Introduction To meet the growing demand for recombinant biotherapeutics in use today, producer cell lines are under constant development to improve their productivity, stability and product quality. Enhancing our understanding of biological responses during cultivation through host-cell omics, complementing conventional bioprocess monitoring and medium-based measurements as proxies for cellular function, may enable immediate adaptation of critical process parameters (CPPs) to ensure efficient and quality-assured production. Methods The design and evaluation of a fast, feasible and representative multi-omics sampling and downstream analysis approach for suspension cultures utilising state-of-the-art methodology and instrumentation were conduced. The resulting workflow allows for analysis of the host-cell proteome, phosphoproteome, metabolome, and secretome. To demonstrate applicability, a fed-batch culture of the openly available, monoclonal antibody-producing cell line NISTCHO was consequently examined for the impact of feeding on cellular processes as indicated by changes in the proteome and reversible phosphorylation of proteins. Sampling was performed 60 min prior to feeding, as well as 15, 60, 90, 120 and 240 min post-feed, in both exponential and stationary growth phases to reflect multiple stages of the bioprocess. Parallel monitoring of nutrient consumption and metabolic excretions was deployed. To fully leverage the high-resolution LC-MS/MS data generated on the Bruker timsTOF pro, curation of the Chinese hamster (Cricetulus griseus) reference proteome, reducing redundancy and improving annotation and coverage, was performed. Additionally, to utilise the statistical power of time-course data, an in-house R package was developed to identify and cluster significant temporal dynamics of features. Results and Discussion A bioprocess-integrated multi-omics sampling and analysis workflow, addressing multiple key challenges, was designed and implemented. Further evaluation included investigation of a NISTCHO fed-batch culture. This work provides comprehensive temporal profiling of protein and protein phosphorylation dynamics in response to changes in nutrient availability around feeding windows distinct to different phases of the bioprocess. Key results range from master regulators of redox homeostasis and metabolism to pinpointing individual regulatory sites of DNA repair enzymes. We emphasise that mass spectrometry can provide an in-depth analysis of cellular function in various product and process-relevant biological settings. The advanced characterisation of an academically available producer cell line and the generation of high-quality bioprocess reference data as part of our DigiTherapeutX project will fuel research efforts towards integrated digitalised production and predictable quality of protein therapeutics. Innovative aspects • Design and implementation of a bioprocess-integrated multi-omics sampling and analysis workflow • Data processing and analysis pipeline includes curated Cricetulus griseus reference proteome and in-house software to handle time-course data • Comprehensive temporal profiling of more than 7000 proteins and 3000 phosphorylation sites in response to feeding distinct to different phases of the NISTCHO fed-batch bioprocess