Towards middle-up analysis of polyclonal antibodies: subclass-specific N-glycosylation profiling of murine immunoglobulin G (IgG) by means of HPLC-MS

Abstract

In recent years, advanced HPLC-MS strategies based on intact protein (“top-down”) or protein subunit (“middle-up/middle-down”) analysis have been implemented for the characterization of therapeutic monoclonal antibodies. Here, we assess feasibility of middle-up/middle-down analysis for polyclonal IgGs exhibiting extensive sequence variability. Specifically, we addressed IgGs from mouse, representing an important model system in immunological investigations. To obtain Fc/2 portions as conserved subunits of IgGs, we made use of the bacterial protease SpeB. For this purpose, we initially determined SpeB cleavage sites in murine IgGs. The resulting Fc/2 portions characteristic of different subclasses were subsequently analysed by ion-pair reversed-phase HPLC hyphenated to high-resolution mass spectrometry. This enabled simultaneous relative quantification of IgG subclasses and their N-glycosylation variants, both of which influence IgG effector functions. To assess method capabilities in an immunological context, we applied the analytical workflow to polyclonal antibodies obtained from BALB/c mice immunized with the grass pollen allergen Phl p 6. The study revealed a shift in IgG subclasses and Fc-glycosylation patterns in total and antigen-specific IgGs from different mouse cohorts, respectively. Eventually, Fc/2 characterization may reveal other protein modifications including oxidation, amino acid exchanges, and C-terminal lysine, and may thus be implemented for quality control of functional antibodies.

Inline electrochemical reduction of NISTmAb for middle-up subunit liquid chromatography-mass spectrometry analysis

“…Localization of PTMs to an individual subunit is also beneficial for the assessment of critical quality attributes. [8][9][10] Subunit production is often carried out by non-biological chemical reaction and/or enzyme digestion. 10,11 Consistent subunit production is essential in middle-up and middledown mass spectrometry workflows requiring effective reduction steps.…”

Proteolytic Profiling of Streptococcal Pyrogenic Exotoxin B (SpeB) by Complementary HPLC-MS Approaches

“…From an analytical point of view, SpeBm efficiently generates Fc/4 and Fc/2 subunits that are readily separated by reversed-phase HPLC and may be examined at high resolution due to their low mass of approximately 12.5 and 25 kDa, respectively. This may also find application in the analysis of biopharmaceuticals, e.g., monoclonal antibodies and polyclonal IgGs as a so-called middle-up/down protease, which aids the characterization of these large biomolecules [ 24 , 36 , 57 ].…”

“…As previously described [ 24 ], polyclonal IgGs were purified using protein G from plasma samples that were obtained as stated in the above paragraphs. For SpeB proteolysis, polyclonal IgGs were diluted in 150 mmol·L −1 ammonium acetate (pH 6.9) to a concentration of 1.0 mg·mL −1 .…”

“…Recombinant SpeBz, SpeBm, and digested human IgG subclasses were analyzed on a Thermo Scientific™ QExactive™ benchtop quadrupole-Orbitrap ® mass spectrometer equipped with an Ion Max™ source with a heated electrospray ionization (HESI) probe (Thermo Fisher Scientific, Bremen, Germany), as well as an MXT715-000—MX Series II Switching Valve (IDEX Health & Science, LLC, Oak Harbor, WA, USA). Mass spectrometric data of IgG subunits were acquired with instrument parameters described earlier [ 24 ]. For SpeBz and SpeBm data acquisition, some parameters were adjusted; the resolution was set to 17,500 at m / z 200 in a mass range of m / z 800 to 3000, and the source heater temperature was lowered to 120 °C.…”

“…Although several studies reported the cleavage of human IgG1 at a specific site in the hinge region [ 17 , 18 , 19 ] and some proposed implications for host immune modulation [ 20 , 21 ], a recent publication states that in vivo human IgG may not be a target of SpeB [ 22 ]. Notably, under reducing conditions, SpeB has been used to generate Fc/2 subunits (C H 2 and C H 3 antibody domains) and Fd subunits (V H and C H 1 domains) from monoclonal human IgG1 antibodies [ 23 ], as well as from polyclonal murine IgGs [ 24 ].…”