WO2014179665A1 - Methods for increasing polypeptide recovery - Google Patents

Abstract

The present invention pertains to a method for purification of a polypeptide of interest from a solution. The present invention also pertains to a method for removing from the solution a cell culture additive, such as dextran sulfate, which is complexed to the polypeptide of interest, such as Neublastin.

Description

METHODS FOR INCREASING POLYPEPTIDE RECOVERY

BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The present invention is directed to a method for purification of a polypeptide of interest from a solution. In particular, the present invention is directed to a method for removing from the solution a cell culture additive, such as dextran sulfate, which is complexed to the polypeptide of interest.

Background Art

[0002] Additives are often added to the cell culture process in order to improve cell growth and cell productivity. For example, dextran sulfate can be added to cell cultures expressing polypeptides in order to prevent the expressed protein from binding to the cells.

[0003] However, the addition of additives can have a negative impact on purification.

For example, certain additives can complex with the polypeptide of interest due to electrostatic interactions. The change in protein charge characteristics significantly suppresses binding of polypeptides of interest to conventional ion exchange chromatography.

[0004] Provided herein are methods for removing cell culture additives that are complexed to a polypeptide of interest, and purifying the polypeptide using a chromatography matrix, thereby improving the yield of the polypeptide in the purification process.

BRIEF SUMMARY OF THE INVENTION

[0005] The present invention pertains to a method of purifying a polypeptide of interest from a solution comprising the polypeptide and a cell culture additive wherein the polypeptide is complexed to the cell culture additive, comprising: (a) separating the cell culture additive from the polypeptide in the complex; (b) contacting the free polypeptide with a chromatographic matrix; and (c) eluting the bound polypeptide from the chromatography matrix, thereby purifying the polypeptide from the solution. [0006] In certain embodiments, the solution is a harvested cell culture fluid (HCCF). In certain embodiments, the polypeptide of interest is positively charged in the solution. In certain embodiments, the cell culture additive is negatively charged in the solution. In a specific embodiment, the polypeptide of interest and the cell culture additive form a complex via an electrostatic bond.

[0007] In certain embodiments, the first step of the purification process comprises separating the cell culture additive from the polypeptide of interest by disrupting the electrostatic bond. In a specific embodiment, the electrostatic bond is disrupted by increasing the conductivity of the solution, such as by addition of sodium chloride (NaCl).

[0008] In certain embodiments, the first step of the purification process comprises separating the cell culture additive from the complex by contacting the complex to a chromatography matrix. In certain embodiments, the chromatography matrix is an anion exchange matrix, such as a Q Sepharose Fast Flow ("QFF") resin.

[0009] In certain embodiments, the second step of the purification process comprises purifying the freed polypeptide with a suitable chromatography matrix. In certain embodiments, the chromatography matrix is a mixed-mode chromatography matrix, such as Capto MMC or Eshmuno HCX. In some embodiments, the chromatography matrix is a cation exchange matrix, such as an SP Sepharose XL ("SPXL") resin. In certain embodiments, the polypeptide of interest is eluted from the chromatography matrix with a buffer comprising a step or linear salt gradient. In a specific embodiment, the buffer comprises NaCl and/or arginine.

[0010] In certain embodiments, the polypeptide of interest is selected from the group consisting of: an antibody, a Transforming Growth Factor (TGF) beta superfamily signaling molecule, an Fc fusion protein, a therapeutic enzyme, a recombinant vaccine, and a clotting factor. In a specific embodiment, the polypeptide of interest is Neublastin.

[Θ011] In a specific embodiment, the cell culture additive is dextran sulfate.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0012] Figure 1. The chromatogram for separation of purified Neublastin from a solution containing dextran sulfate using the SPXL adsorbent. [0013] Figure 2. The size exclusion chromatograms for 0.5 g/L purified Neublastin solutions. Top Panel: solutions containing a) no dextran sulfate, b) 1.5 g/L of 5 kDa dextran sulfate in 100 mM Phosphate, 200 mM NaCl, pH 6.8, and c) 1.5 g/L of 5 kDa dextran sulfate in 100 mM Phosphate, 600 mM NaCl, pH 6.8. Bottom Panel: Purified Neublastin solutions containing 0.25, 1.5, 2.5, and 6 g/L of 5 kD dextran sulfate in 100 mM Phosphate, 600 mM NaCl, pH 6.8.

[0014] Figure 3. SDS-PAGE of the fractions from a Capto MMC run using Neublastin harvested cell culture fluid (HCCF). 20 g of protein was loaded per mL of adsorbent. Lane-2: MW standard, Lane-4: HCCF Load, Lane-6: flow-through, Lane-8: wash, (1 M NaCl, 0.15 M arginine, pH 8.5), 3-fold diluted with RODI water, Lane- 10: elution (1 M NaCl, 0.6 M arginine, pH 8.5), 3-fold diluted with RODI water, Lane-1 1 : NBN drug substance.

10015] Figure 4. SDS-PAGE and Western Blot results of the fractions from a QFF +

SPXL run using Neublastin harvested cell culture fluid in the presence of dextran sulfate. Lane 1 : MW standard, Lane 2: HCCF load, Lane 3 : QFF flow-through, Lane 4: QFF strip, Lane 5: SPXL flow through (diluted 4x), Lane 6: SPXL wash 1, Lane 7: SPXL wash 2, Lane 8: SPXL wash 3, Lane 9: SPXL elution, Lane 10, SPXL strip, Lane 1 1 : NBN drug substance.

[0016] Figure 5. The size exclusion chromato grams of the purified NBN from dextran sulfate (DexS) by QFF+SPXL and the pure NBN drug substance (NBN DS).

[0017] Figure 6. The size exclusion chromatogram for NBN drug substance (NBN DS) or

NBN DS + dextran sulfate (DexS) before and after running through the QFF column. The loading buffer is 75 mM carbonate-bicarbonate, pH = 9.0. Different concentrations of NaCl were used in the loading buffer. Yield is based on SEC peak area.

DETAILED DESCRIPTION OF THE INVENTION I. DEFINITIONS

[0018] Throughout this disclosure, the term "a" or "an" entity refers to one or more of that entity; for example, "a polypeptide," is understood to represent one or more polypeptides. As such, the terms "a" (or "an"), "one or more," and "at least one" can be used interchangeably herein. [0019] Furthermore, "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0020] It is understood that wherever aspects are described herein with the language

"comprising," otherwise analogous aspects described in terms of "consisting of and/or "consisting essentially of are also provided.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Revised, 2000, Oxford University Press, provide one of skill with a general dictionary of many of the terms used in this disclosure.

[0022] Units, prefixes, and symbols are denoted in their Systeme International de Unites

(SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, amino acid sequences are written left to right in amino to carboxy orientation. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0023] The term "polypeptide" as used herein refers a sequential chain of amino acids linked together via peptide bonds. The term is used to refer to an amino acid chain of any length, but one of ordinary skill in the art will understand that the term is not limited to lengthy chains and can refer to a minimal chain comprising two amino acids linked together via a peptide bond. If a single polypeptide is the discrete functioning unit and does require permanent physical association with other polypeptides in order to form the discrete functioning unit, the terms "polypeptide" and "protein" as used herein are used interchangeably. If discrete functional unit is comprised of more than one polypeptide that physically associate with one another, the term "protein" as used herein refers to the multiple polypeptides that are physically coupled and function together as the discrete unit. Thus, as used herein, a "peptide," a "peptide fragment," a "protein," an "amino acid chain," an "amino acid sequence," or any other term used to refer to a chain or chains of two or more amino acids, are generically included in the definition of a "polypeptide," even though each of these terms can have a more specific meaning. The term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term further includes polypeptides which have undergone post-translational or post- synthesis modifications, for example, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.

[0024] The term "isoelectric point (pi)" is the pH at which a particular molecule or surface carries no net electrical charge. The pi of a polypeptide is dependent on the amino acids that make up the polypeptide. At a pH below its pi, the polypeptide carries a net positive charge. At a pH above its pi, the polypeptide carries a net negative charge. A polypeptide can therefore be separated on the basis of its ionization status at a given pH.

[0025] The theoretical pi of a polypeptide can be estimated based on its amino acid sequence. Various algorithms and web-based free tools have been developed for this calculation, e.g. Protein Calculator (at scripps.edu/~cdputnam/protcalc.html) and Compute pI/MW (at expasy.org/compute_pi). The actual pi of a polypeptide can be affected by factors such as post-translational modification. The actual pi can be determined by experimental methods such as isoelectric focusing.

[0026] The term "highly charged," as used herein, refers to the ionization status of a molecule, e.g., a polypeptide with a number of ionizable residues, where the difference between the pi of the molecule and the pH of its surrounding solution is at least about 1.

[0027] "Recombinantly expressed polypeptide" and "recombinant polypeptide" as used herein refer to a polypeptide expressed from a host cell that has been genetically engineered to express that polypeptide. The recombinantly expressed polypeptide can be identical or similar to polypeptides that are normally expressed in the host cell. The recombinantly expressed polypeptide can also be foreign to the host cell, i.e. heterologous to peptides normally expressed in the host cell. Alternatively, the recombinantly expressed polypeptide can be chimeric in that portions of the polypeptide contain amino acid sequences that are identical or similar to polypeptides normally expressed in the host cell, while other portions are foreign to the host cell. Host cells include, but are not limited to, prokaryotic cells, eukaryotic cells, plant cells, yeast cells, animal cells, insect cells, avian cells, and mammalian cells. As used herein, the terms "recombinantly expressed polypeptide" and "recombinant polypeptide" also encompasses an antibody produced by a hybridoma.

[0028] The term "expression" or "expresses" are used herein to refer to transcription and translation occurring within a host cell. The level of expression of a product gene in a host cell can be determined on the basis of either the amount of corresponding mRNA that is present in the cell or the amount of the protein encoded by the product gene that is produced by the cell. For example, mRNA transcribed from a product gene is desirably quantitated by northern hybridization. Sambrook et al, Molecular Cloning: A Laboratory Manual, pp. 7.3-7.57 (Cold Spring Harbor Laboratory Press, 1989). Protein encoded by a product gene can be quantitated either by assaying for the biological activity of the protein or by employing assays that are independent of such activity, such as western blotting, ELISA, HPLC, forteBIO, Bradford assay, absorbance at 280nm, or radioimmunoassay using antibodies that are capable of reacting with the protein. Sambrook et al , Molecular Cloning: A Laboratory Manual, pp. 18.1-18.88 (Cold Spring Harbor Laboratory Press, 1989).

[0029] The term "cell culture additive," as used herein, refers to a compound or molecule which is added to a cell culture medium in order to improve cell survival, cell growth, or cell productivity. The cell culture additive can be charged or not charged. It can be positively charged or negatively charged. In certain embodiments, the cell culture additive is a polyanion ligand. In a specific embodiment, the cell culture additive is dextran sulfate.

[0030] The term "complexed," as used herein, refers to the association of two or more species, generally a polypeptide and a cell culture additive. In certain embodiments, the species adhere to each other via molecular interactions such as van der Waals interaction, electrostatic bond, hydrogen bond, hydrophobic interaction, dipole interaction, short range repulsive interaction, and combination thereo [0031] The terms "culture", "cell culture" and "eukaryotic cell culture" as used herein refer to a eukaryotic cell population that is suspended in a medium (see definition of "medium" below) under conditions suitable to survival and/or growth of the cell population. These terms can refer to the combination comprising the mammalian cell population and the medium in which the population is suspended.

[0032] The terms "medium", "cell culture medium", "culture medium", and "growth medium" as used herein refer to a solution containing nutrients which nourish growing eukaryotic cells. Typically, these solutions provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required by the cell for minimal growth and/or survival. The solution can also contain components that enhance growth and/or survival above the minimal rate, including hormones and growth factors. The solution is formulated to a pH and salt concentration optimal for cell survival and proliferation. The medium can also be a "defined medium" or "chemically defined medium"— a serum-free medium that contains no proteins, hydrolysates or components of unknown composition. Defined media are free of animal-derived components and all components have a known chemical structure. A defined medium can comprise recombinant polypeptides or proteins, for example, but not limited to, hormones, cytokines, interleukins and other signaling molecules.

[0033] A "buffer" is a solution that resists changes in pH by the action of its acid-base conjugate components. Various buffers which can be employed depending, for example, on the desired pH of the buffer are described in Buffers. A Guide for the Preparation and Use of Buffers in Biological Systems, Gueffroy, D., ed. Calbiochem Corporation (1975). In one embodiment, the buffer has a pH in the range from about 2 to about 9, alternatively from about 3 to about 8, alternatively from about 4 to about 7 alternatively from about 5 to about 7. Non-limiting examples of buffers that will control the pH in this range include MES, MOPS, MOPSO, Tris, HEPES, phosphate, acetate, citrate, succinate, and ammonium buffers, as well as combinations of these.

[0034] The term "loading buffer" refers to the buffer, in which the polypeptide being purified is applied to a purification device, e.g. a chromatography column or a filter cartridge. Typically, the loading buffer is selected so that separation of the polypeptide of interest from unwanted impurities can be accomplished. [0035] The term "elution buffer" refers to the buffer, which is typically used to remove

(elute) the polypeptide conjugate from the purification device (e.g. a chromatographic column or filter cartridge) to which it was applied earlier. Typically, the elution buffer is selected so that separation of the polypeptide of interest from unwanted impurities can be accomplished. Often, the concentration of a particular ingredient, such as a particular salt (e.g. NaCl) in the elution buffer is varied during the elution procedure (gradient). The gradient may be continuous or stepwise (interrupted by hold periods).

[0036] The term "chromatography" refers to the process by which a solute of interest, typically a polypeptide, in a mixture is separated from other solutes in a mixture as a result of differences in rates at which the individual solutes of the mixture migrate through a stationary medium under the influence of a moving phase, or in bind and elute processes.

[0037] The term "mixed-mode chromatography" refers to a purification process using mixed mode adsorbents which provide multiple modes of interaction, such as hydrophobic, cation exchange, and hydrogen bonding interaction between the polypeptide of interest and the adsorbent ligands. Commercially available mixed mode chromatography resins include Capto™ MMC, Capto™ MMC ImpRes, Capto Blue, Blue Sepharose™ 6 Fast Flow, Capto™ Adhere, and Capto™ Adhere ImpRes from GE Healthcare Life Sciences or Eshmuno^® HCX from EMD Millipore, or Nuvia™ cPrime from Bio-Rad.

[0038] The terms "cation exchange resin," "cation exchange adsorbent," or "cation exchange matrix" refer to a solid phase which is negatively charged, and which thus has free cations for exchange with cations in an aqueous solution passed over or through the solid phase. A negatively charged ligand attached to the solid phase to form the cation exchange resin may, e.g., be a carboxylate or sulfonate. Commercially available cation exchange resins include carboxy-methyl-cellulose, sulphopropyl (SP) immobilized on agarose (e.g. SP Sepharose™ XL, SP-Sepharose™ Fast Flow, SP Sepharose™ High Performance, CM Sepharose™ Fast Flow, CM Sepharose™ High Performance, Capto™ S, and Capto™ SP ImpRes from GE Healthcare Life Sciences, or Fractogel^® EMD SE HiCap, Fractogel^® EMD SO^3", Fractogel^® EMD COO^", Eshmuno™ S, and Eshmuno™ CPX from EMD Millipore, or UNOsphere™ S and Nuvia™ S from Bio-Rad). f 039] The terms "anion exchange resin," "anion exchange adsorbent," or "anion exchange matrix" are used herein to refer to a solid phase which is positively charged, e.g. having one or more positively charged ligands, such as quaternary amino groups, attached thereto. Commercially available anion exchange resins include DEAE Sepharose™ Fast Flow, Q Sepharose™ Fast Flow, Q Sepharose™ High Performance, Q Sepharose™ XL, Capto™ DEAE, Capto™ Q, and Capto™ Q ImpRes from GE Healthcare Life Sciences, or Fractogel^® EMD TMAE HiCap, Fractogel^® EMD DEAE, and Eshmuno Q from EMD Millipore, or U Osphere™ Q and Nuvia™ Q from Bio-Rad.

[0040] Methods for the determination of yield or purity of a polypeptide are known to those of skill in the art. Yield or purity of a polypeptide may be determined by any suitable, art-recognized method of analysis (e.g., band intensity on a silver stained gel, polyacrylamide gel electrophoresis, ELISA, HPLC and the like). An exemplary method is size-exclusion chromatography (SEC) high-performance liquid chromatography (HPLC), described herein below. Purity may be determined using relative "area under the curve" (AUC) values, which can typically be obtained for peaks in a chromatogram, such as an HPLC chromatogram. Optionally, purities are determined by chromatographic or other means using a standard curve generated using a reference material of known purity. Purity may also be determined on a weight-by-weight basis.

[0041] The term "titer" as used herein refers to the total amount of recombinantly expressed polypeptide or protein produced by a cell culture divided by a given amount of medium volume. Titer is typically expressed in units of milligrams of polypeptide or protein per milliliter of medium or in units of grams of polypeptide or protein per liter of medium.

[0042] The term "antibody" is used to mean an immunoglobulin molecule that recognizes and specifically binds to a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or combinations of the foregoing etc., through at least one antigen recognition site within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab', F(ab')2, and Fv fragments), single chain Fv (scFv) mutants, multispecific antibodies such as bispecific antibodies generated from at least two intact antibodies, monovalent or monospecific antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen determination portion of an antibody, and any other modified immunoglobulin molecule comprising an antigen recognition site so long as the antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively.

[0043] The term "hybridoma" as used herein refers to a cell created by fusion of an immortalized cell derived from an immunologic source and an antibody-producing cell. The resulting hybridoma is an immortalized cell that produces antibodies. The individual cells used to create the hybridoma can be from any mammalian source, including, but not limited to, rat, hamster, pig, rabbit, sheep, pig, goat, and human. The term also encompasses trioma cell lines, which result when progeny of heterohybrid myeloma fusions, which are the product of a fusion between human cells and a murine myeloma cell line, are subsequently fused with a plasma cell. Furthermore, the term is meant to include any immortalized hybrid cell line that produces antibodies such as, for example, quadromas (See, e.g. , Milstein et al, Nature, 537:3053 (1983)).

II. Production and Purification of Polypeptides of Interest

A. Polypeptides of Interest

[0044] Any polypeptide that is expressible in a host cell can be expressed and purified in accordance with the present invention. The polypeptide can be expressed from a gene that is endogenous to the host cell, or from a gene that is introduced into the host cell through genetic engineering. The polypeptide can be one that occurs in nature, or can alternatively have a sequence that was engineered or selected by the hand of man. An engineered polypeptide can be assembled from other polypeptide segments that individually occur in nature, or can include one or more segments that are not naturally occurring.

[0045] A polypeptide of interest often has a desirable biological or chemical activity. For example, the present invention can be employed to purify a pharmaceutically or commercially relevant enzyme, receptor, antibody, hormone, regulatory factor, antigen, binding agent, etc.

[0046] Some polypeptides that are particularly suitable for the purification method of the present invention are those that are highly positively charged or highly negatively charged in a solution with neutral pH. Examples of highly positively charged polypeptides include, but are not limited to, Neublastin.

[0047] The following is a detailed description of some of the polypeptides that may be expressed in a cell culture and purified in accordance with the present invention.

Growth Factors and Other Signaling Molecules

[0048] In some embodiments, the polypeptide of interests comprises a growth factor or a signaling molecule. Growth factors are typically glycoproteins that are secreted by cells and bind to and activate receptors on other cells, initiating a metabolic or developmental change in the receptor cell.

[0049] Non-limiting examples of mammalian growth factors and other signaling molecules include cytokines; epidermal growth factor (EGF); platelet-derived growth factor (PDGF); fibroblast growth factors (FGFs) such as aFGF and bFGF; transforming growth factors (TGFs) such as TGF-alpha and TGF-beta, including TGF-beta I, TGF- beta 2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factor-I and -II (IGF- I and IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins such as CD-3, C0-4, CD-8, and CD- 19; erythropoietin; osteoinductive factors; immunotoxins; a bone morphogenetic protein (BMP); an interferon such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (TLs), e.g., IL-1 to IL-10; tumor necrosis factor (TNF) alpha and beta; insulin A-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; anti-clotting factors such as Protein C; atrial natriuretic factor; lung surfactant; a plasminogen activator, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin, hemopoietic growth factor; enkephalinase; RANTES (regulated on activation normally T- cell expressed and secreted); human macrophage inflammatory protein (MIP-1 -alpha); mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated peptide; neurotrophic factors such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-beta. One of ordinary skill in the art will be aware of other growth factors or signaling molecules that can be expressed in accordance with the present invention. jOOSO] In some embodiments, the polypeptide of interest comprises a TGF-beta superfamily signaling molecule. In certain embodiments, the polypeptide of interest comprises a Neublastin, which is also known as Artemin or Enovin.

Clotting Factors

[0051] In some embodiments, the protein of interest comprises a clotting factor. Clotting factor, as used herein, means any molecule, or analog thereof, which prevents or decreases the duration of a bleeding episode in a subject with a hemostatic disorder. For example, a clotting factor for the invention can be a full-length clotting factor, a mature clotting factor, or a chimeric clotting factor. In other words, it means any molecule having clotting activity. Clotting activity, as used herein, means the ability to participate in a cascade of biochemical reactions that culminates in the formation of a fibrin clot and/or reduces the severity, duration or frequency of hemorrhage or bleeding episode. Examples of clotting factors can be found in U.S. Pat. No. 7,404,956, which is herein incorporated by reference.

[0052] In one embodiment, the clotting factor is Factor VIII, Factor IX, Factor XI, Factor

XII, fibrinogen, prothrombin, Factor V, Factor VII, Factor X, Factor XIII or von Willebrand Factor. The clotting factor can be a factor that participates in the extrinsic pathway. The clotting factor can be a factor that participates in the intrinsic pathway. Alternatively, the clotting factor can be a factor that participates in both the extrinsic and intrinsic pathway.

[0053] In one embodiment, the clotting factor can be a human clotting factor or a non- human clotting factor, e.g., derived from a non-human primate, a pig or any mammal. The clotting factor can be chimeric clotting factor, e.g., the clotting factor can comprise a portion of a human clotting factor and a portion of a porcine clotting factor or a portion of a first non-human clotting factor and a portion of a second non-human clotting factor.

[0054] In another embodiment, the clotting factor can be an activated clotting factor.

Alternatively, the clotting factor can be an inactive form of a clotting factor, e.g., a zymogen. The inactive clotting factor can undergo activation subsequent to being linked to at least a portion of an immunoglobulin constant region. The inactive clotting factor can be activated subsequent to administration to a subject. Alternatively, the inactive clotting factor can be activated prior to administration. [0055] In certain embodiments, the clotting factor is a Factor VIII protein. "Factor VIII protein" or "FVIH protein" as used herein, means functional Factor VIII protein in its normal role in coagulation, unless otherwise specified. Thus, the term FVIII includes variant proteins that are functional. In one embodiment, the FVIII protein is the human, porcine, canine, rat, or murine FVIII protein. A functional FVIII protein can be a fusion protein, such as, but not limited to, a fusion protein comprising a fully or partially B- domain deleted FVIII, at least a portion of an immunoglobulin constant region, e.g., an Fc domain, or both. In some embodiments, a clotting factor is a mature form of Factor VII or a variant thereof Factor VII (FVII, F7; also referred to as Factor 7, coagulation factor VII, serum factor VII, serum prothrombin conversion accelerator, SPCA, proconvertin and eptacog alpha) is a serine protease that is part of the coagulation cascade. FVII includes a Gla domain, two EGF domains (EGF-1 and EGF-2), and a serine protease domain (or peptidase SI domain) that is highly conserved among all members of the peptidase SI family of serine proteases, such as for example with chymotrypsin. FVII occurs as a single chain zymogen, an activated zymogen-like two-chain polypeptide and a fully activated two-chain form.

Antibodies

[0056] In some embodiments, the peptide of interest comprises an antibody or an antibody fragment. Antibodies are proteins that have the ability to specifically bind a particular antigen. Any antibody that can be expressed in a host cell can be used in accordance with the present invention. In one embodiment, the antibody to be expressed and purified is a monoclonal antibody.

[0057] Particular antibodies can be made, for example, by preparing and expressing synthetic genes that encode the recited amino acid sequences or by mutating human germline genes to provide a gene that encodes the recited amino acid sequences. Moreover, these antibodies can be produced, e.g., using one or more of the following methods.

[0058] Numerous methods are available for obtaining antibodies, particularly human antibodies. One exemplary method includes screening protein expression libraries, e.g., phage or ribosome display libraries. Phage display is described, for example, U.S. Pat. No. 5,223,409; Smith (1985) Science 228: 1315-1317; WO 92/18619; WO 91/17271 ; WO 92/20791 ; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; and WO 90/02809, The display of Fab's on phage is described, e.g., in U.S. Pat. Nos. 5,658,727; 5,667,988: and 5,885,793,

[0059] In addition to the use of display libraries, other methods can be used to obtain an antibody. For example, a protein or a peptide thereof can be used as an antigen in a non- human animal, e.g., a rodent, e.g., a mouse, hamster, or rat.

[0060] In one embodiment, the non-human animal includes at least a part of a human immunoglobulin gene. For example, it is possible to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci. Using the hybridoma technology, antigen-specific monoclonal antibodies derived from the genes with the desired specificity can be produced and selected. See, e.g., XENOMOUSE™, Green et al. (1994) Nature Genetics 7: 13-21 , U.S. 2003-0070185, WO 96/34096, and WO 96/33735.

[0061] In another embodiment, a monoclonal antibody is obtained from the non-human animal, and then modified, e.g., humanized or deimmunized. Winter describes an exemplary CDR-grafting method that can be used to prepare humanized antibodies described herein (U.S. Pat. No. 5,225,539). All or some of the CDRs of a particular human antibody can be replaced with at least a portion of a non-human antibody. In one embodiment, it is only necessary to replace the CDRs required for binding or binding determinants of such CDRs to arrive at a useful humanized antibody that binds to an antigen.

[0062] Humanized antibodies can be generated by replacing sequences of the Fv variable region that are not directly involved in antigen binding with equivalent sequences from human Fv variable regions. General methods for generating humanized antibodies are provided by Morrison, S. L. (1985) Science 229: 1202-1207, by Oi et al. (1986) BioTechniques 4:214, and by U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761 ; U.S. Pat. No. 5,693,762; U.S. Pat. No. 5,859,205; and U.S. Pat. No. 6,407,213. Those methods include isolating, manipulating, and expressing the nucleic acid sequences that encode all or part of immunoglobulin Fv variable regions from at least one of a heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art and, for example, can be obtained from a hybridoma producing an antibody against a predetermined target, as described above, from germline immunoglobulin genes, or from synthetic constructs. The recombinant DNA encoding the humanized antibody can then be cloned into ao appropriate expression vector. In one embodiment, the expression vector comprises a polynucleotide encoding a glutamine synthetase polypeptide. (See, e.g., Porter et al, Biotechnol Prog 26(5): 1446-54 (2010).)

[0063] The antibody can include a human Fc region, e.g., a wild-type Fc region or an Fc region that includes one or more alterations. In one embodiment, the constant region is altered, e.g., mutated, to modify the properties of the antibody {e.g., to increase or decrease one or more of: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). For example, the human IgGl constant region can be mutated at one or more residues, e.g., one or more of residues 234 and 237. Antibodies can have mutations in the CH2 region of the heavy chain that reduce or alter effector function, e.g., Fc receptor binding and complement activation. For example, antibodies can have mutations such as those described in U.S. Pat. Nos. 5,624,821 and 5,648,260. Antibodies can also have mutations that stabilize the disulfide bond between the two heavy chains of an immunoglobulin, such as mutations in the hinge region of IgG4, as disclosed in the art (e.g., Angal et al. (1993) Mol. Immunol. 30: 105-08). See also, e.g., U.S. 2005-0037000.

[0064] The antibody can be modified to have an altered glycosylation pattern (i.e., altered from the original or native glycosylation pattern). As used in this context, "altered" means having one or more carbohydrate moieties deleted, and/or having one or more glycosylation sites added to the original antibody. Another means of increasing the number of carbohydrate moieties on the antibodies is by chemical or enzymatic coupling of glycosides to the amino acid residues of the antibody. These methods are described in, e.g., WO 87/05330, and Aplin and Wriston (1981) CRC Crit. Rev. Biochem. 22:259-306. Removal of any carbohydrate moieties present on the antibodies can be accomplished chemically or enzymatically as described in the art (Hakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52; Edge et al (1981) Anal. Biochem. 1 18: 131 ; and Thotakura et al. (1987) Meth. Enzymol. 138:350).

[0065] The antibodies can be in the form of full length antibodies, or in the form of fragments of antibodies, e.g., Fab, F(ab')₂, Fd, dAb, and scFv fragments. Additional forms include a protein that includes a single variable domain, e.g., a camel or camelized domain. See, e.g., U.S. 2005-0079574 and Davies et al. (1996) Protein Eng. 9(6):531-7. [0066] In one embodiment, the antibody is an antigen-binding fragment of a full length antibody, e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment. Typically, the antibody is a full length antibody. The antibody can be a monoclonal antibody or a mono-specific antibody.

[0067] In another embodiment, the antibody can be a human, humanized, CD -grafted, chimeric, mutated, affinity matured, deimmunized, synthetic or otherwise in vitro- generated antibody, and combinations thereof.

[0068] The heavy and light chains of the antibody can be substantially full-length. The protein can include at least one, and preferably two, complete heavy chains, and at least one, and preferably two, complete light chains) or can include an antigen-binding fragment {e.g., a Fab, F(ab')2, Fv or a single chain Fv fragment). In yet other embodiments, the antibody has a heavy chain constant region chosen from, e.g., IgGl , IgG2, IgG3, IgG4, IgM, IgAl , IgA2, IgD, and IgE; particularly, chosen from, e.g., IgGl, IgG2, IgG3, and IgG4, more particularly, IgGl (e.g., human IgGl). Typically, the heavy chain constant region is human or a modified form of a human constant region. In another embodiment, the antibody has a light chain constant region chosen from, e.g., kappa or lambda, particularly, kappa (e.g., human kappa).

Receptors

[0069] In some embodiments, the polypeptide of interest comprises a receptor. Receptors are typically trans-membrane glycoproteins that function by recognizing an extra-cellular signaling ligand. Receptors typically have a protein kinase domain in addition to the ligand recognizing domain, which initiates a signaling pathway by phosphorylatmg target intracellular molecules upon binding the ligand, leading to developmental or metabolic changes within the cell. The receptor can be modified so as to remove the transmembrane and/or intracellular domain(s), in place of which there can optionally be attached an Ig- domain. In one embodiment, receptors to be produced and purified in accordance with the present invention are receptor tyrosine kinases (RTKs). The RTK family includes receptors that are crucial for a variety of functions numerous cell types (see, e.g., Yarden and Ullrich, Ann. Rev. Biochem. 57:433-478, 1988; Ullrich and Schlessinger, Cell 61 :243-254, 1990, incorporated herein by reference). Non-limiting examples of RTf s include members of the fibroblast growth factor (FGF) receptor family, members of the epidermal growth factor receptor (EGF) family, platelet derived growth factor (PDGF) receptor, tyrosine kinase with immunoglobulin and EGF homology domains- 1 (TIE-1) and TIE-2 receptors (Sato et al, Nature 376(6535):70-74 (1995), incorporated herein by reference) and c-Met receptor, some of which have been suggested to promote angiogenesis, directly or indirectly (Mustonen and Alitalo, J Cell Biol. 129:895-898, 1995). Other non-limiting examples of RTK's include fetal liver kinase 1 (FLK-1) (sometimes referred to as kinase insert domain-containing receptor (KDR) (Terman et al, Oncogene 6: 1677-83, 1991) or vascular endothelial cell growth factor receptor 2 (VEGFR-2)), fins-like tyrosine kinase- 1 (Fit- 1 ) (DeVries et al. Science 255;989-991 , 1992; Shibuya et al, Oncogene 5:519-524, 1990), sometimes referred to as vascular endothelial cell growth factor receptor 1 (VEGFR-1), neuropilin-1, endoglin, endosialin, and Axl .

G-Protein Coupled Receptors

[0070] In some embodiments, the polypeptide of interest comprises a G-protein coupled receptor (GPCR). GPCRs are proteins that have seven transmembrane domains. Upon binding of a ligand to a GPCR, a signal is transduced within the cell which results in a change in a biological or physiological property of the cell.

[0071] GPCRs, along with G-proteins and effectors (intracellular enzymes and channels which are modulated by G-proteins), are the components of a modular signaling system that connects the state of intracellular second messengers to extracellular inputs. These genes and gene-products are potential causative agents of disease.

[0072] The GPCR protein superfamily now contains over 250 types of paralogues, receptors that represent variants generated by gene duplications (or other processes), as opposed to orthologues, the same receptor from different species. The superfamily can be broken down into five families: Family I, receptors typified by rhodopsin and the beta2- adrenergic receptor and currently represented by over 200 unique members; Family II, the recently characterized parathyroid hormone/calcitonin/secretin receptor family; Family III, the metabotropic glutamate receptor family in mammals; Family IV, the cAMP receptor family, important in the chemotaxis and development of D. discoideum; and Family V, the fungal mating pheromone receptors such as STE2. Virus proteins

[0073] Additionally, the present invention also provides methods for the purification of virus proteins produced in a cell culture according to methods known to those of skill in the field of virology. The viruses to be purified in accordance with the present invention can be chosen from the range of viruses known to infect the cultured cell type. For instance, when utilizing a mammalian cell culture, viruses can be chosen from the genera of orthomyxoviruses, paramyxoviruses, reoviruses, picornaviruses, flaviviruses, arenaviruses, herpesviruses, poxviruses, coronaviruses and adenoviruses. The virus used can be a wild-type virus, an attenuated virus, a reassortant virus, or a recombinant virus. In addition, instead of actual virions being used to infect the cells with a virus, an infectious nucleic acid clone can be utilized according to infectious clone transfection methods known to those of skill in the field of virology. In one embodiment, the virus produced is an influenza virus.

B. Production of Polypeptides of Interest in a Cell Culture

Cells

[0074] A polypeptide of interest is first expressed and produced in a host cell culture.

Host cells include, but are not limited to, prokaryotic cells, eukaryotic cells, plant cells, yeast cells, animal cells, insect cells, avian cells, and mammalian cells.

[0075] Non-limiting examples of prokaryotic cells that can be used in accordance with the present invention include bacterial cells, such as Gram-negative or Gram-positive bacteria, for example, Escherichia coli.

[0076] Non-limiting examples of mammalian cells that can be used in accordance with the present invention include BALB/c mouse myeloma line (NSO/1 , ECACC No: 851 10503); human retinoblasts (PER.C6 (CruCell, Leiden, The Netherlands)); monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et ah, J Gen Virol, 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells ±DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO- 76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa. ATCC CCL 2); canine kidney cells (MDC , ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI ceils (Mather et al, Annals N. Y. Acad. ScL, 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2). In one embodiment, the present invention is used in the culturing of and expression of polypeptides from CHO cell lines. In a specific embodiment, the CHO cell line is the DG44 CHO cell line. In another specific embodiment, the CHO cell line is a CHO S line. In a specific embodiment, the CHO eel! line comprises a vector comprising a polynucleotide encoding a glutamine synthetase polypeptide, in a further specific embodiment, the CHO cell line expresses an exogenous glutamine synthetase gene. (See, e.g., Porter et al, Biotechnol Prog 26(5): 1446-54 (2010).)

[0077] Additionally, any number of commercially and non-commercially available hybridoma cell lines that express polypeptides or proteins can he utilized in accordance with the present invention.

[0078] The host cells used to produce the polypeptide of interest can be selected or engineered to produce high levels of protein or polypeptide, or to produce large quantities of virus. Often, cells are genetically engineered to produce high levels of protein, for example by introduction of a gene encoding the protein or polypeptide of interest and/or by introduction of control elements that regulate expression of the gene (whether endogenous or introduced) encoding the polypeptide of interest.

[0079] The host cells can also be selected or engineered to modify its posttrans!ational modification pathways. For example, the cells may be selected or engineered to modify a protein glycosylatio pathway.

[0080] The eukaryotic cells can also be selected or engineered to survive in culture for extended periods of time. For example, the cells can be genetically engineered to express a polypeptide or polypeptides that confer extended survival on the cells. In one embodiment, the eukaryotic cells comprise a transgene encoding the BcI-2 polypeptide or a variant thereof, See, e.g., US 7,785,880. In a specific embodiment, the cells comprise a polynucleotide encoding the bcl-xL polypeptide. See, e.g., Chiang GO, Sisk WP. 2005. Biotechnology and Bioengineering 91 (7):779-792.

[0081] The eukaryotic cells can also be selected or engineered to modify its posttranslational modification pathways, in one embodiment, the cells are selected or engineered to modify a protein glycosylation pathway. In a specific embodiment, the cells are selected or engineered to express an aglycosylated protein, e.g., an aglycosylated recombinant antibody. In another specific embodiment, the cells are selected or engineered to express an afucosylated protein, e.g., an afucosylated recombinant antibody.

[0082] The eukaryotic cells can also be selected or engineered to allow culturing in serum free medium.

[0083] The present invention relates to purification methods of polypeptides from a solution such as a cell culture medium. A medium used in the invention can be used in a batch culture, fed-batch culture or a perfusion culture.

[0084] A medium used in the present invention can be a serum-free medium, animal protein-free medium or a chemically-defined medium.

[0085] The present invention also relates to a cell culture composition comprising a medium and cells. In a specific embodiment, a cell culture composition described herein comprises CHO cells. In another specific embodiment, a cell culture composition described herein comprises HEK cells. In another specific embodiment, a cell culture composition described herein comprises hybridoma cells.

[0086] A cell culture composition can comprise cells that have been adapted to grow in serum free medium, animal protein free medium or chemically defined medium. Or it can comprise cells that have been genetically modified to increase their life-span in culture. In one embodiment, the cells have been modified to express an anti-apoptotic gene. In a specific embodiment, the cells have been modified to express the bcl-xL antiapoptotic gene. Additional anti-apoptotic genes that can be used in accordance with the present invention include, but are not limited to, E1B-9K, Aven, Mcl.

[0087] Cell cultures can be cultured in a batch culture, fed batch culture or a perfusion culture. In one embodiment, a cell culture according to a method of the present invention is a serum-free culture. In another embodiment, a cell culture according to a method of the present invention is a chemically defined culture. In a further embodiment, a cell culture according to a method of the present invention is an animal protein free culture.

[0088] The present invention further relates to method of producing a protein or polypeptide of interest, comprising culturing cells capable of producing the protein or polypeptide of interest in a culture comprising a medium described herein; and isolating the protein or polypeptide from the culture. In one embodiment, the protein or polypeptide of interest is a recombinant protein or polypeptide. In one embodiment, the protein or polypeptide of interest is an enzyme, receptor, antibody, hormone, regulatory factor, antigen, or binding agent. In a specific embodiment, the protein is an antibody.

Media

The host cells used to produce the polypeptide of interest may be cultured in a variety of media. The medium may contain e.g., inorganic salts, carbohydrates (e.g., sugars such as glucose, galactose, maltose or fructose), amino acids, vitamins (e.g., B group vitamins (e.g. , B 12), vitamin A vitamin E, riboflavin, thiamine and biotin), fatty acids and lipids (e.g., cholesterol and steroids), proteins and peptides (e.g., albumin, transferrin, fibronectin and fetuin), serum (e.g. , compositions comprising albumins, growth factors and growth inhibitors, such as, fetal bovine serum, newborn calf serum and horse serum), trace elements (e.g. , zinc, copper, selenium and tricarboxylic acid intermediates), hydrolysates (hydrolyzed proteins derived from plant or animal sources), and combinations thereof. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are exemplary nutrient solutions. In addition, any of the media described in Ham and Wallace,(1979) Meth. Enz., 58:44; Barnes and Sato,(1980) Anal. Biochem., 102:255; U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 5,122,469 or 4,560,655; International Publication Nos. WO 90/03430; and WO 87/00195; the disclosures of all of which are incorporated herein by reference, can be used as culture media. Any of these media can be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleosides (such as adenosine and thymidine), antibiotics (such as gentamycin), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range) lipids (such as linoleic or other fatty acids) and their suitable earners, and glucose or an equivalent energy source. In some embodiments the nutrient media is serum-free media, a protein-free media, or a chemically defined media. Any other necessary supplements can also be included at appropriate concentrations that would be known to those skilled in the art. [0090] In one embodiment, the mammalian host cell is a CHO cell and a suitable medium contains a basal medium component such as a DMEM/HAM F-12 based formulation (for composition of DMEM and HAM F12 media, see culture media formulations in American Type Culture Collection Catalogue of Cell Lines and Hybridomas, Sixth Edition, 1988, pages 346-349) with modified concentrations of some components such as amino acids, salts, sugar, and vitamins, and optionally containing glycine, hypoxanthine, and thymidine; recombinant human insulin, hydrolyzed peptone, such as Primatone HS or Primatone RL (Sheffield, England), or the equivalent; a cell protective agent, such as Pluronic F68 or the equivalent pluronic polyol; gentamycin; and trace elements.

[0091] In certain cases, it can be beneficial or necessary to supplement the cell culture with various cell culture additives to improve cell growth or protein production. Non- limiting examples of cell culture additives include hormones and/or other growth factors, particular ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, glucose, and other energy source. In one embodiment, a cell culture additive prevents the binding of a polypeptide of interest to the host cells. In some embodiments, the cell culture additive carries a net charge at neutral pH.

[0092] In certain embodiments, the cell culture additive is a polyanion ligand. In a specific embodiment, the cell culture additive is dextran sulfate. In some embodiments, the dextran sulfate has a molecular weight of about 4 kDa to about 500 kDa, about 4 kDa to about 400 kDa, about 4 kDa to about 300 kDa, about 4 kDa to about 200 kDa, about 4 kDa to about 100 kDa, about 4 kDa to about 50 kDa, about 4 kDa to about 25 kDa, about 4 kDa to about 20 kDa, or about 4 kDa to about 10 kDa. In a specific embodiment, the dextran sulfate has a molecular weight of about 5 kDa. In another specific embodiment, the dextran sulfate has a molecular weight of about 8 kDa. Commercially available dextran sulfate includes, but is not limited to, those with the molecular weight of about 500 kDa (Santa Cruz Biotechnology), about 200 kDa, about 40 kDa, about 9-20 kDa, about 15 kDa, about 6.5-10 kDa, about 5 kDa, and about 4 kDa (Sigma).

[0093] In certain embodiments, the concentration of dextran sulfate or other cell culture additive can be about 0.01 g/L to about 10 g/L, about 0.01 g/L to about 9 g/L, about 0.01 g/L to about 8 g/L, about 0.01 g/L to about 7 g/L, about 0.01 g/L to about 6 g/L, about 0.01 g/L to about 5 g/L, about 0.01 g/L to about 4 g/L, about 0.01 g/L to about 3 g/L, about 0.01 g/L to about 2 g/L, about 0.01 g/L to about 1 g/L, about 0.01 g/L to about 0.5 g/L, about 0.1 g/L to about 10 g/L, about 0.1 g/L to about 9 g/L, about 0.1 g/L to about 8 g/L, about 0.1 g/L to about 7 g/L, about 0.1 g/L to about 6 g/L, about 0.1 g/L to about 5 g/L, about 0.1 g/L to about 4 g/L, about 0.1 g/L to about 3 g/L, about 0.1 g/L to about 2 g/L, about 0.1 g/L to about 1 g/L, about 0.1 g/L to about 0.5 g/L, about 0.2 g/L to about 10 g/L, about 0.2 g/L to about 9 g/L, about 0.2 g/L to about 8 g/L, about 0.2 g/L to about 7 g/L, about 0.2 g/L to about 6 g/L, about 0.2 g/L to about 5 g/L, about 0.2 g/L to about 4 g/L, about 0.2 g/L to about 3 g/L, about 0.2 g/L to about 2 g/L, about 0.2 g/L to about 1 g/L, about 0.2 g/L to about 0.5 g/L, about 0.25 g/L to about 10 g/L, about 0.25 g/L to about 9 g/L, about 0.25 g/L to about 8 g/L, about 0.25 g/L to about 7 g/L, about 0.25 g/L to about 6 g/L, about 0.25 g/L to about 5 g/L, about 0.25 g/L to about 4 g/L, about 0.25 g/L to about 3 g/L, about 0.25 g/L to about 2 g/L, about 0.25 g/L to about 1 g/L, about 0.25 g/L to about 0.5 g/L, about 0.5 g/L to about 10 g/L, about 0.5 g/L to about 9 g/L, about 0.5 g/L to about 8 g/L, about 0.5 g/L to about 7 g/L, about 0.5 g/L to about 6 g/L, about 0.5 g/L to about 5 g/L, about 0.5 g/L to about 4 g/L, about 0.5 g/L to about 3 g/L, about 0.5 g/L to about 2 g/L, or about 0.5 g/L to about 1 g/L.

[0094] In some embodiment, the concentration of dextran sulfate or other cell culture additive in the solution can be about 0.1 g/L, about 0.2 g/L, about 0.25 g/L, about 0.5 g/L, about 1 g/L , about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 5 g/L, about 5.5 g/L, about 6 g/L, about 6.5 g/L, about 7 g/L, about 7.5 g/L, about 8 g/L, about 8.5 g/L, about 9 g/L, about 9.5 g/L, about 10 g/L, about 10.5 g/L, about 1 1 g/L, about 11.5 g/L, or about 12 g/L.

[0095] In some embodiments, a polypeptide of interest produced in a cell culture medium in the presence of a cell culture additive has a higher titer of the polypeptide in the cell culture than the titer of the polypeptide produced in the absence of the cell culture additive.

[0096] In some embodiments, a cell culture medium comprises a polypeptide of interest at a titer of at least about 0.1 g/L, at least about 0.2 g/L, at least about 0.5 g/L, at least about 1 g/L, at least about 2 g/liter, at least about 2.5 g/liter, at least about 3 g/liter, at least about 3.5 g/liter, at least about 4 g/liter, at least about 4.5 g/liter, at least about 5 g/liter. at least about 6 g/liter, at least about 7 g/liter, at least about 8 g/liter, at least about 9 g/liter, or at least about 10 g/liter, or a titer of between about 0.1 g/liter and about 10 g/liter, about 0.2 g/liter and about 10 g/liter, about 0.5 g/liter and about 10 g/liter, about 1 g/liter and about 10 g/liter, about 1.5 g/liter and about 10 g/liter, about 2 g/liter and about

10 g/liter, about 2.5 g/liter and about 10 g/liter, about 3 g/liter and about 10 g/liter, about 4 g/liter and about 10 g/liter, about 5 g/liter and about 10 g/liter, about 0.1 g/liter and about 5 g/liter, about 0.2 g/liter and about 5 g/liter, about 0.5 g/liter and about 5 g/liter, about 1 g/liter and about 5 g/liter, about 1 g/liter and about 4.5 g/liter, or about 1 g/liter and about 4 g/liter.

Cell Culture Processes

[0097] Various methods of preparing mammalian cells for production of proteins or polypeptides by batch and fed-batch culture are well known in the art. A nucleic acid sufficient to achieve expression (typically a vector containing the gene encoding the polypeptide or protein of interest and any operably linked genetic control elements) can be introduced into the host cell line by any number of well-known techniques. Typically, cells are screened to determine which of the host cells have actually taken up the vector and express the polypeptide or protein of interest. Traditional methods of detecting a particular polypeptide or protein of interest expressed by mammalian cells include but are not limited to immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, SDS-PAGE, Western blots, enzyme-linked immunosorbentassay (ELISA), high performance liquid chromatography (HPLC) techniques, biological activity assays and affinity chromatography. One of ordinary skill in the art will be aware of other appropriate techniques for detecting expressed polypeptides or proteins. If multiple host cells express the polypeptide or protein of interest, some or all of the listed techniques can be used to determine which of the cells expresses that polypeptide or protein at the highest levels.

[0098] Once a cell that expresses the polypeptide or protein of interest has been identified, the cell is propagated in culture by any of the variety of methods well-known to one of ordinary skill in the art. The cell expressing the polypeptide of interest is typically propagated by growing it at a temperature and in a medium that is conducive to the survival, growth and viability of the cell. The initial culture volume can be of any size, but is often smaller than the culture volume of the production bioreactor used in the final production of the polypeptide or protein of interest, and frequently cells are passaged several times in bioreactors of increasing volume prior to seeding the production bioreactor. The cell culture can be agitated or shaken to increase oxygenation of the medium and dispersion of nutrients to the cells. Alternatively or additionally, special sparging devices that are well known in the art can be used to increase and control oxygenation of the culture. In accordance with the present invention, one of ordinary skill in the art will understand that it can be beneficial to control or regulate certain internal conditions of the bioreactor, including but not limited to pH, temperature, oxygenation, etc.

[0099] The temperature of the cell culture will be selected based primarily on the range of temperatures at which the cell culture remains viable. For example, during the initial growth phase, CHO cells grow well at 37°C. In general, most mammalian cells grow well within a range of about 25°C to 42°C.

[0100] In order to monitor certain cell culture conditions, it will be necessary to remove small aliquots of the culture for analysis. One of ordinary skill in the art will understand that such removal can potentially introduce contamination into the cell culture, and will take appropriate care to minimize the risk of such contamination.

[0101] As non-limiting example, it can be beneficial or necessary to monitor temperature, pH, cell density, cell viability, integrated viable cell density, lactate levels, ammonium levels, osmolarity, or titer of the expressed polypeptide or protein. Numerous techniques are well known in the art that will allow one of ordinary skill in the art to measure these conditions. For example, cell density can be measured using a hemacytometer, a Coulter counter, or Cell density examination (CEDEX). Viable cell density can be determined by staining a culture sample with Trypan blue. Since only dead cells take up the Trypan blue, viable cell density can be determined by counting the total number of cells, dividing the number of cells that take up the dye by the total number of cells, and taking the reciprocal. HPLC, and instruments such as Biochemistry Analyzer (YSI LifeSciences, Yellow Springs, OH) and BioProfile^® FLEX (Nova Biomedical, Waltham, MA) can be used to determine the levels of lactate, ammonium or the expressed polypeptide or protein. Alternatively, the level of the expressed polypeptide or protein can be determined by standard molecular biology techniques such as coomassie staining of SDS-PAGE gels, Western blotting, Bradford assays, Lowry assays, Biuret assays, and UV absorbance. It can also be beneficial or necessary to monitor the post-translational modifications of the expressed polypeptide or protein, including phosphorylation and glycosylation.

[0102J The practitioner can also monitor the metabolic status of the cell culture, for example, by monitoring the glucose, lactate, ammonium, and amino acid concentrations in the cell culture, as well as by monitoring the oxygen production or carbon dioxide production of the cell culture. For example, cell culture conditions can be analyzed by using NOVA Bioprofile 100 or 400 (NOVA Biomedical, WA). Additionally, the practitioner can monitor the metabolic state of the cell culture by monitoring the activity of mitochondria. In embodiment, mitochondrial activity can be monitored by monitoring the mitochondrial membrane potential using Rhodamine 123. Johnson LV, Walsh ML, Chen LB. 1980. Proceedings of the National Academy of Sciences 77(2):990-994.

C. Removal of Cell Culture Additives and Purification of Polypeptides

[0103] A polypeptide of interest produced in a cell culture can be isolated and purified based on its size, physic-chemical properties, binding affinity, and biological affinity.

[0104] When using recombinant techniques, the polypeptide of interest can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the protein is produced intracellularly, as a first step, the particulate debris, either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration. Where the polypeptide is secreted into the medium, the recombinant host cells may be separated from the cell culture medium by tangential flow filtration, for example.

[0105] Ion exchange chromatography is a chromatographic technique that is commonly used for the purification of charged polypeptides. In ion exchange chromatography, charged patches on the surface of the polypeptides are attracted by opposite charges attached to a chromatography matrix, provided the ionic strength of the surrounding buffer is low. Elution is generally achieved by increasing the ionic strength (i.e. conductivity) of the buffer to compete with the solute for the charged sites of the ion exchange matrix. Changing the pH and thereby altering the charge of the solute is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution).

[0106] A polypeptide with a high pi (e.g., Neublastin, which has a pi of 1 1.3) is highly positively charged at neutral pH, thus is an ideal candidate for purification by cation exchange chromatography. However, a negatively charged cell culture additive (e.g., dextran sulfate) can bind to the positively charged polypeptide, forming a complex that alters the net electrical charge and surface charge distribution of the polypeptide. The change in protein charge characteristics significantly suppresses binding of the protein to the conventional cation exchange matrix, resulting in low binding capacity, thus leading to low yield of the polypeptide in the purification process. Similarly, a polypeptide with a low pi and highly negatively charged in a neutral solution can be complexed to a positively charged cell culture additive, which interferes with the purification of the polypeptide by anion exchange chromatography.

[0107J The present invention provides a method of purifying a polypeptide of interest from a solution comprising the polypeptide and a cell culture additive wherein the polypeptide is complexed to the cell culture additive, comprising: (a) separating the cell culture additive from the polypeptide in the complex; (b) contacting the free polypeptide with a chromatographic matrix; and (c) eluting the bound polypeptide from the chromatography matrix, thereby purifying the polypeptide from the solution.

[0108] In certain embodiments, the solution is a harvested cell culture fluid (HCCF).

HCCF is the supernatant obtained by centrifugation of the cell culture producing the polypeptide of interest, followed by depth filtration to remove the cell debris.

[0109] In certain embodiments, the polypeptide of interest is positively charged in the solution. A polypeptide is positively charged when the pH of the solution is lower than its pi. In certain embodiments, the polypeptide of interest is negatively charged in the solution. A polypeptide is negatively charged when the pH of the solution is higher than its pi. In some embodiments, the polypeptide of interest may not have a net charge in the solution.

[0110] In certain embodiments, the cell culture additive is negatively charged in the solution. In certain embodiments, the cell culture additive is positively charged in the solution. In some embodiments, the cell culture additive may not have a net charge in the solution.

[0111] In a specific embodiment, the polypeptide of interest and the cell culture additive form a complex via an electrostatic bond. In other embodiments, the polypeptide of interest and the cell culture additive form a complex via molecular interactions other than electrostatic bond, [0112] In certain embodiments, the polypeptide of interest is selected from the group consisting of: an antibody, a Transforming Growth Factor (TGF) beta superfamily signaling molecule, an Fc fusion protein, a therapeutic enzyme, a recombinant vaccine, and a clotting factor. In some embodiments, the polypeptide of interest is a TGF-beta superfamily signaling molecule. In other embodiments, the polypeptide of interest is a clotting factor. In a specific embodiment, the polypeptide of interest is Neublastin. In another specific embodiment, the polypeptide of interest is Factor VIII.

[0113] The first step of the presently disclosed purification process is to remove the cell culture additive from the polypeptide of interest in the solution. The second step is to purify the freed polypeptide of interest from the solution.

[0114] In certain embodiments, the first step of the purification process comprises separating the cell culture additive from the polypeptide of interest by disrupting their molecular interaction. In some embodiments, the cell culture additive is separated by disrupting the electrostatic bond. In a specific embodiment, the electrostatic bond is disrupted by increasing the conductivity of the solution.

[0115] In certain embodiments, the conductivity of the solution is increased to greater than about 16 mS/cm, greater than about 20 mS/cm, greater than about 25 mS/cm, greater than about 30 mS/cm, greater than about 35 mS/cm, greater than about 40 mS/cm, greater than about 45 mS/cm, greater than about 50 mS/cm, greater than about 55 mS/cm, or greater than about 60 mS/cm.

[0116] In certain embodiments, the conductivity of the solution is increased to about 20 mS/cm to about 80 mS/cm, about 20 mS/cm to about 70 mS/cm, about 20 mS/cm to about 60 mS/cm, about 20 mS/cm to about 50 mS/cm, about 20 mS/cm to about 40 mS/cm, about 20 mS/cm to about 30 mS/cm, about 30 mS/cm to about 80 mS/cm, about 30 mS/cm to about 70 mS/cm, about 30 mS/cm to about 60 mS/cm, about 30 mS/cm to about 50 mS/cm, about 30 mS/cm to about 40 mS/cm, about 40 mS/cm to about 80 mS/cm, about 40 mS/cm to about 70 mS/cm, about 40 mS/cm to about 60 mS/cm, about 40 mS/cm to about 50 mS/cm, about 50 mS/cm to about 80 mS/cm, about 50 mS/cm to about 70 mS/cm, about 50 mS/cm to about 60 mS/cm, about 60 mS/cm to about 80 mS/cm, or about 60 mS/cm to about 70 mS/cm.

[0117] In some embodiments, the conductivity of the solution is increased to about 20 mS/cm, about 25 mS/cm, about 30 mS/cm, about 35 mS/cm, about 40 mS/cm, about 45 mS/cm, about 50 mS/cm, about 55 mS/cm, about 60 mS/cm, about 65 mS/cm, about 70 mS/cm, about 75 mS/cm, or about 80 mS/cm.

[0118] In certain embodiments, the conductivity of the solution is increased by addition of salts. Non-limiting examples of salts that can be added into the solution or to any buffer used in accordance with the present invention include sodium salts, potassium salts, calcium salts, magnesium salts, barium salts, zinc salts, aluminum salts, ammonium salts, chloride salts, fluoride salts, bromide salts, iodide salts, carbonate salts, nitrate salts, phosphate salts, sulfate salts, acetate salts, and combination thereof. In some embodiments, the conductivity of the solution is increased by addition of sodium chloride (NaCl).

[0119] In certain embodiments, the concentration of the salts in the solution, such as

[NaCl], is increased to greater than about 200 mM, greater than about 250 mM, greater than about 300 mM, greater than about 350 mM, greater than about 400 mM, greater than about 450 mM, greater than about 500 mM, greater than about 550 mM, greater than about 600 mM, greater than about 650 mM, greater than about 700 mM, greater than about 750 mM, or greater than about 800 mM.

[0120] In certain embodiments, the concentration of the salts in the solution, such as

[NaCl], is increased to about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 200 mM to about 400 mM, about 200 mM to about 300 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, about 300 mM to about 500 mM, about 300 mM to about 400 mM, about 400 mM to about 800 mM, about 400 mM to about 700 mM, about 400 mM to about 600 mM, about 400 mM to about 500 mM, about 500 mM to about 800 mM, about 500 mM to about 700 mM, about 500 mM to about 600 mM, about 600 mM to about 800 mM, or about 600 mM to about 700 mM.

[0121] in some embodiments, the concentration of the salts in the solution, such as

[NaCl], is increased to about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, or about 800 mM.

[0122] After the cell culture additive is separated from the polypeptide of interest by increasing conductivity of the solution, the second step comprises purifying the freed polypeptide from the high salt solution by a method suitable for its properties and compatible with high salt concentration in the loading buffer. In certain embodiments, the polypeptide of interest separated from the cell culture additive is contacted with a mixed-mode chromatography matrix. In some embodiments, the mixed-mode chromatography matrix is Capto MMC or Eshmuno HCX.

[0123] In certain embodiments, the polypeptide bound to the mixed-mode matrix is washed and eluted with a salt buffer. The salt concentration and the pH of the buffer can be adjusted and optimized for maximized purity and/or yield. The polypeptide of interest may bind very tightly to the mixed-mode matrix and may not be effectively eluted by changing conductivity or pH of the solution. Therefore, in some embodiments, the buffer further comprises an Elution Modifier in order to improve the elution. "Elution Modifiers" are organic molecules that are known to reduce hydrophobic interaction, hydrogen bonding and electrostatic interactions. In some embodiments, the organic elution modifier is selected from the group consisting of ethylene glycol, propylene glycol, arginine, lysine, histidine, and mixture thereof In one specific embodiment, the organic elution modifier is arginine.

[0124] In some embodiments, the buffer comprises NaCl and/or arginine. In a more specific embodiment, the wash buffer comprises about 1 M NaCl and about 0.15 M arginine, or about 0.5 M arginine alone. In another specific embodiment, the elution buffer comprises about 1 M NaCl and about 0.6 M arginine, or about 0.9 M arginine alone.

[0125] Alternatively, in the first step of the purification method, the cell culture additive can also be removed by capturing it with a suitable chromatography matrix which specifically binds to the cell culture additive but not to the polypeptide of interest.

[0126] In certain embodiments, the cell culture additive is separated from the complex by contacting the complex to a chromatography matrix. To remove a negatively charged additive from a positively charged polypeptide, the chromatography matrix can be an anion exchange matrix. To remove a positively charged additive from a negatively charged polypeptide, the chromatography matrix can be a cation exchange matrix. In some embodiments, the chromatography matrix can be a mixed-mode adsorbent. In a specific embodiment, the anion exchange matrix is a Q Sepharose Fast Flow (QFF) resin. In certain embodiments, the salt concentration such as [NaCl] in the loading buffer, wash buffer, or elution buffer is increased to at least about 200 mM, about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, or about 600 mM.

[0127] After the cell culture additive is captured by the first chromatography matrix, the second step is to purify the polypeptide of interest remaining in the flow-through by subsequent methods based on the properties of the polypeptide. To purify a positively charged polypeptide of interest, a cation exchange matrix can be used. To purify a negatively charged polypeptide of interest, an anion exchange matrix can be used. In a specific embodiment, the cation exchange matrix is an SP Sepharose XL (SPXL) resin. Standard ion exchange chromatography protocols and buffers can be used to optimize the wash and the elution conditions to achieve maximum purity and/or yield of the polypeptide. In some embodiments, the chromatography matrix in the second step can be a mixed-mode adsorbent.

[0128] In certain embodiments, the polypeptide of interest bound to the chromatography matrix, such as a cation exchange matrix, is eluted with a step or linear salt gradient. In certain embodiments, the salt gradient is from 100 mM to about 1000 mM NaCl, or from about 100 mM NaCl to about 900 mM NaCl, or from 100 mM NaCl to about 800 mM NaCl, or from 100 mM NaCl to about 700 mM NaCl, or from 100 mM NaCl to about 600 mM NaCl.

[0129] The eluted polypeptide of interest may be subjected to additional purification steps either prior to, or after, the purification method disclosed herein. Standard methods include but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation or by any other available technique for the purification of proteins (See, e.g., Scopes, Protein Purification Principles and Practice 2nd Edition, Springer- Verlag, New York, 1987; Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, M. P., Simon, M. I., Abelson, J. N. (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997, all incorporated herein by reference). Protease inhibitors such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin can be added at any or all stages in order to reduce or eliminate degradation of the polypeptide or protein during the purification process. Protease inhibitors are particularly desired when cells must be lysed in order to isolate and purify the expressed polypeptide or protein. One of ordinary skill in the art will appreciate that the exact purification technique will vary depending on the character of the polypeptide or protein to be purified, the character of the cells from which the polypeptide or protein is expressed, and the composition of the medium in which the cells were grown.

[0130] The foregoing description is to be understood as being representative only and is not intended to be limiting. Alternative methods and materials for implementing the invention and also additional applications will be apparent to one of skill in the art, and are intended to be included within the accompanying claims.

* * *

[0131] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Molecular Cloning A Laboratory Manual, 2nd Ed., Sambrook et al, ed., Cold Spring Harbor Laboratory Press: (1989); Molecular Cloning: A Laboratory Manual, Sambrook et al, ed., Cold Springs Harbor Laboratory, New York (1992), DNA Cloning, D. N. Glover ed., Volumes I and II (1985); Oligonucleotide Synthesis, M. J. Gait ed., (1984); Mullis et al. U.S. Pat. No: 4,683,195; Nucleic Acid Hybridization, B. D. Hames & S. J. Higgins eds. (1984); Transcription And Translation, B. D. Hames & S. J. Higgins eds. (1984); Culture Of Animal Cells, R. I. Freshney, Alan R. Liss, Inc., (1987); Immobilized Cells And Enzymes, IRL Press, (1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); the treatise, Methods In Enzymology, Academic Press, Inc., N.Y.; Gene Transfer Vectors For Mammalian Cells, J. H. Miller and M. P. Calos eds., Cold Spring Harbor Laboratory (1987); Methods In Enzymology, Vols. 154 and 155 (Wu et al. eds.); Immunochemical Methods In Cell And Molecular Biology, Mayer and Walker, eds., Academic Press, London (1987); Handbook Of Experimental Immunology, Volumes I-IV, D. M. Weir and C. C. Blackwell, eds., (1986); Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989). [0132] All of the references cited above, as well as all references cited herein, are incorporated herein by reference in their entireties.

[0133] The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES Example 1

Dextran Sulfate is Complexed with Neublastin in the Cell Culture Media and Increase of Conductivity in the Solution can Disrupt the Complex

[0134] Neublastin is recombinantly expressed in Chinese Hamster Ovary (CHO) cells for large-scale production. The produced protein is secreted into the cell culture media. During the production phase, dextran sulfate, a highly negatively charged polymer, is added to the Neublastin cell culture in order to prevent Neublastin from binding to the CHO cells, thereby significantly improving cell growth and increasing Neublastin titer.

[0135] However, it has been found that the addition of dextran sulfate caused great loss of Neublastin yield in the subsequent purification process. Previously, Neublastin was readily purified by conventional cation exchange chromatography due to its high pi of 11.3. The established purification process for Neublastin uses SP Sepharose XL ("SPXL," GE Healthcare Life Sciences, Pittsburgh, PA) cation exchange chromatography adsorbent to capture Neublastin from the harvested cell culture fluid (HCCF). The primary mechanism of separation is electrostatic interactions between the highly positively charged Neublastin and the negatively charged ligands on the SPXL adsorbent. After addition of dextran sulfate to the cell culture media, the SPXL purification yield was dramatically decreased from ~74% to -0%.

[0136] The effect of dextran sulfate on the binding of Neublastin to the SPXL adsorbent is shown in Fig. 1. Fig. 1 shows the binding of purified Neublastin to the SPXL adsorbent in the presence of 0.25 g/L of 5 kDa dextran sulfate. The conductivity of the load solution was approximately 16 mS/cm. The chromatogram shows an immediate product breakthrough during the load, suggesting that dextran sulfate interferes with Neublastin binding to the SPXL adsorbent, [0137] The low binding capacity for Neublastin is believed to be a direct result of a change in electrostatic interactions between the protein and adsorbent ligands. The extent of electrostatic interactions is controlled by the conductivity (salt concentration) of the solution and the electrical charge of the protein and adsorbent ligand. The primary reason for the low binding capacity was a change in charge characteristics of Neublastin in the presence of dextran sulfate and not the conductivity of the load solution since there was negligible change in load conductivity after the addition of dextran sulfate. It was believed that the negatively charged dextran sulfate binds to the positively charged Neublastin electrostatically, forming complex structures with very low affinity to the SPXL adsorbent.

[0138] To examine this hypothesis, a series of size exclusion chromatography (SEC) experiments were performed to evaluate the size distribution of the species in the solution. The top panel of Figure 2 shows the size exclusion chromatogram for purified Neublastin solution (100 mM Phosphate, 200 mM NaCl, pH 7) alone (chromatogram a), and in the presence of 1.5 g/L of 5 kDa dextran sulfate (chromatogram b). It is clear from the chromatograms that Neublastin formed larger complex structures with dextran sulfate (corresponding to shorter retention times in the SEC column) when the solution conductivity was low (low NaCl concentration).

[0139] However, when the conductivity of the solution containing purified Neublastin and dextran sulfate was increased to approximately 60 mS/cm (approximately 600 mM NaCl), the peaks corresponding to the complex structures (with residence times below 4.6 min) disappeared in the high conductivity sample and the remaining major peaks overlayed with those of Neublastin alone (chromatogram c). This indicates that the high conductivity has disrupted the complex between Neublastin and dextran sulfate.

[0140] Also shown in the bottom panel of Figure 2 are the size exclusion chromatograms for purified Neublastin in a 600 mM NaCl solution containing dextran sulfate concentrations ranging from 0.25 to 6 g/L. This indicates that increasing salt concentration to 600 mM fully disrupts the Neublastin-dextran sulfate bond over the entire, wide range of dextran sulfate concentration.

[0141] Therefore, these results demonstrate that dextran sulfate is complexed with

Neublastin in the cell culture media, likely via an electrostatic bond, thereby interfering with the binding of Neublastin to the cation exchange resin. The results also indicate that increase of conductivity in the solution can disrupt the complex of Neublastin and dextran sulfate.

Example 2

A Two-Step Method Comprising Removing Dextran Sulfate by Increasing the

Conductivity of the Solution

[0142] In order to improve the purification yield of Neublastin in the presence of dexdran sulfate, a two-step purification method was developed to first clear away the dextran sulfate.

[0143] The first step is to disrupt the bond between Neublastin and dextran sulfate and free the protein from the complex. Considering the electrostatic nature of the bond, an increase in solution conductivity (i.e. , increase in salt concentration) could weaken the electrostatic interactions and form free Neublastin in the solution, as demonstrated in Example 1.

[0144] In the second step of the process, a mixed mode adsorbent, such as Capto MMC, can be used to capture Neublastin from the high conductivity solution and separate it from dextran sulfate. Cation exchange chromatography is not used because the binding capacity of cation exchange adsorbents significantly decreases as the load conductivity increases, with the SPXL adsorbent providing minimal binding at conductivities greater than 20 mS/cm. In contrast, mixed-mode adsorbent provides multiple modes of interaction, e.g., hydrophobic, ion exchange, and hydrogen bonding interactions and can provide high binding capacities at high salt concentrations.

[0145] In a first experiment, the conductivity of a 0.3 g/L purified Neublastin solution containing 0.25 g/L dextran sulfate was adjusted to 60 mS/cm using sodium chloride (600 mM), and loaded onto the Capto MMC column. The Capto MMC adsorbent provided a dynamic binding capacity at 10% breakthrough (DBCio_%) of approximately 30 mg/mL. A similar experiment was performed using a harvested cell culture fluid containing 0.4 g/L Neublastin and 0.25 g/L dextran sulfate. A dynamic binding capacity of greater than 40 mg/mL was achieved. The binding capacity results are summarized in Table 1. Higher levels of dextran sulfate (4-6 g/L) were also evaluated. The capacity of Capto MMC column decreased to 20 mg/mL when the dextran sulfate concentration was increased to 6 g/L. Increasing the column residence time from 4 to 6 minutes helped to partially restore binding capacity of the Capto MMC adsorbent to 26 mg/mL. Similar evaluations were performed using Eshmuno HCX mixed mode adsorbent. The effect of dextran sulfate concentration and column residence time on capacity of the Eshmuno HCX adsorbent is also shown in Table 1. The Capto MMC and Eshmuno HCX adsorbents provided significant binding capacities even in the presence of high concentrations of dextran sulfate, when the conductivity of load solution was adjusted to 60 mS/cm.

Table 1. Dynamic binding capacity of Capto MMC and Eshmuno HCX for Neublastin samples in the presence of 0.25-6 g/L of 5 kDa dextran sulfate when conductivity is

adjusted to 60 mS/cm.

[0146] The Capto MMC adsorbent was further evaluated to determine the wash and elution conditions to obtain high step recovery and high product purity. Neublastin binds to the Capto MMC adsorbent under high conductivity conditions via multiple modes of interactions. As a result, protein binds more tightly to the mixed mode adsorbents compared to adsorbents utilizing a single mode of interaction. Therefore, protein could not be eluted simply by changing conductivity or pH of the solution. Instead, organic elution modifiers such as ethylene glycol, propylene glycol, or arginine were used for effective elution of product.

[0147] Figure 3 shows the SDS PAGE image for the fractions from a chromatography run using Capto MMC column. The wash solution (lane-8) contains 1M NaCl, 0.15 M arginine, and the elution solution (lane-10) contains 1 M NaCl, 0.6 M arginine, Evaluation of different elution conditions suggested that high concentrations of both NaCl and arginine were required for effective removal of impurities and elution of protein from the column, and neither salt nor arginine was effective on its own. The step recovery was approximately 100% based on the MALDI-TOF titer assay, and the Capto MMC column was effective in removing most of the impurities (eluate, lane- 10) except small amounts of low MW (LMW) impurities. These small amounts of impurities may be further removed using SPXL adsorbent. Wash and elution conditions were also evaluated for the Eshmuno HCX adsorbent (data not shown).

[0148] These results clearly demonstrate that the newly developed two-step method is capable of separating dextran sulfate from Neublastin, and provides a high binding capacity for Neublastin using mixed-mode adsorbents, even under conditions of high load conductivity. The level of dextran sulfate in the eluate needs to be determined in the future.

Example 3

An Alternative Two-Step Method Comprising Removing Dextran Sulfate by

An Anion Exchange Resin

[0149] As elucidated above, the reason why dextran sulfate decreases the SPXL purification yield is that negatively charged dextran sulfate binds to positively charged Neublastin, thereby disrupting the interaction between Neublastin and the negatively charged SPX L resin. Therefore, another method to remove dextran sulfate is to capture the negatively charged dextran sulfate with a positively charged resin, such as the Q Sepharose Fast Flow ("QFF," GE Healthcare Life Sciences, Pittsburgh, PA) resin. High salt is present in the QFF flow-through, The conductivity or salt concentration is reduced by 4x dilution with 50 mM phosphate buffer, pH = 7.0. The diluted QFF flow-through can be purified by SPXL. The freed Neublastin can subsequently be purified by conventional cation exchange chromatography.

[0150] In one experiment, Neublastin harvested cel l culture fluid (HCCF) in the presence of 0.25 g/L dextran sulfate was loaded onto a QFF column which had already been equilibrated with NaOH to pH 9.0 and at 500 mM NaCl salt concentration. For example, for a 5mL QFF volume sample 4\ μΙ, NaOH (5M) and 556μί NaCl(5M) were added. The QFF flow-through was subsequently loaded onto SPXL column, washed 3 times with a 50mM Tris, 210 mM NaCl wash buffer at pH8.5, and the bound protein was eluted with a salt buffer (50mM Tris, 500mM NaCl, at pH8.5). As shown in Fig. 4, Neublastin remained in the QFF flow- though (Lane 3) because it is positively charged and does not have strong interaction with positively charged QFF. Because the dextran sulfate was captured by the QFF resin, free Neublastin was able to bind to the SPXL resin, while the other impurities were found in the SPXL flow-through and wash (Lanes 5, 6, 7, and 8).

[0151] As shown in Fig. 5, the SEC chromatogram of purified Neublastin in the presence of dextran sulfate (NBN + DexS) using QFF+SPXL completely overlayed with that of the pure Neublastin (NBN DS). The overall purification yield of Neublastin using QFF+SPXL was 78%, compared with the 2% yield using SPXL for purification only, as shown in Table 2.

Table 2. Purification yield of Neublastin in the presence of dextran sulfate using

FF+SPXL or SPXL onl

[0152] In addition, it was found that increasing concentration of NaCl increased the purification yield of Neublastin from the QFF column. Fig. 6 shows the flow through from the QFF column analyzed by size exclusion chromatography (SEC). When [NaCl] increased from 200 mM to 300 mM, 400 mM, and 500 mM, the yield of Neublastin in the presence of dextran sulfate increased from 27 ± 2%, to 44 ± 1%, 63 ± 0%, and 83% ± 0 %, respectively.

[0153] These results demonstrate that an alternative two-step purification process comprising an anion exchange chromatography to capture dextran sulfate, followed by a cation exchange chromatography to capture Neublastin can greatly increase the purification yield of Neublastin in the presence of dextran sulfate. [0154] The present invention is not to be limited in scope by the specific embodiments described which are intended as single illustrations of individual aspects of the invention, and any compositions or methods which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0155] All documents, articles, publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method of purifying a polypeptide of interest from a solution comprising the polypeptide and a cell culture additive wherein the polypeptide is complexed to the cell culture additive, comprising: (a) separating the cell culture additive from the polypeptide in the complex; (b) contacting the free polypeptide with a chromatographic matrix; and (c) eluting the bound polypeptide from the chromatography matrix, thereby purifying the polypeptide from the solution,

2. The method of claim 1, wherein the polypeptide is positively charged.

3. The method of claim 1 or 2, wherein the cell culture additive is negatively charged.

4. The method of any one of claims 1-3, wherein the complex of the polypeptide and the cell culture additive is formed via an electrostatic bond.

5. The method of claim 4, wherein the ceil culture additive is separated from the polypeptide by disrupting the electrostatic bond.

6. The method of claim 5, wherein the electrostatic bond is disrupted by increasing the conductivity of the solution.

7. The method of claim 6, wherein the conductivity of the solution is increased to greater than about 16 raS/cm.

8. The method of claim 7, wherein the conductivity of the solution is increased to about 20 mS/cm to about 80 mS/cm, about 20 mS/cm to about 70 mS/cm, about 20 mS/cm to about 60 mS/cm, about 20 mS/cm to about 50 mS/cm, about 20 mS/cm to about 40 mS/cm, about 20 mS/cm to about 30 mS/cm, about 30 mS/cm to about 80 mS/cm, about 30 raS/cm to about 70 mS/cm, about 30 mS/cm to about 60 mS/cm, about 30 mS/cm to abou 50 roS/cm, about 30 mS/cm to about 40 mS/cm, about 40 mS/cm to about 80 mS/cm, about 40 mS/cm to about 70 mS/cm, about 40 mS/cm to about 60 mS/cm, about 40 mS/em to about 50 mS/cm, about 50 mS/cm to about 80 mS/cm, about 50 mS/cm to about 70 mS/cm, about 50 mS/cm to about 60 mS/cm, about 60 niS/cni to about 80 mS/cm, or about 60 mS/cm to about 70 mS/cm.

9. The method of claim 7, wherein the conductivity of the solution is increased to about 20 mS/cm, about 25 mS/cm, about 30 mS/cm, about 35 mS/cni, about 40 mS/cm, about 45 mS/cm, about 50 mS/cm, about 55 mS/cm, about 60 mS/cm, about 65 mS/cm, about 70 mS/cm, about 75 mS/cm, or about 80 mS/cnL

10. The method of any one of claims 5-9, wherein the conductivity of the solution is increased by addition of sodium chloride (NaCl).

1 1. The method of claim 10, wherein [NaCl] in the solution is increased to greater than about 200 mM.

12. The method of claim 1 1 , wherein [NaCl] in the solution is increased to about 200 mM to about 800 mM, about 200 mM to about 700 mM, about 200 mM to about 600 mM, about 200 mM to about 500 mM, about 200 mM to about 400 mM, about 200 mM to about 300 mM, about 300 mM to about 800 mM, about 300 mM to about 700 mM, about 300 mM to about 600 mM, about 300 mM to about 500 mM, about 300 mM to about 400 mM, about 400 mM to about 800 mM, about 400 mM to about 700 mM, about 400 mM to about 600 mM, about 400 mM to about 500 mM, about 500 mM to about 800 mM, about 500 mM to about 700 mM, about 500 mM to about 600 mM, about 600 mM to about 800 mM, or about 600 mM to about 700 mM.

13. The method of claim 1 1 , wherein [NaCl] in the solution is increased to about 250 mM, about 300 mM, about 350 mM, about 400 mM, about 450 mM, about 500 mM, about 550 mM, about 600 mM, about 650 mM, about 700 mM, about 750 mM, or about 800 mM.

14. The method of any one of claims 1 -13, wherein the free polypeptide is contacted with a mixed-mode chromatography matrix.

15. The method of claim 14, wherein the mixed-mode chromatography matrix is selected from the group consisting of Capto MMC, Eshmuno HCX, Capto MMC ImpRes, Capto Blue, Nuvia cPrime, Blue Sepharose Fast flow, and Capto Adhere.

16. The method of any one of claims 1-15, wherein the bound polypeptide is washed and eluted with a salt buffer.

17. The method of claim 16, wherein the buffer further comprises an Elution Modifier.

18. The method of claim 17, wherein the Elution Modifier is selected from the group consisting of ethylene glycol, propylene glycol, arginine, lysine, histidine, and mixtures thereof.

19. The method of any one of claims 16-18, wherein the buffer comprises NaCl and arginine, or arginine only.

20. The method of any one of claims 16-19, wherein the wash buffer comprises about 1 M NaCl and about 0.15 M arginine, or about 0.5 M arginine alone.

21. The method of any one of claims 16-20, wherein the elution buffer comprises about 1 M NaCl and about 0.6 M arginine, or about 0.9 M arginine alone.

22. The method of any one of claims 1-13, wherein the cell culture additive is separated from the complex by contacting the complex to a chromatography matrix.

23. The method of claim 22, wherein the chromatography matrix is an anion exchange matrix.

24. The method of claim 23, wherein the anion exchange matrix is a Q Sepharose Fast Flow (QFF) resin, Fractogel TMAE HiCap, Q Sepharose High performance, and Capto Q.

25. The method of any one of claims 22-24, wherein the free polypeptide is contacted with a cation exchange matrix.

26. The method of claim 25, wherein the cation exchange matrix is an SP Sepharose XL (SPXL) resin, SP Sepharose Fast Flow, Capto S, SP Sepharose High Performance, Eshmuno S and Eshmuno CPX.

27. The method of any one of claims 22-26, wherein the bound polypeptide is eluted with a salt buffer.

28. The method of claim 27, wherein the polypeptide is eluted with a step or linear salt gradient.

29. The method of claim 28, wherein the salt gradient is from about 100 mM NaCl to about 1000 mM NaCl, or from about 100 mM NaCl to about 900 mM NaCl, or from 100 mM NaCl to about 800 mM NaCl, or from 100 mM NaCl to about 700 mM NaCl, or from 100 mM NaCl to about 600 mM NaCl,.

30. The method of any one of claims 1-29, wherein the cell culture additive is dextran sulfate.

31. The method of claim 30, wherein the concentration of the dextran sulfate in the solution is about 0.01 g/L to about 10 g/L, about 0.01 g/L to about 9 g/L, about 0.01 g/L to about 8 g/L, about 0.01 g/L to about 7 g/L, about 0.01 g/L to about 6 g/L, about 0.01 g/L to about 5 g/L, about 0.01 g/L to about 4 g/L, about 0.01 g/L to about 3 g/L, about 0.01 g/L to about 2 g/L, about 0.01 g/L to about 1 g/L, about 0.01 g/L to about 0.5 g/L, about 0.1 g/L to about 10 g/L, about 0.1 g/L to about 9 g/L, about 0.1 g/L to about 8 g/L, about 0.1 g/L to about 7 g/L, about 0.1 g/L to about 6 g/L, about 0.1 g/L to about 5 g/L, about 0.1 g/L to about 4 g/L, about 0.1 g/L to about 3 g/L, about 0.1 g/L to about 2 g/L, about 0.1 g/L to about 1 g/L, about 0.1 g/L to about 0.5 g/L, about 0.2 g/L to about 10 g/L, about 0.2 g/L to about 9 g/L, about 0.2 g/L to about 8 g/L, about 0.2 g/L to about 7 g/L, about 0.2 g/L to about 6 g/L, about 0.2 g/L to about 5 g/L, about 0.2 g/L to about 4 g/L, about 0.2 g/L to about 3 g/L, about 0.2 g/L to about 2 g/L, about 0.2 g/L to about 1 g/L, about 0.2 g/L to about 0.5 g/L, about 0.25 g/L to about 10 g/L, about 0.25 g/L to about 9 g/L, about 0.25 g/L to about 8 g/L, about 0.25 g/L to about 7 g/L, about 0.25 g/L to about 6 g/L, about 0.25 g/L to about 5 g/L, about 0.25 g/L to about 4 g/L, about 0.25 g/L to about 3 g/L, about 0.25 g/L to about 2 g/L, about 0.25 g/L to about 1 g/L, about 0.25 g/L to about 0.5 g/L, about 0.5 g/L to about 10 g/L, about 0.5 g/L to about 9 g/L, about 0.5 g/L to about 8 g/L, about 0.5 g/L to about 7 g/L, about 0.5 g/L to about 6 g/L, about 0.5 g/L to about 5 g/L, about 0.5 g/L to about 4 g/L, about 0.5 g/L to about 3 g/L, about 0.5 g/L to about 2 g/L, or about 0.5 g/L to about 1 g/L.

32. The method of any one of claims 1 -31 , wherein the solution is a harvested cell culture fluid (HCCF).

33. The method of any one of claims 1 -32, wherein the polypeptide is recombinantly expressed in a cell culture.

34. The method of claim 33, wherein the polypeptide is expressed in a eukaryotic cell culture.

35. The method of claim 34, wherein the eukaryotic cell is selected from the group consisting of CHO cell, HEK 293 cell, NSO cell, PER C6 cell, HeLa cell, and MDCK cell.

36. The method of claim 35, wherein the eukaryotic cell is CHO cell.

37. The method of any one of claims 1 -36, wherein the polypeptide is selected from the group consisting of: an antibody, a Transforming Growth Factor (TGF) beta superfamily signaling molecule, an Fc fusion protein, a therapeutic enzyme, a recombinant vaccine, and a clotting factor.

38. The method of claim 37, wherein the polypeptide is a TGF-beta superfamily signaling molecule.

39. The method of claim 38, wherein the TGF-beta superfamily signaling molecule is Neublastin.