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European Biopharmaceutical Review

Determination of Absorptivity Constants for Biopharmaceutical Protein Products by Amino Acid Analysis

Early in the development of any pharmaceutical product the absorptivity (as) or extinction coefficient (e) of the molecule is determined. Determination of an absorptivity constant for a small organic molecule is often a minor undertaking. The purified and dried molecule is quantitated by gravimetric means, followed by dissolution in an appropriate buffer or solvent and simple ultraviolet/visible light (UV/VIS) spectrophotometric analysis at its l maximum.

For protein-based biopharmaceuticals, however, this is not as easily accomplished. The limiting step is quantitation of the protein, which often contains contaminating proteins, bound salts, metals and excessive amounts of bound water. Quantitation of purified proteins for determination of absorptivity constants in a defined solution has been accomplished in the past by gravimetric, Kjeldahl and colorimetric techniques (1, 2) and by amino acid analysis. Statistically derived approaches for prediction of the absorptivity constants of proteins are also available.


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By John C Anders, K Chad Bastian and Glenn E Petrie, Research Associates at AAI International John C Anders received his PhD in Biochemistry and Molecular Biology from the University of Florida in 1990. He completed his postdoctoral work in the Department of Immunology at the Walter Reed Army Institute of Research, Washington DC on development of a malaria vaccine. Subsequent military service at the Medical Research Institute of Chemical Defense focused on bringing biotechnological innovations to the treatment and amelioration of the toxic effects of various chemical warfare agents.
Dr Anders now serves as a Senior Research Associate in the Biotechnology Laboratory of AAI International. His current work focuses on various aspects of protein chemistry upon analysis of biopharmaceuticals in a cGMP laboratory environment.

K Chad Bastian received his PhD in Civil Engineering from Purdue University in 1996 and completed a postdoctoral appointment with the US Geological Survey through the University of Kansas Center for Research, Inc.

Dr Bastian is currently employed by AAI International focusing on applications of HPLC/MS and tandem mass spectrometry to pharmaceutical and biopharmaceutical analysis in a GMP environment.

Glenn Petrie received his PhD in Biochemistry from the University of Illinois in September 1983. He began his career at Allied Signal, Research & Technology, where he designed, staffed and managed a $1.5 million speciality analytical laboratory to support a diverse corporate research laboratory.

He is currently employed in the Analytical Division of AAI International, where he provides technical supervision to a staff of PhD, MS and BS scientists. He works closely with clients to define and manage their method development and validation projects.



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John C Anders
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K Chad Bastian
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Glenn E Petrie
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