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Hansen, Henning Gram,Kildegaard, Helene Faustrup,Lee, Gyun Min,Kol, Stefan WILEY‐VCH Verlag 2016 Biotechnology journal Vol.11 No.12
<P><B>Abstract</B></P><P>Accurate titer determination of recombinant proteins is crucial for evaluating protein production cell lines and processes. Even though enzyme‐linked immunosorbent assay (ELISA) is the most widely used assay for determining protein titer, little is known about the accuracy of commercially available ELISA kits. We observed that estimations of recombinant human ø1‐antitrypsin (<I>r</I>ø1AT) titer by Coomassie‐stained SDS‐PAGE gels did not correspond to previously obtained titers obtained by a commercially available ELISA kit. This prompted us to develop two independent quantification assays based on biolayer interferometry and reversed‐phase high‐performance liquid chromatography. We compared the rø1AT titer obtained by these assays with three different off‐the‐shelf ELISA kits and found that the ELISA kits led to inconsistent results. The data presented here show that recombinant protein titers determined by ELISA kits cannot be trusted <I>per se</I>. Consequently, any ELISA kit to be used for determining recombinant protein titer must be validated by a different, preferably orthogonal method.</P>
Hansen, H.G.,Pristovsek, N.,Kildegaard, H.F.,Lee, G.M. Pergamon Press ; Elsevier Science Ltd 2017 Biotechnology advances Vol.35 No.1
<P>Chinese hamster ovary (CHO) cells are the preferred cell factory for the production of therapeutic glycoproteins. Although efforts primarily within bioprocess optimization have led to increased product titers of recombinant proteins (r-proteins) expressed in CHO cells, post-transcriptional bottlenecks in the biosynthetic pathway of r-proteins remain to be solved. To this end, the ectopic expression of transgenes (effector genes) offers great engineering potential. However, studies on effector genes have in some cases led to inconsistent results. Whereas this can in part be attributed to product specificity, other experimental and cellular factors are likely important contributors to these conflicting results. Here, these factors are reviewed and discussed with the objective of guiding future studies on effector genes. (C) 2016 Elsevier Inc. All rights reserved.</P>
Kallehauge, Thomas Beuchert,Kol, Stefan,Rørdam Andersen, Mikael,Kroun Damgaard, Christian,Lee, Gyun Min,Faustrup Kildegaard, Helene WILEY‐VCH Verlag 2016 Biotechnology Journal Vol.11 No.10
<P><B>Abstract</B></P><P>When expressing pharmaceutical recombinant proteins in mammalian cells, the protein is commonly directed through the secretory pathway, in a signal peptide‐dependent manner, to acquire specific post‐translational modifications and to facilitate secretion into the culture medium. One key premise for this is the direction of the mRNA encoding the recombinant protein to the surface of the endoplasmic reticulum (ER) for subsequent protein translocation into the secretory pathway. To evaluate the efficiency of this process in Chinese hamster ovary (CHO) cells, the subcellular localization of recombinant mRNA encoding the therapeutic proteins, erythropoietin (EPO) and Rituximab, was determined. The results show that ER‐directed recombinant mRNAs exhibited an efficient recruitment to the ER when compared to an endogenous ER‐directed mRNA, with no cytoplasmic translation of ER‐directed recombinant proteins observed. These observations indicate that the recombinant mRNA, encoding ER‐directed proteins, follows the same distribution pattern as endogenous mRNA directed towards the ER. Furthermore, the previous established fractionation method proves to be an efficient tool to study not only recombinant mRNA localization, but also recombinant protein trafficking between the ER and cytosol in CHO cells.</P>
One-step generation of triple knockout CHO cell lines using CRISPR/Cas9 and fluorescent enrichment.
Grav, Lise Marie,Lee, Jae Seong,Gerling, Signe,Kallehauge, Thomas Beuchert,Hansen, Anders Holmgaard,Kol, Stefan,Lee, Gyun Min,Pedersen, Lasse Ebdrup,Kildegaard, Helene Faustrup Wiley 2015 Biotechnology Journal Vol.10 No.9
<P>The CRISPR/Cas9 genome editing technology has previously been shown to be a highly efficient tool for generating gene disruptions in CHO cells. In this study we further demonstrate the applicability and efficiency of CRISPR/Cas9 genome editing by disrupting FUT8, BAK and BAX simultaneously in a multiplexing setup in CHO cells. To isolate Cas9-expressing cells from transfected cell pools, GFP was linked to the Cas9 nuclease via a 2A peptide. With this method, the average indel frequencies generated at the three genomic loci were increased from 11% before enrichment to 68% after enrichment. Despite the high number of genome editing events in the enriched cell pools, no significant off-target effects were observed from off-target prediction followed by deep sequencing. Single cell sorting of enriched multiplexed cells and deep sequencing of 97 clones revealed the presence of four single, 23 double and 34 triple gene-disrupted cell lines. Further characterization of selected potential triple knockout clones confirmed the removal of Bak and Bax protein and disrupted fucosylation activity as expected. The knockout cell lines showed improved resistance to apoptosis compared to wild-type CHO-S cells. Taken together, multiplexing with CRISPR/Cas9 can accelerate genome engineering efforts in CHO cells even further.</P>