Case 1:18-cv-01363-CFC Document 79-13 Filed 03/22/19 Page 1 of 16 PageID #:
`9614
`
`ARTICLE
`
`Amino Acid and Glucose Metabolism in Fed-Batch
`CHO Cell Culture Affects Antibody Production and
`Glycosylation
`
`Yuzhou Fan,1,2 Ioscani Jimenez Del Val,3 Christian Mu¨ ller,2 Jette Wagtberg Sen,2
`Søren Kofoed Rasmussen,2 Cleo Kontoravdi,3 Dietmar Weilguny,2
`Mikael Rørdam Andersen1
`1
`Network Engineering of Eukaryotic Cell Factories, Department of Systems Biology,
`Technical University of Denmark, Building 223, 2800 Kgs, Lyngby, Denmark;
`telephone: þ45-45252675; fax: þ45-45884148; e-mail: mr@bio.dtu.dk
`Symphogen A/S, Pederstrupvej 93, 2750, Ballerup, Denmark; telephone: þ45-88382683;
`fax: þ45-45265060; e-mail: dw@symphogen.com
`3
`Center for Process Systems Engineering, Department of Chemical Engineering, Imperial
`College London, London, UK
`
`2
`
`GlcNAc transporter activities, which may be attributed to
`in the cell culture. Furthermore,
`high level of NH
`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. Extracel-
`lular glucose and glutamine concentrations and uptake rates
`were positively correlated with intracellular UDP-Gal
`availability. All these findings are important for optimization
`of fed-batch culture for improving IgG production and
`directing glycosylation quality.
`Biotechnol. Bioeng. 2015;112: 521–535.
`ß 2014 Wiley Periodicals, Inc.
`KEYWORDS: Chinese hamster ovary cells; amino acids;
`glucose; metabolism;
`fed-batch;
`IgG; upstream process
`optimization; glycosylation
`
`þ 4
`
`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, 2006, 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, for example, effectors function,
`
`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 interrela-
`tions could provide insight for obtaining higher immuno-
`globulin 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.
`þ
`and lactate as
`Accumulation of by-products such as NH4
`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 a-1,3-mannosyl-glycoprotein
`2-b-N-acetylglucosaminyltransferase
`(GnTI)
`and UDP-
`
`Correspondence to: M. R. Andersen and D. Weilguny
`Received 5 June 2014; Revision received 12 August 2014; Accepted 5 September 2014
`Accepted manuscript online 12 September 2014;
`Article first published online 10 October 2014 in Wiley Online Library
`(http://onlinelibrary.wiley.com/doi/10.1002/bit.25450/abstract).
`DOI 10.1002/bit.25450
`
`ß 2014 Wiley Periodicals, Inc.
`
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`Case 1:18-cv-01363-CFC Document 79-13 Filed 03/22/19 Page 2 of 16 PageID #:
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`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 produc-
`tion, 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 glycosyla-
`tion-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.,
`2004, 2006) and glutamine (Altamirano et al., 2000, 2001)
`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; Mas-
`trangelo 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 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.
`
`immunogenicity, stability, and clearance rate (Burton and
`Dwek, 2006; Goochee et al., 1991; Jefferis, 2009a,b; 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 characterized (Kornfeld and Kornfeld, 1985). Initially
`in the endoplasmic reticulum (ER), a lipid-linked oligosaccha-
`ride 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, for
`example, 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 a-mannosidase II (ManII)
`activity or when the GlcNAc addition reaction is limited by
`insufficient availability of intracellular UDP-GlcNAc or low a-
`1,3-mannosyl-glycoprotein 2-b-N-acetylglucosaminyltransfer-
`ase (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 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
`
`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
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`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 200 rpm in a
`
`C humidified culture incubator supplied with 5% CO2.
`37
`
`on day 5 and 9 g/L on days 9 and 11. For the 4,384 cell line,
`glucose was added as described above for the 1,030 cell line,
`although it was adjusted to 10 g/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 4,384 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.
`
`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
`
`C, 5% CO2, 200 rpm.
`initial culture volume of 70 mL at 37
`
`C on day 5. All
`The temperature was shifted from 37 to 33.5
`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 osmo-
`lality were measured by Bioprofile 100plus (Nova BioMedi-
`cal, 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 1,030 and
`4,384 cell lines were carried out in two different basal media A
`and B with the corresponding feed media FA and FB.
`In the Aþ FA8 culture (basal media A, feed FA, seeding
`density at 8 105 viable cells/mL), the 1,030 or 4,384 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 8 g/L on days 5 and 7, 10 g/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 4,384 cell culture on days 5, 7, 11, and 13. The culture
`was harvested on day 13 according to viability criteria.
`Only the 1,030 cells were tested in the Aþ FA4 cultivation
`condition. The A þ FA4 culture use same basal media and
`feed as the A þ FA8 culture, but with a different seeding
`density of 4  105 viable cells/mL. The feeding strategy is also
`same as the A þ 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.
`The Bþ 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 1,030 cell line, glucose was adjusted to 6 g/L
`
`Free Amino Acid Analysis
`
`Samples from cell culture were clarified by centrifugation at
`4,500 rpm for 3 min. To precipitate and remove remaining
`proteins, 30 mL 4% sulphosalic acid (Sigma–Aldrich, St.
`Louis, MO) were added into 30 mL clarified sample of the
`supernatant. After centrifugation (12,000g, 5 min), 20 mL of
`the resulting suspension was collected and dried using a
`SpeedVac (Thermo Scientific, Waltham, MA). The dried
`samples were resuspended in 160 mL of
`start buffer,
`containing 0.2 M Trisodium citrate dihydrate (Sigma–Al-
`drich) and 0.65% v/v HNO3 (Sigma–Aldrich) with pH¼ 3.1
`prior to injection 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 (Ex¼
`338 nm, Em¼ 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 postcolumn derivatization and
`fluorescence detection. Eluents used were solvent A (0.2 M
`Trisodium citrate dihydrate, 0.05% v/v phenol, and 5% v/v
`isopropanol, pH adjusted to 3.1 with nitric acid) and solvent B
`(0.21 M sodium borate, 5% v/v isopropanol, pH adjusted to
`10.2 with NaOH). Eluents were prepared freshly and filtered
`by 0.2 mm filter units (Nalgene, Thermo Scientific). Chro-
`matography was carried out using a flow rate at 0.32 mL/min
`
`C with the following
`and a column temperature at 62
`gradient: T0 min¼ 0% B, T150 min¼ 10% B, T28 min¼ 40%
`B, T36 min¼50% B, T40 min¼ 100% B, T52 min¼ 100% B,
`T53 min¼ 0% B. Post column oxidation and derivatization
`
`C in a 50 cm 0.22 mm i.d. coil
`sequentially took place at 62
`with flow of hypoclorite reagent (flow rate¼ 0.3 mL/min) and
`a 150 cm 0.5 mm coil with a flow of OPA reagents (flow
`rate¼ 0.3 mL/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 before) was measured as
`
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`B, T7.1 min¼ 5%, T11 min¼ 5%. The UV detection was
`performed at 215 nm. The different combinations of glycans
`on the IgG was analyzed and quantified according to the peak
`intensity of each isoform in the intact mass spectrum of IgG
`using Bruker Compass Data Analysis 4.2 software (Bruker).
`
`described above. Moreover, the concentration after feeding
`(Cx after) was calculated 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 (qx) from time point t1 to time
`point t2 was calculated from the following equation:
` Ct1
`¼ Ct2
`IVCt2 IVCt1
`
`qx
`
`x before
`
`x after
`
`;
`
`Glycoprofiling of IgG
`IgG glycoprofiling was carried out using GlykoPrep1
`InstantABTM kit (Prozyme, Hayward, CA) for quantifying
`the total of each N-glycans. Digestion, labeling and cleanup of
`N-glycans
`from IgG was performed according to the
`manufacturer’s instructions. Labeled glycans were buffered in
`50% v/v aqueous acetonitrile prior to the HPLC analysis using
`Dionex Ultimate 3000 RSLC System equipped with Ultimate
`3000 RS fluorescence detector (Dionex) and ACQUITY UPLC
`BEH Glycan 1.7 mm, 2.1 150 mm column (Waters). The
`mobile phase used was solvent A (100% acetonitrile; Sigma–
`Aldrich) and solvent B (100 mM ammonium formate with
`pH¼ 4.5; BDH-Merck, Poole, UK). Elution of the sample was
`performed using the following flow rate and gradient:
`T0 min¼ 25% B with flow rate¼ 0.5 min/mL, T5 min¼ 25% B
`with flow rate¼ 0.5 min/mL, T51.5 min¼ 40% B with flow
`rate¼ 0.5 min/mL, T53 min¼ 100% B with flow rate¼ 0.25
`min/mL, T58 min¼ 100% B with flow rate¼ 0.25 min/mL,
`T60 min¼ 25% B with flow rate¼ 0.5 min/mL, T70 min¼ 25% B
`with flow rate¼ 0.5 min/mL, T75 min¼ 25% B with flow
`rate¼ 0.5 min/mL. Fluorescence detection were performed at
`Ex¼ 278 nm and Em¼ 344 nm. Analysis of the chromatogram
`was performed with Chromeleon software (Dionex). Relative
`quantification was performed using peak area and peak
`assignment based on retention time of known standards.
`
`Western Blot Analysis
`
`Cell pellets from culture samples were washed with PBS
`(Invitrogen, Life Technologies, Carlsbad, CA) and lysed in
`RIPA buffer (Thermo scientific) in the presence of protease
`inhibitor cocktail (Thermo scientific) on ice. The lysates were
`sonicated and centrifuged for 2 min at 14,000 G. Total protein
`concentration in the collected supernatant was quantified by
`pierce BCA protein assay kit (Thermo scientific). NuPAGE
`sample loading buffer and reducing buffer (Invitrogen, Life
`Technologies) were added as required for each supernatant
`sample containing same amount of total protein. Samples
`
`C for 10 min prior to loading onto
`were heated at 90
`NuPAGE 4–12% Bis–Tris gel (Invitrogen, Life Technologies).
`Electrophoresis was run at 150 V for 1 h using MES buffer
`(Invitrogen, Life Technologies). Proteins were transferred to
`nitrocellulose membrane using iBlot system (Invitrogen, Life
`Technologies). Membrane was blocked in Odyssey blocking
`buffer (LI-COR) for 1 h (room temperature, 50 rpm), probed
`with goat anti-MGAT1 antibody (1:1,000; Abcam, Cam-
`bridge, UK) and rabbit anti-SLC35A3 antibody (1:1,000;
`Abcam) and rabbit anti b-actin antibody (1:1,000; Cell
`
`C, 50 rpm) and
`Signaling, Danvers, MA) over night (4
`
`in which IVC is the integral of viable cell density.
`
`Nucleotide Sugar Analysis
`
`Cell pellets from 2 mL cell culture samples were collected and
`washed with 2 mL ice-cold 0.9% w/v aqueous NaCl (Sigma–
`
`C, 1,000g, 1 min). They were
`Aldrich) by centrifugation (0
`flash-frozen in liquid nitrogen and stored at 80
`
`C until
`acetonitrile extraction. Under acetonitrile extraction, they
`were then resuspended and incubated in ice-cold 50% v/v
`aqueous acetonitrile (Sigma–Aldrich) on ice for 10 min prior
`
`C, 18,000g, 5 min). Collected supernatant
`to centrifugation (0
`was dried in a SpeedVac (Savant, Thermo Scientific),
`resuspended in 240 mL 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 4,500g for 20 min
`using Multifuge 3SR (Hereaus, Thermo Scientific). The
`supernatant was filtered through a 0.22 mm filter (Millipore,
`Billerica, MA) prior to application onto the self-packed
`MabSelect SuRe ProteinA column, which contains 200 mL of
`MabSelect SuRe protein A resin slurry (GE Healthcare,
`Fairfield, CA) equilibrated with PBS. IgG was captured by the
`column and eluted by 500 mL 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 2.1 5 mm
`column (Waters) in conjunction with micrOTOF-Q II (Bruker,
`Billerica, MA). The flow rate was 0.2 mL/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: T0 min¼ 5% B, T2 min¼ 5% B, T2.1 min¼ 90% B
`T5 min¼ 90% B, T5.1 min¼ 30% B, T6 min¼ 30% B, T7 min¼ 90%
`
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`(glucose and amino acids) were used differently during the
`fed-batch culture and what the IgG titer and glycosylation
`quality were in relation to that.
`
`Culture Behavior and IgG Production
`
`Culture behavior and IgG production constitute essential
`information for assessing media, feeds and overall upstream
`process performance. Figure 1 shows the effect of different
`upstream processes on growth, metabolism and IgG
`production for two model cell lines 1,030 and 4,384. In
`general, the durations of A þ FA4 and A þ FA8 cultures were
`slightly shorter than the B þ FB4 culture. Notably,
`the
`Aþ FA8 culture resulted in faster cell growth and increased
`the integral of viable cells (IVC) for either cell line compared
`to the B þ FB4 culture. More specifically, in Figure 1A, when
`using the B þ FB4 cultivation condition for the 1,030 and
`4,384 cell lines, the peak viable cell concentrations were about
`5 and 15  106 cells/mL on day 11, respectively. In contrast,
`the peak viable cell densities were about 15 and 20  106 cells/
`mL already on day 9 in the A þ FA8 culture for the 1,030 and
`4,384 cell line, respectively. Interestingly, the A þ FA4 culture
`did not exhibit any significant improvement on cell growth
`compared to the B þ FB4 culture. Decline in viability
`
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`9618
`washed with TBS (BioRad, Hercules, CA) with 0.1% Tween
`20 (Millipore, Billerica, MA). After 1 h incubation (room
`temperature, 50 rpm) with IR Dye 800 CW-conjugated
`donkey anti-goat and anti-rabbit antibody (1:5,000; LI-COR,
`Lincoln, NE), the membrane was washed again and subjected
`to fluorescence detection at 800 nm using Odyssey Scanner
`(LI-COR). Bands in Western blot were analyzed using image
`studio lite software (LI-COR).
`
`Results
`The B þ FB4 culture (basal media B, feed FB, seeding density
`at 4  105 viable cells/mL) is the proprietary first-generation
`upstream process developed by Symphogen with optimal
`process parameter fitting with media B and feed FB for most
`of the IgG-producing cell lines generated by Symphogen.
`During in-house upstream optimization, we found media A
`with feed FA has potentially better media capacity for cell
`growth and antibody production in some cell lines if the cell
`culture starts at a suitable seeding density (data not shown).
`Here, we test the Aþ FA4 (basal media A, feed FA, seeding
`density at 4  105 viable cells/mL), A þ FA8 (basal media A,
`feed FA, seeding density at 8  105 viable cells/mL) and
`B þ FB4 cultures in order to demonstrate how the nutrients
`
`Figure 1. Comparison of five fed-batch cultures with different cell lines and cultivation conditions. Viable cell density and integral of viable cells (IVC) versus IgG titer are
`presented in (A) and (B), respectively. Time courses of Glucose (C), Glutamine (D), Glutamate (E), Lactate (F), and Ammonia (G) concentrations are also shown. The error bars
`correspond to one standard deviation calculated from duplicate experiments. In (C), (D), and (E) the data points from Bþ BA4 culture and Aþ FA8 culture on days 3, 4, 6, 8, 10, and 12
`and from Aþ FA4 culture on days 3, 4, 6, 8, 10, 11 are unmeasured pseudo points calculated by assuming the consumption rate is constant between the measured points. Average of
`specific consumption/production rate of certain metabolite was also calculated based on specific consumption/production rate of certain metabolite on each measured time point
`from day 7 to the harvest (same color codes are used for indicating the cultivation conditions).
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`occurred earlier and faster in the A þ FA4 and A þ FA8
`cultures than in the B þ FB4 culture (Fig. S1A). For either the
`the A þ FA8 culture lead to
`1,030 or 4,384 cell
`line,
`considerably higher IgG production than the other cultures,
`as a result of both high cell growth and high specific
`productivity of IgG (Fig. 1B).
`Although the different cultivation conditions are observed
`to influence specific IgG productivity (qp), the average qp of
`each cell
`line is relatively constant and calculated to be
`40.7 pg/cell/day for 1,030 and 14.2 pg/cell/day for 4,384, as
`seen in Figure S3.
`As a consequence of consumption and feeding, glucose
`concentration fluctuated at slightly higher level in the Aþ FA8
`culture compared to the Bþ FB4 culture. Also, quite noticeably,
`glucose largely accumulated during the Aþ FA4 culture
`(Fig. 1C). The accumulation of glutamine after the temperature
`shift in the Aþ FA4 culture reached 3.5 mM at harvest.
`Additionally, the concentration of glutamine was around 1–
`2 mM before feeding in the Aþ FA8 culture for both cell lines
`compared to around 0–1 mM in the Bþ FB4 culture (Fig. 1C).
`Glutamate was generally maintained at a higher concentration
`before feeding in the Aþ FA8 culture (around 3–6 mM) than in
`the Bþ FB4 culture (around 2–4 mM) throughout the fed-
`batch process (Fig. 1E). The average specific glucose and
`glutamine consumption rates from day 7 to harvest were found
`to be generally higher in the Aþ FA8 culture than in the
`Bþ FB4 culture for both cell lines (Fig. 1D and E).
`In terms of by-product formation, the accumulation of
`lactate in the A þ FA8 culture was higher than that in the
`B þ FB4 culture for both cell lines (Fig. 1F). In the Aþ FA4
`culture, both lactate accumulation and its average specific
`production rate were particularly high, which may due to the
`high glucose concentration in this culture (Fig. 1F). Accumu-
`in the Aþ FA8 culture was lower than that in
`þ
`lation of NH4
`the B þ FB4 culture for both cell lines from day 5 to 11
`(Fig. 1G). However, in the Aþ FA4 culture, the accumulation
`þ
`increased dramatically from 2 up to 10 mM during
`of NH4
`the culture, which also demonstrated a particularly high
`þ
`average NH4
`specific production rate (Fig. 1G).
`
`Amino Acid and Glucose Metabolism
`
`To further understand the culture differences among the
`cultivation conditions and cell lines tested, the concentrations
`and specific consumption rates of amino acids and glucose have
`been assessed. In general, amino acid concentrations for 4,384
`cells were more stable in the Aþ FA8 culture than in the
`Bþ FB4 culture between day 5 and 13 (Fig. 2A). Specifically,
`amino acids such as Asp, Ser, Gly, Val, Met, Ile, Leu, Lys, Arg,
`and all the aromatic amino acids (Tyr, Phe, His, and Trp) have a
`clear decline from day 11 onwards in the Bþ FB4 culture.
`However, Ala accumulated in both Aþ FA8 and Bþ FB4
`cultures from day 5 to 13. The majority of amino acids have
`higher concentration on days 5, 9, 11, and 13 in the Bþ FB4
`culture, apart from Glu and Gln on days 5, 9, 11, and 13 and
`Cys, Val, and aromatic amino acids (Tyr, Phe, Trp) on days 11
`and 13. Interestingly, the specific amino acid consumption rates
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`
`Figure 2. Glucose and amino acids metabolisms of 4,384 cell line in A þ FA8 and
`B þ FB4 cultures. (A) Amino acid concentrations on day 5, 9, 11, 13 in the cell culture
`before feeding. All amino acids were measured by free amino acid analysis, except
`glutamine, which was measured by Bioprofile 100 plus. Glutamine, threonine, and
`asparagine eluted as one peak and their concentration cannot be resolved in the
`chromatogram of the free amino acid analysis. (B) Specific consumption rates of amino
`acids: from day 5 after feeding to day 9 before feeding (left point), from day 9 after
`feeding to day 11 before feeding (middle point), from day 11 after feeding to day 13
`before feeding (right point). (C) Specific consumption rate of glucose.
`
`are very much dependent on the concentration of correspond-
`ing amino acids in the culture. It was indicated by the fact that
`the specific amino acid consumption rates (except Glu, Gln, and
`Ala) were higher from day 5 to 13 in the Bþ FB4 culture.
`Conversely, the specific glucose consumption rate was always
`higher in the Aþ FA8 culture from day 5 to 13 (Fig. 2B and C).
`We also found that the specific consumption rates of amino
`acids in the Bþ FB4 culture in early stationary phase (day 9–11)
`were higher than in growth phase (day 5–9) and late stationary
`phase (day 11–13). Conversely, the specific consumption rates
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`of amino acids (except Glu, Gln, Ala, Cys, Tyr, HYP, and ABU)
`in the Aþ FA8 culture are lower in early stationary phase (day
`9–11) than in growth phase (day 5–9) and late stationary phase
`(day 11–13). Furthermore, the specific glucose consumption
`during the cell culture declined after the temperature shift (day
`5) in the Bþ FB4 culture but was stable in the Aþ FA8 culture.
`
`line-dependent (higher in 1,030 cell line than in 4,384 cell line
`under both cultivation conditions), and both species in 1,030
`cell lines were more sensitive to cultivation condition changes.
`Interestingly, the accumulation of UDP-GalNAc, UDP-
`GlcNAc and UDP-GlcA has a rather
`similar
`trend.
`Conversely, UDP-Gal and UDP-Glc accumulate to rather
`lower concentrations compared to UDP-GlcNAc and UDP-
`GalNAc, exhibiting similar profiles. Furthermore,
`their
`concentration was both cell
`line-dependent (higher in
`1,030 cell line than in 4,384 cell line in either cultivation
`condition) and culti