Amino acid and glucose metabolism in fed-batch CHO cell culture affects antibody production and glycosylation Yuzhou Fan1, 2, Ioscani Jimenez Del Val3, Christian Müller2, Jette Wagtberg Sen2, Søren Kofoed Rasmussen2, Cleo Kontoravdi3, Dietmar Weilguny2* and Mikael Rørdam Andersen1* 1Network Engineering of Eukaryotic Cell Factories, Department of Systems Biology, Technical University of Denmark, Building 223, 2800 Kgs. Lyngby, Denmark 2Symphogen A/S, Pederstrupvej 93, 2750 Ballerup, Denmark 3Center for Process Systems Engineering, Department of Chemical Engineering, Imperial College London, South Kensington Campus, London SW7 2AZ, UK *Corresponding author. Address correspondence to Mikael Rørdam Andersen, Department of Systems Biology, Technical University of Denmark, Building 223, 2800 Kgs. Lyngby, Denmark; +4545252675; [email protected] Address correspondence to Dietmar Weilguny, Cell line and Upstream, Symphogen A/S, Pederstrupvej 93, 2750 Ballerup, Denmark; +4588382683; [email protected] Short running title: Process-dependent IgG glycosylation Keywords Chinese hamster ovary cells; amino acids; glucose; metabolism; fed-batch; IgG; upstream process optimization; glycosylation. 1 Abstract Fed-batch Chinese hamster ovary (CHO) cell culture is the most commonly used process for IgG production in the biopharmaceutical industry. Amino acid and glucose consumption, cell growth, metabolism, antibody titer and N-glycosylation patterns are always the major concerns during upstream process optimization, especially media optimization. Gaining knowledge on their interrelations could provide insight for obtaining higher immunoglobulin G (IgG) titer and better controlling glycosylation-related product quality. In this work, different fed-batch processes with two chemically defined proprietary media and feeds were studied using two IgG-producing cell lines. Our results indicate that the balance of glucose and amino acid concentration in the culture is important for cell growth, IgG titer and N-glycosylation. Accordingly, the ideal fate of glucose and amino acids in the culture could be mainly towards energy and recombinant product, respectively. Accumulation of by-products such as NH4+ and lactate as a consequence of unbalanced nutrient supply to cell activities inhibits cell growth. The levels of Leu and Arg in the culture, which relate to cell growth and IgG productivity, need to be well controlled. Amino acids with the highest consumption rates correlate with the most abundant amino acids present in the produced IgG, and thus require sufficient availability during culture. Case-by-case analysis is necessary for understanding the effect of media and process optimization on glycosylation. We found that in certain cases the presence of Man5 glycan can be linked to limitation of UDP-GlcNAc biosynthesis as a result of insufficient extracellular Gln. However, under different culture conditions, high Man5 levels can also result from low α-1,3-mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) and UDP-GlcNAc transporter activities, which may be attributed to high level of NH + in 4 the cell culture. Furthermore, galactosylation of the mAb Fc glycans was found to be limited by UDP-Gal biosynthesis, which was observed to be both cell line and cultivation condition-dependent. Extracellular glucose and glutamine concentrations and uptake rates were positively correlated with 2 intracellular UDP-Gal availability. All these findings are important for optimization of fed-batch culture for improving IgG production and directing glycosylation quality. Introduction In recent decades, the annual global market of recombinant therapeutic proteins has grown significantly from ca. $12 billion in the year 2000 to $33 in 2004 and $99 billion in 2009 (Walsh 2003; Walsh 2006; Walsh 2010). Monoclonal antibodies (mAbs), in particular, which offer novel therapy avenues for cancer, inflammatory diseases, infectious diseases and autoimmune diseases, have had remarkable success in both regulatory approval and global sales (Jimenez Del Val et al. 2010; O'Callaghan and James 2008). Chinese Hamster Ovary (CHO) cells are extensively used for the production of recombinant antibodies as a result of their robust growth and the potential to produce non-immunogenic antibodies with glycosylation patterns similar to humans (Jefferis 2007; Raju 2003). N-linked glycosylation plays a critical role in the biological properties of therapeutic IgG, e.g. effectors function, immunogenicity, stability, and clearance rate (Burton and Dwek 2006; Goochee et al. 1991; Jefferis 2009a; Jefferis 2009b; Raju 2008). Therefore, control of glycosylation is of prime importance to meet regulatory requirements and for quality compliance. Naturally occurring IgG have two conserved N-glycosylation sites at Asn297 with the consensus sequence Asn-X-Ser/Thr on the heavy chains, where X is any amino acid except Pro. The heterogeneity of the glycan structures on each glycosylation site can vary according to their biosynthetic stage from less mature forms (e.g. non-glycosylated and high mannose forms) to more mature forms (e.g. galactosylated and sialylated forms). The process of N-glycosylation, although complicated, has been well characterised (Kornfeld and Kornfeld 1985). Initially in the endoplasmic reticulum (ER), a lipid-linked oligosaccharide 3 precursor (Glc3Man9GlcNAc2-PP-dolichol) is synthesized by transferring N-acetylglucosamine, mannose and glucose residues from UDP-GlcNAc, GDP-mannose and UDP-glucose (the nucleotide sugars synthesized in cytosol and transported into ER), respectively, to a lipid carrier, dolichol phosphate. These precursors are subsequently transferred to the available N-glycosylation sequons present on the nascent polypeptide chain. The three glucose residues present on the now protein- bound oligosaccharide contribute to protein folding via the calnexin-calreticulin cycle. After the cycle has ensured adequate protein folding, all three glucose residues are cleaved from the oligosaccharide (Ellgaard and Helenius 2003). Then, one mannose residue is trimmed in the ER prior to the IgG being translocated to the Golgi apparatus by means of vesicles (Hossler et al. 2009). In the Golgi, the N-linked glycans mature in a step-wise fashion through a number of enzyme- catalyzed reactions where monosaccharide residues are trimmed off or added to the carbohydrate structure. The maturation of glycans is largely dependent on factors such as expression, activity and localization of the glycosidase and glycosyltransferase enzymes (Jassal et al. 2001; Kanda et al. 2006; Mori et al. 2004; Paulson and Colley 1989; Weikert et al. 1999), the intracellular levels and availability of nucleotides and nucleotide sugars, e.g. GDP-Man, UDP-GlcNAc, UDP-Glc, and UDP-Gal (Baker et al. 2001; Hills et al. 2001; Nyberg et al. 1999), and the accessibility of glycosylation sites on the glycoprotein (Holst et al. 1996 ). For example, the Man5 glycans can remain unprocessed due to insufficient -mannosidase II (ManII) activity or when the GlcNAc addition reaction is limited by insufficient availability of intracellular UDP-GlcNAc or low α-1,3- mannosyl-glycoprotein 2-β-N-acetylglucosaminyltransferase (GnTI) activity (Pacis et al. 2011). Glycolysis and glutaminolysis are the key metabolic pathways of CHO cells (Quek et al. 2010). Through glycolysis, CHO cells consume glucose as the main carbon source for energy production and generate lactate as the most common metabolic by-product. Glutaminolysis is the prevalent pathway through which CHO cells assimilate organic nitrogen for biomass synthesis while releasing 4 ammonium as the main by-product (Altamirano et al. 2006; Lu et al. 2005). Fed-batch culture is widely used for the production of recombinant antibodies in industry (Huang et al. 2010). In fed- batch culture, periodic delivery of appropriate feeds provides sufficient nutrients to support cell growth and metabolism and induce a prolonged and productive culture life (Chee Furng Wong et al. 2005). However, accumulation of cellular by-products may inhibit cell growth, threaten culture longevity, reduce antibody production and compromise antibody glycosylation (Chen and Harcum 2005; Dorai et al. 2009; Gawlitzek et al. 2000; Hossler et al. 2009; Li et al. 2012). Understanding the interplay between cell growth, cell metabolism, IgG synthesis and glycosylation and how these factors vary among different cell lines and media composition at the metabolic level will benefit bioprocess optimization, media development and will be useful in identifying screening and engineering targets (Dean and Reddy 2013). Aiming at high titer production and adequate glycosylation-related quality of IgG, different strategies have been proposed to improve CHO cell culture performance. Limiting the feed of glucose (Cruz et al. 1999; Gagnon et al. 2011; Gambhir et al. 1999) and glutamine (Chee Furng Wong et al. 2005), substituting glucose (Altamirano et al. 2006; Altamirano et al. 2004) and glutamine (Altamirano et al. 2001; Altamirano et al. 2000) with alternative nutrients, addition of feed supplements (Gramer et al. 2011), optimization of process parameters such temperature, pH, agitation rate and osmolality (Ahn et al. 2008; Fox et al. 2004; Pacis et al. 2011; Senger and Karim 2003; Trummer et al. 2006) and engineering of metabolic (Fogolin et al. 2004; Kim and Lee 2007; Zhou et al. 2011) and anti-apoptotic (Druz et al. 2013; Mastrangelo et al. 2000) targets have all been attempted. In addition, many efforts have been made on metabolic profiling (Jimenez Del Val et al. 2011; Kochanowski et al. 2008; Sellick et al. 2011), and 13C metabolic flux analysis (Ahn and Antoniewicz 2011; Dean and Reddy 2013; Quek et al. 2010) of CHO cell culture at different 5 growth stages to further understand the interplay between energy, cell growth, protein production and glycosylation in CHO cells. Herein, we present the differences in cell growth, IgG production, nutrient consumption, intracellular nucleotide sugar availability and IgG glycosylation for two IgG-producing cell lines grown in fed-batch cultures with two different chemically-defined proprietary media and feeds. Our results provide an integrative approach to understand the relationship of glucose and amino acid metabolism, nucleotide sugar metabolism, cell growth, IgG production and glycosylation in fed- batch CHO cell culture and give guidance for future process optimization and media development from a metabolic point of view. Material and Methods Cell lines and media Two Symphogen in-house IgG1-producing CHO cell lines (1030 and 4384) were used in this study. Both of them were generated from a dihydrofolate reductase-deficient (DHFR-) CHO DG44 cell line (Urlaub et al. 1983) through methotrexate (MTX) mediated stable transfection with a vector containing DHFR and the genes for antibody heavy and light chains, followed by fluorescence activated cell sorting (FACS) and adaptation to serum-free medium. All basal media and feeds used in this study are proprietary, chemically defined and serum-free. Cells were maintained and expanded in basal media B in shake flask at 200rpm in a 37˚C humidified culture incubator supplied with 5% CO . 2 Fed-batch culture Cells were seeded at a density of 5 ×105 viable cells/ ml for a 2-day passage or 3 ×105 viable cells/ ml for a 3-day passage prior to the inoculation of fed-batch cultures. Cells in fed-batch culture were grown in 500 ml shake flasks with an initial culture volume of 70ml at 37°C, 5% CO , 200rpm. The 2 6 temperature was shifted from 37°C to 33.5°C on day 5. All sampling was carried out before feeding. The culture was harvested when the viability became lower than 60% or on day 14. Viability and viable cell density (VCD) was measured by Vi-CELL XR (Beckman Coulter, Brea, CA). Glucose, glutamine, lactate, ammonium, glutamate, pH and osmolality were measured by Bioprofile 100plus (Nova BioMedical, Waltham, WA). IgG titer was determined by biolayer interferometry using Octet QK384 equipped with Protein A biosensors (ForteBio, Menlo Park, CA) according to the manufacturer's instructions. Duplicates of different fed-batch cultures for the 1030 and 4384 cell lines were carried out in two different basal media A and B with the corresponding feed media FA and FB. In the A+FA8 culture (basal media A, feed FA, seeding density at 8×105 viable cells/ml), the 1030 or 4384 cells were initially seeded at 8×105 viable cells/ml in basal media A. Feed FA (3.3% of the initial culture volume) was added to the culture once a day from day 2 onwards. Glucose was adjusted to 8g/L on days 5 and 7, 10g/L on days 9 and 11. Cell culture was sampled on days 2, 5, 7, 9, 11 and 13 for measuring cell growth, metabolism and IgG expression. Additional sampling for nucleotide sugar measurement was performed on days 2, 5, 9 and 11 and for western blot analysis on days 2 and 11. Samples for amino acid analysis were taken from the 4384 cell culture on days 5, 7, 11 and 13. The culture was harvested on day 13 according to viability criteria. Only the 1030 cells were tested in the A+FA4 cultivation condition. The A+FA4 culture use same basal media and feed as the A+FA8 culture, but with a different seeding density of 4×105 viable cells/ml. The feeding strategy is also same as the A+FA8 process. However, no glucose addition was required in the process. Cell culture was sampled on days 2, 5, 7, 9 and 12 for cell growth, metabolism and IgG expression measurement and was harvested on day 12 according to viability criteria. 7 The B+FB4 culture (basal media B, feed FB, seeding density at 4×105 viable cells/ml) started with an initial culture (cells in basal media B with 13% initial culture volume of feed FB) at a seeding density of 4×105 viable cells/ml. Feed FB (10% of the initial culture volume) was added to the culture on days 2, 5, 7, 9 and 11. For the 1030 cell line, glucose was adjusted to 6g/L on day 5 and 9 g/L on days 9 and 11. For the 4384 cell line, glucose was added as described above for the 1030 cell line, although it was adjusted to 10g/L on day 9. Cell culture was sampled on days 2, 5, 7, 9, 11, 13 and 14 for measuring cell growth, metabolism and IgG expression. Additional sampling for intracellular nucleotide sugar quantification was carried out on days 2, 5, 9 and 11 and for western blot analysis on days 2 and 11. For the 4384 cell line, cell culture was also sampled for amino acid analysis on days 5, 7, 11 and 13. Fed-batch culture was harvested on day 14. Free amino acid analysis Samples from cell culture were clarified by centrifugation at 4500rpm for 3min. To precipitate and remove remaining proteins, 30 µl 4% sulphosalic acid (Sigma-Aldrich, St. Louis, MO) were added into 30 µl clarified sample of the supernatant. After centrifugation (12,000g, 5min), 20µl of the resulting suspension was collected and dried using a SpeedVac (Thermo Scientific, Waltham, MA). The dried samples were resuspended in 160µl of start buffer, containing 0.2M Trisodium citrate dihydrate (Sigma-Aldrich) and 0.65% v/v HNO (Sigma-Aldrich) with pH =3.1 prior to injection 3 into the amino acid analyzer system. The system controlled by Millennium32 software (Waters, Milford, MA) is composed of two M510 pumps (Waters), two regent manager pump (Waters), a M717 refrigerated autosampler (Waters), a M474 fluorescence detector (Ex= 338 nm, Em= 455 nm) (Waters), a column oven (Waters), and a MCI-Gel CK10U column (Mitsubishi Chemical industries, Japan). All chemicals used to prepare the relevant solvents and reagents are purchased from Sigma- Aldrich. Amino acid analysis was performed using cation-exchange chromatography followed by 8 postcolumn derivatization and fluorescence detection. Eluents used were solvent A (0.2M Trisodium citrate dihydrate, 0.05% v/v phenol and 5% v/v isopropanol, pH adjusted to 3.1 with nitric acid) and solvent B (0.21M sodium borate, 5% v/v isopropanol, pH adjusted to 10.2 with NaOH). Eluents were prepared freshly and filtered by 0.2 µm filter units (Nalgene, Thermo Scientific). Chromatography was carried out using a flow rate at 0.32ml/min and a column temperature at 62°C with the following gradient: T =0% B, T =10% B, T =40% B, T 0 min 15 min 28 min 36 =50% B, T = 100% B, T = 100% B, T 0% B. Post column oxidation and min 40 min 52 min 53 min = derivatization sequentially took place at 62°C in a 50cm 0.22mm i.d. coil with flow of hypoclorite reagent (flow rate = 0.3ml/min) and a 150cm 0.5mm coil with a flow of OPA reagents (flow rate = 0.3ml/min). Hypochlorite and OPA reagents can be prepared as described in (Barkholt and Jensen 1989). Peak assignment and integration was done automatically with a user-defined data processing method. Specific metabolic rate The concentration of a certain nutrient or metabolite in the cell culture before feeding (Cx ) was before measured as described above. Moreover, the concentration after feeding (Cx ) was calculated after based on the culture volume and known addition of the proprietary feed at that time point. Additionally, the specific consumption or production rate of certain nutrient or metabolite (q ) from x time point t to time point t was calculated from the following equation: 1 2 , in which IVC is the integral of viable cell density. Nucleotide sugar analysis Cell pellets from 2ml cell culture samples were collected and washed with 2ml ice-cold 0.9% w/v aqueous NaCl (Sigma-Aldrich) by centrifugation (0°C, 1000g, 1min). They were flash-frozen in liquid nitrogen and stored at -80°C until acetonitrile extraction. Under acetonitrile extraction, they 9 were then resuspended and incubated in ice-cold 50% v/v aqueous acetonitrile (Sigma-Aldrich) on ice for 10 min prior to centrifugation (0°C, 18,000g, 5min). Collected supernatant was dried in a SpeedVac (Savant, Thermo Scientific), resuspended in 240µl water and store at -80°C until applying on HPLC for high-performance anion-exchange (HPAEC) analysis as describe in (Jimenez Del Val et al. 2013). IgG purification Harvested cell culture was centrifuged at 4500g for 20 min using Multifuge 3SR (Hereaus, Thermo Scientific). The supernatant was filtered through a 0.22 μm filter (Millipore, Billerica, MA) prior to application onto the self-packed MabSelect SuRe ProteinA column, which contains 200 μl of MabSelect SuRe protein A resin slurry (GE Healthcare, Fairfield, CA) equilibrated with PBS. IgG was captured by the column and eluted by 500 μl of 0.1 M citrate with pH=3.5. The elution was immediately subjected to a buffer exchange procedure by passing through a NAP-5 column (GE Healthcare) equilibrated by a formulation buffer containing 10 mM Citrate (Sigma-Aldrich) and 150 mM NaCl (Sigma-Aldrich) with pH =6.0. IgG concentration was measured using NanoDrop ND-1000 (Thermo Scientific). Purified IgG was stored at -20°C until further analysis. Intact mass analysis of IgG Intact mass analysis of the purified IgG was performed on a LC-MS system using Dionex Ultimate 3000 RSLC System equipped with Ultimate 3000 RS variable wavelength detector (Dionex, Sunnyvale, CA) and Mass Prep micro desalting 2.1x5mm column (Waters) in conjunction with micrOTOF-Q II (Bruker, Billerica, MA). The flow rate was 0.2ml/min. The gradient with solvent A (water with 0.1% formic acid; Sigma-Aldrich) and solvent B (acetonitrile with 0.1% formic acid; Sigma-Aldrich) was as follow: T = 5% B, T = 5% B, T = 90% B T = 90% B, T 0 min 2 min 2.1min 5 min 5.1 min = 30% B, T = 30% B, T = 90% B, T = 5%, T = 5%. The UV detection was 6 min 7 min 7.1 min 11 min performed at 215nm. The different combinations of glycans on the IgG was analyzed and quantified 10