Process development for manufacturing eCD4-Ig

摘要 eCD 4-IG是一种HIV抗体样治疗剂，通过融合CD 4结构域1和2构建， 一种HIV辅助受体模拟磺肽，连接到抗体恒定Fc区的N-和C-末端。 eCD 4-IG将成为治疗和预防HIV感染的新型长效治疗药物。作为 eCD 4-IG是由HIV受体的一部分构建而成的，它能100%中和HIV分离株， 甚至能中和猴免疫缺陷病毒（SIV）。因此，eCD 4-IG对免疫调节具有根本性的障碍。 逃跑这笔赠款将资助细胞系开发和IND使能之间所需的工作 将eCD 4-IG引入临床的研究。在第一阶段，我们将验证关键质量属性（CQA）的检测方法 包括辅受体模拟磺肽的磺基酪氨酸含量，然后显示eCD 4-IG 新细胞系制造的蛋白质符合预定义的CQA。在第二阶段，我们将优化我们的上游工艺， 下游工艺、制剂缓冲液，并生成GLP批次的eCD 4-IG蛋白，用于IND启用 问题研究将优化的上游工艺的特征包括介质和进料选择， 温度变化和补料分批培养与灌注培养。我们的下游过程是基于对流 层析，包括蛋白A亲和层析，阴离子交换（AEX）层析， 和疏水相互作用（HIC）色谱法。这些过程有效地去除了不良的硫酸化 eCD 4-IG、聚集体和宿主细胞蛋白（HCP）。我们将开发一种静脉注射制剂， 和具有可接受的粘度的用于皮下注射的高浓度制剂。生产 在技术转移过程后，用于IND使能研究的GLP蛋白质批次将外包给CMO。 这些是将eCD 4-IG推向临床的关键步骤。

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.