Biopharmaceutical manufacturing is currently the principal growth sector within the pharmaceutical industry. Predominantly formed in mammalian cells, these therapeutic proteins exist as a spectrum of different isoforms and hence pose a unique set of challenges that must be addressed to ensure optimum product quality. To better understand whether the product really is the process, quantitative peptide centred multidimensional liquid chromatography tandem mass spectrometry (LC-MS) based proteomics studies were performed using a Chinese Hamster Ovary (CHO) cell line that expressed an anti-Interleukin-8 IgG1 monoclonal antibody (mAb). Following serum free suspension batch culture under varied bioprocess conditions, quantitative proteomics was completed on the producer CHO cells to elucidate the cellular response to altered culture conditions of pH, temperature and dissolved oxygen. The developed platform was also applied for the analysis of naïve CHO K1 cells following their exposure to spent culture media from the various production runs. Complete characterisation of the expressed mAb was performed, using advanced LC-MS methods including high resolution middle down mass spectrometry; intact protein analysis of critical quality attributes, stable isotope based quantitative glycan analysis and hydrogen deuterium exchange mass spectrometry for structural comparability analysis. In addition data independent LC-MS quantitative proteomics of residual host cell protein impurities was also carried out to evaluate the effect of downstream processing on the quality of the final drug substance. Combined, the findings herein provide a holistic insight into the effect of various upstream and downstream parameters on the quality of therapeutic proteins and facilitate a greater understanding of the molecular mechanisms governing biopharmaceutical production systems, thereby creating a hypothesis for improved future cell line development using various engineering strategies.