Transcriptome and proteome analysis of steady-state in a perfusion CHO cell culture process.

Long-term continuous protein production can be reached by perfusion operation. Through the continuous removal of waste metabolites and supply of nutrients, steady-state (SS) conditions are achieved after a certain transient period, where the conditions inside the reactor are not only uniform in space but also constant in time. Such stable conditions may have beneficial influences on the reduction of product heterogeneities. In this study, we investigated the impact of perfusion cultivation on the intracellular physiological state of a CHO cell line producing a monoclonal antibody (mAb) by global transcriptomics and proteomics. Despite stable viable cell density was maintained right from the beginning of the cultivation time, productivity decrease, and a transition phase for metabolites and product quality was observed before reaching SS conditions. These were traced back to three sources of transient behaviors being hydrodynamic flow rates, intracellular dynamics of gene expression as well as metabolism and cell line instability, superimposing each other. However, 99.4% of all transcripts and proteins reached SS during the first week or were at SS from the beginning. These results demonstrate that the stable extracellular conditions of perfusion lead to SS also of the cellular level.

Proteomic analysis of micro-scale bioreactors as scale-down model for a mAb producing CHO industrial fed-batch platform.

The pharmaceutical production of recombinant proteins, such as monoclonal antibodies, is rather complex and requires proper development work. Accordingly, it is essential to develop appropriate scale-down models, which can mimic the corresponding production scale. In this work, we investigated the impact of the bioreactor scale on intracellular micro-heterogeneities of a CHO cell line producing monoclonal antibodies in fed-batch mode, using a 10 mL micro-bioreactor (ambr™) scale-down model and the corresponding 300 L pilot-scale bioreactor. For each scale, we measured the time evolution of the proteome, which enabled us to compare the impact of the bioreactor scale on the intracellular processes. Nearly absolute accordance between the scales was verified by data mining methods, such as hierarchical clustering and in-detail analysis on a single protein base. The time response of principal enzymes related to N-glycosylation was discussed, emphasizing major dissimilarities between the glycan fractions adorning the heavy chain and the corresponding protein abundance. The enzyme expression displayed mainly a constant profile, whereas the resulting glycan pattern changed over time. It is concluded that the enzymatic activity is influenced by the changing environmental conditions present in the fed-batch processes leading to the observed time-dependent variation.