Development Of Protein Pharmaceuticals Research Articles

Plant expression systems, a budding way to confront chikungunya and Zika in developing countries?

Plant expression systems could be used as biofactories of heterologous proteins that have the potential to be used with biopharmaceutical aims and vaccine design. This technology is scalable, safe and cost-effective and it has been previously proposed as an option for vaccine and protein pharmaceutical development in developing countries. Here we present a proposal of how plant expression systems could be used to address Zika and chikungunya outbreaks through development of vaccines and rapid diagnostic kits.

Plant expression systems, a budding way to confront chikungunya and Zika in developing countries?

Plant expression systems could be used as biofactories of heterologous proteins that have the potential to be used with biopharmaceutical aims and vaccine design. This technology is scalable, safe and cost-effective and it has been previously proposed as an option for vaccine and protein pharmaceutical development in developing countries. Here we present a proposal of how plant expression systems could be used to address Zika and chikungunya outbreaks through development of vaccines and rapid diagnostic kits.

Development of dextran nanoparticles for stabilizing delicate proteins

One of the most challenging problems in the development of protein pharmaceuticals is to deal with stabilities of proteins due to its complicated structures. This study aims to develop a novel approach to stabilize and encapsulate proteins into dextran nanoparticles without contacting the interface between the aqueous phase and the organic phase. The bovine serum albumin, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), β-galactosidase, and myoglobin were selected as model proteins. The proteins were added into an aqueous solution containing the dextran and polyethylene glycol, and then encapsulated into dextran nanoparticles by aqueous-aqueous freezing-induced phase separation. The encapsulation efficiency and recovery of dextran nanoparticles were determined. The dextran nanoparticles loaded with proteins were characterized by scanning electron microscopy and particle size analysis. The protein aggregation was determined by size-exclusion chromatography-high-performance chromatography, and the bioactivity of proteins recovered during formulation steps was determined. The bioactivity of GM-CSF, G-CSF, and β-galactosidase were examined by the proliferation of TF-1 cell, NSF-60 cell, and ortho-nitrophenyl-β-galactoside assay, respectively. The results of bioactivity recovered show that this novel dextran nanoparticle can preserve the protein's bioactivity during the preparation process. LysoSensor™ Yellow/Blue dextran, a pH-sensitive indicator with fluorescence excited at two channels, was encapsulated into dextran nanoparticles to investigate the ability of dextran nanoparticles to resist the acidic microenvironment (pH < 2.5). The result shows that the dextran nanoparticles attenuate the acidic microenvironment in the poly (lactic-co-glycolic acid) microsphere by means of the dilution effect. These novel dextran nanoparticles provided an appealing approach to stabilize the delicate proteins for administration.

Shaking and stirring: Comparison of controlled laboratory stress conditions applied to the human growth hormone

Shaking or stirring in a miniaturized device is often applied in the development of protein pharmaceuticals, serving as a test of stability under physical stress conditions also occurring in the real process. The overall purpose of these “stress tests” is to accelerate protein aggregation that could otherwise take place at a much slower rate, thereby enhancing experimental throughput to speed up determination of critical process parameters of stability. Results are useful to determine critical parameters for formulation development. However, shaking differs from stirring in the forces applied on proteins in solution and therefore, there is the possibility that characteristics of protein stability interrogated with each method may not be the same. We have performed a detailed, time-resolved analysis of aggregation of the human growth hormone (hGH), exposed to stirring and shaking in a well-defined and comparable mini-reactor set-up. We show that aggregation of hGH, which in its native form is a monomer, is not the result of protein deamidation, a chemical protein modification that occurs in hGH under the incubation conditions used. We also show that both under stirring and shaking, hGH appears to precipitate into mainly spherical particles in the presence of only small amounts of soluble protein oligomers. The precipitate appears to contain protein that has a substantial amount of hydrophobic surface exposed. An air–liquid interface, constantly renewed by stirring or shaking, was identified to be mainly responsible for aggregation of hGH under each of the stress conditions applied. In hGH, therefore, controlled shaking and stirring lead to identical conclusions about the overall protein stability and the main critical parameter driving the aggregation, thus informing stabilization strategies and formulation development.

Xenon Hydrate Dissociation Measurements With Model Protein Systems

Effective long-term storage remains a significant challenge to the use and development of protein pharmaceuticals. We have investigated the interactions between clathrate hydrates and model protein solutions to determine the effects on hydrate formation. Here, the dissociation curve and equilibrium conditions for xenon clathrate hydrate with model lysozyme and lactate dehydrogenase (LDH) protein solutions have been studied using calorimetry measurements at pressures ranging from 3 to 20 bar. Sucrose in solution was shown to exhibit small inhibition effects on xenon hydrate formation, shifting the dissociation curve and decreasing the conversion of water to hydrate by 15-26%. The addition of l-histidine buffer and lysozyme at low concentrations did not substantially inhibit hydrate formation. However, small shifts in the dissociation curve were demonstrated for solutions containing LDH. The presence of lysozyme and LDH in solution did not significantly alter the conversion of water to hydrate, indicating that these and similar proteins do not substantially affect the extent of xenon gas hydrate formation. Preliminary experiments were performed for LDH solutions to assess the impact of xenon hydrate formation and dissociation on enzymatic activity, with samples stored in hydrate systems showing small decreases in activity.

The relation between moisture-induced aggregation and structural changes in lyophilized insulin

Long-term stability is a critical factor in the successful development of protein pharmaceuticals. Due to the relative instability of proteins in aqueous solutions, they are formulated frequently and stored as lyophilized powders. Exposure of such powders to moisture constitutes a substantial storage problem leading to aggregation and inactivation. We have investigated the structural consequences of moisture sorption by lyophilized insulin under controlled humidity conditions by employing Fourier transform-infrared (FT-IR) microscopy. Lyophilized insulin samples were stored in humidity chambers under controlled conditions at 50(o)C. Protein aggregation studies were carried out by redissolving the insulin samples and measuring the amount of both soluble protein and insoluble aggregates. Near-UV circular dichroism spectra were collected to assess the tertiary structure. FT-IR microscopy studies were carried out to investigate secondary structural changes in solid-state insulin after incubation at different relative humidities. It was found that sorption of moisture was accompanied by small structural changes in lyophilized insulin at low levels of relative humidity (i.e. 11%). At higher relative humidity levels, structural changes were becoming more pronounced and were characterized by a loss in the alpha-helix and increase in beta-sheet content. The magnitude of the structural changes in tendency paralleled the solid-state instability data (i.e. formation of buffer-insoluble aggregates and loss in tertiary structure upon reconstitution). The results support the hypothesis that water sorption by lyophilized proteins enables structural transitions which can lead to protein aggregation and other deleterious phenomena.

Chemo-Enzymatic Detection of Protein Isoaspartate Using Protein Isoaspartate Methyltransferase and Hydrazine Trapping

Isoaspartate formation is a ubiquitous post-translation modification arising from spontaneous asparagine deamidation or aspartate isomerization. The formation of isoaspartate inserts a methylene group into the protein backbone, generating a "kink", and may drastically alter protein structure and function, thereby playing critical roles in a myriad of biological processes, human diseases, and protein pharmaceutical development. Herein, we report a chemo-enzymatic detection method for the isoaspartate protein, which in particular allows the affinity enrichment of isoaspartate-containing proteins. In the initial step, protein isoaspartate methyltransferase selectively converts isoaspartates into the corresponding methyl esters. Subsequently, the labile methyl ester is trapped by strong nucleophiles in aqueous solutions, such as hydrazines to form hydrazides. The stable hydrazide products can be analyzed by standard proteomic techniques, such as matrix-assisted laser desorption ionization and electrospray ionization mass spectrometry. Furthermore, the chemical trapping step allows us to introduce several tagging strategies for product identification and quantification, such as UV-vis and fluorescence detection through a dansyl derivative. Most significantly, the hydrazide product can be enriched by affinity chromatography using aldehyde resins, thus drastically reducing sample complexity. Our method hence represents the first technique for the affinity enrichment of isoaspartyl proteins and should be amendable to the systematic and comprehensive characterization of isoaspartate, particularly in complex systems.

Quantitation of aggregates in therapeutic proteins using sedimentation velocity analytical ultracentrifugation: Practical considerations that affect precision and accuracy

Aggregation is a major degradation pathway that needs to be characterized and controlled during the development of protein pharmaceuticals. Analytical ultracentrifugation–sedimentation velocity (AUC–SV) is emerging as an important orthogonal tool to size exclusion chromatography to quantitate aggregates. However, the precision and accuracy of modern AUC–SV and the experimental variables that influence these two performance parameters need to be better understood and controlled. To understand the impact of experimental and data analysis variables on the precision, aggregate content in monoclonal antibody preparations was measured by AUC–SV and analyzed by the software program Sedfit. Accuracy and limit of detection were evaluated by spiking a known quantity of a sample enriched in aggregate fraction. The results suggest experimental and data analysis approaches that improve precision and accuracy of aggregate quantitation by AUC–SV. Both precision and accuracy were found to be highly dependent on the quality of the centerpieces as assessed by microscopic examination. The level of precision for quantitating aggregates was found to be approximately ±0.3 to 0.7% over the aggregate content range of approximately 0.6 to 67%. Accuracy, as indicated by approximately 80 to 90% spiked recovery, could be achieved down to aggregate levels as low as approximately 1.5%, whereas the limits of detection and quantitation appear to be approximately 0.2 and 0.6%, respectively.

Peroxide formation in polysorbate 80 and protein stability

Nonionic surfactants are widely used in the development of protein pharmaceuticals. However, the low level of residual peroxides in surfactants can potentially affect the stability of oxidation‐sensitive proteins. In this report, we examined the peroxide formation in polysorbate 80 under a variety of storage conditions and tested the potential of peroxides in polysorbate 80 to oxidize a model protein, IL‐2 mutein. For the first time, we demonstrated that peroxides can be easily generated in neat polysorbate 80 in the presence of air during incubation at elevated temperatures. Polysorbate 80 in aqueous solution exhibited a faster rate of peroxide formation and a greater amount of peroxides during incubation, which is further promoted/catalyzed by light. Peroxide formation can be greatly inhibited by preventing any contact with air/oxygen during storage. IL‐2 mutein can be easily oxidized both in liquid and solid states. A lower level of peroxides in polysorbate 80 did not change the rate of IL‐2 mutein oxidation in liquid state but significantly accelerated its oxidation in solid state under air. A higher level of peroxides in polysorbate 80 caused a significant increase in IL‐2 mutein oxidation both in liquid and solid states, and glutathione can significantly inhibit the peroxide‐induced oxidation of IL‐2 mutein in a lyophilized formulation. In addition, a higher level of peroxides in polysorbate 80 caused immediate IL‐2 mutein oxidation during annealing in lyophilization, suggesting that implementation of an annealing step needs to be carefully evaluated in the development of a lyophilization process for oxidation‐sensitive proteins in the presence of polysorbate. © 2002 Wiley‐Liss Inc. and the American Pharmaceutical Association J Pharm Sci 91:2252–2264, 2002

Regulatory considerations in the development of protein pharmaceuticals.

In 1980, the first investigational new drug (IND) application for a biological product made by utilizing biotechnology was submitted to the U.S. Food and Drug Administration (FDA). It could be argued that biotechnology, as a discipline, was not new. Depending on the definition one selected, biotechnology could be considered as old as traditional beer- or bread-making, as indicated by one of the early definitions: “Biotechnology involves the application of biological systems to technical and industrial processes” (Bonk, 1987). However, as applied to production of pharmaceutical products, it was a new technology for products intended to diagnose and treat diseases or conditions in humans.KeywordsProduct DevelopmentBiological ProductGuidance DocumentLegal EntityBiotechnology ProductThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.