Application of Chinese hamster ovary cell line engineering in the study of lipoprotein lipase and its impact on polysorbate degradation
Date
2016
Authors
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Publisher
University of Delaware
Abstract
Biopharmaceutical products, such as monoclonal antibodies (mAbs) and other recombinant therapeutic proteins, had global sales of $140 billion in 2013. Chinese hamster ovary (CHO) cells are one of the most commonly used cell platforms to express mAbs and recombinant therapeutic proteins. Tools for cell line engineering, such as short interfering RNA (siRNA) interference and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein-9 nuclease (CRISPR/Cas9), which emerged over the last decade, were extensively used to generate recombinant CHO cell lines with desired properties. With the availability of the CHO-K1 and Chinese hamster genome sequences, these tools allow for the rational design of CHO cell engineering through sequence-specific approaches. This thesis describes an application of CHO cell engineering to address the impact of host cell protein (HCP) on formulation stability, a recurring problem observed in the biopharmaceutical industry. One particular HCP, lipoprotein lipase (LPL), is hypothesized to contribute to the degradation of polysorbate 20 (PS20) and polysorbate 80 (PS80), two surfactants used to stabilize recombinant therapeutic proteins in formulation, through enzymatic hydrolysis. The goals of this research are to (i) generate engineered CHO-K1 cell lines with a functional lpl knockout and (ii) study the impact of LPL on polysorbate degradation. Two cell line engineering tools, siRNA interference and CRISPR/Cas9, are applied in this work. Reduction in LPL expression by siRNA interference did not affect cell culture performance and is associated with lower PS80 degradation. The results of this study suggest that LPL may play a significant role in PS80 hydrolysis, which motivates further investigation with a fully functional lpl knockout. Design of experiment methods are applied for optimizing the conditions for the polysorbate degradation assay and conditions are optimized to improve the reproducibility of the assay. These optimized conditions are used in subsequent polysorbate degradation studies. Rational design of targeted gene-editing by CRISPR/Cas9 leads to the generation of five CHO-K1 cell lines with a functional lpl knockout. In this work, incubation with HCPs from the CHO-K1 lpl knockout cell lines was found to reduce polysorbate degradation. The findings from this work support our hypothesis that LPL contributes to polysorbate degradation through enzymatic hydrolysis. The tools and approach used in this thesis demonstrate the application of cell line engineering tools for extensive genetic manipulation and genomic analysis of engineered CHO cell lines and their potential use in addressing biopharmaceutical problems.