E. coli is an attractive host organism for strong recombinant protein expression. It expresses products either as soluble protein or as inclusion bodies (IB). IBs are insoluble, mostly inactive aggregates. However, recent progress enabled the efficient refolding back into their bioactive structure. Targeted IB production processes have been designed based on their characteristic features such as high yields along with purity and their simple separation. More profound process knowledge is needed to reveal interacting parameters required for quality by design grounded process development. This enables strategies for simplifying and accelerating upstream as well as downstream procedures. We present a workflow for gathering deeper process knowledge by a design of experiment approach for improved IGF1 IB formation in relation to impurity concentration. An IB expression maximum of 19.8 mgIGF1·gDCW-1 was found at pH 6.5, 37 °C and an IPTG induction of >45 µmol gDCW-1 for 12 h. Subsequently, three refolding buffers were tested together with a nonwoven anion exchange adsorber filter module. Knowledge-based buffer selection enabled high impurity log reduction values (LRVEndotoxin = 4.9; LRVDNA = 4.8, LRVHCP = 0.1-1) as well as chromatography column guarding potential by using those adsorptive matrices. Furthermore, adsorptive filtration followed by tangential flow filtration proved to be a promising alternative for product concentration.

中文翻译：

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IGF1包含主体：一种基于QbD的过程方法，可实现高效的USP以及DSP早期运行。

大肠杆菌是有吸引力的宿主生物，可实现强大的重组蛋白表达。它以可溶性蛋白或包涵体（IB）形式表达产品。IB是不溶的，大部分是不活泼的聚集体。然而，最近的进展使得能够有效地重新折叠回其生物活性结构。针对目标IB生产工艺的设计基于其特征，例如高产量，纯度高以及分离简单。需要更深入的过程知识，以通过基于设计的过程开发来揭示质量所需的交互参数。这使简化和加速上游以及下游过程的策略成为可能。我们提出了一种通过设计实验方法来收集更深的工艺知识的工作流程，以改进与杂质浓度相关的IGF1 IB的形成。在pH 6.5、37°C​​和IPTG诱导> 45 µmol gDCW-1的条件下，发现IB的最大表达为19.8 mgIGF1·gDCW-1，持续12 h。随后，将三种重折叠缓冲液与非织造阴离子交换吸附过滤器模块一起进行了测试。基于知识的缓冲液选择可实现较高的杂质对数减少值（LRV内毒素= 4.9； LRVDNA = 4.8，LRVHCP = 0.1-1）以及使用这些吸附基质的色谱柱保护潜力。此外，吸附过滤后再进行切向流过滤被证明是产品浓缩的一种有前途的选择。基于知识的缓冲液选择可实现较高的杂质对数减少值（LRV内毒素= 4.9； LRVDNA = 4.8，LRVHCP = 0.1-1）以及使用这些吸附基质的色谱柱保护潜力。此外，吸附过滤后再进行切向流过滤被证明是产品浓缩的一种有前途的选择。基于知识的缓冲液选择可实现较高的杂质对数减少值（LRV内毒素= 4.9； LRVDNA = 4.8，LRVHCP = 0.1-1）以及使用这些吸附基质的色谱柱保护潜力。此外，吸附过滤后再进行切向流过滤被证明是产品浓缩的一种有前途的选择。