HK1230239A1 - Regulating ornithine metabolism to manipulate the high mannose glycoform content of recombinant proteins - Google Patents

Description

Modulation of ornithine metabolism to manipulate high mannose glycoform content of recombinant proteins

This application claims the benefit of U.S. provisional application No. 61/926,481, filed on month 1 and day 13 of 2014, which is incorporated herein by reference in its entirety.

Background

Higher eukaryotes perform a variety of post-translational modifications including methylation, sulfation, phosphorylation, lipid addition, and glycosylation. Many of the secreted proteins, membrane proteins, and proteins that target vesicles and certain intracellular organelles are known to be glycosylated. Glycosylation, which is the covalent attachment of a sugar moiety to a particular amino acid, is one of the most common but important post-translational modifications of recombinant proteins. Protein glycosylation has a variety of functions in cells, including its important role in protein folding and quality control, molecular trafficking and sorting, and cell surface receptor interactions.

N-linked glycosylation involves the addition of an oligosaccharide to an asparagine residue (e.g., Asn-X-Ser/Thr) present in certain recognition sequences in proteins. N-linked glycosylation contains a standard branched structure consisting of mannose (Man), galactose, N-acetylglucosamine and neuraminic acid. High mannose oligosaccharides typically comprise two N-acetylglucosamines and a plurality of 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 mannose 5(Man5), mannose 6(Man6), mannose 7(Man7), mannose 8(Man8), and mannose 9(Man 9).

Although the glycoform of recombinant glycoproteins expressed by Chinese Hamster Ovary (CHO) host cells is largely determined by intrinsic genetic factors, the high mannose glycoform content can also be influenced by cell culture conditions (Pacis, et al, (2011) Biotechnol Bioeng 108, 2348-.

Glycosylation can affect the therapeutic efficacy of recombinant protein drugs. The effects of glycosylation on bioactivity, pharmacokinetics, immunogenicity, solubility, and in vivo clearance of therapeutic glycoproteins have made monitoring and control of glycosylation a key parameter in biopharmaceutical production. The high mannose glycoform content of therapeutic proteins is a key quality attribute that has been found to affect the pharmacokinetic properties of certain therapeutic antibodies (Goetze, et al, (2011) Glycobiology21, 949-59; Yu, et al, (2012) MAbs 4, 475-87). Therefore, a method for controlling the high mannose glycoform content of a therapeutic protein would be beneficial.

There is a need in the pharmaceutical industry to manipulate and control the high mannose glycoform content of recombinant therapeutic glycoproteins, and methods for this would be useful. The present invention provides a method for manipulating the high mannose glycoform content of a recombinant glycoprotein by modulating ornithine metabolism in a host cell.

Summary of The Invention

The present invention provides a method for manipulating the high mannose glycoform content of a recombinant protein comprising culturing a host cell expressing the recombinant protein in a cell culture under conditions which modulate ornithine metabolism in the host cell.

In one embodiment, ornithine metabolism in the host cell is regulated by reducing the accumulation of ornithine 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 a host cell is regulated by adding an arginase inhibitor to the cell culture medium. In another related embodiment, the arginase inhibitor is BEC (S- (2-boronoethyl) -1-cysteine) or DL-a-difluoromethylornithine. In another related embodiment, the arginase inhibitor is BEC (S- (2-boronoethyl) -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 μ M. In another related embodiment, the concentration of the arginase inhibitor is 10 μ M to 2 mM. In yet another related embodiment, the concentration of the arginase inhibitor is 10 μ M. In yet another related embodiment, the concentration of the arginase inhibitor is 0.5 mM. In yet another related embodiment, the concentration of the arginase inhibitor is 1 mM. In yet another related embodiment, the concentration of the arginase inhibitor is 2 mM.

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

In another embodiment, ornithine metabolism in the host cell is regulated by increasing the accumulation of ornithine 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 ornithine, arginine, an ornithine decarboxylase inhibitor, an ornithine transaminase, a nitric oxide synthase inhibitor, or an arginine decarboxylase inhibitor. In yet another related embodiment, ornithine accumulation in the host cell is regulated by the addition of at least 0.6mM ornithine to the cell culture medium. In yet another related embodiment, the concentration of ornithine is from 0.6mM to 14.8 mM. In yet another related embodiment, the concentration of ornithine is from 6mM to 14.8 mM. In yet another related embodiment, the concentration of ornithine is 0.6 mM. In yet another related embodiment, the concentration of ornithine is 6 mM.

In yet another related embodiment, the concentration of ornithine is 14.8 mM. In another embodiment, ornithine accumulation in the host cell is regulated by the addition of at least 8.7mM arginine to the cell culture medium. In yet another related embodiment, the concentration of arginine is 8.7mM 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 embodiment, ornithine accumulation in the host cell is regulated by the addition of an ornithine decarboxylase inhibitor, nitric oxide synthase inhibitor, ornithine transaminase inhibitor, or arginine decarboxylase inhibitor to the cell culture medium. In a related embodiment, ornithine accumulation in the host cell is regulated by the addition of an ornithine decarboxylase inhibitor to the cell culture medium. 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 the host cell is regulated by the addition of an ornithine transaminase inhibitor to the cell culture medium. In yet another embodiment, the ornithine transaminase inhibitor is 5-fluoromethylornithine (F-FMOrn). In yet another related embodiment, the host cell is regulated by the addition of a nitric oxide synthase inhibitor to the cell culture medium. In yet another related embodiment, the nitric oxide synthase inhibitor is 2-ethyl-2-thioisourea or N-nitro-L-arginine and L^G-monomethyl-L-arginine. In yet another related embodiment, the nitric oxide synthase inhibitor is N-nitro-L-arginine and L^G-monomethyl-L-arginine.

In another related embodiment, ornithine accumulation in the host cell is regulated by the addition of an arginine decarboxylase inhibitor to the cell culture medium. In yet another related embodiment, the arginine decarboxylase inhibitor is Asymmetric Dimethylarginine (ADMA).

The present invention provides a method of producing a recombinant protein with reduced high mannose glycoform content comprising culturing a host cell expressing the recombinant protein in a cell culture, wherein ornithine metabolism is regulated by reducing ornithine accumulation in the host cell. In a related embodiment, ornithine accumulation 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 the host cell is reduced by adding an arginase inhibitor to the cell culture medium. In yet another related embodiment, the arginase inhibitor is BEC (S- (2-boronoethyl) -1-cysteine) or DL-a-difluoromethylornithine. In yet another related embodiment, the arginase inhibitor is BEC (S- (2-boronoethyl) -1-cysteine). In yet another related embodiment, the arginase inhibitor is DL-a-difluoromethylornithine. In yet another related embodiment, the arginase inhibitor is at least 10 μ Μ. In yet another related embodiment, the arginase inhibitor is 10 μ M to 2 mM. In yet another related embodiment, the arginase inhibitor is from 10 μ M to 20 μ M. In yet another related embodiment, the arginase inhibitor is 10 μ M. In yet another related embodiment, the arginase inhibitor is 0.5 mM. In yet another related embodiment, the arginase inhibitor is 1 mM. In yet another related embodiment, the arginase inhibitor is 2 mM.

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

The present invention provides a method for producing a recombinant protein with increased high mannose glycoform content comprising culturing a host cell expressing the recombinant protein in a cell culture, wherein ornithine metabolism is regulated by increasing ornithine accumulation in the host cell. In a related embodiment, ornithine accumulation in the host cell is increased by adding ornithine, arginine, an ornithine decarboxylase inhibitor, an ornithine transaminase, a nitric oxide synthase inhibitor, or an arginine decarboxylase inhibitor to the cell culture medium.

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

In another related embodiment, ornithine accumulation in the host cell is increased by culturing the host cell in a cell culture medium comprising at least 8.7mM arginine. In yet another related embodiment, the concentration of arginine is 8.7mM 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 the 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 transaminase inhibitor, or an arginine decarboxylase inhibitor. In another related embodiment, ornithine accumulation in the 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 the host cell is increased by culturing the host cell in a cell culture medium comprising an ornithine transaminase inhibitor. In yet another embodiment, the ornithine transaminase inhibitor is 5-fluoromethylornithine (F-FMOrn).

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

In another embodiment, ornithine accumulation in the host cell is increased by culturing the host cell in a cell culture medium comprising 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 batch culture, fed-batch culture, perfusion culture, or a combination thereof. In yet another related embodiment, the culture is a perfusion culture. In yet another related embodiment, perfusion comprises continuous perfusion. In yet another related embodiment, the rate of perfusion is constant. In yet another related embodiment, perfusion is performed at a rate of less than or equal to 1.0 working volumes per day. In yet another related embodiment, perfusion is achieved by alternating tangential flow.

In another embodiment, the host cell expressing the recombinant protein is cultured in a bioreactor. In yet another related embodiment, the bioreactor has a capacity of at least 500L. In yet another related embodiment, the bioreactor has a capacity of at least 500L to 2000L. In yet another related embodiment, the bioreactor has a capacity of at least 1000L to 2000L. In yet another related embodiment, at least 0.5x10 is used⁶cells/mL inoculate the bioreactor.

In another embodiment, the host cell expressing the recombinant protein is cultured in serum-free cell culture medium. In another related embodiment, the serum-free medium is a perfusion cell culture medium. In yet another related embodiment, the host cell is a mammalian cell. In yet another related embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell.

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.

In another embodiment, the above method further comprises the step of harvesting the recombinant protein produced by the host cell.

In another embodiment, the recombinant protein produced by the host cell is purified and formulated into a pharmaceutically acceptable formulation.

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 formulated in a pharmaceutically acceptable formulation.

Brief Description of Drawings

FIG. 1 overview of ornithine metabolism. ARG, arginine; AZ, anti-enzyme; AZIN, an anti-enzyme inhibitor; P5C, dihydropyrrole-5-carboxylate; ASP, aspartate; ORNT, ornithine transporter; GATM, glycine amidinotransferase; NOS, nitric oxide synthase; OAT, ornithine transaminase; ODC, ornithine decarboxylase; OTC, ornithine transcarbamylase; SMS, spermine synthetase; SRS, spermidine synthase.

Figure 2 identification of ornithine as a metabolic indicator associated with high mannan levels. High mannan levels (% HM) of secreted recombinant monoclonal antibodies from eight different cell lines (cell lines a to H) tested at day 8 (D8, white bar), day 9 (D9, grey bar) and day 10 (D10, black bar) of the fed-batch method evaluated under medium #1(a) or medium #2 (B). Correlation between high mannan levels detected in spent medium (C) and extracellular ornithine levels. Average ornithine levels and average high mannan levels on day 9 from the eight cell lines were compared. The pearson coefficient R, is 0.83.

Figure 3 correlation between high mannose and extracellular ornithine levels: A) high mannose glycoform content detected when cell line H was exposed to eight different production conditions (#1 to # 8). B) Corresponding extracellular relative ornithine levels. C) Correlation between% high mannose glycoform content and extracellular relative ornithine levels. The pearson correlation coefficient R, is 0.78.

FIG. 4 relative mRNA expression levels of arginase 1 from cell pellets collected from eight different cell lines on days 3, 6, 8, 9 and 10. The corresponding high mannose glycoform content is also shown.

FIG. 5 high mannose glycoform content on recombinant glycoproteins expressed by cells grown in cell culture medium containing spermine tetrahydrochloride at concentrations of 0. mu.M, 7. mu.M, 17. mu.M, 35. mu.M and 100. mu.M. Samples were collected on day 5 of the simulated perfusion test. A35 μ M sample served as a control.

FIG. 6 concentration (mg/L) of extracellular ornithine endogenously produced by cell cultures exposed to concentrations of spermine tetrahydrochloride of 0. mu.M, 7. mu.M, 17. mu.M, 35. mu.M and 100. mu.M. Samples were collected on day 5 of the simulated perfusion test. A35 μ M sample served as a control.

FIG. 7 high mannose glycoform content on recombinant glycoproteins expressed by cells grown in cell culture medium containing concentrations of L-ornithine monohydrochloride of 0mM, 0.6mM, 6mM, and 14.8 mM. Samples were collected on day 5 of the simulated perfusion test. The 0mM sample served as a control.

FIG. 8 high mannose glycoform content on recombinant glycoproteins expressed by cell line "I" grown in cell culture medium containing 0.1 g/LL-ornithine monohydrochloride (black bars) or control cell culture without exogenously added ornithine (white bars). Samples were collected on day 6, day 8 and day 12.

FIG. 9 high mannose glycoform content on recombinant glycoproteins expressed by cells grown in cell culture medium containing arginine monohydrochloride at concentrations of 2.2mM, 4.4mM, 6.5mM, 8.7mM, and 17.5 mM. 8.7mM sample served as control. Samples were collected on day 4 of the simulated perfusion test.

FIG. 10 different arginase inhibitors BEC hydrochloride (BEC), DL- α, difluoromethylornithine hydrochloride (DL-a), N in supplemented concentrations of 1. mu.M, 10. mu.M and 20. mu.M^G-high mannose glycoform content on recombinant glycoproteins expressed by cells grown in cell culture medium of Hydroxy-L-arginine monoacetate (NG) and N ω -Hydroxy-norarginine diacetate (Nw). The control contained no inhibitor. Samples were collected on day 4 of the simulated perfusion test.

FIG. 11 high mannose glycoform content on recombinant glycoproteins expressed by cells grown in cell culture medium containing arginase inhibitor BEC hydrochloride (BEC) at concentrations of 0.0mM, 0.01mM, 1.0mM, and 2.0mM, DL-alpha, difluoromethylornithine hydrochloride (DL-a). Samples were collected on day 4 of the simulated perfusion test.

Detailed Description

It was found that the high mannose glycoform content of the expressed recombinant glycoprotein is influenced by the accumulation of ornithine in the host cell and can therefore be manipulated by regulating ornithine metabolism in the host cell.

Ornithine is a non-protein coding amino acid involved in the urea cycle, polyamine synthesis and arginine metabolism. Ornithine is also a precursor of glutamate and proline via ornithine-transaminase (OAT) activity, see figure 1. Defects in OAT in humans result in Gyroid Atrophy (GA) of the choroid and retina, a condition 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 has been shown to reduce plasma ornithine levels and prevent retinal degeneration (Wang T et al, PNAS 2000; 97 (3): 1224-. 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). The synthesis and stability of ODC and polyamine transporter activity are affected by external 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). Increased polyamine biosynthesis has been associated with increased resistance to osmotic stress in plants (Alcazar R et al, Biotechnol Lett 2006; 28: 1867-one 1876). As part of the urea cycle, Ornithine Transcarbamylase (OTC) catalyzes the conversion of ornithine to citrulline. OTC deficiency in humans causes the accumulation of ammonia in the blood (Hopkins et al, arch, disc, 196944: 143-. Ornithine metabolism occurs in cytosol and mitochondria, where OTC and OAT catalyzed metabolic steps occur. The mitochondrial ornithine transporter, orn 1, is required for ornithine import into mitochondria. Mutations in ORNT1 in humans cause hyperornithine-hyperammonemia-homocitrullinuria (HHH) syndrome characterized by elevated ornithine and ammonia plasma levels (Camacho et al, NatGenet (1999); 22: 151-; (Valle D et al, 2001, 1857-1896).

As described herein, the extent of ornithine accumulation in the host cell was found to correlate with the high mannose glycoform content of the expressed recombinant glycoprotein as determined by the extracellular level of ornithine in the cell culture medium. Manipulation of high mannose content can be achieved by regulating ornithine metabolism in the host cell. The present invention provides a method for manipulating the high mannose glycoform content of a recombinant protein comprising culturing a host cell expressing the recombinant protein in a cell culture under conditions which modulate ornithine metabolism in the host cell. Ornithine metabolism may be regulated by reducing or increasing ornithine accumulation in the host cell. The present invention provides a method for producing a recombinant protein with reduced or increased high mannose glycoform content comprising culturing a host cell expressing the recombinant protein in a cell culture that regulates ornithine accumulation in the host cell.

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

Ornithine accumulation in the host cell is the result of altered ornithine metabolism. The extent of ornithine accumulation in the host cell can be regulated by modulating ornithine metabolism. Intracellular metabolite levels may be responsive in extracellular levels (i.e., detected in the cell culture medium). An indicator of ornithine accumulation in a host cell can be made by measuring the amount of ornithine secreted into the cell culture medium. As described herein, a time-course dependent increase in ornithine levels is present in a cell culture medium lacking exogenous ornithine.

"high mannose sugar type content", "high mannan level", and "level of high mannose substances" are used interchangeably and are indicated by the abbreviations "HM", "% HM", "HMN", or "% HMN" and refer to the relative percentages of fine-grained mannose 5(Man5), mannose 6(Man6), mannose 7(Man7), mannose 8(Man8), and mannose 9(Man9) glycan substances.

The level of ornithine secreted into the cell culture medium was found to correlate with the high mannose glycoform content of the recombinant glycoprotein expressed by the host cell in the cell culture. When ornithine accumulation in the host cell is reduced by culturing the host cell in a cell culture medium containing an arginase inhibitor or spermine, the high mannose glycoform content of the expressed glycoprotein is reduced. When ornithine accumulation in a host cell is increased by culturing the host cell in a cell culture medium containing ornithine or arginine, the high mannose glycoform content of the expressed glycoprotein is increased.

The present invention provides a method for modulating ornithine accumulation in a host cell by culturing the host cell in a cell culture medium containing an arginase inhibitor. Arginine is a metabolic precursor of ornithine, and arginase is an enzyme that catalyzes the conversion of arginine to ornithine. It was observed that arginase mRNA expression levels correlated with the amount of ornithine accumulation when comparing the metabolism and expression profiles of different cell lines. Blocking arginase activity with arginase inhibitors could potentially reduce the level of ornithine production. However, the potency of arginase inhibitors may be reduced due to high levels of arginine in the cell culture medium. In addition, there are other metabolic precursors of ornithine (i.e., glutamate and proline, see figure 1) that can contribute to ornithine accumulation.

As described herein, it was found that the high mannose glycoform content of recombinant proteins expressed by cultured host cells can be modulated by the addition of an arginase inhibitor to the cell culture medium. Blocking arginase activity reduces the amount of ornithine production in the host cell, reducing the high mannan level of the expressed recombinant glycoprotein.

Arginase also inhibits Ornithine Transcarbamylase (OTC) (Vissers et al, (1982) J.Gen.Microbio.128: 1235-1247). Blocking arginase activity not only reduces the production of ornithine from arginine, but can potentially release the inhibition of OTC activity, allowing for conversion of ornithine to citrulline, allowing for a further reduction in ornithine accumulation (see, figure 1).

In OTC deficient patients, ammonia levels are elevated. This may also lead to elevated ammonia levels if arginase expression or enhanced activity in host cells expressing recombinant proteins with high levels of high mannans induces OTC inhibition. Elevated intracellular ammonia levels can potentially alter the pH gradient in the Golgi apparatus and cause suboptimal relocation of glycosyltransferases, which leads to higher levels of high mannan due to incomplete glycan branching in the Golgi complex (Campbell et al, (1973) NJM 288 (1): 1-6; Hopkins et al, (1969) architecture of Disease in Childhod 44 (234): 143-.

Suitable inhibitors are known in the art and are available from commercial sources such arginase inhibitors include BEC hydrochloride, DL- α, difluoromethylornithine hydrochloride, N^G-hydroxy-L-arginine and N ω -hydroxy-norarginine. In one embodiment, the arginase inhibitor is BEC (S- (2-boronoethyl) -1-cysteine) or DL-a-difluoromethylornithine. In one embodiment of the present invention, the arginase inhibitor is BEC (S- (2-boronoethyl) -1-cysteine). In one embodiment of the present invention, the arginase inhibitor is DL-a-difluoromethylornithine.

Arginase inhibitors can be added to the cell culture medium at a concentration of at least 10 μ M to reduce the high mannan level of the expressed recombinant protein without significantly affecting productivity. In one embodiment, the concentration of the arginase inhibitor is 10 μ M to 2 mM. In another embodiment, the concentration of the arginase inhibitor is 10 μ M. 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 host cells can also be regulated by culturing the host cells in cell culture media containing spermine, as described in the examples below. Ornithine is the starting point of the polyamine pathway and the synthesis of putrescine, spermidine and spermine, which are polyamines, by the action of Ornithine Decarboxylase (ODC). ODCs can be inactivated directly using exogenously added spermine or by enzyme resistance, see figure 1. As described below, inactivation of ODC can lead to accumulation of ornithine. Inhibition of ODC activity can be relieved by limiting the amount of exogenously added spermine, and ornithine can be metabolized through the polyamine pathway, thus reducing overall ornithine accumulation in the host cell.

Spermine can be added to the cell culture medium at a concentration of less than or equal to 35 μ M to reduce the high mannan level of the expressed recombinant protein without significantly affecting productivity. In one embodiment, the concentration of spermine is 7 μ M to 35 μ M. In one embodiment, the concentration of spermine is 17 μ M to 35 μ M. In one embodiment, the concentration of spermine is 7 μ M to 17 μ M. In another embodiment, the concentration of spermine is 35 μ M. In another embodiment, the concentration of spermine is 17 μ M. In another embodiment, the concentration of spermine is 7 μ M.

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

As described herein, extracellular ornithine levels in conditioned cell culture media were found to correlate with high mannan levels on recombinant glycoproteins. It was found that culturing a host cell expressing a recombinant protein in an ornithine containing cell culture medium produces a recombinant glycoprotein having elevated levels of high mannan.

As described above, ornithine accumulation in cell culture media is likely to reflect alterations in ornithine metabolism that can result in similar ammonia accumulation to patients carrying defective ornithine metabolism genes (e.g., OTC defect or orn 1 mutation). However, the cellular mechanism behind the ammonia-induced increase in high mannose is unknown, and it has been shown that changes in the pH gradient in the golgi apparatus can lead to suboptimal relocation of glycosyltransferases. These changes can lead to a reduction in the effectiveness of the glycosylase to complete glycan branches and thus to higher levels of high mannan levels.

Another possibility is that ornithine accumulation potentially induces disturbances in redox homeostasis (Zanatta et al, (2013) Life sciences 93 (4): 161-. Ornithine is clearly associated with high mannose, suggesting the possibility of modulating high mannose glycoform content through cellular redox status. Ornithine may increase the level of lipid oxidation. Because many glycosylation-regulating enzymes are lipid membrane-bound, alterations in lipid oxidation caused by ornithine accumulation can potentially alter the integrity and activity of glycosylation-regulating enzymes in the golgi and ER, and subsequently affect high mannose glycoform content.

Ornithine may be added to the cell culture medium at a concentration of at least 0.6mM to elevate the high mannan level of the expressed recombinant protein without significantly affecting productivity. In one embodiment, the concentration of ornithine is from 0.6mM to 14.8 mM. In one embodiment, the concentration of ornithine is from 6mM to 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 a metabolic precursor of ornithine. It was found that high mannan levels were elevated in recombinant proteins expressed in host cells cultured in cell culture medium containing exogenous arginine. Increasing the amount of exogenous arginine increases the amount of metabolic precursors available for ornithine synthesis, thus increasing the level of ornithine in the host cell.

The present invention provides for the regulation of ornithine accumulation in host cells by the addition of arginine at a concentration of at least 8.7mM to elevate high mannan levels of expressed recombinant proteins without significantly affecting productivity. In one embodiment, the concentration of arginine is 8.7mM to 17.5 mM. In another embodiment, the concentration of arginine is 8.7 mM. In another embodiment, the concentration of arginine is 17.5 mM.

Ornithine accumulation can be regulated by the addition of an ornithine decarboxylase inhibitor (ODC), ornithine transaminase (OAT), Nitric Oxide Synthase (NOS), or Arginine Decarboxylase (ADC) to the cell culture medium, thus providing a means to regulate ornithine metabolism and ornithine accumulation in the host cell to manipulate the high mannose glycoform content 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 α -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 for the manipulation of high mannose glycoform content of recombinant proteins by augmenting cell culture with these inhibitors to increase ornithine accumulation in host cells.

Ornithine accumulation can be regulated by blocking the activity of enzymes regulating arginine accumulation (fig. 1). By small molecule inhibitors such as 2-ethyl-2-thioisourea and N-nitro-L-arginine and L^GThe inhibition of nitric oxide synthase activity by monomethyl-L-arginine and/or the inhibition of arginine decarboxylase activity by Asymmetric Dimethylarginine (ADMA) increases the flux of ornithine conversion from arginine. The present invention provides for the manipulation of high mannose glycoform content of recombinant proteins by augmenting cell culture with these inhibitors to increase ornithine accumulation in host cells.

In one embodiment of the invention, the cell culture medium provided is a serum-free cell culture medium. In one embodiment, the cell culture medium is a perfusion cell culture medium.

As used herein, the term "cell culture medium" (also referred to as "medium", "cell culture medium", "tissue culture medium") refers to any nutrient solution used to grow cells, such as animal or mammalian cells, and which typically provides at least one or more components from the group consisting of: an energy source (typically in the form of a carbohydrate, such as glucose); one or more of all essential amino acids, and typically 20 essential amino acids, plus cysteine; vitamins and/or other organic compounds that are often required at low concentrations; lipids or free fatty acids; and trace elements such as inorganic compounds or naturally occurring elements that are typically required at very low concentrations, often in the micromolar range.

Depending on the needs of the cells to be cultured and/or the desired cell culture parameters, the nutrient solution may optionally be supplemented with additional components to optimize cell growth, such as hormones and other growth factors, e.g., insulin, transferrin, epidermal growth factor, serum, and the like; salts such as calcium, magnesium and phosphate and buffers such as HEPES; nucleosides and bases, such as adenosine, thymidine, hypoxanthine; and protein and tissue hydrolysates, such as hydrolyzed animal proteins (peptones or peptone mixtures, which can be obtained as by-products from animals, refined gelatin or plant substances); antibiotics, such as gentamicin; cytoprotective or surface-active agents, e.g.F68; polyamines such as putrescine, spermidine or spermine (see, e.g., WIPO publication No. WO 2008/154014) and pyruvate (see, e.g., U.S. patent No. 8053238).

Cell culture media include those commonly employed in and/or known for any cell culture method, such as, but not limited to, batch, extended batch, fed-batch, and/or perfusion, or continuous culture of cells.

The cell culture media components can be completely milled into a powder media formulation; partially milled with liquid supplement, added to cell culture medium as needed; or the nutrients may be added to the cell culture in a completely liquid form.

"basal" (or batch) cell culture medium refers to a cell culture medium that is typically used to initiate a cell culture and is sufficient to completely support the cell culture.

"growth" cell culture medium refers to cell culture medium that is typically used in a cell culture during an exponential growth phase ("growth phase") and is sufficient to fully support the cell culture during this phase. The growing cell culture medium may also contain a selection agent that confers resistance or viability to the selectable marker incorporated into the host cell line. Such selection agents include, but are not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, bleomycin, methionine sulfoximine, methotrexate, glutamine-free cell culture media, cell culture media lacking glycine, hypoxanthine, and thymidine, or lacking thymidine alone.

By "production" cell culture medium is meant cell culture medium that is typically used in cell culture during the transition phase and production phase at the end of exponential growth and protein production instead and is sufficient to fully maintain the cell density, cell viability and/or product titer required for these phases.

"perfusion" cell culture medium refers to a cell culture medium that is typically used in cell cultures maintained by perfusion culture or continuous culture methods and is sufficient to fully support the cell culture during this process. The perfusion cell culture medium formulation may be concentrated or more concentrated than the base cell culture medium formulation to accommodate the method used to remove the spent medium. Perfusion cell culture media can be used during 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 identified or known to be consumed during the production phase of the cell culture. The concentrated medium can be based on almost any cell culture medium formulation. Such concentrated feed media may contain, for example, about 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 12X, 14X, 16X, 20X, 30X, 50X, 100X, 200X, 400X, 600X, 800X, or even 1000X of their normal amounts of some or all of the components of the cell culture medium.

Cell cultures may also be supplemented with independently concentrated feeds of specific nutrients that may be difficult to prepare or rapidly depleted in the cell culture. Such nutrients may be amino acids such as tyrosine, cysteine, and/or cystine (see, e.g., WIPO publication No. 2012/145682). In one embodiment, a concentrated tyrosine solution is fed separately 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 fed independently to a cell culture grown in a cell culture medium lacking tyrosine, cystine, or cysteine. The separate feeding may be started before or at the beginning of the production phase. The independent feeding may be achieved by fed-batch of cell culture medium on the same or different days as the concentrated feed medium. The independent feed may also be perfused on the same or different days as the perfusion medium.

In certain embodiments, the cell culture medium is serum free and/or free of animal-derived products or components. In certain embodiments, the cell culture medium is chemically defined, wherein all chemical components are known.

As understood by practitioners, animal or mammalian cells are cultured in a medium suitable for culturing the particular cell, and this can be determined by one of skill in the art without undue experimentation. Commercially available media can be utilized and include, but are not limited to, Iscove's modified Dulbecco's medium, RPMI 1640, minimum essential medium-alpha. (MEM-alpha), Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, alpha MEM, Eagle's basal medium with Earle's BSS, high glucose DMEM with glutamate, high glucose DMEM without glutamate, low glucose DMEM without glutamate, DMEM with glutamate: f121: 1. GMEM (Glasgow's MEM), GMEM containing glutamate, Grace's complete insect Medium, Grace's insect Medium without FBS, Ham's F-10 with glutamate, Ham's F-12 with glutamate, IMDM with HEPES and without glutamate, IP41 insect Medium, 15(Leibovitz) (2X) without glutamate or phenol Red, 15(Leibovitz) without glutamate, McCoy's 5A modified Medium, Medium 199, MEM Eagle (2X) without glutamate or phenol Red, MEM Eagle-Earle BSS with glutamate, MEM MEAGE-Earle BSS without glutamate, MEM Eagle-Handse BSS without glutamate, NCTC-109 with glutamate, Richch's CM with glutamate, RPMI 1640 with HEPES, glutamate and/or penicillin-streptomycin, RPMI 1640 with glutamate, RPMI 1640 without glutamate, Schneider's insect medium or any other medium known to the person skilled in the art formulated for a specific cell type. Supplementary components or ingredients (including optional components) may be added to the aforementioned exemplary media at appropriate concentrations or amounts as needed or desired, and will be known and practiced by those of ordinary skill in the art using routine skill.

In one embodiment of the invention provides a host cell is a mammalian cell. In one embodiment, the host cell is a Chinese Hamster Ovary (CHO) cell.

The cell lines (also referred to as "host cells") used in the present invention are genetically engineered to express polypeptides of commercial or scientific value. Cell lines are typically derived from lineages that originate from primary cultures that can be maintained in culture for an indefinite period of time. The cells may contain an expression vector (construct), such as a plasmid or the like, introduced, for example, by transformation, transfection, infection or injection, with a coding sequence or portion thereof encoding a protein for expression and production in culture. Such expression vectors contain the necessary elements for the transcriptional and translational insertion of the coding sequence. Methods well known and practiced by those skilled in the art can be used to construct expression vectors containing sequences encoding the proteins and polypeptides produced, as well as appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo gene recombination. In j.sambrook et al, 2012,Molecular Cloning，A Laboratory Manualcold Spring Harbor Press, Plainview, N.Y. version 4 or any previous version; ausubel et al, 2013,Current Protocols in Molecular Biology，John Wiley&sons, New York, n.y, or any previous version; kaufman, R.J.,Large Scale Mammalian Cell Culture，1990 describe such techniques, all of which are incorporated herein for any purposeText.

Animal cells, mammalian cells, cultured cells, animal or mammalian host cells, recombinant host cells, and the like are all terms used for cells that can be cultured according to the methods of the invention. Such cells are typically cell lines obtained or derived from mammals and are capable of growing and surviving when in monolayer culture or suspension culture in media containing appropriate nutrients and/or other factors such as those described herein. Cells are generally selected that can express and secrete the protein or can be molecularly engineered to express and secrete large quantities of a particular protein, more specifically the glycoprotein of interest, into the culture medium. It will be appreciated that the protein produced by the host cell may be endogenous or homologous to the host cell. Alternatively, the protein is heterologous, i.e., heterogeneous, to the host cell, e.g., a human protein produced and secreted by a Chinese Hamster Ovary (CHO) host cell. In addition, mammalian proteins, i.e., those originally obtained or derived from a mammalian organism, can be obtained by the methods of the present invention and can be secreted by the cells into the culture medium.

The methods of the invention can be used in the culture of a variety of cells. In one embodiment, the cultured cells are eukaryotic cells, such as plant and/or animal cells. The cell may be a mammalian cell, a fish cell, an insect cell, an amphibian cell, or an avian cell. A wide variety of mammalian cell lines suitable for growth in culture are available from the american germplasm preservation center (Manassas, Va.) and other depositories as well as commercial suppliers. Cells that can be used in the methods of the invention include, but are not limited to, MK2.7 cells, PER-C6 cells, Chinese hamster ovary Cells (CHO) such as CHO-K1(ATCC CCL-61), DG44(Chasin et al, 1986, Som. cell Molec. Genet., 12: 555-; monkey kidney cells (CV1, ATCC CCL-70); conversion by SV40Monkey kidney CV1 cells (COS cells, COS-7, ATCC CRL-1651); HEK 293 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, kidney epithelial cells, and retinal epithelial cells), as well as established cell lines and strains thereof (e.g., human embryonic kidney cells (e.g., 293 cells or subcloned 293 cells for growth in suspension culture, Graham et al, 1977, J.Gen.Virol., 36: 59), baby hamster kidney cells (BHK, ATCCCL-10), mouse support cells (TM4, Mather, 1980, biol.Reprod., 23: Aca. 251), human cervical cancer cells (HELA, ATCC-2), canine kidney cells (ATCC 2, CCL-34W cells (human lung cells), ATCC CCL-75); human liver cancer cells (HEP-G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCCCL-51); buffalo rat hepatocytes (BRL 3A, ATCC CRL-1442); TRI cells (Mather, 1982, Annals NYAcad.Sci., 383: 44-68); MCR 5 cells; FS4 cells; PER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCK cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa229 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, 2RA cells, WISH cells, BS-C-I cells, LLC-MK cells₂Cells, clone M-3 cells, 1-10 cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PK₁Cells, PK (15) cells, GH₁Cells, GH₃Cells, L2 cells, LLC-RC256 cells, MH₁C₁Cells, XC cells, MDOK cells, VSW cells, and TH-I, B1 cells or derivatives thereof), fibroblasts from any tissue or organ (including but not limited to heart, liver, kidney, colon, intestine, esophagus, stomach, neural tissue (brain, spinal cord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue (lymph nodes, adenoids, tonsil, 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, citrullineAcidemia 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 cells, WI-38 cells, WI-26 cells, MiCl₁Cells, CV-1 cells, COS-3 cells, COS-7 cells, Vero 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 cell, HSDM₁C₃Cells, KLN205 cells, McCoy cells, mouse L cells, line 2071 (mouse L) cells, L-M line (mouse L) cells, L-MTK (mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1 cells, Swiss/3T3 cells, Indian motac cells (Indian muttac cells), SIRC cells, C cells_IICells and Jensen cells or derivatives thereof) or any other cell type known to those skilled in the art.

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

In one embodiment of the invention, the host cell expressing the recombinant protein is cultured in a bioreactor. In another embodiment of the invention, the bioreactor has a capacity of at least 500L. In a related embodiment, the bioreactor has a capacity of at least 500L to 2000L. In yet another related embodiment, the bioreactor has a capacity of at least 1000L to 2000L. In one embodiment of the invention, the cells are cultured by culturing with at least 0.5X10 in serum-free medium⁶cells/mL were seeded into the bioreactor to establish. In one embodiment, the invention further comprises the step of harvesting the recombinant protein produced by the host cell. In one embodiment of the invention, it is contemplated that the recombinant protein produced by the host cell is purified and formulated into a pharmaceutically acceptable formulation.

For purposes of understanding and not limitation, the skilled practitioner will appreciate that cell culture and culture operations for protein production can include three general types; i.e., batch culture, extended culture, fed-batch culture, perfusion culture, or combinations thereof. In batch culture, cells are initially cultured in culture medium and such medium is not removed, replaced or supplemented, i.e., the cells are not "fed" with fresh medium during or before the end of the culturing operation. The desired product is harvested at the end of the cultivation operation.

For fed-batch culture, the culture run time is increased during the run by supplementing the medium with fresh medium once or more times daily (or continuously), i.e., "feeding" the cells with new medium ("feed medium") during the culture period. Fed-batch culture may include various feeding regimens and times as described above, e.g., daily, every other day, every third day, etc., more than once per day, or less than once per day, etc. In addition, fed-batch culture can be continuously fed with a feed medium. The desired product is then harvested at the end of the cultivation/production operation.

Perfusion culture is a culture in which a cell culture receives fresh perfusion medium and removes spent medium. The perfusion of fresh medium into the cell culture and removal of spent medium may be continuous, stepwise, intermittent, or a combination of any or all of these. The perfusion rate may range from less than one working volume per day to many working volumes per day. Preferably, the cells remain in the culture and the removed spent medium is substantially free of cells or has significantly fewer cells than the culture. Recombinant proteins expressed by cell cultures can also be retained in culture or removed with spent media. Removal of spent media can be accomplished by several means including centrifugation, sedimentation, or filtration, see, e.g., Voisard et al (2003), Biotechnology and Bioengineering 82: 751-65. The preferred filtration method is alternating tangential flow filtration. The alternating tangential flow is maintained by pumping the culture medium through the hollow fiber filter assembly using an ATF device. See, for example, U.S. Pat. nos. 6,544,424; furey (2002) Gen. Eng. News.22(7), 62-63. The filter separates particles based on size or molecular weight. Depending on the application, the filter may be selected based on pore size or molecular weight cut-off (MWCO) value. Filters include membrane filters, ceramic filters and metal filters and may be of any shape, including spiral wound or spiral tubular or sheet form.

The term "perfusion flow rate" is the amount of culture medium that passes through (added to and removed from) the bioreactor in a given time, typically expressed as a fraction or multiple of the working volume. "working volume" refers to the amount of bioreactor volume used for cell culture. In one embodiment, the perfusion flow rate is one working volume per day or less.

Cell culture can be carried out under conditions for small-scale to large-scale production of recombinant proteins, using culture vessels and/or culture devices conventionally used for animal or mammalian cell culture. Those skilled in the art will appreciate that tissue culture dishes, T-flasks and spinner flasks are typically used on a bench scale. For culturing on larger scale equipment, such as, but not limited to, fermentor tank culture devices, airlift culture devices, fluidized bed bioreactors, hollow fiber bioreactors, roller bottle cultures, stirred tank bioreactor systems, packed bed culture devices, and single use disposable bags or any other suitable device known to those skilled in the art may be used. Microcarriers may or may not be used with roller bottle or stirred tank bioreactor systems. The system may be operated in batch mode, fed-batch mode or perfusion/continuous mode. Furthermore, the culture device or system may be equipped with additional devices, such as cell separators using filters, gravity, centrifugal force, or the like.

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

The term "growth phase" of a cell culture refers to the exponential cell growth phase (i.e., log phase) during which cells typically divide rapidly. The cells are maintained in the growth phase for a period of about one day, or about two days, or about three days, or about four days or more. For example, the duration of time that the cells are maintained in the growth phase will vary based on the cell type and cell growth rate as well as the culture conditions.

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 at which culture conditions can be controlled to support a switch 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, osmolality, vitamins, amino acids, sugars, peptones, ammonium, and salts.

The term "production phase" of a cell culture refers to the period of time during which cell growth has stabilized. Logarithmic cell growth is usually terminated before or during this phase, and protein production takes over. Fed-batch and perfusion cell culture methods supplement the cell culture medium or provide fresh medium in order to reach and maintain the cell density, cell viability and product titer required at this stage. The production phase can be carried out on a large scale. Large scale cell culture can be maintained in volumes of at least about 100, 500, 1000, 2000, 3000, 5000, 7000, 8000, 10,000, 15,000, 20,000 liters. In a preferred embodiment, the production phase is carried out in a 500L, 1000L and/or 2000L bioreactor.

Typically, cell culture prior to final production culture is subjected to two prior stages, seed and inoculum training. The seed training phase (N-X) occurs at a small scale where cells expand rapidly in number. Training in inoculaStage (N-1), the cells are further expanded to produce an inoculum for a production bioreactor, such as at least 0.5x10⁶Individual cells/mL of inoculum. Seeds and N-1 training can be produced by any culture method, typically batch cell culture. > 15x10⁶An N-1 cell density of one cell/mL is typical for seeding production bioreactors. The higher N-1 cell density can reduce or even eliminate the time required to reach the desired cell density in the production bioreactor. The preferred method for achieving higher N-1 cell densities is perfusion culture using alternating tangential flow filtration. N-1 cell cultures grown by perfusion methods using alternating tangential flow filtration can provide any desired density (such as > 90x 10)⁶Density of individual cells/mL or more). N-1 cell cultures can be used to produce rapid perfusion inoculum cultures or can be used as rolling seed stock cultures that are maintained to inoculate multiple production bioreactors. The inoculation density may have a positive effect on the recombinant protein level produced. Product levels tend to increase with increasing seeding density. The improvement in titer is not only limited by the higher seeding density but is likely to be influenced by the metabolic state and cell cycle state of the cells 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 viable cells in a given volume of culture medium as determined by standard viability assays, such as the trypan blue dye exclusion method. The term "hematocrit" (PCV), also known as "percent hematocrit" (% 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. dec20: 95 (6): 1228-33). The hematocrit is a function of cell density and cell diameter; the increase in cell volume may be caused by an increase in cell density or cell diameter or both. The hematocrit is a measure of the level of solids in a cell culture.

During production, the growth phase may be present at a higher temperature than the production phase. For example, the growth phase may be present at a first temperature set point of about 35 ℃ to about 38 ℃, and the production phase may be present at a second temperature set point of about 29 ℃ to 37 ℃, optionally about 30 ℃ to about 36 ℃ or about 30 ℃ to about 34 ℃.

Furthermore, chemical inducers of protein production, such as caffeine, butyrate and/or hexamethylene diethylamide (HMBA), may be added simultaneously with, before or after the temperature change. If the inducers are added after the temperature change, they may be added one hour to five days after the temperature change, optionally one to two days after the temperature change. When the cells produce the desired protein, the cell culture can be maintained for days or even weeks.

Another method of maintaining cells in a desired physiological state entails inducing cell growth arrest by exposing the cell culture to low L-asparagine conditions (see, e.g., WIPO publication No. WO 2013/006479). Cell growth arrest can be achieved and maintained by media containing limited concentrations of L-asparagine and maintaining low concentrations of L-asparagine in the cell culture. Maintaining a concentration of L-asparagine of 5mM or less can be used to maintain the cells in a growth arrested state, whereby productivity is increased.

Cell cycle inhibitors, compounds known or suspected to regulate cell cycle progression and associated transcriptional processes, DNA repair, differentiation, senescence and apoptosis associated therewith are also useful for inducing cell growth arrest. Cell cycle inhibitors that interact with a cycle clock, such as Cyclin Dependent Kinase (CDK), are as useful as those molecules that interact with proteins from other pathways, such as AKT, mTOR, and other pathways that directly or indirectly affect the cell cycle.

Cell culture conditions suitable for the process of the invention are those typically used or known for batch, fed-batch or perfusion (continuous) culture of cells, or any combination of these methods, wherein pH, dissolved oxygen (O) are noted₂) And carbon dioxide (CO)₂) Stirring and humidity and temperature.

The methods of the invention can be used to culture cells expressing a recombinant protein of interest. The expressed recombinant proteins may be secreted into the culture medium from which they may be recovered and/or collected. In addition, proteins from such cultures or components (e.g., from the culture medium) can be purified or partially purified using known methods and products known in the art and/or obtained from commercial suppliers. The purified protein may then be "formulated" (meaning buffer exchanged into a pharmaceutically acceptable formulation), sterilized, packaged in bulk, and/or packaged for use by the end user. Pharmaceutically acceptable formulations may 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, 18 th edition 1995, mack publishing Company, Easton, PA.

In one embodiment of the invention a recombinant protein is provided which is a glycoprotein. In one embodiment of the invention a recombinant protein is provided selected from the group consisting of a human antibody, a humanized antibody, a chimeric antibody, a recombinant fusion protein or a cytokine. Recombinant proteins produced by the methods of the invention are also provided. In one embodiment, the corresponding recombinant protein is formulated in a pharmaceutically acceptable formulation.

As used herein, "peptide," "polypeptide," and "protein" are used interchangeably throughout and refer to a molecule comprising two or more amino acid residues joined to each other by peptide bonds. Peptides, polypeptides and proteins also include modifications including, but not limited to, glycosylation to produce glycoproteins, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation.

As used herein, the term "glycoprotein" refers to peptides and proteins, including antibodies, having at least one oligosaccharide side chain comprising mannose residues. The glycoprotein may be homologous to the host cell, or may be heterologous to the host cell utilized, i.e., heterogeneous, such as, for example, a human glycoprotein produced by a Chinese Hamster Ovary (CHO) host cell. Such glycoproteins are commonly referred to as "recombinant glycoproteins". In certain embodiments, the glycoprotein expressed by the host cell is directly secreted into the culture medium.

Proteins may have scientific or commercial value, including protein-based drugs. Proteins include antibodies, fusion proteins, and cytokines, among others. Peptides, polypeptides, and proteins can be produced by recombinant animal cell lines using cell culture methods, and can be referred to as "recombinant peptides", "recombinant polypeptides", and "recombinant proteins". The expressed protein may be produced intracellularly or secreted into the culture medium from which it may be recovered and/or collected.

Non-limiting examples of mammalian proteins that can be conveniently produced by the methods of the present invention include proteins comprising an amino acid sequence that is identical or substantially similar to all or part of one of the following proteins: tumor Necrosis Factor (TNF), flt3 ligand (WO 94/28391), erythropoietin, thrombopoietin, calcitonin, IL-2, angiopoietin-2 (Maison pierre et al (1997),Science277 (5322): 55-60), ligands of receptor activators of NF-. kappa.B (RANKL, WO 01/36637), Tumor Necrosis Factor (TNF) -related apoptosis-inducing ligand (TRAIL, WO 97/01633), thymic stromal 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. Pat. No. 6,204,363), epidermal growth factor, keratinocyte growth factor, megakaryote growth and development factor, RANTES, human fibrinogen-like 2 protein (FGL 2; NCBI accession No. NM-00682; R ü eg and Pyrela (1995),Gene160: 257-62), growth hormone, insulin, insulinotropic hormone, insulin-like growth factor, parathyroid hormone, interferons including α -interferon, gamma-interferon and consensus interferon (consensus interferon) (U.S. Pat. Nos. 4,695,623 and 4,897471), nerve growth factor, brain-derived neurotrophic factor, synaptotagmin-like protein (SLP 1-5), neurotrophic factor-3, glucagon, interleukin, colony stimulating factor, lymphotoxin- βLeukemia inhibitory factor and oncostatin-M. A description of the proteins which can be produced according to the process of the invention can be found, for example, inHuman Cytokines：Handbook for Basic and Clinical Research, brochure(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); andThe cell Handbook, volume 1 and volume 2(Thompson and Lotze, ed., academic Press, San Diego, Calif., 2003).

In addition, the methods of the invention will be used to produce proteins comprising all or part of the amino acid sequence of a receptor for any of the above-described proteins, antagonists for such receptors or any of the above-described proteins, and/or proteins substantially similar to such receptors or antagonists. These receptors and antagonists include: two forms of tumor necrosis factor receptor (TNFR, also known as p55 and p75, U.S. Pat. No. 5,395,760 and U.S. Pat. No. 5,610,279), interleukin-1 (IL-1) receptor (type I and type II; European patent No. 0460846, U.S. Pat. No. 4,968,607 and U.S. Pat. No. 5,767,064), IL-1 receptor antagonist (U.S. Pat. No. 6,337,072), IL-1 antagonist or inhibitor (U.S. Pat. Nos. 5,981,713, 6,096,728 and 5,075,222), IL-2 receptor, IL-4 receptor (European patent No. 0367566 and U.S. Pat. No. 5,856,296), IL-15 receptor, IL-17 receptor, IL-18 receptor, Fc receptor, granulocyte-macrophage colony stimulating factor receptor, granulocyte colony stimulating factor receptor, receptor for oncostatin-M and receptor for leukemia inhibitory factor, receptor activator of NF- κ B receptor (RANK, WO 01/36637 and U.S. patent No. 6,271,349), osteoprotegerin (U.S. patent No. 6,015,938), receptors for TRAIL (including TRAIL receptors 1, 2, 3, and 4), and receptors containing death domains, such as Fas or apoptosis-inducing receptor (AIR).

Other proteins that can be produced using the present invention include proteins comprising all or part of the amino acid sequence of a differentiation antigen (also known as CD proteins) or their ligands or proteins substantially similar to either. In thatLeukocyte Typing VI(Proceedings of theVIth International WorSuch antigens are disclosed in kshop and Conference, Kishimoto, Kikutani et al, eds, Kobe, Japan, 1996). Similar CD proteins are disclosed in later seminars. Examples of such antigens include CD22, CD27, CD30, CD39, CD40, and additional ligands (CD27 ligand, CD30 ligand, etc.). Several CD antigens are members of the TNF receptor family, which also includes 41BB and OX 40. The ligand is typically a member of the TNF family, as are 41BB ligand and OX40 ligand.

The invention can also be used to produce enzymatically active proteins or their ligands. Examples include proteins comprising all or part of one of the following proteins or their ligands or proteins substantially similar to either: including members of the disintegrin and metalloprotease domain families of TNF-alpha convertases, various kinases, glucocerebrosidase, superoxide dismutase, tissue plasminogen activator, factor VIII, factor IX, apolipoprotein E, apolipoprotein A-I, globin, IL-2 antagonists, alpha-1 antitrypsin, ligands for any of the above enzymes, and a myriad of other enzymes and their ligands.

The term "antibody" includes reference to glycosylated and non-glycosylated immunoglobulins of any isotype or subclass or antigen binding regions thereof that compete with intact antibodies for specific binding, including human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies, monoclonal antibodies, polyclonal antibodies, and oligomers or antigen-binding fragments thereof, unless otherwise specified. Also included are antibodies having antigen binding fragments or antigen binding regions (such as Fab, Fab ', F (ab')₂Fv, diabodies, Fd, dAb, macroantibodies, single chain antibody molecules, Complementarity Determining Region (CDR) fragments, scFv, diabodies, triabodies, tetrabodies, and polypeptides comprising at least a portion of an immunoglobulin sufficient for binding a specific antigen to a target polypeptide). The term "antibody" includes, but is not limited to, those antibodies that are produced, expressed, produced, 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, recognitionThose antibodies including, but not limited to, any one or a combination of the proteins described above and/or antigens CD2, CD3, CD4, CD8, CD11a, CD14, CD18, CD20, CD22, CD23, CD25, CD33, CD40, CD44, CD52, CD80(B7.1), CD86(B7.2), CD147, IL-1 α, IL-1 β, IL-2, IL-3, IL-7, IL-4, IL-5, IL-8, IL-10, IL-2 receptor, IL-4 receptor, IL-6 receptor, IL-13 receptor, IL-18 receptor subunit, FGL2, PDGF-B and analogs thereof (see, U.S. Pat. Nos. 5,272,064 and 5,149,792), VEGF, TGF- β, liver- β, TGF-receptor (see U.S. Pat. No. 6,235), BlL 3, PDG-B, and analogs thereof (see also, U.S. Pat. No. 6,235), interferon-gamma, VEGF-B, interferon (see also known as interferon-4, interferon-gamma; see, interferon-4; see also known as interferon-4; interferon-gamma; see, interferon-7),Cytokine Growth Factor Rev19-25, C5 complement, IgE, tumor antigen CA125, tumor antigen MUC1, PEM antigen, LCG (which is an expressed gene product associated with lung cancer), HER-2, HER-3, tumor-associated glycoprotein TAG-72, SK-1 antigen, a tumor-associated epitope present at elevated levels in the serum of patients with colon and/or pancreatic cancer, a cancer-associated epitope or protein expressed on breast, colon, squamous cell, prostate, pancreas, lung and/or renal cancer cells and/or melanoma, glioma or neuroblastoma cells, tumor necrosis center, integrin α β, integrin VLA-4, B2 integrin, TRAIL receptor 1, 2, 3 and 4, RANK ligand, TNF- α, adhesion molecule VAP-1, epithelial cell adhesion molecule (EpCAM), adhesion molecule-3 (ICAM-3), leukocyte integrin, platelet adhesion protein gCLIG/CTLA, TNF-IIIA, TNF-adhesion molecule, TNF-1, human lymphokine receptor antigen (TNF-lymphokine receptor antigen), human lymphokine receptor antigen (HLA-10), human immunodeficiency virus (TNF-alpha-TNF-gamma-TNF-receptor antigen), human lymphokine (TNF-binding protein, human immunodeficiency virus receptor (TNF-alpha-10), human immunodeficiency virus) variants thereof, and human immunodeficiency virus (TNF-alpha-10) variants thereof, which are specific examples of which are not shown hereinAdalimumab, bevacizumab, infliximab, abciximab, alemtuzumab, basilizumab, basiliximab, belimumab, brerunumab, conatinumab, pemetrexed, cetuximab, conatumumab, denozumab (denosumab), eculizumab, gemtuzumab ozogamicin, golimumab (golimumab), ibritumomab, labezumab, mabuzumab, matuzumab, metralizumab, motavizumab, moruzumab-CD 3, natalizumab, palivizumab, ofazumab, ofamumab, omalizumab, omab, omalizumab, agovacizumab, oguevizumab (oregovacb), palivizumab, panitumumab, pemuzumab, rituximab, rozumab, tuzumab ozitumumab, tuzumab, tuximab, tuveltuzumab, tuveltuvelutvavub, tuzumab, tacrolic, dolizumab, dol, dolizumab (dol, dolizumab), Zalutumumab and zalutumumab.

The invention may also be used to produce recombinant fusion proteins comprising, for example, any of the above proteins. For example, the methods of the invention can be used to produce recombinant fusion proteins comprising one of the above proteins plus a multimerization domain such as a leucine zipper, a coiled coil, an immunoglobulin, or the Fc portion of a substantially similar protein. See, for example, WO 94/10308; lovejoy et al (1993),Science259: 1288-1293; harbury et al (1993),Science262: 1401 to 05; harbury et al (1994),Nature371：80-83；etc. (1999) of the above-mentioned compounds,Structure7: 255-64. Specifically included in such recombinant fusion proteins are proteins in which a portion of the receptor is fused to the Fc portion of an antibody, such as etanercept (a p75 TNFR: Fc) and belief (CTLA 4: Fc). Chimeric proteins and chimeric polypeptides as well as fragments or portions or mutants, variants or analogs of any of the foregoing proteins and polypeptides are also included in suitable proteins, polypeptides and peptides that can be produced by the methods of the invention.

Although the terms used in this application are standard in the art, the definitions of certain terms are also provided herein to ensure a clear and definite meaning of the claims. Units, prefixes, and symbols may be denoted in their SI accepted form. Recitation of ranges of values herein are inclusive of the numbers defining the range and include and support each integer within the defined range. Unless otherwise indicated, the methods and techniques described herein are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See, e.g., Sambrook et al molecular cloning: a Laboratory Manual, 3 rd edition, 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 but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference. The description in one embodiment of the invention may be combined with other embodiments of the invention.

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

Examples

Example 1

Extracellular ornithine levels were found to be associated with high mannose glycoform content. Eight CHO cell lines expressing recombinant antibodies 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 proprietary cell culture media (medium #1 and medium #2) each without ornithine. Samples of spent media were removed and subjected to large scale metabolomics analysis on days 8, 9 and 10 of culture. The% HM was determined using the Endo-H rCE-SDS method, followed by replacement by the HILIC method described below. The relative levels of ornithine in the spent media were determined by large scale metabolomics analysis in which media components were separated by liquid chromatography and detected by high resolution spectrometry. The components are identified by matching the fragment spectra to a library of spectra of known compounds. The relative abundance of each component was determined from the peak areas of the mass spectra signals. FIGS. 2A and 2B show the high glycaemic levels (% HM) of secreted monoclonal antibodies of cell lines A-H in media #1 and media #2 from day 8, day 9 and day 10. Figure 2C shows the correlation between high mannose% and extracellular ornithine levels. The correlation was determined by comparing all 8 cell lines (represented by squares) using data from day 9 samples. The results show a strong correlation between high mannose glycoform content and extracellular ornithine levels.

Cell line H was then grown in a 3L bioreactor in fed-batch culture. The duration of the culture was 12 days. Four perfusion feeds of 7%, 9% and 9% were performed on day 3, day 5, day 7 and day 9. Further, a 50% glucose solution was added daily as needed starting on day 3 to maintain a glucose concentration of 2g/L or more. After 4 days of growth, at 15 × 10⁵Each cell/ml was inoculated into a production bioreactor. Cells were maintained in growth medium until the production phase was initiated. 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, betaine was supplemented to the production feed medium at a concentration of 24mM on day 0. No further betaine supplementation is provided. Four perfusion feeds of 7%, 9% and 9% were performed on day 3, day 5, day 7 and day 9. Further, a 50% glucose solution was added daily as needed starting on day 3 to maintain a glucose concentration of 2g/L or more.

In conditions #3 and #4, the removal of copper sulfate was tested. Copper sulfate was removed from the production basal medium powder. Condition #3 served as a control, copper sulfate stock was added to the basal medium. In condition #4, no copper sulfate was added to any of the media, creating a copper deficient culture environment. For conditions #3 and #4, both were treated with the same perfusion "feed" medium containing copper.

In conditions #5 to #8, high osmolality and low osmolality were tested. In conditions #5 and #6, cells were fed with 90% production batch medium, i.e., less than 10% of the nutrients were provided, which caused the cells to experience reduced osmolarity. In condition #6, the cell culture medium was restored to the control level of-300 mOsm by titration with NaCl. In conditions #7 and #8, cells were fed with 85% feed medium, where the medium in condition #8 was returned to control levels by titration with NaCl.

Samples of spent media were removed and subjected to large-scale metabolomics analysis on days 3, 6, 8, 9 and 10 of culture.

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

Example 2

Arginase 1 mRNA expression levels were determined on selected days during a 10 day fed-batch production culture using 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 upregulate higher levels of high mannose in cell lines in a time-course dependent manner, see figure 4. This suggests that specific targeting with arginase to block arginase activity and reduce the amount of ornithine production can be used to reduce high mannan levels.

Example 3

This example demonstrates that manipulation of the high mannose glycoform content of a recombinant glycoprotein is addressed by controlling ornithine accumulation in a host cell expressing the recombinant glycoprotein.

Cell lines, cell cultures and media

Cell line H was used in this study. Cells were maintained in 3L conical flasks (Corning Life Sciences, Lowell, Mass.) with a 1L working volume and 5% CO at 36 ℃₂Cultured under standard humid conditions and automated CO at 70rpm₂The incubator was shaken (ThermoFisher Scientific, Waltham, Mass.). Cells were subcultured every four days in selective growth medium containing Methotrexate (MTX) at a concentration of 500nM, and then transfected, seeded and cultured in growth medium for four days, followed by seeding in 24-well plates for the experiments described below.

Small-scale simulated perfusion

Modified mock perfusion in 24-well plates (Axygen, Union City, CA) was used to assess the effect of spermine, arginine, ornithine and arginase concentrations on High Mannose (HMN) modulation. Perfusion medium without arginine preparation was used for small-scale simulated perfusion experiment #3 (arginine) according to experimental designAcid concentration study.) in a small-scale simulated perfusion experiment #4, all of the arginase inhibitors were added to the perfusion medium four arginase inhibitors were purchased from EMD Millipore corporation (Billerica, MA), BEC hydrochloride, DL- α, difluoromethylornithine hydrochloride, N^G-hydroxy-L-arginine monoacetate and N ω -hydroxy-norarginine diacetate.

Briefly, CHO cells were used in the range of 10x10⁶cell/mL-20X 10⁶Target density of individual cells/mL was seeded in plates with 3mL working volume per well. Cells were incubated at 36 ℃ with 5% CO₂85% relative humidity, and shaking at 225rpm in a 50-mm orbital diameter Kuhner incubator (Kuhner AG, Basel, Switzerland) for 3 or 4 days. Every 24 hours, cells were centrifuged at 200xg for 5 minutes (Beckman Coulter, break, CA) to collect spent media, and then each well was replenished with 3mL of fresh media. The collected spent media was then analyzed for titer, key metabolites, and% high mannose (% HMN) if necessary. Cells were then harvested and cell number and cell viability were determined.

Cell growth, metabolite and antibody titer analysis

Viable cell density and viability were determined using a Cedex cell counter (Roche Innovative, beileffed, Germany). Metabolites including glucose, lactate, ammonia, glutamine, glutamate were obtained from Nova bioprofile Flex (Nova Biomedical, Waltham, MA). Antibody concentration in spent media was determined using an affinity protein a Ultra Performance Liquid Chromatography (UPLC) (Waters Corporation, Milford, MA) assay equipped with a 50mm x4.6mm i.d. poros a/20 protein a column (Life Technologies, Carlsbad, CA). After sample injection, the column was washed by Phosphate Buffered Saline (PBS) at pH 7.1 to remove CHO host cell proteins. The bound antibody was then eluted in acidic PBS buffer (pH 1.9) and detected by Ultraviolet (UV) absorbance at 280nm to quantify the antibody concentration.

HILIC glycan profile

Antibodies to different N-glycans were analyzed by hydrophilic interaction liquid chromatography (HILIC). The purified antibody was digested by N-glycosidase F (New England BioLabs, Ipswich, Mass.) at 37 ℃ for 2 hours to release glycans. Released glycans were labeled with 2-aminobenzoic acid and cleaned using GlycoClean S cartridges (Prozyme, Heyward, CA). The purified glycans were then desalted and reconstituted in water for assay. HILIC chromatography was performed using UPLC (Waters Corporation, Milford, Mass.) with a 100mm x2.1mm i.d BEH glycan column and the eluted glycans were detected, identified and quantified by a fluorescence detector based on the different elution times of the different glycans.

Small-scale simulated perfusion experiment # 1: concentration study of spermine

Five different concentrations of spermine were tested in this study. Perfusion cell culture media containing 0. mu.M, 7. mu.M, 17. mu.M, 35. mu.M and 100. mu.M spermine tetrahydrochloride (speramine 4HCl) were tested. Perfusion medium containing 35 μ M spermine served as control. The results from the day 5 samples show a decrease in% HMN with decreasing spermine concentration, see figure 5. The reduction/consumption of spermine did not affect the potency. The reduction of HM level was achieved by a reduction in ornithine level when the amount of spermine in the medium was reduced. As shown in fig. 6, the amount of ornithine decreased with decreasing concentration of spermine.

Small-scale simulated perfusion experiment # 2: ornithine concentration study

Four different concentrations of L-ornithine monohydrochloride were tested. Perfusion cell culture medium containing 14.8mM, 6mM, 0.6mM and 0 (control) mM L-ornithine monohydrochloride (Sigma-Aldrich, St. Louis, Mo.) was used. The results show that as ornithine concentration decreases,% HMN decreases, see fig. 7. The second experiment was performed in a 2L bioreactor using cell line I. Cell line I expressed IgG2 antibody and was grown under fed-batch conditions. In one bioreactor, the medium received a single supplementation of 0.1g/L L-ornithine monohydrochloride on day 0 of culture, and the second bioreactor served as a control without ornithine. The culture was maintained in cell culture medium containing soy hydrolysate for 12 days. Perfusion feed medium containing soy hydrolysate was fed on day 4 and day 8.

Glycan profiling was performed by peptide mapping. The antibody was digested by trypsin using a method similar to that described by Ren et al (2009) anal. biochem.39212-21). Specifically, approximately 50. mu.g to 70. mu.g of each antibody was denatured with 7.0M guanidine hydrochloride, 6mM Dithiothreitol (DTT) dissolved in 0.2M tris buffer (pH 7.5) and reduced for 30 minutes at 37 ℃. Each denatured/reduced sample was alkylated with 14mM iodoacetic acid for 25 minutes at 25 ℃, followed by quenching the reaction by addition of 8mM DTT. The reduced/alkylated antibody samples were then exchanged with Pierce detergent clean-up spin columns (Thermo Fisher Scientific inc., Rockford, Il) to 0.1M tris buffer pH 7.5 according to the manufacturer's suggested protocol. The buffer exchanged samples were incubated with 3.5. mu.g trypsin for 60 minutes at 37 ℃. Digestion was quenched by the addition of 2.2 μ Ι _ of 10% acetic acid. About 12-17 μ g of digested antibody was injected for analysis.

Digested antibodies were analyzed using an Agilent 1260 HPLC system directly linked to a Thermo Scientific LTQ-Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc., Rockford, Il.). The proteolyzed peptide was separated on a 2.1X150mm, 1.7 μ particle Waters BEH 300C 18 column (Waters Corporation, Milford, Mass.) at 40 ℃ 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. The peptide was eluted with a gradient of 0.5-40% B over 90 minutes, followed by column washing and re-equilibration. The mass spectrometer was set up for a full MS scan with 120,000 resolution in an orbital trap followed by five data-dependent CID MS/MS scans with dynamic exclusion in a linear trap. Automated data analysis of glycan spectra was performed using a mass analyzer (see, Zhang, (2009) Analytical Chemistry 81: 8354-8364).

The results again show that as ornithine concentration decreases,% HMN decreases, see fig. 8.

Small-scale simulated perfusion experiment # 3: arginine concentration study

Five different concentrations of arginine were tested in this study. Perfusion cell culture media containing 3.686g/L, 1.38g/L, 0.92g/L, and 0.46g/L arginine were tested. Perfusion medium containing 1.843g/L arginine was used as a control. The results show that as the arginine concentration decreases,% HM decreases, see fig. 9.

Small-scale simulated perfusion experiment # 4: arginase inhibitor study

In the first series of experiments, four commercially available arginase inhibitors, BEC hydrochloride, DL- α, difluoromethylornithine hydrochloride, N-acetyl-D-ornithine hydrochloride, were added to the cell culture at three different concentrations, 1. mu.M, 10. mu.M and 20. mu.M^GThe control contained no inhibitor the experiments concluded that the inhibitors BEC and DL- α were most effective at reducing% HM (FIG. 10).

A second series of experiments was performed using BEC and DL-alpha inhibitors. BEC inhibitors were tested in perfusion cell culture medium at concentrations of 0 (control), 10. mu.M and 0.5 mM. DL-alpha inhibitors were tested in perfusion medium at concentrations of 0 (control), 10. mu.M, 1.0mM and 2.0 mM. The% HM was confirmed to decrease with increasing concentrations of both inhibitors, see figure 11.

Claims (56)

1. A method for manipulating the high mannose glycoform content of a recombinant protein comprising culturing a host cell expressing the recombinant protein in a cell culture under conditions that modulate ornithine metabolism in the host cell.

2. The method of claim 1, wherein ornithine metabolism in the host cell is regulated by reducing ornithine accumulation in the host cell.

3. The method of claim 2, wherein ornithine accumulation in the host cell is regulated by culturing the host cell in a cell culture medium containing an arginase inhibitor or spermine.

4. The method of claim 3, wherein ornithine accumulation in the host cell is regulated by the addition of an arginase inhibitor to the cell culture medium.

5. The method of claim 4, wherein the arginase inhibitor is BEC (S- (2-boronoethyl) -l-cysteine) or DL-a-difluoromethylornithine.

6. The method of claim 4, wherein the arginase inhibitor is BEC (S- (2-boronoethyl) -l-cysteine).

7. The method of claim 4, wherein the arginase inhibitor is DL-a-difluoromethylornithine.

8. The method of claim 4, wherein the concentration of the arginase inhibitor is at least 10 μ M.

9. The method of claim 4, wherein the concentration of the arginase inhibitor is 10 μ M to 2 mM.

10. The method of claim 4, wherein the concentration of the arginase inhibitor is 10 μ M.

11. The method of claim 4, wherein the concentration of the arginase inhibitor is 0.5 mM.

12. The method of claim 4, wherein the concentration of the arginase inhibitor is 1 mM.

13. The method of claim 4, wherein the concentration of the arginase inhibitor is 2 mM.

14. The method of claim 3, wherein ornithine accumulation in the host cell is regulated by the addition of 35 μ M or less spermine to the cell culture medium.

15. The method according to claim 14, wherein the concentration of spermine is 7 μ Μ to 35 μ Μ.

16. The method according to claim 14, wherein the concentration of spermine is 17 μ Μ to 35 μ Μ.

17. The method according to claim 14, wherein the concentration of spermine is 7 μ Μ to 17 μ Μ.

18. The method according to claim 14, wherein the concentration of spermine is 35 μ M.

19. The method according to claim 14, wherein the concentration of spermine is 17 μ M.

20. The method according to claim 14, wherein the concentration of spermine is 7 μ M.

21. The method of claim 1, wherein ornithine metabolism in the host cell is regulated by increasing ornithine accumulation.

22. The method of claim 1 or 21, wherein ornithine accumulation in the host cell is regulated by culturing the host cell in a cell culture medium containing ornithine, arginine, an ornithine decarboxylase inhibitor, an ornithine transaminase, a nitric oxide synthase inhibitor, or an arginine decarboxylase inhibitor.

23. The method of claim 22, wherein ornithine accumulation in the host cell is regulated by the addition of at least 0.6mM ornithine to the cell culture medium.

24. The method of claim 22, wherein the concentration of ornithine is from 0.6 to 14.8 mM.

25. The method of claim 22, wherein the concentration of ornithine is from 6 to 14.8 mM.

26. The method of claim 22, wherein the concentration of ornithine is 0.6 mM.

27. The method of claim 22, wherein the concentration of ornithine is 6 mM.

28. The method of claim 22, wherein the concentration of ornithine is 14.8 mM.

29. The method of claim 22, wherein ornithine accumulation in the host cell is regulated by the addition of at least 8.7mM arginine to cell culture media.

30. The method of claim 22, wherein the concentration of arginine is 8.7mM to 17.5 mM.

31. The method of claim 22, wherein the concentration of arginine is 8.7 mM.

32. The method of claim 22, wherein the concentration of arginine is 17.5 mM.

33. A method of producing a recombinant protein having a reduced high mannose glycoform content comprising culturing a host cell expressing the recombinant protein in a cell culture, wherein ornithine metabolism is modulated by reducing ornithine accumulation in the host cell.

34. A method of producing a recombinant protein having increased high mannose glycoform content comprising culturing a host cell expressing the recombinant protein in a cell culture, wherein ornithine metabolism is regulated by increasing ornithine accumulation in the host cell.

35. The method of any one of claims 1, 33, or 34, wherein the host cell expressing the recombinant protein is cultured in batch culture, fed-batch culture, perfusion culture, or a combination thereof.

36. The method of claim 35, wherein the culture is a perfusion culture.

37. The method of claim 36, wherein perfusing comprises continuous perfusion.

38. The method of claim 35, wherein the rate of perfusion is constant.

39. The method of claim 35, wherein the perfusing is performed at a rate of less than or equal to 1.0 working volumes per day.

40. The method according to claim 35, wherein the perfusion is achieved by alternating tangential flow.

41. The method of claim 1, 33, or 34, wherein the host cell expressing the recombinant protein is cultured in a bioreactor.

42. The method of claim 41, wherein the bioreactor has a capacity of at least 500L.

43. The method of claim 41, wherein the bioreactor has a capacity of at least 500L to 2000L.

44. The method of claim 41, wherein the bioreactor has a capacity of at least 1000L to 2000L.

45. The method of claim 41, wherein at least 0.5x10 is used⁶cells/mL were seeded into the bioreactor.

46. The method of claim 1, 33, or 34, wherein the host cell expressing the recombinant protein is cultured in serum-free cell culture medium.

47. The method according to claim 46, wherein the serum-free medium is a perfusion cell medium.

48. The method of claim 1, 33 or 34, wherein the host cell is a mammalian cell.

49. The method of claim 1, 33, or 34, wherein the host cell is a Chinese Hamster Ovary (CHO) cell.

50. The method of claim 1, 33, or 34, wherein the recombinant protein is a glycoprotein.

51. The method of claim 1, 33 or 34, wherein 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.

52. The method of claim 1, 33, or 34, further comprising the step of harvesting the recombinant protein produced by the host cell.

53. The method of claim 1, 33, or 34, wherein the recombinant protein produced by the host cell is purified and formulated into a pharmaceutically acceptable formulation.

54. A recombinant protein produced by the method of claim 1, 33, or 34.

55. The recombinant protein according to claim 54, which is purified.

56. The recombinant protein according to claim 54, formulated in a pharmaceutically acceptable formulation.