Study On The Charge Heterogeneity Of Monoclonal Antibody Produced In RCHO Cell Cultures

Recently,with the continuous expansion of the antibody drug market,large-scale cultivation of mammalian cells producing antibody drugs has become the core technology of the biopharmaceutical industry.As glycoproteins,antibody drugs will undergo a series of post-translational modifications and degradations,leading to the generation of heterogeneity.Charge heterogeneity,a critical quality attribution(CQA),plays important roles in the efficacy and safety of the drugs.Due to the lacking in understanding of charge heterogeneity and its relationship with the cell culture process,quality control of antibody drugs is usually a problem during manufacturing.Therefore,a comprehensive understanding of the relationship among the clinical characteristics,quality attributions and cell culture process,especially the generation of charge variants and its relationship with cell culture process,is important to establish a high yield,high quality and robust cell culture process.In this paper,the charge heterogeneity characteristic of a monoclonal antibody(mAb)produced by an rCHO cell line was firstly investigated via a series of quality analysis methods.By cell-free assay and establishment of kinetic models,the generation process of acidic charge variants and basic charge variants(lysine variant)were then deeply investigated.Besides,the effects of culture temperature and medium components on the mAb charge variation were also determined,which laid the foundation for the establishment of cell culture process and effective control of mAb charge heterogeneity.Firstly,the previously established rCHO cell fed-batch culture process,including cell growth,mAb production and charge distribution were investigated.It is found that the peak cell density reached 10.5×106 cells/ml,the final mAb concentration and specific production rate were 3181 mg/L and 28.21 mg/109cells/day,respectively.Weak cation exchange chromatography(WCX)results showed that the mAb produced contained high levels of both acidic and basic charge variants,which were 27.7%and 34.9%respectively at the end of the culture.The mAb charge heterogeneity was then characterized by a panel of analytical techniques.The generation site of charge variants was determined by papain digestion and the results revealed that the modifications leading to the charge heterogeneity were located in both Fab and Fc fragments.Then,three charge fractions,including acidic,main and basic were separated and collected by WCX and the aggregate content,reduced fragment content,glycosylation pattern,binding affinity and CDC bioactivity of the three fractions were detected.It is found that the aggregate,reduced fragment and sialylation glycan contents of the acidic fraction were much higher than those of the basic fraction,suggesting that aggregate,reduced fragment and sialic acid are important sources of mAb acidic variants.Besides,bioactivity detection results showed that charge heterogeneity led to a significant decrease in both binding affinity and CDC bioactivity.Therefore,charge heterogeneity is a CQA and it is necessary to get insights into the generation of mAb charge variants and its relationship with cell culture process.Regarding the high levels of acidic variants,a comprehensive understanding of the generation of acidic variants during cell culture process was obtained by cell-free assay and establishment of kinetic model.Cell-free assay results indicated that the acidic variants generation takes place inside and outside of the cells,consisting of both intracellular and extracellular processes.Besides,the extracellular process was believed to be a spontaneous process without enzyme catalysis which was affected by temperature and medium composition.Lowering culture temperature could significanlty slow down the extracellular acidic variants generation.Additionally,kinetics study revealled that the extracellular process is a first-order reaction and the Eapp value estimated from the Arrhenius equation suggested that the extracellular process might be mainly attributed to asparagine deamidation.Furthermore,a kinetic model of acidic variants generation was established,indicating that the extracellular process plays a dominant role(74.9%)in acidic variants generation.Besides,it is also found that the acidic variant level when secreted(Qa)was rapidly increased at the late stage of the culture,which was found to be associated with the increase of aggregate and fragment contents.Since lowering culture temperature could effectively decrease the extracellular acidic variants generation,the effects of culture temperature on rCHO cell growth,mAb production and charge distribution was investigated.It is found that lowering culture temperature inhibited mAb production and affected mAb charge distribution while had no significant effect on cell growth.MAb production and specific productivity were decreased from 3532 mg/L and 29.15 mg/109cells/day to 2436 mg/L and 12.29 mg/109cells/day,respectively,when the temperature was decreased from 37℃ to 32℃.For the charge distribution,while lowering culture temperature decreased the acidic variants level,the basic variants level was increased and the main species level was not affected.When the temperature was decreased from 37℃ to 32℃,the acidic variants level was decreased by 9%and the basic variants level was increased by 7%.Additionally,the temperature shift time also had remarkable effects on mAb production and charge distribution.An earlier temperature shift led to a lower mAb concentration,the mAb concentration obtained when the temperature was shifted on day 4 was 66.2%of the mAb concentration when shifted on day 10.On the other hand,with the delay of temperature shift time,mAb acidic variants level was increased while the basic variants level was decreased,the main species level remained unchanged.The acidic and basic variants levels were increased and decreased by 4%,respectively,when the shift time was delayed from day 4 to day 10.Considering the high levels of basic variants during the rCHO cell cultures,lysine variation of the mAb was detected by the basic carboxypeptidase digestion.It is found that the lysine variant level was as high as 16.8%at the end of the culture,which led to the high level of basic variants.Therefore,it is an effective approach to decrease the basic variants level by controlling the lysine variant level.Accordingly,the effects of culture temperature,metal ions(copper and zinc)and basic amino acids(arginine and lysine)on the C-terminal lysine processing and mAb lysine variant level were investigated.Lowering culture temperature significantly increased the level of lysine variant.RT-PCR results indicated that CpB and CpH are two basic carboxypeptidases responsible for the C-terminal lysine processing.Lowering culture temperature significantly decreased the expression of CpB,which finally affected mAb C-terminal lysine processing and lysine variant level.On the other hand,the medium copper and zinc ions played important roles in rCHO cell growth,mAb production and charge distribution.Copper and zinc,as the inhibitor and cofactor of basic carboxypeptidases,can inhibit and improve the activity of basic carboxypeptidases,respectively,and affect the C-terminal lysine processing and lysine variant level further.Besides,it is also found that high levels of lysine variant were associated with high concentrations of basic amino acids in our previous study.Product inhibition effect of arginine and lysine on the basic carboxypeptidases was evident from the notable intra-and extracellular arginine and lysine concentrations comparable with Ki values(inhibition constant)of basic carboxypeptidases.Cell-free assay further confirmed the presence of the product inhibition effect in CHO cells.Finally,the C-terminal lysine processing during cell culture process was further investigated by cell-free assay and the establishment of kinetic model.It is found that the C-terminal lysine processing consists of both intracellular and extracellular process and the extracellular C-terminal lysine processing is a first-order reaction.The rate coefficient(k)and processing rate were both increased during the cell culture process while the intracellular process rate was decreased.Besides,it is also found that the intracellular process plays a dominant role(93.6%)in the C-terminal lysine processing by the calculation of the kinetic model.Through this research,the generation of mAb charge variants during cell culture process was comprehensively investigated,the understanding of the relationship between mAb charge variation and cell culture process was also enriched,which laid the foundation for the establishment of cell culture process and effective control of mAb charge heterogeneity.