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Nathan Lewis, PhD
KEYNOTE: A Systems Approach To Engineering Protein Production in Mammalian Cells
University of California, San Diego
Alan Dickson, PhD
KEYNOTE: Modifying CHO using ‘Omics’ and Ideas From Novel Platforms
University of Manchester
The majority of biotherapeutics are produced in mammalian cells. To facilitate cell line development, we are mapping the pathways involved in mammalian cell growth and protein production. We have used these with genome editing techniques to develop cell lines with improved traits for protein production.
A toolbox of vector elements and novel engineered CHO cell lines were developed, which resulted in an increase of titer and improved product quality. By integrating these vector and cell line engineering technologies, we are aiming for further reducing time lines during cell line development.
Adaptation of CRISPR/Cas9, a bacterial genome defense system, for eukaryotic molecular genetics has ushered in a new phase of the genomics revolution. Here, I will present the framework of a CRISPR/Cas9-based platform for drug target discovery and validation and how this platform can be exploited for single gene to genome-scale experimentation.
At CFB we have used our high throughput cell engineering pipeline to generate a panel of engineered CHO cells with improved properties for the production of recombinant therapeutic proteins.
We now have a large collection of cell lines generating tailored glycoprofiles, and we have used these to produce a therapeutic protein with a defined N-glycan profile, matching the current plasmas derived product.
Our studies revealed that pyruvate kinase muscle-1 (PKM1) expression correlates with lactogenic behaviour in CHO cells and deletion of PKM1 or PKM gene in CHO cells reduces lactate production and results in a metabolic shift in these cells.
For certain deletion configurations, this resulted in an increase productivity, which is likely due to the observed metabolic shift.
Expression vector design plays a key role for efficient recombinant protein expression in Chinese Hamster Ovary cell lines. Here we evaluated vector design strategies including signal peptide and codon editing choices as well as vector construction approaches that accelerated the transition of lead molecules from Research and optimized clinical cell line development.
Cell line engineering techniques like CRISPR/Cas9 can efficiently alter the glycosylation of recombinant proteins by modifying the gene expression of glycosyltransferases, glycosidases or proteins involved in transport and metabolism of glycosylation precursors in producer cell lines. N‑glycosylation is particularly important for the pharmacokinetics of recombinant glycoprotein therapeutics and can prolong dosing intervals to the benefit for patients, especially when administration is performed intravenously.
Recombinant glycoproteins produced in non-human cells may carry glycostructures antigenic to humans. Using the human embryonic kidney FreeStyle 293-F (HEK 293-F) cell line as expression platform offers the advantage of human-type glycosylation patterns. Therapeutic coagulation factor VII (FVII) can prevent and treat bleeding episodes in patients with congenital FVII deficiency, Hemophilia A or B with inhibitors to Factor VIII or Factor IX and acquired hemophilia. However, FVII expressed in the HEK 293-F cell line carries considerable levels of terminal N-acetylgalactosamine (GalNAc) on its N-glycans.
In order to lower binding of FVII to the hepatic asialoglycoprotein receptor (ASGP-R), and thus reduce FVII clearance, two GalNAc transferases were knocked-out in the HEK 293-F cell line by CRISPR/Cas9. Effects of B4GalNT3 and B4GalNT4 single or double knock-outs on released N-glycan level of purified FVII were analyzed by hydrophilic interaction liquid chromatography coupled with mass spectrometry. Our results revealed a successful reduction of terminal GalNAc accompanied by an increase in terminal galactosylation and, beneficially, N-glycan sialylationl. N-glycan profiles were correlated with ASGP‑R binding measured by surface plasmon resonance and pharmacokinetics assayed in a rat model. Reduced GalNAc levels and increased sialylation led to lower ASGP-R binding and improved FVII recovery in vivo by more than 50 %. Using the example of FVII expressed in a modified HEK 293-F cell line, we demonstrated that glycoengineering via cell line design is a straightforward and rational approach to optimize the glycosylation profile of protein therapeutics using state-of-the-art gene-editing tools.