High sensitivity LC-MS profiling of antibody-drug conjugates with difluoroacetic acid ion pairing

Abstract

Reversed-phase liquid chromatography (RPLC) separations of proteins using optical detection generally use trifluoroacetic acid (TFA) because it is a strong, hydrophobic acid and a very effective ion-pairing agent for minimizing chromatographic secondary interactions. Conversely and in order to avoid ion suppression, analyses entailing mass spectrometry (MS) detection is often performed with a weaker ion-pairing modifier, like formic acid (FA), but resolution quality may be reduced. To gain both the chromatographic advantages of TFA and the enhanced MS sensitivity of FA, we explored the use of an alternative acid, difluoroacetic acid (DFA). This acid modifier is less acidic and less hydrophobic than TFA and is believed to advantageously affect the surface tension of electrospray droplets. Thus, it is possible to increase MS sensitivity threefold by replacing TFA with DFA. Moreover, we have observed DFA ion pairing to concomitantly produce higher chromatographic resolution than FA and even TFA. For this reason, we prepared and used MS-quality DFA in place of FA and TFA in separations involving IdeS digested, reduced NIST mAb and a proprietary antibody-drug conjugate (ADC), aiming to increase sensitivity, resolution and protein recovery. The resulting method using DFA was qualified and applied to two other ADCs and gave heightened sensitivity, resolution and protein recovery versus analyses using TFA. This new method, based on a purified, trace metal free DFA, can potentially become a state-of-the-art liquid chromatography-MS technique for the deep characterization of ADCs.

Quantitative Measurement of Melittin in Asian Honeybee Venom Using a New Method Including UPLC-QqTOF-MS

“…In the context of our study, we considered it necessary to adjust the UPLC conditions for analyzing the melittin in bee venom, especially in terms of the choice of additives for the mobile phase, because additives can aid in increasing separation [42], in addition to improving the response of high-level buffers. Common mobile phase additives include formic acid, trifluoroacetic acid (TFA), ammonium formate, and ammonium hydroxide, where formic acid and TFA are commonly used to enhance the ionization efficiency of peptides and proteins in the LC separation [43][44][45].…”

Effect of difluoroacetic acid and biological matrices on the development of a liquid chromatography–triple quadrupole mass spectrometry method for determination of intact growth factor proteins

“…Nevertheless, the use of TFA in LC–MS systems is discouraged due to its suppressive effect on ESI efficiency [44]. To potentially achieve good LC separations without compromising MS sensitivity, DFA was investigated as an alternate volatile ion‐pairing additive [21]. To the best of our knowledge, the effectiveness of DFA as an ion‐pairing reagent for LC–MS analysis of intact proteins has not been investigated in detail and reported in the literature.…”

“…Recently, difluoroacetic acid (DFA) was promoted as a promising alternative acidic mobile phase modifier for the separation of drugs, peptides, and antibody–drug conjugates [20,21]. DFA effectively lowers pH to suppress deleterious silanol interactions; it is also a strong ion‐pairing agent suitable to improve RP‐LC separations, but it does not decrease MS sensitivity as much as TFA.…”

“…DFA effectively lowers pH to suppress deleterious silanol interactions; it is also a strong ion‐pairing agent suitable to improve RP‐LC separations, but it does not decrease MS sensitivity as much as TFA. Several studies have evaluated the effect of mobile phase additives, with a focus on TFA, for the analysis of peptides and proteins [22–25], but only one recent study dealt specifically with the use of DFA for improving LC–MS characterization of antibody–drug conjugates [21]. The authors have shown that it was possible to increase MS sensitivity two to three times and to improve the recovery as well as chromatographic resolution of subunit‐digested monoclonal antibody by replacing TFA with DFA as a mobile phase modifier.…”

Sample preparation and fractionation techniques for intact proteins for mass spectrometric analysis

“…While significant research efforts were focused on intact protein separations from the 1970s through the 1990s [112–118], there has not been an emphasis on interfacing with MS detection (LC–MS) until more recently. Indeed, the realization of more effective volatile ion pair reagents, such as difluoroacetic acid, for efficient and sensitive RP‐LC–MS analysis of intact proteins are only just now being explored [27,119].…”