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.     

表达抗-CD25单克隆抗体的GS-NS0骨髓瘤细胞无血清培养及代谢特性

抗-CD25单克隆抗体作为免疫抑制剂拥有广阔的市场前景和巨大的经济价值。本实验以表达抗?CD25单克隆抗体的GS-NS0细胞为研究对象,开发了支持其大规模培养和抗体表达的无血清低蛋白培养基,批培养最大活细胞密度和最大抗体浓度分别达3×106cells/mL和300mg/L以上,比商业无血清培养基(Excell 620+0.2% primatone)分别提高了100%和46%。通过批培养实验,研究了细胞的生长、葡萄糖和氨基酸代谢、以及产物表达特点,并揭示了批培养过程中初始葡萄糖浓度对GS-NS0细胞生长与代谢的影响规律。为优化GS-NS0细胞培养过程和抗CD25单抗成功迈向产业化提供了重要的科学依据。     

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.     

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.     

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.     

Application of a nonradioactive method of measuring protein synthesis in industrially relevant chinese hamster ovary cells

Due to the high medical and commercial value of recombinant proteins for clinical and diagnostic purposes, the protein synthesis machinery of mammalian host cells is the subject of extensive research by the biopharmaceutical industry. RNA translation and protein synthesis are steps that may determine the extent of growth and productivity of host cells. To address the problems of utilization of current radioisotope methods with proprietary media, we have focused on the application of an alternative method of measuring protein synthesis in recombinant Chinese hamster ovary (CHO) cells. This method employs puromycin as a nonradioactive label which incorporates into nascent polypeptide chains and is detectable by western blotting. This method, which is referred to as SUnSET, successfully demonstrated the expected changes in protein synthesis in conditions that inhibit and restore translation activity and was reproducibly quantifiable. The study of the effects of feed and sodium butyrate addition on protein synthesis by SUnSET revealed an increase following 1 h feed supplementation while a high concentration of sodium butyrate was able to decrease translation during the same treatment period. Finally, SUnSET was used to compare protein synthesis activity during batch culture of the CHO cell line in relation to growth. The results indicate that as the cells approached the end of batch culture, the global rate of protein synthesis declined in parallel with the decreasing growth rate. In conclusion, this method can be used as a “snapshot” to directly monitor the effects of different culture conditions and treatments on translation in recombinant host cells. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1043–1049, 2013     

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.     

Responses of GS-NS0 Myeloma cells to osmolality: Cell growth, intracellular mass metabolism, energy metabolism, and antibody production

The influence of osmolality on growth, metabolism, and antibody production of mammalian cells has been widely reported in the past. However, more information about the responses of GS-NS0 Myeloma cells to osmolality, especially regarding the intracellular mass and energy metabolism, has not been available in detail. Fed-batch cultures started at different osmolalities in the range of 280∼370 mOsm/kg were designed to investigate the effects. As the osmolality and cell status changed during the process, cell performance was evaluated in the comparable periods with similar growth rates, nutrition concentrations, and relatively consistent environments. Metabolic flux analysis indicated most of extra consumed glucose at higher osmolalities flowed into lactate formation pathway. The proportion of glucose flux flowed into glycolysis pathway remained approximately 90% and the need of glucose for biomass synthesis was constantly. Also, more than 88% of the glutamine was used in biomass synthesis and the absolute flux remained constant. The specific consumption rate of glutamine declined significantly when cells were cultured in hypo-osmolality (276 mOsm/kg) and a portion of glutamine was synthesized from glutamate. Furthermore, cells were in the state of high energy production at osmolality of 276 mOsm/kg. More glucose flowed into TCA circle with the high efficiency of energy production to meet the demand. Thus, the IVC, the specific antibody production rate, and maximal antibody concentration in fed-batch culture started at 280 mOsm/kg decreased by 35, 36, and 48% compared to those in the culture started at 330 mOsm/kg.     

Assembly and plasma membrane targeting of recombinant immunoglobulin chains in plants with a murine immunoglobulin transmembrane sequence

The cDNA encoding a full-length murine immunoglobulin 1 heavy chain with its native leader sequence, transmembrane and intracellular domains was introduced into transgenic plants. Transformed plants expressed the recombinant polypeptide, but, in contrast to plants expressing the heavy chain without transmembrane sequence, the protein appeared to be associated with a plant cell membrane. Extraction of the membrane-associated heavy chain required the presence of a non-ionic detergent, and immunofluorescence studies of protoplasts demonstrated surface expression of membrane Ig heavy chain on up to 40% of the cells from a transgenic leaf. In plants expressing both the membrane Ig heavy chain and its partner light chain, functional antibody was also localised to the plant cell membrane and retention of the heavy chain at this site appeared to have no effect on the efficiency of antibody assembly. This approach of localising and accumulating recombinant antibody in cell membranes may have a number of applications, including passive immunisation against plant pathogens.     

Glycosylation causes an apparent block in translation of immunoglobulin heavy chain

Analysis of nascent heavy chains isolated from MPC11 (gamma 2b heavy chains) and MOPC 21 (gamma 1 heavy chains) mouse myeloma cells demonstrates an accumulation of nascent heavy chains which are slightly smaller in mass (approximately 35,000 daltons) than nascent heavy chains which have just been glycosylated (approximately 38,000 daltons). The accumulation of 35,000-dalton nascent heavy chain appears to be a consequence of the glycosylation process since tunicamycin, an inhibitor of glycosylation, abolishes the apparent translational block manifested by the accumulation of 35,000-dalton nascent chains. Tunicamycin also causes a 15 to 25% increase n the relative rate of synthesis of heavy chain compared to the corresponding rate of synthesis of the nonglycosylated light chain synthesized by the same cell. These results suggest that the translation block, caused by the glycosylation process, of heavy chain synthesis contributes to the imbalance of heavy chain and light chain biosynthesis observed in malignant and normal lymphoid cells.     

Kinetic analysis of hybridoma growth and monoclonal antibody production in semicontinuous culture

Hybridoma I.13.17 was grown in semicontinuous culture in an attempt to investigate the steady-state concentrations of key components of monoclonal antibody (MAb) synthesis (e.g., intracellular MAb, IgG messenger RNAs) at different dilution rates between 0.008 and 0.055 h(-1). There was a general trend of increasing steady-state levels of total cytoplasmic RNA, total cell-associated MAb or cytoplasmic MAb, DNA synthesis rate, cellular metabolic activity, heavy (H-) and light (L-) chain IgG mRNAs with the increase in dilution rates. Increase in the half-lives of H- and L-chain mRNAs with increase in dilution rates may be sufficient to account for their increasing levels found under the same conditions. The specific growth rate was profoundly affected by the dilution rate, particularly near the lower end of the dilution rate range. Linear relationships were observed between the steady-state amounts of total cell-associated MAb and the relative levels of H- and L-chain mRNAs. Material balances on intracellular MAb demonstrated an increasing percentage of antibody not released into the growth medium (e.g., stored within the cell or anchored to the cell membrane) with increasing dilution rate. The MAb production rate per cell decreased significantly with the increase in dilution rates. No correlation was found between the relative levels of H- or L-chain mRNAs and the specific MAb production rate. Possible implications of rate-limiting steps in MAb synthesis and secretion are discussed.