Abstract
When analyzing large complex protein biopharmaceuticals, ion-pairing agents imparting low pH are widely used as mobile phase additives to improve the chromatographic performance. However, one of the most effective additives in RPLC and HILIC, trifluoroacetic acid (TFA), is known as a strong suppressor of the MS signal and limits its use in hyphenated techniques. In this study, we evaluated a wide range of acidic additives to find alternatives to TFA that provided comparable chromatographic performance and improved MS sensitivity. It was observed that stronger acidic additives were required for intact level analysis compared to subunit level analysis and that the additive nature had a larger impact on the chromatographic performance in HILIC mode compared to RPLC. Therefore, four additives were identified as valuable alternatives to TFA in RPLC mode, namely, difluoroacetic acid (DFA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), and methanesulfonic acid (MSA). Only one of these additives provided acceptable performance in HILIC mode, namely, TClAA.After evaluation of the MS performance, TClAA was discarded due to the apparent loss of intensity in both RPLC-MS and HILIC-MS mode. Together, these results demonstrate that for HILIC-MS analysis TFA remains the gold standard additive. However, DFA was found as promising alternative to TFA for RPLC-MS analysis and could play an important role in the development of methods for the characterization of the increasingly complex protein biopharmaceuticals.
Highlights
Over the past decades, biopharmaceuticals, and among them, monoclonal antibodies, have emerged as an important class of therapeutics [1,2]
With the aim to find alternative mobile phase additives providing suitable chromatographic performance and acceptable MS sensitivity compared to trifluoroacetic acid (TFA), fourteen acidic compounds were evaluated in this study, namely formic acid (FA), acetic acid (AA), MFA, difluoroacetic acid (DFA), chloroacetic acid (ClAA), dichloroacetic acid (DClAA), trichloroacetic acid (TClAA), bromoacetic acid (BrAA), dibromoacetic acid Sigma- 1.48 2.14 (DBrAA), tribromoacetic acid Sigma- 0.72 2.08 (TBrAA), iodoacetic acid (IAA), bromodichloroacetic Sigma- 0.05 acid (BrDClAA), chlorodibromoacetic Sigma- 0.13 acid (ClDBrAA), and methanesulfonic acid (MSA)
ClDBrAA, BrDClAA, and MFA were discarded, since they cost more than 100 USD for less than 1 g of product and were not economically competitive on the basis of the amount of acid required for the preparation of the mobile phases
Summary
Biopharmaceuticals, and among them, monoclonal antibodies (mAbs), have emerged as an important class of therapeutics [1,2]. The large size of the mAbs results in a slow diffusivity and mass transfer of the analytes: severe band broadening may be experienced, as well as peak tailing [16] Together, these phenomena negatively affect the chromatographic performance. By acting as ion-pairing agents, acidic additives can form neutral ion-pairs with the basic groups of the protein, namely histidine, lysine and arginine and thereby enhance the hydrophobic character of the analyte [13] This will lead to better retention and improved peak shapes during analysis. Being a strong acid (pKa 1⁄4 0.3), TFA is a very effective ion-pairing agent that provides symmetrical and narrow peak shapes for proteins analyzed in RPLC and HILIC. We have attempted to find alternatives to TFA that can provide excellent chromatographic performance without neglecting the MS sensitivity For this purpose, fourteen derivatives of acetic acid were considered in addition to MSA. Volatility was assessed for selected additives and the performance was investigated in MS to evaluate their relevance in LC-MS
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