Development of predictive tools and formulations for improved stability and delivery

项目描述：工业界传统上开发生物活性重组蛋白和肽制剂，以通过进行制剂前研究来稳定和递送此类药物，这些研究确定了药物浓度、赋形剂、缓冲液和其他制剂条件，以提供适合目的的制剂（即具有所需稳定性并且可以以活性形式递送而不会降解生物活性药物）。传统上，这种配方的开发是使用实验设计方法来筛选大量潜在的变量，并为特定产品“确定”“合适的”配方。这种方法通常对于已建立的生物治疗剂（例如单克隆抗体（mAb））效果良好，其中历史知识被用作开发新分子的起点，尽管即使在该领域中对于一些分子，也可能难以将分子稳定在所需浓度。对于非mAb生物治疗药物，由于分子的不同性质，缺乏这些方面的历史数据，以及我们对稳定性、重组蛋白/肽和制剂之间关系的理解一般有限，因此设计适当的制剂可能更具挑战性。 在本项目中，我们将采用加速和长期稳定性研究，研究两类基于非mAb的生物治疗药物（肽和重链融合蛋白），以及残留宿主细胞蛋白和辅料条件对后续稳定性的影响。学生将用CHO或E表示。大肠杆菌细胞模拟肽和重链融合蛋白（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）。学生将在整个纯化过程中使用质谱、ELISA和基于凝胶的方法来确定剩余的宿主细胞蛋白的量，并将其与标准加速稳定性研究中分子的后续稳定性联系起来（根据最终产品监管机构批准的要求）通过DSC、CD、颗粒形成分析、尺寸排阻色谱法进行监测，基于细胞的活性测定（适当时）和质谱法（里程碑3）。然后，在MedImmune使用标准工业程序对纯化材料进行配方变量的实验设计分析，以开发和检测生物素产品的配方（里程碑4）。还将进行计算机模拟分析，以突出可能向分子中引入不稳定性的潜在氨基酸或区域，以及使用实验设计配方筛选产生的与稳定性相关的定点突变体（里程碑5）。拟定的工作计划将提高我们对蛋白质稳定化分子机制的理解。我们还注意到，在英国很难招募到生物治疗（而不是小分子）方面训练有素的配方科学家，这是生物加工的一个领域。该项目将培训一名配方科学家，并进一步加深我们对阻止许多关键治疗靶分子稳定配方的问题的理解。该项目是两个研究所在配方研究方面成功合作的博士生项目的直接结果。

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.