Transcriptome study and identification of potential marker genes related to the stable expression of recombinant proteins in CHO clones.

BACKGROUND: Chinese hamster ovary (CHO) cells have become the host of choice for the production of recombinant proteins, due to their capacity for correct protein folding, assembly, and posttranslational modifications. The most widely used system for recombinant proteins is the gene amplification procedure that uses the CHO-Dhfr expression system. However, CHO cells are known to have a very unstable karyotype. This is due to chromosome rearrangements that can arise from translocations and homologous recombination, especially when cells with the CHO-Dhfr expression system are treated with methotrexate hydrate. The present method used in the industry for testing clones for their long-term stability of recombinant protein production is empirical, and it involves their cultivation over extended periods of time prior to the selection of the most suitable clone for further bioprocess development. The aim of the present study was the identification of marker genes that can predict stable expression of recombinant genes in particular clones early in the development stage. RESULTS: The transcriptome profiles of CHO clones with stable and unstable recombinant protein production were investigated over 10-weeks of cultivation, using a DNA microarray. We identified 14 genes that were differentially expressed between the stable and unstable clones already at 2 weeks from the beginning of the cultivation. Their expression was validated by reverse-transcription quantitative real-time PCR (RT-qPCR). Furthermore, the k-nearest neighbour algorithm approach shows that the combination of the gene expression patterns of only five of these 14 genes is sufficient to predict stable recombinant protein production in clones in the early phases of cell-line development. CONCLUSIONS: The exact molecular mechanisms that cause unstable recombinant protein production are not fully understood. However, the expression profiles of some genes in clones with stable and unstable recombinant protein production allow prediction of such instability early in the cell-line development stage. We have thus developed a proof-of-concept for a novel approach to eliminate unstable clones in the CHO-Dhfr expression system, which saves time and labour-intensive work in cell-line development.

Transcriptomic variation between different Chinese hamster ovary cell lines.

OBJECTIVES: To identify transcription markers that uniquely determine specific Chinese hamster ovary (CHO) cell lines and can be used for the identification of cell lines in the process of biopharmaceutical cell-line development. RESULTS: Five CHO cell lines with different origins were extensively characterised at the transcriptomic level and the results were compared to their karyotype characterisation. The analysed cell lines differ in their karyotype but, due to the genome instability observed during parental and recombinant cell-line establishment, karyotyping is not the preferred method for accurate identification of the various CHO cell lines. Marker genes unique to a specific cell line were identified by microarrays, and their expression was validated by reverse-transcription quantitative real-time PCR. The analysed cell lines can be differentiated by the presence/absence of detectable marker gene expression. Additionally, the similarity of the transcriptional profiles is dependent on cell-line history but independent of the manipulation steps involved in the recombinant cell-line development process. CONCLUSIONS: Certain transcripts can be used as markers for the identification of a CHO cell line undergoing recombinant development and thus represent a powerful tool for ensuring the maintenance of high quality standards.

Murine monoclonal antibodies directed against human recombinant Macrophage Migration Inhibitory Factor.

Macrophage Migration Inhibitory Factor (MIF) is a crucial component of the immune system acting together with glucocorticosteroids to regulate immunity and inflammation. Understanding of its many putative functions and action mechanisms is still ambiguous. Due to the newest findings that a local MIF expression is up regulated in allograft rejection and in glomerulonephritis, an interest in MIF research is increasing and is focused on possibilities of anti-MIF treatment.In the present work new murine monoclonal antibodies (MAbs) directed against human recombinant MIF (hrMIF) are described. hrMIF protein used for the immunisation was tested for its biological activity and has evident macrophage migration inhibitory activity. The selected MAbs were purified and further characterised. They recognised MIF in a Western blot experiment after a native IEF. Anti-MIF MAb designated as Ml inhibited MIF activity in the test, which was performed in the 48 well Boyden chamber system. It is presumed that Ml MAb could be used as a potential therapeutic agent.

Erythropoietin unfolding: thermodynamics and its correlation with structural features.

Human erythropoietin (EPO) is a glycoprotein hormone considered to be the principal regulator of red blood cell formation. Although its recombinant version (rEPO) has been widely used for treatment of various anemias and its biological effects are relatively well-known, we know little about its biophysical properties and their relation to its structure. To gain a fuller understanding of the structural and functional properties of rEPO on the molecular level we followed its thermal and urea-induced unfolding at different pH (3.1-9.4) and urea concentrations (0-8 M) using spectropolarimetry, UV absorption, intrinsic emission fluorescence, and differential scanning calorimetry. Our results show that under a variety of conditions rEPO undergoes thermal or urea-induced denaturation that may be considered as a reversible two-state process characterized by unusually high (thermal) or moderate (urea-induced) extent of the residual structure. The highest thermal stability of the protein observed in aqueous solutions at physiological pH appears to be due to the largest difference in the extent of structure in the denatured and native state at this pH. The comparison between experimentally determined energetics of rEPO denaturation and its structure-based calculations indicates that the parametrization of thermodynamic quantities in terms of changes in solvent accessible nonpolar and polar surface areas resulting from protein unfolding can be successfully used provided that these changes are estimated from combination of experimentally determined deltaC(o)p and deltaH(o) values and not calculated from the structure of the protein's folded and assumingly fully unfolded state.