M. Derouazi

100-liter transient transfection

This is the first report of two successful 100 l scale transienttransfections in a standard stirred bioreactor. More than half a gram of a monoclonal antibody (IgG) were produced in less than 10 days using a technology called large-scale transient gene expression(LS-TGE). Suspension adapted HEK 293 EBNA SF cells were transfectedwithin a 150 l (nominal) bioreactor by a modified calcium phosphateco-precipitation method with more than 75 mg of plasmid DNA per run.A mixture of three different plasmids, one encoding for the heavychain of a human recombinant immunoglobulin, the other for the corresponding light chain and a third one for the green fluorescent protein (GFP, 2–4% of DNA in transfection cocktail)were co-transfected. The GFP vector was chosen to monitor transfection efficiency. Expression of GFP could be registered asearly as 20 h after DNA addition, using fluorescence microscopy. We demonstrate that transient transfection can be done at the100 l scale, thus providing a new tool to produce hundreds of milligrams or even gram amounts of recombinant protein. Akey advantage of LS-TGE resides in its speed. In the presentedcases, the entire production process for the synthesis of halfa gram of a recombinant antibody, including DNA preparationand necessary expansion of cells prior to transfection, wasexecuted in less than a month. Having an established transfection/expression process allows to run productioncampaigns for any given protein, within one facility, with onesingle host cell line and therefore only one single seed train. Without any need to create and maintain stable cell lines, expression of new r-proteins is not only faster and more economical but also more flexible.

Calcium phosphate transfection generates mammalian recombinant cell lines with higher specific productivity than polyfection

Transfection with polyethylenimine (PEI) was evaluated as a method for the generation of recombinant Chinese hamster ovary (CHO DG44) cell lines by direct comparison with calcium phosphate‐DNA coprecipitation (CaPO4) using both green fluorescent protein (GFP) and a monoclonal antibody as reporter proteins. Following transfection with a GFP expression vector, the proportion of GFP‐positive cells as determined by flow cytometry was fourfold higher for the PEI transfection as compared to the CaPO4 transfection. However, the mean level of transient GFP expression for the cells with the highest level of fluorescence was twofold greater for the CaPO4 transfection. Fluorescence in situ hybridization on metaphase chromosomes from pools of cells grown under selective pressure demonstrated that plasmid integration always occurred at a single site regardless of the transfection method. Importantly, the copy number of integrated plasmids was measurably higher in cells transfected with CaPO4. The efficiency of recombinant cell line recovery under selective pressure was fivefold higher following PEI transfection, but the average specific productivity of a recombinant antibody was about twofold higher for the CaPO4‐derived cell lines. Nevertheless, no difference between the two transfection methods was observed in terms of the stability of protein production. These results demonstrated the feasibility of generating recombinant CHO‐derived cell lines by PEI transfection. However, this method appeared inferior to CaPO4 transfection with regard to the specific productivity of the recovered cell lines. Biotechnol. Bioeng.

DNA Delivery by Microinjection for the Generation of Recombinant Mammalian Cell Lines

Gene transfer methods for producing recombinant cell lines are often not very efficient. One reason is that the recombinant DNA is delivered into the cell cytoplasm and only a small fraction reaches the nucleus. This chapter describes a method for microinjecting DNA directly into the nucleus. Direct injection has several advantages including the ability to deliver a defined copy number into the nucleus, the avoidance of DNAses that are present in the cell cytoplasm, and the lack of a need for extensive subcloning to find the recombinant cells. The procedure is described for two cell lines, CHO DG44 and BHK-21, using green fluorescent protein as a reporter gene. However, this method could easily be adapted to other cells lines and using other recombinant genes.

Gene Transfer to a Animal Cells in Culture by Microinjection: A Novel Tool to Create Recombinant Cell Lines?

We are using microinjection of plasmid DNA into the cytoplasm and nucleus of Chinese hamster ovary (CHO) cells to better understand the mechanisms involved in DNA degradation and integration within the cell and to identify the requirements for the establishment of highly productive cell lines. Microinjection is an excellent tool for these studies since it allowed us to control the timing and the location of DNA transferred into single cells.

Towards an Affinity-Based Capture System for the Isolation of High Expression Cells Using a Co-Expressed Surface Protein

Identification, selection and expansion of the highest producing mammalian cell lines is tedious and often dependent on chance. We are attempting to establish a novel cell identification and selection system, based on the expression of a tagged plasma membrane protein that serves as a capturing device for rare cells overproducing a co-expressed recombinant protein. Here we studied the feasibility of this approach with transiently transfected CHO cells in suspension culture. Cells expressing an epitope-tagged plasma membrane protein were selected with antibody-coated protein A beads. We found that with increased expression of the tagged protein the level of the co-expressed recombinant protein was reduced. However, the data do support the notion that immobilized antibodies can be employed to select cells displaying an epitope-tagged protein.

Stability and Cytogenetic Characterization of Recombinant CHO Cell Lines Established by Microinjection and Calcium Phosphate Transfection

Chinese hamster ovary cells (CHO) are widely used for the stable production of recombinant proteins. Typically, recombinant cell lines are characterized for the stability of protein expression over a period corresponding to the time needed to scale-up the culture and harvest the product (e.g. 2 to 3 months), for the number of plasmid copies integrated into the host genome, and for the quality and quantity of the recombinant protein. In this study we extended the characterization to the cytogenetic level. Sixteen recombinant CHO cell lines were established using calcium phosphate transfection and microinjection as DNA transfer methods. For each cell line we observed by fluorescence in situ hybridization a single integration site regardless of the gene delivery method, the topology of the DNA (circular or linear), or the integrated plasmid copy number (between 1 and 50). Integration was not targeted to a specific chromosome. Chromosomal rearrangements were observed in about half of these cell lines. This phenomenon occurred independently of the gene transfer method. Interestingly the rearrangements were not on the chromosome where the plasmid integrated. We observed rearrangements between chromosomes and chromosomal imbalances.

Scalable, Serum-free, High Efficiency Transfection of CHO Cells using an Economical DNA Transfer Vehicle

Reports on large-scale transient gene expression (TGE) in mammalian cells focus on two transfection technologies. Polyethyleneimine (PEI) and calcium phosphate (CaPi) mediated transfection have both been shown to work at scales beyond 10 liters. Unfortunately, both approaches yield higher recombinant protein (r-protein) levels in the presence of serum than in its absence. Since serum is a major cost factor and generally a hindrance to protein purification, our goal is to develop a productive large-scale TGE process in the absence of serum.