Detection and reduction of microaggregates in insulin preparations |
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Authors: | Caryn L. Heldt Mirco Sorci David Posada Amir Hirsa Georges Belfort |
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Affiliation: | 1. Howard P. Isermann Department of Chemical Engineering and The Center of Biotechnology and Interdisciplinary Studies, 110 8th Street, Troy, New York 12180, USA;2. telephone: 518‐276‐6948;3. fax: 518‐276‐4030;4. Department of Mechanical, Aerospace, and Nuclear Engineering, Rensselaer Polytechnic Institute, Troy, New York |
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Abstract: | Insulin is an important biotherapeutic protein, and it is also a model protein used to study amyloid diseases, such as Alzheimer's and Parkinson's. The preparation of the protein can lead to small amounts of aggregate in the solution, which in turn may lead to irreproducible in vitro results. Using several pre‐treatment methods, we have determined that pH cycling and diafiltration of the insulin removes microaggregates that may be present in the solution. These microaggregates were not detectable with traditional biochemical methods, but using small‐angle neutron scattering, we were able to show that pH cycling reduces the radius of gyration of the insulin. Diafiltration removes the aggregates by size and pH cycling dissolves the aggregates by adjusting the pH through the pI of the protein. Pre‐treating the insulin with either pH cycling or diafiltration allowed reproducible kinetics of fibrillation for the insulin protein. Microaggregates are a common problem in protein production, formulation, and preparation; here we show that they are the main cause for inconsistent behavior and how pH cycling and diafiltration can mitigate this problem. Biotechnol. Bioeng. 2011; 108:237–241. © 2010 Wiley Periodicals, Inc. |
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Keywords: | insulin amyloid microaggregates diafiltration pH cycling |
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