Aggregation of proteins and its prevention by carbohydrate excipients: albumins and gamma-globulin.
Moisture-induced (2-10 microL added to 10 mg) aggregation of solid-state albumin and gamma-globulin was investigated by incubation at 37 degrees C for 24 h. The insoluble aggregates were centrifuged from a reconstituted solution, dissolved in a solution containing denaturant and reducing agent, and analysed by a Bio-Rad protein assay kit. Of the three albumins used, maximum aggregation (8.2%) was observed with bovine serum albumin that was essentially fatty-acid free. The maximum aggregation observed with gamma-globulin was 7.0%. A bell-shaped curve for percent aggregation was observed with increasing moisture content and was especially prominent for bovine serum albumin. When mixed with carbohydrate excipients in a 1:1 ratio, aggregation was reduced for both bovine serum albumin and gamma-globulin by all four of the following excipients used: Emdex, dextrose, trehalose and hydroxypropyl beta-cyclodextrin. For bovine serum albumin, the aggregation was reduced about sixfold, with Emdex being the most effective excipient. The likely mechanism of the resulting aggregation was covalent linkages formed due to intermolecular thiol disulphide interchange.
Katakam
M
M
Department of Pharmacal Sciences, School of Pharmacy, Auburn University, AL 36849-5503, USA.
Banga
A K
AK
D001665:Binding Sites; D002021:Buffers; D002241:Carbohydrates / Q000737:chemistry*; D003505:Cyclodextrins / Q000737:chemistry; D005079:Excipients / Q000737:chemistry*; D005453:Fluorescence; D005612:Freeze Drying; D005947:Glucose / Q000737:chemistry; D006801:Humans; D009930:Organic Chemicals; D010084:Oxidation-Reduction; D011489:Protein Denaturation; D012709:Serum Albumin / Q000737:chemistry / Q000378:metabolism*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism*; D013696:Temperature; D014199:Trehalose / Q000737:chemistry; D014867:Water; D047392:beta-Cyclodextrins; D005719:gamma-Globulins / Q000378:metabolism*
D002021:Buffers; D002241:Carbohydrates; D003505:Cyclodextrins; D005079:Excipients; D009930:Organic Chemicals; D012709:Serum Albumin; D047392:beta-Cyclodextrins; D005719:gamma-Globulins; D014867:Water; D012710:Serum Albumin, Bovine; C038962:EMDEX; D014199:Trehalose; D005947:Glucose; C031215:betadex