Methods for monitoring CHAPS-assisted refolding and diafiltration of recombinant protein

Abstract

The use of the recombinant human growth factor bone morphogenetic protein 2 (rhBMP-2) is important for the cure of bone defects as well as in the field of dentistry. For the production of this protein a recombinant approach in the host E. coli is pursued in this study. Like many recombinant proteins that are produced in the host E. coli, rhBMP-2 aggregates in the cell in form of inclusion bodies. These dense particles contain the misfolded monomer, which then needs to be refolded correctly into the active homodimer. Since the unit operation refolding is a complex process, the development of an analytical method for the monitoring of the monomer/dimer ratio would allow revealing a better process control and hence an efficient process design. If time resolved in-line analytics could be established, the control could be conducted at any point of time. Moreover, the detection of the detergent CHAPS, which is an additive in the refolding in order to keep rhBMP-2 solubilized, is desired during refolding and the subsequent diafiltration/ultrafiltration (UF) step. The formation of CHAPS micelles could lead to errors when it comes to analytical measurements that are based on size, due to unknown aggregation behavior of rhBMP-2 inside CHAPS micelles. In addition, a determination of the CHAPS concentration after the UF/DF unit operation could reveal the detergent as a possible in-process impurity. The aim of this work was to develop a SEC-HPLC method for the quantification of monomer and dimer of rhBMP-2 during the refolding process. Here we show the evaluation of 4 columns for separation and quantification of rhBMP-2 variants. Different columns, buffer compositions and pH were screened in order to enable direct access to the refolding state. In the scope of this work no final HPLC method could be developed to reach this aim. Furthermore a method for fast and easy detection of the potential critical process impurity CHAPS was aimed to be developed. This aim was pursued successfully and a method for the detection of micelle formation of CHAPS could be developed. The fluorescence measurement can be used for the design of filtration steps involving CHAPS to efficiently detect this process impurity.