CHO-DG44 Cells Research Articles

A quantitative proteomic analysis of cellular responses to high glucose media in Chinese hamster ovary cells.

A goal in recombinant protein production using Chinese hamster ovary (CHO) cells is to achieve both high specific productivity and high cell density. Addition of glucose to the culture media is necessary to maintain both cell growth and viability. We varied the glucose concentration in the media from 5 to 16 g/L and found that although specific productivity of CHO-DG44 cells increased with the glucose level, the integrated viable cell density decreased. To examine the biological basis of these results, we conducted a discovery proteomic study of CHO-DG44 cells grown under batch conditions in normal (5 g/L) or high (15 g/L) glucose over 3, 6, and 9 days. Approximately 5,000 proteins were confidently identified against an mRNA-based CHO-DG44 specific proteome database, with 2,800 proteins quantified with at least two peptides. A self-organizing map algorithm was used to deconvolute temporal expression profiles of quantitated proteins. Functional analysis of altered proteins suggested that differences in growth between the two glucose levels resulted from changes in crosstalk between glucose metabolism, recombinant protein expression, and cell death, providing an overall picture of the responses to high glucose environment. The high glucose environment may enhance recombinant dihydrofolate reductase in CHO cells by up-regulating NCK1 and down-regulating PRKRA, and may lower integrated viable cell density by activating mitochondrial- and endoplasmic reticulum-mediated cell death pathways by up-regulating HtrA2 and calpains. These proteins are suggested as potential targets for bioengineering to enhance recombinant protein production.

High-yield preparation of recombinant human α-thrombin for therapeutic use

In this study, we established stable cell lines producing 1.5mg/mL recombinant human prothrombin in 400-L fed-batch culture, using CHO DG44 cells as a host cell line. And we also established a recombinant human α-thrombin purification process that produces a purified product suitable for use as a biopharmaceutical, by using recombinant ecarin from CHO DG44 cells, achieving a total yield of approximately 27% of prothrombin in culture medium. The establishment of stable cell lines with high expression levels, long-term passage stability and satisfactory scale-up are essential to ensure the stable supply of biopharmaceuticals. Furthermore, biopharmaceuticals must be of high quality to assure safety and effectiveness in target applications. We had previously reported that recombinant human prethrombin-2 expression level in a stable cell line established using the mouse myeloma cells, Sp2/0-Ag14, reached 200μg/mL using animal-free materials in 50-L fed-batch culture. However, the productivity was insufficient to completely replace α-thrombin in human plasma preparations. By employing CHO DG44 cells as a host cell line, we had established a stable cell line and achieved significant improvements in quality, productivity of recombinant human α-thrombin manufacture suitable for use as a biopharmaceutical.

Recombinant human lactoferrin-Fc fusion with an improved plasma half-life

Lactoferrin (LF), an 80-kDa iron-binding glycoprotein found in mammalian exocrine secretions, has potential therapeutic efficacy due to its extensive health-promoting effects. However, LF is rapidly cleared from the circulation (∼12.6min half-life for recombinant human LF [rhLF] in rats), which limits its therapeutic potential. Therefore, to improve plasma stability, we developed a recombinant human LF (hLF)-immunoglobulin G1 (IgG1) fragment crystallizable domain (Fc) fusion (hLF-hinge-CH2-CH3) expressed in a Chinese Hamster Ovary cell (CHO) expression system and evaluated the in vitro bioactivities and pharmacokinetic properties of the purified fusion. CHO DG44 cells were transfected with an expression vector coding for recombinant hLF-hinge-CH2-CH3. Iron binding, Caco-2 uptake, and thermal stability were investigated in vitro, and pharmacokinetic parameters were investigated in vivo. hLF-hinge-CH2-CH3 was significantly expressed in CHO cells (∼100mg/l culture), was readily purified, and exhibited 98.3% of the non-fused rhLF iron-binding activity. Caco-2 uptake and thermal stability were improved for hLF-Fc fusion relative to rhLF. Moreover, hLF-hinge-CH2-CH3 demonstrated a plasma half-life that was 9.1-fold longer than that of rhLF as well as longer than that of the PEGylated bovine LFs that we previously developed. Thus, CHO-derived hLF-hinge-CH2-CH3, with enhanced pharmacokinetic properties, is a promising candidate drug for potential parenteral administration.

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro

To evaluate the biochemical features and activities of a glyco-engineered form of the anti-human epidermal growth factor receptor monoclonal antibody (EGFR mAb) cetuximab in vitro. The genes encoding the Chinese hamster bisecting glycosylation enzyme (GnTIII) and anti-human EGFR mAb were cloned and coexpressed in CHO DG44 cells. The bisecting-glycosylated recombinant EGFR mAb (bisec-EGFR mAb) produced by these cells was characterized with regard to its glycan profile, antiproliferative activity, Fc receptor binding affinity and cell lysis capability. The content of galactose-α-1,3-galactose (α-Gal) in the bisec-EGFR mAb was measured using HPAEC-PAD. The bisec-EGFR mAb had a higher content of bisecting N-acetylglucosamine residues. Compared to the wild type EGFR mAb, the bisec-EGFR mAb exhibited 3-fold higher cell lysis capability in the antibody-dependent cellular cytotoxicity assay, and 1.36-fold higher antiproliferative activity against the human epidermoid carcinoma line A431. Furthermore, the bisec-EGFR mAb had a higher binding affinity for human FcγRIa and FcγRIIIa-158F than the wild type EGFR mAb. Moreover, α-Gal, which was responsible for cetuximab-induced hypersensitivity reactions, was not detected in the bisec-EGFR mAb. The glyco-engineered EGFR mAb with more bisecting modifications and lower α-Gal content than the approved therapeutic antibody Erbitux shows improved functionality in vitro, and requires in vivo validations.

Production of anti TNF-α antibodies in eukaryotic cells using different combinations of vectors carrying heavy and light chains.

Tumor necrosis factor-α (TNF-α) plays a key role in rheumatoid arthritis and some other autoimmune diseases. Therapy with anti-TNF-α recombinant antibodies (Ab) appears to be highly effective. Production of new hyper-producing eukaryotic cell lines can decrease the treatment cost, which currently is very high. However, due to the complexity of protein transcription, translation, processing, and secretion in mammalian cells, the stages at which antibody expression is affected are still poorly determined. The aim of this work was to compare the productivity of two cell lines developed in CHO DG44 cells, deficient in dihydrofolate reductase, transfected with vectors carrying either heavy (H) or light (L) chains of chimeric antibody under different combinations of selective elements. Both H and L chains were cloned either in pOptiVEC or pcDNA3.3 vectors and different combinations were used to produce HL and LH cell lines. We have shown that Ab production has been low and comparable between HL and LH cells until selection on methotrexate (MTX) when LH but not HL cells have responded with 3.5 times increased productivity. Flow cytometry analysis has demonstrated that intracellular concentration of full size Abs in LH cells was 5.6 times higher than in HL ones due to higher amount of H chain synthesis. No differences in viability between HL and LH cells have been found. We have concluded that the expression of H chain in the pOptiVEC vector, which is responsible for MTX resistance, has led to the suppression of H chain synthesis and limitation in full Ab assembly.

Comparison of Internal Ribosome Entry Site (IRES) and Furin-2A (F2A) for Monoclonal Antibody Expression Level and Quality in CHO Cells

Four versions of tricistronic vectors expressing IgG1 light chain (LC), IgG1 heavy chain (HC), and dihydrofolate reductase (DHFR) in one transcript were designed to compare internal ribosome entry site (IRES) and furin-2A (F2A) for their influence on monoclonal antibody (mAb) expression level and quality in CHO DG44 cells. LC and HC genes are arranged as either the first or the second cistron. When using mAb quantification methods based on the detection antibodies against HC Fc region, F2A-mediated tricistronic vectors appeared to express mAb at higher levels than the IRES-mediated tricistronic vectors in both transient and stable transfections. Further analysis revealed that more than 40% of products detected in stably transfected pools generated using the two F2A-mediated tricistronic vectors were aggregates. LC and HC from the F2A stably transfected pools were not properly processed, giving rise to LC+F2A+HC or HC+F2A+LC fusion proteins, LC and HC polypeptides with F2A remnants, and incorrectly cleaved signal peptides. Both IRES-mediated tricistronic vectors express mAb with correct sizes and signal peptide cleavage. Arrangement of LC as the first cistron in the IRES-mediated tricistronic vectors exhibits increased mAb expression level, better growth, and minimized product aggregation, while arrangement of HC as first cistron results in low expression, slower growth, and high aggregation. The results obtained will be beneficial for designing vectors that enhance mAb expression level and quality in mammalian cells.

Immunoglobulin Fc domain fusion to apolipoprotein(a) kringle V significantly prolongs plasma half-life without affecting its anti-angiogenic activity

Angiogenesis is crucial for tumor growth and metastasis. Blocking this process is, therefore, a potentially powerful approach for the treatment of cancer. Human apolipoprotein(a) kringle V (rhLK8) is an angiogenesis inhibitor and is currently under development as an anti-cancer therapeutic. However, a relatively short in vivo half-life limits its widespread clinical use. This study was performed to evaluate whether fusion of an Fc domain to rhLK8 can extend plasma half-life. RhLK8-Fc fusion protein was expressed in CHO DG44 cells as a dimer and was readily purified by protein G affinity chromatography. The anti-angiogenic activity of rhLK8-Fc was similar to that of rhLK8, as determined by migration and tube formation assays with endothelial cells in vitro and a chorioallantoic membrane assay in vivo. Pharmacokinetic profiles in mice after single intravenous administration of rhLK8 or rhLK8-Fc showed that Fc fusion significantly increased the elimination half-life (t(½)) and the systemic exposure (AUC(inf)) of the protein, in parallel with a significant decrease in total clearance (CL). These data suggest that Fc fusion to rhLK8 is a powerful strategy for extending the plasma half-life of rhLK8 without affecting its anti-angiogenic activity, and could thus improve the clinical applicability of rhLK8.

DNA delivery with hyperbranched polylysine: A comparative study with linear and dendritic polylysine

PEI and polylysine are among the most investigated synthetic polymeric carriers for DNA delivery. Apart from their practical use, these 2 classes of polymers are also of interest from a fundamental point of view as they both can be prepared in different architectures (linear and branched/dendritic) and in a wide range of molecular weights, which is attractive to establish basic structure–activity relationships. This manuscript reports the results of an extensive study on the influence of molecular weight and architecture of a library of polylysine variants that includes linear, dendritic and hyperbranched polylysine. Hyperbranched polylysine is a new polylysine-based carrier that is structurally related to dendritic polylysine but possesses a randomly branched structure. Hyperbranched polylysine is attractive as it can be prepared in a one-step process on a large scale. The performance of these 3 classes of polylysine analogs was evaluated by assessing eGFP and IgG production in transient gene expression experiments with CHO DG44 cells, which revealed that protein production generally increased with increasing molecular weight and that at comparable molecular weight, the hyperbranched analogs were superior as compared to the dendritic and linear polylysines. To understand the differences between the gene delivery properties of the hyperbranched polylysine analogs on the one hand and the dendritic and linear polylysines on the other hand, the uptake and trafficking of the corresponding polyplexes were investigated. These experiments allowed us to identify (i) polyplex–external cell membrane binding, (ii) free, unbound polylysine coexisting with polyplexes as well as (iii) polymer buffer capacity as three possible factors that may contribute to the superior transfection properties of the hyperbranched polylysines as compared to their linear and dendritic analogs. Altogether, the results of this study indicate that hyperbranched polylysine is an interesting, alternative synthetic gene carrier. Hyperbranched polylysine can be produced at low costs and in large quantities, is partially biodegradable, which may help to prevent cumulative cytotoxicity, and possesses transfection properties that can approach those of PEI.

Production of a Neutralizing Mouse-Human Chimeric Antibody against Botulinum Neurotoxin Serotype E

A mouse-human chimeric antibody that can neutralize botulinum neurotoxin serotype E (BoNT/E) was developed. Variable regions of heavy and light chains obtained using a mouse hybridoma clone (E9-4) cDNA, which was selected on the basis of neutralizing activity against BoNT/E, were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and a mouse-human chimeric antibody (EC94) was purified to examine binding and neutralizing activity against BoNT/E. EC94 exhibited the same levels of binding activities against BoNT/E as those of a parent mouse monoclonal antibody and neutralized more than 4,000 LD(50)/mg antibody. This chimeric antibody seems to be a useful candidate for infant botulism in which the use of passive immunotherapy is not planned so as to avoid serious events such as anaphylactic shock. We designed shuffling chimeric antibodies with replacement of V(H) or V(L) of EC94 with that of a chimeric antibody (AC24) that possessed neutralizing activity against BoNT/A. These shuffling antibodies did not exhibit neutralizing activity against either BoNT/E or BoNT/A.

Identifying the CHO Secretome using Mucin-type O-Linked Glycosylation and Click-chemistry

Chinese hamster ovary cells (CHO) are the most common cell line used in the production of therapeutic proteins. Understanding the complex pattern of secreted host cell proteins (HCP) that are released by CHO cells will facilitate the development of new recombinant protein production processes. In this study, we have adapted the N-azido-galactosamine (GalNAz) metabolic labeling method to enable the mass spectrometry identification and quantification of secreted proteins in cell culture media. CHO DG44 and CHO-S cells were cultured in media containing GalNAz, which was metabolically incorporated into mucin-type O-linked glycans of secreted proteins. These proteins were effectively enriched using click-chemistry from the cell culture media, allowing for the analysis of secreted proteins across multiple days of cell growth. When compared to the standard method for secretome analysis, the GalNAz method not only increased the total number of proteins identified but dramatically improved the quality of data by decreasing the number of background proteins (cytosolic or nuclear) to essentially zero.

Characterization of neutralizing mouse‐human chimeric and shuffling antibodies against botulinum neurotoxin A

Mouse-human chimeric monoclonal antibodies that could neutralize botulinum neurotoxins were developed and an attempt was made to establish mouse hybridoma cell clones that produced monoclonal antibodies that neutralized botulinum neurotoxin serotype A (BoNT/A). Four clones (2-4, 2-5, 9-4 and B1) were selected for chimerization on the basis of their neutralizing activity against BoNT/A and the cDNA of the variable regions of their heavy (V(H)) and light chains (V(L)) were fused with the upstream regions of the constant counterparts of human kappa light and gamma 1 heavy chain genes, respectively. CHO-DG44 cells were transfected with these plasmids and mouse-human chimeric antibodies (AC24, AC25, AC94 and ACB1) purified to examine their binding and neutralizing activities. Each chimeric antibody exhibited almost the same capability as each parent mouse mAb to bind and neutralize activities against BoNT/A. From the chimeric antibodies against BoNT/A, shuffling chimeric antibodies designed with replacement of their V(H) or V(L) domains were constructed. A shuffling antibody (AC2494) that derived its V(H) and V(L) domains from chimeric antibodies AC24 and AC94, respectively, showed much higher neutralizing activity than did other shuffling antibodies and parent counterparts. This result indicates that it is possible to build high-potency neutralizing chimeric antibodies by selecting and shuffling V(H) and V(L) domains from a variety of repertoires. A shuffling chimeric antibody might be the best candidate for replacing horse antitoxin for inducing passive immunotherapy against botulism.

Comparative Study on the In Vitro Cytotoxicity of Linear, Dendritic, and Hyperbranched Polylysine Analogues

Lysine-based polycations are widely used as nonviral carriers for gene delivery. This manuscript reports the results of a comparative study on the in vitro cytotoxicity of a library of three structural polylysine variants, namely, linear polylysine (LPL), dendritic polylysine (DPL), and hyperbranched polylysine (HBPL). The aim of this study was to identify possible effects of polymer molecular weight and architecture on both immediate and delayed cytotoxicity and also to provide a mechanistic understanding for possible differences. Acute cytotoxicities were evaluated using cell viability assays with CHO DG44 cells. At comparable molecular weights, the EC(50) values for the LPL analogues were ∼5-250 times higher as compared to the DPL and HBPL samples. For low molecular weight polycations, osmotic shock was found to be an important contributor to immediate cell death, whereas for the higher molecular weight analogues, direct cell membrane disruption was identified to play a role. Delayed cytotoxicity (≥3 h) was assessed by identifying several of the hallmark events that characterize apoptosis, including phosphatidyl serine translocation, mitochondrial membrane depolarization, cytoplasmic cytochrome C release, and caspase 3 activation. At comparable molecular weights, apoptosis was found to be more pronounced for DPL and HBPL as compared to LPL. This difference was ascribed to the fact that LPL is completely enzymatically degradable, in contrast to DPL and HBPL, which also contain ε-peptidic bonds and are only partially degradable. Because their toxicity profiles are similar, HBPL is an interesting (i.e., synthetically easily accessible and inexpensive) alternative to DPL for the nonviral delivery of DNA.

Hyperbranched Polylysine: A Versatile, Biodegradable Transfection Agent for the Production of Recombinant Proteins by Transient Gene Expression and the Transfection of Primary Cells

The feasibility of a new transfection agent, HBPL, for the production of recombinant IgG antibody via TGE as well as for the transfection of primary cells is studied. Under the conditions investigated, transfection of CHO-DG44 cells using HBPL results in IgG yields that are comparable to those obtained with PEI. In experiments with CHO-K1 cells and MEFs the use of HPBL allows to achieve transfection efficiencies comparable to or better than those obtained with PEI or Fugene®. HBPL-mediated transfection does not require complex pre-formation, works well in serum-containing media and is biodegradable, which may prevent cumulative cytotoxicity and facilitates downstream processing.

Recombinant Fc-IL-18BPc Isoform Inhibits IL-18-Induced Cytokine Production

IL-18 is a pro-inflammatory cytokine that is produced from T cells and NK cells. IL-18 has been implicated in the pathogenesis of various inflammatory and cardiovascular diseases. IL-18 binding protein (IL-18BP) is a natural inhibitor of IL-18 that possesses higher affinity to IL-18 than that of the IL-18 receptor alpha chain on the cell surface. Human isoform a and c among four isoforms of IL-18BPs have an inhibitory effect on IL-18-induced cytokines whereas mouse IL-18BP isoforms exist only in two isoforms: c and d. Fc-fusion protein is a molecule in which the immunoglobulin Fc is fused genetically to a protein of interest, such as an extracellular domain of a receptor, ligand, or enzyme. In this study, we expressed and purified human Fc-IL-18BPa and c isoforms from CHO-DG44 cells and their biological activities were compared to each other. This is the first time that expressed recombinant human Fc-IL-18BPc has been examined for its biological activity on IL-18-induced IFNγ in human PBMC and IL-6 in A549/IL-18Rβ.

Reduced glutamine concentration improves protein production in growth-arrested CHO-DG44 and HEK-293E cells

For most cultivated mammalian cells, glutamine is an essential medium component. However, glutamine consumption results in the production of ammonia, a cytotoxic byproduct. Here we investigated the effect of glutamine reduction on recombinant protein production and ammonia accumulation in transiently transfected CHO and HEK-293E cells maintained under conditions of growth arrest. Maximum transient recombinant protein yields were observed in HEK-293E cultures without glutamine and in CHO cultures with 2 mM glutamine. The initial concentration of glutamine correlated with the level of ammonia accumulation in each culture. For both a stable CHO-derived cell line and a polyclonal population of recombinant CHO cells grown under conditions of mild hypothermia, the highest volumetric protein productivity was observed in cultures without glutamine. Here, the level of ammonia accumulation also corresponded to the initial glutamine concentration. Our data demonstrate that reduction of glutamine in the medium is an effective approach to improve protein production in both transiently and stably transfected mammalian cells when applying conditions that reduce or arrest the growth of these cells.

Increasing antibody yield and modulating final product quality using the FreedomTM CHO-STM production platform

Background Cell line development (CLD) is a critical step in the generation of biotherapeutics, but it is still hindered by several pain points, including the lengthy and laborintensive workflow needed to isolate desirable clones, lack of reproducibility, as well as potential protein quality issues. Over the last decade, antibody titers in mammalian cell culture systems in excess of 3 g/L have been achieved through the use of novel media and feeds. However, it is still a challenge to consistently and rapidly create a stable cell line and a cell culture process capable of supporting both high antibody yield and acceptable post-translational modifications while managing the effort required for execution of the workflow. The goal of the study was to develop a robust and reproducible stable cell line workflow to generate scalable high-producing clones in less than 6 months, with industry-standard titers and desirable product quality using minimal effort. Using CHO-STM as the host cell line, we first evaluated if a single medium could be used for the entire CLD workflow, therefore avoiding the issues and complications of changing media during this process. We investigated if a formulation previously shown to increase titer as a production medium could in fact be used for all CLD steps, from transfection to stable pool isolation all the way through to clone productivity, without compromising titers or performance. The same rich production medium was used in limiting dilution cloning and compared to a lean cloning medium prototype. Furthermore, robustness of the workflow was verified by testing multiple molecules. We also explored reducing effort by streamlining all the steps of the workflow. Finally, we assessed top clone scalability and expressed product quality. We tested whether clones chosen only by titers responded well to scale-up and process development in a model bioreactor setting. In addition, product glycosylation from these clones was compared to the same molecule produced in CHO DG44 cells, a well-characterized production platform.

Determination of intracellular antibody production, cell density, and viability of recombinant CHO-DG44 cells using the MACSQuant® Analyzer

Expression of recombinant antibodies in CHO cells is a state-of-the-art procedure in research and industry. Generation of cell lines producing high amounts of antibodies is one of the major tasks to increase process efficiency. The establishment of clones is often achieved by methotrexate (MTX)–mediated gene amplification in CHO-DG44 cells. Evaluation of MTX-mediated amplification is usually facilitated by intracellular staining of CHO cells. We used the MACSQuant® Analyzer to determine the fraction of antibody-producing cells following staining of intracellular IgG with fluorochrome-conjugated antibodies. Additionally, the instrument was utilized for the rapid and reproducible determination of cell density and viability.

Transposon mediated co-integration and co-expression of transgenes in CHO-DG44 cells

Background Transposon systems mediate stable integration of exogenous DNA elements into a host cell genome, and have been successfully used in mammalian cells for the generation of stable cell lines. The piggyBac (PB) transposon system has been shown to have several advantages over the other transposon system available [1-3]. It has also been shown to generate stable cell lines at significantly higher frequency than the conventional transfections [3]. Here, we investigated the efficiency of the piggyBac (PB) transposon to facilitate the co-expression of multiple artificial transposons, each bearing a single transgene and the puromycin resistance gene for selection. Green fluorescent protein (eGFP), red fluorescent protein (mKate), and a human IgG1 antibody were used as model proteins [4]. The effect of the stringency of selection on pool productivity was determined with increasing concentrations of puromycin. The duration of selection necessary for the generation of recombinant cell pools was also tested by selecting for a period of either 5 or 10 days.

The use of filler DNA for improved transfection and reduced DNA needs in transient gene expression with CHO and HEK cells

Background Transient gene expression (TGE) is a rapid method for the production of recombinant proteins. Protein productivity in TGE has improved significantly over the past decade, reaching 300 mg/L and 1 g/L in CHO DG44 (CHO) and HEK 293E (HEK) cells, respectively [1,2]. However, the amount of plasmid DNA needed for transfection remains relatively high, contributing significantly to the overall cost of the TGE process. In order to reduce the amount of plasmid DNA in TGE, we examined the possibility of partially replacing it with herring sperm DNA (non-coding “filler” DNA) in transfections of CHO and HEK cells.

Application of the novel and convenient IR/MAR gene amplification technology to the production of recombinant protein pharmaceuticals

Amplification of DHFR gene in CHO cells by selection of MTx has been widely applied to the establishment of stable cell lines that efficiently produce recombinant protein pharmaceuticals. However, the DHFR/MTx technology was highly time-and labor-consuming. On the other hand, we had found that a plasmid bearing a mammalian replication initiation region (IR) and a matrix attachment region (MAR) initiates gene amplification in mammalian cells, and it quite efficiently generate the chromosomal HSR and/or the extrachromosomal DMs [1,2]. This is a completely original technology of gene amplification, and we have revealed the mechanism why and how such plasmid may mimic gene amplification [2,3]. Now, we aimed to adopt this technology to the industrial production of recombinant protein pharmaceuticals. We constructed plasmids with or without IR/MAR, with several promoters for drug resistant gene, antibody gene or Fc receptor gene, and with various orientations of these elements. The plasmid DNA with several physical structures were transfected to human COLO 320DM or hamster CHO DG44 cells, with or without another DNA. The transformed cells were selected by various conditions. The polyclonal transformants or the cloned cells were evaluated by the protein production (ELISA), as well as by the structure of amplified region (FISH). As a result, the usage of IR/MAR technology enabled us to obtain cells, in which the introduced genes were amplified to a few hundreds to thousands copies per cells as DMs or HSR of various size and shape (Figure ​(Figure1),1), which depended both on the vector constructs and the host cell lines. Such stable cells with amplified genes could be obtained within one month, and the protein production was increased more than a hundred-fold compared with the case without IR/MAR. A cell clone showed the specific production rate that reached almost the highest reported for antibody protein (45 pg/cell/day). Furthermore, we have found several novel ways that further improve the protein production level. For example, the combination of the IR/MAR and the DHFR/MTx technologies synergistically work and far more rapidly and easily generate the cells of higher production rate than previously. Figure 1 The IR/MAR plasmid can generates DMs (A), HSR (B), Ladder-HSR (C), and Fine ladder-HSR (D). Among the metaphase chromosome spread, we detected the plasmid sequence by FISH in green. Gene expression was generally higher from DMs than HSR. However, DMs ... In conclusion, the IR/MAR technology is a novel highly-competitive technology for use in recombinant protein production, and it further has potentials for improvement.