Identification and quantitation of hinge cysteinylation on endogenous IgG2 antibodies from human serum

Figures & data

Figure 1. Three structural isoforms of IgG2 (A, A/B, and B) and their associated disulfide connections depicted with red lines. Modified and used with permission of American Chemical Society, from ref.;¹⁵ permission conveyed through Copyright Clearance Center, Inc

Figure 2. Depictions of doubly cysteinylated and classically linked IgG2-A forms. “Cys” denotes a terminal cysteine molecule that is disulfide bonded to a cysteine on the antibody. Modified and used with permission of American Chemical Society, from ref.;¹⁵ permission conveyed through Copyright Clearance Center, Inc

Figure 3. SIM experiments of mAb1-Y Lys-C peptide maps. (a) Extracted ion chromatograms of doubly cysteinylated and classically linked IgG-A hinge Lys-C dipeptides (10 ppm mass tolerance for extraction). (b) Corresponding MS1 spectra using Orbitrap detection (60 000 resolution). (c) Corresponding MS1 spectra that are zoomed-in on the mass features of interest

Figure 4. PRM experiments of mAb1-Y Lys-C peptide maps. Sequences of (a) doubly cysteinylated and (b) classically linked IgG2-A hinge Lys-C dipeptides annotated with b- and y-ion fragmentation sites. (c) Corresponding annotated PRM spectra using CID fragmentation and Orbitrap detection (30 000 resolution, 10 ppm mass tolerance for assignments). (d) Corresponding extracted ion chromatograms of precursor/b8 product ion transitions (10 ppm mass tolerance for extraction)

Figure 5. Deconvoluted mass spectra of a recombinant IgG cocktail sample (including IgG1, IgG2, and IgG4 antibodies) that has been subjected to Protein G pulldown or anti-IgG2 pulldown. The heterogeneity within each recombinant IgG subclass is predominantly attributable to Fc glycosylation

Figure 6. PRM analysis of doubly cysteinylated IgG2 standards (prepared by co-mixing mAb1-Y and mAb1-Z) showing a linear response

Table 1. PRM repeatability analysis with a 0.3% doubly cysteinylated IgG2-A control sample (prepared by co-mixing mAb1-Y and mAb1-Z). Nine replicates in total were analyzed

Statistic	%2Cys
Mean	0.3%
Standard deviation	0.03%
S/N	10

Figure 7. PRM experiments of endogenous IgG2 Lys-C peptide maps. (a) PRM spectra using CID fragmentation and Orbitrap detection (30 000 resolution, 10 ppm mass tolerance for assignments). (b) Corresponding extracted ion chromatograms of precursor/b8 product ion transitions (10 ppm mass tolerance for extraction). Depicted here is data of endogenous IgG2s affinity purified from a single human subject, as an example (in total 10 human subject samples were analyzed)

Table 2. Relative abundances of doubly cysteinylated IgG2-A form (%2Cys) in endogenous IgG2s purified from healthy human serum. XIC_{doubly cysteinylated} and XIC_{classically linked} are the extracted ion chromatogram peak areas of the precursor/product ion transitions for the doubly cysteinylated and classically linked IgG2-A Lys-C dipeptides, respectively. Demographic information corresponding to each human subject is included. There was no obvious correlation between hinge cysteinylation levels and age, gender or race, granted the sample size was small

Gender	Race	Age	XICclassically linked	XICdoubly cysteinylated	%2Cys
Male	White	52	21214823	96558	0.5
Male	Hispanic	66	15023572	139367	0.9
Male	White	59	9312008	78597	0.8
Male	Black	58	15631974	94021	0.6
Male	White	44	17710870	159266	0.9
Female	White	44	25823084	160364	0.6
Female	Hispanic	63	17255965	204560	1.2
Female	White	63	23764328	142695	0.6
Female	White	58	15456256	72583	0.5
Female	Black	63	17672827	229390	1.3
			Average		0.8
			Standard Deviation		0.3

Table 3. Comparison of endogenous exposure and dosed exposure (hypothetical) to doubly cysteinylated IgG2-A. Included are the mean values for parameters relevant to the exposure calculations

	Endogenous		Dosed therapeutic (hypothetical)
Parameter	Mean value	Ref	Mean value	Ref
IgG serum concentration	11.8 mg/mL	ref.^Citation31	0.1 mg/mL*	n/a
%IgG2	22%	ref.^Citation25	100%	n/a
%IgG2-A (of IgG2)	7.2%	ref.^Citation19	25%	n/a
%2Cys (of IgG2-A)	0.8%	This study	0.5%	n/a
Doubly cysteinylated IgG2-A exposure in serum	1.5 µg/mL		0.125 µg/mL

*Assuming a hypothetical IgG2 therapeutic dose of 250 mg and an average human serum volume of 2.5L.

Table 4. Parent/b8-product ion transitions used for %2Cys quantitation of PRM experiments. The three b8-product ions listed represent the second, third, and fourth isotopes

		Parent ion			b8-product ion
Lys-C IgG2-A hinge dipeptide	Monoisotopic mass	m/z	z	Isolation width	m/z	z	Mass tolerance
Classically linked	5350.5911 Da	1071.9241	+5	3 Da	1169.8431, 1170.1743, 1170.5077	+3	10 ppm
Doubly cysteinylated	5590.6160 Da	1119.9241	+5	3 Da	1249.8461, 1250.1804, 1250.5151	+3	10 ppm

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