EA047861B1 - REGULATION OF ORNITHINE METABOLISM TO CONTROL THE CONTENT OF HIGH-MANNOSE GLYCOFORMS OF RECOMBINANT PROTEINS - Google Patents

Description

This application claims priority to U.S. Provisional Application No. 61/926,481, filed January 13, 2014, which is incorporated herein by reference in its entirety.

State of the art of the invention

Various post-translational modifications, including methylation, sulfation, phosphorylation, lipid attachment, and glycosylation, are performed by higher eukaryotes. Many secreted proteins, membrane proteins, and proteins targeted to vesicles or some intracellular organelles are known to be glycosylated. Glycosylation, the covalent attachment of sugar residues to specific amino acids, is one of the most common post-translational modifications of recombinant proteins. Protein glycosylation has several functions in the cell, including its important role in protein folding and quality control, molecular migration and sorting, and cell surface receptor interactions.

N-linked glycosylation, which involves the addition of oligosaccharides to an asparagine residue, is found in some protein recognition sequences (e.g., Asn-X-Ser/Thr). N-linked glycoproteins contain standard branched structures composed of mannose (Man), galactose, N-acetylglucosamine, and neuraminic acids. High-mannose oligosaccharides typically contain two N-acetylglucosamines with multiple mannose residues (5 or more). Glycoproteins produced in mammalian cell culture may contain varying levels of these high-mannose (HM or HMN) glycoforms, such as Mannose5 (Man5), Mannose6 (Man6), Mannose7 (Man7), Mannose8 (Man8), and Mannose9 (Man9).

While the glycoforms of the recombinant glycoprotein expressed by the Chinese hamster ovary (CHO) host cell are largely determined by intrinsic genetic factors, the content of high mannose glycoforms is also influenced by cell culture conditions (Pacis, et al., (2011) Biotechnol Bioeng 108, 2348-2358).

Glycosylation can influence the therapeutic efficacy of recombinant protein drugs. The impact of glycosylation on the potency, pharmacokinetics, immunogenicity, solubility, and in vivo clearance of therapeutic glycoproteins has made monitoring and control of glycosylation a critical parameter for biopharmaceutical manufacturing. The content of high-mannose glycoforms of therapeutic proteins is a critical quality parameter that has been shown to influence the pharmacokinetic properties of some therapeutic antibodies (Goetze, et al., (2011) Glycobiology 21, 949-59; Yu, et al., (2012) MAbs 4, 475-87). Therefore, methods to control the content of high-mannose glycoforms of therapeutic proteins will be of practical importance.

There is a need in the pharmaceutical industry to manage and control the content of high mannose glycoforms of therapeutic glycoproteins, and methods for doing so would be desirable. The present invention provides a method for managing the content of high mannose glycoforms of recombinant glycoproteins by regulating ornithine metabolism in host cells.

The essence of the invention

The invention provides a method for controlling the content of high-mannose glycoforms of a recombinant protein, comprising culturing a host cell expressing the recombinant protein in a cell culture under conditions that regulate ornithine metabolism in the host cell.

In one embodiment of the invention, ornithine metabolism in a host cell is regulated by reducing ornithine accumulation in the host cell. In a related embodiment, ornithine accumulation in the host cell is regulated by culturing the host cell in a cell culture medium containing an arginase inhibitor or spermine. In another related embodiment, ornithine accumulation in the host cell is regulated by adding an arginase inhibitor to the cell culture medium. In another related embodiment, the arginase inhibitor is BEC (L-(2-boroethyl)-1-cysteine) or DL-a-difluoromethylornithine. In another related embodiment, the arginase inhibitor is BEC (L-(2-boroethyl)-1-cysteine). In another related embodiment, the arginase inhibitor is DL-a-difluoromethylornithine. In another related embodiment, the concentration of the arginase inhibitor is at least 10 μM. In another related embodiment, the concentration of the arginase inhibitor is from 10 μM to 2 mM. In another related embodiment, the concentration of the arginase inhibitor is 10 μM. In another related embodiment, the concentration of the arginase inhibitor is 0.5 mM. In another related embodiment, the concentration of the arginase inhibitor is 1 mM. In another related embodiment, the concentration of the arginase inhibitor is 2 mM.

In another embodiment, the accumulation of ornithine in the host cell is controlled by adding 35 μM or less spermine to the cell culture medium. In a related embodiment, the spermine concentration is from 7 μM to 35 μM. In another related embodiment, the spermine concentration is from 17 μM to 35 μM. In another related embodiment, the spermine concentration is from 7 μM to 17 μM. In another related embodiment, the spermine concentration is 35 μM. In another related embodiment, the spermine concentration is 17 μM. In another related embodiment, the spermine concentration is 7 μM.

- 1 047861

In another embodiment, ornithine metabolism in a host cell is regulated by increasing the accumulation of ornithine in the host cell. In a related embodiment, the accumulation of ornithine in the host cell is regulated by culturing the host cell in a cell culture medium comprising ornithine, arginine, an ornithine decarboxylase inhibitor, an ornithine aminotransferase inhibitor, a nitric oxide synthase inhibitor, or an arginine decarboxylase inhibitor. In another related embodiment, the accumulation of ornithine in the host cell is regulated by adding at least 0.6 mM ornithine to the cell culture medium. In another related embodiment, the concentration of ornithine is 0.6-14.8 mM. In another related embodiment, the concentration of ornithine is 6-14.8 mM. In another related embodiment, the concentration of ornithine is 0.6 mM. In another closely related embodiment, the ornithine concentration is 6 mM.

In another related embodiment, the ornithine concentration is 14.8 mM. In another related embodiment, the accumulation of ornithine in the host cell is controlled by adding at least 8.7 mM arginine to the cell culture medium. In another related embodiment, the arginine concentration is from 8.7 mM to 17.5 mM. In another related embodiment, the arginine concentration is 8.7 mM. In another related embodiment, the arginine concentration is 17.5 mM.

In another embodiment, ornithine accumulation in the host cell is controlled by adding an ornithine decarboxylase inhibitor, a nitric oxide synthase inhibitor, an ornithine aminotransferase inhibitor, or an arginine decarboxylase inhibitor to the cell culture medium. In a related embodiment, ornithine accumulation in the host cell is controlled by adding an ornithine decarboxylase inhibitor to the cell culture medium. In another related embodiment, the ornithine decarboxylase inhibitor is alpha-difluoromethylornithine (DMFO). In another related embodiment, the ornithine decarboxylase inhibitor is piperonyl butoxide (PBO).

In another related embodiment, the accumulation of ornithine in the host cell is regulated by adding an ornithine aminotransferase inhibitor to the cell culture medium. In yet another related embodiment, the ornithine aminotransferase inhibitor is 5-fluoromethylornithine (F-FMOm). In yet another related embodiment, the host cell is regulated by adding a nitric oxide synthase inhibitor to the cell culture medium. In yet another related embodiment, the nitric oxide synthase inhibitor is 2-ethyl-2-thiopseudourea or N-nitro-G-arginine and G°-monomethyl-G-arginine. In yet another related embodiment, the nitric oxide synthase inhibitor is N-nitro-G-arginine and G°-monomethyl-G-arginine.

In another related embodiment, ornithine accumulation in the host cell is regulated by adding an arginine decarboxylase inhibitor to the cell culture medium. In another related embodiment, the arginine decarboxylase inhibitor is asymmetric dimethylarginine (ADMA).

The invention provides a method for producing a recombinant protein in which the content of high-mannose glycoforms is reduced, comprising culturing a host cell that expresses the recombinant protein in a cell culture, wherein the ornithine metabolism is regulated by reducing the accumulation of ornithine in the host cell. In a related embodiment, the accumulation of ornithine in the host cell is reduced by culturing the host cell in a cell culture medium containing an arginase inhibitor or spermine.

In a related embodiment, ornithine accumulation in a host cell is reduced by adding an arginase inhibitor to the cell culture medium. In another related embodiment, the arginase inhibitor is BEC (B-(2-boroethyl)-1-cysteine) or DL-a-difluoromethylornithine. In another related embodiment, the arginase inhibitor is BEC (B-(2-boroethyl)-1-cysteine). In another related embodiment, the arginase inhibitor is DL-a-difluoromethylornithine. In another related embodiment, the arginase inhibitor is at least 10 μM. In another related embodiment, the arginase inhibitor is 10 μM to 2 mM. In another related embodiment, the arginase inhibitor is 10 μM to 20 μM. In another related embodiment, the arginase inhibitor is 10 μM. In another related embodiment, the arginase inhibitor is 0.5 mM. In another related embodiment, the arginase inhibitor is 1 mM. In another related embodiment, the arginase inhibitor is 2 mM.

In an embodiment, the accumulation of ornithine in the host cell is reduced by culturing the host cell in a cell culture medium containing 35 μM or less spermine in the cell culture medium. In another related embodiment, the spermine concentration is from 7 μM to 35 μM. In another related embodiment, the spermine concentration is from 17 μM to 35 μM. In another related embodiment, the spermine concentration is from 0.07 mL/L to 0.17 mL/L. In another related embodiment, the spermine concentration is 35 μM. In another related embodiment, the spermine concentration is 17 μM. In another related embodiment, the spermine concentration is 7 μM.

- 2 047861

The invention provides a method for producing a recombinant protein in which the content of high-mannose glycoforms is increased, comprising culturing a host cell that expresses the recombinant protein in a cell culture, wherein the ornithine metabolism is regulated by increasing the accumulation of ornithine in the host cell. In a related embodiment, the accumulation of ornithine in the host cell is increased by adding ornithine, arginine, an ornithine decarboxylase inhibitor, an ornithine aminotransferase inhibitor, a nitric oxide synthase inhibitor, or an arginine decarboxylase inhibitor to the cell culture medium.

In another related embodiment, the accumulation of ornithine in the host cell is increased by culturing the host cell in a cell culture medium containing at least 0.6 mM ornithine. In yet another related embodiment, the ornithine concentration is 0.6-14.8 mM. In yet another related embodiment, the ornithine concentration is 6-14.8 mM. In yet another related embodiment, the ornithine concentration is 0.6 mM. In yet another related embodiment, the ornithine concentration is 6 mM. In yet another related embodiment, the ornithine concentration is 14.8 mM.

In another related embodiment, the accumulation of ornithine in the host cell is increased by culturing the host cell in a cell culture medium containing at least 8.7 mM arginine. In yet another related embodiment, the concentration of arginine is from 8.7 mM to 17.5 mM. In yet another related embodiment, the concentration of arginine is 8.7 mM. In yet another related embodiment, the concentration of arginine is 17.5 mM.

In another related embodiment, ornithine accumulation in a host cell is increased by culturing the host cell in a cell culture medium comprising an ornithine decarboxylase inhibitor, a nitric oxide synthase inhibitor, an ornithine aminotransferase inhibitor, or an arginine decarboxylase inhibitor. In another related embodiment, ornithine accumulation in a host cell is increased by culturing the host cell in a cell culture medium comprising an ornithine decarboxylase inhibitor. In yet another related embodiment, the ornithine decarboxylase inhibitor is alpha-difluoromethylornithine (DMFO). In yet another related embodiment, the ornithine decarboxylase inhibitor is piperonyl butoxide (PBO).

In another related embodiment, ornithine accumulation in a host cell is increased by culturing the host cell in a cell culture medium containing an ornithine aminotransferase inhibitor. In yet another related embodiment, the ornithine aminotransferase inhibitor is 5-fluoromethylornithine (F-FMOrn).

In another related embodiment, ornithine accumulation in a host cell is increased by culturing the host cell in a cell culture medium containing a nitric oxide synthase inhibitor. In yet another related embodiment, the nitric oxide synthase inhibitor is 2-ethyl-2-thiopseudourea or N-nitro-G-arginine and LG-monomethyl-L-arginine.

In another embodiment, ornithine accumulation in a host cell is increased by culturing the host cell in a cell culture medium containing an arginine decarboxylase inhibitor. In yet another related embodiment, the arginine decarboxylase inhibitor is asymmetric dimethylarginine (ADMA).

In another embodiment, the host cell expressing the recombinant protein is cultured in a batch culture, a fed-batch culture, a flow culture, or a combination thereof. In another related embodiment, the culture is a flow culture. In another related embodiment, the perfusion comprises continuous perfusion. In another related embodiment, the perfusion rate is constant. In another related embodiment, the perfusion is performed at a rate less than or equal to 1.0 working volume per day. In another related embodiment, the perfusion is achieved by alternating tangential flows.

In another embodiment, the host cell expressing the recombinant protein is cultured in a bioreactor. In another related embodiment, the bioreactor has a volume of at least 500 L. In another related embodiment, the bioreactor has a volume of at least 500 L to 2,000 L. In another related embodiment, the bioreactor has a volume of at least 1,000 L to 2,000 L. In another related embodiment, the bioreactor is inoculated with at least 0.5x106 cells/mL.

In another embodiment, the host cell expressing the recombinant protein is cultured in a serum-free cell culture medium. In another related embodiment, the serum-free cell culture medium is a flow-through cell culture medium. In yet another related embodiment, the host cells are mammalian cells. In yet another related embodiment, the host cells are Chinese hamster ovary (CHO) cells.

In another embodiment, the recombinant protein is a glycoprotein. In another embodiment, the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine.

- 3 047861

In another embodiment, the above-mentioned methods further comprise the step of collecting the recombinant protein produced by the host cell.

In another embodiment, the recombinant protein produced by the host cell is purified and provided in a pharmaceutically acceptable dosage form.

In another embodiment, a recombinant protein produced by any of the above methods is provided. In a related embodiment, the recombinant protein is purified. In another embodiment, the recombinant protein is provided in a pharmaceutically acceptable dosage form.

Brief description of graphic materials

Fig. 1 - General idea of ornithine metabolism. ARG - arginase; AZ - antizyme; AZIN - antizyme inhibitor; P5C - pyrroline-5-carboxylate; ASP - aspartate; ORNT - ornithine transporter; GATM - glycine aminotransferase; NOS - nitric oxide synthase; OAT - ornithine aminotransferase; ODC - ornithine decarboxylase; OTC - ornithine transcarbamylase; SMS - spermine synthase; SRS - spermidine synthase.

Fig. 2 - Identification of ornithine as a metabolic marker associated with high-mannose glycan levels. High-mannose glycan levels (%HM) of secreted recombinant monoclonal antibodies from eight different cell lines (cell lines A through H) detected on days 8 (D8, white bars), 9 (D9, gray bars), and 10 (D10, black bars) of a fed-batch process measured in medium #1 (A) or medium #2 (B). Correlation between high-mannose glycan levels and extracellular ornithine levels measured in the spent culture (C). On average, ornithine levels and high-mannose glycans from the eight cell lines were compared on day 9. Pearson's correlation coefficient, R=0.83.

Fig. 3 - Correlation between high-mannose levels and extracellular ornithine levels: A) Content of high-mannose glycoforms measured when the H cell line was subjected to eight different culture conditions (#1 - #8). B) Corresponding relative extracellular ornithine levels. C) Correlation between % content of high-mannose glycoforms and relative extracellular ornithine levels. Pearson's correlation coefficient, R=0.78.

Fig. 4 - Relative levels of arginase-1 mRNA expression in cell pellets collected from 8 different cell lines at days 3, 6, 8, 9 and 10. The corresponding content of high-mannose glycoforms is also shown.

Fig. 5 - Content of high-mannose glycoforms in recombinant proteins expressed by cells grown in cell culture medium containing spermine tetrahydrochloride at concentrations of 0, 7, 17, 35, and 100 μM. Samples were collected on day 5 of the control perfusion analysis. Samples of 35 μM served as a control.

Fig. 6 - Concentration of extracellular ornithine (mg/L) produced endogenously by cells of cultures exposed to spermine tetrahydrochloride at concentrations of 0, 7, 17, 35 and 100 μM. Samples were collected on day 5 of the control perfusion analysis. Samples of 35 μM served as a control.

Fig. 7 - Content of high-mannose glycoforms in recombinant proteins expressed by cells grown in cell culture medium containing L-ornithine monohydrochloride at concentrations of 0, 0.6, 6, and 14.8 mM. Samples were collected on day 5 of the control perfusion analysis. Samples of 0 mM served as controls.

Fig. 8 - Content of high-mannose glycoforms in recombinant proteins expressed by cell line I grown in cell culture medium containing 0.1 g/l L-ornithine monohydrochloride (black bars) or a control cell culture containing no exogenously added ornithine (white bars). Samples were collected on days 6, 8, and 12.

Fig. 9 - Content of high-mannose glycoforms in recombinant proteins expressed by cells grown in cell culture medium containing arginine monohydrochloride at concentrations of 2.2, 4.4, 6.5, 8.7, and 17.5 mM. The 8.7 mM sample served as a control. Samples were collected on day 4 of the control perfusion analysis.

Fig. 10 - Content of high-mannose glycoforms in recombinant proteins expressed by cells grown in cell culture medium supplemented with various arginase inhibitors. BEC hydrochloride (BEC), DL-a-difluoromethylornithine hydrochloride (DL-a), K'-hydroxy-E-arginine monoacetate salt (NG) and Nω-hydroxy-nor-arginine diacetate salt (Nw) at concentrations of 1, 10 and 20 μM. The control did not contain the inhibitor. Samples were collected on day 4 of the control perfusion analysis.

Fig. 11 - Content of high-mannose glycoforms in recombinant proteins expressed by cells grown in cell culture medium containing the arginase inhibitor BEC hydrochloride (BEC) at concentrations of 0.0, 0.01, and 0.5 mM and DL-a-difluoromethylornithine hydrochloride (DL-a) at concentrations of 0.0, 0.01, 1.0, and 2.0 mM. Samples were collected on day 4 of the control perfusion analysis.

- 4 047861

Detailed description of the essence of the invention

It was found that the content of high-mannose glycoforms of the expressed recombinant glycoprotein was influenced by ornithine accumulation in the host cell and, as a result, could be controlled by regulating ornithine metabolism in the host cell.

Ornithine is a non-protein-coding amino acid that is involved in the urea cycle, polyamine synthesis, and arginine metabolism. Ornithine is also a precursor of glutamate and proline via ornithine 5-aminotransferase (OAT) activity, see Fig. 1. In humans, OAT deficiency results in choroidal-retinal gyratory atrophy (GA), a disorder characterized by retinal degeneration and plasma ornithine accumulation (Takki K et al., Br J Ophthalmol. 1974; 58(11): 907-16). In a mouse model of OAT deficiency, an arginine-restricted diet was shown to decrease plasma ornithine levels and prevent retinal degeneration (Wang T et al., PNAS 2000; 97(3): 1224-1229). Ornithine decarboxylase (ODC), which catalyzes the conversion of ornithine to putrescine, is the rate-limiting enzyme of the polyamine biosynthetic pathway (Pegg A, JBC. 2006; 281(21): 14529-14532). ODC synthesis and stability, as well as polyamine transporter activity, are dependent on environmental osmotic conditions (Munro G et al., BBA 1975; 411(2): 263-281; Tohyama et al., Eur J Biochem. 1991; 202(3):1327-1331; Michell J et al., 1998; 329:453-459). Enhanced polyamine biosynthesis is associated with increased tolerance to osmotic stress in plants (Alcazar R et al., Biotechnol Lett 2006; 28:1867-1876). The conversion of ornithine to citrulline is catalyzed by ornithine transcarbamylase (OTC) as part of the urea cycle. OTC deficiency in humans results in accumulation of ammonia in the blood (Hopkins et al., Arch.Dis.Childh., 1969 44:143-148). Ornithine metabolism occurs in both the cytosol and mitochondria, with OTC- and OAT-catalyzed metabolic steps occurring in the mitochondria. The mitochondrial ornithine transporter ORNT1 is required for ornithine import into the mitochondria. In humans, mutations in ORNT1 are responsible for hyperornithinemia, hyperammonemia, homocitrullinuria (HHH) syndrome, which is characterized by elevated plasma ornithine and ammonia levels (Camacho et al., Nat Genet (1999); 22:151-158); (Valle D et al, 2001, 1857-1896).

As described herein, the degree of ornithine accumulation in a host cell, as measured by extracellular ornithine levels in cell culture medium, correlated with the content of high mannose glycoforms of the expressed recombinant glycoproteins. Control of high mannose content can be achieved by regulating ornithine metabolism in a host cell. The invention provides a method for controlling the content of high mannose glycoforms of a recombinant protein, comprising culturing a host cell expressing the recombinant protein in a cell culture under conditions that regulate ornithine metabolism in the host cell. Ornithine metabolism can be controlled by decreasing or increasing ornithine accumulation in a host cell. The present invention provides methods for producing recombinant proteins in which the content of high mannose glycoforms is reduced or increased, comprising culturing a host cell that expresses the recombinant protein in a cell culture, wherein the accumulation of ornithine in the host cell is regulated.

Ornithine metabolism refers to the chemical or enzymatic reactions and pathways involved in the biosynthesis, transport, catabolic processes, and metabolic transformations of ornithine. The urea cycle, polyamine synthesis, creatine synthesis, and the mitochondrial ornithine catabolism pathway are examples of ornithine metabolism. An overview is shown in Fig. 1.

Ornithine accumulation in the host cell is a consequence of altered ornithine metabolism. The degree of ornithine accumulation in the host cell can be controlled by regulating ornithine metabolism. Intracellular metabolite levels can be reflected in extracellular levels (i.e., measured in the cell culture medium). An indicator of ornithine accumulation in the host cell can be obtained by measuring the amount of ornithine secreted into the cell culture medium. The dynamic increases in ornithine levels described herein were detected in cell culture without the addition of exogenous ornithine.

High mannose glycoform content, high mannose glycan level, and high mannose species levels are used interchangeably and are abbreviated as HM, %HM, HMN, or %HMN and refer to the relative percentage content of the combined glycan species mannose 5 (Man5), mannose 6 (Man6), mannose 7 (Man7), mannose 8 (Man8), and mannose 9 (Man9).

The level of ornithine secreted into the cell culture medium was found to correlate with the content of high-mannose glycoforms of the recombinant glycoproteins expressed by the host cells in cell culture. When host cell ornithine accumulation was reduced by culturing the host cell in cell culture medium containing arginase inhibitor or spermine, the content of high-mannose glycoforms of the recombinant glycoproteins expressed was reduced. When host cell ornithine accumulation was increased by culturing the host cell in cell culture medium containing

- 5 047861 ornithine or arginine, the content of high-mannose glycoforms of recombinant expressed glycoproteins was increased.

The present invention provides a method for regulating the accumulation of ornithine in a host cell by culturing the host cell in a cell culture medium containing an arginase inhibitor.

Arginine is the metabolic precursor of ornithine, and arginase is the enzyme that catalyzes the conversion of arginine to ornithine. It has been noted that arginase mRNA expression levels correlate with the amount of ornithine accumulation when comparing metabolic and expression profiles of different cell lines. By blocking arginase activity with an arginase inhibitor, it is possible to potentially reduce the levels of ornithine production. However, the efficacy of arginase inhibitors may be compromised by high levels of arginine in the cell culture medium. Additionally, there are other metabolic precursors of ornithine (e.g., glutamate and proline, see Fig. 1) that may contribute to ornithine accumulation.

As described herein, it was found that the content of high-mannose glycoforms of recombinant glycoproteins expressed by a cultured host cell could be controlled by adding an arginase inhibitor to the cell culture medium. Blocking arginase activity reduced the amount of ornithine production in the host cells and decreased the level of high-mannose glycans of the expressed recombinant glycoproteins.

Arginase also inhibits ornithine transcarbamylase (OTC) (Vissers et al., (1982) J. Gen. Microbio. 128:1235-1247). Blocking arginase activity not only reduces ornithine production from arginine, but could potentially relieve the suppression of OTC activity by allowing ornithine to be converted to citrulline, resulting in further reduction of ornithine accumulation (see Fig. 1).

Patients with OTC deficiency have elevated ammonia levels. When increased expression or activity of arginase in host cell lines expressing recombinant protein with high levels of high mannose glycans induces OTC inhibition, this also leads to increased ammonia levels. Increased intracellular ammonia levels may alter the pH gradient across the Golgi apparatus and cause suboptimal redistribution of glycosyltransferases, resulting in increased levels of high mannose glycans due to incomplete glycan branching within the Golgi complex (Campbell et al, (1973) NJM 288 (1):1–6; Hopkins et al, (1969) Archive of Disease in Childhood 44 (234):143–148; Muhling et al, (2001) Amino Acids 21 (3):303–318; Park et al., (2000) J. Biotechnol 81 (2):129–140; Rivinoja et al., (2009) J. Cell Physiol. 220 (1):144–154; and Axelsson et al., (2001) Glycobiology 11(8):633-644).

Suitable arginase inhibitors are known in the art and are available from commercial sources. Such arginase inhibitors include BEC hydrochloride, DL-a-difluoromethylornithine hydrochloride; X'-hydroxy-E-arginine, and Nω-hydroxy-nor-arginine. In one embodiment, the arginase inhibitor is BEC (X-(2-boroethyl)-1-cysteine) or DL-a-difluoromethylornithine. In one embodiment, the arginase inhibitor is BEC (N-(2-boroethyl)-1-cysteine). In one embodiment, the arginase inhibitor is DL-a-difluoromethylornithine.

Arginase inhibitors can be added to the cell culture medium at a concentration of at least 10 μM to reduce the levels of high mannose glycans of the expressed recombinant proteins without significantly affecting productivity. In one embodiment, the concentration of the arginase inhibitor is from 10 μM to 2 mM. In another embodiment, the concentration of the arginase inhibitor is 10 μM. In another embodiment, the concentration of the arginase inhibitor is 0.5 mM. In another embodiment, the concentration of the arginase inhibitor is 1 mM. In another embodiment, the concentration of the arginase inhibitor is 2 mM.

Ornithine accumulation in the host cell can also be regulated by culturing the host cell in cell culture medium containing spermine, as described in the examples below. Ornithine, through the action of ornithine decarboxylase (ODC), is the starting point for the polyamine pathway and the synthesis of the polyamines putrescine, spermidine and spermine. Exogenously supplied spermine can be used to inactivate ODC directly or via antizyme, see Fig. 1. Inactivation of ODC can lead to ornithine accumulation, as described below. By limiting the amount of exogenously supplied spermine, the suppression of ODC activity can be relieved and ornithine can be metabolized via the polyamine pathway, resulting in a reduction in the amount of ornithine accumulation in the host cell.

Spermine may be added to the cell culture medium at a concentration of less than or equal to 35 μM to reduce the levels of high mannose glycans of the expressed recombinant proteins without significantly affecting productivity. In one embodiment, the concentration of spermine is from 7 μM to 35 μM. In one embodiment, the concentration of spermine is from 17 μM to 35 μM. In one embodiment, the concentration of spermine is from 7 μM to 17 μM. In another embodiment, the concentration of spermine is 35 μM. In another embodiment, the concentration of spermine is 17 μM. In another embodiment, the concentration

- 6 047861 spermine is 7 μM.

Another method for regulating ornithine metabolism is to increase ornithine accumulation. In one embodiment, the invention provides for regulating ornithine accumulation in a host cell by culturing the host cell in a cell culture medium containing ornithine, arginine, an ornithine decarboxylase inhibitor, an ornithine aminotransferase inhibitor, a nitric oxide synthase inhibitor, or an arginine decarboxylase inhibitor.

As described herein, levels of extracellular ornithine in cell culture medium correlated with levels of high-mannose glycans of recombinant glycoproteins. It was found that culturing host cells expressing recombinant proteins in cell culture medium containing ornithine resulted in the formation of recombinant glycoproteins having increased levels of high-mannose glycans.

As described above, the accumulation of ornithine in the cell culture medium likely reflects alterations in ornithine metabolism that may result in ammonia accumulation similar to that seen in patients with defective ornithine metabolism genes (e.g., OTC deficiency or ORNT1 mutation). While the cellular mechanism underlying the ammonia-induced increase in high-mannose glycosyl forms is unknown, it has been hypothesized that alterations in the pH gradient within the Golgi apparatus may result in suboptimal redistribution of glycosyltransferases. These alterations may result in decreased availability of glycosylation enzymes to complete glycan branching and, consequently, result in elevated levels of high-mannose glycans.

Another possible scenario is that ornithine accumulation potentially causes a disturbance in redox homeostasis (Zanatta et al., (2013) Life sciences 93(4): 161-168). Ornithine correlates positively with high-mannose glycans, suggesting that the content of high-mannose glycosylates is regulated by the redox state of the cell. Ornithine may increase lipid oxidation levels. Since many glycosylation regulatory enzymes are membrane-bound, changes in lipid oxidation caused by ornithine accumulation could potentially affect the integrity and activity of glycosylation regulatory enzymes in the Golgi and ER and subsequently affect the content of high-mannose glycosylates.

Ornithine can be added to the cell culture medium at a concentration of at least 0.6 mM to increase the levels of high mannose glycans of the expressed recombinant proteins without significantly affecting productivity. In one embodiment, the concentration of ornithine is 0.6-14.8 mM. In one embodiment, the concentration of ornithine is 6-14.8 mM. In another embodiment, the concentration of ornithine is 0.6 mM. In another embodiment, the concentration of ornithine is 6 mM. In another embodiment, the concentration of ornithine is 14.8 mM.

Arginine is the metabolic precursor of ornithine. High mannose glycan levels have been found to be increased in recombinant proteins expressed by cells grown in cell culture medium containing exogenous arginine. Increasing the amount of exogenous arginine increases the amount of metabolic precursor available for ornithine synthesis, thereby increasing ornithine levels in host cells.

The present invention provides for the regulation of ornithine accumulation in host cells by adding arginine to a cell culture medium at a concentration of at least 8.7 mM to increase the levels of high mannose glycans of expressed recombinant proteins without significantly affecting productivity. In one embodiment, the arginine concentration is from 8.7 mM to 17.5 mM. In another embodiment, the arginine concentration is 8.7 mM. In another embodiment, the arginine concentration is 17.5 mM.

Ornithine accumulation can be regulated by adding inhibitors of ornithine decarboxylase (ODC), ornithine aminotransferase (OAT), nitric oxide synthase (NOS), or arginine decarboxylase (ADC) to the cell culture medium, thereby providing the possibility of regulating ornithine metabolism and ornithine accumulation in the host cell to control the content of high-mannose glycoforms of the recombinant protein.

Ornithine accumulation can be increased by blocking the activity of ornithine metabolizing enzymes such as ODC and OAT (see Fig. 1). Small molecule inhibitors specific for ODC such as alpha-difluoromethylornithine (DFMO) and piperonyl butoxide (PBO) are commercially available. OAT is blocked by 5-fluoromethylornithine (5-FMOrn) T (Daune et al., 1988, Biochem J. 253:481-488). The present invention provides supplementation of cell culture with these inhibitors to enhance ornithine accumulation in host cells to control the content of high mannose glycoforms of recombinant protein.

Ornithine accumulation can be regulated by blocking the activity of enzymes that regulate arginine accumulation (Fig. 1). Inhibition of nitric oxide synthase activity by small molecule inhibitors such as 2-ethyl-2-thiopseudourea and N-nitro-G-arginine, and LG

- 7 047861 monomethyl-G-arginine. and/or arginine decarboxylase activity via asymmetric dimethylarginine (ADMA) can enhance the conversion flux of arginine to ornithine. The present invention provides for the addition of these inhibitors to cell culture to enhance ornithine accumulation in the host cell to control the content of high mannose glycoforms of the recombinant protein.

In one embodiment of the invention, it is envisaged that the cell culture medium is a serum-free cell culture medium. In one embodiment, the cell culture medium is a flow-through cell culture medium.

As used herein, the terms cell culture medium (also called culture medium, cell culture medium, tissue culture medium) refer to any nutrient solution used to grow cells, such as animal or mammalian cells, and which typically provides at least one or more of the following: an energy source (usually in the form of carbohydrates such as glucose); one or more of the essential amino acids, and typically the twenty essential amino acids, plus cysteine; vitamins and/or other organic substances, typically required in low concentrations; fats or free fatty acids; and trace elements, such as inorganic compounds or natural elements, which are typically required in very low concentrations, typically in the micromolar range.

The nutrient solution may optionally be supplemented with additional components for optimizing cell growth, such as hormones and other growth factors, such as insulin, transferrin, epidermal growth factor, serum, and the like; salts, such as calcium, magnesium and phosphates, and buffers, such as HEPES; nucleosides and bases, such as adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysate, such as hydrolyzed animal or vegetable protein (peptone or peptone mixtures, which can be obtained from animal by-products, purified gelatin or vegetable raw materials); antibiotics, such as gentamicin; cell protectants or surfactants, such as Pluronic® F68; polyamines, such as putrescine, spermidine or spermine (see, for example, WIPO Publication No. WO 2008/154014) and pyruvate (see, for example, US Patent No. 8,053,238), depending on the requirements of the cells in culture and/or the necessary cell culture parameters.

Cell culture media include those commonly used in and/or known for use in any cell culture process, such as, but not limited to, semi-continuous, extended semi-continuous, fed-batch, and/or continuous or continuous cell culture.

The cell culture medium components can be completely ground into a powder medium; partially ground with liquid supplements added to the cell culture medium as needed; or the nutrients can be added in completely liquid form to the cell culture.

Basic (or batch) cell culture medium refers to cell culture medium that is typically used to initiate cell culture and is complete enough to support cell culture.

Cell culture growth medium refers to a cell culture medium that is typically used in cell cultures during the exponential growth phase, and is complete enough to maintain the cell culture during this phase. The cell culture growth medium may also contain agents that provide survival or resistance to selectable markers included in the host cell line. Such selection agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfoximine, methotrexate, glutamine-free cell culture medium, glycine-free cell culture medium, hypoxanthine and thymidine, or thymidine alone.

Production cell culture medium refers to cell culture medium that is typically used in cell cultures during the transition and production phases, after exponential growth has been completed when protein production begins, and is sufficiently complete to maintain the desired cell density, viability, and/or productivity during these phases.

Perfusion cell culture medium refers to a cell culture medium that is commonly used in cell cultures to support perfusion or continuous culture processes and is complete enough to support cell culture during these processes. Perfusion cell culture medium formulations may be enriched and more concentrated than basal cell culture medium formulations to accommodate the method used to remove spent medium. Perfusion cell culture medium may be used during both the growth phase and the production phase.

The concentrated cell culture medium may contain some or all of the nutrients required to maintain the cell culture; in particular, the concentrated medium may contain nutrients defined as or known to be consumed during

- 8 047861 cell culture production phases. The concentrated medium may be based on any composition of the cell culture medium. Such a concentrated nutrient medium may contain some or all of the constituents of the cell culture medium, for example, in a concentration of about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X or even about 1000X of their normal amount.

Cell cultures may also be supplemented with independent concentrated portions of certain nutrients that may be difficult to prepare or are rapidly consumed in cell cultures. Such nutrients may be amino acids, such as tyrosine, cysteine and/or cystine (see, for example, WIPO Publication No. 2012/145682). In one embodiment, a concentrated solution of tyrosine is independently fed to a cell culture grown in a cell culture medium containing tyrosine, such that the concentration of tyrosine in the cell culture does not exceed 8 mM. In another embodiment, a concentrated solution of tyrosine and cystine is independently fed to a cell culture grown in a cell culture medium lacking tyrosine, cystine or cysteine. The independent feeding may begin before or at the beginning of the production step. Independent feeds can be performed by feeding the cell culture medium on the same or different days as those performed for the concentrated growth medium. Independent feeds can also be infused on the same or different days as the perfusion medium.

The cell culture medium, in certain embodiments, is serum-free and/or does not contain animal products or ingredients. The cell culture medium, in certain embodiments, is chemically defined, such that all chemical components are known.

It will be clear to those skilled in the art that the animal or mammalian cells are cultured in a medium suitable for culturing the particular cell and which can be determined by one skilled in the art without undue experimentation. Commercially available culture media may be used, including, but not limited to, Dulbecco's Modified Medium, RPMI 1640, Minimum Essential Medium alpha (MEM-alpha), Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, alpha MEM, Eagle's Basic Medium with Erle's BSS, High Glucose DMEM with Glutamine, High Glucose DMEM without Glutamine, Low Glucose DMEM without Glutamine, DMEM:F12 1:1 with Glutamine, GMEM (Glasgow MEM), GMEM with Glutamine, Gray's Complete Insect Medium, Gray's Insect Medium without FBS, Hama F-10 with Glutamine, Hama F-12 with Glutamine, IMDM with HEPES and Glutamine, IMDM with HEPES and No Glutamine, IP41 Insect Medium, 15 (Leibowitz)(2X) without glutamine or phenol red, 15 (Leibowitz) without glutamine, Modified McCoy's Medium 5A, Medium 199, MEM Eagle's without glutamine or phenol red (2X), MEM Eagle-Earle BSS with glutamine, MEM Eagle-Earle BSS without glutamine, MEM Eagle-Hanks BSS without glutamine, NCTC-109 with glutamine, Ritchers' Medium CM with glutamine, RPMI 1640 with HEPES, glutamine and/or penicillin-streptomycin, RPMI 1640 with glutamine, RPMI 1640 without glutamine, Shader's Medium for insect cells, or any other medium known to one of skill in the art that is designed for particular cell types. To the above exemplary media, additional components or ingredients, including additional components, may be added in appropriate concentrations or amounts as necessary or desired and as known to one skilled in the art using standard skills.

In one embodiment, the host cells are mammalian cells. In one embodiment, the host cells are Chinese hamster ovary (CHO) cells.

The cell lines (also referred to as host cells) used in the present invention are genetically modified to express a commercially or scientifically desirable polypeptide. The cell lines are typically derived from a cell generation obtained from a main culture, and can be maintained in culture indefinitely. The cells can contain expression vectors (constructs), such as plasmids and the like, introduced, for example, by transformation, transfection, infection or injection, that contain coding sequences, or portions thereof, coding for the proteins to be expressed and produced during cultivation. Such expression vectors contain the necessary elements for transcription and translation of the inserted coding sequence. Methods that are well known and practiced by those skilled in the art can be used to construct expression vectors containing sequences coding for the proteins and polypeptides to be produced, as well as the corresponding transcription and translation control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described in J. Sambrook et al., 2012, Molecular Cloning, A Laboratory Manual, ^4th edition Cold Spring Harbor Press, Plainview, NY, or any previous edition; F. M. Ausubel et al., 2013, Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY, or any previous edition; Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, all of which are incorporated herein for all purposes.

- 9 047861

Animal cells, mammalian cells, cultured cells, animal or mammalian host cells, host cells, recombinant cells, recombinant host cells, and the like are suitable for cells that can be cultured according to the methods of the present invention. Such cells are typically cell lines derived or produced from mammals and are capable of growing and surviving when placed in a monolayer or suspension culture on a medium containing appropriate nutrients and/or other factors, such as those described herein. The cells are typically selected to be able to express and secrete proteins, or to be molecularly modified to express or secrete a large amount of a particular protein, more specifically a desired glycoprotein, into the growth medium. It is understood that the protein produced by the host cell may be endogenous or homologous to the host cell. In addition, the protein is heterologous, i.e., introduced into a host cell, for example, a human protein produced and expressed by Chinese hamster ovary (CHO) host cells. In addition, mammalian proteins, i.e., originally obtained or produced from a mammalian organism, are obtained by the methods of the present invention and can be secreted by the cells into the culture medium.

The methods of the present invention can be used in the culture of various cells. In one embodiment, the cultured cells are eukaryotic cells, such as plant and/or animal cells. The cells can be mammalian cells, fish cells, insect cells, amphibian cells, or avian cells. Various mammalian cell lines suitable for growth in culture are available from the American Culture Collection (Manassas, VA) and other depositories, as well as commercial suppliers. Cells that can be used in the processes of the invention include, but are not limited to, MK2.7 cells, PER-C6 cells, Chinese hamster ovary (CHO) cells such as CHO-K1 (ATCC CCL-61), DG44 (Chasm et al., 1986, Som. Cell Molec. Genet., 12:555-556; Kolkekar et al., 1997, Biochemistry, 36:10901-10909; and WO 01/92337 A2), dihydrofolate reductase negative CHO cells (CHO/-DHFR, Urlaub and Chasm, 1980, Proc. Natl. Acad. Sci. USA, 77:4216), and dp12.CHO cells (U.S. Patent No. 5,721,121); monkey kidney cells (CV1, ATCC CCL-70); monkey kidney cells CV1 transformed with SV40 (COS cells, COS-7, ATCC CRL-1651); HEK 293 cells, and Sp2/0 cells, 5L8 hybridoma cells, Daudi cells, EL4 cells, HeLa cells, HL-60 cells, K562 cells, Jurkat cells, THP-1 cells, Sp2/0 cells, primary epithelial cells (e.g., keratinocytes, cervical epithelial cells, bronchial epithelial cells, tracheal epithelial cells, renal epithelial cells, and retinal epithelial cells) and established cell lines and strains thereof (e.g., human embryonic kidney cells (e.g., 293 cells, or 293 cells subcloned for growth in suspension culture, Graham et al., 1977, J. Gen. Virol., 36:59); baby hamster kidney cells (BHK, ATCC CCL-10); mouse Sertoli cells (TM4, Mather, 1980, Biol. Reprod., 23:243-251); human cervical carcinoma cells (HELA, ATCC CCL-2); canine kidney cells (MDCK, ATCC CCL-34); human lung cells (W138, ATCC CCL-75); human hepatoma cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL-51); Buffalo rat liver cells (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NY Acad. Sci., 383:44-68); MCR 5 cells; FS4 cells; retinal PER-C6 cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229 cells, HeLa S3 cells, Hep-2 cells, KB cells, LS 180 cells, LS 174T cells, NCI-H-548 cells, RPMI 2650 cells, SW-13 cells, T24 cells, WI-28 VA13 cells, 2RA cells, WISH cells, BS-CI cells, LLC-MK2 cells, Clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK1 cells, PK(15) cells, GH1 cells, GH ₃ cells, L2 cells, LLC-RC 256 cells, MH1C1 cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells or derivatives thereof), fibroblast cells from any tissue or organ (including, but not limited to, heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissues (brain, spinal cord), lung, vascular tissues (arteries, veins, capillaries), lymphoid tissues (lymph gland, adenoid, gland, bone marrow and blood), spleen, and fibroblasts and fibroblast-like cell lines (e.g., TRG-2 cells, IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempsey cells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit 529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells, Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5, WI-38 cells, WI26 cells, MiCl1 cells, CV-1 cells, COS-1 cells, COS-3 cells, COS-7 cells, African green monkey kidney cells (VERO-76, ATCC CRL-1587; VERO, ATCC CCL-81); DBS-FrhL-2 cells, BALB/3T3 cells, F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells, NOR-10 cells, C3H/IOTI/2 cells, HSDM1C3 cells, KLN205 cells, McCoy cells, mouse L cells, strain 2071 (mouse L cells), LM strain (mouse L cells), L-MTK (mouse L cells), NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, CII cells, and Jensen cells, or derivatives thereof) or other cell types known to one of skill in the art.

The cells may be suitable for adherent, monolayer or suspension culture, transfection and expression of proteins such as antibodies. The cells may be used in a semi-continuous,

- 10 047861 periodic with feeding and flow or continuous methods of cultivation.

In one embodiment, a host cell expressing a recombinant protein is cultured in a bioreactor. In another embodiment, the bioreactor has a volume of at least 500 L. In another related embodiment, the bioreactor has a volume of at least 500 L to 2000 L. In another related embodiment, the bioreactor has a volume of at least 1000 L to 2000 L. In one embodiment of the present invention, the cell culture is obtained by introducing them into the bioreactor using at least 0.5 x 10 ⁶ cells / ml in a serum-free culture medium. In one embodiment, the invention also includes the step of collecting the recombinant protein produced by the host cell. In one embodiment, the recombinant protein produced by the host cell is purified and provided in a pharmaceutically acceptable dosage form.

For purposes of understanding, but not limitation, it will be understood by one skilled in the art that cell cultures and culture cycles for protein production may include three basic types, namely, semi-continuous, extended semi-continuous, fed-batch, flow-through, or a combination thereof. In a semi-continuous culture, cells are initially cultured in a medium, and this medium is not removed, replaced, or supplemented, i.e., the cells are not fed with fresh medium, during or before the end of the culture cycle. The desired product is harvested at the end of the culture cycle.

In fed-batch cultures, the growth cycle is extended by replenishing the culture medium one or more times per day (or continuously) with fresh medium during the cycle, i.e., the cells are fed with new medium (feed medium) during the culture period. Fed-batch cultures may include various feeding schedules and times as described above, such as daily, every other day, every two days, etc., more than once per day, or less frequently than once per day, etc. Also, fed-batch cultures may be supplemented with feed medium continuously. The desired product is then harvested at the end of the culture/production cycle.

A continuous culture is one in which the cell culture is supplied with fresh perfusion medium and spent medium is removed. The perfusion of fresh medium into the cell culture and the removal of spent medium may occur continuously, in stages, intermittently, or in combinations of any or all of these. Perfusion rates may range from less than one working volume per day to multiple working volumes per day. Preferably, the cells are retained in the culture and the spent medium that is removed contains substantially no cells or significantly fewer cells than the culture. Recombinant proteins expressed in the cell culture may also be retained in the culture or removed with the spent medium. Removal of spent medium may be achieved by a variety of means including centrifugation, sedimentation, or filtration, See, e.g., Voisard et al., (2003), Biotechnology and Bioengineering 82:751-65. A preferred filtration method is alternating tangential flow filtration. Alternating tangential flow is achieved by pumping the media through hollow fiber filter modules using an ATF device. See, for example, U.S. Patent No. 6,544,424; Furey (2002) Gen. Eng. News. 22 (7), 62-63. Filters separate particles based on size or molecular weight. Depending on the application, filters can be selected based on pore size or molecular weight cutoff value (Nominal Molecular Weight Cutoff (MWCO)). Filters include membrane filters, ceramic filters, and metal filters and can be of any shape including spiral or tubular or sheet form.

The term perfusion flow rate means the amount of medium that passes through (added and removed) the bioreactor, usually expressed as a fraction or several working volumes, over a period of time. Working volume refers to the volume of the bioreactor used to culture the cells. In one embodiment, the perfusion flow rate is one working volume per day or less.

The cell culture can be implemented in conditions for small and large scale production of recombinant proteins using culture vessels and/or culture apparatuses that are traditionally used in animal or mammalian cell culture. It should be clear to those skilled in the art that Petri dishes, T-flasks and stirred flasks are typically used in laboratory scale. For large scale cultivation, equipment such as, but not limited to, fermenter bioreactors, airlift bioreactors, fluidized bed bioreactors, hollow fiber bioreactors, spinner flask cultures, stirred tank bioreactors, filter media bioreactors and disposable bags or any other suitable devices known to those skilled in the art can be used. Microcarriers may or may not be used in combination with spinner flasks or stirred tank bioreactors. The systems can be used in semi-continuous, fed-batch or continuous/flow cultivation modes. In addition, the culture apparatus or system can be equipped with additional apparatus such as a cell separator based on filters, gravity, centrifugal force, etc.

- 11 047861

Recombinant protein production can be accomplished in multi-step culture processes. In multi-step processes, cells are cultured in two or more distinct stages. For example, cells may be cultured first in one or more growth phases, under conditions that enhance cell proliferation and viability, followed by a production phase, under conditions that enhance protein production. In commercial processes for the production of recombinant proteins in mammalian cells, there are typically multiple, e.g., at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more growth phases that occur in different culture vessels (N-x - N-1) prior to the final production culture. The growth and production phases may be preceded by (or separated into) one or more transition phases. The production phase may be carried out on a large scale.

The term growth phase of a cell culture refers to the period of exponential cell growth (e.g., log phase), in which cells typically multiply rapidly. Cells remain in the growth phase for about one day, or about two days, or about three days, or about four days, or longer than four days. The length of time that cells remain in the growth phase will depend on the cell type and rate of cell growth and the culture conditions, for example.

The term transition phase refers to the period of time between the growth phase and the production phase. Generally, the transition phase is the time during which environmental conditions can be controlled to support the transition from the growth phase to the production phase. Various cell culture parameters that can be controlled include, but are not limited to, one or more of: temperature, osmolarity, vitamins, amino acids, sugars, peptones, ammonium, and salts.

The term production phase of cell culture refers to the period of time when cell growth reaches a plateau. Logarithmic cell growth typically ends before or during this phase and protein production begins. Fed-batch and flow-through cell culture processes supplement the cell culture medium or provide fresh medium to achieve and maintain the desired cell density, viability and product titer at this stage. The production stage can be carried out on a large scale. Large-scale cell cultures can be maintained in a volume of at least about 100, 500, 1000, 2000, 3000, 5000, 7000, 8000, 10000, 15000, 20000 liters. In a preferred embodiment, the production stage is carried out in bioreactors with a volume of 500 L, 1000 L and/or 2000 L.

Typically, cell cultures that precede the final production culture go through two preceding stages, seed and inoculation fermenter systems. The seed fermenter (N-X) phase occurs at small scale, where cells rapidly increase in number. In the inoculation fermenter (N-1) phase, cells grow to create an inoculum for the production bioreactor, such as inocula with at least 0.5x106 cells/mL. Seed and N-1 systems can be produced by any culture method, typically semi-continuous cell culture. N-1 cell densities > 15x106 cells/mL are typical for seed bioreactors. Higher N-1 cell densities can reduce or even eliminate the time required to achieve the desired cell density in a production bioreactor. The preferred method for achieving higher N-1 cell densities is continuous flow culture using tangential flow filtration. N-1 cell culture grown using a tangential flow filtration perfusion process can provide cells at any desired density, such as >90x106 cells/mL or more. N-1 cell culture can be used to create a single bolus inoculation culture or can be used as a rotating seed stock culture that is maintained to inoculate multiple production bioreactors. Inoculation density can have a positive effect on the level of recombinant protein production. Product levels tend to increase with increasing inoculation density. The improvement in titer is not solely related to increasing inoculation density, but is likely to be influenced by the metabolism and cell cycle state of the cells being placed in production.

The term cell density refers to the number of cells in a given volume of culture medium. Viable cell density refers to the number of living cells per unit volume of culture medium, as determined by standard viability assays (e.g., trypan blue dye exclusion). The term pelleted cell volume (PCV), also known as percent pelleted cell volume (%PCV), is the ratio of the volume occupied by cells to the total volume of the cell culture, expressed as a percentage (see Stettler, et al., (2006) Biotechnol Bioeng. Dec 20:95(6):1228-33). The pelleted cell volume is a function of cell density and cell diameter; an increase in packed cell volume can result from an increase in either cell density or cell diameter, or both. The pelleted cell volume is an indication of the level of density in the cell culture.

During the manufacturing process, the growth phase may occur at a higher temperature than the production phase. For example, the growth phase may occur at a first set temperature of about

- 12 047861

35°C to about 38°C, and the production phase may take place at a first predetermined temperature of from about 29°C to about 37°C, optionally from about 30°C to about 36°C, or from about 30°C to about 34°C.

In addition, chemical inducers of protein biosynthesis, such as caffeine, butyrate, and/or hexamethylenebisacetamide (HMBA), may be added simultaneously, before, or after the temperature change. If the inducers are added after the temperature change, they may be added from one hour to five days after the temperature change, optionally from one to two days after the temperature change. Cell cultures may be maintained for several days or even weeks while the cells produce the desired protein(s).

Another method for maintaining cells in a desired physiological state is to induce growth arrest in cells when growing a cell culture under conditions of low concentrations of L-asparagine (see, for example, WIPO Publication No. WO2013/006479). Growth arrest can be achieved and maintained through a culture medium containing a limiting concentration of L-asparagine and by maintaining a low concentration of L-asparagine in the cell culture. Maintaining the concentration of L-asparagine at 5 mM or less can be used to maintain cells in a growth arrest state, thereby increasing productivity.

Cell cycle inhibitors - compounds that precisely or likely regulate the progression of the cell cycle and the associated processes of transcription, DNA repair, differentiation, senescence and apoptosis are also useful for inducing cell growth arrest. Cell cycle inhibitors that interact with cycle processes, such as cyclin-dependent kinases (CDKs), are in demand, as are molecules that interact with proteins from other regulatory pathways, such as AKT, mTOR, and other pathways that directly or indirectly affect the cell cycle.

Cell culture conditions suitable for the methods of the present invention are those commonly used and known in batch, fed-batch, or continuous cell culture, or any combination of these methods, with attention to pH, dissolved oxygen ( _O2 ), and carbon dioxide (CO2), agitation, humidity, and temperature.

The methods of the invention can be used to culture cells that express the desired recombinant proteins. The expressed recombinant proteins can be secreted into the culture medium, from which they can be recovered and/or collected. In addition, the proteins can be purified or partially purified from such a culture medium or component (e.g., from the culture medium) using known processes and products known in the art and/or available from commercial suppliers. The purified proteins can then be formulated, which means exchanging the buffer with a pharmaceutically acceptable formulation, sterilizing, bulk packaging, and/or packaging for the end user. Pharmaceutically acceptable formulations can include diluents, carriers, solubilizers, emulsifiers, preservatives, and/or adjuvants. The preparation of pharmaceutically acceptable formulations is within the skill of the art and includes those described in Remington's Pharmaceutical Sciences, 18th ed. 1995, Mack Publishing Company, Easton, PA.

In one embodiment, the invention provides that the recombinant protein is a glycoprotein. In one embodiment, the invention provides that the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein, or a cytokine. It is also provided that the recombinant protein is created by the method of the present invention. In one embodiment, the recombinant protein is provided in a pharmaceutically acceptable dosage form.

As used herein, the terms peptide, polypeptide, and protein are used interchangeably and refer to a molecule consisting of two or more amino acid residues joined together by peptide bonds. Peptides, polypeptides, and proteins also include modifications including, but not limited to, glycosylation to form glycoproteins, lipid addition, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and DCP-ribosylation.

As used herein, the term glycoprotein refers to peptides and proteins, including antibodies, having at least one oligosaccharide side chain containing mannose residues. Glycoproteins may be homologous to the host cell or may be heterologous, i.e. introduced into the host cell used, such as, for example, human glycoprotein produced by a Chinese hamster ovary (CHO) host cell. Such glycoproteins are typically referred to as recombinant glycoproteins. In some embodiments, glycoproteins expressed by the host cell are secreted directly into the medium.

Proteins may have scientific or commercial interest, including, protein-based drugs. Proteins include, among others, antibodies, chimeric proteins, and cytokines. Peptides, polypeptides, and proteins can be produced from recombinant animal cell lines using culture techniques.

- 13,047,861 cells and may be referred to as a recombinant peptide, a recombinant polypeptide, and a recombinant protein. The protein(s) expressed may be produced intracellularly or secreted into a culture medium from which it may be recovered and/or collected.

Non-limiting examples of mammalian proteins that may be advantageously produced by the methods of the present invention include proteins comprising amino acid sequences identical to or substantially similar in whole or in part to one of the following proteins: tumor necrosis factor (TNF) flt3 ligand (WO 94/28391), erythropoietin, thrombopoietin, calcitonin, IL-2, angiopoietin-2 (Maisonpierre et al. (1997), Science 277(5322):55-60), ligand for the receptor activator of factor NF-kappa B (RANKL, WO 01/36637), tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL, WO 97/01633), thymic stromal-derived lymphopoietin, granulocyte colony-stimulating factor, granulocyte-macrophage colony-stimulating factor (GM-CSF, Australian Patent No. 588819), mast cell growth factor, stem cell growth factor (U.S. Patent No. 6204363), epidermal growth factor, keratinocyte growth factor, megakaryotic growth and development factor, chemokine released by T cells upon activation (RANTES), human fibrinogen-like protein 2 (FGL2; NCBI Accession No. NM_00682; Ruegg and Pytela (1995) Gene 160:257-62) growth hormone, insulin, insulinotropin, insulin-like growth factors, parathyroid hormone, interferons including α-interferons, γ-interferon, and consensus interferons (U.S. Patents 4,695,623 and 4,897,471), nerve growth factor, brain-derived neurotrophic factor, synaptotagmin-like proteins (SLP 1-5), neurotrophin-3, glucagon, interleukins, colony-stimulating factors, lymphotoxin-β, leukemia inhibitory factor, and oncostatin-M. Descriptions of proteins that can be produced according to the methods of the invention can be found in, for example, Human Cytokines: Handbook for Basic and Clinical Research, all volumes (Aggarwal and Gutterman, eds. Blackwell Sciences, Cambridge, MA, 1998); Growth Factors: A Practical Approach (McKay and Leigh, eds., Oxford University Press Inc., New York, 1993); and The Cytokine Handbook, Vols. 1 and 2 (Thompson and Lotze eds., Academic Press, San Diego, CA, 2003).

In addition, the methods of the invention will be useful for producing proteins comprising all or part of the amino acid sequence of a receptor for any of the above proteins, an antagonist of such a receptor or any of the above proteins, and/or proteins substantially similar to such receptors or antagonists. These receptors and antagonists include: both forms of the tumor necrosis factor receptor (TNFR, also known as p55 and p75, U.S. Patent No. 5,395,760 U.S. Patent No. 5,610,279), interleukin-1 (IL-1) receptors (types I and II; U.S. Patent No. 0,460,846, U.S. Patent No. 4,968,607, and U.S. Patent No. 5,767,064), IL-1 receptor antagonist (U.S. Patent No. 6,337,072), IL-1 antagonists or inhibitors (U.S. Patents Nos. 5,981,713, 6,096,728, and 5,075,222), IL-2 receptors, IL-4 receptors (U.S. Patent No. 0,052,000), and IL-5 receptors (U.S. Patent No. 0,052,000). 367,566 and U.S. Patent No. 5,856,296), IL-15 receptors, IL-17 receptors, IL-18 receptors, Fc receptors, granulocyte-macrophage colony-stimulating factor receptor, granulocyte colony-stimulating factor receptor, oncostatin-m and leukemia inhibitory factor receptor, NF-kappa B receptor activator (RANK, WO 01/36637 and U.S. Patent No. 6,271,349), osteoprotegerin (U.S. Patent No. 6,015,938), TRAIL (TNF-dependent apoptosis-inducing ligand) receptors (including TRAIL receptors 1, 2, 3 and 4), and receptors that contain death domains such as Fas or apoptosis-inducing receptor (AIR).

Other proteins that can be produced using the present invention include proteins containing all or part of the amino acid sequence of differentiation antigens (referred to as CD proteins) or ligands thereof, and proteins substantially similar to any of these. Such antigens are disclosed in Leukocyte Typing VI (Proceedings of the VIth International Workshop and Conference, Kishimoto, Kikutani et al., eds., Kobe, Japan, 1996). Similar CD proteins are disclosed in subsequent workshops. Examples of such antigens include CD22, CD27, CD30, CD39, CD40, and ligands therefor (CD27 ligand, CD30 ligand, etc.). Some CD antigens are members of the TNF receptor family, which also includes 41BB and OX40. The ligands are often members of the TNF family, such as 41BB ligand and OX40 ligand.

Enzymatically active proteins or ligands thereof can also be prepared using the present invention. Examples include proteins comprising all or part of the following proteins or ligands thereof or a protein substantially similar to one of these: members of the dysenterygrin and metalloproteinase domain families, including TNF-alpha converting enzyme, various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein A-I, globins, IL-2 antagonist, alpha-1 antitrypsin, ligands for any of the above enzymes, as well as a variety of other enzymes and ligands thereof.

The term antibody includes references to both glycosylated and non-glycosylated immunoglobulins of any isotype or subclass, or to the antigen-binding region thereof that competes with the intact antibody for specific binding, unless otherwise indicated, including human, humanized, chimeric, multi-specific, monoclonal, polyclonal and oligomers or antigen-binding fragments thereof. Also included are proteins having antigen binding

- 14 047861 a binding fragment or portion such as Fab, Fab', F(ab')2, Fv, diabodies, Fd, dAb, maxibodies, single chain antibody molecules, complementarity determining region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies and polypeptides comprising at least a portion of an immunoglobulin that is sufficient to produce a specific antigen binding target polypeptide. The term antibody includes, but is not limited to, those produced, expressed, created or isolated by recombinant means, such as antibodies isolated from a host cell transfected to express the antibody.

Examples of antibodies include, but are not limited to, those that recognize any one or a combination of proteins, including, but not limited to, the above-mentioned proteins and/or the following antigens: CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80 (B7.1), CD86 (B7.2), CD147, IL-1a, ΗΠ-1β, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL2 receptor subunits, IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor, FGL2, PDGF-β, and analogs thereof (see U.S. Pat. Nos. 5272064 and 5149792), vascular endothelial growth factor (VEGF), platelet-derived growth factor (TGF), TGF-e2, TGF-β!. epidermal growth factor (EGF) receptor (see U.S. Patent No. 6,235,883), VEGF receptor, hepatocyte growth factor, osteoprotegrin ligand, interferon gamma, B lymphocyte stimulator (BlyS, also known as BAFF, THANK, TALL-1, and zTNF4; see Do and ChenKiang (2002), Cytokine Growth Factor Rev. 13(1): 19-25), C5 complement, IgE, CA125 tumor antigen, MUC1 tumor antigen, PEM antigen, LCG (which is a gene product expressed in association with lung cancer), HER-2, HER-3, tumor-associated glycoprotein TAG-72, SK-1 antigen, tumor-associated epitopes that are present at elevated levels in the serum of patients with colon cancer and/or pancreatic cancer, cancer-associated epitopes or proteins expressed in breast, colorectal, squamous cell, prostate, pancreatic, lung and/or kidney cancer cells and/or in melanoma, glioma, or neuroblastoma cells, the necrotic core of a tumor, integrin alpha 4 beta 7, integrin VLA-4, integrins B2, TRAIL receptors 1, 2, 3, and 4, receptor activator of nuclear factor kappa B (RANK), RANK ligand, TNF-α, VAP-1 adhesion molecule, epithelial cell adhesion molecule (EpCAM), intercellular adhesion molecule-3 (ICAM-3), leukointegrin adhesion, platelet glycoprotein gp IIb/IIIa, cardiac myosin heavy chain, parathyroid hormone, rNAPc2 (which is an inhibitor of cellular adhesion factor factor-VIIa), major histocompatibility complex (MHC) I, carcinoembryonic antigen (CEA), alpha-fetoprotein (AFP), tumor necrosis factor (TNF), CTLA-4 (which is a cytotoxic T lymphocyte-associated antigen), Fc-y-1 receptor, HLA-DR 10 beta, HLA-DR antigen, sclerostin, L-selectin, respiratory syncytial virus, human immunodeficiency virus (HIV), hepatitis B virus (HBV), Streptococcus mutansr, and Staphlycoccus aureus. Specific examples of known antibodies that can be produced using the methods of the present invention include, but are not limited to, adalimumab, bevacizumab, infliximab, abciximab, alemtuzumab, bapineuzumab, basiliximab, belimumab, briakinumab, canakinumab, certolizumab pegol, cetuximab, conatumumab, denosumab, eculizumab, gemtuzumab ozogamicin, golimumab, ibritutomab tiuxetan, labetuzumab, mapatumumab, matuzumab, mepolizumab, motavizumab, muromonab-CD3, natalizumab, nimotuzumab, ofatumumab, omalizumab, oregovomab, palivizumab, panitumumab, pemtumomab, pertuzumab, ranibizumab, rituximab, rovelizumab, tocilizumab, tositumomab, trastuzumab, ustekinumab, vedolizomab, zalutumumab and zanolimumab.

The invention can also be used to produce recombinant chimeric proteins, including, for example, any of the above proteins. For example, recombinant chimeric proteins comprising one of the above proteins plus a multimerization domain, such as a leucine zipper, a coiled coil, an immunoglobulin Fc fragment, or a similar protein, can be produced using the methods of the present invention. See, for example, WO94/10308; Lovejoy et al. (1993), Science 259:1288-1293; Harbury et al. (1993), Science 262:1401-05; Harbury et al. (1994), Nature 371:80-83; Hakansson et al. (1999), Structure 7:255-64. In particular, among other recombinant chimeric proteins, proteins are included in which the receptor region is fused with the Fc region of an antibody, such as etanercept (p75 TNFR:FC) and belatacept (CTLA4:Fc). Chimeric proteins and polypeptides, as well as fragments or regions, or mutants, variants, analogs of any of the above-mentioned proteins and polypeptides are also included among the suitable proteins, polypeptides and peptides that can be obtained by the methods of the present invention.

While the terminology used in this application is standard in the art, certain terms are defined herein to provide clarity and certainty in the meaning of the claims. Units, prefixes, and symbols may be denoted in their conventional SI form. Numerical ranges recited herein include the numbers defining the range and include any integer within the stated range. The methods and techniques described herein are generally performed in accordance with conventional methods well known in the art and as described in various general and more specific references cited and discussed herein.

- 15 047861 unless otherwise indicated. See, for example, Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001) and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990). All documents or portions of documents cited in this application, including, without limitation, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference. What is described in an embodiment of the present invention may be combined with other embodiments of the present invention.

The present invention is not limited in scope to the specific embodiments described herein, which serve as a single illustration of individual aspects of the invention, and functionally equivalent methods and components are within the scope of the present invention. Moreover, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Examples

Example 1

Extracellular ornithine levels were found to correlate with high mannose glycoform content. Eight recombinant antibody-expressing CHO cell lines with high mannose glycoform content ranging from < 5% to > 20% were selected for this experiment (cell line A - cell line H). Cells were grown in 10-day fed-batch culture in shake flasks using two different specialized cell culture media, each lacking ornithine (medium #1 and #2). Spent media were sampled at days 8, 9, and 10 of culture and subjected to extensive metabolomics analyses. %HM (high mannose glycans) was determined using the Endo-H rCE-SDS assay, later replaced by the hydrophilic interaction liquid chromatography (HILIC) assay described below. Relative ornithine levels in spent medium were determined by large-scale metabolomics analyses in which medium components were separated by liquid chromatography and determined by high-resolution spectrometry. Components were identified by matching their fragmentation spectra to a library of spectra of known compounds. The relative abundance of each component was determined from the peak area of its mass spectrometric signals. High-mannose glycan (%HM) levels of secreted recombinant monoclonal antibodies from cell lines A-H on days 8, 9, and 10 in medium #1 and medium #2 are shown in Fig. 2A and B. Fig. 2C shows the relationship between %HM and extracellular ornithine levels. The correlation was determined by comparing all 8 cell lines (represented as squares) using data from 9-day samples. The results obtained indicate a strong correlation between the content of high-mannose glycoforms and extracellular ornithine levels.

The H cell line was then grown in fed-batch culture in a 3 L bioreactor.

The growth period was 12 days. Four bolus feeds of 7%, 9%, 9%, and 9% were administered on days 3, 5, 7, and 9. In addition, 50% glucose was added daily starting on day 3 as required to maintain glucose concentrations above 2 g/L. Production bioreactors were inoculated with 15 x 10 ⁵ cells/mL after 4 days of growth. Cells were maintained in the growth medium until the start of the production phase. Eight different process conditions were then compared. Condition 1 served as a control. No changes were made to the production feed medium.

In condition #2, the production feed medium was supplemented with 24 mM betaine on day 0. No further betaine supplementation was performed. Four bolus feeds of 7%, 9%, 9%, and 9% were performed on days 3, 5, 7, and 9. In addition, 50% glucose solution was added daily beginning on day 3 as required to maintain glucose concentrations above 2 g/L.

In Conditions 3 and 4, copper sulfate removal was tested. Copper sulfate was removed from the production medium powder. Condition 3 served as a control, a stock solution of copper sulfate was added to the basal medium. In Condition 4, copper sulfate was not added to either medium, creating a copper-deficient medium. Both Conditions 3 and 4 were treated with the same copper-containing bolus feeder medium.

In conditions #5-#8, high and low osmolarity were tested. In conditions #5 and #6, cells were fed with 90% production base medium, i.e., 10% less nutrients were provided, resulting in cells experiencing reduced osmolarity. In condition #6, the cell culture medium was returned to the control level, ~300 mOsm, by titration with NaCl. In conditions #7 and #8, cells were fed with 85% feed medium, with the medium in condition #8 returned to the control level by titration with NaCl.

- 16 047861

Samples of the spent medium were collected on days 3, 6, 8, 9 and 10 of cultivation and subjected to large-scale metabolomics analyses.

Again, there was a significant correlation between extracellular ornithine levels and the high-mannose glycoform content. Fig. 3A shows the percentage levels of high-mannose glycans determined for the H cell line exposed to 8 different bioreactor conditions (#1-#8). Fig. 3B shows the corresponding extracellular ornithine levels. Fig. 3C shows the relationship between % high-mannose glycans and extracellular ornithine levels. The correlation was determined by comparing all 8 conditions (represented as squares) using data from 9-day samples.

Example 2

Arginase 1 mRNA expression levels were measured at selected days during 10 days of fed-batch semi-continuous culture in the eight cell lines described in Example 1.

mRNA expression levels were assessed using the QuantiGene Multiplex Assay kit (Affymetrix, Inc., Santa Clara, CA) according to the manufacturer's instructions.

Arginase 1, the enzyme that catalyzes the conversion of arginine to ornithine, was found to be activated in cell lines with elevated levels of high mannose glycans in a time-dependent manner, see Fig. 4. This suggests that specific targeting of arginase inhibitors to block arginase activity and reduce ornithine production could be used to reduce levels of high mannose glycans.

Example 3

This example demonstrates control of the content of high-mannose glycoforms of recombinant glycoproteins by regulating the accumulation of ornithine in a host cell expressing the recombinant glycoprotein.

Cell lines, cell cultures and media

The H cell lines used in this study were cultured in 3 L Erlenmeyer shake flasks (Corning Life Sciences, Lowell, MA) with 1 L working volume and cultured under standard humidified conditions at 36°C, 5% CO ₂ , and shaking at 70 rpm in an automated CO ₂ incubator (Thermo Fisher Scientific, Waltham, MA). Cells were subcultured in selective growth medium containing 500 nM methotrexate (MTX) every four days and then sequentially transferred, inoculated, and cultured in growth medium for four days before seeding into 24-well plates for the experiments described below.

Small-scale control perfusion

A modified control perfusion in a 24-well deepwell plate (Axygen, Union City, CA) was used to evaluate the effects of spermine, arginine, ornithine, and arginase concentrations on high mannose glycan (HMN) modulation. An arginine-free formulation of the perfusion medium was used for small-scale control perfusion experiment #3 (arginine concentration study) according to the experimental design. In small-scale control perfusion experiment #4, all arginase inhibitors were added to the perfusion medium. The four arginase inhibitors: BEC hydrochloride, DL-α-difluoromethylornithine hydrochloride, NGhydroxy-L-arginine monoacetate salt, and Nω-hydroxy-nor-arginine diacetate salt were purchased from EMD Millipore Corporation (Billerica, MA).

Briefly, CHO cells were plated at a target density of 10-20x106 cells/mL, 3 mL working volume for each well. Cells were cultured at 36°C, 5% CO2, 85% relative humidity, and rocked at 225 rpm in a 50 mm Kuhner orbital incubator (Kuhner AG, Basel, Switzerland) for 3 or 4 days. Every 24 h, cells were centrifuged at 200xg for 5 min (Beckman Coulter, Brea, CA) to collect spent medium, and each well was then replenished with 3 mL of fresh medium. Collected spent medium was analyzed for titer, key metabolites, and % high mannose glycans (%HMN) (if appropriate). Cells were then harvested, and cell number and viability were measured.

Cell growth, metabolite and antibody titer assays

Viable cell density and viability were determined using a Cedex cell counter (Roche Innovative, Bielefeld, Germany). Metabolites including glucose, lactate, ammonia, glutamine, glutamate were obtained from NovaBioprofile Flex (Nova Biomedical, Waltham, MA). Antibody concentration in the spent medium was determined using Protein A affinity ultra-performance liquid chromatography (UHPLC) (Waters Corporation, Milford, MA) equipped with a 50 mm x 4.6 mm POROS A/20 protein A column (Life Technologies, Carlsbad, CA). After sample injection, the column was washed with phosphate-buffered saline (PBS) pH 7.1 to remove host cell CHO proteins. The bound antibodies were then eluted in acidic PBS buffer (pH 1.9) and detected by UV absorbance at 280 nm to quantify the antibody concentration.

- 17 047861

Glycan mapping using HILIC

Different N-glycan species of antibodies were analyzed by hydrophilic interaction liquid chromatography (HILIC). Purified antibodies were treated with N-glucosidase F (New England BioLabs, Ipswich, MA) at 37°C for 2 h to release glycans. The released glycans were labeled with 2-aminobenzoic acid and purified using GlycoClean S cartridges (Prozyme, Hayward, CA). The purified glycans were then desalted and dissolved in water for analysis. HILIC chromatography was performed on a 100 mm x 2.1 mm BEH Glycan column using UHPLC (Waters Corporation, Milford, MA) and the eluted glycans were detected, identified and quantified using a fluorescence detector based on the different elution times of the different glycans.

Small-scale control perfusion experiment #1:

Spermine concentration test

In this study, 5 different concentrations of spermine were tested. Perfusion medium containing 0, 7, 17, 35 and 100 μM spermine tetrahydrochloride (spermine 4HCl) was tested. Perfusion medium containing 35 μM spermine served as a control. Results from 5 day old samples demonstrate that as spermine concentration decreased, %HMN decreased, see Fig. 5. Spermine reduction/depletion did not affect the titer. The reduction in HM was achieved by reducing ornithine levels, by reducing the amount of spermine in the medium. As shown in Fig. 6, the amount of ornithine decreased with decreasing spermine concentration.

Small-Scale Control Perfusion Experiment #2: Ornithine Concentration Study

Four different concentrations of L-ornithine monohydrochloride were tested. Perfusion medium containing 14.8, 6, 0.6, and 0 (control) mM L-ornithine monohydrochloride (Sigma-Aldrich, St. Louis, MO) was used. The results showed that as the ornithine concentration increased, %HMN also increased, see Fig. 7. A second experiment was performed using cell line I in 2-liter bioreactors. Cell line I expressed the IgG2 antibody and was grown in fed-batch culture in one bioreactor, the medium received a single supplement of 0.1 g/L L-ornithine monohydrochloride on day 0 of culture, and the second bioreactor served as an ornithine-free control. Cultures were maintained for 12 days in cell culture medium containing soy hydrolysates. Bolus medium-feeding containing soybean hydrolysate was administered on days 4 and 8.

Glycan profiling was performed using peptide mapping. Antibodies were trypsinized in a manner similar to that described by Ren et al. (2009) Anal. Biochem. 392 12–21. Specifically, about 50–70 μg of each antibody was denatured and reduced with 7.0 M guanidine monochloride, 6 mM dithiothreitol (DTT) in 0.2 M Tris buffer (pH 7.5) at 37°C for 30 min. Each denatured/reduced sample was alkylated with 14 mM iodoacetic acid at 25°C for 25 min, followed by quenching with 8 mM DTT. Reduced/alkylated antibody samples were then transferred to 0.1 M Tris buffer at pH 7.5 using detergent-removing Pierce spin columns (Thermo Fisher Scientific Inc., Rockford, IL) according to the manufacturer's suggested protocol. The buffered sample was incubated at 37°C with 3.5 μg trypsin for 60 min. The enzymatic digestion was quenched by the addition of 2.2 μl 10% acetic acid. About 12-17 μg of digested antibody was injected for analysis.

Enzyme-digested antibodies were analyzed using an Agilent 1260 HPLC system directly coupled to a Thermo Scientific LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc., Rockford, IL). Proteolytic peptides were separated on a Waters BEH 300 C18 column (Waters Corporation, Milford, MA) 2.1 x 150 mm, 1.7 μ particles at 40°C with a flow rate of 0.2 mL/min. Mobile phase A was 0.02% TFA in water, and mobile phase B was 0.018% TFA in acetonitrile. Peptides were eluted with a gradient of 0.5-40% B over 90 min, followed by washing and re-equilibration of the column. The mass spectrometer was set up for a full scan orbitrap MS at 120,000 resolution, followed by five data-dependent collision-induced dissociation (CID) linear trap MS/MS scans with dynamic isolation. Automated data analysis for glycan profiling was performed using MassAnalyzer (see Zhang, (2009) Analytical Chemistry 81: 8354–8364).

The results again showed that as the ornithine concentration increased, %HMN also increased, see Fig. 8.

Small-Scale Control Perfusion Experiment #3: Arginine Concentration Study

In this study, 5 different concentrations of arginine were tested. Perfusion culture medium containing 3.686, 1.38, 0.92 and 0.46 g/L arginine was tested. Perfusion culture containing 1.843 g/L arginine served as a control. The results showed that when the concentration of arginine increased, %HM also increased, see Fig. 9.

Small-Scale Control Perfusion Experiment #4: Arginase Inhibitor Studies

Two series of experiments were conducted with the arginase inhibitor. In the first series of experiments

-

Claims (26)

Four commercially available arginase inhibitors: BEC hydrochloride, DL-α-difluoromethylornithine hydrochloride, N'-hydroxy-E-arginine monoacetate (NG) salt and Nω-hydroxy-nor-arginine diacetate salt were added to the cell cultures at three different concentrations: 1, 10 and 20 μM. The control did not contain any inhibitor. From this experiment, it was concluded that BEC and DL-α inhibitors were the most effective in reducing %HM (Fig. 10). The second series of experiments was performed using two inhibitors: BEC and DL-α. The BEC inhibitor was tested at concentrations of 0 (control), 10 μM, and 0.5 mM in the perfusion medium. The DL-α inhibitors were tested at concentrations of 0 (control), 10 μM, 1.0 mM, and 2.0 mM in the perfusion medium. It was shown that %HM decreased with increasing concentrations of both inhibitors, see Fig. 11. CLAUSE OF INVENTION 1. A method for reducing the content of high-mannose glycoforms of a recombinant protein, comprising culturing a host cell expressing the recombinant protein in a culture medium containing 35 μM or less spermine. 2. The method according to claim 1, wherein the concentration of spermine is from 7 μM to 35 μM. 3. The method according to claim 1, wherein the concentration of spermine is from 17 μM to 35 μM. 4. The method according to claim 1, wherein the concentration of spermine is from 7 μM to 17 μM. 5. The method according to claim 1, wherein the concentration of spermine is 35 μM. 6. The method according to claim 1, wherein the concentration of spermine is 17 μM. 7. The method according to claim 1, wherein the concentration of spermine is 7 μM. 8. The method of claim 1, wherein the host cell expressing the recombinant protein is cultured in a batch culture, a fed-batch culture, a flow culture, or a combination thereof. 9. The method according to claim 8, wherein the culture is a flow culture. 10. The method according to claim 9, wherein the perfusion comprises continuous perfusion. 11. The method according to claim 9, wherein the perfusion rate is constant. 12. The method according to claim 9, wherein the perfusion is carried out at a rate less than or equal to 1.0 working volume per day. 13. The method according to claim 9, wherein perfusion is achieved by alternating tangential flows. 14. The method according to claim 1, wherein the host cell expressing the recombinant protein is cultured in a bioreactor. 15. The method according to claim 14, wherein the bioreactor has a volume of at least 500 l. 16. The method according to claim 14, wherein the bioreactor has a volume of at least 500 L to 2000 L. 17. The method according to claim 14, wherein the bioreactor has a volume of at least 1000 L to 2000 L. 18. The method according to claim 14, wherein the bioreactor is inoculated with at least 0.5x10 ⁶ cells/ml. 19. The method according to claim 1, wherein the host cell expressing the recombinant protein is cultured in a serum-free cell culture medium. 20. The method according to claim 19, wherein the serum-free cell culture medium is a flow-through cell culture medium. 21. The method according to claim 1, wherein the host cells are mammalian cells. 22. The method of claim 1, wherein the host cells are Chinese hamster ovary (CHO) cells. 23. The method according to claim 1, wherein the recombinant protein is a glycoprotein. 24. The method according to claim 1, wherein the recombinant protein is selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant chimeric protein, or a cytokine. 25. The method according to claim 1, further comprising the step of collecting the recombinant protein produced by the host cell. 26. The method according to claim 1, wherein the recombinant protein produced by the host cell is purified and provided in a pharmaceutically acceptable dosage form. -