Generation and characterization of a superior host cell line for biomanufacturing
© Ritter et al. 2015
Published: 14 December 2015
Chinese hamster ovary (CHO) cells are the most widely used host for large scale production of recombinant therapeutic proteins exhibiting high productivities in the gram per liter range. Although these cells have been extensively characterized and optimized, a demand to further increase the performance towards higher productivity and clonal stability exists. Up to date, the clone selection process is mainly based on phenotypic screens like titer measurements and growth data instead of more reliable biomarkers. Recently, the genomes of Chinese hamster as well as of different CHO cell lines were sequenced, assembled and annotated [1–3]. This new information gives the opportunity for functional analysis of the transcriptome and allows rational designs for the identification of biomarkers for clone selection and targets for cell line engineering.
Material and methods
CHO-K1a and CHO-C8DEL cell lines were cultivated in shake flasks in a non-humidified shaker cabinet at 150 rpm, 10% CO2 at 36.5°C in suspension in proprietary, chemically defined culture media. Cell viabilities and growth rates were monitored by means of an automated system (ViCell, Beckman Coulter).
For transient expression plasmid DNA and polyethylenimine "Max" (Polysciences, Eppelheim, Germany) were diluted into OptiMEM medium (Life Technologies) and added to the cells after complex formation. The titer of the model protein in the supernatant was measured by Protein A HPLC on day 3 and day 6 post transfection.
Cells were stably transfected by electroporation (Amaxa Nucleofection system, Lonza, Germany) with an expression plasmid encoding for a human monoclonal antibody according to the manufacturer's instructions.48 h after transfection, selection was started by addition of geneticin (G418). As soon as cells recovered to a viability of above 80%, a second selection step was applied by passaging the cells into G418-free medium containing 1 µM methotrexate (MTX). When the cells resumed logarithmic growth, cell cultivation was continued in MTX-containing medium throughout single cell sorting, screening and productivity assessments. Cells were stained using a FITC-labeled mouse IgG-anti-human IgG (BD Pharmingen) and single cell sorting was performed using a FACS-Aria II instrument (Becton Dickinson). The 5% most strongly fluorescent cells were gated and sorted as single cells into 96-well plates. Volumetric productivities of G418 and MTX selected pools and clones were determined by Protein A HPLC in cell culture supernatants.
Analysis of gene expression profiles of 5-7 high and 5-7 low monoclonal antibody producing CHO-K1a clones of three different monoclonal antibody projects surprisingly revealed that the telomeric region of chromosome 8 including several genes is often lost in transfected, high antibody producing CHO-K1a derived clones. Likewise surprisingly, a high correlation between the loss of this telomeric region and increased productivity as well as clonal stability could be identified in CHO-K1a clones.
With our study we contribute to the knowledge of how the recently published CHO genomes [1–3] can be used to meet the growing demand of industry to further develop CHO cell lines and to increase their performance for the production of new therapeutic proteins in short time periods.
We compared gene expression profiles of high and low antibody producing CHO-K1a clones and identified a subset of genes, localized at the telomeric region of chromosome 8, which have a role regarding productivity and production stability. These genes can be used as biomarkers for selecting high and stable producing cell lines. In addition, we generated and characterized a new parental CHO cell line lacking this telomeric region, which displays several advantageous characteristics for cell line development and manufacturing.
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