Amgen, UCLA, and University of Arizona Use BioResolve RP mAb Polyphenyl Columns for Rapid LC-MS Screening of Membrane Proteins

As a chromatography scientist at Waters, I often get the opportunity to travel to different laboratories around the world and to meet with various types of researchers. This allows me to stay up to date on the newest scientific trends and topics, particularly in the fast growing world of biopharmaceutical analysis. In June of this year, I visited Amgen Inc. at their campus in Thousand Oaks, California. There, I met with scientists in the Amgen Discovery Research group who were working on the development of platform methods for membrane protein screening and were in the process of publishing their findings in Analytical Chemistry. Surprisingly, when the author, Jennifer Lippens, presented her research to us, I saw that the column used in her platform LC-MS method was the BioResolve™ RP mAb Polyphenyl Column, which was a column Waters released last year and a technology that I worked extensively to develop.

Reprinted (adapted) with permission from Lippens JL, Egea PF, Spahr C, Vaish A, Keener JE, Marty MT, et al. Rapid LC-MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins. Anal Chem 2018.  Copyright(2018) American Chemical Society.

This paper, “Rapid LC−MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins”, can be found in the Journal of Analytical Chemistry online. It discusses the need for a high-throughput, platform LC-MS method for fast and accurate molecular weight determination of membrane proteins, which are thought to be involved in diseases such as cancer and thus may be exploitable for  therapeutic treatments. The method development took many factors into consideration, where one especially important element was column selection.

When I spoke to the author, she told me that they had investigated many different columns (C3, C4, C8, and cyano) but were dissatisfied with the results as the chemistries did not perform well with very hydrophobic membrane proteins since the proteins would irreversibly ‘stick’ to the stationary phase. This was especially prevalent with the C8 chemistries explored, as the bonding is too retentive for a high-throughput method. Then, with one last attempt to use a recently introduced, novel column technology  – the BioResolve RP mAb Polyphenyl Column – she happened upon a breakthrough. Not only does the particle design allow the use of UPLC pressures and higher throughput, this polyphenyl bonding is theorized to work well for her method because of its high coverage, structurally rigid bonding that minimizes the heterogeneity of protein adsorption. Moreover, the unique bonding allowed the use of both 0.1% formic acid (FA) and 0.1% trifluoroacetic acid (TFA) modified mobile phases, lower temperatures (of 65 °C), and the chance to maximize chromatographic resolution, enhance MS sensitivity, and minimize carryover.

As shown in Figure 2 from the journal article (below), the polyphenyl column was the only column able to separate the formylated and non-formylated aquaporinZ membrane protein. Using this column, the method gave highly reproducible results. Not only was this method tested for membrane proteins, a bispecific antibody construct was also tested. Evaluation of this bispecific antibody construct, which links the targeting regions of two antibodies, shows that this rapid LC-MS method for screening membrane proteins may also serve for the analysis of other hydrophobic protein species.

Reprinted (adapted) with permission from Lippens JL, Egea PF, Spahr C, Vaish A, Keener JE, Marty MT, et al. Rapid LC-MS Method for Accurate Molecular Weight Determination of Membrane and Hydrophobic Proteins. Anal Chem 2018.  Copyright(2018) American Chemical Society.

On the whole, it was exciting to see the potential that the Waters BioResolve RP mAb Polyphenyl Column demonstrated through this new LC-MS method for membrane proteins. Its amenability to milder elution conditions and higher pressures brings new options to scientists.  Waters is currently continuing to work on improving elution conditions with the release of a new mobile phase additive based on a purified, LC-MS quality difluoroacetic acid (IonHance DFA). This additive works to optimize a method to have the enhanced MS sensitivity of FA and high chromatographic resolution of TFA. It will be interesting to see if methods such as those described in this article can be improved even further with the use of LC-MS quality DFA.