Kopp Julian, Slouka Christoph, Strohmer Daniel, Kager Julian, Spadiut Oliver, Herwig Christoph
Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, Institute of Chemical Engineering, Vienna University of Technology, 1060 Vienna, Austria.
Research Division Biochemical Engineering, Institute of Chemical Engineering, Vienna University of Technology, 1060 Vienna, Austria.
Microorganisms. 2018 Nov 25;6(4):116. doi: 10.3390/microorganisms6040116.
The Gram-negative bacterium is the host of choice for producing a multitude of recombinant proteins relevant in the pharmaceutical industry. Generally, cultivation is easy, media are cheap, and a high product titer can be obtained. However, harsh induction procedures combined with the usage of IPTG (isopropyl β-d-1 thiogalactopyranoside) as an inducer are often believed to cause stress reactions, leading to intracellular protein aggregates, which are so known as so-called inclusion bodies (IBs). Downstream applications in bacterial processes cause the bottleneck in overall process performance, as bacteria lack many post-translational modifications, resulting in time and cost-intensive approaches. Especially purification of inclusion bodies is notoriously known for its long processing times and low yields. In this contribution, we present screening strategies for determination of nclusion body bead size in an -based bioprocess producing exclusively inclusion bodies. Size can be seen as a critical quality attribute (CQA), as changes in inclusion body behavior have a major effect on subsequent downstream processing. A model-based approach was used, aiming to trigger a distinct inclusion body size: Physiological feeding control, using q as a critical process parameter, has a high impact on inclusion body size and could be modelled using a hyperbolic saturation mechanism calculated in form of a cumulated substrate uptake rate. Within this model, the sugar uptake rate of the cells, in the form of the cumulated sugar uptake-value, was simulated and considered being a key performance indicator for determination of the desired size. We want to highlight that the usage of the mentioned screening strategy in combination with a model-based approach will allow tuning of the process towards a certain inclusion body size using a q based control only. Optimized inclusion body size at the time-point of harvest should stabilize downstream processing and, therefore, increase the overall time-space yield. Furthermore, production of distinct inclusion body size may be interesting for application as a biocatalyst and nanoparticulate material.
革兰氏阴性菌是生产众多制药行业相关重组蛋白的理想宿主。一般来说，培养容易，培养基便宜，并且可以获得高产品滴度。然而，人们通常认为，严苛的诱导程序与使用异丙基-β-D-硫代半乳糖苷（IPTG）作为诱导剂会引发应激反应，导致细胞内蛋白质聚集体，即所谓的包涵体（IBs）。细菌工艺中的下游应用是整个工艺性能的瓶颈，因为细菌缺乏许多翻译后修饰，导致方法耗时且成本高昂。尤其是包涵体的纯化，其处理时间长且产率低是出了名的。在本论文中，我们展示了在一个专门生产包涵体的基于[未提及具体名称]的生物工艺中，用于确定包涵体珠粒大小的筛选策略。大小可被视为关键质量属性（CQA），因为包涵体行为的变化对后续下游加工有重大影响。我们采用了基于模型的方法，旨在引发特定的包涵体大小：以q作为关键工艺参数的生理补料控制，对包涵体大小有很大影响，并且可以使用以累积底物摄取率形式计算的双曲线饱和机制进行建模。在该模型中，以累积糖摄取值形式模拟细胞的糖摄取率，并将其视为确定所需大小的关键性能指标。我们想强调的是，将上述筛选策略与基于模型的方法结合使用，仅通过基于q的控制就能使工艺朝着特定的包涵体大小进行调整。收获时优化的包涵体大小应能稳定下游加工，从而提高整体时空产率。此外，生产特定大小的包涵体作为生物催化剂和纳米颗粒材料可能会很有意义。
