title: Preparation of excipient-free recombinant human tissue-type plasminogen activator by lyophilization from ammonium bicarbonate solution: an investigation of the two-stage sublimation phenomenon.
abstract: Dry, excipient-free recombinant human tissue-type plasminogen activator (tPA) powder was prepared by lyophilization from ammonium bicarbonate solution. Ammonium bicarbonate sublimes into ammonia, water, and carbon dioxide upon lyophilization, without causing measurable harm to the protein. There were approximately 4 mol of residual ammonium ion per mole of lyophilized tPA. Under certain lyophilization conditions, a large pressure increase in the lyophilizer chamber occurred, presenting a pressure control problem. Microscopy and sublimation rate measurements on the frozen matrix revealed that ice sublimation occurred first, followed by the sublimation of ammonium bicarbonate. Analysis of the sectioned frozen matrix indicated that the bicarbonate salt was evenly distributed throughout the vial, suggesting that the delay of ammonium bicarbonate sublimation was not due to hindrance by ice. In the two-stage process, ice sublimation proceeded according to zero-order kinetics, whereas ammonium bicarbonate sublimation followed a grain-burning (2/ 3-order) model and was governed by a higher activation enthalpy. In most cases, the sublimation rate of ammonium bicarbonate in the presence of tPA was lower than that in the absence of the protein. Sublimation activation enthalpy for ammonium bicarbonate in the presence of tPA was 26.1 +/- 3.8 kcal/mol, which was approximately 10 kcal/mol greater than that for the tPA-free system. Consistent with a prediction from our kinetic modeling, a 6-h extension of primary drying enabled us to conduct lyophilization while maintaining pressure control.
authors.affiliation: Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, CA 94080, USA.
authors.forename: D E
authors.initials: DE
authors.lastname: Overcashier
authors.forename: D A
authors.initials: DA
authors.lastname: Brooks
authors.forename: H R
authors.initials: HR
authors.lastname: Costantino
authors.forename: C C
authors.initials: CC
authors.lastname: Hsu
mesh_terms: D001639:Bicarbonates / Q000737:chemistry; D005079:Excipients; D005612:Freeze Drying; D006801:Humans; D007700:Kinetics; D011994:Recombinant Proteins / Q000138:chemical synthesis; D012996:Solutions; D013816:Thermodynamics; D010959:Tissue Plasminogen Activator / Q000138:chemical synthesis*
chemical_list: D001639:Bicarbonates; D005079:Excipients; D011994:Recombinant Proteins; D012996:Solutions; C027043:ammonium bicarbonate; D010959:Tissue Plasminogen Activator