Small HCPs in a 12 kDa Protein Drug Analyzed by GeLC-MS/MS

The content of low molecular weight host cell proteins (HCPs) in purified protein drugs is often difficult to evaluate, due to their low immunogenicity and poor ability to be visualized in gel-based total protein stains. The proteome of commonly used expression organisms, such as E. coli and Chinese Hamster cells, contains 30-40% proteins with a molecular weight below 20kDa, and these are easily missed in both in gel separations, Western blots and ELISA quantitation of the total HCP-content. To provide unbiased analysis of small as well as larger HCPs, we introduce the use of a mass spectrometry-based orthogonal method, well known from proteomics, called GeLC-MS/MS.

Here, we analyze an in process protein drug, a 12 kDa protein produced in E. coli, as well as the corresponding null cell lysate, using 1D-PAGE and nano-flow LC-MS/MS (GeLC-MS/MS) to achieve high coverage of small HCPs.