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

生物制药，即用作药物的蛋白质，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步骤，包括重组表达、通过称为柱色谱的技术分离和开发最佳最终制剂。从所有其他污染物质中分离生物药物通常涉及许多步骤。所用的柱色谱法利用了靶蛋白和固定在支持物（树脂）上的另一种分子之间的特异性相互作用。理想情况下，从柱中洗涤所有污染物质，并改变条件以从树脂中释放蛋白质。最佳结合、洗涤和释放步骤通常需要显著改变周围溶液的性质，例如pH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阶段所达到的浓度。目前尚不清楚不同的溶液条件和蛋白质浓度对蛋白质质量的影响。事实上，它们可能导致非常不希望的效应，如非特异性聚集。科学家分离生物药物遇到的另一个关键问题是不需要的污染物的结合，这降低了柱分离靶蛋白的能力。树脂的定期更换显著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，我们的目标是调查这些问题，以期改善隔离协议使用一种专门的技术称为衰减全反射傅里叶变换红外（ATR-FTIR）光谱。这种技术使我们能够在不同条件下在静态液滴和动态系统中对靶蛋白分子进行化学成像（拍摄化学照片），这些系统类似于用于大规模分离的系统。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降低材料的使用寿命。我们预计这些实验的结果将为分离方案的优化提供信息，以最大限度地减少柱上靶蛋白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.