Biopharmaceutical protein products have revolutionized treatment of diseases and are one of the fastest growing segments of the pharmaceutical industry. The production and purification of biopharmaceutical proteins using cellular expression systems is challenging, due to the heterogeneity of the product and complexity of parameters influencing the process. Close monitoring of Critical Quality attributes (CQAs), - attributes of the product itself or impurities that are introduced through the process known to influence efficacy, safety or stability of the final product, - is necessary.
Host cell proteins (HCPs), proteins introduced by the expression system, are especially challenging to monitor and to deplete trough purification, due to a wide range of physicochemical properties like molecular weight or isoelectric points, which can often be similar to the properties of the product. The industrial standard to monitor HCP are enzyme-linked immunosorbent assays (ELISA). These assays are targeted to specific proteins of interest and only detect immunoreactive HCP species. In contrast mass spectrometry (MS) is a non-targeted approach and allows unbiased identification and quantification of HCPs population down to single HCPs. Monitoring HCP populations within the bioreactor would increase the understanding on the influence of processing conditions on their distribution and quantity, especially regarding specific HCPs that have been identified as critical for product quality and safety.
Here, we present method development to monitor and relatively quantify HCP directly from clarified cell culture fluid (CCCF) samples using a bottom-up MS approach. To optimise the method, we investigated the influence of matrix interference and influence of different sample preparation protocols, pre-treatment, liquid chromatography and MS parameters, MS acquisition techniques and the use of different software tools for data analysis. Methods and protocols were evaluated for robustness, sensitivity, ease of use, time requirements and transferability for use in an industrial environment. To mirror the highest possible complexity and the effect of high abundant proteins compared to the HCPs, the method development was carried out using CCCF samples obtained from a hybridoma cell line, which contains foetal calf serum in the culture media.
The optimized method will be used to characterise and relatively quantify HCP from industrial CCCF samples over a time course in a timely manner and should be easily transferable to other applications.