Characterization of monoclonal antibody size variants containing extra light chains

Figures & data

Figure 1. Overlaid SEC UV traces of an IgG1 mAb and three lots of MAb-A materials shown in expanded scale to enable visualization of small peaks. IgG1 mAb sample has a typical SEC profile, while MAb-A contains an unusual peak, labeled Peak 1.

Figure 2. Overlaid SEC UV trace (____) with MALS calculated molar mass (…….) of MAb-A reference material.

Figure 3. Overlaid chromatograms of MAb-A reference material and a purified SEC Peak 1 fraction shown in full scale and expanded scale in insert.

Figure 4. ESI-TOF Deconvoluted masses and theoretical masses for A) MAb-A Lot A SEC Peak 1 fraction and B) MAb-A Lot B SEC Peak 1 fraction.

Table 1. Comparison of theoretical masses with ESI-TOF MS observed masses

Composition	Theoretical mass (Da)	Observed masses in Lot A Peak 1 (Da)	Observed masses in Lot B Peak 1 (Da)
Intact MAb-A 2H2L	148809
2H3L+Cys	172755	172761 (major)	172758
2H3L+GSH	172941	172942	172941 (major)
2H3L+GSH^+Hexose	173103		173105
2H4L	196461	196464	196461

L (S-S linked): 23826 Da, Cys: 121 Da, GSH: 307 Da. The intact mass of 148809 Da is the MAb-A containing G0F glycan (asialo-, agalacto-, biantennary, core-substituted with fucose).

Figure 5. Electropherograms of non-reduced (A) and reduced (B) MAb-A reference material (Ref) and SEC Peak 1 fraction obtained using Agilent 2100 Bioanalyzer with Protein 250 Kit. NG: non-glycosylated heavy chain.

Figure 6. SDS PAGE Sypro Ruby stained gel image of non-reduced and reduced MAb-A samples, SEC monomer and Peak 1 fractions.

Figure 7. Overlaid total ion chromatograms (TIC) of Lys-C peptide maps of non-reduced MAb-A Lot B, non-reduced Lot B Peak 1, and reduced Lot B Peak 1. H14(Ox.M252)-S-S-H18 is the disulfide linked heavy chain peptide containing oxidized methionine 252 (Ox. M252). H24-S-S-H28(Ox.M428) is the disulfide linked heavy chain peptide containing oxidized methionine 428 (Ox. M428). L6(clipped)-S-S-L12 is the disulfide linked light chain peptide containing a non-specific clip.

Table 2. Comparison of peptides’ EIC peak areas of Lys-C digested non-reduced MAb-A Lot B and Lot B Peak 1 Fraction

Peptide^a	Normalized^b peak area
	MAb-A Lot B	Lot B Peak 1	Peak 1/ MAb-A
H25	1.00	1.00	1.0
H6	1.61	1.60	1.0
H26	1.83	1.81	1.0
L5	1.70	2.40	1.4
L8	0.90	1.20	1.3
L9	1.22	1.80	1.5
H7-S-S-H8	1.28	1.31	1.0
H13-S-S-H13 (inter-chain)	0.15	0.14	0.9
H14-S-S-H18	1.01	0.78	1.0 ^c
H14(ox.M252)-S-S-H18	0.02	0.22
H24-S-S-H28	0.50	0.41
H24-S-S-H28(ox.M428)	0.01	0.11	1.0 ^c
L13-S-S-H12 (inter-chain)	0.58	0.57	1.0
L6-S-S-L12	0.91	1.01
L6(clipped)-S-S-L12	0.00	0.33	1.5^c

^a H, heavy chain, L, light chain, -S-S-, disulfide linked peptides. ^bNormalized by dividing the peak area of peptide H25 in the same sample. ^cThe ratio of sums of expected and oxidized or clipped peptides.

Figure 8. Tandem mass spectrum of the precursor ion of doubly charged disulfide linked L13-S-S-L13 at m/z 811.33. y1-S is the y1 ion without N-terminal serine.

Table 3. Relative EIC peak areas of different forms of light chain C-terminal peptide in non-reduced MAb-A and Peak 1 fraction

Peptide^a	Extracted ion M2H^+	Relative EIC peak area (%)^b
		MAb-A Lot B	Lot B Peak 1
L13-S-S-H12	631.25	100	100
L13-S-S- L13	811.33	0.1	16.5
L13-S-S-GSH	559.21	0.2	9.9
L13-S-S-Cys	466.17	0.0	4.9
L13-NEM	469.19	0.5	1.1
L13	406.67	0.0	0.0
Sum		100.8	132.4

^a The samples were treated with NEM and digested with Lys-C. L13, light chain N-terminal peptide with Cys at the N-terminus; -S-S-, disulfide link. ^bNormalized by dividing the peak area of peptide L13-S-S-H12 in the same sample.

Table 4. Potency test results of SEC monomer and peak 1 fractions collected from MAb-A Lot B

Sample description	Relative potency (%)
	Replicate 1	Replicate 2	Average
MAb-A Lot B	100.9	99.6	99
MAb-A SEC Monomer Peak	101.8	97.6	100
MAb-A SEC Peak 1	49.4	50.2	50

Figure 9. Proposed major structures of SEC Peak 1 components through proposed disulfide linkages (structures due to non-specific binding in LC variable region are not shown). The blue lines represent heavy chains and the orange lines represent light chains.