Weiß, Christine Hilde (2022)
Impact of iron on CHO metabolism and recombinant protein production.
Technische Universität Darmstadt
doi: 10.26083/tuprints-00021046
Ph.D. Thesis, Primary publication, Publisher's Version
Text
Dissertation_Christine Hilde Weiß_Impact of iron on CHO metabolism and recombinant protein production_final.pdf Copyright Information: CC BY-SA 4.0 International - Creative Commons, Attribution ShareAlike. Download (3MB) |
Item Type: | Ph.D. Thesis | ||||
---|---|---|---|---|---|
Type of entry: | Primary publication | ||||
Title: | Impact of iron on CHO metabolism and recombinant protein production | ||||
Language: | English | ||||
Referees: | Kolmar, Prof. Dr. Harald ; Preuß, Prof. Dr. Karlheinz D. | ||||
Date: | 2022 | ||||
Place of Publication: | Darmstadt | ||||
Collation: | vi, 134 Seiten | ||||
Date of oral examination: | 16 February 2022 | ||||
DOI: | 10.26083/tuprints-00021046 | ||||
Abstract: | Iron is an essential transition metal required in cell culture medium (CCM) due to its vital role in many cellular processes such as energy metabolism, deoxyribonucleic acid (DNA) biosynthesis or antioxidant functions. However, due to its redox capabilities, iron can also catalyze Fenton reactions favoring the formation of reactive oxygen species (ROS) that may cause severe cellular damages. This study sought to investigate the impact of iron in CCM on Chinese hamster ovary (CHO) cell line performance, on critical quality attributes (CQAs) of different recombinant proteins, on messenger ribonucleic acid (mRNA) expression levels of genes involved in iron homeostasis, and on intracellular iron or labile iron pool (LIP) levels, whereby for the last two readouts a method development was performed prior to analyzing the project relevant samples. Besides the successful establishment of a ferrozine-based assay for detecting total intracellular iron amount and the promising results obtained upon testing the fluorescent probe RhoNox-1 for detecting changes in LIP concentrations, none of the other two (fluorescent) probes tested during method development, namely calcein and TRX-PURO, were able to determine LIP amounts within CHO cells. Since those probes rely on a rather high LIP concentration present within cells, a low LIP present within CHO cells either due to a limited, saturated or low iron uptake, or due to an immediate distribution or usage of iron within the cells once taken up was thus suggested. Data also revealed that iron raw material impurities are strongly impacting cell performance and CQAs. Whereas manganese was identified as the main impurity improving cell performance and altering protein glycosylation level within Cellvento® 4CHO and 4Feed fed-batch platform with manganese presenting additionally an opposite effect on cell culture compared to iron, copper impurity contributed to an overall increased cell performance of the tested CHOZN® cell line in EX-CELL® Advanced CHO Fed-Batch-Medium platform. Usage of low impurity iron raw materials is therefore crucial to decouple the effects of iron and its trace element impurities by controlling and adjusting each element concentration independently and thereby guarantee the run of consistent and stable cell culture processes. Among the different iron sources tested within CCM during a fed-batch experiment, non-chelated iron sources caused a faster decrease in measured iron concentration within the supernatant and led to a higher detected iron amount present within the cell pellets taken during the course of the fed-batch process compared to the tested chelated iron sources, whereas no cell growth was obtained upon ferric chloride (FeCl3) usage. At a first glance, data suggest an increased uptake efficiency for CHO cells upon usage of non-chelated iron sources, however, differences might have rather come from a faster iron precipitate formation within CCM upon usage of non-chelated iron sources, additionally, since mRNA expression levels of genes involved in iron uptake did not indicate for a difference between chelated and non-chelated iron sources. The removal of possible iron precipitates prior to intracellular iron measurement as well as the investigation of the fate of iron in CCM seems thus to be crucial to understand iron-related uptake mechanisms. |
||||
Alternative Abstract: |
|
||||
Status: | Publisher's Version | ||||
URN: | urn:nbn:de:tuda-tuprints-210460 | ||||
Classification DDC: | 500 Science and mathematics > 540 Chemistry | ||||
Divisions: | 07 Department of Chemistry > Clemens-Schöpf-Institut > Fachgebiet Biochemie | ||||
Date Deposited: | 31 Mar 2022 12:22 | ||||
Last Modified: | 04 Aug 2022 09:25 | ||||
URI: | https://tuprints.ulb.tu-darmstadt.de/id/eprint/21046 | ||||
PPN: | 494267828 | ||||
Export: |
View Item |