Recovery of Chinese hamster ovary host cell proteins for proteomic analysis

Abstract

Identification and characterization of Chinese hamster ovary (CHO) host cell protein (HCP) impurities by proteomic techniques can aid bioprocess design and lead to more efficient development and improved biopharmaceutical manufacturing operations. Recovery of extracellular CHO HCP for proteomic analysis is particularly challenging due to the relatively low protein concentration and complex composition of media. In this article, we report the development of optimized protocols that improve proteome capture for CHO HCP. Eleven precipitation protocols were screened for protein recovery and optimized for a subset of precipitants by a design of experiments (DOE) approach. Because total protein recovery does not fully replicate a proteomics experiment, or detect non-protein agents that may interfere with proteomic methods, a subset of precipitation conditions were compared by two-dimensional electrophoresis and liquid chromatography coupled with mass spectrometry, with optimized recovery shown to differ between the two proteomic methods. This work demonstrates broadly applicable methods that can be applied as initial steps to optimize sample preparation of any sample type for proteomic analysis, and presents optimized precipitation protocols for extracellular CHO HCP recovery, which can vary appreciably between gel-based and shotgun proteomic methods.

Keywords: Chinese hamster ovary cells; Design of experiments optimization; Extracellular host cell proteins; Proteomics; Sample preparation.

Figure 1

(A) Total extracellular CHO HCP recovery by Bradford assay following precipitation according to various protocols and resolubilization for 2-DE and shotgun proteomics. Error bars represent the standard error of the mean for duplicate experiments. (B) SDS-PAGE comparison of protein recovery from different precipitation methods following resolubilization for shotgun proteomics.

Figure 2

Central composite design regression coefficients of coded values (Table 1) for total extracellular CHO HCP recovery following precipitation by (A) TCA with resolubilization for 2-DE (model significance p = 0.0033, R² = 0.689) and shotgun proteomics (model significance p = 0.0036, R² = 0.636), (B) methanol with resolubilization for 2-DE (model significance p = 0.0296, R² = 0.868) and shotgun proteomics (model significance p < 0.0001, R² = 0.599), and (C) acetone with resolubilization for 2-DE (model significance p = 0.0110, R² = 0.575) and shotgun proteomics (model significance p = 0.0391, R² = 0.490). Total protein recovery from central composite design as function of (D) TCA concentration, (E) methanol concentration, and (F) acetone concentration. Error bars represent standard error of the coefficient estimate (A–C) or the spread of the data (D–F). All experiments performed in duplicate with the exception of methanol with resolubilization for 2-DE (single experiment) and shotgun (triplicate experiments) proteomics. Statistical significance determined by t-test and denoted as ** p < 0.05 and * p < 0.1.

Figure 3

(A) Concentration recovery as a function of extent of concentration for two filter loadings. Precipitation recovery as a function of initial extracellular CHO HCP concentration following resolubilization for (B) 2-DE, and (C) shotgun proteomics. Overall protein recovery for concentration and precipitation operations, with respect to initial extracellular CHO HCP concentration, following resolubilization for (D) 2-DE, and (E) shotgun proteomics. (F) Precipitation recovery dependence on salt concentration with error bars representing the spread of the data from duplicate (TCA, acetone) or triplicate (methanol) experiments.

Figure 4

(A) Total number of protein spots and overall spot volume detected by ImageMaster v5.0 software following 2-DE analysis of extracellular CHO HCPs precipitated by four different optimized methods. Representative 2-DE images of extracellular CHO HCPs prepared by (B) TCA and (C) methanol precipitations. Molecular weight (MW) and isoelectric point (pI) labels approximated from the location of seven proteins previously identified by MALDI-TOF/TOF. (D) Total number of peptides and proteins identified from shotgun analysis of extracellular CHO HCPs precipitated by four different optimized methods. Error bars represent standard error of the mean from duplicate experiments.

Figure 5

Representative 2-DE image of extracellular CHO HCPs prepared by TCA precipitation including identification of proteins from 19 spots. Molecular weight (MW) and isoelectric point (pI) labels approximated from the location of seven proteins previously identified by MALDI-TOF/TOF.