Production Technology for Recombinant Intravenous Immunoglobulin

重组静脉免疫球蛋白生产技术

• 批准号: 8976337
• 负责人: David Scott Johnson
• 金额: $ 22.5万
• 依托单位: GIGAGEN, INC.
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-24 至 2016-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8976337
• 关键词: Animals; Antibodies; Antibody Repertoire; Autoimmune Process; Biological; Biological Assay; Blood; Blood donor; Capital; Caring; Cells; Chronic Lymphocytic Leukemia; DNA; DNA Library; Diabetes Mellitus; Diagnostic; Doctor of Philosophy; Dose; Drug Kinetics; Engineering; Escherichia coli; Factor XIa; Fertilization in Vitro; Frequencies; Genetic; Genomics; Human; IgA Deficiency; Immune; Immune System Diseases; Immunoglobulin G; Immunoglobulin Variable Region; In Vitro; Insulin; Intravenous Immunoglobulins; Investments; Label; Lead; Libraries; Licensing; Light; Linear Regressions; Methods; Microfluidics; Modality; Molecular; Patients; Pharmaceutical Preparations; Phase; Physicians; Pichia; Population; Preparation; Production; Proteins; Recombinants; Replacement Therapy; Resources; Reverse Transcriptase Polymerase Chain Reaction; Risk; Sales; Serum; Serum Proteins; Small Business Innovation Research Grant; Surveys; System; Technology; Testing; Therapeutic; Thrombocytopenic Purpura; Time; Toxicology; United States National Institutes of Health; Variant; Vasculitis; Viral; Yeasts; aging population; antigen binding; antitumor drug; base; congenital immunodeficiency; cost; glycosylation; hepatitis A virus antibodies; innovation; new technology; next generation sequencing; novel therapeutics; protein expression; public health relevance; tumor

 DESCRIPTION (provided by applicant): Production Technology for Recombinant IVIg Organization: GigaGen Inc. PI: David S. Johnson, Ph.D. The Specific Aim of this SBIR Phase I project is to develop a natural repertoire antibody protein expression system that will form the basis of a recombinant intravenous immunoglobulin (rIVIg) therapeutic product. IVIg is a pool of proteins isolated from the sera of thousands of donors. The FDA has approved IVIg therapy for six indications, including idiopathic (immune) thrombocytopenic purpura (ITP), Kawasaki's vasculitis, B cell chronic lymphocytic leukemia (CLL), and primary immunodeficiencies (Orange et al., 2006). IVIg sales are $7 billion worldwide and growing at 8-10% per year, due to an aging population and ever-expanding off-label modalities (Taylor & Shapiro, 2013). Unfortunately, current methods for IVIg production threaten continued expansion of IVIg therapy because of supply chain risk, impurities and contamination, and batch-to-batch variation. In this Phase I project, we will take steps to demonstrate that we can express GigaMuneTM natural human repertoire DNA libraries a stable yeast expression system. Analogous to Genentech 30 years ago (Russo 2003), our primary technology innovation is to use natural Ig repertoire DNA libraries expressed in a humanized Pichia yeast production system (Li et al., 2006) to replace a resource-limited biological drug with a recombinant alternative. To make rIVIg, we will first use GigaGen GigaMuneTM technology to capture and re- create expressed Ig repertoires from >1000 blood donors. GigaMuneTM uses advanced microfluidics and genomics to generate RT-PCR libraries from millions of single cells per donor, with native IgG subtypes and pairing between heavy and light chain. We will then stably express the DNA repertoires en masse in engineered Pichia to produce massively polyclonal engineered rIVIg protein product. Our rIVIg will have natural repertoire genetics, engineerable content, programmable glycosylation, low production cost, and consistent and predictable production. We will accomplish the Specific Aim by performing the following tasks: (i) Engineer a system for subcloning GigaLink(tm) DNA libraries en masse with native IgG isotype intact; (ii) Optimize GigaLink(tm) DNA library delivery and stable display in a Pichia yeast production system; and (iii) Use next-generation sequencing (NGS) and Ig assays to assess several cell passages for uniformity and isotype content. We will be successful if we achieve the following metrics: (i) Use NGS to show that the Ig clone frequencies of the >107 diversity GigaLink(tm) libraries are maintained when subcloned and stably expressed in the yeast production system (linear regression; a=0.05, power=0.8); and (ii) Use NGS and antigen binding assays to demonstrate <10% CV between cell passages and time points (one-proportion z-test; a=0.05, power=0.8). Phase I will demonstrate that we can reproducibly produce high-diversity GigaLink(tm) protein libraries in a yeast expression platform. In Phase II, we will take steps to build a GMP production facility and perform toxicology and pharmacokinetic studies on our rIVIg preparations. At first, rIVIg will simply substitute for conventional IVIg, especially for patients who are deficient in antibodies (i.e., hypogammaglobulinemia or humoral deficiencies). Later, our ability to engineer the content of the DNA library will open up broad new applications, such as IgA deficiency and polyclonal anti-tumor therapeutics.

 描述(由申请人提供)：重组IVIg组织的生产技术：GigaGen Inc.PI：David S.Johnson，Ph.D.这项SBIR第一阶段项目的具体目标是开发一种天然的抗体蛋白表达系统，它将构成重组静脉免疫球蛋白(RIVIg)治疗产品的基础。IVIG是从数千名捐赠者的血清中分离出来的蛋白质池。FDA已批准IVIg治疗六种适应症，包括特发性(免疫性)血小板减少性紫癜(ITP)、川崎氏血管炎、B细胞慢性淋巴细胞白血病(CLL)和原发免疫缺陷(Orange等人，2006年)。由于人口老龄化和不断扩大的标签外模式，IVIG在全球的销售额为70亿美元，并以每年8%-10%的速度增长(Taylor&Shapiro，2013)。不幸的是，由于供应链风险、杂质和污染以及批次之间的差异，目前的IVIg生产方法威胁着IVIg疗法的继续扩展。在这个第一阶段的项目中，我们将采取措施证明我们可以表达GigaMuneTM天然人类谱系DNA文库的一个稳定的酵母表达系统。与30年前的Genentech(Russo 2003)类似，我们的主要技术创新是使用人源化毕赤酵母生产系统中表达的天然Ig谱系DNA文库(Li等人，2006)，用重组替代品取代资源有限的生物药物。为了制作rIVIg，我们将首先使用GigaGen GigaMuneTM技术来捕获并重新创建1000名献血者的表达Ig谱系。GigaMuneTM使用先进的微流体和基因组学技术，从每个捐赠者的数百万个单细胞中产生RT-PCR文库，具有天然的免疫球蛋白亚型，并在重链和轻链之间配对。然后，我们将在工程化毕赤酵母中稳定表达DNA谱带，以生产大规模的多克隆工程化rIVIg蛋白产品。我们的RIVIg将拥有天然的曲目遗传学，可工程的内容，可编程的糖基化，低生产成本，以及一致和可预测的生产。我们将通过执行以下任务来实现这一具体目标：(I)设计一个整体亚克隆GigaLink(Tm)DNA文库的系统，同时保持本地免疫球蛋白同型的完整性；(Ii)优化GigaLink(Tm)DNA文库的交付和稳定 在毕赤酵母生产系统中展示；以及(Iii)使用下一代测序(NGS)和免疫球蛋白(Ig)分析来评估几代细胞的一致性和同型含量。如果我们达到以下指标，我们就会成功：(I)使用NGS来证明&gt；107多样性GigaLink(Tm)文库的Ig克隆频率在亚克隆并在酵母生产系统中稳定表达时保持不变(线性回归；a=0.05，POWER=0.8)；以及(Ii)使用NGS和抗原结合试验来证明细胞传代和时间点之间的&lt；10%变异系数(一比例z检验；a=0.05，POWER=0.8)。第一阶段将展示我们可以在酵母表达平台中重复生产高多样性的GigaLink(Tm)蛋白质文库。在第二阶段，我们将采取措施建立GMP生产设施，并对我们的RIVIg制剂进行毒理学和药代动力学研究。首先，rIVIg将简单地替代传统的IVIg，特别是对于抗体缺乏的患者(即低丙种球蛋白血症或体液缺陷)。稍后，我们设计DNA文库内容的能力将打开广泛的新应用，例如IgA缺乏症和多克隆抗肿瘤治疗。