Inhibition of glutamine‐dependent autophagy increases t‐PA production in CHO Cell fed‐batch processes

Abstract

Understanding the cellular responses caused by metabolic stress is crucial for the design of robust fed-batch bioprocesses that maximize the expression of recombinant proteins. Chinese hamster ovary cells were investigated in chemically defined, serum-free cultures yielding 10(7) cells/mL and up to 500 mg/L recombinant tissue-plasminogen activator (t-PA). Upon glutamine depletion increased autophagosome formation and autophagic flux were observed, along with decreased proliferation and high viability. Higher lysosomal levels correlated with decreased productivity. Chemical inhibition of autophagy with 3-methyl adenine (3-MA) increased the t-PA yield by 2.8-fold. Autophagy-related MAP1LC3 and LAMP2 mRNA levels increased continuously in all cultures. Analysis of protein quality revealed that 3-MA treatment did not alter glycan antennarity while increasing fucosylation, galactosylation, and sialylation. Taken together, these findings indicate that inhibition of autophagy can considerably increase the yield of biotechnology fed-batch processes, without compromising the glycosylation capacity of cells. Monitoring or genetic engineering of autophagy provides novel avenues to improve the performance of cell culture-based recombinant protein production.

Multiplexed Digital mRNA Expression Analysis Profiles System‐Wide Changes in mRNA Abundance and Responsiveness of UPR‐Specific Gene Expression Changes During Batch Culture of Recombinant Chinese Hamster Ovary Cells

“…While the NanoString nCounter has the capacity to quantify a large subset of mRNAs belonging to varied and linked pathways, we have used the current studies to define mRNAs that are most likely to provide an informative fingerprint of cellular state. The smaller subset offers the potential for exploitation to interrogate a range of cellular challenges such as sodium butyrate treatment, hyperosmotic conditions, or elevated ammonium concentrations . Despite the profiling observed in this study, further work will be needed to refine the association between mRNA expression patterns and cellular phenotypes (and to provide deeper understanding of molecular descriptions of cellular phenotype).…”

Regulatory Coordination between Two Major Intracellular Homeostatic Systems

“…The exact role of glutamine in regulation of autophagy is complex and multifactorial. Glutamine deprivation in perfused rat liver resulted in a significant increase in autophagy (87)(88)(89). In contrast, in intestinal epithelial cells, glutamine administration increased autophagy under basal and stress conditions (75).…”

Host–Guest Chemistry of Aromatic‐Amide‐Linked Bis‐ and Tris‐Calix[4]pyrroles with Bis‐Carboxylates and Citrate Anion

“…Glutamine limitation has been shown to reduce ammonium ion accumulation in culture 26–28. However, maintaining low glutamine levels during fed‐batch culture may not be practical or robust at industrial scales since it complicates the process and can reduce cell growth and product titers and may also induce autophagy in the cells 46. An alternative approach to reduce ammonia production, would be to use substitutes that reduce or even eliminate the need for glutamine in the medium.…”

“…Since the feeding was begun only after cell densities had reached ∼1 × 10 6 cells/mL, the feeding was initiated at different times for different supplements. Finally, glucose (Sigma) was added to the fed‐batch cultures to maintain ≥3 g/L44, 46, 50 on an as needed basis.…”

“…t‐PA levels in fed‐batch cultures have been reported up to 45 mg/L using serum‐free medium40, 44 and up to 250 mg/L of t‐PA using serum containing medium 45. Recently, we have reported over 500 mg/L fed‐batch production using inhibition of autophagy by 3‐methyl adenine 46. Here we report, the development of a fed‐batch feed media supplemented with plant hydrolysates and specific amino acids (AAs) along with the benefits to be obtained from combinations and sequencing of alternative medium glutamine replacement strategies.…”