Advances in Cell Line Development To Enhance Biotherapeutic Production

生物治疗学是在活宿主中产生的产品。对于大多数生物治疗药，也被删节至生物制剂细胞系are the bedrock of the manufacturing process. Among biologics withregulatory approval，制造平台涵盖细菌（例如，大肠杆菌), yeast (e.g.,S. cerevisiae），昆虫和哺乳动物细胞系。在哺乳动物细胞中产生的生物制剂，主要平台，绝大多数，around 70%，在中国仓鼠卵巢中（CHO）基于细胞的系统。CHO细胞是哺乳动物宿主的主力，因为它们易于通过编码蛋白质治疗的转基因（通常是单克隆抗体）转染。CHO细胞还以类似人蛋白的方式糖基化蛋白质，使其与具有良好活性且适合人类的蛋白质疗法的产生。此外，从过程的角度来看，它们易于以悬浮格式和高密度生长，从而增强了生产的生物制剂量。由于它们是仓鼠起源，因此CHO细胞不太可能传播人类病毒。尽管CHO细胞处于生物治疗生产的最前沿，但也使用了其他细胞系，例如鼠髓瘤来源NS0线and the mouse spleen myeloma hybridomaSP2/0线.

Moreover, mammalian host cells are employed to generate a broad spectrum of therapeutic classes.Most biologics are protein therapeutics, particularly monoclonal antibodies. Nevertheless, there is also a rapidly growing repertoire ofnon-protein therapeutics哺乳动物细胞用于制造，包括肽，核酸，碳水化合物和疫苗。作为主要的生物制剂类别，已经非常重点是优化蛋白质疗法，例如产量和翻译后修饰，但是对其他类别的研究也是活跃的和正在进行的，例如碳水化合物生物学。

Controlling glycans: Engineering cell lines to optimize carbohydrate biologics

苏珊·沙夫斯坦（Susan Sharfstein），纽约州立大学理工学院纳米级科学与工程学院的纳米生物科学教授，试图了解如何了解culture conditionsand cell physiology influence the production of therapeutic proteins and carbohydrates in mammalian cell lines. She leverages rational design and “omics”工具识别这些因素，其目的是工程细胞系和培养条件，以优化蛋白质和碳水化合物治疗剂的生产。

这manufacturing process for protein therapeutics begins by transfecting the host cell with instructions to make the protein. It is a templated process, whereby the instructions specify the sequence of amino acids needed to generate the protein therapeutic.In contrast,糖基化, the formation of carbohydrate (or “glycosidic”) bonds to a molecule, either protein or carbohydrate, is a non-templated process, generated by a multi-enzyme biosynthetic pathway. Therefore, glycosylation results in a heterogeneous mixture of alternative glycoforms of the glycoprotein or carbohydrate. All mammalian cells, includingCHO细胞, generate glycosylated proteins. However, the biosynthetic pathways that dictate glycosylation are presently not fully understood, making it difficult to control the heterogeneity of the resultant glycoprotein or carbohydrate biologics.

糖型在效力，稳定性和血浆半衰期方面有所不同，从而影响糖蛋白生物学的功效和剂量要求。它们的免疫原性也有所不同，这可能会影响患者不良反应的速度。因此，对工程哺乳动物细胞系引起了兴趣，可以将糖蛋白或碳水化合物生物学的产生转向最佳糖型。这Sharfstein laboratoryis particularly interested in the carbohydrate drug肝素，是世界上使用最广泛的抗凝药物。Sharfstein解释说：“肝素是一种屠宰场的产品，就像胰岛素是在重组DNA技术的出现之前允许生产重组人类胰岛素之前。”“我们的目标是创建一个新的范式，其中碳水化合物药物制造（例如，肝素）移至同一水平，在CGMP（当前良好的制造实践）条件下以生物工程产品的形式生产。”

不幸的是，肝素主要由肥大细胞产生，肥大细胞不适合培养。幸运的是，密切相关和结构相似heparan sulfateis produced in nearly all mammalian cells, including cells that are amenable to extended cell culture, such as CHO cells and mastocytoma cells, a tumorigenic variant of mast cells. To alter the glycosylation patterns of heparan sulfates, it is necessary to engineer their multi-enzyme biosynthetic pathway. To address this, in a study in which Sharfstein was an author, the researchers reasoned that multi-gene manipulation of the glycosylation biosynthetic pathway might generate a heparan sulfate with anticoagulant properties emulating that of heparin. In this “multiplex”研究小组基因组策略设计了小鼠肥大细胞瘤细胞系，以超过药物级肝素的抗凝活性产生高度硫酸盐硫酸盐。将转录组学分析用于比较肥大细胞与肥大细胞细胞中糖基化生物合成途径的酶的表达。该研究从肥大细胞中上调或下调的糖基化生物合成途径中发现了几种关键酶。当这些酶过表达或交替被CRISPR/CAS 9在肥大细胞瘤细胞中淘汰时，这些细胞会产生具有高抗凝活性的乙par醇硫酸盐。

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“Big data” to solve big problems in biologics productivity from cell lines

In addition to engineering cell lines to shift production to favored glycoforms, Sharfstein and her laboratory are also applying omics techniques to enhance protein therapeutic productivity. Presently, host cells transfected with the gene to express a protein therapeutic, under control of a promotor, are amplified and treated with a selection reagent. Single clones are isolated from the pool of cells, profiled for stability and protein therapeutic productivity, and the top clone is finally selected. So, it is a time consuming and iterative selection process. Although workable, it might be more time efficient if enhanced protein therapeutic productivity could be engineered into cell lines in a more systematic and targeted manner.

Working in the CHO cell line, a suite of omics methods, including transcriptomic, proteomic, phosphoproteomic and epigenomics, was applied to compare low- versus high-yielding CHO clones producing a monoclonal antibody under the control of the cytomegalovirus (CMV).这study发现转录因子cAMP响应元件结合蛋白1（CREB1）与CHO克隆中的CMV启动子相互作用，具有较高的单克隆抗体生产率。CREB1必须被磷酸化才能变得活跃。因此，核蛋白质组和磷蛋白酶调节CHO细胞中重组蛋白的最终生产力。Sharfstein讨论了研究结果，“此外，我们有大量数据表明，低生产力克隆的能量途径，核糖体和蛋白质体活性有所不同。”

Once omics analysis pinpoints actionable targets that influence clone productivity, these characteristics can be engineered into cell lines to develop high-yielding clones. “In theory, we can leverage our omics discoveries and modeling to rationally design and engineer cell lines to interface with bioprocess development. With the development of CRISPR technology, it becomes much easier to knock-in or knock-out biological activities to yield better, more productive host cells, and we anticipate this will become a large aspect of future cell line development,” concluded Sharfstein.

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Stable transgene insertion intodhfrAuxotroph Cho细胞：选择过程

Lawrence Chasin，哥伦比亚大学生物科学系名誉教授，使用computational methodsin his research to uncover the complex mechanisms of mRNA splicing, including in CHO cells. More recently, he and his实验室对工程CHO细胞系感兴趣，以帮助选择高收益克隆。“这选择过程对于高收益，稳定表达的Cho克隆可能是冗长且劳动密集型的。它可以首先将药物抗性基因引入正常的CHO细胞或将生物合成缺乏的基因纠正为补充营养性CHO细胞。”该过程的Chasin解释说。辅助营养物是突变的生物或细胞，如果不外源添加特定的营养素就无法生存，而正常菌株不需要生存，因为它通过生物合成途径内源性产生营养。“一种用于重组蛋白产生的广泛使用的CHO细胞特异性缺乏二氢叶酸还原酶（dhfr）基因，它在不添加甘氨酸，低黄嘌呤和胸苷的情况下使菌株无法生存，这是缩写为GHT的营养培养基的混合物，” Chasin继续说道。“ CHO细胞被感兴趣的基因转染，编码蛋白质治疗，并与dhfrgene, both of which are integrated into the genome atdiverse locations跨越各种克隆。获得在缺乏GHT的增长介质中生存的能力的CHO细胞已稳定融合dhfralong with the gene of interest.”

Following this phase, single clones are isolated that express a high level of DHFR with a concomitant high level of the protein interest, usually because they harbor a high number of gene copies. The highest-yielding clones with the best growth characteristics are then selected to manufacture the protein therapeutic. “The yield is dependent on the位置集成到基因组中的基因, which is a stochastic process left to chance,” Chasin elaborated. “Clones that have integrated multiple copy numbers of the gene can also have higher expression levels. Although a stochastic process as well, the copy number selection can be enhanced by treating cells with the DHFR inhibitor, methotrexate. Cells with a sufficiently high number ofdhfrgenes will synthesize enough DHFR to overcome the inhibitor and so will be selected, along with a comparably high number of the neighboring therapeutic transgene.” Subsequent selection for even more copies ofdhfr基因利用基因扩增，这是一种可以自发发生在CHO细胞中的现象。高收益克隆是由多发池扩增产生的，甲氨蝶呤剂量增加。这样，具有超过100份的克隆dhfrgene can be isolated. “The disadvantages of this strategy are that it is time consuming, and that high gene copy numbers do not always insure high levels of expression. An alternative that also works is a targeted integration of the selectable gene into a site(s) on the genome that allows very high levels of gene expression,” explained Chasin.

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多型营养性CHO细胞：精美的选择方法

Chasin和他的团队最近还创建了multiauxotrophicCHO细胞系（CHO8A），可用于在一个Fell Scoop中选择多个集成转基因的副本，从而避免了对几轮选择的要求。这种方法还规定了使用合成试剂或抗生素治疗细胞的需求，这是有利的。“我们在生物合成途径中删除了一系列编码为RNA和DNA的嘌呤和嘧啶的构建块的酶促步骤编码的基因。这导致了一个可营养的CHO细胞系，cho8a，缺乏在没有五种天然营养物质的情况下生存的能力。” Chasin描述了这种新方法。“然后，我们将缺陷的CHO8A细胞与八个质粒池共转化，每个质粒都含有转基因以及不同的救援基因（即编码一种嘌呤或嘧啶生物合成酶之一单克隆抗体轻或重链。”

Only transfected clones that integrate all eight genes will survive and grow in popular growth media lacking purines and pyrimidines. Furthermore, the probability of eight integrations automatically selects transfectant clones that have accumulated a high copy number (40-120) of total plasmids. “As a result, we found that the expression of the neighboring monoclonal antibody gene reached levels comparable to those used in manufacturing. Another use of this multiauxotrophic CHO cell line would be the ability to select up to 8 different proteins or protein subunits at once. Furthermore, this strategy could be applied to other cell lines as well,” concluded Chasin of their approach.

当被问及生物治疗生产细胞系开发中未来的研究方向时，Chasin回答说，目前的生产力瓶颈现在似乎是分泌的限制。“能够明显更高分泌的宿主细胞的基因工程和选择方法应降低制造成本，这是工业和社会的重要目标。”