Mass spectrometry for structural characterization of therapeutic antibodies

Abstract

Antibodies, also known as immunoglobulins, have emerged as one of the most promising classes of therapeutics in the biopharmaceutical industry. The need for complete characterization of the quality attributes of these molecules requires sophisticated techniques. Mass spectrometry (MS) has become an essential analytical tool for the structural characterization of therapeutic antibodies, due to its superior resolution over other analytical techniques. It has been widely used in virtually all phases of antibody development. Structural features determined by MS include amino acid sequence, disulfide linkages, carbohydrate structure and profile, and many different post-translational, in-process, and in-storage modifications. In this review, we will discuss various MS-based techniques for the structural characterization of monoclonal antibodies. These techniques are categorized as mass determination of intact antibodies, and as middle-up, bottom-up, top-down, and middle-down structural characterizations. Each of these techniques has its advantages and disadvantages in terms of structural resolution, sequence coverage, sample consumption, and effort required for analyses. The role of MS in glycan structural characterization and profiling will also be discussed.

Mass measurement and top-down HPLC/MS analysis of intact monoclonal antibodies on a hybrid linear quadrupole ion trap-orbitrap mass spectrometer

“…A program developed at Amgen, MassAnalyzer, was used to automatically assign the fragment ions [34,35] and to prepare the fragment coverage map [27]. It was also used to calculate the theoretical average masses of the antibodies and their fragments, using atomic weights of elements from organic sources listed in reference [3].…”

“…Using nonreduced peptide mapping of collected fractions, the peaks were previously identified as having the same amino acid sequence and glycosylation, but different disulfide structure at the hinge [37,38]. The measured masses were within 2-4 Da from the theoretical mass of 147,250 Da using atomic weights of elements from organic sources listed in Zhang et al [3].…”

“…This has resulted in a strong need for a highthroughput methods for analysis of different antibody drug candidates. Mass spectrometry has become one of the most powerful techniques for the structural characterization of monoclonal antibodies (mAbs) [3]. Traditionally, structural characterization of mAbs has been performed by a "bottom-up" approach after first digesting them to peptides [4 -6].…”

“…Discovery of the Orbitrap analyzer [26] and its first commercialization as a hybrid linear quadrupole ion trap-Orbitrap mass spectrometer (LTQ Orbitrap, Thermo Scientific, Bremen, Germany) provided many laboratories with the opportunity to achieve high mass resolution without a superconducting magnet and its special and expensive maintenance. The LTQ-Orbitrap was used for top-down analysis of intact monoclonal IgG antibodies [27] and their light and heavy chains [18] during direct infusion as well as on-line with HPLC [3]. In addition, top-down (tandem mass spectroscopy, MS/MS) analysis of antibody chains was performed using a q-TOF mass spectrometer during direct infusion [28], and on-line with HPLC using in-source N-terminal fragmentation on an orthogonal-TOF instrument [29].…”

The impact of trisulfide modification of antibodies on the properties of antibody-drug conjugates manufactured using thiol chemistry

“…Each subclass of IgG molecules has welldefined homogenous disulfide bond structures; however, free cysteine residues, scrambled disulfide bonds and the presence of trisulfide bonds and thioether bonds have also been widely reported. [24][25][26] More recently, trisulfide bonds have been reported in all 4 subclasses of human IgG molecules. [27][28][29][30][31] In a recent study of ADC development using inter-chain cysteinedirected linker chemistry as described above, trisulfide bonds have been reported to affect the reduction step by TCEP during the antibody-drug conjugation process and affect average DAR value.…”