title: Moisture-induced solid state instabilities in alpha-chymotrypsin and their reduction through chemical glycosylation.
abstract: BACKGROUND
  Protein instability remains the main factor limiting the development of protein therapeutics. The fragile nature (structurally and chemically) of proteins makes them susceptible to detrimental events during processing, storage, and delivery. To overcome this, proteins are often formulated in the solid-state which combines superior stability properties with reduced operational costs. Nevertheless, solid protein pharmaceuticals can also suffer from instability problems due to moisture sorption. Chemical protein glycosylation has evolved into an important tool to overcome several instability issues associated with proteins. Herein, we employed chemical glycosylation to stabilize a solid-state protein formulation against moisture-induced deterioration in the lyophilized state.
  
  
  RESULTS
  First, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme alpha-chymotrypsin (alpha-CT) under controlled humidity conditions. Results showed that alpha-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, alpha-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that alpha-CT structural dynamics increased at increasing RH. The magnitude of the structural changes in tendency parallels the solid-state instability data (i.e., formation of buffer-insoluble aggregates, inactivation, and loss of native conformation upon reconstitution). To determine if these moisture-induced instability issues could be ameliorated by chemical glycosylation we proceeded to modify our model protein with chemically activated glycans of differing lengths (lactose and dextran (10 kDa)). The various glycoconjugates showed a marked decrease in aggregation and an increase in residual activity after incubation. These stabilization effects were found to be independent of the glycan size.
  
  
  CONCLUSIONS
  Water sorption leads to aggregation, inactivation, and structural changes of alpha-CT as has been similarly shown to occur for many other proteins. These instabilities correlate with an increase in protein structural dynamics as a result of moisture exposure. In this work, we present a novel methodology to stabilize proteins against structural perturbations in the solid-state since chemical glycosylation was effective in decreasing and/or preventing the traditionally observed moisture-induced aggregation and inactivation. It is suggested that the stabilization provided by these chemically attached glycans comes from the steric hindrance that the sugars conveys on the protein surface therefore preventing the interaction of the protein internal electrostatics with that of the water molecules and thus reducing the protein structural dynamics upon moisture exposure.
authors.affiliation: Department of Chemistry, University of Puerto Rico, Río Piedras Campus, PO Box 23346, San Juan 00931-3346, Puerto Rico.
authors.forename: Giselle M
authors.initials: GM
authors.lastname: Flores-Fernández
authors.forename: Miraida
authors.initials: M
authors.lastname: Pagán
authors.forename: Mariangely
authors.initials: M
authors.lastname: Almenas
authors.forename: Ricardo J
authors.initials: RJ
authors.lastname: Solá
authors.forename: Kai
authors.initials: K
authors.lastname: Griebenow
mesh_terms: D002918:Chymotrypsin / Q000737:chemistry*; D003911:Dextrans / Q000138:chemical synthesis; D004795:Enzyme Stability; D005612:Freeze Drying; D006001:Glycoconjugates / Q000138:chemical synthesis; D006031:Glycosylation; D006813:Humidity; D007700:Kinetics; D014867:Water / Q000737:chemistry
chemical_list: D003911:Dextrans; D006001:Glycoconjugates; D014867:Water; D002918:Chymotrypsin; C117608:alpha-chymotrypsin