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WO2020053661A1 - Procédés de purification d'anticorps émanant du lait de mammifères non humains transgéniques comprenant l'utilisation de chitosane - Google Patents

Procédés de purification d'anticorps émanant du lait de mammifères non humains transgéniques comprenant l'utilisation de chitosane Download PDF

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Publication number
WO2020053661A1
WO2020053661A1 PCT/IB2019/001016 IB2019001016W WO2020053661A1 WO 2020053661 A1 WO2020053661 A1 WO 2020053661A1 IB 2019001016 W IB2019001016 W IB 2019001016W WO 2020053661 A1 WO2020053661 A1 WO 2020053661A1
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Prior art keywords
milk
protein
composition
chitosan solution
chitosan
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PCT/IB2019/001016
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English (en)
Inventor
Nicholas C. MASIELLO
Abdessatar Chtourou
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Laboratoire Francais Du Fractionnement Et Des Biotechnologies
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Publication of WO2020053661A1 publication Critical patent/WO2020053661A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/18—Ion-exchange chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8125—Alpha-1-antitrypsin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/81—Protease inhibitors
    • C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
    • C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
    • C07K14/8121—Serpins
    • C07K14/8128—Antithrombin III
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6432—Coagulation factor Xa (3.4.21.6)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the disclosure relates, at least in part, to methods for purifying proteins from the milk of non-human transgenic mammals.
  • the methods described herein relate to the purification of proteins produced in the milk of non-human transgenic mammals using an acidic chitosan solution.
  • Proteins such as antibodies, are used for a large number of industrial and
  • proteins are produced using recombinant expression systems, such as unicellular organisms (bacteria or yeasts), insect cells (baculovirus system/insect cells), or transgenic plants.
  • recombinant expression systems such as unicellular organisms (bacteria or yeasts), insect cells (baculovirus system/insect cells), or transgenic plants.
  • 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).
  • 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.
  • 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.
  • 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.
  • the cation exchange chromatography comprises a resin comprising a sulphopropyl ligand.
  • 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.
  • 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.
  • precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk.
  • recovering the clarified milk comprises a centrifugation step or a filtration step.
  • the filtration step is performed using depth filtration.
  • the filtration is performed using a cloth filter.
  • the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter.
  • the filtration is performed using reverse flow mode.
  • the centrifugation step is performed at a speed of about 4000-12000 xg for 5-60 minutes.
  • the depth filtration removes particles having a size greater than 1 pm.
  • the filtration is performed by filtering the clarified milk through a cloth filter, followed by filtering the milk through a depth filter.
  • the method further comprises subjecting the protein to affinity chromatography.
  • the affinity chromatography comprises Protein A chromatography.
  • the method further comprises subjecting the protein to viral inactivation.
  • the milk is raw milk, whole milk, or decreamed milk.
  • 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.
  • the protein is a therapeutic protein. In some embodiments, the protein is a human protein.
  • 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.
  • the anti-TNFa antibody has the same sequence amino acid sequence as adalimumab.
  • the antibody is an Fc fragment.
  • the therapeutic protein is antithrombin or alpha-antitrypsin.
  • the Fc fusion protein comprises an Fc fragment and a coagulation protein.
  • the coagulation protein is Factor X.
  • the purity of the protein is about 90%, 91% 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%.
  • 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.
  • the purification method of step (c) produces a clarified milk comprising the protein.
  • 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
  • purifying the protein from the milk further comprises subjecting the protein to Fc aptamer column chromatography.
  • the composition comprises the protein and a pharmaceutical acceptable carrier.
  • the method further comprises recovering the clarified milk.
  • 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.
  • 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.
  • the cation exchange chromatography column comprises a resin comprising a quaternary amine ligand.
  • 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.
  • the cation exchange chromatography comprises a resin comprising a sulphopropyl ligand.
  • 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.
  • 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.
  • recovering the clarified milk comprises centrifugation or filtration.
  • the filtration is depth filtration.
  • the filtration is performed using a cloth filter.
  • the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter.
  • the filtration is performed using reverse flow mode.
  • the centrifugation is performed at a speed of about 4000-12000 xg for 5-60 minutes.
  • the depth filtration removes particles having a size beyond greater than 1 pm.
  • the filtration is performed by filtering the clarified milk through a cloth filter, followed by filtering the milk through a depth filter.
  • the method further comprises subjecting the protein to affinity chromatography.
  • the affinity chromatography comprises Protein A chromatography.
  • the method further comprises subjecting the protein to viral inactivation.
  • the milk is raw milk, whole milk, or decreamed milk.
  • 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.
  • the protein is a therapeutic protein. In some embodiments, the protein is a human protein.
  • the therapeutic protein is an antibody, Fc-fusion protein, antithrombin, or alpha-antitrypsin.
  • the antibody is a monoclonal antibody.
  • 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.
  • the Fc fusion protein comprises an Fc fragment and a coagulation protein.
  • the coagulation protein is Factor X.
  • 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 use of chitosan for precipitating protein and lipid from transgenically produced milk in a method for preparing a composition comprising a protein.
  • the precipitated protein and lipid produces a clarified milk.
  • the clarified milk is subjected to anion exchange
  • 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
  • the 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.
  • the anion exchange chromatography column comprises a resin comprising a quaternary amine ligand.
  • 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.
  • the chitosan is comprised in an acidic chitosan solution.
  • the acidic chitosan solution comprises medium molecular weight (MMW) chitosan.
  • the acidic chitosan solution comprises high molecular weight (HMW) chitosan.
  • the acidic chitosan solution comprises ultra-high molecular weight (UHMW) chitosan.
  • the acidic chitosan solution has a pH between about 4.6 to about 6.5.
  • 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.
  • 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.
  • precipitating the milk with the acidic chitosan solution comprises adding about 0.5 gram to 3 grams of chitosan per liter of milk.
  • the milk is raw milk, whole milk, or decreamed milk.
  • 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.
  • the protein is a therapeutic protein. In some embodiments, the protein is a human protein.
  • the therapeutic protein is an antibody, Fc fusion protein, antithrombin, or alpha-antitrypsin.
  • the antibody is a monoclonal antibody.
  • 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
  • the therapeutic protein is antithrombin or alpha-antitrypsin.
  • the Fc fusion protein comprises an Fc fragment and a coagulation protein.
  • the coagulation protein is Factor X.
  • the precipitated protein and lipid is subjected to centrifugation or filtration.
  • the filtration is depth filtration.
  • the filtration is performed using a cloth filter.
  • the cloth filter is a polyester felt bag filter or a pleated polyester felt bag filter.
  • the filtration is performed using reverse flow mode.
  • the centrifugation is performed at a speed of about 4000-12000 xg for 5-60 minutes.
  • the depth filtration removes particles having a size beyond 1 pm.
  • the filtration is performed by filtering the clarified milk through a cloth filter, followed by filtering the milk through a depth filter.
  • the composition comprising the protein is subjected to affinity chromatography.
  • the affinity chromatography comprises Protein A chromatography.
  • the composition comprising the protein is subjected to viral inactivation.
  • 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%.
  • 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”)
  • 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
  • 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
  • HPLC-SEC performance liquid chromatography- size exclusion chromatography
  • Fig. 5 shows a table indicating the experimental parameters tested using Design- Expert® 10.
  • 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.
  • proteins e.g ., therapeutic proteins, such as antibodies
  • 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.
  • 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
  • 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.
  • 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.
  • extraction and purification of the proteins from milk remains one of the limiting steps of this expression system due to the complexity of milk.
  • milk constituents can be classified into three categories.
  • the whey consists of carbohydrates, proteins (e.g., lactalbumin,
  • 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.
  • 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.
  • the method further involves subjecting the clarified milk to anion exchange chromatography.
  • the clarified milk comprising the protein or a composition comprising the protein is further subjected to cation exchange chromatography and/or affinity chromatography.
  • the methods described herein relate to producing and purifying a protein from the milk of a transgenic non-human mammal.
  • the methods may be used to produce and purify any proteins that may be transgenically produced.
  • the protein is a therapeutic protein.
  • 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.
  • 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).
  • the therapeutic protein is a human protein.
  • 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.
  • the protein is antithrombin.
  • 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
  • the protein is an Fc fusion protein comprising an Fc domain and a coagulation protein.
  • the Fc fusion protein comprises an Fc domain and Factor X.
  • the methods described herein are for production and purification of antibodies or fragments thereof.
  • 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
  • CDR complementarity determining regions
  • FR framework regions
  • 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 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.
  • the antibody is an Fc fragment.
  • the antibodies are of the isotype IgG, IgA or IgD.
  • 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.
  • the antibodies are bispecific or multispecific antibodies.
  • the antibodies of the present disclosure can be modified to be in the form of a bispecific antibody, or a multispecific antibody.
  • 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.
  • agent e.g., a protein, peptide, or protein or peptide complex
  • 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.
  • 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.
  • 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.
  • the heavy chain constant is comprised of at least two domains.
  • the heavy chain constant region is comprised of CH2 and CH3 domains.
  • 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.
  • the antibodies can be chimeric or humanized antibodies.
  • 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.
  • 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.
  • 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.
  • 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.
  • the antibody is a full-length antibody.
  • the full-length antibody comprises a heavy chain and a light chain.
  • 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.
  • the anti-HER2 antibody has the same amino acid sequence as trastuzumab.
  • 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.
  • the anti-CD20 antibody has the same amino acid sequence as ublituximab.
  • 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.
  • the anti-EGFR antibody has the same amino acid sequence as cetuximab (Erbitux).
  • 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.
  • the anti-TNF-alpha antibody has the same amino acid sequence as infliximab / Remicade (Centocor), adalimumab / Humira (Abbott), or golimumab / Simponi (Centocor).
  • the anti-TNF-alpha antibody has the same amino acid sequence as adalimumab.
  • 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.
  • 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.
  • the antibody consists of the heavy chain sequence of SEQ ID NO: 1 and the light chain sequence of SEQ ID NO: 2.
  • 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.
  • sequences are based on the sequences of adalimumab published in US Patent 6,090,382.
  • the sequences of adalimumab are those as published in US Patent 6,090,382, incorporated by reference herein in its entirety.
  • 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.
  • the anti-TNF-alpha antibody disclosed herein is further purified.
  • Fc fusion protein refers to an Fc domain fused to a biologically active protein.
  • 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).
  • the Fc domain comprises two heavy chain constant domains.
  • 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
  • any subtype e.g., IgGl, IgG2, IgG3,
  • the Fc domain is an IgGl Fc domain.
  • 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.
  • 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.
  • 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.
  • amino acid sequence of a non-limiting example of an Fc domain is provided in SEQ ID NO: 10:
  • nucleic acid sequence of a non-limiting example of an Fc domain is provided in SEQ ID NO: 11:
  • 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.
  • the polypeptide is attached directly to the Fc domain.
  • 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 (G 4 S) n , n being comprised between 1 and 5.
  • n 3
  • the linker sequence is SEQ ID NO: 13: GGGGSGGGGSGGGGS.
  • 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.
  • 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.
  • 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.
  • the milk comprising the protein is precipitated with an acidic chitosan solution.
  • 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.
  • 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.
  • the chitosan is preferably extract from chitin present in mushrooms.
  • 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.
  • the acidic chitosan solution comprises high molecular weight chitosan.
  • the acidic chitosan solution comprises medium molecular weight chitosan.
  • 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).
  • the acidic chitosan solution is used to precipitate milk lipids and caseins from the milk, thereby clarifying the milk comprising the protein.
  • 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.
  • an acidic pH e.g ., less than 7.0
  • the term“acidic” refers to a pH that is less than 7.0.
  • the acidic chitosan solution has a pH between about 2.0 to 4.0.
  • 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.
  • 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.
  • 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.
  • the acidic chitosan solution is added to the milk until the solution“breaks.”
  • the term“break” refers to the precipitation of the milk lipids and caseins.
  • the acidic chitosan solution is added to the milk until the milk lipids and/or caseins precipitate.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • the acidic chitosan solution is added to the milk resulting in a pH value of about 6.0 to 6.5.
  • 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.
  • 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.
  • 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.
  • the clarified milk is subjected to phase separation to recover the clarified milk (whey) containing the protein and remove precipitated milk proteins and lipids.
  • the phase separation is performed by centrifugation or filtration (e.g ., cloth filtration, or depth filtration).
  • the phase separation is performed by centrifugation of the clarified milk.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the phase separation is performed by filtration of the clarified milk.
  • 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.
  • 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.
  • a porous filtration medium e.g., a depth filter
  • 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.
  • 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.
  • the depth filter has a nominal retention rating of about 3 pm to about 15 pm.
  • the depth filter has a nominal retention rating of about 6 pm to about 12 pm.
  • the depth filtration is performed using a Pall K700 depth filter (PALL Life Sciences).
  • 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.
  • 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.
  • the equilibration buffer comprises Tris. In some embodiments, the equilibration buffer comprises 20 mM Tris at pH 8.0.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cloth filter is Grade #90 cheese cloth.
  • the cloth filter is a propylene felt bag filter having a pore size between 1 -200pm, preferably lpm.
  • the cloth filter is a propylene pleated felt bag filter.
  • 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.
  • 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.
  • the clarified milk is subjected to chromatography.
  • the clarified milk is subjected to anion exchange chromatography.
  • 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.
  • 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.
  • 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.
  • 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).
  • the clarified milk is applied to the anion exchange chromatography column and the protein is recovered from the anion exchange
  • ligand 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.
  • the anion exchange chromatography comprises resin comprise a quartemary amine ligand.
  • the resin comprising the quarternary amine ligand is a Q Sepharose Fast Flow anion exchanger (GE Healthcare Life Sciences). In some
  • 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)).
  • a filter comprising quartemary amine ligand e.g., a SARTOBIND® Q membrane adsorber (Sartorius Stedim Plastics GmbH)
  • anion exchange chromatography removes impurities and/or contaminating proteins (e.g., undesired proteins and other negatively charged molecules) from the clarified milk.
  • 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.
  • the anion exchange chromatography removes lactalbumin, lactoglobulin, albumin, and/or lactoferrin from the clarified milk.
  • 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.
  • the antibodies are not retained by the ligand and pass through the column.
  • the clarified milk is diluted with a buffer at a particular pH prior to anion exchange chromatography.
  • the pH of the clarified milk is about 6.
  • 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.
  • 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.
  • 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
  • Samples from the anion exchange chromatograph 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.
  • 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.
  • 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.
  • the method comprises further subjecting the protein or a composition comprising the protein to further purification such as by affinity chromatography.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the cation exchange chromatography comprises resin comprise a sulphopropyl ligand.
  • the resin comprising the sulphopropyl ligand is a SP-Sepharose Fast Flow cation exchange resin (GE Healthcare Life Sciences).
  • 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.
  • 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.
  • the impurities and/or contaminating molecules are not retained by the ligand and pass through the column.
  • the proteins are recovered from the cation exchange chromatography column by elution using an appropriate buffer.
  • 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.
  • Positive charges e.g., positively charged amino acids within a polypeptide, such as the antibodies
  • Samples from the cation exchange chromatograph 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.
  • 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.
  • the method further involves subjecting the clarified milk to chromatography.
  • the method further involves subjecting the clarified milk to affinity chromatography.
  • 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.
  • 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.
  • the resin contains a binding partner that specifically interacts with a component in the mixture, thereby removing the component from the mixture.
  • 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).
  • 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).
  • the affinity chromatography is used to remove free chain antibodies (e.g ., free heavy chains, free light chains).
  • 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.
  • the desired components e.g., the proteins
  • the desired components 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.
  • 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.
  • a molecule e.g., ligand
  • a 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) is immobilized on a resin used in the affinity chromatography.
  • the desired proteins are antibodies.
  • 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.
  • 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.
  • the Protein A is immobilized on agarose beads (e.g., SP-Sepharose).
  • the affinity chromatography is an Fc aptamer column chromatography.
  • the clarified milk is subjected to chromatography.
  • the clarified milk is subjected to Fc aptamer column chromatography.
  • 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.
  • “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.
  • 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.
  • SEQ ID NO: 7 Aptamer sequence referred to as SEQ ID NO: 7 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 5 herein:
  • SEQ ID NO: 6 Aptamer sequence referred to as SEQ ID NO: 1 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 6 herein:
  • SEQ ID NO: 7 Aptamer sequence referred to as SEQ ID NO: 2 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 7 herein:
  • SEQ ID NO: 19 Primer sequence referred to as SEQ ID NO: 19 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 8 herein:
  • SEQ ID NO: 20 Primer sequence referred to as SEQ ID NO: 20 in PCT publication WO 2018/019538 Al, referred to as SEQ ID NO: 9 herein:
  • 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.
  • 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.
  • the method further involves subjecting the clarified milk to chromatography.
  • 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.
  • the protein or clarified milk comprising the protein are subjected to affinity chromatography.
  • 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.
  • the viral inactivation is used to inactivate viruses, such as enveloped viruses.
  • the antibody or clarified milk comprising the protein are subjected viral inactivation and subsequently subjected to affinity purification.
  • the viral inactivation comprises treatment of the clarified milk or composition comprising the protein with an organic solvent and detergent.
  • the organic solvent is tri-n-butyl phosphate and the detergent is polysorbate 80.
  • 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.
  • 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.
  • the viral inactivation comprises treatment the clarified milk or composition comprising the protein with a glycol, such as propylene glycol.
  • 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.
  • the viral inactivation comprises subjecting the clarified milk or composition comprising the protein to nanofiltration for removal of viruses, such as non- enveloped viruses.
  • the acidic chitosan solution is prepared using an acidic buffer to dissolve the chitosan.
  • acidic buffers include, without limitation, acetic acid, citric acid, hydrochloric acid, sodium acetate, ethanoic acid.
  • 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.
  • the acidic chitosan solution is prepared using an acidic buffer comprising 100 mM acetic acid.
  • 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,
  • the anion exchange chromatography is performed using a sodium phosphate buffer.
  • 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.
  • the sodium phosphate buffer has a pH of about 7.0.
  • 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.
  • 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,
  • the sodium phosphate buffer has a pH of about 8.0.
  • 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
  • the equilibration buffer is a sodium phosphate buffer.
  • 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.
  • the equilibration buffer comprises 20 mM sodium phosphate.
  • 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.
  • the affinity chromatography comprises washing the affinity chromatography column with one or more wash buffers.
  • the wash buffer is a phosphate buffered solution (PBS).
  • the wash buffer is a sodium acetate buffer.
  • 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.
  • the wash buffer comprises 100 mM sodium acetate.
  • 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.
  • the wash buffer is a phosphate buffered solution having a pH of about 7.0.
  • the wash buffer further comprises a salt.
  • 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.
  • the affinity chromatography comprises recovering the protein from the affinity chromatography column with an elution buffer.
  • the elution buffer is a glycine buffer.
  • the glycine buffer has a
  • the elution buffer comprises 100 mM glycine. In some embodiments, the elution buffer has a pH about 2.0,
  • 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.
  • the milk is contacted with an ethylenediaminetetraacetic acid (EDTA) solution prior to precipitating the milk comprising the protein with an acidic chitosan solution.
  • EDTA ethylenediaminetetraacetic acid
  • 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.
  • the EDTA solution comprises 40 mM EDTA.
  • 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.
  • 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 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.
  • 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.
  • some vectors such as yeast artificial chromosomes (YACs)
  • YACs yeast artificial chromosomes
  • 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.
  • 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.
  • a 3'- sequence which can be derived from a naturally secreted milk protein, can be added to improve stability of mRNA.
  • 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.
  • the promoters are milk-specific promoters.
  • 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)
  • WAP whey acidic protein
  • 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).
  • LTR long terminal repeat
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • the disclosure provides a method for the production of a protein, comprising:
  • the disclosure provides a method of
  • 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.
  • “ungulate” is of or relating to a hoofed typically herbivorous quadruped mammal, including, without limitation, sheep, goats, cattle and horses.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • hCG human chorionic gonadotropin
  • lactation 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.
  • 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.
  • the protein is a therapeutic protein.
  • the therapeutic protein is a coagulation protein.
  • the therapeutic protein is an Fc fusion protein.
  • the protein is an antibody or an Fc fragment of an antibody.
  • 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.
  • 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.
  • the milk is fresh milk.
  • fresh milk refers to milk that has been freshly collected and has not been frozen.
  • the fresh milk comprising an antibody is clarified, as described herein.
  • fresh milk is harvested and subjected to one or steps of the purification process in the same day.
  • 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.
  • the milk e.g., fresh or previously frozen
  • the milk is warmed to ambient temperature prior to precipitation with the acidic chitosan solution.
  • the milk e.g., fresh or previously frozen
  • 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.
  • the milk is whole milk.
  • 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.
  • the raw milk has not been subjected to pasteurization.
  • 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.
  • the milk is decreamed milk.
  • the milk is defatted raw milk.
  • the milk is skimmed milk.
  • 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.
  • proteins produced as described herein have enhanced
  • proteins produced by methods described herein are of higher purity compared to proteins produced by other methods.
  • the transgenically produced proteins that are subsequently isolated and purified are at least 90 - 99.99% pure.
  • 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.
  • 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.
  • “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).
  • 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%.
  • 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%.
  • 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.
  • 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.
  • the disclosure provides methods comprising administering proteins produced using the methods described herein to a subject in need thereof.
  • 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.
  • the subject has a coagulation disorder.
  • the subject has a hereditary deficiency in a coagulation protein.
  • the subject has an acquired deficiency in a coagulation protein.
  • 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.
  • the subject has an inflammatory disorder or autoimmune disorder.
  • 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.
  • the disclosure provides methods for administering proteins produced using the methods described herein to a subject in need thereof.
  • 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,
  • 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.
  • 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 include Alzheimer, cancer and depression.
  • aspects of the invention relate to administering effective amounts of a protein, or compositions comprising an antibody.
  • methods comprise
  • the transgenic protein is purified, for example using the methods of purification described herein.
  • the subject has an inflammatory condition or an autoimmune condition.
  • 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.
  • 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 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.
  • compositions including pharmaceutical compositions, which comprise transgenically produced and purified protein and a
  • compositions comprise proteins produced in transgenic non-human mammals.
  • compositions which comprise an amount of a protein produced by the methods described herein and a pharmaceutically acceptable vehicle, diluent or carrier.
  • the compositions provided herein are used for in vivo applications, such as treatment of a disease or disorder.
  • 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.
  • diluents examples include 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
  • 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 route of administration varies depending on the response desired.
  • the proteins or compositions herein may be administered to a subject via oral, parenteral or topical administration.
  • the compositions herein are administered by intravenous infusion.
  • 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.
  • 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:
  • Example 2 Chitosan clarification of milk from transgenic goats (100 mL scale)
  • PES polyethersulfone
  • Example 3 Chitosan clarification of milk from transgenic goats (200 mL scale)
  • 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)
  • 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.
  • 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.
  • 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
  • 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.
  • an SP Sepharose cation exchange chromatography column to remove free heavy chains and light chains.
  • 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%.
  • 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
  • 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.
  • Example 8 Preparation of Protein A-purified anti-TNF-a antibodies with chitosan precipitation
  • Example 9 Optimization of parameters of the acidic chitosan precipitation process
  • time post-chitosan addition ranged from 5 to 30 minutes
  • amount of chitosan ranged from 10% to 15%
  • temperature during the chitosan precipitation ranged from 10°C to 37°C.
  • the hold time after chitosan addition 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.
  • 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
  • Heparin affinity (% high affinity) was performed using the below conditions:
  • 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
  • Buffer Al 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 5.8 + l50mM NaCl + 5 mM MgCl 2 using a flow rate at l.OmF/min
  • Buffer Bl 20 mM imidazole pH 7.5 + 150 mM NaCl + 5 mM MgCI 2
  • Load 5 mL chito san-clarified milk.
  • 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 MgCl 2 . Using a flow rate of 1.0 mL/min.
  • Buffer B l 20 mM imidazole pH 7.5 + 150 mM NaCl + 5 mM MgCh
  • 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.
  • 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).

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Abstract

La présente invention concerne des procédés de production d'anticorps et de compositions comprenant des anticorps émanant du lait de mammifères non humains transgéniques, impliquant la précipitation à l'aide d'une solution de chitosane acide.
PCT/IB2019/001016 2018-09-13 2019-09-13 Procédés de purification d'anticorps émanant du lait de mammifères non humains transgéniques comprenant l'utilisation de chitosane WO2020053661A1 (fr)

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Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US5598369A (en) 1994-06-28 1997-01-28 Advanced Micro Devices, Inc. Flash EEPROM array with floating substrate erase operation
WO1997012901A1 (fr) * 1995-10-05 1997-04-10 Immucell Corporation Procede d'isolement d'immunoglobuline a partir de petit lait
US5747031A (en) * 1995-10-05 1998-05-05 Immucell Corporation Process for isolating immunoglobulins in whey
US5843705A (en) 1995-02-21 1998-12-01 Genzyme Transgenic Corporation Transgenically produced antithrombin III
US5945577A (en) 1997-01-10 1999-08-31 University Of Massachusetts As Represented By Its Amherst Campus Cloning using donor nuclei from proliferating somatic cells
US6090382A (en) 1996-02-09 2000-07-18 Basf Aktiengesellschaft Human antibodies that bind human TNFα
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US20030201230A1 (en) * 1999-02-22 2003-10-30 Kopf Henry B. Purification of biological substances
WO2007138199A2 (fr) 2006-05-31 2007-12-06 Lfb Biotechnologies Composition de facteur vii recombinant ou transgenique, chaque molecule de facteur vii possedant deux sites de n-glycosylation a motifs glycanniques definis
US7867491B2 (en) 2007-05-30 2011-01-11 Genexine Co., Ltd. Immunoglobulin fusion proteins
WO2012090067A1 (fr) 2010-12-30 2012-07-05 Lfb Biotechnologies Glycols en tant qu'agents d'inactivation de pathogènes
US8431132B2 (en) 2006-01-27 2013-04-30 Novagen Holding Corporation Recombinant human EPO-FC fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
WO2017217930A1 (fr) * 2016-06-15 2017-12-21 Agency For Science, Technology And Research Procédé d'amélioration des performances de procédés de chromatographie pour la purification de protéines
WO2018019538A1 (fr) 2016-07-28 2018-02-01 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Aptamères anti-immunoglobuline g et leurs utilisations

Patent Citations (20)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5545807A (en) 1988-10-12 1996-08-13 The Babraham Institute Production of antibodies from transgenic animals
US5591669A (en) 1988-12-05 1997-01-07 Genpharm International, Inc. Transgenic mice depleted in a mature lymphocytic cell-type
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5545806A (en) 1990-08-29 1996-08-13 Genpharm International, Inc. Ransgenic non-human animals for producing heterologous antibodies
US5598369A (en) 1994-06-28 1997-01-28 Advanced Micro Devices, Inc. Flash EEPROM array with floating substrate erase operation
US7928064B2 (en) 1995-02-21 2011-04-19 Gtc Biotherapeutics, Inc. Transgenically produced antithrombin III
US5843705A (en) 1995-02-21 1998-12-01 Genzyme Transgenic Corporation Transgenically produced antithrombin III
US7019193B2 (en) 1995-02-21 2006-03-28 Gtc Biotherapeutics, Inc. Treatments using transgenic goat produced antithrombin III
US6441145B1 (en) 1995-02-21 2002-08-27 Genzyme Transgenics, Inc. Transgenically produced Antithrombin III
US5747031A (en) * 1995-10-05 1998-05-05 Immucell Corporation Process for isolating immunoglobulins in whey
WO1997012901A1 (fr) * 1995-10-05 1997-04-10 Immucell Corporation Procede d'isolement d'immunoglobuline a partir de petit lait
US6090382A (en) 1996-02-09 2000-07-18 Basf Aktiengesellschaft Human antibodies that bind human TNFα
US5945577A (en) 1997-01-10 1999-08-31 University Of Massachusetts As Represented By Its Amherst Campus Cloning using donor nuclei from proliferating somatic cells
US20030201230A1 (en) * 1999-02-22 2003-10-30 Kopf Henry B. Purification of biological substances
US8431132B2 (en) 2006-01-27 2013-04-30 Novagen Holding Corporation Recombinant human EPO-FC fusion proteins with prolonged half-life and enhanced erythropoietic activity in vivo
WO2007138199A2 (fr) 2006-05-31 2007-12-06 Lfb Biotechnologies Composition de facteur vii recombinant ou transgenique, chaque molecule de facteur vii possedant deux sites de n-glycosylation a motifs glycanniques definis
US7867491B2 (en) 2007-05-30 2011-01-11 Genexine Co., Ltd. Immunoglobulin fusion proteins
WO2012090067A1 (fr) 2010-12-30 2012-07-05 Lfb Biotechnologies Glycols en tant qu'agents d'inactivation de pathogènes
WO2017217930A1 (fr) * 2016-06-15 2017-12-21 Agency For Science, Technology And Research Procédé d'amélioration des performances de procédés de chromatographie pour la purification de protéines
WO2018019538A1 (fr) 2016-07-28 2018-02-01 Laboratoire Francais Du Fractionnement Et Des Biotechnologies Aptamères anti-immunoglobuline g et leurs utilisations

Non-Patent Citations (18)

* Cited by examiner, † Cited by third party
Title
ASHKENAZI ET AL., INT. REV. IMMUNOL., vol. 10, 1993, pages 219 - 227
AUSAR ET AL., J. DAIRY SCI., vol. 84, 2000, pages 361 - 369
BECK ET AL., MABS, vol. 3, 2011, pages 415 - 416
CARTER, EXP CELL RES, vol. 317, 2011, pages 1261 - 1269
CHAMOW ET AL., TRENDS BIOTECHNOL., vol. 14, 1996, pages 52 - 60
CLARK ET AL., BIOTECHNOLOGY, vol. 7, 1989, pages 487 - 492
CZAJKOWSKY ET AL., EMBO MOL MED, vol. 4, 2012, pages 1015 - 1028
DITULLIO, BIOTECHNOLOGY, vol. 10, 1992, pages 74 - 77
E. CASAL ET AL: "Use of Chitosan for Selective Removal of [beta]-Lactoglobulin from Whey", JOURNAL OF DAIRY SCIENCE., vol. 89, no. 5, 1 May 2006 (2006-05-01), US, pages 1384 - 1389, XP055660709, ISSN: 0022-0302, DOI: 10.3168/jds.S0022-0302(06)72206-8 *
GORDON ET AL., BIOTECHNOLOGY, vol. 5, 1987, pages 1183 - 1187
HWANG D-C ET AL: "SELECTIVE PRECIPITATION AND REMOVAL OF LIPIDS FROM CHEESE WHEY USING CHITOSAN", JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY, AMERICAN CHEMICAL SOCIETY, BOOKS AND JOURNALS DIVISION, US, vol. 43, no. 1, 1 January 1995 (1995-01-01), pages 33 - 37, XP000485665, ISSN: 0021-8561, DOI: 10.1021/JF00049A008 *
HWANG ET AL., J. AGRIC. FOOD CHEM., vol. 43, 1995, pages 33 - 37
KIM ET AL., EUR. J. IMMUNOL., vol. 24, 1994, pages 2429 - 2434
LEE ET AL., FOOD RESEARCH INTERNATIONAL, vol. 42, 2009, pages 733 - 8
LOW ET AL., HUMAN REPRODUCTION, vol. 20, no. 7, 2005, pages 1805 - 1813
MONTILLA ET AL., J. DAIRY SCI, vol. 89, 2005, pages 1384 - 1389
SOULIER ET AL., FEBS LETTS, vol. 297, 1992, pages 13
WALDMANN, SCIENCE, vol. 252, 1991, pages 1657

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