Extension of human GCSF serum half-life by the fusion of albumin binding domain

Abstract

Granulocyte colony stimulating factor (GCSF) can decrease mortality of patients undergo chemotherapy through increasing neutrophil counts. Many strategies have been developed to improve its blood circulating time. Albumin binding domain (ABD) was genetically fused to N-terminal end of GCSF encoding sequence and expressed as cytoplasmic inclusion bodies within Escherichia coli. Biological activity of ABD-GCSF protein was assessed by proliferation assay on NFS-60 cells. Physicochemical properties were analyzed through size exclusion chromatography, circular dichroism, intrinsic fluorescence spectroscopy and dynamic light scattering. Pharmacodynamics and pharmacokinetic properties were also investigated in a neutropenic rat model. CD and IFS spectra revealed that ABD fusion to GCSF did not significantly affect the secondary and tertiary structures of the molecule. DLS and SEC results indicated the absence of aggregation formation. EC50 value of the ABD-GCSF in proliferation of NFS-60 cells was 75.76 pg/ml after 72 h in comparison with control GCSF molecules (Filgrastim: 73.1 pg/ml and PEG-Filgrastim: 44.6 pg/ml). Animal studies of ABD-GCSF represented improved serum half-life (9.3 ± 0.7 h) and consequently reduced renal clearance (16.1 ± 1.4 ml/h.kg) in comparison with Filgrastim (1.7 ± 0.1 h). Enhanced neutrophils count following administration of ABD-GCSF was comparable with Filgrastim and weaker than PEG-Filgrastim treated rats. In vitro and in vivo results suggested the ABD fusion as a potential approach for improving GCSF properties.

Figure 1

Schematic view of the expression cassette.

Figure 2

ABD-GCSF protein. (a) Protein expression: M: Protein Mw marker; #1, 3: Recombinant bacterial lysates before induction; #2, 4: Recombinant bacterial lysates after induction. (b) Protein purification: #1: Initial sample (IS); #2: Flow through (FT) sample; #3: Washing sample; M: Protein Mw marker; #4–8: Eluted samples. (c) Western blotting: M: Protein Mw marker; #1, 2: Bacterial lysates before and after induction; #3, 4: Eluted proteins. The full-size original gels and the blot are presented in supplementary Fig. S1a,b,c, respectively.

Figure 3

Size exclusion chromatography of Filgrastim and ABD-GCSF. Molecular weight marker includes the following proteins: (1) vitamin B12 (1.350 kDa); (2) horse myoglobin (17 kDa); (3) chicken ovalbumin (44 kDa); (4) bovine c-globulin (158 kDa); (5) bovine thyroglobulin (670 kDa); (5). Last peak obtained at retention time of 22 min represents excipients from the dilution buffer.

Figure 4

Dynamic light scattering measurements of ABD-GCSF and PEG-Filgrastim in comparison with Filgrastim. Data presented as mean ± SD. *Considered P values less than 0.05 in comparison to Filgrastim protein.

Figure 5

Fluorescence emission spectra of GCSF derivatives.

Figure 6

CD spectra of GCSF derivatives. (a) Far-UV spectrum (190–250 nm). (b) Near-UV spectrum (250–350 nm). Filgrastim and ABD-GCSF are represented in green and black lines, respectively.

Figure 7

Dose response curves for GCSF derivatives on NFS-60 cells. Data are given as mean ± SD values of triplicate wells.

Figure 8

ELISA showing HSA binding of ABD-GCSF. The graph represents the mean ± SD of duplicates.

Figure 9

Mean of (a) neutrophil and (b) WBC counts in neutropenic rats after receiving single doses of GCSF derivatives. Data are means ± SE of 3 random rats/group.

Figure 10

Plasma concentration–time profile of Filgrastim, PEG-Filgrastim and ABD-GCSF in rats. 5 rats/group subcutaneously received GCSF derivatives. All points report the mean ± SD of animals.