Glycine rich sequences with pharmacokinetic enhancing properties of PEG polymers

描述(申请人提供)：以蛋白质为基础的生物制药市场正在迅速扩大。然而，许多蛋白质的临床应用受到其血清半衰期短的限制，需要频繁注射。最常见的改善血清半衰期的方法是聚乙二醇化。聚乙二醇(PEG)与治疗性蛋白质的化学偶联通常会导致产品混合物中包含不活跃的异构体，并降低产品的整体效力。蛋白质的化学聚乙二醇化大大增加了制造成本，需要精确的过程控制和分析分析，以确保产品成分的重现性。目前的项目描述了模仿化学聚乙二醇的物理化学性质的多肽链(称为rPEG)。RPEG是亲水性的，具有很大的流体动力学半径。最重要的是，rPEGS可以与治疗性蛋白重组融合，产生同质产品。RPEGS与生物制药的融合有望提供类似于化学聚乙二醇化的好处(血清半衰期长，免疫原性降低)，同时提供更好的产品效力、均质性，并显著降低制造成本。该项目的第一阶段非常成功，证明了rPEG技术的可行性。开发出了非常接近于化学聚乙二醇性质的rPEG序列。我们证明了一个20 kDa的rPEG序列的表观分子量为180 kDa。将rPEG与模型蛋白GFP融合，使其在大鼠的血清半衰期从1-3h增加到约10h，与化学聚乙二醇化的效果相似。此外，这种rPEG-GFP融合只在大鼠身上引起了非常弱的免疫反应。我们的第二阶段目标是将rPEG技术应用于人类生长激素(HGH)。HGH目前用于治疗儿童侏儒症。2006年销售额超过30亿美元。由于其快速的血浆消除，hGH治疗需要每天注射。目前还没有人生长激素的长效形式被批准，由于形成了含有非活性异构体的混合物，人生长激素的化学聚乙二醇化反应的成功有限。我们的目标是开发生产、纯化和配制rPEG-hGH的方法。最终的产品将在体外和体内进行彻底的表征。由此产生的数据包将使rPEG-hGH进入临床开发。项目期间开发的方法和数据将验证rPEG技术，并使其能够广泛应用于其他蛋白质药物。 与公共卫生相关：许多生物药物的使用受到其血清半衰期短的限制，这需要频繁注射。该项目的目标是开发模仿聚乙二醇特性的重组多肽链(称为rPEG)。这些rPEG可以直接与蛋白质药物融合，以增加它们的血清半衰期。我们将通过开发一种治疗儿童侏儒症的长效人类生长激素来验证rPEG技术。

DESCRIPTION (provided by applicant): The market for protein-based biopharmaceuticals is rapidly expanding. However, the clinical utility of many proteins is limited by their short serum half-life, requiring frequent injections. The most common approach to improve serum half-life is PEGylation. The chemical conjugation of Polyethylene glycol (PEG) to therapeutic proteins typically results in product mixtures that include inactive isomers and reduce the overall potency of the product. The chemical PEGylation of proteins significantly increases manufacturing costs, requiring precise process control and analytical assays to ensure reproducibility of product composition. The current project describes polypeptide chains (called rPEGs) that mimic the physicochemical properties of chemical PEG. rPEGs are hydrophilic and have very large hydrodynamic radii. Most importantly, rPEGs can be recombinantly fused to therapeutic proteins resulting in homogeneous products. The fusion of rPEGs to biopharmaceutical is expected to provide benefits that are similar to chemical PEGylation (long serum half-life, reduced immunogenicity) while offering improved product potency, homogeneity, and significantly reduced manufacturing costs. Phase I of this project was extremely successful and demonstrated the feasibility of the rPEG technology. rPEG sequences that closely mimic the properties of chemical PEG were developed. We demonstrated that a 20 kDa rPEG sequence has an apparent molecular weight of 180 kDa. Fusion of rPEG to the model protein GFP increased its serum half-life in rats from 1-3 h to approximately 10 h, similar to the effect of chemical PEGylation. Furthermore, this rPEG-GFP fusion elicited only a very weak immune response in rats. Our phase II goal is to apply rPEG technology to human growth hormone (hGH). hGH is currently used for the treatment of dwarfism in children. 2006 sales exceeded $3B. Due to its rapid plasma elimination, hGH treatment requires daily injections. No long-acting form of hGH has been approved and chemical PEGylation of hGH had limited success due to the formation of mixtures containing inactive isomers. We aim to develop methods for the production, purification, and formulation of rPEG-hGH. The resulting product will be thoroughly characterized in vitro and in vivo. The resulting data package will allow rPEG- hGH to enter clinical development. The methods and data developed during the project will validate rPEG technology and enable its broad application to other protein pharmaceuticals. PUBLIC HEALTH RELEVANCE: The utility of many biopharmaceuticals is limited by their short serum half-life, which requires frequent injections. The goal of this project is to develop recombinant peptide chains (called rPEG) that mimic the properties of polyethylene glycol. These rPEGs can be directly fused to protein pharmaceuticals to increase their serum half-life. We will validate rPEG technology by developing a long-acting version of human growth hormone for the treatment of dwarfism in children.