悬浮培养重组中国仓鼠卵巢细胞CHO-C12表达抗EGFRvⅢ嵌合抗体C12研究
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摘要
嵌合抗体C12具有对EGFRvⅢ表达阳性肝癌的潜在治疗价值。本论文对表达该C12抗体的的rCHO-C12细胞进行了无血清悬浮培养驯化,并对其在体外培养的生长代谢特性及在生物反应器中的流加补料和灌注培养工艺进行了初步研究。
1)采用直接降血清方法对rCHO-C12细胞进行了无血清驯化。rCHO-C12细胞完全适应EX302无血清培养条件后,最大细胞密度可达到6×106cells/mL;降血清后对数生长期内抗体C12的比产率略有降低；同时对该rCHO-C12细胞在EX302(JRH),CHO-CD (Gibco), SAF-CHO-G-001(清大有为)三种培养基中的生长曲线的比较,CHO-CD (Gibco)对rCHO-C12具有更好的细胞培养性能(如细胞存活率和最大活细胞密度),但从经济和实验性能比较,EX302更适合于该CHO细胞在生物反应器内的大规模培养。
2)比较了rCHO-C12细胞分别在摇瓶和5L生物反应器分批培养时的生长代谢及抗体表达特性。结果表明：(1)该细胞在生物反应器中培养时细胞生长最大密度为4×106cells/mL, C12抗体最高表达量达到190mg/L,其均约为摇瓶批培养细胞密度和抗体表达水平的2/3；(2)在生物反应器中批培养时葡萄糖的限制浓度在6mmol/L左右,而谷氨酰胺限制性浓度为1mmol/L左右。同时发现部分氨基酸如Asp、Glu、Asn、Cys、Thr、Met、Trp、Ser、Ile、Leu、Lys比消耗速率较大,并且Asp、Glu、Asn在细胞生长期耗竭,这可能是影响细胞在生物反应器中最大活细胞密度及抗体表达量的一个重要因素。这为建立氨基酸平衡流加培养提供了基础。
3)建立了rCHO-C12细胞生物反应器中流加培养和灌注培养等方式高效表达C12抗体。结果表明：(1)氨基酸平衡流加培养和灌注培养有利于维持高细胞存活率,以延长培养周期；(2)与流加培养方式相比,灌注培养的底物的细胞得率((YVx/Gluc和YVx/Glun)和底物形成抗体C12的得率系数(YAb/gluc和YAb/glun)均最大,分别为13.87×108cells/g、291.94×108cells/g、44.72mg/g和721.40 mg/g,而且灌注培养获得的总抗体量最多,达到1854mg,而氨基酸平衡流加培养达到的总抗体浓度最大,为282mg/L；同时,发现灌注培养代谢副产物乳酸和氨的最大浓度仅分别为15mmol/L和3.8mmol/L,低于其它培养方式。因此,灌注培养方式是rCHO-C12细胞表达抗体C12的最佳培养方式。
The mouse-human chimeric anti-EGFRvIII antibody C12 was expressed by recombinant CHO-C12 cells, which has potentials to be used as therapeutics solely or conbination with 5-fluorouracil(5-FU) for the treatment of EGFRvIII-positive Hepatocellular carcinoma (HCC). In this thesis,the adaption of rCHO-C12 cells growth to serum-free midium was carried out and culture characterastics of rCHO-C12 cells were investigated. Additionally, three bioprocesses including batch, fed-batch and perfusion bioprocesses have been developed for antibody C12 production in suspension culture.
1) Direct adaption of rCHO-C12 cells growth to EX302 serum-free medium was established. The peak cell density of 6×106cells/mL was achieved when rCHO-C12 cells was cultured in EX302 serum-free medium. It was observed that the specific antibody C12 formation rate of rCHO-C12 cells cultivated in serum-free medium was slighly lower than that obtained with serum medium at exponential phase. The serum-free medium CHO-CD is better for rCHO-C12 cells with higher performances in viable cell density among the three test media CHO-CD (Gibco), EX302(JRH), SAF-CHO-G-001 (Qingdayouwei,China).Economically and experimentally, EX302 were more suitable for CHO-C12 cells large-scale culture in bioreactor.
2) Culure performances like cell growth characterization and C12 production of rCHO-C12 cells cultured in shake flasks culutre and in a 5L bioreactor were investigated. The results showed that the maximum cell density and highest antibody concentration of rCHO-C12 cells batch-cultured in a 5L bioreactor were 4×106cells/mL and 190mg/L, respectively, which were approximately 70% of the values obtained in shake flasks culture. Also, the limited glucose and glutamine concentrations in bioreactor were 6mmol/L and 1mmol/L for rCHO-C12 cells growth, respectively. Regarding metabolic by-products lactate, ammonia and alanine accumulation in bioreator were remarkably lower than that in shake flasks. In addition, the specific consumption rate of the following consuming amino acids: Asp, Glu, Asn, Cys, Thr, Met, Trp, Ser, Ile, Leu in bioreactor culture were higher than that in shake flasks except to Ile and Leu. Especially, three amino acids including Asp、Glu、Asn was consumeded to depletion at early growth phase, resulting in limitation in cell growth and antibody production. Based on these results, a balanced amino-acid fed-batch bioprocess was developed.
3) Fed-batch and perfusion bioprocesses in bioreator were successfully developed for antibody C12 production. The results showed that (1) compared with the batch culture mode, fed-batch and perfusion culture modes could effectively maintain higher cells viability so as to extend the cell culture duration; (2) The maximum mean yield coefficients like YVx/Gluc of 13.87×108cells/g,YVx/Glun of 291.94×108cells/g, YAb/gluc of 44.72mg/g and YAb/glun of 721.40 mg/g were achieved in perfusion culture mode with the highest total amount of antibody C12 of 1854mg, however, the maximum C12 Ab concentration of 282mg/L was reached in balanced amino acid fed-batch bioprocess. Besides, it was observed that the metabolic by-products lactic acid of 15mmol/L and ammonia of 3.8 mmol/L were lowest for perfusion culture among three developed culture processes. Therefore, perfusion culture mode could be the optimum bioprocess for antibody C12 production by rCHO-C12 cells.
1)采用直接降血清方法对rCHO-C12细胞进行了无血清驯化。rCHO-C12细胞完全适应EX302无血清培养条件后,最大细胞密度可达到6×106cells/mL;降血清后对数生长期内抗体C12的比产率略有降低；同时对该rCHO-C12细胞在EX302(JRH),CHO-CD (Gibco), SAF-CHO-G-001(清大有为)三种培养基中的生长曲线的比较,CHO-CD (Gibco)对rCHO-C12具有更好的细胞培养性能(如细胞存活率和最大活细胞密度),但从经济和实验性能比较,EX302更适合于该CHO细胞在生物反应器内的大规模培养。
2)比较了rCHO-C12细胞分别在摇瓶和5L生物反应器分批培养时的生长代谢及抗体表达特性。结果表明：(1)该细胞在生物反应器中培养时细胞生长最大密度为4×106cells/mL, C12抗体最高表达量达到190mg/L,其均约为摇瓶批培养细胞密度和抗体表达水平的2/3；(2)在生物反应器中批培养时葡萄糖的限制浓度在6mmol/L左右,而谷氨酰胺限制性浓度为1mmol/L左右。同时发现部分氨基酸如Asp、Glu、Asn、Cys、Thr、Met、Trp、Ser、Ile、Leu、Lys比消耗速率较大,并且Asp、Glu、Asn在细胞生长期耗竭,这可能是影响细胞在生物反应器中最大活细胞密度及抗体表达量的一个重要因素。这为建立氨基酸平衡流加培养提供了基础。
3)建立了rCHO-C12细胞生物反应器中流加培养和灌注培养等方式高效表达C12抗体。结果表明：(1)氨基酸平衡流加培养和灌注培养有利于维持高细胞存活率,以延长培养周期；(2)与流加培养方式相比,灌注培养的底物的细胞得率((YVx/Gluc和YVx/Glun)和底物形成抗体C12的得率系数(YAb/gluc和YAb/glun)均最大,分别为13.87×108cells/g、291.94×108cells/g、44.72mg/g和721.40 mg/g,而且灌注培养获得的总抗体量最多,达到1854mg,而氨基酸平衡流加培养达到的总抗体浓度最大,为282mg/L；同时,发现灌注培养代谢副产物乳酸和氨的最大浓度仅分别为15mmol/L和3.8mmol/L,低于其它培养方式。因此,灌注培养方式是rCHO-C12细胞表达抗体C12的最佳培养方式。
The mouse-human chimeric anti-EGFRvIII antibody C12 was expressed by recombinant CHO-C12 cells, which has potentials to be used as therapeutics solely or conbination with 5-fluorouracil(5-FU) for the treatment of EGFRvIII-positive Hepatocellular carcinoma (HCC). In this thesis,the adaption of rCHO-C12 cells growth to serum-free midium was carried out and culture characterastics of rCHO-C12 cells were investigated. Additionally, three bioprocesses including batch, fed-batch and perfusion bioprocesses have been developed for antibody C12 production in suspension culture.
1) Direct adaption of rCHO-C12 cells growth to EX302 serum-free medium was established. The peak cell density of 6×106cells/mL was achieved when rCHO-C12 cells was cultured in EX302 serum-free medium. It was observed that the specific antibody C12 formation rate of rCHO-C12 cells cultivated in serum-free medium was slighly lower than that obtained with serum medium at exponential phase. The serum-free medium CHO-CD is better for rCHO-C12 cells with higher performances in viable cell density among the three test media CHO-CD (Gibco), EX302(JRH), SAF-CHO-G-001 (Qingdayouwei,China).Economically and experimentally, EX302 were more suitable for CHO-C12 cells large-scale culture in bioreactor.
2) Culure performances like cell growth characterization and C12 production of rCHO-C12 cells cultured in shake flasks culutre and in a 5L bioreactor were investigated. The results showed that the maximum cell density and highest antibody concentration of rCHO-C12 cells batch-cultured in a 5L bioreactor were 4×106cells/mL and 190mg/L, respectively, which were approximately 70% of the values obtained in shake flasks culture. Also, the limited glucose and glutamine concentrations in bioreactor were 6mmol/L and 1mmol/L for rCHO-C12 cells growth, respectively. Regarding metabolic by-products lactate, ammonia and alanine accumulation in bioreator were remarkably lower than that in shake flasks. In addition, the specific consumption rate of the following consuming amino acids: Asp, Glu, Asn, Cys, Thr, Met, Trp, Ser, Ile, Leu in bioreactor culture were higher than that in shake flasks except to Ile and Leu. Especially, three amino acids including Asp、Glu、Asn was consumeded to depletion at early growth phase, resulting in limitation in cell growth and antibody production. Based on these results, a balanced amino-acid fed-batch bioprocess was developed.
3) Fed-batch and perfusion bioprocesses in bioreator were successfully developed for antibody C12 production. The results showed that (1) compared with the batch culture mode, fed-batch and perfusion culture modes could effectively maintain higher cells viability so as to extend the cell culture duration; (2) The maximum mean yield coefficients like YVx/Gluc of 13.87×108cells/g,YVx/Glun of 291.94×108cells/g, YAb/gluc of 44.72mg/g and YAb/glun of 721.40 mg/g were achieved in perfusion culture mode with the highest total amount of antibody C12 of 1854mg, however, the maximum C12 Ab concentration of 282mg/L was reached in balanced amino acid fed-batch bioprocess. Besides, it was observed that the metabolic by-products lactic acid of 15mmol/L and ammonia of 3.8 mmol/L were lowest for perfusion culture among three developed culture processes. Therefore, perfusion culture mode could be the optimum bioprocess for antibody C12 production by rCHO-C12 cells.
引文
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