Purity Analysis and Identity Confirmation of Proteins Using Optimized Reversed Phase Chromatography LC / Exact Mass Tof-MS Systems

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Beth L. Gillece-Castro;Ziling Lu;Thomas E. Wheat;and Jeffrey R. Mazzeo [Waters]
Second Symposium on the Practical Applications of Mass Spectrometry in the Biotechnology and Pharmaceutical Industries, Practical MS
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Practical MS
RNase A, cytochrome c, transferrin, hemoglobin, and enolase
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Since chemical degradation or alteration of proteins may affect the biological properties and any chemical modification will change the molecular weight of the protein, it is attractive to consider using measurement of that property of the protein to determine the degree of modification. Determination of molecular weight using mass spectrometry techniques is a fast and accurate tool for the characterization of recombinant proteins. The success of this analytical approach is contingent high signal-to-noise spectra that can be interpreted without prior knowledge of the sample composition. The measurement is, therefore, facilitated by first separating the several protein species that may be present in the sample so that they may be individually identified and quantitated. Reverse phase chromatography provides an aqueous environment for protein separations based primarily on the hydrophobicity of the surface of the protein at the pH of the mobile phase. Acidic pH values have the effect of denaturing the proteins so that the effective surface interacting with the hydrophobic stationary phase includes all of the amino acid side chains. Under these conditions, we have studied the behavior of proteins on non-porous and fully porous particles. The base particles were silica, polymeric, and bridged-ethyl hybrid chemistries. The hydrophobic ligands C4, C8, C18, and Phenyl have each been evaluated for their retention and resolution capabilities with protein mixtures. The optimized LC analyses were combined with automated high resolution MS for the determination of molecular weights. The selected examples of analysis include the changes likely to be encountered in stability testing, such as, oxidation and deamidation. The approach is applied to the separation of IgG light chains from heavy chains and the measurement of the glycoform distribution of IgG and other proteins.

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