Cell-culture growth conditions resulting in the oxidation of a recombinant antigen-binding fragment

Jeffrey R Fishpaugh,Cheng Zhao,Michelle A Stemler,Khandaker Siddiquee,Bill Zeck,Steven P Allen

doi:10.1186/s40643-019-0270-8

Jeffrey R Fishpaugh, Cheng Zhao + Show 4 more

Open Access

https://doi.org/10.1186/s40643-019-0270-8

Copy DOI

Journal: Bioresources and Bioprocessing	Publication Date: Sep 17, 2019
Citations: 4	License type: open-access

Affiliation: Abbott Fund

Abstract

Use of Quality-by-Design (QbD) tools is becoming an important part of the bioprocessing industry when developing a process for manufacturing operations to ensure the robustness and reproducibility of the biologic product. In the present study, a QbD tool, Design of Experiments (DOE), was utilized to optimize a bioprocess for the production of a CHO recombinant antigen-binding fragment (rFab) in small-scale bioreactors. DOE studies evaluated percent dissolved oxygen, temperature, and feeding strategy specific to this Chinese Hamster Ovary (CHO) clone. It was determined that these factors influenced cell viability, yield of the recombinant protein, and metabolic byproduct formation. To ensure the quality of the target molecule in the cell-culture process, small-scale purifications and analytical evaluation of the target molecule were completed prior to cell-culture scale-up to ensure that oxidation of the rFab, presence of free light chain, and truncation of thiol group were not observed. Analysis of the purified rFab by mass spectrometry indicated that rFab oxidation occurred under poor cell-culture conditions. PCR profile array results also revealed increased transcription of the oxidative genes Superoxide Dismutase 3, Myeloperoxidase, Dual Oxidase Like 2, Nuclear Receptor Coactivator 7, NADPH Oxidase Organizer 1, Mitochondria Uncouple Protein 3, Eosinophil Peroxidase, Lactoperoxidase Like, Serum Albumin Like, and Glutathione S-Transferase Pi 1 in this CHO strain. The present study suggests a mechanism and pathway for the oxidation of an rFab molecule during cell-culture bioprocess optimization. The present study also demonstrated the importance of utilizing the QbD tool of DOE to optimize the cell-culture bioprocess prior to scaling up into the large-scale production bioreactor.

Highlights

Use of Quality-by-Design (QbD) tools leads to the identification of both critical process parameters (CPPs) and critical quality attributes (CQAs) and is becoming an important part of the bioprocessing industry when developing and executing a process in manufacturing operations (Nagashima et al 2013; Torkashvand and Vaziri 2017; Lipsitz et al 2016; Horvath et al 2010)
It has been reported that there are several enzymes associated with generation of reactive oxygen species (ROS), such as NAPDH oxidases (NOXs) and dual oxidase (DUOX), that are highly expressed in cells under oxidative stress (Filomeni et al 2015; Schieber and Chandel 2014)
We describe the development of a Chinese Hamster Ovary (CHO) expressed recombinant antigen-binding fragment (rFab) cell-culture bioprocess leading to a purified molecule that is used in a diagnostic product

Summary

Introduction

Use of Quality-by-Design (QbD) tools leads to the identification of both critical process parameters (CPPs) and critical quality attributes (CQAs) and is becoming an important part of the bioprocessing industry when developing and executing a process in manufacturing operations (Nagashima et al 2013; Torkashvand and Vaziri 2017; Lipsitz et al 2016; Horvath et al 2010). Both molecules are subject to oxidation, another major concern for maintaining the quality of the product (Karu et al 1995; Nelson and Reichert 2009; Yan et al 2009; Moritz and Stracke 2017). Oxidative stress has been associated with high rates of aeration, mixing speeds, temperature, osmolality, pH, and overexpression of the target protein resulting in the production of reactive oxygen species (ROS) inside the mammalian cells (Filomeni et al 2015; Schieber and Chandel 2014). Common reactive oxygen species are hydrogen peroxide, superoxide radicals, peroxynitrite and hydroxide radicals The presence of these ROSs results in DNA damage and oxidation of the expressed target molecule. It has been reported that there are several enzymes associated with generation of ROSs, such as NAPDH oxidases (NOXs) and dual oxidase (DUOX), that are highly expressed in cells under oxidative stress (Filomeni et al 2015; Schieber and Chandel 2014)

Methods

Results

Conclusion