Evaluation of the Influenza A Replicon for Transient Expression of Recombinant Proteins in Mammalian Cells

Recombinant protein expression in mammalian cells has become a very important technique over the last twenty years. It is mainly used for production of complex proteins for biopharmaceutical applications. Transient recombinant protein expression is a possible strategy to produce high quality material for preclinical trials within days. Viral replicon based expression systems have been established over the years and are ideal for transient protein expression. In this study we describe the evaluation of an influenza A replicon for the expression of recombinant proteins. We investigated transfection and expression levels in HEK-293 cells with EGFP and firefly luciferase as reporter proteins. Furthermore, we studied the influence of different influenza non-coding regions and temperature optima for protein expression as well. Additionally, we exploited the viral replication machinery for the expression of an antiviral protein, the human monoclonal anti-HIV-gp41 antibody 3D6. Finally we could demonstrate that the expression of a single secreted protein, an antibody light chain, by the influenza replicon, resulted in fivefold higher expression levels compared to the usually used CMV promoter based expression. We emphasize that the influenza A replicon system is feasible for high level expression of complex proteins in mammalian cells

Genome-wide analysis of recombinant protein expression in Chinese hamster ovary cells

Chinese hamster ovary (CHO) Zellen sind derzeit die Arbeitstiere der biopharmazeutischen Industrie für die Produktion von rekombinanten therapeutischen Proteinen, hauptsächlich, weil sie dazu befähigt sind, in Suspension zu wachsen und komplexe Proteine mit human-ähnlicher Glykosylierung zu sekretieren. Obwohl CHO Zellen schon seit über 25 Jahren dafür verwendet werden, sind die zellulären Prozesse, welche die Synthese und Sekretion von rekombinanten Proteinen steuern und limitieren, noch sehr schlecht verstanden. Zudem stellen andere Expressionssysteme wie die Hefe Pichia pastoris eine interessante Alternative dar. Um die Fähigkeit von CHO Zellen und P. pastoris rekombinante Proteine zu sekretieren zu vergleichen, wurden Zelllinien bzw. Stämme entwickelt, die zwei unterschiedlich komplexe Proteine produzieren und diese in vergleichbaren Bioprozessen kultiviert. Unsere Ergebnisse zeigen, dass die Sekretionsmaschinerie von CHO Zellen wesentlich effizienter ist. Besonders die Sekretion des komplexeren Proteins war in P. pastoris stark beeinträchtigt. Um die rekombinante Proteinproduktion in CHO Zellen auf einer physiologischen Ebene zu untersuchen und um Genexpressionsdaten für einen speziesübergreifenden Vergleich mit P. pastoris zu generieren, wurden niedrig- und hochproduzierende CHO Zelllinien sowie ein Nichtproduzent unter stationären Bedingungen kultiviert und die Gesamtgenexpression mittels eines neuen CHO-spezifischen Microarrays gemessen. Zusätzlich wurde auch die Expression von microRNAs analysiert. MicroRNAs sind kurze (22 Nukleotide) nicht-kodierende RNAs, die eine wichtige Rolle bei der Regelung der Genexpression in höheren Eukaryoten spielen. Daher wird ihnen ein großes Potential als Ziel für die gentechnische Optimierung (Cell Engineering) von CHO Zellen zugeschrieben. In der vorliegenden Arbeit wurden etliche Gene und microRNAs identifiziert, die möglicherweise an der rekombinanten Proteinproduktion in CHO Zellen beteiligt sind und daher potenzielle Ziele für Cell Engineering zur Verbesserung bioprozessrelevanter Eigenschaften, wie Produktivität und Langlebigkeit darstellen. Allerdings deuten unsere Ergebnisse auch darauf hin, dass die Reaktion von CHO Zellen auf die Expression von Fremdproteinen stark produkt- und/oder klon-spezifisch ist.Chinese hamster ovary (CHO) cells are currently the workhorses of the biopharmaceutical industry for the production of recombinant therapeutic proteins, mainly because of their ability to grow in suspension and to secrete complex proteins with human-like glycosylation. However, although CHO cells have been used for more the 25 years, the cellular processes that control and limit recombinant protein synthesis and secretion are still poorly understood. In addition, other expression systems such as the yeast Pichia pastoris are about to enter this field. To compare their capability for recombinant protein secretion, high producing CHO cell lines and P. pastoris strains expressing two proteins of different complexity were established and cultivated in comparable bioprocesses. Our results indicate that the protein secretion machinery of CHO cells is much more efficient, and especially the secretion of the more complex protein was strongly impaired in P. pastoris. To investigate recombinant protein production in CHO cells at a physiological level and to provide transcriptomics data for a cross-species comparison with P. pastoris, low and high producing CHO cell lines as well as a non-producer were cultivated under steady-state conditions and global gene expression was profiled using a novel CHO-specific microarray. In addition, the expression of microRNAs was analyzed. MicroRNAs are short (22 nucleotides) non-coding RNAs that play an important role in the regulation of gene expression in higher eukaryotes and therefore may have great potential as cell engineering targets. In the present study, we identified several genes and microRNAs that might be involved in recombinant protein production in CHO cells and consequently may constitute potential targets for cell engineering to improve bioprocess-relevant properties such as productivity or longevity. However, our results also suggest that the reaction of CHO cells to heterologous protein expression is strongly product and/or clone-specific.by Andreas Michael MaccaniAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Univ. für Bodenkultur, Diss., 2014OeBB(VLID)193168

Plasmids and plasmid amounts used for various experiments.

<p>pEGFP-N1, pcDNA luc, pRC-LC and pRC-HC carry the CMV immediate-early promoter, pGL3-control and pRL carry SV40 promoter elements, pTripolis and pBi-NP drive bidirectional transcription (mRNA and vRNA) of the influenza polymerases and the NP protein. pMono-EGFP, pMono-lucNS, pMono-LC, pMono-HC, pMono-lucM and pMono-lucPB1 drive monodirectional (vRNA) transcription of the gene of interest. AB stands for antibody, LC stands for light chain experiments.</p

Schematic drawings of plasmids used to generate the influenza replicon based expression system.

<p>pTripolis and pBi-NP generate the influenza replicon (mRNA/protein and vRNA) whereas pMono plasmids with various genes of interest or reporter genes drive transcription of vRNA.</p

EGFP expression.

<p>HEK-293 cells were either tranfected with pEGFP-N1 (CMV) or with the influenza replicon and pMono-EGFP (Flu Replicon). Pictures were taken 56 and 98 hours post transfection. Non-transfected cells were used as negative control (Neg. Control).</p

Activity of the influenza replicon in a human, a canine and a rodent cell line.

<p>HEK -293, MDCK or CHO-K1 cells were either tranfected with pcDNA-luc (CMV) or with the influenza replicon and pMono-lucNS (Influenza Replicon). The HEK-293 negative control has been transfected with pMono-lucNS and pBi-NP only. Additionally, pRL, coding for <i>Renilla</i> luciferase, was cotransfected in all cases. In case of MDCK and CHO-K1, untransfected cells have been used as negative controls and can therefore not be normalized, absolute values can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013265#s2" target="_blank">Results</a> section. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Data represents arithmetic mean values and standard deviation.</p

Light chain expression.

<p>HEK-293 cells were either tranfected with pRC-LC (CMV) or with the influenza replicon and pMono-LC (Flu Replicon). Light chain concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and standard deviation.</p

Influence of different NCRs on the expression level

<p>. HEK-293 cells were tranfected with the influenza replicon and either pMono-lucNS (NCR NS) or pMono-lucM (NCR M) or pMono-lucPB1 (NCR PB1) or with pMono-lucNS and pBi-NP only (Neg. Contr.). Additionally, pRL, coding for <i>Renilla</i> luciferase, was cotransfected in all cases. Cells were harvested 48 hours post transfection and subjected to luciferase assay. Firefly values have been normalized to <i>Renilla</i> values. Data represents arithmetic mean values and standard deviation.</p

FACS analysis of cells transfected with either pEGFP-N1 (CMV) or the influenza replicon system and pMono-EGFP (Flu Replicon) 77 hours post transfection.

<p>Cells transfected with the replicon system show two very distinct populations in the forward light scatter (EGFP), one completely negative, the other highly positive. pEGFP-N1 driven EGFP expressions shows a broad distribution spanning from completely negative to moderate and high expressing cells.</p

Expression of a the monoclonal anti-gp41 antibody 3D6.

<p>HEK-293 cells were either tranfected with pRC-LC and pRC-HC (CMV HC∶LC = 1∶1, CMV HC∶LC = 10∶1) or with the influenza replicon and pMono-LC and pMono-HC (Flu Replicon HC∶LC = 1∶1, Flu Replicon HC∶LC = 10∶1). Heavy chain to light chain ratios of 1∶1 and 10∶1 were tested. Antibody concentration in the culture supernatant was monitored for 101 hours by ELISA. Data represents arithmetic mean values and standard deviation.</p