Ughade Santosh, Rana Sunil, Nadeem Mohd, Kumthekar Rupali, Mahajani Sanjay, Bhambure Rahul
Chemical Engineering and Process Development Division, CSIR - National Chemical Laboratory, Pune 411008, India.
Academy of Scientific and Innovative Research (AcSIR), Ghaziabad 201002, India.
ACS Omega. 2024 Jan 5;9(3):3204-3216. doi: 10.1021/acsomega.3c04463. eCollection 2024 Jan 23.
protein refolding is one of the critical unit operations in manufacturing recombinant peptides expressed using as host cells. This study is focused on designing size exclusion chromatography-assisted refolding process for biosimilar recombinant parathyroid hormone. Inclusion bodies (IBs) of recombinant parathyroid hormone were solubilized at higher pH, and refolding was performed using size exclusion chromatography. In the first part of the investigation, DoE-based empirical optimization was performed to achieve a higher refolding yield for a biosimilar recombinant parathyroid hormone. The effect of solubilized inclusion body (IB) feed volume, concentration of IBs, and residence time on refolding of recombinant teriparatide was studied using the Box-Behnken design. Size exclusion chromatography (SEC)-assisted refolding was performed at 8 °C at pH 10.5 by using 20 mM Tris buffer. The maximum refolding yield of 98.12% was achieved at feed volume (12.5% of CV) and 20 mg/mL inclusion body (IB) concentration with a residence time of 50 min and a purity of 66.1% based on densitometric analysis using SDS-PAGE. In the latter part of the investigation, the general rate mechanistic model framework for size exclusion chromatography was developed and validated with the experimental results. The developed model helped in the accurate prediction of the elution volumes and product yield. The developed model also helps to predict the elution performance of a scalable column a priori. Post refolding, the formation of the native peptide structure was examined using various orthogonal analytical tools to study the protein's primary, secondary, and tertiary structures. The developed hybrid process development approach is a valuable tool toachieve high-yield, scalable refolding conditions for recombinant proteins without disulfide bonds.
蛋白质重折叠是使用[具体宿主细胞]作为宿主细胞表达的重组肽生产过程中的关键单元操作之一。本研究聚焦于设计用于生物类似物重组甲状旁腺激素的尺寸排阻色谱辅助重折叠工艺。重组甲状旁腺激素的包涵体在较高pH值下溶解，并使用尺寸排阻色谱进行重折叠。在研究的第一部分，基于实验设计的经验优化用于实现生物类似物重组甲状旁腺激素更高的重折叠产率。使用Box-Behnken设计研究了溶解的包涵体（IB）进料体积、IB浓度和停留时间对重组特立帕肽重折叠的影响。尺寸排阻色谱（SEC）辅助重折叠在8℃、pH 10.5条件下使用20 mM Tris缓冲液进行。基于SDS-PAGE的光密度分析，在进料体积（柱体积的12.5%）、20 mg/mL包涵体（IB）浓度、停留时间50分钟和纯度66.1%的条件下，实现了98.12%的最大重折叠产率。在研究的后半部分，建立了尺寸排阻色谱的通用速率机理模型框架，并通过实验结果进行了验证。所建立的模型有助于准确预测洗脱体积和产物产率。所建立的模型还有助于先验预测可扩展柱的洗脱性能。重折叠后，使用各种正交分析工具检查天然肽结构的形成，以研究蛋白质的一级、二级和三级结构。所开发的混合工艺开发方法是一种有价值的工具，可实现无二硫键重组蛋白的高产率、可扩展重折叠条件。
