Profiling of host cell proteins by two‐dimensional difference gel electrophoresis (2D‐DIGE): Implications for downstream process development

Abstract

Host cell proteins (HCPs) constitute a major group of impurities for biologic drugs produced using cell culture technology. HCPs are required to be closely monitored and adequately removed in the downstream process. However, HCPs are a complex mixture of proteins with significantly diverse molecular and immunological properties. An overall understanding of the composition of HCPs and changes in their molecular properties upon changes in upstream and harvest process conditions can greatly facilitate downstream process design. This article describes the use of a comparative proteomic profiling method viz. two-dimensional difference gel electrophoresis (2D-DIGE) to examine HCP composition in the harvest stream of CHO cell culture. The effect of upstream process parameters such as cell culture media, bioreactor control strategy, feeding strategy, and cell culture duration/cell viability on HCP profile was examined using this technique. Among all the parameters studied, cell viability generated the most significant changes on the HCP profile. 2D-DIGE was also used to compare the HCP differences between monoclonal antibody producing and null cell cultures. The HCP species in production cell culture was found to be well represented in null cell culture, which confirms the suitability of using the null cell culture for immunoassay reagent generation. 2D-DIGE is complimentary to the commonly used HCP immunoassay. It provides a direct comparison of the changes in HCP composition under different conditions and can reveal properties (pI, MW) of individual species, whereas the immunoassay sensitively quantifies total HCP amount in a given sample.

Analysis of host-cell proteins in biotherapeutic proteins by comprehensive online two-dimensional liquid chromatography/mass spectrometry

“…The generic ELISA kits that are commercially available for monitoring HCPs are less specific than the process specific immunoassays, and do not offer complete coverage for all the HCPs present in a sample. Other techniques such as two-dimensional gel electrophoresis coupled to fluorescent staining 12,13 is only semi-quantitative, has a limited dynamic range (3 to 4 orders of magnitude), and requires additional techniques (e.g., mass spectrometry) for HCP identification.…”

“…In addition to HCP monitoring experiments, samples spiked with 13 C 15 N-isotopically labeled peptides were also analyzed using the constructed MRM methods for the absolute quantification of three selected HCPs (clusterin, elongation factor 1-α and glyceraldehyde-3-phosphate dehydrogenase). The isotopically labeled peptide analogs (one peptide for each HCP) were spiked in the six PTG1 samples before the samples were digested with trypsin (see the Experimental Section).…”

“…The performance of the MRM assay was evaluated by analyzing 21 HCPs in 20 min with an average peak area RSD of 13.3%. Absolute quantification using 13 C 15 N-labeled peptides analogs as internal standards for three selected HCPs was performed on two LC-MRM platforms. The results were compared with TOF-based, 2D-LC/ MS E quantification method using the Hi3 peptide method.…”

“…For absolute protein quantification by the MRM approach, three 13 C 15 N-isotopically labeled peptides (Sigma Aldrich, St. Louis, MO) were spiked into 250 μL of PTG1 sample before the digestion. The final concentration for all three isotopically labeled peptides spiked in the PTG1 digest was 20 nM.…”

“…The experimental conditions for the MRM experiment (peptide sequences/transitions, collision energy and dwell times) are presented in Table S1. The absolute quantification for three selected HCPs after spiking three 13 C 15 Nisotopically labeled peptides (one peptide for each HCP) was also performed for all six PTG1 preparations using the same setup and method described above.…”