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Measuring and Increasing Protein Solubility
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High concentration protein delivery is difficult to achieve for several protein pharmaceuticals due to low solubility. In this review, we discuss different types of low protein solubility, including low in vitro solubility, which is relevant to the formulation of protein pharmaceuticals. We also discuss different methods of measuring protein solubility with an emphasis on the method of inducing amorphous precipitation using ammonium sulfate. Finally, we discuss strategies for increasing protein solubility, including site-directed mutagenesis. Evidence from solubility-changing mutations in the literature indicate that some hydrophilic residues (aspartic acid, glutamic acid, and serine) contribute significantly more favorably to protein solubility than other hydrophilic residues (asparagine, glutamine, threonine, lysine, and arginine). These findings should prove useful especially in cases where protein structure is not known. In these cases, instead of targeting hydrophobic residues that are often buried, one could target hydrophilic residues that do not contribute favorably to protein solubility and replace them with hydrophilic residues that contribute more favorably.
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... Moreover, Glu can also increase the helicity of proteins, which is crucial for the binding and folding of proteins [75]. The analysis of amino acid substitutions by Trevino and colleagues revealed that the Glu residue significantly contributes to protein solubility compared to other residues, such as Asp, Ser, Gln, Thr and Arg [76,77]. In a mAb formulation, equimolar concentrations of Asp-Glu can increase mAb stability at weakly acidic and neutral pH due to Arg-to-Glu interactions in the solution [78]. ...
As the demand for immunotherapy to treat and manage cancers, infectious diseases and other disorders grows, a comprehensive understanding of amino acids and their intricate role in antibody engineering has become a prime requirement. Naturally produced antibodies may not have the most suitable amino acids at the complementarity determining regions (CDR) and framework regions, for therapeutic purposes. Therefore, to enhance the binding affinity and therapeutic properties of an antibody, the specific impact of certain amino acids on the antibody’s architecture must be thoroughly studied. In antibody engineering, it is crucial to identify the key amino acid residues that significantly contribute to improving antibody properties. Therapeutic antibodies with higher binding affinity and improved functionality can be achieved through modifications or substitutions with highly suitable amino acid residues. Here, we have indicated the frequency of amino acids and their association with the binding free energy in CDRs. The review also analyzes the experimental outcome of two studies that reveal the frequency of amino acids in CDRs and provides their significant correlation between the outcomes. Additionally, it discusses the various bond interactions within the antibody structure and antigen binding. A detailed understanding of these amino acid properties should assist in the analysis of antibody sequences and structures needed for designing and enhancing the overall performance of therapeutic antibodies.
... budesonide, formoterol, etc.) chemical drugs and biotherapeutics (e.g. proteins and peptides, etc.) to the targeted regions in airway [99,[150][151][152], for the treatment of local (e.g. asthma, COPD, lung cancer etc.) and systemic diseases (e.g. ...
... El efecto significativo del pH y la fuerza iónica presente sobre la solubilidad de las proteínas y en el empleo de los intercambiadores catiónicos se corresponde con lo reportado en las literaturas. (8,18) La proteína RBDhis (aa 319-541) tiende a presentar menor estabilidad a medida que se disminuye el pH de 7 a 5. Esto puede estar asociado a que con el aumento del número de grupos cargados en una proteína (a un pH alejado del punto isoeléctrico (pI= 8,4)), el aumento de los efectos electrostáticos dentro de la proteína desestabiliza la conformación plegada debido a que la densidad de carga en la proteína plegada es mayor que en el estado Fig. 1. Superficie respuesta estimada de la condición de adsorción de la etapa de captura a partir de la combinación de los factores que maximiza el criterio de deseabilidad. ...
The urgent need to develop and produce safe and effective vaccines to guarantee the reduction of the spread of the type 2 coronavirus that causes severe acute respiratory syndrome, led the Center for Molecular Immunology and the Finlay Vaccine Institute to develop two vaccines and one candidate vaccine to combat the 2019 coronavirus pandemic. As part of the establishment of the production process, experiments were carried out on the possible steps of the purification process of the receptor binding domain molecule (aa 319-541) with a view to its subsequent technological transfer on an industrial scale. This molecule is fused with a hexahistidine tag at its C-terminal end and has nine cysteine residues in its sequence that form four intramolecular disulfide bonds; leaving a free cysteine that allows two molecules to be obtained: dimeric and monomeric, which constitute the antigens of the SOBERANA®02 and SOBERANA®Plus vaccines and the SOBERANA 01 vaccine candidate. The best adsorption conditions of the chromatographic matrices of affinity for metal chelates, cationic exchange and molecular exclusion were determined. The performance of the process was evaluated on a pilot scale and the molecule was characterized according to its physical-chemical and biological properties. The results obtained showed a 60.02 ± 5.15% total recovery of the protein of interest with more than 98% purity in both molecules, an efficient removal of contaminants and an antigenicity greater than 90% referred to the control monomer of the domain receptor binding with 99% purity; which demonstrates that the established process is efficient in obtaining a product with the required quality.
... El efecto significativo del pH y la fuerza iónica presente sobre la solubilidad de las proteínas y en el empleo de los intercambiadores catiónicos se corresponde con lo reportado en las literaturas. (8,18) La proteína RBDhis (aa 319-541) tiende a presentar menor estabilidad a medida que se disminuye el pH de 7 a 5. Esto puede estar asociado a que con el aumento del número de grupos cargados en una proteína (a un pH alejado del punto isoeléctrico (pI= 8,4)), el aumento de los efectos electrostáticos dentro de la proteína desestabiliza la conformación plegada debido a que la densidad de carga en la proteína plegada es mayor que en el estado Fig. 1. Superficie respuesta estimada de la condición de adsorción de la etapa de captura a partir de la combinación de los factores que maximiza el criterio de deseabilidad. ...
La urgente necesidad de desarrollar y producir vacunas seguras y efectivas para garantizar la reducción de la propagación del coronavirus de tipo 2 causante del síndrome respiratorio agudo severo, hizo que el Centro de Inmunología Molecular y el Instituto Finlay de Vacunas, desarrollaran dos vacunas y un candidato vacunal contra la COVID-19, que tienen como componente la molécula del dominio de unión al receptor (aa 319-541) del virus. Para establecer el proceso productivo, se realizaron experimentos en los posibles pasos del proceso de purificación de la molécula del dominio de unión al receptor (aa 319-541), con vistas a su posterior transferencia tecnológica a escala industrial. Dicha molécula está fusionada con una etiqueta de hexahistidina en su extremo C-terminal y presenta nueve residuos de cisteína en su secuencia que forman cuatro enlaces disulfuros intramoleculares, quedando una cisteína libre que permite obtener dos moléculas: dimérica y monomérica, antígenos que forman parte de las vacunas SOBERANA®02 y SOBERANA®Plus y el candidato vacunal SOBERANA 01. Se determinaron las mejores condiciones de adsorción de las matrices cromatográficas de afinidad por quelatos metálicos, intercambio catiónico y exclusión molecular. Se evaluó el desempeño del proceso a escala piloto y se caracterizó la molécula de acuerdo a sus propiedades físico-químicas y biológicas. Los resultados obtenidos mostraron un 60,02 ± 5,15% de recuperación total de la proteína de interés, con más del 98% de pureza en ambas moléculas, una eficiente remoción de contaminantes y una antigenicidad mayor del 90% referido al monómero control del dominio de unión al receptor con 99% de pureza, lo que demuestra que el proceso establecido es eficiente en la obtención de un producto con la calidad requerida.
... Despite a protein being expressed in a soluble and folded state, the protein in its isolated state may still form aggregates when possessing low intrinsic, in vitro solubility [24,25]. The taCA enzyme, known for its low in vitro solubility, aggregates under low-salt buffer conditions [18]. ...
Carbonic anhydrase (CA), an enzyme catalyzing the reversible hydration reaction of carbon dioxide (CO2), is considered a promising biocatalyst for CO2 reduction. The α-CA of Thermovibrio ammonificans (taCA) has emerged as a compelling candidate due to its high thermostability, a critical factor for industrial applications. However, the low-level expression and poor in vitro solubility have hampered further utilization of taCA. Recently, these limitations have been addressed through the fusion of the NEXT tag, a marine-derived, intrinsically disordered small peptide that enhances protein expression and solubility. In this study, the solubility and stability of NEXT-taCA were further investigated. When the linker length between the NEXT tag and the taCA was shortened, the expression level decreased without compromising solubility-enhancing performance. A comparison between the NEXT tag and the NT11 tag demonstrated the NEXT tag’s superiority in improving both the expression and solubility of taCA. While the thermostability of taCA was lower than that of the extensively engineered DvCA10, the NEXT-tagged taCA exhibited a 30% improvement in long-term thermostability compared to the untagged taCA, suggesting that enhanced solubility can contribute to enzyme thermostability. Furthermore, the bioprospecting of two intrinsically disordered peptides (Hcr and Hku tags) as novel solubility-enhancing fusion tags was explored, demonstrating their performance in improving the expression and solubility of taCA. These efforts will advance the practical application of taCA and provide tools and insights for enzyme biochemistry and bioengineering.
... [21] [22] [23] [24] [25] [26] [27] [10] [28] [29] [30,31] 6 2 2 [6] [32] ...
In silico protein research has been a focus for a long time, while its recent combination with machine learning gives great contributions to related areas. This review mainly focuses on four major fields of in silico protein research that combine with machine learning, which are molecular dynamics, structure prediction, property prediction and molecule design. Molecular dynamics depends on force field parameters, which is necessary for accurate results. Machine learning can help to construct satisfied force field parameters. In molecular dynamics, machine learning can also help to do the free energy calculation by a relatively low cost. Structure prediction is usually predicting the structure of given sequence. Structure prediction is of high complexity and data amount, which is what machine learning good at. By the help of machine learning, scientists have gained great achievements in 3D structure prediction of proteins. On the other hand, predicting proteins’ properties based on their known information is also important to protein research. The most challenging, however, is molecule design. Though drug-like small molecule design and protein design have achieved much in recent years, this field is still remain to be explored. This review focuses on the above three areas and make an outlook to in silico protein research with machine learning.
... The development of high-concentration biologic formulations can be challenging due to their physical instability at higher concentrations, including limited solubility, phase separation, opalescence, irreversible aggregation, 33,112 high solution viscosity during manufacturing, 113,114 intermolecular PPIs, 115,116 and clinical performance issues such as bioavailability and immunogenicity. 12,29,117 As with other protein-based biotherapeutics, biologics are subject to physical instability throughout development, including during processing, formulation, storage, and distribution. ...
Biologic drugs are used to treat a variety of cancers and chronic diseases. While most of these treatments are administered intravenously by trained healthcare professionals, a noticeable trend has emerged favoring subcutaneous (SC) administration. SC administration of biologics poses several challenges. Biologic drugs often require higher doses for optimal efficacy, surpassing the low volume capacity of traditional SC delivery methods like autoinjectors. Consequently, high concentrations of active ingredients are needed, creating time-consuming formulation obstacles. Alternatives to traditional SC delivery systems are therefore needed to support higher-volume biologic formulations and to reduce development time and other risks associated with high-concentration biologic formulations. Here, we outline key considerations for SC biologic drug formulations and delivery and explore a paradigm shift: the flexibility afforded by low-to-moderate-concentration drugs in high-volume formulations as an alternative to the traditionally difficult approach of high-concentration, low-volume SC formulation delivery.
