Evaluation of feed strategy for high quality biosimilar IgG production in CHO cell fed-batch process

Chinese Hamster Ovary (CHO) cell lines are grown in cultures with varying asparagine and glutamine concentrations, but further study is needed to characterize the interplay between these amino acids. By following 13C-glucose, 13C-glutamine, and 13C-asparagine tracers using metabolic flux analysis (MFA), CHO cell metabolism was characterized in an industrially relevant fed-batch process under glutamine supplemented and low glutamine conditions during early and late exponential growth. For both conditions MFA revealed glucose as the primary carbon source to the tricarboxylic acid (TCA) cycle followed by glutamine and asparagine as secondary sources. Early exponential phase CHO cells prefer glutamine over asparagine to support the TCA cycle under the glutamine supplemented condition, while asparagine was critical for TCA activity for the low glutamine condition. Overall TCA fluxes were similar for both conditions due to the trade-offs associated with reliance on glutamine and/or asparagine. However, glutamine supplementation increased fluxes to alanine, lactate and enrichment of glutathione, N-Acetyl-Glucosamine (NAG) and pyrimidine-containing-molecules. The late exponential phase exhibited reduced central carbon metabolism dominated by glucose, while lactate reincorporation and aspartate uptake were preferred over glutamine and asparagine. These 13C studies demonstrate that metabolic flux is process time dependent and can be modulated by varying feed composition.

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Perfusion reduces bispecific antibody aggregation via mitigating mitochondrial dysfunction-induced glutathione oxidation and ER stress in CHO cells

Scientific Reports ◽

10.1038/s41598-020-73573-4 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Pritam Sinharoy ◽

Aaron H. Aziz ◽

Natalia I. Majewska ◽

Sanjeev Ahuja ◽

Michael W. Handlogten

Keyword(s):

Er Stress ◽

Mitochondrial Dysfunction ◽

Batch Process ◽

Aggregate Formation ◽

Fed Batch ◽

Cho Cell ◽

Perfusion Bioreactors ◽

Aggregation Formation ◽

Glutathione Oxidation ◽

Intracellular Redox

Abstract One major challenge observed for the expression of therapeutic bispecific antibodies (BisAbs) is high product aggregates. Aggregates increase the risk of immune responses in patients and therefore must be removed at the expense of purification yields. BisAbs contain engineered disulfide bonds, which have been demonstrated to form product aggregates, if mispaired. However, the underlying intracellular mechanisms leading to product aggregate formation remain unknown. We demonstrate that impaired glutathione regulation underlies BisAb aggregation formation in a CHO cell process. Aggregate formation was evaluated for the same clonal CHO cell line producing a BisAb using fed-batch and perfusion processes. The perfusion process produced significantly lower BisAb aggregates compared to the fed-batch process. Perfusion bioreactors attenuated mitochondrial dysfunction and ER stress resulting in a favorable intracellular redox environment as indicated by improved reduced to oxidized glutathione ratio. Conversely, mitochondrial dysfunction-induced glutathione oxidation and ER stress disrupted the intracellular redox homeostasis, leading to product aggregation in the fed-batch process. Combined, our results demonstrate that mitochondrial dysfunction and ER stress impaired glutathione regulation leading to higher product aggregates in the fed-batch process. This is the first study to utilize perfusion bioreactors as a tool to demonstrate the intracellular mechanisms underlying product aggregation formation.

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Transcriptome Analysis of CHO Cell Size Increase During a Fed-Batch Process

Biotechnology Journal ◽

10.1002/biot.201800156 ◽

2018 ◽

Vol 14 (3) ◽

pp. 1800156 ◽

Cited By ~ 2

Author(s):

Xiao Pan ◽

Abdulaziz A. Alsayyari ◽

Ciska Dalm ◽

Jos A. Hageman ◽

René H. Wijffels ◽

...

Keyword(s):

Cell Size ◽

Transcriptome Analysis ◽

Batch Process ◽

Size Increase ◽

Fed Batch ◽

Cho Cell

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Transcriptome analysis for the scale-down of a CHO cell fed-batch process

Journal of Biotechnology ◽

10.1016/j.jbiotec.2018.05.012 ◽

2018 ◽

Vol 279 ◽

pp. 61-72 ◽

Cited By ~ 7

Author(s):

Abdulaziz A. Alsayyari ◽

Xiao Pan ◽

Ciska Dalm ◽

Jochem W. van der Veen ◽

Nienke Vriezen ◽

...

Keyword(s):

Transcriptome Analysis ◽

Batch Process ◽

Fed Batch ◽

Cho Cell ◽

Scale Down

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Production of Rhamnolipid Biosurfactant from Fed Batch Culture by Pseudomonas aeruginosa using Multiple Substrates

Current Nutrition & Food Science ◽

10.2174/1573401314666181107100127 ◽

2020 ◽

Vol 16 (6) ◽

pp. 928-933

Author(s):

Jujjavarapu S. Eswari

Keyword(s):

Pseudomonas Aeruginosa ◽

Batch Process ◽

Time Interval ◽

Fed Batch ◽

Multiple Substrates ◽

Surface Active Agents ◽

Limiting Nutrients ◽

Limiting Nutrient ◽

Surface Active ◽

Rhamnolipid Biosurfactant

Objective: Biosurfactants are the surface active agents which are used for the reduction of surface and interfacial tensions of liquids. Rhamnolipids are the surfactants produced by Pseudomonas aeruginosa. It requires minimum nutrition for its growth as it can also grow in distilled water. The rhamnolipids produced by Pseudomonas aeruginosa are extra-cellular glycolipids consisting of L-rhamnose and 3-hydroxyalkanoic acid. Methods: The fed-batch method for the rhamnolipid production is considered in this study to know the influence of the carbon, nitrogen, phosphorous substrates as growth-limiting nutrients. Pulse feeding is employed for limiting nutrient addition at particular time interval to obtain maximum rhamnolipid formation from Pseudomonas aeruginosa compared with the batch process. Results: Out of 3 fed batch strategies constant glucose fed batch strategy shows best and gave maximum rhamnolipid concentration of 0.134 g/l.

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