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Novel Bioconjugation Strategy Using Elevated Hydrostatic Pressure: A Case Study for the Site-Specific Attachment of Polyethylene Glycol (PEGylation) of Recombinant Human Ciliary Neurotrophic Factor
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2017-10-31 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00531 Qi Wang 1, 2 , Chun Zhang 1 , Fangxia Guo 1, 2 , Zenglan Li 1, 2 , Yongdong Liu 1 , Zhiguo Su 1, 3
Bioconjugate Chemistry ( IF 4.0 ) Pub Date : 2017-10-31 00:00:00 , DOI: 10.1021/acs.bioconjchem.7b00531 Qi Wang 1, 2 , Chun Zhang 1 , Fangxia Guo 1, 2 , Zenglan Li 1, 2 , Yongdong Liu 1 , Zhiguo Su 1, 3
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In this paper, we reported a novel strategy for the site-specific attachment of polyethylene glycol (PEGylation) of proteins using elevated hydrostatic pressure. The process was similar to the conventional one except the reactor was under elevated hydrostatic pressure. The model protein was recombinant human ciliary neurotrophic factor (rhCNTF), and the reagent was monomethoxy-polyethylene glycol–maleimide (mPEG–MAL). PEGylation with mPEG (40 kDa)–MAL at pH 7.0 under normal pressure for 5 h achieved a less than 5% yield. In comparison, when the pressure was elevated, the PEGylation yield was increased dramatically, reaching nearly 90% at 250 MPa. Furthermore, the following phenomena were observed: (1) high-hydrostatic-pressure PEGylation (HHPP) could operate at a low reactant ratio of 1:1.2 (rhCNTF to mPEG–MAL), while the conventional process needs a much-higher ratio. (2) Short and long chains of PEG gave a similar yield of 90% in HHPP, while the conventional yield for the short chain of the PEG was higher than that of the long chain. (3) The reaction pH in the range of 7.0 to 8.0 had almost no influence upon the yield of HHPP, while the PEGylation yield was significantly increased by a factor of three from pH 7.0 to 8.0 at normal pressure. Surface accessibility analysis was performed using GRASP2 software, and we found that Cys17 of rhCNTF was located at the concave patches, which may have steric hindrance for the PEG to approach. The speculated benefit of HHPP was the facilitation of target-site exposure, reducing the steric hindrance and making the reaction much easier. Structure and activity analysis demonstrated that the HHPP product was comparable to the PEGylated rhCNTF prepared through a conventional method. Overall, this work demonstrated that HHPP, as we proposed, may have application potentials in various conjugations of biomacromolecules.
中文翻译：
使用升高的静水压力的新型生物共轭策略：重组人睫状神经营养因子的聚乙二醇（PEG化）的特定位点附着的案例研究。
在本文中，我们报道了使用升高的静水压力对蛋白质进行聚乙二醇（PEG化）的位点特异性附着的新策略。该方法与常规方法相似，除了反应器处于升高的静水压力下。模型蛋白是重组人睫状神经营养因子（rhCNTF），试剂是单甲氧基-聚乙二醇-马来酰亚胺（mPEG-MAL）。在常压下，在pH 7.0下用mPEG（40 kDa）–MAL进行PEG化5小时，收率不到5％。相比之下，当压力升高时，聚乙二醇化产率急剧增加，在250 MPa时达到近90％。此外，还观察到以下现象：（1）高静水压力PEG化（HHPP）可以在1：1.2的低反应物比率（rhCNTF与mPEG–MAL）下运行，而传统工艺需要更高的比例。（2）PEG的短链和长链在HHPP中的产率相似，为90％，而PEG的短链的常规产率高于长链。（3）反应pH在7.0至8.0范围内对HHPP的产率几乎没有影响，而聚乙二醇化产率在常压下从pH 7.0至8.0显着增加了三倍。使用GRASP2软件进行了表面可及性分析，我们发现rhCNTF的Cys17位于凹形斑块上，这可能对PEG的进入造成空间位阻。HHPP的推测好处是促进了目标部位的暴露，减少了空间位阻，并使反应更加容易。结构和活性分析表明，HHPP产品与通过常规方法制备的PEG化rhCNTF相当。总的来说，这项工作表明，如我们所提出的，HHPP在生物大分子的各种缀合中可能具有应用潜力。
更新日期：2017-11-01
中文翻译：
使用升高的静水压力的新型生物共轭策略：重组人睫状神经营养因子的聚乙二醇（PEG化）的特定位点附着的案例研究。
在本文中，我们报道了使用升高的静水压力对蛋白质进行聚乙二醇（PEG化）的位点特异性附着的新策略。该方法与常规方法相似，除了反应器处于升高的静水压力下。模型蛋白是重组人睫状神经营养因子（rhCNTF），试剂是单甲氧基-聚乙二醇-马来酰亚胺（mPEG-MAL）。在常压下，在pH 7.0下用mPEG（40 kDa）–MAL进行PEG化5小时，收率不到5％。相比之下，当压力升高时，聚乙二醇化产率急剧增加，在250 MPa时达到近90％。此外，还观察到以下现象：（1）高静水压力PEG化（HHPP）可以在1：1.2的低反应物比率（rhCNTF与mPEG–MAL）下运行，而传统工艺需要更高的比例。（2）PEG的短链和长链在HHPP中的产率相似，为90％，而PEG的短链的常规产率高于长链。（3）反应pH在7.0至8.0范围内对HHPP的产率几乎没有影响，而聚乙二醇化产率在常压下从pH 7.0至8.0显着增加了三倍。使用GRASP2软件进行了表面可及性分析，我们发现rhCNTF的Cys17位于凹形斑块上，这可能对PEG的进入造成空间位阻。HHPP的推测好处是促进了目标部位的暴露，减少了空间位阻，并使反应更加容易。结构和活性分析表明，HHPP产品与通过常规方法制备的PEG化rhCNTF相当。总的来说，这项工作表明，如我们所提出的，HHPP在生物大分子的各种缀合中可能具有应用潜力。
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