Protein modification during antiviral heat bioprocessing

• Published in: Biotechnology and bioengineering 2000-01, Vol.67 (2), p.177-188
• Main Authors: Smales, C. Mark, Pepper, Duncan S., James, David C.
• Format: Article
• Language: English
• Subjects: advanced glycation end products; antiviral heat treatment; Biological and medical sciences; Biotechnology; Carbohydrates - chemistry; Chromatography, High Pressure Liquid - methods; Drug Contamination - prevention & control; Drug products; Enzymes; Fundamental and applied biological sciences. Psychology; Health. Pharmaceutical industry; Heat treatment; Hot Temperature; Hydrolysis; Industrial applications and implications. Economical aspects; Liquid chromatography; lysozyme; Mass Spectrometry - methods; Muramidase - chemistry; Other active biomolecules; Peptide Mapping; Production of active biomolecules; protein formulation; protein glycation; Proteins - chemistry; Sucrose - chemistry; Sugars; therapeutic protein bioprocessing; Thermal effects; Viruses
• ISSN: 0006-3592; 1097-0290
• DOI: 10.1002/(SICI)1097-0290(20000120)67:2<177::AID-BIT7>3.0.CO;2-3

Heat treatment is routinely used in the preparation of therapeutic protein biopharmaceuticals as a means of viral inactivation. However, in undertaking virucidal heat treatments, a balance must be found between the bioprocessing conditions, virus kill, and the maintenance of protein integrity. In this study, we utilize a simple model protein, hen egg‐white lysozyme, to investigate the relationship between antiviral bioprocess conditions (protein formulation and temperature) and the extent and type of protein modification. A variety of industrially relevant wet‐ and dry‐heat treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Although there was no evidence of lysozyme aggregation or crosslinking during any of the heat treatments, using liquid chromatography–electrospray ionization–mass spectroscopy (LC‐ESI‐MS) and peptide mapping we show that protein modifications do occur with increasingly harsh heat treatment. Modifications were predominantly found after wet‐heat treatment, the major covalent modification of lysozyme under these conditions being glycation of Lys97, by either glucose or fructose derived from hydrolyzed sucrose. The extent of sucrose hydrolysis was itself dependent on both the duration of heat treatment and formulation composition. Advanced glycation end products (AGEs) and additional unidentified products were also present in protein samples subjected to extended heat treatment. AGEs were derived primarily from initial glycation by fructose and not glucose. These findings have implications for the improvement of bioprocesses to ensure protein product quality. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 177–188, 2000.