EDTA (ethylenediaminetetraacetic acid) is used in biopharmaceutical processes to remove metal impurities that may be bound to the protein. It is a water-soluble hexadentate chelating agent which is widely used to bind to metal ions such as iron and calcium. It is one of several excipients added to the buffer used in the ultrafiltration-diafiltration step. The excipient concentration in the diafiltration buffer is particularly important because its composition will be used for the protein formulation. During diafiltration, the buffer is exchanged with the protein solution for more than 7 diavolumes. Excipient concentrations between the buffer and final protein concentration can change because of the attractive charge between EDTA and the monoclonal antibody (Gibbs-Donnan effect) as the protein is filtered across a 30 kDa membrane. This causes the EDTA concentration to increase over the ultrafiltration-diafiltration step. Based on the calculation and estimation, the proposed EDTA concentration was used in the buffer for the first intent. However, we observed the EDTA concentration was consistently low in the buffer which resulted in a low concentration in the final bulk drug substance across small- and large-scale batches. A root-cause analysis was performed to investigate process and analytical causes. The analytical platform EDTA assay method was adjusted based on the findings and additional process development work was performed, and eventually a project-specific approach was implemented to achieve the accurate detection of EDTA in formulation buffer, in-process and BDS samples.

Investigation of low concentration of EDTA excipient in diafiltration buffer used in a monoclonal antibody purification process

ACS Fall 2021

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