Protein‐free transfection of CHO host cells with an IgG‐fusion protein: Selection and characterization of stable high producers and comparison to conventionally transfected clones

Abstract

In order to improve the current techniques of cell cultivation in the absence of serum, we have developed a protein-free transfection protocol for CHO cells, based on the Nucleofector technology. After starting with a heterogeneous pool of primary transfectants which express the fusion protein EpoFc, we isolated single clones and compared them with parallel clones generated by lipofection in serum-dependent cultivation. Our intensive characterization program was based on determination of specific productivity (q(p)) and analysis of genetic parameters. In two nucleofection experiments, transfection with 5 microg of DNA resulted in best productivities of the primary cell pools. After subcloning, the q(p) could be raised up to 27 pg x cells(-1) x day(-1). While the serum-dependent transfectants exhibited specific productivities up to 57 pg x cells(-1) x day(-1) in serum-dependent cultivation, a significant decrease that resulted in the range of q(p) of the protein-free transfectants was observed after switching to protein-free conditions. Investigation of genetic parameters revealed higher mRNA levels and gene copy numbers (GCN) for the protein-free adapted serum-dependent transfectants. Therefore, we assume that problems during protein-free adaptation (PFA) lead to a less efficient translation machinery after serum deprivation. We describe the generation of stable-producing recombinant CHO clones by protein-free transfection of a protein-free adapted host cell line, which reduces the risk of adverse clonal changes after PFA. The main advantage of this approach is the earlier predictability of clone behavior, which makes the generation of production clones by protein-free transfection, a viable and highly efficient strategy for recombinant cell line development.

Next-generation sequencing of the Chinese hamster ovary microRNA transcriptome: Identification, annotation and profiling of microRNAs as targets for cellular engineering

“…The experimental verification, whether overexpression of miRNAs that are repressed in serum-free adapted cell lines can restore some of the growth characteristics observed for CHO cells grown in the presence of serum is currently ongoing. Of further interest from a biotechnological perspective are miRNA transcription signatures that are specific to recombinant protein producing CHO cell lines, as these clonal cell lines are the result of gene amplification (Lattenmayer et al, 2007) and selection of clones with high specific recombinant protein production. Hence, the differential regulation of cgr-miR-21 in recombinant CHO cells is of high interest, not least, since human miR-21 is known to play an important role the regulation of cell growth and apoptosis (Krichevsky and Gabriely, 2009).…”

“…Serum-free adapted CHO-DUXB11 cells were cultivated in 1:1 DMEM/Ham's F12 media supplemented with 4 mM l -Gln, 0.25% soy peptone, 0.1% Pluronic F68 and 1x PF and HT supplement. The recombinant DUXB11 cells were transfected with an Erythropoietin-Fc fusion protein (Lattenmayer et al, 2007) and cultivated in the same medium with the addition of 0.19 μM methotrexate (MTX).…”

Stable overexpression of miR-17 enhances recombinant protein production of CHO cells

“…The recombinant CHO-EpoFc cell line was established as previously described (Lattenmayer et al, 2007) and was later adapted to growth in serum-free and l -glutamine free CD CHO medium (Gibco, Invitrogen, Carlsbad, CA, USA) supplemented with 0.096 μM MTX and 1 ml anti clumping agent (Gibco, Invitrogen, Carlsbad, CA, USA) per 500 ml medium (Taschwer et al, 2012). Cell cultures were cultivated in conical flasks with a working volume of 30 ml at 37 °C in a humidified atmosphere containing 7% carbon dioxide and constant shaking at 140 rpm.…”

Advances in Mammalian Cell Line Development Technologies for Recombinant Protein Production

“…Another reason for cloning the cells is that high producing clones occur rarely in the heterogeneous cell populations after transfection and gene amplification [24,25,26,27,28]. Hence, in situations where high recombinant protein productivity is essential for product commercial viability (for example, the production of monoclonal antibody therapeutics) thousands of clones have to be screened to obtain a set of production clones.…”