Site-selective fatty acid chain conjugation of the N-terminus of the recombinant human granulocyte colony-stimulating factor

Abstract

The clinical application of the recombinant human granulocyte colony-stimulating factor (rhG-CSF) is restricted by its short serum half-life. Herein, site-selective modification of the N-terminus of rhG-CSF with PAL-PEG₃-Ph-CHO was used to develop a long-acting rhG-CSF. The optimized conditions for rhG-CSF modification with PAL-PEG₃-Ph-CHO were: reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH₃ in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PAL-PEG₃-Ph-CHO to rhG-CSF of 6:1, temperature of 20°C, and reaction time of 12 h, consequently, achieving a PAL-PEG₃-Ph-rhG-CSF product yield of 70.8%. The reaction mixture was purified via preparative liquid chromatography, yielding the single-modified product PAL-PEG₃-Ph-rhG-CSF with a HPLC purity exceeding 95%. The molecular weight of PAL-PEG₃-Ph-rhG-CSF was 19297 Da by MALDI-TOF-MS, which was consistent with the theoretical value. The circular dichroism analysis revealed no significant change in its secondary structure compared to unmodified rhG-CSF. The PAL-PEG₃-Ph-rhG-CSF retained 82.0% of the in vitro biological activity of unmodified rhG-CSF. The pharmacokinetic analyses showed that the serum half-life of PAL-PEG₃-Ph-rhG-CSF was 7.404 ± 0.777 h in mice, 4.08 times longer than unmodified rhG-CSF. Additionally, a single subcutaneous dose of PAL-PEG₃-Ph-rhG-CSF presented comparable in vivo efficacy to multiple doses of rhG-CSF. This study demonstrated an efficacious strategy for developing long-acting rhG-CSF drug candidates.

Keywords: fatty acid chain conjugation; long-acting rhG-CSF; recombinant human granulocyte colony-stimulating factor; rhG-CSF; serum half-life; site-selective modification.

FIGURE 1

Synthesis routes of PAL-PEG₃-Ph-CHO (A) and PAL-PEG₃-Ph-rhG-CSF (B).

FIGURE 2

RP-HPLC analysis (A) and SDS-PAGE analysis (B) of the reaction mixture of the modification of rhG-CSF with PAL-PEG₃-Ph-CHO. For RP-HPLC analysis (A), (a) NaCNBH₃, (b) Tween 20, (c) rhG-CSF, (d) PAL-PEG₃-Ph-CHO, and (e) Reaction mixture obtained at 10 h. For SDS-PAGE analysis (B), lane 1: Protein maker, lane 2-9: the reaction mixture respectively obtained at 4–12 h. The bands corresponded to rhG-CSF and PAL-PEG₃-Ph-rhG-CSF could be identified based on the result in Figure 4B. The other reaction conditions: rhG-CSF (1 mg/mL) and PAL-PEG₃-Ph-CHO (mole ratio of PAL-PEG₃-Ph-CHO to rhG-CSF 5:1) were reacted in acetate buffer (20 mmol/L, pH 4.5) containing 3% (w/v) Tween 20 and 30 mM NaCNBH₃ at 20°C.

FIGURE 3

Effects of Tween 20 concentration (A), pH (B), temperature (C) and the molar ratio of PAL-PEG₃-Ph-CHO to rhG-CSF (D) and the reaction time (E) on the conjugation of rhG-CSF with PAL-PEG₃-Ph-CHO. The standard condition for the analysis of each of the four parameters (Tween 20 concentrations, pH, temperature and the molar ratios of PAL-PEG₃-Ph-CHO to rhG-CSF) was as follows: Tween 20 concentration of 5% (w/v) and 30 mM NaCNBH₃ in acetate buffer (20 mmol/L, pH 4.5), mole ratio of PAL-PEG₃-Ph-CHO to rhG-CSF of 5:1, pH 4.5, temperature 20°C and reaction time 10 h. The effect of reaction time was investigated at reaction solvent system of 3% (w/v) Tween 20 and 30 mM NaCNBH₃ in acetate buffer (20 mmol/L, pH 5.0), molar ratio of PAL-PEG₃-Ph-CHO to rhG-CSF of 6:1, temperature of 20°C. Data were expressed as mean ± SD (n = 3). Values with different letters (a, b, c and d) were significantly statistically different from each other (one-way ANOVA followed by Tukey’s multiple comparison, p < 0 .05). Values without significant difference in each comparison share the same letter.

FIGURE 4

(A) Purification of reaction mixture of the modification of rhG-CSF with PAL-PEG₃-Ph-CHO by preparative liquid chromatography; (B) SDS-PAGE analysis of rhG-CSF and purified PAL-PEG₃-Ph-rhG-CSF.Lane 1: protein marker, lane 2: rhG-CSF, Lane 3: purified PAL-PEG₃-Ph-rhG-CSF; (C) Purity analysis of PAL-rhG-CSF by RP-HPLC.

FIGURE 5

Circular dichroism (CD) of rhG-CSF and PAL-PEG₃-Ph-rhG-CSF.

FIGURE 6

In vitro bioactivities of rhG-CSF and PAL-PEG₃-Ph-rhG-CSF.

FIGURE 7

Pharmacokinetic analysis of rhG-CSF and PAL-PEG₃-Ph-rhG-CSF.

FIGURE 8

Effects of rhG-CSF and PAL-PEG₃-Ph-rhG-CSF on the WBC counts in CTX treated mice. PAL-PEG₃-Ph-rhG-CSF are single injections; rhG-CSF is divided into 5 days, 0.1 mg/kg or 0.2 mg/kg per day, a total of 0.5 mg/kg or 1.0 mg/kg. The values of *p < 0.05, **p < 0.01 and ***p < 0.001 were considered significant for intergroup comparisons with the normal control group.