植物-Cell Cultures and Cell Lines for Recombinant Protein Expression

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Cell cultures derived from mammalian and bacterial cell lines are the conventional production systems in bioprocessing. But they also have their limitations. Media for mammalian cultures in particular are notoriously expensive, and traditional cell cultures can be highly sensitive to growing conditions. During the late 1980s and into the 1990s, plants and plant-derived cell cultures were introduced as alternative cell-culture systems (1,2)。Although transgenic plants (genetically modified) once looked promising in the early 2000s, the cost and manufacturing complexity of such systems were hurdles that proved to be too high to negotiate, so research in transgenic plants faded. Nonetheless, progress had been made in using plant cell cultures for producing antibodies and proteins (3)。In the past 10 years, plant cells increasingly have been considered as cost-effective expression systems for recombinant and animal-free proteins, emergency vaccines, immunotherapies, and other products that could benefit from plant glycans (4)。Extensive research has focused on the optimization of protein expression, particularly addressing internal factors such as expression cassettes, protein targeting, and coexpression of protease inhibitors as well as nutritional and physical cultivation parameters affecting plant growth and fitness (4,5)。

2012年,美国食品药品监督管理局(FDA)批准了由以色列基于以色列的生物制药公司Protalix Biolalix Biotalix Biothapeutics和合作伙伴辉瑞开发的1型Gaucher病的长期酶替代疗法。该产品是该疾病的第一个植物性表达的酶替代疗法。这也是该公司的Procellex蛋白质制造系统衍生出的第一个植物细胞表达的药物。我们要求Einat Brill Almon(Protalix产品开发高级副总裁)和Eyal Rubin(Protalix高级副总裁兼首席财务官)讨论使用植物细胞系的好处以及如何开发此类平台。

Advantages of Using Plant-Cell Culture
Why did Protalix choose to pursue plant-cell expression over mammalian-cell systems?Protalix选择植物细胞作为1990年代初期表达重组蛋白的潜在平台。我们的目标是将新颖的生产平台引入新兴行业,以生产重组人类蛋白作为生物治疗学。当时,蛋白质表达的主要生产平台是微生物和哺乳动物细胞。细菌细胞缺乏进行复杂翻译后修饰的基本能力,例如糖基化,二硫键和正确的折叠,这是许多生物疗法蛋白具有功能所必需的。当时,可以产生复杂蛋白质的哺乳动物细胞需要一组相对复杂且昂贵的生长条件,例如富含动物衍生成分的复杂培养基。这些系统还需要具有狭窄操作范围的高度控制的环境条件。

植物cells are a promising production platform because they can make complex proteins with posttranslational modifications, and they are simple and cost effective to culture. Plant cells are relatively easy to culture in vitro, and they are tolerant to a wide range of environmental conditions. They also grow nicely at room temperature without the need for supplementing cultures with CO2 or other gas. Their growing media are composed of simple minerals and sugar, and no complex animal-derived components are required (a regulatory advantage). Plant-cell systems are not susceptible to viral contamination in production, which can ensure continuous and reliable supply.

植物细胞培养是否需要特定的生物反应器系统和其他设备?植物cells are less demanding in terms of growing conditions and media composition than mammalian cells are. A plant-cell system also is more robust. Plant cells can be cultivated in bioreactors of different types and sizes. At Protalix, plant cells are grown in suspension culture in 10–800-L bioreactors. Plant cells can be cultivated in single-use or multiuse bioreactors. Our bioreactors are disposable, and the 800-L bioreactors can be scaled out to meet large capacity needs. Our plant-cell system requires a higher footprint of bioreactor area, but maintenance of that equipment is negligible.

What plant species are proving to be most useful and/or successful for gene expression?来自许多植物物种(例如胡萝卜,番茄,大米,拟南芥和烟草)的细胞可用于表达活跃且正确折叠的复杂人蛋白。对于有用且成功的生产平台,您将选择在培养物中剧烈生长的细胞(时间很高)并产生相对“光滑”的悬浮液(某些植物细胞往往在培养中聚集)。理想的选择也应该适合遗传操作。

After we produced out first approved product in carrot cells, we selected tobacco BY2 (Nicotiana tabacum明亮的黄色2)细胞为我们的首选生产宿主。BY2细胞在培养中迅速生长,易于通过不同的表达系统(包括植物病毒的复制子)在遗传上转化。在植物细胞的世界中,BY2细胞是主力的,就像中国仓鼠卵巢(CHO)细胞适用于哺乳动物细胞平台一样。在过去的50年中,BY2细胞在世界各地的实验室中都用于植物细胞生物学的基础研究和应用研究。因此,它们具有高度的特征。

Screening and Analysis
How does clone screening compare between plant and mammalian cells, and what attributes should upstream scientists be mindful about when screening plant-cell clones?The main factors that should be considered when cloning plant cells are their tendency to grow in small clumps and their reluctance to survive and propagate while not surrounded (or accompanied) by other cells (e.g., “social” cells). Although mammalian cells can be counted and diluted to ≤1 cell/well easily and precisely to ensure single-cell culture, this approach is not straightforward with plant cells. To circumvent those limitations, cell clumps can be separated enzymatically into single cells or protoplasts and cloned with the application of a feeder-culture. This approach uses either a cell-free conditioned medium or nontransformed cells (that eventually will not survive the antibiotic-supplemented medium) as a supporting feeder. Similar to cloning mammalian cells, the process of cloning should be repeated more than once (subcloning) to verify clonality of the selected cell lines.

What can upstream scientists do to ensure and even augment cell viability?Biomanufacturers should provide and control proper growing conditions in terms of culture-medium composition, ambient temperature, proper agitation, and frequent subculturing. Viability through upstream production is ensured by implementing in-process control measures. That includes measuring cell concentrations and some medium properties at various control points, ensuring that process results comply with preset specifications.

What analytical tests are most important for plant-cell–expressed proteins?Characterization and quality control release tests for plant-cell–expressed proteins are no different than for proteins produced with other cell systems. A protein needs to be safe, efficacious, and consistent from batch to batch. Thus, as with proteins from other production systems, all in-process controls (IPCs) and all critical quality attributes (CQAs) are measured.

基于植物的系统对当前的基因编辑方法的反应如何?基于植物的系统对定期间隔短的短滴体重复序列(CRISPR),与CRISPR相关的蛋白9(CAS9)基因编辑以及其他基因– Knockin/Out Technologies的簇具有高度反应性。这些技术通常与植物和植物细胞成功使用。

Addressing Regulatory Concerns
What regulatory concerns are unique to proteins produced using plant-based systems?Like other eukaryotic organisms, plants produce glycoproteins with N-glycans attached to asparagine residues, although the glycans differ in structure from those of mammals. Plant glycans can contain an α(1,3)-fucose linked to the proximalN-acetylglucosamine (GlcNAc) residue and/or β(1,2)-xylose residues attached to the bisecting mannose of the glycan core, which are not present in mammalian proteins. The immunogenic effect of plant glycan moieties has been the basis of much debate.

Recent publications and clinical experience with taliglucerase alfa and pegunigalsidase alfa produced at Protalix did not cause concerns over plant-derived glycans. Taliglucerase alfa has been approved by regulatory authorities, and pegunigalsidase alfa has been submitted to the FDA for biologics license application (BLA) approval.

What regulatory concerns related to proteins produced by mammalian cells can be bypassed by using plant-cell systems?植物-cell systems intrinsically do not carry the risk of infection by or transmission of human or other animal pathogens. Because the culture media used with plant cells lack mammalian-derived components, the risk of transmission of mammalian viruses or prions associated with use of such components in mammalian-cell culture media is eliminated. Moreover, mammalian viruses cannot infect nor propagate in plant-cell cultures. These factors are not only important to safety, but they also reduce operational costs significantly compared with mammalian expression systems. That has been recognized by all regulatory agencies. Thus, Protalix is exempt from testing for viruses.

植物-Cell Expression Platform
What can you tell us about Elelyso (taliglucerase alfa) and the ProCellEx platform used to manufacture it?Elelyso (taliglucerase alfa) is a recombinant human β-glucocerebrosidase that is used as an enzyme replacement therapy (ERT) and is approved as a parenteral treatment for Gaucher patients. The Protalix ProCellEx platform is a proprietary bioreactor system consisting of large flexible polyethylene bags into which culture medium and air from a central system are supplied under sterile conditions. That central air supply also serves as an agitation method for suspended cells, reducing mechanical forces that otherwise could harm the cells. In this system, bioreactors are assembled serially within a confined cleanroom facility. That enables the culture of thousands of liters of transformed plant cells that are expressing a recombinant protein of interest at an industrial scale and at economical costs.

The system allows for horizontal scale out (by adding more bioreactors of the same size). That translates into two substantial advantages: a smooth transition from pilot to commercial scale with little need for making adjustments for comparability of the upstream processes; and a flexible production that quickly and cost-effectively reacts to fluctuations in the market. The protein yields achieved from our second product are about 20-fold compared with that of our first product. We expect future products to reach even higher yields.

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3Ma JK, et al. Generation and Assembly of Secretory Antibodies in Plants.Science268(5211)1995:716–719;https://doi.org/10.1126/science.7732380。

4Schillberg S, et al. Critical Analysis of the Commercial Potential of Plants for the Production of Recombinant Proteins.前面。植物科学。10, 2019; https://doi.org/10.3389/fpls.2019.00720.

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布莱恩·加沙尔(Brian Gazaille)is associate editor (brian.gazaille@informa.com), 和Maribel Riosis managing editor atbeplay官方注册;maribel.rios@informa.com.Einat Brill阿尔蒙是产品开发高级副总裁,Eyal Rubin是Protalix Biotherapeutics的高级副总裁兼首席财务官,2 Snunit Street, Science Park, POB 455, Carmiel 2161401, Israel;http://www.protalix.com.

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