Diversity in host clone performance within a Chinese hamster ovary cell line

Much effort has been expended to improve the capabilities of individual Chinese hamster ovary (CHO) host cell lines to synthesize recombinant therapeutic proteins (rPs). However, given the increasing variety in rP molecular types and formats it may be advantageous to employ a toolbox of CHO host cell lines in biomanufacturing. Such a toolbox would contain a panel of hosts with specific capabilities to synthesize certain molecular types at high volumetric concentrations and with the correct product quality (PQ). In this work, we examine a panel of clonally derived host cell lines isolated from CHOK1SV for the ability to manufacture two model proteins, an IgG4 monoclonal antibody (Mab) and an Fc‐fusion protein (etanercept). We show that these host cell lines vary in their relative ability to synthesize these proteins in transient and stable pool production format. Furthermore, we examined the PQ attributes of the stable pool‐produced Mab and etanercept (by N‐glycan ultra performance liquid chromatography (UPLC) and liquid chromatography ‐ tandem mass spectrometry (LC‐MS/MS), respectively), and uncovered substantial variation between the host cell lines in Mab N‐glycan micro‐heterogeneity and etanercept N and O‐linked macro‐heterogeneity. To further investigate the capabilities of these hosts to act as cell factories, we examined the glycosylation pathway gene expression profiles as well as the levels of endoplasmic reticulum (ER) and mitochondria in the untransfected hosts. We uncovered a moderate correlation between ER mass and the volumetric product concentration in transient and stable pool Mab production. This work demonstrates the utility of leveraging diversity within the CHOK1SV pool to identify new host cell lines with different performance characteristics. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1187–1200, 2015     

Analysis of dynamic changes in the proteome of a Bcl-XL overexpressing Chinese hamster ovary cell culture during exponential and stationary phases

Mammalian cell cultures used for biopharmaceutical production undergo various dynamic biological changes over time, including the transition of cells from an exponential growth phase to a stationary phase during cell culture. To better understand the dynamic aspects of cell culture, a quantitative proteomics approach was used to identify dynamic trends in protein expression over the course of a Chinese hamster ovary (CHO) cell culture for the production of a recombinant monoclonal antibody and overexpressing the antiapoptotic gene Bcl-xl. Samples were analyzed using a method incorporating iTRAQ labeling, two-dimensional LC/MS, and linear regression calculations to identify significant dynamic trends in protein abundance. Using this approach, 59 proteins were identified with significant temporal changes in expression. Pathway analysis tools were used to identify a putative network of proteins associated with cell growth and apoptosis. Among the differentially expressed proteins were molecular chaperones and isomerases, such as GRP78 and PDI, and reported cell growth markers MCM2 and MCM5. In addition, two proteins with growth-regulating properties, transglutaminase-2 and clusterin, were identified. These proteins are associated with tumor proliferation and apoptosis and were observed to be expressed at relatively high levels during stationary phase, which was confirmed by western blotting. The proteomic methodology described here provides a dynamic view of protein expression throughout a CHO fed-batch cell culture, which may be useful for further elucidating the biological processes driving mammalian cell culture performance.     

Development of a serum-free culture medium for the large scale production of recombinant protein from a Chinese hamster ovary cell line

A serum-free medium, WCM5, has been developed for the large scale propagation of CHO (Chinese hamster ovary) cells which express recombinant protein using dihydrofolate reductase as a selectable marker. WCM5 was prepared by supplementing Iscoves medium without lecithin, albumin or transferrin with a number of components which were shown to benefit growth. WCM5 medium contained 5 mg l^–1 human recombinant insulin (Nucellin) but was otherwise protein-free. CHO 3D11^* cells which had been engineered to express a humanised antibody, CAMPATH^*-1H, were routinely grown using serum-containing medium. From a seeding density of 10⁵ cells ml^–1, cells grown in static culture with serum reached a maximal cell density of 6.5×10⁵ cells ml^–1 after 6 days in culture and produced a maximal antibody concentration of 69 mg l^–1 after 11 days in culture. CHO 3D11^* cells grown with serum were washed in serum-free medium then cultured in WCM5 medium. Following a period of adaptation the cell growth and product yield was superior to that achieved with serum-containing medium. CHO cells producing CAMPATH-1H grown in an 8000 l stirred bioreactor seeded with 2×10⁵ cells ml^–1 reached a maximal viable cell density of 2.16×10⁶ cells ml^–1 after 108 h in culture and a maximal antibody concentration of 131.1 mg l^–1 after 122 h in culture.Abbreviations CHO Chinese hamster ovary - dhfr dihydrofolate reductase - dhfr dihydrofolate reductase deficient - MTX methotrexate - H hypoxanthine - T thymidine - T/V trypsin versene - F12 Hams F12 medium - NEAA non essential amino acids     

Glucose-limited chemostat culture of Chinese hamster ovary cells producing recombinant human interferon-gamma

A Chinese hamster ovary (CHO) cell line expressing recombinant human interferon-gamma (IFN-gamma) was grown under glucose limitation in a chemostate at a constant dilution rate of 0.015 h(-1) with glucose feed concentrations of 2.75 mM and 4.25 mM. The changes in cell concentration that accompanied changes in the glucose feed concentration indicated that the cells were glucose-limited. The cell yield on glucose remained constant, but there was a decline in residual glucose concentration and a reduced lactate yield from glucose in the latter stages of the culture. The consumption rates for many of the essential amino acids were increased later in the culture. The volumetric rate of interferon-gamma production was maintained throughout the course of this culture, indicating that IFN-gamma expression was stable under these conditions. However, the specific rate of IFN-gamma production was significantly lower at the higher glucose feed concentration. Under glucose limitation, the proportion of fully glycosylated IFN-gamma produced by these cells was less than that produced in the early stages of batch cultures. The proportion of fully glycosylated IFN-gamma increased during transient periods of glucose excess, suggesting that the culture environment influences the glycosylation of IFN-gamma.     

胞外唾液酸酶造成工程中国仓鼠卵巢细胞株所产人源重组促红素唾液酸含量降低

为了对工程中国仓鼠卵巢(CHO)细胞所产人源重组促红素(rhEPO)的N-糖基化特点进行考察,静置培养工程细胞后,通过等电聚焦和凝集素共沉淀对培养上清中的rhEPO进行分析,并对无血清培养上清中乳酸脱氢酶(LDH)和唾液酸酶活性进行检测,发现这株CHO细胞可以表达唾液酸含量较高的rhEPO蛋白。但是随着培养时间的延长,细胞的存活率逐渐降低,死亡的细胞将胞内的唾液酸酶释放到胞外,唾液酸酶的降解作用会造成N-糖链分枝末端的唾液酸占有率降低,导致rhEPO蛋白糖基化形态的变化。所使用的方法及得到的结果为进一步对工业过程进行分析提供了参考。     

Stable expression of recombinant human coagulation factor XIII in protein-free suspension culture of Chinese hamster ovary cells

The recombinant a and bsubunits for human coagulation factor XIII were transfected into Chinese hamster ovary (CHO) cells. CHO cells were amplified and selected with methotrexate in adherent cultures containing serum, and CHO 1-62 cells were later selected in protein-free medium. To develop a recombinant factor XIII production process in a suspension culture, we have investigated the growth characteristics of CHO cells and the maintenance of factor XIII expression in the culture medium. Suspension adaptation of CHO cells was performed in protein-free medium, GC-CHO-PI, by two methods, such as serum weaning and direct switching from serum containing media to protein-free media. Although the growth of CHO cells in suspension culture was affected initially by serum depletion, cell specific productivity of factor XIII showed only minor changes by the direct switching to protein-free medium during a suspension culture. As for the long-term stability of factor XIII, CHO 1-62 cells showed a stable expression of factor XIII in protein-free condition for 1000 h. These results indicate that the CHO 1-62cells can be adapted to express recombinant human factor XIII in a stable maimer in suspension culture using a protein-free medium. Our results demonstrate that enhanced cell growth in a continuous manner is achievable for factor XIII production in a protein-free medium when a perfusion bioreactor culture system with a spin filter is employed. This revised version was published online in August 2006 with corrections to the Cover Date.     

Cell cycle phase dependent productivity of a recombinant Chinese hamster ovary cell line

A Chinese Hamster Ovary cell line, CHO1-15₅₀₀, producing recombinant human tissue type plasminogen activator (tPA) via the dihydrofolate reductase (DHFR) amplification system, was studied in batch culture. In this system both DHFR and tPA are under the control of the strong constitutive viral SV40 early promoter. Employing the cumulative viable cell-hour approach, the specific productivity of tPA had maxima in the lag (0.065 pg cell⁻¹ h⁻¹) and early decline (0.040 pg cell⁻¹ h⁻¹) population growth phases. The viable population was assigned into four subpopulations (G1, S, G2/M phase, and Apoptotic cells) using flow cytometric analysis. As expected, intracellular DHFR was maximally expressed during the S cell cycle phase. The production of tPA, however, was found to be a direct linear function of the G1 phase, with a subpopulation specific productivity of 0.080 pg c-h⁻¹. Productivity maxima in the lag and early decline corroborate the flow cytometric data, indicative that this recombinant tPA production occurs primarily in the G1 cell cycle phase, not the S phase. This suggests that endogenous regulatory mechanisms are important controlling influences on the production of recombinant tPA in this ovarian cell line. Productivity from recombinant cell lines cannot be inferred from either the plasmid construct or the host cell alone. Elucidation of the relationship between expression of recombinant protein and the cell cycle phases of the host cell is a major component of the characterization of the animal cell production system. This information facilitates rational process design, including operating mode, modelling and control, and medium formulation.     

A new Chinese hamster ovary cell line expressing α2,6-sialyltransferase used as universal host for the production of human-like sialylated recombinant glycoproteins

Chinese hamster ovary (CHO) cells are widely employed to produce glycosylated recombinant proteins. Our group as well as others have demonstrated that the sialylation defect of CHO cells can be corrected by transfecting the α2,6-sialyltransferase (α2,6-ST) cDNA. Glycoproteins produced by such CHO cells display both α2,6- and α2,3-linked terminal sialic acid residues, similar to human glycoproteins. Here, we have established a CHO cell line stably expressing α2,6-ST, providing a universal host for further transfections of human genes. Several relevant parameters of the universal host cell line were studied, demonstrating that the α2,6-ST transgene was stably integrated into the CHO cell genome, that transgene expression was stable in the absence of selective pressure, that the recombinant sialyltransferase was correctly localized in the Golgi and, finally, that the bioreactor growth parameters of the universal host were comparable to those of the parental cell line. A second step consisted in the stable transfection into the universal host of cDNAs for human glycoproteins of therapeutic interest, i.e. interferon-γ and the tissue inhibitor of metalloproteinases-1. Interferon-γ purified from the universal host carried 40.4% α2,6- and 59.6% α2,3-sialic acid residues and showed improved pharmacokinetics in clearance studies when compared to interferon-γ produced by normal CHO cells.     

Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium

Summary A new synthetic medium (referred to as GC₃) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and selenium (1×10⁻⁷ M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium) also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading. Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium.     

Culture media optimization for Chinese hamster ovary cell growth and expression of recombinant varicella-zoster virus glycoprotein E

Herpes zoster (HZ) is caused by reactivation of varicella-zoster virus (VZV) latent in the sensory ganglia and causes severe pain, often leading to postherpetic neuralgia (PHN). Two prophylactic vaccines against HZ are currently licensed for human use, a live attenuated vaccine and a subunit vaccine containing recombinant VZV glycoprotein E (gE) as antigen. The latter has superior protective efficacy against HZ and PHN. During HZ subunit vaccine development, we obtained Chinese hamster ovary (CHO) cell clones expressing VZV gE. This study was performed to optimize culture media conditions for CHO cell growth and gE production. Using a high-throughput culture system, three CHO cell clones were cultured in microtiter plates containing 24 different basal media, and three basal media were selected. The clone with the highest gE expression was fed-batch cultured in each of the three basal media in combination with 13 different feed media. A pair of media, BalanCD CHO Growth A and EX-CELL Advanced CHO Feed 1, with the highest productivity was selected for gE production. Scale-up fed-batch cultures of the selected clone cultured in a wave bag bioreactor containing the optimized media yielded 2440 mg gE protein/L culture, a 11.5-fold increase compared to original culture conditions (batch culture in CD OptiCHO medium). The optimized media condition is used to produce VZV gE antigen for an HZ subunit vaccine, which is under phase I clinical trial. This study would provide valuable insights on culture media optimization for CHO cells expressing a recombinant vaccine antigen.

     

Efficient expression of stable recombinant human insulin-like growth factor-1 fusion with human serum albumin in Chinese hamster ovary cells

Insulin-like growth factor-1 (IGF-1) plays a crucial role in cell development, differentiation, and metabolism, and has been a potential therapeutic agent for many diseases. Chinese hamster ovary (CHO) cells are widely used for production of recombinant therapeutic proteins, but the expression level of IGF-1 in CHO cells is very low (1,500?µg/L) and the half-life of IGF-1 in blood circulation is only 4.5?min according to previous studies. Therefore, IGF-1 was fused to long-circulating serum protein human serum albumin (HSA) and expressed in CHO cells. After 8-day fed-batch culture, the expression level of HSA–IGF-1 reached 100?mg/L. The fusion protein HSA–IGF-1 was purified with a recovery of 35% using a two-step chromatographic procedure. According to bioactivity assay, the purified HSA–IGF-1 could stimulate the proliferation of NIH3T3 cells in a dose-dependent fashion and promote the cell-cycle progression. Besides this, HSA–IGF-1 could bind to IGF-1 receptor on cell membrane and activate the intracellular PI3K/AKT signaling pathway. Our study suggested that HSA fusion technology carried out in CHO cells not only provided bioactivity in HSA–IGF-1 for further research but also offered a beneficial strategy to produce other similar cytokines in CHO cells.     

Expression and purification of recombinant human angiopoietin-2 produced in Chinese hamster ovary cells

Angiopoietin-2 (Ang2) is a complex regulator of vascular remodeling that plays a role in both blood vessel sprouting and blood vessel regression through its receptor Tie2. Recombinant Chinese hamster ovary (rCHO) cell lines expressing a high level (20 microg/mL) of recombinant human Ang2 protein (rhAng2) with an amino-terminal FLAG-tag was constructed by transfecting the expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and the subsequent gene amplification in medium containing stepwise increments in methotrexate level such as 0.02, 0.08, and 0.32 microM. The rhAng2 secreted from rCHO cells was purified at a purification yield of 53.6% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng2 as a homodimeric glycoprotein form. Furthermore, rhAng2 binds to the Tie2 receptor and phosphorylates Tie2 in a concentration-dependent manner. Therefore, our rhAng2 could be useful for clarifying biological effect of exogenous Ang2 in the future.     

Biphasic culture strategy based on hyperosmotic pressure for improved humanized antibody production in Chinese hamster ovary cell culture

Hyperosmotic pressure increased specific antibody productivity (q(Ab)) of recombinant Chinese hamster ovary (rCHO) cells (SH2-0.32) and it depressed cell growth. Thus, the use of hyperosmolar medium did not increase the maximum antibody concentration substantially. To overcome this drawback, the feasibility of biphasic culture strategy was investigated. In the biphasic culture, cells were first cultivated in the standard medium with physiological osmolality (294 mOsm/kg) for cell growth. When cells reached the late exponential growth phase, the spent standard medium was replaced with the fresh hyperosmolar medium (522 mOsm/kg) for antibody production. The q(Ab) in growth phase with the standard medium was 2.1 microg per 10(6) cells/d, whereas the q(Ab) in antibody production phase with the hyperosmolar medium was 11.1 microg per 10(6) cells/d. Northern blot analysis showed a positive relationship between the relative contents of intracellular immunoglobulin messenger ribonucleic acid and q(Ab). Because of the enhanced q(Ab) and the increased cell concentration in biphasic culture, the maximum antibody concentration obtained in biphasic culture with 522 mOsm/kg medium exchange was 161% higher than that obtained in batch culture with the standard medium. Taken together, the simple biphasic culture strategy based on hyperosmotic culture is effective in improving antibody production of rCHO cells.     

Expression and purification of recombinant human Angiopoietin-1 produced in Chinese hamster ovary cells

Angiopoietin-1 (Ang1) is an essential molecule for blood vessel formation. In an effort to produce large quantities of Ang1, recombinant Chinese hamster ovary (rCHO) cells expressing a high level of recombinant human Ang1 protein (rhAng1) with an amino terminal FLAG-tag were constructed by transfecting the expression vector into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in a medium containing step-wise increments of methotrexate, such as 0.02, 0.08, and 0.32 μM. The rhAng1 secreted from rCHO cells was purified at a purification yield of 18.4% from the cultured medium using an anti-FLAG M2 agarose affinity gel. SDS-PAGE and Western blot analyses showed that rCHO cells secret rhAng1 as heterogeneous multimers. Moreover, rhAng1 expressed in rCHO cells is biologically active in vitro as demonstrated by its ability to bind to the Tie2 receptor and to phosphorylate Tie2. Therefore, the rhAng1 produced from CHO cells could be useful for clarifying the biological effects of exogenous rhAng1 in the future.     

Production of human mutant biologically active hepatocyte growth factor in Chinese hamster ovary cells

Hepatocyte growth factor (HGF) is a potent multifunctional cytokine that affects proliferation, migration, and morphogenesis of various cells. HGF is secreted as an inactive single-chain precursor protein and activated by the cleavage of serine proteases to form heterodimers. In our current study, the cleavage site of HGF was blocked by replaced Arg 494 of Glu (R494E) that resulted in the single-chain HGF (R494E) unable to be cleaved by serine proteases. We established Chinese hamster ovary (CHO) cells overexpressing HGF (R494E), the expression of HGF (R494E) achieved 12?mg/L and was similar to a previously reported study. The recombinant protein was then purified from culture medium using a two-step chromatographic procedure that resulted in about a 40% recovery rate. The purified HGF (R494E) was obtained as a single-chain active protein. It concluded that HGF (R494E) exhibited a biologically active protein and the overexpressing CHO cell line supplied sufficient material for future studies. The R494E replacement of the cleavage site would be beneficial to the utility of other similar therapeutic proteins.