Characterization of chimeric antibody producing CHO cells in the course of dihydrofolate reductase-mediated gene amplification and their stability in the absence of selective pressure

Recombinant Chinese hamster ovary (CHO) cells expressing a high-level of chimeric antibody against S surface antigen of hepatitis B virus were obtained by co-transfection of heavy and light chain cDNA expression vectors into dihydrofolate reductase (dhfr)-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level such as 0.02, 0.08, 0.32, 1.0, and 4.0 microM. The highest producer (HP) subclone was isolated from each MTX level and was characterized with respect to cell growth and antibody production in the corresponding level of MTX. The specific growth rate of the HP subclone was inversely proportional to the MTX level. On the other hand, its specific antibody productivity (qAb) rapidly increased with increasing MTX level up to 0.08 microM, and thereafter, it gradually increased to 20 microg/10(6) cells/day at 4 microM MTX. Southern blot analysis showed that the enhanced qAb at higher MTX level resulted from immunoglobulin (Ig) gene amplification. The stability of the HP subclones isolated at 0.02, 0.08, 0.32, and 1.0 microM MTX in regard to antibody production was investigated during long-term culture in the absence of MTX. The qAb of all subclones significantly decreased during the culture. However, the relative extent of decrease in qAb was variable among the subclones. The HP subclone isolated at 1 microM MTX was most stable and could retain 59% of the initial qAb after 80 days of cultivation. Southern blot analysis showed that this decrease in qAb of the subclones resulted mainly from the loss of Ig gene copies during long-term culture. Despite the decreased qAb, the HP subclone isolated at 1 microM MTX could maintain high volumetric antibody productivity over three months because of improved cell growth rate during long-term culture.     

t-PA cDNA在CHO细胞中的高效稳定表达

我们曾报道t-PA mRNA非翻译区序列对其表达有明显的抑制作用,在此基础上,通过对5′-UTR及3′-UTR的改造,使t-PA在COS-7细胞中的表达水平提高30倍左右。将t-PA表达质粒用电击法转染中国仓鼠卵巢细胞二氢叶酸还原酶缺陷株(CHO-dhfr),经过混合加压及筛选,在CHO细胞中高效表达了t-PA,表达水平达到5000～6000 IU/10~6细胞/24hr。重组t-PA具有与天然t-PA相同的分子量及酶活性。经过8个月连续传代,表达水平未下降,表明细胞株是稳定的,其主要指标均符合工程细胞株的要求。     

Clonal variability within dihydrofolate reductase-mediated gene amplified Chinese hamster ovary cells: stability in the absence of selective pressure

Recombinant Chinese hamster ovary (rCHO) cells expressing a high level of chimeric antibody were obtained by cotransfection of heavy- and light-chain cDNA expression vectors into dihydrofolate reductase-deficient CHO cells and subsequent gene amplification in medium containing stepwise increments in methotrexate (MTX) level up to 1.0 microM. To determine the clonal variability within the amplified cell population in regard to antibody production stability, 20 subclones were randomly isolated from the amplified cell population at 1.0 microM MTX (CS13-1.0 cells). Clonal analysis showed that CS13-1.0 cells were heterogeneous with regard to specific growth rate (mu) and specific antibody productivity (qAb), although they were derived from a single clone. The mu and qAb of 20 subclones were in the range of 0.51 to 0.72 day-1 and 10.9 to 19.1 microgram/10(6) cells/day, respectively. During 8 weeks of cultivation in the absence of selective pressure, the mu of most subclones did not change significantly. On the other hand, their qAb decreased significantly. Furthermore, the relative decrease in qAb varied among subclones, ranging from 30% to 80%. Southern and Northern blot analyses showed that this decreased qAb resulted mainly from the loss of amplified immunoglobulin (Ig) gene copies and their respective cytoplasmic mRNAs. For the sake of screening convenience, an attempted was made to correlate the initial properties of subclones (such as mu, qAb, and Ig gene copies) with their antibody production stability during long-term culture. Among these initial properties examined, only qAb of subclones could help to predict their stability to some extent. The subclones with high qAb were relatively stable with regard to antibody production during long-term culture in the absence of selective pressure (P < 0. 005, ANOVA). Taken together, the clonal heterogeneity in an amplified CHO cell population necessitates clonal analysis for screening stable clones with high qAb.     

SV40载体在COS7细胞中的复制及外源基因的表达

为评价SV4 0载体 COS7稳定表达系统的可能应用价值 ,将含人组织型纤溶酶原激活剂 (t PA)编码区的SV4 0载体pctPA转染COS7细胞 ,并以G4 18选择培养 2 8d ,得到稳定的抗性细胞库 .Southern印迹和溶圈法分别分析该细胞库在随后的细胞传代中细胞内附加体拷贝数及t PA表达水平的变化 .在经选择培养 2 8d的COS7细胞库中 ,质粒pctPA以染色体外附加体的形式存在 (30 0拷贝 细胞 ) ,t PA的表达水平为 1 1μg d(10 6细胞 ) .在随后 3个月的动态观察中 ,随着细胞传代 ,该COS7细胞库tPA的表达水平虽逐渐递减 ,但在第 1个月内可保持在 1μg d(10 6细胞 )以上 .基于SV4 0载体 COS7稳定表达系统无需筛选克隆 ,在稳定的抗性细胞库形成后的 1个月内可能保持目的基因的高水平表达 ,因而较适合于需同时制备多种重组蛋白的实验 .     

Cell cycle-dependent DHFR and t-PA production in cotransfected, MTX-amplified CHO cells revealed by dual-laser flow cytometry

The cell cycle-dependent regulation of the cellular dihydrofolate reductase content (DHFR) and tissue plasminogen activator (t-PA) production and secretion in plasmid-amplified cells was investigated in the DHFR-negative CHO cells transfected with the plasmid pSV-tPA.dhfr. This plasmid, carrying the dhfr and t-PA gene under control of different promotors, was amplified by serial passages in 5 microM methotrexate (MTX) for dhfr gene amplification. The intracellular amount of DHFR was quantitated in viable cells by MTX-FITC labeling and flow cytometric analysis of the FITC fluorescence. In comparison with the original CHO cells, the pSVtPA.dhfr-amplified cells showed a greater than 230-fold increase in MTX-FITC fluorescence. Using dual laser flow cytometry (uv: vital cell cycle with Hoechst 33342; 488 nm: DHFR with MTX-FITC), we show a maximum increase in the intracellular DHFR content during G1 and/or at G1/S transition (100 to 157%), followed by a continuous increase to 200% during S and G2/M. To determine t-PA production CHO cells were sorted from G1-, early/late S-, and G2/M-phase. After 1-, 2-, and 4-h incubation periods, t-PA production was quantitated using a sensitive t-PA ELISA technique. We found that t-PA production and secretion (2-h assay), unlike the expression of DHFR, increased continuously from relatively 100% in G1 to 127% in early S and reached its maximum of 159% in late S, whereas in G2/M-phase it decreased to 118%. Our results show that in pSVtPA.dhfr-coamplified CHO cells gene products DHFR and t-PA both exhibit different cell cycle-correlated accumulation and secretion, respectively, indicating that the brightest MTX-FITC-positive cells (G2/M) do not display the highest t-PA secretion rate.     

Expression of erythroid differentiation factor (EDF) in Chinese hamster ovary cells

Plasmid DNA containing EDF subunit cDNA and mouse dihydrofolate reductase (DHFR) cDNA was transfected into CHO DHFR- cells by the calcium-phosphate method. DHFR positive transformants secreted recombinant EDF (r-EDF) constitutively in an active form and accumulated it in the conditioned medium. Furthermore, cells which were resistant to methotrexate (MTX : 0.5 microM) secreted r-EDF up to 1 microgram/ml. r-EDF was identical to natural EDF (n-EDF) produced by human acute monocytic leukemia cell line, THP-1, as regards its dimeric structure and a biological activity.     

Tissue plasminogen activator (t-PA) production by a high density culture of weakly adherent human embryonic kidney cells using a polyurethane-foam packed-bed culture system

Weakly adherent cells of the 293 line attached well to the internal surface of polyurethane foam (PUF) and grew to the high density of 6.83 × 10⁷ cells/cm³ PUF in stationary culture. The maximum productivity of tissue plasminogen activator (t-PA) was 0.158 IU/10⁶ cells per day. The productivity decreased at the stationary phase of cell growth, so we designed a PUF-plate packed-bed culture system for high density culture and continuous production of t-PA. A maximal cell density of 3.24 × 10⁷ cells/cm³ PUF and a t-PA productivity of 0.326 IU/10⁶cells per day were obtained in 25-day perfusion cultures. Although the cell density decreased to half that in PUF stationary culture, the t-PA productivity increased twofold and was maintained for 25 culture days.     

Increased t-PA yields using ultrafiltration of an inhibitory product from CHO fed-batch culture

Fed-batch operation for the production of t-PA using Chinese Hamster Ovary (CHO) cells was optimized using serial and parallel experimentation. The feed, an isotonic concentrate, was improved to obtain 2- to 2.5-fold increases in integrated viable cell days versus batch. With a low glucose inoculum train, the viability index was further increased up to 4.5-fold. Hydrolysates were substituted for the amino acid portion of the concentrate with no significant change in fed-batch results. The concentrate addition rate was based on a constant 4 pmol/cell.day glucose uptake rate that maintained a relatively constant glucose concentration (approximately 3 mM). Increased viable cell indices did not lead to concomitant increases in t-PA concentrations compared to batch. The fed-batch concentrate and feeding strategy were shown to be effective in hybridoma culture, where a 4-fold increase in viable cell index yielded a 4-fold increase in antibody concentration. The half-life of t-PA decreased from 43 to 15 days with decreasing cell viability (from 92% to 71%), but this was not sufficient to explain the apparent t-PA threshold. Instead, the CHO results were explained by a reduction in t-PA production at higher extracellular t-PA concentrations that limited the fed-batch maximum at 35 mg/L for the cell line investigated. Analysis of both the total and t-PA mRNA levels revealed no response to increasing extracellular t-PA concentrations upon exogenous additions. Instead, intracellular t-PA levels were increased, revealing a possible secretory pathway limitation. A new reactor configuration was developed using an acoustic filter to retain the cells in the reactor while an ultrafiltration module stripped t-PA from the clarified medium before the permeate was returned to the reactor. By adding this harvesting step, the t-PA fed-batch production was increased over 2-fold, up to a yield of 80 mg/L.     

Generation of recombinant CHO(dhfr-) cell lines by single selection for dhfr+ transformants.

In order to establish a mammalian cell expression system with a minimum of selection steps and a stable expression of microgram amounts of recombinant protein (human tissue-type plasminogen activator mutants and chimeric proteins) per 10(6) cells per day, we investigated Chinese hamster ovary cells and the dihydrofolate reductase-deficient Chinese hamster ovary cell line CHO(dhfr-). The 1tPA expression vector pCMVtPA was cotransfected either with the SV40 enhancer sequence containing dhfr expression vector pMT2 or with the enhancerless dhfr expression vector pAdD26SV(A) into CHO(dhfr-) cells. With both dhfr expression plasmids, selection for dhfr+ transformants followed by single dilution cloning was sufficient to generate cell lines with a production level of up to 4.6 micrograms tPA/10(6) cells.day. This approach is useful if gene amplification procedures are time-consuming and impracticable because of a large number of recombinant proteins. In order to establish CHO cell lines with a tPA expression level as high as that in the case of CHO(dhfr-) cells, repeated dilution cloning is necessary.     

Endogenous thymidine and hypoxanthine are a source of error in evaluating methotrexate cytotoxicity by clonogenic assays using undialyzed fetal bovine serum

None of 13 fresh human tumor samples of various histology cloned in a two-layer agar culture system with 20% undialyzed fetal bovine serum (FBS) showed sensitivity to three antifolates, methotrexate (MTX), trimetrexate and 5,8-dideazaisofolic acid (IAHQ), even after continuous exposure to the highest concentrations (100 microM) for 21 days. In order to investigate this lack of antifolate drug effect, we compared the toxicity of continuous MTX exposure in the human colon carcinoma cell line HCT-8, cloned in a thymidineless medium (RPMI 1640) supplemented with 10% horse serum (HS), 10% fetal bovine serum (FBS), 20% FBS or 20% dialyzed FBS. In the presence of native FBS, when the minimum clone size was set at 30 cells/colony, the survival of HCT-8 cells reached a plateau at approximately 60% of untreated control after exposure to MTX concentrations between 0.1 microM and 100 microM. Only when the minimum clone size was set at 2 X 10(3) cells/colony was the sensitivity of HCT-8 cells to the antimetabolite comparable to that obtained in HS or dialyzed FBS (ED50 values in the range of 0.01 microM). MTX protection experiments indicated that even very small concentrations of thymidine and hypoxanthine together were sufficient to reproduce the pattern of sensitivity to MTX observed under culture conditions with undialyzed FBS. We conclude that for a proper evaluation of MTX cytotoxicity in clonogenic assays, dialyzed FBS and thymidine-less media should be employed; if native FBS is an absolute requirement for growth, only very large colonies (at least 10 cell divisions) should be scored.     

Human tissue-type plasminogen activator. Production in continuous serum-free cell culture and rapid purification.

A simplified procedure for the production and purification of human tissue-type plasminogen activator (t-PA) is described. Bowes-melanoma cells were maintained in continuous serum-free culture. The cell nutrient consisted of Dulbecco's modified Eagle's medium (DMEM) supplemented with insulin (5 mg/litre), transferrin (5 mg/litre), progesterone (1 nM), cortisol (10 nM), aprotinin (2 X 10(4) units/litre) and a mixture of trace elements. t-PA accumulated in the culture medium at a rate of 40 units/day per ml and was harvested every third day. Cell losses during each harvest, leading to a steady decline of enzyme yields, were compensated for by treating the cells with 5% (v/v) fetal-bovine serum in DMEM every 6-8 weeks. t-PA was rapidly purified by a combination of cation-exchange chromatography and gel filtration. The procedure yielded mainly single-chain t-PA of a specific activity of 80 000 to 100 000 units/mg.     

Continuous production of tissue plasminogen activator from recombinant CHO cells in a depth filter perfusion system

Summary A depth filter perfusion system (DFPS), equipped with a 40-m polypropylene depth filter for cell immobilization, was used for the continuous production of tissue plasminogen activator (t-PA) from recombinant Chinese hamster ovary cells. Final cell density in the DFPS with oxygen control was 1.8×10⁷ cells/mL of the total working volume and maximum t-PA productivity was 2.63 mg/L/day. Dissolved oxygen concentration in the filter matrix was successfully controlled by air sparging and stable operation was possible for more than 20 days.     

组织型纤溶酶原激活剂（t—PA）突变体工程细胞株生物学特性分析

The morphology of t-PA mutant engineered cell line FSGGI48 is similar to that of CHO-dhfr- cell in polygon. When aminopterin (MTX) concentration is 5 x 10(-6) mol/L, a part of cells become slim, but they are still in polygon. Therefore, the morphology of FSGGI48 cell line is normal. The expression level is 4000 IU/10(6) cells/24 h in serum-free medium. The cell line is bequeathed for three months in vitro and revived after freezing three months, the greater part of cells are stable. The expression level is 3000-4000 IU/10(6) cells/24 h. The assay of tumorigenesis shows that the cells, cellular DNA and purified products don't form tumors in nude mice. The cell line hasn't been contaminated by mycoplasma. The results of chromosomal analysis show that the chromosomal number of CHO-dhfr- cell is 20 and the abnormal rate is 6%. FSGGI48 cell line is same as CHO-dhfr- cell in chromosomal number and the abnormal rate is 15%-18%, it is normal in scope. Therefore, the engineered cell line FSGGI48 is excellent.     

组织型纤溶酶原激活剂cDNA表达质粒的构建及其在CHO细胞中的表达

人组织型纤溶酶原激活剂(t-PA)cDNA克隆片段。采用两种方式构建成真核表达质粒。第一:切除t-PA3＇端非编码区序列后插入SR启动子和SV_(40)晚期Poly(A)终止信号之间,形成pMGZ6001质粒;第二:将3＇端部分切除并带有Poly(A)加尾信号的t-PA片段插入由金属硫蛋白MT启动子调控的载体中,分别组建成含大T抗原与不含大T抗原的两个表达质粒pMGZ6002和pMGZ6003。这三种质粒用磷酸钙共沉淀法和电穿孔法转染CHO-dhfr细胞,阳性克隆细胞均能合成并分泌rt-PA。其分子量约68kD,并能与t-PA单克隆抗体特异结合,溶解纤维蛋白。阳性克隆经MTX选择培养扩增基因,培液中rt-PA表达水平可达3000IU/(10~6细胞·48h)。     

Enhancement of tissue plasminogen activator production from human normal fibroblast IMR-90 cells by limitation of cell growth

Summary Tissue plasminogen activator (t-PA) production induced by proteose peptone from IMR-90 cells was investigated. Cells monolayered on plastic surfaces had a higher ability to produce t-PA per unit cell compared to those grown tri-dimensionally on ceramic pieces. Furthermore, confluent monolayers of the cells, which suffered contact inhibition and resulted in limited growth, were available for t-PA production. Repeated batch production with microcarriers, on which the cells were almost confluent monolayers similar to those in T-flasks, was performed. Utilization of the cells, which had limited serum in the growth phase, resulted in an increase in production. Moreover, dilution of the basal components of the medium at initiation of the production phase markedly promoted t-PA production. The volumetric productivity was stable for 30 days at 100 IU/cm³ per day. The cells were then mostly retained on microcarriers. Thus, an effective and scalable method of t-PA production by normal fibroblast cells was developed. Offprint requests to: S. Mitsuda     

Cultivation of immortalized human hepatocytes HepZ on macroporous CultiSpher G microcarriers

Cultivation of the new immortalized hepatocyte cell line HepZ was performed with a 1:1 mixture of DMEM and Ham's F12 media containing 5% FCS. The cells were grown in their 40th passage in 100 mL and 1 L volumes in spinner flasks and in a bioreactor, respectively. For the production of adherently growing HepZ cells macroporous CultiSpher G gelatin microcarriers were used in various concentrations from 1 to 3 g/L. The cells were seeded in a density of 2 x 10(5) cells/mL when using a microcarrier concentration of 1 g/L and 5 x 10(5) cells/mL at a microcarrier concentration of 3 g/L. After 7 days of cultivation a maximum cell concentration of 4.5 x 10(6) cells/mL was obtained in the spinner culture using a microcarrier concentration of 1 g/L. With bubble-free aeration and daily medium exchange from day 7, 7.1 x 10(6) cells/mL were achieved in the bioreactor using a microcarrier concentration of 3 g/L. The cells exhibited a maximum specific growth rate of 0.84 per day in the spinner system and 1.0 per day in the bioreactor, respectively. During the growth phase the lactate dehydrogenase (LDH) activity rose slightly up to values of 200 U/L. At the end of cultivation the macroporous carriers were completely filled with cells exhibiting a spherical morphology whereas the hepatocytes on the outer surface were flat-shaped. Concerning their metabolic activity the cells predominantly consumed glutamine and glucose. During the growth phase lactate was produced up to 19.3 mM in the spinner culture and up to 9.1 mM in the bioreactor. Maximal oxygen consumption was 1950 nmol/(10(6) cells. day). HepZ cells resisted a 4-day long chilling period at 9.5 degrees C. The cytochrome P450 system was challenged with a pulse of 7 microgram/mL lidocaine at a cell density of 4.5 x 10(6) cells/mL. Five ng/mL monoethylglycinexylidide (MEGX) was generated within 1 day without phenobarbital induction compared to 26 ng/mL after a preceded three day induction period with 50 microgram/mL of phenobarbital indicating hepatic potency. Thus, the new immortalized HepZ cell line, exhibiting primary metabolic functions and appropriate for a mass cell cultivation, suggests its application for a bioartificial liver support system.     

Production of human tissue-type plasminogen activator in different mammalian cell lines using an Epstein-Barr virus vector.

Human tissue-type plasminogen activator (t-PA) cDNA inserted into an Epstein-Barr virus (EBV) derived expression vector was transfected into human HeLa, 293, K-562 and hamster CHO-K1 cells and the expression of t-PA was studied. The best t-PA producing cell clones were found among CHO-K1 cells (up to 11 micrograms d-1 per 10(6) cells). However, HeLa and 293 cells were most efficiently transfected, e.g. about 70% of the selected cell clones were t-PA positive. The vector DNA copy numbers correlated with the mRNA levels and the protein levels for all cell lines analysed, with the exception for the K-562 cell line, where the production of t-PA was very low. The results obtained indicated that the highest expression levels were achieved in low density cultures.     

Characterization of a fusion protein consisting of amino acids 1 to 263 of tissue-type plasminogen activator and amino acids 144 to 411 of urokinase-type plasminogen activator

A hybrid human cDNA was constructed by splicing of a cDNA fragment of tissue-type plasminogen activator (t-PA), encoding 5'-untranslated, the pre-pro region and amino acids Ser1-Thr263, with a cDNA fragment of urokinase-type plasminogen activator (u-PA), encoding amino acids Leu144-Leu411. The cDNA fragments were obtained from full length t-PA cDNA, cloned from Bowes melanoma poly(A)+ mRNA, and from full length u-PA cDNA, cloned from CALU-3 lung adenocarcinoma poly(A)+ mRNA. The hybrid (t-PA/u-PA) cDNA was expressed in Chinese hamster ovary cells and the translation product purified from the conditioned cell culture media. On SDS-gel electrophoresis under reducing conditions, the protein migrated as a single band with approximate Mr 70,000. On immunoblotting, it reacted both with rabbit antisera raised against human t-PA and against human u-PA. The urokinase-like amidolytic activity of the protein was only 320 IU/mg but increased to 43,000 IU/mg after treatment with plasmin, which resulted in conversion of the single-chain molecule (t-PA/scu-PA) to a two-chain molecule (t-PA/tcu-PA). The specific activity of the protein on fibrin plates was 57,000 IU/mg by comparison with the International Reference Preparation for Urokinase. Both the single-chain hybrid (t-PA/scu-PA) and the two-chain plasmin derivative (t-PA/tcu-PA) bound specifically to fibrin, albeit more weakly than t-PA. The t-PA/tcu-PA hybrid had a higher selectivity for fibrin than tcu-PA, measured in a system composed of a whole human 125I-fibrin-labeled plasma clot immersed in human plasma. Both hybrid proteins activated plasminogen directly with Km = 1.5 microM and k2 = 0.0058 s-1 for t-PA/scu-PA and with Km = 80 microM and k2 = 5.6 s-1 for t-PA/tcu-PA. CNBr-digested fibrinogen stimulated the activation of plasminogen with t-PA/tcu-PA (Km = 0.20 microM and k2 = 1.2 s-1). It is concluded that these t-PA/u-PA hybrid proteins combine, at least to some extent, the fibrin-affinity of t-PA with the enzymatic properties of u-PA (either scu-PA or tcu-PA), which in some assays result in improved fibrin-mediated plasminogen activation.     

Amplification of multicistronic plasmids in the human 293 cell line and secretion of correctly processed recombinant human protein C

We have constructed multicistronic vectors containing the cDNAs for murine dihydrofolate reductase (DHFR), hygromycin phosphotransferase (HyPR), and human protein C (HPC), an antithrombotic factor. Using a sequential selection protocol with hygromycin (Hy) and methotrexate (MTX), we demonstrate the selective amplification of the murine dhfr cDNA in the adenovirus-transformed human kidney cell line 293, and the coamplification of the cDNA for HPC. Such recombinant 293 cell lines secreted HPC at levels as high as 25 micrograms/10(6) cells/day. In addition, we found that the complex vitamin K-dependent posttranslational modification of gamma-carboxylation of glutamate was not limiting at these high secretion levels, although the proteolytic processing of the protein was slightly reduced. Further, the HPC secreted from the gene-amplified cell lines had full anticoagulant activity when compared to plasma-derived HPC.