Effects Of Tyrosine On RCHO Cell Culture Performance And Monoclonal Antibody Production And Its Mechanism

Recently,with the rapid development of antibody drug industry,mammalian cell culture producing antibody drugs has become the core technology of the biopharmaceutical industry.Under the guidance of the QbD concept,establishing a high-yield and high-quality and robust cell culture process is an inevitable requirement for the development of antibody drug industry.The rational design of nutrient composition of medium is an important guarantee to realize this requirement.Therefore,it is very important to understand the eff-ect of tyrosine or other nutrients on cell growth and production.In this paper,the decease of viable cell density and productivity during rCHO cell culture will be key point of process performance study.Firstly,the effects of tyrosine on fed-batch culture performance of rCHO cells will be studied,as well as the causes of cell death type and its mechanism by tyrosine limitation.Then,the effects of tyrosine concentrations on antibody productivity will be studied basing on two aspects,which are extracellular culture environment and intracellular antibody synthesis.Finally,combining the change of cell extracellular and intracellular environment and antibody crucial quality attribution,and the effects of tyrosine limitation on antibody synthesis and its mechanism will be further explored,which laid the foundation for optimization the supply of key nutrients such as tyrosine strategy and the establishment of robust cell culture process.Firstly,the previously established rCHO cell fed-batch culture process,including cell growth,culture pH and mAb production,were investigated.It is found that viable cell density,cell viability,and antibody productivity rapid declined to zero during the late phase of fed-batch cultures.and cells died along with culture pH decreasing to below 6.5.Therefore,3 process parameters and 18 key components of medium was researched the influence on the performance of fed-batch cultures.It was found that the antibody concentration increased only when the amount of tyrosine was fed.The effects of tyrosine on cell culture performance was further investigated by single factor gradient experiment.The results show that when the cumulative amount of tyrosine reached 4.3 mmol/L or above,the viable cell density,cell viability,antibody productivity and culture pH could be maintained relatively in the late stage of fed-batch cultures.However,when the cumulative amount of tyrosine was as low as 1.9 mmol/L or below,both cell growth and antibody production during fed-batch cultures were severely restrained,and culture pH was also significantly reduced.This indicated that tyrosine was the key factor affecting cell growth,antibody production and culture pH in the late stage of fed-batch cultures.Regarding cell death rapidly during tyrosine limitation culture,two typical fed-batch cultures xwhich gained high or low tyrosine concentration(Ctrl and Tyr-Limit)were chosen for further research,and extracellular and intracellular tyrosine concentrations and cell growth state were investigated.The results show that tyrosine concentration decreased to limitation level on day 7 of the Tyr-Limit culture.In Tyr-Limit culture the cell activity gradually decreased to zero from day 9 to 12.Cells showed clumps and morphological incompleteness after day 10 in Tyr-Limit culture.The viable cell density and cell viability decreased to zero rapidly during 6 hours after day 11 of Tyr-Limit culture.However,cells were kept in a relatively stable state during the Ctrl culture.The results show that tyrosine depletion might lead to irreversible damage of cells and eventually cell death.Further investigation on four cell death types in typical fed-batch cultures including autophagy,apoptosis,necrosis and mitosis revealed that there was no abnormality in G2/M phase checkpoints associated with mitotic disaster under the tyrosine depletion condition.The proportion of necrotic cells was also higher than that of the control experiment.But,when tyrosine was depleted,the intracellular LC3? and LC3?/LC3I levels increased gradually,and were significantly higher than the control experiment.At the same time,the positive rate of MDC reached 31.7%and 53.8%on day 8 and day 11 of the Ctrl culture,respectively,which was much higher than that on day 3 and the whole control experiment(about 5%).This indicates that cell death occurring during Tyr-Limit culture might be dominated by autophagy.On this basis,the meida exchange experiments were used to control the concentrations of tyrosine between 0-1.8 mmol/L.The effects of tyrosine concentrations on cell growth were investigated.The results show that when the concentration of tyrosine was as low as 0.1 mmol/L or less,the cell growth was limited.When the concentration of tyrosine was further reduced to 0.01 mmol/L or less,the cells directly die rapidly.At the same time,when the concentration of tyrosine was controlled between 0-1.0 mmol/L,the lower the concentration of tyrosine,the higher the level of autophagy.Moreover,this autophagy was associated with mTOR signaling pathway by a tyrosine depletion signal,which activated mTOR-mediated ULK protein and then promoted autophagy.The cell death along with culture pH rapid decrease in the late of fed-batch culture.The results show that the main reason for the rapid decrease of the culture pH was not the gradual accumulation of lactic acid,but mainly the cell autophagy caused by the tyrosine depletion.Although the low culture pH led to the gradual death of cells,it was quite different from the rapid death of cells in the late stage of Tyr-Limit culture.The decrease in the culture pH had no significant effect on apoptosis,but also had a certain inhibitory effect on autophagy.In addition,it has been found that when the culture pH droped below 6.5,cell necrosis was promoted.In this paper,we investigated the difference in the antibody expression in the late stage of typical fed-batch cultures.It was found that the low concentration of tyrosine not only reduced the specific rate of antibody production,but also caused a significant change in the quality of the antibody,such as increased fragmentation contents,decreased aggregation contents,increased lysine variant contents,and altered charge variant composition.Furthermore,the effects of tyrosine concentration on the specific rate of antibody production were analyzed from the asp ects of cell extracellular culture environment and intracellular antibody synthesis.It is found that the increase of the osmotic pressure,lactate concentration and ammonia concentration in the culture environment were only the results of changes in cell metabolism caused by the tyrosine depletion,while the intracellular antibody translation process was the main limitation step.Comparing the required tyrosine supply rate(0.008 pmol/cell/day)to maintain the specific rate of antibody production in the late culture,it was found that there was the substrate limitation in the antibody translation process in the Tyr-Limit culture,and the inhibitted mTOR pathway and the declined energy efficiency further limitted the antibody translation process,which ultimately led to a significant reduction in antibody translation efficiency.At the same time,experiments have found that the tyrosine depletion also affected the synthesis processing and modification of antibodies,resulting in changes of CQAs such as antibody fragmentation,aggregation and charge variants.The tyrosine depletion led to an increase in the antibody fragment content,which was mainly due to the low concentration of tyrosine in the culture supernatant,,which leads to an increase in the reducing ability of the culture supernatant,and cell death to release glutathione and thioredoxase.The tyrosine depletion caused a significant decrease in the polymer content of the antibody,mainly due to the accumulation of cystine and cysteine during the tyrosine depletion culture.The tyrosine depletion also resulted in a significant increase in lysine variant content,which may be related to down-regulation of CpB and CpH mRNA transcription levels during Tyr-Limit culture.Through the research in this paper,on the one hand,the key roles of tyrosine in the high-density f'ed-batch culture of rCHO cells were deeply understood.On the other hand,the tyrosine depletion on the cell growth and antibody production were further understood.The understanding can provide scientific guidance for the rational design of the nutrient composition of medium and the optimization of the supply strategy of key nutrients such as tyrosine.