Abstract
Unfavorable process conditions lead to adverse cultivation states, limited cell growth and thus hamper biotherapeutic protein production. Oxygen deficiency or hyperosmolality are among the most critical process conditions and therefore require continuous monitoring. We established a novel sensor CHO cell line with the ability to automatically sense and report unwanted process conditions by the expression of destabilized fluorescent proteins. To this end, an inducible real-time system to detect hypoxia by hypoxia response elements (HREs) of vascular endothelial growth factor (VEGF) origin reporting limitations by the expression of destabilized green fluorescent protein (GFP) was created. Additionally, we established a technique for observing hyperosmolality by exploiting osmotic response elements (OREs) for the expression of unstable blue fluorescent protein (BFP, FKBP-BFP), enabling the simultaneous automated supervision of two bioprocess parameters by using a dual sensor CHO cell line transfected with a multiplexable monitoring system. We finally also provided a fully automated in-line fluorescence microscopy-based setup to observe CHO cells and their response to varying culture conditions. In summary, we created the first CHO cell line, reporting unfavorable process parameters to the operator, and provided a novel and promising sensor technology accelerating the implementation of the process analytical technology (PAT) initiative by innovative solutions.
Highlights
Unfavorable process conditions lead to adverse cultivation states, limited cell growth and hamper biotherapeutic protein production
For example, leads to the stabilization of hypoxia-inducing factor 1α (HIF-1α), which dimerizes with its cofactor HIF-1β to interact with hypoxia response elements (HREs) and activates the expression of protective genes such as erythropoietin (EPO) or vascular endothelial growth factor A (VEGF-A) (Fig. 1B) to perpetuate cellular physiological functions[17–20]
The hypoxia response pathway is mainly controlled by hypoxia-inducing factor-1α (HIF-1α), which is degraded during normoxia
Summary
Unfavorable process conditions lead to adverse cultivation states, limited cell growth and hamper biotherapeutic protein production. We established a novel sensor CHO cell line with the ability to automatically sense and report unwanted process conditions by the expression of destabilized fluorescent proteins To this end, an inducible real-time system to detect hypoxia by hypoxia response elements (HREs) of vascular endothelial growth factor (VEGF) origin reporting limitations by the expression of destabilized green fluorescent protein (GFP) was created. Osmolality of more than 0.42 osm/kg[8] can negatively influence a bioprocess, as hyperosmolality is reported to reduce growth and cellular viability and impair product quality, as increased salt concentrations may promote aggregation or change the glycosylation pattern of a b iotherapeutic[9–11] To avoid these drawbacks, continuous monitoring of critical process parameters is required by p O2 probes for oxygen and manual offline measurements for osmolality to detect and adjust deviations and sustain a successful bioprocess. We were able to demonstrate independent functionality of the introduced monitoring systems and to set up an in-line automatically functional fluorescence microscope-based method to enable observation of CHO cells close to real time
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