Cell engineering strategies to enhance production of next-generation biologics

Lead Research Organisation: University of Edinburgh
Department Name: Sch of Biological Sciences

Abstract

The Chinese Hamster Ovary (CHO) cell is the most widely used industrial expression system, generating ~70% of biopharmaceuticals (including multiple monoclonal antibodies) with a market value >$100 billion. However, many 'difficult-to-express' biologics - including novel molecules such as bi- and tri-specific antibodies - give unpredictably lower titres and additional complexities, requiring extensive cell line and process development. Productivity can be compromised by transgene suppression and bottlenecks in translation, trafficking, processing or secretion. The aim of this project is to use a combination of synthetic biology, genetic engineering tools, and modern 'omics platforms (genomics, metabolomics and proteomics) to discover and address bottlenecks in the production of novel biologics in CHO cells.

The student will learn cutting edge gene editing tools (including CRISPR/Cas9), synthetic biology tools and metabolomics and proteomics platforms.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509644/1 01/10/2016 30/09/2021
2015180 Studentship EP/N509644/1 01/02/2018 31/08/2021 Anna Mastela
 
Description The Chinese Hamster Ovary (CHO) cell lines are the workhorse of the biopharmaceutical industry for generating therapeutic recombinant proteins. Despite great improvements in bioprocessing, many 'difficult-to-express' biologics - including novel antibody formats - yield in unpredictably low titres and/or have decreased quality. This may be caused by bottlenecks in transcription, translation, folding, and/or secretion.

The project aims to characterise potential expression bottlenecks in CHO cells producing different antibody formats by combined synthetic biology and multi-omics approaches. This will hopefully contribute to identification of novel cellular stress biomarkers as well as targets for CHO cell line engineering for enhanced expression of DTE proteins.

Design of a cell system for comparative multi-omics analyses of expression bottlenecks in CHO cells was based on targeted integration of genes of interest (GOIs) at the pre-selected genomic location. During course of the project, an improved method for targeted integration of transgenes in CHO-K1 cell line was developed.

Targeted integration was achieved by directing GOI into a desired genomic location by creating a double-strand break (DSB) using the CRISPR/Cas9 system. Following on the formation of a DSB and in presence of a repair template (a donor plasmid with GOI flanked by homology arms which sequences corresponded to the targeted genomic site) knock-in of GOIs was achieved.

To increase efficiency of targeted integration, a donor plasmid linearization in vivo was employed. The same sequence recognised by the sgRNA that was used for generation of a DSB at the desired genomic location was also incorporated upstream of 5' homology arm to linearize the donor template upon transfection into the cell.

Additionally, a dual-fluorescence selection system that involved incorporation of two orthogonal fluorescent markers into the repair template - the first present within the region that was knocked-in during targeted integration, and the second located outside of the desired integration region - enabled augmented selection of cells that integrated DNA cargo only at the desired location through an efficient counter-selection of cells with off-target integrations.

This strategy allowed to integrate up to ~6.5 kb of DNA at the pre-selected genomic location, which is - to the best of our knowledge - the largest DNA sequence incorporated in the CHO genome using CRISPR/Cas9-directed targeted integration.

Moreover, specialist skills in synthetic biology and omics - including modular DNA assembly, CRISPR/Cas9-based cell line engineering, and mass spectrometry - were gained by the student
Exploitation Route We propose that the improved targeted integration strategy can be adapted for use in gene and cell engineering applications, irrespective of cell lines and model organisms, in both academic and industrial settings.
Sectors Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology