WO2019116096A1 - Production of fc fragments - Google Patents

Abstract

The present disclosure provides cells and transgenic non-human mammals for the production of Fc fragments compositions and uses thereof.

Description

PRODUCTION OF FC FRAGMENTS

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. § 119(e) of U. S. Provisional Application Serial No. 62/599,445, filed on December 15, 2017, the entire disclosure of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The disclosure relates, at least in part, to methods for producing Fc fragments.

BACKGROUND OF THE INVENTION

Production of therapeutic molecules can involve recombinant expression in cell culture, transgenic expression in animals, and extraction from natural sources. To ensure safety and efficacy of these molecules for administration to subjects, the therapeutic molecules are purified to remove any impurities or potentially harmful contaminants.

SUMMARY OF THE INVENTION

Aspects of the present invention provide methods of producing a fragment

crystallizable (Fc) fragment comprising providing a transgenic non-human mammal that has been modified to express an antibody comprising an Fc fragment in the mammary gland; harvesting the antibody comprising the Fc fragment from milk produced by the mammary gland of the transgenic non-human mammal; and isolating the Fc fragment from the antibody, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to (i) hydrophobic interaction chromatography; (ii) affinity chromatography; and (iii)

hydroxyapatite chromatography.

Aspects of the present invention provide methods of producing an Fc fragment, the method comprising providing a transgenic non-human mammal that has been modified to express an Fc fragment; harvesting the Fc fragment from the milk produced by the mammary gland of the transgenic non-human mammal; and isolating the Fc fragment from the antibody, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to (i) hydrophobic interaction chromatography; (ii) affinity chromatography; and (iii)

hydroxyapatite chromatography. In some embodiments, isolating the Fc fragment further comprises (a) obtaining an antibody comprising an Fc fragment and one or more additional fragment; and (b) digesting the antibody of (a) to produce an Fc fragment and one or more additional fragments. In some embodiments, the hydrophobic interaction chromatography separates the Fc fragment from one or more additional fragments of the antibody. In some embodiments, the hydrophobic interaction chromatography comprises applying the Fc fragment and one or more additional fragments to a hydrophobic interaction chromatography column; and recovering the Fc fragment from the hydrophobic interaction chromatography column.

In some embodiments, the affinity chromatography comprises applying the Fc fragment to an affinity chromatography column; recovering the Fc fragment from the affinity chromatography column; and adding a salt to the Fc fragment recovered from the affinity chromatography column. In some embodiments, the salt is sodium chloride. In some embodiments, the sodium chloride is added to a concentration of 170 mM.

In some embodiments, the antibody is digested prior to hydrophobic interaction chromatography. In some embodiments, the digestion is performed by an enzyme. In some embodiments, the enzyme is a cysteine protease. In some embodiments, the cysteine protease is papain. In some embodiments, the papain is immobilized on a solid support.

In some embodiments, the affinity chromatography comprises Protein L affinity chromatography. In some embodiments, the hydroxyapatite chromatography comprises applying the Fc fragment to a hydroxyapatite chromatography column; and recovering a purified Fc fragment from the affinity chromatography column. In some embodiments, the hydroxyapatite chromatography is performed using a hydroxyapatite chromatography column comprising a hydroxyapatite resin. In some embodiments, the hydroxyapatite resin comprises macroporous spherical beads of hydroxyapatite.

In some embodiments, the transgenic non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, or llama. In some embodiments, the transgenic non-human mammal is also transgenic for the expression of a sialyl transferase.

In some embodiments, obtaining the antibody or Fc fragment comprises purifying the antibody or Fc fragment. In some embodiments, the antibody or Fc fragment is purified using affinity chromatography. In some embodiments, the Fc fragment is purified using affinity chromatography. In some embodiments, the affinity chromatography comprises Protein A affinity chromatography. In some embodiments, the antibody isotype is IgE, IgG, IgA, IgM or IgD. In some embodiments, the antibody isotype is IgG. In some embodiments, the antibody is Herceptin.

In some embodiments, the purity of the isolated Fc fragment is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%. In some embodiments, the purity of the isolated Fc fragment is assessed by high performance liquid chromatography, SDS-PAGE gel electrophoresis, or contaminant protein EFISA.

In some embodiments, the hydrophobic interaction chromatography is performed using a hydrophobic chromatography column comprising an organic polymer resin. In some embodiments, the organic polymer resin is phenyl organic polymer resin. In some embodiments, the hydrophobic interaction column is eluted using a salt buffer. In some embodiments, the elution of the hydrophobic interaction column is performed using a decreasing gradient of the salt buffer concentration.

In some embodiments, the Fc fragment has anti-inflammatory properties. In some embodiments, the Fc fragment is used to treat a subject with an autoimmune condition or an inflammatory condition.

Aspects of the present invention provide methods of producing an Fc fragment comprising providing a mammary epithelial cell that has been modified to express an antibody comprising an Fc fragment in the mammary gland; harvesting the antibody comprising the Fc fragment from the mammary epithelial cell of the transgenic mammal; and isolating the Fc fragment from the antibody, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to (a) hydrophobic interaction chromatography; (b) affinity chromatography; and (c) hydroxyapatite chromatography.

Aspects of the present invention provide methods of producing an Fc fragment, the method comprising providing a mammary epithelial cell that has been modified to express an Fc fragment; harvesting the Fc fragment from the mammary epithelial cell; and isolating the Fc fragment, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to (a) hydrophobic interaction chromatography; (b) affinity chromatography; and (c) hydroxyapatite chromatography.

In some embodiments, isolating the Fc fragment further comprises (a) obtaining an antibody comprising an Fc fragment and one or more additional fragment; and (b) digesting the antibody of (a) to produce an Fc fragment and one or more additional fragments. In some embodiments, the hydrophobic interaction chromatography separates the Fc fragment from one or more additional fragments of the antibody. In some embodiments, the hydrophobic interaction chromatography comprises applying the Fc fragment and one or more additional fragments to a hydrophobic interaction chromatography column; and recovering the Fc fragment from the hydrophobic interaction chromatography column.

In some embodiments, the affinity chromatography comprises applying the Fc fragment to an affinity chromatography column; recovering the Fc fragment from the affinity chromatography column; and adding a salt to the Fc fragment recovered from the affinity chromatography column. In some embodiments, the salt is sodium chloride. In some embodiments, the sodium chloride is added to a concentration of 170 mM.

In some embodiments, the antibody is digested prior to hydrophobic interaction chromatography. In some embodiments, the digestion is performed by an enzyme. In some embodiments, the enzyme is a cysteine protease. In some embodiments, the cysteine protease is papain. In some embodiments, the papain is immobilized on a solid support.

In some embodiments, the affinity chromatography comprises Protein L affinity chromatography.

In some embodiments, the hydroxyapatite chromatography comprises applying the Fc fragment to a hydroxyapatite chromatography column; and recovering a purified Fc fragment from the affinity chromatography column. In some embodiments, the hydroxyapatite chromatography is performed using a hydroxyapatite chromatography column comprising a hydroxyapatite resin. In some embodiments, the hydroxyapatite resin comprises

macroporous spherical beads of hydroxyapatite.

In some embodiments, the mammary gland epithelial cell is from a transgenic non human mammal. In some embodiments, the transgenic non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, or llama.

In some embodiments, the mammary gland epithelial cell has also been modified to express a sialyl transferase.

In some embodiments, obtaining the antibody or Fc fragment comprises purifying the antibody or Fc fragment. In some embodiments, the antibody or Fc fragment is purified using affinity chromatography. In some embodiments, the affinity chromatography comprises Protein A affinity chromatography.

In some embodiments, the antibody isotype is IgE, IgG, IgA, IgM or IgD. In some embodiments, the antibody isotype is IgG. In some embodiments, the antibody is Herceptin.

In some embodiments, the purity of the isolated Fc fragment is at least 95%, 96%,

97%, 98%, 99%, 99.5% or 99.9%. In some embodiments, the purity of the isolated Fc fragment is assessed by high performance liquid chromatography, SDS-PAGE gel electrophoresis, or contaminant protein ELISA.

In some embodiments, the hydrophobic interaction chromatography is performed using a hydrophobic chromatography column comprising an organic polymer resin. In some embodiments, the organic polymer resin is phenyl organic polymer resin.

In some embodiments, the hydrophobic interaction column is eluted using a salt buffer. In some embodiments, the elution of the hydrophobic interaction column is performed using a decreasing gradient of the salt buffer concentration.

In some embodiments, the Fc fragment has anti-inflammatory properties. In some embodiments, the Fc fragment is used to treat a subject with an autoimmune condition or an inflammatory condition.

Aspects of the present invention provide purified Fc fragments produced by any of the methods described herein.

Aspects of the present invention provide methods comprising administering a therapeutically effective amount of an Fc fragment produced in a transgenic non-human mammal to a subject in need thereof. In some embodiments, the subject has an inflammatory condition or an autoimmune condition.

Each of the limitations of the invention can encompass various embodiments of the invention. It is, therefore, anticipated that each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the Figures. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are not intended to be drawn to scale. The Figures are illustrative only and are not required for enablement of the disclosure. For purposes of clarity, not every component may be labeled in every drawing. In the drawings: Figure 1 shows a non-limiting example of a work flow for digestion of transgenically- produced Herceptin/trastuzumab, followed by purification of resulting Fc fragments.

Additional features of non-limiting embodiments are provided in boxes.

Figure 2 shows a non-limiting example of a chromatogram from a SP Sepharose Big Bead chromatography step of an Fc fragment production process as described in Example 2.

Figure 3 shows a non-limiting example of a chromatogram from a Protein A chromatography step of an Fc fragment production process as described in Example 2.

Figure 4 shows a non-limiting example of a chromatogram from a Q Sepharose chromatography step of an Fc fragment production process as described in Example 2.

Figure 5 shows a non-limiting example of a chromatogram from a hydrophobic interaction chromatography step of an Fc fragment production process using a Tosoh Phenyl 600M column as described in Example 3.

Figure 6 shows a non-limiting example of a chromatogram from a Protein L affinity chromatography step of an Fc fragment production process as described in Example 3.

Figure 7 A shows a non-limited example of a chromatogram of Grade 1 Fc fragments. Figure 7B shows a non-limiting example of a chromatogram of Grade 2 Fc fragments.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein are methods, cells and transgenic animals for the production of antibodies containing an Fc fragment, and methods of isolating and purifying the Fc fragment from such antibodies. Also disclosed herein are methods, cells and transgenic animals for the production, isolation, and purification of Fc fragments. Production of many therapeutic molecules relies on recombinant expression in cells or organisms and/or extraction from natural sources. For example, Intravenous immunoglobulin (IVIG) contains anti

inflammatory IgG antibodies and is extracted from human serum. The anti-inflammatory properties of the heterogeneous IVIG are attributed to the Fc fragment of the antibodies

(Samuelsson et al., Science (200l)29l(5503):484-6). Described herein are alternative methods for the production of Fc fragments. The methods described herein result in a surprisingly high level of purity of the isolated Fc fragment, for example by reducing the abundance of other antibody fragments and other impurities, and overcome difficulties of traditional purification methods for isolating Fc fragments. The methods described herein also allow for purification of a higher capacity and higher yield recovery of Fc fragments, which may be advantageous for purification of larger quantities of Fc fragments, such as for industrial purification. The purified Fc fragments described herein may also have improved activity, such as in reducing inflammation and/or autoimmunity.

This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of“including,” “comprising,” or“having,”“containing,”“involving,” and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Fc fragments

Aspects of the invention relate to methods of producing a fraction crystallizable (Fc) fragment. As used herein, an“Fc fragment” refers to the portion of an immunoglobulin that interacts with cell surface Fc receptors. An Fc fragment comprises two polypeptide fragments and may be covalently linked by one or more disulfides. Each of the two polypeptide fragments may comprise one or more heavy chain constant domains selected from CH2, CH3, and CH4. In some embodiments, the Fc fragment comprises heavy chain constant domains CH2 and CH3. Fc fragments from immunoglobulins of any isotype ( e.g ., IgG, IgA, IgD, IgE, IgM) can be compatible with aspects of the invention. In some embodiments, the Fc fragment is an IgG Fc fragment. In some embodiments, the Fc fragment comprises the sequence provided by SEQ ID NO: 1.

The amino acid sequence of the Fc fragment of trastuzumab is provided in SEQ ID

NO:l:

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTI SKAKGQPREPQVYTLPPSRDELTKNQVSLT CLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK An Fc fragment associated with the invention may comprise one or more N-glycans at the Fc-gamma glycosylation site in the heavy chain (Asn297) of the Fc fragment. A variety of glycosylation patterns can occur at the Fc gamma glycosylation site. Oligosaccharides found at this site include galactose, N-acetylglucosamine (GlcNac), mannose, sialic acid, N- acetylneuraminic acid (NeuAc or NANA), N-glycolylneuraminic (NGNA) and fucose. N- glycans found at the Fc gamma glycosylation site generally have a common core structure consisting of an unbranched chain of a first N-acetylglucosamine (GlcNAc), which is attached to the asparagine of the antibody, a second GlcNAc that is attached to the first GlcNac and a first mannose that is attached to the second GlcNac. Two additional mannoses are attached to the first mannose of the GlcNAc-GlcNAc-mannose chain to complete the core structure, providing two“arms” for additional glycosylation. In addition, fucose residues can be attached to the N-linked first GlcNAc.

Aspects of the invention relate to isolation of Fc fragments from antibodies comprising Fc fragments. Any antibody comprising an Fc fragment can be compatible with aspects of the invention. Some aspects relate to isolation of Fc fragments that are not derived from antibodies. Fc fragments can be native Fc fragments, meaning the Fc fragment comprises the native or natural amino acid sequence of the region of the antibody from which the Fc fragment is isolated. In some embodiments, a native Fc fragment is not further modified, prior to or after isolation of the Fc fragment relative to the antibody from which the Fc fragment is isolated. In other embodiments, Fc fragments can be variant Fc fragments. Variant Fc fragments include Fc fragments which contain an amino acid sequence that differs from the native or natural amino acid sequence of the region of the antibody from which the Fc fragment is isolated. For example, the amino acid sequence can be mutated ( e.g ., through one or more substitutions, insertions, and/or deletions of amino acid residues). A variant Fc fragment may have been modified prior to or after isolation of the Fc fragment from the antibody. In some embodiments, the variant Fc fragments comprises additional glycosylation moieties compared to a native Fc fragment.

In some embodiments, a native Fc fragment is an IgG, IgA, IgD, IgE or IgM native Fc fragment. In some embodiments, a variant Fc fragment is an IgG, IgA, IgD, IgE or IgM variant Fc fragment.

As used herein, the term“antibody” refers to a polypeptide comprising at least two heavy (H) chains and two light (L) chains. The terms“antibody” and“immunoglobulin” are used interchangeably herein and are equivalent. Each heavy chain of an antibody is comprised of a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region is comprised of at least three domains, CH1, CH2, CH3, and optionally CH4. Each light chain of an antibody is comprised of a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed

complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order:

FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions within the Fc fragment of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system ( e.g ., effector cells) and the first component (Clq) of the classical complement system.

In some embodiments the antibodies are of the isotype IgG, IgA or IgD. In further embodiments, the antibodies are selected from the group consisting of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD and IgE or have immunoglobulin constant and/or variable domains of IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgAsec, IgD or IgE. In other embodiments, the antibodies are bispecific or multispecific antibodies. According to an alternative embodiment, the antibodies of the present disclosure can be modified to be in the form of a bispecific antibody, or a multispecific antibody. The term“bispecific antibody” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has two different binding specificities which bind to, or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector cell. The term“multispecific antibody” is intended to include any agent, e.g., a protein, peptide, or protein or peptide complex, which has more than two different binding specificities which bind to, or interact with (a) a cell surface antigen, (b) an Fc receptor on the surface of an effector cell, and (c) at least one other component. Accordingly, the disclosure includes, but is not limited to, bispecific, trispecific, tetraspecific, and other multispecific antibodies which are directed to cell surface antigens, and to Fc receptors on effector cells.

In other embodiments, the antibodies are heavy chain antibodies. The term“heavy chain antibody” refers to a polypeptide that has two heavy chains and no light chains. Each of the heavy chains of the heavy chain antibody is comprised of a heavy chain constant (CH) region and a heavy chain variable (VH) region. In some embodiments, the heavy chain constant is comprised of at least two domains. In some embodiments, the heavy chain constant region is comprised of CH2 and CH3 domains.

The term“antibodies” also encompasses different types of antibodies, e.g., recombinant antibodies, monoclonal antibodies, humanized antibodies or chimeric antibodies, or a mixture of these.

In some embodiments, the antibodies are recombinant antibodies. The term “recombinant antibody,” as used herein, is intended to include antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from an animal that is transgenic for another species’ immunoglobulin genes, antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial antibody library, or antibodies prepared, expressed, created or isolated by any other means that involves splicing of immunoglobulin gene sequences to other DNA sequences.

In yet other embodiments, the antibodies can be chimeric or humanized antibodies. As used herein, the term“chimeric antibody” refers to an antibody that combines parts of a non-human (e.g., mouse, rat, rabbit) antibody with parts of a human antibody. As used herein, the term“humanized antibody” refers to an antibody that retains only the antigen binding CDRs from the parent antibody in association with human framework regions (see, Waldmann, 1991, Science 252:1657). Such chimeric or humanized antibodies retaining binding specificity of the murine antibody are expected to have reduced immunogenicity when administered in vivo for diagnostic, prophylactic or therapeutic applications according to the disclosure.

In certain embodiments, the antibodies are human antibodies. The term“human antibody,” as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the disclosure may include amino acid residues not encoded by human germline

immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). Human antibodies are generated using transgenic mice carrying parts of the human immune system rather than the mouse system. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. patents 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous ( e.g ., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals results in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies are prepared according to standard hybridoma technology. These monoclonal antibodies have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (HAMA) responses when administered to humans. The human antibodies, like any of the antibodies provided herein can be monoclonal antibodies.

In some embodiments, the antibody is a full-length antibody. In some embodiments the full-length antibody comprises a heavy chain and a light chain. In some embodiments, the antibody is an anti-HER2 antibody. In some embodiments, the heavy chain comprises SEQ ID NO:2 and the light chain comprises SEQ ID NOG. In some embodiments, the antibody includes an Fc portion comprising SEQ ID NO: 1. In some embodiments, the antibody is trastuzumab.

In some embodiments, the antibody consists of the heavy chain sequence of SEQ ID NOG and the light chain sequence of SEQ ID NOG. In some embodiments, the heavy chain sequence is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOG and/or the light chain sequence is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NOG.

In certain embodiments, the Fc fragment of the antibody consists of the sequence of SEQ ID NO :l. In some embodiments, the Fc fragment is at least 70%, 71%, 72%, 73%,

74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:l. It should be appreciated that any antibody could be compatible with aspects of the invention.

The heavy chain of trastuzumab is provided in SEQ ID NOG:

MEFGLSWLFLVAILKGVQCEVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA

RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVT

VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL S SWTVP S S S LGTQTY I CNVNHKP SNTKVDKKVEPKSCDKTHTCPP CPAPELLGGP SVFLFPPKPKDT LMI SRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNS TYRWSVLTVLHQDWLNGKE YKCKVSNKALPAP I EKT I SKAKGQPREPQVYTLPP SRDELTKNQVS LTCLVKGFYP SD IAVEWE SNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKS LS LSPGK

The light chain of trastuzumab is provided in SEQ ID NO:3:

MDMRVPAQLLGLLLLWLRGARCD I QMTQSP S S LSASVGDRVT I TCRASQDVNTAVAWYQQKP GKAPKL LI YSASFLYS GVP SRF SGSRSGTDFTLT I S SLQPEDFATYYCQQHYTTPP TFGQGTKVE I KRTVAAP S VF IFPP SDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS GNSQESVTEQDSKD STYS LS STLTLS KADYEKHKVYACEVTHQGLS SPVTKSFNRGEC

Purification of antibodies or Fc fragments from transgenic animals

In one aspect, antibodies are purified from transgenic non-human mammals. In some embodiments, the antibodies are secreted into the milk of the transgenic non-human mammals. The antibodies can be purified from the milk of transgenic non-human mammals such that the antibodies are substantially pure. In some embodiments, substantially pure includes substantially free of contaminants. Such purification can result in an intermediate product that is further processed.

In one aspect, antibodies comprising Fc fragments are purified from a mammary epithelial cell that has been modified to express an antibody comprising an Fc fragment. The antibodies can be purified from a mammary epithelial cell such that the antibodies are substantially pure.

In one aspect, Fc fragments are purified from a mammary epithelial cell that has been modified to express an Fc fragment. The Fc fragments can be purified from a mammary epithelial cell such that the Fc fragments are substantially pure.

In some embodiments, substantially pure includes substantially free of contaminants. Such purification can result in an intermediate product that is further processed.

Antibodies comprising Fc fragments that are harvested from the milk of a transgenic non-human mammal or from a mammary epithelial cell can be purified using any suitable means known in the art to generate an intermediate product. Similarly, Fc fragments that are harvested from the milk of a transgenic non-human mammal or from a mammary epithelial cell can be purified using any suitable means known in the art to generate an intermediate product. In some embodiments, the antibody or Fc fragment is purified using a milk clarification process. In some embodiments, the antibody or Fc fragment is purified using a cream separator. Cream separators and use thereof are well known in the art. In some embodiments, the antibody or Fc fragment is purified using column chromatography. Column chromatography is well known in the art (see, e.g., Current Protocols in Essential Laboratory Techniques Unit 6.2 (2008) for general chromatography methods). In some embodiments, the antibody or Fc fragment is purified using a cream separator followed by column chromatography. In some embodiments, the antibody or Fc fragment is purified using a cream separator followed by more than one step of column chromatography.

In some embodiments, the antibody or Fc fragment is removed from decreamed milk in a step involving a resin, such as agarose beads (e.g., Sepharose beads). In some embodiments, the agarose beads are modified with sulphopropyl (SP) cation exchange groups. In some embodiments, the agarose beads have a particle size of 100-300 pm. In some embodiments, the agarose beads are SP Sepharose Big Beads (GE Healthcare Life Sciences; Pittsburgh, PA). In some embodiments, the SP Sepharose Big Beads are washed with a phosphate buffer.

In some embodiments, an antibody or an Fc fragment is purified using protein-G and/or protein-A affinity chromatography (see, e.g., Carter (2011) Exp Cell Res 317:1261- 1269, incorporated by reference herein). In some embodiments, the antibodies or the Fc fragments are purified by immunoprecipitation (see, e.g., Current Protocols in Cell Biology Unit 7.2 (2001)).

In some embodiments, the antibody or an Fc fragment is further subjected to one or more additional chromatography steps. In some embodiments, the antibody or an Fc fragment obtained from the affinity chromatography is subjected to ultrafiltration/diafiltration followed by one or more additional chromatography steps. In some embodiments, the additional chromatography step is ion exchange chromatography. In some embodiments, the ion exchange chromatography involves a resin, such as Q Sepharose Fast Flow.

In some aspects, antibodies comprising Fc fragments or Fc fragments are exposed to a detergent during purification. Use of detergents in polypeptide purification methods are well known in the art and may aid in dissolving cell membranes, solubilizing polypeptides, maintaining polypeptides in solution, and/or denaturing polypeptides. Non-limiting examples of detergents include, without limitation, sodium dodecyl sulfate (SDS), triton X-100, 3-[(3- cholamidopropyl)dimethylammonio]- l-propanesulfonate (CHAPS), 3-[(3- cholamidopropyl)dimethylammonio] -2-hydroxy- 1 -propanesulfonate (CHAPS O) , NP-40, Tween 20, Tween 80, octyl glucoside, and octyl thioglucoside.

In some embodiments, the antibodies or Fc fragments obtained, for example from the milk of transgenic non-human mammals, are subjected to a process involving sequentially subjecting the antibody or Fc fragments to hydrophobic interaction chromatography, affinity chromatography, and hydroxyapatite chromatography, as described herein.

Isolation and purification of Fc fragments

i. Hydrophobic interaction chromatography

Aspects of the invention provide methods of isolating an Fc fragment from an antibody by subjecting the antibody to hydrophobic interaction chromatography, followed by further purification such as by affinity chromatography and hydroxyapatite chromatography. Other aspects of the invention provide methods of isolating an Fc fragment by subjecting the Fc fragment sequentially to hydrophobic interaction chromatography, affinity

chromatography, and hydroxyapatite chromatography. As used herein,“hydrophobic interaction chromatography” (HIC) refers to a method of separating components ( e.g ., antibodies, Fc fragments) in a mixture based on reversible interactions between the components and an immobilized ligand within a column, wherein hydrophobic amino acid residues within a polypeptide interact with a hydrophobic ligand contained within the column. Components can be eluted from the column by altering concentrations of a buffer, such as by applying a decreasing concentration gradient of a salt buffer. Molecules within a mixture can have different hydrophobicity characteristics, so interaction with the ligand of the column can be disrupted at different concentrations of the buffer. In some embodiments, samples from the elution of the column are collected at each concentration of the buffer for further analysis and/or purification. In some embodiments, only the portion of the elution that comprises or is suspected to comprise the Fc fragment is collected for further analysis and/or purification.

As used herein,“resin” refers to a matrix, such as a matrix comprising beads attached to a ligand. Selection of an appropriate resin will be familiar to one of ordinary skill in the art and depends on characteristics of the components of the mixture, which will be applied to the resin. In some embodiments, the resin used for an HIC column comprises an organic polymer ligand. Non-limiting examples of organic polymer resins include phenyl, ether, butyl, hexyl, or polypropylene glycol resin. In some embodiments the organic polymer resin is phenyl organic polymer resin (e.g. Tosoh Phenyl 600M). It should be appreciated that a variety of resins can be compatible with aspects of the invention. For example, the resin can be a resin produced by Tosoh Bioscience LLC, King of Prussia, PA. In some embodiments, the resin is Tosoh Phenyl 600M, Tosoh Phenyl 650CM, Tosoh Butyl 600M, or Tosoh PPG 600M.

The antibodies, fragments thereof, or Fc fragments, can be eluted from the column by applying a buffer at various concentrations ( e.g ., using a step gradient). In some

embodiments, samples from the elution are collected at each concentration of the buffer for further analysis. In some embodiments, only the portion of the elution that comprises or is suspected to comprise the Fc fragment is collected for further analysis.

The elution samples comprising the antibody, fragments thereof, or Fc fragment, can be further analyzed or assessed for purity by any method known in the art including, without limitation, Western blotting, protein electrophoresis, protein staining, high performance liquid chromatography or mass spectrometry.

In some embodiments, antibodies, fragments thereof, or Fc fragments eluted from the HIC column are subjected to ultrafiltration/diafiltration. In some embodiments, antibodies, fragments thereof, or Fc fragments eluted from the HIC column are subjected to

ultrafiltration/diafiltration into a salt buffer. In some embodiments, the salt buffer is 50 mM NaCl.

In some embodiments, the concentration of the antibodies, fragments thereof, or Fc fragments eluted from the HIC column may be adjusted. In some embodiments, the concentration of the antibodies, fragments thereof, or Fc fragments eluted from the HIC column may be adjusted to a concentration of approximately 20 mg/mL or less. ii. Antibody digestion

In some embodiments of the invention, an antibody is digested prior to subjecting the antibody to HIC. The antibody can be digested by any method known in the art, including, without limitation, enzymatic, chemical, or mechanical digestion methods. In some embodiments, the digestion of the antibody is performed by an enzyme. Non-limiting examples of enzymes for use in digesting antibodies include cysteine proteases such as papain and ficin, aspartate proteases such as pepsin, and serine proteases such as trypsin. In preferred embodiments, the antibody is digested at a site between the Fc fragment and the additional fragment of the antibody. In preferred embodiments, the enzyme does not digest the Fc fragment but separates the Fc fragment from the additional fragment of the antibody.

In some embodiments, the digestion results in production of an Fc fragment and an additional fragment. In some embodiments, the additional fragment is a Fab fragment, a Fab’ fragment, a F(ab’)2 fragment or a scFv fragment.

Digestion may be performed at a temperature in which the enzyme is active. An appropriate duration of the digestion will be evident to one of ordinary skill in the art and can be determined using routine methods known in the art. In some embodiments, the enzyme used to digest an antibody is immobilized on a solid support, or in a free form, or in any other form that is compatible with methods described herein. As used herein,“immobilized on a solid support” refers to a ligand ( e.g ., polymer, enzyme) that is attached to a resin, for example agarose beads.

In some embodiments, the antibody digestion is performed for at least 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, or more. In some embodiments, the antibody digestion is performed overnight.

In some embodiments, a salt buffer may be added to the digested antibody, for example prior to HIC. In some embodiments, sodium sulfate is added to the digested antibody to a concentration of 0.6 M. iii. Affinity chromatography

Aspects of the invention relate to subjecting an antibody containing an Fc fragment sequentially to HIC, affinity chromatography, and hydroxyapatite chromatography. Other aspects relate to subjecting an Fc fragment sequentially to HIC, affinity chromatography, and hydroxyapatite chromatography. In some embodiments, the HIC elution portions that comprise or are suspected to comprise the Fc fragment are subjected to affinity

chromatography. As used herein,“affinity chromatography” refers to a method of separating components from a mixture based on interaction of a component in a mixture with a molecule immobilized, for example on resin, such as agarose beads.

The interaction between the components and the immobilized molecule may be based on any interaction known in the art, such as hydrogen bonding, ionic interaction, disulfide bridges, hydrophobic interactions. In some embodiments, the resin contains a binding partner that specifically interacts with a component in the mixture, thereby removing the component from the mixture. In some embodiments, the resin contains an immobilized receptor and the ligand of the receptor is removed from a mixture of components based on the interaction between the receptor and ligand. Affinity chromatography methods may be used for any of a number of purposes. For example, affinity chromatography may be used to remove desired components from a mixture. As another example, affinity chromatography may be used to remove undesired components from a mixture (e.g., contaminants).

In some embodiments, affinity chromatography may be used to remove contaminants from a sample containing Fc fragments, for example a sample containing Fc fragments that was previously subjected to HIC. In some embodiments, the affinity chromatography is used to remove undigested antibodies, Fab fragments, Fab’ fragments, or F(ab’)2 fragments. In some embodiments, a resin used in the affinity chromatography interacts with undigested antibodies, Fab fragments, Fab’ fragments, or F(ab’)2 fragments, thereby removing these molecules from the mixture. In some embodiments, the desired component (e.g., the Fc fragments) remain in the solution and are not removed by the resin of the affinity

chromatography. In some embodiments, the Fc fragments are in the flow-through and are collected from the affinity chromatography step.

Any molecule that interacts with undigested antibodies, Fab fragments, Fab’ fragments, and/or F(ab’)2 fragments and does not interact with Fc fragments or has less affinity for Fc fragments (relative to the affinity to undigested antibodies, Fab fragments,

Fab’ fragments, and/or F(ab’)2 fragments) may be used in the step of affinity

chromatography. In some embodiments, a molecule that interacts with undigested antibodies, Fab fragments, Fab’ fragments, and/or F(ab’)2 fragments and does not interact with Fc fragments or has less affinity for Fc fragments (relative to the affinity to undigested antibodies, Fab fragments, Fab’ fragments, and/or F(ab’)2 fragments) is immobilized on a resin used in the affinity chromatography. In some embodiments, the molecule is Protein L, which interacts with the light chain of an antibody (or Fab fragments, Fab’ fragments or F(ab’)2 fragments).

In some embodiments, the affinity chromatography is performed using Protein L resin and the Fc fragments remain in the flow-through from the affinity chromatography column. Protein L may be immobilized on any resin used in affinity chromatography. In some embodiments, the Protein L is immobilized on agarose beads (e.g., SP-Sepharose).

Following affinity chromatography, salt or a buffer containing salt may be added to the flow-through containing the Fc fragments, as described herein. In some embodiments, a salt buffer may be added to the receptacle collecting the Fc fragments from the affinity chromatography. In some embodiments, sodium chloride is added to the receptacle collecting the Fc fragments. In some embodiments, sodium chloride is added to the receptacle collecting the Fc fragments to a final concentration of between about 50-150 mM. In some embodiments, sodium chloride is added to the receptacle collecting the Fc fragments to a final concentration of about 100 mM.

In some embodiments, Fc fragments obtained from an affinity chromatography step may be immediately processed by hydroxyapatite chromatography. iv. Hydroxyapatite chromatography

Aspects of the invention relate to subjecting an antibody containing an Fc fragment sequentially to HIC, affinity chromatography, and hydroxyapatite chromatography. Other aspects relate to subjecting an Fc fragment sequentially to HIC, affinity chromatography, and hydroxyapatite chromatography. In some embodiments, the salt concentration of a solution containing the Fc fragments obtained in the flow-through from the affinity chromatography is increased and the Fc fragments are subjected to hydroxyapatite chromatography.

As used herein,“hydroxyapatite chromatography” refers to a chromatography method using a hydroxylated calcium phosphate matrix, referred to as hydroxyapatite and having the chemical formula Caio(P0₄)₆(OH)₂. The term hydroxyapatite, as used in the art, may also be referred to as“HA” or“HAP.” Unlike many other forms of chromatography that utilize a ligand immobilized on a matrix, hydroxyapatite functions as both the matrix and the ligand. The calcium ions and phosphate ions of the hydroxyapatite contribute to interaction (e.g., calcium metal affinity, phosphoryl cation exchange) with components (e.g., biomolecules, such as antibodies or antibody fragments) in a mixture.

The hydroxyapatite chromatography may comprise at least the steps of applying a solution comprising Fc fragments to a hydroxyapatite chromatography column. The hydroxyapatite chromatography column may contain any form of hydroxyapatite known in the art suitable for use in chromatography. In some embodiments, the hydroxyapatite chromatography column contains ceramic hydroxyapatite (also referred to as“cHA”), crystalline hydroxyapatite, or composite hydroxyapatite. In some embodiments, the hydroxyapatite chromatography column contains ceramic hydroxyapatite in the form of macroporous spherical beads. In some embodiments, the ceramic hydroxyapatite is a Type I or Type II ceramic hydroxyapatite.

In some embodiments, the ceramic hydroxyapatite has a mean particle size of about

20 pm, 40 pm, or 80 pm. In some embodiments, the ceramic hydroxyapatite has a mean particle size of 39 pm. In some embodiments, the ceramic hydroxyapatite is CaPure-HA™ (Tosoh Biosciences). Additional commercial sources of ceramic hydroxyapatite will be evident to one of ordinary skill in the art.

In some embodiments, a solution containing Fc fragments is applied to a

hydroxyapatite chromatography column and purified Fc fragments are recovered from the hydroxyapatite chromatography column. In some embodiments, one or more wash buffers are applied to the hydroxyapatite chromatography column, for example, prior to recovering the purified Fc fragments. In some embodiments, an elution buffer ( e.g ., a sodium phosphate buffer) is applied to the hydroxyapatite chromatography column to elute the Fc fragments and purified Fc fragments are collected. In some embodiments, the purified Fc fragments collected from the hydroxyapatite chromatography are subsequently subjected to at least one ultrafiltration or diafiltration.

Use of hydroxyapatite in the purification process allows for high selectivity and resolution of desired products (e.g., Fc fragment) from a mixture. In some embodiments, the hydroxyapatite chromatography removes contaminants from a sample containing Fc fragments. In some embodiments, hydroxyapatite chromatography removes unpaired Fc fragments (Fc fragments without the cysteine-cysteine disulfide bond). In some

embodiments, hydroxyapatite chromatography removes undigested and/or partially digested antibodies from the Fc fragments.

iv. Buffer conditions

It should be appreciated that a variety of solutions, such as buffers, can be compatible with aspects of the invention. In some embodiments, digestion of an antibody comprising an Fc fragment occurs in a tromethamine (tris) buffer. In some embodiment the buffer is a tris- phosphate buffer. In other embodiments, the buffer is a phosphate buffer. In some embodiments, the buffer has a concentration of between 1 mM and 100 mM, between 2 mM and 50 mM, or between 5 mM and 20 mM. In some embodiments, the buffer concentration is less than 1 mM. In some embodiments, the buffer concentration is more than 100 mM. In some embodiments, the buffer has a concentration of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,

39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 mM or more. In some embodiments, the buffer concentration is approximately 20 mM. It should be appreciated that the buffer concentration is dependent on the nature of the buffer that is being used.

In some embodiments, the pH of the buffer is between pH 6 and pH 9 or between pH 6.5 and pH 7.5. For example, in some embodiments, the pH is approximately 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4 or 7.5. In some embodiments, the pH of the buffer is approximately 7.0. If needed, acid (such as HC1) or base (such as NaOH) can be added to the buffer to attain the desired pH. Ethylenediaminetetraacetic acid (EDTA) may be added to the buffer to chelate multivalent cations. In some embodiments, the EDTA concentration is between 1 mM and 100 mM, between 2 mM and 50 mM, or between 5 mM and 20 mM. In some embodiments, the EDTA concentration is approximately 10 mM.

In some embodiments, the HIC column is equilibrated prior to addition of a sample (e.g., digested antibodies). In some embodiments, the HIC column is equilibrated prior to addition of a sample using an equilibration buffer. In some embodiments, the equilibration buffer comprises 0.6 M sodium sulfate. In some embodiments, the equilibration buffer comprises sodium sulfate and sodium phosphate. In some embodiments, the equilibration buffer comprises 0.6 M sodium sulfate, 20 mM sodium phosphate, and has pH 7.0.

In some embodiments, the HIC column is eluted using a salt buffer or concentration gradient thereof. In some embodiments, the salt buffer is a sodium sulfate buffer. In some embodiments, the concentration range of a salt buffer used to elute a HIC column can be from 0.1 M to 1 M, or between 0.3 M and 0.8 M, or between 0.4 M to 0.6 M. In some embodiments, the HIC column is eluted using a salt buffer at a concentration of 0.4 M. In some embodiments, the HIC column is eluted using a sodium sulfate buffer at a concentration of 0.4 M. In some embodiments, the HIC column is eluted using a buffer comprising 20 mM sodium phosphate, 0.4 M sodium sulfate, and has pH 7.0.

In some embodiments, a salt is added to a buffer following affinity chromatography with Protein L. Fc fragments, when present at high concentrations, may precipitate or aggregate under low salt conditions. Therefore, in some embodiments, salt may be added to a buffer to reduce precipitation or aggregation of Fc fragments. In some embodiments, the salt is added to a buffer to a concentration of at least 100 mM. In some embodiments, the salt is added to a buffer to a concentration of about 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 210 mM, 220 mM, 230 mM, 240 mM, 250 mM or higher. In some embodiments, the salt is sodium chloride. In some embodiments, the salt is sodium chloride and is added to the buffer to a concentration of about 170 mM.

In some embodiments, the HA column is equilibrated using a salt buffer. In some embodiments, the salt buffer is a low salt buffer. In some embodiments, the low salt buffer has a concentration of less than 10 mM. In some embodiments, the HA column is equilibrated using a salt buffer comprising sodium phosphate. In some embodiments, the HA column is equilibrated using a salt buffer comprising sodium phosphate at a concentration of about 5 mM and having pH 6.5. In some embodiments, the HA column is washed using a salt buffer. In some embodiments, the salt buffer comprises sodium chloride. In some embodiments, the salt buffer has a concentration of about 150-200 mM. In some

embodiments, the HA column is washed using a salt buffer. In some embodiments, the salt buffer comprises sodium chloride at a concentration of about 170 mM.

In some embodiments, the HA column is eluted using a salt buffer. In some embodiments, the salt buffer is a sodium phosphate buffer. In some embodiments, the HA column is eluted using a salt buffer comprising sodium phosphate at a concentration of about 200 mM and having pH 6.5.

Constructs for the generation of transgenic animals expressing antibodies

Some aspects of the invention relate to producing primary cell lines containing a construct ( e.g ., encoding an Fc fragment or an antibody comprising an Fc fragment) for use in producing transgenic goats by nuclear transfer. The constructs can be transfected into primary goat skin epithelial cells, which are clonally expanded and fully characterized to assess transgene copy number, transgene structural integrity and chromosomal integration site. As used herein,“nuclear transfer” refers to a method of cloning wherein the nucleus from a donor cell is transplanted into an enucleated oocyte.

Coding sequences for proteins of interest (e.g., an Fc fragment or an antibody comprising an Fc fragment) can be obtained from any suitable source including by screening libraries of genomic material or reverse-translated messenger RNA derived from the animal of choice (such as an equine), obtained from sequence databases such as NCBI, GenBank, or by obtaining the sequences of the antibody or Fc fragment, etc. The sequences can be cloned into an appropriate plasmid vector and amplified in a suitable host organism, like E. coli.

After amplification of the vector, the DNA construct can be excised, purified from the remains of the vector and introduced into expression vectors that can be used to produce transgenic animals. The transgenic animals will have the desired transgenic protein integrated into their genome.

After amplification of the vector, the DNA construct can also be excised with the appropriate 5' and 3' control sequences, purified away from the remains of the vector and used to produce transgenic animals that have integrated into their genome the desired expression constructs. Conversely, with some vectors, such as yeast artificial chromosomes (YACs), it is not necessary to remove the assembled construct from the vector; in such cases the amplified vector may be used directly to make transgenic animals. The coding sequence can be operatively linked to a control sequence, which enables the coding sequence to be expressed in the milk of a transgenic non-human mammal.

A DNA sequence which is suitable for directing production of an Fc fragment or an antibody comprising an Fc fragment in the milk of transgenic animals can carry a 5 '-promoter region derived from a naturally-derived milk protein. This promoter is consequently under the control of hormonal and tissue- specific factors and is most active in lactating mammary tissue. In some embodiments, the promoter is a caprine beta casein promoter. The promoter can be operably linked to a DNA sequence directing the production of a protein leader sequence, which directs the secretion of the transgenic protein across the mammary epithelium into the milk. In some embodiments, a 3 '-sequence, which can be derived from a naturally secreted milk protein, can be added to improve stability of mRNA.

As used herein, a“leader sequence” or“signal sequence” is a nucleic acid sequence that encodes a protein secretory signal, and, when operably linked to a downstream nucleic acid molecule encoding a transgenic protein directs secretion. The leader sequence may be the native human leader sequence, an artificially-derived leader, or may be obtained from the same gene as the promoter used to direct transcription of the transgene coding sequence, or from another protein that is normally secreted from a cell, such as a mammalian mammary epithelial cell.

In some embodiments, the promoters are milk-specific promoters. As used herein, a “milk- specific promoter” is a promoter that naturally directs expression of a gene in a cell that secretes a protein into milk ( e.g ., a mammary epithelial cell) and includes, for example, the casein promoters, e.g., oc-casein promoter (e.g., alpha S-l casein promoter and alpha S2- casein promoter), b-casein promoter (e.g., the goat beta casein gene promoter (DiTullio, BIOTECHNOLOGY 10:74-77, 1992, incorporated by reference herein), g-casein promoter, K- casein promoter, whey acidic protein (WAP) promoter (Gordon et al. (1987) BIOTECHNOLOGY 5: 1183-1187, incorporated by reference herein), b-lactoglobulin promoter (Clark et al., BIOTECHNOLOGY (1989) 7: 487-492, incorporated by reference herein) and oc-lactalbumin promoter (Soulier et al., FEBS LETTS. (1992) 297:13,

incorporated by reference herein). Also included in this definition are promoters that are specifically activated in mammary tissue, such as, for example, the long terminal repeat (LTR) promoter of the mouse mammary tumor virus (MMTV).

As used herein, a coding sequence and regulatory sequence are said to be“operably joined” when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. In order for the coding sequences to be translated into a functional protein the coding sequences are operably joined to regulatory sequences. Two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame- shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region is operably joined to a coding sequence if the promoter region is capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired polypeptide (e.g., Fc fragment or antibody).

As used herein, a“vector” may be any of a number of nucleic acids into which a desired sequence may be inserted, such as by restriction and ligation, for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium, or just a single time per host as the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted, such as by restriction and ligation, such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells, which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., b-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

Mammary epithelial cells and transgenic animals for production of Fc fragments

In one aspect, the disclosure provides mammary gland epithelial cells that express an antibody comprising an Fc fragment. In another aspect, the disclosure provides mammary gland epithelial cells that express an Fc fragment. In some embodiments, the disclosure provides a transgenic non-human mammal comprising mammary gland epithelial cells that express the antibody comprising an Fc fragment. In other embodiments, the disclosure provides a transgenic non-human mammal comprising mammary gland epithelial cells that express the Fc fragment.

In one aspect, the disclosure provides a method for the production of an Fc fragment or an antibody comprising an Fc fragment, comprising:

(a) transfecting non-human mammalian cells with a transgene DNA construct encoding an Fc fragment or an antibody comprising an Fc fragment;

(b) selecting cells in which said transgene DNA construct has been inserted into the genome of the cells; and

(c) performing a first nuclear transfer procedure to generate a non-human transgenic mammal heterozygous for the Fc fragment or the antibody comprising an Fc fragment, and that can express the Fc fragment or the antibody comprising an Fc fragment in its milk.

In one aspect, the disclosure provides a method of

(a) providing a non-human transgenic mammal engineered to express an Fc fragment or an antibody comprising an Fc fragment, (b) expressing the Fc fragment or the antibody comprising the Fc fragment in the milk of the non-human transgenic mammal; and

(c) isolating the Fc fragment or the antibody comprising the Fc fragment produced in the milk.

Transgenic animals can also be generated according to methods known in the art (See e.g., U.S. Patent No. 5,945,577, incorporated by reference herein). Animals suitable for transgenic expression, include, but are not limited to goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, or llama. Suitable animals also include bovine, caprine, and ovine, which relate to various species of cows, goats, and sheep, respectively. Suitable animals also include ungulates. As used herein,“ungulate” is of or relating to a hoofed typically herbivorous quadruped mammal, including, without limitation, sheep, goats, cattle and horses. In one embodiment, the animals are generated by co-transfecting primary cells with separate constructs. These cells are then used for nuclear transfer. Alternatively, if micro-injection is used to generate the transgenic animals, the constructs may be injected.

Cloning will result in a multiplicity of transgenic animals - each capable of producing an Fc fragment or an antibody comprising an Fc fragment or other gene construct of interest. The production methods include the use of the cloned animals and the offspring of those animals. In some embodiments, the cloned animals are caprines, bovines or mice. Cloning also encompasses the nuclear transfer of fetuses, nuclear transfer, tissue and organ transplantation and the creation of chimeric offspring.

One step of the cloning process comprises transferring the genome of a cell that contains the transgene encoding the Fc fragment construct or the antibody construct into an enucleated oocyte. As used herein,“transgene” refers to any piece of a nucleic acid molecule that is inserted by artifice into a cell, or an ancestor thereof, and becomes part of the genome of an animal which develops from that cell. Such a transgene may include a gene which is partly or entirely exogenous ( i.e ., foreign) to the transgenic animal, or may represent a gene having identity to an endogenous gene of the animal.

Suitable mammalian sources for oocytes include goats, sheep, cows, rabbits, guinea pigs, mice, hamsters, rats, non-human primates, etc. Preferably, oocytes are obtained from ungulates, and most preferably goats or cattle. Methods for isolation of oocytes are well known in the art. Essentially, the process comprises isolating oocytes from the ovaries or reproductive tract of a mammal, e.g., a goat. A readily available source of ungulate oocytes is from hormonally-induced female animals. For the successful use of techniques such as genetic engineering, nuclear transfer and cloning, oocytes may preferably be matured in vivo before these cells may be used as recipient cells for nuclear transfer, and before they were fertilized by the sperm cell to develop into an embryo. Metaphase II stage oocytes, which have been matured in vivo, have been successfully used in nuclear transfer techniques.

Essentially, mature metaphase II oocytes are collected surgically from either non-super ovulated or super ovulated animals several hours past the onset of estrus or past the injection of human chorionic gonadotropin (hCG) or similar hormone.

One of the tools used to predict the quantity and quality of the recombinant protein expressed in the mammary gland is through the induction of lactation (Ebert KM, 1994). Induced lactation allows for the expression and analysis of protein from the early stage of transgenic production rather than from the first natural lactation resulting from pregnancy, which is at least a year later. Induction of lactation can be done either hormonally or manually.

In one aspect the disclosure provides mammary gland epithelial cells and transgenic non-human mammals that produce an Fc fragment or an antibody comprising an Fc fragment. Mammary gland epithelial cells and transgenic non-human mammals according to aspects of the invention express nucleic acid sequences encoding the antibody. In some embodiments, the nucleic acid sequences comprise a sequence encoding the Fc fragment set forth in SEQ ID NO: 1.

Production of Fc fragments

Aspects of the invention relate to transgenic Fc fragments. In some embodiments, the transgenic Fc fragment is purified.

In some aspects, Fc fragments produced as described herein in transgenic non-human mammals or in mammary epithelial cells have altered characteristics compared to Fc fragments produced by other methods. For example, Fc fragments produced as described herein can exhibit altered glycosylation and/or sialylation compared to Fc fragments produced by other methods. Fc fragments produced as described herein can exhibit increased half-lives and/or stability compared to Fc fragments produced by other methods. Fc fragments produced as described herein can also exhibit enhanced anti-inflammatory properties when administered to a subject compared to Fc fragments produced by other methods. In one aspect, the disclosure provides recombinant or transgenically produced antibodies wherein the Fc fragments are subsequently isolated and purified from the antibodies. In another aspect, the disclosure provides transgenically produced Fc fragments that are subsequently isolated and purified. Such Fc fragments and compositions comprising recombinant or transgenically produced Fc fragments can exhibit glycosylation and/or sialylation. For example, in some embodiments, Fc fragments produced in mammary epithelial cells of a non-human mammal, and then isolated and purified, have increased levels of glycosylation and/or sialylation when compared to Fc fragments not produced in mammary gland epithelial cells. In some embodiments, the Fc fragments not produced in mammary gland epithelial cells are produced in cell culture. As used herein, Fc fragments “produced in cell culture” when compared to Fc fragments produced in mammary epithelial cells, refers to Fc fragments produced in standard production cell lines ( e.g ., CHO cells or baculovirus-Sf9 cells) but excluding mammary epithelial cells. In some embodiments, Fc fragments produced in mammary epithelial cells of a non-human mammal, and then isolated and purified, have increased levels of glycosylation and or sialylation when compared to Fc fragments isolated from IVIG.

In some embodiments the methods above further comprise steps for inducing lactation of the transgenic non-human mammal. In some embodiments the methods further comprise additional isolation and/or purification steps. In yet other embodiments the methods further comprise steps for comparing the glycosylation pattern of the Fc fragments produced in cell culture, e.g. non-mammary cell culture. In further embodiments, the methods further comprise steps for comparing the glycosylation pattern of the Fc fragments obtained to Fc fragments produced by non-mammary epithelial cells. Such cells can be cells of a cell culture. Experimental techniques for assessing the glycosylation pattern of the Fc fragments are known to those of ordinary skill in the art. Such methods include, e.g., liquid

chromatography mass spectrometry, tandem mass spectrometry, and Western blot analysis.

In some aspects, the Fc fragments disclosed herein are generated by producing an antibody comprising an Fc fragment in a transgenic non-human mammal or in mammary epithelial cells. In other embodiments, the Fc fragments disclosed herein are generated by producing the Fc fragments in a transgenic non-human mammal or in mammary epithelial cells. In some embodiments, it may be advantageous to increase the sialylation level of the Fc fragments. The sialylation levels of the Fc fragments can be increased for instance by subjecting the Fc fragment or the antibody comprising the Fc fragment to sialyl transferases. The Fc fragment or the antibody comprising the Fc fragment can be subjected to sialyl transferases in vitro or in vivo. The Fc fragment or the antibody comprising the Fc fragment can be sialylated in vitro by subjecting the Fc fragment or the antibody comprising the Fc fragment to a sialyl transferase and the appropriate saccharide -based substrate. The Fc fragment or the antibody comprising the Fc fragment can be sialylated in vivo by producing a sialyl transferase in the mammary gland or mammary epithelial cells.

In some aspects, the disclosure provides methods for the production in the mammary gland of transgenic non-human mammals, or in mammary epithelial cells, of an Fc fragment or an antibody comprising an Fc fragment with increased levels of alpha-2, 6-sialylation. Fc fragments that exhibits increased sialylation may exhibit increased anti-inflammatory properties.

In one aspect, the disclosure provides transgenic non-human mammals (and mammary epithelial cells) that are transgenic for the production in the mammary gland of an Fc fragment or an antibody comprising an Fc fragment, and that are also transgenic for the production of sialyl transferase. The Fc fragments produced by such animals and cells are expected to have increased levels of terminal alpha-2, 6-sialic acid linkages.

In one aspect, the disclosure provides methods of treating a subject comprising administering to a subject the Fc fragments that have increased levels of terminal alpha-2, 6- sialic acid linkages.

The Fc fragment can be obtained, in some embodiments, by harvesting the Fc fragment or the antibody comprising the Fc fragment, from the milk of a transgenic mammal produced as provided herein or from an offspring of said transgenic mammal. In some embodiments, the Fc fragment is produced at a level of at least 1 gram per liter of milk produced. For example, in some embodiments, methods described herein allow for production of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47,

48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 grams per liter of an Fc fragment. In some embodiments, the antibody is produced at a level of at least 1 gram per liter of milk produced. For example, in some embodiments, methods described herein allow for production of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,

41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,

66, 67, 68, 69 or 70 grams per liter of an antibody. In some aspects, the Fc fragments produced as described herein have enhanced characteristics compared to Fc fragments produced by other methods. For example, in some embodiments, Fc fragments produced by methods described herein are of higher purity compared to Fc fragments produced by other methods. In some embodiments, the transgenically produced Fc fragments that are subsequently isolated and purified are at least 95 - 99.99% pure. In some embodiments, the transgenically produced Fc fragments that are subsequently isolated and purified are at least 90.0, 91.0, 92.0, 93.0, 94.0, 95.0, 96.0, 97.0, 98.0, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 99.91, 99.92, 99.92, 99.93, 99.94. 99.95, 99.96, 99.97, 99.98, or 99.99% pure. In preferred embodiments, the transgenically produced Fc fragments that are subsequently isolated and purified are at least 99.99% pure. In some embodiments, the Fc fragments isolated and purified from the transgenically produced antibodies are at least 95 - 99.99% pure. In some embodiments, the Fc fragments isolated and purified from the transgenically produced antibodies are at least 90.0, 91.0, 92.0, 93.0, 94.0, 95.0, 96.0, 97.0, 98.0, 99.0, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9, 99.91, 99.92, 99.92, 99.93, 99.94. 99.95, 99.96, 99.97, 99.98, or 99.99% pure.

Purity of Fc fragments produced by any of the methods described herein may be assessed by any technique known to those of skill in the art, including, without limitation, Western blotting, protein electrophoresis, protein staining, high performance liquid chromatography, mass spectrometry, contaminant protein ELISA, etc.

Fc fragments produced as described herein can also be produced with enhanced efficiency compared to Fc fragments produced by other methods. As used herein,“enhanced efficiency” refers to a higher percent yield of Fc fragments relative to the starting material.

In some embodiments, the starting material refers to the starting material of the production process ( e.g ., raw milk), and percent yield of proteins relative to the starting material is the total or overall percent yield. In some embodiments, the starting material refers to the starting material of a specific step of the production process. In some embodiments, the percent yield of Fc fragments isolated and purified from the transgenically produced antibodies is 5-15%. In some embodiments, the percent yield of Fc fragments isolated and purified from the transgenically produced antibodies is at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%. In some embodiments, the percent yield of Fc fragments isolated and purified from transgenically produced Fc fragments is 50-90%, preferably 60- 80%. In some embodiments, the percent yield of Fc fragments isolated and purified from the transgenically produced Fc fragments is at least 50, 55, 60, 65, 70, 75, 80, 85, 90%.

Fc fragments produced as described herein may also have enhanced activity compared to Fc fragments produced methods. As used herein“enhanced activity” refers to an enhanced ability to reduce inflammation and/or autoimmunity relative to Fc fragments produced by other methods. In some embodiments, the activity of the Fc fragments isolated and purified from transgenically produced antibodies is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more compared to Fc fragments produced by other methods. In some embodiments, the activity of the Fc fragments isolated and purified from

transgenically produced Fc fragments is enhanced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more compared to Fc fragments produced by other methods.

Methods of treatment

In some aspects, the disclosure provides methods of administering an Fc fragment or composition comprising an Fc fragment to a subject in need thereof. Methods for

determining whether a subject is in need of a treatment comprising an Fc fragment are known in the art. For example, in some embodiments, a subject in need of a treatment comprising administering an Fc fragment or composition comprising an Fc fragment is a subject having an autoimmune condition or an inflammatory condition. Non-limiting examples of autoimmune conditions and/or inflammatory conditions that could be treated by practice of the invention described herein will be apparent to one of ordinary skill in the art. Non limiting examples are specifically incorporated by reference from U.S. Patent No. 8,349,793 and PCT publication WO2013/034738.

Non-limiting examples of autoimmune conditions include Acute disseminated encephalomyelitis (ADEM), Addison's disease, Agammaglobulinemia, Alopecia areata, Amyotrophic lateral sclerosis (Also Lou Gehrig's disease; Motor Neuron Disease),

Ankylosing Spondylitis, Antiphospholipid syndrome, Antisynthetase syndrome, Atopic allergy, Atopic dermatitis, Autoimmune aplastic anemia, Autoimmune cardiomyopathy, Autoimmune enteropathy, Autoimmune hemolytic anemia, Autoimmune hepatitis,

Autoimmune inner ear disease, Autoimmune lymphoproliferative syndrome, Autoimmune peripheral neuropathy, Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmune progesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmune urticarial, Autoimmune uveitis, Balo disease/Balo concentric sclerosis, Behcet's disease, Berger's disease, Bickerstaff s encephalitis, Blau syndrome, Bullous pemphigoid, Castleman's disease, Celiac disease, Chagas disease, Chronic inflammatory demyelinating

polyneuropathy, Chronic recurrent multifocal osteomyelitis, Chronic obstructive pulmonary disease, Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome, Cold agglutinin disease, Complement component 2 deficiency, Contact dermatitis, Cranial arteritis, CREST syndrome, Crohn's disease, Cushing's Syndrome, Cutaneous leukocytoclastic angiitis, Dego's disease, Dercum's disease, Dermatitis herpetiformis, Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemic sclerosis, Dressler's syndrome, Drug-induced lupus, Discoid lupus erythematosus, Eczema, Endometriosis, Enthesitis-related arthritis, Eosinophilic fasciitis, Eosinophilic gastroenteritis, Eosinophilic pneumonia, Epidermolysis bullosa acquisita, Erythema nodosum, Erythroblastosis fetalis, Essential mixed cryoglobulinemia, Evan's syndrome, Fibrodysplasia ossificans progressive, Fibrosing alveolitis (or Idiopathic pulmonary fibrosis), Gastritis, Gastrointestinal pemphigoid, Glomerulonephritis,

Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome (GBS), Hashimoto's encephalopathy, Hashimoto's thyroiditis, Henoch- Schonlein purpura, Herpes gestationis aka Gestational Pemphigoid, Hidradenitis suppurativa, Hughes-Stovin syndrome,

Hypogammaglobulinemia, Idiopathic inflammatory demyelinating diseases, Idiopathic pulmonary fibrosis, Idiopathic thrombocytopenic purpura (See Autoimmune

thrombocytopenic purpura), IgA nephropathy, Inclusion body myositis, Chronic

inflammatory demyelinating polyneuropathy, Interstitial cystitis, Juvenile idiopathic arthritis aka Juvenile rheumatoid arthritis, Kawasaki's disease, Lambert-Eaton myasthenic syndrome, Leukocytoclastic vasculitis, Lichen planus, Lichen sclerosus, Linear IgA disease (LAD), Lupoid hepatitis aka Autoimmune hepatitis, Lupus erythematosus, Majeed syndrome, Meniere's disease, Microscopic polyangiitis, Miller-Fisher syndrome, Mixed connective tissue disease, Morphea, Mucha-Habermann disease aka Pityriasis lichenoides et

varioliformis acuta, Multiple sclerosis, Myasthenia gravis, Myositis, Narcolepsy,

Neuromyelitis optica (also Devic's disease), Neuromyotonia, Occular cicatricial pemphigoid, Opsoclonus myoclonus syndrome, Ord's thyroiditis, Palindromic rheumatism, PANDAS (pediatric autoimmune neuropsychiatric disorders associated with streptococcus),

Paraneoplastic cerebellar degeneration, Paroxysmal nocturnal hemoglobinuria (PNH), Parry Romberg syndrome, Parsonage-Turner syndrome, Pars planitis, Pemphigus vulgaris, Pernicious anaemia, Perivenous encephalomyelitis, POEMS syndrome, Polyarteritis nodosa,

Polymyalgia rheumatic, Polymyositis, Primary biliary cirrhosis, Primary sclerosing cholangitis, Progressive inflammatory neuropathy, Psoriasis, Psoriatic arthritis, Pyoderma gangrenosum, Pure red cell aplasia, Rasmussen's encephalitis, Raynaud phenomenon, Relapsing polychondritis, Reiter's syndrome, Restless leg syndrome, Retroperitoneal fibrosis, Rheumatoid arthritis, Rheumatic fever, Sarcoidosis, Schizophrenia, Schmidt syndrome another form of APS, Schnitzler syndrome, Scleritis, Scleroderma, Serum Sickness, Sjogren's syndrome, Spondyloarthropathy, Still's disease, Stiff person syndrome, Subacute bacterial endocarditis (SBE), Susac's syndrome, Sweet's syndrome, Sydenham chorea see PANDAS, Sympathetic ophthalmia, Systemic lupus erythematosus see Lupus erythematosus, Takayasu's arteritis, Temporal arteritis (also known as "giant cell arteritis"), Thrombocytopenia, Tolosa- Hunt syndrome, Transverse myelitis, Ulcerative colitis, Undifferentiated connective tissue disease, Undifferentiated spondyloarthropathy, Urticarial vasculitis, Vasculitis, Vitiligo and Wegener's granulomatosis.

Non-limiting examples of inflammatory conditions include Ankylosing Spondylitis (AS), Antiphospholipid Antibody Syndrome (APS), Gout, Inflammatory Arthritis Center, Myositis, Rheumatoid Arthritis, Scleroderma, Sjogren's Syndrome, Systemic Lupus

Erythematosus (SLE, Lupus), Vasculitis, Appendicitis, Bursitis, Colitis, Cystitis, Dermatitis, Infective meningitis, Tonsillitis, Asthma, Pneumonia, Phlebitis, RSD/CRPS, Rhinitis, Tendonitis, Tonsillitis, Vasculitis, pruritus, skin inflammation, psoriasis, atopic dermatitis, allergic contact dermatitis, irritant contact dermatitis, and seborrhoeic dermatitis,

keratinopathy, inflammatory bowel disease, ulcerative colitis, Crohn's disease, multiple sclerosis, osteoarthritis, Hashimoto's thyroidis, myasthenia gravis, diabetes type I or II, inflammatory lung injury, inflammatory liver injury, inflammatory glomerular injury, keratoconjunctivitis, an inflammatory disease of the joints, skin, or muscle, acute or chronic idiopathic inflammatory arthritis, a demyelinating disease, chronic obstructive pulmonary disease, interstitial lung disease, interstitial nephritis and chronic active hepatitis.

Pharmaceutical compositions, dosage, and administration

Aspects of the invention relate to administering effective amounts of a Fc fragment, or compositions comprising a Fc fragment. In some embodiments, methods comprise administering a therapeutically effective amount of a transgenic Fc fragment to a subject in need thereof. In some embodiments, the transgenic Fc fragment is purified, for example using the methods of purification described herein. In some embodiments, the subject has an inflammatory condition or an autoimmune condition. As used herein, a“therapeutically effective amount” or an“effective amount” refers to an amount of Fc fragment or composition comprising an Fc fragment that is effective to influence a condition. For example, in some embodiments, an effective amount is an amount that is sufficient for reducing at least one symptom associated with inflammation and/or autoimmunity. Determining an effective amount depends on such factors as toxicity and efficacy of the composition. These factors will differ depending on other factors such as potency, relative bioavailability, subject body weight, severity of adverse side-effects and preferred mode of administration. Toxicity may be determined using methods well known in the art. Efficacy may be determined utilizing the same guidance. Efficacy of an Fc fragment that reduces inflammation and/or autoimmunity, for example, can be in some embodiments measured by quantifying the amount of an inflammatory cytokine, the presence or quantity of inflammatory cells, amount of specific antibodies, or characteristics such as redness or swelling. An effective amount can be readily determined by one of ordinary skill in the art.

Dosage may be adjusted appropriately to achieve desired levels, local or systemic, depending upon the mode of administration. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that subject tolerance permits. In some embodiments, multiple doses per day can be used to achieve appropriate systemic levels of a product or composition. Appropriate systemic levels can be determined by, for example, measurement of the subject’s peak or sustained plasma level of the Fc fragment. “Dose” and“dosage” are used interchangeably herein.

In some aspects, the disclosure provides compositions, including pharmaceutical compositions, which comprise transgenically produced and purified Fc fragments and a pharmaceutically acceptable vehicle, diluent or carrier. In some embodiments, the pharmaceutical compositions comprise Fc fragments produced in transgenic non-human mammals. In some embodiments, the pharmaceutical compositions comprise Fc fragments produced in mammary epithelial cells.

In some embodiments, the compositions provided herein are used for in vivo applications, such as treatment of a disease or disorder. Depending on the intended mode of administration in vivo the compositions used may be in the dosage form of solid, semi-solid or liquid such as, e.g., tablets, pills, powders, capsules, gels, ointments, liquids, suspensions, or the like. Preferably, the compositions are administered in unit dosage forms suitable for single administration of precise dosage amounts. The compositions may also include, depending on the formulation desired, pharmaceutically acceptable carriers or diluents, which are defined as aqueous-based vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the Fc fragment. Examples of such diluents are distilled water, physiological saline, Ringer's solution, dextrose solution, and Hank's solution. The same diluents may be used to reconstitute a lyophilized recombinant protein of interest. Effective amounts of such diluent or carrier are amounts which are effective to obtain a

pharmaceutically acceptable formulation in terms of solubility of components, biological activity, etc. In some embodiments the compositions provided herein are sterile.

Administration during in vivo treatment may be by any number of routes, including oral, parenteral, intramuscular, intranasal, sublingual, intratracheal, inhalation, ocular, vaginal, and rectal. Intracapsular, intravenous, and intraperitoneal routes of administration may also be employed. The skilled artisan recognizes that the route of administration varies depending on the response desired. For example, the Fc fragments or compositions herein may be administered to a subject via oral, parenteral or topical administration. In one embodiment, the compositions herein are administered by intravenous infusion.

The compositions, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Pharmaceutical formulations for parenteral administration include aqueous solutions of the active

compositions in water soluble form. Additionally, suspensions of the active compositions may be prepared as appropriate oily injection suspensions. Alternatively, the active compositions may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.

The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compositions, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249:1527-1533, 1990, which is incorporated herein by reference.

Therapeutics may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

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. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. The methods and techniques of the present disclosure are generally performed according to conventional methods well- known in the art. Generally, nomenclatures used in connection with, and techniques of biochemistry, enzymology, molecular and cellular biology, 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.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference, in particular for the teaching that is referenced hereinabove. However, the citation of any reference is not intended to be an admission that the reference is prior art.

EXAMPLES

Example 1: Generation of transgenic goats that produce Herceptin/trastuzumab

Transgenic goats were generated that include the nucleic acid sequence encoding the trastuzumab antibody in their genome. The goats producing trastuzumab were generated using traditional microinjection techniques (See e.g., US 7,928,064, incorporated by reference herein). The cDNA encoding the heavy and light chain (SEQ ID NO:4 and SEQ ID NO:5) were ligated with the beta casein expression vector to yield constructs BC2601 HC and BC2602 LC. In these plasmids, the nucleic acid sequence encoding trastuzumab is under the control of a promoter facilitating the expression of trastuzumab in the mammary gland of the goats. The prokaryotic sequences were removed and the DNA microinjected into pre implantation embryos of the goat. These embryos were then transferred to pseudo pregnant females. The progeny that resulted were screened for the presence of the transgenes. Those that carried both chains were identified as transgenic founders.

A nucleic acid sequence encoding the heavy chain of trastuzumab is provided in SEQ ID NO:4:

ATGGAGTTCGGCCTGAGCTGGCTGTTCCTGGTGGCCATCCTGAAGGGCGTGCAGTGCGAGGTGCAGCTGGTCGAG

AGCGGAGGAGGACTGGTCCAGCCTGGCGGCAGCCTGAGACTGAGCTGCGCCGCCAGCGGCTTCAACATCAAGGAC

ACCTACATCCACTGGGTGCGCCAGGCTCCAGGGAAAGGGCTCGAATGGGTGGCCAGGATCTACCCCACCAACGGC

TACACCAGATACGCCGACAGCGTGAAGGGCAGGTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTG

CAGATGAACAGCCTGAGGGCCGAGGACACCGCCGTGTACTACTGCAGCAGATGGGGTGGGGATGGCTTCTACGCC

ATGGACTACTGGGGGCAGGGCACACTGGTCACAGTCTCCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTG

GCTCCTTCCTCTAAATCCACAAGCGGCGGCACCGCTGCCCTGGGCTGCCTGGTGAAGGACTACTTCCCCGAGCCC

GTGACCGTGTCTTGGAACTCTGGCGCCCTGACCTCCGGCGTGCACACCTTCCCCGCCGTGCTGCAGAGCAGCGGC

CTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCTCTTCCTCTCTCGGAACACAGACCTACATCTGCAACGTGAAC

CACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGAGCCCAAGAGCTGCGACAAGACCCATACATGTCCTCCC

TGTCCTGCTCCTGAGCTGCTGGGCGGACCCTCCGTGTTCCTGTTCCCCCCCAAGCCCAAGGACACCCTGATGATC

AGCAGGACCCCCGAGGTGACCTGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGAAGTTCAACTGGTAC

GTGGACGGCGTGGAGGTGCACAACGCCAAGACCAAGCCCAGAGAGGAGCAGTACAACAGCACCTACAGGGTGGTG

TCCGTGCTGACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAAGTCTCCAACAAGGCCCTG

CCAGCCCCCATCGAAAAGACCATCAGCAAGGCCAAGGGCCAGCCTCGCGAGCCCCAGGTGTACACCCTGCCCCCC

TCCCGCGACGAGCTGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTCTACCCCAGCGATATCGCC

GTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACAAGACCACCCCCCCTGTGCTGGACAGCGACGGCAGC

TTCTTCCTGTACAGCAAGCTGACCGTGGACAAGAGCAGGTGGCAGCAGGGAAATGTCTTTTCCTGTTCCGTCATG

CATGAAGCTCTGCACAACCACTACACCCAGAAGTCCCTGAGCCTGAGCCCCGGCAAGTGATAG A nucleic acid sequence encoding the light chain of trastuzumab is provided in SEQ ID NO:5:

ATGACTCAGTCTCCTTCTTCCCTCTCCGCCAGCGTGGGCGACAGAGTGACCATCACCTGCAGGGCCAGCCAGGAC GTGAACACCGCCGTGGCCTGGTATCAGCAGAAGCCCGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTC CTGTACAGCGGCGTGCCCAGCAGGTTCAGCGGCAGCAGAAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTG CAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCCCCACCTTCGGCCAGGGCACCAAG GTGGAGATCAAGAGGACCGTGGCCGCTCCCAGCGTGTTCATCTTCCCCCCCAGCGACGAGCAGCTGAAGTCCGGC ACCGCCTCCGTGGTGTGCCTGCTGAACAACTTCTACCCCCGCGAGGCCAAGGTGCAGTGGAAGGTGGACAACGCC CTGCAGAGCGGCAACAGCCAGGAGAGCGTCACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAGCACC CTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAGGTGACCCACCAGGGCCTGTCCAGC CCCGTGACCAAGAGCTTCAACAGGGGCGAGTGCTGA

When age appropriate, the founder animals were bred. Following pregnancy and parturition the goats were milked. For example, Goat N0366 had been through two natural lactations producing approximately 1.5-2F of milk per day for 200 days, resulting in 40-70 g/F of Herceptin/trastuzumab.

Example 2: Antibody clarification

Trastuzumab produced in the milk of transgenic goats was isolated using the antibody purification process briefly outlined in Table 1.

Table 1: Antibody Purification Process

Milk de-creaming process

Milk that was harvested from transgenic goats was subjected to a clarification process. Briefly, frozen milk (8-10 L) was thawed overnight at room temperature. Then, the milk was warmed to a temperature of 40°C in a water bath. The de-creamer was turned on dry and allowed to achieve maximum speed, and the 40°C milk was immediately poured in the de-creamer bowl. The skimmed milk was collected into a tared tank liner, and the fat was collected into a separate container and discarded. The mass of the skim milk was recorded for each of 16 runs and presented in Table 2.

Table 2: Process Load Material

⁺Milk from three goats was used in the process.

*The estimate of product (trastuzumab) mass is based on a 50 g/L milk concentration, which is the theoretical average concentration. This is based on reverse phase HPLC assay of 40-60 g/L of trastuzumab per goat over the course of their milk production. To calculate the estimate of grams, the skim milk (kg) is multiplied by 50 g/L. SP Sepharose Big Beads Chromatography

A 30 L SP Big Beads (GE Healthcare) column was used on the AKTA Ready System at a linear velocity of 150 - 300 cm/hr. To prepare for the SP load, the de-creamed milk was diluted with 5 volumes of water to equal the pH and conductivity of the equilibration buffer. The pH was manually adjusted to 6, as needed, and the conductivity was equal to or less than 2.3 mS/cm.

The SP Big Bead column was first sanitized with 2 column volumes of 0.1 N NaOH. To reduce the time to pH the column, 1 column volume of 100 mM Na phosphate pH 6.5 was used before it was equilibrated with 20 mM Na phosphate pH 6.75.

The de-creamed milk was loaded and then washed with 2 column volumes of the equilibration buffer. The product was eluted with PBS pH 6.75 and cleaned with 2 column volumes of 0.5 N NaOH/l M NaCl in up-flow mode. Table 3 presents the method and column information for the final optimized method. An example of a chromatogram using SP Sepharose Big Beads is shown in Figure 2.

Table 3: Column and Method using SP Big Beads

The product was concentrated to ~40g/L using 1.5 m² of Tangenx 30 kDa

ultrafiltration membranes. The SP elution was optionally concentrated and frozen. The estimated recovery from the 16 runs was 98% based on the“Estimate of grams” from Table 2. A total of 6.6 kg of antibody was eluted from the SP Big Beads process step, as shown in see Table 4. Data not recorded in the batch is indicated as“N/A.” Table 4: SP Big Bead Process Step Data

Protein A Chromatography

The GE recombinant Protein A Sepharose Fast Flow column was run on the AKTA Ready System at a linear velocity of 150 cm/hr (100 cm/hr during the load). To safeguard the Protein A, a 0.8 + 0.2 pm filter was used to filter the load material prior to the

chromatography step.

From storage, the column was equilibrated with PBS pH 7.0 before loading. The column was loaded with 20-25 grams of trastuzumab per liter of resin. The column was washed with PBS, a low pH acetate buffer, and then PBS again to remove as much impurities as possible before the elution. The product was eluted with 100 mM glycine pH 3.0 and immediately neutralized with 2 M Tris pH 8 at approximately a l/20th volume. Table 5 presents the method and column information for the final optimized method.

Table 5: Column and Method using Protein A chromatography

The concentration of the load and elution fractions were determined by A280 using a general antibody extinction coefficient (€ 2x0 = 1.4). Due to the limited amount of resin, the Protein A column was run 83 times, four runs per day. The clean-in-place (CIP) cleaning was performed once approximately every 15 cycles due to the long term caustic instability of the Protein A media. The column was also undersized for the campaign. Figure 3 shows an example of a Protein A chromatogram. As shown in Table 6, the average recovery was 89% and yielded approximately 5.3 kg. Data not recorded in the batch is indicated as“N/A.”

Table 6: Protein A Process Data

Ultrafiltration/Diafiltration 1: Post- Protein A Chromatography

The ultrafiltration system was equilibrated with 20mM Tris pH 8.0. The concentrated Protein A eluate was diafiltered against 5 diavolumes of 20mM Tris pH 8.0 buffer. The product was recovered from the 2m² of Pall Omega 10 kDa ultrafiltration membranes, filtered with a Sartorius Sartopore 2 0.8/0.2 um and frozen at -80°C. Table 7 presents the ultrafiltration/diafiltration system information.

Table 7: Ultrafiltration/Diafiltration System

The UF/DF system was cleaned with a 0.5N NaOH, 400ppm bleach solution that was warmed to approximately 40-45 °C. First, a recirculation was performed for approximately 15 minutes. It was drained and another recirculation of 0.5N NaOH, 400ppm bleach for approximately 30 minutes. It was flushed and stored in 0.1 N NAOH until next use.

There were 21 Post Protein A UF/DF runs. One UF/DF was performed for every 4 cycles of Protein A. The UF/DF runs were split into two cycles per day. The average recovery was 96%. The final concentration was approximately 60 g/L, see Table 8.

Table 8: Ultrafiltration/Diafiltration Process Data

Q Sepharose Flow Through Chromatography

The Q Sepharose was run on the AKTA Ready System at a linear velocity of 100-200 cm/hr, see Table 9. The diafiltered Protein A eluate was thawed overnight at room temperature then directly loaded onto an equilibrated Q Sepharose Fast Flow column with an inline pre-filter. Prior to exposure to product, the column was cleaned with 0.1 N NaOH and a pre-conditioning step with 200 mM Na phosphate, pH 6.5 to neutralize the cleaning solution and equilibrated with 20 mM Tris, pH 8.0. During the load, the flow through was collected when the absorbance rose above the established baseline. After loading, the column was washed with 20 mM Tris, pH 8.0 and collected until the absorbance returned to the baseline. The column was cleaned with 0.5 N NaOH, 1.0 M NaCl and stored overnight in 10 mM NaOH. See Table 9 presents the method and column information. Figure 4 shows an example Q Sepharose chromatogram.

The recovery average of the 12 total runs was 86%. A total of 4.4 kg of antibody was eluted from the Q Sepharose process step; see Table 10. Data not recorded in the batch is indicated as“N/A.” A load capacity study of the Q Sepharose was performed at lab scale. It was determined that the Q Sepharose load capacity can be increased to 200g/L.

Table 9: Q Sepharose column and method information

Table 10: Q Sepharose Process Data

Ultrafiltration/Diafiltration 2: Post-Q Sepharose chromatography

The system was equilibrated with PBS pH 7.0 buffer. The Q Sepharose flow through was divided into two parts and processed separately (2 cycles). The Q Sepharose flow through was diafiltered against 5 diavolumes of PBS pH 7.0 buffer. The product was recovered from the 2m² of Pall Omega lOkDa ultrafiltration membranes. After both runs the product was filtered with a Sartorius Sartopore 2 XLM 0.8/0.2 um and referred as purified mAb. The mAb was stored at 2-8°C for enzymatic antibody digestion. Table 11 presents the ultrafiltration/diafiltration system information.

The UF/DF system was cleaned with a 0.5 N NaOH, 400 ppm bleach solution that was warmed to approximately 40-45 °C. First, a recirculation was performed for

approximately 15 minutes. It was drained and another recirculation of 0.5N NaOH, 400 ppm bleach was performed for approximately 30 minutes. It was flushed and stored in 0.1 N NAOH until next use.

As shown in Table 12, the average recovery for the 12 post-Q Sepharose Q UF/DF samples processed was 101%. The average final concentration was approximately 60 g/L. Data not recorded in the batch is indicated as“N/A.”

Table 11: Ultrafiltration/Diafiltration system information

Table 12: Ultrafiltration/Diafiltration 2 Process Data

Example 3: Digestion and Purification of Herceptin/trastuzumab

Antibodies obtained using the process presented in Example 2 were subjected to an additional purification process shown in Table 13 to obtain isolated Fc fragments. Briefly, the purified antibodies were digested overnight then loaded onto a Phenyl hydrophobic interaction chromatography column to separate the different fragments of the antibody, recovering the Fc portion. The material was then diafiltered into a low ionic strength buffer with pH 6.5 to remove the residual Fab and undigested antibody through the combined SP Sepharose Fast Flow + Protein L affinity chromatography step. The last chromatography step of hydroxyapatite chromatography using CaPure, separates the half Fc fragments (Fc fragments with improper disulfide bonds) from the whole, intact Fc fragments. The whole Fc fragments were diafiltered into PBS, concentrated to lOOg/L, and filtered using a 0.22pm filter before storage at -80°C. Table 13: Fc fragment purification process

Antibody Digestion

A total of 2 Phenyl column loads were to be digested per day. It was ensured that the material and all subsequent reagents were filtered at 0.22 pm for this overnight process. Reagents were used within approximately 24 hours of manufacture and stored at 2-8°C before use (if not used immediately). The day before the phenyl column was run; 480 grams were taken from the Q Sepharose UF/DF and warmed to approximately 37°C. A 1/10 total volume of 100 mM cysteine / 100 mM EDTA pH 7.0 was pumped into to the warmed bag.

Next, 0.16 mg of papain per gram of antibody was pumped to the bag. The digest was incubated overnight at a constant temperature of 37°C (+/-3). The total weight was recorded on the bag to help to calculate the dilution the next day. A total of 4.75 kg of mAb was digested, as shown in Table 14. Hydrophobic Interaction Chromatography

The Hydrophobic Interaction Chromatography was performed using Tosoh Phenyl 600M on the AKTA Ready System and run at a linear velocity of 50 - 140 cm/hr. The digested diafiltered Q-Sepharose flow-through was diluted 1.13:1 (w/w) with 20 mM sodium phosphate, 1.2 M sodium sulfate pH 7.0 prior to loading. The conductivity of the load matched the equilibration buffer, approximately 69 mS/cm. The column was first sanitized with 0.1 N NaOH followed by 0.5 CV of H₂0 before conditioning the column with 20 mM sodium phosphate, 600 mM sodium sulfate pH 7.0. After loading, the digested material was washed with 5 CV of equilibration buffer to wash the Fab fragments off the column. The Fc fragments were eluted with 5 CV of 20 mM sodium phosphate, 400 mM sodium sulfate pH 7.0. Lastly, the column was stripped of all the undigested material with 0.1 N NaOH and stored in 10 mM NaOH until next run. Table 14 presents the method and column information, and Figure 5 shows an example of a chromatogram from the hydrophobic interaction chromatography.

As shown in Table 15, the average protein recovery was 21% and the yield was 980 g of Fc fragments. Data not recorded in the batch is indicated as“N/A.”

Table 14: Process and Method using Tosoh Phenyl 600 M Column

Table 15: Phenyl Column Process Data

Ultrafiltration/Diafiltration 3 - Post Hydrophobic Interaction Chromatography

The system was equilibrated with lOmM sodium phosphate, 50mM NaCl pH7.0 buffer. The elution from the hydrophobic interaction chromatography (HIC) was diafiltered against 7 diavolumes of lOmM sodium phosphate, 50mM NaCl pH7.0 buffer. It was determined that the final concentration should not exceed 22g/L. The conductivity was < 6.5mS/cm. The product was recovered, and the mass of the product was obtained. The product was loaded onto the SP-Sepharose Fast Flow - Protein L combined column, with a 0.2 um pre-filter, immediately following UF/DF. Table 16 presents the

ultrafiltration/diafiltration system information.

Table 16: Ultrafiltration/diafiltration system information.

The UF/DF system was cleaned with a 0.5N NaOH, 400 ppm bleach solution that was warmed to approximately 40-45 °C. First, a recirculation was performed for approximately 15 minutes. It was drained and another recirculation of 0.5N NaOH, 400ppm bleach was performed for approximately 30 minutes. It was flushed and stored in 0.1N NAOH until next use.

The average recovery from the 6 post-HIC UF/DF runs shown in Table 17 was 98%, and the concentrations were between 40-50 g/L.

Table 17: Ultrafiltration/Diafiltration 3 Process Data

Affinity Chromatography

An affinity chromatography step was performed to remove the residual Fab fragments. The affinity chromatography using Protein L was run on the AKTA Pilot System at a linear velocity of 100 150 cm/hr. A SP-Sepharose fast flow column was used as a pre column and was approximately twice the size of the Protein L column. The sample was diluted 1:1 (v/v) with 5mM sodium phosphate pH 6.5 immediately before loading. The conductivity was less than 5 mS/cm. The Protein L column capacity was 440 g/L, and SP- Sepharose fast flow column capacity was 184 g/L. Both columns were cleaned with 0.1 N NaOH, contact time not to exceed 15 minutes due to caustic instability of the Protein-L media. The pH of the columns were neutralized using 200 mM sodium phosphate pH 6.5 before equilibrating with 5 mM sodium phosphate pH 6.5, as a flow though step.

The wash was collected immediately after the absorbance was above the baseline. The columns were cleaned in 0.1 N NaOH, brought to a neutral pH, and then stored in 20% ethanol. The method and column information is presented in Table 18. Figure 6 shows an example chromatograph from the Protein L affinity chromatography.

As shown in Table 19, the average recovery from the 7 total runs was 82% and the yield was 800 grams. Data not recorded in the batch is indicated as“N/A.” To minimize issues with stability during the load and elution the precipitated Fc was removed from the bag and re- solubilized into a concentrated salt solution. The concentration of the post-HIC Phenyl UF/DF was lowered to 20 g/L. The Fc fragment load was not diluted for the Protein L affinity chromatography until the system was equilibrated. In addition, salt was added immediately to the elution (not to exceed l5mS/cm) to prevent Fc precipitation during 2 - 8 °C storage.

Table 18: Affinity Chromatography Method and Process

Table 19: Protein L Affinity Chromatography Process Data

Hydroxyapatite Chromatography

The Hydroxyapatite Chromatography was performed using Tosoh CaPure run on the AKTA Ready System at a linear velocity of 140 cm/hr (lOOcm/hr during the strip). The column was loaded with sample that had been diluted 1:1 with 5 mM sodium phosphate pH 6.5. The conductivity was less than or equal to 12 mS/cm to prevent FT absorbance. Prior to exposure to product, the column was cleaned with 0.1 N NaOH, then pre-conditioned with 200 mM sodium phosphate, pH 6.5 to neutralize the cleaning solution and equilibrated with 5mM sodium phosphate pH 6.5. After loading, it was washed with 5mM sodium phosphate, 170 mM NaCl pH 6.5 to remove the half Fc fragments. After 6 CV, the column was eluted with 200 mM sodium phosphate pH 6.5 to elute the whole Fc. The column was cleaned and stored in 0.1 N NaOH. The method and column information is presented in Table 20.

Capacity was also determined for the media and it is not recommend to exceed 9g/L.

Table 20: Hydroxyapatite Chromatography Method and Process

As shown in Table 21, the average recovery was 55%, and the total yield was approximately 470 g. By day 4, the protein had majorly precipitated out of solution in the load bags from the Protein L / SP elution. The process load bags were folded to create a funnel so that the heavier precipitated material would settle out. After a few hours, the precipitated Fc fragment material was then slowly drained out and set aside. The precipitated Fc fragment material was diluted, and salt was added until the material went back into solution. The bags with the material that was in solution were e processed, and the re solubilized Fc fragment material was segregated for further evaluation. Table 21: Hydroxyapatite Chromatography Process Data

The samples indicated as“Grade 1” were the unadulterated Fc fragment material that remained in solution. The samples indicated as“Grade 2” were the Fc fragment material that had precipitated under low ionic strength buffer. Because it was re-solubilized into a higher salt buffer, it was diluted 1:1 immediately before loading onto the CaPure column. The

Elution peaks from the CaPure were equivalent. The only difference on the chromatogram is the conductivity of the load. Figures 7A and 7B show examples of chromatograms for Grade 1 and Grade 2 material, respectively. Ultrafiltration/Diafiltration 4

The eluate from the hydroxyapatite chromatography was diafiltered against 6 diavolumes of PBS pH 7.0 buffer and concentrated to approximately 100 g/L. The formulated drug substance was filtered using a 0.8 mhi + 0.2 mhi Sartopore 2 XLG sterile filter and stored at -80°C.

The ultrafiltration/diafiltration system was cleaned with a 0.5 N NaOH, 400 ppm bleach solution that was warmed to approximately 40-45 °C. First, a recirculation was performed for approximately 15 minutes. It was drained and another recirculation of 0.5N NaOH, 400 ppm bleach was performed for approximately 30 minutes. The system was flushed and stored in 0.1 N NAOH until the next use. Table 22 presents the method and membrane information.

Two final ultrafiltration/diafiltration steps were performed on the Grade 1 and Grade 2 material, shown in Table 23. The final buffer was PBS pH 7.0 buffer. Grade 1 yielded 310 g and Grade 2 yielded 170 g which makes the total yield just under 500g.

Table 22: Ultrafiltration/Diafiltration 4 Method and Membrane

Table 23: Ultrafiltration/Diafiltration 4- Process Data

The production of recombinant Fc fragments derived from transgenic goat milk was successfully performed. The overall recovery was 7%. The methods resulted in the production of a Grade 1 batch of Fc fragment material that remained in solution and a Grade 2 batch of resolubilized Fc fragment material.

Claims

CLAIMS What is claimed is:

1. A method of producing a fragment crystallizable (Fc) fragment, the method comprising

providing a transgenic non-human mammal that has been modified to express an antibody comprising an Fc fragment in the mammary gland;

harvesting the antibody comprising the Fc fragment from milk produced by the mammary gland of the transgenic non-human mammal; and

isolating the Fc fragment from the antibody, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to

(i) hydrophobic interaction chromatography;

(ii) affinity chromatography; and

(iii) hydroxyapatite chromatography.

2. A method of producing an Fc fragment, the method comprising

providing a transgenic non-human mammal that has been modified to express an Fc fragment;

harvesting the Fc fragment from the milk produced by the mammary gland of the transgenic non-human mammal; and

isolating the Fc fragment from the antibody, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to

(i) hydrophobic interaction chromatography;

(ii) affinity chromatography; and

(iii) hydroxyapatite chromatography.

3. The method of claims 1, wherein isolating the Fc fragment further comprises

(a) obtaining an antibody comprising an Fc fragment and one or more additional fragment; and

(b) digesting the antibody of (a) to produce an Fc fragment and one or more additional fragments.

4. The method of claim 1 or 3, wherein the hydrophobic interaction chromatography separates the Fc fragment from one or more additional fragments of the antibody.

5. The method of any one of claims 1-4, wherein the hydrophobic interaction chromatography comprises

applying the Fc fragment and one or more additional fragments to a hydrophobic interaction chromatography column; and

recovering the Fc fragment from the hydrophobic interaction chromatography column.

6. The method of any one of claims 1-5, wherein the affinity chromatography comprises applying the Fc fragment to an affinity chromatography column;

recovering the Fc fragment from the affinity chromatography column; and adding a salt to the Fc fragment recovered from the affinity chromatography column.

7. The method of claim 6, wherein the salt is sodium chloride.

8. The method of claim 7, wherein the sodium chloride is added to a concentration of 170 mM.

9. The method of any one of claims 1 or 3-8, wherein the antibody is digested prior to hydrophobic interaction chromatography.

10. The method of claim 9, wherein the digestion is performed by an enzyme.

11. The method of claim 10, wherein the enzyme is a cysteine protease.

12. The method of claim 11, wherein the cysteine protease is papain.

13. The method of claim 12, wherein the papain is immobilized on a solid support.

14. The method of any one of claims 1-13, wherein the affinity chromatography comprises Protein L affinity chromatography.

15. The method of any one of claims 1-14, wherein the hydroxyapatite chromatography comprises applying the Fc fragment to a hydroxyapatite chromatography column; and

recovering a purified Fc fragment from the affinity chromatography column.

16. The method of claim 15, wherein the hydroxyapatite chromatography is performed using a hydroxyapatite chromatography column comprising a hydroxyapatite resin.

17. The method of claim 16, wherein the hydroxyapatite resin comprises macroporous spherical beads of hydroxyapatite.

18. The method of any one of claims 1-17, wherein the transgenic non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, or llama.

19. The method of any one of claims 1-18, wherein the transgenic non-human mammal is also transgenic for the expression of a sialyl transferase.

20. The method of any one of claims 3-19, wherein obtaining the antibody or Fc fragment comprises purifying the antibody or Fc fragment.

21. The method of claim 20, wherein the antibody or Fc fragment is purified using affinity chromatography.

22. The method of claim 21, wherein the Fc fragment is purified using affinity

chromatography.

23. The method of claim 22, wherein the affinity chromatography comprises Protein A affinity chromatography.

24. The method of any one of claims 1-23, wherein the antibody isotype is IgE, IgG, IgA, IgM or IgD.

25. The method of claim 24, wherein the antibody isotype is IgG.

26. The method of claim 25, wherein the antibody is Herceptin.

27. The method of any one of claims 1-26, wherein the purity of the isolated Fc fragment is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.

28. The method of claim 27, wherein the purity of the isolated Fc fragment is assessed by high performance liquid chromatography, SDS-PAGE gel electrophoresis, or contaminant protein ELISA.

29. The method of any one of claims 1-28, wherein the hydrophobic interaction chromatography is performed using a hydrophobic chromatography column comprising an organic polymer resin.

30. The method of claim 29, wherein the organic polymer resin is phenyl organic polymer resin.

31. The method of any one of claims 1-30, wherein the hydrophobic interaction column is eluted using a salt buffer.

32. The method of claim 31, wherein the elution of the hydrophobic interaction column is performed using a decreasing gradient of the salt buffer concentration.

33. The method of any one of claims 1-32, wherein the Fc fragment has anti

inflammatory properties.

34. The method of any one of claims 1-33, wherein the Fc fragment is used to treat a subject with an autoimmune condition or an inflammatory condition.

35. A method of producing an Fc fragment, the method comprising

providing a mammary epithelial cell that has been modified to express an antibody comprising an Fc fragment in the mammary gland;

harvesting the antibody comprising the Fc fragment from the mammary epithelial cell of the transgenic mammal; and isolating the Fc fragment from the antibody, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to

(a) hydrophobic interaction chromatography;

(b) affinity chromatography; and

(c) hydroxyapatite chromatography.

36. A method of producing an Fc fragment, the method comprising

providing a mammary epithelial cell that has been modified to express an Fc fragment;

harvesting the Fc fragment from the mammary epithelial cell; and

isolating the Fc fragment, wherein isolating the Fc fragment comprises subjecting the antibody sequentially to

(a) hydrophobic interaction chromatography;

(b) affinity chromatography; and

(c) hydroxyapatite chromatography.

37. The method of claims 35, wherein isolating the Fc fragment further comprises

(a) obtaining an antibody comprising an Fc fragment and one or more additional fragment; and

(b) digesting the antibody of (a) to produce an Fc fragment and one or more additional fragments.

38. The method of claim 35 or 37, wherein the hydrophobic interaction chromatography separates the Fc fragment from one or more additional fragments of the antibody.

39. The method of any one of claims 35-38, wherein the hydrophobic interaction chromatography comprises

applying the Fc fragment and one or more additional fragments to a hydrophobic interaction chromatography column; and

recovering the Fc fragment from the hydrophobic interaction chromatography column.

40. The method of any one of claims 35-39, wherein the affinity chromatography comprises

applying the Fc fragment to an affinity chromatography column;

recovering the Fc fragment from the affinity chromatography column; and adding a salt to the Fc fragment recovered from the affinity chromatography column.

41. The method of claim 40, wherein the salt is sodium chloride.

42. The method of claim 41, wherein the sodium chloride is added to a concentration of 170 mM.

43. The method of any one of claims 35 or 37-42, wherein the antibody is digested prior to hydrophobic interaction chromatography.

44. The method of claim 43, wherein the digestion is performed by an enzyme.

45. The method of claim 44, wherein the enzyme is a cysteine protease.

46. The method of claim 45, wherein the cysteine protease is papain.

47. The method of claim 46, wherein the papain is immobilized on a solid support.

48. The method of any one of claims 35-47, wherein the affinity chromatography comprises Protein L affinity chromatography.

49. The method of any one of claims 35-48, wherein the hydroxyapatite chromatography comprises applying the Fc fragment to a hydroxyapatite chromatography column; and

recovering a purified Fc fragment from the affinity chromatography column.

50. The method of claim 49, wherein the hydroxyapatite chromatography is performed using a hydroxyapatite chromatography column comprising a hydroxyapatite resin.

51. The method of claim 50, wherein the hydroxyapatite resin comprises macroporous spherical beads of hydroxyapatite.

52. The method of any one of claims 35-51, wherein the mammary gland epithelial cell is from a transgenic non-human mammal.

53. The method of claim 52, wherein the transgenic non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse, or llama.

54. The method of any one of claims 35-53, wherein the mammary gland epithelial cell has also been modified to express a sialyl transferase.

55. The method of any one of claims 37-49, wherein obtaining the antibody or Fc fragment comprises purifying the antibody or Fc fragment.

56. The method of claim 55, wherein the antibody or Fc fragment is purified using affinity chromatography.

57. The method of claim 56, wherein the affinity chromatography comprises Protein A affinity chromatography.

58. The method of any one of claims 35-57, wherein the antibody isotype is IgE, IgG, IgA, IgM or IgD.

59. The method of claim 58, wherein the antibody isotype is IgG.

60. The method of claim 59, wherein the antibody is Herceptin.

61. The method of any one of claims 35-60, wherein the purity of the isolated Fc fragment is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%.

62. The method of claim 61, wherein the purity of the isolated Fc fragment is assessed by high performance liquid chromatography, SDS-PAGE gel electrophoresis, or contaminant protein ELISA.

63. The method of any one of claims 35-62, wherein the hydrophobic interaction chromatography is performed using a hydrophobic chromatography column comprising an organic polymer resin.

64. The method of claim 63, wherein the organic polymer resin is phenyl organic polymer resin.

65. The method of any one of claims 35-64, wherein the hydrophobic interaction column is eluted using a salt buffer.

66. The method of claim 65, wherein the elution of the hydrophobic interaction column is performed using a decreasing gradient of the salt buffer concentration.

67. The method of any one of claims 35-66, wherein the Fc fragment has anti

inflammatory properties.

68. The method of any one of claims 35-67, wherein the Fc fragment is used to treat a subject with an autoimmune condition or an inflammatory condition.

69. A purified Fc fragment produced by the method of any one of the preceding claims.

70. A method comprising administering a therapeutically effective amount of an Fc fragment produced in a transgenic non-human mammal to a subject in need thereof.

71. The method of claim 70, wherein the subject has an inflammatory condition or an autoimmune condition.