Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, New York.
Department of Chemical Engineering, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts.
Biotechnol Bioeng. 2018 Aug;115(8):2048-2060. doi: 10.1002/bit.26718. Epub 2018 May 4.
In this study, we describe a new approach for the characterization of process-related impurities along with an in silico tool to generate orthogonal, integrated downstream purification processes for biological products. A one-time characterization of process-related impurities from product expression in Pichia pastoris was first carried out using linear salt and pH gradients on a library of multimodal, salt-tolerant, and hydrophobic charge induction chromatographic resins. The Reversed-phase ultra-performance liquid chromatography (UPLC) analysis of the fractions from these gradients was then used to generate large data sets of impurity profiles. A retention database of the biological product was also generated using the same linear salt and pH gradients on these resins, without fraction collection. The resulting two data sets were then analyzed using an in silico tool, which incorporated integrated manufacturing constraints to generate and rank potential three-step purification sequences based on their predicted purification performance as well as whole-process "orthogonality" for impurity removal. Highly ranked sequences were further examined to identify templates for process development. The efficacy of this approach was successfully demonstrated for the rapid development of robust integrated processes for human growth hormone and granulocyte-colony stimulating factor.
在这项研究中，我们描述了一种新的方法，用于对与工艺相关的杂质进行特征描述，以及一种用于生成生物制品正交、集成下游纯化工艺的计算工具。我们首次使用多模态、耐盐和疏水电荷诱导色谱树脂库，对毕赤酵母表达产物中的与工艺相关的杂质进行一次性特征描述，采用线性盐和 pH 梯度。然后使用这些梯度的馏分的反相超高效液相色谱 (UPLC) 分析来生成杂质谱的大数据集。还使用相同的线性盐和 pH 梯度在这些树脂上生成生物制品的保留数据库，而无需馏分收集。然后使用一种计算工具对这两个数据集进行分析，该工具将综合制造约束纳入其中，以根据预测的纯化性能以及整个过程的“正交性”来生成和对潜在的三步纯化序列进行排序，以去除杂质。对排名较高的序列进行进一步检查，以确定用于工艺开发的模板。这种方法已成功应用于快速开发人生长激素和粒细胞集落刺激因子的稳健集成工艺。
