Eng. Life Sci. 2015, 0, 1–10 www.els-journal.com J´ er´ emy Rimbon * Andr´ es S´ anchez-Kopper * Andreas Wahl Ralf Takors Institute of Biochemical Engineering, University of Stuttgart, Stuttgart, Germany Research Article Monitoring intracellular protein degradation in antibody-producing Chinese hamster ovary cells Intracellular proteolysis in mammalian cells is a native cellular strategy to recycle proteins and peptides. Whether or not this mechanism may hamper monoclonal antibody (mAb) formation in Chinese hamster ovary (CHO) cells was the key driver for this study. Exponentially growing, anti-interleukin (IL)-8 producing CHO cells were fed with 13 C-labeled L-lysine. The fate of the labeling signal was tracked in intracellular peptides, which were the proteolytic fragments of the mAb. Signal anal- ysis was performed in samples after cell disruption, anion exchange SPE and Q-ToF mass detection. Four degradation peptides were found, with HYTQKSLSLSPGK and HYTQKSLSLSPG containing two and one L-lysine unit (K), respectively. La- beling dynamics were used for model-based identification of the degradation rate in four biological replicates. Degradation rates of 22–25 pmol/10 8 cells/h were esti- mated, representing about 3% of the net mAb production. Hence, intracellular mAb degradation occurs even under the rather smooth production conditions installed. Keywords: Batch bioreactor / Intracellular degradation / Monoclonal antibody / Proteolysis / Recombinant protein  Additional supporting information may be found in the online version of this article at the publisher’s web-site Received: September 29, 2014; revised: February 13, 2015; accepted: March 19, 2015 DOI: 10.1002/elsc.201400103 1 Introduction The production of heterologous proteins is one of the key ap- plications of mammalian cells, predominately Chinese hamster ovary (CHO), today [1]. According to recent market figures, monoclonal antibody (mAb) production alone summed up to 38 billion US$ in 2009 [2]. Currently, biopharmaceutical man- ufacturers worldwide are yielding to optimize production per- formance using volumetric and cell-specific productivities as optimization parameters. This issue addresses the optimization of external factors, such as culture conditions, and internal factor such as expression or secretion systems, likewise. In this context, the questions may arise: how much mAb is already degraded inside the cells before it could be successfully exported into the culture medium? Is intracellular protein degradation a promis- ing target for maximizing cell-specific productivities further? Correspondence: Prof. Ralf Takors (ralf.takors@ibvt.uni- stuttgart.de), Institute of Biochemical Engineering, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany Abbreviations: CHO, Chinese hamster ovary; mAb, monoclonal antibody About 60 years ago, first evidences for intracellular protein degradation were found by Schoenheimer and Urey detecting heavy nitrogen ( 15 N) labeled amino groups (-NH 2 ) in multiple amino acids after labeled tyrosine was fed to rats [3]. The further discovery of the lysosome by Christian de Duve identified organelles able to degrade intracellular and extracellular proteins [4]. In lysosomes, acidic conditions are installed, enabling the protease-based degradation of fused pro- teins (macroautophagy), or even the digestion of whole or- ganelles such as mitochondria, ER membranes, glycogen bod- ies, and other cytoplasmic (macroautophagy) under starvation conditions [5]. However, the discovery of lysosomes could not adequately explain phenomena like different half-lives of proteins [6], inhibitor-specific impacts on protein degradation [7], and the energy dependency of degradation [8]. Only after the discovery of ubiquitin-proteasome activities, the missing links became visible. It was found that ubiquitin (or ATP-dependent proteolysis factor 1, APF1 as called before its identification), is covalently conjugated to protein substrates by ∗ Both authors contributed equally to this work. C  2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 1