Chromatographic Method For Purification Research Articles

Reaction Optimization of Strontium Perchlorate Catalyzed Novel Protocol for Stereoselective Synthesis of Dihydropyrimidinones.

Hydrated strontium perchlorate [Sr(ClO4)2.3H2O] acts as a very strong oxidizing and dehydrating agent. Until now, it could not be reported as a catalyst in dehydration mechanism-based organic synthetic reactions. Therefore, it is important to find whether it could be an effective catalyst for one-pot multicomponent reactions (MCRs). The main objective of the present work is the development of a novel process for the synthesis of 1,4-dihydropyrimidinones through the one-pot multicomponent strategy using hydrated Sr(ClO4)2 as a catalyst. Furthermore, it includes process optimization, stereoselectivity, and spectroscopic characterization of the synthesized compounds. Conventional and microwave-supported synthesis of 1,4-dihydropyrimidinones using 20 mol % of hydrated Sr(ClO4)2 catalyst via the one-pot solvent-free reaction was discovered as a new catalytic MCR methodology. The box-Behnken design approach and advanced analytical techniques were used for process optimization and reaction analysis. The results confirmed that hydrated Sr(ClO4)2; works as an efficient catalyst for one-pot multicomponent organic synthesis under both conventional and microwave heating. It is an effective catalyst for laboratory synthesis of 1,4-dihydropyrimidinones stereoselectively with moderate to excellent yield without any undesirable effect. Microwave heating provided the desired product within 1-4 minutes. Moreover, this method provides easy isolation of the pure products simply by recrystallization, and without the use of a chromatographic purification method. The simplicity and neutrality of reaction conditions, easy post-reaction workup, higher satisfactory to excellent yield, effectiveness, the diversity of substrates, etc. render the hydrated Sr(ClO4)2 catalyst-based protocol for the stereoselective synthesis of 1,4-dihydropyrimidinones as a highly efficient method. Furthermore, it has been found to be safe un-der laboratory reaction conditions and no undesirable issues have been faced during the process.

An improved workflow for the development of MS-compatible liquid chromatography assay purity and purification methods by using automated LC Screening instrumentation and in silico modeling.

The development of liquid chromatography UV and mass spectrometry (LC-UV-MS) assays in pharmaceutical analysis is pivotal to improve quality control by providing critical information about drug purity, stability, and presence and identity of byproducts and impurities. Analytical method development of these assays is time-consuming, which often causes it to become a bottle neck in drug development and poses a challenge for process chemists to quickly improve the chemistry. In this study, a systematic and efficient workflow was designed to develop purity assay and purification methods for a wide range of compounds including peptides, proteins, and small molecules with MS-compatible mobile phases (MP) by using automated LC screening instrumentation and in silico modeling tools. Initial LC MPs and chromatography column screening experiments enabled quick identification of conditions which provided the best resolution in the vicinity of the target compounds, which is further optimized using computer-assisted modeling (LC Simulator from ACD/Labs). The experimental retention times were in good agreement with the predicted retention times from LC Simulator (ΔtR < 7%). This workflow presents a practical workflow to significantly expedite the time needed to develop optimized LC-UV-MS methods, allowing for a facile, automatic method optimization and reducing the amount of manual work involved in developing new methods during drug development.

A two-step chromatographic purification method for Ni for its isotopic analysis by MC-ICP-MS

The new purification procedure for Ni isotope analysis presented here is both economical and time-efficient, improving the ability for further investigation of Ni isotope geochemistry in various geological and environmental systems.

Comparison of Miniaturized Chromatographic Columns and 96-Well Plates for Automated Antibody Purification under Economic and Sustainable Aspects

Therapeutic antibodies are an area of major interest in treating infectious diseases, autoimmune diseases, and cancer. Efficient purification strategies are required to discover and produce antibodies, representing a significant cost driver. Miniaturization and automation of the chromatographic purification process may contribute to cost reduction. In this article, miniaturized chromatographic columns and 96-well filter plates are used to purify an IgG antibody from cell cultures and are compared in terms of quality, cost, and sustainability. The validation results of the two miniaturized chromatographic antibody purification methods indicate high efficacy and are comparable except for minor differences in yield. The lowest cost per sample can be obtained with the miniaturized chromatographic columns, as annual throughput can be maximized with the miniaturized plates. By evaluating sustainability, significant reductions in laboratory waste produced during antibody purification can be shown, along with an increase in employee safety by using the automation system. Overall, the comparison of results shows that, in the development of new purification strategies, the type of implementation has a decisive influence on the outcomes in terms of recovery, cost-effectiveness, and sustainability.

Chimeric Human Papillomavirus-16 Virus-like Particles Presenting HIV-1 P18I10 Peptide: Expression, Purification, Bio-Physical Properties and Immunogenicity in BALB/c Mice.

Human papillomavirus (HPV) vaccines based on HPV L1 virus-like particles (VLPs) are already licensed but not accessible worldwide. About 38.0 million people were living with HIV in 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against both viruses are an urgent need. In this study, the HIV-1 P18I10 CTL peptide from the V3 loop of HIV-1 gp120 glycoprotein was inserted into the HPV16 L1 protein to construct chimeric HPV:HIV (L1:P18I10) VLPs. Instead of the traditional baculovirus expression vector/insect cell (BEVS/IC) system, we established an alternative mammalian 293F cell-based expression system using cost-effective polyethylenimine-mediated transfection for L1:P18I10 protein production. Compared with conventional ultracentrifugation, we optimized a novel chromatographic purification method which could significantly increase L1:P18I10 VLP recovery (~56%). Chimeric L1:P18I10 VLPs purified from both methods were capable of self-assembling to integral particles and shared similar biophysical and morphological properties. After BALB/c mice immunization with 293F cell-derived and chromatography-purified L1:P18I10 VLPs, almost the same titer of anti-L1 IgG (p = 0.6409) was observed as Gardasil anti-HPV vaccine-immunized mice. Significant titers of anti-P18I10 binding antibodies (p < 0.01%) and P18I10-specific IFN-γ secreting splenocytes (p = 0.0002) were detected in L1:P18I10 VLP-immunized mice in comparison with licensed Gardasil-9 HPV vaccine. Furthermore, we demonstrated that insertion of HIV-1 P18I10 peptide into HPV16 L1 capsid protein did not affect the induction in anti-L1 antibodies. All in all, we expected that the mammalian cell expression system and chromatographic purification methods could be time-saving, cost-effective, scalable platforms to engineer bivalent VLP-based vaccines against HPV and HIV-1.

Automated Synthesis of [18F]Flumazenil Application in GABAA Receptor Neuroimaging Availability for Rat Model of Anxiety.

Clinical studies have demonstrated that the γ-aminobutyric acid type A (GABAA) receptor complex plays a central role in the modulation of anxiety. Conditioned fear and anxiety-like behaviors have many similarities at the neuroanatomical and pharmacological levels. The radioactive GABA/BZR receptor antagonist, fluorine-18-labeled flumazenil, [18F]flumazenil, behaves as a potential PET imaging agent for the evaluation of cortical damage of the brain in stroke, alcoholism, and for Alzheimer disease investigation. The main goal of our study was to investigate a fully automated nucleophilic fluorination system, with solid extraction purification, developed to replace traditional preparation methods, and to detect underlying expressions of contextual fear and characterize the distribution of GABAA receptors in fear-conditioned rats by [18F]flumazenil. A carrier-free nucleophilic fluorination method using an automatic synthesizer with direct labeling of a nitro-flumazenil precursor was implemented. The semi-preparative high-performance liquid chromatography (HPLC) purification method (RCY = 15-20%) was applied to obtain high purity [18F]flumazenil. Nano-positron emission tomography (NanoPET)/computed tomography (CT) imaging and ex vivo autoradiography were used to analyze the fear conditioning of rats trained with 1-10 tone-foot-shock pairings. The anxiety rats had a significantly lower cerebral accumulation (in the amygdala, prefrontal cortex, cortex, and hippocampus) of fear conditioning. Our rat autoradiography results also supported the findings of PET imaging. Key findings were obtained by developing straightforward labeling and purification procedures that can be easily adapted to commercially available modules for the high radiochemical purity of [18F]flumazenil. The use of an automatic synthesizer with semi-preparative HPLC purification would be a suitable reference method for new drug studies of GABAA/BZR receptors in the future.

CIM monolithic chromatography as a useful tool for endotoxin reduction and purification of bacteriophage particles supported with PAT analytics

Bacteriophages represent immense potential as therapeutic agents. Many of the most compelling applications of bacteriophages involve human therapy, some pertinent to gene therapy, others involving antibiotic replacement. Phages themselves are considered safe for humans. However, phage lysates may contain many kinds of harmful by-products, especially endotoxins of gram-negative bacteria and protein toxins produced by many pathogenic bacterial species. In bacteriophage research and therapy, most applications ask for highly purified phage suspensions, as such it is crucial to reduce proteins, endotoxins, DNA and other contaminants.In this article we present an efficient two-step chromatographic purification method for P. aeruginosa bacteriophage PP-01, using Convective Interaction Media (CIM®) monoliths, that is cGMP compliant and easy to scale-up for most stringent production of the therapeutic phage. First chromatographic step on CIMmultus OH resulted in 100% bacteriophage recovery with a reduction of 98 % protein and more than 99 % DNA content. Polishing was conducted using three different column options, CIMmultus with QA, H-Bond and PrimaS ligands. For PP-01 bacteriophage all three different options worked, but multimodal ligands H-Bond and PrimaS outperformed traditional QA in endotoxin removal (7 log step reduction). Additionally, an HPLC analytical method was developed to estimate phage concentration and impurity profile in different in-process samples. The HPLC method shows good correlation with drop assay titration, provides useful insights and can be run very fast with just 20 min per sample analysis.

Escherichia coli as a New Platform for the Fast Production of Vault-like Nanoparticles: An Optimized Protocol

Vaults are protein nanoparticles that are found in almost all eukaryotic cells but are absent in prokaryotic ones. Due to their properties (nanometric size, biodegradability, biocompatibility, and lack of immunogenicity), vaults show enormous potential as a bio-inspired, self-assembled drug-delivery system (DDS). Vault architecture is directed by self-assembly of the "major vault protein" (MVP), the main component of this nanoparticle. Recombinant expression (in different eukaryotic systems) of the MVP resulted in the formation of nanoparticles that were indistinguishable from native vaults. Nowadays, recombinant vaults for different applications are routinely produced in insect cells and purified by successive ultracentrifugations, which are both tedious and time-consuming strategies. To offer cost-efficient and faster protocols for nanoparticle production, we propose the production of vault-like nanoparticles in Escherichia coli cells, which are still one of the most widely used prokaryotic cell factories for recombinant protein production. The strategy proposed allowed for the spontaneous encapsulation of the engineered cargo protein within the self-assembled vault-like nanoparticles by simply mixing the clarified lysates of the producing cells. Combined with well-established affinity chromatography purification methods, our approach contains faster, cost-efficient procedures for biofabrication in a well-known microbial cell factory and the purification of "ready-to-use" loaded protein nanoparticles, thereby opening the way to faster and easier engineering and production of vault-based DDSs.

Optimized Sample Preparation and Microscale Separation Methods for High-Sensitivity Analysis of Hydrophilic Peptides.

The optimization of solid-phase extraction (SPE) purification and chromatographic separation is usually neglected during proteomics studies. However, the effects on detection performance are not negligible, especially when working with highly glycosylated samples. We performed a comparative study of different SPE setups, including an in-house optimized method and reversed-phase chromatographic gradients for the analysis of highly glycosylated plasma fractions as a model sample for glycopeptide analysis. The in-house-developed SPE method outperformed the graphite-based and hydrophilic interaction liquid chromatography (HILIC) purification methods in detection performance, recovery, and repeatability. During optimization of the chromatography, peak distribution was maximized to increase the peptide detection rate. As a result, we present sample purification and chromatographic separation methods optimized for the analysis of hydrophilic samples, the most important of which is heavily N-glycosylated protein mixtures.

Binding of Coxsackievirus A21 procapsids to immobilized glutathione depends on cell culture conditions during infection

A prototype strain of Coxsackievirus A21 (CVA21) is under clinical evaluation as an oncolytic virus immunotherapy. To improve scalability of the manufacturing process, an affinity chromatography purification method was developed using immobilized glutathione resin that captured infectious CVA21 virions from cell culture harvests with high recovery and impurity clearance. Unexpectedly, the binding of empty CVA21 procapsids depended on production cell culture conditions during infection including temperature, presence of serum in the media, and production cell line. At 37 °C and 2% serum during infection, procapsids flowed-through while infectious virions bound and were recovered at >95% yield in the chromatography elution. However, at sub-physiological temperature or after removal of serum at infection, both procapsids and mature virions bound and co-eluted from the immobilized glutathione ligand. This work may improve the understanding of CVA21 capsid assembly and presents an efficient purification method that may be applied to picornaviruses that interact with intracellular GSH.

Ion Exchange Chromatographic Methods for Purification of Therapeutic Antibodies.

Ion Exchange Chromatography has been a critical unit operation for manufacturing of therapeutic antibodies. Cation and anion exchange chromatography are used extensively to remove process-related as well as product-related impurities to obtain the final product. In this chapter, we describe the methods for separating and purifying charge variants and aggregates for manufacturing of monoclonal antibodies. The methods related to removal of host cell impurities such as host cell DNA and host cell proteins are also described. With minimal modifications, the protocols described here can be employed to purify any monoclonal antibody.

Process development and scale-up optimization of the SARS-CoV-2 receptor binding domain\u2013based vaccine candidate, RBD219-N1C1

A SARS-CoV-2 RBD219-N1C1 (RBD219-N1C1) recombinant protein antigen formulated on Alhydrogel® has recently been shown to elicit a robust neutralizing antibody response against SARS-CoV-2 pseudovirus in mice. The antigen has been produced under current good manufacturing practices (cGMPs) and is now in clinical testing. Here, we report on process development and scale-up optimization for upstream fermentation and downstream purification of the antigen. This includes production at the 1-L and 5-L scales in the yeast, Pichia pastoris, and the comparison of three different chromatographic purification methods. This culminated in the selection of a process to produce RBD219-N1C1 with a yield of >400 mg per liter of fermentation with >92% purity and >39% target product recovery after purification. In addition, we show the results from analytical studies, including SEC-HPLC, DLS, and an ACE2 receptor binding assay that were performed to characterize the purified proteins to select the best purification process. Finally, we propose an optimized upstream fermentation and downstream purification process that generates quality RBD219-N1C1 protein antigen and is fully scalable at a low cost.Key points• Yeast fermentation conditions for a recombinant COVID-19 vaccine were determined.• Three purification protocols for a COVID-19 vaccine antigen were compared.• Reproducibility of a scalable, low-cost process for a COVID-19 vaccine was shown.Graphical abstract

Oncolytic virus purification with periodic counter-current chromatography.

Virus-based biologicals are one of the most promising biopharmaceuticals of the 21st century medicine and play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. Oncolytic virus manufacturing scale can range from 5 L in research and development up to 50 L for clinical studies and reach hundreds of liters for commercial scale. The inherent productivity and high integration potential of periodic counter-current chromatography (PCC) offer a transversal solution to decrease equipment footprint and the reduction of several non-value-added unit operations. We report on the design of an efficient PCC process applied to the intermediate purification of oncolytic adenovirus. The developed ion-exchange chromatographic purification method was carried out using a four-column setup for three different scenarios: (i) variation in the feedstock, (ii) potential use of a post-load washing step to improve virus recovery, and(iii) stability during extended operation. Obtained virus recoveries (57%-86%) and impurity reductions (>80% DNA, and >70% total protein) match or overcome batch purification. Regarding process stability and automation, our results show that not only the dynamic control strategy used is able to suppress perturbations in the sample inlet but also allows for unattended operation in the case of ion exchange capture.

Air-assisted dispersive liquid phase microextraction coupled chromatography quantification for purification of therapeutic lectin from aloe vera – A potential COVID-19 immune booster

The present research focuses on devising air assisted liquid phase microextraction coupled chromatographic quantification method for purification of therapeutic lectin using switchable low transition temperature mixtures. Four switchable low transition temperature mixtures were formed by mixing benzyl trimethyl ammonium chloride with sodium salts of various heavy chain fatty acids in the molar ratio of 1:1. Thermophysical properties (Density, Refractive index and surface tension) were determined as a function of temperature. Air-assisted dispersive liquid-phase microextraction with switchable solvents was carried out to obtain lectin rich concentrate in top phase with an extraction efficiency of (85% w/w). The purified lectin was quantified by size exclusion chromatography with the retention time of 2.3 min and resolution of 6.2 with NTU of 13.2. The ultrapure fraction of lectin was obtained with anion exchange chromatography yielding antioxidant activity of 40.2 mg GAE/g lectin and haemagglutination activity of 45.36 HU/g in the isolate. The method of dispersive liquid-phase microextraction provides a high selective one-pot extraction and partial purification of the therapeutic lectin from aloe vera.

Preparative isolation of 1,1-diphenyl-2-picrylhydrazyl inhibitors from Ribes himalense using medium-pressure and two-dimensional reversed-phase/reversed-phase liquid chromatography guided by an online HPLC-1, 1-diphenyl-2-picrylhydrazyl assay.

The lack of suitable chromatographic purification methods makes it a challenge to effectively isolate the chemical components of traditional Tibetan medicines. Ribes himalense is a rarely studied Tibetan medicine, reputed to have free radical-scavenging effects. In the present work, we utilized it as a model herb to highlight an approach for the separation of 1,1-diphenyl-2-picrylhydrazyl inhibitors via medium-pressure chromatography and two-dimensional reversed-phase/reversed-phase interaction liquid chromatography under the guidance of an online high-performance liquid chromatography-1,1-diphenyl-2-picrylhydrazyl assay. Finally, we obtained two free radical inhibitors (>95% purity) from the R. himalense extract. This is the first report of the rapid isolation of these free radical inhibitors from R. himalense. This method can be useful in quality standard assessment and further pharmacological activity research, and may be used as a reference for the composition research of various natural products.

Convenient Synthesis of Novel Chalcone and Pyrazoline Sulfonamide Derivatives as Potential Antibacterial Agents

Novel chalcone sulfonamide derivatives were synthesized starting from benzophenones and aldehydes in 3 steps. Also, (E)-3-(4-chlorophenyl)-1-(4-methoxyphenyl)prop-2-en-1-one was converted to different pyrazoline in four steps. The synthesis of sulfonamides was carried out under solvent-free conditions at room temperature and the products were obtained in high purity after a simple work-up without using any chromatography method for purification. All of the products were evaluated for their in vitro antibacterial activity against Staphylococcus aureus and Escherichia coli.

Impact of impurities in bromocresol green indicator dye on spectrophotometric total alkalinity measurements

Abstract. Due to its accurate and precise character, spectrophotometric pH detection is a common technique applied in measurement methods for carbonate system parameters. However, impurities in the used pH indicator dyes can influence the measurements quality. During our work described here, we focused on impacts of impurities in the pH indicator dye bromocresol green (BCG) on spectrophotometric seawater total alkalinity (AT) measurements. In order to evaluate the extent of such influences, purified BCG served as a reference. First, a high-performance liquid chromatography (HPLC) purification method for BCG was developed as such a method did not exist at the time of this study. An analysis of BCG dye from four different vendors with this method revealed different types and quantities of impurities. After successful purification, AT measurements with purified and unpurified BCG were carried out using the novel autonomous analyzer CONTROS HydroFIA® TA. Long-term measurements in the laboratory revealed a direct influence of impurity types and quantities on the drift behavior of the analyzer. The purer the BCG, the smaller was the AT increase per measurement. The observed drift is generally caused by deposits in the optical pathway mainly generated by the impurities. However, the analyzers drift behavior could not be fully overcome. Furthermore, we could show that a certain impurity type in some indicator dyes changed the drift pattern from linear to nonlinear, which can impair long-term deployments of the system. Consequently, such indicators are impractical for these applications. Laboratory performance characterization experiments revealed no improvement of the measurement quality (precision and bias) by using purified BCG as long as the impurities of the unpurified dye do not exceed a quantity of 2 % (relationship of peak areas in the chromatogram). However, BCG with impurity quantities higher than 6 % provided AT values which failed fundamental quality requirements. In conclusion, to gain optimal AT measurements especially during long-term deployments, an indicator purification is not necessarily required as long as the purchased dye has a purity level of at least 98 % and is free of the named impurity type. Consequently, high-quality AT measurements do not require pure but the purest BCG that is purchasable.

Structural features, selenization modification, antioxidant and anti-tumor effects of polysaccharides from alfalfa roots

Hot water extraction and chromatographic purification methods were used to extract and purify two polysaccharides (RAPS-1 and RAPS-2) from the roots of alfalfa. Subsequently, RAPS-2 was modified using the HNO3/Na2SeO3 method to obtain Se-RAPS-2. The structural features, antioxidant and in vitro anti-tumor activities of the three polysaccharides were evaluated. The structural analysis revealed that RAPS-1 (Mw = 10.0 kDa) was composed of rhamnose, xylose, arabinose, galacturonic acid, mannose and glucose, whereas RAPS-2 (Mw = 15.8 kDa) consisted of rhamnose, xylose, galacturonic acid, mannose, glucose and galactose. RAPS-1 contained 1 → 2, 1 → 4, 1 → 3, and 1 → 6 or 1 → glycosidic bonds; however, while RAPS-2 lacked 1 → 4 glycosidic linkages. The molecular weight of Se-RAPS-2 was 11.0 kDa less than that of RAPS-2. The results of activities demonstrated that Se-RAPS-2 displayed superior antioxidant activity and inhibitory effect in HepG2 cells than RAPS-1 and RAPS-2.

Membrane Protein Production and Purification from Escherichia coli and Sf9 Insect Cells.

A major obstacle to studying membrane proteins by biophysical techniques is the difficulty in producing sufficient amounts of materials for functional and structural studies. To overexpress the target membrane protein heterologously, especially an eukaryotic protein, a key step is to find the optimal host expression system and perform subsequent expression optimization. In this chapter, we describe protocols for screening membrane protein production using bacterial and insect cells, solubilization screening, large-scale production, and commonly used affinity chromatography purification methods. We discuss general optimization conditions, such as promoters and tags, and describe current techniques that can be used in any laboratory without specialized expensive equipment. Especially for insect cells, GFP fusions are particularly useful for localization and in-gel fluorescence detection of the proteins on SDS-PAGE. We give detailed protocols that can be used to screen the best expression and purification conditions for membrane protein study.

Marine-derived polygalactofucan and its β-2-deoxy-amino-substituted glucopyranan composite attenuate 3-hydroxy-3-methylglutaryl-CoA reductase: prospective natural anti-dyslipidemic leads

Adverse side effects reported for the use of statin drugs provided insights to develop potential anti-dyslipidemic derivatives from natural origin. The objective of the work was to develop the marine-derived polysaccharides attenuating 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), and as prospective natural anti-dyslipidemic leads. Physical and chromatographic purification methods were used to isolate the polygalactofucan from the marine macroalga Sargassum wightii and β-(2-deoxy)-amino-substituted glucopyrananan from a marine crustacean. Glycosidic linkage analysis by the process of methylation was used for structural elucidation of the polygalactofucan, and the methylated and partially methylated alditol acetates were characterized using extensive spectroscopic experiments. The polysaccharide composite constituting the titled polysaccharide motifs showed significant HMGCR inhibitory potential (IC90 0.12 mg mL−1) and an increase in HMG-CoA/mevalonate ratio (1.68 mg dL−1) compared with the high-fat diet (HFD)-treated animals (1.04 mg dL−1), which recognized its hypo-lipidemic efficacy. In vivo results demonstrated about 70% reduction in the triglyceride levels with the concomitant increase (~39%) of hepatic lipoprotein lipase (LPL) activity in the HFD-fed Wistar rats treated with 500 mg kg−1 body weight. The results illustrated the use of marine-derived polygalactofucan composite as potential anti-dyslipidemic agent.