Improving biopharmaceutical production in microbial systems: Engineering GlycoPEGylation in E.coli

我们的目标是在细菌大肠杆菌（Escherichia coli）（E.大肠杆菌），其可以被纯化，然后被有效地修饰以改善其生物和物理特性，从而改善整体有效性。虽然CA。在2006年至2010年期间批准的真正新的生物制药（基于蛋白质的药物）中，有30%采用了E。大肠杆菌中，有机会改善这种宿主系统。例如，较小的蛋白质或蛋白质片段如抗体片段可以在E.大肠杆菌与哺乳动物或植物细胞系统相比，由于相对便宜的生长要求、高细胞密度和高蛋白产量，虽然作为药物有效，但这些蛋白质的较小尺寸意味着它们在人体中具有较高的清除率（即药物通过肾脏清除），从而降低了剂量的整体功效。该项目建立在生产后修饰的概念上，以增加这些类型蛋白质的循环半衰期（药物在体内持续时间更长）。一种惰性的合成聚合物聚乙二醇（PEG）通常用于工业中，并将在这里使用-其与药物的连接称为PEG化。靶蛋白IFN-a2 b（称为细胞因子的干扰素医学家族的成员）将作为该项目的范例“药物”，因为它是一种众所周知且广泛生产的治疗剂。此外，它以前已经被聚乙二醇化，它的选择得到了金砖四国工业合作伙伴（龙沙和富士Diosynth）的支持。优化聚乙二醇化过程受到了广泛关注，因为效率直接转化为制造成本（高效率意味着降低制造成本）。传统的方法导致了随机聚乙二醇化，这意味着许多不同的蛋白质形式，降低了生产率（并增加了成本）。随后提出了几种定点方法，包括从大肠杆菌中纯化蛋白质的方法。大肠杆菌中，然后两种酶（生物催化剂）在大肠杆菌外的一个单独的过程中使用后，蛋白质已制成（在体外）添加糖（酶1），然后糖-PEG（酶2）。该过程被称为糖聚乙二醇化。这个项目建立在这个概念和利用新发现的能力E。利用另一种微生物的酶机制（BRIC 1资助），使大肠杆菌中的蛋白质糖基化（将糖基添加到细胞中的蛋白质中）。通过在蛋白质靶点上设计一个糖（糖基化）连接位点，我们已经证明了E。大肠杆菌可以识别并添加一个特定的糖的网站（与IFN-a2 b和其他蛋白质，如GFP）。我们建议，这种修饰的蛋白质可以被纯化，然后在细胞外加入PEG的一步反应中使用。这需要一种特殊的酶，这种酶将被设计和优化。我们将使用尖端生物工程技术的组合，现在被认为是合成生物学新兴领域的一部分，来操纵E。大肠杆菌中表达IFN-α 2b修饰蛋白。我们将采用内部代谢工程策略（向前和向后/反向）来提高产量。为了提高PEG化效率，IFN-a2 b中的糖接受位点将被改变以优化添加的糖的酶识别。为了快速转化为工业，优化的细胞系统和蛋白质将在生物反应器中进行测试，我们已经证明，生物反应器增加了E.杆菌我们希望深入了解这种产品的生产（可制造性）有多容易，我们将用E.对于质量控制，将使用多种工具的组合测试修饰的IFN-a2 b的生物物理稳定性。此外，将在整个过程中对现有的定点糖基聚乙二醇化方法进行成本比较。

We aim to produce an example therapeutic protein (medicine) in the bacterium Escherichia coli (E. coli) that can be purified and then efficiently modified to improve its biological and physical characteristics and thus overall effectiveness. Although ca. 30% of the genuinely new biopharmaceuticals (protein-based medicines) approved between 2006-10 employed E. coli, there is an opportunity to improve this host system. For example, smaller proteins or protein fragments such as antibody fragments can be made more efficiently in E. coli compared to mammalian or plant cell systems due to relatively inexpensive growth requirements, high cell densities and high protein yields. Although effective as medicines, the smaller size of these proteins means they have a higher clearance rate in humans (ie the drug is removed by the kidneys), reducing overall efficacy of the dose. This project builds on the concept of post-production modification to increase the circulatory half-life of these type of proteins (the drug lasts longer in the body). An inert, synthetic polymer, polyethylene glycol (PEG) is commonly used in industry and will be employed here - its attachment to the drug is known as PEGylation. The target protein IFN-a2b (a member of the interferon family of medicine known as cytokines) will serve as the exemplar 'drug' for this project as it is a well understood and widely manufactured therapeutic agent. In addition, it has been PEGylated previously and it's selection has been supported by BRIC industrial partners (Lonza and Fuji Diosynth). Optimising the process of PEGylation has received a lot of attention as the efficiency directly translates into manufacturing costs (high efficiency means reduced manufacturing costs). Traditional methods have led to random PEGylation that means many different protein forms are made, reducing productivity (and increasing costs). Several site-directed methods were subsequently proposed including one where the protein is purified from E. coli and then two enzymes (biological catalysts) are used in a separate process outside E .coli after the protein has been made (in vitro) to add a sugar (enzyme 1) and then sugar-PEG (enzyme 2). The process is referred to as glycoPEGylation. This project builds on this concept and exploits the newly discovered ability of E. coli to glycosylate proteins (add sugar groups to the protein in the cell) using enzymatic machinery from another microorganism (BRIC1 - funded). By designing a sugar (glycosylation) attachment site into the protein target, we have shown that the sugar-adding (glycosylation) machinery in E. coli can recognise and add a specific sugar to the site (with IFN-a2b and other proteins such as GFP). We propose that this modified protein can be purified and then used in a one step reaction outside the cell where PEG is added. The requires a specific enzyme that will be designed and optimised.We will use a combination of cutting edge biological engineering techniques, now considered part of an emerging field known as synthetic biology, to manipulate E. coli to produce the modified protein target IFN-a2b. We will employ in-house metabolic engineering strategies (forward and backward/inverse) to improve yields. To improve PEGylation efficiency, the sugar acceptance site in IFN-a2b will be varied to optimise enzyme recognition of the added sugar. For rapid translation to industry the optimised cell system and protein will be tested in bioreactors which we have already shown increases antibody fragment production yields in E. coli. We wish to gain insight into how easy this product would be to manufacture (manufacturability) and we will design fermentation with E. coli and discuss this with BRIC members.For quality control, the modified IFN-a2b will be tested for biophysical stability throughout using a combination of tools. Also, cost comparisons to the existing site-directed glycoPEGylation methodology, will be performed throughout.