During drug development, the removal of impurities and purification of a final drug product is absolutely essential in order to ensure the safety and efficacy of a therapeutic drug. Of particular concern for biologics are impurities that can stem from host cell proteins. Because biologics are developed through cell culture and fermentation within a host cell, proteins from this host cell can be co-purified with the final biologic. These host cell proteins or HCPs can cause the final product to have undesired side-effects such as eliciting an immune response in patients taking the drug, or affecting the drug’s stability or efficacy. As a result, regulating agencies require drug companies to monitor levels of HCPs during the development and purification of a biologic and to remove HCPs to an acceptable level in the final biotherapeutic product.
One of the most common methods for detecting and measuring HCPs is through the use of an ELISA developed using polyclonal antibodies raised against the host cells used in the fermentation process. Although these assays can take up to several months to develop, they typically are capable of detecting a majority of HCPs. However, “a majority” simply isn’t good enough. Often the assay can be dominated by highly abundant and highly immunogenic proteins. Lower abundant/immunogenic proteins can be more difficult to detect. Additionally, some proteins may not elicit an immune response at all in the animals used to generate the ELISA and can, therefore, go completely undetected. This can be particularly troublesome if the protein does have an immunogenic response in humans, as it can cause serious adverse reactions in patients taking the biologic. As a result, orthogonal techniques such as 1D and 2D SDS-PAGE, DIGE, and LC-MS/MS are often employed to supplement ELISA and fully characterize HCPs in a biologic preparation.1
Enter SWATH® Acquisition! SWATH Acquisition on a TripleTOF® 6600 System provides an unbiased method for detecting low-level HCPs – even those that were previously unknown or did not generate an immunogenic response in animal studies. The technique is comprehensive, sensitive, fast, and reproducible, enabling the profiling and identification of the HCP complement of 1000s of proteins at a sub-ppm level in a single 1-hour run. However, rather than being used as a supplement to ELISA, SWATH Analysis is an alternative to ELISA for fully characterizing and monitoring HCPs.
SWATH Acquisition works by collecting MS and MS/MS data across the entire data range in an unbiased data-independent fashion. Because of the speed, sensitivity, and dynamic range of the TripleTOF System, and the intelligent design of the SWATH workflow, the full HCP complement can be detected at sub-ppm levels in 1 hour. And contrary to other LC-MS/MS techniques employed for HCP analysis, no complicated and extensive LC fractionation is required. As described by SCIEX researchers in a recent webinar and also in this informative slide presentation, this can have profound effects on the efficiency and overall cost of a biologic through process development, with time and cost savings of 20x for a typical study.
Additionally, because SWATH Acquisition is unbiased, the approach provides substantial benefits over other MS strategies by capturing comprehensive, quantitative MS and MS/MS information for every analyte in the sample. Thus, sequence variants and low-level modifications to the biologic can also be captured in the same data set. In fact, protein contaminants from any source can be detected along with the HCPs. Because of the comprehensive nature of the SWATH data file, the digital record that is created can be used as a digital archive of the current state of a sample. Any protein contaminant concerns that may emerge in the future can be tracked over time by mining the data retrospectively.
The method is mature and has been used extensively in the microflow regime where the LC details have been optimized for easy implementation. The workflow can also support high flow and nanoflow chromatography where it may be advantageous to perform HCP analysis in the early development stage when there is not as much product available for characterization.
To learn more about SWATH Acquisition on a SCIEX TripleTOF® 6600 System for HCP analysis and how it can save you valuable time and money during biotherapeutic development, view the webinar or presentation today.
- Fengqiang Wang, Ph.D., Daisy Richardson, Mohammed Shameem “Host-Cell Protein Measurement and Control”, June 01, 2015, BioPharm International, Volume 28, Issue 6, pg 32–38