University of Nebraska-Lincoln Biological Process Development Facility, College of Engineering, University of Nebraska-Lincoln, Lincoln, NE 68588-0668, USA.
Biotechnol Prog. 2011 Jul;27(4):1036-47. doi: 10.1002/btpr.631. Epub 2011 May 31.
Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A-chain (RTA), which is an N-glycosidase enzyme, and the B-chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1-33/44-198 protein, herein denoted RVEa™) expressed in Escherichia coli using a codon-optimized gene was shown to be non-toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale-up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)-compliant production of RVEc™ at the 40 L scale. The average yield of the final purified bulk RVEc™ is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc™ was >99% pure by three HPLC methods and SDS-PAGE. The intact mass and peptide mapping analysis of RVEc™ confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc™ was immunoprotective against lethal ricin challenge and elicited neutralizing anti-ricin antibodies in 95-100% of the vaccinated mice.
蓖麻毒素是一种强效毒素，也是一种潜在的生物恐怖主义武器，目前尚无特效解毒剂或疫苗可用。该全毒素由两条通过单个二硫键连接的多肽链组成：A 链（RTA），它是一种 N-糖苷酶酶，B 链（RTB），是一种与哺乳动物靶细胞表面半乳糖基部分结合的凝集素多肽。先前（McHugh 等人），使用密码子优化基因在大肠杆菌中表达的重组截短形式的 RTA（rRTA1-33/44-198 蛋白，此处表示为 RVEa™）被证明是无毒、稳定的，并能在小鼠中抵抗蓖麻毒素的攻击。在这里，我们描述了在 12L 发酵规模上的工艺开发和放大，以及在 40L 规模上符合现行良好生产规范（cGMP）的 RVEc™的生产。最终纯化的 RVEc™的平均产量约为湿细胞重量的 16g/kg 或发酵液的 1.2g/L。RVEc™通过三种 HPLC 方法和 SDS-PAGE 法的纯度>99%。RVEc™的完整质量和肽图分析证实了产物的身份，并且与不存在翻译后修饰一致。效力测定表明，RVEc™对致死性蓖麻毒素攻击具有免疫保护作用，并在 95-100%的接种小鼠中引起中和抗蓖麻毒素抗体。
