WO2014110440A1

WO2014110440A1 - Methods for improved production and recovery of recombinant proteins from eukaryotic cell cultures

Info

Publication number: WO2014110440A1
Application number: PCT/US2014/011139
Authority: WO
Inventors: Alan Gilbert; William C. YANG; Yao-ming HUANG; Jiuyi LU
Original assignee: Biogen Idec Ma Inc.
Priority date: 2013-01-10
Filing date: 2014-01-10
Publication date: 2014-07-17
Also published as: EP2943582A1

Abstract

The present disclosure is directed to methods to improve the yields of proteins recombinantly produced in eukaryotic cells comprising the addition of anionic substances such as valproate, malate, succinate, fumarate, citrate (e.g., ferric citrate), polyanionic compounds (e.g., polysulfated compounds such dextran sulfate or polyvinyl sulfate), and combinations thereof, wherein the addition of such compounds reduces the binding of recombinant proteins to the cell surface, therefore increasing the amount of soluble protein that can recovered from the culture medium. In addition, the protein yields can be improved according to the disclosed methods by preventing or reducing cell growth inhibition caused by the binding of the expressed recombinant proteins (e.g., neublastin, or antibodies or antigen-binding fragments thereof) to the cell surface. The disclosed anionic substances can also be used during the harvesting phase of a protein production process to release the expressed recombinant proteins from the cell surface prior to protein purification.

Description

METHODS FOR IMPROVED PRODUCTION AND RECOVERY OF RECOMBINANT PROTEINS FROM EUKARYOTIC CELL CULTURES

BACKGROUND

[0001] Recombinant protein production is the preferred method for producing proteins from medical or industrial uses. To maximize protein yields, it is crucial to optimize the protein production process. One problem encountered when using mammalian cells as production hosts is that recombinant proteins secreted to the medium often adhere to the cell membrane. This binding of the recombinant product to the cell membrane can influence the production yield by, e.g. , reducing protein titer in the medium and in certain cases having deleterious effects on cellular growth. When recombinant proteins are produced in culture media containing additives such animal serum or other protein preparations, the proteins and peptides in these additives tend to bind to nonspecific binding sites on the cell surface, minimizing the binding of recombinant protein product to the cell surface. However, to minimize contamination from animal proteins, most manufacturing methods for therapeutic recombinant proteins use low protein, no protein, or serum free cell culture media. Under these cell culture conditions, nonspecific protein binding to cell membrane tends to be higher, significantly hindering protein production by, for example, decreasing cell growth rates and cell viability, and/or decreasing the recovery of recombinant protein.

[0002] Nonspecific binding of recombinant proteins to cell surface membranes can involve several physicochemical mechanisms. Proteins bind to the membranes phospholipid bilayer as a result of hydrophobic interactions between the bilayer and exposed nonpolar residues at the surface of the protein. Nonspecific binding can also be the result of protein-protein and/or protein-lipid electrostatic interactions. For example, any positively charged protein will be attracted to a negatively charged membrane by nonspecific electrostatic interactions. In some cases, the release of the recombinant protein from the cell membranes can be increased by modifying the culture medium. Such modifications can be physiological, or non-physiological increases in salt concentration, e.g., NaCl accompanied by the addition detergent and/or by adjusting a specific pH can in some cases release the bound proteins. [0003] Some of the methods described in the literature require, for example, the use of sorbitol and high salt concentrations (see, e.g., Berman et al, Mol. Cell Biol. Res. Com. 4:337-344 (2001)); detergents and high salt concentrations (see, e.g., Vaandrager et al, J. Biol. Chem. 271 : 7025-7029 (1996)); low pH and high salt concentrations (see, e.g. , Denys et al, Biochem J., 336: 689-697 (1998); Zuber et al , J. Cell Physiology 170:217- 227 (1997)); organic solvents such as butanol and detergents (see, e.g., Grass et al , Infection and Immunity 72:219-228 (2004)); or high salt concentrations (see., e.g. , Mounier et al, J. Biol. Chem. 279:25024-25038 (2004)). Ionic substances known to facilitate the release of proteins bound to the cell surface include NH₄Acetate, MgCl₂, KH₂P0₄, Na₂S0₄, KC1, CaCl₂, amino acids, or mixtures of peptides and/or amino acids (see, e.g. , Intl. Publ. No. WO2006103258)

[0004] Neurturin, persephin, and neublastin (also known as artemin and enovin) comprise the GDNF ligand family of neurotrophic factors. The most recently discovered GDNF ligand family member is neublastin, which promotes the outgrowth and survival of neurons of the peripheral and central nervous system (Baudet et al, Development, 127:4335 (2000); Rosenblad et al, Mol. Cell. Neurosci. 15: 199 (2000)). These proteins contain a high number of positively charged amino acid residues, mostly arginines, and isoelectric points ranging from about 9 for GDNF to about 12 for neublastin and neurturin.

[0005] In general, and specifically for the recombinant production of therapeutic proteins

(including the GDNF ligand family of neurotrophic factors, antibodies, clotting factors, etc.), it is essential for the economy of the product to optimize the manufacturing process to achieve a high productivity. Thus, it is apparent that methods to increase the recovery of recombinant proteins in eukaryotic or mammalian expression systems are highly desirable, particularly methods for serum-free production of proteins which are needed for medical applications.

BRIEF SUMMARY

[0006] The present disclosure provides methods to improve the production of recombinant proteins in eukaryotic cells. These methods comprise the addition of anionic substances such as (i) carboxylic acids, for example, valproate (e.g., sodium valproate), malate, succinate, fumarate, citrate {e.g. , ferric citrate or sodium citrate), (ii) polyanionic 2014/011139

- 3 - compounds (e.g. , polysulfated compounds such dextran sulfate or polyvinyl sulfate), and (iii) combinations thereof, wherein the addition of such compounds reduces the binding of recombinant proteins to the cell surface. In turn, the reduced protein binding to the cell surface increases the amount of soluble protein that can be recovered from the culture medium. In addition, protein yields can be improved according to the disclosed methods by preventing or reducing cell growth inhibition caused by the binding of the expressed recombinant proteins (e.g., neublastin or antibodies or antigen-binding fragments thereof) to the cell surface.

[0007] The disclosed anionic substances can also be applied after cell culture, for example during the harvesting phase of a recombinant protein's production process, to release the expressed recombinant proteins from the cell surface prior to protein purification. In some aspects, protein attachment to different surfaces can be reduced or protein release can be effected during protein purification steps (e.g. , during chromatography) by employing the disclosed anionic substances and combinations thereof.

[0008] The present disclosure provides a method for increasing recombinant protein recovery from eukaryotic cells, comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from valproate, malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to increase recovery of the recombinant protein relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance. In some aspects, recovery of recombinant protein is increased by at least about 10% to at least about 100% relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

{0009] Also provided is a method for increasing recombinant protein production from eukaryotic cells, comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from valproate, malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to increase production of the recombinant protein relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance. In some aspects, production of recombinant protein is increased by at least about 10% to at least about 50% relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0010] The present disclosure also provides a method for decreasing recombinant protein attachment to the surface of eukaryotic cells comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from valproate, malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to decrease attachment of the recombinant protein to the eukaryotic cells' surface relative to the attachment of the same recombinant protein to eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance. In some aspects, attachment of recombinant protein to the eukaryotic cells' surface is decreased by at least about 10% to at least about 90% relative to the attachment of the same recombinant protein to eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0011] Also provided is a method for reducing recombinant protein-induced inhibition of eukaryotic cell growth comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from valproate, malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to reduce recombinant protein-induced growth inhibition relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance. In some aspects, growth inhibition by the recombinant protein is decreased by at least about 9

- 5 -

7% to at least about 21% relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0012] The present disclosure also provides a method for increasing the viability of eukaryotic cells expressing a recombinant protein comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein, arid (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from valproate, malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to increase eukaryotic cell viability relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in chemically defined cell culture medium but not subjected to the at least one anionic substance. In some aspects, the viability of eukaryotic cells expressing a recombinant protein is increased by at least about 5% relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0013] In some aspects of the methods disclosed above, the eukaryotic cells are mammalian eukaryotic cells. In other aspects, the mammalian eukaryotic cells are CHO cells or HEK293 cells. In some aspects, the mammalian eukaryotic cells are Chinese Hamster Ovary (CHO) cells. In specific aspects, the CHO cells are CHO DG44. In some aspects, the cell culture medium has less than 10% of mammalian serum (by volume). In other aspects, the cell culture medium contains no more than about 1% mammalian serum by volume. In other aspects, the cell culture medium is serum free. In other aspects, the cell culture medium is protein free.

[0014] In some aspects of the methods disclosed above, the recombinant protein is a

GDNF ligand family protein. In other aspects, the GDNF ligand family protein is selected from the group consisting of GDNF, neublastin, neurturin, persephin, or a fragment or variant thereof. In certain aspects, the GDNF family ligand is neublastin or a fragment or variant thereof. In some aspects, the neublastin or a fragment or variant thereof is human neublastin. In other aspects, the recombinant protein is a basic protein. In certain aspects, the basic protein has a pi of at least 10. In some aspects, the basic protein has a pi of at least 1 1. In some aspects, the basic protein comprises at least 10% basic amino acids. In other aspects, the basic protein is an arginine rich protein. In some aspects, the arginine rich protein comprises at least 12% arginine amino acids.

[0015] In other aspects of the methods disclosed above, the polyanionic compound is a polysulfated or a polysulfonated compound. In some aspects, the polysulfate compound is a polysulfated saccharide. In other aspects, the polysulfated saccharide is a dextran sulfate. In other aspects, the dextran sulfate has an average molecular weight of about 5,000 Dalton. In some aspects, the polysulfated compound is polyvinyl sulfate. In other aspects, the citrate is sodium citrate or ferric citrate. In other aspects, the concentration of ferric citrate is in a range from about ImM to about lOOmM. In some aspects, the concentration of ferric citrate is about 50 mM. In some aspects, the ferric citrate is added to raise its concentration to at least 2mM. In other aspects, the concentration of sodium citrate is in a range from about ImM to about lOOmM. In some aspects, the concentration of sodium citrate is about 100 mM. In other aspects, sodium citrate is added to raise its concentration to at least 2mM. In some aspects, the concentration of succinate is in a range from about ImM to about l OOmM. In other aspects, the concentration of succinate is about 100 mM. In some aspects, succinate is added to raise its concentration to at least 2mM. In some aspects, the concentration of fumarate is in a range from about ImM to about lOOmM. In other aspects, the concentration of fumarate is about 100 mM. In some aspects, fumarate is added to raise its concentration to at least 2mM. In some aspects, the concentration of malate is in a range from about ImM to about lOOmM. In other aspects, the concentration of malate is about 100 mM. In some aspects, malate is added to raise its concentration to at least 2 mM.

[0016] In other aspects, the concentration of polysulfated compound is in a range from about O.Olg/L to about lg/L. In some aspects, the polysulfated compound is dextran sulfate. In other aspects, the polysulfated compound is polyvinyl sulfate. In some aspects, the concentration of dextran sulfate is about 0.1 g/L. In other aspects, dextran sulfate is added to raise its concentration to at least 0.25g/L. In some aspects, the anionic substance comprises a polyanionic compound and a citrate. In other aspects, the polyanionic compound is dextran sulfate and the citrate is ferric citrate.

[0017] In some aspects of the methods disclosed above, the anionic substance is added during the induction phase but not during the proliferation phase. In other aspects, the concentration of anionic substance is kept constant during cultivation. In some aspects, the concentration of anionic substance is increased or decreased during cultivation. In other aspects, the anionic substance is added 1 to 4 weeks prior to the separation of the recombinant protein. In some aspects, the anionic substance is added 1 to 7 days prior to the separation of the recombinant protein. In other aspects, the anionic substance is added 1 to 24 hours prior to the separation of the recombinant protein. In some aspects, the anionic substance is added 1 to 3 hours prior to the separation of the recombinant protein. In other aspects, the anionic substance is added 1 to 60 minutes prior to the separation of the recombinant protein. In some aspects, the eukaryotic cells are grown and maintained at a density of at least 10⁵ cells per ml. of culture medium. In other aspects, the anionic substance is added to the cell culture medium at inoculation and/or during the production phase. In some aspects, the eukaryotic cells are grown in a fed batch process. In other aspects, the eukaryotic cells are grown in a continuous process.

[0018] In some aspects of the methods disclosed above, the culture medium further contains a non-physiological concentration of an ionic substance selected from the group consisting of NH₄ acetate, MgCl₂, KH₂P0₄, NaS0₄, KC1, CaCl₂, an amino acid, and a mixture of peptides and/or amino acids. In some aspects eukaryotic cells are grown under hyperosmolar conditions. In other aspects, the osmolarity of the cell culture medium is between about 250 and about 600 mOsm. In some aspects, the anionic substance or combination thereof is added to reach the equilibration balance within recombinant protein and eukaryotic cell surface, sufficient to disrupt ionic binding and release bound recombinant proteins from the eukaryotic cell surface without destroying the cell. In some aspects, at last two anionic substances are added.

[0019] In some aspects, the methods disclosed above further comprise at least one of the following steps: (a) isolating the recombinant protein from the culturing medium; (b) separating the culture medium from the cultivated eukaryotic cells, resulting in two separate fractions, a fraction of cultivated eukaryotic cells and a fraction of liquid medium; (c) contacting or resuspending the fraction of cultivated eukaryotic cells with a release composition comprising a sufficient amount of at least one anionic substance selected from malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof to release the recombinant protein from the eukaryotic cell surface; (d) separating the release composition from the eukaryotic cells, resulting in two separate fractions, a fraction of eukaryotic cells and a fraction of release composition comprising the recombinant protein released from the eukaryotic cell surface; (e) isolating the recombinant protein from the fraction of release composition; and, (f) suspending the fraction of eukaryotic cells in culture medium and reculturing. In some aspects, the separation of the culture medium or the release in steps (d) or (f) composition from the cultivated cells comprises at least a technique selected from the group consisting of centrifugation, filtration, diafiltration, tangential filtration, dead end filtration, micro filtration, electrical fields, magnetic fields, and ultrafiltration. In some aspects, the isolation of the recombinant protein in steps (c) or (g) comprises at least a technique selected from the group consisting of immuno-affinity chromatography, affinity chromatography, protein precipitation, buffer exchanges, ionic exchange chromatography, hydrophobic interaction chromatography, mixed mode hydrophobic/ion exchange chromatography media, chelating chromatography, carbohydrate affinity like lectin or heparin affinity chromatography, size-exclusion chromatography, electrophoresis, dialysis, different precipitation agents such as polyethylene glycol, ammonium sulfate, ethanol, hydroxyl apatite adsorption, and filter membrane adsorption.] In some aspects, the methods disclosed above comprise collecting the recombinant protein. The present disclosure also provides a recombinant protein obtained by any of the methods disclosed herein. Also provided is a pharmaceutical composition comprising a recombinant protein obtained by any one of the methods disclosed herein, and a pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition comprises neublastin, a neublastin fragment, or a variant thereof.

] Also provided is a method for increasing recombinant protein recovery from eukaryotic cells, comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound, to increase recovery of the recombinant protein relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined cell culture medium but not subjected to valproate and the at least one polyanionic compound. In some aspects, recovery of recombinant protein is increased by at least about 10% to at least about 100% relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound..

[0022] Also provided is method for increasing recombinant protein production from eukaryotic cells, comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound, to increase production of the recombinant protein relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound, in some aspects, production of recombinant protein is increased by at least about 10% to at least about 50% relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

[0023J Also provided is a method for decreasing recombinant protein attachment to the surface of eukaryotic cells comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound, to decrease attachment of the recombinant protein to the eukaryotic cells' surface relative to the attachment of the same recombinant protein to eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound . In some aspects, attachment of recombinant protein to the eukaryotic cells' surface is decreased by at least about 10% to at least about 90% relative to the attachment of the same recombinant protein to eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

[0024] Also provided is method for reducing recombinant protein-induced inhibition of eukaryotic cell growth comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound to reduce 9

- 10 - recombinant protein-induced growth inhibition relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound. In some aspects, growth inhibition be recombinant protein is decreased by at least about 7% to at least about 21% relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

[0025] Also provided is method for increasing the viability of eukaryotic cells expressing a recombinant protein comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound to increase eukaryotic cell viability relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound. In some aspects, the viability of eukaryotic cells expressing a recombinant protein is increased by at least about 5% relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

[0026] In some aspects, valproate and at least one polyanionic compound are applied to cell cultures comprising a chemically defined medium where the eukaryotic cells are mammalian eukaryotic cells. In some aspects, the mammalian eukaryotic cells are CHO cells or HEK293 cells. In some aspects the chemically defined cell culture medium has less than 10% of mammalian serum (by volume). In other aspects, the chemically defined cell culture medium is serum free. In some aspects, the chemically defined cell culture medium is protein free.

[0027] In some aspects, valproate is applied to cell cultures with at least one polyanionic compound, wherein the polyanionic compound is a polysulfated or a polysultbnated compound. In some aspects, the polysulfate compound is a polysulfated saccharide, In some aspects, the polysulfated saccharide is a dextran sulfate. In some aspects, the dextran sulfate has an average molecular weight of about 5,000 Dalton. In some aspects, the valproate is sodium valproate. In some aspects, the concentration of sodium valproate is in a range from about ImM to about l OOmM. In some aspects, the concentration of sodium valproate is lower than 1 mM. In some aspects, the concentration of sodium valproate is about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some aspects, sodium valproate is added to raise its concentration to at least 2mM.

[0028] In some aspects, valproate is applied to cell cultures with at least one polyanionic compound, wherein the polyanionic compound is a polysulfated compound in a concentration range from about 0.01 g/L to about 1 g/L. In some aspects, the polysulfated compound is dextran sulfate. In some aspects, dextran sulfate is added to raise its concentration to at least 0.25 g/L. In some aspects, dextran sulfate is added to raise its concentration to at least 1 g/L.

[0029] In some aspects, the anionic substance comprising valproate and at least one polyanionic compound (e.g., dextran sulfate) is (i) added during the induction phase but not during the proliferation phase; and/or (ii) added to the chemically defined cell culture medium at inoculation and/or during the production phase; and/or (iii) added 1 to 4 weeks, 1 to 7 days, 1 to 24 hours, 1 to 3 hours, or 1 to 60 minutes prior to the separation of the recombinant protein. In some aspects, the concentration of anionic substance is (i) kept constant during cultivation, or (ii) increased or decreased during cultivation.

[0030] In some aspects, the anionic substance comprising valproate and at least one polyanionic compound (e.g., dextran sulfate) is added to eukaryotic cells that are grown in a fed batch process or in a continuous process. In some aspects, the eukaryotic cells are grown and maintained at a density of at least 10⁵ cells per ml. of chemically defined culture medium. In some aspects, the chemically defined culture medium further contains a non-physiological concentration of an ionic substance selected from the group consisting of NH₄ acetate, MgCl₂, K¾P0₄, NaS0₄, KC1, CaCl₂, an amino acid, and a mixture of peptides and/or amino acids. In some aspects, the at least one anionic substance or combination thereof is added to reach the equilibration balance within recombinant protein and eukaryotic cell surface, sufficient to disrupt ionic binding and release bound recombinant proteins from the eukaryotic cell surface without destroying the cell.

[0031 ] In some aspects, the methods comprising subjecting cells to a sufficient amount of at least one anionic substance comprising valproate and at least one polyanionic compound disclosed herein, further comprise at least one of the following steps: (c) isolating the recombinant protein from the culturing medium; (d) separating the culture medium from the cultivated eukaryotic cells, resulting in two separate fractions, a fraction of cultivated eukaryotic cells and a fraction of liquid medium; (e) contacting or resuspending the fraction of cultivated eukaryotic cells with a release composition comprising a sufficient amount of at least one anionic substance comprising valproic acid and a polyanionic compound, and combinations thereof to release the recombinant protein from the eukaryotic cell surface; (f) separating the release composition from the eukaryotic cells, resulting in two separate fractions, a fraction of eukaryotic cells and a fraction of release composition comprising the recombinant protein released from the eukaryotic cell surface; (g) isolating the recombinant protein from the fraction of release composition; and, (h) suspending the fraction of eukaryotic cells in culture medium and reculturing. In some aspects, the separation of the culture medium or the release in steps (d) or (f) composition from the cultivated cells comprises at least a technique selected from the group consisting of centrifugation, filtration, diafiltration, tangential filtration, dead end filtration, micro filtration, electrical fields, magnetic fields, and ultrafiltration. In some aspects, the isolation of the recombinant protein in steps (c) or (g) comprises at least a technique selected from the group consisting of immuno-affinity chromatography, affinity chromatography, protein precipitation, buffer exchanges, ionic exchange chromatography, hydrophobic interaction chromatography, mixed mode hydrophobic/ion exchange chromatography media, chelating chromatography, carbohydrate affinity like lectin or heparin affinity chromatography, size-exclusion chromatography, electrophoresis, dialysis, different precipitation agents such as polyethylene glycol, ammonium sulfate, ethanol, hydroxyl apatite adsorption, and filter membrane adsorption. In some aspects, the method(s) further comprise collecting the recombinant protein. The present disclosure also provides recombinant protein obtained by any of the methods disclosed herein comprising subjecting cultured cells to a sufficient amount of valproate and at least one polyanionic compound. Also provided are pharmaceutical compositions comprising at least one recombinant protein (e.g., neublastin or an antibody or antigen- binding fragment thereof) obtained by any one of the methods disclosed herein comprising subjecting cultured cells to a sufficient amount of valproate and at least one polyanionic compound, and a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES FIG. l shows the neublastin titer in samples comprising either neublastin- expressing 902 cells or N65 cells in cell culture medium after treating the cells with an additional amount of neublastin (Drug Substance; DS) to a 42 mg/L final concentration (except control samples 1 and 21). Samples were also incubated with various anionic substances (except control samples 2 and 22). The assayed conditions were: (1) 902 cells with 50 mM Na₂S0₄ without addition of neublastin, and without addition of anionic substance; (2) 902 cells with added neublastin, but without treatment with anionic substance; (3) 902 cells with added neublastin, treated with 5 mg/ml Arginine, (4) 902 cells with added neublastin, treated with 50 mM NaCl, 5mM Tris pH 7; (5) 902 cells with added neublastin, treated with 100 mM malate; (6) 902 cells with added neublastin, treated with 100 mM succinate; (7) 902 cells with added neublastin, treated with 50 mM Na₂S0₄, 5 mM Tris pH 7; (8) 902 cells with added neublastin, treated with 50 mM Na₂S0₄, 5 mM Tris pH 7, at 36°C; (9) 902 cells with added neublastin, treated with 50 mM Na₂S0₄; (10) 902 cells with added neublastin, treated with 100 mM fumarate; (1 1) 902 cells with added neublastin, treated with 50 mM K₂S0₄, 5 mM Tris pH 7; (12) 902 cells with added neublastin, treated with 50 mM ferric citrate; (13) 902 cells with added neublastin, treated with 100 mM sodium citrate, pH 7; (14) 902 cells with added neublastin, treated with 0.01 g/L dextran sulfate; (15) 902 cells with added neublastin, treated with 0.1 g/L dextran sulfate; (16) 902 cells with added neublastin, treated with lg/L dextran sulfate; (17) 902 cells with added neublastin, treated with 1 mM citrate; (18) 902 cells with added neublastin, treated with 10 mM citrate; (19) 902 cells with added neublastin, treated with 100 mM citrate; (20) 902 cells; (21) NS65 cells without addition of neublastin, and without addition of anionic substance; (22) N65 cells with added neublastin, but without treatment with anionic substance; (23) N65 cells with added neublastin, treated with 0.01 g/L dextran sulfate; (24) N65 cells with added neublastin, treated with 0.1 g/L dextran sulfate; (25) N65 cells with added neublastin, treated with l g/L dextran sulfate; (26) N65 cells with added neublastin, treated with ImM citrate; (27) N65 cells with added neublastin, treated with lOmM citrate; and (28) N65 cells with added neublastin, treated with lOOmM citrate. Cells were incubated with neublastin in medium supplemented with anionic substances at the concentrations above. The cells/neublastin/medium mixtures were all diluted by 10% using a concentrated stock 2014/011139

- 14 - solution of lOx anionic substance. Accordingly, the 50 mM K₂S0₄, 5 mM Tris pH 7 treatment was accomplished, for example, by adding 500 mM K₂S0₄, 50 mM Tris pH 7 at 10% of the final volume of cells plus the buffer.

[0033] FIG. 2A shows the viable cell density of N65 cells which were untreated (neat) received a 1% addition of empty drug substance buffer, or received at 1% addition of drug substance (neublastin). FIG. 2B shows the viable cell density of 902 cells which were untreated (neat) received a 1% addition of empty drug substance buffer, or received at 1% addition of drug substance (neublastin).

[0034] FIG. 3 shows viable cell density of neublastin expressing 902 cells supplemented with a 0%, 1%, 3%, or 5% neublastin addition by volume. Cells were cultivated for 3 days in basal medium containing ferric citrate, dextran sulfate, or dextran sulfate and ferric citrate. The black bar represents viable cell density after addition of 1 % drug substance without any anionic substance additives.

[0035] FIG. 4 shows viable cell density of neublastin expressing 902 cells maintained in medium containing ferric citrate or dextran sulfate with or without added neublastin (NBN). Also shown are viable cell densities for control samples maintained in medium without anionic substance additives (dextran sulfate or ferric citrate) (positive control: no anionic substance additives, no added neublastin; negative control: no anionic substance additives, added neublastin).

[0036] FIG. 5 shows viable cell density of neublastin expressing 902 cells maintained in fed batch mode for 17 days. Cultures contained (i) platform medium alone (control); (ii) basal medium enriched with 2mM ferric citrate, (iii) basal medium enriched with 2mM ferric citrate and feed medium enriched with 2.4mM ferric citrate, or (iv) basal medium enriched with 2mM ferric citrate and 0.25 g/L dextran sulfate.

[0037] FIG. 6 shows relative titer of neublastin produced using 902 cells in the presence of dextran sulfate. 902 cells were cultivated in CM3 basal medium and operated in fed batch mode for 13 days. Fed batch medium contained either 0 g/L, 0.25 g/L, 1.25 g/L, or 2.5 g/L dextran sulfate. Titer was measured by ELISA using a proprietary antibody specific for neublastin.

[0038] FIG. 7 shows relative titer of neublastin produced using 902 cells maintained in fed batch mode in bioreactors for 17 days. The titer is relative to the titer produced by 902 cells cultivated in CM3 basal medium. Cultures in CM3 basal medium enriched with 2.3mM ferric citrate in basal medium produce higher titers. Relative titer from cultures treated with 100 mM sodium citrate at harvest are also reported as anionic treated titer.

[0039] FIG. 8 shows the effect of valproate (VPA) on cell line A growth (FIG. 8A), viability (FIG. 8B), harvest titer (FIG. 8C), and titer v. IVC (FIG. 8D). · = Control;■ I mM VPA; A= 2mM VPA;▼ = 3mM VPA.

[0040] FIG. 9 shows the effect of VPA on cell line B growth (FIG. 9A), viability (FIG.

9B), harvest titer (FIG. 9C), and titer v. IVC (FIG. 9D). · = Control;■ = ImM VPA; A= 2mM VPA;▼ - 3mM VPA.

[0041] FIG. 10 shows the synergism between VPA concentration and day of addition for cell line A. Concentration and day of addition were not statistically significant individually accordingly to the factorial experiment, but the two-factor interaction was significant (p<0.03). The modeled red lines show that higher concentrations of VPA added later in the culture duration yield higher titers.

[0042] FIG. 1 1 shows that VPA increases harvest titer in cell line A fed-batch bioreactors. Effect of VPA on cell line A growth (FIG. 1 1 A), viability (FIG. 1 I B), harvest titer (FIG. 1 1 C), and titer v. IVC (FIG. 1 ID). 1 g/L dextran sulfate was added on Day 0 and 3.5mM VPA was added on Day 9. · = Control;■ = DS; A = VPA; ▼ = VPA + dextran sulfate.

[0043] FIG. 12 shows that VPA increases harvest titer in cell line B fed-batch bioreactors. Effect of VPA on cell line A growth (FIG. 12 A), viability (FIG. 12B), harvest titer (FIG. 12C), and titer v. IVC (FIG. 12D). 1 g/L dextran sulfate was added on Day 9 and 3.5mM VPA was added on Day 12. ® = Control;▼ = VPA + dextran sulfate.

[0044] FIG. 13 shows the structures of exemplary monocarboxylic (valproic), dicarboxylic (fumaric, malic, succinic, tartaric, maleic), and tricarboxylic acids (citric, isocitric).

DETAILED DESCRIPTION

[0045] The present disclosure provides methods to improve the recombinant production of proteins in eukaryotic cells. These methods comprise the addition of anionic substances such as (i) carboxylic acids, for example, valproate (e.g., sodium valproate), malate, succinate, fumarate, citrate (e.g., ferric citrate or sodium citrate), (ii) polyanionic compounds (e.g. , polysulfated compounds such dextran sulfate or polyvinyl sulfate), and T/US2014/011139

- 16 -

(iii) combinations thereof, to cell expressing recombinant proteins wherein the addition of such compounds reduces the binding of the recombinant proteins to the cell surface. The release of membrane-bound recombinant protein increases the amount of soluble protein that can subsequently be recovered. In addition, protein yields can be improved according to the disclosed methods by preventing or reducing cell growth inhibition caused by the binding of the expressed recombinant protein (e.g., neublastin or an antibody or antigen- binding fragment thereof) to the cell surface. The disclosed anionic substances can be applied not only during cell culture, but also after cell culture, for example during the harvesting phase of a recombinant protein's manufacturing process, to release the expressed recombinant proteins from the cell surface prior to protein purification.

Definitions

[0046] It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. The terms "a" (or "an"), as well as the terms "one or more," and "at least one" can be used interchangeably herein.

[0047] 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).

[0048] 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.

[0049] 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 Biornedicine 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_f [0050] 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 or 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. Amino acids are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, are referred to by their commonly accepted single-letter codes.

[0051] The term "sequence" as used to refer to a protein sequence, a peptide sequence, a polypeptide sequence, or an amino acid sequence means a linear representation of the amino acid constituents in the polypeptide in an amino-terminal to carboxyl-terminal direction in which residues that neighbor each other in the representation are contiguous in the primary structure of the polypeptide.

[0052] By a "protein" or "polypeptide" is meant any sequence of two or more amino acids linearly linked by amide bonds (peptide bonds) regardless of length, post-translation modification, or function. As used herein, the term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides." "Polypeptide," "peptide," and "protein" are used interchangeably herein. Thus, peptides, dipeptides, tripeptides, or oligopeptides are included within the definition of "polypeptide," and the term "polypeptide" can be used instead of, or interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids.

[0053] A polypeptide can be derived from a natural biological source or produced by recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. A polypeptide can be generated in any manner, including by chemical synthesis. Also included as polypeptides of the present invention are fragments, derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. [0054] The term "fragment" when referring to polypeptides and proteins of the present invention include any polypeptides or proteins which retain at least some of the properties of the reference polypeptide or protein. For example, in the case of GDNF ligand family proteins, e.g., neublastin, the term fragment would refer to any polypeptide or protein which retains at least some of the neurotrophic properties of the reference polypeptide or protein. In the case of antibodies, the term fragment would refer to any polypeptide of protein which retains the antigen-binding specificity of the parent antibody. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments.

[0055] The term "variant" as used herein refers to a polypeptide sequence that differs from that of a parent polypeptide sequence by virtue of at least one amino acid modification. Variants can occur naturally or be non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions, or additions.

[0056] "Derivatives" of polypeptides or proteins of the invention are polypeptides or proteins which have been altered so as to exhibit additional features not found on the native polypeptide or protein. Also included as "derivatives" are those peptides that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. A polypeptide or amino acid sequence "derived from" a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence.

[0057] Polypeptides derived from another peptide can have one or more mutations relative to the starting polypeptide, e.g. , one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. In some aspects, the polypeptide comprises an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting polypeptide. In some aspects, the variant will have an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g. , over the length of the variant molecule. In one aspect, there is one amino acid difference between a starting polypeptide sequence and the sequence derived therefrom. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e. same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity.

[0058] A polypeptide which is "isolated" is a polypeptide which is in a form not found in nature. Isolated polypeptides include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some aspects, a polypeptide which is isolated is substantially pure.

[0059] A "recombinant" polypeptide or protein refers to a polypeptide or protein produced via recombinant DNA technology. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable teclmique. The polypeptides disclosed herein, e.g., GNDF ligand family proteins such as neublastin, or antibodies and antigen- binding fragment thereof, can be recombinantly produced using methods known in the art. Alternatively, the proteins and peptides disclosed herein can be chemically synthesized.

[0060] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g. , lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g. , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. , threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another aspect, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

[0061] Non-conservative substitutions include those in which (i) a residue having an electropositive side chain (e.g. , Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g. , Glu or Asp), (ii) a hydrophilic residue (e.g. , Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g. , Ala, Leu, He, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, lie or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala, Ser) or no side chain (e.g. , Gly).

[0062] Other substitutions can be readily identified by workers of ordinary skill. For example, for the amino acid alanine, a substitution can be taken from any one of D- alanine, glycine, beta-alanine, L-cysteine and D-cysteine. For lysine, a replacement can be any one of D-lysine, arginine, D-arginine, homo-arginine, methionine, D-methionine, ornithine, or D- ornithine. Generally, substitutions in functionally important regions that may be expected to induce changes in the properties of isolated polypeptides are those in which: (i) a polar residue, e.g., serine or threonine, is substituted for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, or alanine; (ii) a cysteine residue is substituted for (or by) any other residue; (iii) a residue having an electropositive side chain, e.g., lysine, arginine or histidine, is substituted for (or by) a residue having an electronegative side chain, e.g., glutamic acid or aspartic acid; or (iv) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having such a side chain, e.g., glycine. The likelihood that one of the foregoing non-conservative substitutions may alter functional properties of the protein is also correlated to the position of the substitution with respect to functionally important regions of the protein: some non-conservative substitutions may accordingly have little or no effect on biological properties.

[0063] The term "percent sequence identity" between two polypeptide or polynucleotide sequences refers to the number of identical matched positions shared by the sequences over a comparison window, taking into account additions or deletions (i.e. , gaps) that must be introduced for optimal alignment of the two sequences. A matched position is any position where an identical nucleotide or amino acid is presented in both the target and reference sequence. Gaps presented in the target sequence are not counted since gaps are not nucleotides or amino acids. Likewise, gaps presented in the reference sequence are not counted since target sequence nucleotides or amino acids are counted, not nucleotides or amino acids from the reference sequence.

[0064J The percentage of sequence identity is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity. The comparison of sequences and determination of percent sequence identity between two sequences may be accomplished using readily available software both for online use and for download. Suitable software programs are available from various sources, and for alignment of both protein and nucleotide sequences. One suitable program to determine percent sequence identity is bl2seq, part of the BLAST suite of program available from the U.S. government's National Center for Biotechnology Information BLAST web site (blast.ncbi.nlm.nih.gov). B12seq performs a comparison between two sequences using either the BLASTN or BLASTP algorithm. BLASTN is used to compare nucleic acid sequences, while BLASTP is used to compare amino acid sequences. Other suitable programs are, e.g. , Needle, Stretcher, Water, or Matcher, part of the EMBOSS suite of bioinformatics programs and also available from the European Bioinformatics Institute (EBI) at www.ebi.ac.uk/Tools/psa.

[0065] Different regions within a single polynucleotide or polypeptide target sequence that aligns with a polynucleotide or polypeptide reference sequence can each have their own percent sequence identity. It is noted that the percent sequence identity value is rounded to the nearest tenth. For example, 80.1 1, 80.12, 80.13, and 80.14 are rounded down to 80.1, while 80.15, 80.16, 80.17, 80.18, and 80.19 are rounded up to 80.2. It also is noted that the length value will always be an integer.

[0066] In certain aspects, the percentage identity "X" of a first amino acid sequence to a second sequence amino acid is calculated as 100 x (Y/Z), where Y is the number of amino acid residues scored as identical matches in the alignment of the first and second sequences (as aligned by visual inspection or a particular sequence alignment program) and Z is the total number of residues in the second sequence. If the length of a first sequence is longer than the second sequence, the percent identity of the first sequence to the second sequence will be higher than the percent identity of the second sequence to the first sequence.

[0067] One skilled in the art will appreciate that the generation of a sequence alignment for the calculation of a percent sequence identity is not limited to binary sequence- sequence comparisons exclusively driven by primary sequence data. Sequence alignments can be derived from multiple sequence alignments. One suitable program to generate multiple sequence alignments is ClustalW2, available from www.clustal.org. Another suitable program is MUSCLE, available from www.drive5.com/muscle/. ClustalW2 and MUSCLE are alternatively available, e.g. , from the EBI.

[0068] It will also be appreciated that sequence alignments can be generated by integrating sequence data with data from heterogeneous sources such as structural data (e.g. , cry stallo graphic protein structures), functional data (e.g., location of mutations), or phylogeiietic data. A suitable program that integrates heterogeneous data to generate a multiple sequence alignment is T-Coffee, available at www.tcoffee.org, and alternatively available, e.g. , from the EBI. It will also be appreciated that the final alignment used to calculate percent sequence identity may be curated either automatically or manually.

[0069] "About," as used herein for a range, modifies both ends of the range. Thus, "about

10-20" means "about 10 to about 20."

[0070] As used herein, a "sufficient amount" or "an amount sufficient to" achieve a particular result refers to an amount of an anionic substance that is effective to produce a desired effect. In some aspects, a sufficient amount is an amount of anionic substance that can reduce recombinant protein binding to the cell surface by at least 10% with respect to a cell not treated with anionic substance. In some aspects, a sufficient amount is an amount of anionic substance that can reduce growth inhibition caused by a recombinant protein binding to the cell surface by at least 10% with respect a cell not treated with the anionic substance. In some aspects, a sufficient amount is an amount of anionic substance that can increase survival of a eukaryotic cell expressing a recombinant protein by at least 10% with respect to a cell not treated with the anionic substance. A sufficient amount can be determined empirically and in a routine manner, in relation to the stated purpose.

[0071] The term "increased" with respect to a functional characteristic is used to indicate that the relevant functional characteristic is significantly increased relative to that of a 11139

- 23 - reference, as determined under comparable conditions. In some aspects, the functional characteristic is binding of a recombinantly expressed protein to the cell surface. In other aspects, the functional characteristic is cell growth or cell survival. In some aspects, the increase in the functional characteristic (e.g. , increased cell survival due to decreased binding of recombinant protein, e.g., neublastin, to the cell surface) is, e.g., at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%), at least about 75%), at least about 80%, at least about 85%, at least about 90%), at least about 95%, at least about 96%, at least about 97%>, at least about 98%, or at least about 99%o higher relative to a reference (for example the survival of cells not treated with the ionic substances disclosed herein), as determined under comparable conditions. In some aspects, the increase in the functional characteristic (e.g. , increased cell survival or increased cell growth) is, e.g. , an at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8- fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30- fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70- fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold increase relative to a reference (for example when compared to the survival or growth of cells not treated with the ionic substances disclosed herein), as determined under comparable conditions.

The term "decreased" with respect to a functional characteristic is significantly decreased relative to that of a reference, as determined under comparable conditions. In some aspects, the functional characteristic is binding of a recombinantly expressed protein to the cell surface. In other aspects, the functional characteristic is cell growth or cell survival. In some aspects, the decrease in the functional characteristic (e.g., binding of a recombinant protein, e.g., neublastin, to the cell surface) is, e.g. , at least about 5%, at least about 10%, at least about 15%), at least about 20%, at least about 25%, at least about 30%), at least about 35%>, at least about 40%, at least about 45%, at least about 50%, at least about 55%), at least about 60%), at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% lower relative to a reference (for example when compared to the binding of the recombinant protein, e.g., neublastin, to the cell surface when cells are not treated with the ionic substances disclosed herein), as determined under comparable conditions. In some aspects, the decrease in the functional characteristic (e.g., binding of the recombinant protein, e.g., neublastin, to the cell surface) is, e.g. , at least about 2-fold, at least about 3- fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, or at least about 100-fold lower relative to a reference (for example when compared to the binding of the recombinant protein, e.g., neublastin, to the cell surface when cells are not treated with the ionic substances disclosed herein), as determined under comparable conditions.

As used herein, the terms "cell" and "eukaryotic cell" are used interchangeably and refer to a cell or a population of cells harboring or capable of harboring a recombinant nucleic acid, generally in a vector. Eukaryotic cells can be, for example, fungal cells (e.g. , yeast cells such as Saccharomyces cerevisiae, Pichia pastoris, or Schizosaccharomyces pombe), and various animal cells, such as insect cells (e.g. , Sf-9) or mammalian cells (e.g., HEK293F, CHO, COS- 7, NIH-3T3). As used herein, the terms "cell" and "eukaryotic cell" are intended to encompass a singular "cell" as well as plural "cells." The terms "cell" and "eukaryotic cell" include but are not limited to cells obtained from plants, mammals, birds (avian), insects, fish, amphibians, reptiles and the like. The methods disclosed herein can be applied to mammalian cells, in particular, to cells of human origin which can be primary cells derived from a tissue sample, diploid cell strains, transformed cells (stably or transiently transformed), or established cell lines (e.g., CHO), each of which can optionally be genetically altered. The methods disclosed herein can also be applied to mammalian cells such as hybridonias, CHO cells, COS cells, VERO cells, HeTa cells, 294 cells, PER-C6 cells, K562 cells, MOLT-4 cells, Ml cells, NS-1 cells, MDBK ceils, MDCK cells, MRC-5 cells, WI-38 cells, WEHI cells, SP2/0 cells, BHK cells (including BHK-21 cells), primary and/or immortalized lymphocytes, macrophages, dendritic cells, keratinocytes, hepatocytes, neural cells, renal cells, fibroblasts, endothelial cells, tumor cells, epithelial cells, hematopoietic stem cells, mesenchymal stem cells, embryonic stem cells, stem cells of neuronal, hepatic, renal, dermal, endothelial, epithelial, and mesothelial original, and derivatives thereof. In particular, the methods disclosed herein can be applied to cells and stem cells expressing recombinant proteins or viruses. The methods disclosed herein can also be applied to insect cells. Insect cells particularly suitable for recombinant protein expression include those derived from Spodoptera species {e.g. , Sf9 or Sf21 , derived from Spodoptera frugiperdd) or Trichoplusa species {e.g. , HIGH FIVE ™ or MG1 , derived from Trichoplusa ni).

The term "vector" means a construct, which is capable of delivering, and in some aspects, expressing, one or more gene(s) or sequence(s) of interest in a host cell, e.g., an eukaryotic host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells. As used herein, the term "vector" is intended to encompass a singular "vector" as well as plural "vectors." As discussed above, the methods of the present disclosure can be applied to eukaryotic cells, in particular to eukaryotic cells that have been genetically engineered to express a polypeptide of commercial or scientific interest. By genetically engineered is meant that the cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and/or otherwise altered {e.g. , by homologous recombination and gene activation or fusion of a recombinant cells with a non- recombinant cells) so as to cause the cell to express a desired recombinant polypeptide. Methods and vectors for genetically engineering cells and/or cell lines to express a polypeptide of interest are well known to those of skill in the art; for example, various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel at al. , eds. (Wiley & Sons, New York, 1988, and quarterly updates) and Sambrook et al, Molecular Cloning: A Laboratory Manual (Cold Spring Laboratory Press, 1989).

The terms "cell culture," "culture," or "cultivation," as used herein refer to the maintenance of cells under artificial, in vitro conditions favoring growth and/or differentiation and/or continued viability, in an active or quiescent state, of the cells. Cultivation is assessed by the number of viable cells/ml culture medium. Cell viability can be determined routinely using methods known in the art. Cells can be plated according to the experimental conditions determined by the investigator. The optimal T U 2014/011139

- 26 - plating and culture conditions for a given cell type can be determined by one of ordinary skill in the art using only routine experimentation. The cell seeding densities for each experimental condition can be optimized for the specific culture conditions being used. Mammalian cells are typically cultivated in a cell incubator at about 37°C, while the optimal temperatures for cultivation of avian, nematode, and insect cells are typically lower and are well-known to those of ordinary skill in the art. The incubator atmosphere can be humidified for cultivation of animal cells, and can contain about 3-10% carbon dioxide in air.

[0076] Cell cultured in closed or batch culture systems typically undergo complete medium exchange (i.e., replacing spent media with fresh media) about every 2-3 days, or more or less frequently as required by the specific cell type. Cells cultured in perfusion culture systems (e.g., in bioreactors or fermenters) can receive fresh media on a continuously recirculating basis. Cells can also be cultivated in a shaker, stir-tank, air-lift, or perfusion culture. Some cell types (e.g. , hybridomas, cell or lymphoid and myeloid origin) are inherently able to grow in suspension culture. Other cells (e.g. , CHO, 293, HeLa, BHK) which may originally be cultivated in adherent culture can be induced to grow in suspension culture.

[0077] The term "cell culture" when applied to a population of cells refers to a population of cells undergoing "culture," "cultivation," or "cell culture" are defined above.

[0078] The terms "chemically defined cell culture medium," "culture medium," or

"medium" (plural media in each case) refer to a nutritive solution for cultivating eukaryotic cells and can be used interchangeably. A cell culture medium is composed of a number of ingredients and these ingredients can vary from medium to medium. The term refers to a culture medium used for cell culture, e.g. , basal medium, without the addition of a sufficient amount of at least one anionic substance of the present disclosure, e.g., dextran sulfate, ferric citrate, or a combination thereof.

[0079] The term "sufficient amount" as used herein refers to an amount of anionic substance of the present disclosure, e.g. , a valproate such as sodium valproate, a citrate such as ferric citrate, dextran sulfate, , or a combination thereof, sufficient to produce a desired effect such as increasing recombinant protein production, increasing recombinant protein recovery, decreasing recombinant protein attachment to the surface of eukaryotic cells. P T/US2014/011139

- 27 -

[0080] As used herein, the term "inoculum" refers to a volume of eukaryotic cells harvested from growing in a culture medium for addition (i.e. , "inoculation") to a culture medium at the beginning of a "production phase," during which or at the end of which the recombinant protein of interested is harvested. As used herein, the term "seeding" refers to addition or inoculation of growing cells into a culture medium at the beginning of the production phase.

[0081] The term "batch culture" refers to a culture allowed to progress from inoculation to conclusion without re-feeding the culture cells with fresh medium.

[0082] The term "suspension culture" as used herein refers to cell culture in which the majority or all of the cells in a culture vessel are present in suspension, and the minority or none of the cells in the culture vessel are attached to the vessel surface or to another surface within the vessel. In some aspects, a "suspension culture" can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% of the cells, or at least about 99% of the cells in the culture vessel in suspension, not attached to a surface on or in the culture vessel. The methods disclosed herein are suitable for either monolayer or suspension culture, transfection, and cultivation of cells, expression of recombinant proteins in monolayer or suspension culture, and for post-harvesting steps requiring the separation of a recombinantly expressed protein from the cell surface or other surface to which the recombinant protein of interest may be attached (e.g., a separation membrane or a chromatographic substrate).

[0083] The term "contacting" as used herein refers to the placing of cells in vitro into a vessel (e.g., a culture vessel) with a medium (e.g., a cell culture medium in which the cells are to be cultivated, which can contain one or more anionic substances of the present disclosure, or a buffer solution, which can contain one or more anionic substances of the present disclosure, to be used as release composition). The term "contacting" encompasses, for example, mixing cells with medium, pipetting medium onto cells in a vessel, submerging cells in medium, etc.

[0084] T he term "culture vessel" as used herein refers to a glass, plastic or metal container that can. provide an aseptic environment for culturing cells. Typically, in vitro cell culture is performed under sterile, controlled temperature and atmospheric conditions in tissue culture pates (e.g., 10 cm plates. 96 well plates, etc.), or other adherent culture (e.g., on microcarrier beads), or in suspension culture such as in roller bottles. Cultures 2014/011139

- 28 - can be grown in shaker flasks, small scale bioreactors, and/or large scale bioreactors. A bioreactor is a device used to culture animal cells in which environmental conditions such as temperature, atmosphere, agitation, and/or pH can be monitored and adjusted. A number of companies (e.g., ABS Inc., Wilmington, DE; Cell Trends, Inc., Middletown, MD; etc.) also offer cell culture services on a contract basis.

[0085] Cell cultures, e.g., mammalian cell cultures (adherent or non-adherent and growing or growth arrested), can be small scale cultures, such as for example in 100 ml containers having about 30 ml of media, 250 ml containers having about 80 to 90 ml of media, 250 ml containers having about 150 to 200 ml of media. Alternatively, the cultures can be large scale such as for example 100 ml containers having about 300 to 1000 ml of media, 3000 ml containers having about 500 to 3000 ml of media, 8000 ml container having about 2000 to about 8000 ml of media, and 15000 ml container having about 4000 to about 15000 ml of media. Large scale cultures can also be in bioreactors.

[0086] In some aspects, the methods of the present disclosure can be applied to perfused cell cultures. Perfused cell cultures are typically cultured continuously and can be grown for as little as about 5 days and for as long as about 9 months or longer, but are typically cultured for about 25 days. Thus, in some aspects, the anionic substances of the present disclosure can be included in perfused culture media either continuously or intermittently over the course of the perfused culture run.

[0087] The term "protein-free medium" refers to a culture medium which contains no exogenous proteins or peptides (e.g. , no serum proteins such as serum albumin or attachment factors, nutritive proteins such as growth factors, or metal ion carrier proteins such a transferrin or ceruloplasmin). A protein-free medium is distinguished from low- protein and essentially protein-free media, both of which contain low amounts of proteins and/or peptides. The term "serum-free medium" as used herein refers a medium containing suitable supplements except any kind of serum (e.g., fetal bovine serum (FBS), calf serum, horse serum, goat serum, human serum, etc.). The methods of the present disclosure can be applied to cell cultures grown using any medium capable of supporting the growth of animal cells in culture. Thus, the methods of the present disclosure are broadly applicable to animal cells in culture and the choice of medium is not crucial to the invention, [0088] The term "basal medium" as used herein means a medium that supports growth of certain single-celled organisms and cells that do not require special media additives. Typical basal medium components are known in the art and include salts, amino acids, vitamins, and a carbon source (e.g. , glucose). Other components that do not change the basic characteristic of the medium but are otherwise desirable can also be included, such as the pH indicator phenol red. For example, Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM/FJ 2) is a basal medium commonly used to make suitable growth media for mammalian cell culture. Cell culture media suitable for the application of the methods disclosed herein are commercially available from ATCC (Manassas, VA). For example, any one or a combination of the following basal media can be used: RMPI- 1640 Medium, Dulbecco's Modified Eagle's Medium, Minimum Essential Medium Eagle, F-12K Medium, Iscove's Modified Dulbecco's Medium, etc.

[0089] Often, depending upon the requirements of the particular cell line used, medium also contains a serum additive such as Fetal Bovine Serum, or as serum replacement. Examples of serum-replacement (for serum-free growth of cells) are TCH™ and TM- 235™ (Celox, St. Paul, MN). When defined medium that is serum-free and/or peptone- free is used, the medium is usually highly enriched for amino acids and trace elements (see, e.g. , U.S. Pat. Nos. 5,122,469; and 5,633,162).

[0090] Serum adds to the expense of cell culture and in addition there are serious regulatory concerns about viral contamination in serum, and further, removing serum proteins from downstream processing is burdensome. As such, in some aspects the methods of the present disclosure are applied to cell cultures in which the cell culture medium is serum free or essentially free of serum and the recombinant polypeptide producing cell lines have been selected for growth without serum (see, e.g. , Rasmussen et al , Cytotechnology 28:31-42 (1998)).

[0091] Essentially serum free is meant to include very low amounts of serum in the culture media. This includes less than about 20% serum, less that about 1% serum, less than about 0.5% serum, and less than about 0.25% serum.

[0092] The term "yield" as used herein refers to the amount of recombinant protein expressed by cultured cells, and can be measured, for example, in terms of grams of recombinant protein produced/ml medium. If the recombinant protein is secreted by the cells, the recombinant protein can be isolated from the culture medium by methods known to those of ordinary skill in the art. The amount of recombinant protein expressed by the protein and present in the culture medium can readily be determined by those of ordinary skill in the art.

[0093] The term "pharmaceutical composition" as used herein refers to a preparation which is in such form as to permit the biological activity of the active ingredient to be effective, and which contains no additional components which are unaeceptably toxic to a subject to which the composition would be administered. Such composition can be sterile.

Anionic Substances

[0094] The present disclosure provides methods comprising the addition of anionic substances to a cell culture medium or release composition to increase the production of recombinant proteins, e.g., by reducing binding of the recombinant protein to the cell surface, releasing the recombinant basic protein from the cell surface, increasing cell growth and/or cell survival during cultivation, or reducing binding of the recombinant protein to other surfaces. The term "release composition" as used herein refers to a solution comprising at least one the anionic substances disclosed herein that can be added to cells expressing a recombinant protein after the cells have been separated from the culture medium (e.g., via filtration or centrifugation), wherein the addition of the solution reduces the amount of recombinant protein attached to the surface of the cells. Generally, the use of a release composition comprises separating the cells from the culture medium via centrifugation or another suitable cell separation technique, resuspending the cells in a buffer solution and adding at least one of the anionic substances disclosed herein (in this case the resulting solution is the release composition) or resuspending the cells in release composition (buffer solution plus at least of the anionic substances disclosed herein), wherein the addition of the release composition causes the release of the recombinant protein from the cell surface. A release composition can also be used to reduce or prevent the binding of the recombinant protein of interest to other surfaces (e.g. , the surface of a filtration membrane or a chromatographic substrate) during downstream processing.

[0095] In some aspects, the anionic substances disclosed herein comprise more than one negatively charged group, e.g. , sulfate, sulfonate, carboxylate, perfluorosulfonate, phosphate, phosphonate groups. As used herein, the term used to refer to an anionic substance, e.g. , "fumarate," refers to the free acid form (i.e., fumaric acid) as well as salts and esters (i. e. , fumarates). [0096] In some aspects, the anionic substance is a monocarboxylate, e.g., valproate, propanoate, acetate, butyrate, valerate, hexanoate, heptanoate, pyruvate, acetoacetate, levulate, etc. In some aspects, the valproate is sodium valproate. As used herein, the term anionic substance does not include amino acids, although those compounds can posses one or two carboxylate groups.

[0097] In some aspects, the anionic substance is a dicarboxylate, e.g., fumarate, succinate or malate. Other dicarboxylates known in the art are, e.g. , oxalacetates, maleates, - ketoglutarates, muconates, traumates, terephthalates, isophthalates, oxalates, malonates, glutarates, adipates, pimelates, suberates, azelates, sebacates, etc. In some aspects, the anionic substance is a tricarboxylate, e.g. , citrate. Other tricarboxylates are, e.g., isocitrates, oxalosuccinates, aconitates, etc. In some aspects, the citrate is, e.g. , ferric citrate or sodium citrate.

[0098] In some aspects, the anionic substance comprises (i) an alkanoic acid or salt thereof (e.g., valproic acid or sodium valproate), (ii) a polyanionic compound (e.g., a dextran sulfate), or a (iii) a combination thereof, wherein the alkanoic acid or salt thereof acts synergistically with the polyanionic compound to reach the desired effects (i.e., increasing recombinant protein recovery, and/or increasing recombinant protein production, and/or decreasing recombinant protein attachment to the surface of eukaryotic cells, and/or reducing recombinant protein-induced inhibition of eukaryotic cell growth, and/or increasing the viability of eukaryotic cells expressing a recombinant protein). In some aspects, the alkanoic acid comprises a straight alkanoic acid, a branched-chain alkanoic acid, a saturated alkanoic acid, an unsaturated alkanoic acid, or a combination thereof (see, for example, U.S. Pat. No. 5,705,364, which is herein incorporated by reference in its entirety). In some aspects, the alkanoic acid comprises from one to ten carbon atoms. In some aspects, the alkanoic acid comprises from three to six carbon atoms. In some aspects the alkanoic acid is butyric acid. In some aspects, the alkanoic acid is valproic acid. In some aspect the alkanoic acid salt is sodium butyrate. In other aspects, the alkanoic acid salt is sodium valproate.

[0099] In some aspects, the anionic substance can be a polyanionic compound. In some aspects, the polyanionic compound comprises a polymeric scaffold (e.g. , a polysaccharide scaffold or a polyvinyl scaffold) comprising a plurality of substituent anionic groups (e.g. , sulfate or sulfonate). In some aspects, the polyanionic compound is a poly sulfated U 2014/011139

- 32 - compound. In other aspects, the polysulfated compound is polyvinyl sulfate. In certain aspects, the polyanionic compound is a polysulfonated compound. In specific aspects, the polyanionic compound can be, for example, heparin, heparin sulfate, chondroitin sulfate, keratan sulfate, dermatan sulfate, sulfated chitin, sulfated chitosan, sulfated alginic acid, pentosan polysulfate, sulfated cyclodextrins, polystyrene sulfonate, sulfated polyvinyl alcohol, polyvinyl sulfate, or polyethylene sulfonate.

30] In some specific aspects, the polyanionic compound can be a sulfated polysaccharide. In some aspects, the sulfated polysaccharide can be a polysulfated saccharide. As used herein, the term "sulfated polysaccharide" means a sulfated material having more than ten units of simple sugar. In some aspects, the sulfated polysaccharide is an alpha(l ,6) linked polysaccharide. The sulfated polysaccharides disclosed herein also have a percent of sulfur that is sufficient to decrease recombinant protein attachment to the surface of eukaryotic cells without significant toxicity. The term sulfate polysaccharide encompasses homo- and heteropoly saccharides. In some aspects, a sulfated polysaccharide is a homopolysaccharide such as dextran sulfate or cellulose sulfate, with monomeric units consisting of either aldo-, deoxyaldo-, keto- or deoxyketopentoses, including, but not restricted to, arabinose, ribose, deoxyribose, galactose, fructose, sorbose, rhamnose and fucose, joined by either alpha- or beta- linkages. The polymer can be linear or branched, with free hydroxyl groups of the monomeric units maximally or partially sulfated. The monomeric unit can be further modified, for example, by the presence of carboxyl, amino and ester groups.

I I ] As used herein, the term "dextran" means a polysaccharide containing a backbone of D-glucose units linked predominantly alpha-D(l ,6), composed exclusively of alpha-D- glucopyranosyl units differing only in degree of branching and chain length. As used herein, the term "dextran sulfate" refers to an alpha- 1 ,6-polyglucose polysulfated saccharide containing up to three sulfate groups per glucose molecule of varying molecular weight ranges, e.g. , 4,000-500,000 Da. In some aspects, the dextran sulfate contains at least about 10%, at least about 1 1%, at least about 12%>, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%), at least about 19%, or at least about 20%> sulfur. In some aspects, the dextran sulfate has a molecular weight of at least about 3,000 Da; at least about 4,000 Da; at least about 5,000 Da; at least about 6,000 Da; at least about 7,000 Da; at least about 8,000 Da; at least about 9,000 Da; or at least about 10,000 Da. In other aspects, the dextran sulfate has a molecular weight of at least about 20,000 Da; at least about 25,000 Da; at least about 30,000 Da; at least about 35,000 Da; at least about 40,000 Da; at least about 45,000 Da; at least about 50,000 Da; at least about 55,000 Da; at least about 60,000 Da; at least about 65,000 Da; at least about 70,000 Da; at least about 75,000 Da; at least about 80,000 Da; at least about 85,000; at least about 90,000 Da; at least about 95,000 Da; or at least about 100,000 Da. In some other aspects, the dextran sulfate has a molecular weight of at least about 1 10,000 Da; at least about 120,000 Da; at least about 130,000 Da; at least about 140,000 Da; at least about 150,000 Da; at least about 160,000 Da; at least about 170,000 Da; at least about 180,000 Da; at least about 190,000 Da; at least about 200,000 Da; at least about 210,000 Da; at least about 220,000 Da; at least about 230,000 Da; at least about 240,000 Da; at least about 250,000 Da; at least about 260,000 Da; at least about 270,000 Da; at least about 280,000 Da; at least about 290,000 Da; at least about 300,000 Da; at least about 310,000 Da; at least about 320,000 Da; at least about 330,000 Da; at least about 340,000 Da; at least about 350,000 Da; at least about 360,000 Da; at least about 370,000 Da; at least about 380,000 Da; at least about 390,000 Da; at least about 400,000 Da; at least about 410,000 Da; at least about 420,000 Da; at least about 430,000 Da; at least about 440,000 Da; at least about 450,000 Da; at least about 460,000 Da; at least about 470,000 Da; at least about 480,000 Da; at least about 490,000 Da; at least about 500,000 Da; at least about 510,000 Da; at least about 520,000 Da; at least about 530,000 Da; at least about 540,000 Da; at least about 550,000 Da; at least about 560,000 Da; at least about 570,000 Da; at least about 580,000 Da; at least about 590,000 Da; at least about 600,000 Da.

In some specific aspects, the anionic substance is a polyvinyl sulfate. In other aspects, the anionic substance disclosed herein comprises at least one monocarboxylate, dicarboxylate or tricarboxylate (see, e.g., FIG. 13), in combination with one or more polyanionic compounds. For example, in some aspects the anionic substance comprises a tricarboxylate in combination with a polysulfate. In some aspects, the anionic substance comprises a citrate and a dextran sulfate. In some specific aspects, the anionic substance comprises ferric citrate and dextran sulfate. In specific aspects, the anionic substance comprises a valproate and a dextran sulfate. In some specific aspects, the anionic substance comprises sodium valproate and dextran sulfate. [0103] in some aspects, the concentration of valproate is in a range from about ImM to about lOOraM. In some aspects, the concentration of valproate is at least about I mM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. in some aspects, the concentration of valproate is at least about 1 5 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM,_. at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM. at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. in a specific aspect, the concentration of valproate is about 3 mM. In a specific aspect, the concentration of valproate is about 2 mM. In another specific aspect, the concentration of valproate is about 10 mM. In another specific aspect, the concentration of valproate is about 50 mM. In another specific aspect, the concentration of valproate is about 100 mM. In a specific aspect, a concentrated valproate solution is added to the culture medium or release composition to raise the valproate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 mM, at least 50 mM or at least 100 mM.

[0104] In some aspects, the concentration of sodium valproate is in a range from about

ImM to about l OOmM. In some aspects, the concentration of sodium valproate is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In some aspects, the concentration of sodium valproate is at least about 15 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In a specific aspect, the concentration of sodium valproate is about 1 mM. In a specific aspect, the concentration of sodium valproate is about 2 mM. In another specific aspect, the concentration of sodium valproate is about 10 mM. In another specific aspect, the concentration of sodium valproate is about 50 mM. In another specific aspect, the concentration of sodium valproate is about 100 mM. In a specific aspect, a concentrated sodium valproate solution is added to the culture medium or release composition to raise the sodium valproate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 mM, at least 50 mM or at least 100 mM.

In some aspects, the concentration of citrate is in a range from about ImM to about lOOmM. In some aspects, the concentration of citrate is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 mM, at least about

6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In some aspects, the concentration of citrate is at least about 15 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In a specific aspect, the concentration of citrate is about 1 mM. In a specific aspect, the concentration of citrate is about 2 mM. In another specific aspect, the concentration of citrate is about 10 mM. In another specific aspect, the concentration of citrate is about 50 mM. In another specific aspect, the concentration of citrate is about 100 mM. In a specific aspect, a concentrated citrate solution is added to the culture medium or release composition to raise the citrate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 mM, at least 50 mM or at least 100 mM.

In some aspects, the citrate is sodium citrate or ferric citrate. In some aspects, the concentration of ferric citrate is in a range from about ImM to about lOOmM. In some aspects, the concentration of ferric citrate is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 mM, at least about 6 mM, at least about

7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In some aspects, the concentration of ferric citrate is at least about 15 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM. at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In a specific aspect, the concentration of ferric citrate is about 1 mM. In a specific aspect, the concentration of ferric citrate is about 2 mM. In another specific aspect, the concentration of ferric citrate is about 10 mM. In another specific aspect, the concentration of ferric citrate is about 50 mM. In another specific aspect, the concentration of ferric citrate is about 100 mM. In a specific aspect, a concentrated ferric citrate solution is added to the culture medium or release composition to raise the ferric citrate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 mM, at least 50 mM or at least 100 mM.

In some aspects, the concentration of sodium citrate is in a range from about ImM to about lOOmM. In some aspects, the concentration of sodium citrate is at least about 1 rnM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In some aspects, the concentration of sodium citrate is at least about 15 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In a specific aspect, the concentration of sodium citrate is about 1 mM. In a specific aspect, the concentration of sodium citrate is about 2 mM. In another specific aspect, the concentration of sodium citrate is about 10 mM. In another specific aspect, the concentration of sodium citrate is about 50 mM. In another specific aspect, the concentration of sodium citrate is about 100 mM. In a specific aspect, a concentrated sodium citrate solution is added to the culture medium or release composition to raise the sodium citrate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 rnM, at least 50 mM or at least 100 mM.

In some aspects, the concentration of succinate is in a range from about ImM to about lOOmM. In some aspects, the concentration of succinate is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In some aspects, the concentration of succinate is at least about 15 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 2014/011139

- 37 - mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In a specific aspect, the concentration of succinate is about 1 mM. In a specific aspect, the concentration of succinate is about 2 mM. In another specific aspect, the concentration of succinate is about 10 mM. In another specific aspect, the concentration of succinate is about 50 mM. In another specific aspect, the concentration of succinate is about 100 mM. In a specific aspect, a concentrated succinate solution is added to the culture medium or release composition to raise the succinate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 mM, at least 50 mM or at least 100 mM.

[0109] In some aspects, the concentration of fumarate is in a range from about ImM to about lOOmM. In some aspects, the concentration of fumarate is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 mM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In some aspects, the concentration of fumarate is at least about 15 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In a specific aspect, the concentration of fumarate is about 1 mM. In a specific aspect, the concentration of fumarate is about 2 mM. In another specific aspect, the concentration of fumarate is about 10 mM. In another specific aspect, the concentration of fumarate is about 50 mM. In another specific aspect, the concentration of fumarate is about 100 mM. In a specific aspect, a concentrated fumarate solution is added to the culture medium or release composition to raise the fumarate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 mM, at least 50 mM or at least 100 mM.

[0110] In some aspects, the concentration of malate is in a range from about ImM to about l OOmM. In some aspects, the concentration of malate is at least about 1 mM, at least about 2 mM, at least about 3 mM, at least 4 mM, at least about 5 rnM, at least about 6 mM, at least about 7 mM, at least about 8 mM, at least about 9 mM, or at least about 10 mM. In some aspects, the concentration of malate is at least about 15 mM, at least 20 mM, at least about 25 mM, at least about 30 mM, at least about 30 mM, at least about 35 mM, at least about 40 mM, at least about 45 mM, at least about 50 mM, at least about 55 mM, at least about 60 mM, at least about 65 mM, at least about 70 mM, at least about 75 mM, at least about 80 mM, at least about 85 mM, at least about 90 mM, at least about 95 mM, or at least about 100 mM. In a specific aspect, the concentration of malate is about 1 mM. In a specific aspect, the concentration of malate is about 2 mM. In another specific aspect, the concentration of malate is about 10 mM. In another specific aspect, the concentration of malate is about 50 mM. In another specific aspect, the concentration of malate is about 100 mM. In a specific aspect, a concentrated malate solution is added to the culture medium or release composition to raise the malate concentration of the culture medium or release composition to at least 1 mM, at least 2 mM, at least 10 mM, at least 50 mM or at least 100 mM. In some aspects, the concentration of polysulfated compound, e.g., dextran sulfate or polyvinyl sulfate, is in a range from about O.Olg/L to about lg/L. In some aspects, the concentration of polysulfated compound, e.g., dextran sulfate or polyvinyl sulfate, is at least about 0.01 g/L, or at least about 0.02 g/L, or at least about 0.03 g/L, or at least about 0.04 g/L, or at least about 0.04 g/L, or at least 0.05 g/L, or at least about 0.06 g/L, or at least about 0.07 g/L, or at least about 0.08 g/L, or at least about 0.09 g/L, or at least about 0.1 g/L. In some aspect, the concentration of polysulfated compound, e.g., dextran sulfate or polyvinyl sulfate, is at least about 0.15 g/L, or at least about 020 g/L, or at least about 0.25 g/L, or at least about 0.30 g/L, or at least about 0.35 g/L, or at least about 0.40 g/L, or at least about 0.45 g/L, or at least about 0.50 g/L, or at least about 0.55 g/L, or at least about 0.60 g/L, or at least about 0.65 g/L, or at least about 0.70 g/L, or at least about 0.75 g/L, or at least about 0.80 g/L, or at least about 0.85 g/L, or at least about 0.90 g/L, or at least about 0.95 g/L, or at least about 1 g/L.

In a specific aspect, the concentration of polysulfated compound, e.g., dextran sulfat or polyvinyl sulfate, is about 0.1 g/L. In another specific aspect, the concentration of polysulfated compound, e.g., dextran sulfate or polyvinyl sulfate, is about 0.25 g/L. In another specific aspect, the concentration of polysulfated compound, e.g., dextran sulfate or polyvinyl sulfate, is about 0.50 g/L. In another specific aspect, the concentration of polysulfated compound, e.g., dextran sulfate or polyvinyl sulfate, is about 1 g/L. In a specific aspect, a concentrated polysulfated compound, e.g., dextran sulfate or polyvinyl sulfate, is added to the culture medium or release composition to raise the malate concentration of the culture medium or release composition to at least 0.1, at least 0.25, at least 0.50, or at least 1 g/L.

[0112] In some aspects, the anionic substance of the present disclosure can be used in combination with an alkaline or earth alkaline salt such as a salt of the Hofmeister series comprising, for example, as anions P0₄ ^3", S0₄ ^2", CH₃COO^", CI^", Br^", NO^3", C104^", Γ, SCN^" and, for example, as cations NH⁴⁺, Rb⁺, K⁺, Na⁺ _; Cs⁺, Li⁺, Zn²⁺, Mg²⁺, Ca²⁺, Mn²⁺, Cu²⁺ and Ba²⁺. Particularly salts present in combination with the anionic substance of the present disclosure can be selected, for example, from (NH₄)₃P0₄, (NH₄)₂HP0₄, (NH₄)H₂P0₄, (NH₄)₂S0₄, NH₄CH₃COO, NH₄C1, NH₄Br, NH₄N0₃, NH₄C10₄, NH I, NH₄SCN, Rb₃P0₄, Rb₂HP0₄, RbH₂P0₄, Rb₂S0₄, Rb₄CH₃COO, Rb₄Cl, Rb₄Br, Rb₄N0₃, Rb₄C10₄, Rb₄I, Rb₄SCN, K₃P0₄, K₂HP0₄, KH₂P0₄, K₂S0₄, KCH₃COO, KC1, Br, KN0₃, KC10₄, KI, KSCN, Na₃P0₄, Na₂HP0₄, NaH₂P0₄, Na₂S0₄, NaCH₃COO, NaCl, NaBr, NaN0₃, NaC10₄, Nal, NaSCN, ZnCl₂, Cs₃P0₄, Cs₂HP0₄, CsH₂P0₄, Cs₂S0₄, CsCH₃COO, CsCl, CsBr, CsN0₃, CsC10₄, Csl, CsSCN, Li₃P0₄, Li₂HP0₄, LiH₂P0₄, Li₂S0₄, LiCH₃COO, LiCl, LiBr, LiN0₃, L1CIO4, Lil, LiSCN, Cu₂S0₄, Mg₃(P0₄)₂, Mg₂HP0₄, Mg(H₂P0₄)₂, Mg₂S0₄, Mg(CH₃COO)₂, MgCl₂, MgBr₂, Mg(N0₃)₂, Mg(C10₄)₂, Mgl₂, Mg(SCN)₂, MnCl₂, Ca₃(P0₄)₂, Ca₂HP0₄, Ca(H₂P0₄)₂, CaS0₄, Ca(CH₃COO)₂, CaCl₂, CaBr₂, Ca(N0₃)₂, Ca(C10₄)₂, Cal₂, Ca(SCN)₂, Ba₃(P0₄)₂, Ba₂HP0₄, Ba(H₂P0₄)₂, BaS0₄, Ba(CH₃COO)₂, BaCl₂, BaBr₂, Ba(N0₃)₂, Ba(C10₄)₂, Bal₂, Ba(SCN)₂, and combinations thereof.

[0113] In some aspects, the anionic substance of the present disclosure is used in combination with an additional ionic substance, for example, NH₄ acetate, MgCl₂, KH₂P0₄, NaS0₄, KC1, CaCl₂, one or more amino acids, and a mixture of one or more peptides and/or amino acids.

[0114] In some aspects, the anionic substance disclosed herein is used at a physiological concentration. In some aspects, the anionic substance disclosed herein is used at a non- physiological concentration. In some aspects, the anionic substance disclosed herein is used as part of a combination of anionic substances, wherein at least one of the anionic substances is used at a non-physiological concentration. In other aspects, the anionic substance disclosed herein is used as part of a combination comprising one of the salts disclosed above, wherein at least one salt is used at a non-physiological concentration. In some aspects, at least two anionic substances are used simultaneously. In some aspects, at least two anionic substances are used sequentially.

[0115] In some aspect, the anionic substance comprises a polyanionic compound and a citrate. In some specific aspects, the anionic substance comprises citrate at a concentration of about ImM to about 10 mM and dextran sulfate at a concentration of about 0.1 g/L to about 0.5 g/L.

[0116] In some aspects, the polyanionic compound is dextran sulfate and the citrate is ferric citrate. In some specific aspects, the anionic substance comprises ferric citrate at a concentration of about ImM to about 10 mM and dextran sulfate at a concentration of about 0.1 g/L to about 0.5 g/L. In some specific aspects, the anionic substance comprises ferric citrate at a concentration of about 2mM and dextran sulfate at a concentration of about 0.25 g/L.

[0117] In other aspect, the polyanionic compound is dextran sulfate and the citrate is sodium citrate. In some specific aspects, the anionic substance comprises sodium citrate at a concentration of about ImM to about 10 mM and dextran sulfate at a concentration of about 0.1 g/L to about 0.5 g/L. In some specific aspects, the anionic substance comprises sodium citrate at a concentration of about 2mM and dextran sulfate at a concentration of about 0.25 g/L.

[0118] In some aspect, the anionic substance comprises a polyanionic compound and a valproate. In some specific aspects, the anionic substance comprises a valproate at a concentration of about ImM to about 10 mM and dextran sulfate at a concentration of about 0.1 g/L to about 0.5 g/L. In some specific aspects, the anionic substance comprises a valproate at a concentration of about 2mM and dextran sulfate at a concentration of about 0.25 g/L.

[0119] In some aspects, the polyanionic compound is dextran sulfate and the valproate is sodium valproate. In some specific aspects, the anionic substance comprises sodium valproate at a concentration of about ImM to about 10 mM and dextran sulfate at a concentration of about 0.1 g/L to about 0.5 g/L. In some specific aspects, the anionic substance comprises sodium valproate at a concentration of about 2niM and dextran sulfate at a concentration of about 0.25 g/L.

[0120] In some aspects, the anionic substances disclosed herein, e.g., sodium citrate, ferric citrate, valproate, or dextran sulfate, alone or in combination, can be added to freshly formulated basal medium or release composition, or can be formulated in a solution of basal medium or release composition. The anionic substances disclosed herein can be prepared as lx to 1 ,000 formulations, e.g., lx, l Ox, l OOx, 500x or l OOOx formulation, which are then diluted appropriately into the culture medium or release composition to provide a l x final formulation in the complete culture medium or release composition.

[0121] To formulate a culture medium or release composition to use according to the methods of the present disclosure, an anionic substance of the present disclosure (e.g. , ferric citrate, sodium citrate, sodium valproate, or dextran sulfate) can be added to a culture medium or release composition in an amount effective to increase the production of recombinant protein, e.g., by reducing binding of the recombinant protein to the cell surface, releasing the recombinant basic protein from the cell surface, increasing cell growth and/or cell survival during cultivation, or reducing binding of the recombinant protein to other surfaces. The method to dissolve the anionic substances into the culture medium or release composition vary and can be determined by one of ordinary skill in the art with no more than routine experimentation. Typically, the anionic substances can be added to the culture medium or release composition in any order.

[0122] If the anionic substances are prepared as concentrated solutions, an appropriate amount of the concentrated solution is combined with the appropriate amount of diluent (e.g., culture medium) to produce a l x formulation. As will be readily apparent to one of ordinary skill in the art, the concentration of a given anionic substance disclosed herein can be increased or decreased beyond the ranges disclosed above, and the effect of the increased or decreased concentration on the production of the recombinant protein can be determined using only routine experimentation. In some specific aspects, the culture media and release compositions comprising the anionic substances disclosed herein contain no or only small amounts of non-ionic detergents. In some specific aspects, the culture media and release compositions comprising the anionic substances disclosed herein are free of non-ionic detergents.

[0123] In some aspects, two or more anionic substances disclosed herein are mixed with each other to exert the same effect as when only one anionic substance is added to the culture medium or release composition. For example, a carboxylate (e.g., a monocarboxylate such as valproate, a dicarboxylate such as fumarate or malate, or a tricarboxylate such as citrate, e.g.,) can be mixed with a polysulfated compound, e.g., a dextran sulfate. In some aspects, the valproate is sodium valproate. In some aspects, the citrate is sodium citrate or ferric citrate. The concentration of a mixture of anionic substances needed to reach the desired effects (i.e. , increasing recombinant protein recovery, and/or increasing recombinant protein production, and/or decreasing recombinant protein attachment to the surface of eukaryotic cells, and/or reducing recombinant protein-induced inhibition of eukaryotic cell growth, and/or increasing the viability of eukaryotic cells expressing a recombinant protein) is mainly dependent on two factors, the number of anionic substances and the concentration of each anionic substance. Thus, if more anionic substances are mixed, less concentration of each is needed to reach the maximum desired effect. In principle, this can be calculated on a mathematical basis. However, in specific cases, the anionic substances can exert synergistic effect which lower the required concentration of anionic substances compared with a theoretical estimation.

GDNF Family Proteins

[0124] The methods disclosed herein can be applied to the recombinant production of

GDNF ligand family proteins. The GDNF ligand family of proteins comprises neublastin, GDNF, neurturin, and persephin. In some aspects, GDNF ligand family proteins are produced by recombinant DNA techniques. For example, a nucleic acid molecule encoding a GDNF ligand family protein can be inserted into a vector, e.g. , an expression vector, and the nucleic acid can be introduced into an eukaryotic cell. Suitable eukaryotic cells include, e.g. , mammalian cells (such as human cells or CHO cells), fungal cells, yeast cells, and insect cells. When expressed in a recombinant cell, the cell is preferably cultured under conditions allowing for expression of a GDNF ligand family protein. The GDNF ligand family protein can be recovered from a cell suspension if desired. As used herein, "recovered" means that the recombinant protein is removed from those components of a cell or culture medium, or release composition, in which it is present prior to the recovery process.

[0125] Variant GDNF ligand family proteins can be constructed using any of several methods known in the art. The production and use of GDFN family proteins, variants, and derivatives thereof is disclosed, for example, in International Patent Publications WO2007/103182, WO2002/072826, WO2000/001815, WO2004/108760, WO2002/060929, WO2006/023781, WO2002/078730, WO2007/042040, WO2009/002193, WO2007/019860, WO2000/004050, WO 1997/008196, WOl 997/033911, WO2000/018799, WO2004/069176, WO2004/094592,

WO2007/100898, WO2008/137574, WO2006/023782, WO2007/098283, and WO2009/059755, all of which are incorporated herein by reference in their entireties.

[0126] Amino acid sequences of human, mouse, and rat neublastin, GDNF, neurturin, and persephin as well as their respective pre pro forms are shown in TABLE 1.

TABLE 1 : Amino Acid Sequences of GDNF Family Proteins

proteins, truncated forms, or any other post-translationally modified protein. It is assumed that a bioactive neublastin is in the dimerized form for each neublastin variant, because dimer formation is required for activity.

[0128] Little to no activity is observed in a monomeric neublastin polypeptide. A bioactive neublastin polypeptide includes a dimerized polypeptide that, in the presence of a cofactor (such as GFRa3 r RET), binds to GFRct3 or to a complex of GFRa3 and RET, induces dimerization of RET, and autophosphorylation of RET. Accordingly, the terms "neublastin" or a "neublastin polypeptide," as used herein, refer to a polypeptide which possesses neurotrophic activity (e.g. , as described in WO 00/01815). The terms "neublastin" or a "neublastin polypeptide," as used herein, encompass wild-type neublastins, truncated neublastins (i. e. , neublastin fragments), neublastin variants, and derivative or modified neublastins.

[0129] Any method for detecting neublastin binding to GFRa3 or to a complex of

GFRa3 and RET can be used to evaluate the biological activity a neublastin polypeptide. Exemplary assays for detecting the ternary complex-binding ability of a neublastin polypeptide are described in WOOO/001815 and in Example 7 of U.S. Patent No. 8,263,553. A neublastin polypeptide can also be assessed to evaluate its ability to trigger the neublastin signaling cascade. For example, the Kinase Receptor Activation (KIRA) assay described in Example 6 of U.S. Patent No. 8,263,553 can be used to assess the ability of a neublastin polypeptide to induce RET autophosphorylation {see also, Sadick et al, 1996, Anal. Biochem., 235(2):207). Exemplary neublastin polypeptides (wild-type, truncated, variants, and derivative or modified neublastins) are disclosed below:

1. Wild-type Neublastins

[0130] In some aspects, the neublastin polypeptide is a mature wild type human neublastin polypeptide 1 13 amino acids in length (SEQ ID NO: 1). The following "wild- type" neublastin amino acid ("aa" or "AA") sequences are exemplary of those that are useful in the methods and compositions of the present disclosure:

(i) AA-8o-AA,4o of SEQ ID NO:5 ("wild type" human prepro),

(ii) AA-4i-AAi4o of SEQ ID NO:5 (pro human),

(iii) AAi-AA,4o of SEQ ID NO:5 (mature 140AA (SEQ ID NO: 17); hereafter

"NBN140"),

(iv) AA₂5-AA₁₄o of SEQ ID NO:5 (mature 116AA (SEQ ID NO: 18); hereafter

"NBN1 16"),

(v) AA₂₈-AAi4o of SEQ ID NO:5 (mature 113AA (SEQ ID NOT); hereafter

"NBN1 13"),

(vi) AA-₈₀-AAi44 of SEQ ID NO:6 (murine prepro),

(vii) AA, -AAi44 of SEQ ID NO:6 (murine mature-144 AA),

(viii) AAi-AA₂₂₄ of SEQ ID NO:7 (rat prepro),

(ix) AA-₈₁-AA₂₂₄ of SEQ ID NO: 7 (rat mature-144 A A),

(x) Peptides with a C-terminal sequence set forth in AA107-AA140 of SEQ ID NO:5, or in particular AA76-AA140 of SEQ ID NO:5, and which retain the 7 Cys residues characteristic of the GDNF family and of the TGF-β super family.

[0131] In one aspect, the neublastin polypeptide contains (seven) cysteines conserved as in SEQ ID NO:5 at positions 43, 70, 74, 107, 108, 136 and 138. These seven conserved cysteine residues are known within the TGF-β superfamily to form three intramonomeric disulfide bonds (located, e.g. , in SEQ ID NO:5 between cysteine residues 43-108, 70-136, and 74-138) and one intermonomeric disulfide bond (located, e.g. , in SEQ ID NO:5 between cysteine residues 107-107), which together with the extended beta strand region constitutes the conserved structural motif for the TGF-p superfamily. See, e.g. , Daopin et al , Proteins 1993, 17:176-192.

2. Truncated Neublastins

[0132] Neublastin polypeptides useful in the present disclosure also include truncated forms of the full length neublastin protein, i.e., neublastin fragments. In such truncated molecules, one or more amino acids have been deleted from the N-terminus and/or the C- terminus. In some aspects, one or more amino acids have been deleted from the N- terminus. In others aspects, one or more amino acids have been deleted from the C- terminus.

[0133 J Although the mature human neublastin polypeptide NBN113 (SEQ ID NOT) consists of the carboxy terminal 1 13 amino acids of pre pro neublastin (SEQ ID NO:5), not all of the 1 13 amino acids are required to achieve useful neublastin biological activity.

[0134] The truncated neublastin polypeptides described herein include a polypeptide sequence that encompasses the seven cysteine residues conserved in the mature neublastin sequence. In certain preferred embodiments, the truncated neublastin polypeptide includes at least the 85 carboxy terminal amino acids of mature NBN1 13 neublastin polypeptide.

[0135] Examples of truncated neublastin forms include:

(i) the 1 12AA polypeptide sequence designated herein as NBN1 12, which possesses the carboxy terminal 1 12 amino acids of a mature neublastin polypeptide, e.g. , amino acids 29-140 of SEQ ID NO:5 (SEQ ID NO: 19);

(ii) the 1 11 AA polypeptide sequence designated herein as NBN1 1 1, which possesses the carboxy terminal 1 1 1 amino acids of a mature neublastin polypeptide, e.g. , amino acids 30-140 of SEQ ID NO:5 (SEQ ID NO:20);

(iii) the 1 10AA polypeptide sequence designated herein as NBN1 10, which possesses the carboxy terminal 1 10 amino acids of a mature neublastin polypeptide, e.g., amino acids 31-140 of SEQ ID NO:5 (SEQ ID NO:21);

(iv) the 109AA polypeptide sequence designated herein as NBN109, which possesses the carboxy terminal 109 amino acids of a mature neublastin polypeptide, e.g. , amino acids 32-140 of SEQ ID NO:5 (SEQ ID NO:22); (v) the 108AA polypeptide sequence designated herein as NBN108, which possesses the carboxy terminal 108 amino acids of a mature neublastin polypeptide, e.g. , amino acids 33-140 of SEQ ID NO:5 (SEQ ID NO:23);

(vi) the 107AA polypeptide sequence designated herein as NBN107, which possesses the carboxy terminal 107 amino acids of a mature neublastin polypeptide, e.g. , amino acids 34-140 of SEQ ID NO:5 (SEQ ID NO:24);

(vii) the 106AA polypeptide sequence designated herein as NB 106, which possesses the carboxy terminal 106 amino acids of a mature neublastin polypeptide, e.g., amino acids 35-140 of SEQ ID NO:5 (SEQ ID NO:25);

(viii) the 105AA polypeptide sequence designated herein as NBN105, which possesses the carboxy terminal 105 amino acids of a mature neublastin polypeptide, e.g., amino acids 36-140 of SEQ ID NO:5 (SEQ ID NO:26);

(ix) the 104AA polypeptide sequence designated herein as NBN104, which possesses the carboxy terminal 104 amino acids of a mature neublastin polypeptide, e.g., amino acids 37-140 of SEQ ID NO:5 (SEQ ID NO:27);

(x) the 103 AA polypeptide sequence designated herein as NBN103, which possesses the carboxy terminal 103 amino acids of a mature neublastin polypeptide, e.g. , amino acids 38-140 of SEQ ID NO:5 (SEQ ID NO:28);

(xi) the 102AA polypeptide sequence designated herein as NBN102, which possesses the carboxy terminal 102 amino acids of a mature neublastin polypeptide, e.g. , amino acids 39-140 of SEQ ID NO:5 (SEQ ID NO:29);

(xii) the 101 AA polypeptide sequence designated herein as NBN101, which possesses the carboxy terminal 101 amino acids of a mature neublastin polypeptide, e.g., amino acids 40-140 of SEQ ID NO:5 (SEQ ID NO:30);

(xiii) the 100AA polypeptide sequence designated herein as NBNIOO, which possesses the carboxy terminal 100 amino acids of a mature neublastin polypeptide, e.g. , amino acids 41-140 of SEQ ID NO:5 (SEQ ID NO:31); and,

(xiv) the 99AA polypeptide sequence designated herein as NBN99, which possesses the carboxy terminal 99 amino acids of a mature neublastin polypeptide, e.g. , amino acids 42-140 of SEQ ID NO:5 (SEQ ID NO:32), [0136] It is understood that the truncated forms of neublastin disclosed herein (e.g. , the

1 12AA through 99AA forms) have neurotrophic activity.

[0137] In some aspects, the truncated neublastin polypeptide comprises the 99AA, 100

AA, 101AA, 102AA, 103AA, 104AA, 105AA, 106AA, 107AA, 108AA, 109AA, l l OAA, l l l AA, or 1 12AA carboxy terminal amino acids of NBN 1 13 (i. e. , NBN99, NBN100, NBN101 , NBN102, NBN103, NBN 104, NBN 105, NBN106, NBN107, NBN108, NBN109, NBN1 10, NBN1 1 1 or NBN1 12, respectively). In other aspects, the truncated neublastin polypeptide consists or essentially consists of the 99AA, 100AA, 101AA, 102AA, 103AA, 104AA, 105AA, 106AA, 107AA, 108AA, 109AA, 1 10AA, 1 1 1AA, or 1 12AA carboxy terminal amino acids of mature 1 13 neublastin polypeptide (i. e. , NBN99, NBN100, NBN101 , NBN102, NBN103, NBN104, NBN105, NBN106, NBN107, NBN108, NBN109, NBN1 10, NBN1 1 1 or NBN1 12, respectively).

[0138] The sequences can also be found in the murine and rat neublastin polypeptides as the carboxy terminal 99AA, 100AA, 101AA, 102AA, 103AA, 104AA, 105AA, 106AA, 107AA, 108AA, 109AA, 1 10AA, 1 1 1AA, or 1 12AA, respectively, in SEQ ID NOS:6 and 7. These examples of truncated neublastin forms are bioactive (referred to "bioactive truncated neublastin polypeptides") as they have been demonstrated herein to have neurotrophic activity. As stated above, neublastin dimerization is required for bioactivity, as little to no activity is observed with the neublastin monomeric polypeptide.

3. Variant Neublastins with Substantial Similarity or Identity

[0139] A variant neublastin polypeptide can also vary in sequence from the corresponding wild-type polypeptide. In particular, certain amino acid substitutions can be introduced into the neublastin sequence without appreciable loss of a neublastin biological activity. The neublastin polypeptides disclosed herein also include those neublastin polypeptides that have an amino acid sequence with substantial similarity or identity to the various prepro, pro, mature and truncated "neublastin" polypeptides set forth above.

[0140] In exemplary aspects, a variant neublastin polypeptide (i) contains one or more amino acid substitutions, and (ii) is at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical or similar to the neublastin polypeptides in SEQ ID NOS:5, 6, 7 or 17-32. The degree to which a candidate polypeptide shares homology with a neublastin polypeptide of the present disclosure is determined as the degree of similarity or identity between two amino acid sequences. A high level of sequence identity indicates a likelihood that the first sequence is derived from the second sequence. Amino acid sequence identity requires identical amino acid sequences between two aligned sequences. Thus, a candidate sequence sharing 70% amino acid identity with a reference sequence, requires that, following alignment, 70% of the amino acids in the candidate sequence are identical to the corresponding amino acids in the reference sequence.

[0.141 ] Accordingly, the instant disclosure encompasses polypeptides exhibits a degree of sequence identity of at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% with the amino acid sequences presented herein as SEQ ID NO:5 (human neublastin), SEQ ID NOS:6 and 7 (rodent neublastin), or SEQ ID NOS: 17- 32 (mature and truncated neublastins), as determined using any one of the programs described above.

[0142] As noted above, the neublastin polypeptides disclosed herein include variant polypeptides. In the context of this disclosure, the term "variant polypeptide" includes a polypeptide (or protein) having an amino acid sequence that differs from the sequences presented as SEQ ID NO:5 (human neublastin), or SEQ ID NOS:6 and 7 (rodent neublastin), or SEQ ID NOS: 17-32 (mature and truncated neublastins), at one or more amino acid positions. Such variant polypeptides include the modified polypeptides described above, as well as conservative substitutions, splice variants, isoforms, homologues from other species, and polymorphisms.

[0143] Also provided by the invention are multimeric polypeptides that include a variant neublastin polypeptide. The multimeric polypeptides are preferably provided as purified multimeric polypeptides. Examples of multimeric complexes include, e.g. , dimeric complexes. The multimeric complex can be provided as a heteromeric or homomeric complex. Thus, the multimeric complex can be a heterodimeric complex including one variant neublastin polypeptide and one non-variant neublastin or a heterodimeric complex including two or more variant neublastin polypeptides.

[0144] Typically, biological similarity, as referred to above, reflects substitutions on the wild type sequence with conserved amino acids. For example, one would expect conservative amino acid substitutions to have little or no effect on the biological activity, particularly if they represent less than 10% of the total number of residues in the polypeptide or protein. In some aspects, conservative amino acid substitutions represent changes in less than 5% of the polypeptide or protein, in particular, less than 2% of the polypeptide or protein. For example, when calculated in accordance, e.g. , with human NBNl 13, specific conservative substitutions would represent fewer than three amino acid substitutions in the wild type mature amino acid sequence. In a particularly aspect, there is a single amino acid substitution in the mature sequence, wherein both the substituted and replacement amino acid are non-cyclic.

[0145] Modifications of the primary amino acid sequence of neublastin may result in proteins which have substantially equivalent activity as compared to the unmodified counterpart polypeptide, and thus may be considered functional analogs of the parent proteins. Such modifications may be deliberate, e.g. , as by site-directed mutagenesis, or they may occur spontaneously, and include splice variants, isoforms, homologues from other species, and polymorphisms. Such functional analogs are also contemplated.

[0146] Moreover, modifications of the primary amino acid sequence may result in proteins which do not retain the biological activity of the parent neublastin polypeptide, including dominant negative forms, etc. A dominant negative protein may interfere with the wild-type protein by binding to, or otherwise sequestering regulating agents, such as upstream or downstream components, that normally interact functionally with the polypeptide. Such dominant negative forms are also contemplated.

[0147] In some aspects, the neublastin polypeptides disclosed herein comprise mutations at one or more residues expected to result in a variant neublastin polypeptide having reduced or absent heparin binding ability as compared to wild type neublastin. Accordingly, a variant neublastin polypeptide can contain an amino acid substitution, relative to SEQ ID NO: l (NBNl 13), at (i) an arginine residue at one or more of positions

48, 49, or 51 , and/or (ii) one or more of Ser 46, Ser 73, Gly 72, Arg 39, Gin 21 , Ser 20, Arg 68, Arg 33, His 32, Val 94, Arg 7, Arg 9, or Arg 14.

[0148] Unless otherwise stated, any reference herein to an arginine amino acid by position number refers to the numbering of residues relative to SEQ ID NO: l (NBN l 13). A neublastin amino acid residue designated for substitution (e.g., an arginine residue at position 48, 49, and/or 51) can be substituted with a non-conservative amino acid residue (e.g., glutamic acid) or a conservative or amino acid residue. Substitution of Arg48, Arg

49, and/or Arg51 with a non-conservative amino acid can result in a variant neublastin 11139

- 52 - polypeptide that has reduced heparin binding activity but retains (or even shows enhance) neublastin biological activity. Exemplary amino acids that can be substituted an amino acid residue identified herein (e.g. , an arginine residue at position 48, 49, and/or 51 ) include glutamic acid, aspartic acid, and alanine. In addition to the specific arginine substitutions disclosed above, a neublastin polypeptide can also contain one or more additions, substitutions, and/or deletions at other amino acid positions.

Exemplary Arg-substituted variants of NBN1 13, NBN104, and NBN99 neublastin

polypeptides are disclosed below (equivalent variants can be produced, for example, using any of the neublastin orthologs, variants, and truncations described above):

(i) Arg48-NBN1 13 variant (SEQ ID NO:33)

AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCERA RSPHDLSLASLLGAGALRPPPGSRPVSQFCCRPTRYEAVSFMDVNSTWRT VDRLSATACGCLG

(ii) Arg49-NBN1 13 variant (SEQ ID NO:34)

AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCREA RSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWRT VDRLSATACGCLG

(iii) Arg51 -NBN1 13 variant (SEQ ID NO:35)

AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRR AESPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWR TVDRLSATACGCLG

(iv) Arg48-Arg48-NBN1 13 variant (SEQ ID NO:36)

AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCEEA RSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWRT VDRLSATACGCLG

(v) Arg48 - Arg49 -NBN99 (SEQ ID NO:37)

GCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCEEARSPHDLSLASLLGAG ALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWRTVDRLSATACGCLG

(vi) Arg48-Arg49-NBN104 (SEQ ID NO:38)

AAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCEEARSPHDLSLA SLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWRTVDRLSATAC GCLG

(vii) Arg49-Arg51 -NBN 1 13 (SEQ ID NO:39)

AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCREA ESPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWRT VDRLSATACGCLG

(viii) Arg48- Arg51 -NBN 1 13 (SEQ ID NO:40)

AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCERA ESPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNST RT VDRLSATACGCLG

In some embodiments, one or more of the arginines at position 14, position 39,

position 68, or the asparagine at position 95, in the amino acid sequence of the NBN1 13 neublastin polypeptide, is replaced by an amino acid other than arginine or asparagine. In some aspects, the wild-type type amino acid is replaced with lysine or cysteine.

[0151] In some aspects, the altered residues in the variant neublastin polypeptide are chosen to facilitate coupling of a polymer such as a polyalkylene glycol polymer at the location of the modified amino acid. Preferred sites of modification of a neublastin polypeptide are those at solvent accessible regions in the neublastin polypeptide. Such sites can be chosen based on inspection of the crystal structure of the related neurotrophic factor, GDNF, whose crystal structure is described in Eigenbrot et al , Nat. Struct. Biol. 4:435-38 (1997). Sites can also be chosen based on the structural-functional information provided for persephin/neublastin chimeric proteins. These chimeras are described in Baloh et al , J. Biol. Chem. 275:3412-20 (2000). Modification sites can also be chosen based on the structure of human neublastin alone or in complexes with its receptor GFRa3 (Wang et al , Structure 14: 1083-1092 (2006); PDB codes: 2 ASK, 2GYZ, 2GYR, and 2GHO).

4. Derivative or Modified Neublastins

[0152] Neublastin polypeptides of the present disclosure also include chimeric polypeptides or cleavable fusion polypeptides in which another polypeptide (a "heterologous polypeptide") is fused at the N-tenminus or the C-terminus of the neublastin polypeptide or fragment thereof. A chimeric polypeptide may be produced by fusing a nucleic acid sequence (or a portion thereof) encoding another polypeptide to a nucleic acid sequence (or a portion thereof) encoding a neublastin polypeptide. Techniques for producing chimeric polypeptides are standard techniques well known in the art. Such techniques usually require joining the sequences such that they are in the same reading frame, and expression of the fused polypeptide under the control of the same promoter(s) and terminator.

[0153] "Heterologous polypeptide," as used when referring to an amino acid sequence, refers to a sequence that originates from a source foreign to the particular host cell, or, if from the same host cell, is modified from its original form. Exemplary heterologous sequences include a heterologous signal sequence (e.g. , native rat albumin signal sequence, a modified rat signal sequence, or a human growth hormone signal sequence) or a sequence used for purification of a variant neublastin polypeptide (e.g., a histidine tag).

[0154] The heterologous signal sequence peptides can be selected, for example, from the group consisting of a growth factor signal peptide, a hormone signal peptide, a cytokine signal peptide and an immunoglobulin signal peptide (IgSP). Thus, examples of signal peptides are signal peptides selected from the group consisting of TGFp signal peptides, GDF signal peptides, IGF signal peptides, BMP signal peptides, neurotrophin signal peptides, PDGF signal peptide and EGF signal peptide, signal peptides selected from a hormone signal peptide, said hormone being selected from the group consisting of growth hormone, insulin, ADH, LH, FSH, ACTH, MSH, TSH, T3, T4, and DHEA, or an interleukin signal peptide. In one aspect, the signal peptide is selected from the group consisting of neurturin signal peptide, GDNF signal peptide, persephin signal peptide, and NGF signal peptide. In another aspect, the signal peptide is selected from the group consisting of albumin signal peptide, modified albumin signal peptide, and growth hormone signal peptide, such as a signal peptide selected from the group consisting of rat albumin signal peptide, modified rat albumin signal peptide, and human growth hormone signal peptide, such as rat albumin signal peptide and human growth hormone signal peptide.

[0155] Thus, in some aspects, the secreted neublastin polypeptide is fused to a native rat albumin signal peptide. In other aspects, the secreted neublastin polypeptide is linked to a modified rat albumin signal sequence. In yet other aspects, the secreted neublastin polypeptide is fused to a human growth hormone signal sequence.

[0156] In yet another aspect, the signal peptide is an immunoglobulin signal peptide, such as the immunoglobulin heavy chain signal peptide. In particular, an immunoglobulin signal peptide can be a signal peptide selected from the group consisting of mouse IgSP, rat IgSP, porcine IgSP, simian IgSP, human IgSP, such as mouse IgSP or human IgSP. In some aspects, the signal peptide is a synthetic signal peptide.

[0157] The present disclosure also contemplates neublastin fusion proteins, such as Ig- fusions, as described, e.g., in U.S. Pat. Nos. 5,434, 131 ; 5,565,335; 5,541 ,087; and 5,726,044, each herein incorporated by reference, or serum albumin fusions.

[0158] The modified neublastin polypeptides can also be N-glycosylated polypeptides. In one embodiment, the Asn residue at position 122 of SEQ ID NO: 5 is glycosylated. GDNF Polypeptides

[0159] In some aspects, the methods of the present disclosure can be applied to the production of recombinant GDNF polypeptides. The terms "GDNF" or a "GDNF polypeptide," as used herein, refer to a polypeptide which possesses neurotrophic activity, The terms "GDNF" or a "GDNF polypeptide," as used herein, encompass wild-type GDNFs, truncated GDNFs (i.e. , GDNF fragments), GDNF variants, and derivative or modified GDNFs.

[0160] Mature wild type human GDNF is 134 amino acids in length (SEQ ID NO:2).

Polypeptides having the amino acid sequence of SEQ ID NO:2 or biologically active variants thereof can be used in the methods described herein. A variant GDNF polypeptide can contain one or more additions, substitutions, and/or deletions, as detailed in the following sections. A variant GDNF polypeptide can vary in length from the corresponding wild- type polypeptide. Although the mature human GDNF polypeptide (SEQ ID NO:2) consists of the carboxy terminal 134 amino acids of pre pro GDNF (SEQ ID NO: 8), not all of the 134 amino acids are required to achieve useful GDNF biological activity (e.g. , amino terminal truncation is permissible). A variant GDNF polypeptide can also vary in sequence from the corresponding wild-type polypeptide. In particular, certain amino acid substitutions can be introduced into the GDNF sequence without appreciable loss of a GDNF biological activity. In exemplary aspects, a variant GDNF polypeptide (i) can contain one or more amino acid substitutions, and (ii) can be at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:2. A variant GDNF polypeptide differing in sequence from SEQ ID NO:2 can include one or more amino acid substitutions (conservative or non-conservative), one or more deletions, and/or one or more insertions.

Neurturin Polypeptides

[0161] In some aspects, the methods of the present disclosure can be applied to the production of recombinant neurturin polypeptides. The terms "neurturin" or a "neurturin polypeptide," as used herein, refer to a polypeptide which possesses neurotrophic activity. The terms "neurturin" or a "neurturin polypeptide," as used herein, encompass wild-type neurturins, truncated neurturins (i. e., neurturin fragments), neurturin variants, and derivative or modified neurturins. Mature wild type human neurturin is 102 amino acids in length (SEQ ID N0:3). Polypeptides having the amino acid sequence of SEQ ID NO:3 or biologically active variants thereof can be used in the methods described herein. A variant neurturin polypeptide can contain one or more additions, substitutions, and/or deletions. A variant neurturin polypeptide can vary in length from the corresponding wild-type polypeptide. Although the mature human neurturin polypeptide (SEQ ID NO:3) consists of the carboxy terminal 102 amino acids of pre pro neurturin (SEQ ID NO:l 1), not all of the 102 amino acids are required to achieve useful neurturin biological activity (e.g., amino terminal truncation is permissible).

[0162] A variant neurturin polypeptide can also vary in sequence from the corresponding wild-type polypeptide. In particular, certain amino acid substitutions can be introduced into the neurturin sequence without appreciable loss of a neurturin biological activity. In exemplary aspects, a variant neurturin polypeptide (i) can contains one or more amino acid substitutions, and (ii) can be at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:3. A variant neurturin polypeptide differing in sequence from SEQ ID NO: 3 can include one or more amino acid substitutions (conservative or non- conservative), one or more deletions, and/or one or more insertions.

Persephin Polypeptides

[0163] In some aspects, the methods of the present disclosure can be applied to the production of recombinant persephin polypeptides. The terms "persephin" or a "persephin polypeptide," as used herein, refer to a polypeptide which possesses neurotrophic activity. The terms "persephin" or a "persephin polypeptide," as used herein, encompass wild-type persephins, truncated persephins (i.e. , persephin fragments), persephin variants, and derivative or modified persephins. Mature wild type human persephin is 96 amino acids in length (SEQ ID NO:4). Polypeptides having the amino acid sequence of SEQ ID NO:4 or biologically active variants thereof can be used in the methods described herein. A variant persephin polypeptide can contain one or more additions, substitutions, and/or deletions, as detailed in the following sections.

[0164] A variant persephin polypeptide can vary in length from the corresponding wild- type polypeptide. Although the mature human persephin polypeptide (SEQ ID NO:4) consists of the carboxy terminal 96 amino acids of pre pro persephin (SEQ ID NO: 14), not all of the 96 amino acids are required to achieve useful persephin biological activity (e.g., amino terminal truncation is permissible). U 2014/011139

- 57

[0165] A variant persephin polypeptide can also vary in sequence from the corresponding wild-type polypeptide. In particular, certain amino acid substitutions can be introduced into the persephin sequence without appreciable loss of a persephin biological activity. In exemplary aspects, a variant persephin polypeptide (i) can contain one or more amino acid substitutions, and/or (ii) can be at least 70%, 80%, 85%, 90%, 95%, 98% or 99% identical to SEQ ID NO:4. A variant persephin polypeptide differing in sequence from SEQ ID NO:4 may include one or more amino acid substitutions (conservative or non- conservative), one or more deletions, and/or one or more insertions.

[0166] A GDNF ligand family protein (e.g. , a neublastin polypeptide, a GDNF polypeptide, a neurturin polypeptide, or a persephin polypeptide described herein) can be a derivative or modified polypeptide, i.e., it optionally contains one or more heterologous moieties in addition to a GDNF ligand family protein. The term "heterologous," as used when referring to a moiety comprising an amino acid sequence, refers to a sequence that originates from a source foreign to the particular host cell, or, if from the same host cell, is modified from its original form. Exemplary heterologous sequences include a heterologous signal sequence (e.g. , native rat albumin signal sequence, a modified rat signal sequence, or a human growth hormone signal sequence) or a sequence used for purification of a GDNF ligand family protein (e. g. , a histidine tag). In some cases, the heterologous moieties are non-protein molecules, for example, PEG.

Improved Production of Basic Proteins

[0167] The methods disclosed here are particularly useful to increase the production yield of recombinant basic proteins. The methods disclosed herein can increase the production yield of recombinant basic proteins, for example, by reducing binding of the recombinant protein to the cell surface, releasing the recombinant basic protein from the cell surface, increasing cell growth and/or cell survival during cultivation, or reducing binding of the recombinant protein to other surfaces during harvesting or other downstream steps (e.g., reduce binding to chromatographic supports or filtration membranes). In some aspects, the recombinant protein is a not basic protein comprising at least one basic protein domain.

[0168] In some aspects, the recombinant protein is a basic protein with a pi of at least about 8, at least about 8.5, at least about 9, at least about 9.5, at least about 10, at least about 10.5, at least about 1 1 , at least about 1 1.5, at least about 12, or at least about 12.5. In some aspects, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 1 1%, at least about 12%», at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least 24% or at least 25% of the amino acids in the recombinant protein are basic amino acids. In some aspects, more that 25% of the amino acids in the recombinant protein are basic amino acids.

In some aspects, at least about 10%, at least about 1 1%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least 24%) or at least 25% of the amino acids in the recombinant protein are arginine. In some aspects, more than 25% of the amino acids in the recombinant protein are arginine.

In some aspects, the recombinant protein comprises at least one basic domain, i.e., a protein domain with a basic pi. In some aspects, the recombinant protein comprises at least two basic domains. In some aspects, the recombinant protein comprises more than two basic domains. In some aspects, at least about 5%, at least about 6%, at least about 1%, at least about 8%, at least about 9%, at least about 10%, at least about 1 1%, at least about 12%), at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%), at least about 22%, at least about 23%, at least 24% or at least 25% of the amino acids in at least one basic domain in the recombinant protein are basic amino acids. In some aspects, more that 25% of the amino acids in a basic in the recombinant protein are basic amino acids. In some aspects, at least about 10%, at least about 1 1%, at least about 12%), at least about 13%), at least about 14%), at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%o, at least about 23%, at least 24% or at least 25%o of the amino acids in a basic domain in the recombinant protein are arginine. In some aspects, more than 25% of the amino acids in a basic domain in the recombinant protein are arginine. Improved Production of Natural and Recombinant Proteins

[0172] The methods of the present disclosure can also be applied to the production of naturally synthesized proteins which are encoded by genes of the cultivated cell as well as recombinant proteins secreted by cells. The methods of the instant disclosure can be applied not only to proteins or human and animal origin, but also proteins from other sources such as plant, insects, etc., and mutated, artificial, synthetic, fusion or chimeric proteins.

[0173] The methods of the present disclosure can be applied, for example, to polypeptide- based drugs, also known as biologies. In some aspects, the polypeptides are expressed as extracellular products, which can be either secreted into the culture medium, or membrane-associated proteins (for example, peripheral membrane proteins). Recombinant proteins that can be produced using the methods disclosed herein include but are not limited to peptide hormones, growth factors, cytokines, antibodies, etc.

[0174] The method of the present disclosure can also be applied to the production antibodies or fragments thereof. The term "antibody" is used herein in its broadest sense and includes, e.g. , monoclonal antibodies, polyclonal antibodies, multivalent antibodies, multispecific antibodies, chimeric antibodies, and humanized antibodies. The term "antibody" includes whole antibodies. The term "antibody" also refers to a protein comprising at least two immunoglobulin heavy (H) chains and two immunoglobulin light (L) chains inter-connected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI , CH2 and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL.

[0175] The term "antigen-binding fragment" refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full- length antibody. Examples of binding fragments encompassed within the term " antigen- binding fragment" of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; 14 011139

- 60 -

(iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al. , (1989) Nature 341 :544-546), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0176] The term "recombinant human antibody", as used herein, includes all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as (a) antibodies isolated from an animal {e.g. , a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In certain aspects, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0177] The term "antibody" as used herein also includes "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad, Sci. USA 57:6851-6855 (1984)).

[0178] The methods to prepare and improve production {e.g. , to increase production of purified product, increase yield, increase recovery, increase the stability or batch to batch consistency of the product, etc.) disclosed herein can be applied to any surface associating protein (e.g. , a cell surface associating protein, or a protein non-specifically associating to other surfaces) that function based on surface change and/or has a high pi, wherein the addition of a sufficient amount of at least one anionic substance disclosed herein (e.g., ferric citrate, dextran sulfate, or a combination of ferric citrate and dextran sulfate) can disrupt the interaction between the protein and the surface.

Methods to Increase Production of Recombinant Proteins during Cell Culture

[0179] The present disclosure provides a method for increasing recombinant protein recovery from eukaryotic cells, comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein (e.g., neublastin, or an antibody or antigen-binding fragment thereof) in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance disclosed herein (e.g., a valproate such a sodium valproate, a citrate such as sodium citrate or ferric citrate, or dextran sulfate; a combination of a citrate such as ferric citrate and dextran sulfate; or a combination of a valproate such as sodium valproate and dextran sulfate) to increase recovery of the recombinant protein relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0180] In some aspects, the anionic substance is, for example, a valproate (e.g., sodium valproate), a malate, a succinate, a fumarate, a citrate (e.g., sodium citrate or ferric citrate), a polyaniohic compound (e.g., dextran sulfate), or a combination thereof. In a specific aspect, the anionic substance is ferric citrate. In a specific aspect, the concentration of ferric citrate is about 1 mM, 2 mM, 3 mM, 4 niM, or 5 mM. In a specific aspect, the anionic substance is sodium citrate. In a specific aspect, the U 2014/011139

- 62 - concentration of sodium citrate is about 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM. In a specific aspect, the anionic substance is valproate. In some aspects, the valproate is sodium valproate. In a specific aspect, the concentration of sodium valproate is about 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM. In another specific aspect, the anionic substance is dextran sulfate. In a specific aspect, the concentration of dextran sulfate is about 0.25 g/L. In another specific aspect, the anionic substance is a combination of ferric citrate and dextran sulfate. In some specific aspects, the anionic substance comprises ferric citrate at a concentration of about 2mM and dextran sulfate at a concentration of about 0.25 g/L. In another specific aspect, the anionic substance is a combination of a valproate and dextran sulfate. In some specific aspects, the anionic substance is a combination of sodium valproate and dextran sulfate. In some specific aspects, the anionic substance comprises sodium valproate at a concentration of about 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM and dextran sulfate at a concentration of about 1 g/L.

[0181] In some aspects, the recovery of recombinant protein is increased by at least about

10%, by at least about 15%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 35%, by at least about 40%, by at least about 45%, by at least about 50%, by at least about 55%, by at least about 60%, by at least about 65%, by at least about 70%, by at least about 75%, by at least about 80%, by at least about 85%, by at least about 90%, by at least about 95%, or by at least about 100% relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0182] Also disclosed is a method for increasing recombinant protein production from eukaryotic cells, comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein (e.g. , neublastin, or an antibody or antigen-binding fragment thereof) in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance disclosed herein (e.g., a valproate, such as sodium valproate; a citrate, such as sodium citrate or ferric citrate; dextran sulfate; a combination of a citrate such as ferric citrate or sodium citrate, and dextran sulfate; or a combination of a valproate such as sodium valproate, and dextran sulfate), to increase production of the recombinant protein relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0183] In some aspects, the anionic substance is, for example, a valproate (e.g., sodium valproate), a malate, a succinate, a fumarate, a citrate (e.g., sodium citrate or ferric citrate), a polyanionic compound (e.g. , dextran sulfate), or a combination thereof. In a specific aspect, the anionic substance is ferric citrate. In a specific aspect, the concentration of ferric citrate is about 2mM. In another specific aspect, the anionic substance is dextran sulfate. In a specific aspect, the concentration of dextran sulfate is about 0.25 g/L. In another specific aspect, the anionic substance is a combination of ferric citrate and dextran sulfate. In some specific aspects, the anionic substance comprises ferric citrate at a concentration of about 2mM and dextran sulfate at a concentration of about 0.25 g/L.

[0184] In some aspects, the anionic substance is valproate. In a specific aspect, the valproate is sodium valproate. In some specific aspects, the concentration of sodium valproate is about 1 mM, 2 rnM, 3 mM, 4 mM or 5 mM. In some aspects, the anionic substance is a combination of valproate (e.g., sodium valproate) and dextran sulfate. In some specific aspects, the anionic substance comprises sodium valproate at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM or about 5 mM, and dextran sulfate at a concentration of about 1 g/L.

[0185] In some aspects, the production of recombinant protein is increased by at least about 10%, by at least about 15%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 35%, by at least about 40%, by at least about 45%, or by at least about 50% relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0186] Also disclosed is a method for decreasing recombinant protein attachment to the surface of eukaryotic cells comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein (e.g., neublastin, or an antibody or antigen-binding fragment thereof) in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance disclosed herein (e.g. , a valproate such as sodium valproate, a citrate such as ferric citrate or sodium citrate, dextran sulfate, a combination of a citrate such as ferric citrate and dextran sulfate, or a combination of a valproate such as sodium valproate and dextran sulfate), to decrease attachment of the recombinant protein to the eukaryotic cells' surface relative to the attachment of the same recombinant protein to eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance. In some aspects, the anionic substance is, for example, a valproate, a malate, a succinate, a fumarate, a citrate, a polyanionic compound (e.g., dextran sulfate), or a combination thereof. In a specific aspect, the anionic substance is ferric citrate. In a specific aspect, the concentration of ferric citrate is about 2mM. In another specific aspect, the anionic substance is dextran sulfate. In a specific aspect, the concentration of dextran sulfate is about 0.25 g/L. In another specific aspect, the anionic substance is a combination of ferric citrate and dextran sulfate. In some specific aspects, the anionic substance comprises ferric citrate at a concentration of about 2 mM and dextran sulfate at a concentration of about 0.25 g/L. in some aspects the anionic substance is a valproate. In some aspects the valproate is sodium valproate. In some aspects, the anionic substance is a combination of sodium valproate and dextran sulfate. In some specific aspects, the anionic substance comprises sodium valproate at a concentration of about 1 mM, 2 mM, 3 mM, 4 mM, or 5 mM, and dextran sulfate at about 1 g/L.

In some aspects, attachment of recombinant protein to the eukaryotic cells' surface is decreased by at least about 10%, by at least about 20%, by at least about 30%, by at least about 40%, by at least about 50%, by at least about 60%, by at least about 70%, by at least about 80%, or by at least about 90% relative to the attachment of the same recombinant protein to eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

The present disclosure also provides a method for reducing recombinant protein- induced inhibition of eukaryotic cell growth comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein (e.g. , neublastin, or an antibody or antigen- binding fragment thereof) in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance disclosed herein (e.g. , a valproate such as sodium valproate, a citrate such as ferric citrate or sodium citrate, a dextran sulfate, a combination of a citrate such as ferric citrate and dextran sulfate, or a combination of a valproate such as sodium valproate and dextran sulfate) to reduce recombinant protein- induced growth inhibition relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

In some aspects, the anionic substance is, for example, a valproate (e.g., sodium valproate), a malate, a succinate, a fumarate, a citrate (e.g., sodium citrate or ferric citrate), a polyanionic compound (e.g. , dextran sulfate), or a combination thereof. In a specific aspect, the anionic substance is ferric citrate. In a specific aspect, the concentration of ferric citrate is about 2mM. In another specific aspect, the anionic substance is dextran sulfate. In a specific aspect, the concentration of dextran sulfate is about 0.25 g/L. In another specific aspect, the anionic substance is a combination of ferric citrate and dextran sulfate. In some specific aspects, the anionic substance comprises ferric citrate at a concentration of about 2 mM and dextran sulfate at a concentration of about 0.25 g/L. In a specific aspect, the anionic substance is sodium valproate. In another specific aspect, the concentration of sodium valproate is about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In a specific aspect, the anionic substance is a combination of sodium valproate and dextran sulfate. In some aspects, the anionic substance comprises sodium valproate at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM, and dextran sulfate at a concentration of about 1 g L.

In some aspects, growth inhibition be recombinant protein is decreased by at least about 5%, by at least about 7%, by at least about 9%, by at least about 1 1 %, by at least about 13%, by at least about 15%, by at least about 17%, by at least about 19%, or by at least about 21% relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

The present disclosure also provides a method for increasing the viability of eukaryotic cells expressing a recombinant protein comprising (a) cultivating eukaryotic cells capable of expressing the recombinant protein (e.g.. neublastin, or an antibody or antigen-binding fragment thereof) in a chemically defined cell culture medium, and (b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance disclosed herein (e.g. , a valproate T/US2014/011139

- 66 - such as sodium valproate, a citrate such as ferric citrate, dextran sulfate, a combination of a citrate such as ferric citrate and dextran sulfate, or a combination of a valproate such as sodium valproate and dextran sulfate) to increase eukaryotic cell viability relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in chemically defined cell culture medium but not subjected to the at least one anionic substance. In some aspects, the anionic substance is, for example, a valproate (e.g., sodium valproate), a malate, a succinate, a fumarate, a citrate (e.g., sodium citrate or ferric citrate), a polyanionic compound (e.g., dextran sulfate), or a combination thereof. In a specific aspect, the anionic substance is ferric citrate. In a specific aspect, the concentration of ferric citrate is about 2 mM. In another specific aspect, the anionic substance is dextran sulfate. In a specific aspect, the concentration of dextran sulfate is about 0.25 g/L. In another specific aspect, the anionic substance is a combination of ferric citrate and dextran sulfate. In some specific aspects, the anionic substance comprises ferric citrate at a concentration of about 2 mM and dextran sulfate at a concentration of about 0.25 g/L. In some aspects, the anionic substance is a valproate. In some aspects the valproate is sodium valproate. In some aspects, the concentration of is about 1 rnM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM. In some aspects, the anionic substance is a combination of sodium valproate and dextran sulfate. In some aspects, the anionic substance comprises sodium valproate at a concentration of about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM, and dextran sulfate at a concentration of about l g/L.

[0192] In some aspects, the viability of eukaryotic cells expressing a recombinant protein is increased by at least about 5%, by at least about 10%, by at least about 15%, by at least about 20%, by at least about 25%, by at least about 30%, by at least about 35%, by at least about 40%, by at least about 45%, by at least about 50%, by at least about 55%), by at least about 60%, by at least about 65%>, by at least about 70%, by at least about 75%, by at least about 80%>, by at least about 85%, by at least about 90%, by at least 95%>, or by at least about 21% relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in chemically defined cell culture medium but not subjected to the at least one anionic substance.

[0193] In some aspects, the eukaryotic cells used in the above disclosed method are mammalian eukaryotic cells. In some aspects, the mammalian eukaryotic cells are primary cells or immortalized cells such as kidney, bladder, lung, liver cardiac muscle, smooth muscle, ovary or gastrointestinal cells. In certain aspects, the cells are human embryonic kidney cells such as HEK293, HEK293T, HEK293F or HEK293H. In other aspects, the cells are Chinese hamster ovary (CHO) cells. In one specific aspect, the CHO cells are CHO DG44 cells. In other aspects, the cells are hybridomas or insect cells.

[0194] In some specific aspects, cells are adapted to be cultivated under serum-free conditions. Accordingly, in some specific aspects, the cell culture medium is serum free. In other aspect, the cell culture medium has less than 10% of mammalian serum (by volume). In other aspect, the cell culture medium has less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1 % of mammalian serum (by volume). In one specific aspect, the cell culture medium contains no more than about 1% mammalian serum by volume. In another specific aspect, the cell culture medium is protein free.

[0195] In some specific aspects, the increasing or adjusting of the concentration of the anionic substance in the cell suspension (e.g. , cells in the cell culture medium or a suspension of cells isolated from the culture medium) is effected by adding to the cell suspension a concentrated solution comprising at least one anionic substance of the present disclosure in an amount sufficient to cause at least one of the above disclosed effects (i.e. , increasing recombinant protein recovery, and/or increasing recombinant protein production, and/or decreasing recombinant protein attachment to the surface of eukaryotic cells, and/or reducing recombinant protein-induced inhibition of eukaryotic cell growth, and/or increasing the viability of eukaryotic cells expressing a recombinant protein).

[0196] In other aspects, the culture medium or release composition are formulated with at least one anionic substance of the present disclosure iri an amount sufficient to cause at least one of the above disclosed effects (i. e. , increasing recombinant protein recovery, and/or increasing recombinant protein production, and/or decreasing recombinant protein attachment to the surface of eukaryotic cells, and/or reducing recombinant protein- induced inhibition of eukaryotic cell growth, and/or increasing the viability of eukaryotic cells expressing a recombinant protein). In yet other aspects, the increasing or adjusting of the concentration of the anionic substance in the cell suspension (e.g. , cells in the cell 9

- 68 - culture medium or a suspension of cells isolated from the culture medium) is effected by adding to the cell suspension at least one anionic substance of the present disclosure in solid form or as a slurry in an amount sufficient to cause at least one of the above disclosed effects (i. e. , increasing recombinant protein recovery, and/or increasing recombinant protein production, and/or decreasing recombinant protein attaclinient to the surface of eukaryotic cells, and/or reducing recombinant protein-induced inhibition of eukaryotic cell growth, and/or increasing the viability of eukaryotic cells expressing a recombinant protein).

In some aspects, the anionic substance can be added 1 to 4 weeks prior to the separation of the recombinant protein from a cell suspension (e.g. , cells in the cell culture medium or suspension of cells isolated from the culture medium). For example, the anionic substance can be added in 1 about week, in about 2 weeks, in about 3 weeks, or in about 4 weeks prior to the separation of the recombinant protein. In other aspects, the anionic substance can be added 1 to 7 days prior to the separation of the recombinant protein. For example, the anionic substance can be added about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days prior to the separation of the recombinant protein. In some other aspects, the anionic substance can be added 1 to 24 hours prior to the separation of the recombinant protein. In other aspects, the anionic substance can be added 1 to 3 hours prior to the separation of the recombinant protein. For example, the anionic substance can be added about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23 or 24 hours prior to the separation of the recombinant protein. In other aspects, the anionic substance can be added 1 to 60 minutes prior to the separation of the recombinant protein. For example, the anionic substance can be added about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes prior to the separation of the recombinant protein.

In some aspects, the anionic substance is added in one step to reach the desired final concentration. In other aspects, the anionic substance is added in a plurality of discrete steps to reach the desired final concentration. In some aspects, the final concentration of the anionic substance can be reached gradually during a 1 to 4 weeks period prior to the separation of the recombinant protein from a cell suspension (e.g. , cells in the cell culture medium or suspension of cells isolated from the culture medium). For example, the final concentration of the anionic substance can be reached gradually in about, about 2 week, about 3 weeks, or about 4 weeks prior to the separation of the recombinant protein. In other aspects, the final concentration of the anionic substance can be reached gradually during a 1 to 7 days period prior to the separation of the recombinant protein. For example, the final concentration of the anionic substance can be reached gradually during a period of about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, or about 7 days prior to the separation of the recombinant protein.

In some other aspects, the final concentration of the anionic substance can be reached gradually during a 1 to 24 hours period prior to the separation of the recombinant protein. In other aspects, the final concentration of the anionic substance can be reached gradually during a 1 to 3 hours period prior to the separation of the recombinant protein. For example, the final concentration of the anionic substance can be reached gradually during a 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours period prior to the separation of the recombinant protein. In other aspects, the final concentration of the anionic substance can be reached gradually during a 1 to 60 minutes period prior to the separation of the recombinant protein. For example, the final concentration of the anionic substance can be reached gradually during a period of about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 or 60 minutes prior to the separation of the recombinant protein. In some aspects, the anionic substance can be added using dialysis or filtration, e.g., diafiltration.

In some aspects, the cells are grown and maintained at a density of at least 10⁵

cells per ml of culture medium. In certain aspects, cells are grown and maintained at a density between at least 10⁵ and at least 5xl0⁶ cells per ml of culture medium. The cell seeding densities for each experimental condition can be optimized for the specific culture conditions being used. For monolayer culture in plastic culture vessels, an initial seeding density of 1 -5 x 10⁵ cells/cm² can be generally used, while for suspension cultivation a higher seeding density (e.g., 5-20 x 10⁵ cells/cm²) can be generally used. Mammalian cells are typically cultivated in a cell incubator at about 37°C. The incubator atmosphere can be humidified and contain about 3-10% carbon dioxide in air. In general, 8-10% carbon dioxide in air is used, although cultivation of certain cell lines can require as much as 20% carbon dioxide in air for optimal results. Cells in closed or batch culture generally undergo complete medium exchange (i.e. , replacing spent media with fresh media) when the cells reach a density of about 1.5-2.0 x 10⁶ cells/ml. Cells in perfusion culture (e.g. , in bioreactors or fermenters) receive fresh media on a continuously recirculating basis.

[0201] Most of cultured cells have a fairly wide tolerance to osmotic pressure. An osmolarity of approximately 290 mOsm is considered optimal for the culture of mammalian cells in vitro, although normal osmolarity can be different for other species. In practice, osmolarities between 260 mOsm and 320 mOsm are quite acceptable for most mammalian cell cultures. In some aspects, the anionic substances of the present disclosure are used in culture media or release compositions with osmolarities adjusted between about 250 mOsm and about 600 mOsm. In other aspects, the osmolarity is adjusted between 265 to about 280 mOsm. In other aspects, the osmolarity is adjusted between about 265 mOsm and about 275 Osm. If desired, the osmolarity can be increased by adding a suitable salt, e.g., NaCl, or other suitable substance such as sucrose, mannitol or polyethylene glycol. In some aspects, cells are cultivated under hyperosmolar conditions.

[0202] In some aspects, the cultivation process includes a growth or proliferation phase and an induction phase. During the growth phase, the cells are cultured in conditions that promote cell growth. During the induction phase, the cells are cultured in conditions that promote production of the recombinant protein interest, which is produced by the cells in responds to physical (e.g. , temperature change) or chemical change (e.g. , addition of a chemical compound) that triggers recombinant protein production. In some aspects, the anionic substance is added during the induction phase but not during the growth or proliferation phase. In other aspects, the concentration of anionic substance is kept constant during cultivation. In other aspects, the concentration of anionic substance is increased or decreased during cultivation.

[0203] In some aspects, the eukaryotic cells are grown in a fed batch process comprising several phases, namely a growth phase and a production. In some aspects, the anionic substance is added to the cell culture medium at inoculation and/or during the growth phase and/or during the production phase. The term "inoculation" refers to the addition of cells to the cell culture medium to begin the culture process. In other aspect, the anionic substance is added after inoculation, e.g., during the growth phase or the production phase. [0204] In other aspects, the eukaryotic cells are grown in a continuous process. Under these experimental conditions, cell can be continuously cultivated in the presence of at least one the anionic substances disclosed herein, such that recombinant protein is continuously released from the cell surface. The recombinant protein can then removed from the culture medium using, for example, a continuous centrifuge or diafiltration over a micro filter membrane. This approach allows the protection of sensitive protein from proteolytic degradation, or protection of concentration sensitive proteins from aggregation, due to the fast removal of the recombinant protein from the cell culture.

[0205] In some aspects, the concentration of anionic substance is non-physiological, i.e., a concentration of the anionic substance which is higher than the concentration in the cell or in the cell culture medium under normal cultivation conditions. In some aspects, the anionic substance or combination thereof is added to reach the equilibration balance within recombinant protein and eukaryotic cell surface, sufficient to disrupt ionic binding and release bound recombinant proteins from the eukaryotic cell surface without destroying the cell.

[0206] In some aspects, the concentration of anionic substance is selected in such a manner that the viability of the cells is maintained. In addition, the viability of the cells can be maintained by harvesting the recombinant protein (e.g. , neublastin, or an antibody or antigen-binding fragment thereof) and replacing the cell culture medium with fresh culture medium, to enable a cyclic production process of the recombinant protein. The recombinant protein can be harvested directly from the culture medium, and/or it can be recovered after the application of a release composition to cell isolates from the cell culture, e.g. , after centrifugation, and resuspended in a release composition comprising at least of the anionic substances disclosed herein. Using this approach, recombinant protein could be isolated from the cell culture medium, and additional amount of recombinant protein can be recovered after being released from the cell surface after the application of the release composition.

[0207] Accordingly, the present disclosure also provides a method in which any of the methods disclosed above further comprises at least one of the following steps:

(a) isolating the recombinant protein (e.g., neublastin, or an antibody or antigen-binding fragment thereof) from the culturing medium; 4 011139

- 72 -

(b) separating the culture medium from the cultivated eukaryotic cells, resulting in two separate fractions, a fraction of cultivated eukaryotic cells and a fraction of liquid medium;

(c) contacting or resuspending the fraction of cultivated eukaryotic cells with a release composition comprising a sufficient amount of at least one anionic substance selected from valproate, malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof to release the recombinant protein from the eukaryotic cell surface.

(d) separating the release composition from the eukaryotic cells, resulting in two separate fractions, a fraction of eukaryotic cells and a fraction of release composition comprising the recombinant protein released from the eukaryotic cell surface;

(e) isolating the recombinant protein from the fraction of release composition; and,

(f) suspending the fraction of eukaryotic cells in culture medium and reculturing.

Recombinant Protein Purification

[0208] The recombinant protein of interest (e.g. , neublastin, or an antibody or antigen- binding fragment thereof) can be recovered from the cultured medium after release from the cell surface, or from a release buffer applied to the cultured cells after harvesting. In some aspects, the anionic substances disclosed herein can be used during cultivation and/or during harvest and/or during purification. Generally, after separating the culture medium or release buffer containing the recombinant protein of interest from the cells and other particulate debris, e.g. , by centrifugation, filtration, diafiltration, tangential filtration, dead end filtration, microfiltration, electric fields, magnetic fields, or ultrafiltration, the recombinant protein can be purified from contaminant soluble proteins and polypeptides.

[0209] The following procedures exemplary of suitable recombinant protein purification procedures: imniuno-affinity chromatography, affinity chromatography, protein precipitation (e.g., using ethanol, polyethylene glycol, or ammonium sulfate), buffer exchanges, ionic exchange chromatography, hydrophobic interaction chi omaiography, mixed mode hydrophobic/ion exchange chromatography media, chelating chromatography, carbohydrate affinity (e.g. , lectin or heparin affinity chromatography), size-exclusion chromatography, electrophoresis (e.g. , SDS-PAGE), dialysis, hydroxyl apatite adsorption, filter membrane adsorption, gel filtration, HPLC, ultrafiltration, diafiltration, etc. In some aspects, recombination protein purification can be conducted using different precipitation agent, such as polyethylene glycol, ammonium sulfate, or ethanol.

[0210J One skilled in the art will appreciate that numerous methods known in the art can be applied to the purification of the recombinant protein of interest, and that such methods can require modification to account for changes in the character of the recombinant protein upon expression in recombinant cell culture. In some aspects of the present disclosure, a GDNF ligand family protein (e.g. , a neublastin polypeptide, GDNF polypeptide, neurturin polypeptide, or persephin polypeptide) is purified using at least one of the techniques disclosed above. In specific aspects of the present disclosure, the purified recombinant protein is neublastin. In some aspects of the present disclosure, an antibody or antigen-binding fragment is purified using at least one of the techniques disclosed above.

Pharmaceutical Compositions

[0211] A recombinant protein produced according to the methods disclosed herein can be incorporated into a pharmaceutical composition containing a therapeutically effective amount of the recombinant protein and a pharmaceutically acceptable carrier. In some specific aspects, the recombinant protein is a GDNF ligand family protein. In other aspects, the protein is an antibody or antigen-binding fragment thereof. Accordingly, a GDNF ligand family protein (e.g., a neublastin polypeptide, GDNF polypeptide, neurturin polypeptide, or persephin polypeptide) or an antibody or antigen-binding fragment thereof produced according to the methods disclosed herein, can be incorporated into a pharmaceutical composition. In some aspects, the pharmaceutical composition comprises a therapeutically effective amount of a GDNF ligand family protein, or combination thereof. In other aspects, the pharmaceutical composition comprises a therapeutically effective of an antibody or antigen-binding fragment thereof, or a combination thereof. In addition to one or more recombinant proteins produced according to the method disclosed here ( e.g. , GDNF ligand family protein, or antibodies or antigen-binding fragments thereof), a pharmaceutical composition can contain other pharmaceutically active compounds, e.g. , heparin or heparan sulfate, and also contain one or more adjuvants, excipients, carriers, and/or diluents. 14 011139

[0212] Acceptable diluents, carriers and excipients typically do not adversely affect a recipient's homeostasis (e.g. , electrolyte balance). Acceptable carriers include biocompatible, inert or bioabsorbable salts, buffering agents, oligo- or polysaccharides, polymers, viscosity-improving agents, preservatives and the like. One exemplary carrier is physiologic saline (0.15 M NaCl, pH 7.0 to 7.4). Another exemplary carrier is 50 mM sodium phosphate, 100 mM sodium chloride. Further details on techniques for formulation and administration of pharmaceutical compositions can be found in, e.g. , Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).

Examples

Example 1

Release on Neublastin from the Cell Surface

[0213] To determine whether treatment with polyanionic molecules could release recombinantly expressed neublastin from the cell surface, a wide variety of polyanionic and non-polyanionic compounds and experimental conditions was used. Neublastin expressing cells (cell lines denoted as N65 and 902) were cultivated at 36°C in shake flasks, shaking at 125rpm in an incubator maintained at 5% C02. Both N65 and 902 cell lines were based on Chinese Hamster Ovary (CHO) DG44 host cells. Neublastin expression was induced based on standard selection procedures using DHFR coexpression. Cells were routinely grown in chemically defined proprietary CM3 medium as described in Huang et al , Biotechnology Progress 26: 1400-1410 (2010). Cells were routinely maintained in 1 L and 3L shake flasks with 200 mL and 1L working volumes, respectively. No additives were included in the basal medium except for ferric citrate, which was included at a 0.2 mM concentration.

[0214] Neublastin expressing cells were split into 10 ml aliquots in 15 ml Corning centrifuge tubes. Neublastin drug substance (DS) at 4.2 mg/ml was spiked into the 10 ml aliquots at 1% of the total culture volume. Additives as indicated in FIG. 1 were added to the cultures using stock solutions at least 10 fold concentrated from the final targeted concentration. The additives and experimental conditions corresponding to the experimental data shown in FIG. 1 were: : (1) 902 cells with 50 mM Na₂S0₄ without addition of neublastin, and without addition of anionic substance; (2) 902 cells with added neublastin, but without treatment with anionic substance; (3) 902 cells with added neublastin, treated with 5 mg/mg Arginine, (4) 902 cells with added neublastin, treated with 50 mM NaCl, 5mM Tris pH 7; (5) 902 cells with added neublastin, treated with 100 niM malate; (6) 902 cells with added neublastin, treated with 100 mM succinate; (7) 902 cells with added neublastin, treated with 50 mM Na₂S0 , 5 mM Tris pH 7; (8) 902 cells with added neublastin, treated with 50 mM Na₂S0₄, 5 mM Tris pH 7, at 36°C; (9) 902 cells with added neublastin, treated with 50 mM Na₂S0₄; (10) 902 cells with added neublastin, treated with 100 mM fumarate; (11) 902 cells with added neublastin, treated with 50 mM K₂S0₄, 5 mM Tris pH 7; (12) 902 cells with added neublastin, treated with 50 mM ferric citrate; (13) 902 cells with added neublastin, treated with 100 mM sodium citrate, pH 7; (14) 902 cells with added neublastin, treated with 0.01 g/L dextran sulfate; (15) 902 cells with added neublastin, treated with 0.1 g/L dextran sulfate; (16) 902 cells with added neublastin, treated with lg/L dextran sulfate; (17) 902 cells with added neublastin, treated with 1 mM citrate; (18) 902 cells with added neublastin, treated with 10 mM citrate; (19) 902 cells with added neublastin, treated with 100 mM citrate; (20) 902 cells; (21) N65 cells without addition of neublastin, and without addition of anionic substance; (22) N65 cells with added neublastin, but without treatment with anionic substance; (23) N65 cells with added neublastin, treated with 0.01 g/L dextran sulfate; (24) N65 cells with added neublastin, treated with 0.1 g/L dextran sulfate; (25) N65 cells with added neublastin, treated with lg/L dextran sulfate; (26) N65 cells with added neublastin, treated with ImM citrate; (27) N65 cells with added neublastin, treated with l OmM citrate; and (28) N65 cells with added neublastin, treated with l OOmM citrate.

[0215] Cells were incubated with neublastin in medium supplemented with anionic substances at the concentrations shown above. The cells/neublastin/medium mixtures were all diluted by 10% using a concentration stock solution of lOx anionic substance. Thus, for example, the treatment with 50 mM K₂S0₄, 5 mM Tris pH 7 was accomplished by adding 500 mM K₂S0₄, 50 mM Tris pH 7 at 10% of the final volume of cells plus the buffer.

[0216] Cultures were allowed to incubate for up to 1 hour with or without gentle shaking at ambient temperature or at 36°C. After the incubation period, the cultures were centrifuged and the supernatants were analyzed for the presence of neublastin. The presence of neublastin was detected using a 2-dimensional liquid chromatography assay (2DLC). The 2DLC assay is incompatible with dextran sulfate.

[0217] Addition of anionic compounds, particularly sodium citrate and ferric citrate, were effective in releasing neublastin from the cell surface. Sulfates and polysulfates (e.g. , dextran sulfate) were also effective, their effect being more pronounced than the effect observed when sodium chloride was used. Dextran sulfate was observed to bind to neublastin, preventing accurate titer measurements.

Example 2

Neublastin-Induced Cell Growth Inhibition

[0218] Neublastin expressing cells (cell lines denoted as N65 and 902) were cultivated at

36°C in shake flasks, shaking at 125rpm in an incubator maintained at 5% C02. Cultures were run in duplicate. Each condition was either untreated with neublastin (neat), received a 1% addition of empty neublastin drug substance buffer, or received at 1 % addition of neublastin drug substance (40 mg/L). Samples were taken daily and analyzed for cell growth. All cell lines tested showed a decrease in growth upon addition of neublastin drug substance only (FIG. 2).

[0219] Cells were grown in chemically defined proprietary basal medium, CM3, as described in Huang et al. , Biotechnology Progress 26: 1400-1410 (2010). Briefly, cells were inoculated at 2xl0⁵ cells/mL for 902 cells, and at 3xl0⁵ cells/mL for N65 cells in 125 mL shake flasks with 30 mL working volumes. Cell counts were performed using a CEDEX counter (Innovatis AG, Bielefeld, Germany).

Example 3

Addition of Dextran Sulfate and/or Ferric Citrate Relieves

Neublastin-Induced Cell Growth Inhibition

[0220] Neublastin expressing cells, 902, were cultivated at 36°C in shake flasks, shaking at 125rpm in an incubator maintained at 5% C02. Cultures were supplemented with a 0%, 1%, 3%, or 5% neublastin (Drug Substance) addition by volume. Cells were cultivated for 3 days in basal medium containing anionic substances, namely, ferric citrate, dextran sulfate, or dextran sulfate and ferric citrate. Ferric citrate was used at 2.3 mM and dextran sulfate at 0.25 g/L. The black bar in FIG. 3 indicates the inhibition observed for 1% neublastin addition without the addition of anionic substances. Growth inhibition by neublastin was relieved upon inclusion of anionic substances to the culture medium. Higher growth was observed even when 5% neublastin was added along with anionic substances to the medium as compared to 1 % neublastin added to the medium but without the addition of any anionic substances (FIG. 3).

[0221] Cells were grown in chemically defined proprietary basal medium, CM3, as described in Huang et al, Biotechnology Progress 26: 1400-1410 (2010). Briefly, cells were inoculated at 2x10³ cells/mL in 125 niL shake flasks with 30 mL working volumes of CM3 medium containing the specified concentration of anionic substance. Cell counts were performed using a CEDEX counter (Innovatis AG, Bielefeld, Germany).

Example 4

Addition of Dextran Sulfate or Ferric Citrate Increases High Peak Cell Densities in Shake Flask cultures

[0222] Neublastin expressing cells (902 cells) were cultivated at 36°C in shake flasks, shaking at 125rpm in an incubator maintained at 5% C02. Cultures were maintained in a fed batch mode for 10 days. Samples consisted of cell cultivated without the addition of exogenous neublastin (NBN) and without treatment with anionic substances (positive control), cells cultivated with exogenous NBN but without treatment with anionic substances (negative control), cells cultivated with dextran sulfate (with or without exogenous NBN), and cells cultivated with ferric citrate (with or without exogenous NBN) (FIG. 4). Ferric citrate was used at 2.3mM and dextran sulfate was used at 0.25 g/L. Cultures maintained in medium containing ferric citrate or dextran sulfate grew to higher peak viable cells densities than cultures not containing additives. This was true regardless of whether exogenous neublastin was added.

[0223] Cells were grown in chemically defined proprietary basal medium, CM3, as described in Huang et al , Biotechnology Progress 26: 1400-1410 (2010). Briefly, cells were inoculated at 2xl0⁵ cells/mL in 125 mL shake flasks with 30 mL working volumes of CM3 medium containing the specified concentration of anionic substance. Cell counts were performed using a CEDEX counter (Innovatis AG, Bielefeld, Germany). Example 5

Addition of Ferric Citrate or Ferric Citrate/Dextran Sulfate Increases Viable Cell Density in Bioreactor Cell Cultures

[0224] Neublastin expressing cells (902 cells) were cultivated at 36°C in a 5L bioreactor.

Cultures were maintained in fed batch mode for 17 days. Cultures containing (i) 2.3 mM ferric citrate in basal medium, (ii) 2.3 mM ferric citrate in basal medium and 2.4 mM in feed medium, or (iii) 2.3 mM ferric citrate and 0.25 g/L dextran sulfate in basal medium all grew to higher peak viable cell densities than control cultures (FIG. 5).

[0225] Cells were cultivated in bioreactors and fed according to the fed batch protocol described in Huang, et al. (2010). Daily cell counts and viability were determined using a CEDEX counter (Innovatis AG, Bielefeld, Germany).

Example 6

Addition of Dextran Sulfate to Feed Increases Productivity in Shake Flask Cultures

[0226] Neublastin expressing cells (902 cells) were cultivated at 36°C in shake flasks, shaking at 125rpm in an incubator maintained at 5% C02. Cultures were maintained in a fed batch mode for 13 days. Basal medium consisted of CM3 medium supplemented with 0.25 g/L dextran sulfate. Feed medium was supplemented with either 0 g/L, 0.25 g/L, 1.25 g/L, or 2.5 g/L dextran sulfate (FIG. 6). Supernatant titer was measured using ELISA and an antibody specific to neublastin. Titer increases in response to increased dextran sulfate added to the culture despite similar cell growth. Consequently, feeding dextran sulfate lead to an increased cell specific productivity.

Example 7

Increase of Ferric Citrate in Basal Medium or Addition of Sodium Citrate at Harvest Increases Titer in Bioreactor Cell Cultures

[0227] Neublastin expressing cells (902 cells) were cultivated at 36°C in a 5L bioreactor.

Cultures were maintained in fed batch mode for 17 days. Cells were cultivated in bioreactors and fed according to the fed batch protocol described in Huang, et al. (2010). Daily cell counts and viability were determined using a CEDEX counter (Innovatis AG, Bielefeld, Germany). Sodium citrate was added to cell culture samples as described above. Titer was measured using 2DLC from the supernatant before and after the citrate treatment.

[0228] Cultures containing (i) 2.3 mM ferric citrate in basal medium produced over 70% more titer than cells grown in CM3, and (ii) 100 mM citrate addition at harvest resulted in 50 to over 150% increase in titer compared to CM3 supernatant (FIG. 7). While increasing the concentration of ferric citrate in the basal medium increased cell growth, such growth increase alone was not sufficient to account for the increase in titer. Therefore, the increase in titer indicated that the addition of anionic substance during harvest caused an increase in productivity by releasing neublastin from the cell surface.

Example 8

Addition of Valproate (VP A) to CHO Cell Fed-batch Cultures Improves Monoclonal Antibody Titers

[0229] Improving the productivity of a biopharmaceutical CHO fed-batch cell culture can enable cost savings and more efficient manufacturing capacity utilization. In this study, the effect of valproate (VPA, valproic acid, 2-propylpentanoic acid), a branched monocarboxylic acid, on the productivity of three of our CHO cell lines that stably express monoclonal antibodies was examined. Fed-batch shake flask VPA titrations on the three different CHO cell lines yielded cell line-specific results. Cell line A responded highly positively, cell line B responded mildly positively, and cell line C did not respond. Factorial experiments were then performed to identify the optimal VPA concentration and day of addition for cell line A. After identifying the optimal conditions for cell line A, verification experiments were performed in fed-batch bioreactors for cell line A and B. These experiments confirmed that a high dose of VPA late in the culture could increase harvest titer >20% without greatly changing antibody aggregation or charge heterogeneity. These results indicated that VPA can function as a small molecule enhancer of protein production for biopharmaceutical CHO cell culture processes.

[0230] The majority of today's biopharmaceutical products are made using Chinese hamster ovary (CHO) cell culture with host cells that stably express a recombinant therapeutic protein-of-interest. Since cell culture is the first step in any biologic manufacturing process, improving the productivity of the upstream process plays a key role in reducing the cost of and increasing the manufacturing capacity for bringing biologic drugs to market_* 14 011139

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[0231] In addition to cell line engineering (Kramer et al. (2010) Appl. Microbiol.

Biotechnol. 88:425-436; Kim et al. (2012) Appl. Microbiol. Biotechnol. 93 :917-930) and media and process optimization (Li et al. (2010) rnAbs 2:466-479) approaches for increasing productivity, small molecule enhancers can also be used. One small molecule approach for rapidly increasing CHO cell productivity is the administration of carboxylic acids such as hydroxamic acids (Allen et al. (2008) Biotechnol. Bioeng. 100:1 193-1204) or alkanoic acids (Liu et al. (2001) J. Biosci. Bioeng. 91 :71-75) to CHO cell cultures, the most widely-used of which is sodium butyrate (Mimura et al. (2001) J. Immunol. Methods. 247:205-216; Jiang et al. (2008) Biotechnol. Bioeng. 100:189-194; Jeon et al. (2007) J. Microbiol. Biotechnol. 17:1036-1040; Hendrick et al. (2001) Cytotechnology . 36:71-83; Kantardjieff et al. (2010) J. Biotech. 145:143-159).

[0232] The instant study focused on the effects of valproic acid (VPA, 2-propylpentanoic acid) on the productivity of stably-transfected CHO cells. VPA is a branched-chain carboxylic acid. VPA has been shown to improve recombinant protein expression in transiently-transfected CHO (Backliwal et al. (2008) Biotechnol. Bioeng. 101 : 182-189) and HEK293 (Backliwal et al. (2008) Nucl. Acids Res. 36.Έ96-Ε98) cells. US 2009/0023186 Al disclose the use of VPA to improve recombinant protein production in cell culture processes that use stable gene expression.

[0233] Sodium valproate, a salt form of VPA, was used to increase the antibody harvest titers in our CHO cell lines. Cell-specific effects of VPA in a fed-batch shake flask model using three CHO cell lines were evaluated, identifying two out of the three cell lines as responsive to VPA. The shake flask model was then used t in a factorial experiment to determine the optimal concentration and timing of VPA administration for the most responsive cell line. Finally, the optimal VPA concentration and day of addition was verified in a fed-batch bioreactor model and showed that VPA can increase antibody harvest titers over 20% in bioreactors without compromising product quality.

Materials and Methods

[0234] Cell lines and media: Three different CHO cell lines producing three different monoclonal antibodies were used in this study. The stable expression system used by cell lines A, B, and C was based on DHFR amplification (Pallavicini et al. (1990) Mol. Cell. Biol. 10:401-404; Gandor et al. (1995) FEBS Lett. 377:290-294). Cells in this study were all grown using proprietary chemically-defined basal and feed media (Huang et al. (2010) Biotech. Progress 26: 1400-1410; Kshirsagar et al. (2012) Biotechnol. Bioeng. 109:2523- 2532). Dextran sulfate (DS) with an average molecular weight of 5,000 Da and sodium valproate (Sigma-Aldrich, St. Louis, MO) were used as cell culture media supplements in this study.

[0235] Seed cultures: All three cell lines were thawed and grown as previously described

(Huang et al. (2010) Biotech. Progress 26: 1400-1410; Kshirsagar et al. (2012) Biotechnol. Bioeng. 109:2523-2532). Cells were passaged in 1L or 3L shake flasks (Corning, NY) every 3-4 days using chemically-defined basal media with incubator settings of 36°C and 5% C0₂.

[0236] Fed-batch shake flask cultures: The fed-batch shake flask cultures for cell lines A,

B, and C were performed in 500-mL shake flasks (Corning, NY) with 75-rnL working volumes in a humidified INFORS incubator (INFORS AG, Bottmingen, Switzerland) at 35°C and 5% C0₂. Agitation was set at 150 rpm. All cells were seeded at 4 x 10⁵ vc/mL in chemically-defined basal media containing 1 g/L dextran sulfate. Feed media were administered on day 3 and day 5 onwards daily until the day before harvest and culture termination. The feed amount was calculated as a pre-determined fixed percentage based on current culture volume. Glucose stock solution was added as necessary. ImM, 2mM, or 3mM VPA was added on Day 8 of the culture.

[0237] A rotatable central composite design fed-batch shake flask experiment to optimize

VPA concentration and timing of addition for cell line A was designed using Design- Expert 8 statistical software (Stat-Ease, Minneapolis, MN). The concentration of VPA ranged from 0 to 4mM and the timing of VPA addition ranged from day 4 to day 10. Since the VPA concentration and timing affected cell growth, growth-based feeding was implemented for this particular experiment. Feeding was proportional to the integral of viable cells (IVC), which was determined from the area under the viable cell density curve and is estimated by using a sum of trapezoids approximation across the desired time interval.

[0238] Fed-batch bioreactor cultures: The fed-batch production bioreactors for cell lines

A and B were performed in 5-L Applikon bioreactors with 2.5-L initial working volumes. Like the fed-batch shake flask experiments, all cells were seeded at 4 x 10⁵ vc/mL in chemically-defined basal media with or without lg/L dextran sulfate. For cell line A, lg/L dextran sulfate was present on day 0 for conditions containing dextran sulfate. For 14011139

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glycan structures were labeled with 2-aminobenzamide (2-AB). Labeled glycans were then analyzed using HILIC-UPLC.

Results and Discussion

[0242] Enhancement of CHO cell productivity by VPA varies from cell line to cell line:

VPA titrations were performed on cell lines A, B, and C. lmM, 2mM, or 3mM VPA was added to fed-batch shake flask cultures on day 8. Experiments were performed using fed- batch shake flasks containing 1 g/L dextran sulfate. 1 g/L dextran sulfate was added on Day 0 and varying concentrations of VPA were added on Day 8. A slightly toxic effect of VPA on cell growth was observed on all cell lines; VCD and cell viability declined in the days following VPA addition (FIG. 8A, FIG. 8B, FIG. 9 A, FIG. 9B).

[0243] VPA increased the titer and specific productivity of our stably-transfected CHO cells, confirming the results observed in transiently-transfected CHO cells (Backliwal et al. (2008) Biotechnol. Bioeng. 101 : 182-189). However, the effect of VPA on productivity varied from cell line A to C. VPA improved the harvest titer of cell line A by 15% (FIG. 8C) and harvest titer of cell line B by 10% (FIG. 9C). There was also an increase in specific productivity due to the depressed growth (FIG. 8D, FIG. 9D). The cell-specific effect of VPA could be a manifestation of specific membrane properties..

[0244] Enhancement of CHO cell productivity by VPA is a function of concentration and timing: Since VPA was observed to be a cytotoxic compound in the titration experiments for cell lines A, B, and C, the optimum concentration and timing of VPA administration was next determined. Cell line A was used as the model cell line since it had the largest response in magnitude to VPA.

[0245] A 2-factor central composite design examining the interaction of VPA concentration (OmM to 4mM) and addition timing (day 4 to day 10) was used to determine the response of VCD, viability, IVC, harvest titer, and specific productivity as a function of VPA concentration day of addition. In agreement with the titration experiments, toxicity with increased concentrations of VPA was observed.

[0246] Though adding low concentrations of VPA early in the culture increased specific productivity, it also led to growth inhibition. This growth inhibition reduced the cell mass available to make protein. Thus, the harvest titer on day 15 for the early VPA addition condition was similar to that of the control. The best time to minimize the effects of a growth-inhibiting, cytotoxic compound were later in the culture. Indeed, this was observed in the factorial experiment as there is a reduction in toxicity when VPA is added later in the culture duration (FIG. 10).

[0247] The majority of the models were one-factor effects, except that of harvest titer.

VPA concentration and day of addition were insignificant as single factors in determining harvest titer, but their two-factor interaction was statistically significant. The results suggested that the maximal harvest titer could be obtained by adding a high doses of VPA later in the culture.

[0248] VPA addition in bioreactors increases antibody titers without changing product quality: Based on the results of the fed-batch shake flask design-of-experiment study, confirmatory fed-batch bioreactor experiments were performed where the effect of VPA on the growth, viability, and productivity of cell line A were examined. Addition of VPA was again slightly cytotoxic, reducing growth and viability (FIG. 1 1 A, FIG. 1 IB). The bioreactor experiments confirmed the results of the shake flask experiments, showing that VPA increased the harvest titer compared to the control (FIG. 1 1C). VPA also improved the specific productivity of cell line A in bioreactors (FIG. 1 1).

[0249] VPA effect was dependent of dextran sulfate presence in the culture media: The effect of VPA in bioreactors was completely dependent on the presence of dextran sulfate. The administration of VPA alone had little effect on harvest titer, but when VPA was combined with dextran sulfate, it increased harvest titer by greater than 20% (FIG. 1 1C). The effect of VPA on bioreactors was tested with or without dextran sulfate because dextran sulfate was present by default in the shake flask model as a means to reduce cell aggregation. Dextran sulfate was not present in the bioreactor model because automated cell counting data showed that the agitation supplied by the impeller was sufficient to break apart cell aggregates. These observations suggest that dextran sulfate interacts with the VPA and the cells in such a way that it dislodges a pool of protein that is adsorbed on the surface of the cell.

[0250] Confirmatory bioreactor experiments were also performed with cell line B. In these experiments, the lessons-learned from cell line A were applied. Accordingly, the addition of VPA from day 9 to day 12 in cultures containing dextran sulfate was delayed. Addition of VPA and dextran sulfate again reduced growth and viability (FIG. 12 A, FIG. 12B). However, the delayed addition strategy yielded a 20% increase in cell line B's harvest titer (FIG. 12C, FIG. 12D). This increase was higher than that observed in cell 9

- 85 - line B shake flasks, which suggested that delaying the time of VPA addition can generate higher titers.

The most encouraging result from the bioreactor experiments was discovering that the increase in titer obtained through VPA addition did not come at the expense of antibody product quality for cell lines A and B, as show in TABLE 2. For cell line A, 1 g/L dextran sulfate was added on day 0 and VPA was added on day 9. For cell line B, 1 g/L dextran sulfate was added on day 9 and VPA was added on day 12. Day 15 harvest material was purified via protein A chromatography. Protein A eluates from the fed-batch bioreactor experiments were tested for impurities via GXII protein profiling, charge heterogeneity via ICIEF, and N-glycan profile via HILIC-UPLC. The VPA+DS conditions did not result in significant product quality differences compared to the control conditions for cell lines A and B.

TABLE 2: Addition of VPA yielded comparable antibody product quality.

GXII Charge Heterogeneity N-glycan Profile

Cell

Condition %Major

Line %Acidic %Main %Basic %G0F %G1F %G2F

Peak

A Control 94.8% 45.0% 50.0% 5.0% 65.8% 13.0% 2.0%

A DS 96.8% 50.4% 45.8% 3.8% 60.7% 24.1% 3.5%

A VPA 97.7% 43.5% 52.3% 4.2% 68.1% 16.6% 2.2%

A VPA+DS 96.2% 43.6% 51.8% 4.6% 68.1% 16.8% 2.3%

B Control 98.5% 47.2% 51.8% 0.9% 54.5% 34.2% 5.2%

B VPA+DS 97.8% 54.0% 45.1 % 0.9% 51.1 % 34.7% 5.4% ] The ability to generate a product quality profile that is comparable to an existing process greatly increases the commercial attractiveness of VPA. Process changes that increase the productivity of clinical or commercial program must result in critical quality attribute profiles that ultimately do not change the protein-of-interest's efficacy and safety, such as antibody aggregation, charge heterogeneity, and glycosylation. VPA did not change the aggregation, charge heterogeneity, or the glycosylation profile of the antibodies produced by cell line A or B. VPA's ability to maintain the key product quality attributes of the corresponding control process makes it an attractive and flexible cell culture lever for not only improving the titer of new products in early stage development, but also that of legacy products where product quality comparability is important. Conclusion

[0253] The instant study demonstrated that addition of VPA and dextran sulfate to CHO cells stably expressing monoclonal antibodies increased harvest titers by up to 20% without compromising antibody product quality, though the magnitude of the increase was dependent on the cell line. Factorial experiments were performed to identify the optimal concentration and timing of VPA addition for one CHO cell line in a shake flask model and then verified the results in a bioreactor model. In doing this, it was also determined that the addition of dextran sulfate further improved overall cell culture performance in a bioreactor model.

[0254] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0255] The present invention has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0256] The foregoing description of the specific aspects will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and/or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance. [0257] The breadth and scope of the present invention should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0258] The claims in the instant application are different than those of the parent application or other related applications. The Applicant therefore rescinds any disclaimer of claim scope made in the parent application or any predecessor application in relation to the instant application. The Examiner is therefore advised that any such previous disclaimer and the cited references that it was made to avoid, may need to be revisited. Further, the Examiner is also reminded that any disclaimer made in the instant application should not be read into or against the parent application.

[0259] All publications, patents and patent applications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains, and are herein incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in their entireties.

Claims

WHAT IS CLAIMED IS:

A method for increasing recombinant protein recovery from eukaryotic cells, comprising:

(a) cultivating eukaryotic cells capable of expressing the recombinant protein in a chemically defined cell culture medium, and

(b) subjecting the cells to a sufficient amount of at least one anionic substance selected from malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to increase recovery of the recombinant protein relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined cell culture medium but not subjected to the at least one anionic substance.

The method of claim 1, wherein recovery of recombinant protein is increased by at least about 10% to at least about 100% relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

A method for increasing recombinant protein production from eukaryotic cells, comprising:

(b) subjecting the cells to a sufficient amount of at least one anionic substance selected from malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to increase production of the recombinant protein relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

4. The method of claim 3, wherein production of recombinant protein is increased by at least about 10% to at least about 50% relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

5. A method for decreasing recombinant protein attachment to the surface of eukaryotic cells comprising:

(b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to decrease attachment of the recombinant protein to the eukaryotic cells' surface relative to the attachment of the same recombinant protein to eukaryotic cells grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

6. The method of claim 5, wherein attachment of recombinant protein to the eukaryotic cells' surface is decreased by at least about 10% to at least about 90% relative to the attachment of the same recombinant protein to eukaryotic cells grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

7. A method for reducing recombinant protein-induced inhibition of eukaryotic cell growth comprising:

(b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to reduce recombinant protein-induced growth inhibition relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

8. The method of claim 7, wherein growth inhibition be recombinant protein is decreased by at least about 7% to at least about 21% relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

9. A method for increasing the viability of eukaryotic cells expressing a recombinant protein comprising:

(b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of at least one anionic substance selected from malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof, to increase eukaryotic cell viability relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

10. The method of claim 9, wherein the viability of eukaryotic cells expressing a recombinant protein is increased by at least about 5% relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in the same chemically defined culture medium but not subjected to the at least one anionic substance.

1 1. The method of any one of claims 1 to 10, wherein the eukaryotic cells are a mammalian eukaryotic cells.

12. The method of claim 11, wherein the mammalian eukaryotic cells are CHO cells or HEK293 cells.

13. The method of claim 12, wherein the mammalian eukaryotic cells are Chinese Hamster Ovary (CHO) cells.

The method of claim 13, wherein the CHO cells are CHO DG44.

15. The method of any one claims 1 to 14, wherein chemically defined cell culture medium has less than 10% of mammalian serum (by volume).

16. The method of claim 15, wherein the chemically defined cell culture medium contains no more than about 1% mammalian serum by volume.

17. The method of any one of claims 1 to 14, wherein the chemically defined cell culture medium is serum free.

18. The method of any one of claims 1 to 15, wherein the chemically defined cell culture medium is protein free.

19. The method of any one of claims 1 to 18, wherein the recombinant protein is a GDNF ligand family protein.

20. The method of claim 19, wherein the GDNF ligand family protein is selected from the group consisting of GDNF, neublastin, neurturin, and persephin.

21. The method of claim 20, wherein the GDNF family ligand is neublastin.

22. The method of claim 21 , wherein the neublastin is human neublastin.

23. The method of any one of claims 1 to 18, wherein the recombinant protein is a basic protein.

24. The method of claim 23, wherein the basic protein has a pi of at least 10.

25. The method of claim 24, wherein the basic protein has a pi of at least 1 1.

26. The method of claim 23, wherein the basic protein comprises at least 10% basic amino acids.

27. The method of claim 23, wherein the basic protein is an arginine rich protein.

28. The method of claim 27, wherein the arginine rich protein comprises at least 12% arginine amino acids.

29. The method of any one of claims 1 to 28, wherein the polyanionic compound is a polysulfated or a polysulfonated compound.

30. The method of claim 29, wherein the polysulfate compound is a polysulfated saccharide.

31. The method of claim 30, wherein polysulfated saccharide is a dextran sulfate.

32. The method of claim 31 , wherein the dextran sulfate has an average molecular weight of about 5,000 Dalton.

33. The method of claim 29, wherein the polysulfated compound is polyvinyl sulfate.

34. The method of claims 1 to 29, wherein the citrate is sodium citrate or ferric citrate.

35. The method of claim 34, wherein the concentration of ferric citrate is in a range from about ImM to about lOOmM.

36. The method of claim 35, wherein the concentration of ferric citrate is about 50 mM.

37. The method of claim 34, wherein ferric citrate is added to raise its concentration to at least 2mM.

38. The method of claim 34, wherein the concentration of sodium citrate is in a range from about ImM to about lOOmM.

39. The method of claim 38, w herein the concentration of sodium citrate is about 100 mM,

40. The method of claim 34. wherein sodium citrate is added to raise its concentration to at least 2m M.

41. The method of any one of claims i to 29, wherein the concentration of succinate is in a range from about I rnM to about lOOmM.

42. The method of claim 41, wherein the concentration of succinate is about 100 mM.

43. The method of any one of claims 1 to 29, wherein succinate is added to raise its concentration to at least 2mM.

44. The method of any one of claims 1 to 29, wherein the concentration of fumarate is in a range from about IniM to about 1 OOmM.

45. The method of claim 44, wherein the coiicentraLion of fumarate is about 100 mM,

46. The method of any one of claims 1 to 29, wherein fumarate is added to raise its concentration to at least 2mM.

47. The method of any one of claims I to 29, wherein the concentration of malate is in a range from about lmM to about lOOmM. 8. The method of claim 47, wherein the concentration of malate is about 100 mM,

49. The method of any one of claims 1 to 29, wherein malate is added to raise its concen trati on to at least 2m i.

50. The method of any one of claims 1 to 29, wherein the concentration of polysulfated compound is in a range from about O.Olg L to about ig/L.

51. The method of claim 50, wherein the polysulfated compound is dextran sulfate.

52. The method of claim 50, wherein the polysulfated compound is polyvinyl sulfate.

53. The method of claim 51 , wherein the concentration of dextran sulfate is about 0.1 g/L.

54. The method of claim 51 , wherein dextran sulfate is added to raise its concentration to at least 0.25g/L.

55. The method of claim 1, wherein the anionic substance comprises a polyanionic compound and a citrate.

56. The method of claim 55, wherein the polyanionic compound is dextran sulfate and the citrate is ferric citrate.

57. The method of any one of claims 1 to 56, wherein the anionic substance is added during the induction phase but not during the proliferation phase.

58. The method of any one of claims 1 to56, wherein the concentration of anionic substance is kept constant during cultivation.

59. The method of any one of claims 1 to56, wherein the concentration of anionic substance is increased or decreased during cultivation.

60. The method of any one of claims 1 to 56, wherein the anionic substance is added 1 to 4 weeks prior to the separation of the recombinant protein.

61. The method of any one of claims 1 to 56, wherein the anionic substance is added 1 to 7 days prior to the separation of the recombinant protein.

62. The method of any one of claims 1 to 56, wherein the anionic substance is added 1 to 24 hours prior to the separation of the recombinant protein.

63. The method of any one of claims 1 to 56, wherein the anionic substance is added 1 to 3 hours prior to the separation of the recombinant protein.

64. The method of any one of claims 1 to 56, wherein the anionic substance is added 1 to 60 minutes prior to the separation of the recombinant protein.

65. The method of any one of claims 1 to 56, wherein the eukaryotic cells are grown and maintained at a density of at least 10⁵ cells per ml. of chemically defined culture medium.

66. The method of any one of claims 1 to 56, wherein the anionic substance is added to the chemically defined cell culture medium at inoculation and/or during the production phase.

67. The method of any one of claims 1 to 56, wherein the eukaryotic cells are grown in a fed batch process.

68. The method of any one of claims 1 to 56, wherein the eukaryotic cells are grown in a continuous process.

69. The method of any one of claims 1 to 56, wherein the chemically defined culture medium further contains a non-physiological concentration of an ionic substance selected from the group consisting of NH₄ acetate, MgCl₂, KH₂P0₄, NaS0₄, KC1, CaCl₂, an amino acid, and a mixture of peptides and/or amino acids.

70. The method of any one of claims 1 to 56, wherein eukaryotic cells are grown under "^""" hyperosmolar conditions.

71. The method of any one of claims 1 to 56, wherein the osmolarity of the chemically defined cell culture medium is between about 250 and about 600 mOsm,

72. The method of any one of claims 1 to 56, wherein the at least one anionic substance or combination thereof is added to reach the equilibration balance within recombinant protein and eukaryotic cell surface, sufficient to disrupt ionic binding and release bound recombinant proteins from the eukaryotic cell surface without destroying the cell.

73. The method of claim 1, wherein at last two anionic substances are added.

74. The method of any one of claims 1 to 73, further comprising at least one of the following steps:

(c) isolating the recombinant protein from the culturing medium;

(d) separating the culture medium from the cultivated eukaryotic cells, resulting in two separate fractions, a fraction of cultivated eukaryotic cells and a fraction of liquid medium;

(e) contacting or resuspending the fraction of cultivated eukaryotic cells with a release composition comprising a sufficient amount of at least one anionic substance selected from malate, succinate, fumarate, citrate, polyanionic compounds, and combinations thereof to release the recombinant protein from the eukaryotic cell surface;

(f) separating the release composition from the eukaryotic cells, resulting in two separate fractions, a fraction of eukaryotic cells and a fraction of release composition comprising the recombinant protein released from the eukaryotic cell surface;

(g) isolating the recombinant protein from the fraction of release composition; and,

(h) suspending the fraction of eukaryotic cells in culture medium and reculturing.

75. The method of claim 74, wherein the separation of the culture medium or the release in steps (d) or (f) composition from the cultivated cells comprises at least a technique selected from the group consisting of centrifugation, filtration, diafiltration, tangential filtration, dead end filtration, micro filtration, electrical fields, magnetic fields, and """ultrafiltration. ^"""

76. The method of claim 74, wherein the isolation of the recombinant protein in steps (c) or (g) comprises at least a technique selected from the group consisting of immuno-affmity chromatography, affinity chromatography, protein precipitation, buffer exchanges, ionic exchange chromatography, hydrophobic interaction chromatography, mixed mode hydrophobic/ion exchange chromatography media, chelating chromatography, carbohydrate affinity like lectin or heparin affinity chromatography, size-exclusion chromatography, electrophoresis, dialysis, different precipitation agents such as polyethylene glycol, ammonium sulfate, ethanol, hydroxyl apatite adsorption, and filter membrane adsorption.

77. The method of any one of claims 1 to 76, comprising collecting the recombinant protein.

78. A recombinant protein obtained by the method of claim 77.

79. A pharmaceutical composition comprising the recombinant protein of claim 78 and a pharmaceutically acceptable carrier.

80. The pharmaceutical composition of claim 79, comprising neublastin.

81. A method for increasing recombinant protein recovery from eukaryotic cells, comprising:

(b) subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound, to increase recovery of the recombinant protein relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined cell culture medium but not subjected to valproate and the at least one polyanionic compound.

82. The method of claim 1, wherein recovery of recombinant protein is increased by at least about 10% to at least about 100% relative to the recovery of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the polyanionic compound.

83. A method for increasing recombinant protein production from eukaryotic cells, comprising:

(b) subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound, to increase production of the recombinant protein relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

84. The method of claim 3, wherein production of recombinant protein is increased by at least about 10% to at least about 50% relative to the amount of the same recombinant protein that would be produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

85. A method for decreasing recombinant protein attachment to the surface of eukaryotic cells comprising:

(b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound, to decrease attachment of the recombinant protein to the eukaryotic cells' surface relative to the attachment of the same recombinant protein to eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

86. The method of claim 85, wherein attachment of recombinant protein to the eukaryotic cells' surface is decreased by at least about 10% to at least about 90% relative to the attachment of the same recombinant protein to eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

87. A method for reducing recombinant protein-induced inhibition of eukaryotic cell growth comprising:

(b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of valproate and at least one polyanionic compound to reduce recombinant protein-induced growth inhibition relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

88. The method of claim 87, wherein growth inhibition be recombinant protein is decreased by at least about 7% to at least about 21% relative to the growth inhibition caused by the same recombinant protein produced by the same eukaryotic cells grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

89. A method for increasing the viability of eukaryotic cells expressing a recombinant protein comprising:

(b) prior to separating the recombinant protein from the eukaryotic cells, subjecting the cells to a sufficient amount of of valproate and at least one polyanionic compound to increase eukaryotic cell viability relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in the same chemically defined culture medium but not subjected to valproate and the at least one polyanionic compound.

90. The method of claim 89, wherein the viability of eukaryotic cells expressing a recombinant protein is increased by at least about 5% relative to the viability of the same eukaryotic cells expressing the same recombinant protein grown in the same chemically defmed culture medium but not subjected to valproate and the at least one polyanionic compound.

91. The method of any one of claims 81 to 90, wherein the eukaryotic cells are mammalian eukaryotic cells.

92. The method of claim 91, wherein the mammalian eukaryotic cells are CHO cells or HE 293 cells.

93. The method of any one claims 1 to 92, wherein chemically defined cell culture medium has less than 10% of mammalian serum (by volume).

94. The method of any one of claims 81 to 93, wherein the chemically defined cell culture medium is serum free.

95. The method of any one of claims 81 to 94, wherein the chemically defined cell culture medium is protein free.

96. The method of any one of claims 81 to 95, wherein the polyanionic compound is a polysulfated or a polysulfonated compound.

97. The method of claim 96, wherein the polysulfate compound is a polysulfated saccharide.

98. The method of claim 97, wherein the polysulfated saccharide is a dextran sulfate.

99. The method of claim 98, wherein the dextran sulfate has an average molecular weight of about 5,000 Dalton.

100. The method of claims 89 to 99, wherein the valproate is sodium valproate.

101. The method of claim 100, wherein the concentration of sodium valproate is in a range from about ImM to about lOOmM.

102. The method of claim 101, wherein the concentration of sodium valproate is lower than 1 mM.

103. The method of claim 102, wherein the concentration of sodium valproate is about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM.

104. The method of claim 100, wherein sodium valproate is added to raise its concentration to at least 2mM.

105. The method of any one of claims 81 to 97, wherein the concentration of polysulfated compound is in a range from about 0.01 g/L to about 1 g/L.

106. The method of claim 105, wherein the polysulfated compound is dextran sulfate.

107. The method of claim 106, wherein dextran sulfate is added to raise its concentration to at least 0.25 g/L.

108. The method of claim 106, wherein dextran sulfate is added to raise its concentration to at least 1 g/L.

109. The method of any one of claims 81 to 108, wherein the anionic substance is

(i) added during the induction phase but not during the proliferation phase;

(ii) added to the chemically defined cell culture medium at inoculation and/or during the production phase;

(iii) added 1 to 4 weeks prior to the separation of the recombinant protein;

(iv) added 1 to 7 days prior to the separation of the recombinant protein;

(v) added 1 to 24 hours prior to the separation of the recombinant protein;

(vi) added 1 to 3 hours prior to the separation of the recombinant protein; or (vii) added 1 to 60 minutes prior to the separation of the recombinant protein. 10. The method of any one of claims 81 to 109, wherein the concentration of anionic substance is

(i) kept constant during cultivation, or

(ii) increased or decreased during cultivation. 11. The method of any one of claims 81 to 110, wherein the eukaryotic cells are grown in a fed batch process or in a continuous process. 12. The method of any one of claims 81 to 1 11, wherein the eukaryotic cells are grown and maintained at a density of at least 10^s cells per ml. of chemically defined culture medium. 13. The method of any one of claims 81 to 1 12, wherein the chemically defined culture medium further contains a non-physiological concentration of an ionic substance selected from the group consisting of NH₄ acetate, MgCl₂, KH₂P0₄, NaS0₄, KC1, CaCl₂, an amino acid, and a mixture of peptides and/or amino acids. 14. The method of any one of claims 81 to 1 13, wherein the at least one anionic substance or combination thereof is added to reach the equilibration balance within recombinant protein and eukaryotic cell surface, sufficient to disrupt ionic binding and release bound recombinant proteins from the eukaryotic cell surface without destroying the cell. 15. The method of any one of claims 81 to 114, further comprising at least one of the following steps:

(a) isolating the recombinant protein from the culturing medium;

(c) contacting or resuspending the fraction of cultivated eukaryotic cells with a release composition comprising a sufficient amount of at least one anionic substance comprising valproic acid and a polyanionic compound, and combinations thereof to release the recombinant protein from the eukaryotic cell surface;

(e) isolating the recombinant protein from the fraction of release composition: and,

1 16. The method of claim 1 15, wherein the separation of the culture medium or the release in steps (d) or (f) composition from the cultivated cells comprises at least a technique selected from the group consisting of centrifugation, filtration, diafiltration, tangential filtration, dead end filtration, micro filtration, electrical fields, magnetic fields, and ultrafiltration.

1 1 7. The method of claim 1 1 5, wherein the isolation of the recombinant protein in steps (c) or (g) comprises at least a technique selected from the group consisting of immuno-affinity chromatography, affinity chromatography, protein precipitation, buffer exchanges, ionic exchange chromatography, hydrophobic interaction chromatography, mixed mode hydrophobic/ion exchange chromatography media, chelating chromatography, carbohydrate affinity like lectin or heparin affinity chromatography, size-exclusion chromatography, electrophoresis, dialysis, different precipitation agents such as polyethylene glycol, ammonium sulfate, ethanol, hvdroxyl apatite adsorption, and filter membrane adsorption.

1 18. The method of any one of claims 1 to 1 17, comprising collecting the recombinant protein.

1 19. A recombinant protein obtained by the method of claim 1 1 8.

120. A phannaceutical composition comprising the recombinant protein of claim 1 19 and a pharmaceutically acceptable carrier. The pharmaceutical composition of claim 119, comprising an antibody or antigen-binding fragment thereof.