Protein inactivation and aggregation are serious drawbacks in the encapsulation of proteins in bioerodible polymers by water-in-oil-in-water (w/o/w) encapsulation. The model protein alpha-chymotrypsin was employed to investigate whether its stabilization towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of non-aggregated and active protein. Due to the formation of amorphous aggregates alpha-chymotrypsin is an excellent sensor to probe unfolding events. Furthermore, its enzymatic activity is highly sensitive towards the presence of organic solvents. alpha-Chymotrypsin in aqueous solution showed substantial aggregation and activity loss when it was homogenized with @entity17058 due to adsorption to the interface. Its w/o/w encapsulation in @entity7050 ( @entity7050 ) microspheres caused formation of 35% non-covalent aggregates and reduced the specific activity by 14%. Screening for efficient excipients revealed that co-dissolving the protein with maltose and @entity968 ( @entity970 , M(w) 5000) in the first aqueous phase reduced interface-induced @entity6960 and inactivation. Employing these excipients during encapsulation led to a reduction in alpha-chymotrypsin inactivation (10%) and aggregation (12%). Optimizing the effect of @entity970 by also dissolving the excipient in the organic phase prior to encapsulation further decreased the amount of non-covalent aggregates to 7% and loss in activity to 5%. The data obtained demonstrate that the w/o emulsification step is the main stress-factor in the w/o/w encapsulation procedure but subsequent encapsulation steps also cause some @entity6960 .

Stabilization of alpha-chymotrypsin at the @entity17058 /water interface and upon water-in-oil-in-water encapsulation in XXXX microspheres.

@entity17058 :: ('CHEBI:15767', 'Chemical') :: ['CH(2)Cl(2)'] @entity7050 :: ('MESH:C043435', 'Chemical') :: ['PLGA', 'poly(lactic-co-glycolic)acid'] @entity6960 :: ('MESH:D001796', 'Disease') :: ['protein aggregation'] @entity970 :: ('CHEBI:46793', 'Chemical') :: ['PEG'] @entity968 :: ('MESH:D011092', 'Chemical') :: ['polyethylene glycol']
@entity7050 :: (MESH:C043435,Chemical) :: ['PLGA']
