(175bb) Model-Based Design of Non-affinity Chromatofocusing Capture Method for On-demand Production of Biologics

For personalized medicines, national emergencies, military operations, and related applications there is a growing need to produce single-dose levels of therapeutic proteins at the point of care. To satisfy this need, the “biologically-derived medicines on demand (Bio-MOD)” device has been developed, which is an integrated and portable bioprocessing system. A cell-free expression system is utilized in this device to produce a single dose of therapeutic-grade biologics in only a few hours. In this study, the technique of chromatofocusing is investigated as a non-affinity capture method to produce a totally tag-less biologic in the Bio-MOD device. Chromatofocusing is an ion-exchange technique that usually employs a polyampholyte buffer, but in this study a simple mixture of adsorbed and unadsorbed buffering species is used for creating a retained, self-generated pH gradient inside the column. Since the pH gradient is internally generated with respect to the column, a simple step change is employed between the starting and elution buffers. This gradient in turn causes a focusing effect so that highly resolved protein bands are produced. In addition to a high resolution, the major advantages of this technique are a short separation time, a high selectivity, a high capacity, and simple and cost-effective operation. However, it is generally difficult to select the specific buffer compositions used for a particular protein mixture. To account for this, a computer simulation method is developed not only to design the proper buffer composition for each product protein produced in the Bio-MOD device, but also to simulate, monitor and optimize the separation process. Results of this study indicate that a purity of more than 98% is achieved for a protein expressed in a cell-free system during a single non-affinity-based capture step starting from a 0.5% purity in the harvest. Also, the computer simulation method for the purification process developed in this study facilitates the use of Quality by design (QbD) and Process Analytical Technology (PAT) to ensure final product quality.