Surface adsorption of recombinant human interferon-gamma in lyophilized and spray-lyophilized formulations.
Recombinant human interferon-gamma (rhIFN-gamma) was lyophilized or spray-lyophilized in 9.5% trehalose, +/- 0.12% polysorbate 20 in 10 mM potassium phosphate, pH 7.5. We measured recovery of soluble protein after spraying, freeze-thawing, and drying and reconstitution. Infrared spectroscopy showed rhIFN-gamma secondary structure to be native-like in all dried powders. Powders were characterized using electron spectroscopy for chemical analysis, time-of-flight secondary ion mass spectroscopy, X-ray diffraction, and gas adsorption isotherms. rhIFN-gamma adsorbed at air/liquid interfaces during spraying, and to ice/liquid interfaces during lyophilization. The concentration of rhIFN-gamma at ice/liquid interfaces was approximately one-fourth that adsorbed at air/liquid interfaces. Addition of 0.12% polysorbate 20 reduced the concentration of rhIFN-gamma at both interfaces. Time-of-flight secondary ion mass spectroscopy detected polysorbate 20 on surfaces of lyophilized powders. Lyophilized samples dried more slowly but reconstituted more quickly than spray-lyophilized samples. rhIFN-gamma aggregated after nebulization, but aggregation decreased in 0.12% polysorbate 20. Addition of 0.12% polysorbate 20 reduced protein surface adsorption and decreased but did not completely prevent aggregation. Insignificant aggregation occurred after exposure to ice/liquid interfaces, but subsequent drying and reconstitution caused aggregation. The majority of the aggregation is due to adsorption at air-liquid and solid-air interfaces formed during spray-lyophilization or lyophilization.
Department of Chemical Engineering, Center for Pharmaceutical Biotechnology, University of Colorado, Engineering Center, Room ECCH 111, Boulder 80309-0424, USA.
Serena D
SD
Webb
Stephen L
SL
Golledge
Jeffrey L
JL
Cleland
John F
JF
Carpenter
Theodore W
TW
Randolph
D002626:Chemistry, Pharmaceutical; D005612:Freeze Drying; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry*; D011136:Polysorbates / Q000737:chemistry; D011208:Powders; D011994:Recombinant Proteins / Q000737:chemistry; D013501:Surface-Active Agents / Q000737:chemistry*
D011136:Polysorbates; D011208:Powders; D011994:Recombinant Proteins; D013501:Surface-Active Agents; D007371:Interferon-gamma