Improving Mass Spectral Quality of Monoclonal Antibody Middle-Up LC-MS Analysis by Shifting the Protein Charge State Distribution

Abstract

Monoclonal antibodies (mAbs) are experiencing accelerated development in the pharmaceutical industry. Utilization of middle-up LC-MS methodology can provide detailed characterization of mAbs via reduction and/or enzymatic cleavage of the mAb into smaller protein fragments. However, under typical LC-MS conditions, these fragments, especially the more heterogeneous heavy chain, can present charge state distributions (CSD) featuring a severe interference in the low mass-to-charge (m/z) region in the mass spectrum, adversely impacting spectral quality of these proteins and ultimately the deconvoluted mass spectrum. Here, we introduce a novel method to characterize protein fragments by partially reducing mAbs and using acidic mobile phases (MPs) with a trace amount of base additive. Gas-phase charge stripping occurs with the basic MP additive, causing the CSD to shift to a higher m/z region resulting in high-quality mass spectra with enhanced resolution of protein charge states. Subsequently, high-quality deconvoluted spectra and accurate mass measurement of the fragments are achieved. This method has been applied to the intact mass measurement of mAbs and antibody drug conjugates.

Simple Approach for Improved LC–MS Analysis of Protein Biopharmaceuticals via Modification of Desolvation Gas

“…In a recent study, Ding et al have found the intact mass analysis of reduced monoclonal antibodies can be greatly improved by adding a trace amount of base additive into mobile phases. 16 To evaluate if similar improvements can be achieved via desolvation gas modification, reduced NISTmAb was used as a test article. Triethylamine (TEA), a frequently used charge reducing reagent, was diluted to 1% (v/v) using acetonitrile (ACN) and used to deliver the base vapor into the desolvation gas.…”

“…By shifting the charge state envelope toward a high m / z region, the MS spectrum can be greatly simplified due to the increased spacing between two adjacent charge states. In a recent study, Ding et al have found the intact mass analysis of reduced monoclonal antibodies can be greatly improved by adding a trace amount of base additive into mobile phases . To evaluate if similar improvements can be achieved via desolvation gas modification, reduced NISTmAb was used as a test article.…”

“…We also demonstrated the utility of this approach by successfully deciphering a highly heterogeneous protein molecule that cannot be resolved by the standard method. Again, unlike many previous reports where charge reduction was achieved by either modifying the mobile phases or postcolumn addition of the weak bases, , the new device enables the charge reduction via the desolvation gas, thus eliminating concerns over chromatographic performance as well as retaining the practicality for routine analysis.…”

“…We have also demonstrated that the same device can be utilized to modify the desolvation gas with weak base, in order to improve the LC–MS based intact mass analysis of protein biopharmaceuticals via charge reduction. Charge reduction strategies have already been widely exploited in many intact mass applications by either simplifying the MS spectra of complex protein samples or by better preserving noncovalent interactions during native MS experiments. − During evaluation of the developed device, we found that charge reduction strategies can also be utilized to stabilize highly charged protein analytes during the MS analysis and prevent them from undesired fragmentation and decay, thus leading to overall improved spectral quality. We also demonstrated the utility of this approach by successfully deciphering a highly heterogeneous protein molecule that cannot be resolved by the standard method.…”

Unambiguous Sequence Characterization of a Monoclonal Antibody in a Single Analysis Using a Nonspecific Immobilized Enzyme Reactor

“…In the year 2017 alone, ten antibodies were approved by the FDA as a Novel Drug . These mAbs represent a treatment for a wide variety of targets, such as autoimmune diseases to cancers. − The advent of this therapeutic modality has driven the need to fully characterize mAbs. − However, mAbs prove much more challenging to characterize than small-molecule drugs.…”