RESUMEN
This review is in support of the development of selective, precise, fast, and validated capillary electrophoresis (CE) methods. It follows up a similar article from 1998, Wätzig H, Degenhardt M, Kunkel A. "Strategies for capillary electrophoresis: method development and validation for pharmaceutical and biological applications," pointing out which fundamentals are still valid and at the same time showing the enormous achievements in the last 25 years. The structures of both reviews are widely similar, in order to facilitate their simultaneous use. Focusing on pharmaceutical and biological applications, the successful use of CE is now demonstrated by more than 600 carefully selected references. Many of those are recent reviews; therefore, a significant overview about the field is provided. There are extra sections about sample pretreatment related to CE and microchip CE, and a completely revised section about method development for protein analytes and biomolecules in general. The general strategies for method development are summed up with regard to selectivity, efficiency, precision, analysis time, limit of detection, sample pretreatment requirements, and validation.
Asunto(s)
Electroforesis Capilar , Electroforesis por Microchip , Electroforesis Capilar/métodos , Proteínas , Preparaciones FarmacéuticasRESUMEN
Nucleic acids are the blueprint of life. They are not only the construction plan of the single cell or higher associations of them, but also necessary for function, communication and regulation. Due to the pandemic, the attention shifted in particular to their therapeutic potential as a vaccine. As pharmaceutical oligonucleotides are unique in terms of their stability and application, special delivery systems were also considered. Oligonucleotide production systems can vary and depend on the feasibility, availability, price and intended application. To achieve good purity, reliable results and match the strict specifications in the pharmaceutical industry, the separation of oligonucleotides is always essential. Besides the separation required for production, additional and specifically different separation techniques are needed for analysis to determine if the product complies with the designated specifications. After a short introduction to ribonucleic acids (RNAs), messenger RNA vaccines, and their production and delivery systems, an overview regarding separation techniques will be provided. This not only emphasises electrophoretic separations but also includes spin columns, extractions, precipitations, magnetic nanoparticles and several chromatographic separation principles, such as ion exchange chromatography, ion-pair reversed-phase, size exclusion and affinity.
Asunto(s)
Ácidos Nucleicos , Oligonucleótidos , Oligonucleótidos/análisis , Cromatografía por Intercambio Iónico/métodos , Cromatografía Líquida de Alta Presión/métodos , Preparaciones FarmacéuticasRESUMEN
Today proteins are possibly the most important class of substances. Yet new tasks for proteins are still often solved by trial-and-error approaches. However, in some areas these euphemistically called "screening approaches" are not suitable. E.g. stability tests just take too long and therefore require a more strategic, target-orientated concept. This concept is available by grouping proteins according to their physicochemical properties and then pulling out the right drawer for new tasks. These properties include size, then charge and hydrophobicity as well as their patchinesses, and the degree of order. In addition, solubility, the content of (free) enthalpy, aromatic-amino-acid- and α/ß-frequency as well as helix capping, and corresponding patchiness, the number of specific motifs and domains as well as the typical concentration range can be helpful to discriminate between different groups of proteins. Analyzing correlations will reduce the necessary amount of parameters and additional ones, which may be still undiscovered at the present time, can be identified looking at protein subgroups with similar physicochemical properties which still behave heterogeneously. Step-by-step the methodology will be improved. Possibly protein stability will be the driver of this process, but all other areas such as production, purification and analytics including sample pre-treatment and the choice of appropriate separation conditions for e.g. chromatography and electrophoresis will profit from a rational strategy.
Asunto(s)
Proteínas/análisis , Aminoácidos/análisis , Cromatografía Liquida/métodos , Interacciones Hidrofóbicas e Hidrofílicas , Espectrometría de Masas/métodos , Proteínas/química , Solubilidad , TermodinámicaRESUMEN
Purification of recombinant proteins is often a challenging matter because high purity and high recovery are desired. If the expressed recombinant protein is also in a complex matrix, such as from the silkworm expression system, purification becomes more challenging. Even if purification from the silkworm expression system is troublesome, it benefits from a high capacity for the production of recombinant proteins. In this study, magnetic nanoparticles (MNPs) were investigated as a suitable tool for the purification of proteins from the complex matrix of the silkworm fat body. The MNPs were modified with nickel so that they have an affinity for His-tagged proteins, as the MNP purification protocol itself does not need special equipment except for a magnet. Among the three different kinds of investigated MNPs, MNPs with sizes of 100 nm to 200 nm and approximately 20 nm-thick nickel shells were the most suitable for our purpose. With them, the total protein amount was reduced by up to at least approximately 77.7%, with a protein recovery of around 50.8% from the silkworm fat body. The minimum binding capacity was estimated to be 83.3 µg protein/mg MNP. Therefore, these MNPs are a promising tool as a purification pretreatment of complex sample matrices.
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Bombyx/metabolismo , Cromatografía de Afinidad/métodos , Cuerpo Adiposo/química , Nanopartículas de Magnetita/química , Animales , Escherichia coli/genética , Magnetismo , Níquel , Fenómenos Físicos , Proteínas RecombinantesRESUMEN
The material properties of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its proteins are discussed. We review the viral structure, size, rigidity, lipophilicity, isoelectric point, buoyant density and centrifugation conditions, stability against pH, temperature, UV light, gamma radiation, and susceptibility to various chemical agents including solvents and detergents. Possible inactivation, downstream, and formulation conditions are given including suitable buffers and some first ideas for quality-control methods. This information supports vaccine development and discussion with competent authorities during vaccine approval and is certainly related to drug-targeting strategies and hygienics. Several instructive tables are given, including the pI and grand average of hydropathicity (GRAVY) of SARS-CoV-1 and -2 proteins in comparison. SARS-CoV-1 and SARS-CoV-2 are similar in many regards, so information can often be derived. Both are unusually stable, but sensitive at their lipophilic membranes. However, since seemingly small differences can have strong effects, for example, on immunologically relevant epitope settings, unevaluated knowledge transfer from SARS-CoV-1 to SARS-CoV-2 cannot be advised. Published knowledge regarding downstream processes, formulations and quality assuring methods is, as yet, limited. However, standard approaches employed for other viruses and vaccines seem to be feasible including virus inactivation, centrifugation conditions, and the use of adjuvants.
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Betacoronavirus/química , Proteínas Virales/química , Vacunas Virales/farmacología , Animales , Betacoronavirus/efectos de los fármacos , Betacoronavirus/efectos de la radiación , Desinfectantes/farmacología , Electroforesis , Calor , Humanos , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , SARS-CoV-2 , Rayos Ultravioleta , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/farmacología , Vacunas Virales/inmunología , Inactivación de Virus/efectos de la radiaciónRESUMEN
The silkworm, Bombyx mori, is a promising expression system for the production of recombinant proteins, but the purification of these proteins is not easy because of the large amount of host proteins present. To investigate purity, recovery and scale-up ability of the purification of recombinant proteins expressed in silkworm larval hemolymph without any affinity tags, we used mCherry, a red fluorescence protein, as a model. The host cell proteins could be greatly reduced using a three-step chromatography protocol consisting of hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC) and heparin chromatography after heat pretreatment. The thermal treatment had the greatest impact on the removal of host cell extracellular proteins and increasing purity. There were still some minor traces of host cell proteins in the purified sample, which showed that the purification of recombinant proteins from the silkworm hemolymph was still challenging. The proposed protocol and affinity tag purification reduced the overall protein content by 99.84% and 99.95%, respectively, while the amount of DNA was reduced by 98.41% and 99.53%, respectively. Purities of our proposed protocol based on SDS-PAGE and capillary electrophoresis (CE) analyses were 85.45% and 43.60%, respectively, while those of Strep-tag affinity purification were 100% or 63.69%, respectively. Using densitometry, the overall recovery was calculated was 5.78%, which was higher than 4.09% using Strep-tag affinity purification. This proposed protocol, mainly based on thermal treatment, HIC, SEC and HiTrap Heparin HP column chromatography, is applicable to an upscalable purification for the silkworm expression system without employing affinity tag chromatography process.
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Bombyx/química , Cromatografía de Afinidad/métodos , Hemolinfa/química , Larva/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Animales , Bombyx/metabolismo , Electroforesis en Gel de Poliacrilamida , Larva/metabolismo , Oligopéptidos/genética , Oligopéptidos/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genéticaRESUMEN
A three-stage chromatography protocol for the purification of human papillomavirus-like particles (HPV-LPs) from the silkworm-based Bombyx mori nucleopolyhedrovirus bacmid expression system was developed. For host cell DNA separation, anion exchange chromatography was used after screening for a suitable stationary phase. Using the two separation principles of cation exchange chromatography and metal affinity of ceramic hydroxyapatite (CHT) as a second stage, the amount of baculovirus in the sample was reduced to less than the detection limit of qPCR. The CHT separation was optimized with respect to the elution buffer used; 150-600â¯mM sodium phosphate, pHâ¯7.2, resulted in the highest recovery of HPV-LPs. Using heparin chromatography, it was possible to reduce the sample volume and to thus highly concentrate the target protein during the separation of contaminating proteins. During the second purification stage, over 99.3% of the DNA was removed, and no infectious baculoviruses remained. After concentration by heparin column chromatography, over 99.9% of the DNA and protein had been removed. The purity achieved by this method exceeds that obtained by DDDDK-tag-based affinity chromatography and sucrose gradient ultracentrifugation, which were used as comparative purification methods. The 3-stage purification of HPV-LPs from silkworm fat bodies described here was a proof of concept and is a scalable method, but the overall yield remains to be improved.
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Bombyx/genética , Nucleopoliedrovirus/metabolismo , Papillomaviridae/aislamiento & purificación , Virión/aislamiento & purificación , Cultivo de Virus/métodos , Animales , Bombyx/metabolismo , Cromatografía por Intercambio Iónico , Nucleopoliedrovirus/genética , Papillomaviridae/química , Virión/químicaRESUMEN
Virus-like particles (VLPs) are a promising and developing option for vaccination and gene therapy. They are also interesting as shuttles for drug targeting. Currently, several different gene expression systems are available, among which the silkworm expression system is known for its mass production capacity. However, cost-effective purification with high purity of the target protein is a particular bottleneck for this system. The present review evaluates the advances in the purification of VLPs, especially from silkworm larval hemolymph. Beginning with applicable pre-treatments for VLPs over to chromatography methods and quality control of the purified VLPs. Whereupon the main focus is on the different chromatography approaches for the purification, but the structure of the VLPs and their intended use for humans make also the quality control important. Within this, the stability of the VLPs which has to be considered for the purification is as well discussed.