Polysome profiling of mAb producing CHO cell lines links translational control of cell proliferation and recombinant mRNA loading onto ribosomes with global and recombinant protein synthesis

Abstract

mRNA translation is a key process determining growth, proliferation and duration of a Chinese hamster ovary (CHO) cell culture and influences recombinant protein synthesis rate. During bioprocessing, CHO cells can experience stresses leading to reprogramming of translation and decreased global protein synthesis. Here we apply polysome profiling to determine reprogramming and translational capabilities in host and recombinant monoclonal antibody-producing (mAb) CHO cell lines during batch culture. Recombinant cell lines with the fastest cell specific growth rates were those with the highest global translational efficiency. However, total ribosomal capacity, determined from polysome profiles, did not relate to the fastest growing or highest producing mAb cell line, suggesting it is the ability to utilize available machinery that determines protein synthetic capacity. Cell lines with higher cell specific productivities tended to have elevated recombinant heavy chain transcript copy numbers, localized to the translationally active heavy polysomes. The highest titer cell line was that which sustained recombinant protein synthesis and maintained high recombinant transcript copy numbers in polysomes. Investigation of specific endogenous transcripts revealed a number that maintained or reprogrammed into heavy polysomes, identifying targets for potential cell engineering or those with 5′ untranslated regions that might be utilized to enhance recombinant transcript translation.

Application of Imaging Flow Cytometry for the Characterization of Intracellular Attributes in Chinese Hamster Ovary Cell Lines at the Single‐Cell Level

“…We note that the cell line with the highest HC mRNA content on day 3 did not have the highest titer. This cell line also had a high amount of LC mRNA on day 6, suggesting that above a certain transcript threshold, there might be potential bottlenecks further down the translðation and secretion pathways …”

“…CHO cell lines stably expressing a recombinant immunoglobulin G1κ (IgG1κ) were grown in proprietary medium supplemented with 50 µ m methionine sulfoximine (Sigma‐Aldrich) and routinely subcultured as previously described …”

“…The IC polypeptide was assessed using standard western analysis . Membranes were incubated overnight with an anti‐IgG (FC) antibody conjugated to horseradish peroxidase (HRP) or anti‐κ conjugated to HRP diluted at 1:10 000 (both from The Binding Site).…”

“…Total RNA was extracted using an RNeasy kit and treated with DNase (Qiagen). Quantitative reverse transcription PCR (qRT‐PCR) was performed as previously reported …”

Towards Enhancing Therapeutic Glycoprotein Bioproduction: Interventions in the PI3K/AKT/mTOR Pathway

“…Mounting biochemical evidence indicates a correlation between mTORC1 activity and cell line productivity. Comparisons between low and high productivity cells suggest a correlation between productivity and polysome formation, a hallmark of mTORC1 activity (Godfrey et al, 2017). The eIF4E/4E-BP1 stoichiometry positively correlates with cell productivity (Josse et al, 2016).…”