%0 Journal Article %1 ISI:000357679500005 %A Rimbon, Jeremy %A Sanchez-Kopper, Andres %A Wahl, Andreas %A Takors, Ralf %C 111 RIVER ST, HOBOKEN 07030-5774, NJ USA %D 2015 %I WILEY-BLACKWELL %J ENGINEERING IN LIFE SCIENCES %K myown %N 5, SI %P 499-508 %R 10.1002/elsc.201400103 %T Monitoring intracellular protein degradation in antibody-producing Chinese hamster ovary cells %U https://doi.org/10.1002/elsc.201400103 %V 15 %X 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 C-13-labeled L-lysine. The fate of the labeling signal was tracked in intracellular peptides, which were the proteolytic fragments of the mAb. Signal analysis 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. Labeling 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 estimated, representing about 3\% of the net mAb production. Hence, intracellular mAb degradation occurs even under the rather smooth production conditions installed.

@article{ISI:000357679500005, abstract = {{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 C-13-labeled L-lysine. The fate of the labeling signal was tracked in intracellular peptides, which were the proteolytic fragments of the mAb. Signal analysis 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. Labeling 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 estimated, representing about 3\% of the net mAb production. Hence, intracellular mAb degradation occurs even under the rather smooth production conditions installed.}}, added-at = {2018-06-08T13:04:13.000+0200}, address = {{111 RIVER ST, HOBOKEN 07030-5774, NJ USA}}, affiliation = {{Takors, R (Reprint Author), Univ Stuttgart, Inst Biochem Engn, Allmandring 31, D-70569 Stuttgart, Germany. Rimbon, Jeremy; Sanchez-Kopper, Andres; Wahl, Andreas; Takors, Ralf, Univ Stuttgart, Inst Biochem Engn, D-70569 Stuttgart, Germany.}}, author = {Rimbon, Jeremy and Sanchez-Kopper, Andres and Wahl, Andreas and Takors, Ralf}, author-email = {{ralf.takors@ibvt.uni-stuttgart.de}}, biburl = {https://puma.ub.uni-stuttgart.de/bibtex/2e452c273847e1eb78b7fcf03d9e310a8/ralftakors}, da = {{2018-01-26}}, doc-delivery-number = {{CM4UC}}, doi = {{10.1002/elsc.201400103}}, eissn = {{1618-2863}}, funding-acknowledgement = {{company Bohringer Ingelheim (Biberach, Germany); German Academic Exchange Service (DAAD, Bonn, Germany); Instituto Tecnologico de Costa Rica (ITCR, Cartago, Costa Rica)}}, funding-text = {{The work was partially co-funded by the company Bohringer Ingelheim (Biberach, Germany) and co-supported by the German Academic Exchange Service (DAAD, Bonn, Germany) and the Instituto Tecnologico de Costa Rica (ITCR, Cartago, Costa Rica). We would like to kindly thank the cell culture technology group in Bielefeld for providing the strain CHO-K1.}}, interhash = {a8d27542b93a9527a3b545b187365f18}, intrahash = {e452c273847e1eb78b7fcf03d9e310a8}, issn = {{1618-0240}}, journal = {{ENGINEERING IN LIFE SCIENCES}}, journal-iso = {{Eng. Life Sci.}}, keywords = {myown}, keywords-plus = {{GENOME-WIDE ANALYSIS; CODED AFFINITY TAGS; MASS-SPECTROMETRY; MAMMALIAN-CELLS; METABOLISM; CULTURE; AMINOTRANSFERASE; TRANSLATION; PROTEASOMES; COMPONENTS}}, language = {{English}}, month = {{JUL}}, number = {{5, SI}}, number-of-cited-references = {{51}}, pages = {{499-508}}, publisher = {{WILEY-BLACKWELL}}, research-areas = {{Biotechnology \& Applied Microbiology}}, times-cited = {{3}}, timestamp = {2018-06-08T11:04:13.000+0200}, title = {{Monitoring intracellular protein degradation in antibody-producing Chinese hamster ovary cells}}, type = {{Article}}, unique-id = {{ISI:000357679500005}}, url = {https://doi.org/10.1002/elsc.201400103}, usage-count-last-180-days = {{0}}, usage-count-since-2013 = {{9}}, volume = {{15}}, web-of-science-categories = {{Biotechnology \& Applied Microbiology}}, year = {{2015}} }