Littérature scientifique sur le sujet « Transposons, CHO cells, recombinant proteins, antibodies »

Recently, many biopharmaceuticals have been developed such as cytokines, growth factors, and antibodies. These recombinant proteins are mostly expressed by CHO cells. However, the culture medium of CHO cells requires the addition of serum, which can contain unknown biological substances such as viruses, or requires the addition of expensive growth factors. To avoid the risks of biological ingredients and to decrease the cost of biopharmaceutical production, we developed a non-protein and lipid medium adopted (NPLAd) CHO cell line using the adapted culture method. Our results indicated that autocrine EGF production and insulin addition are essential for NPLAd CHO cell growth. However, the rate of cell proliferation of NPLAd CHO cells was decreased compared with original CHO-K1 cells. The proliferation of NPLAd CHO cells was improved by GM3 addition, suggesting increased signaling efficiency of autocrine factors. No difference was found in the growth rate between original CHO-K1 and NPLAd CHO cells supplemented with insulin and GM3. The productivity of recombinant protein in NPLAd CHO cells was verified using secreting luciferase reporter system. As a result, luciferase activity in NPLAd CHO cells showed more than three times higher than in the original CHOK1 cells. The results suggested that this cell line could be useful for biopharmaceutical recombinant protein.

ABSTRACTVAR2CSA, a member of thePlasmodium falciparumerythrocyte membrane protein 1 (PfEMP1) family, is a leading candidate for use in vaccines to protect first-time mothers from placental malaria (PM). VAR2CSA, which is comprised of a series of six Duffy binding-like (DBL) domains, binds chondroitin sulfate A (CSA) on placental syncytiotrophoblast. Several recombinant DBL domains have been shown to bind CSA. In order to identify and develop recombinant proteins suitable for clinical development, DBL2X and DBL3X, as well as their respective third subdomain (S3) from the FCR3 parasite clone, were expressed inEscherichia coli, refolded, and purified. All but DBL3X-S3 recombinant proteins bound to CSA expressed on Chinese hamster ovary (CHO)-K1 cells but not to CHO-pgsA745 cells, which are CSA negative as determined by flow cytometry. All but DBL3X-S3 bound to CSA on chondroitin sulfate proteoglycan (CSPG) as determined by surface plasmon resonance (SPR) analysis. Purified IgG from rats and rabbits immunized with these four recombinant proteins bound homologous and some heterologous parasite-infected erythrocytes (IE). Using a novel flow cytometry inhibition-of-binding assay (flow-IBA), antibodies against DBL3X-S3 inhibited 35% and 45% of IE binding to CSA on CHO-K1 cells compared to results for soluble CSA (sCSA) and purified multigravida (MG) IgG, respectively, from areas in Tanzania to which malaria is endemic. Antibodies generated against the other domains provided little or no inhibition of IE binding to CSA on CHO-K1 cells as determined by the flow cytometry inhibition-of-binding assay. These results demonstrate for the first time the ability to identify antibodies to VAR2CSA DBL domains and subdomains capable of inhibiting VAR2CSA parasite-IE binding to CSA by flow cytometry. The flow cytometry inhibition-of-binding assay was robust and provided an accurate, reproducible, and reliable means to identify blocking of IE binding to CSA and promises to be significant in the development of a vaccine to protect pregnant women.

Phosphatidylglycerophosphate (PGP) synthase catalyses the committed step in the biosynthesis of phosphatidylglycerol and cardiolipin in mammalian cells. Recently we isolated a Chinese hamster ovary (CHO) PGS1 cDNA encoding PGP synthase. In the present study we purified this PGP synthase to near-homogeneity from the mitochondrial fraction of CHO-K1 cells; the final enzyme preparation gave a single 60kDa protein on SDS/PAGE. Polyclonal antibodies raised against a recombinant CHO PGS1 protein cross-reacted with the purified 60kDa protein and with CHO membrane proteins of 60kDa and 62kDa that increased after transfection with the PGS1 cDNA. The 60 and 62kDa protein levels in a PGP synthase-defective mutant of CHO-K1 cells were markedly lower than those in CHO-K1 cells. These results indicated that the purified 60kDa protein was PGP synthase encoded by the PGS1 gene. In addition we found that the purified PGP synthase had no PGP phosphatase activity, indicating that phosphatidylglycerol was produced from CDP-diacylglycerol through two steps catalysed by distinct enzymes, PGP synthase and PGP phosphatase.

Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants. The major proteins secreted by these cells consisted of latent TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.

Recombinant type 1 transforming growth factor beta (TGF-beta) was expressed to high levels in CHO cells by using dihydrofolate reductase (dhfr) gene amplification. The expression plasmid was derived from the pSV2 vectors and contained, in tandem, the simian TGF-beta and mouse dhfr cDNAs. Transcription of both cDNAs was controlled by the simian virus 40 early promoter. Stepwise selection of transfected CHO cells in increasing concentrations of methotrexate yielded cell lines that expressed amplified TGF-beta nucleic acid sequences. The expression plasmid DNA was amplified greater than 35-fold in one of the methotrexate-selected transfectants. The major proteins secreted by these cells consisted of latent TGF-beta and TGF-beta precursor polypeptides, as judged by immunoblots by using site-specific anti-peptide antibodies derived from various regions of the TGF-beta precursor. Levels of recombinant TGF-beta protein secreted by these cells approached 30 micrograms/24 h per 10(7) cells and required prior acidification for optimal activity; nonacidified supernatants were approximately 1% as active as acidified material. Antibodies directed toward sequences present in the mature growth factor readily identified a proteolytically processed recombinant TGF-beta which, on sodium dodecyl sulfate-polyacrylamide gels, comigrated with highly purified natural TGF-beta. In addition to mature recombinant TGF-beta, site-specific antibodies demonstrated the existence of larger TGF-beta precursor polypeptides. The availability of biologically active recombinant type 1 TGF-beta and precursor forms should provide a means to examine the structure, function, and potential in vivo therapeutic use of this growth factor.

Background: Recombinant anti-vascular endothelial growth factor (VEGF) monoclonal antibodies and Fc-fusion proteins have been widely used for the effective treatment of retinal neovascular diseases. In this regard, VEGFR-Fc fusions, which act as strong VEGF inhibitors, have been approved for the treatment of age-related macular degeneration (AMD) and diabetic macular edema (DME). Production of monoclonal antibodies and Fc-fusion proteins relies on mammalian host systems such as Chinese hamster ovary (CHO) cells. Application of genomic regulatory elements including scaffold/matrix attachment regions (SAR/MARs) can profoundly affect recombinant protein expression in CHO cells. Methods: To construct the VEGFR-Fc expression vectors, the enhanced green fluorescent protein (EGFP) gene was replaced by the VEGFR-Fc coding sequence in pEGFP-SAR-puro and pEGFP-puro vectors. Recombinant plasmids were transfected to CHO-K1 cells using TurboFect transfection reagent. VEGFR-Fc expression was evaluated in transiently transfected cells as well as stable cell pools and clones using an enzyme-linked immunosorbent assay (ELISA). Results: IFN-SAR showed no significant effect on transient expression of VEGFR-Fc during 72 h of culture. However, a 2.2-fold enhancement in VEGFR-Fc fusion protein titer was observed in IFN-SAR containing stable cell pools. Further evaluation of the VEGFR-Fc expression level in single-cell clones also indicated that clones with the highest VEGFR-Fc expression belonged to the pools transfected with IFN-SAR construct. Conclusion: Our results indicate that the incorporation of IFN-SAR in expression vector can increase the expression of VEGFR-Fc in stable cell pools as well as single-cell clones. In contrast, transient expression of the fusion protein was not affected by IFN-SAR. More studies are needed to investigate the mechanism underlying this effect, including the analysis of mRNA expression and gene copy number in stable cell pools as well as clonal cells.

As indicated by an ever-increasing number of FDA approvals, biotherapeutics constitute powerful tools for the treatment of various diseases, with monoclonal antibodies (mAbs) accounting for more than 50% of newly approved drugs between 2014 and 2018 (Walsh, 2018). The pharmaceutical industry has made great progress in developing reliable and efficient bioproduction processes to meet the demand for recombinant mAbs. Mammalian cell lines are preferred for the production of functional, complex recombinant proteins including mAbs, with Chinese hamster ovary (CHO) cells being used in most instances. Despite significant advances in cell growth control for biologics manufacturing, cellular responses to environmental changes need to be understood in order to further improve productivity. Metabolomics offers a promising approach for developing suitable strategies to unlock the full potential of cellular production. This review summarizes key findings on catabolism and anabolism for each phase of cell growth (exponential growth, the stationary phase and decline) with a focus on the principal metabolic pathways (glycolysis, the pentose phosphate pathway and the tricarboxylic acid cycle) and the families of biomolecules that impact these circuities (nucleotides, amino acids, lipids and energy-rich metabolites).

We have used fragments of the TSH receptor (TSHR) expressed in E. coli as glutathione S-transferase fusion proteins to produce rabbit polyclonal antibodies and a panel (n=5) of monoclonal antibodies to the extracellular fragment of the TSHR. The binding characteristics of the antibodies to linear, conformational, glycosylated and unglycosylated forms of the receptor in different assay systems have been investigated. The reactivity of these antibodies with the TSHR was assessed by Western blotting with both native and recombinant human TSHR expressed in CHO cells, immunoprecipitation of 35S-labelled full-length TSHR produced in an in vitro transcription/ translation system, immunoprecipitation of 125I-TSH/TSHR complexes, inhibition of 125I-TSH binding to the TSHR and fluorescence activated cell sorter (FACS) analysis of binding to CHO-K1 cells expressing the TSHR on their cell surface. Fab fragments of monoclonal antibodies were isolated, labelled with 125I and used to determine the affinity constants of these antibodies with receptor, bound and free Fab being separated by polyethylene glycol (PEG) precipitation. Rabbit polyclonal and mouse monoclonal antibodies reacted with the TSHR in Western blotting and one monoclonal antibody (3C7) was able to inhibit 125I-TSH binding to native human TSHR (74% inhibition), recombinant human TSHR (84% inhibition) and porcine TSHR (65% inhibition). Affinity constant values for TSHR monoclonal antibody Fab fragments calculated using Scatchard analysis were about 10(7) M(-1). Four out of five monoclonal antibodies reacted in FACS analysis with TSHR expressed on the surface of CHO-K1 cells. The FACS unreactive monoclonal (3C7) bound well to detergent solubilised TSH receptors and this emphasised the importance of using a combination of FACS analysis and radioactively-labelled probes in analysis of the TSH receptor. The monoclonal antibodies produced in this study were found to be of relatively low affinity but proved useful for detection of the receptor by Western blotting and by FACS analysis.

Cell-free protein synthesis (CFPS) is an emerging tool for the rapid production of difficult-to-express proteins as well as for identifying protein synthesis bottlenecks. In CFPS, the biotic phase is substituted by extracts of living cells devoid of any of their own genetic material. The main advantage is that these systems delineate cell growth from recombinant protein production, enabling the expression of targets that would otherwise place too big a burden on living cells. We have conducted a techno-economic analysis of a CFPS system to produce monoclonal antibodies (mAbs) using extracts of Chinese hamster ovary (CHO) cells. We compare the performance of the CFPS system with two alternative production strategies: stable and transient gene expression in CHO cells. Our assessment shows that the viability of CFPS for mAb production requires a significant increase in the product yield and the recycling of high-cost components such as DNA. Nevertheless, CFPS shows significant promise for personalized medicine applications, providing a platform for on-demand production and simplified supply chains.

Abstract In biopharmaceutical production, Chinese hamster ovary (CHO) cells derived from Cricetulus griseus remain the most commonly used host cell for recombinant protein production, especially antibodies. Over the last decade, in-depth multi-omics characterization of these CHO cells provided data for extensive cell line engineering and corresponding increases in productivity. However, exosomes, extracellular vesicles containing proteins and nucleic acids, are barely researched at all in CHO cells. Exosomes have been proven to be a ubiquitous mediator of intercellular communication and are proposed as new biopharmaceutical format for drug delivery, indicator reflecting host cell condition and anti-apoptotic factor in spent media. Here we provide a brief overview of different separation techniques and subsequently perform a proteome and regulatory, non-coding RNA analysis of exosomes, derived from lab-scale bioreactor cultivations of a CHO-K1 cell line, to lay out reference data for further research in the field. Applying bottom-up orbitrap shotgun proteomics and next-generation small RNA sequencing, we detected 1395 proteins, 144 micro RNA (miRNA), and 914 PIWI-interacting RNA (piRNA) species differentially across the phases of a batch cultivation process. The exosomal proteome and RNA data are compared with other extracellular fractions and cell lysate, yielding several significantly exosome-enriched species. Key points • First-time comprehensive protein and miRNA characterization of CHO exosomes. • Isolation protocol and time point of bioprocess strongly affect quality of extracellular vesicles. • CHO-derived exosomes also contain numerous piRNA species of yet unknown function.

Proteine ricombinanti e anticorpi sono reagenti chiave per lo sviluppo di saggi immunodiagnostici. Tuttavia proteine che hanno modifiche post-transcrizionali complesse o più subunità, come gli anticorpi, sono raramente espresse in microrganismi. Infatti, per ottenere una corretta conformazione sono necessarie le modifiche post-transcrizionali tipiche delle cellule di mammifero. Negli ultimi anni sono stati sviluppati diversi metodi per il trasferimento genico, ma nonostante ciò è ancora difficile ottenere linee cellulari stabili e altamente produttive. Le strategie convenzionali si basano sull’integrazione spontanea di DNA episomale e perciò hanno una bassa efficienza di generazione di cloni e, spesso, una scarsa espressione del transgene a causa di effetti posizionali e del silenziamento di transgeni integrati come concatameri.Per superare questi limiti, ho valutato le potenzialità dell’utilizzo dei trasposoni piggyBac come nuovo metodo per veicolare transgeni. I sistemi basati sui trasposoni sfruttano la capacità dell’enzima trasposasi di catalizzare in trans l’integrazione del transgene in regioni genomiche attivamente trascritte. Per studiare questo sistema e confrontarlo con i vettori tradizionali ho clonato, in entrambi i plasmidi, due proteine modello, il fibroblast growth factor 23 (hFGF23) e un anticorpo ricombinante per la produzione in cellule CHO (chinese hamster ovary). I risultati ottenuti hanno dimostrato che la trasposizione aumenta la frequenza di generazione di linee cellulari stabili di circa 15 volte rispetto alla trasfezione di plasmidi standard. Inoltre, lo screening dei cloni è facilitato perchè la generazione di linee cellulari stabili è più rapida e la frequenza di cloni produttivi è maggiore. Infine ho dimostrato che la frequenza di cloni altamente produttivi è influenzata dalla forza del promotore clonato nel PB. Grazie a questo sistema, ho generato linee cellulari in grado di esprimere l’hFGF23 con una resa media di 35 mg/L in colture batch. La proteina purificata è stata correttamente riconosciuta da un test immunodiagnostico (DiaSorin) con risultati paragonabili ad un hFGF23 ricombinante commerciale. In seguito ho valutato l'utilizzo del sistema dei trasposoni per la generazione di anticorpi ricombinanti. Dopo l'identificazione delle regioni variabili della catena pesante (HC) e leggera (LC) di una immunoglobulina IgG2a murina sviluppata con la tecnologia dell’ibridoma, ho generato un anticorpo chimerico IgG1. A tal scopo, le sequenze variabili sono state clonate a monte delle sequenze costanti della rispettive catene e poi inserite o in vettori standard o nei trasposoni per la co-espressione in cellule CHO. I risultati ottenuti hanno evidenziato i vantaggi dell’uso del sistema dei trasposoni PB per integrare stabilmente due transgeni con un’unica trasfezione, mantenendo un rapporto molare appropriato come richiesto per la corretta formazione di anticorpi (sbilanciato a favore di LC). Le integrazioni casuali, tipiche delle trasfezioni con plasmidi standard, hanno creato difficoltà nella co-espressione dei transgeni, infatti l’80% dei cloni aveva una produttività molto bassa. Al contrario, le integrazioni mediate dalla trasposasi PB hanno aumentato il numero di cloni altamente produttivi. Infine, l'immunoglobulina IgG1 chimerica è stata purificata e ha mostrato affinità e prestazioni immunochimiche paragonabili a quelle dell'anticorpo IgG2a prodotto dall’ibridoma originale, confermando le potenzialità del nostro sistema. In conclusione, questa tesi dimostra che il sistema basato sui trasposoni PB può essere considerato una rapida ed efficace alternativa al metodo standard per la generazione di linee cellulari stabili capaci di produrre alte rese di reagenti critici per applicazioni diagnostiche.
Recombinant proteins and antibodies are the key reagents for development of diagnostic immunoassays. Recombinant proteins are commonly produced in both prokaryotic and eukaryotic microorganism because they allow high productivity with rapidity and low costs. However, complex proteins that contain posttranslational modifications, several disulphide bonds or multiple subunits, such as antibodies, are challenging to be expressed in these hosts. Indeed, to obtain properly folded and functional complex biomolecules it is required the posttranscriptional metabolic machinery only available in mammalian cells. Although different approaches for gene transfer have been developed in the last 15 years, it is still difficult to obtain stable, high-producing cell lines for industrial applications. Conventional methods, based on spontaneous integration of episomal DNA, often result in low efficiency of clone establishment and in low transgene expression mainly due to plasmid concatemers silencing and/or positional effects. To overcome these limitations, in my thesis project, I evaluated the potentiality of using an improved PiggyBac (PB) transposon system as new molecular tool for transgene delivery. Transposon-based approaches rely on the ability of transposase enzyme to catalyze single transgene integration into actively transcribed regions of genome. In order to assess the suitability of PB transposon vectors compared to conventional methods, two different model proteins, the human fibroblast growth factor 23 (hFGF23) and one mouse recombinant antibody, have been cloned into both expression plasmids and produced in CHO (Chinese Hamster Ovary) cells. A preliminary comparison between the two expression systems demonstrated that PB transposition increased the frequency of stable cell lines generation up to 10-15 fold compared to standard plasmid transfection. Cell lines establishment was faster and the frequency of high-producing clones was enhanced, thus reducing the extent of clones screening to recover the best performing cell lines. In addition, I also evaluated that changing PB promoter strength affected the frequency of high-producing clones. Taking advantages from these results, I was able to generate CHO cell lines expressing hFGF23 protein with an average yield of 35 mg/L in batch culture. The obtained purified protein was correctly detected by an automated chemiluminescence immunoassay (DiaSorin) with results comparable to a commercial available mammalian recombinant hFGF23 protein and it resulted biologically active when tested in a cell proliferation assay. Then I evaluated the application of PB transposon system for the generation of recombinant antibodies. After identification of heavy and light chain variable regions from an IgG2a mouse immunoglobulin developed by hybridoma technology, I have generated a chimeric IgG1 antibody by cloning mouse variable regions upstream of mouse heavy and light chain constant sequences. The ensuing full length sequences were cloned into standard vectors and transposon for co-expression in CHO cells. In these set of experiments, my results highlighted the advantages of using PB transposon to stably integrate, in one transfection step, two different transgenes with an appropriate molar ratio (light/heavy chain ratio unbalanced in favour of light chian), as required for proper antibody assembly. Random integrations, typical of standard plasmid transfections, showed difficulties in fine tuning of co-transfected transgenes expression, resulting in 80% of clones with very low productivity. In contrast, integrations mediated by PB transposase increased the number of high producing clones. The chimeric IgG1 immunoglobulin, purified from the best producing clone, showed affinity and immunochemical performances comparable to that of the parental hybridoma IgG2a antibody, confirming the potentiality of our system. In conclusion, my work demonstrates that the PB transposon system is a quick and powerful alternative to standard method for generation of stable, high-producing recombinant mammalian cell lines to generate critical reagents useful for diagnostic applications.