Host cell protein dynamics in the supernatant of a mAb producing CHO cell line

Abstract

The characterization of host cell protein (HCP) content during the production of therapeutic recombinant proteins is an important aspect in the drug development process. Despite this, key components of the HCP profile and how this changes with processing has not been fully investigated. Here we have investigated the supernatant HCP profile at different times throughout culture of a null and model GS-CHO monoclonal antibody producing mammalian cell line grown in fed-batch mode. Using 2D-PAGE and LC-MS/MS we identify a number of intracellular proteins (e.g., protein disulfide isomerise; elongation factor 2; calreticulin) that show a significant change in abundance relative to the general increase in HCP concentration observed with progression of culture. Those HCPs that showed a significant change in abundance across the culture above the general increase were dependent on the cell line examined. Further, our data suggests that the majority of HCPs in the supernatant of the cell lines investigated here arise through lysis or breakage of cells, associated with loss in viability, and are not present due to the secretion of protein material from within the cell. SELDI-TOF and principal components analysis were also investigated to enable rapid monitoring of changes in the HCP profile. SELDI-TOF analysis showed the same trends in the HCP profile as observed by 2D-PAGE analysis and highlighted biomarkers that could be used for process monitoring. These data further our understanding of the relationship between the HCP profile and cell viability and may ultimately enable a more directed development of purification strategies and the development of cell lines based upon their HCP profile.

Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry

“…First, mAbs could be engineered to minimize 'sticky' patches resulting from unique variable region sequences and presumably responsible for much product-specific HCP binding. Second, it is crucial to minimize cell lysis during cell culture/harvest because this represents the major source of HCPs in harvest fluid 28,29 as well as the major source of HCP profile variability. 30 Third, effective oncolumn wash conditions, capable of disrupting HCP interactions Limited numbers of CHO cell proteins have been identified in prior reports, either based on MS analysis of 2D gel spots (e.g., references [13][14][15][16][17] or direct LC-MS of lysed cells.…”

Rapid high-throughput characterisation, classification and selection of recombinant mammalian cell line phenotypes using intact cell MALDI-ToF mass spectrometry fingerprinting and PLS-DA modelling

“…Using the CLC workflow described above, a naïve CLC (here after referred to as CLC1) was initially undertaken (see process outlined in Figure 1A) to generate recombinant GS-CHO cell lines expressing the IgG4 monoclonal antibody cB72.3 that is routinely used at Lonza as a model system (Smales et al, 2004;Tait et al, 2012). After the initial transfection procedure and FACS sorting, 119 unique cell lines were taken from the initial transfection stage to the multi-well plate stage whereby cell samples were collected for MALDI-ToF analysis.…”

Toward the complete characterization of host cell proteins in biotherapeutics via affinity depletions, LC-MS/MS, and multivariate analysis

“…[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] In this methodology, proteins are first digested enzymatically. The resulting peptides are then separated (usually by liquid chromatography), and subsequently detected by mass/ charge via a mass spectrometer.…”