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Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

ABSTRACT: Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ion channels and multipass membrane proteins as well as cytotoxic proteins. To enhance the production of "difficult-to-express" proteins, alternative technologies were developed, mainly based on translationally active cell lysates. These so called "cell-free" protein synthesis systems enable an efficient production of different classes of proteins. Eukaryotic cell-free systems harboring endogenous microsomal structures for the synthesis of functional membrane proteins and posttranslationally modified proteins are of particular interest for future applications. Therefore, we present current developments in cell-free protein synthesis based on translationally active CHO cell extracts, underlining the high potential of this platform. We present novel results highlighting the optimization of protein yields, the synthesis of various "difficult-to-express" proteins and the cotranslational incorporation of non-standard amino acids, which was exemplarily demonstrated by residue specific labeling of the glycoprotein Erythropoietin and the multimeric membrane protein KCSA.

SUBMITTER: Thoring L

PROVIDER: S-EPMC5042383 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

Thoring Lena L Wüstenhagen Doreen A DA Borowiak Maria M Stech Marlitt M Sonnabend Andrei A Kubick Stefan S

PloS one 20160929 9

Nowadays, biotechnological processes play a pivotal role in target protein production. In this context, Chinese Hamster Ovary (CHO) cells are one of the most prominent cell lines for the expression of recombinant proteins and revealed as a safe host for nearly 40 years. Nevertheless, the major bottleneck of common in vivo protein expression platforms becomes obvious when looking at the production of so called "difficult-to-express" proteins. This class of proteins comprises in particular several ...[more]

PMID: 27684475

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Project description:Over the past decade, global interest in the development of therapeutic monoclonal antibodies (mAbs) has risen rapidly. As therapeutic agents, antibodies have shown marked efficacy in combatting a range of cancers and immune diseases with high target specificity and low toxicity (Carla Lucia et al. in PLoS ONE 6:e24071, 2011; Donaghy in MAbs 8:659-671, 2016; Nasiri et al. in J Cell Physiol 9:6441-6457, 2018; Teo et al. in Cancer Immunol Immunother 61:2295-2309, 2012). Recent advances in cell culture technology, such as high-throughput clone screening, have facilitated antibody production at concentrations exceeding 10 g/L (Chen et al. in BMC Immunol 19:35, 2018; Huang et al. in Biotechnol Prog 26:1400-1410, 2010; Lu et al. in Biotechnol Bioeng 110:191-205, 2013; Singh et al. in Biotechnol Bioeng 113:698-716, 2016). As titers have improved, the industry has begun to focus on the adjustment of target antibody quality profiles to improve efficacy. Cell lines, culture media, and culture conditions impact protein quality (Van Beers and Bardor in Biotechnol J 7:1473-1484, 2012). Optimization of critical quality attributes (CQAs), such as charge variants, can be achieved through bioprocess development and is the preferred approach as changes to the cell line or growth media used is considered unfavorable by regulatory bodies (Gawlitzek et al. in Biotechnol Bioeng 103:1164-1175, 2009; Jordan et al. in Cytotechnology 65:31-40, 2013; Pan et al. in Cytotechnology 69:39-56, 2016). In this study, the effect of process control and ion supplementation on charge variants of mAbs produced by Chinese hamster ovary (CHO) cells was investigated. Results of this study demonstrated that the concentration of Zn2+, duration of culturing, and temperature affect charge variants of a given mAb. Under the optimum conditions of 3L bioreactors, the most significant was that Zn2 + and temperature shift could further improve the quality of antibody. The main peak increased by 12%, and the acid peak decreased by 16%. At the same time, there was no significant loss of titer. This study provided supporting evidence for methods to improve charge variants arising during mAb production.

Dataset Information

Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

Publications

Cell-Free Systems Based on CHO Cell Lysates: Optimization Strategies, Synthesis of "Difficult-to-Express" Proteins and Future Perspectives.

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