A serum-free medium (SFM-MDM) without any animal-driven protein was developed for the production of recombinant antibody in suspension cultures of recombinant Chinese hamster ovary (CHO) cell line, $CS^*13-1.00$. Even though small amount of medium components were added to the basal medium, their inhibitory effect on cell aggregation made single-cell suspension cultures possible. Through fortification of several vitamins, final recombinant antibody concentration and specific antibody productivity were considerably enhanced. Srum-free formulation for a rCHO cell line can be used as a starting or basal serum-free medium for the development of highly effective cell culture bioprocess using recombinant CHO cells to produce recombinant pharmaceutical proteins. To modify basal protein-free medium, two statistical methods was utilized. Concentrations of vitamins and protein hydrolysates were optimized by using the experimental design method including the Plackett-Burman method. Through computer software, $Design-Expert^\textregistered$, optimal ratio of two components was predicted. 33 % of A and 67% of B were decided as proper ratio to enhance cell growth and recombinant antibody production.
Effects of various culture temperature and pH on cell growth and protein production were investigated in the cultivation of $CS^*13-1.00$ cells. When culture temperature was shifted periodically between 37 ℃ and 33 ℃ every 24 hour, integral of viable cell density was increased significantly compared to the control at 37 ℃ at given pH conditions, resulting in increase of final concentration of recombinant antibody. Glucose uptake rate and lactate production rate increased in pH dependent manner as pH set value increased in a range of 6.8 to 7.2. When the pH was adjusted at pH 7.0 and 7.2 with a periodic shift of temperature, final antibody concentrations were 3.5 and 3.9-fold higher than the control, respectively. Also, When culture pH was shifted from 7.0 to 6.8, integral of viable cell density and antibody concentration was $18.04\times10^6$ cells d and 174.36 mg l-1, respectively. Through results, pH adjustment by dual phase (shift to low level) could be a promising strategy for enhancing production of target proteins and reducing byproduct accumulation in further culture process. In fed-batch culture, with the feeding strategy for consideration of specific glucose consumption rate cell viability was maintained with relatively high level after exponential growth phase was passed and maximum cell density $(2.78cells ml^{-1})$ was 84% higher, resulting in 2.85 and 2.84 times higher integral of viable cell density and final antibody concentration, respectively, compared with batch culture. With periodic shift of temperature, cell viability was preserved with level more than 60% for about 400 hours, integral of viable cell density was 3.58 times higher than the batch. Final concentration was $265.33mgl^{-1}$, which was much higher than that obtained from the batch culture (4.04 times) and the previous fed-batch culture (1.42 times). Also, with high initial cell density, cell density was increased directly from initial phase of culture and maximum specific growth rate was $1.11d^{-1}$ and volumetric antibody productivity was dramatically increased to $25.50mgl^{-1}d^{-1}$ which was 3.25 times higher than the batch culture.
Mathematical model equations were established for prediction of culture behavior of $CS^*13-1.00$ in batch and fed-batch culture. Strategy of culture temperature shift to low level was optimized to modify batch equation. Antibody concentration was high when the shift point was culture time of 48 or 72 hour, mid-exponential growth phase. Also, effect of high initial cell density on cell growth and recombinant antibody production was also predicted in fed-batch culture. Volumetric productivity could be increased with high initial cell density through decrease of culture period.
재조합 CHO 세포 $(CS^*13-1.00)$ 의 부유 배양을 통해 재조합 항체를 생산하기 위해 동물 유래 단백질이 포함되지 않은 무혈청 배지를 개발하였다. 배양 중 세포들이 뭉치는 것을 막기 위해 몇 가지 배지 성분들을 소량 투여하여 단일 세포로서 부유배양이 가능하게 하였으며 비타민의 첨가로 재조합 항체의 최종 생산량과 비생산성을 향상 시켰다. Plackett-Burman 법과 컴퓨터 프로그램인 Design-Expert를 이용한 통계적 방법들을 통해 무혈청 배지내의 비타민이나 단백질 가수분해물의 농도를 변화시켰다.
배양 온도와 pH의 변화가 세포 성장과 재조합 항체 생산에 미치는 영향에 관한 연구를 수행하였다. 배양 온도를 37℃ 에서 33℃ 로 24시간마다 주기적으로 변화 시켰을 때 누적 생존 세포수와 최종 항체 생산량이 37℃ 에서 계속 배양했을 때보다 크게 증가하였다. 배양 pH가 6.6에서 7.4로 증가함에 따라 포도당 소모 속도 및 젖산 생산 속도는 증가하였고 최종항체 생산량은 pH를 조절해주지 않은 경우보다 pH 7.0과 7.2에서 각각 3.5배와 3.9배 증가하였다. 배양 도중 조절 pH를 바꾸는 연구에서 pH가 7.0에서 6.8로 바뀌었을 때 누적 생존 세포수와 항체 생산량이 증가하는 것을 확인하였다.
생물 반응기를 이용한 CHO 세포의 유가식 배양에 관한 연구에서는 비 포도당 소모 속도를 고려하여 배양 중 배지를 공급하였다. 최대 세포 농도는 회분식 배양과 대비하여 84%이상 증가하였고 세포 농도가 최대로 증가한 이후에도 상당한 기간 동안 비교적 높게 유지되어 누적 생존 세포수와 최종 항체 농도는 회분식 배양에서 보다 각각 2.9배와 2.8배 증가하였다. 앞서 긍정적인 효과를 보였던 주기적 온도 변화 전략을 유가식 배양에 적용한 결과 세포 생존률은 대략 400시간 동안 60% 이상으로 유지되었고 누적 생존 세포수 및 최종 항체의 농도는 회분식 배양 대비 각각 3.6배, 4.0배 증가하였다. 또한 초기 세포 농도를 높게 하여 유가식 배양을 수행한 결과 비 성장 속도가 크게 증가하였으며 회분식 배양 대비 부피 생산성은 3.3배 이상 증가한 것을 확인 하였다.
$CS^*13-1.00$ 세포의 회분식, 유가식 배양시의 다양한 상황을 예측하기 위한 수학적 모델을 구현하였다. 배양 중 온도를 33℃ 로 처음 낮추는 시점은 모델 시뮬레이션 결과 48시간과 72시간 대가 가장 적절한 것으로 나타났다. 유가식 배양에서 다양한 초기 세포 농도가 세포 성장 전반과 재조합 항체 생산에 미치는 영향에 대해서 알아보았고 높은 초기 세포 농도가 부피 생산성을 증가시키는 원인 또한 규명하였다.
