Development of predictive tools and formulations for improved stability and delivery of recombinant protein formulations for bio therapeutic use

行业目前雇用配方科学家调查一系列配方前研究，以确定各种蛋白质和单克隆抗体进入市场时所需的配方成分和浓度。然而，目前确定保存、稳定和递送的最佳配方的方法严重依赖于试错方法。所描述的工作计划将测试这样一个假设：“通过研究蛋白质序列，使用分子动力学计算来预测聚集，通过使用配方试剂矩阵和模型蛋白质的条件，将有可能预测改进的配方策略和特定重组蛋白质产品的组成。”此外，提出的工作计划将提高我们对蛋白质稳定的分子机制的理解。我们注意到，训练有素的配方科学家相对较少，这是金砖四国计划强调的需要关注的生物加工领域。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。学生将首先使用MedImmune提供的模型单克隆抗体来研究以下目标：(1)使用硅分子动力学模拟来预测抗体序列cdr的变化如何影响抗体聚集的理论倾向(2)通过实验测试预测的硅聚集，以确定该方法在抗体开发早期突出潜在麻烦的序列或残基方面的有效性(3)使用DSC，光散射和质谱方法为配方研究建立高通量筛选方法，以确定给定治疗蛋白的“最佳”配方(4)使用核磁共振STD分析来确定配方中的辅料如何与模型蛋白表面相互作用(5)通过适当的分析确定不仅对稳定性和聚集的影响，而且对活性的影响(6)调查治疗产品中糖型分布之间的任何联系。聚合、形成和稳定性/活性。研究中使用的初始单克隆抗体将在MedImmune上进行表征并易于获得。其他治疗性蛋白质产品也可用于研究，如果他们需要。当二硫键将这些单个结构域结合在一起时，抗体的单个亚基可用于质谱分析。该提案的结果将是：(1)发展预测治疗性蛋白质聚集的计算机方法，(2)单克隆蛋白稳定的详细配方和“通用”配方的开发，(3)提高对配方状态下蛋白质完整性的影响和决定、保存、稳定和维持的机制的理解。(4)进一步发展分析方法以监测单克隆抗体的稳定性和真实性。

Industry currently employs formulation scientists who investigate a range of pre-formulation studies to determine formulation compositions and concentrations as required for various proteins and monoclonal antibodies as they come to market. However, current methods for determining the best formulation(s) for preservation, stability and delivery rely heavily on trial-and-error approaches. The described programme of work will test the hypothesis that 'by investigating protein sequence, use of molecular dynamic calculations to predict aggregation, and by employing a matrix of formulation reagents and conditions with model proteins, it will be possible to predict improved formulation strategies and compositions for specific recombinant protein products'. In addition, the proposed programme of work will improve our understanding of the molecular mechanisms by which proteins may be stabilised. We note that trained formulation scientists are relatively rare and that this is an area of Bioprocessing that has been highlighted through the BRIC programme as in need of attention. This project will train a formulation scientist and further our understanding of the issues preventing stable formualtion 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. The student will initially use model monoclonal antibodies provided by MedImmune to investigate the following objectives: (1) use in silico molecular dynamic simulations to predict how changes to the CDRs of antibody sequences influences the theroetical propensity of antibodies to aggregation (2) test the predicted in silico aggregation experimentally to determine how effective this approach is in highlighting potentially troublesome sequences or residues early n the antibody development stage (3) use DSC, light scattering and mass spectrometry approaches to set up a high throughput screening method for formulation studies to determine the 'best' formulation for a given therapeutic protein (4) use NMR STD analysis to determine how excipients in formulations interact with the surface of a model protein (5) determine effects not only on stability and aggregation but activity through appropriate assays (6) investigate any link between the distribution of glycoforms within a therapeutic product, aggregation, formualtion and stability/activity. The initial monoclonal antibodies used in the study will have been characterised at MedImmune and be readily available. Additional therapeutic prtein products are also availabe for study should they be required. The individual subunits of the antibodies are amenable to mass spectrometry when the disulphide bonds holding these individual domains together are reduced. The outcomes of this proposal will be (1) the development of in silico methods to predict agggregation of therapeutic proteins, (2) detailed formulations for the stabilisation of monoclonals and the development of 'generic' formulations, (3) an improved understanding of the effects on, and the mechanisms that determine , preservation, stabilisation and the maintenance of protein integrity in the formulated state, and (4) further development of analytical methodology to monitor stabilisation and authenticity of monoclonal antibodies.