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.

Keywords: ADC; DAR; DFA; Difluoroacetic acid; FA; IdeS digestion; LC-MS; MS sensitivity; NIST mAb; TFA; antibody-drug conjugate; disulfide isoforms; drug-to-antibody ratio; formic acid; mAb; metal adducts; monoclonal antibody; peak capacity; potassium; protein recovery; reversed-phase chromatography; salt adducts; sodium; subunit profiling; trifluoroacetic acid.

Figure 1.

(a) The influence of sodium and potassium content on mass spectral quality as depicted by an overlay of the deconvoluted mass spectra of the NIST mAb light chain subunit obtained using as-received DFA and distilled DFA and (b) ICP-MS quantitation for as-received (reagent-grade) versus distilled DFA. Separations were performed with a high coverage phenyl-bonded superficially porous silica 450 Å, 2.7 μm, 2.1 × 50 mm column using a flow rate of 0.2 mL/min, column temperature of 80°C, and 0.25 μg mass loads.

Figure 2.

(a) UV chromatograms of NIST mAb subunits obtained using 0.1% FA, DFA, or TFA-modified mobile phases, and (b) total ion current chromatograms (TICs) of NIST mAb subunits obtained using 0.1% FA, DFA, or TFA-modified mobile phases. Separations were performed with a high coverage phenyl-bonded superficially porous silica 450 Å, 2.7 μm, 2.1 × 50 mm column using a flow rate of 0.2 mL/min, column temperature of 80°C, and 0.25 μg mass loads.

Figure 3.

(a) Representation of the different possible drug load distributions of cysteine-conjugated ADCs.¹⁸ Subunits from a cysteine-linked auristatin-conjugated antibody as separated with (b) a C4-bonded organosilica 300 Å fully porous stationary phase, 0.6 mL/min flow rate, 80°C temperature, 0.1% TFA-modified mobile phases, and 90:10 acetonitrile/IPA eluent versus (c) a method consisting of a phenyl bonded 2.7 µm superficially porous 450 Å stationary phase, 0.6 mL/min flow rate, 70°C temperature, and 0.15% DFA-modified mobile phases.

Figure 4.

MS peak intensities of oxidized and extended C-terminal Fc/2 subunits from a cysteine-linked auristatin conjugated as determined by the (a) method consisting of a C4-bonded organosilica 300 Å fully porous stationary phase, 0.6 mL/min flow rate, 80°C temperature, 0.1% TFA-modified mobile phases, and 90:10 acetonitrile/IPA eluent versus (b) the new method consisting of a phenyl bonded 2.7 µm superficially porous 450 Å stationary phase, 0.6 mL/min flow rate, 70°C temperature, and 0.15% DFA-modified mobile phases.

Figure 5.

Qualification of the proposed method using 0.15% DFA-modified mobile phases with different cysteine-linked ADCs. (a) Subunits from a cysteine-linked auristatin conjugated antibody previously used in the proposed method. (b) Subunits from a commercially available cysteine linked, dansyl-cadaverine-SMCC conjugated ADC mimic. (c) Subunits from a second cysteine-linked ADC, separated using higher temperature. Separations were performed with a 2.1 × 150 mm column packed with a high coverage phenyl bonded 2.7 µm superficially porous 450 Å stationary phase using a flow rate of 0.6 mL/min, column temperature of 70°C or 80°C, and 1 μg mass loads.