Perfusion process combining low temperature and valeric acid for enhanced recombinant factor VIII production.

Perfusion operation mode remains the preferred platform for production of labile biopharmaceuticals (e.g., blood factors) and is also being increasingly adopted for production of stable products (e.g., monoclonal antibodies). Regardless of the product, process development typically aims at maximizing production capacity. In this work, we investigated the impact of perfusion cultivation conditions on process productivity for production of human factor VIII (FVIII). Recombinant CHO cells were cultivated in bioreactors coupled to inclined settlers and the effects of reducing the temperature to 31°C with or without valeric acid (VA) supplementation were evaluated. Increases in cell specific productivity (qp ) up to 2.4-fold (FVIII concentration) and up to 3.0-fold (FVIII biological activity) were obtained at 31°C with VA compared to the control at 37°C. Biological activity is the most important quality attribute for FVIII and was positively affected by mild hypothermia in combination with the chemical inducer. The low temperature conditions resulted in enhanced product transcript levels, suggesting that the higher qp is related to the increased mRNA levels. Furthermore, a high-producer subclone was evaluated under the perfusion conditions optimized for the parental clone (31°C with VA), yielding increases in qp of 6-fold and 15-fold compared to the parental clone cultivated under the same condition and at 37°C, respectively. The proposed perfusion strategy enables increased product formation without increasing production costs, being potentially applicable to perfusion production of other CHO-derived biopharmaceuticals. To the best of our knowledge, this is the first report showing the benefits of perfusion combining mild hypothermia with VA supplementation.

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.

Establishment of a CpG island microarray for analyses of genome-wide DNA methylation in Chinese hamster ovary cells.

Optimizing productivity and growth rates of recombinant Chinese hamster ovary (CHO) cells requires insight into the regulation of cellular processes. In this regard, the elucidation of the epigenetic process of DNA methylation, known to influence transcription by a differential occurrence in CpG islands in promoter regions, is increasingly gaining importance. However, DNA methylation has not yet been investigated on a genomic scale in CHO cells and suitable tools have not existed until now. Based on the genomic and transcriptomic CHO data currently available, we developed a customized oligonucleotide microarray covering 19598 CpG islands (89 % of total bioinformatically identified CpG islands) in the CHO genome. We applied our CHO-specific CpG island microarray to investigate the effect of butyrate treatment on differential DNA methylation in CHO cultures in a time-dependent approach. Supplementation of butyrate is known to enhance cell specific productivities in CHO cells and leads to alterations of epigenetic silencing events. Gene ontology clusters regarding, e.g., chromatin modification or DNA repair, were significantly overrepresented 24 h after butyrate addition. Functional classifications furthermore indicated that several major signaling systems such as the Wnt/ß-catenin pathway were affected by butyrate treatment. Our novel CHO-specific CpG island microarray will provide valuable information in future studies of cellular processes associated with productivity and product characteristics.

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.

Affibody-mediated retention of the epidermal growth factor receptor in the secretory compartments leads to inhibition of phosphorylation in the kinase domain.

Abnormal activity of the epidermal growth factor receptor (EGFR) is associated with various cancer-related processes and motivates the search for strategies that can selectively block EGFR signalling. In this study, functional knockdown of EGFR was achieved through expression of an affibody construct, (ZEGFR:1907)(2-)KDEL, with high affinity for EGFR and extended with the amino acids KDEL to make it resident in the secretory compartments. Expression of (ZEGFR:1907)(2-)KDEL resulted in 80% reduction ofthe cell surface level of EGFR, and fluorescent staining for EGFR and the (ZEGFR:1907)(2-)KDEL construct showed overlapping intracellular localisation. Immunocapture of EGFR from cell lysates showed that an intracellular complex between EGFR and the affibody construct had been formed, further indicating aspecific interaction between the affibody construct and EGFR. Surface depletion of EGFR led to a dramatic decrease in the amount of kinase domain phosphorylated EGFR, coincident with a significant decrease in the proliferation rate.