mRNA stability and antibody production in CHO cells: Improvement through gene optimization

The productivity of stably transfected cell lines is of critical importance for the manufacturing of therapeutic proteins. Various methods have been successfully implemented to increase the production output of mammalian cell cultures. Increasing evidence suggests that optimization of the gene coding sequences of an expression vector can improve specific cell line yield of the recombinant protein. Here we demonstrate that gene optimization substantially enhances antibody production in Chinese hamster ovary cells. When gene optimization was applied to the heavy and light chain genes of a therapeutic antibody, we observed increased antibody production in transient transfection. Elevated heavy chain mRNA level was associated with the increase of antibody production. Further analysis suggested that the increased antibody expression is attributable to enhanced mRNA stability resulting from gene optimization. Gene optimization also led to increased antibody production in stable clones.     

The effect of dissolved oxygen on the production and the glycosylation profile of recombinant human erythropoietin produced from CHO cells

Human recombinant erythropoietin (rHuEPO) was produced from Chinese hamster ovary (CHO) cells transfected with the human EPO gene. The cells were grown in batch cultures in controlled bioreactors in which the set-points for dissolved oxygen varied between 3% and 200%. The cell-specific growth rate and final cell yield was significantly lower under hyperoxic conditions (200% DO). However, there was no significant difference in growth rates at other oxygen levels compared to control cultures run under a normoxic condition (50% DO). The specific productivity of EPO was significantly lower at a DO set-point of 3% and 200% but maintained a consistently high value between 10% to 100% DO. The EPO produced under all conditions as analyzed by two-dimensional electrophoresis showed a molecular weight range of 33 to 37 kDa and a low isoelectric point range of 3.5 to 5.0. This corresponds to a highly glycosylated and sialylated protein with a profile showing at least seven distinct isoforms. The glycan pattern of isolated samples of EPO was analyzed by weak anion exchange (WAX) HPLC and by normal-phase HPLC incorporating sequential digestion with exoglycosidase arrays. Assigned structures were confirmed by mass spectrometry (MALDI-MS). The most prominent glycan structures were core fucosylated tetranntenary with variable sialylation. However, significant biantennary, triantennary, and non-fucosylated glycans were also identified. Detailed analysis of these glycan structures produced under variable dissolved oxygen levels did not show consistently significant variations except for the ratio of fucosylated to non-fucosylated isoforms. Maximum core fucosylation (80%) was observed at 50% and 100% DO, whereas higher or lower DO levels resulted in reduced fucosylation. This observation of lower fucosylation at high or low DO levels is consistent with previous data reported for glycoprotein production in insect cells.     

The effect of osmolarity on metabolism and morphology in adhesion and suspension chinese hamster ovary cells producing tissue plasminogen activator

The effects of constant osmolarity, between 300 and500 mOsm/kg, on the metabolism of Chinese HamsterOvary (CHO) cells producing tissue plasminogenactivator (tPA) were compared between adhesion andsuspension cultures. In both suspension and adhesionculture, the specific rates of glucose consumption(_G), lactate production (q_L), and tPAproduction (q_tPA) increased as osmolarityincreased, while these rates decreased when osmolaritywas higher than the respective critical levels. However, specific growth rate () decreased withincrease in osmolarity and this slope grew steeper inthe osmolarity range higher than the critical level. The decrease in in the adhesion culture was morerapid than that in the suspension culture. Thecritical osmolarity for adhesion culture (400 mOsm/kg)was lower than that for suspension culture (450 mOsm/kg). These results indicated that the adhesionculture was more sensitive to increase of osmolaritythan the suspension culture, while the specific ratesobtained from the adhesion cultures were in general1.5- to 3-fold higher than those obtained from thesuspension cultures. Cell volume increased asosmolarity increased in both the suspension andadhesion cultures, as reported previously forsuspension culture of hybridoma cells, but there wasno morphological change in the suspension culture. Incontrast, cell height decreased and cell adhesion areamarkedly increased as osmolarity increased in theadhesion culture. This morphological change inadhesion cultures may be one reason for the highersensitivity of adherent cells to the increase ofosmolarity than suspended cells.     

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ – a study of an industrial mAb producing CHO‐S cell line

There are few studies defining CHO host cell proteins (HCPs) and the flux of these throughout a downstream purification process. Here we have applied quantitative iTRAQ proteomics to follow the HCP profile of an antibody (mAb) producing CHO‐S cell line throughout a standard downstream purification procedure consisting of a Protein A, cation and anion exchange process. We used both 6 sample iTRAQ experiment to analyze technical replicates of three samples, which were culture harvest (HCCF), Protein A flow through and Protein A eluate and an 8 sample format to analyze technical replicates of four sample types; HCCF compared to Protein A eluate and subsequent cation and anion exchange purification. In the 6 sample iTRAQ experiment, 8781 spectra were confidently matched to peptides from 819 proteins (including the mAb chains). Across both the 6 and 8 sample experiments 936 proteins were identified. In the 8 sample comparison, 4187 spectra were confidently matched to peptides from 219 proteins. We then used the iTRAQ data to enable estimation of the relative change of individual proteins across the purification steps. These data provide the basis for application of iTRAQ for process development based upon knowledge of critical HCPs.     

The gel microdrop secretion assay: Identification of a low productivity subpopulation arising during the production of human antibody in CHO cells

The long-term stability of high-level expression is the mostimportant factor to consider when choosing cell lines for the expression of recombinant proteins. Declining volumetricyields in large-scale fermentation can be caused by changes affecting the cell population as a whole such as loss in viability, depletion of nutrients or accumulation of metabolites affecting cell growth. Alternatively, geneticinstability may lead to the outgrowth of a less productive,metabolically favored sub-population. Currently a variety ofparameters are measured to monitor the condition of cells infermenters including glucose uptake, lactate accumulation andoxygen consumption; in addition, periodic viable cell countsallow the determination of the growth rate and viability of the population. All of these methods measure the condition ofthe cell population as a whole and changes must involve a significantly large proportion of the total culture in orderto be detectable. Here we report on a method that allows theevaluation of the productivity of individual cells. Using the gel microdrop secretion assay, we detected the appearance ofa sub-population of cells with lower productivity. Subsequentanalysis of the culture confirmed the existence of lower productivity cells with a lower vector copy number. Therefore,the single cell secretion assay proved to be a rapid method todetect and isolate a low productivity variant of the producer cell line.     

Overexpression of mutant cell division cycle 25 homolog B (CDC25B) enhances the efficiency of selection in Chinese hamster ovary cells

The effects of mutant cell division cycle 25 homolog B (CDC25B) overexpression on the generation of cells producing a monoclonal antibody were investigated in Chinese hamster ovary (CHO) cells. Mutant CDC25B (m-CDC25B) expression plasmids were transfected into CHO DG44-derived cells producing a monoclonal antibody, and the frequency of highly producing cells was assessed following gene amplification in the presence of 250 nM methotrexate. Most of the clones obtained from the m-CDC25B-overexpressing cells had higher antibody titers than did mock-transfected control cells. This arose from either higher transgene copy numbers or higher mRNA expression levels for the antibody. However, the high mRNA expression levels were not always accompanied by increases in transgene copy numbers. Our results suggest that cells producing high levels of a monoclonal antibody can be selected efficiently using m-CDC25B overexpression.     

An optimized method for extraction and quantification of nucleotides and nucleotide sugars from mammalian cells

Glycosylation is a critical attribute of therapeutic proteins given its impact on the clinical safety and efficacy of these molecules. The biochemical process of glycosylation is inextricably dependent on metabolism and ensuing availability of nucleotides and nucleotide sugars (NSs) during cell culture. Herein, we present a comprehensive methodology to extract and quantify these metabolites from cultured cells. To establish the full protocol, two methods for the extraction of these compounds were evaluated for efficiency, and the requirement for quenching and washing the sample was assessed. A chromatographic method based on anion exchange has been optimized to separate and quantify eight nucleotides and nine NSs in less than 30 min. Degradation of nucleotides and NSs under extraction conditions was evaluated to aid in selection of the most efficient extraction protocol. We conclude that the optimized chromatographic method is quick, robust, and sensitive for quantifying nucleotides and NSs. Furthermore, our results show that samples taken from cell culture should be treated with 50% v/v acetonitrile and do not require quenching or washing for reliable extraction of nucleotides and NSs. This comprehensive protocol should prove useful in determining the impact of nucleotide and NS metabolism on protein glycosylation.     

Transient transfection of mammalian cells with DNA of the plant-pathogenic Ti-plasmid and expression of marker and resident sequences

Summary The large Ti-plasmid from Agrobacterium tumefaciens strain C58 has been used for transfection experiments with mammalian cells. In DNA from Tupaia baby fibroblasts Ti-plasmid sequences could be identified by filter hybridization as long as four weeks after transfection including two cell passages. The hybridization signals decreased rapidly after addition of the Ti-plasmid DNA-coprecipitate to the cells. The signals were often not detected any more after the first day, but were visible one week after transfection. Nuclei prepared from Ti-plasmid-transfected cells hybridized to pTi-specific RNA. With the chloramphenicol acetyl transferase-gene as marker no discrimination in DNA uptake was found between the Ti-plasmid and much smaller plasmids. According to the number of nuclei with homology to pTi-sequences it is assumed that about 0.2% of the cells carry Ti-plasmid DNA in the nucleus. Analysis of RNA isolated from cells transfected with cloned segments of the Ti-plasmid revealed that the TDNA region of the Ti-plasmid was predominantly transcribed.Abbreviations CAT Chloramphenicol Acetyl Transferase - NPT Neomycin Phosphotransferase - SDS Sodium Dodecylsulfate - TK Thymidine Kinase     

The effect of UDP-glucuronosyltransferase 1A1 expression on the mutagenicity and metabolism of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine in CHO cells

UDP-glucuronosyltransferase proteins (UGT) catalyze the glucuronidation of both endogenous and xenobiotic compounds. In previous studies, UGT1A1 has been implicated in the detoxification of certain food-borne carcinogenic-heterocyclic amines. To determine the importance of UDP-glucuronosyltransferase 1A1 (UGT1A1) in the biotransformation of the cooked-food carcinogen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), genetically modified CHO cells that are nucleotide excision repair-deficient, and express cytochrome P4501A2 (UV5P3 cell line) were transfected with a cDNA plasmid of human UGT1A1 to establish the UDP-glucuronosyltransferase 1A1 expressing 5P3hUGT1A1 cell line. Expression of the UGT1A1 gene was verified by screening neo gene expressing clonal isolates (G-418 resistant) for their sensitivity to cell killing from PhIP exposure. Five of 11 clones were chosen for further analysis due to their resistance to cell killing. Western blot analysis was used to confirm the presence of the UGT1A1 and CYP1A2 proteins. All five clones displayed a 52-kDa protein band, which corresponded to a UGT1A1 control protein. Only four of the clones had a protein band that corresponded to the CYP1A2 control protein. Correct fragment size of the cDNAs in the remaining four clones was confirmed by RT-PCR and quantification of the mRNA product was accomplished by real-time RT-PCR. Expression of UGT1A1 in the transfected cells was 10⁴–10⁵-fold higher relative to the UV5P3 parental cells. One clone (#14) had a 10-fold higher increase in expression at 1.47 × 10⁵ over the other three clones. This clone was also the most active in converting N-hydroxy-PhIP to N-hydroxy-PhIP glucuronide conjugates in microsomal metabolism assays. Based on the D₅₀ values, the cytotoxic effect of PhIP was decreased 350-fold in the 5P3hUGT1A1 cells compared to the UV5P3 control cells. In addition, no significant increase in mutation frequency was observed in the transfected cells. These results clearly indicate that UGT1A1 plays a critical role in PhIP biotransformation, providing protection against PhIP-mediated cytotoxicity and mutagenicity.     

Use of a Plackett-Burman statistical design to determine the effect of selected amino acids on monoclonal antibody production in CHO cells

Culture media design is central to the optimization of monoclonal antibody (mAb) production. Although general strategies do not currently exist for optimization of culture media, the combined use of statistical design and analysis of experiments and strategies based on simple material balances can facilitate culture media design. In this study, we evaluate the effect of selected amino acids on the growth rate and monoclonal antibody production of a Chinese hamster ovary DG-44 (CHO-DG44) cell line. These amino acids were selected based on their relative mass fraction in the specific mAb produced in this study, their consumption rate during bioreactor experiments, and also through a literature review. A Plackett-Burman statistical design was conducted to minimize the number of experiments needed to obtain statistically relevant information. The effect of this set of amino acids was evaluated during exponential cell culture (considering viable cell concentration and the specific growth rate as main output variables) and during the high cell-density stage (considering mAb final concentration and specific productivity as relevant output variables). For this particular cell line, leucine (Leu) and arginine (Arg) had the highest negative and positive effects on cell viability, respectively; Leu and threonine (Thr) had the highest negative effect on growth rate, and valine (Val) and Arg demonstrated the highest positive impact on mAb final concentration. Results suggest the pertinence of a two-stage strategy for amino acid supplementation, with a mixture optimized for cell growth and a different amino acid mixture for mAb production at high density.     

Induction and expression of mutations in mammalian cells in the absence of DNA synthesis and cell division

The induction of mutations by the alkylating agent ethyl methanesulfonate (EMS) was determined with Chinese hamster ovary cells maintained in serum-free medium to arrest DNA synthesis and cell division. The arrested cultures were treated with EMS and maintained in serum-free medium for various time intervals post-treatment before serum containing medium was added to initiate DNA synthesis and cell division. The concentration-dependent increase in 6-thioguanine-resistant mutants in the arrested cultures was similar to that found with exponentially dividing cultures when serum was added to the arrested cultures immediately after the EMS treatment; the time course of phenotypic expression was also similar with both cultures. In addition, maintenance of the arrested cultures in serum-free medium for up to 18 days post-treatment resulted in no change in the mutant frequency. This suggests that the mutagenic damage is not removed in these arrested cultures. Furthermore, maintenance of the arrested state for increasing time intervals before serum addition results in decreases in the time necessary for maximum phenotypic expression. Cultures maintained in serum-free medium for 16 days after mutation treatment show complete expression of the mutations with no need for subculture. This last result suggests that the mutagenic damage induced by EMS in Chinese hamster ovary cells is not removed and that this damage results in both the induction and expression of mutation in the absence of DNA replication.     

The effect of ammonia on the O-linked glycosylation of granulocyte colony-stimulating factor produced by chinese hamster ovary cells

Ammonium ion concentrations ranging from 0 to 10 mM are shown to significantly reduce the sialylation of granuiocyte colony-stimulating factor (G-CSF) produced by recombinant Chinese hamster ovary cells. Specifically, the degree of completion of the final reaction in the O-linked glycosylation pathway, the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine, is reduced by NH(4) (+) concentrations of as low as 2 mM. The effect of ammonia on sialylation is rapid, sustained, and does not affect the secretion rate of G-CSF. Additionally, the effect can be mimicked using the weak base chloroquine, suggesting that the effect is related to the weak base characteristics of ammonia. In support of this hypothesis, experiments using brefeldin A suggest that the addition of sialic acid in an alpha(2,6) linkage to N-acetylgalactosamine occurs in the trans-Golgi compartment prior to the trans-Golgi network, which would be expected under normal conditions to have a slightly acidic pH in the range from 6.5 to 6.75. Ammonium ion concentrations of 10 mM would be expected to reduce significantly the differences in pH between acidic intracellular compartments and the cytoplasm. The pH-activity profile for the CHO O-linked alpha(2,6) sialytransferase using monosialylated G-CSF as a substrate reveals a twofold decrease in enzymatic activity across the pH range from 6.75 to 7.0.Mathematical modeling of this sialylation reaction supports the hypothesis that this twofold decrease in sialyltransferase activity resulting from an ammoniainduced increase in trans-Golgi pH could produce the observed decrease in G-CSF sialylation. (c) 1995 John Wiley & Sons, Inc.     

Differential effect of exocytic SNAREs on the production of recombinant proteins in mammalian cells

Mammalian cells play a dominant role in the industrial production of biopharmaceutical proteins. However, the productivity of producer cells is often hindered by a bottleneck in the saturated secretory pathway, where a sophisticated mechanism of vesicle trafficking is mediated by numerous proteins and their complexes, among which are the cross‐kingdom conserved SNAREs [soluble NSF (N‐ethylmaleimide‐sensitive factor) receptor]. The SNAREs assemble into complexes by means of four interactive α‐helices and, thus, trigger the fusion of transport vesicles with the respective target membranes. We report that the transgenic expression of exocytic SNAREs, which control the fusion of secretory vesicles to the plasma membrane, differentially impacts the secretory capacity of HEK‐293, HeLa, and CHO‐K1 cells. While other exocytic SNAREs have no effect or a negative effect, SNAP‐23 [synaptosome‐associated protein of 23 kDa] and VAMP8 [vesicle‐associated membrane protein 8] specifically increase the production of recombinant proteins when they are ectopically and stably expressed in mammalian cells. The targeted and effective intervention in the secretory capacity of SNARE proteins is a novel engineering strategy, which could lead to the development of new therapies by increasing the production of biopharmaceutical proteins or by boosting the secretion of cell implants in cell therapy initiatives. Biotechnol. Bioeng. 2011; 108:611–620. © 2010 Wiley Periodicals, Inc.     

Effect of cloned gene dosage on cell growth and hepatitis B surface antigen synthesis and secretion in recombinant CHO cells

The effect of cloned gene copy number on growth and product formation has been studied in sufficient detail using a Chinese hamster overy (CHO) cell line producing recombinant hepatits B surface antigen (HbsAg). Batch culture experiments were carried out in T flasks in order to characterize cell growth and HbsAg secretion in various clones carrying different numbers of HbsAg gene copies integrated into CHO cell chromosomes. Specific growth rates were found to decrease with increasing gane copy number. Secreted HbsAg concentration and specific HbsAg secretion rates were found to increase with increase in gene copy number. Gene copy numbers in each clone determined using Southern hybridizations were positively correlated with intracellular dihydrofolate reductase (dhfr) content using a flow cytometric assay. The mRNA levels quantitated using Northern hybridization followed by autroadiography and densitometry also gave the same trends. The flow cytometry experiments show that while parental cells were quite homogeneous with respect to intracellular dhfr content, the amplified clones exhibit a great deal of heterogeneity in dhfr content. Pulse-chase experiments show that the efficiency of HbsAg secretion (defined here as the fraction of initially labeled HbsAg that is secreted into the extracellular medium at the end of a 23.5-h chase) decreases and also that the intracellular HbsAg degradation increases with increasing gene copy number.