For the optimization of recombinant protein production in Chinese hamster ovary (CHO) cells, the effects of various environmental factors such as temperature, pH on cell growth and protein quality were systematically investigated using CHO cell lines producing erythropoietin (EPO), humanized anti-4-1BB antibody, therapeutic antibody and follicle stimulating factor (FSH).
To determine the effect of low culture temperature on erythropoietin (EPO) production in recombinant Chinese hamster ovary (rCHO) cells, rCHO cells producing EPO (LGE10-9-27) were cultivated at 30, 33, and 37℃, respectively. At a culture temperature lower than 37℃, cell growth was suppressed, but cell viability remained high for a longer culture period. When the culture temperature was lowered from 37℃ to 33℃, more than 2.5-fold increase in the maximum EPO concentration was achieved. This enhanced EPO production at 33℃ was not just because of the extended culture longevity with the decreased release of proteolytic enzymes from dead cells, but mainly because of enhanced specific EPO productivity ($q_{EPO}$). The $q_{EPO}$ at 33℃ was $0.35 ± 0.08 μg/10^6$ cells/hr, which was approximately 4-fold higher than that at $37℃. Although the highest $q_{EPO}$ of $0.49 ± 0.14 μg/10^6$ cells/hr was obtained at 30℃, the maximum EPO concentration was lowest because the detrimental effect of lowering culture temperature on cell growth outweighed its beneficial effect on $q_{EPO}$. Like $q_{EPO}$, the relative EPO mRNA content increased by lowering culture temperature, indicating that the increased transcription level of EPO was responsible in part for the enhanced $q_{EPO}$ at low culture temperature. The quality of EPO produced at 33℃ in regard to isoform pattern, sialic acid content and in vivo biological activity was comparable to or even better than that produced at 37℃. Taken together, the results obtained here demonstrate the potential of the application of the low culture temperature to the commercial EPO production in rCHO cells.
To clarify the beneficial effect of low culture temperature on specific productivity (q), rCHO cells producing anti-4-1BB antibody (LGA31-56) were cultivated at three different temperatures, 30, 33, and 37℃. Lowering the culture temperature led to suppressed cell growth, cell cycle arrest in $G_0/G_1$ phase, and improved cell viability for a longer period. However, antibody production as well as $q_{Ab}$ was not increased at low culture temperature. The maximum antibody concentration and qAb at $37℃ were $110.6 ± 2.6 μg/mL$ and $0.43 ± 0.03μg/10^6$ cells/hr, respectively, while those at 30℃ were $28.3 ± 3.8 μg/mL$ and $0.44 ± 0.07 μg/10^6$ cells/hr, respectively. Northern blot analysis revealed that lowering the culture temperature did not increase the transcription level of heavy and light chains. These results were quite in contrast with the improved production of EPO, which is expressed in the same CHO host and driven by the same CMV promoters, by lowering the temperature. Therefore, the results obtained imply that the beneficial effect of low culture temperature on recombinant protein production in rCHO cells is cell line-specific.
To understand the different responses of recombinant rCHO cells to low culture temperature regarding q, 12 parental clones and their corresponding amplified clones producing a therapeutic antibody were cultivated at 32℃ and 37℃. The specific growth rate (μ) of all clones, including both parental and amplified clones, decreased by 30-63% at 32℃ , compared to 37℃. In contrast, their specific antibody productivity $(q_{Ab})$ was significantly enhanced at 32℃. Furthermore, the degree of $q_{Ab}$ enhancement at 32℃ varied a lot from 4-fold to 25-fold among the parental clones with different integration sites of the antibody gene. At 32℃, most of the amplified clones, regardless of methotrexate (MTX) levels, also showed enhanced $q_{Ab}$, but to a lesser extent than their parental clones. However, clone #14 amplified at 0.32 μM MTX (#14-0.32) and clone #20 amplified at 1 mM MTX (clone #20-1.00), unlike their parental clones, did not show enhanced $q_{Ab}$ at 32℃. Thus, it was found that the enhancing effect of low culture temperature on q of rCHO cells depends on clones. Taken together, the results obtained here emphasize the importance of clonal selection for the successful application of low culture temperature to the enhanced antibody production in rCHO cells.
To investigate the effect of culture pH in the range of 6.85 to 7.80 on cell growth and EPO production at 32.5℃ and 37.0℃, serum-free suspension cultures of rCHO were performed in a bioreactor with pH control. Lowering culture temperature from 37.0℃ to 32.5℃ suppressed cell growth, but cell viability remained high for a longer culture period. Regardless of culture temperature, the highest μ and maximum viable cell concentration were obtained at pH 7.20 and pH 7.00, respectively. Like μ, specific consumption rates of glucose and glutamine decreased at 32.5℃, compared to 37.0℃. In addition, they increased with increasing culture pH. Culture pH at 32.5℃ affected $q_{EPO}$ in a different fashion from that at 37℃. At 37℃, the $q_{EPO}$ was fairly constant in the pH range of 6.85 to 7.80, while the $q_{EPO}$ at 32.5℃ was significantly influenced by culture pH. The highest $q_{EPO}$ was obtained at pH 7.00 and 3 32.5℃ and its value was approximately 1.5-fold higher than that at pH 7.00 and 37.0℃. The proportion of acidic EPO isoforms, which is a critical factor for high in vivo biological activity of EPO, was highest in the stationary phase of growth, regardless of culture tempe and pH. Although cell viability rapidly decreased in death phase at both 32.5℃ and 37.0℃, the significant degradation of produced EPO, probably by the action of proteases released from lysed cells, was only observed at 37.0℃. Taken together, through the optimization of culture temperature a a more than 2.7-fold increase in maximum EPO concentratcompared with that at 37.0℃. These results demonstrate the importance of optimization of culture temperature and pH for enhancing EPO production in serum-free, suspension culture of rCHO cells.
The feasibility of biphasic culture strategy for the enhancement of volumetric productivity was also investigated. In monophasic cultures, rCHO cells producing EPO showed 3-fold increase in EPO production when grown at 32.5℃, pH 7.00 compared to 37.0℃, pH 7.20. However, the operation time was almost 3-fold longer at 32.5℃, pH 7.00 than at 37.0℃, pH 7.20 due to the growth suppression at 32.5℃, pH 7.00, which led volumetric productivity to only 1.4-fold increase. To increase volumetric productivity, biphasic cultures were applied where CHO cells were cultivated at growth-preferable conditions of 37.0℃, pH 7.20 for 3 days or 4 days and cultivated further at production-preferable conditions of 32.5℃, pH 7.00. Biphasic culture with a shift of conditions on day 4 resulted in about 50% reduction in operation time compared to monophasic culture at 32.5℃, pH 7.00. However, volumetric productivity marginally increased because biphasic culture did not yield far higher EPO production. To examine whether the addition of carbon and nitrogen sources at shifting time in biphasic cultures resulted in further increase in volumetric productivity, we performed biphasic cultures with the addition of glucose and soy peptone. Although biphasic culture with the addition of both glucose and soy peptone at shifting time resulted in 34% increase in modified volumetric productivity compared to biphasic culture without addition, this improvement of modified volumetric productivity seems still unsatisfactory to apply biphasic culture strategy for the production of EPO while considering much dead time of biphasic culture due to more frequent batch cycles. Consequently, for applying biphasic culture, more competitive and refined biphasic culture strategies that can yield significantly high volumetric productivity are needed.
To clarify that adaptation to low culture temperature resulted in alleviating growth suppression without decreasing q, rCHO cells producing EPO and FSH were serially cultured at 32℃ and 37℃ as a control using spinner flask. Through adaptation, m of both CHO cells increased and its value of $0.019 hr^{-1}$ for CHO-EPO cells and 0.011 $hr^{-1}$ for CHO-FSH cells remained constant after 15 generation. These results suggest that the growth suppression of CHO cells at low culture temperature could be alleviated by an adaptation. However, for both CHO-EPO and CHO-FSH cells, the q decreased through an adaptation to low culture temperature. This decrease of q through an adaptation was not caused by genetic instability. Because the q of both CHO cells decreased through an adaptation, adaptation to low culture temperature is not likely to be applicable for enhanced recombinant protein production although the alleviation of growth suppression was observed.
The effect of simultaneous application of stressful conditions on cell growth and EPO production was investigated. A single stressful culture condition induced by hypoosmotic stress (210 mOsm/kg), low culture temperature (32℃), or NaBu addition (1 mM) resulted in a 1.8 to 2.2-fold enhancement of qEPO of rCHO cells, compared to normal culture condition (37℃ and 310 mOsm/kg). Simultaneous application of these stressful conditions further enhanced qEPO up to approximately 5-fold. However, the quality of EPO was affected by stressful culture conditions. The proportion of acidic isoforms of EPO under a single stressful condition was 2.8-13.8% lower than that under normal culture condition. Simultaneous application of the stressful conditions further decreased the portion of acidic isoforms, but not significantly. Despite 5-fold enhancement of $q_{EPO}$, the portion of acidic isoforms under the simultaneous application of stressful culture conditions was 12.9-21.6% lower than that under normal culture condition. Taken together, these results suggest the potential of simultaneous application of different stressful culture conditions to the production phase of two-stage culture, where cell growth and production phases are separated, for improved EPO production.
CHO 세포에서 재조합 단백질의 생산에 미치는 환경 인자들의 영향 CHO 세포에서 재조합 단백질의 생산을 최적화하기 위하여 환경 인자들이 CHO 세포의 성장과 재조합 단백질의 생산에 미치는 영향을 연구하였다. 저온 배양이 EPO의 생산에 미치는 영향을 알아보기 위하여 EPO를 생산하는 CHO세포를 30, 33, 37°C에서 배양하였다. 37°C보다 낮은 온도에서의 배양은 세포성장을 저해하였지만 세포 생존 율도 저온 배양에서 높았다. 33°C에서 배양하였을 때 EPO생산량도 2.5배 이상 증가하였고 EPO의 단위세포 당 생산속도도 4배 가까이 증가하였다. 이러한 EPO의 단위세포 당 생산속도의 증가는 세포 내 EPO의 mRNA양의 증가에 기인하는 것이었다. 33°C에서 배양하였을 때 EPO의 품질도 감소하지 않고 오히려 37°C배양보다 우수함을 IEF 분석과 생화학적 활성의 측정을 통하여 확인하였다. 항4-1BB 항체를 생산하는 CHO세포의 경우, 저온에서 배양한 결과 세포의 성장이 저해되는 것은 EPO를 생산하는 CHO세포의 경우와 같았지만 항4-1BB 항체의 생산량과 단위세포 당 항4-1BB 항체의 생산속도는 증가하지 않았다. 이와 같이 저온배양을 하였을 때, CHO세포의 단위세포 당 단백질 생산속도가 CHO세포에 따라 다른 현상을 이해하기 위하여 치료용 항체를 생산하는 12개의 parental CHO세포 클론과 amplified CHO세포 클론을 저온배양(32°C) 하였다. 그 결과 CHO세포 클론에 따라 저온배양에서의 단위세포 당 단백질 생산속도가 다르다는 것을 확인하였다. 따라서 저온배양을 재조합 단백질의 생산을 증가시키는데 적용하기 위하여는 먼저 클론 선택과정을 통하여 저온배양 시 단위세포 당 단백질 생산속도가 증가하는 클론을 선택하여야 한다. pH가 EPO의 생산에 미치는 영향을 알아보기 위하여 EPO를 생산하는 CHO세포를 생물반응기에서 pH 6.85-7.8 과 온도 32.5, 37°C 조건 하에서 배양하였다. 그 결과 pH는 CHO세포의 성장과 EPO생산에 큰 영향을 미치는 환경 인자임을 확인하였다. 저온배양 시 세포성장을 저해하였으나 세포 생존 율은 높았다. 배양온도와 관계없이 세포 비성장 속도는 pH 7.2에서 최대였으나 최대세포농도는 pH 7.0에서 최대였다. EPO의 단위세포 당 생산속도는 37°C의 경우 pH와 관계없이 일정하였으나 32.5°C의 경우 pH 7.0에서 최대였고 그 값은 37°C 배양에 비하여 약 1.5배 높았다. EPO의 품질을 IEF분석을 통하여 확인한 결과 세포성장 정지기에서 EPO의 acidic isoform이 최대였고 이것은 수확시기가 세포성장 정지기임을 의미한다. Acidic isofom은 세포 사멸기에 감소하였는데 특히 37°C배양에서 그 정도가 32.5°C배양보다 심하였다. 32.5°C, pH 7.0에서 배양하였을 때 EPO의 생산양은 37°C, pH 7.2에서 배양하였을 때에 비하여 약 3배정도 증가하지만 운전시간이 3배정도 길어지기 때문에 EPO의 부피생산성은 불과 1.4배 증가하였다. 따라서 EPO의 부피생산성을 증가 시키기 위하여는 EPO의 생산 양을 증가시킴과 동시에 운전시간도 줄여야 함을 알 수 있었다. CHO세포배양을 세포성장에 최적인 37°C, pH 7.2 조건에서 일정시간 동안 배양한 후 배양조건을 EPO생산에 최적인 32.5°C, pH 7.0로 바꾼 후 배양을 하는 Biphasic 배양 방식의 효용성을 알아보았다. Biphasic배양 방식은 운전시간을 32.5°C, pH 7.0에서 배양하는 것에 비하여 약 1/2로 줄일 수 있었으나 EPO의 생산양은 크게 증가 시키지 못하여 EPO의 부피생산성도 증가시키지 못하였다. 따라서 Biphasic 배양방식의 적용을 위하여는 운전주기를 줄임과 동시에 EPO의 생산양도 크게 증가시키는 배양전략을 개발하여야 한다. CHO세포를 32°C에 적응 시킴으로써 저온배양에서 단위세포 당 단백질 생산속도를 유지하면서 세포성장 속도를 증가 시킬 수 있는지를 조사하였다. EPO와 FSH를 생산하는 CHO세포를 32°C에 적응시킨 결과 저온배양에서의 세포성장 속도는 증가되었다. 그러나 동시에 단위세포 당 EPO와 FSH의 생산속도는 적응되지 않은 세포에 비하여 1/2 정도로 감소하였다. 따라서 저온배양에 CHO세포를 적응시키는 것은 단위세포 당 단백질 생산속도의 감소 시키므로 단백질의 부피생산성 증가에 도움이 되지 않을 것으로 판단된다. EPO를 생산하는 CHO세포에 여러 종류의 스트레스 환경 인자 (저온, 뷰티르산염, 고삼투압, 저삼투압)를 동시에 적용하였을 때 세포성장과 EPO생산을 조사하였다. 단일 스트레스 환경 하에서 세포성장은 저해되었고 여러 종류의 스트레스 환경 인자가 동시에 적용되었을 때에는 단일 스트레스 환경에서 보다 세포성장이 더욱 저해되었다. EPO의 단위세포 당 생산속도는 고삼투압 환경 하에서는 정상환경 하에 비하여 크게 증가하지 않았으나 나머지 단일 스트레스 환경 하에서는 2배 이상 증가하였다. 여러 종류의 스트레스 환경 인자가 동시에 적용되었을 때에는 단위세포 당 생산속도는 단일 스트레스 환경에서 보다 더욱 증가되었다. 따라서 EPO생산 단계에서 여러 종류의 스트레스 환경 인자를 동시에 적용하면 부피생산성은 크게 증가 될 것으로 기대된다.
