Date Degree Awarded

Spring 5-18-2019

Degree Type

Restricted to Claremont Colleges Dissertation

Degree Name

PHD in Applied Life Sciences

First Thesis/Dissertation Advisor

Parviz Shamlou

Second Thesis/Dissertation Advisor

Cameron Bardliving

Third Thesis/Dissertation Advisor

Terri Christianson

Abstract

Difficult-to-express (DTE) recombinant proteins like multi-specific proteins, DTE monoclonal antibodies and lysosomal enzymes, have seen difficulties in manufacturability using Chinese hamster ovary (CHO) cells and other mammalian cells as production platforms. CHO cells are preferably used for protein production because of their innate ability to secrete human-like recombinant proteins with post-translational modification, resistance to viral infection and familiarity with drug regulators. However, despite huge progress made in engineering CHO cells for high volumetric productivity, expression of DTE proteins like recombinant lysosomal sulfatase represent one of the poorly understood proteins. Furthermore, there are growing interest in the use of microRNAs (miRNAs) to engineer CHO cells expressing DTE proteins to improve cell performance of relevant bioprocess phenotypes. Therefore, we sought to understand miRNA expression profiles in CHO cell lines stably expressing DTE lysosomal protein and examined the effect of microRNA-engineering of these cell lines on protein expression. Firstly, we utilized next generation sequencing (NGS) technology for an integrated mRNA and microRNA profiling of three CHO cell lines (including parental cell) stably expressing a DTE lysosomal protein cultivated in a biphasic fed-batch mode within a 5L Dasgip bioreactor. With exception to the parental cell line, the other two cell lines differ by their productivity and were identified as low and high producers. Following RNA-seq and small RNA-seq data analyses by the Bioinformatics group at BioMarin, among other analyses, correlation and differential analyses identified 500 mRNA and 35 miRNAs that were differentially expressed in these cell lines over cultivation period. This study identified miRNAs that are potential targets for engineering of CHO cells for the enhancement of DTE protein expression. Secondly, in an independent study, we identified miR-23a and miR-377 as miRNAs targeting sulfatase modifying factor 1 (SUMF1) by using in silico prediction tools as SUMF1 is an activator of sulfatases. Transient inhibition of endogenous miR-23a/miR-377 significantly enhanced recombinant sulfatase enzyme specific activity in CHO cells without affecting cell growth. Though, inhibition of miR-23a/miR-377 had no significant effect on the mRNA and protein levels of SUMF1, overexpression of miR-23a/377 significantly reduced both the mRNA and protein levels of SUMF1. In summary, our data demonstrates the importance of using miRNA to optimize protein expression in CHO cells secreting DTE recombinant lysosomal proteins.

Rights Information

© 2019 Ifeanyi Michael Amadi (Keck Graduate Institute/BioMarin)

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