Pharmacokinetic properties of IgG and various Fc fusion proteins in mice

Figures & data

Figure 1. Schematic composition of the various proteins. Carbohydrates are shown as hexagonal gray symbols.

Figure 2. Pharmacokinetics of IgG1 and scFv-Fc fusion proteins. a) Anti-CEA antibody molecules. b) Anti-EGFR antibody molecules. CD1 mice received a single injection of 25 µg protein and serum concentrations were determined by ELISA.

Table 1 Biochemical and pharmacokinetic properties

Protein	Mr[kDa]	Sr[nm]	Tm[°C]	pI	t1/2α[h]	t1/2β[h]	AUC [%h]
anti-EGFR:
IgG1	146	5.5	n.d.	8.3	4.8 ± 1.9	218 ± 27	6129 ± 682
scFv-Fc	104	4.3	66/76	8.1	2.7 ± 0.8	112 ± 23	3257 ± 562
anti-CEA:
IgG1 (CHO)	148	5.6	58/76	6.4	7.0 ± 3.0	222 ± 8	7945 ± 222
scFv-Fc	104	4.8	46/76	6.0	2.5 ± 0.9	104 ± 6	3966 ± 828
scDb-Fc	157	5.5	51	7.2	2.8 ± 1.4	140 ± 35	4143 ± 1378
scFv-scCLCH1-Fc	151	5.6	54/76	6.5	3.2 ± 0.6	53 ± 11	2349 ± 403
scFv	27	2.5	46	5.9	0.2 ± 0.1	0.6 ± 0.2	16 ± 4
scDb	53	2.6	51	7.7	0.4 ± 0.1	1.3 ± 0.2	56 ± 15
scDb-CH2	67	n.d.	50	7.6	0.6 ± 0.1	0.8 ± 0.1	80 ± 20

n.d., not determined

Figure 3. a) SDS-PAGE analysis of IgG and Fc fusion proteins (lanes 1 and 7, IgG1 produced in CHO cells; lanes 2 and 8, scFv-Fc; lanes 3 and 9, scDb-Fc; lanes 4 and 10, scFv-scCLCH1-Fc; lanes 5 and 11, IgG1 produced in HEK293 cells; lanes 6 and 12, scDb-CH2) analyzed under non-reducing (1–6) or reducing (7–12) conditions. Proteins were stained with Coomassie blue. b) Size-exclusion chromatography of IgG and Fc fusion proteins as indicated.

Figure 4. ELISA of binding of IgG and Fc fusion proteins to immobilized CEA. Bound proteins were detected with an anti-Fc HRP-conjugated antibody.

Figure 5. a) Thermal melting points of IgG and Fc fusion proteins determined by dynamic light scattering using 1°C intervals and 2 min equilibration time. A drastic increase in mean count rates is defined as melting point and indicated by dashed lines. b) In vitro serum stability of IgG and Fc fusion proteins. Proteins were incubated with mouse serum at 37°C for up to 7 d and active protein was determined by ELISA (IgG and Fc fusion proteins with an anti-Fc antibody, scDb-CH2 either with an anti-His-tag antibody or protein L).

Figure 6. a) QCM measurements of binding of IgG and Fc fusion proteins to immobilized mouse FcRn (at a density of 50 Hz) at pH 6.0 or pH 7.4. For comparison, binding curves are shown for 100 nM protein, except for scDb-CH2 which was analyzed at 1 µM. b) Analysis of dissociation of proteins bound to mouse FcRn (at pH 6.0) by shifting pH to 7.4. c) Analysis of dissociation of IgG and scFv-Fc fusion protein to human FcRn at pH 6.0 by shifting pH to 7.4.

Table 2 Binding to mouse FcRn

		kon	koff	KD
Protein		[M^−1s^−1]	[s^−1]	[nM]
IgG (CHO)		1.5 × 10^5	1.6 × 10^−2	108
scFv-Fc		1.7 × 10^5	1.8 × 10^−2	107
scDb-Fc		2.0 × 10^5	1.6 × 10^−2	82
scFv-scCLCH1-Fc		1.7 × 10^5	1.2 × 10^−2	121

Figure 7. a) Pharmacokinetics of IgG and Fc fusion proteins. b) Pharmacokinetics of scFv as well as scDb and scDb-CH2. CD1 mice received a single injection of 25 µg protein and serum concentrations were determined by ELISA. c) Statistical analysis of terminal half-lives and AUCs of the IgGs and Fc fusion proteins.