Principal component analysis (PCA) clusters the bioreactor runs into three separate groups using three components, which describes 55 % of the variance: days 0-3, days 4-9, and days 10-14 (see Figure ). These groupings correlate with cellular phases identified from bioreactor stats such as viable cell count and total cell count (Figure ) and are well described in the literature. Broadly these bioreactor phases correlate with stages of the cell cycle, namely: cellular growth, stationary phase and cell death. We observe a sigmoidal curve of total cell count, with proliferation occurring steadily between days 0-8 and cell viability remaining high until day 10 where it begins to drop as the cells enter the stress phase. Product titre is measurable from day 4 and increases steadily until harvesting on day 14. While the PCA shows grouping into three broad metabolic states, there is a clear observable progression of the protein expression, where each day has a set of proteins which are most abundant (Figure ). Pathway analysis of the data revealed that pathways relating to proliferation, DNA replication and energy production changed significantly between key time points (days 1, 4, 7 and 14) which cover the metabolic transitions between the gross cellular phases (see SI). Proteins which relate to these pathways, were observed to undergo high log fold change (LFC) across the bioreactor runs, and were identified as being of high importance through prior knowledge of CHO cell metabolism, were selected as targets (Figure ). Eight proteins (Figure ), of which seven were observed with both instruments, were selected for targeted assays which will then be representative of the global changes that occur in the bioreactor across batch production. The chosen peptides are from the following key proteins that have been shown to differentially regulate across production: DNA replication licensing factor MCM2 (MCM2), proliferating cellular nuclear antigen (PCNA) inform on cell proliferation ; lactate dehydrogenase chain A (LDHA) and mitochondrial carnitine/acylcarnitine transferase (MCAT), provide insights to energy production ; Glutathione S-transferase Mu 7 (GSTM7) and lysosome associated membrane protein 1 (LAMP1), both report on the effects of oxidative stress and autophagy ; Protein-glutamine gamma-glutamyltransferase 2 (TGM2) plays a key role in apoptosis as well as other processes. Additionally Clusterin (CLUS), a chaperone, was chosen since it is commonly observed co-eluting with mAbs during protein-A purification and binds non-specifically to IgG Fab and Fc regions. The rationale for choosing these proteins is that they all represent different critical processes during the cell cycle and follow observable trends of quantification. MRM assays are more robust and simpler and would provide a go-to method for routine use in industrial settings. Triple quadrupoles have higher sensitivity than discovery platforms, although here we have selected peptides where the abundance and change in abundance are well within the sensitivity of most modern LC-MS instruments.