Interface-induced protein unfolding and aggregation

2004 2004

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Abstract (summary)

Protein inactivation and aggregation at the water/CH2Cl 2 interface is one of the most detrimental events hindering the encapsulation of structurally unperturbed proteins into poly(lactide-co-glycolide) (PLG) microspheres for their clinical application as sustained release dosage forms. Herein, we investigated the inactivation and aggregation of the model proteins hen egg-white lysozyme and α-chymotrypsin at this interface and devised methods to prevent both events. When both enzymes were exposed to the water/CH2Cl2 interface in a procedure simulating the first emulsification step used in the water-in-oil-in-water (w/o/w) microencapsulation technique, protein aggregation occurred, loss in specific activity, and structural perturbations. The aggregates formed contained intermolecular β-sheets and were non-covalent in nature. We demonstrated that protein inactivation was caused by the irreversible formation of an unfolded or denatured protein species, which was able to leave the interface and accumulate in the aqueous phase.

It has been shown that by co-dissolving various sugars and polyhydric alcohols with lysozyme in the first aqueous buffer, interface-induced lysozyme aggregation and inactivation can be minimized. An excipient effect on the conformational stability of lysozyme has been excluded for the first time in this investigation to contribute to preventing lysozyme unfolding and aggregation. α-Chymotrypsin in aqueous solution showed substantial aggregation and activity loss when it was homogenized with CH2Cl2 due to adsorption to the interface. Screening for efficient excipients revealed that co-dissolving the protein with maltose and polyethylene glycol (PEG, Mw 8000) in the first aqueous phase reduced interface-induced protein aggregation and inactivation.

Then, we investigate whether stabilizing the proteins towards the major stress factors in the w/o/w encapsulation procedure would allow for the encapsulation and release of structurally unperturbed, non-aggregated, and active protein. When lysozyme and α-chymotrypsin were encapsulated in poly(lactic- co-glycolic)acid (PLGA) microspheres without stabilizing additives, they showed substantial loss in activity and aggregation. Herein, it was found that those excipients, which were efficient in preventing interface-induced structural perturbations were also efficient in minimizing lyophilization-induced structural perturbations. The efficient excipients identified also reduced structural perturbations upon lysozyme or α-chymotrypsin encapsulation in PLGA microspheres and this led to reduced protein inactivation and aggregation. However, lysozyme inactivation was completely prevented only by employing the efficient excipients in the second aqueous phase also. Employing maltose and polyethylene glycol (PEG, Mw 8000) during encapsulation of α-chymotrypsin led to a reduction in α-chymotrypsin inactivation and aggregation. Optimizing the effect of PEG by also dissolving the excipient in the organic phase prior to encapsulation further decreased the amount of non-covalent aggregates and loss in activity. In summary, protein aggregation and inactivation were minimized by rationally selecting excipients efficient in stabilizing proteins against the major stress factors of w/o/w encapsulation. (Abstract shortened by UMI.)

Indexing (details)

Analytical chemistry
0487: Biochemistry
0491: Pharmacology
0486: Analytical chemistry
Identifier / keyword
Pure sciences; Dichloromethane; Protein aggregation; Protein unfolding
Interface-induced protein unfolding and aggregation
Perez Rodriguez, Caroline
Number of pages
Publication year
Degree date
School code
DAI-B 65/11, Dissertation Abstracts International
Place of publication
Ann Arbor
Country of publication
United States
0496130501, 9780496130504
Griebenow, Kai
University of Puerto Rico, Rio Piedras (Puerto Rico)
University location
United States -- Puerto Rico
Source type
Dissertations & Theses
Document type
Dissertation/thesis number
ProQuest document ID
Database copyright ProQuest LLC; ProQuest does not claim copyright in the individual underlying works.
Document URL
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