Table 3.

Light chain peptides identified with possible serine to asparagine substitution in RAZUMAB batches. Variant peptides of RAZUMAB light chain are shown along with the possible position of the serine to asparagine (Ser → Asn) substitution. The LC peptide number after LysC cleavage (#), the corresponding amino acid range, and the peptide sequence are shown for each entry. The possible positions of the Ser → Asn substitution are indicated in red italic and in red bold underlined when position is confirmed. Additional peptide properties are included such as retention time (RT), mass-to-charge ratio (m/z), charge, observed mass (Da), and mass delta calculated as the difference between the observed and expected precursor masses divided by the expected precursor mass multiplied by 10⁶ (ppm). The percentage of Ser → Asn misincorporation is determined for each variant peptide as the volume of the variant peptide cluster divided by the sum of the volumes of the variant and its corresponding native peptide m/z. Results for 2 independent experiments are shown in which batch 1 was analyzed altogether with batch 2 or batch 5. Batches 2 and 5 were analyzed only in separate experiments. Values for batch 1 are reported for both experiments to assess inter-experiment reproducibility. Coefficient of variation (CV) is calculated for each ion based on the estimates obtained for batch 1 in these 2 independent experiments. *potential overestimation due to the presence of substantial sodium adduct. ‡ Signature ions for partial Ser → Asn substitution were detected for each of the 4 serine residues of peptide L-L9, indicating that the variant ion at 1081.49 m/z is a mixture of several isobaric peptides with partial misintegration of Asn for Ser at all 4 sites as observed in the chimeric MS/MS spectrum.

									Misincorporation (%)
									Experiment 1		Experiment 2
#	Range	Sequence	Ser→Asn [position]	RT [min]	m/z [Th]	Charge	Mass [Da]	Mass Delta [ppm]	batch 1	batch 2	batch 1	batch 5	CV batch 1 (%)
L-L1	1–39	DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQK	30	80.1	1463.38	3	4387.12	3.0	1.5	2.0	1.2	1.7	15
L-L1	1–39	DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQK	14	81.1	1463.37	3	4387.09	−2.9	1.5*	2.1*	2.4*	3.2*	33
L-L4	46–103	VLYIFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTK	76 or 77	93.5	1608.51	4	6430.03	−3.5	5.0	6.6	5.4	7.2	6
L-L6	108–126	RTVAAPSVFIFPPSDEQLK	114	70.8	1065.08	2	2128.14	3.3	0.4	0.5	0.4	0.5	10
L-L7	127–145	SGTASVVCLLNNFYPREAK	131	78.3	1077.05	2	2152.08	1.2	0.5	0.7	0.6	0.7	12
L-L9	150–169	VDNALQSGNSQESVTEQDSK	156, 159, 162, and 168‡	30.8	1081.49	2	2160.96	−6.8	2.2	3.1	1.9	1.9	11
L-L10	170–183	DSTYSLSSTLTLSK	176	50.9	765.39	2	1528.76	0.7	0.6	0.7	0.5	0.7	7
L-L10	170–183	DSTYSLSSTLTLSK	177	52.8	765.39	2	1528.76	−1.1	2.9	4.0	2.6	3.5	6
L-L13	191–207	VYACEVTHQGLSSPVTK	202 or 203	42.6	951.97	2	1901.93	−0.7	0.7	0.9	0.6	0.7	18