Effects of lysosomal biotherapeutic recombinant protein expression on cell stress and protease and general host cell protein release in Chinese hamster ovary cells.

Damiano Migani,C Mark Smales,Daniel G Bracewell

doi:10.1002/btpr.2455

Damiano Migani, C Mark Smales + Show 1 more

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https://doi.org/10.1002/btpr.2455

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Abstract

Recombinant human Acid Alpha Glucosidase (GAA) is the therapeutic enzyme used for the treatment of Pompe disease, a rare genetic disorder characterized by GAA deficiency in the cell lysosomes (Raben et al., Curr Mol Med. 2002; 2:145–166). The manufacturing process for GAA can be challenging, in part due to protease degradation. The overall goal of this study was to understand the effects of GAA overexpression on cell lysosomal phenotype and host cell protein (HCP) release, and any resultant consequences for protease levels and ease of manufacture. To do this we first generated a human recombinant GAA producing stable CHO cell line and designed the capture chromatographic step anion exchange (IEX). We then collected images of cell lysosomes via transmission electron microscopy (TEM) and compared the resulting data with that from a null CHO cell line. TEM imaging revealed 72% of all lysosomes in the GAA cell line were engorged indicating extensive cell stress; by comparison only 8% of lysosomes in the null CHO had a similar phenotype. Furthermore, comparison of the HCP profile among cell lines (GAA, mAb, and Null) capture eluates, showed that while most HCPs released were common across them, some were unique to the GAA producer, implying that cell stress caused by overexpression of GAA has a molecule specific effect on HCP release. Protease analysis via zymograms showed an overall reduction in proteolytic activity after the capture step but also revealed the presence of co‐eluting proteases at approximately 80 KDa, which MS analysis putatively identified as dipeptidyl peptidase 3 and prolyl endopeptidase. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:666–676, 2017

Highlights

Host cell protein (HCP) release during the production of therapeutic proteins is a critical quality attribute that must be monitored and reduced to acceptable levels during the production of a biotherapeutic protein.[1]
We provide evidence that overexpression of a lysosomal therapeutic recombinant protein impacts on the lysosomal phenotype of a CHO cell line
We achieved this by comparing cell lysosomal phenotype and HCP profile of three CHO cell lines: a lysosomal biotherapeutic protein producer (GAA), a monoclonal antibody (mAb) producer and a null

Summary

Introduction

Host cell protein (HCP) release during the production of therapeutic proteins is a critical quality attribute that must be monitored and reduced to acceptable levels during the production of a biotherapeutic protein.[1] Being able to control HCP release during harvest, and their removal via downstream processing, is an important component of the manufacturing of such molecules and must be reported to Additional Supporting Information may be found in the online version of this article. As a wide range of recombinant products are negatively affected by the presence of proteases such as reported for mAbs,[4] fusion proteins,[5] coagulation factors[6] and based on personal communication with BioMarin who have manufacturing experience of GAA therapeutic enzyme products, we suspect GAA in certain conditions to be potentially affected

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