WO2020053661A1

WO2020053661A1 - Methods of purifying antibodies from the milk of transgenic non-human mammals comprising the use of chitosan

Info

Publication number: WO2020053661A1
Application number: PCT/IB2019/001016
Authority: WO
Inventors: Nicholas C. MASIELLO; Abdessatar Chtourou
Original assignee: Laboratoire Francais Du Fractionnement Et Des Biotechnologies
Priority date: 2018-09-13
Filing date: 2019-09-13
Publication date: 2020-03-19

Abstract

The present disclosure relates to methods of producing antibodies and compositions comprising antibodies from the milk of transgenic non-human mammals, involving precipitation using an acidic chitosan solution.

Description

METHODS OF PURIFYING ANTIBODIES FROM THE MILK OF TRANSGENIC NON-HUMAN MAMMALS COMPRISING THE USE OF CHITOSAN

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 62/730,605, filed September 13, 2018, entitled“Methods of Purifying Antibodies from the Milk of Transgenic Non-Human Mammals,” the entire contents of which are incorporated by reference herein.

FIELD OF THE INVENTION

The disclosure relates, at least in part, to methods for purifying proteins from the milk of non-human transgenic mammals. Specifically, the methods described herein relate to the purification of proteins produced in the milk of non-human transgenic mammals using an acidic chitosan solution.

BACKGROUND OF THE INVENTION

Proteins, such as antibodies, are used for a large number of industrial and

pharmaceutical applications, such as diagnosis and therapy. To sustain high levels of production, many proteins are produced using recombinant expression systems, such as unicellular organisms (bacteria or yeasts), insect cells (baculovirus system/insect cells), or transgenic plants. However, these expression systems have many limitations, such as protein misfolding, the inability to produce certain complex proteins, and limited protein post- translational modification ( e.g ., glycosylation).

SUMMARY OF THE INVENTION

Aspects of the present disclosure provide methods of producing a protein comprising (a) providing a transgenic non-human mammal that has been modified to express a protein in the mammary gland; (b) harvesting milk produced in the mammary gland of the transgenic non-human mammal; and (c) purifying the protein from the milk, wherein purifying the protein comprises precipitating the milk comprising the protein with an acidic chitosan solution to produce a clarified milk comprising the protein.

In some embodiments, purifying the protein from the milk further comprises subjecting the clarified milk to anion exchange chromatography. In some embodiments, purifying the antibody from the milk further comprises subjecting the protein to cation exchange chromatography. In some embodiments, purifying the antibody from the milk further comprises subjecting the protein to Fc aptamer column chromatography. In some embodiments, the method further comprises recovering the clarified milk produced in step (c). In some embodiments, the anion exchange chromatography comprises applying the clarified milk to an anion exchange chromatography column; and recovering the protein from the anion exchange chromatography column. In some embodiments, the anion exchange chromatography column comprises a resin comprising a quaternary amine ligand.

In some embodiments, the cation exchange chromatography comprises applying the clarified milk to a cation exchange chromatography column; and recovering the protein from the cation exchange chromatography column. In some embodiments, the cation exchange chromatography comprises a resin comprising a sulphopropyl ligand.

In some embodiments, the acidic chitosan solution comprises medium molecular weight (MMW) chitosan. In some embodiments, the acidic chitosan solution comprises high molecular weight (HMW) chitosan. In some embodiments, the acidic chitosan solution comprises ultra-high molecular weight (UHMW) chitosan. In some embodiments, the acidic chitosan solution has a pH between about 4.6 to about 6.5. In some embodiments, the acidic chitosan solution is prepared by dissolving about 10 g of chitosan per liter in 100 mM of acetic acid having a pH about 4.6 to about 6.5.

In some embodiments, precipitating the milk with the acidic chitosan solution comprises adding about 10 to about 15 % (w/w) of the acidic chitosan solution to the milk.

In some embodiments, precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk. In some embodiments, recovering the clarified milk comprises a centrifugation step or a filtration step. In some embodiments, the filtration step is performed using depth filtration. In some embodiments, the filtration is performed using a cloth filter. In some embodiments, the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter. In some embodiments, the filtration is performed using reverse flow mode. In some embodiments, the centrifugation step is performed at a speed of about 4000-12000 xg for 5-60 minutes. In some

embodiments, the depth filtration removes particles having a size greater than 1 pm. In some embodiments the filtration is performed by filtering the clarified milk through a cloth filter, followed by filtering the milk through a depth filter.

In some embodiments, the method further comprises subjecting the protein to affinity chromatography. In some embodiments, the affinity chromatography comprises Protein A chromatography. In some embodiments, the method further comprises subjecting the protein to viral inactivation.

In some embodiments, the milk is raw milk, whole milk, or decreamed milk.

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 a goat. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is a human protein. In some embodiments, the therapeutic protein is an antibody, Fc fusion protein, antithrombin, or alpha-antitrypsin. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an anti-TNFa antibody, anti-CD20 antibody, anti-EGFR antibody, or anti-HER2 antibody. In some embodiments, the anti-TNFa antibody has the same sequence amino acid sequence as adalimumab. In some embodiments, the antibody is an Fc fragment. In some embodiments, the therapeutic protein is antithrombin or alpha-antitrypsin. In some embodiments, the Fc fusion protein comprises an Fc fragment and a coagulation protein. In some embodiments, the coagulation protein is Factor X.

In some embodiments, the purity of the protein is about 90%, 91% 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

Aspects of the present disclosure provide compositions comprising a protein produced by a method comprising a) providing a transgenic non-human mammal that has been modified to express the protein in its mammary gland; b) recovering milk from the mammary gland of said transgenic non-human mammal; and c) purifying said protein by a purification method comprising a step of precipitation of said milk with chitosan. In some embodiments, the purification method of step (c) produces a clarified milk comprising the protein.

In some embodiments, purifying the protein from the milk further comprises subjecting the clarified milk to anion exchange chromatography. In some embodiments, purifying the protein from the milk further comprises subjecting the protein to cation exchange

chromatography. In some embodiments, purifying the protein from the milk further comprises subjecting the protein to Fc aptamer column chromatography. In some

embodiments, the composition comprises the protein and a pharmaceutical acceptable carrier. In some embodiments, the method further comprises recovering the clarified milk. In some embodiments, the anion exchange chromatography comprises applying the clarified milk to an anion exchange chromatography column; and recovering the protein from the anion exchange chromatography column. In some embodiments, the anion exchange

chromatography column comprises a resin comprising a quaternary amine ligand. In some embodiments, the cation exchange chromatography comprises applying the clarified milk to a cation exchange chromatography column; and recovering the protein from the cation exchange chromatography column. In some embodiments, the cation exchange chromatography comprises a resin comprising a sulphopropyl ligand.

In some embodiments, precipitating the milk with the acidic chitosan solution comprises adding about 10% to about 15% (w/w) of the acidic chitosan solution to the milk. In some embodiments, precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk.

In some embodiments, recovering the clarified milk comprises centrifugation or filtration. In some embodiments, the filtration is depth filtration. In some embodiments, the filtration is performed using a cloth filter. In some embodiments, the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter. In some embodiments, the filtration is performed using reverse flow mode. In some embodiments, the centrifugation is performed at a speed of about 4000-12000 xg for 5-60 minutes. In some embodiments, the depth filtration removes particles having a size beyond greater than 1 pm. In some embodiments the filtration is performed by filtering the clarified milk through a cloth filter, followed by filtering the milk through a depth filter. In some embodiments, the method further comprises subjecting the protein to affinity chromatography. In some embodiments, the affinity chromatography comprises Protein A chromatography. In some embodiments, the method further comprises subjecting the protein to viral inactivation.

In some embodiments, the milk is raw milk, whole milk, or decreamed milk.

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 a goat. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is a human protein. In some embodiments, the therapeutic protein is an antibody, Fc-fusion protein, antithrombin, or alpha-antitrypsin. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an anti-TNFa antibody, anti-CD20 antibody, anti-EGFR antibody, or anti-HER2 antibody. In some embodiments, the anti-TNFa antibody has the same amino acid sequence as adalimumab. In some embodiments, the antibody is an Fc fragment. In some

embodiments, the therapeutic protein is antithrombin or alpha-antitrypsin. In some embodiments, the Fc fusion protein comprises an Fc fragment and a coagulation protein. In some embodiments, the coagulation protein is Factor X.

Aspects of the present disclosure provide use of chitosan for precipitating protein and lipid from transgenically produced milk in a method for preparing a composition comprising a protein. In some embodiments, the precipitated protein and lipid produces a clarified milk.

In some embodiments, the clarified milk is subjected to anion exchange

chromatography. In some embodiments, the clarified milk is subjected to cation exchange chromatography. In some embodiments, the clarified milk is subjected to Fc aptamer column chromatography. In some embodiments, the composition comprises the protein and a pharmaceutical acceptable carrier. In some embodiments, the anion exchange

chromatography comprises applying the precipitated protein and lipid to an anion exchange chromatography column; and recovering the composition comprising the protein from the anion exchange chromatography column. In some embodiments, the anion exchange chromatography column comprises a resin comprising a quaternary amine ligand. In some embodiments, the cation exchange chromatography comprises applying the clarified milk to a cation exchange chromatography column; and recovering the protein from the cation exchange chromatography column.

In some embodiments, the chitosan is comprised in an acidic chitosan solution. In some embodiments, the acidic chitosan solution comprises medium molecular weight (MMW) chitosan. In some embodiments, the acidic chitosan solution comprises high molecular weight (HMW) chitosan. In some embodiments, the acidic chitosan solution comprises ultra-high molecular weight (UHMW) chitosan. In some embodiments, the acidic chitosan solution has a pH between about 4.6 to about 6.5.

In some embodiments, the acidic chitosan solution is prepared by dissolving about 10 g of chitosan per liter in 100 mM of acetic acid having a pH about 4.6 to about 6.5. In some embodiments, precipitating the milk with the acidic chitosan solution comprises adding about 10 to about 15 % (w/w) of the acidic chitosan solution to the milk. In some embodiments, precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk.

In some embodiments, the milk is raw milk, whole milk, or decreamed milk.

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 a goat. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is a human protein. In some embodiments, the therapeutic protein is an antibody, Fc fusion protein, antithrombin, or alpha-antitrypsin. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is an anti-TNFa antibody, anti-CD20 antibody, anti-EGFR antibody, or anti-HER2 antibody. In some embodiments, the anti-TNFa antibody has the same amino acid sequence as adalimumab. In some embodiments, the antibody is an Fc fragment. In some

In some embodiments, the precipitated protein and lipid is subjected to centrifugation or filtration. In some embodiments, the filtration is depth filtration. In some embodiments, the filtration is performed using a cloth filter. In some embodiments, the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter. In some embodiments, the filtration is performed using reverse flow mode. In some embodiments, the centrifugation is performed at a speed of about 4000-12000 xg for 5-60 minutes. In some embodiments, the depth filtration removes particles having a size beyond 1 pm. In some embodiments the filtration is performed by filtering the clarified milk through a cloth filter, followed by filtering the milk through a depth filter. In some embodiments, the composition comprising the protein is subjected to affinity chromatography. In some embodiments, the affinity chromatography comprises Protein A chromatography. In some embodiments, the composition comprising the protein is subjected to viral inactivation.

In some embodiments, the purity of the protein in the composition is about 90%, 91% 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

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 combination 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:

Fig. 1 shows a representative stained gel of the chitosan-clarified milk filtrate following depth filtration. Fane 1 shows the molecular weight markers (“MW Markers”), and lane 2 shows the filtrate following depth filtration (“DF Filtrate”). The arrows indicate the presence of beta-lactoglobulin (“Beta-lac”) and alpha-lactoglobulin (“Alpha-lac”).

Fig. 2A shows a representative trace from a preparation of chitosan-clarified milk filtrate obtained from a goat following anion exchange chromatography using a Q Sepharose Big Bead column. Fig. 2B shows a representative stained gel of samples at the indicated stages of preparation. Fane 1 shows milk obtained from a goat; lane 2 shows chitosan- clarified milk filtrate following depth filtration; lane 3 shows the flow through from anion exchange chromatography using a Q Sepharose Big Bead column; and lane 4 shows proteins stripped from the Q Sepharose Big Bead column.

Fig. 3A shows a representative trace from a preparation of chitosan-clarified milk applied to MiniChrome-8 Tosoh AF-rProtein A-650F column, following by wash using Buffers Al and A2, and elution of the anti-TNFa antibodies using Buffer Bl. Fig. 3B shows a representative stained non-reducing SDS-PAGE gel of samples at the indicated stages of producing anti-TNFa antibodies from milk obtained from transgenic goats. Fane 1 shows the starting material (“ST”); lane 2 shows the flow through from a MiniChrome-8 Tosoh AF- rProtein A-650F column; lane 3 shows the wash using Buffers Al and A2; and lane 4 shows the elution using Buffer Bl. Fig. 3C shows a representative trace of an anti-TNFa antibody preparation using high performance liquid chromatography-size exclusion chromatography (HPFC-SEC). The anti-TNFa antibody preparation was obtained from the chromatography run shown in Fig. 3A. Fig. 4A shows a representative trace from a preparation of chitosan-clarified milk applied to a Tosoh AF-rProtein A-650F column followed by elution of the anti-TNFa antibodies. Fig. 4B shows a representative stained non-reducing SDS-PAGE gel of samples at the indicated stages of producing anti-TNFa antibodies from milk obtained from transgenic goats. Lane 1 shows the molecular weight ladder, lane 2 shows the loading material

(“Load”), lanes 3-5 show samples obtained from separate Tosoh AF-rProtein A-650F columns, and lane 6 shows pooled samples following tangential flow chromatography. Fig. 4C shows a representative trace of an anti-TNFa antibody preparation using high

performance liquid chromatography- size exclusion chromatography (HPLC-SEC). The anti- TNFa antibody preparation was obtained from the chromatography run shown in Fig. 4A.

Fig. 5 shows a table indicating the experimental parameters tested using Design- Expert® 10. A model of the turbidity (A₄oo) of clarified milk precipitated with an acidic chitosan solution and filtered through a cloth filter ( i.e ., cheese cloth), has been assessed, as well as the volume recovered through the cloth filter, the filterability (0.2 pm) of filtrate through the cloth filter {i.e., cheese cloth), and the curd size of curds obtained from

precipitating milk with an acidic chitosan solution (assessed on a visual scale from 1 to 10). The varying parameters were the amount of chitosan (%w/w), time, and temperature.

Fig. 6A shows a representative trace from a preparation of chitosan-clarified milk filtrate containing Fc fragments following chromatography using an Fc aptamer column. Fig. 6B shows a representative stained gel of samples at the indicated stages of preparation. Lane 1 shows the starting material; lane 2 shows the flow through from the chromatography using an Fc aptamer column; and lane 3 shows proteins eluted from the column.

DETAILED DESCRIPTION OF THE INVENTION

Production of proteins ( e.g ., therapeutic proteins, such as antibodies) using

recombinant expression systems presents many challenges and limitations, such as protein misfolding, the inability to produce certain complex proteins, incomplete posttranslational modification (e.g., glycosylation), and different glycosylation profiles than those found on human proteins. In view of these limitations, many recombinant expression systems for the production of proteins currently focus on expression in mammalian cell culture, which are costly and have limited yields. Production of proteins ( e.g ., antibodies) in the milk of non-human mammals (e.g., cows, rabbits or goats) has been evaluated, and the gross cost of producing a recombinant protein in the transgenic milk is estimated to be at least 5-fold lower, more particularly 5- to lO-fold lower than the cost of its production in the CHO cell line. In this approach, the proteins are expressed in the mammary epithelial cells of the transgenic non-human mammal. The protein is thus secreted into the milk of the transgenic non-human mammals and recovered from the milk by extraction and purification methods. Although ample amounts of proteins are produced in the milk of transgenic non-human mammals, extraction and purification of the proteins from milk remains one of the limiting steps of this expression system due to the complexity of milk. In particular, milk constituents can be classified into three categories. The whey consists of carbohydrates, proteins (e.g., lactalbumin,

lactoglobulin, albumins, and immunoglobulins from blood), minerals, and water-soluble vitamins. The lipid phase (or cream), consists essentially of lipids in the form of fat globule emulsions having a diameter of approximately 2 pm to 12 pm. Finally, the colloidal micellar phase consists of casein proteins and phosphocalcic salts, which form colloidal micellar complexes, capable of reaching diameters of approximately 0.5 pm, and are frequently in the form of aggregates (“clusters”) of tricalcium phosphate.

Described herein are methods of producing and purifying a protein from the milk of a transgenic non-human mammal. The methods disclosed herein result in a surprisingly high level of purity and high level of recovery (yield) of the isolated protein and overcome many difficulties of conventional purification methods for isolating recombinant proteins from milk. The methods described herein involve providing a transgenic non-human mammal that has been modified to express a protein in the mammary gland, harvesting milk produced by the mammary gland of the transgenic non-human mammal, and purifying the protein. The disclosure further relates to methods of purifying the protein from the milk comprising precipitating the milk comprising the protein with an acidic chitosan solution to produce clarified milk comprising the protein. In some embodiments, the method further involves subjecting the clarified milk to anion exchange chromatography. In some embodiments, the clarified milk comprising the protein or a composition comprising the protein is further subjected to cation exchange chromatography and/or affinity chromatography.

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.

Proteins

The methods described herein relate to producing and purifying a protein from the milk of a transgenic non-human mammal. In general, the methods may be used to produce and purify any proteins that may be transgenically produced. In some embodiments, the protein is a therapeutic protein. In some embodiments, the protein is a therapeutic protein that may be produced, purified, and then used for therapeutic application ( e.g ., administered to a subject to treat a disease or disorder). Production of a protein in the mammary gland of transgenic non-human mammals has the advantage of allowing for the production of large amounts of protein. A variety of proteins can be produced in the mammary gland and transgenic production in non-human mammals has been used to produce human therapeutic proteins, such as serum proteins. An example of a transgenically produced serum protein is Atryn®, a transgenically produced antithrombin, which has been approved for use in both the US and Europe (See e.g., US 5,843,705, US 6,441,145, US 7,019,193 and US 7,928,064).

In some embodiments, the therapeutic protein is a human protein. In some embodiments, the therapeutic protein is an antibody, an Fc fragment, an Fc fusion protein, a coagulation protein, or an Fc fusion protein comprising a coagulation protein fused to an Fc fragment. In some embodiments, the protein is antithrombin. In some embodiments, the protein is alpha-antitrypsin.

Non-limiting examples of coagulation proteins include Factor I (fibrinogen), Factor II (prothrombin), Factor III (tissue factor), Factor IV, Factor V, Factor VI, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII (Hageman factor), Factor XIII, von

Willebrand factor, prekallikrein, high-molecule weight kininogen, fibronectin, antithrombin, heparin, protein C, protein S, protein Z, plasminogen, tissue plasminogen activator, urokinase, plasminogen activator inhibitor- 1, plasminogen activator inhibitor-2. In some embodiments, the protein is an Fc fusion protein comprising an Fc domain and a coagulation protein. In some embodiments, the Fc fusion protein comprises an Fc domain and Factor X.

Antibodies

In some embodiments, the methods described herein are for production and purification of antibodies or fragments thereof. 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 carboxyterminus 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.

Antibodies generally contain a fragment crystallizable (“Fc”) domain and two fragment antigen binding (“Fab”) domains. The term antibody, as used herein, encompasses not only full length polyclonal and monoclonal antibodies, but also, for example, antigen binding fragments thereof (such as Fab, Fab’, F(ab’)2, Fv), single chain (scFv), mutants thereof, humanized antibodies, recombinant antibodies, chimeric antibodies, or a mixture of these. In some embodiments, the antibody is an Fc fragment.

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, for example 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, for example 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.

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. Anti-HER2 antibodies bind HER2 and have been used as a therapeutic, for example, in cancers characterized by the expression of HER2. In some embodiments, the anti-HER2 antibody has the same amino acid sequence as trastuzumab.

In some embodiments, the antibody is an anti-CD20 antibody. Anti-CD20 antibodies bind CD20 and have been used as a therapeutic, for example, in cancers characterized by the expression of CD20. In some embodiments, the anti-CD20 antibody has the same amino acid sequence as ublituximab.

In some embodiments, the antibody is an anti-EGFR antibody. Anti-EGFR antibodies bind EGFR and have been used as a therapeutic, for example, in cancers characterized by aberrant expression of EGFR. In some embodiments, the anti-EGFR antibody has the same amino acid sequence as cetuximab (Erbitux).

In some embodiments, the antibody is an anti-TNF-alpha antibody. Anti-TNF-alpha antibodies, also referred to herein as“anti-TNFoc antibodies,” bind TNF-alpha and have been used as a therapeutic in a variety of diseases characterized by dysregulation of TNF-alpha, including inflammatory disorders. In some embodiments, the anti-TNF-alpha antibody has the same amino acid sequence as infliximab / Remicade (Centocor), adalimumab / Humira (Abbott), or golimumab / Simponi (Centocor). In some embodiments, the anti-TNF-alpha antibody has the same amino acid sequence as adalimumab. In some embodiments, the anti-TNF-alpha antibody includes a heavy chain which comprises SEQ ID NO: 1. In some embodiments, the anti-TNF-alpha antibody includes a light chain which comprises SEQ ID NO: 2. In some embodiments, the anti-TNF-alpha antibody includes a heavy chain which comprises SEQ ID NO: 1 and a light chain which comprises SEQ ID NO: 2. In some embodiments, the anti-TNF-alpha antibody includes a heavy chain which consists of SEQ ID NO: 1. In some embodiments, the anti-TNF-alpha antibody includes a light chain that consists of SEQ ID NO: 2. In some embodiments, the anti-TNF-alpha antibody includes a heavy chain which consists of SEQ ID NO: 1 and a light chain that consists of SEQ ID NO: 2. In some embodiments, the anti-TNF-alpha antibody has the same amino acid sequence as adalimumab.

In some embodiments, the antibody consists of the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2. 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 NO: 1 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 NO: 2.

The sequences are based on the sequences of adalimumab published in US Patent 6,090,382. In some embodiments, the sequences of adalimumab are those as published in US Patent 6,090,382, incorporated by reference herein in its entirety.

It should further be appreciated that in some embodiments, the disclosure also includes antibodies that are based on the sequence of adalimumab but that include mutations that provide the antibodies with additional beneficial desired properties related to bioavailability, stability etc. In some embodiments, the anti-TNF-alpha antibody disclosed herein is further purified.

Fc-fusion proteins

Aspects of the invention relate to producing and purifying Fc fusion proteins. As used herein, the term“Fc fusion protein” refers to an Fc domain fused to a biologically active protein.

As used herein, an“Fc domain” or“Fc fragment” refers to the portion of an immunoglobulin molecule that interacts with cell surface Fc receptors. An Fc domain can comprise one or more heavy chain constant domains (CH). In some embodiments, the Fc domain comprises two heavy chain constant domains. In some embodiments, the Fc domain comprises heavy chain constant domains CH2 and CH3. Fc domains from immunoglobulins of any isotype ( e.g ., IgG, IgA, IgM, IgE, IgD) and any subtype (e.g., IgGl, IgG2, IgG3,

IgG4) can be compatible with aspects of the invention. In some embodiments, the Fc domain is an IgGl Fc domain.

Fusion of Fc domains to biologically active proteins is known in the art (see, e.g.,

U.S. Patent No. 8,431,132, U.S. Patent No. 7,867,491, Czajkowsky et al. (2012) EMBO Mol Med 4:1015-1028; Beck et al. (2011) MAbs 3:415-416; Low et al. (2005) Human

Reproduction 20(7):1805-1813; Ashkenazi et al. (1993) Int. Rev. Immunol. 10:219-227; Chamow et al. (1996) Trends Biotechnol. 14:52-60; Kim et al. (1994) Eur. J. Immunol. 24:2429-2434. Fc domains can be obtained via routine technology, e.g., PCR amplification from a suitable source. An Fc domain can be naturally occurring or synthetic. In some embodiments, an Fc domain is derived from a human, primate, bovine, porcine, caprine, ovine, rodent or canine mammal. More particularly, an Fc domain is derived from a mammalian source including, without limitation, human or other primate, dog, cat, horse, cow, pig, sheep, goat, rabbit, mouse or rat.

In some embodiments, the Fc domain comprises the sequence of SEQ ID NO: 10. In certain embodiments, the Fc domain consists of the sequence of SEQ ID NO: 10. In some embodiments, the Fc domain 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: 10.

The amino acid sequence of a non-limiting example of an Fc domain is provided in SEQ ID NO: 10:

The nucleic acid sequence of a non-limiting example of an Fc domain is provided in SEQ ID NO: 11:

In some embodiments, the nucleic acid encoding the Fc domain comprises SEQ ID NO: 11. In certain embodiments, the nucleic acid encoding the Fc domain consists of SEQ ID NO: 11. In some embodiments, the nucleic acid sequence encoding the Fc domain is at least 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% or 99% identical to SEQ ID NO: 11.

The Fc domain can be covalently linked to a polypeptide. In some embodiments, the polypeptide is attached directly to the Fc domain. For example, a polypeptide can be attached to the flexible hinge region of the Fc domain. A linker region can also be included connecting the polypeptide to the Fc domain, as would be understood by one of ordinary skill in the art. An example of a linker sequence is provided by the sequence (GGGGS)_n (SEQ ID NO: 12) or (G₄S)_n, n being comprised between 1 and 5. Preferably, n=3, and the linker sequence is SEQ ID NO: 13: GGGGSGGGGSGGGGS.

In some embodiments, the Fc fusion protein comprises an Fc domain and a therapeutic protein. In some embodiments, the Fc fusion protein comprises an Fc domain and a coagulation protein or portion thereof. In some embodiments, the Fc fusion protein comprises an Fc domain and Factor X. In some embodiments, the Fc fusion protein comprises an Fc domain and alpha antitrypsin.

Purification of proteins from milk of transgenic non-human mammals

/. Precipitation with acidic chitosan solution Aspects of the invention provide methods for producing a protein comprising precipitating the milk collected from a transgenic non-human mammal to produce clarified milk comprising the protein. As used herein, the term“clarification” or“clarified” or “clarify” refers to the separation of the whey from the micellar phases (essentially casein proteins and phosphocalcic salts) and lipidic (or cream) phases of the milk. Clarification can also eliminate some whey proteins by precipitation.“Clarified milk” (also called whey) is a clear milk from which the lipids (cream) and some of proteins ( e.g caseins, and sometimes, according to the methods described herein, certain whey proteins) have been removed.

As described herein, the milk comprising the protein is precipitated with an acidic chitosan solution. In some embodiments, contacting the milk comprising the protein with an acidic chitosan solution precipitates the caseins present in the milk, thereby producing clarified milk (whey) comprising the protein.

As used herein, the term“chitosan” refers to linear polysaccharides comprising random D-glucosamine and N-acetyl-D-glucosamine subunits, which are linked by b-(1 4)- linkages. Chitosan can be obtained from chitin present in shells of crustaceans, such as shrimp, and may be extracted from chitin, for example by deacetylation. Alternatively, chitosan may be extracted from chitin present in mushrooms, for example by deacetylation.

In some embodiments, the chitosan is preferably extract from chitin present in mushrooms.

In general, chitosan can be classified based on the molecular size range of the polysaccharide chains. Ultra-High molecular weight (UHMW) chitosan has a molecular weight of more than 10000 kDa; high molecular weight (HMW) chitosan has a molecular weight of approximately 310 kDa - 375 kDa; medium molecular weight (MMW) chitosan has a molecular weight of approximately 190 kDa -310 kDa; and low molecular weight (LMW) chitosan has a molecular weight of approximately 50 kDa -190 kDa. In some embodiments, the acidic chitosan solution comprises high molecular weight chitosan. In some embodiments, the acidic chitosan solution comprises medium molecular weight chitosan. In some

embodiments, the acidic chitosan solution comprises low molecular weight chitosan.

Chitosan solutions have been used in the food industry to precipitate b-lactoglobulin, caseins, and milks from whey (Montilla et al. J. Dairy Sci (2005) 89: 1384-1389; Ausar et al. J. Dairy Sci. (2000) 84: 361-369; Hwang et al. J. Agric. Food Chem. (1995) 43: 33-37). Furthermore, chitosan has been found to form complexes with whey proteins, such as a- lactalbumin and b-lactoglobulin (Lee et al. Food Research International (2009) 42: 733-8).

In some embodiments, the acidic chitosan solution is used to precipitate milk lipids and caseins from the milk, thereby clarifying the milk comprising the protein. In some embodiments, the chitosan solution is prepared by dissolving chitosan in a buffer having an acidic pH ( e.g ., less than 7.0) to solubilize the chitosan. As used herein, the term“acidic” refers to a pH that is less than 7.0. In some embodiments, the acidic chitosan solution has a pH between about 2.0 to 4.0. In some embodiments, the acidic chitosan solution has a pH between of about 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0. In some embodiments, the acidic chitosan solution has a pH of about 3.0.

In some embodiments, the chitosan solution is prepared by dissolving chitosan in an acidic buffer. In some embodiments, the chitosan solution is prepared by dissolving chitosan in an acidic buffer at a ratio of about 10 grams of chitosan per liter of the acidic buffer. In some embodiments, the acidic buffer is acetic acid.

In some embodiments, the chitosan solution is prepared by dissolving about 10 grams of chitosan in an acidic buffer at a ratio of approximately 10 grams of chitosan per liter of 100 mM acetic acid having a pH of about 3.5 to 4.5.

Precipitating the milk comprising the protein involves contacting the milk with the acidic chitosan solution such that the milk lipids and caseins precipitate. In some

embodiments, the acidic chitosan solution is added to the milk until the solution“breaks.” As used herein, the term“break” refers to the precipitation of the milk lipids and caseins. In some embodiments, the acidic chitosan solution is added to the milk until the milk lipids and/or caseins precipitate. For example, the acidic chitosan solution may be gradually added to the milk (e.g., while mixing/stirring) until the solution breaks and precipitate is observed. Once the solution is observed to break (precipitate), mixing/stirring is terminated.

In some embodiments, the acidic chitosan solution is added to the milk at a ratio of about 100 mL to about 500 mL of the acidic chitosan solution per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk at a ratio of about 100 mL to about 300 mL, about 150 mL to about 250 mL, about 200 mL to about 300 mL, about 200 mL to about 400 mL, or about 300 mL to about 500 mL of the acidic chitosan solution per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk at a ratio of about 100 mL of the acidic chitosan solution per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk at a ratio of about 200 mL of the acidic chitosan solution per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk at a ratio of about 300 mL of the acidic chitosan solution per liter of milk.

In some embodiments, the acidic chitosan solution is added to the milk at a ratio of about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270,

280, 290, or 300 mL of the acidic chitosan solution per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that about 0.5 to 5.0 grams of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that about 0.5 to 3.0 grams, about 0.5 to 1.5 grams, about 1.0 to 2.5 grams, about 1.0 to 2.0 grams, about 2.0 to 3.0 grams, about 2.0 to 4.0 grams, or about 3.0 to 5.0 grams of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that about 1.5 grams of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that about 1.0 gram of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that about 2.0 grams of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that about 2.5 gram of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that 3.0 grams of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that 0.5, 0.6, 0.7, 0.76, 0.8, 0.9, 1.0, 1.1, 1.125, 1.2, 1.25, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, or 3.0 grams of chitosan is added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that 0.8 grams of chitosan are added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that 1.2 grams of chitosan are added per liter of milk. In some embodiments, the acidic chitosan solution is added to the milk such that 1.1 grams of chitosan are added per liter of milk. In some embodiments, about 15% (v/v) of a 10 g/L acidic chitosan solution is added to the milk.

In some embodiments, the acidic chitosan solution is added to the milk resulting in a pH value of about 4.6 to 8.0. In some embodiments, the acidic chitosan solution is added to the milk resulting in a pH value of about 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,

5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8,

7.9, or 8.0. In some embodiments, the acidic chitosan solution is added to the milk resulting in a pH value of about 6.0 to 6.5.

In some embodiments, the acidic chitosan solution is added to the milk resulting in between about 10-20% chitosan in the milk-chitosan solution. In some embodiments, the acidic chitosan solution is added to the milk resulting in between about 10-15% chitosan in the milk-chitosan solution. In some embodiments, the acidic chitosan solution is added to the milk resulting in between about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% chitosan in the milk-chitosan solution. In some embodiments, lOg/L of acidic chitosan solution is added to the milk in a concentration of 15% volume/volume. In some embodiments, the acidic chitosan solution and milk are incubated for at least 5 minutes to about 18 hours prior to separating the solid and liquid phases of the clarified milk. In some embodiments, the acidic chitosan solution and milk are incubated for 5 mins, 10 mins, 15 mins, 20 mins, 25 mins, 30 mins, 35 mins, 40 mins, 45 mins, 50 mins, 55 mins, 60 mins, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 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, 21 hrs, 22 hrs, 23 hrs, 24 hrs, or 25 hrs.

In some embodiments, the precipitation is performed at a temperature between about 20°C and 40°C. In some embodiments, the precipitation is performed at a temperature between about 22°C and 37°C. In some embodiments, the precipitation is performed at a temperature between about 20°C, 2l°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 3l°C, 32°C, 33°C, 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, and 40°C.

Aspects of the protein purification comprise precipitating the milk comprising the protein to separate the solid phase (precipitate) and liquid phases of the clarified milk. In some embodiments, the clarified milk is subjected to phase separation to recover the clarified milk (whey) containing the protein and remove precipitated milk proteins and lipids. In some embodiments, the phase separation is performed by centrifugation or filtration ( e.g ., cloth filtration, or depth filtration).

In some embodiments, the phase separation is performed by centrifugation of the clarified milk. In general, the centrifugation is performed at a speed and for a period of time such that the solid (precipitate), which comprises precipitated milk proteins, is pelleted. In some embodiments, the centrifugation is performed at a speed of about 4000-12000 xg. In some embodiments, the centrifugation is performed at a speed of about 4000 xg. In some embodiments, the centrifugation is performed at a speed of about 5000 xg. In some embodiments, the centrifugation is performed at a speed of about 6000 xg. In some embodiments, the centrifugation is performed at a speed of about 7000 xg. In some embodiments, the centrifugation is performed at a speed of about 8000 xg. In some embodiments, the centrifugation is performed at a speed of about 9000 xg. In some embodiments, the centrifugation is performed at a speed of about 10000 xg. In some embodiments, the centrifugation is performed at a speed of about 12000 xg.

In some embodiments, the centrifugation is performed for 5-60 minutes. In some embodiments, the centrifugation is performed for 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 minutes. In some embodiments, the centrifugation is performed for 5 minutes. In some embodiments, the centrifugation is performed for about 10 minutes. In some embodiments, the centrifugation is performed for about 20 minutes. In some embodiments, the centrifugation is performed for about 30 minutes. In some embodiments, the

centrifugation is performed for about 40 minutes. In some embodiments, the centrifugation is performed for about 50 minutes. In some embodiments, the centrifugation is performed for about 60 minutes.

In some embodiments, the clarified milk is subjected to phase separation to remove the precipitate ( e.g ., precipitated milk proteins and lipids) and recover the clarified milk (whey) containing the protein. In some embodiments, the phase separation is performed by filtration of the clarified milk. In some embodiments, the phase separation is performed by depth filtration of the clarified milk. In general, the depth filtration is performed such that the solid (precipitate), which comprises precipitated milk proteins, is removed from the clarified milk comprising the desired protein. As used herein, the term“depth filtration” or“dead end” filtration refers to the process of filtering of solution using a porous filtration medium (e.g., a depth filter) that traps solids of a certain size and allows liquid (e.g., clarified milk comprising the protein) to flow through.

Depth filters comprise a matrix of randomly oriented, bonded fibers that retain particles; the size of particles retained for a depth filter is referred to as the nominal retention rating of the filter. In some embodiments, the depth filter removes solid particles having a size that is greater than about 3 pm, 4 pm, 5 pm, 6 pm, 7 pm, 8 pm, 9 pm, 10 pm, 11 pm, 12 pm, 13 pm, 14 pm, 15 pm, or 16 pm. In some embodiments, the depth filter has a nominal retention rating of about 3 pm to about 15 pm. In some embodiments, the depth filter has a nominal retention rating of about 6 pm to about 12 pm. In some embodiments, the depth filtration is performed using a Pall K700 depth filter (PALL Life Sciences). In some embodiments, the depth filtration is performed using a Pall T2600 depth filter (PALL Life Sciences). In some embodiments, the depth filtration is performed using a Pall K250P depth filter (PALL Life Sciences). In some embodiments, the depth filtration removes particles having a size (e.g., diameter) greater than about 1 pm.

In some embodiments, the precipitated milk is diluted with equilibration buffer prior to depth filtration. In some embodiments, the precipitated milk is diluted about l-fold, 2- fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or lO-fold with equilibration buffer prior to depth filtration. In some embodiments, the equilibration buffer comprises Tris. In some embodiments, the equilibration buffer comprises 20 mM Tris at pH 8.0.

In some embodiments, the phase separation is performed by filtration of the clarified milk. In some embodiments, the filtration is performed by filtering the clarified milk through a filter. In some embodiments, the filter has a pore size of about 200 to 1000 pm. In some embodiments, the filter has a pore size of 200 pm, 250 pm, 300 pm, 350 pm, 400 pm, 450 pm, 500 pm, 550 pm, 600 pm, 650 pm, 700 pm, 750 pm, 800 pm, 850 pm, 900 pm, 950 pm or 1000 pm. In some embodiments, the filter is a cloth filter ( e.g ., cheese cloth). In general cloth filters can be classified into grades based on the thread count of the cloth. For example, Grade #10 has 20 x 12 threads per inch (vertical by horizontal); Grade #40 has 24 x 20 threads per inch; Grade # 50 has 28 x 24 threads per inch; Grade #60 has 32 x 28 threads per inch; and Grade #90 has 44 x 36 threads per inch. The process of filtering of the solution through the cloth filter that traps solids of a certain size and allows liquid (e.g., clarified milk comprising the protein) to flow through. In some embodiments, the cloth filter is Grade #90 cheese cloth. In some embodiments, the cloth filter is a propylene felt bag filter having a pore size between 1 -200pm, preferably lpm. In some embodiments, the cloth filter is a propylene pleated felt bag filter. In some embodiments the filtration is performed by filtering the clarified milk through a cloth filter, followed by filtering the milk through a depth filter. In some embodiments the filtration is performed by filtering the clarified milk through a propylene felt bag filter having a pore size of lpm, followed by filtering the milk through a depth filter followed by filtering the milk through a 0.2 pm filter. In some embodiments, the bag filtration is performed using a reverse flow mode.

II. Anion exchange chromatography

Aspects of the invention relate to purifying a protein from milk obtained from a transgenic non-human mammal comprising precipitating the milk with an acidic chitosan solution to produce clarified milk comprising the protein. In some embodiments, the clarified milk is subjected to chromatography. In some embodiments, the clarified milk is subjected to anion exchange chromatography. In some embodiments, the method comprises further subjecting the protein or a composition comprising the protein to further purification such as by cation exchange chromatography and/or affinity chromatography. In some embodiments, the methods of purifying a protein from milk obtained from a transgenic non-human mammal consist of precipitating the milk with an acidic chitosan solution to produce clarified milk, and subjecting the clarified milk to an anion-exchange chromatography.

As used herein,“anion exchange chromatography” refers to a method of separating components from a mixture based on charge interactions between components in a mixture and an immobilized ligand within a column. In particular, anion exchange chromatography comprises a positively charged ligand immobilized for example on a resin, such as agarose beads, which has an affinity for net negatively charged molecules. The relative binding affinity of the positively charged ligand and the net negatively charged molecules is based on the strength of the negative charge (e.g., stronger negative charges results in increased binding affinity). In some embodiments, the clarified milk is applied to the anion exchange chromatography column and the protein is recovered from the anion exchange

chromatography column.

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. Non-limiting examples of ligands that may be immobilized on a resin for use in anion exchange chromatography, as described herein, include diethylaminoethanol (DEAE), polyethyleneimine, quaternized polyethyleneimine, and quaternary amine. In some embodiments, the anion exchange chromatography comprises resin comprise a quartemary amine ligand. In some embodiments, the resin comprising the quarternary amine ligand is a Q Sepharose Fast Flow anion exchanger (GE Healthcare Life Sciences). In some

embodiments, the anion exchange chromatography is performed, followed by filtration using a filter comprising quartemary amine ligand (e.g., a SARTOBIND® Q membrane adsorber (Sartorius Stedim Plastics GmbH)).

In general, anion exchange chromatography removes impurities and/or contaminating proteins (e.g., undesired proteins and other negatively charged molecules) from the clarified milk. In some embodiments, the proteins are antibodies. Antibodies, such as the anti-TNF- alpha antibodies described herein, have a basic isoelectric point (e.g., pi ~9), whereas many milk proteins from the transgenic non-human mammal have a lower isoelectric point and are anionic. In some embodiments, the anion exchange chromatography removes lactalbumin, lactoglobulin, albumin, and/or lactoferrin from the clarified milk. In some embodiments, the anion exchange chromatography comprises a resin containing an immobilized positively charged ligand and interaction between the ligand and net negatively charged components of the clarified milk are retained in the column by the ligand. In some embodiments, the antibodies are not retained by the ligand and pass through the column.

In some embodiments, the clarified milk is diluted with a buffer at a particular pH prior to anion exchange chromatography. In some embodiments, the pH of the clarified milk is about 6. In some embodiments, the clarified milk is diluted with a buffer at a particular pH that is above the isoelectric point (pi) of proteins and/or other undesired components prior to anion exchange chromatography. In some embodiments, the clarified milk is diluted with a buffer at a pH about 7.5, 8.0, 8.5, 9.0, 9.5 or higher prior to anion exchange chromatography. In some embodiments, the clarified milk is diluted with a buffer at a pH 8.5 to 9.5 prior to anion exchange chromatography. Negative charges ( e.g ., negatively charged amino acids within a polypeptide, other negatively charged molecules) interact with the positively charged ligand and are retained within the anion exchange chromatography column.

Samples from the anion exchange chromatograph (e.g., flow through samples comprising the protein) 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.

III. Cation exchange chromatography

Aspects of the invention relate to purifying a protein from milk obtained from transgenic non-human mammal comprising precipitating the milk with an acidic chitosan solution to produce clarified milk comprising the protein. In some embodiments, the method involves subjecting the clarified milk to anion exchange chromatography. Other aspects of the invention relate to purifying a protein from milk obtained from transgenic non-human mammal comprising precipitating the milk with an acidic chitosan solution to produce clarified milk comprising the protein, subjecting the clarified milk to anion exchange chromatography, and subjecting the protein or a composition comprising the protein to cation exchange chromatography. In some embodiments, the method comprises further subjecting the protein or a composition comprising the protein to further purification such as by affinity chromatography. In some embodiments, the methods of purifying a protein from milk obtained from a transgenic non-human mammal consist of precipitating the milk with an acidic chitosan solution to produce clarified milk, and subjecting the clarified milk to an cation-exchange chromatography.

As used herein,“cation exchange chromatography” refers to a method of separating components from a mixture based on charge interactions between components in a mixture and an immobilized ligand within a column. In contrast to anion exchange chromatography, which involves a positively charged ligand, cation exchange chromatography comprises a negatively charged ligand immobilized for example on a resin, such as agarose beads, which have an affinity for net positively charged molecules. The relative binding affinity of the negatively charged ligand and the net positively charged molecules is based on the strength of the positive charge (e.g., stronger positive charges results in increased binding affinity). In some embodiments, the clarified milk, a protein, or a composition comprising a protein is applied to the cation exchange chromatography column and the protein is recovered from the cation exchange chromatography column.

Selection of an appropriate resin for use in the cation exchange chromatography 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. Non-limiting examples of ligands that may be immobilized on a resin for use in cation exchange chromatography, as described herein, include carboxy-methyl-cellulose, sulphopropyl (SP), and sulphonyl. In some embodiments, the cation exchange chromatography comprises resin comprise a sulphopropyl ligand. In some embodiments, the resin comprising the sulphopropyl ligand is a SP-Sepharose Fast Flow cation exchange resin (GE Healthcare Life Sciences).

In general, cation exchange chromatography removes impurities and/or contaminating molecules ( e.g ., undesired proteins, free heavy chains or free light chains of an antibody) from the clarified milk or a composition comprising the protein. In some embodiments, the cation exchange chromatography comprises a resin containing an immobilized negatively charged ligand and interaction between the ligand and net positively charged components of the clarified milk or composition (e.g., the desired proteins) are retained in the column by the ligand. In some embodiments, the impurities and/or contaminating molecules are not retained by the ligand and pass through the column. In some embodiments, the proteins are recovered from the cation exchange chromatography column by elution using an appropriate buffer.

In some embodiments, the clarified milk or composition comprising the proteins is diluted with a buffer at a particular pH prior to cation exchange chromatography. In some embodiments, the clarified milk or composition comprising the proteins is diluted with a buffer at a particular pH that is below the isoelectric point (pi) of the proteins prior to cation exchange chromatography. In some embodiments, the clarified milk is diluted with a buffer at a pH about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0 prior to cation exchange chromatography. In some embodiments, the clarified milk is diluted with a buffer at a pH about 8 prior to cation exchange chromatography. Positive charges (e.g., positively charged amino acids within a polypeptide, such as the antibodies) interact with the negatively charged ligand and are retained within the cation exchange chromatography column.

Samples from the cation exchange chromatograph (e.g., flow through samples comprising the antibody) 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.

IV. Affinity chromatography

Aspects of the invention relate to purifying a protein from milk obtained from a transgenic non-human mammal comprising precipitating the milk with an acidic chitosan solution to produce clarified milk comprising the protein. In some embodiments, the method further involves subjecting the clarified milk to chromatography. In some embodiments, the method further involves subjecting the clarified milk to affinity chromatography. In some embodiments, the methods of purifying a protein from milk obtained from a transgenic non human mammal consist of precipitating the milk with an acidic chitosan solution to produce clarified milk, and subjecting the clarified milk to an affinity chromatography. Other aspects related to precipitating the milk with an acidic chitosan solution to produce clarified milk comprising the protein, subjecting the clarified milk to anion exchange chromatography, and subjecting the protein to cation exchange chromatography. In some embodiments, the protein or clarified milk comprising the protein 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, and 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 a protein of interest, e.g. a therapeutic protein. Contaminants can be undesired proteins from the host non-human transgenic mammal or undesired forms of the protein of interest (degradation products, e.g. free heavy or light chains if the protein is an antibody). In some embodiments, the affinity chromatography is used to remove free chain antibodies ( e.g ., free heavy chains, free light chains). In some embodiments, the affinity chromatography is used to remove undesired components, such as solvents and/or detergents, for example solvents and/or detergents used in viral inactivation. In some embodiments, the desired components (e.g., the proteins) are bound by the resin of the affinity chromatography column and the undesired components remain in the solution and flow through the resin of the affinity chromatography column. In some embodiments, the proteins are bound (retained) by the resin and are collected by elution from the affinity chromatography column using an appropriate buffer.

Any molecule that interacts with the desired proteins and does not interact with the undesired components or has less affinity for the undesired components (relative to the affinity to the desired proteins) may be used in the step of affinity chromatography. In some embodiments, a molecule (e.g., ligand) that interacts with the desired proteins and does not interact with the undesired components or has less affinity for the undesired components (relative to the affinity to the desired proteins) is immobilized on a resin used in the affinity chromatography. In some embodiments, the desired proteins are antibodies. In some embodiments, the molecule is Protein A, which interacts with the Fc fragment of antibodies. Protein- A affinity chromatography known in the art, for example in Carter (2011) Exp Cell Res (317): 1261-1269.

In some embodiments, the affinity chromatography is performed using Protein A resin and the antibodies are retained by the resin and the undesired components remain in the flow through from the affinity chromatography column. Protein A may be immobilized on any resin used in affinity chromatography. In some embodiments, the Protein A is immobilized on agarose beads (e.g., SP-Sepharose).

In some embodiments, the affinity chromatography is an Fc aptamer column chromatography. In some embodiments, the clarified milk is subjected to chromatography.

In some embodiments, the clarified milk is subjected to Fc aptamer column chromatography. In some embodiments, the methods of purifying a protein from milk obtained from a transgenic non-human mammal consist of precipitating the milk with an acidic chitosan solution to produce clarified milk, and subjecting the clarified milk to an Fc aptamer column chromatography. As used herein,“Fc aptamers” refer to synthetic single-stranded

polynucleotides typically comprising 20 to 150 nucleotides in length and are able to bind with a high affinity to an Fc fragment, including antibodies comprising Fc fragments, isolated Fc fragments, and Fc-fusion proteins. Examples of Fc aptamers compatible with use in the methods described herein are known in the art, e.g. , in PCT Publication WO 2018/019538 Al. Examples of aptamers that can be used to bind and elute a protein comprising an Fc fragment, e.g. an isolated Fc fragment or an Fc fusion protein, is the A6-4 aptamer disclosed in the PCT publication WO 2018/019538 Al and corresponding to the aptamer of sequence SEQ ID NO: 7 flanked by its primers of SEQ ID NO: 19 and SEQ ID NO: 20 of WO 2018/019538 Al. Examples of aptamers that can be used for removing goat IgG are A6-2 (SEQ ID NO: 1 flanked by its primers of SEQ ID NO: 19 and SEQ ID NO: 20 of WO 2018/019538 Al) and A6-8 (SEQ ID NO: 2 flanked by its primers of SEQ ID NO: 19 and SEQ ID NO: 20 of WO 2018/019538 Al) aptamers.

The nucleic acid sequences of aptamers disclosed in PCT publication WO

2018/019538 Al are provided below.

Aptamer sequence referred to as SEQ ID NO: 7 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 5 herein:

AAGTTTGTGG GGCTGGGGTT CGGTCCTGGC ACAAATTCGT

Aptamer sequence referred to as SEQ ID NO: 1 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 6 herein:

CCCAGCCTCA TCTCACGGCA TAGTCTCGCC ACACTGGAA

Aptamer sequence referred to as SEQ ID NO: 2 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 7 herein:

CACGGTATAG TCTCGCCCAG TGCCCTTTGT 5 TGGACTTCCT

Primer sequence referred to as SEQ ID NO: 19 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 8 herein:

GGGTCAATGC CAGGTCTC

Primer sequence referred to as SEQ ID NO: 20 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 9 herein:

ATCGGCTCGC AAGCAGTC

In some embodiments, the aptamer comprises SEQ ID NO: 5, flanked by primers set forth by SEQ ID NO: 8 and SEQ ID NO: 9. In some embodiments, the aptamer comprises SEQ ID NO: 6, flanked by primers set forth by SEQ ID NO: 8 and SEQ ID NO: 9. In some embodiments, the aptamer comprises SEQ ID NO: 7, flanked by primers set forth by SEQ ID NO: 8 and SEQ ID NO: 9.

V. Viral inactivation

Aspects of the invention relate to purifying a protein from milk obtained from transgenic non-human mammal comprising precipitating the milk with an acidic chitosan solution to produce clarified milk comprising the protein. In some embodiments, the method further involves subjecting the clarified milk to chromatography. In some embodiments, the method further involves subjecting the clarified milk to anion exchange chromatography. Other aspects relate to precipitating the milk with an acidic chitosan solution to produce clarified milk comprising the protein, subjecting the clarified milk to anion exchange chromatography, and subjecting the protein to cation exchange chromatography. In some embodiments, the protein or clarified milk comprising the protein are subjected to affinity chromatography. In some embodiments, the protein or clarified milk comprising the protein are subjected viral inactivation to inactivate any viruses ( e.g ., from the transgenic non-human mammal) that may be present. In some embodiments, the viral inactivation is used to inactivate viruses, such as enveloped viruses. In some embodiments, the antibody or clarified milk comprising the protein are subjected viral inactivation and subsequently subjected to affinity purification.

In some embodiments, the viral inactivation comprises treatment of the clarified milk or composition comprising the protein with an organic solvent and detergent. In some embodiments, the organic solvent is tri-n-butyl phosphate and the detergent is polysorbate 80. In some embodiments, the viral inactivation step comprises adding solvent to the clarified milk or composition comprising the protein to a final concentration of about 0.3% v/v. In some embodiments, the viral inactivation step comprises adding detergent to the clarified milk or composition comprising the protein to a final concentration of about 1% v/v.

Methods of inactivation viruses in a composition comprising a therapeutic protein that is produced in the mammary gland of transgenic non-human mammal using solvents and detergents are known in the art. See, e.g. PCT Publication No. WO 2007/138199A2, which is incorporated by reference herein.

In some embodiments, the viral inactivation comprises treatment the clarified milk or composition comprising the protein with a glycol, such as propylene glycol. In some embodiments, the viral inactivation step comprises adding a glycol to the clarified milk or composition comprising the protein to a final concentration of about 40-50% v/v. See, e.g., PCT Publication No. WO 2012/090067.

In some embodiments, the viral inactivation comprises subjecting the clarified milk or composition comprising the protein to nanofiltration for removal of viruses, such as non- enveloped viruses.

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, as described herein, the acidic chitosan solution is prepared using an acidic buffer to dissolve the chitosan. Examples of acidic buffers include, without limitation, acetic acid, citric acid, hydrochloric acid, sodium acetate, ethanoic acid. In some embodiments, the buffer has a concentration between 50 mM and 500 mM, between 50 mM and 250 mM, or between 50 mM and 150 mM. In some embodiments, the buffer concentration is about 100 mM. In some embodiments, the acidic chitosan solution is prepared using an acidic buffer comprising 100 mM acetic acid. In some embodiments, the pH of the acidic buffer is between pH 3.5 and pH 6.5. For example, in some embodiments, the pH of the acidic buffer is approximately 3.5, 3.6, 3.7, 3.8, 3.9, 4.0,

4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1,

6.2, 6.3, 6.4, or 6.5.

In embodiments, the anion exchange chromatography is performed using a sodium phosphate buffer. In some embodiments, the sodium phosphate buffer has a pH about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In some embodiments, the sodium phosphate buffer has a pH of about 7.0.

In some embodiments, the anion exchange chromatography comprises diluting the clarified milk with an equilibration buffer. In some embodiments, the anion exchange chromatography comprises washing the anion exchange chromatography column with the equilibration buffer. In some embodiments, the equilibration buffer is a Tris buffer. In some embodiments, the Tris buffer has a concentration between 1 mM and 100 mM Tris, between 10 mM and 50 mM, between 10 mM and 30 mM, between 15 mM and 25 mM. In some embodiments, the equilibration buffer comprises 20 mM Tris. In some embodiments, the equilibration buffer has a pH about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2,

8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, or 9.0. In some embodiments, the sodium phosphate buffer has a pH of about 8.0. In some embodiments, the cation exchange chromatography comprises diluting the clarified milk or a composition comprising the protein with an equilibration buffer. In some embodiments, the cation exchange chromatography comprises washing the cation exchange chromatography column with the equilibration buffer. In some embodiments, the

equilibration buffer is a sodium phosphate buffer. In some embodiments, the sodium phosphate buffer has a concentration between 1 mM and 100 mM, between 10 mM and 50 mM, between 10 mM and 30 mM, between 15 mM and 25 mM. In some embodiments, the equilibration buffer comprises 20 mM sodium phosphate. In some embodiments, the equilibration buffer has a pH about 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.

In some embodiments, the affinity chromatography comprises washing the affinity chromatography column with one or more wash buffers. In some embodiments, the wash buffer is a phosphate buffered solution (PBS). In some embodiments, the wash buffer is a sodium acetate buffer. In some embodiments, the sodium acetate buffer has a concentration between 50 mM and 500 mM, between 50 mM and 250 mM, between 50 mM and 150 mM, between 75 mM and 150 mM. In some embodiments, the wash buffer comprises 100 mM sodium acetate. In some embodiments, the wash buffer has a pH about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 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 wash buffer is a phosphate buffered solution having a pH of about 7.0.

In some embodiments, the wash buffer further comprises a salt. In some

embodiments, the salt is added to a buffer to a concentration of about 500 mM. In some embodiments, the salt is added to a buffer to a concentration of about 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM or higher. In some embodiments, the salt is sodium chloride. In some embodiments, the wash buffer further comprises 500 mM sodium chloride.

In some embodiments, the affinity chromatography comprises recovering the protein from the affinity chromatography column with an elution buffer. In some embodiments, the elution buffer is a glycine buffer. In some embodiments, the glycine buffer has a

concentration between 50 mM and 500 mM, between 50 mM and 250 mM, between 50 mM and 150 mM, between 75 mM and 150 mM. In some embodiments, the elution buffer comprises 100 mM glycine. In some embodiments, the elution buffer has a pH about 2.0,

2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, or 4.0. In some embodiments, the elution buffer has a pH of 3.0. In some embodiments, the elution buffer is neutralized following elution of the protein from the affinity chromatography column.

In some embodiments, the milk is contacted with an ethylenediaminetetraacetic acid (EDTA) solution prior to precipitating the milk comprising the protein with an acidic chitosan solution. In some embodiments, the EDTA solution has a concentration between 10 mM and 100 mM, between 15 mM and 85 mM, between 25 mM and 75 mM, or between 30 mM and 50 mM. In some embodiments, the EDTA solution comprises 40 mM EDTA.

Constructs for the sene ration of transgenic animals expressing proteins

Some aspects of the invention relate to producing primary cell lines containing a construct ( e.g ., encoding a protein) for use in producing transgenic non-human mammals by nuclear transfer. The constructs can be transfected into primary non-human mammal 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 antibody) 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, 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 antibody 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., a- 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 a- 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 ., an 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.

Transgenic non-human mammals Aspects of the present disclosure provide a transgenic non-human mammal comprising mammary gland epithelial cells that express a protein.

In one aspect, the disclosure provides a method for the production of a protein, comprising:

(a) transfecting non-human mammalian cells with a transgene DNA construct encoding a protein;

(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 protein, and that can express the protein in its milk.

In one aspect, the disclosure provides a method of

(a) providing a non-human transgenic mammal engineered to express a protein,

(b) expressing the protein in the milk of the non-human transgenic mammal; and

(c) isolating the protein 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 antibody 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 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 transgenic non-human mammals that produce a protein (e.g., therapeutic proteins). Transgenic non-human mammals according to aspects of the invention express nucleic acid sequences encoding the protein. In some embodiments, the protein is a therapeutic protein. In some embodiments, the therapeutic protein is a coagulation protein. In some embodiments, the therapeutic protein is an Fc fusion protein. In some embodiments, the protein is an antibody or an Fc fragment of an antibody. In some embodiments, the nucleic acid sequences encode an anti-TNF-alpha antibody and comprise a sequence encoding the heavy chain forth in SEQ ID NO: 3 and light chain set forth in SEQ ID NO: 4.

Harvesting milk from transgenic non-human mammals Aspects of the present invention provide methods of harvesting milk produced by the mammary gland of the transgenic non-human mammal that has been modified to express a protein in the mammary gland. In some embodiments, the milk is harvested from a single transgenic non-human mammal. In some embodiments, the milk is harvested from multiple transgenic non-human mammal and pooled into one sample for purification. In some embodiments, the milk is collected from a goat. In some embodiments, the milk is harvested from a single goat or a population of goats. In some embodiments, the milk is harvested from multiple goats and pooled into one sample for purification. In some embodiments, the milk is harvested from a cow or population of cows. In some embodiments, the milk is harvested from a rabbit or population of rabbits. The milk is harvested from a pig or population of pigs.

In some embodiments, the milk is fresh milk. As used herein, the term“fresh milk” refers to milk that has been freshly collected and has not been frozen. In some embodiments, the fresh milk comprising an antibody is clarified, as described herein. In some

embodiments, fresh milk is harvested and subjected to one or steps of the purification process in the same day. Alternatively, in some embodiments, the milk is harvested and frozen prior to purification. Frozen milk may be thawed, for example, in a tepid water bath or at ambient temperature ( e.g ., about 20°C-25°C), prior to purification.

In some embodiments, the milk (e.g., fresh or previously frozen) is warmed to ambient temperature prior to precipitation with the acidic chitosan solution. In some embodiments, the milk (e.g., fresh or previously frozen) is warmed to the temperature of about 37°C prior to precipitation with the acidic chitosan solution..

In some embodiments, purification of the protein from the milk is performed within 24 hours of harvesting the milk. In some embodiments, purification of the protein from the milk is performed within 48 hours of harvesting the milk. In some embodiments, purification of the protein from the milk is performed within 72 hours of harvesting the milk.

In some embodiments, the milk is whole milk. In some embodiments, the milk is raw milk. The terms“whole milk” and“raw milk” can be used interchangeably and refer to milk that has not been previously subjected to a separation process therefore comprises the constituents of milk harvested from a mammal, including components in the whey and colloidal micellar phrase, e.g., caseins and the b-lactoglobulin. In some embodiments, the raw milk has not been subjected to pasteurization.

Methods of separating milk into one or more constituents or separating one or more components from milk are known in the art. For example, the process of cremation generally involves centrifugation to separate the skim milk, including whey and caseins, from the cream ( e.g ., the lipid phase). Removal of the cream (lipids) results in“decreamed milk,” also referred to as“defatted raw milk” or“skimmed milk,” which includes the protein components of milk, including proteins in the whey and in the colloidal micellar phase. In some embodiments, the milk is decreamed milk. In some embodiments, the milk is defatted raw milk. In some embodiments, the milk is skimmed milk.

Production of proteins

The proteins can be obtained, in some embodiments, by harvesting the protein from the milk of a transgenic mammal produced as provided herein or from an offspring of said transgenic mammal.

In some aspects, the proteins produced as described herein have enhanced

characteristics compared to proteins produced by other methods. For example, in some embodiments, proteins produced by methods described herein are of higher purity compared to proteins produced by other methods. In some embodiments, the transgenically produced proteins that are subsequently isolated and purified are at least 90 - 99.99% pure. In some embodiments, the transgenically produced proteins that are subsequently isolated and purified are at least 90, 91, 92, 93, 94 95, 96, 97, 98, 99, 99.5, 99.9 or 99.99% pure.

Purity of proteins 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.

Proteins produced as described herein can also be produced with enhanced efficiency compared to proteins produced by other methods. As used herein,“enhanced efficiency” refers to a higher percent yield of proteins relative to the starting material. The starting material can refer to the starting material of the production process (e.g. raw milk) and the percent yield of proteins relative to the starting material is called total or overall percent yield. The starting material can also refer to the starting material of a specific step of the production process (e.g. clarified milk). In some embodiments, the overall percent yield of proteins purified from milk obtained from transgenic non-human mammals that have been modified to produce the antibodies is 60 - 80%. In some embodiments, the overall percent yield of proteins purified from milk obtained from transgenic non-human mammals that have been modified to produce the proteins is at least 60, 65, 70, 75, or 80%. In some

embodiments, the overall percent yield of proteins purified from milk obtained from transgenic non-human mammals is assessed by Protein A-HPLC analysis. In some embodiments, the overall percent yield of proteins purified from milk obtained from transgenic non-human mammals that have been modified to produce the proteins is at least 60, 65, 70, 75, or 80% as assessed by Protein A-HPLC analysis. In some embodiments, the percent yield of proteins purified from milk obtained from transgenic non-human mammals that have been modified to produce the antibodies is calculated directly using the quantity of the protein of interest in the starting material and its quantity in the final product. In some embodiments, the percent yield of proteins purified from milk obtained from transgenic non human mammals that have been modified to produce the antibodies is calculated at each step of the production process (the starting material being the final product of the preceding step) and the overall percent yield is calculated by multiplying the yields at each step of the purification process.

Methods of treatment

In one aspect, the disclosure provides methods comprising administering proteins produced using the methods described herein to a subject in need thereof.

In some embodiment, the subject has cancer. In some embodiment, the subject has a cancer characterized by the expression of HER2. In some embodiment, the subject has a cancer characterized by the expression of CD20. In some embodiment, the subject has a cancer characterized by the expression of EGFR.

In some other embodiments, the subject has a coagulation disorder. In some embodiment, the subject has a hereditary deficiency in a coagulation protein. In some embodiment, the subject has an acquired deficiency in a coagulation protein. In some embodiment, the coagulation protein is Factor I (fibrinogen), Factor II (prothrombin), Factor III (tissue factor), Factor IV, Factor V, Factor VI, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII (Hageman factor), Factor XIII, von Willebrand factor, prekallikrein, high-molecule weight kininogen, fibronectin, antithrombin, heparin, protein C, protein S, protein Z, plasminogen, tissue plasminogen activator, urokinase, plasminogen activator inhibitor- 1, plasminogen activator inhibitor-2.

In some embodiment, the subject has an inflammatory disorder or autoimmune disorder. In some embodiment, the inflammatory disorder or autoimmune disorder is rheumatoid arthritis, psoriasis, Crohn’s disease, juvenile idiopathic arthritis, ankylozing spondylitis, ulcerative colitis, chronic inflammation, hepatitis, Behcet’s disease, Wegener’s granulomatosis, or sarcoidosis.

In one aspect, the disclosure provides methods for administering proteins produced using the methods described herein to a subject in need thereof. In some embodiments, the subject has an immune disorder or disorder associated with inflammation. Immune disorders and disorders associated with inflammation include but are not limited, to adult respiratory distress syndrome, arteriosclerosis, asthma, atherosclerosis, cholecystitis, cirrhosis, Crohn's disease, diabetes mellitus, emphysema, hypereosinophilia, inflammation, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, rheumatoid arthritis, scleroderma, colitis, systemic lupus erythematosus, lupus nephritis, diabetes mellitus, inflammatory bowel disease, celiac disease, an autoimmune thyroid disease, Addison's disease, Sjogren’s syndrome, Sydenham's chorea, Takayasu's arteritis, Wegener's granulomatosis, autoimmune gastritis, autoimmune hepatitis, cutaneous autoimmune diseases, autoimmune dilated cardiomyopathy, multiple sclerosis, myocarditis, myasthenia gravis, pernicious anemia, polymyalgia, psoriasis, rapidly progressive glomerulonephritis, rheumatoid arthritis, ulcerative colitis, vasculitis,

autoimmune diseases of the muscle, autoimmune diseases of the testis, autoimmune diseases of the ovary and autoimmune diseases of the eye, acne vulgari, asthma, autoimmune diseases, celiac disease, chronic prostatitis, glomerulonephritis, hypersensitivities, inflammatory bowel diseases, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, sarcoidosis, transplant rejection, vasculitis, and interstitial cystitis.

In one aspect, the disclosure provides methods for administering any one of proteins or compositions described herein to a subject in need thereof. In some embodiments, a subject in need of treatments is a subject with a disease characterized by a dysregulation of TNF levels. Disease characterized by a dysregulation of TNF levels, in addition to the inflammatory and immune disorders discussed above, include Alzheimer, cancer and depression.

Pharmaceutical compositions

Aspects of the invention relate to administering effective amounts of a protein, or compositions comprising an antibody. In some embodiments, methods comprise

administering a therapeutically effective amount of a transgenic protein to a subject in need thereof. In some embodiments, the transgenic protein 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 an antibody or composition comprising an antibody 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 a protein 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 antibody.

“Dose” and“dosage” are used interchangeably herein.

In some aspects, the disclosure provides compositions, including pharmaceutical compositions, which comprise transgenically produced and purified protein and a

pharmaceutically acceptable vehicle, diluent or carrier. In some embodiments, the pharmaceutical compositions comprise proteins produced in transgenic non-human mammals.

In one aspect, the disclosure provides pharmaceutical compositions which comprise an amount of a protein produced by the methods described herein and a pharmaceutically acceptable vehicle, diluent or carrier.

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. 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 antibody. 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 proteins 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.

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 anti-TNF-a antibodies

Transgenic goats were generated that include the nucleic acid sequence encoding the human anti-TNF-a antibody in their genome. The goats producing anti-TNF-a antibodies were generated using traditional microinjection techniques (See e.g., U.S. 7,928,064). The cDNA encoding the heavy and light chain (SEQ ID NO: 3) and (SEQ ID NO: 4) were ligated with the beta casein expression vector to yield constructs that induce the expression of the antibodies. In these plasmids, the nucleic acid sequences encoding is the antibody are under the control of a promoter facilitating the expression of the antibody in the mammary gland of the goats. The prokaryotic sequences were removed and the DNA microinjected into pre implantation embryos of the goats. These embryos were then transferred to pseudo pregnant females. The progeny that resulted were screened for the presence of the transgene. Those that carried the both chains were identified as transgenic founders.

A nucleic acid sequence encoding the heavy chain of the anti-TNF-a antibody is provided in SEQ ID NO.: 3:

A nucleic acid sequence encoding the light chain of the anti-TNF-a antibody is provided in SEQ ID NO.: 4:

When age appropriate, the founder animals were bred. Following pregnancy and parturition the goats were milked. The milk collected from these transgenic goats generates approximatively 15 g/L of anti-TNF-a antibodies.

Example 2: Chitosan clarification of milk from transgenic goats (100 mL scale)

Milk was collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands. Approximately 105 mL of milk containing anti-TNF-a antibodies was brought to room temperature, and 28.5 mL of chitosan dissolved in acetic acid (solution containing 2.7 g/L chitosan) was added, resulting in a pH value of about 6.0. The milk and chitosan solution were reacted for 5 minutes, followed by centrifugation at 10,000 xg for 5 minutes to pellet the casein and lipids from the milk. The supernatant, containing the anti-TNF-a antibodies, was removed and filtered through a depth filter, for example having retention pore size of 2.7 pm and a volumetric throughput of 228 L/m², and a

polyethersulfone (PES) filter having a pore size of 0.45 pm and a volumetric throughput of 169 L/m², resulting in a 86% yield of anti-TNF-a antibodies, as assessed by Protein A-HPLC analysis.

Example 3: Chitosan clarification of milk from transgenic goats (200 mL scale)

Milk was collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands. Approximately 200 mL of milk containing anti-TNF-a antibodies was combined with 25 mL of chitosan solution (containing 10 mg/mL chitosan). The mixture was then filtered through a depth filter having a pore size between 15-45 pm ( ._<?., Pall T2600 depth filter) and a volumetric throughput of > 22 L/m² (no pressure was observed) resulting in a 70% yield of anti-TNF-a antibodies, as assessed by Protein A-HPLC analysis. The filtrate appeared slightly hazy, suggesting that a smaller pore size may be effective.

As shown in Fig. 1, the filtrate from depth filtration still contained some beta- lactoglobulin and alpha-lactoglobulin, indicating that these proteins were not fully precipitated by chitosan treatment.

Example 4: Chitosan clarification of milk from transgenic goats (200 mL scale)

Milk was collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands. Approximately 200 mL of milk containing anti-TNF-a antibodies was mixed on a stir plate and approximately 22.5 mL of the acidic chitosan solution (containing 10 mg/mL chitosan) was added slowly. Once a precipitation reaction occurred, addition of the chitosan solution was terminated. The reaction was held for 5 minutes, and the mixture was applied to a depth filter having pore size of 2-8 pm ( i.e ., Pall K250P). 225 mL of the mixture was passed through the filter at 0.2 PSI. The percent recovery of the anti-TNF-a antibodies was determined to be 66%, and the filtrate was observed to be very clear. Alternative filters, notably a cheese cloth, were used and a recovery up to 90% was observed.

Example 5: Purification of chitosan clarified milk using Anion Exchange Chromatography Milk collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands was precipitated using chitosan and subjected to depth filtration. Approximately 100 mg of the depth filtrate was diluted 1:8 with equilibration buffer (20 mM Tris pH8) prior to applying the sample to a Q Sepharose Big Bead column (X/K 16/22) at a flow rate of 5 mL/min using sodium phosphate buffers at pH 7 (Fig. 2A). The flow through was collected based on absorbance at 280 nm. Collection continued into the post-load washing with equilibration buffer until the UV absorbance returned to baseline. Approximately 113 mL volume was collected at 1.0 g/L of antibodies, with 100% recovery.

Following collection, the column was stripped with 2 M sodium chloride or directly cleaned with 0.1 N sodium hydroxide / 1 M sodium chloride.

Samples of the depth filtrate loading material, flow through, and strip were analyzed by SDS-PAGE gel (Fig. 2B). The product was estimated to be at least 99% pure (estimated 99.9% pure). Chitosan precipitation of the milk did not appear to affect performance of the anion exchange chromatography. The process removed most goat milk proteins ( e.g ., albumin, beta-lactalbumin, alpha-lactalbumin), however minor trace amounts of lactoferrin remained in the solution.

The identification of contaminating goat proteins can be determined along with the abundance of each of the contaminants. Additionally, the amount of free immunoglobulin heavy and light chains can be estimated and optionally removed using, for example, a SP Sepharose cation exchange chromatography column.

Example 6: Chitosan precipitation and viral clearance of milk from transgenic goats

Milk is collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands and is precipitated using a chitosan solution to remove caseins. The precipitant is removed by depth filtration. The filtration may optionally be subjected to anion exchange chromatography using a Q Sepharose anion exchange chromatography column to remove most contaminating goat proteins, DNA, RNA, and endotoxins, as described in Example 5.

The solution is subsequently subjected to a viral clearance step, such as a

solvent/detergent (S/D) treatment. For example, Tween/tri-(n-butyl) phosphate may be added to approximately 10 mL of the chitosan-clarified milk while mixing (as described PCT Publication WO 2007/138199A2).

Antibodies obtained further to the viral clearance step may optionally be subjected to chromatography using an SP Sepharose cation exchange chromatography column to remove free heavy chains and light chains. Briefly, the flow through from the Q Sepharose anion exchange chromatography column comprising the antibody was equilibrated with 20 mM sodium phosphate having a pH between 6.8-8.0 and then directly applied to the SP Sepharose cation exchange chromatography column. The column was washed with equilibration buffer, and the antibodies were eluted using 200 mM sodium chloride in 20 mM sodium phosphate. The recovery of the purified antibodies was estimated to be approximately 75%.

Example 7: Preparation of Protein A-purified anti-TNF-a antibodies with chitosan precipitation

Milk collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands was precipitated using chitosan and subjected to depth filtration. As shown in Fig. 3A, the clarified milk was applied to a Protein A-resin column ( e.g ., MiniChrome-8 Tosoh AF-rProtein A 650F column) using the below conditions:

Buffer Al: PBS pH 7.0. Flow at 2.0mL/min

Buffer A2: 0.1M Na acetate pH 5.5 + 0.5M NaCl

Buffer Bl: 0.1M glycine pH 3.0

Load: 25mL depth filtrate (l2mg/mL, about 300mg). Add 25mL PBS.

The column was washed with Buffer Al, then Buffer A2, and then eluted with Buffer Bl. The eluate was neutralized with 1/10 volume 1M Tris pH 7.5 and resulting 16.0 mL of product at 18.6 mg/mL anti-TNFa antibodies (297.6 mg total).

The eluted product was analyzed by NR-SDS-PAGE (Fig. 3B) and HPLC-SEC (Fig.

3C).

Example 8: Preparation of Protein A-purified anti-TNF-a antibodies with chitosan precipitation

In a larger scale purification, 2 liters preparations of milk collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands were utilized. Approximately 225 mL of chitosan solution (10 mg/mL in 100 mM acetic acid) was added slowly to the milk with stirring and mixed until precipitation was observed. The reaction proceeded for 10 minutes after which the precipitate was pelleted by centrifugation (12,000 xg for 60 minutes). The supernatant was removed and filtered through a 0.2 pm filter. The resulting clarified milk had a volume of approximately 1850 mL containing 14.2 mg/mL anti-TNFa antibodies (26.3 g, 84% recovery).

Approximately 12.7 g of anti-TNFa antibodies obtained above, were subjected to further purification using Protein A-resin (Tosoh AF-rPA HC-650F, having a 60 g/L capacity, 150 cm/hr) (Fig. 4A). Approximately 12730 mg of product was eluted, resulting in recovery of 90% to >99% after the Protein A chromatography. The overall recovery of the process was about 77.3%. The product was further subjected to tangential flow filtration using 10 mM Na acetate, pH 5. Samples from various steps of purification were analyzed by NR-SDS-PAGE gel (loading material from the Protein A column, samples during the Protein A chromatography, and following tangential flow filtration). The final product was determined to be approximately 99.5% pure, as assessed by HPLC-SEC (Fig. 4C).

Example 9: Optimization of parameters of the acidic chitosan precipitation process To determine and optimize parameters of the acidic chitosan precipitation process, different parameters were varied (Fig. 5A). Briefly, the time post-chitosan addition ranged from 5 to 30 minutes; the amount of chitosan (%w/w) ranged from 10% to 15%, and the temperature during the chitosan precipitation ranged from 10°C to 37°C. Other parameters remained constant: 42mL of milk from a single pool was used for the experiments; the acidic chitosan solution contained 10 g/L chitosan in 100 mM acetic acid; the reaction was mixed at 500 rpm using a magnetic stir bar; and the precipitated samples were passed through a cloth filter ( i.e ., cheese cloth 4-ply #90 Grade) in a 6” by 6” funnel over a 15 minute drain time.

The volume recovered through the cheese cloth was measured and the turbidity assessed. The filterability of the cheese cloth filtrate (ability to be filtered through a 0.2 pm filter) was also assessed. Finally, the size of the curds formed in the chitosan/milk mixture was also measured on a visual scale fro. 1 to 10. Results are shown in Fig. 5A. Models may be used to predict outcome of precipitation based on the parameters assessed (chitosan to milk ratio and temperature) (Figs. 5B-5E).

In conclusion, the hold time after chitosan addition (up to 45min) did not affect the responses assessed, whereas the milk to chitosan ratio and the temperature of the precipitation did affect the process, and these parameters appeared to interact with each other. In particular, the process involving low temperature milk may be improved by adding higher amounts of chitosan or with the process involving lower amounts chitosan may be improved by adding higher temperatures. Chitosan precipitation was efficient in the temperature range of approximately 22 - 37 °C with 10-15 % chitosan solution.

Example 10: Purification of antithrombin from milk using chitosan precipitation

Milk collected from transgenic non-human mammals that were engineered to express antithrombin in the mammary glands is precipitated using chitosan and subjected to further purification as set forth in the experiments below:

Experiment 1 (Exp #1): Gl-style spin clarification comprises a centrifugation step at 12,000 xg for 60 minutes

Experiment 2a (Exp #2a): 200 mL milk + 30 mL 10 mg/mL medium molecular weight chitosan, pH 6, spin clarified

Experiment 2b (Exp #2b): Exp #2a pH-adjusted to pH 7 to improve antithrombin stability with phosphate and Tris buffers and filtered

The experiments above resulted in yields (relative to Exp #1 set at 100%) of 89% for Exp.# 2a and 85% for Exp.# 2b. Sepharose/Heparin purification was used to isolate the antithrombin for analysis using the below conditions:

5mL SP Sepharose HiTrap + 8mL Heparin HyperD

SP column inline for equilibration, load, wash 1 (removed for wash 2 and elution)

Equilibration and Wash Buffer: PBS pH7.4

Wash 2: 20mM phosphate, 300 mM NaCl pH7

Elution Buffer: 20mM phosphate, 2.5M NaCl pH 7

Reverse Phase (RP) Purity (oxidation) was performed using the below conditions:

Waters Xbridge BEH300 C4 2.lx250mm column

Mobile phase A - 0.1% TFA in water

Mobile phase B - 0.08% TFA in acetonitrile

Size exclusion chromatography to assess aggregation was performed using the below conditions:

Tosoh TSK-gel G3000swxl column

Buffer - AT Formulation

Heparin affinity (% high affinity) was performed using the below conditions:

lmF GE Heparin HP HiTrap

Mobile phase A - 50 mM Tris, 10 mM citrate pH7.4

Mobile phase B - 50 mM Tris, 10 mM citrate, 3M NaCl, pH7.4

Example 11: Purification of Fc fragments from goat milk using chitosan precipitation and aptamer column chromatography

Milk collected from transgenic goats that were engineered to express Fc in the mammary glands was precipitated using chitosan and subjected to chromatography using an Fc aptamer column. Briefly, the column was equilibrated using Buffer Al, described below. As shown in Fig. 6A, the chitosan clarified whey was diluted with 1 volume of 100 mM MES pH 5.8 and 1 volume Buffer Al and applied to the column using the below conditions:

Buffer Al: 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.8 + l50mM NaCl + 5 mM MgCl₂ using a flow rate at l.OmF/min

Buffer Bl: 20 mM imidazole pH 7.5 + 150 mM NaCl + 5 mM MgCI₂ Load: 5 mL chito san-clarified milk.

The column was then washed with Buffer Al and Fc fragments were eluted using Buffer B l. This resulted in 8.5 mL of product at 1.37 mg/mL Fc fragments (11.6 mg total). The eluted product was analyzed by SDS-PAGE (Fig. 6B). The yield of Fc fragments that can be reached is at least 80%. The final product is estimated to be more than 98% pure, as assessed for example by HPLC-SEC.

Example 12: Purification of Factor X-Fc fragments from milk using chitosan precipitation and aptamer column chromatography

Milk collected from transgenic animals that are engineered to express Fc in the mammary glands can be precipitated using chitosan and subjected to chromatography using an Fc aptamer column. Briefly, the column is equilibrated using Buffer Al, described below. The chitosan clarified whey is dialyzed with 20 mM MES pH 5.8 +150 mM NaCl + 2 mM EGTA. One volume of Buffer Al is added prior to loading the column, then applied to the column using the below conditions:

Buffer Al: 20 mM MES pH 5.8 + l50mM NaCl + 5 mM MgCl₂. Using a flow rate of 1.0 mL/min.

Buffer B l: 20 mM imidazole pH 7.5 + 150 mM NaCl + 5 mM MgCh

Load: 5 mL chito san-clarified milk.

The column is then washed with Buffer Al and Factor X-Fc is eluted using Buffer B 1. The eluted product may be analyzed by SDS-PAGE.

Example 13: Precipitation with acidic chitosan solution and phase separation to remove the precipitate and recover the clarified milk containing the protein

Milk collected from transgenic goats that were engineered to express anti-TNF-a antibodies in the mammary glands was precipitated using chitosan and subjected to filtration on different filters.

Results obtained using different kinds of filters (depth filter, cloth filter or sequence cloth filter-depth filter) show that the phase separation is efficient no matter the type of filter, resulting in a clear or perfectly clear clarified milk (Table 1).

Pleated polyester felt bags have the advantage to increase the capacity of the bag (compared to non-pleated polyester felt bags) by increasing surface area in the same footprint. Pleated polyester felt bags in reverse flow have the advantage to increase the capacity of the filter. A specific sequence of polyester felt bag followed by depth filtration is of particular interest because the polyester felt bag enables the removal of precipitate, before finalizing the separation on depth filter (which can in this case be smaller since most of precipitate has already been removed by the cloth filter step).

Table 1

Claims

What is claimed is: CLAIMS

1. A method of producing a protein, the method comprising:

(a) providing a transgenic non-human mammal that has been modified to express a protein in the mammary gland;

(b) harvesting milk produced in the mammary gland of the transgenic non-human mammal; and

(c) purifying the protein from the milk, wherein purifying the protein comprises precipitating the milk comprising the protein with an acidic chitosan solution to produce a clarified milk comprising the protein.

2. The method of claim 1, wherein purifying the protein from the milk further comprises subjecting the clarified milk to anion exchange chromatography.

3. The method of claim 1 or 2, wherein purifying the antibody from the milk further comprises subjecting the protein to cation exchange chromatography.

4. The method of claim 1, wherein purifying the antibody from the milk further

comprises subjecting the protein to Fc aptamer column chromatography.

5. The method of any one of claims 1-4, further comprising recovering the clarified milk produced in step (c).

6. The method of any one of claims 2-3 or 5, wherein the anion exchange

chromatography comprises applying the clarified milk to an anion exchange chromatography column; and recovering the protein from the anion exchange chromatography column.

7. The method of claim 6, wherein the anion exchange chromatography column

comprises a resin comprising a quaternary amine ligand.

8. The method of any one of claims 3 or 5-7, wherein the cation exchange

chromatography comprises applying the clarified milk to a cation exchange chromatography column; and recovering the protein from the cation exchange chromatography column.

9. The method of claim 8, wherein the cation exchange chromatography comprises a resin comprising a sulphopropyl ligand.

10. The method of any one of claims 1-9, wherein the acidic chitosan solution comprises medium molecular weight (MMW) chitosan.

11. The method of any one of claims 1-9, wherein the acidic chitosan solution comprises high molecular weight (HMW) chitosan.

12. The method of any one of claims 1-9, wherein the acidic chitosan solution comprises ultra-high molecular weight (UHMW) chitosan.

13. The method of any one of claims 1-9, wherein the acidic chitosan solution has a pH between 2 to 4.

14. The method of any one of claims 1-13, wherein the acidic chitosan solution is

prepared by dissolving about 10 g of chitosan per liter in 100 mM of acetic acid having a pH about 4.6 to about 6.5.

15. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 10% to about 15% (w/w) of the acidic chitosan solution to the milk.

16. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk.

17. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1.0 gram to 2.5 grams of chitosan per liter of milk.

18. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1.0 gram to 2.0 grams of chitosan per liter of milk.

19. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1 gram of chitosan per liter of milk.

20. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1.5 gram of chitosan per liter of milk.

21. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 2 gram of chitosan per liter of milk.

22. The method of any one of claims 1-14, wherein precipitating the milk with the acidic chitosan solution comprises adding about 2.5 gram of chitosan per liter of milk.

23. The method of any one of claims 1-22, wherein recovering the clarified milk

comprises a centrifugation step or a filtration step.

24. The method of any one of claims 1-22, wherein recovering the clarified milk

comprises a filtration step.

25. The method of claim 24, wherein the filtration step is performed using depth

filtration.

26. The method of claim 24, wherein the filtration step is performed using a cloth filter.

27. The method of claim 26, wherein the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter.

28. The method of claim 24, wherein the filtration step first uses cloth filtration followed by the use of depth filtration.

29. The method of any one of claims 1-22, wherein recovering the clarified milk

comprises a centrifugation step.

30. The method of claim 29, wherein the centrifugation step is performed at a speed of about 4000-12000 xg for 5-60 minutes.

31. The method of claim 25, wherein the use of depth filtration removes particles having a size greater than 1 pm.

32. The method of any one of claims 1-31, further comprising subjecting the protein to affinity chromatography.

33. The method of claim 32, wherein the affinity chromatography comprises Protein A chromatography.

34. The method of any one of claims 1-33, further comprising subjecting the protein to viral inactivation.

35. The method of any one of claims 1-34, wherein the milk is raw milk, whole milk, or decreamed milk.

36. The method of any one of claims 1-35, wherein the transgenic non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

37. The method of claim 36, wherein the transgenic non-human mammal is a goat.

38. The method of any one of claims 1-37, wherein the protein is a therapeutic protein.

39. The method of any one of claims 1-38, wherein the protein is a human protein.

40. The method of claim 38, wherein the therapeutic protein is an antibody, Fc fusion protein, antithrombin, or alpha-antitrypsin.

41. The method of claim 38, wherein the therapeutic protein is a monoclonal antibody.

42. The method of claim 38, wherein the therapeutic protein is an anti-TNFa antibody, anti- CD20 antibody, anti-EGFR antibody, or anti-HER2 antibody.

43. The method of claim 38, wherein the therapeutic protein is an anti-TNFa antibody that has the same amino acid sequence as adalimumah.

44. The method of claim 38, wherein the therapeutic protein is an Fc fragment.

45. The method of claim 38, wherein the therapeutic protein is antithrombin or alpha- antitrypsin.

46. The method of claim 38, wherein the therapeutic protein is a Fc fusion protein that comprises an Fc fragment and a coagulation protein.

47. The method of claim 46, wherein the coagulation protein is Factor X.

48. The method of any one of claims 1-47, wherein the purity of the protein is about 90%, 91% 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

49. A composition comprising a protein produced by a method comprising:

a) providing a transgenic non-human mammal that has been modified to express the protein in its mammary gland;

b) recovering milk from the mammary gland of said transgenic non-human

mammal; and

c) purifying said protein by a purification method comprising a step of

precipitation of said milk with an acidic chitosan solution .

50. The composition of claim 49, wherein the purification method of step (c) produces a clarified milk comprising the protein.

51. The composition of claims 49 or 50, wherein purifying the protein from the milk further comprises subjecting the clarified milk to anion exchange chromatography.

52. The composition of any one of claims 49-51, wherein purifying the protein from the milk further comprises subjecting the protein to cation exchange chromatography.

53. The composition of claim 49, wherein purifying the protein from the milk further comprises subjecting the protein to Fc aptamer column chromatography.

54. The composition of any one of claims 49-53, wherein the composition comprises the protein and a pharmaceutical acceptable carrier.

55. The composition of any one of claims 49-54, further comprising recovering the

clarified milk.

56. The composition of any one of claims 51-52 or 54-55, wherein the anion exchange chromatography comprises applying the clarified milk to an anion exchange chromatography column; and recovering the protein from the anion exchange chromatography column.

57. The composition of claim 56, wherein the anion exchange chromatography column comprises a resin comprising a quaternary amine ligand.

58. The composition of any one of claims 52 or 54-57, wherein the cation exchange

59. The composition of claim 58, wherein the cation exchange chromatography comprises a resin comprising a sulphopropyl ligand.

60. The composition of any one of claims 49-59, wherein the acidic chitosan solution comprises medium molecular weight (MMW) chitosan.

61. The composition of any one of claims 49-59, wherein the acidic chitosan solution comprises high molecular weight (HMW) chitosan.

62. The composition of any one of claims 49-59, wherein the acidic chitosan solution comprises ultra-high molecular weight (UHMW) chitosan.

63. The composition of any one of claims 49-59, wherein the acidic chitosan solution has a pH between 2 to 4.

64. The composition of any one of claims 49-63, wherein the acidic chitosan solution is prepared by dissolving about 10 g of chitosan per liter in 100 mM of acetic acid having a pH about 4.6 to about 6.5.

65. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 10% to about 15% (w/w) of the acidic chitosan solution to the milk.

66. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk.

67. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1.0 gram to 2.5 grams of chitosan per liter of milk.

68. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1.0 gram to 2.0 grams of chitosan per liter of milk.

69. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1 gram of chitosan per liter of milk.

70. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 1.5 gram of chitosan per liter of milk.

71. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 2 gram of chitosan per liter of milk.

72. The composition of any one of claims 49-64, wherein precipitating the milk with the acidic chitosan solution comprises adding about 2.5 gram of chitosan per liter of milk.

73. The composition of any one of claim 55-72, wherein recovering the clarified milk comprises a centrifugation step or a filtration step.

74. The composition of claim 73, wherein recovering the clarified milk comprises a

filtration step.

75. The composition of claim 74, wherein the filtration step is performed using depth filtration.

76. The composition of claim 74, wherein the filtration is performed using a cloth filter.

77. The composition of claim 76, wherein the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter.

78. The composition of claim 74, wherein the filtration step first uses cloth filtration

followed by the use of depth filtration.

79. The composition of claim 73, wherein recovering the clarified milk comprises a

centrifugation step.

80. The composition of claim 79, wherein the centrifugation is performed at a speed of about 4000-12000 xg for 5-60 minutes.

81. The composition of claim 75, wherein the use of depth filtration removes particles having a size greater than 1 pm.

82. The composition of any one of claims 49-81, further comprising subjecting the protein to affinity chromatography.

83. The composition of claim 82, wherein the affinity chromatography comprises Protein A chromatography.

84. The composition of any one of claims 49-83, further comprising subjecting the

protein to viral inactivation.

85. The composition of any one of claims 49-84, wherein the milk is raw milk, whole milk, or decreamed milk.

86. The composition of any one of claims 49-85, wherein the transgenic non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

87. The composition of claim 86, wherein the transgenic non-human mammal is a goat.

88. The composition of any one of claims 49-87, wherein the protein is a therapeutic protein.

89. The composition of any one of claims 49-87, wherein the protein is a human protein.

90. The composition of claim 88, wherein the therapeutic protein is an antibody, Fc- fusion protein, antithrombin, or alpha-antitrypsin.

91. The composition of claim 88, wherein the therapeutic protein is a monoclonal

antibody.

92. The composition of claims 90 or 91, wherein the therapeutic protein is an anti-TNFa antibody, anti-CD20 antibody, anti-EGFR antibody, or anti-HER2 antibody.

93. The composition of claim 92, wherein the anti-TNFa antibody has the same amino acid sequence as adalimumab.

94. The composition of claim 88, wherein the therapeutic protein is an Fc fragment.

95. The composition of claim 88, wherein the therapeutic protein is antithrombin or

alpha- antitryp sin .

96. The composition of claim 88, wherein the therapeutic protein is an Fc fusion protein comprising an Fc fragment and a coagulation protein.

97. The composition of claim 96, wherein the coagulation protein is Factor X.

98. The composition of any one of claims 49-97, wherein the purity of the protein is about 90%, 91% 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.

99. A method of preparing a composition comprising a protein, wherein the method uses chitosan for precipitating the protein and a lipid from transgenically produced milk.

100. The method of claim 99, wherein the precipitated protein and lipid produces a

clarified milk.

101. The method of claim 100, wherein the clarified milk is subjected to anion exchange chromatography.

102. The method of claim 100 or 101, wherein the clarified milk is subjected to cation exchange chromatography.

103. The method of claim 100, wherein the clarified milk is subjected to Fc aptamer

column chromatography.

104. The method of any one of claims 99-103, wherein the composition comprises the protein and a pharmaceutical acceptable carrier.

105. The method of claim 101 or 104, wherein the anion exchange chromatography comprises applying the precipitated protein and lipid to an anion exchange chromatography column; and recovering the composition comprising the protein from the anion exchange chromatography column.

106. The method of claim 105, wherein the anion exchange chromatography column comprises a resin comprising a quaternary amine ligand.

107. The method claim 102 or 104, wherein the cation exchange chromatography

comprises applying the clarified milk to a cation exchange chromatography column; and recovering the protein from the cation exchange chromatography column.

108. The method of any one of claims 99-107, wherein the chitosan is comprised in an acidic

chitosan solution.

109. The method of claim 108, wherein the acidic chitosan solution comprises medium molecular weight (MMW) chitosan.

110. The method of claim 108, wherein the acidic chitosan solution comprises high

molecular weight (HMW) chitosan.

111. The method of claim 108, wherein the acidic chitosan solution comprises ultra-high molecular weight (UHMW) chitosan.

112. The method of any one of claims 108-111, wherein the acidic chitosan solution has a pH between 2 to 4.

113. The method of any one of claims 108-112, wherein the acidic chitosan solution is prepared by dissolving about 10 g of chitosan per liter in 100 mM of acetic acid having a pH about 4.6 to about 6.5.

114. The method of any one of claims 108-113, wherein precipitating the milk with the acidic chitosan solution comprises adding about 10 to about 15 % (w/w) of the acidic chitosan solution to the milk.

115. The method of any one of claims 108-113, wherein precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk.

116. The method of any one of claims 99-115, wherein the milk is raw milk, whole milk, or decreamed milk.

117. The method of any of claims 99-116, wherein the transgenically produced milk is

produced in a transgenic non-human mammal.

118. The method of claim 117, wherein the transgenic non-human mammal is a goat, sheep, bison, camel, cow, pig, rabbit, buffalo, horse, rat, mouse or llama.

119. The method of claim 117, wherein the transgenic non-human mammal is a goat.

120. The method of any one of claims 99-119, wherein the protein is a therapeutic protein.

121. The method of any one of claims 99-120, wherein the protein is a human protein.

122. The method of claim 120, wherein the therapeutic protein is an antibody, Fc fusion protein, antithrombin, or alpha-antitrypsin.

123. The method of claim 120, wherein the therapeutic protein is a monoclonal antibody.

124. The method of claim 120 or 123, wherein the therapeutic protein is an anti-TNFa antibody, anti-CD20 antibody, anti-EGFR antibody, or anti-HER2 antibody.

125. The method of claim 124, wherein the anti-TNFa antibody has the same amino acid sequence as adalimumah.

126. The method of claim 120, wherein the therapeutic protein is an Fc fragment.

127. The method of claim 120, wherein the therapeutic protein is antithrombin or alpha- antitrypsin.

128. The method of claim 120, wherein the therapeutic protein is an Fc fusion protein comprising an Fc fragment and a coagulation protein.

129. The method of claim 128, wherein the coagulation protein is Factor X.

130. The method of any one of claims 99-129, wherein the precipitated protein and lipid are subjected to a centrifugation step or a filtration step.

131. The method of claim 130, wherein the precipitated protein and lipid are subjected to a filtration step.

132. The method of claim 131, wherein the filtration step is performed using depth

filtration.

133. The method of claim 131, wherein the filtration step is performed using a cloth filter.

134. The method of claim 130, wherein the precipitated protein and lipid are subjected to a centrifugation step.

135. The method of claim 134, wherein the centrifugation is performed at a speed of about 4000-12000 xg for 5-60 minutes.

136. The method of claim 132, wherein the use of depth filtration removes particles having a size beyond 1 pm.

137. The method of any one of claims 99-136, wherein the composition comprising the protein is subjected to affinity chromatography.

138. The method of claim 137, wherein the affinity chromatography comprises Protein A chromatography.

139. The method of any one of claims 99-138, wherein the composition comprising the protein is subjected to viral inactivation.

140. The method of any one of claims 99-139, wherein the purity of the protein in the

composition is about 90%, 91% 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.