Antibody characterization using novel ERLIC-MS/MS-based peptide mapping

Figures & data

Figure 1. Representative gradient 2 and the base peak chromatogram of the denosumab tryptic peptides on ERLIC-MS/MS.

Figure 2. 2D presentation of ERLIC-MS/MS based peptide mapping of denosumab tryptic peptides (gradient 2).

Table 1. The characterization of major variant peptides of denosumab (gradient 2).

Amino Acids		Peptide/Modifications	pI Value^a	R.T (min)	Modification Percentage^b
Terminal Peptides
LC-[1–18]		EIVLTQSPGTLSLSPGER	4.3	49.38	0.16
	E1	N-p-Glu formation	↑	43.50
HC-[1–19]		EVQLLESGGGLVQPGGSLR	4.3	47.37	0.06
	E1	N-p-Glu formation	↑	41.57
HC- [441–448]		SLSLSPGK	10.0	32.95	99.03
	K448	Loss of C-Lys	6.0	28.63
Methionine Oxidation
HC- [77–87]		NTLYLQMNSLR	9.8	31.2	0.14
	M85	M Oxidation	–	35.2
HC- [99–126]		DPGTTVIMSWFDPWGQGTLVTVSSASTK	3.9	45.5	2.53
	M106	M Oxidation	–	47.7
HC- [250–256]		DTLMISR	6.7	40.1	4.30
	M253	M Oxidation	–	44.5
HC-[357–361]		EEMTK	4.3	61.4	4.13
	M359	M Oxidation	–	67.1
HC- [394–410]		TTPPMLDSDGSFFLYSK	3.9	48.6	2.59
	M398	M Oxidation	–	51.8
Asparagine Deamidation
HC-[372–393]		GFYPSDIAVEWESNGQPENNYK	3.7	65.13	96.21
	N385	Asn to isoAsp	↓	71.35	0.54
	N390	Asn to isoAsp	↓	71.12	0.16
	N390	Asn to Asp	↓	70.58	0.77
	N385	Asn to Suc	–	63.08	0.28
	N390	Asn to Suc	–	62.49	2.04
Glycosylation
HC-[294–302]		EEQFNSTFR	4.3	55.20	1.46
	N298	[G0]	↓	70.75	7.76
	N298	[G0F]	↓	70.90	64.59
	N298	[G1F]	↓	71.10	14.63
	N298	[G2F]	↓	71.26	1.00
	N298	[G0F-GlcNAc]	↓	70.66	1.85
	N298	[M5]	↓	70.88	8.70

^aSymbol arrow “↑” means value increases; symbol arrow “↓” means value decreases.

^bRelative percentage is an approximation based on integrated peak area within 10 ppm of theoretical mass.

Figure 3. Representative chromatograms and mass spectra of terminal peptides (gradient 2). (A) The extracted ion chromatograms of N-terminal peptide of heavy chain; (B) The MS2 mass spectra of N-terminal peptide of heavy chain.

Figure 4. Representative chromatograms and mass spectra of methionine-containing peptides H17 and its oxidized form (gradient 2).

Figure 5. (A) The separation of non-deamidated and deamidated peptides of “GFYPSDIAVEWESNGQPENNYK” in gradient 1. (B) The MS2 fragment ion mass spectra analysis of labelled peaks.

Figure 6. The extracted ion chromatograms of major glycopeptides in monoclonal antibody denosumab (gradient 2).