minutes. In contrast, WT IgG3 has a high propensity for aggregation. Even at pH 5.5, more proportion of aggregation is observed. At pH 3.,5, a progressive increase of percentage of aggregation is observed with longer incubation time (5% aggregation following 10 mins versus 11% following 60 mins at pH3.5). Protein A mutation in IgG3 did not affect its stability in acidic conditions. Acid-induced aggregation was however prevented following incubation at pH 3.4 for 60 min at 37°C when the R409K mutation was introduced into the IgG3 SpA proficient Fc (Figure 19). Aggregate formation at low pH was effectively prevented and occurred to the same extent as that of IgG1. Our results suggested that the amino acid 409 within the CH3 domain is therefore involved in the novel aggregate formation of feline IgG3 and its susceptibility to aggregation at low pHs. Protein A binding affinity validation of aggregation stabilised mutant To ensure that the addition of the R409K mutation did not impact on its ability to be purified, binding affinity to Protein A Biacore 8K (Cytiva) was assessed as described in Example 3. The results show that mutating this additional amino acid (R409K) within the IgG3-SpA+ Fc does not significantly impact on its protein A binding affinity (KD: IgG3-SpA+ =7.91e-009M, KD: IgG3- SpA+ve + R409K=1.21e-008M) (Figure 20). Complement Dependent Cytotoxicity (CDC) Activity in aggregation stabilised mutant To ensure R409K mutation did not affect the natural effector function of the IgG, CDC activity was assessed as described in Example 4. CDC assay was performed using human CD20 expressing MS4 cells and feline chimeric IgG1-WT, IgG3-WT, IgG3-SpA +ve, and IgG3- SpA+ve + R409K antibodies containing the Ofatumumab variable region. Antibodies were used at serial 1:3 dilutions ranging from 30µg / ml to 0.0003 µg / ml. No CDC activity was demonstrated following addition of the R409K mutation, showing that the mutation had no gain of function effect on IgG3 which naturally lacks effector function (Figure 21). SPR based FcγR1a (CD64) binding validation of feline IgG3- SpA+ve + R409K mutant To further confirm the R409K mutation had no effect on the IgGs natural cellular effector functions, binding affinity of feline IgGs to Fc gamma 1 Receptor (CD64), the transmembrane protein which binds to the Fc tail of IgG to allow elicitation of effector function, was assessed using Biacore 8K (Cytiva). Briefly, Protein A Sensor Chip (Cytiva) was docked into Biacore 8K, equilibrated for 30’ at RT and then Running Buffer (10mM HEPES pH7.4150mM NaCl 3mM EDTA and 0.005% Tween20) was applied to the SPR chip surface. Variable region B, feline IgG1-WT, IgG3-WT, IgG3-SpA +ve, and IgG3- SpA+ve + R409K chimeric antibodies were diluted into running buffer at 6nM concentration. These have been immobilised using 90sec association at 10uL / min as capturing step, followed by injection of running buffer to remove any unbound product. Human CD64 / FCGR1A Protein (from Stratech - Catalogue Number: 10256-H08H-SIB) was diluted in Running Buffer at 150nM with 1:2 further dilutions down to 9.375nM. Kinetics were assessed using multi-cycle kinetics with capture step method (60sec association – 450sec dissociation) followed by regeneration step (0.1M Glycine pH2.2 contact time 60sec FR 30uL / min). Kinetics quantification have been performed using Biacore Insight following standard analyses methods. As a confirmation of the reduced CDC activity of the IgG3-WT and mutant antibodies, affinity for FcγR1a of IgG3 SpA+ and IgG3- SpA+ve + R409K was reduced compared to that of WT IgG1 (Figure 22A). SPR based FcRn binding validation of feline IgG3- SpA+ve + R409K mutant Feline Antibodies (IgG3-WT, IgG3-SpA +ve, and IgG3- SpA+ve + R409K as described above, with reference to Figure 20) binding affinity to Fc neonatal receptor was assessed as described in previously to ensure the addition of the R409K mutant did not affect the half-life of the antibody. Affinity to FcRn of IgG3 SpA +ve, and IgG3- SpA+ve + R409K was comparable to that of WT IgG-3 counterpart (Figure 22B). In vivo half life validation of feline IgG3- SpA+ve + R409K mutant In vivo half-life validation of wild type and mutant variants are performed in Sprague Dawley rats. Feline IgG1-WT, IgG3-WT, IgG3-SpA +ve, and IgG3- SpA+ve + R409K are injected (intraperitoneal or intravenous) into such rats and serum titre of the injected antibodies measured over time. A small amount of blood (20-50ul) is collected from saphenous vein at defined time points (Blood collection at day 0, 1 HR, 6 HR, 1 Day, 2 Day, 4 Day, 8 Day, 21, Day 35, Day 49 post injection). Serum is separated from cellular components by centrifuging the blood (7000 rcf for 5’). Feline IgGs in rat serum is quantified by using anti feline IgG ELISA (i.e.Abcam, ab190523) enabling pharmacokinetic profile (pK) to be determined. The experimental plan is highlighted in Figure 23. Similar half-life values are observed for the IgG3 WT (t1 / 2 = 298.8hrs) and the protein A proficiency mutant either alone (IgG3 SpA+3) or in combination with the R409K mutation (IgG3 SpA+3 R409K) (t1 / 2 = 335.8hr, 299.9hr respectively) (Figure 27). Taken together with the FcRN SPR binding data both the SpA+3 (V309L, T315K) and the R409K mutation do not affect serum half-life of the antibody compared to its WT counterpart. Sequences Table 1 Amino Acid Sequences

[0002] Table 2 – single cycle kinetics )

[0003] *Note for dIgG-Cwt unreliable fitting / values were obtained. Table 3 - Amount of protein recovered from optimised MabSelect SuRe LX method with % of each species quantified by HSCX in Table 4 – Amount of protein recovered from optimised MabSelect SuRe LX method with concentration, volume and total yield shown per samples in different columns Table 5 – Results from SPR Protein A binding affinity measurements of homodimers and heterodimers from different samples B Table 6 – Binding affinity measurements from heterodimer peak of different samples measured using SPR are shown

Claims

CLAIMS 1. A binding molecule comprising at least one variant IgG Fc domain comprising at least one amino acid substitution that reduces binding affinity to Protein A relative to the wild-type IgG domain, wherein the binding molecule is a companion animal binding molecule.

2. The binding molecule according to claim 1, wherein the variant IgG Fc domain comprises one or more of the following amino acid substitutions, a. an amino acid substitution at position 252, b. an amino acid substitution at position 253, c. an amino acid substitution at position 309, d. an arginine at position 311, i.e.311R, e. an amino acid substitution at position 315, f. an amino acid substitution at position 433, g. an amino acid substitution at position 435, and / or h. an amino acid substitution at 436, wherein the positions are numbered according to the EU numbering system.

3. The binding molecule according to claim 1 or 2, wherein said IgG domain comprises: a. an amino acid substitution at one or more of position 252, 253 and / or 315 according to EU numbering; b. an amino acid substitution at one or more of position 252, 253, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; c. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; d. an amino acid substitution at one or more of position 252, 311 and / or 315 according to EU numbering and wherein the amino acid substitution at position 311 comprises 311R; e. an amino acid substitution at one or more of position 252 and / or 315 according to EU numbering; f. an arginine at position 311 according to EU numbering; g. an amino acid substitution at one or more of position 253 and / or 435 according to EU numbering; h. an amino acid substitution at position 435 according to the EU numbering system; i. an amino acid substitution at position 436 according to EU numbering; or j. an amino acid substitution at one or more of position 309 and / or 315 according to EU numbering.

4. The binding molecule according to any one of claims 2 or 3, wherein the amino acid substitution at position 252 comprises 252V or 252R, the amino acid substitution at position 253 comprises 253T or 253D, the amino substitution at position 309 comprises 309E or 309V or 309Q, the amino acid substitution at position 311 comprises 311R , the amino acidsubstitution at position 315 comprises 315S or 315D or 315T, the amino acid substitution at position 433 comprises 433A, the amino acid substitution at position 435 comprises 435R or 435Q or 435A, the amino acid substitution at position 436 comprises 436F.

5. The binding molecule according to any preceding claim, wherein the IgG domain is canine and is selected from a canine IgG-A Fc domain, a canine IgG-B Fc domain, or a canine IgG- D Fc domain.

6. The binding molecule according to any one of claims 1 to 4, wherein the IgG domain is feline and is selected from a feline IgG1a Fc domain, a feline IgG1b Fc domain, or a feline IgG2 Fc domain.

7. The binding molecule of any preceding claim, wherein the variant IgG Fc domain is a CH2 or CH3 domain.

8. The binding molecule according to claim 5, wherein the variant IgG Fc domain is a canine IgG-A domain and comprises one or more of the following amino acid substitutions R252V, I253T I253D, E309V, E309Q, Q311R, T315S, T315D Q433A, H435R, H435A, and / or Y436F.

9. The binding molecule according to claim 5, wherein the variant IgG Fc domain is a canine IgG-B domain and comprises one or more of the following amino acid substitutions L252V, L252R, I253T, I253D, G309E, G309V, G309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435A, and / or Y436F.

10. The binding molecule according to claim 5, wherein the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following amino acid substitutions R252V, I253T, I253D, E309V, E309Q, Q311R, T315S, T315D, Q433A, H435R, H435A, and / or Y436F.

11. The binding molecule of claim 6 wherein the variant IgG Fc domain is a feline IgG1a and comprises one or more of the following amino acid substitutions S252V, S252R, I253T, I253D, L309V, L309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435Q, H435A, and or H436F 12. The binding molecule of claim 6 wherein the variant IgG Fc domain is a feline IgG1b and comprises one or more of the following amino acid substitutions S252V, S252R, I253T, I253D, L309V, L309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435Q, H435A, and or H436F 13. The binding molecule of claim 6 wherein the variant IgG Fc domain is a feline IgG2 and comprises one or more of the following amino acid substitutions S252V, S252R, I253T, I253D, L309V, L309Q, Q311R, K315T, K315S, K315D, H433A, H435R, H435Q, H435A, and or H436F.

14. The binding molecule of claim 5 wherein the canine IgG-B domain comprises the following amino acid substitutions a. I253T, L252V and K315S; b. I253T, L252V, K315S and Q311R;c. L252V and K315S, or d. I253T and H433A 15. The binding molecule of any preceding claim, wherein the variant IgG Fc domain comprises one of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO:42, SEQ ID NO: 43, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, or a sequence with at least 90% or 95% sequence identity thereto.

16. A binding molecule comprising at least one variant IgG Fc domain comprising an amino acid substitution at position 315 which increases binding affinity to Protein A relative to the wild- type IgG domain according to EU numbering, wherein the binding molecule is a companion animal binding molecule.

17. The binding molecule according to claim 16, wherein the variant IgG Fc domain further comprises an amino acid substitution at one or more of any of positions 252, 253, 309, 433 according to EU numbering 18. The binding molecule according to claim 16 or 17, wherein the variant IgG Fc domain comprises: a. an amino acid substitution at one or more of position 252, 253 and / or 315 according to EU numbering; or b. an amino acid substitution at one or more of position 252, 315, and / or 433 according to EU numbering.

19. The binding molecule according to any one of claims 17 or 18, wherein the amino acid substitution at position 252 comprises 252L, the amino acid substation at position 253 comprises 253I, the amino acid substitution at position 309 comprises 309L, the amino acid substitution at position 315 comprises 315K, the amino acid substitution at position 433 comprises 433H.

20. The binding molecule according to any one of claims 16 to 19, wherein the IgG Fc domain is canine and is selected from a canine IgG-A Fc domain, a canine IgG-C Fc domain or a canine IgG-D Fc domain.

21. The binding molecule according to any one of claims 16 to 19, wherein the IgG Fc domain is feline and is selected from a feline IgG1a Fc domain, a feline IgG1b Fc domain, or a feline IgG3 Fc domain.

22. The binding molecule according to claim 20, wherein the variant IgG Fc domain is a canine IgG-A domain and comprises one or more of the following amino acid substitutions R252L, E309L, T315K, Q433H.

23. The binding molecule according to claim 20, wherein the variant IgG Fc domain is a canine IgG-C domain and comprises one or more of the following amino acid substitutions V252L, T253I, G309L, S315K.

24. The binding molecule according to claim 20, wherein the variant IgG Fc domain is a canine IgG-D domain and comprises one or more of the following amino acid substitutions R252L, E309L, T315K, Q433H.

25. The binding molecule of claim 21 wherein the variant IgG Fc domain is a feline IgG1b and comprises the following amino acid substitution S252L.

26. The binding molecule of claim 21 wherein the variant IgG Fc domain is a feline IgG2 and comprises the amino acid substitution S252L.

27. The binding molecule of claim 21 wherein the variant IgG Fc domain is a feline IgG3 and comprises one or more of the following amino acid substitutions S252L, V309L, T315K.

28. The binding molecule of claim 20 wherein the variant IgG Fc domain is a canine IgG-A Fc domain and comprises R252L, T315K and Q433H.

29. The binding molecule of claim 20 wherein the variant IgG Fc domain is a canine IgG-C Fc domain and comprises V252L, T253I and S315K.

30. The binding molecule of claim 20 wherein the variant IgG Fc domain is a canine IgG-D Fc domain and comprises R252L, T315K and Q433H.

31. The binding molecule of claim 21, wherein the variant IgG Fc domain in a feline IgG3 Fc and comprises V309L and / or T315K.

32. The binding molecule of any one or claims 16 to 31, wherein the variant IgG Fc domain comprises one of SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO:18, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 35 or a sequence with at least 90% or 95% sequence identity thereto.

33. The binding molecule of any of the preceding claims, wherein the IgG domain is a CH2 or CH3 domain.

34. A binding molecule comprising at least one variant IgG heavy chain comprising an amino acid substitution in the CH3 domain and / or in the hinge domain that increases stability relative to the wild-type IgG heavy chain, wherein the binding molecule is a companion animal binding molecule.

35. The binding molecule according to claim 34, wherein the variant IgG heavy chain comprises an amino acid substitution at position 409 according to EU numbering.

36. The binding molecule according to claim 34 or 35 wherein the amino acid substitution at position 409 comprises 409K.

37. The binding molecule according to any one of claims 34 to 36, wherein the variant IgG heavy chain is selected from a feline IgG-1b heavy chain, a feline IgG-2 heavy chain or a feline IgG-3 heavy chain.

38. The binding molecule according to claim 34, wherein the variant IgG heavy chain comprises amino acid substitution at one or more of positions 219, 220, 221, 222, 223, 224, 225, and / or 226 according to EU numbering.

39. The binding molecule according to claim 38, wherein the variant IgG heavy chain is a feline IgG2 heavy chain.

40. The binding molecule according to claim 38 or 39, wherein the variant IgG heavy chain comprises one or more of the following amino acid substitutions, A219D, S220H, T221P, I222P, E223G, S224P, T225aP, G225bC, E225cD, G226C.

41. The binding molecule according to any one of claims 38 to 40, wherein the IgG heavy chain comprises: a. A219D, S220H, T221P, I222P, E223G, S224P, T225aP, G225bC, E225cD, and G226C, or b. G225bC, E225cD and G226C 42. The binding molecule of any of the preceding claims, wherein the protein is a heterodimeric protein.

43. The binding molecule of any one of claims 34 to 42, wherein the IgG domain comprises one of SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41, or a sequence with at least 90% or 95% sequence identity thereto.

44. A pharmaceutical composition comprising the binding molecule of any of the preceding claims.

45. A nucleic acid encoding a binding molecule of any of the preceding claims.

46. A vector comprising a nucleic acid according to claim 45.

47. A host cell comprising a nucleic acid according to claim 45 or a vector according to claim 46.

48. A kit comprising a protein of any one of claims 1 to 43 or a pharmaceutical composition according to claim 44.

49. A binding molecule according to any one of claims 1 to 43 wherein the variant IgG Fc domain or variant IgG heavy chain domain is non-immunogenic or substantially non-immunogenic in a companion animal.

50. A method for purifying a heterodimeric protein comprising a binding molecule according to any of claims 1 to 15, comprising a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the first polypeptide comprises a variant IgG Fc domain comprising a decreased affinity for Protein A, and wherein the first polypeptide has lower affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide; and b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range.

51. A method for purifying a heterodimeric protein comprising a binding molecule according to any one of claims 16 to 32, comprising a. loading an affinity matrix with a mixture of multimeric proteins comprising (i) a heterodimer comprising the first polypeptide and a second polypeptide wherein the firstpolypeptide comprises a variant IgG Fc domain comprising an increased affinity for Protein A, and wherein the first polypeptide has greater affinity for the affinity matrix than does the second polypeptide (ii); and a first homodimer comprising two copies of said second polypeptide; and b. eluting and collecting the heterodimer from the affinity matrix in a buffer comprising a chaotropic agent and having a first pH range, wherein the first homodimer elutes from the affinity matrix in the buffer at a second pH range.

52. The method according to claims 50 or 51, wherein the mixture of multimeric proteins comprises a second homodimer comprising two copies of the second polypeptide.

53. The method according to any one of claims 50 to 52, wherein the affinity matrix comprises a Protein A ligand affixed to a substrate.

54. The method according to any one of claims 50 to 53, comprising the step of applying a pH gradient to the loaded affinity matrix of step (a).

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