Moisture-induced solid state instabilities in α-chymotrypsin and their reduction through chemical glycosylation

Abstract

BackgroundProtein 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.ResultsFirst, we investigated the consequences of moisture sorption on the stability and structural conformation of the model enzyme α-chymotrypsin (α-CT) under controlled humidity conditions. Results showed that α-CT aggregates and inactivates as a function of increased relative humidity (RH). Furthermore, α-CT loses its native secondary and tertiary structure rapidly at increasing RH. In addition, H/D exchange studies revealed that α-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.ConclusionWater sorption leads to aggregation, inactivation, and structural changes of α-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.

Influence of Reactive Oxygen Species on the Enzyme Stability and Activity in the Presence of Ionic Liquids

“…Figure 5a shows the CD spectrum of CT in buffer solutions depicting distinguished maxima at 254, 289 and 296 nm, corresponding to the presence of Trp residues of CT. These results are very well correlated with earlier reported results by various research groups [29], [49], [54]. Decrease in intensity of peaks can be probably due to the treatment of CT with plasma jet; changes being more prominent at 286 nm.…”

“…The far-UV CD spectrum (200–240 nm) of the enzyme indicated that CT has a peculiar secondary structure in presence of all of the above co-solvents discussed. CT is a type of all-β protein characterized by a CD spectrum, which resembles that of a random coil conformation [29], [54]. Crystal structure data showed that this kind of protein consists of antiparallel pleated β-sheets which are either highly distorted or form very short irregular strands [29].The same was reflected with APPJ action as well.…”

The influence of residual water on the solid-state properties of freeze-dried fibrinogen

“…Increased water content also favors a higher conformational mobility of the protein molecule [11], making it more susceptible to chemical modifications. It was reported that increased water levels could lead to higher intermolecular disulfide exchange reactions forming covalent aggregates [10,11,13,16]. From a physical perspective, the residual water can act as a medium and plasticizer, resulting in decreased glass transition temperatures and increased crystallization rates [18][19][20].…”

“…The water content of lyophilized protein formulations often determines the chemical and physical stability of the solid state of protein powders [3,9]. The general trend is that the product integrity improves with decreasing water content, and various studies are available reporting on the effect of increased residual water amounts on physical and chemical instabilities [9][10][11], including recent publications on moisture-induced aggregation of freeze-dried proteins [12][13][14][15][16]. Breen et al reported growing Asp isomerization with increasing water content [10].…”

“…In addition, intermolecular S-S shuffling in response to increasing residual water content was investigated [12][13][14]. For example, hydrogendeuterium exchange studies showed that structural dynamics of a-chymotrypsin increased with a raise in relative humidity [16].…”