Process development for manufacturing eCD4-Ig

摘要 ECD4-Ig是一种针对HIV的抗体样疗法，它是通过融合CD4域1和2构建的，并且 抗体恒定Fc区N-末端和C-末端的HIV辅助受体模拟磺肽。 ECD4-Ig将加入治疗和预防艾滋病毒感染的新兴长效疗法类别。AS 作为由部分HIV受体构建的结果，eCD4-Ig可以中和100%的HIV分离株，并且 甚至中和猴免疫缺陷病毒(SIV)。因此，eCD4-Ig构成了基本的障碍 逃走。这笔赠款将资助在细胞系开发和IND使能之间所需的工作 将eCD4-Ig带入临床的研究。在第一阶段，我们将验证关键质量属性(CQA)的分析 包括磺基酪氨酸含量的辅受体模拟磺基多肽，然后显示eCD4-Ig 由新的细胞系制造的蛋白质符合预先定义的CQA。在第二阶段，我们将优化我们的上游流程， 下游加工，配方缓冲，并产生一批用于IND的GLP批次的eCD4-Ig蛋白 学习。将进行优化的上游流程的功能包括媒体和源选择、 温度漂移，以及分批补料培养与灌流培养。我们的下游过程是基于对流的 层析，包括蛋白A亲和层析，阴离子交换(AEX)层析， 疏水相互作用(HIC)层析。这些过程有效地去除了劣质的硫酸盐 ECD4-Ig、聚集体和宿主细胞蛋白(HCP)。我们将开发一种静脉注射的配方， 以及一种可接受粘度的高浓度皮下注射配方。制作一部 GLP大量用于IND研究的蛋白质将在技术转让过程后外包给CMO。 这些是将eCD4-Ig引入临床的关键的下一步。

ABSTRACT eCD4-Ig is an antibody-like therapeutic for HIV that was constructed by fusing CD4 domains 1 and 2, and an HIV coreceptor-mimetic sulfopeptide, to the N- and C-termini of the constant Fc region of an antibody. eCD4-Ig will join an emerging class of long-acting therapeutics for treating and preventing HIV infection. As a consequence of being built from parts of HIV’s receptors, eCD4-Ig neutralizes 100% of HIV isolates, and even neutralizes simian immunodeficiency virus (SIV). Thus, eCD4-Ig presents fundamental barriers to escape. This grant will fund the work that is needed in between cell line development and IND-enabling studies to bring eCD4-Ig to the clinic. In Phase I, we will validate assays for critical quality attributes (CQAs) including the sulfotyrosine content of the coreceptor-mimetic sulfopeptide, and then show that the eCD4-Ig protein made by new cell lines meets pre-defined CQAs. In Phase II, we will optimize our upstream process, downstream process, formulation buffers, and generate a GLP lot of eCD4-Ig protein for IND-enabling studies. Features of the upstream process that will be optimized include media and feed selection, a temperature shift, and fed-batch versus perfusion culture. Our downstream process is based on convective chromatography, including for Protein A affinity chromatography, anion exchange (AEX) chromatography, and hydrophobic interaction (HIC) chromatography. These processes efficiently remove poorly sulfated eCD4-Ig, aggregates, and host cell protein (HCP). We will develop a formulation for intravenous injection, and a high-concentration formulation with acceptable viscosity for subcutaneous injection. Production of a GLP lot of protein for IND-enabling studies will be outsourced to a CMO after a technology transfer process. These are the essential next steps towards bringing eCD4-Ig to the clinic.