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

Abstract

(2014) Comprehensive tracking of host cell proteins during monoclonal antibody purifications using mass spectrometry, mAbs, 6:3, 659-670,

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

“…10 and mAbs2014. 6 "ProtAdepl" and "ProtLdepl" represent the highest counts of HCPs identified in this report by either Protein A or Protein L depletion, respectively. The methodology presented in this article was able to detect »100 times more HCPs compared to other techniques that analyzed samples with low starting HCP concentrations (< 10 ppm range).…”

“…6 However, by using the analysis workflow described herein, our data shows that even though commercial grade biologics go through extensive multi-step purification processes, a large number of HCPs (albeit low abundant) can still be present in the final drug product. Furthermore, the most abundant host cell proteins only represent a small portion of the total HCP abundance.…”

“…[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.…”

“…[4][5][6]11,14 Since HCPs are several orders of magnitude lower in concentration compared to the protein therapeutic, 2-dimensional LC separation helps increase the sensitivity of HCP detection. HCP quantitation is then achieved typically by spiking in one or more protein standards at known concentrations.…”

“…HCP quantitation is then achieved typically by spiking in one or more protein standards at known concentrations. [4][5][6]11,20 While these methodologies have substantially increased the depth of HCP characterization, often no proteins (or very few) are detected in final, commercial-grade drug products. Another problem is that the protein standards used will likely have different response factors in the mass spectrometer compared to that of host cell impurities, thus confounding quantitation of specific HCP.…”