LncRNA analysis of mAb producing CHO clones reveals marker and engineering potential.

Long non-coding RNAs (lncRNAs) are a potential new cell line engineering tool for improvement of yield and stability of CHO cells. In this study, we performed RNA sequencing of mAb producer CHO clones to study the lncRNA and protein coding transcriptome in relation to productivity. First, a robust linear model was used to identify genes correlating to productivity. To unravel specific patterns in expression of these genes, we employed weighted gene coexpression analysis (WGCNA) to find coexpressed modules, looking both for lncRNAs and coding genes. There was little overlap in the genes associated with productivity between the two products studied, possibly due to the difference in absolute range of productivity between the two mAbs. Therefore, we focused on the product with higher productivity and stronger candidate lncRNAs. To evaluate their potential as engineering targets, these candidate lncRNAs were transiently overexpressed or deleted by stable CRISPR Cas9 knock out both in a high and a low productivity subclone. We found that the thus achieved expression level of the identified lncRNAs, as confirmed by qPCR, does correlate well to productivity, so that they represent good markers that may be used for early clone selection. Additionally, we found that the deletion of one tested lncRNA region decreased viable cell density (VCD), prolonged culture time and increased cell size, final titer and specific productivity per cell. These results demonstrate the feasibility and usefulness of engineering lncRNA expression in production cell lines.

Utilization of non-AUG initiation codons in a flow cytometric method for efficient selection of recombinant cell lines.

Here we describe a method that couples flow cytometric detection with the attenuated translation of a reporter protein to enable efficient selection of CHO clones producing high levels of recombinant proteins. In this system, a small cell surface reporter protein is expressed from an upstream open reading frame utilizing a non-AUG initiation (alternate start) codon. Due to the low translation initiation efficiency of this alternate start codon, the majority of translation initiation events occur at the first AUG of the downstream open reading frame encoding the recombinant protein of interest. While translation of the reporter is significantly reduced, the levels are sufficient for detection using flow cytometric methods and, in turn, predictive of protein expression from the gene of interest since both ORFs are translated from the same mRNA. Using this system, CHO cells have been sorted to obtain enriched pools producing significantly higher levels of recombinant proteins than the starting cell population and clones with significantly better productivity than those generated from limiting dilution cloning. This method also serves as an effective screening tool during clone expansion to enable resources to be focused solely on clones with both high and stable expression.

Accelerated clone selection for recombinant CHO CELLS using a FACS-based high-throughput screen.

Flow cytometry was partnered with a nonfluorescent reporter protein for rapid, early stage identification of clones producing high levels of a therapeutic protein. A cell surface protein, not normally expressed on CHO cells, is coexpressed, as a reporter, with the therapeutic protein and detected using a fluorescently labeled antibody. The genes encoding the reporter protein and the therapeutic protein are linked by an IRES, so that they are transcribed in the same mRNA but are translated independently. Since they each arise from a common mRNA, the reporter protein's expression level accurately predicts the relative expression level of the therapeutic protein for each clone. This method provides an effective process for generating recombinant cell lines producing high levels of therapeutic proteins, with the benefits of rapid and accurate 96-well plate clone screening and elimination of unstable clones at an earlier stage in the development process. Furthermore, because this method does not rely on the availability of an antibody specific for the therapeutic protein being expressed, it can be easily implemented into any cell line development process.