Susceptibility of rhDNase I to glycation in the dry-powder state.
The accumulated data on the glycation process in rhDNase I, formulated with lactose and stored in the dry-powder state, indicates that this protein becomes covalently modified with lactose at five of the six lysines (2, 50, 77, 157, 260) and to a lesser extent on the amino terminus. Analysis of the three-dimensional protein structure indicates that the reported requirements for the specificity of site reactivity, site accessibility, and the presence of a proton donor/acceptor group near the reaction site, are maintained in this protein. A chemical reaction in the dry-powder state may become permissible simply due to the close packing and resultant high concentrations of the reactant molecules. The reaction between reducing sugar and protein in the dried state indicates that the arrangement of molecules within the dry-powder particle allows for direct contact between all the reactants, including contacts between protein molecules, which may contribute to the completion of the covalent reaction at a surface-accessible reactive site in which the required surrounding microenvironment for self-catalysis is available on only 35% of the individual molecules. These findings should indicate that the use of reducing sugars in the formulation of an excipient for the stabilization of dried protein is not advisable, as reaction clearly occurs between the reducing sugar and protein because of the potentially reactive nature of the proteins' accessible amino groups.
Quan
C P
CP
Analytical Chemistry Department, Genentech, Inc., South San Francisco, California 94080, USA. cpq@gene.com
Wu
S
S
Dasovich
N
N
Hsu
C
C
Patapoff
T
T
Canova-Davis
E
E
D000595:Amino Acid Sequence; D003850:Deoxyribonuclease I / Q000737:chemistry*; D003890:Desiccation; D005947:Glucose / Q000737:chemistry*; D007785:Lactose / Q000737:chemistry; D008969:Molecular Sequence Data; D011208:Powders; D011994:Recombinant Proteins / Q000737:chemistry
D011208:Powders; D011994:Recombinant Proteins; D003850:Deoxyribonuclease I; D005947:Glucose; D007785:Lactose