Cation exchange versus multimodal cation exchange resins for antibody capture from CHO supernatants: Identification of contaminating Host Cell Proteins by mass spectrometry

Purification process of recombinant monoclonal antibodies with mixed mode chromatography

“…Aggregation of antibodies at an acid pH is known to occur during purification and must be limited in order to avoid any undesirable immunological responses [25]. We consider that there are three ways to overcome this problem: (i) buffering of the elution fraction with 1 M Tris-HCl pH 8.5 to raise the pH [26], (ii) addition of arginine to the elution buffer [27], (iii) the use of sodium citrate as an alternative elution buffer: [28]. These three possibilities were tested in the column.…”

“…Conditions for binding and elution were tested in microplates as described previously [23,28]. Optimal conditions were found to be 50 mM sodium citrate buffer pH 4 (6 mS/cm) for equilibration and 200 mM sodium phosphate buffer at pH 7.5 and 21 mS/cm for elution.…”

Quantitative definition and monitoring of the host cell protein proteome using iTRAQ – a study of an industrial mAb producing CHO‐S cell line

“…For example, Pezzini et al demonstrated how conditions for ‘optimal’ mixed mode chromatography purification of mAbs from CHO cell culture harvest material can be determined by utilizing Design of Experiment modeling approaches combined with mass spectrometry analysis to identify those HCPs co‐purifying with the target mAb. The differences in selectivity and efficiency of classical versus multimodal cation exchange chromatography for mAb purification with respect to those HCPs retained in the mAb fraction have also been demonstrated by mass spectrometry . A comparison of the HCP profile of three null CHO cell lines using ELISA, 2D‐PAGE and LC‐MS/MS approaches indicated that the HCPs in different feedstocks for downstream processing were not as diverse as might have been expected .…”

“…However, there are limitations to the ELISA approach, therefore the use of orthogonal methods to support process development and validation is recommended . The major orthogonal approaches that have emerged to monitor/measure HCPs are 2D‐PAGE , particularly 2D‐DIGE , and mass spectrometry . These approaches give information on the specific nature/identification of those HCPs present during up‐stream and downstream processing, allowing clearance and risk to be assessed on the nature of those HCPs present, rather than on the combined concentration of HCPs .…”

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.…”