Multiplexed Middle-Down Mass Spectrometry as a Method for Revealing Light and Heavy Chain Connectivity in a Monoclonal Antibody

Abstract

Pairing light and heavy chains in monoclonal antibodies (mAbs) using top-down (TD) or middle-down (MD) mass spectrometry (MS) may complement the sequence information on single chains provided by highthroughput genomic sequencing and bottom-up proteomics, favoring the rational selection of drug candidates. The 50 kDa F(ab) subunits of mAbs are the smallest structural units that contain the required information on chain pairing. These subunits can be enzymatically produced from whole mAbs and interrogated in their intact form by TD/MD MS approaches. However, the high structural complexity of F(ab) subunits requires increased sensitivity of the modern TD/MD MS for a comprehensive structural analysis. To address this and similar challenges, we developed and applied a multiplexed TD/MD MS workflow based on spectral averaging of tandem mass spectra (MS/MS) across multiple liquid chromatography (LC)−MS/MS runs acquired in reduced or full profile mode using an Orbitrap Fourier transform mass spectrometer (FTMS). We first benchmark the workflow using myoglobin as a reference protein, and then validate it for the analysis of the 50 kDa F(ab) subunit of a therapeutic mAb, trastuzumab. Obtained results confirm the envisioned benefits in terms of increased signal-to-noise ratio of product ions from utilizing multiple LC−MS/MS runs for TD/MD protein analysis using mass spectral averaging. The workflow performance is compared with the earlier introduced multiplexed TD/MD MS workflow based on transient averaging in Orbitrap FTMS. For the latter, we also report on enabling absorption mode FT processing and demonstrate its comparable performance to the enhanced FT (eFT) spectral representation.

Direct measurement of light and heavy antibody chains using ion mobility and middle-down mass spectrometry

“…1 Bottom-up approaches digest the mAb into peptides before analysis, 6 top-down MS methods analyze intact molecules, 7-10 and middle-down procedures are performed by measuring the mass and subsequent fragmenting of large pieces or subunits from mAbs (typically 25-50 kDa) that are more suitable for state-of-the-art liquid chromatography tandem MS (LC-MS/MS) methods and techniques. [11][12][13][14][15][16] The subunits or parts of the polypeptide chain can be obtained through the chemical reduction of disulfide bonds, yielding free Lc and Hc, 17 or by using a specific enzymatic proteolysis (i.e., digestion with IdeS or IdeZ) that usually generates F(ab') 2 (∼100 kDa) and Fc (∼50 kDa) pieces. 18 The S-S bonds in these pieces can be further reduced, resulting in three ∼25 kDa subunits: one Lc and two portions of Hc named Fc/2 and Fd.…”

Middle-down approach: a choice to sequence and characterize proteins/proteomes by mass spectrometry

“…GingisKHAN™ 278 Upper hinge region of human immunoglobulin G1 S. no. Chemicals Site specicity of cleavage…”

“…138 In a very recently published study, Tsybin and co-workers have reported about multiplexed TD/MD MS workow, which combines both TD and MD approaches, particularly for characterization of monoclonal antibodies. 278…”

Characterization of therapeutic proteins by cation exchange chromatography-mass spectrometry and top-down analysis

“…7,10,23,29,35 With a few exceptions, 45 RP separation of protein charge variants with one modification is typically relatively poor, 46,47 but RP LC-MS is sensitive and easily amenable to top-down analysis. 43,[48][49][50][51] This is because proteins elute in the RP mobile phase in an unfolded and highly charged state, 52 which enables ionization prior to entering the mass analyzer. 53,54 RP LC-MS has therefore been widely used for the characterization of therapeutic proteins and antibodies.…”