Development of predictive tools and formulations for improved stability and delivery

项目描述：行业传统上开发生物治疗重组蛋白和肽制剂，通过进行制剂前研究，确定药物浓度、赋形剂、缓冲液和其他制剂条件，以提供适合用途的制剂（即具有所需的稳定性，并且可以在不降解生物活性药物的情况下以活性形式递送），以稳定和递送此类药物。传统上，这种配方的开发是使用实验方法设计来筛选大量潜在变量并“确定”特定产品的“合适”配方。这种方法通常适用于已建立的生物治疗药物，例如单克隆抗体（mab），在单克隆抗体中，历史知识被用作开发新分子的起点，尽管即使在该领域，对某些分子来说，将分子稳定在所需的浓度也很困难。对于非单抗生物治疗药物，由于分子的不同性质，缺乏历史数据，以及我们对稳定性，重组蛋白/肽和配方之间关系的一般有限理解，设计合适的配方可能更具挑战性。在这个项目中，我们将研究两类非单抗生物治疗药物，多肽和重链融合蛋白，以及残留宿主细胞蛋白和赋形条件对后续稳定性的影响，使用加速和长期稳定性研究。学生将在CHO或大肠杆菌细胞模型中表达多肽和重链融合蛋白（里程碑1），然后使用亲和和离子交换色谱法纯化这些蛋白（里程碑2）。在整个纯化过程中，学生将使用质谱法、ELISA和基于凝胶的方法来确定宿主细胞蛋白的残留量，并将其与标准加速稳定性研究（根据最终产品监管机构批准的要求）下分子的后续稳定性联系起来，这些研究由DSC、CD、颗粒形成分析、尺寸排除色谱、基于细胞的活性测定（如适用）和质谱法（里程碑3）监测。然后，纯化的材料将在MedImmune使用标准工业程序进行配方变量的实验设计分析，以开发和测试生物治疗产品的配方（里程碑4）。还将进行硅分析，以突出潜在的氨基酸或可能引入不稳定分子的区域，并使用实验设计配方筛选这些产生的位点定向突变和与稳定性相关的突变（里程碑5）。拟议的工作计划将提高我们对稳定蛋白质的分子机制的理解。我们还注意到，在英国很难招募到训练有素的生物治疗制剂科学家（与小分子相反），这是一个生物处理领域。该项目将培养一名配方科学家，并进一步了解阻碍一些关键治疗靶分子稳定配方的问题。该项目是两个研究所在配方研究方面成功合作博士研究生的直接结果。

Project Description:Industry traditionally develops biotherapeutic recombinant protein and peptide formulations to stabilise and deliver such drugs by undertaking pre-formulation studies that identify drug concentration, excipient, buffer and other formulation conditions that provide a formulation fit for purpose (i.e. one that has the required stability and can be delivered in an active form without degradation of the bioactive drug). Traditionally the development of such formulations is undertaken using design of experiment approaches to screen a large number of potential variables and 'identify' a 'suitable' formulation for a specific product. This approach generally works well for established biotherapeutics, for example monoclonal antibodies (mAbs), where historical knowledge is used as a starting point in the development of a new molecule, although even in this field for some molecules it can be difficult to stabilise the molecule at the desired concentration. For non-mAb biotherapeutics it can be more challenging to design appropriate formulations due to the divergent nature of the molecules, lack of historical data on these and our general limited understanding of the relationship between stability, recombinant proteins/peptides and formulations. In this project we will investigate two classes of non-mAb based biotherapeutics, peptides and heavy chain fusion proteins, and the influence of residue host cell proteins and excipient conditions on subsequent stability using accelerated and long term stability studies. The student will express in CHO or E. coli cells model peptide and heavy chain fusion proteins (milestone 1) and then purify these using affinity and ion exchange chromatography (milestone 2). The student will use mass spectrometry, ELISA and gel based approaches throughout the purification process to determine the amounts of host cell protein remaining and relate this to subsequent stability of the molecule under standard accelerated stability studies (as required for regulator approval of final products) monitored by DSC, CD, particle formation analysis, size exclusion chromatography, cell based activity assays (where appropriate) and mass spectrometry (milestone 3). The purified materials will then be subjected to design of experiments analysis of formulation variables at MedImmune using standard industrial procedures to develop and test formulations for biotherapeutic products (milestone 4). In silico analysis will also be undertaken to highlight potential amino acids or areas that might introduce instability into the molecules and site directed mutants of these produced and related to stability using the design-of-experiments formulation screen (milestone 5)The proposed programme of work will improve our understanding of the molecular mechanisms by which proteins may be stabilised. We also note that trained formulation scientists in biotherapeutics (as opposed to small molecules) are difficult to recruit in the UK and that this is an area of Bioprocessing. This project will train a formulation scientist and further our understanding of the issues preventing stable formulation of a number of key therapeutic target molecules. This project arises as a direct result of a successful collaborative PhD studentship between the two institutes in formulation studies.