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.     

Effect of amplification ofdhfr andlac Z genes on growth and β-galactosidase expression in suspension cultures of recombinant CHO cells

Studies were conducted to characterize the effect of gene amplification and foreign gene expression on recombinant CHO cell growth. Chinese hamster ovary (CHO) cells were transfected with an expression vector containing the gene for dihydrofolate reductase (dhfr) and the gene for human β-interferon (β-IFN) or thelac Z gene which codes for β-galactosidase (β-gal). The recombinant genes in these CHO cells were amplified stepwise by growth in 0, 10⁻⁷, and 10⁻⁶ M methotrexate (MTX), and the β-gal expressing cells were adapted to suspension culture. Flow cytometric methods (FCM) were used to measure the distribution of amplifieddhfr gene content and foreign β-gal gene expression in the cell populations. A biochemical assay for β-gal was also used. Beta-gal expression was found to increase with increasing gene amplification. The growth rate of recombinant CHO cells at 10⁻⁷ M MTX was found to be 20% lower than that of recombinant CHO cells in MTX-free medium, and the cell growth rate at 10⁻⁶ M MTX was 20% lower than that of recombinant CHO cells at 10⁻⁷ M MTX. There was no effect of 10⁻⁵ M MTX on the growth of CHO-DG44 (dhfr-) cells. The reduction of growth rate in recombinant CHO cells is therefore thought to be mainly due to the effect ofdhfr and foreign gene amplification and increased β-galactosidase expression.     

Evaluating the interaction between UCOE and DHFR‐linked amplification and stability of recombinant protein expression

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry. In the creation of mammalian cell lines plasmid DNA carrying the gene‐of‐interest integrates randomly into the host cell genome, which results in variable levels of gene expression between cell lines due to gene silencing mechanisms. In addition, cell lines often show unstable protein production during long‐term culture. This means that a large number of clones need to be screened in order to isolate stable, high producing cell lines making mammalian cell line development a long and laborious process. In this study an expression platform incorporating a Ubiquitous Chromatin Opening Element (UCOE; which are proposed to maintain chromatin in an open state) has been utilised for the expression of eGFP in CHO cells. Cell lines containing a UCOE vector, showed a significantly higher and more consistent eGFP expression than the non‐UCOE cell lines without DHFR amplification. To further improve recombinant protein production cell lines were amplified with methotrexate (MTX). UCOE cell lines showed improved growth in MTX therefore amplification to 250 nM MTX was achieved following a one‐step amplification procedure. However, non‐UCOE cell lines showed higher levels of eGFP production following MTX amplification. In addition, UCOE cell lines did not improve stability during long‐term culture in the absence of selective pressure. Stable eGFP production was achieved for all cell lines when MTX is present. Finally, UCOE cell lines displayed more consistent response to external stimuli than non‐UCOE cell lines, suggesting that UCOE cell lines are less prone to clonal variability. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1014–1025, 2015     

Expression of an anchored urokinase in the apical endothelial cell membrane. Preservation of enzymatic activity and enhancement of cell surface plasminogen activation.

A mutant single chain urokinase plasminogen activator (scu-PA) was constructed by the addition of an apical membrane targeting signal from decay accelerating factor to the scu-PA carboxyl terminus. Bovine aortic endothelial cells (EC) were transduced with the mutant scu-PA. Metabolic labeling, immunoprecipitation, and gel electrophoresis revealed that the mutant scu-PA was present in a single-chain form at the EC surface. Immunohistochemistry and enzyme-linked immunosorbent assay before and after treatment of EC with phosphotidylinositol-specific phospholipase C confirmed that scu-PA was attached to the EC surface by a glycosyl-phosphotidylinositol anchor. Approximately 10(6) anchored scu-PA molecules/cell were present; however, anchoring was not 100% efficient, with scu-PA released into the medium as well. Selective biotinylation of the apical and basolateral surfaces revealed that anchored scu-PA was polarized to the apical surface. Apically anchored scu-PA could be converted by plasmin to two-chain urokinase, with a normal specific activity (140,000 IU/mg) as measured with the chromogenic substrate S-2444. Expression of anchored scu-PA resulted in an increase in EC surface plasminogen activator activity, as compared with the activity of either untransduced EC or EC transduced with a wild type scu-PA. These experiments demonstrate: 1) apical membrane targeting can be accomplished in EC; 2) scu-PA can be anchored to the EC surface with preservation of enzymatic activity; 3) EC surface plasminogen activator activity is significantly increased by the presence of anchored scu-PA. Cell surface targeted plasminogen activators may eventually be useful in the prevention and treatment of intravascular thrombosis.     

Interaction of single-chain urokinase-type plasminogen activator with human endothelial cells

The interaction of urokinase-type plasminogen activators with receptors on the surface of endothelial cells may play an important role in the regulation of fibrinolysis and cell migration. Therefore, we investigated whether human umbilical vein endothelial cells (HUVEC) express receptors for single-chain urokinase (scu-PA) on the cell surface and examined the effect of such binding on plasminogen activator activity. Binding of 125I-labeled scu-PA to HUVEC, performed at 4 degrees C, was saturable, reversible, and specific (k+1 4 +/- 1 X 10(6) min-1 M-1, k-1 6.2 +/- 1.4 X 10(-3) min-1, Kd 2.8 +/- 0.1 nM; Bmax 2.2 +/- 0.1 X 10(5) sites/cell; mean +/- S.E.). Binding of radiolabeled scu-PA was inhibited by both natural and recombinant wild-type scu-PA, high molecular weight two-chain u-PA (tcu-PA), catalytic site-inactivated tcu-PA, an amino-terminal fragment of u-PA (amino acids 1-143), and a smaller peptide (amino acids 4-42) corresponding primarily to the epidermal growth factor-like domain. Binding was not inhibited by low molecular weight urokinase or by a recombinant scu-PA missing amino acids 9-45. Cell-bound scu-PA migrated at its native molecular mass on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the presence of plasminogen, scu-PA bound to endothelial cells generated greater plasmin activity than did scu-PA in the absence of cells. In contrast, when tcu-PA was added directly to HUVEC, sodium dodecyl sulfate-stable complexes formed with cell or matrix-associated plasminogen activator inhibitors with a loss of plasminogen activator activity. These studies suggest that endothelial cells in culture express high affinity binding sites for the epidermal growth factor domain of scu-PA. Interaction of scu-PA with these receptors may permit plasminogen activator activity to be expressed at discrete sites on the endothelial cell membrane.     

Ubiquitous Chromatin Opening Elements (UCOEs) effect on transgene position and expression stability in CHO cells following methotrexate (MTX) amplification

The requirement for complex therapeutic proteins has resulted in mammalian cells, especially CHO cells, being the dominant host for recombinant protein manufacturing. In creating recombinant CHO cell lines, the expression vectors integrate into various parts of the genome leading to variable levels of expression and stability of protein production. This makes mammalian cell line development a long and laborious process. Therefore, with the intention to accelerate process development of recombinant protein production in CHO systems, UCOEs are utilized to diminish instability of production by maintaining an open chromatin surrounding in combination with MTX amplification. Chromosome painting and FISH analysis were performed to provide detailed molecular evaluation on the location of amplified genes and its relationship to the productivity and stability of the amplified cell lines. In summary, cell lines generated with vectors containing UCOEs retained stable GFP expression with MTX present (but instability was observed in the absence of MTX). UCOE cell lines displayed a higher frequency of integration into >1 chromosome than non‐UCOE group. Cell populations were more homogenous in terms of transgene location at the end of Long‐term culture (LTC). Overall our findings suggest variation in eGFP fluorescence may be attributed to changes in transgene integration profile over LTC.     

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.     

Characterization of a recombinant fusion protein of the finger domain of tissue-type plasminogen activator with a truncated single chain urokinase-type plasminogen activator

Human recombinant single chain urokinase-type plasminogen activator (recombinant scu-PA) and a hybrid between human tissue-type plasminogen activator (t-PA) and scu-PA, obtained by ligation of cDNA fragments encoding the NH2-terminal region (amino acids 1-67) of t-PA and the COOH-terminal region (amino acids 136-411) of scu-PA, were expressed in a mammalian cell system. The proteins were purified from conditioned culture media containing 2% fetal calf serum by chromatography on zinc chelate-Sepharose, immunoadsorption chromatography on an insolubilized murine monoclonal antibody directed against urokinase, benzamidine-Sepharose chromatography, and Ultrogel AcA 44 gel filtration. Between 180 and 230 micrograms of the purified proteins were obtained per liter of conditioned medium, with a yield of approximately 18% and a purification factor of 720-1900. On sodium dodecyl sulfate gel electrophoresis under reducing conditions, the proteins migrated as single bands with approximate Mr 50,000 for recombinant scu-PA and Mr 43,000 for the t-PA/scu-PA hybrid. Following conversion to urokinase with plasmin, the proteins had a specific amidolytic activity comparable to that of natural scu-PA. Both proteins activated plasminogen directly with Km = 0.53 and 1.4 microM and k2 = 0.0034 and 0.0027 s-1, respectively. Both proteins did not bind specifically to fibrin and had a comparable degree of fibrin selectivity as measured in a system composed of a whole human 125I-fibrin-labeled plasma clot suspended in human plasma. It is concluded that this chimeric protein, consisting of the NH2-terminal "finger-like" domain of t-PA and the COOH-terminal region of scu-PA, has very similar enzymatic properties as compared to scu-PA, but has not acquired the fibrin affinity of t-PA.     

Stable, recombinant expression of human insulin-like growth factor binding protein-1 (hIGFBP-1) in Chinese hamster ovary (CHO) cells

Stable expression of human insulin-like growth factor of binding protein-1 (hIGFBP-1)at high levels has been achieved in Chinese hamster ovary (CHO) cells by co-transfection and subsequent co-amplification of expression vectors containing the hIGFBP-1 cDNA and a dihydrofolate reductase (DHFR) cDNA gene into DHFR-deficient cells. Stepwise selection of the DHFR⁺ transformants in increasing concentrations of methotrexate (MTX) generated cells which had high copy numbers of the hIGFBP-1 gene (around 100 copies in cells amplified in medium containing 100 nM MTX). Expression of hIGFBP-1 in mixed clones was found to increase with increasing copy number and an apparent correlation between intra- and extracellular levels of hIGFBP-1 produced by these cells was observed. It was further observed that continuous cultivation over eight months in medium supplemented with 100 nM MTX increased the production of hIGFBP-1 25 times. The productivity did not increase further after five more months cultivation in MTX containing medium. A subcloning of this cell line gave clones with an even higher productivity. Further amplification in 500 nM or 1 uM MTX did not increase the hIGFBP-1 production. This revised version was published online in July 2006 with corrections to the Cover Date.     

Performance study of perfusion cultures for the production of single-chain urokinase-type plasminogen activator (scu-PA) in a 2.5 l spin-filter bioreactor

A perfusion-control strategy based on cellular consumption rates of oxygen and glucose was established for the production of single-chain urokinase-type plasminogen activator (scu-PA). Employing this strategy, the influences of microcarrier types and the culture media on culture performances were evaluated. In the control perfusion culture, which used a solid microcarrier and a 1% fetal bovine serum (FBS) medium, viable cell density reached 3.1?×?10⁷?cells?ml^?1. However, formation of large, heterogeneous aggregates (500–1,000?μm) resulted in a gradual decrease in viable cell density to less than 1.0?×?10⁷?cells?ml^?1. Accordingly, declines in the production of urokinase-type plasminogen activator (u-PA) and in the scu-PA portion of u-PA were observed. In the serum-free media, cell growth and u-PA production were suppressed 2–3?times, but were significantly enhanced when a porous microcarrier, Cultispheer G, was used. The cell-growth profile showed a continuous increase in cell density, reaching 5.1?×?10⁷?cells?ml^?1, and the production of u-PA remained stable throughout the culture (1586?±?247?IU?ml^?1). The values of all the parameters associated with cell growth and u-PA production were fairly comparable to or even higher than those in the control culture. Moreover, a 13% higher scu-PA portion of u-PA was observed in the serum-free culture, regardless of the microcarrier type, compared with scu-PA portion of u-PA in the control culture.     

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.     

Comparative Assessment on the Expression Level of Recombinant Human Follicle-Stimulating Hormone (FSH) in Serum-Containing Versus Protein-Free Culture Media

Production of recombinant pharmaceutical proteins has made a great contribution to modern biotechnology. At present, quick advances in protein expression lead to the enhancement of product quantity and quality as well as reduction in timescale processing. In the current study, we assessed the expression level of recombinant human follicle-stimulating hormone (rhFSH) in adherent and suspension Chinese hamster ovary (CHO) cell lines by cultivation in serum-containing and chemically defined, protein-free media. The expression cassette entailing FSH subunits was transfected to CHO/dhfr- and CHO DG44 cell lines, and gene amplification was achieved using dihydrofolate reductase (DHFR)/methotrexate (MTX) system. Afterward, the expression level of rhFSH was studied using real-time PCR, Western blotting and ELISA. Our achievements revealed that stepwise increase in MTX [up to 2000 nano-molar (nM)] leads to boost the expression level of rhFSH mRNA in both cell lines, although DG44 have better results, as mRNA expression level reached 124.8- and 168.3-fold in alpha and beta subunits, respectively. DG44 cells have also the best protein production in 2000 nM MTX, which reached 1.7-fold in comparison with that of the mock group. According to the above results and many advantages of protein-free media, DG44 is preferable cell line for future steps.     

The optimization of serum-free medium for the production of the scu-PA by the addition of algal extracts

The addition of 2.8 g/ml algal extracts enhanced both scu-PA production and cell growth in a serum-free medium, compared to a conventional serum-free medium for the cultivation of recombinant CHO cells. The growth rate and scu-PA production were relatively lower in the serum-free medium than 5% serum containing medium: however, specific scu-PA production rate was higher in the serum-free medium due to the long-term period of cultivation (3.66×10^–4 vs. 2.48×10^–4 IU/cell/day). Overall scu-PA production rate was also greater in an enforced serum-free medium as 25,000 IU/day over 50 d of perfusion cultivation. The conversion ratio of scu-PA to tcu-PA was greatly reduced in the serum-free medium during perfusion cultivation (10% compared to 20% conversion in a serum containing medium).     

A mouse hybrid cell line that supports gene expression from a variety of promoters in amplifiable vectors

Summary A hybrid cell line was constructed by fusion of mouse L-cells with an NIH3T3 cell line derivative containing a hybrid gene consisting of the mouse immunoglobulin kappa (IgK) variable gene promoter linked to theEscherichia coli gpt gene. Such hybrids grew to a much higher density compared to either of the parental cell lines. The utility of this cell line as a host to express foreign genes was tested by the expression of TGF-β cDNA using the cytomegalovirus promoter. The vector also contained the human dihydrofolate reductase (DHFR) gene driven by SV40 early promoter, to allow for the amplification of the transfected gene. Initial transformants, selected at 100 nM methotrexate (MTX), were subsequently selected for resistance to a higher concentration of MTX (2 μM). Such clones expressed an increased level of TGF-β when compared to the initial transformants. Both the initial transformants and the clones with the amplified DHFR gene produced TGF-β in an acid-activatable precursor form. This mouse hybrid host cell line also allowed the expression of foreign genes cloned in an eukaryotic expression vector with the mouse IgK variable region promoter and human growth hormone as the reporter gene, whereas such vectors did not function in CHO cells. The mouse hybrid cell line was also found to be capable of being used with a broad range of promoters.     

Optimization of pro-urokinase secretion from recombinant Saccharomyces cerevisiae

Secretion of a nonglycosylated form of human pro-urokinase, also known as single-chain urinary plasminogen activator (scu-PA), from Saccharomyces cerevisiae is described. A "supersecreting" yeast strain harboring multiple copies of integrated plasmids was grown batchwise and at constant respiratory quotient (RQ) in 20-L fermenters. Because the promoters used to drive expression of the pro-urokinase genes are not tightly regulated, secretion into the culture supernatant was growth associated. Although the final cell density achieved in the perturbed-batch fermentation (45 g dry wt/L) was less than that observed in the RQ-controlled culture (77 g dry wt/L), the scu-PA titer in the perturbed-batch fermentation (1863 IU/mL) was nearly twice that attained at constant RQ (1108 IU/mL). The effects on cell growth and scu-PA titer of other process variables (pH, temperature, phosphate concentration, and medium composition) are also discussed.     

Mechanisms of unintended amino acid sequence changes in recombinant monoclonal antibodies expressed in Chinese Hamster Ovary (CHO) cells

An amino acid sequence variant is defined as an unintended amino acid sequence change and contributes to product heterogeneity. Recombinant monoclonal antibodies (MAbs) are primarily expressed from Chinese Hamster Ovary (CHO) cells using stably transfected production cell lines. Selections and amplifications with reagents such as methotrexate (MTX) are often required to achieve high producing stable cell lines. Since MTX is often used to generate high producing cell lines, we investigated the genomic mutation rates of the hypoxanthine–guanine phosphoribosyltransferase (HGPRT or HPRT) gene using a 6‐thioguanine (6‐TG) assay under various concentrations of MTX selection in CHO cells. Our results show that the 6‐TG resistance increased as the MTX concentration increased during stable cell line development. We also investigated low levels of sequence variants observed in two stable cell lines expressing different MAbs. Our data show that the replacement of serine at position 167 by arginine (S167R) in the light chain of antibody A (MAb‐A) was due to a genomic nucleotide sequence change whereas the replacement of serine at position 63 by asparagine (S63N) in the heavy chain of antibody B (MAb‐B) was likely due to translational misincorporation. This mistranslation is codon specific since S63N mistranslation is not detectable when the S63 AGC codon is changed to a TCC or TCT codon. Our results demonstrate that both a genomic nucleotide change and translational misincorporation can lead to low levels of sequence variants and mistranslation of serine to asparagine can be eliminated by substituting the TCC or TCT codon for the S63 AGC codon without impacting antibody productivity. Biotechnol. Bioeng. 2010;107: 163–171. © 2010 Wiley Periodicals, Inc.