Diao Jinpian, Young Lincoln, Zhou Peng, Shuler Michael Louis
Genentech, Inc., 1 DNA Way, South San Francisco, California 94080, USA.
Biotechnol Bioeng. 2008 May 1;100(1):72-81. doi: 10.1002/bit.21751.
Biopharmaceutical production would benefit from rapid methods to optimize production of therapeutic proteins by screening host cell line/vector combination, culture media, and operational parameters such as timing of induction. Miniaturized bioreactors are an emerging research area aiming at improving the development speed. In this work, a 3 mm thick mini-bioreactor including two 12 mm wide culture chambers connected by a 5 mm wide channel is described. Active mixing is achieved by pressure shuttling between the two chambers. Gas-liquid phase exchange for oxygen and carbon dioxide is realized by molecular diffusion through 50 microm thick polymethylpentene membranes. With this unique design, a velocity difference between the middle area and the side areas at the interfaces of the culture chambers and the connecting channel is created, which enhances the mixing efficiency. The observed mixing time is on the order of 100 s. The combination of high permeability toward oxygen of polymethylpentene membranes and fluid movement during active pressure shuttling enables higher volumetric oxygen transfer coefficients, 5.7 +/- 0.4-14.8 +/- 0.6 h(-1), to be obtained in the mini-bioreactors than the values found in traditional 50 mL spinner flasks, 2.0-2.5 h(-1). Meanwhile, the calculated volume averaged shear stress, in the range of 10(-2)-10(-1) N/m(2), is within the typical tolerable range of animal cells. To demonstrate the applicability of this mini-bioreactor to culture suspended animal cells, the insect cell, Spodoptera frugiperda, is cultured in mini-bioreactors operated under a K(L)a value of 14.8 +/- 0.6 h(-1) and compared to the same cells cultured in 50 mL spinner flasks operated under a K(L)a value of 2.2 h(-1). Sf-21 cells cultured in the mini-bioreactors present comparable length of lag phases and growth rates to their counterparts cultured in 50 mL spinner flasks, but achieve a higher maximum cell density of 5.3 +/- 0.9 x 10(6) cell/mL than the value of 3.4 +/- 0.4 x 10(6) cell/mL obtained by cells cultured in 50 mL spinner flasks. Sf-21 cells infected with SEAP-baculovirus produce a maximum SEAP concentration of 11.3 +/- 0.7 U/mL when cultured in the mini-bioreactor. In contrast, infected Sf-21 cells cultured in 50 mL spinner flasks produce a maximum SEAP concentration of 7.4 +/- 0.9 U/mL and onset of production is delayed from 18 h in minibioreactor to 40 h in spinner flasks.
生物制药生产将受益于快速方法，通过筛选宿主细胞系/载体组合、培养基和操作参数（如诱导时间）来优化治疗性蛋白质的生产。小型生物反应器是一个新兴的研究领域，旨在提高开发速度。在这项工作中，描述了一种3毫米厚的微型生物反应器，它包括两个通过5毫米宽的通道连接的12毫米宽的培养室。通过在两个腔室之间进行压力切换来实现主动混合。氧气和二氧化碳的气液相交换通过50微米厚的聚甲基戊烯膜的分子扩散来实现。通过这种独特的设计，在培养室和连接通道的界面处的中间区域和侧面区域之间产生了速度差，这提高了混合效率。观察到的混合时间约为100秒。聚甲基戊烯膜对氧气的高渗透性与主动压力切换过程中的流体运动相结合，使得在微型生物反应器中能够获得比传统50毫升旋转瓶（2.0 - 2.5 h⁻¹）更高的体积氧传递系数，即5.7 ± 0.4 - 14.8 ± 0.6 h⁻¹。同时，计算得到的体积平均剪切应力在10⁻² - 10⁻¹ N/m²范围内，处于动物细胞的典型耐受范围内。为了证明这种微型生物反应器对悬浮动物细胞培养的适用性，将昆虫细胞草地贪夜蛾在K(L)a值为14.8 ± 0.6 h⁻¹的微型生物反应器中培养，并与在K(L)a值为2.2 h⁻¹的50毫升旋转瓶中培养的相同细胞进行比较。在微型生物反应器中培养的Sf - 21细胞与在50毫升旋转瓶中培养的细胞相比，具有相当的延迟期长度和生长速率，但达到了更高的最大细胞密度，为5.3 ± 0.9×10⁶ 细胞/毫升，而在50毫升旋转瓶中培养的细胞获得的值为3.4 ± 0.4×10⁶ 细胞/毫升。感染了SEAP - 杆状病毒的Sf - 21细胞在微型生物反应器中培养时产生的最大SEAP浓度为11.3 ± 0.7 U/毫升。相比之下，在50毫升旋转瓶中培养的感染Sf - 21细胞产生的最大SEAP浓度为7.4 ± 0.9 U/毫升，并且生产开始时间从微型生物反应器中的18小时延迟到旋转瓶中的40小时。
