Sustained release of pharmaceutical proteins from biocompatible polymers offers new opportunities in the treatment and prevention of disease. The manufacturing of such sustained-release dosage forms, and also the release from them, can impose substantial stresses on the chemical integrity and native, three-dimensional structure of proteins. Recently, novel strategies have been developed towards elucidation and amelioration of these stresses. Non-invasive technologies have been implemented to investigate the complex destabilization pathways that can occur. Such insights allow for rational approaches to protect proteins upon encapsulation and release from bioerodible systems. Stabilization of proteins when utilizing the most commonly employed procedure, the water-in-oil-in-water (w/o/w) double emulsion technique, requires approaches that are based mainly on either increasing the thermodynamic stability of the protein or preventing contact of the protein with the destabilizing agent (e.g. the water/oil interface) by use of various additives. However, protein stability is still often problematic when using the w/o/w technique, and thus alternative methods have become increasingly popular. These methods, such as the solid-in-oil-in-oil (s/o/o) and solid-in-oil-in-water (s/o/w) techniques, are based on the suspension of dry protein powders in an anhydrous organic solvent. It has become apparent that protein structure in the organic phase is stabilized because the protein is "rigidified" and therefore unfolding and large protein structural perturbations are kinetically prohibited. This review focuses on strategies leading to the stabilization of protein structure when employing these different encapsulation procedures.
In this work we establish the relationship between chemical glycosylation and protein thermodynamic, kinetic, and colloidal stability. While there have been reports in the literature that chemical glycosylation modulates protein stability, mechanistic details still remain uncertain. To address this issue, we designed and coupled monofunctional activated glycans (lactose and dextran) to the model protein alpha-chymotrypsin (alpha-CT). This resulted in a series of glycoconjugates with variations in the glycan size and degree of glycosylation. Thermodynamic unfolding, thermal inactivation, and temperature-induced aggregation experiments revealed that chemical glycosylation increased protein thermodynamic (Delta G(25 degrees C)), kinetic (t(1/2)(45 degrees C)), and colloidal stability. These results highlight the potential of chemical glycosylation with monofunctional activated glycans as a technology for increasing the long-term stability of liquid protein formulations for industrial and biotherapeutic applications.
PET imaging with radiolabeled drugs provides information on tumor uptake and dose-dependent target interaction to support selection of an optimal dose for future efficacy testing. In this immuno-PET study of the anti–human epidermal growth factor receptor (HER3) mAb GSK2849330, we investigated the biodistribution and tumor uptake of 89 Zr-labeled GSK2849330 and evaluated target engagement as a function of antibody mass dose. Methods: 89 Zr-GSK2849330 distribution was monitored in 6 patients with HER3-positive tumors not amenable to standard treatment. Patients received 2 administrations of 89 Zr-GSK2849330. Imaging after tracer only was performed at baseline; dose-dependent inhibition of 89 Zr-GSK2849330 uptake in tumor tissues was evaluated 2 wk later using increasing doses of unlabeled GSK2849330 in combination with the tracer. Up to 3 PET scans (2 hours post infusion [p.i.] and days 2 and 5 p.i.) were performed after tracer administration. Biodistribution and tumor targeting were assessed visually and quantitatively using SUV. The 50% and 90% inhibitory mass doses (ID 50 and ID 90 ) of target-mediated antibody uptake were calculated using a Patlak transformation. Results: At baseline, imaging with tracer showed good tumor uptake in all evaluable patients. Predosing with unlabeled mAb reduced the tumor uptake rate in a dose-dependent manner. Saturation of 89 Zr-mAb uptake by tumors was seen at the highest dose (30 mg/kg). Despite the limited number of patients, an exploratory ID 50 of 2 mg/kg and ID 90 of 18 mg/kg have been determined. Conclusion: In this immuno-PET study, dose-dependent inhibition of tumor uptake of 89 Zr-GSK2849330 by unlabeled mAb confirmed target engagement of mAb to the HER3 receptor. This study further validates the use of immuno-PET to directly visualize tissue drug disposition in patients with a noninvasive approach and to measure target engagement at the site of action, offering the potential for dose selection.
Fourier transform infrared (FTIR) spectroscopy has emerged as a powerful tool to guide the development of stable lyophilized protein formulations by providing information on the structure of proteins in amorphous solids. The underlying assumption is that IR spectral changes in the amide I and III region upon protein dehydration are caused by protein structural changes. However, it has been claimed that amide I IR spectral changes could be the result of water removal per se. Here, we investigated whether such claims hold true. The structure of horseradish peroxidase (HRP) and poly(ethylene glycol)-modified HRP (HRP-PEG) has been investigated under various conditions (in aqueous solution, the amorphous dehydrated state, and dissolved/suspended in toluene and benzene) by UV-visible (UV-Vis), FTIR, and resonance Raman spectroscopy. The resonance Raman and UV-Vis spectra of dehydrated HRP-PEG dissolved in neat toluene or benzene were very similar to that of HRP in aqueous buffer, and thus the heme environment (heme iron spin, coordination, and redox state) was essentially the same under both conditions. Therefore, the three-dimensional structure of HRP-PEG dissolved in benzene and toluene was similar to that in aqueous solution. The amide I IR spectra of HRP-PEG in aqueous buffer and of dehydrated HRP-PEG dissolved in neat benzene and toluene were also very similar, and the secondary structure compositions (percentages of alpha-helices and beta-sheets) were within the standard error the same. These results are irreconcilable with recent claims that water removal per se could cause substantial amide I IR spectral changes (M. van de Weert, P.I. Haris, W.E. Hennink, and D.J. Crommelin. 2001. Anal. Biochem. 297:160-169). On the contrary, amide I IR spectral changes upon protein dehydration are caused by perturbations in the secondary structure.
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