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

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

James A Madsen et al. MAbs. 2015.

Abstract

Host cell protein (HCP) impurities are generated by the host organism during the production of therapeutic recombinant proteins, and are difficult to remove completely. Though commonly present in small quantities, if levels are not controlled, HCPs can potentially reduce drug efficacy and cause adverse patient reactions. A high resolution approach for thorough HCP characterization of therapeutic monoclonal antibodies is presented herein. In this method, antibody samples are first depleted via affinity enrichment (e.g., Protein A, Protein L) using milligram quantities of material. The HCP-containing flow-through is then enzymatically digested, analyzed using nano-UPLC-MS/MS, and proteins are identified through database searching. Nearly 700 HCPs were identified from samples with very low total HCP levels (< 1 ppm to ∼ 10 ppm) using this method. Quantitation of individual HCPs was performed using normalized spectral counting as the number of peptide spectrum matches (PSMs) per protein is proportional to protein abundance. Multivariate analysis tools were utilized to assess similarities between HCP profiles by: 1) quantifying overlaps between HCP identities; and 2) comparing correlations between individual protein abundances as calculated by spectral counts. Clustering analysis using these measures of dissimilarity between HCP profiles enabled high resolution differentiation of commercial grade monoclonal antibody samples generated from different cell lines, cell culture, and purification processes.

Keywords: affinity; host cell proteins; mass spectrometry; monoclonal antibody; proteomics.

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Figures

Figure 1.

Figure 1.

Schematic diagram of the experimental workflow.

Figure 2.

Figure 2.

Counts of HCP identities versus total HCP content (ppm measurement by ELISA) as reported herein, as well as in previous LC-MS-based HCP studies. The previous works are labeled as follows: AnalBiochem2012, mAbs2012, RapCommMS2014, BiotechProg2013, PLoSOne2013, and mAbs2014. “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).

Figure 3.

Figure 3.

CHO cell lysate before and after Protein A depletion: (a) overlap of identified CHO proteins (b) correlation between abundances of individual HCPs detected in both samples.

Figure 4.

Figure 4.

Jaccard distances and log base 10 of statistical significances of Spearman correlations between all samples analyzed in this study. Pairwise comparisons that have darker shades of purple have more HCP overlap (as seen in the Jaccard distance heat map) and are more significantly correlated with respect to individual protein abundances (as seen in the log10Pr(≥ρ) heat map); those that are more peach-colored, have less overlap and are less correlated, respectively.

Figure 5.

Figure 5.

Illustration of how method resolution increases with increasing number of HCPs detected (using Spearman correlation).

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