DoE based integration approach of upstream and downstream processing regarding HCP and ATPE as harvest operation

• A new optimization focus of USP development towards lower HCP content is necessary.
• We described the influences of single media components on the impurity profile.
• We developed a medium resulting in increased IgG and decreased HCP production.
• Harvest operations influence strongly the HCP spectrum entering DSP operations.
• ATPE harvest results in cell separation and HCP reduction without product loss.

Conventional USP optimization concepts have led to increasing product titers and significantly raised impurity profiles. Varying compositions of cultivation broth present additional challenges in downstream processing. This work addresses the resulting problems by combining methodical and technological approaches.The methodical approach is based on the integration of upstream and downstream process development by extending the range of USP optimization. In addition to high product titer, cell numbers and product quality, the composition and concentration of host cell proteins were taken into account. The feasibility of this approach was demonstrated within this work. A model CHO cell line producing IgG was used to develop a medium, which generates higher titers and cell numbers as well as a lower amount of HCP. The new composition of HCP was designed to simplify subsequent purification steps. Experiments were statistically planned and the results were validated, all resulting in higher product titers but lower HCP levels.In the next step, the influence of the cultivation and cell harvest operations on the impurities was investigated. Centrifugation, filtration and aqueous two phase extraction were evaluated regarding their ability to separate cells and HCP without product loss. ATPE distinguished itself as the cell harvest operation of choice by high cell separation efficiency, high yields, and a substantial removal of HCP which could not be met by centrifugation and filtration.