Christian Doppler Laboratory for Mechanistic and Physiological Methods for Improved Bioprocesses, TU Wien, Gumpendorfer Straße 1a, 1060 Vienna, Austria.
SANDOZ GmbH, Mondseestrasse 11, 4866 Unterach, Austria.
J Pharm Biomed Anal. 2020 Sep 5;188:113412. doi: 10.1016/j.jpba.2020.113412. Epub 2020 Jun 4.
Biopharmaceutical drug substances are generally produced using fermentation technology and are subsequently purified in the following downstream process. For the determination of critical quality attributes (CQAs), such as target protein titer and purity, monitoring tools are required before quality control analysis. We herein present a novel reversed phase liquid chromatography method (RPLC), which enables facile and robust protein quantification during upstream and downstream processing of intracellularly produced proteins in E. coli. The overall goal was to develop a fast, robust and mass spectrometry compatible method which can baseline resolve and quantify each protein of interest. Method development consisted of three steps, oriented on an Analytical Quality by Design (AQbD) workflow: (i) the stationary phase as primary parameter was chosen based on state-of-the art technology thus minimizing protein on-column adsorption and providing high efficiency, (ii) secondary parameters (i.e. gradient conditions and column temperature) were optimized applying chromatographic modeling, and (iii) the established Method Operable Design Region (MODR) was challenged and confirmed during robustness testing, performed in-silico and experimentally by a Design of experiment (DoE) based approach. Finally, we validated the RPLC method for pivotal validation parameters (i.e. linearity, limit of quantification, and repeatability) and compared it for protein quantification against a well-established analytical methodology. The outcome of this study shows (i) a protocol for RPLC development using an AQbD principle for new method generation and (ii) a highly versatile RPLC method, suited for quick and straightforward recombinant protein titer measurement being applicable for the detection of a broad range of proteins.
生物制药原料药通常采用发酵技术生产，随后在以下下游工艺中进行纯化。对于关键质量属性（CQAs）的测定，例如目标蛋白滴度和纯度，在质量控制分析之前需要监测工具。我们在此介绍了一种新颖的反相液相色谱法（RPLC），该方法可在大肠杆菌中胞内产生的蛋白质的上游和下游处理过程中实现简便、稳健的蛋白质定量。总体目标是开发一种快速、稳健且与质谱兼容的方法，能够基线分辨和定量每个感兴趣的蛋白质。方法开发包括三个步骤，以分析质量设计（AQbD）工作流程为导向：（i）基于最先进的技术选择固定相作为主要参数，从而最小化蛋白质在柱上的吸附并提供高效率，（ii）优化二级参数（即梯度条件和柱温）应用色谱建模，（iii）建立的方法可操作设计区域（MODR）在稳健性测试中受到挑战并得到确认，通过基于实验设计（DoE）的方法进行了计算机模拟和实验验证。最后，我们针对关键验证参数（即线性、定量下限和重复性）验证了 RPLC 方法，并将其与一种成熟的分析方法进行了蛋白质定量比较。该研究的结果表明：（i）一种使用 AQbD 原则生成新方法的 RPLC 开发方案，（ii）一种高度通用的 RPLC 方法，适用于快速简便的重组蛋白滴度测量，可用于检测广泛范围的蛋白质。
