High performance liquid chromatography (HPLC) based direct and simultaneous estimation of excipients in...

Excipients are critical components of a biotherapeutic product as they have a direct impact on the product's stability and hence on its safety and efficacy. A typical biotherapeutic drug product has a variety of excipients and these need to be maintained within very tight specifications in order to ensure consistency in product quality. Hence, there is always a need for estimation of excipient concentration in the drug product. Deviations can occur during formulation process either due to inaccurate buffer preparation or during ultrafiltration/diafiltration of high protein concentration solutions. This paper reports on development of two high performance liquid chromatography (HPLC) based methods for direct and simultaneous estimation of excipients in biopharmaceutical products. Both the methods are modified such that protein is retained in the guard column resulted in clear separation of excipients without any baseline drifts. Ion exclusion chromatography (IEC) and ion pairing chromatography (IPC) have been successfully optimized for analysing 17 commonly used excipients. Operating conditions such as temperature, type of ion pairing agent, and mobile phase strength have been optimized. The proposed methods have been successfully validated as per regulatory guidelines with respect to linearity, accuracy, matrix interference, precision, limit of detection (LOD), and limit of quantification (LOQ). The methods have been successfully used for analysing excipients in a variety of biotherapeutics including insulin, granulocyte colony stimulating factor, rituximab, and ranibizumab. Further, utility of the proposed methods for process development has been demonstrated for designing an ultrafiltration/diafiltration process for one of the mAb products.