Sandra Klausing

Methods in mammalian cell line engineering: from random mutagenesis to sequence-specific approaches

Due to the increasing demand for recombinant proteins, the interest in mammalian cell culture, especially of Chinese hamster ovary cells, grows rapidly. This is accompanied by the desire to improve cell lines in order to achieve higher titers and a better product quality. Until recently, most cell line development procedures were based on random integration and gene amplification, but several methods for targeted genetic modification of cells have been developed. Some of those are homologous recombination, RNA interference and zinc-finger nucleases. Especially the latter two have evolved considerably and will soon become a standard for cell line engineering in research and industrial application. This review presents an overview of established as well as new and promising techniques for targeted genetic modification of mammalian cells.

The mucin MUC1 modulates the tumor immunological microenvironment through engagement of the lectin Siglec-9

Siglec-9 is a sialic-acid-binding lectin expressed predominantly on myeloid cells. Aberrant glycosylation occurs in essentially all types of cancers and results in increased sialylation. Thus, when the mucin MUC1 is expressed on cancer cells, it is decorated by multiple short, sialylated O-linked glycans (MUC1-ST). Here we found that this cancer-specific MUC1 glycoform, through engagement of Siglec-9, ‘educated’ myeloid cells to release factors associated with determination of the tumor microenvironment and disease progression. Moreover, MUC1-ST induced macrophages to display a tumor-associated macrophage (TAM)-like phenotype, with increased expression of the checkpoint ligand PD-L1. Binding of MUC1-ST to Siglec-9 did not activate the phosphatases SHP-1 or SHP-2 but, unexpectedly, induced calcium flux that led to activation of the kinases MEK-ERK. This work defines a critical role for aberrantly glycosylated MUC1 and identifies an activating pathway that follows engagement of Siglec-9.

Bioreactor cultivation of CHO DP-12 cells under sodium butyrate treatment - comparative transcriptome analysis with CHO cDNA microarrays.

BackgroundSodium butyrate (NaBu) is not only known to inhibitproliferation but also to increase the specific productiv-ity in cultivation of Chinese hamster ovary (CHO) cells[1] – the most commonly used mammalian cell line forpharmaceutical protein production [2]. So far, little isknown about the underlying mechanisms and genes thatare affected by butyrate treatment. Besides the proteo-mic approach to unravel proteins involved in the pro-cesses, the analysis of transcriptomes presents anotherpromising method. Here we show an application of ourCHO cDNA microarray to identify genes associatedwith increased productivity during cultivation of CHOcells under sodium butyrate treatment.Materials and methodsFour batch cultivations of CHO DP-12 cells (clone #1934, ATCC CRL-12445) were performed in 2 L bior-eactor systems under pO

Enhancing cell growth and antibody production in CHO cells by siRNA knockdown of novel target genes

Background Seven out of the ten top-selling biopharmaceuticals in 2011 are produced in Chinese Hamster Ovary (CHO) cells [1]. This tremendous commercial interest makes the development and application of strategies for cell line optimization, like gene overexpression or knockdown to enhance cell specific productivity and cellular growth, highly interesting. In this work, we investigated the knockdown effect of novel target genes by siRNA as a powerful tool for CHO cell line engineering.

Medium development beyond production media: Chemically defined media for transfection and single cell cultivation

Background State-of-the-art medium and feed development has proven its potential for increasing bioprocess efficiency many times. By the application of such advanced chemically defined and animal component-free formulations, in combination with recent cell lines, the yield of mammalian cell cultures was pushed to the binary gram per liter level. In this work, the current progress in designing special application media for transfection and single cell growth is presented. In the context of medium development, these techniques pose specific challenges which differ from production media. However, interest in such specialized products is high. On one side, clone selection is a key factor for robust and effective processes. On the other side, applications for transient transfection range from purposes in R&D, over the production of pre-clinical material to personalized medicine or vaccine production. In terms of upscaling, efficient transient gene expression (TGE) processes require a bifunctional medium supporting both cell growth and transfection.

First CpG island microarray for genome-wide analyses of DNA methylation in Chinese hamster ovary cells: new insights into the epigenetic answer to butyrate treatment

Background Optimizing productivity and growth of recombinant Chinese hamster ovary (CHO) cells requires insight and intervention in regulatory processes. This is to some extent accomplished by several ‘omics’ approaches. However, many questions remain unanswered and bioprocess development is therefore still partially empirical. In this regard, the analysis of DNA methylation as one of the earliest cellular regulatory levels is increasingly gaining importance. This epigenetic process is known to influence transcriptional events when it occurs at specific genomic regions with high CpG frequencies, called CpG islands (CGIs). Being methylated, CGIs attract proteins with methyl-DNA binding domains (MBD proteins) that in turn can interact with chromatin modifying complexes, thereby leading to a transcriptionally inactive state of the associated gene [1]. In CHO cells, DNA methylation has yet only been investigated in gene-specific approaches, e.g. regarding the CMV promoter [2]. To analyze differential DNA methylation in CHO cultures on a genomic scale, we developed a microarray covering 19,598 CGIs in the CHO genome. We applied it to elucidate the effect of butyrate on CHO DP-12 cultures, as this short chain fatty acid (SCFA) is known to elicit epigenetic responses by inhibiting histone-deacetylases [3].

Introducing a new chemically defined medium and feed for hybridoma cell lines

Background Hybridoma technology was established in the 2nd half of the 20th century and in the view of current protein production it might seem old-fashioned. Despite, it is commonly used to produce monoclonal antibodies (mAbs) for R & D, clinical diagnostics or medical applications and the demand for mAbs produced by hybridomas is still high. However, compared to CHO, only a few serum-free hybridoma media are available and even less suppliers for chemically defined products are on the market. In this work, a new chemically defined medium and feed were developed to bring hybridoma processes to the next level and to target the existing gap in the market.