Evaluation of protein modification during anti-viral heat bioprocessing by electrospray ionization mass spectrometry.
During the preparation of therapeutic plasma and recombinant protein biopharmaceuticals heat-treatment is routinely applied as a means of viral inactivation. However, as most proteins denature and aggregate under heat stress, it is necessary to add thermostabilizing excipients to protein formulations destined for anti-viral heat-treatment in order to prevent protein damage. Anti-viral heat-treatment bioprocessing therefore requires that a balance be found between the bioprocessing conditions, virus kill and protein integrity. In this study we have utilized a simple model protein, beta-lactoglobulin, to investigate the relationship between virucidal heat-treatment conditions (protein formulation and temperature) and the type and extent of protein modification in the liquid state. A variety of industrially relevant heat-treatments were undertaken, using formulations that included sucrose as a thermostabilizing excipient. Using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) we show here that protein modifications do occur with increasingly harsh heat-treatment. The predominant modification under these conditions was protein glycation by either glucose or fructose derived from hydrolyzed sucrose. Advanced glycation end products and additional unidentified products were also present in beta-lactoglobulin protein samples subjected to extended heat-treatment. These findings have implications for the improvement of anti-viral heat-treatment bioprocesses to ensure the safety and efficacy of protein biopharmaceuticals. CopyrightCopyright 2001 John Wiley & Sons, Ltd.
Research School of Biosciences, University of Kent, Canterbury CT2 7NJ, Kent, UK. c.m.smales@ukc.ac.uk
C M
CM
Smales
D S
DS
Pepper
D C
DC
James
D001798:Blood Proteins / Q000737:chemistry*; D002853:Chromatography, Liquid; D004340:Drug Contamination; D005632:Fructose / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006361:Heating; D006358:Hot Temperature; D007202:Indicators and Reagents; D007782:Lactoglobulins / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013242:Sterilization; D014780:Viruses
D001798:Blood Proteins; D006023:Glycoproteins; D007202:Indicators and Reagents; D007782:Lactoglobulins; D011994:Recombinant Proteins; D005632:Fructose; D005947:Glucose