Title

Mammalian Cell Line Development Platform for Recombinant Protein Production: Expanding the Protein Expression Toolbox for Research and Drug Discovery Applications

Graduation Date

5-2017

Document Type

Master's Thesis

Degree Name

Master of Science

Department or Program

Biological Sciences

Department or Program Chair

Maggie Louie, Ph.D.

First Reader

Vishal Agrawal, Ph.D.

Second Reader

Ekaterina Kalashnikova, Ph.D.

Abstract

Recombinant proteins have revolutionized the biomedical industry, providing therapeutics for life-threatening diseases and protein reagents for research applications. BioMarin Pharmaceutical Inc. develops recombinant protein therapeutics to treat rare diseases including lysosomal storage disorders (LSDs), a group of about 50 individually rare disorders together affecting 1 in 8,000 live births. With an increase in the number of novel therapeutics in our drug discovery pipeline, there is a high demand to produce a variety of recombinant proteins for early-stage drug development projects. In order to equip our protein production process with the tools and capability for diverse protein expression, it is valuable to expand our expression toolbox with high-expressing platforms. The goal of this project is to expand to our current expression platforms by developing a murine myeloma based expression system with SP2/0 cells as a host. Since the SP2/0 cell line is amongst the most commonly used cell lines for therapeutic and reagent protein production, developing a SP2/0 expression system may offer additional benefits to our recombinant protein production needs including: expression of difficultto-express proteins, improving titers, and extending recombinant cell line stability. A lysosomal enzyme therapeutic candidate is expressed in the SP2/0 cells as a proof-of-concept for developing this protein expression platform. To this end, we have shown that SP2/0 cells can be grown to a high density in commercially available serum-free media with a doubling time of less than twenty four hours. A clone isolation strategy was used to pick the top clone expressing high levels of recombinant protein. Using the highest expressing clone, we developed a high yielding bioprocess at a two liter scale to demonstrate the utility of this system for generating recombinant proteins at large scale. Furthermore, the therapeutic properties of the recombinant protein expressed in SP2/0 cells are similar to the recombinant protein expressed in Chinese hamster ovary (CHO) cell lines, demonstrating similar uptake into diseased cells (Kuptake values) and binding affinity to the receptor responsible for drug mediated cellular uptake. Thus, the SP2/0 expression system proves to be a valuable addition to our expression toolbox for the production of research-grade protein therapeutics for cell-based assays.

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