PEGylation of therapeutic proteins has long been recognized as a safe and effective approach to enhance pharmacokinetic properties of proteins by increasing the in-vivo half-life and thereby the bioavailability. Despite all the benefits linked to PEGylation, high cost of PEGylated products has hindered accessibility of these products to patients. Continuous processing offers a solution to this predicament with its proven capability to improve economics without sacrificing product quality. In this study, we report the development of an integrated continuous PEGylation and purification process for a therapeutic protein, PEG-GCSF. The methodology to achieve this consisted of developing the batch PEGylation and purification protocols followed by their conversion into an integrated continuous process. A batch process involving rapid and highly productive PEGylation (reaction completion within one hour of reaction time) followed by cation exchange chromatography was developed. Enabling technologies like coiled flow inversion reactor, inline concentrator and counter-current chromatography, were utilized for the successful conversion of the batch process to continuous mode. The final integrated continuous process consisted of continuous PEGylation in a coiled flow inverter reactor followed by four column continuous counter-current cation exchange chromatography. Continuous chromatography was performed in a novel displacement mode, wherein all the multi-PEGylated impurities were removed in the loading flow-through and the pure mono-PEGylated protein was obtained in a single step salt elution. In combination with our previously established GCSF manufacturing train, the end-to-end continuous manufacturing process starting from inclusion bodies to unformulated PEG-GCSF drug substance was successfully run for 12 h. All attributes were found to be consistent over the period of operation with product purity > 99 % and high molecular weight impurities < 0.5 %. We hope that the current study will lay the foundation for implementation of continuous processing as a method to improve manufacturability of PEGylated therapeutic proteins.

长期以来，人们一直认为治疗性蛋白质的PEG化是一种安全有效的方法，可通过增加体内半衰期并从而提高其生物利用度来增强蛋白质的药代动力学特性。尽管具有与聚乙二醇化有关的所有好处，但聚乙二醇化产品的高成本阻碍了这些产品对患者的可及性。连续加工以其在不牺牲产品质量的前提下提高经济效益的成熟能力为这种困境提供了解决方案。在这项研究中，我们报告了治疗性蛋白质PEG-GCSF的集成连续PEG化和纯化工艺的发展。实现这一目标的方法包括开发批处理的PEG化和纯化方案，然后将其转化为集成的连续过程。开发了一种批处理方法，该方法涉及快速且高产的PEG化（反应时间在一小时内完成反应），然后进行阳离子交换色谱分析。诸如旋流转化反应器，在线浓缩器和逆流色谱法之类的使能技术被用于成功地将间歇过程转化为连续模式。最终的综合连续过程包括：在螺旋流式逆变器反应器中进行连续PEG化，然后进行四柱连续逆流阳离子交换色谱分析。以新颖的置换模式进行连续色谱分析，其中所有的多聚乙二醇化杂质均在上样流中被去除，并且在单步盐洗脱中获得了纯的单聚乙二醇化蛋白。结合我们先前建立的GCSF制造流程，从包涵体到未配制的PEG-GCSF原料药的端到端连续制造过程成功运行了12小时。发现所有属性在操作期间都是一致的，产品纯度> 99％，高分子量杂质<0.5％。我们希望当前的研究将为实施连续加工奠定基础，以提高PEG化治疗性蛋白的可制造性。0.5％。我们希望当前的研究将为实施连续加工奠定基础，以提高PEG化治疗性蛋白的可制造性。0.5％。我们希望当前的研究将为实施连续加工奠定基础，以提高PEG化治疗性蛋白的可制造性。