Medrano Giuliana, Dolan Maureen C, Condori Jose, Radin David N, Cramer Carole L
Arkansas Biosciences Institute, Arkansas State University, State University, AR, USA.
Methods Mol Biol. 2012;824:535-64. doi: 10.1007/978-1-61779-433-9_29.
Plant-based expression technologies for recombinant proteins have begun to receive acceptance for pharmaceuticals and other commercial markets. Protein products derived from plants offer safer, more cost-effective, and less capital-intensive alternatives to traditional manufacturing systems using microbial fermentation or animal cell culture bioreactors. Moreover, plants are now known to be capable of expressing bioactive proteins from a diverse array of species including animals and humans. Methods development to assess the quality and performance of proteins manufactured in plants are essential to support the QA/QC demands as plant-produced protein products transition to the commercial marketplace. Within the pharmaceutical arena, process validation and acceptance criteria for biological products must comply with the Food and Drug Administration (FDA) and ICH Q6B guidelines in order to initiate the regulatory approval process. Detailed product specifications will also need to be developed and validated for plant-made proteins for the bioenergy, food, chemical synthesis, or research reagent markets.We have, therefore, developed assessment methods for important qualitative and quantitative parameters of the products and the manufacturing methods utilized in plant-based production systems. In this chapter, we describe a number of procedures to validate product identity and characteristics including mass analyses, antibody cross-reactivity, N-terminal sequencing, and bioactivity. We also address methods for routine assessment of yield, recovery, and purity. The methods presented are those developed for the synthesis and recovery of the avian cytokine, chicken interleukin-12 (ChIL-12), produced in the leaves of Nicotiana benthamiana. The ChIL-12 protein used as a model for this chapter includes a C-terminal histidine epitope (HIS-tag) and, thus, these methods may be directly applicable to other HIS-tagged proteins produced in plants. However, the overall strategy presented using the ChIL-12(HIS) example should provide the basis of standard procedures for assessing the quality of other plant-based protein products and manufacturing systems.
用于重组蛋白的基于植物的表达技术已开始在制药和其他商业市场中得到认可。与使用微生物发酵或动物细胞培养生物反应器的传统制造系统相比，源自植物的蛋白质产品提供了更安全、更具成本效益且资本密集度更低的替代方案。此外，现在已知植物能够表达包括动物和人类在内的多种物种的生物活性蛋白。随着植物生产的蛋白质产品向商业市场过渡，开发评估植物中生产的蛋白质质量和性能的方法对于满足质量保证/质量控制要求至关重要。在制药领域，生物制品的工艺验证和验收标准必须符合美国食品药品监督管理局（FDA）和国际人用药品注册技术协调会（ICH）Q6B指南，以便启动监管审批程序。还需要为生物能源、食品、化学合成或研究试剂市场的植物制造蛋白制定并验证详细的产品规格。因此，我们开发了评估基于植物的生产系统中产品和制造方法的重要定性和定量参数的方法。在本章中，我们描述了一些验证产品身份和特性的程序，包括质量分析、抗体交叉反应性、N端测序和生物活性。我们还介绍了产量、回收率和纯度的常规评估方法。所介绍的方法是为在本氏烟草叶片中合成和回收禽细胞因子鸡白细胞介素-12（ChIL-12）而开发的。本章用作模型的ChIL-12蛋白包含一个C端组氨酸表位（HIS标签），因此，这些方法可能直接适用于植物中产生的其他带HIS标签的蛋白。然而，以ChIL-12（HIS）为例介绍的总体策略应为评估其他基于植物的蛋白质产品和制造系统的质量提供标准程序的基础。
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