title: Microheterogeneity in frozen protein solutions.
abstract: In frozen and lyophilized systems, the biological to be stabilized (e.g. therapeutic protein, biomarker, drug-delivery vesicle) and the cryo-/lyo-protectant should be co-localized for successful stabilization. During freezing and drying, many factors cause physical separation of the biological from the cryo-/lyo-protectant, called microheterogeneity (MH), which may result in poor stabilization efficiency. We have developed a novel technique that utilized confocal Raman microspectroscopy in combination with counter-gradient freezing to evaluate the effect of a wide range of freezing temperatures (-20<TF<0°C) on the MH generated within a frozen formulation in only a few experiments. The freezing experiments conducted with a model system (albumin and trehalose) showed the presence of different degrees of MH in the freeze-concentrated liquid (FCL) in all solutions tested. Mainly, albumin tended to accumulate near the ice interface, where it was physically separated from the cryoprotectant. In frozen 10wt% trehalose solutions, heterogeneity in FCL was relatively low at any TF. In frozen 20wt% trehalose solutions, the optimum albumin to trehalose ratio in the FCL can only be ensured if the solution was frozen within a narrow range of temperatures (-16<TF<-10°C). In the 30wt% trehalose solutions, freezing within a much more narrow range (-12<TF<-10°C) was needed to ensure a fairly homogeneous FCL. The method developed here will be helpful for the development of uniformly frozen and stable formulations and freezing protocols for biological as MH is presumed to directly impact stability.
authors.affiliation: Biostabilization Laboratory, Mechanical Engineering Department, College of Science and Engineering, University of Minnesota, Minneapolis, MN 55455, United States.
authors.forename: Alan
authors.initials: A
authors.lastname: Twomey
authors.affiliation: Heat and Mass Transfer Laboratory, Mechanical Engineering Department, Kyushu University, Fukuoka, Japan.
authors.forename: Kosaku
authors.initials: K
authors.lastname: Kurata
authors.affiliation: Heat and Mass Transfer Laboratory, Mechanical Engineering Department, Kyushu University, Fukuoka, Japan.
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Nagare
authors.affiliation: Heat and Mass Transfer Laboratory, Mechanical Engineering Department, Kyushu University, Fukuoka, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takamatsu
authors.affiliation: Biostabilization Laboratory, Mechanical Engineering Department, College of Science and Engineering, University of Minnesota, Minneapolis, MN 55455, United States. Electronic address: aaksan@umn.edu.
authors.forename: Alptekin
authors.initials: A
authors.lastname: Aksan
mesh_terms: D000418:Albumins / Q000737:chemistry; D002138:Calibration; D003451:Cryoprotective Agents; D004355:Drug Stability; D005612:Freeze Drying; D005615:Freezing; D011506:Proteins / Q000737:chemistry*; D012996:Solutions; D013059:Spectrum Analysis, Raman; D014199:Trehalose / Q000737:chemistry
chemical_list: D000418:Albumins; D003451:Cryoprotective Agents; D011506:Proteins; D012996:Solutions; D014199:Trehalose