Development Of Serum-free Medium For DHFR-CHO Cells And Investigation On The Roles Of Some Key Medium Components

The emergence of monoclonal antibody (mAb) drugs hold great promise in treating many stubborn diseases, such as cancer, autoimmune diseases, and fulminating infectious diseases. They turn out to be the fastest evolving biomedicine in recent years, demonstrating a huge marketing potenial. Due to the generally high doses (tens to hundreds of milligrams) required in treatments, both capacity and efficiency of antibody production become two critical factors in dictating the ultimate success of their industrialization. Since bioreactor-based large-scale animal cell culture represents the core technology nowadays in the field of manufacturing antibody drugs, how to meet the demands for higher speed, efficiency and quality has become the theme for its current and future development. Among this, the development of serum-free medium and investigation on its key components is one of the critical steps in process development and optimization of large-scale animal cell culture, which eventually leads to rationally optimized culture medium formula and feed strategies, thus enabling antibody production at lower cost and higher efficency. Towards this end, in the present study, statistical design methodologies and high throughput technologies were utilized to develop serum-free medium formula and several different basal media were established to accommodate growth and antibody production of DHFR-CHO cells; based on these, a few key medium components including ferric citrate and hypoxanthine/thymidine were studied in details concerning their effects on cell growth and antibody production, with the objective to further optimize serum-free medium formula and feed strategies. These studies certainly lay the foundation for developing efficient and cost-effective animal cell culture process.First of all, a high throughput technology was established by sequential application of three statistical methodologies including Plackett-Burman design, method of steepest ascent, and central composite design, and five serum-free medium formula were successfully developed, which could stably support DHFR-CHO cells long-term subcultures. Based on the further optimization of three key medium components (ferric citrate, hypoxanthine/thymidine, and taurine), an optimized serum-free medium formulation (OptM) was ultimately formed. Moreover, the results of batch culture in OptM medium demonstrated that cell growth and antibody production were very comparable to the commercial serum-free medium (HyClone(?) SFM4CHOTM), with a maximum viable cell density of 3.93×106 cells/ml and a final antibody concentration of 251 mg/L, respectively. To further cognize the roles of some key medium components in serum-free medium and optimize the subsequent fed-batch culture process, the present work investigated the influences of ferric citrate and hypoxanthine/thymidine on cell growth, metabolism, antibody production and the underlying mechanisms.It was found that cell growth and antibody production could not be well supported when cells cultivated in the absence of ferric citrate, even when transferrin-Fe was maintained at a very high concentration (400 mg/L). Thus, the result revealed that ferric citrate was a very important component in serum-free medium, playing an irreplaceable role in supporting cell growth, which could not be provided by transferrin-Fe. This unexpected phenomenon was predominantly due to the different uptake rate of Fe and could be explained in two aspects:(1) with the same molar amount of Fe, ferric citrate showed a 6-fold higher uptake rate than transferrin-Fe; (2) the transferrin receptor on the cell membrane was not sensitive to the change of intracellular Fe concentration, which could be demonstrated by the un-altered mRNA level when the intracellular Fe concentration was quite low and sufficient Fe was provided solely by transferrin-Fe in the culture medium. Additionally, it was found that the concentration range of 50-125 mg/L of ferric citrate was more suitable for both cell growth and antibody production and the concentration at 75 mg/L turned out to be the optimum. On the other hand, cells were much easier to suffer from cellular apoptosis when exposed to too low (0 and 25 mg/L) or too high (250 and 500 mg/L) concentrations of ferric citrate. Furthermore, ferric citrate had barely effect on the specific antibody production rate and it fulfilled its function by the formation of ferric-citate chelated complex rather than ferric ion and citric acid solely.By comparison between a weak (citric acid) and a strong iron chelator (EDTA), it was found that citric acid was superior to EDTA in respect of iron uptake rate. Furthermore, ferric ion was also found to be much easier to uptake than ferrous ion in DHFR-CHO cells, which led to improved cell growth and antibody production.A complete investigation of hypoxanthine/thymidine (H&T) on cell growth, metabolism, antibody production and physiological characteristics were also performed. As a result, cell growth and antibody production were found to be dependent upon H&T concentration. Specifically, H&T at a low concentration (10 mg/L hypoxanthine and 2 mg/L thymidine, denoted as H10T2) stimulated initial cell growth, while showing no effect on specific antibody production rate (qmAb); on the other hand, at a high concentration (100 mg/L hypoxanthine and 20 mg/L thymidine, denoted as H100T20), H&T tended to inhibit cell growth, but obviously promoted qmAb.The highest antibody volumetric concentration was achieved in H10T2 culture, with a 25.7% enhancement of antibody concentration compared to H0T0 culture, mainly through elevated integrated viable cell concentration (IVCC). By analyzing the metabolic rates of glucose, lactate, ammonia and amino acids, it was found that these metabolic rates were relatively much higher in early period of culture (0-4 d) than those of late period; metabolic rates were much lower in H10T2 culture than those in H100T20 culture with highest glucose utilization rate in H10T2 culture and prominent elevated total intracellular protein (TIP) in H100T20 culture. In addition, the accelerated cell growth behavior in H10T2 medium during the first 2 d may be partially explained by the higher proportion of cells in S phase before 2 d.To further recognize the stability upon H&T addition during long-term subcultures (40 generation), cell growth, antibody production, antibody light chain/heavy chain mRNA level and cellular mutagenesis were all taken account into investigation. It was found that cell growth stability was not altered upon H&T addition; Adversely, the supplementation of H&T could obviously stimulate cell growth. In respect of qmAb, cells under both H&T and methotrexate (MTX) culture conditions showed similar stable antibody production during the first 20 subcultures, followed by a gradual decline in the gmAb, with a 40% drop at the 40th generation. As it showed, H&T addition increased antibody production by some extents (20-25%) instead of accelerating decay kinetics in qmAb.Furthermore, the decreased qmAb at the 40th generation of long-term subculture correlated well with the decreased transcript level of intracellular heavy chain (HC) of mAb. Further analyzing the cell mutagenesis regarding the hypoxanthine-guanine phosphoribosyltransferase (HPRT), it was demonstrated that the DHFR-CHO cells experienced a spontaneous mutation (5.75-fold) in the course of long-term cultivation and this HPRT spontaneous mutation could be repressed by H&T addition to a less extent (2.75-fold). On the other hand, treatment of MTX provoked the mutation rate (10.5-fold), and this induced mutagenesis could not be reversed by H&T addition.Finally, a fed-batch culture using a biphasic addition strategy of H&T was established in bioreactor based on the further concentration optimization of ferric citrate and citric acid. Compared with the initial batch culture, the developed fed-batch culture in 2 L bioreactor significantly increased maximum viable cell density (6.45×106 cells/ml) and final antibody concentration (632 mg/L).Taken collectively, a serum-free medium for DHFR-CHO cells producing anti-CD20 antibody was successfully developed with independent intellectual property rights, and an economic and efficient biphasic fed-batch culture process was established, which laid a solid foundation for the industrialized production of the monoclonal antibody. In addition, the roles of ferric citrate and hypoxanthine/thymidine in cell growth and antibody production were firstly reported in the present work, which might be very informative to other investigators.