A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization
A new site-specific monoPEGylated filgrastim derivative prepared by enzymatic conjugation: Production and physicochemical characterization
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DOI：
10.1016/j.jconrel.2012.06.026
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发表时间：
2012-12-28
影响因子：
10.8
通讯作者：
Caliceti, Paolo
中科院分区：
文献类型：
--
作者：
Scaramuzza, Silvia;Tonon, Giancarlo;Caliceti, Paolo
我们描述了重组形式非格司亭（甲硫氨酰人粒细胞集落刺激因子，rh-Met-G-CSF）的新型单聚乙二醇化衍生物 BK0026 的制备和表征，该衍生物是通过微生物转谷氨酰胺酶催化反应，通过酶促位点特异性 20 kDa PEG 与谷氨酰胺 135 残基缀合而制备的。 BK0026 通过单一阳离子交换层析步骤纯化至临床级，并通过一组物理化学分析进行表征。 NH2 末端序列和肽图分析表明 BK0026 和非聚乙二醇化非格司亭的一级结构没有差异。圆二色性和荧光光谱显示高级蛋白质结构的保存。通过内切蛋白酶 Glu-C 肽图谱结合质谱分析和 PEG 化肽的 NH2 末端序列，鉴定了谷氨酰胺 135 上的单个缀合位点。通过多种分析方法测定了 BK0026 的纯度以及与产品和工艺相关的污染物，结果表明，BK0026 在 4-8°C 储存时可稳定保存 2 年以上。非格司亭酶促聚乙二醇化的优点是谷氨酰胺 135 结合的绝对特异性以及在非常温和的反应条件下的高聚乙二醇化产率。新的位点特异性单聚乙二醇化非格司亭是临床前和临床研究的有前途的候选药物，旨在开发一种长期治疗化疗期间肿瘤患者中性粒细胞减少症的方法。 (c) 2012 Elsevier B.V. 保留所有权利。
We describe the preparation and characterization of a new monoPEGylated derivate of a recombinant form of filgrastim (methionyl human granulocite colony stimulating factor, rh-Met-G-CSF), BK0026, prepared by enzymatic site-specific 20 kDa PEG conjugation to glutamine 135 residue by microbial transglutaminase catalyzed reaction. BK0026 was purified to a clinical grade by a single cation exchange chromatography step and characterized by using a panel of physicochemical analyses. NH2-terminal sequence and peptidemapping demonstrated no differences between the primary structure of BK0026 and the non-PEGylated filgrastim. The circular dichroism and fluorescence spectroscopy showed the preservation of high order protein structure. The single conjugation site on glutamine 135 was identified by endoproteinase Glu-C peptide mapping combined with mass spectrometry analysis and NH2-terminal sequence of the PEGylated peptides. BK0026 purity as well as product- and process-related contaminants was determined by several analytical methods, which showed that BK0026 is stable for more than 2 years when stored at 4-8 degrees C. The advantages of enzymatic PEGylation of filgrastim are the absolute specificity of glutamine 135 conjugation combined with high PEGylation yields under very mild reaction conditions. The new site specific monoPEGylated filgrastim is a promising candidate for preclinical and clinical studies aimed at developing a long-lasting treatment of neutropenia in oncological patients under chemotherapy treatments. (c) 2012 Elsevier B.V. All rights reserved.