Purification Development, Genzyme Corporation, Framingham, Massachusetts 01701, USA.
Biotechnol Bioeng. 2012 May;109(5):1217-27. doi: 10.1002/bit.24406. Epub 2011 Dec 26.
In this article, we describe a hydrophobic interaction chromatography (HIC) method to remodel the carbohydrates on recombinant human β-glucocerebrosidase (GCR) and the use of hydroxyl ethyl starch (HES) an ethylated starch polymer, to improve this process. GCR is a therapeutic protein used in the treatment of Gaucher disease, a life threatening condition in which patients lack sufficient functional levels of this enzyme. Gaucher disease is the most common inherited lysosomal storage disorder resulting in hepatomegaly, splenomegaly, and bone and lung pathology due to the accumulation of glucosylceramide in the lysosomes of macrophages (Beutler and Grabowski, 2001). The oligosaccharide remodeling of GCR, performed on HIC using three enzymes that remove sugars, increases macrophage uptake through the mannose receptor and thereby lowers its therapeutic dose versus unmodified GCR (Furbish et al., 1981; Van Patten et al., 2007). In this article we describe findings that the addition of HES lowered the amounts of three deglycosylating enzymes needed for remodeling GCR. HES also stabilized the activity of α-glucosidase, α-galactosidase, and GCR under conditions in which these three enzymes rapidly lose activity in the absence of this polymer. Circular dichroism (CD) and second derivative UV spectroscopy revealed that the secondary and tertiary structure of α-glucosidase was unchanged while for GCR there was a slight compaction of the secondary structure but no apparent affect on the tertiary structure. The thermal stability of both GCR and α-glucosidase were enhanced by HES as both molecules showed an increased transition midpoint (T(m)).
本文描述了一种疏水相互作用色谱（HIC）方法，用于重塑重组人β-葡糖脑苷脂酶（GCR）上的碳水化合物，并使用羟乙基淀粉（HES）这种乙基化淀粉聚合物来改进该过程。GCR 是一种治疗蛋白，用于治疗戈谢病，这是一种危及生命的疾病，患者缺乏这种酶的足够功能性水平。戈谢病是最常见的遗传性溶酶体贮积病，由于葡萄糖脑苷脂在巨噬细胞溶酶体中的积累，导致肝肿大、脾肿大以及骨骼和肺部病理（Beutler 和 Grabowski，2001）。使用三种去除糖的酶在 HIC 上对 GCR 进行寡糖重塑，通过甘露糖受体增加巨噬细胞摄取，从而降低其治疗剂量，与未修饰的 GCR 相比（Furbish 等人，1981；Van Patten 等人，2007）。本文描述了发现添加 HES 降低了用于重塑 GCR 的三种去糖基化酶的用量。在没有这种聚合物的情况下，HES 还能稳定α-葡萄糖苷酶、α-半乳糖苷酶和 GCR 的活性，在这些酶迅速失活的条件下。圆二色性（CD）和二级导数紫外光谱表明，α-葡萄糖苷酶的二级和三级结构没有变化，而 GCR 的二级结构略有紧缩，但对三级结构没有明显影响。HES 增强了 GCR 和 α-葡萄糖苷酶的热稳定性，因为这两种分子的转变中点（T(m)）都增加了。
