Systematic Strategies for Developing Peptide Maps

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Ziling Lu;Beth L. Gillece-Castro;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
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Biopharmaceuticals are often characterized by using peptide maps to detect important structural features that can be related to biological properties. The same mapping methods are ultimately used for monitoring the safety and efficacy of the protein during process development, production, and so on. The peptide maps must, therefore, be developed so that the entire protein sequence is covered. The map must also accommodate all the important structural variants of the protein. Development of such a map is a complicated and time-consuming process. Because there are so many experimental variables that affect retention of peptides, a systematic method development strategy is required. The process can be divided into column selection and operating procedures. In this study, we evaluate the effects of column chemistry, particle size, pore size, and column dimension on peptide chromatography. Pairs of columns were compared to evaluate the effect of each variable independently. The effect of each parameter was monitored by identifying specific peptides in complex digests, using LC-MS. This description of the effects of column properties on peptide resolution can be used to narrow the choices for column selection as the first step in systematically developing a peptide map for use in biopharmaceutical analysis.

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