Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal 'C-tag'.
Accelerating the clinical development of protein-based vaccines for malaria by efficient purification using a four amino acid C-terminal 'C-tag'.
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DOI：
10.1016/j.ijpara.2016.12.001
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发表时间：
2017-06
影响因子：
4
通讯作者：
Draper SJ
中科院分区：
文献类型：
--
作者：
Jin J;Hjerrild KA;Silk SE;Brown RE;Labbé GM;Marshall JM;Wright KE;Bezemer S;Clemmensen SB;Biswas S;Li Y;El-Turabi A;Douglas AD;Hermans P;Detmers FJ;de Jongh WA;Higgins MK;Ashfield R;Draper SJ
四个氨基酸的"C-标签"的融合允许PfRH5疟疾疫苗的纯化。实现了40 - 45%的总工艺产率和非常高的产物纯度（> 99%）。His6标记的和C标记的PfRH5是构象的并且结合至basigin。C-tag将促进难以生产的抗原的临床翻译。定制生物制造工艺的开发仍然是转化研究的关键瓶颈，特别是当概念验证I/II期临床试验需要少量新产品时。在这些情况下，快速且相对便宜地开发生物制造过程而不对产品质量或安全性造成风险的能力通过允许免疫原设计中的新抗原或概念更快速地进入人体测试而提供了巨大的优势。这些生产和纯化方面的挑战在开发用于困难寄生虫病的重组蛋白疫苗时尤其明显，恶性疟原虫疟疾就是一个主要例子。为此，我们之前已经报道了使用ExpreS2黑腹果蝇Schneider 2稳定细胞系系统表达用于疟疾的新型蛋白疫苗，然而，非常低的总工艺产率（通常<5%的六组氨酸标记的蛋白回收率）意味着初始纯化策略不适合于这种疫苗的放大和临床生物制造。在这里，我们描述了一种新的亲和纯化方法，非常适合于纯化编码恶性疟原虫网织红细胞结合蛋白同系物5的相同蛋白质-目前是下一代疫苗评估的主要抗原，旨在防止血液阶段寄生虫侵入红细胞。该纯化系统利用称为"C-tag'"的C-末端标签，其由四种氨基酸谷氨酸-脯氨酸-谷氨酸-丙氨酸（E-P-E-A）组成，其在与骆驼科单链抗体（称为NbSyn2）偶联的CaptureSelect ™亲和树脂上选择性纯化。该短C-标签与恶性疟原虫网织红细胞结合蛋白同系物5的C-末端融合在温和条件下直接从澄清、浓缩的Schneider 2细胞上清液的单步纯化中实现了> 85%的回收率和> 70%的纯度。生物化学和免疫学分析表明，C-标记和六组氨酸标记的恶性疟原虫网织红细胞结合蛋白同源物5蛋白是相当的。C-tag技术有可能成为当前符合良好生产规范的平台的基础，这可以大大提高基于蛋白质的新型产品进入临床测试的速度和便利性。
Fusion of a four amino acid ‘C-tag’ allows purification of a PfRH5 malaria vaccine. Overall process yield of 40–45% and very high product purity (>99%) was achieved. His6-tagged and C-tagged PfRH5 are conformational and bind to basigin. C-tag will facilitate the clinical translation of difficult-to-produce antigens. Development of bespoke biomanufacturing processes remains a critical bottleneck for translational studies, in particular when modest quantities of a novel product are required for proof-of-concept Phase I/II clinical trials. In these instances the ability to develop a biomanufacturing process quickly and relatively cheaply, without risk to product quality or safety, provides a great advantage by allowing new antigens or concepts in immunogen design to more rapidly enter human testing. These challenges with production and purification are particularly apparent when developing recombinant protein-based vaccines for difficult parasitic diseases, with Plasmodium falciparum malaria being a prime example. To that end, we have previously reported the expression of a novel protein vaccine for malaria using the ExpreS2Drosophila melanogaster Schneider 2 stable cell line system, however, a very low overall process yield (typically <5% recovery of hexa-histidine-tagged protein) meant the initial purification strategy was not suitable for scale-up and clinical biomanufacture of such a vaccine. Here we describe a newly available affinity purification method that was ideally suited to purification of the same protein which encodes the P. falciparum reticulocyte-binding protein homolog 5 – currently the leading antigen for assessment in next generation vaccines aiming to prevent red blood cell invasion by the blood-stage parasite. This purification system makes use of a C-terminal tag known as ‘C-tag’, composed of the four amino acids, glutamic acid – proline – glutamic acid – alanine (E-P-E-A), which is selectively purified on a CaptureSelect™ affinity resin coupled to a camelid single chain antibody, called NbSyn2. The C-terminal fusion of this short C-tag to P. falciparum reticulocyte-binding protein homolog 5 achieved >85% recovery and >70% purity in a single step purification directly from clarified, concentrated Schneider 2 cell supernatant under mild conditions. Biochemical and immunological analysis showed that the C-tagged and hexa-histidine-tagged P. falciparum reticulocyte-binding protein homolog 5 proteins are comparable. The C-tag technology has the potential to form the basis of a current good manufacturing practice-compliant platform, which could greatly improve the speed and ease with which novel protein-based products progress to clinical testing.