Real‐time quantification and supplementation of bioreactor amino acids to prolong culture time and maintain antibody product quality
Real‐time quantification and supplementation of bioreactor amino acids to prolong culture time and maintain antibody product quality
复制标题
实时定量和补充生物反应器氨基酸，以延长培养时间并保持抗体产品质量
DOI：
10.1002/btpr.2894
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发表时间：
2019
影响因子：
2.9
通讯作者：
C. Agarabi
中科院分区：
文献类型：
--
作者：
David N. Powers;Yifan Wang;Erica J. Fratz;Sai Rashmika Velugula;Brittany Chavez;Phillip Angart;Nicholas Trunfio;Seongkyu Yoon;C. Agarabi
对生物反应器中的细胞培养物进行真实的实时监测可以加快响应速度，以纠正潜在的批次失效扰动，这些扰动通常在批次完成之前未被发现并导致失效。目前，分析技术致力于生物反应器参数的真实的实时监测，如pH值、溶解氧和温度，营养物质如葡萄糖和谷氨酰胺，或代谢产物如乳酸盐。尽管氨基酸作为治疗性蛋白质产品的结构单元的重要性，但除了谷氨酰胺之外，它们的浓度通常不被测量。在这里，我们提出了一项关于氨基酸监测，补充策略以及这些技术如何影响细胞生长曲线和产品质量的研究。我们使用初步生物反应器运行通过确定初始氨基酸消耗模式来建立基线，其结果用于选择在生物反应器中耗尽的氨基酸库。将这些氨基酸合并成混合物，在后续运行期间补充到生物反应器中，使用我们开发的基于质谱的在线方法监测其浓度，以快速评估粗生物反应器培养基中的氨基酸浓度。我们发现这些混合物可以延长培养寿命，逆转导致批次死亡的活细胞密度下降。此外，我们评估了这些策略如何影响蛋白质产品质量，例如聚糖谱。氨基酸消耗数据与主成分分析中的最终聚糖谱一致，以确定哪些氨基酸与聚糖结果最密切相关。
Real‐time monitoring of cell cultures in bioreactors can enable expedited responses necessary to correct potential batch failure perturbations which may normally go undiscovered until the completion of the batch and result in failure. Currently, analytical technologies are dedicated to real‐time monitoring of bioreactor parameters such as pH, dissolved oxygen, and temperature, nutrients such as glucose and glutamine, or metabolites such as lactate. Despite the importance of amino acids as the building blocks of therapeutic protein products, other than glutamine their concentrations are not commonly measured. Here, we present a study into amino acid monitoring, supplementation strategies, and how these techniques may impact the cell growth profiles and product quality. We used preliminary bioreactor runs to establish baselines by determining initial amino acid consumption patterns, the results of which were used to select a pool of amino acids which gets depleted in the bioreactor. These amino acids were combined into blends which were supplemented into bioreactors during a subsequent run, the concentrations of which were monitored using a mass spectrometry based at‐line method we developed to quickly assess amino acid concentrations from crude bioreactor media. We found that these blends could prolong culture life, reversing a viable cell density decrease that was leading to batch death. Additionally, we assessed how these strategies might impact protein product quality, such as the glycan profile. The amino acid consumption data were aligned with the final glycan profiles in principal component analysis to identify which amino acids are most closely associated with glycan outcomes.