Aggregation of rhDNase occurred during the compression of KBr pellets used for FTIR spectroscopy.
OBJECTIVE
To determine if a protein changes when it is compressed into a KBr pellet for FTIR spectroscopy measurement in the solid state, using recombinant human deoxyribonuclease I (rhDNase) as an example.


METHODS
Lyophilized rhDNase with KBr compressed at different pressures were analyzed by FTIR spectroscopy, size exclusion HPLC and enzymatic activity assay. Different protein/KBr weight ratios and residual water contents were studied for their possible effects on aggregation.


RESULTS
Depending on the pressure, a loss of enzymatic activity accompanied by an increase in soluble high molecular weight aggregates of the protein (up to approximately 15%) was demonstrated. Aggregation was reduced to less than 5% by a suitable dilution of the protein in KBr (1 in 1000). In contrast, water content variability (1-11 wt. %) did not affect aggregation.


CONCLUSIONS
The findings emphasize the importance to examine for protein integrity when using the KBr method for FTIR sample preparation. Protein aggregation may be minimized by optimizing the sample preparation condition such as changing the protein/KBr weight ratio.
Chan
H K
HK
Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080, USA.
Ongpipattanakul
B
B
Au-Yeung
J
J
D001965:Bromides / Q000737:chemistry*; D003850:Deoxyribonuclease I / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D004355:Drug Stability; D006801:Humans; D017680:Potassium Compounds / Q000737:chemistry*; D011312:Pressure; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D014867:Water / Q000737:chemistry
D001965:Bromides; D017680:Potassium Compounds; D011994:Recombinant Proteins; D014867:Water; D003850:Deoxyribonuclease I; C039004:potassium bromide