Characterization of the stability of recombinant protein production in the GS-NS0 expression system

The GS-NS0 system is an important mammalian expression system used largely within industry for the high-level expression of recombinant proteins for therapeutic use. It is essential that the productivity of this system remains stable throughout culture expansion for the successful long-term production of recombinant proteins. Here we present a study of the stability of recombinant protein production from unamplified GS-NS0 cell lines over extended period of continuous culture. The cell lines used in this study were generated by the transfection of NS0 cells with DNA encoding for a secreted recombinant protein and by two subsequent rounds of limiting dilution cloning prior to analysis of stability. The stability of recombinant protein production was assessed at intervals over a period of 134 days using repeated batch culture in shake flasks. Heterogeneous stability was identified. The productivity of some clones remained consistent throughout 134 days of continuous culture. Others exhibit rapid and progressive loss of productivity. Analysis of the causal relationships underlying stability indicates that the initial transfectant determines the susceptibility to loss or retention of productivity. Selection of production clones on the basis of growth and productivity alone will not predict stability during long-term culture. Our research indicates that stable high-producing clones can readily be obtained from use of the GS-NS0 system in the absence of amplification but there may be molecular features of the original transfectants that could serve as very important predictive indicators of the stability of recombinant protein production.     

Comparative proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NS0) cells. Four homogeneous NS0 cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab datasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production.     

Stability of recombinant protein production in the GS-NS0 expression system is unaffected by cryopreservation

Mammalian cells form a very important part of the repertoire of production systems available to scientists involved in the production of recombinant proteins. During the production of therapeutic proteins it is vital for regulatory approval of products that no phenotypic or genetic changes are observed in the cell line or product. As part of the generation and development of therapeutic protein production, cell lines have to be frozen at various stages to create cell banks. If cryopreservation and revival of frozen stocks were to give rise to any phenotypic changes in the cells, this would again be detrimental to the further development of that particular cell line. This study uses one of the most industrially important expression systems, the GS-NS0 expression system, to examine the effect of cryopreservation on the growth and productivity profile of cell lines that exhibit differential degrees of stability during prolonged (production) culture periods. Results show that cryopreservation and revival procedures do not alter the stability characteristics of cell lines. This type of information is of great value in definition of protocols for cell line development.     

Functional proteomic analysis of GS-NS0 murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We previously compared changes in individual protein abundance between the proteomes of GS-NS0 cell lines with varying rates of cell-specific recombinant monoclonal antibody production (qMab). Here we extend analyses of our proteomic dataset to statistically determine if particular cell lines have distinct functional capabilities that facilitate production of secreted recombinant Mab. We categorized 79 proteins identified by mass spectrometry according to their biological function or location in the cell and statistically compared the relative abundance of proteins in each category between GS-NS0 cell lines with varying qMab. We found that the relative abundance of proteins in ER chaperone, non-ER chaperone, cytoskeletal, cell signaling, metabolic, and mitochondrial categories were significantly increased with qMab. As the GS-NS0 cell line with highest qMab also had an increased intracellular abundance of unassembled Mab heavy chain (HC), we tested the hypothesis that the increased ER chaperone content was caused by induction of an unfolded protein response (UPR) signaling pathway. Immunoblot analyses revealed that spliced X-box binding protein 1 (XBP1), a marker for UPR induction, was not detectable in the GS-NS0 cells with elevated qMab, although it was induced by chemical inhibitors of protein folding. These data suggest that qMab is functionally related to the abundance of specific categories of proteins that together facilitate recombinant protein production. We infer that individual cells within parental populations are more functionally equipped for high-level recombinant protein production than others and that this bias could be used to select cells that are more likely to achieve high qMab.     

Metabolic control of recombinant monoclonal antibody N-glycosylation in GS-NS0 cells

Variable N-glycosylation at Asn(297) in the Fc region of recombinant therapeutic immunoglobulin G (IgG) molecules, specifically terminal galactosylation and sialylation, may affect both pharmacokinetic behavior and effector functions of recombinant therapeutic antibodies. We investigated the hypothesis that IgG Fc glycosylation can be controlled by manipulation of cellular nucleotide-sugar metabolism. In control cultures, N-glycans associated with the Fc domain of a recombinant humanized IgG1 produced by GS-NS0 cells in culture were predominantly biantennary, variably beta-galactosylated (average 0.3 mol galactose complex N-glycan(-1)) structures with no bisecting N-acetylglucosamine residues, sialylation, or alpha1,3-linked galactosylation evident. However, a variable proportion (5% to 15%) of high-mannose (Man5 to Man9) oligosaccharides were present. To manipulate the cellular content of the nucleotide sugar precursor required for galactosylation, UDP-Gal, we included either 10 mM glucosamine or 10 mM galactose in the culture medium. In the case of the former, a 17-fold increase in cellular UDP-N-acetylhexosamine content was observed, with a concomitant reduction (33%) in total UDP-hexose, although the ratio of UDP-Glc:UDP-Gal (4:1) was unchanged. Associated with these alterations in cellular UDP-sugar content was a significant reduction (57%) in the galactosylation of Fc-derived oligosaccharides. The proportion of high-mannose-type N-glycans (specifically Man5, the substrate for N-acetylglucosaminyltransferase I) at Asn(297) was unaffected. In contrast, inclusion of 10 mM galactose in culture specifically stimulated UDP-Gal content almost five-fold. However, this resulted in only a minimal, insignificant increase (6%) in beta1,4-galactosylation of Fc N-glycans. Sialylation was not improved upon the addition of the CMP-sialic acid (CMP-SA) precursor N-acetylmannosamine (20 mM), even with an associated 44-fold increase in cellular CMP-SA content. Analysis of recombinant IgG1 Fc glycosylation during batch culture showed that beta1,4-linked galactosylation declined slightly during culture, although, in the latter stages of culture, the release of proteases and glycosidases by lysed cells were likely to have contributed to the more dramatic drop in galactosylation. These data demonstrate: (i) the effect of steric hindrance on Fc N-glycan processing; (ii) the extent to which alterations in cellular nucleotide-sugar content may affect Fc N-glycan processing; and (iii) the potential for direct metabolic control of Fc N-glycosylation.     

Proteomic analysis of enriched microsomal fractions from GS-NS0 murine myeloma cells with varying secreted recombinant monoclonal antibody productivities

The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high-level antibody secretion. We used GS-NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid-exponential growth phase. These were analysed by 2-D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up-regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell-engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study.     

A rapid selection/amplification procedure for high-level expression of recombinant protein in a metal-amplifiable mammalian expression system

We describe a strategy for the selection and amplification of foreign gene expression in Chinese hamster ovary (CHO) cells employing a metallothionein gene-containing expression vector. This report describes an amplification procedure that results in an enrichment of clones exhibiting high levels of recombinant protein production and reduces the labour required for screening recombinant cell lines.     

Inhibition of glutamine-dependent autophagy increases t-PA production in CHO cell fed-batch processes

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.     

The use of serum-free medium for the production of functionally active humanised monoclonal antibody from NS0 mouse myeloma cells engineered using glutamine synthetase as a selectable marker

A protein-free growth medium (W38 medium) had previously been developed for the NS0 mouse myeloma cell line which is cholesterol-auxotrophic. This paper describes the development of a protein-free growth medium for NS0 cells expressing humanised monoclonal antibody using GS (glutamine synthetase) as a selectable marker. Several GS-engineered NS0 cell lines expressing humanised monoclonal antibody grew in a modification of W38 medium which maintained GS-selection, supplemented with cholesterol, phosphatidylcholine and -cyclodextrin. Further studies showed that additional glutamic acid, asparagine, ribonucleosides and choline chloride improved cell growth. Amino acid analysis identified a number of amino acids that were being depleted from the culture medium. NS0 cell lines 9D4 and 2H5 expressing CAMPATH-1H^* were adapted to enable them to grow serum-free in the absence of cholesterol and -cyclodextrin. Cholesterol-independent 9D4 (9D4.CF) cells grown in shake flask culture using an enriched protein-free medium (WNSD medium), supplemented with human recombinant insulin (Nucellin), reached a maximum cell density to 1.86×10⁶ cells ml^–1 producing 76.6 mg l^–1 of antibody. CAMPATH-1H antibody produced using serum-free medium was found to be functionally activein vitro in the Antibody Dependant Cellular Cytotoxicity (ADCC) assay.Abbreviations C cholesterol - CD cyclodextrin - dhfr dihydrofolate reductase - F68 Pluronic F68 - GS glutamine synthetase - MSX methionine sulphoximine - P phosphatidylcholine - PC-FBS phosphatidylcholine, cholesterol and foetal bovine serum - RPMI RPMI 1640 medium - ADCC Antibody-dependant cellular cytotoxicity     

Defined protein and animal component-free NS0 fed-batch culture

A chemically defined protein and animal component-free fed-batch process for an NS0 cell line producing a human IgG(1) antibody has been developed. The fed-batch feed profile was optimised in a step-wise manner. Depletion of measurable compounds was determined by direct analysis. The cellular need for non-measurable compounds was tested by continued culturing of cell suspension, removed from the bioreactor, in shake-flasks supplemented with critical substances. In the final fed-batch culture, 8.4 x 10(6) viable cells mL(-1) and 625 mg antibody L(-1) was obtained as compared to 2.3 x 10(6) cells mL(-1) and 70 mg antibody L(-1) in batch. The increase in cell density, in combination with a prolonged declining phase where antibody formation continued, resulted in a 6.2-fold increase in total cell yield, a 10.5-fold increase in viable cell hours and an 11.4-fold increase in product yield. These improvements were obtained by using a feed with glucose, glutamine, amino acids, lipids, sodium selenite, ethanolamine and vitamins. Specifically, supplementation with lipids (cholesterol) had a drastic effect on the maximum viable cell density. Calcium, magnesium and potassium were not depleted and a feed also containing iron, lithium, manganese, phosphorous and zinc did not significantly enhance the cell yield. The growth and death profiles in the final fed-batch indicated that nutrient deprivation was not the main cause of cell death. The ammonium concentration and the osmolality increased to potentially inhibitory levels, but an imbalance in the supply of growth/survival factors may also contribute to termination of the culture.     

Isolation, characterization and recombinant protein expression in Veggie-CHO: A serum-free CHO host cell line

The dihydrofolate reductase-deficient Chinese hamster ovary cell line, DXB11-CHO, commonly used as a host cell for the production of recombinant proteins requires 7.5% serum-supplementation for optimal growth. Regulatory issues surrounding the use of serum in clinical production processes and the direct and indirect costs of using serum in large-scale production and recovery processes have triggered efforts to derive serum-independent host cell lines. We have successfully isolated a serum-free host that we named Veggie- CHO. Veggie-CHO was generated by adapting DXB11-CHO cells to growth in serum-free media in the absence of exogenous growth factors such as Transferrin and Insulin-like growth factor, which we have previously shown to be essential for growth and viability of DXB11- CHO cells. Veggie-CHO cells have been shown to maintain an average doubling time of 22 hr in continuous growth cultures over a period of three months and have retained the dihydrofolate reductase -deficient phenotype of their parental DXB11-CHO cells. These properties and the stability of its serum-free phenotype have allowed the use of Veggie- CHO as host cells for transfection and amplified expression of recombinant proteins. We describe the derivation a serum-free recombinant cell line with an average doubling time of 20 hr and specific productivity of 2.5 Units recombinant Flt-3L protein per 10e6 cells per day. This revised version was published online in August 2006 with corrections to the Cover Date.     

Dynamic analysis of GS-NS0 cells producing a recombinant monoclonal antibody during fed-batch culture

In this study we have analyzed the dynamic covariation of the mammalian cell proteome with respect to functional phenotype during fed-batch culture of NS0 murine myeloma cells producing a recombinant IgG(4) monoclonal antibody. GS-NS0 cells were cultured in duplicate 10 L bioreactors (36.5 degrees C, 15% DOT, pH 7.0) for 335 h and supplemented with a continuous feed stream after 120 h. Cell-specific growth rate declined continuously after 72 h of culture. Cell-specific recombinant monoclonal antibody production rate (qP) varied sixfold through culture. Whilst qP correlated with relative recombinant heavy chain mRNA abundance up to 216 h, qP subsequently declined, independent of recombinant heavy chain or light chain mRNA abundance. GS-NS0 cultures were sampled at 48 h intervals between 24 and 264 h of culture for proteomic analyses. Total protein abundance and nascent polypeptide synthesis was determined by 2D PAGE of unlabeled proteins visualized by SYPRO Ruby and autoradiography of (35)S-labeled polypeptides, respectively. Covariation of nascent polypeptide synthesis and abundance with biomass-specific cell growth, glucose and glutamate consumption, lactate and Mab production rates were then examined using two partial least squares regression models. Most changes in polypeptide synthesis or abundance for proteins previously identified by mass spectrometry were positively correlated with biomass-specific growth rate. We conclude that the substantial transitions in cell physiology and qP that occur during culture utilize a relatively constant complement of the most abundant host cell machines that vary primarily with respect to induced changes in cell growth rate.     

Molecular analysis of successful cell line selection in transfected GS-NS0 myeloma cells

The production of recombinant proteins from mammalian cells is now an essential part of biotechnology. However, despite this importance, the detailed characteristics of good producing cell lines remain largely unknown. The industrially important GS-NS0 mammalian expression system is able to produce large amounts of protein from relatively few copies of recombinant genes. This makes GS-NS0 cell lines ideal candidates to study the consequence of recombinant plasmid transfection in mammalian cells. This study investigated the molecular features of a panel of 17 randomly chosen GS-NS0 cell lines engineered to produce a recombinant antibody. The research analysed antibody production via enzyme-linked immunosorbent assay (ELISA), and investigated the molecular features of the transfectants by Northern, Southern and copy number analysis. The cell lines generated produced a range of antibody concentrations. In addition, for transfectants defined as producers of recombinant antibody there was a positive correlation between specific productivity and heavy chain mRNA expression. The use of Northern and Southern analysis allowed determination of the functional integrity of the transfected plasmid. Over 50% of the transfectants studied had molecular defects at the level of mRNA and/or cDNA. Cell lines were identified with suspected defects in the regulatory regions of transfected genes in addition to cell lines which lacked recombinant genes. Also, "false-positive" cell lines were generated which were able to overcome the GS selection pressure without producing any recombinant antibody. This article discusses these findings in relation to vector design.     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.     

Relationship between cell size, cell cycle and specific recombinant protein productivity

Centrifugal elutriation was used to produce cell cycle enrichedfractions of four commercially relevant recombinant cell lines,chosen to allow for variation in properties due to construct,expression system and parent cell type, from normally growingheterogeneous batch cultures. As these fractions had identicalculture histories and had not been subjected to any insult orstress which was likely to have adversely affected cellularmetabolism, they were ideal for further study of cellularproperties. Specific productivity, cell size and cell cyclestate of replicate elutriated fractions were measured for eachcell line. Results showed that cell size was the major cellulardeterminant of productivity for all cell lines examined. Productformation was not restricted to any particular cell cycle phaseand in all cases, production occurred irrespective of cell cyclephase. Specific productivity was lowest when the majority ofcells in the fraction were G₁, intermediate when themajority of cells in the fraction were S phase and greater whenthe majority of cells in the fraction were in G₂/M. However, the evidence suggests that size is the major cellulardeterminant of productivity; the apparent relationship betweencell cycle and productivity is secondary and can simply beascribed to the increasing size of cells as they progress thoughthe cell cycle. Thus, in addition to cell density and viabilitycell size is the cellular parameter which should be incorporatednot only into mathematical models of recombinant mammalian cellproduction processes but also into process monitoring andcontrol strategies.     

Derivation and characterization of cholesterol-independent non-GS NS0 cell lines for production of recombinant antibodies

Presented is an antibody production platform based on the fed-batch culture of recombinant NS0-derived cell lines. NS0 host cells, obtained from the European Collection of Cell Cultures (ECACC, Salisbury, UK, Part No. 85110503), were first adapted to grow in a protein-free, cholesterol-free medium. The resulting host cell line was designated NS0-PFCF (protein-free, cholesterol-free). The five production cell lines presented here were generated using a common protocol consisting of transfection by electroporation and subcloning. The NS0-PFCF host cell line was transfected using a single expression vector containing the Escherichia coli xanthine-guanine phosphoribosyl transferase gene (gpt), and the antibody heavy and light chain genes driven by the CMV promoter. The five cell lines were chosen after one to three rounds of iterative subcloning, which resulted in a 19-64% increase in antibody productivity when four mother-daughter cell pairs were cultured in a fed-batch bioreactor process. The production cell lines were genetically characterized to determine antibody gene integrity, nucleotide sequences, copy number, and the number of insertion sites in the NS0 cell genome. Genetic characterization data indicate that each of the five production cell lines has a single stably integrated copy of the antibody expression vector, and that the antibody genes are correctly expressed. Stability of antibody production was evaluated for three of the five cell lines by comparing the early stage seed bank with the Working Cell Bank (WCB). Antibody productivity was shown to be stable in two of three cell lines evaluated, while one of the cell lines exhibited a 20% drop in productivity after passaging for approximately 4 weeks. These five NS0-derived production cell lines were successfully cultured to produce antibodies with acceptable product quality attributes in a standardized fed-batch bioreactor process, consistently achieving an average specific productivity of 20-60 pg/cell-day, and a volumetric productivity exceeding 120 mg/L-day (Burky et al., 2006). In contrast to the commonly available NS0 host cell line, which requires serum and cholesterol for growth, and the commonly used expression vector system, which uses a proprietary glutamine synthetase selection marker (GS-NS0), these NS0 cells are cholesterol-independent, grow well in a protein-free medium, use a non-proprietary selection marker, and do not require gene amplification for productivity improvement. These characteristics are advantageous for use of this NS0 cell line platform for manufacturing therapeutic antibodies.     

长效重组蛋白药物发展动态

部分重组蛋白药物存在半衰期短的缺陷,临床给药频率高,且大多为注射给药,严重影响患者使用依从性。长效重组蛋白药物是近年来生物技术药物发展的重要趋势之一。对蛋白分子进行改造或修饰,延长重组蛋白药物的半衰期,实现长效以减少给药频率主要通过4种方式:化学修饰、构建突变体、蛋白融合、糖基化修饰。针对上述4种长效化方式及已上市相关产品进行了综述。展望未来,紧跟国外先进技术和质量标准发展,进一步提高国产长效重组蛋白药物质量水平,推进国内相关产品标准升级,推动创新长效重组蛋白药物开发及专利布局是未来几年国内该领域的发展方向。     

Scale-up of the adenovirus expression system for the production of recombinant protein in human 293S cells

Human 293S cells, a cell line adapted to suspension culture, were grown to 5×10⁶ cells/mL in batch with calcium-free DMEM. These cells, infected with new constructions of adenovirus vectors, yielded as much as 10 to 20% recombinant protein with respect to the total cellular protein content. Until recently, high specific productivity of recombinant protein was limited to low cell density infected cultures of no more than 5×10⁵ cells/mL. In this paper, we show with a model protein, Protein Tyrosine Phosphatase 1C how high product yield can be maintained at high cell densities of 2×10⁶ cells/mL by a medium replacement strategy. This allows the production of as much as 90 mg/L of active recombinant protein per culture volume. Analysis of key limiting/inhibiting medium components showed that glucose addition along with pH control can yield the same productivity as a medium replacement strategy at high cell density in calcium-free DMEM. Finally, the above results were reproduced in 3L bioreactor suspension culture thereby establishing the scalability of this expression system. The process we developed is used routinely with the same success for the production of various recombinant proteins and viruses.Abbreviations CFDMEM calcium-free DMEM - CS bovine calf serum - hpi hours post-infection - J+ enriched Joklik medium - MLP major late promoter - MOI multiplicity of infection (# of infectious viral particle/cell) - q specific consumption rate (mole/cell.h) - pfu plaque forming unit (# of infectious viral particle) - Y yield (g/E6 cells or mole/cell)     

Effects of glutamine and asparagine on recombinant antibody production using CHO‐GS cell lines

A unique and nontraditional approach using glutamine and asparagine supplements for CHO‐glutamine synthetase (GS) cell lines was studied. In our experiments, we found that a decrease in pH and an increase in cell death occurred in production phase of a GS cell line, leading to reduced antibody expression and lower antibody yields. The experimental results and the statistical analysis (ANOVA) indicated that additions of glutamine and asparagine in the basal and feed media were effective to buffer the cell culture pH, reduce lactate generation, maintain a higher cell viability profile, and improve antibody productivity. In bench‐top bioreactors, glutamine and asparagine supplementation helped to prevent cell death, improve antibody yield, and reduce base usage. Glutamine is normally excluded from culture media for GS cell lines to prevent the bypass of selection pressure. In this study, however, the addition of glutamine did not affect cell population homogeneity, protein quality, or decrease antibody yield of two GS cell lines. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1457–1468, 2014