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Bioprocess Engineering and Supporting Technologies
Biotechnology and Bioengineering DOI 10.1002/bit.25534
A Robust Method for Increasing Fc Glycan High Mannose Level of Recombinant Antibodies†
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Chung-Jr Huang1*, Henry Lin1,2, Jerry (Xiaoming) Yang1,3
Cell Science & Technology, Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand
Oaks, CA 91320
Running Title: A Robust Method for High Mannose Modulation
*Corresponding Author: Chung-Jr Huang, Mail Stop: 18S-1-A, One Amgen Center Dr., Amgen Inc., Thousand Oaks, CA, 91320, Tel: (805) 447-7695, E-mail: [email protected]
2: Present address: Boehringer Ingelheim Fremont, Inc., 6701 Kaiser Drive, Fremont, CA 94555, USA 3: Present address: DZM Biotech Ltd.
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/bit.25534]
Additional Supporting Information may be found in the online version of this article.
This article is protected by copyright. All rights reserved Received September 3, 2014; Revision Received December 15, 2014; Accepted December 23, 2014
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Abstract
High mannose (HM) glycoforms on antibody Fc glycan are recognized as critical quality attributes for therapeutic antibody products. Methods to control HM have been largely empirical, and it is challenging to target a desired HM level during antibody process development. A novel and robust method to increase antibody HM glycoforms is demonstrated in this study using multiple antibodies and cell lines without adversely impacting cell culture performance, including viable cell density, viability, and protein titer. This approach utilizes mannose as a carbon source and the ratio of mannose to total hexose (glucose and mannose) in feed media determines the extent of HM glycan content of an antibody expressed in cell culture. Scale-up of this strategy from 3-mL small scale plate to bioreactor (1.5 L) is also demonstrated with comparable results. Further full glycan map analysis shows that HM increase predominantly correlates with the decrease in G0F glycan, with minimum impact on other glycoforms. Possible hypotheses for the HM glycan modulation using mannose as carbon source are also discussed. Three pathways, including GDP-mannose biosynthesis, early protein glycosylation and UDP-N-acetyl-glucosamine biosynthesis, might be involved and contribute to this HM modulation. This article is protected by copyright. All rights reserved
Keywords: Chinese hamster ovary (CHO) cells, Recombinant antibody, Antibody Fc glycans, High mannose level modulation
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Introduction
IgG antibodies produced in mammalian cell cultures contain varying levels of high mannose (HM) glycoforms such as Man5, Man6, Man7, Man8, and Man9. The HM level on the antibody Fc glycan is a critical quality attribute that affects pharmacokinetic properties and effector function of therapeutic antibodies (Goetze et al., 2011; Yu et al., 2012). Glycoforms of an antibody expressed by Chinese hamster ovary (CHO) cell are largely determined during the processes of cell line generation and clone selection. However, HM content can also be affected by cell culture conditions (Pacis et al. 2011). It is common in the therapeutic antibody industry to seek a desired range of HM content for an antibody product due to process changes, scale-up, improvements, or the need to match existing antibody quality attributes. Thus far, methods applied for manipulating HM content of an antibody in cell culture include changes to medium compositions, osmolarlity, pH, and temperature (Ahn et al., 2008; Wong et al., 2005; Pacis et al., 2011; Yu et al., 2012). In general, these methods are effective only to certain cell lines, molecule types, and culture medium. Additionally, some methods can also alter antibody productivity, cell culture performance, and other antibody quality attributes. The effectiveness of these methods is also limited regarding modulating HM to a predetermined level during the cell culture processes. Therefore, a robust and predictive HM regulation method will immensely benefit process development for therapeutic proteins and antibodies.
In this study, we demonstrate a robust method that modulates HM content of several therapeutic antibodies
by applying mannose as a carbon source in the cell culture process. It was observed that the HM content of antibodies was proportional to the increase of mannose to total hexose (M:H) ratio in the cell culture medium. This effect was also universally applied to all tested antibodies (cell lines), with the magnitude of HM increase being cell-line–and molecule-specific. In this study, the mannose pathway and its impact on glycosylation in CHO cells are discussed, and the hypotheses of modulating HM by adding mannose as a carbon source are also discussed.
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Material and Methods Cell lines, cell culture, and media Serum-free adapted DXB-11 CHO cell lines expressing different recombinant antibodies were used in this
study. Cells were maintained in 3-L Erlenmeyer shake flasks (Corning Life Sciences, Lowell, MA) with a 1-L working volume and cultivated under standard humidified conditions at 36 °C, 5% CO2, and shaken at 70 rpm in an automatic CO2 incubator (Thermo Fisher Scientific, Waltham, MA). All cells were subcultured in the medium containing different concentrations of methotrexate (MTX) every 4 days, and were transferred, inoculated, and cultured in proprietary chemically-defined medium 1 for 4 days before seeding in 24–deep-well plates or inoculation in bioreactors. Another proprietary chemically-defined medium 2 was used as the control medium in the study. In the first different carbon source screening experiment, 96% of the glucose in the medium 2 was substituted with the tested carbon sources, including mannose, galactose, fructose, and maltose (Sigma-Aldrich, St. Louis, MO). In other studies, glucose in medium 2 was substituted with mannose according to the experiment design (Table 1).
Small-scale mock perfusion cell culture A newly developed mock perfusion experiments in 24–deep-well plates (Axygen, Union City, CA) were
used to evaluate effects of different carbon sources and M:H ratios on HM modulation. This high throughput method was developed as a small scale model for high cell density (HCD) perfusion bioreactor culture. HCD perfusion bioreactor culture is characterized by high cell density and medium perfusion, with the majority of protein is produced at production phase. Therefore, high seed density is used in this model with culture medium being exchanged every day to support high cell density growth to mimic the performance of HCD perfusion bioreactor cultures. Briefly, different cell lines were seeded in the plate at the targeted density ranging from 20–30 x 106 cells/mL with 3-mL working volumes for each well. To reach such a high seed density, cells were centrifuged and concentrated from above-mentioned seed subcultures. The cells then were cultivated at 36 °C, 5% This article is protected by copyright. All rights reserved
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CO2, 85% relative humidity and shaken in incubator (Kuhner AG, Basel, Switzerland) for 3 or 4 days. Every 24 hours, cells were centrifuged at 200xg for 5 minutes (Beckman Coulter, Brea, CA) to collect the spent media, and each well was then replenished with 3 mL fresh medium. The collected spent medium was further analyzed for titer, key metabolites, and HM level (when necessary). When cells were harvested, cell counts and viability were measured. All experimental conditions were performed in duplicate.
Perfusion cultivation in bioreactors A bioreactor experiment studying effects of different glucose:mannose ratios on HM level for cell line 1
was performed using three 3-L bioreactors (Applikon Biotechnology, Foster City, CA) with 1.5L working volume. The bioreactors were equipped with hollow fiber cartridges (GE Healthcare Biosciences, Pittsburgh, PA) which retain the produced mAb inside the bioreactor. In brief, cells were seeded in the bioreactors containing 1.5 L medium 1. An automated bioprocessing software (DeltaV™) was used for data acquisition and parameter control. During the cell culture processes, cells were initially grown at 36 °C then temperature shifted, once the desired cell density was reached. The dissolved oxygen level was maintained at 40% of air saturation and supplemented with pure oxygen when needed. Bioreactor pH was maintained at 7.0 by CO2 or 1 M sodium carbonate addition. At day 3, three types of medium 2 media containing different concentrations of glucose and mannose (M:H ratios of 0, 0.5 and 0.94), were fed into the bioreactors, and the permeate through the hollow fiber was also collected at the same rate. Samples were taken from three bioreactors and analyzed daily during the run.
Cell growth, antibody titer, and metabolite analysis Viable cell density and viability were determined using the Cedex cell counter (Roche Innovative,
Beilefeld, Germany). Antibody concentrations in the spent medium were determined using Protein A ultraperformance liquid chromatography (UPLC) (Waters Corporation, Milford, MA) equipped with a 50 mm x 4.6 mm i.d. POROS A/20 protein A column (Life Technologies, Carlsbad, CA). After sample was injected, the column was washed with phosphate-buffered saline (PBS) pH = 7.1 to remove CHO host cell proteins. Bound antibodies were This article is protected by copyright. All rights reserved
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then eluted in acidic PBS buffer (pH = 1.9) and detected by UV absorbance at 280 nm to quantify antibody concentration. For experiments conducted using 24–deep-well plates, each condition was performed in duplicate and an averaged value was presented. For bioreactor samples, metabolites including glucose, lactate, ammonia, and glutamine were analyzed using the NovaBioprofile Flex (Nova Biomedical, Waltham, MA).
High throughput Fc HM content assay A high-throughput microchip capillary electrophoresis (CE) method was used to determine HM levels of
recombinant antibodies directly from the spent medium of the 24–deep-well plate samples (Chen et al., 2008). In brief, samples were digested with endoglycosidase H (Endo H) for 2 hours at 37 °C. After digestion, antibodies were denatured at 70 °C for 10 minutes and injected in LabChip GXII (Caliper Life Sciences, Hopkinton, MA) to quantify non-glycosylated heavy chain (NGHC). Samples not digested by Endo H were used as controls. The % of NGHC from the control was subtracted by the %NGHC of the adjusted sample to yield the %HM value. For bioreactor samples, antibodies were first purified by affinity protein A MediaScout MiniChrom column (ATOLL, Weingarten, Germany) before the HM assay. The purified proteins were digested with Endo H and denatured as previously described. The percentages of NGHC from digested samples and controls were determined using a ProteomeLab PA800 Capillary Electrophoresis system (Beckman Coulter, Brea, CA), and two NGHC values were used to determine the HM level. The HM level was normalized and the presented.
HILIC whole Fc glycan map assay Different N-glycan species in antibodies were analyzed by hydrophilic-interaction liquid chromatography
(HILIC) for samples collected from bioreactors (Melmer et al., 2010). The purified antibodies were digested by Nglycosidase F (New England BioLabs, Ipswich, MA) at 37 °C for 2 hours to release the glycans. The released glycans were labeled with 2-aminobenzoic acid and cleaned up by GlycoClean S cartridges (Prozyme, Hayward, CA). Purified glycans were then desalted and reconstituted in water for the assay. HILIC was performed with a 100 mm x 2.1 mm i.d. BEH Glycan column using UPLC (Waters Corporation, Milford, MA), and the eluted glycans This article is protected by copyright. All rights reserved
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were detected, identified, and qualified by a fluorescence detector based on elution time. The percentage of each detected glycoforms were normalized and presented.
Results
Mannose utilization maintains cell growth and protein production but increases HM content in cell line 1 (mAb1) To explore the effects of different carbon sources on cell growth, antibody production and quality attributes
such as HM, five different sugars, including glucose, mannose, galactose, fructose and maltose, were evaluated using a small-scale (24–deep-well plate) mock perfusion model. As shown in Figure 1A, when galactose, fructose, and maltose were metabolized by cell line 1, cell growth was significantly inhibited. The viability and normalized antibody titer were also adversely impacted (Figure 1B, C). After analysis of antibody Fc glycan quality attributes, however, the normalized HM level were found to be between 165-168 in these cultures, about five-fold higher than that of the glucose control (34.8) (Figure 1D). Interestingly, cell line 1 cultured with mannose as a carbon source yielded comparable growth, viability, and normalized antibody titer as glucose control (Figures 1A–C). In this culture, viable cell density (VCD) continued to increase and reached 520 x 105 cells/mL with a viability of 94% at day 3, and mAb1 was produced at a normalized titer of 0.97. However, mannose culture also had a high normalized HM level of 100 (Figure 1D). This indicates that the presence of mannose in the medium affects antibody Fc glycan processing while maintaining equivalent growth and protein production when compared to glucose-grown culture.
HM content in mAb1 can be modulated with various mannose:glucose ratios Based on the previous plate study, we hypothesized that mannose concentration in the medium could
influence HM levels in mAb1 antibody. CHO cells may uptake mannose at similar rate as glucose, and various ratios of mannose:glucose could potentially yield different levels of HM while having no impact on cell growth or protein production. To test this hypothesis, the glucose in the medium was replaced with different amount of This article is protected by copyright. All rights reserved
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mannose, and a mock perfusion experiment was performed to verify the hypothesized HM modulation effects (Table 1). When glucose was replaced by 25, 50, 75, and 96% of mannose in the medium, the normalized HM level increased from 35.9 to 48.1, 63, 82.1, and 100, respectively, for mAb1(Figure 2D). The growth, viability, and normalized antibody titer were not affected by changing the mannose concentration in the media (Figure 2A-2C). These results demonstrate that various mannose:glucose ratios can linearly modulate the HM level without impacting cell growth and productivity.
The HM modulation effect is applicable and predictable for different cell lines (mAbs) To test whether the mannose-associated HM modulation effect is universally applicable, four other cell
lines, producing mAb 2, 3, 4, 5 were cultivated in media containing various mannose:glucose ratios as in the previous experiment. After harvest, samples were analyzed, and normalized HM level was plotted against the ratio of mannose to total hexose (M:H) in the medium. All four cell lines have elevated HM at high M:H ratios, as previously observed for cell line 1 (mAb1) (Figure 3). Thus, this modulation effect applied to all the cell clines we tested, a phenomenon that is striking especially when considering that these cell lines inherently have different HM levels for their respective antibodies. For example, the cell line 2 produces mAb2 with normalized HM level of 6 in the control medium (glucose only), and the HM increases to 13.2 at an M:H ratio of 0.94. Alternatively, cell line 5 produced mAb5 that has normalized 104.6 HM level in the control medium. When the M:H ratio is 0.94, the normalized HM increased to 207.2. Moreover, a strong linear correlation between normalized HM and M:H ratio was seen for all cell lines. This indicates that HM can be modulated by controlling the M:H ratio in the medium. In other words, when cells are cultivated in the media with a certain M:H ratio, the HM level of the produced antibodies becomes predictable.
The HM modulation effect is also scalable in bioreactors To verify the scalable aspect of HM modulation, a 1.5L bioreactor run using the mAb1 production process
was conducted. Starting at day 3, three bioreactors were perfused with media containing different M:H ratios of 0 This article is protected by copyright. All rights reserved
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(the control), 0.5, and 0.94. During the cultivation period, samples were taken daily, and cell counts, cell viability, and metabolites were measured. Figure 4 shows the time course for cell growth, normalized antibody yield, and normalized HM level from three bioreactors. When cells were grown in perfusion media with an M:H ratio of 0.94, the Day 15 normalized VCD reached 100 with 78% viability and a normalized titer of 0.89, which were all equivalent to the control. Cell growth, viability, and titer were all slightly lower for an M:H ratio of 0.5 (Figure A, B and C). For HM content, differentiated dynamic profiles were observed (Figure 4D). The control had 31.7 normalized HM level at day 6, which then gradually increased to 39.9 at day 15. When the M:H ratio was 0.5, a normalized HM of 32.5 was also observed at day 6, but increased about two-fold to 63.4 at day 15. Interestingly, the M:H ratio of 0.94 initially produced high normalized HM of 67.8 and reached 100 at day 9. The normalized HM value plateaued at around 100 throughout the rest of the cultivation period. These results indicated that the HM modulation effect is applicable at bioreactor process and scale. This effect was also predictable with an Rsquared value of 0.98 (Figure 5). Moreover, a good correlation was also observed between the small scale mock perfusion and perfusion bioreactor. Figure 5 compares the HM modulation effect between small scale mock perfusion and the Day 15 normalized HM levels from bioreactor for mAb1. The slopes of the trend lines were 59.24 and 62.58 for mock perfusion and bioreactor, respectively, demonstrating that the correlation between HM and M:H ratio was scale-independent.
Full glycan map analysis with HM modulation To understand the overall impact on antibody Fc glycan by mannose modulation, purified mAb1 from the
bioreactor process was analyzed with a full glycan map assay. As shown in Figure 6, certain glycans, including galactosylated (G1F+G2F), M3G0F and sialylated forms were not affected by different M:H ratios in the medium. High M:H ratios predominantly increased HM and decreased G0F. For example, M:H ratio of 0.94 had the highest normalized HM level of 100 and the lowest normalized G0F level of 132. In addition, increased M:H ratios produced slightly higher amounts of afucosylated and hybrid Fc glycans. A few existing unidentified glycan forms also increased to a small extent. The percentage of each HM species was also identified and compared (Figure 7). This article is protected by copyright. All rights reserved
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With an increase in M:H ratio of the medium, the levels of Man8, Man7, Man6, and Man5 glycans all significantly increased when compared to the control. Man5 was found to be the major HM species in the three conditions. In addition, the percentage increase in Man6, Man7 and Man8 species were higher than that of Man5 species at higher M:H ratios. These results suggest that high M:H ratios slow down early glycan processing steps, leading to elevated levels of all HM species. With increase in the H:M ratio, more primitive HM species were found in the produced antibodies.
Discussion
A robust method for HM modulation Mannose and its derivatives, such as N-acetylmannosamine, have been supplemented into the cell culture
medium to study their effects on cellular metabolism and N-glycans of recombinant proteins (Altamirano et al., 2013; Butler, 2006; Wong et al., 2010). The replacement of glucose by mannose enhances volumetric productivity of t-PA and reduces specific oxygen consumption rate, without impacting t-PA sialylation level (Berrios, 2011). Addition of mannose with other N-glycan precursors does not increase site-occupancy of t-PA but shows to increase sialylation of recombinant glycoproteins (Butler, 2006; Gawlitzek et al., 2009). Wong and colleagues also reported that including the N-acetylmannosamine and uridine in the medium result in 36% increase in INF-γ sialylation (Wong et al. 2010). In this study, we found that mannose also increases %HM of Fc glycan in mAbs produced by CHO cells, without impacting cell culture performance. Furthermore, the ratios of mannose to total hexose in the culture medium modulate Fc glycan HM levels. Different cellular process parameters and production medium components have been reported to affect HM level of recombinant proteins produced in CHO cells. However, these methods are not robust for commercial cell culture processes. Lowering temperatures (< 32 °C) have been shown to increase certain HM species and reduce antennary structure and sialylation of recombinant erythropoietin (Ahn et al., 2008). Recently, Pacis et al. demonstrated that antibody Man5 level can be increased more than two-fold from 12% to 28% by increasing medium osmolarity and culture duration time (Pacis et al.,
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2011). Although moderate linear correlations may be found between medium osmolarity and Man5 level, high osmolarity media could negatively impact cell growth, viability, and protein titer (Zhu et al., 2005). The time course HM profiles observed in three bioreactors is much of an interesting observation. The result
indicates that HM upregulation by mannose may exhibit a saturation effect in cell line 1. However, it is yet to be determined whether similar dynamics apply to different cell lines, as inherent HM values (in glucose) are different across these cells lines (Figure 4). Traditionally, when cells are cultured in flasks or plates, physical parameters and nutritional components are not well controlled. Hence, scale variations regarding cell growth, protein production, and glycosylation pattern are commonly observed in cell culture processes (Humphrey, 1998). In this study, we found a good correlation between the 24–deep-well plate mock perfusion and bioreactor on HM modulation effects (Figure 5). The high cell density inoculation and daily medium exchange might contribute to this correlation, further validating that the developed method showed in this study is a good scale-down model for perfusion bioreactor.
Hypothesis of underlying mechanisms In mammalian cells, different glucose transporters (GLUTs) are responsible for the import of different
carbohydrates to provide an energy source for cell growth and metabolism. At least 14 GLUTs have been identified, and their expression profiles in cells are highly tissue-specific. Different GLUTs were shown to transport different sugar substrates with distinct affinities and transport capacities (Augustin, 2010). When galactose, fructose, and maltose were used as carbon sources, reduced cell growth and protein production observed in cell line 1 mock perfusion cultures were likely due to slow uptake of galactose, fructose, and maltose by GLUTs. For CHO cells, high uptake rates of glucose and mannose by GLUTs result in high cell growth and elevated levels of by-products such as lactate and ammonia. In contrast, poor transport of galactose and fructose reduces cell growth and by-product accumulation (Altamirano et al., 2000; Wlaschin and Hu, 2007). We hypothesize that slow transport of galactose, fructose, and maltose in the cell line 1 is responsible for the increase in HM level of mAb1 antibody. Although once inside the cells, these sugars convert to glucose or glycolytic intermediates, such as This article is protected by copyright. All rights reserved
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glucose-6-phosphate and fructose-6-phsophate (F6P), the supply of these intermediates for energy production and for glycan precursors remains insufficient due to the low sugar transport. Such a glucose limitation-like state increases HM and aglycosylation of recombinant proteins. For example, low glucose concentration in the media increases the levels of hybrid and HM glycan forms on recombinant interferon gamma (Wong et al., 2005). Since cells preferentially use glucose for energy production, low nucleotide triphosphate pools and reduced intracellular levels of UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-N-acetyl-galactosamine were found in glucosestarved cells (Nyberg et al., 1999). This depletion would eventually affect complex glycan processing due to the shortage of nucleotide sugars, producing more HM-type proteins. In glucose-limiting culture, the insufficient supply of UDP-GlcNAc is further deteriorated by glutaminolysis, in which glutamine, a key amino acid for UDPGlcNAc biosynthesis, is channeled into energy production (Hayter et al., 1992; Nyberg et al., 1999). This was also the case in this study, where glutamine was low or depleted in galactose, fructose, and maltose cultures (data not shown) and further explains the five-fold increase of HM in these cultures. Unlike other tested carbon sources, mannose can be transported and metabolized as efficiently as glucose is
in CHO cells. However, increased HM level is also observed in mannose culture. Upon intake, intracellular mannose is phosphorylated by hexokinase to mannose-6-phosphate (M6P), which can then be converted by phosphomannose isomerase (MPI) to F6P to enter the glycolytic cycle (Figure 8) (Herman, 1971). This study and others suggest that this pathway is highly efficient for energy generation in CHO cells (Altamirano et al., 2000; Berrios et al., 2011); hence, intracellular nucleotide triphosphate pools and intracellular UDP-GlcNAc levels in mannose culture should be comparable to those of glucose-grown cultures. Glutamine levels were also equivalent among three bioreactor cultures (Figure S1). Together, these results suggest that mechanisms other than energy related substrate limitation lead to HM increase in mannose-grown cultures. When cells are grown in glucose-only media, cellular mannose for glycan biosynthesis is derived entirely from glucose (Alton et al., 1998; Gao et al., 2005). This suggests that early glycan processing steps are impacted, resulting in HM increase in mannose culture. The cause may be related to upstream mannose metabolism or increased influx of GDP-mannose for glycan biosynthesis. At this point, it was hypothesized that the presence of mannose or its intermediates such as M6P are This article is protected by copyright. All rights reserved
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responsible for HM upregulation. Elucidation of different mannose metabolite levels by metabolite profiling and tracing of the fluxes of these metabolites would provide data to validate this hypothesis. To further investigate the mechanism of HM upregulation, glycan maps of samples from three bioreactors
were elucidated (Figure 6 and 7). High M:H ratios slightly increase the levels of afucosylated complex, unknown and hybrid glycans. GDP-mannose is known to convert to GDP-fucose by GDP-mannose 4,6-dehydratase (GMD) and two other enzymes in the de novo fucosylation pathway. GDP-fucose is also a potent inhibitor of GMD (Becker and Lowe, 2003). High mannose concentration in the medium should theoretically increase the intracellular GDP-mannose and GDP-fucose levels that lead to low afucosylation (due to increased substrate supply). Therefore, the increased afucosylated complex observed in this study may be due to strong feedback inhibition of GDP-fucose to GMD that eventually decrease intracellular GDP-fucose level or by unidentified mechanisms. The galactosylation and sialylation of mAb1 are similar upon the addition of mannose in the culture media. The decrease of G0F correlates with HM increase. These results indicated that mainly the early glycosylation pathway (before G0F) is affected. Moreover, analyses of HM species showed that increased M:H ratios concurrently increased the levels of different HM species, including Man8, Man7, Man6, and Man5. Although Man5 is the major HM species in all three conditions, the levels of other primitive HM species increases significantly with high M:H ratios. The HM species accumulation profile further suggests that two glycosylation steps, HM trimming by -mannosidase I (-MAN I) and UDP-GlcNAc addition by UDP-GlcNAc transferase (GnT1), are inhibited, increasing the HM level in mAb1 (Figure 8). The first step for cells to utilize intracellular mannose is to convert mannose to M6P, a key metabolite
bridging energy generation (glycolysis) and N-glycan biosynthesis (Alton et al., 1998). Elevated intracellular M6P levels have been demonstrated in mouse embryonic fibroblasts when 10 mM mannose was supplemented in the media (Gao et al., 2011). Therefore, it is reasonable to theorize that elevated M6P levels should also be found in high M:H ratio cultures, suggesting a possible flux increase for N-glycan biosynthesis. As illustrated in Figure 8, three pathways, GDP-mannose biosynthesis, early glycosylation, and UDP-GlcNAc biosynthesis, may have been involved in the HM upregulation observed in this study. Increased levels of primitive HM species suggest that the This article is protected by copyright. All rights reserved
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increased concentration of mannose or its intermediates (M6P, mannose-1-phosphate [M1P], or GDP-Mannose) may directly or indirectly inhibit -MANI activity. High Man5 levels also suggest that the UDP-GlcNAc biosynthesis pathway might also be involved. However, our data suggest that the elevated Man5 or HM level observed in mannose condition is not related to UDP-GlcNAc limitation caused by glucose starvation (Figure 2 and Table S1). Instead, it is more likely that mannose or its intermediates affect UDP-GlcNAc biosynthesis (by enzyme inhibition), its transportation, or GnT1 activity (Figure 8). Understanding the transcriptional and expressional levels of these key enzymes as well as their enzymatic activities will provide further insight into determining the precise inhibition mechanism(s) involved.
Conclusions
When multiple antibody cell lines were cultivated in mannose-containing media, cell growth and antibody
production were equivalent to those of glucose-grown cultures. However, HM level was increased, and different M:H ratios were shown to linearly modulate the HM of these antibody molecules. The HM modulation effect was also demonstrated in perfusion bioreactors, and there was a good correlation between small-scale mock perfusion and bioreactor scale. Hence, the HM modulation effect appears to be predictive and scalable, making this method an effective tool for increasing or controlling HM to desired levels. Glycan map data further suggest that the early glycosylation steps might be affected and thereby contribute to the accumulation of HM species. It is hypothesized that the elevated levels of mannose or its intermediates involved in glycan biosynthesis play a crucial role in HM modulation. Although other methods have been proposed for HM regulation, they were cell-line– and moleculedependent. In addition, these methods tend to impact other antibody quality attributes in addition to HM. To our knowledge, the method presented here is the first that can robustly control HM level of recombinant proteins without compromising cell growth and antibody titer. A robust and predictable method for HM modulation is demonstrated in this study. In the future, we plan to pair this method of increasing HM with those that effectively decrease HM to allow for bidirectional tuning of HM to the desired level.
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Acknowledgments
The authors thank the ATO High Throughput Lab for their supports with titer (Dazy Johnson) and HM
assay (Janice Chen, Mee Ko, Susan Callahan, and Tuong-Vi Don), Bruce Mason from Analytical Sciences for glycan map analysis, and the ATO media group for media preparation. The authors also thank Szilan Fodor, Chetan Goudar and Natalia Gomez for invaluable review of and suggestions for this work.
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Herman RH. 1971. Mannose metabolism. I. Am J Clin Nutr 24(4):488-98. Humphrey A. 1998. Shake Flask to Fermentor: What Have We Learned? Biotechnology Progress 14(1):3-7. Melmer M, Stangler T, Schiefermeier M, Brunner W, Toll H, Rupprechter A, Lindner W, Premstaller A. 2010. HILIC analysis of fluorescence-labeled N-glycans from recombinant biopharmaceuticals. Anal Bioanal Chem 398(2):905-14.
Nyberg GB, Balcarcel RR, Follstad BD, Stephanopoulos G, Wang DI. 1999. Metabolic effects on recombinant interferon-gamma glycosylation in continuous culture of Chinese hamster ovary cells. Biotechnol Bioeng 62(3):336-47.
Pacis E, Yu M, Autsen J, Bayer R, Li F. 2011. Effects of cell culture conditions on antibody N-linked glycosylation-what affects high mannose 5 glycoform. Biotechnol Bioeng 108(10) 2348-58
Wlaschin KF, Hu WS. 2007. Engineering cell metabolism for high-density cell culture via manipulation of sugar transport. J Biotechnol 131(2):168-76.
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Wong NSC, Wati L, Nissom PM, Feng HT, Lee MM, Yap MGS. 2010 An investigation of intracellular glycosylation activities in CHO cells: Effects of nucleotide sugar precursor feeding. Biotechnol Bioeng 107 (2):321–36.
Wong DCF, Wong KTK, Goh LT, Heng CK,Yap MGS. 2005. Impact of dynamic online fed-batch strategies on metabolism, productivity and N-glycosylation quality in CHO cell cultures. Biotechnol Bioeng 89(2):16477.
Yu M, Brown D, Reed C, Chung S, Lutman J, Stefanich E, Wong A, Stephan JP, Bayer R. 2012. Production, characterization, and pharmacokinetic properties of antibodies with N-linked Mannose-5 glycans. MAbs 4(4):475-87.
Zhu MM, Goyal A, Rank DL, Gupta SK, Vanden Boom T, Lee SS. 2005. Effects of elevated pCO2 and osmolality on growth of CHO cells and production of antibody-fusion protein B1: a case study. Biotechnol Prog 21(1):70-7.
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Figure Legends
Figure 1. Effects of alternative carbon sources on (A) cell growth, (B) viability, (C) antibody production, and (D) HM level of cell line 1 (mAb1) cells. Glucose and four alternative sugars, including mannose, galactose, fructose and maltose were tested. Each condition was performed in duplicate. In this experiment, for HM level, duplicate samples were mixed and analyzed (endo H method). Averaged values are normalized and presented. HM level is normalized to mannose condition. Figure 2. Effects of the titration of medium glucose by mannose on cell culture performance and Fc glycan HM levels of mAb1. (A) Cell growth, (B) viability, (C) normalized antibody titer, and (D) normalized HM level. Each condition was performed in duplicate. Duplicate samples were collected and analyzed (endo H method) for HM level. HM level is normalized to M:H ratio of 0.94 condition. Figure 3. Impact of different M:H ratio media on HM levels of different cell lines and recombinant antibodies (mAbs). Additional four different cell lines, including (B) cell line 2 (C) cell line 3, (D) cell line 4, and (E) cell line 5, were investigated for HM modulation by various H:M ratios in the culture medium. Each condition was performed in duplicate. Samples were collected to determine HM level of different molecules using the endo H method. HM level is normalized to mAb1 of M:H = 0.94. Figure 4. Effects of different M:H ratio media on HM levels and cell culture performance of cell line 1 cultured in 3L bioreactors (1.5 L working volume). (A) Normalized cell growth, (B) viability, (C) normalized antibody titer, (D) normalized HM level. HM level was assayed using the Endo H method. Both VCD and HM are normalized to the condition of M:H =0.94 (Day 15). Figure 5. Correlation comparison between small-scale mock perfusion and 3-L bioreactor regarding HM modulation by various M:H ratio media. Open square: mock perfusion; black triangle: bioreactor. HM level is normalized to M:H ratio of 0.94 from the perfusion bioreactor. Figure 6. Comparison of mAb1 Fc glycan maps from three bioreactors perfused with media containing different M:H ratios (0, 0.5, and 0.94). Day 15 samples were collected and the purified antibodies were used for assay. A This article is protected by copyright. All rights reserved
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full glycan map of each sample was analyzed using the HILIC method. Each glycoform is normalized to HM level of M:H=0.94.
Figure 7. Impact of three different M:H ratio media on the levels of detected HM glycan species, including Man8, Man7, Man6, and Man5, in mAb1. Man 9 specie was not detected or the peak area was negligible. The HILIC method was used to assay HM glycan species. Each HM glycoform is normalized to HM level of M:H = 0.94. Figure 8. Possible mechanisms that can contribute to HM modulation in using high M:H ratio media for cell culture processes. Three pathways, including GDP-mannose biosynthesis, early glycosylation, and UDP-GlcNAc biosynthesis, may be involved in the increased accumulation of different HM species, thereby leading to high HM level in the produced antibodies. (HK: Hexokinase; MPI: mannose 6 phosphate isomerase; PMM2: Phosphomannomutase; GMPP: mannose-1-phosphate guanylyltransferase; ALGP: mannosyltransferase; DPM1: dolichol-phosphate mannosyltransferase; M5-DLO-Flippase: Man5GlcNAc2-PP-dolichol Flippase; MPD-Flippase: Mannose-phosphate-dolichol Flippase; OST: Oligosaccharide transferase; GFAT: fructose-6-phosphateamidotransferase; GNA: Glucosamine-6-phosphate-N-acetyltransferase; AGM1: phospho-N-acetylglucosamine mutase; UAP: UDP-GlcNAc pyrophosphorylase; MAN: mannosidase; GnT1: α-1,3-mannosyl-glycoprotein 2-β-Nacetylglucosaminyltransferase
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Table 1. Experimental conditions for using five different culture media containing various percentages of glucose and mannose to study their HM modulation effects on multiple recombinant antibodies.
Media
Glucose (%)
Mannose (%)
Mannose/ *Total Hexose (M:H) ratio
1
100
0
0
2
75
25
0.25
3
50
50
0.5
4
25
75
0.75
5
6
94
0.94
* Total hexose is the sum of glucose and mannose in the culture medium.
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Bioprocess Engineering and Supporting Technologies
Biotechnology and Bioengineering DOI 10.1002/bit.25534
A Robust Method for Increasing Fc Glycan High Mannose Level of Recombinant Antibodies†
1
Chung-Jr Huang1*, Henry Lin1,2, Jerry (Xiaoming) Yang1,3
Cell Science & Technology, Process and Product Development, Amgen Inc., One Amgen Center Drive, Thousand
Oaks, CA 91320
Running Title: A Robust Method for High Mannose Modulation
*Corresponding Author: Chung-Jr Huang, Mail Stop: 18S-1-A, One Amgen Center Dr., Amgen Inc., Thousand Oaks, CA, 91320, Tel: (805) 447-7695, E-mail: [email protected]
2: Present address: Boehringer Ingelheim Fremont, Inc., 6701 Kaiser Drive, Fremont, CA 94555, USA 3: Present address: DZM Biotech Ltd.
†
This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: [10.1002/bit.25534]
Additional Supporting Information may be found in the online version of this article.
This article is protected by copyright. All rights reserved Received September 3, 2014; Revision Received December 15, 2014; Accepted December 23, 2014
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Abstract
High mannose (HM) glycoforms on antibody Fc glycan are recognized as critical quality attributes for therapeutic antibody products. Methods to control HM have been largely empirical, and it is challenging to target a desired HM level during antibody process development. A novel and robust method to increase antibody HM glycoforms is demonstrated in this study using multiple antibodies and cell lines without adversely impacting cell culture performance, including viable cell density, viability, and protein titer. This approach utilizes mannose as a carbon source and the ratio of mannose to total hexose (glucose and mannose) in feed media determines the extent of HM glycan content of an antibody expressed in cell culture. Scale-up of this strategy from 3-mL small scale plate to bioreactor (1.5 L) is also demonstrated with comparable results. Further full glycan map analysis shows that HM increase predominantly correlates with the decrease in G0F glycan, with minimum impact on other glycoforms. Possible hypotheses for the HM glycan modulation using mannose as carbon source are also discussed. Three pathways, including GDP-mannose biosynthesis, early protein glycosylation and UDP-N-acetyl-glucosamine biosynthesis, might be involved and contribute to this HM modulation. This article is protected by copyright. All rights reserved
Keywords: Chinese hamster ovary (CHO) cells, Recombinant antibody, Antibody Fc glycans, High mannose level modulation
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Introduction
IgG antibodies produced in mammalian cell cultures contain varying levels of high mannose (HM) glycoforms such as Man5, Man6, Man7, Man8, and Man9. The HM level on the antibody Fc glycan is a critical quality attribute that affects pharmacokinetic properties and effector function of therapeutic antibodies (Goetze et al., 2011; Yu et al., 2012). Glycoforms of an antibody expressed by Chinese hamster ovary (CHO) cell are largely determined during the processes of cell line generation and clone selection. However, HM content can also be affected by cell culture conditions (Pacis et al. 2011). It is common in the therapeutic antibody industry to seek a desired range of HM content for an antibody product due to process changes, scale-up, improvements, or the need to match existing antibody quality attributes. Thus far, methods applied for manipulating HM content of an antibody in cell culture include changes to medium compositions, osmolarlity, pH, and temperature (Ahn et al., 2008; Wong et al., 2005; Pacis et al., 2011; Yu et al., 2012). In general, these methods are effective only to certain cell lines, molecule types, and culture medium. Additionally, some methods can also alter antibody productivity, cell culture performance, and other antibody quality attributes. The effectiveness of these methods is also limited regarding modulating HM to a predetermined level during the cell culture processes. Therefore, a robust and predictive HM regulation method will immensely benefit process development for therapeutic proteins and antibodies.
In this study, we demonstrate a robust method that modulates HM content of several therapeutic antibodies
by applying mannose as a carbon source in the cell culture process. It was observed that the HM content of antibodies was proportional to the increase of mannose to total hexose (M:H) ratio in the cell culture medium. This effect was also universally applied to all tested antibodies (cell lines), with the magnitude of HM increase being cell-line–and molecule-specific. In this study, the mannose pathway and its impact on glycosylation in CHO cells are discussed, and the hypotheses of modulating HM by adding mannose as a carbon source are also discussed.
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Material and Methods Cell lines, cell culture, and media Serum-free adapted DXB-11 CHO cell lines expressing different recombinant antibodies were used in this
study. Cells were maintained in 3-L Erlenmeyer shake flasks (Corning Life Sciences, Lowell, MA) with a 1-L working volume and cultivated under standard humidified conditions at 36 °C, 5% CO2, and shaken at 70 rpm in an automatic CO2 incubator (Thermo Fisher Scientific, Waltham, MA). All cells were subcultured in the medium containing different concentrations of methotrexate (MTX) every 4 days, and were transferred, inoculated, and cultured in proprietary chemically-defined medium 1 for 4 days before seeding in 24–deep-well plates or inoculation in bioreactors. Another proprietary chemically-defined medium 2 was used as the control medium in the study. In the first different carbon source screening experiment, 96% of the glucose in the medium 2 was substituted with the tested carbon sources, including mannose, galactose, fructose, and maltose (Sigma-Aldrich, St. Louis, MO). In other studies, glucose in medium 2 was substituted with mannose according to the experiment design (Table 1).
Small-scale mock perfusion cell culture A newly developed mock perfusion experiments in 24–deep-well plates (Axygen, Union City, CA) were
used to evaluate effects of different carbon sources and M:H ratios on HM modulation. This high throughput method was developed as a small scale model for high cell density (HCD) perfusion bioreactor culture. HCD perfusion bioreactor culture is characterized by high cell density and medium perfusion, with the majority of protein is produced at production phase. Therefore, high seed density is used in this model with culture medium being exchanged every day to support high cell density growth to mimic the performance of HCD perfusion bioreactor cultures. Briefly, different cell lines were seeded in the plate at the targeted density ranging from 20–30 x 106 cells/mL with 3-mL working volumes for each well. To reach such a high seed density, cells were centrifuged and concentrated from above-mentioned seed subcultures. The cells then were cultivated at 36 °C, 5% This article is protected by copyright. All rights reserved
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CO2, 85% relative humidity and shaken in incubator (Kuhner AG, Basel, Switzerland) for 3 or 4 days. Every 24 hours, cells were centrifuged at 200xg for 5 minutes (Beckman Coulter, Brea, CA) to collect the spent media, and each well was then replenished with 3 mL fresh medium. The collected spent medium was further analyzed for titer, key metabolites, and HM level (when necessary). When cells were harvested, cell counts and viability were measured. All experimental conditions were performed in duplicate.
Perfusion cultivation in bioreactors A bioreactor experiment studying effects of different glucose:mannose ratios on HM level for cell line 1
was performed using three 3-L bioreactors (Applikon Biotechnology, Foster City, CA) with 1.5L working volume. The bioreactors were equipped with hollow fiber cartridges (GE Healthcare Biosciences, Pittsburgh, PA) which retain the produced mAb inside the bioreactor. In brief, cells were seeded in the bioreactors containing 1.5 L medium 1. An automated bioprocessing software (DeltaV™) was used for data acquisition and parameter control. During the cell culture processes, cells were initially grown at 36 °C then temperature shifted, once the desired cell density was reached. The dissolved oxygen level was maintained at 40% of air saturation and supplemented with pure oxygen when needed. Bioreactor pH was maintained at 7.0 by CO2 or 1 M sodium carbonate addition. At day 3, three types of medium 2 media containing different concentrations of glucose and mannose (M:H ratios of 0, 0.5 and 0.94), were fed into the bioreactors, and the permeate through the hollow fiber was also collected at the same rate. Samples were taken from three bioreactors and analyzed daily during the run.
Cell growth, antibody titer, and metabolite analysis Viable cell density and viability were determined using the Cedex cell counter (Roche Innovative,
Beilefeld, Germany). Antibody concentrations in the spent medium were determined using Protein A ultraperformance liquid chromatography (UPLC) (Waters Corporation, Milford, MA) equipped with a 50 mm x 4.6 mm i.d. POROS A/20 protein A column (Life Technologies, Carlsbad, CA). After sample was injected, the column was washed with phosphate-buffered saline (PBS) pH = 7.1 to remove CHO host cell proteins. Bound antibodies were This article is protected by copyright. All rights reserved
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then eluted in acidic PBS buffer (pH = 1.9) and detected by UV absorbance at 280 nm to quantify antibody concentration. For experiments conducted using 24–deep-well plates, each condition was performed in duplicate and an averaged value was presented. For bioreactor samples, metabolites including glucose, lactate, ammonia, and glutamine were analyzed using the NovaBioprofile Flex (Nova Biomedical, Waltham, MA).
High throughput Fc HM content assay A high-throughput microchip capillary electrophoresis (CE) method was used to determine HM levels of
recombinant antibodies directly from the spent medium of the 24–deep-well plate samples (Chen et al., 2008). In brief, samples were digested with endoglycosidase H (Endo H) for 2 hours at 37 °C. After digestion, antibodies were denatured at 70 °C for 10 minutes and injected in LabChip GXII (Caliper Life Sciences, Hopkinton, MA) to quantify non-glycosylated heavy chain (NGHC). Samples not digested by Endo H were used as controls. The % of NGHC from the control was subtracted by the %NGHC of the adjusted sample to yield the %HM value. For bioreactor samples, antibodies were first purified by affinity protein A MediaScout MiniChrom column (ATOLL, Weingarten, Germany) before the HM assay. The purified proteins were digested with Endo H and denatured as previously described. The percentages of NGHC from digested samples and controls were determined using a ProteomeLab PA800 Capillary Electrophoresis system (Beckman Coulter, Brea, CA), and two NGHC values were used to determine the HM level. The HM level was normalized and the presented.
HILIC whole Fc glycan map assay Different N-glycan species in antibodies were analyzed by hydrophilic-interaction liquid chromatography
(HILIC) for samples collected from bioreactors (Melmer et al., 2010). The purified antibodies were digested by Nglycosidase F (New England BioLabs, Ipswich, MA) at 37 °C for 2 hours to release the glycans. The released glycans were labeled with 2-aminobenzoic acid and cleaned up by GlycoClean S cartridges (Prozyme, Hayward, CA). Purified glycans were then desalted and reconstituted in water for the assay. HILIC was performed with a 100 mm x 2.1 mm i.d. BEH Glycan column using UPLC (Waters Corporation, Milford, MA), and the eluted glycans This article is protected by copyright. All rights reserved
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were detected, identified, and qualified by a fluorescence detector based on elution time. The percentage of each detected glycoforms were normalized and presented.
Results
Mannose utilization maintains cell growth and protein production but increases HM content in cell line 1 (mAb1) To explore the effects of different carbon sources on cell growth, antibody production and quality attributes
such as HM, five different sugars, including glucose, mannose, galactose, fructose and maltose, were evaluated using a small-scale (24–deep-well plate) mock perfusion model. As shown in Figure 1A, when galactose, fructose, and maltose were metabolized by cell line 1, cell growth was significantly inhibited. The viability and normalized antibody titer were also adversely impacted (Figure 1B, C). After analysis of antibody Fc glycan quality attributes, however, the normalized HM level were found to be between 165-168 in these cultures, about five-fold higher than that of the glucose control (34.8) (Figure 1D). Interestingly, cell line 1 cultured with mannose as a carbon source yielded comparable growth, viability, and normalized antibody titer as glucose control (Figures 1A–C). In this culture, viable cell density (VCD) continued to increase and reached 520 x 105 cells/mL with a viability of 94% at day 3, and mAb1 was produced at a normalized titer of 0.97. However, mannose culture also had a high normalized HM level of 100 (Figure 1D). This indicates that the presence of mannose in the medium affects antibody Fc glycan processing while maintaining equivalent growth and protein production when compared to glucose-grown culture.
HM content in mAb1 can be modulated with various mannose:glucose ratios Based on the previous plate study, we hypothesized that mannose concentration in the medium could
influence HM levels in mAb1 antibody. CHO cells may uptake mannose at similar rate as glucose, and various ratios of mannose:glucose could potentially yield different levels of HM while having no impact on cell growth or protein production. To test this hypothesis, the glucose in the medium was replaced with different amount of This article is protected by copyright. All rights reserved
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mannose, and a mock perfusion experiment was performed to verify the hypothesized HM modulation effects (Table 1). When glucose was replaced by 25, 50, 75, and 96% of mannose in the medium, the normalized HM level increased from 35.9 to 48.1, 63, 82.1, and 100, respectively, for mAb1(Figure 2D). The growth, viability, and normalized antibody titer were not affected by changing the mannose concentration in the media (Figure 2A-2C). These results demonstrate that various mannose:glucose ratios can linearly modulate the HM level without impacting cell growth and productivity.
The HM modulation effect is applicable and predictable for different cell lines (mAbs) To test whether the mannose-associated HM modulation effect is universally applicable, four other cell
lines, producing mAb 2, 3, 4, 5 were cultivated in media containing various mannose:glucose ratios as in the previous experiment. After harvest, samples were analyzed, and normalized HM level was plotted against the ratio of mannose to total hexose (M:H) in the medium. All four cell lines have elevated HM at high M:H ratios, as previously observed for cell line 1 (mAb1) (Figure 3). Thus, this modulation effect applied to all the cell clines we tested, a phenomenon that is striking especially when considering that these cell lines inherently have different HM levels for their respective antibodies. For example, the cell line 2 produces mAb2 with normalized HM level of 6 in the control medium (glucose only), and the HM increases to 13.2 at an M:H ratio of 0.94. Alternatively, cell line 5 produced mAb5 that has normalized 104.6 HM level in the control medium. When the M:H ratio is 0.94, the normalized HM increased to 207.2. Moreover, a strong linear correlation between normalized HM and M:H ratio was seen for all cell lines. This indicates that HM can be modulated by controlling the M:H ratio in the medium. In other words, when cells are cultivated in the media with a certain M:H ratio, the HM level of the produced antibodies becomes predictable.
The HM modulation effect is also scalable in bioreactors To verify the scalable aspect of HM modulation, a 1.5L bioreactor run using the mAb1 production process
was conducted. Starting at day 3, three bioreactors were perfused with media containing different M:H ratios of 0 This article is protected by copyright. All rights reserved
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(the control), 0.5, and 0.94. During the cultivation period, samples were taken daily, and cell counts, cell viability, and metabolites were measured. Figure 4 shows the time course for cell growth, normalized antibody yield, and normalized HM level from three bioreactors. When cells were grown in perfusion media with an M:H ratio of 0.94, the Day 15 normalized VCD reached 100 with 78% viability and a normalized titer of 0.89, which were all equivalent to the control. Cell growth, viability, and titer were all slightly lower for an M:H ratio of 0.5 (Figure A, B and C). For HM content, differentiated dynamic profiles were observed (Figure 4D). The control had 31.7 normalized HM level at day 6, which then gradually increased to 39.9 at day 15. When the M:H ratio was 0.5, a normalized HM of 32.5 was also observed at day 6, but increased about two-fold to 63.4 at day 15. Interestingly, the M:H ratio of 0.94 initially produced high normalized HM of 67.8 and reached 100 at day 9. The normalized HM value plateaued at around 100 throughout the rest of the cultivation period. These results indicated that the HM modulation effect is applicable at bioreactor process and scale. This effect was also predictable with an Rsquared value of 0.98 (Figure 5). Moreover, a good correlation was also observed between the small scale mock perfusion and perfusion bioreactor. Figure 5 compares the HM modulation effect between small scale mock perfusion and the Day 15 normalized HM levels from bioreactor for mAb1. The slopes of the trend lines were 59.24 and 62.58 for mock perfusion and bioreactor, respectively, demonstrating that the correlation between HM and M:H ratio was scale-independent.
Full glycan map analysis with HM modulation To understand the overall impact on antibody Fc glycan by mannose modulation, purified mAb1 from the
bioreactor process was analyzed with a full glycan map assay. As shown in Figure 6, certain glycans, including galactosylated (G1F+G2F), M3G0F and sialylated forms were not affected by different M:H ratios in the medium. High M:H ratios predominantly increased HM and decreased G0F. For example, M:H ratio of 0.94 had the highest normalized HM level of 100 and the lowest normalized G0F level of 132. In addition, increased M:H ratios produced slightly higher amounts of afucosylated and hybrid Fc glycans. A few existing unidentified glycan forms also increased to a small extent. The percentage of each HM species was also identified and compared (Figure 7). This article is protected by copyright. All rights reserved
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With an increase in M:H ratio of the medium, the levels of Man8, Man7, Man6, and Man5 glycans all significantly increased when compared to the control. Man5 was found to be the major HM species in the three conditions. In addition, the percentage increase in Man6, Man7 and Man8 species were higher than that of Man5 species at higher M:H ratios. These results suggest that high M:H ratios slow down early glycan processing steps, leading to elevated levels of all HM species. With increase in the H:M ratio, more primitive HM species were found in the produced antibodies.
Discussion
A robust method for HM modulation Mannose and its derivatives, such as N-acetylmannosamine, have been supplemented into the cell culture
medium to study their effects on cellular metabolism and N-glycans of recombinant proteins (Altamirano et al., 2013; Butler, 2006; Wong et al., 2010). The replacement of glucose by mannose enhances volumetric productivity of t-PA and reduces specific oxygen consumption rate, without impacting t-PA sialylation level (Berrios, 2011). Addition of mannose with other N-glycan precursors does not increase site-occupancy of t-PA but shows to increase sialylation of recombinant glycoproteins (Butler, 2006; Gawlitzek et al., 2009). Wong and colleagues also reported that including the N-acetylmannosamine and uridine in the medium result in 36% increase in INF-γ sialylation (Wong et al. 2010). In this study, we found that mannose also increases %HM of Fc glycan in mAbs produced by CHO cells, without impacting cell culture performance. Furthermore, the ratios of mannose to total hexose in the culture medium modulate Fc glycan HM levels. Different cellular process parameters and production medium components have been reported to affect HM level of recombinant proteins produced in CHO cells. However, these methods are not robust for commercial cell culture processes. Lowering temperatures (< 32 °C) have been shown to increase certain HM species and reduce antennary structure and sialylation of recombinant erythropoietin (Ahn et al., 2008). Recently, Pacis et al. demonstrated that antibody Man5 level can be increased more than two-fold from 12% to 28% by increasing medium osmolarity and culture duration time (Pacis et al.,
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2011). Although moderate linear correlations may be found between medium osmolarity and Man5 level, high osmolarity media could negatively impact cell growth, viability, and protein titer (Zhu et al., 2005). The time course HM profiles observed in three bioreactors is much of an interesting observation. The result
indicates that HM upregulation by mannose may exhibit a saturation effect in cell line 1. However, it is yet to be determined whether similar dynamics apply to different cell lines, as inherent HM values (in glucose) are different across these cells lines (Figure 4). Traditionally, when cells are cultured in flasks or plates, physical parameters and nutritional components are not well controlled. Hence, scale variations regarding cell growth, protein production, and glycosylation pattern are commonly observed in cell culture processes (Humphrey, 1998). In this study, we found a good correlation between the 24–deep-well plate mock perfusion and bioreactor on HM modulation effects (Figure 5). The high cell density inoculation and daily medium exchange might contribute to this correlation, further validating that the developed method showed in this study is a good scale-down model for perfusion bioreactor.
Hypothesis of underlying mechanisms In mammalian cells, different glucose transporters (GLUTs) are responsible for the import of different
carbohydrates to provide an energy source for cell growth and metabolism. At least 14 GLUTs have been identified, and their expression profiles in cells are highly tissue-specific. Different GLUTs were shown to transport different sugar substrates with distinct affinities and transport capacities (Augustin, 2010). When galactose, fructose, and maltose were used as carbon sources, reduced cell growth and protein production observed in cell line 1 mock perfusion cultures were likely due to slow uptake of galactose, fructose, and maltose by GLUTs. For CHO cells, high uptake rates of glucose and mannose by GLUTs result in high cell growth and elevated levels of by-products such as lactate and ammonia. In contrast, poor transport of galactose and fructose reduces cell growth and by-product accumulation (Altamirano et al., 2000; Wlaschin and Hu, 2007). We hypothesize that slow transport of galactose, fructose, and maltose in the cell line 1 is responsible for the increase in HM level of mAb1 antibody. Although once inside the cells, these sugars convert to glucose or glycolytic intermediates, such as This article is protected by copyright. All rights reserved
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glucose-6-phosphate and fructose-6-phsophate (F6P), the supply of these intermediates for energy production and for glycan precursors remains insufficient due to the low sugar transport. Such a glucose limitation-like state increases HM and aglycosylation of recombinant proteins. For example, low glucose concentration in the media increases the levels of hybrid and HM glycan forms on recombinant interferon gamma (Wong et al., 2005). Since cells preferentially use glucose for energy production, low nucleotide triphosphate pools and reduced intracellular levels of UDP-N-acetyl-glucosamine (UDP-GlcNAc) and UDP-N-acetyl-galactosamine were found in glucosestarved cells (Nyberg et al., 1999). This depletion would eventually affect complex glycan processing due to the shortage of nucleotide sugars, producing more HM-type proteins. In glucose-limiting culture, the insufficient supply of UDP-GlcNAc is further deteriorated by glutaminolysis, in which glutamine, a key amino acid for UDPGlcNAc biosynthesis, is channeled into energy production (Hayter et al., 1992; Nyberg et al., 1999). This was also the case in this study, where glutamine was low or depleted in galactose, fructose, and maltose cultures (data not shown) and further explains the five-fold increase of HM in these cultures. Unlike other tested carbon sources, mannose can be transported and metabolized as efficiently as glucose is
in CHO cells. However, increased HM level is also observed in mannose culture. Upon intake, intracellular mannose is phosphorylated by hexokinase to mannose-6-phosphate (M6P), which can then be converted by phosphomannose isomerase (MPI) to F6P to enter the glycolytic cycle (Figure 8) (Herman, 1971). This study and others suggest that this pathway is highly efficient for energy generation in CHO cells (Altamirano et al., 2000; Berrios et al., 2011); hence, intracellular nucleotide triphosphate pools and intracellular UDP-GlcNAc levels in mannose culture should be comparable to those of glucose-grown cultures. Glutamine levels were also equivalent among three bioreactor cultures (Figure S1). Together, these results suggest that mechanisms other than energy related substrate limitation lead to HM increase in mannose-grown cultures. When cells are grown in glucose-only media, cellular mannose for glycan biosynthesis is derived entirely from glucose (Alton et al., 1998; Gao et al., 2005). This suggests that early glycan processing steps are impacted, resulting in HM increase in mannose culture. The cause may be related to upstream mannose metabolism or increased influx of GDP-mannose for glycan biosynthesis. At this point, it was hypothesized that the presence of mannose or its intermediates such as M6P are This article is protected by copyright. All rights reserved
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responsible for HM upregulation. Elucidation of different mannose metabolite levels by metabolite profiling and tracing of the fluxes of these metabolites would provide data to validate this hypothesis. To further investigate the mechanism of HM upregulation, glycan maps of samples from three bioreactors
were elucidated (Figure 6 and 7). High M:H ratios slightly increase the levels of afucosylated complex, unknown and hybrid glycans. GDP-mannose is known to convert to GDP-fucose by GDP-mannose 4,6-dehydratase (GMD) and two other enzymes in the de novo fucosylation pathway. GDP-fucose is also a potent inhibitor of GMD (Becker and Lowe, 2003). High mannose concentration in the medium should theoretically increase the intracellular GDP-mannose and GDP-fucose levels that lead to low afucosylation (due to increased substrate supply). Therefore, the increased afucosylated complex observed in this study may be due to strong feedback inhibition of GDP-fucose to GMD that eventually decrease intracellular GDP-fucose level or by unidentified mechanisms. The galactosylation and sialylation of mAb1 are similar upon the addition of mannose in the culture media. The decrease of G0F correlates with HM increase. These results indicated that mainly the early glycosylation pathway (before G0F) is affected. Moreover, analyses of HM species showed that increased M:H ratios concurrently increased the levels of different HM species, including Man8, Man7, Man6, and Man5. Although Man5 is the major HM species in all three conditions, the levels of other primitive HM species increases significantly with high M:H ratios. The HM species accumulation profile further suggests that two glycosylation steps, HM trimming by -mannosidase I (-MAN I) and UDP-GlcNAc addition by UDP-GlcNAc transferase (GnT1), are inhibited, increasing the HM level in mAb1 (Figure 8). The first step for cells to utilize intracellular mannose is to convert mannose to M6P, a key metabolite
bridging energy generation (glycolysis) and N-glycan biosynthesis (Alton et al., 1998). Elevated intracellular M6P levels have been demonstrated in mouse embryonic fibroblasts when 10 mM mannose was supplemented in the media (Gao et al., 2011). Therefore, it is reasonable to theorize that elevated M6P levels should also be found in high M:H ratio cultures, suggesting a possible flux increase for N-glycan biosynthesis. As illustrated in Figure 8, three pathways, GDP-mannose biosynthesis, early glycosylation, and UDP-GlcNAc biosynthesis, may have been involved in the HM upregulation observed in this study. Increased levels of primitive HM species suggest that the This article is protected by copyright. All rights reserved
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increased concentration of mannose or its intermediates (M6P, mannose-1-phosphate [M1P], or GDP-Mannose) may directly or indirectly inhibit -MANI activity. High Man5 levels also suggest that the UDP-GlcNAc biosynthesis pathway might also be involved. However, our data suggest that the elevated Man5 or HM level observed in mannose condition is not related to UDP-GlcNAc limitation caused by glucose starvation (Figure 2 and Table S1). Instead, it is more likely that mannose or its intermediates affect UDP-GlcNAc biosynthesis (by enzyme inhibition), its transportation, or GnT1 activity (Figure 8). Understanding the transcriptional and expressional levels of these key enzymes as well as their enzymatic activities will provide further insight into determining the precise inhibition mechanism(s) involved.
Conclusions
When multiple antibody cell lines were cultivated in mannose-containing media, cell growth and antibody
production were equivalent to those of glucose-grown cultures. However, HM level was increased, and different M:H ratios were shown to linearly modulate the HM of these antibody molecules. The HM modulation effect was also demonstrated in perfusion bioreactors, and there was a good correlation between small-scale mock perfusion and bioreactor scale. Hence, the HM modulation effect appears to be predictive and scalable, making this method an effective tool for increasing or controlling HM to desired levels. Glycan map data further suggest that the early glycosylation steps might be affected and thereby contribute to the accumulation of HM species. It is hypothesized that the elevated levels of mannose or its intermediates involved in glycan biosynthesis play a crucial role in HM modulation. Although other methods have been proposed for HM regulation, they were cell-line– and moleculedependent. In addition, these methods tend to impact other antibody quality attributes in addition to HM. To our knowledge, the method presented here is the first that can robustly control HM level of recombinant proteins without compromising cell growth and antibody titer. A robust and predictable method for HM modulation is demonstrated in this study. In the future, we plan to pair this method of increasing HM with those that effectively decrease HM to allow for bidirectional tuning of HM to the desired level.
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Acknowledgments
The authors thank the ATO High Throughput Lab for their supports with titer (Dazy Johnson) and HM
assay (Janice Chen, Mee Ko, Susan Callahan, and Tuong-Vi Don), Bruce Mason from Analytical Sciences for glycan map analysis, and the ATO media group for media preparation. The authors also thank Szilan Fodor, Chetan Goudar and Natalia Gomez for invaluable review of and suggestions for this work.
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References
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Figure Legends
Figure 1. Effects of alternative carbon sources on (A) cell growth, (B) viability, (C) antibody production, and (D) HM level of cell line 1 (mAb1) cells. Glucose and four alternative sugars, including mannose, galactose, fructose and maltose were tested. Each condition was performed in duplicate. In this experiment, for HM level, duplicate samples were mixed and analyzed (endo H method). Averaged values are normalized and presented. HM level is normalized to mannose condition. Figure 2. Effects of the titration of medium glucose by mannose on cell culture performance and Fc glycan HM levels of mAb1. (A) Cell growth, (B) viability, (C) normalized antibody titer, and (D) normalized HM level. Each condition was performed in duplicate. Duplicate samples were collected and analyzed (endo H method) for HM level. HM level is normalized to M:H ratio of 0.94 condition. Figure 3. Impact of different M:H ratio media on HM levels of different cell lines and recombinant antibodies (mAbs). Additional four different cell lines, including (B) cell line 2 (C) cell line 3, (D) cell line 4, and (E) cell line 5, were investigated for HM modulation by various H:M ratios in the culture medium. Each condition was performed in duplicate. Samples were collected to determine HM level of different molecules using the endo H method. HM level is normalized to mAb1 of M:H = 0.94. Figure 4. Effects of different M:H ratio media on HM levels and cell culture performance of cell line 1 cultured in 3L bioreactors (1.5 L working volume). (A) Normalized cell growth, (B) viability, (C) normalized antibody titer, (D) normalized HM level. HM level was assayed using the Endo H method. Both VCD and HM are normalized to the condition of M:H =0.94 (Day 15). Figure 5. Correlation comparison between small-scale mock perfusion and 3-L bioreactor regarding HM modulation by various M:H ratio media. Open square: mock perfusion; black triangle: bioreactor. HM level is normalized to M:H ratio of 0.94 from the perfusion bioreactor. Figure 6. Comparison of mAb1 Fc glycan maps from three bioreactors perfused with media containing different M:H ratios (0, 0.5, and 0.94). Day 15 samples were collected and the purified antibodies were used for assay. A This article is protected by copyright. All rights reserved
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full glycan map of each sample was analyzed using the HILIC method. Each glycoform is normalized to HM level of M:H=0.94.
Figure 7. Impact of three different M:H ratio media on the levels of detected HM glycan species, including Man8, Man7, Man6, and Man5, in mAb1. Man 9 specie was not detected or the peak area was negligible. The HILIC method was used to assay HM glycan species. Each HM glycoform is normalized to HM level of M:H = 0.94. Figure 8. Possible mechanisms that can contribute to HM modulation in using high M:H ratio media for cell culture processes. Three pathways, including GDP-mannose biosynthesis, early glycosylation, and UDP-GlcNAc biosynthesis, may be involved in the increased accumulation of different HM species, thereby leading to high HM level in the produced antibodies. (HK: Hexokinase; MPI: mannose 6 phosphate isomerase; PMM2: Phosphomannomutase; GMPP: mannose-1-phosphate guanylyltransferase; ALGP: mannosyltransferase; DPM1: dolichol-phosphate mannosyltransferase; M5-DLO-Flippase: Man5GlcNAc2-PP-dolichol Flippase; MPD-Flippase: Mannose-phosphate-dolichol Flippase; OST: Oligosaccharide transferase; GFAT: fructose-6-phosphateamidotransferase; GNA: Glucosamine-6-phosphate-N-acetyltransferase; AGM1: phospho-N-acetylglucosamine mutase; UAP: UDP-GlcNAc pyrophosphorylase; MAN: mannosidase; GnT1: α-1,3-mannosyl-glycoprotein 2-β-Nacetylglucosaminyltransferase
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Table 1. Experimental conditions for using five different culture media containing various percentages of glucose and mannose to study their HM modulation effects on multiple recombinant antibodies.
Media
Glucose (%)
Mannose (%)
Mannose/ *Total Hexose (M:H) ratio
1
100
0
0
2
75
25
0.25
3
50
50
0.5
4
25
75
0.75
5
6
94
0.94
* Total hexose is the sum of glucose and mannose in the culture medium.
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