Application of ATR-FTIR imaging to industrial scale production of therapeutic antibodies

生物制药，即将蛋白质用作药物，是治疗一系列疾病的新兴领域。这些分子的大规模生产需要许多离散的步骤，包括重组表达，通过柱层析技术进行分离，以及开发最佳的最终配方。将生物药品从所有其他污染物质中分离出来通常涉及许多步骤。柱层析法利用目标蛋白和固定在载体树脂上的另一分子之间的特定相互作用。理想情况下，所有的污染物质都从色谱柱中冲洗出来，并改变条件，以便从树脂中释放蛋白质。最佳的结合、洗涤和释放步骤通常要求周围溶液的性质发生重大变化，例如pH值和离子强度。此外，可以将非常高浓度的蛋白质结合到这些树脂上。所达到的浓度可能高于生产过程中任何其他点所达到的浓度。目前还不清楚不同的溶液条件和蛋白质浓度对蛋白质质量的确切影响。事实上，它们可能导致非常不受欢迎的效果，例如非特异性聚集。科学家分离生物制药遇到的另一个关键问题是不需要的污染物物质的结合，这降低了色谱柱分离目标蛋白质的能力。定期更换树脂大大增加了分离过程的成本，从而增加了生物制药生产的成本。在这里，我们的目的是研究这些问题，以期使用一种称为衰减全反射-傅里叶变换红外（ATR-FTIR）光谱的专门技术来改进隔离协议。这项技术允许我们在静态液滴和类似于大规模分离的动态系统中，在不同条件下对目标蛋白质分子进行化学成像（拍摄化学照片）。我们还可以直接对使用过的树脂进行成像，以了解哪些污染分子会减少材料的使用寿命。我们预计这些实验的结果将为分离方案的优化提供信息，以最大限度地减少靶蛋白在柱上的损失，并增加树脂寿命。

Biopharmaceuticals, proteins used as drugs, are an emerging area in the treatment of a range of diseases. The large scale production of these molecules requires a number of discrete steps including recombinant expression, isolation by a technique called column chromatography and development of an optimal final formulation. The isolation of the biopharmaceuticals from all other contaminating material usually involves a number of steps. The column chromatography used exploits a specific interaction between the target protein and another molecule immobilized on a support, the resin. Ideally all the contaminating material is washed from the column and the conditions are changed in order to release the protein from the resin. Optimal binding, washing and release steps often require significant changes in the properties of the surrounding solution, for example pH and ionic strength. In addition, it is possible to bind very high concentrations of the protein onto these resins. The concentrations reached are likely to be higher than those achieved at any other point in the production process. It is not known exactly what effects the different solution conditions and protein concentrations have on the quality of the protein. Indeed they may result in highly undesirable effects such as non-specific aggregation. Another key issue that scientists isolating biopharmaceuticals encounter is the binding of unwanted contaminant materials which reduces the ability of the column to isolate the target protein. Regular replacement of the resin significantly increases the cost of the isolation process and thus of biopharmaceutical production. Here we aim to investigate these issues with a view to improving isolation protocols using a specialized technique called Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. This technique allows us to chemically image (take chemical photographs of) the target protein molecules under different conditions both in static droplets and in dynamic systems similar to those used for large scale isolation. We can also directly image used resin with a view to understanding what contaminating molecules reduce the useful lifetime of the material. We anticipate that the results of these experiments will inform optimization of isolation protocols to both minimize target protein losses on-column and increase resin lifetime.