Decreasing drug development timeline via upstream process intensification A scalable, high-intensity perfusion process was developed at Boehringer Ingelheim, Fremont Inc which is 10× more productive for producing recombinant proteins than comparative fed batch processes in the same 14-day run duration. By eliminating wasteful cell bleed we were able to achieve cell densities up to five times greater than standard "steady state" perfusion culture previously used. In order to sustain such large cell masses at manageable media exchange rates, concentrated media feeds were developed which effectively allow for optimization of nutrient delivery and dilution rate. We believe this system is scalable up to 1kL; the process has already been demonstrated successfully at the pilot scale (100L), where bioreactor productivities averaging over 5 g/L/day have been demonstrated. We begin development with new cell lines for the high intensity perfusion process by adapting spin-tube and shake flask models that others have used for fed batch. These methods are used to test for important control parameters to allow full development in a 2L bioreactor. AMBR250 bioreactors can be used, though not optimal, as will be discussed. Due to the simplicity of the process design, the integrated downstream is developed at small scale using classical batch chromatographic techniques, including high throughput process development and standard chromatographic steps. The virus inactivation step is developed by accounting for viscosity and titration of the product and buffer in the Protein A elution peak, which differ slightly from product to product. With these simple development techniques, we believe the highly productive process could be commercially viable at Phase Ⅰ, with limited to no Phase Ⅲ process development.
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机译：通过上游工艺强化缩短药物开发时间线Boehringer Ingelheim，Fremont Inc.开发了一种可扩展的高强度灌注工艺，在相同的14天运行时间内，该蛋白的生产重组蛋白的能力比对比补料分批工艺高10倍。通过消除浪费的细胞流血，我们能够获得比以前使用的标准“稳态”灌注培养高出五倍的细胞密度。为了以可控制的培养基交换速率维持如此大的细胞团，开发了浓缩培养基进料，其有效地使营养物递送和稀释率最优化。我们认为该系统可扩展至1kL;该工艺已经在中试规模（100L）上得到了成功的证明，其中生物反应器的生产率平均已超过5 g / L /天。我们通过适应其他人用于补料批次的旋转管和摇瓶模型，开始开发用于高强度灌注过程的新细胞系。这些方法用于测试重要的控制参数，以允许在2L生物反应器中完全显影。如将要讨论的那样，可以使用AMBR250生物反应器，尽管不是最佳的。由于工艺设计的简单性，使用经典的批量色谱技术（包括高通量工艺开发和标准色谱步骤）以小规模开发了集成下游产品。通过考虑蛋白A洗脱峰中产物和缓冲液的粘度和滴定度来开发病毒灭活步骤，这在产物之间略有不同。通过这些简单的开发技术，我们相信高生产率的工艺在第一阶段可能具有商业可行性，并且仅限于无第三阶段的工艺开发。
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