Prolonged circulation of recombinant human granulocyte-colony stimulating factor by covalent linkage to albumin through a heterobifunctional polyethylene glycol.

Purpose: Recombinant human granulocyte-colony stimulating factor (rhG-CSF) was covalently conjugated to both rat and human serum albumin (RSA and HSA respectively) to increases the circulating half life (t1/2) of rhG-CSF.

Methods: Conjugates of RSA (MW 67,000) and HSA (MW 66,000) were prepared by linking the two proteins through a heterobifunctional maleimido-carboxyl polyethylene glycol (PEG) and were tested in the rat. The conjugates were injected intravenously (IV) at the equivalent dose of 50 micrograms/kg of rhG-CSF, and white blood cell (WBC) counts and plasma concentrations of drug were determined. A comparison of pharmacokinetic parameters was made between rhG-CSF, the conjugates RSA-PEG-rhG-CSF and HSA-PEG-rhG-CSF, and a non-covalent mixture of rhG-CSF and HSA.

Results: The albumin-rhG-CSF conjugates are eliminated more slowly from the circulation. The clearance values are reduced from 0.839 +/- 0.121 ml/min/kg for rhG-CSF to 0.172 +/- 0.013 ml/min/kg for RSA-PEG-rhG-CSF and 0.141 +/- 0.005 ml/min/kg for HSA-PEG-rhG-CSF. WBC counts increased in both absolute number and duration as compared to rhG-CSF alone. The albumin rhG-CSF conjugates had enhanced serum stability relative to free rhG-CSF. The rate of degradation of the albumin conjugates incubated in rat serum at 37 degrees C decreased five fold.

Conclusions: The results from the study show that specific conjugation of rhG-CSF to albumin decreases plasma clearance in vivo, causes increased WBC response, and increases serum stability as compared to free rhG-CSF.