Impact of Fc N-linked glycans on in vivo clearance of an immunoglobulin G1 antibody produced by NS0 cell line

Figures & data

Figure 1. Glycan profiles of mAb1 by UPLC (a); inset shows enlarged figure. (b) Structures of glycans; asterisks indicate galactose-alpha-1,3-galactose linkages (b). G, galactose; GN, N-acetyl-glucosamine; M, mannose; f, fucose; NGc, N-glycolylneuraminic acid

Figure 2. Purification scheme for mAb1 from human serum

Figure 3. Comparison of UPLC glycan profiles. (a) mAb1; (b) human serum; (c) mAb1 from human serum purified only by protein A and immobilized anti-mAb1 affinity columns; (d) mAb1 from human serum purified by protein A, immobilized anti-mAb1 affinity, and WCX columns. Arrows in (c) indicate glycan species present in serum proteins, which may interfere with mAb1 glycan profiles. After two-step purification, the interference peaks from serum proteins were not completely removed (c), and major glycan species of mAb1 co-eluted with glycans from serum. The peak marked with an asterisk (c, d) showed a specific glycan peak observed only in serum glycoproteins and used to confirm purification efficiency. After three-step purification, this major serum glycan interference was significantly reduced (d), and both glycan profiles before (a) after (d) were comparable

Figure 4. Comparison of glycan profiles. (a) mAb1 without purification; (b) mAb1 purified from PBS buffer with three-step purification; and (c) mAb1 purified from serum with three-step purification. Overlay of three profiles demonstrated that interference by serum glycoproteins in mAb1 glycan profile was negligible after three-step purification

Figure 5. Comparison of WCX chromatograms (a) and glycan profiles (b) among non-purified mAb1 and degraded mAb1 stressed at 25℃ or 40℃ for 3 months and spiked in and purified from serum

Table 1. Glycan analysis of degraded mAb1

Samples	Major glycans				Gal-α-Gal							NGNA						M5
	G0f	G1f (alpha 1,6)	G1f (alpha 1,3)	G2f	G1f-GN+G	G1f+G (b)	G1f+G (a)	G2f+G(a)	G2f+2 G	G2f+G(b) co-eluted with G1f+NGc(a)	G2f+G+ NGc	G1f+NGc(a) co-eluted with G2f+G(b)	G1f-GN+NGc	G1f+NGc(b)	G2f+NGc	G2f+G+ NGc	G2f+2NGc
Non-purified mAb1	26.8	30.4	9.3	12.0	0.7	1.1	0.4	1.8	1.5	1.9	1.2	1.9	1.0	0.4	1.8	1.2	0.0	1.1
Stressed at 25℃	27.3	29.7	9.2	12.1	0.6	0.9	0.1	1.7	1.1	2.0	1.1	2.0	1.1	0.5	2.0	1.1	0.1	1.0
Stressed at 40℃	28.9	31.1	9.6	12.6	0.6	0.9	0.2	1.5	1.0	1.5	0.7	1.5	0.9	0.4	1.4	0.7	0.0	1.0

Table 2. Recovered mAb1 from three-step purification process

Patient No.	Recovered mAb1 (µg)
	T0	TF
Patient 1	33.9	27.9
Patient 2	30.3	28.5
Patient 3	44.4	35.1
Patient 4	37.8	30.9
Patient 5	36.6	25.2
Patient 6	30.3	23.4
Patient 7	39.9	24.0
Process control	26.1

100 µg of mAb1 in each clinical serum samples was processed.

Process control: 100 µg of mAb1 purified from 0.5 mL of serum.

Figure 6. Overlay of glycan profiles of purified mAb1 from clinical study patient. (a) patient 1 at T0; (b) patient 1 at TF. No significant differences were observed between two samples, especially for minor glycan species

Table 3. Glycan analysis of mAb1 purified from 14 clinical sera

Sample Name	Major glycans				Gal-α-Gal										NGNA							M5
	G0f	G1f (alpha 1,6)	G1f (alpha 1,3)	G2f	G1f-GN+G		G1f+G(b)	G1f+G(a)	G2f+G(a)		G2f+2 G	G2f+G(b); co-eluted with G1f+NGc(a)	G2f+G+ NGc		G1f+NGc(a); co-eluted with G2f+G(b)	G1f-GN+NGc	G1f+NGc(b)	G2f+NGc	G2f+G+ NGc		G2f+2NGc
Patient 1 T0	37.3	30.0	8.3	8.5		0.2	1.7	0.3		1.2	0.9	1.9		1.7	1.9	0.6	1.3	2.7		1.7	0.1	0.4
Patient 1 TF	38.5	30.2	8.5	8.6		0.2	1.4	0.3		1.2	0.8	1.6		1.4	1.6	0.5	1.2	2.5		1.4	0.1	0.2
Patient 2 T0	34.7	31.0	8.7	9.1		0.2	1.9	0.3		1.5	1.0	1.8		1.8	1.8	0.5	1.3	2.8		1.8	0.1	0.4
Patient 2 TF	33.3	31.9	9.2	9.9		0.2	1.8	0.3		1.6	1.1	1.8		1.7	1.8	0.5	1.2	2.7		1.7	0.1	0.3
Patient 3 T0	37.9	29.7	8.3	8.2		0.3	1.8	0.3		1.2	0.9	1.8		1.7	1.8	0.6	1.3	2.7		1.7	0.1	0.5
Patient 3 TF	39.2	30.6	8.6	8.5		0.2	1.5	0.3		1.2	0.9	1.5		1.3	1.5	0.5	1.1	2.2		1.3	ND	0.2
Patient 4 T0	26.5	33.8	9.4	11.2		0.2	2.2	0.4		2.0	1.3	2.2		2.4	2.2	0.6	1.4	3.6		2.4	0.1	0.4
Patient 4 TF	26.7	33.8	10.0	11.7		0.2	1.9	0.4		1.9	1.2	1.9		1.9	1.9	0.7	1.1	2.9		1.9	ND	0.2
Patient 5 T0	37.7	29.5	8.5	8.5		0.3	1.8	0.3		1.2	1.0	1.8		1.7	1.8	0.7	1.3	2.6		1.7	ND	0.4
Patient 5 TF	39.0	29.8	8.8	8.7		0.2	1.5	0.3		1.1	0.8	1.5		1.2	1.5	0.6	1.0	2.0		1.2	ND	0.2
Patient 6 T0	37.5	29.9	8.5	8.5		0.2	1.7	0.3		1.3	0.9	1.8		1.7	1.8	0.6	1.3	2.7		1.7	0.1	0.4
Patient 6 TF	37.8	30.4	8.6	8.7		0.2	1.6	0.3		1.2	0.8	1.7		1.5	1.7	0.6	1.2	2.4		1.5	0.1	0.2
Patient 7 T0	38.4	29.6	8.4	8.3		0.2	1.7	0.3		1.2	0.9	1.7		1.5	1.7	0.6	1.2	2.5		1.5	0.1	0.4
Patient 7 TF	39.9	30.0	8.7	8.4		0.2	1.6	0.3		1.2	0.8	1.5		1.3	1.5	0.5	1.0	2.1		1.3	ND	0.3

Various mAb1 DP lots were administered among patients.

ND, not detected.

Figure 7. Comparison of major glycans (G0f, G1f, G2f) between T0 and TF. T0 (black): samples taken from patients after receiving final dose on d 337; TF (gray) samples taken from patients on d 365 (4 weeks of final dose)

Figure 8. Comparison of Gal-α-Gal and NGNA between T0 and TF. T0 (black): samples taken from patients after receiving final dose on d 337; TF (gray) samples taken from patients on d 365 (4 weeks of final dose)

Figure 9. Changes in levels of Mannose 5 between T0 and TF. T0 (black): samples taken from patients after receiving final dose on d 337; TF (gray) samples taken from patients on d 365 (4 weeks of final dose)