Search Results (32)

Whey, a large-scale dairy industry by-product, can be converted into whey protein concentrate (WPC), providing a cost-effective nutrient-rich substrate for microbial fermentation. We investigated protease production by Aspergillus oryzae using WPC as the sole substrate in submerged fermentation. Following fermentation, the protease was purified sequentially from the crude extract by salting-out, which yielded a substantial purification factor (~39), and subsequent ion-exchange chromatography. The non-adsorbed chromatographic fraction showed the highest protease activity (92.6 U/mL) and revealed one main protein band ~45 kDa via SDS-PAGE. Enzyme characterization demonstrated activity across neutral-to-alkaline conditions, optimal at pH 9.0 and 37 °C, with stability maintained between 30 °C and 37 °C. The enzyme was classified as a serine protease based on strong inhibition by PMSF and SDS; its activity was also inhibited by Zn²⁺, Mg²⁺, and K⁺, but enhanced by Ca²⁺. This work validates WPC as an efficient substrate for protease production by A. oryzae and presents a promising strategy for valorizing industrial by-products through sustainable biotechnology. Full article

Background/Objectives: Pharmaceutical companies are aware of the ongoing effort to satisfy the increasing global demand for therapeutic-grade monoclonal antibodies (mAbs), an especially difficult challenge for poor and developing countries. We present a simple, economical, single-step purification approach at neutral pH for polyclonal human IgG (hIgG), which does not require any expensive ligands, chromatography columns, polymers, or membranes. Methods/Results: Instead, porous precipitates of commercial, recyclable aromatic [bathophenanthroline:cation] complexes were found to efficiently capture impurity proteins from CHO cells or E. coli lysate while maintaining the majority of the highly concentrated hIgG (5–15 mg/mL) in the supernatant. [(Batho)₃:Zn²⁺] complexes were the most promising, resulting in hIgG with a purity of ≈95%, by SDS-PAGE. This purified hIgG is monomeric (by dynamic light scattering, DLS) and preserves the native secondary structure (by far UV circular dichroism spectroscopy, CD). The process yield is >90% (by densitometry) and is maintained after a 100-fold increase in the reaction volume, which required only proportional increases in reagents. Conclusions: Although Protein A chromatographic columns, the industry gold standard, have a limited binding capacity, are costly, and require familiarity with column maintenance, we are attempting, by our efforts, to help to produce a more efficient, simple, and economical purification platform. Full article

The mixotrophic dinoflagellate Akashiwo sanguinea is known to have acute toxic effects on multiple marine organisms, while the composition and chemical properties of its toxins remain unclear. In this study, we established a method for separation and purification of A. sanguinea toxins using chromatographic techniques. The acetone extract of A. sanguinea exhibited higher hemolytic activity and shorter incubation time compared to methanol and ethyl acetate extracts. Five fractions were obtained by solid-phase extraction (SPE), of which SPE3 (acetone/water ratio 3:2) and SPE4 (acetone/water ratio 4:1) exhibited the highest hemolytic activities and allelopathic effects. Further purification on SPE3 and SPE4 using reverse-phase high-performance liquid chromatography (RP-HPLC) coupled with a diode array detector (DAD) resulted in 11 subfractions, among which Fr4-5 displayed the strongest hemolytic activity. Nearly all active subfractions exhibited higher hemolytic activities incubated under light than those in the dark (p < 0.05), suggesting that A. sanguinea can produce both photosensitive and non-photosensitive toxins, with the former being the primary contributors to its hemolytic activity. Molecular characterization by UV-Vis, FTIR, and HRMS/MS analysis revealed that the structural features of Fr4-5 were highly consistent with porphyrin analogs and could be derived from chlorophyll c-related precursors. These findings highlight that the photosensitive toxins in A. sanguinea may serve dual roles in stress adaptation and ecological competition, potentially contributing to the formation of the blooms. Full article

In this study, we assess an alternative Fc-fusion protein purification method that does not rely on chromatographic media or ligands. Recombinant human acetylcholinesterase, fused to the Fc domain of human IgG1 (henceforth, AChE-Fc), was purified with precipitated aromatic complexes composed of the bathophenanthroline (henceforth, batho) chelator with either Zn²⁺ or Cu²⁺ ions (i.e., [(batho)₃:Zn²⁺] or [(batho)₂:Cu²⁺]) in the presence of polyethylene glycol 6000 (PEG-6000). In a three-step purification process conducted at pH 7, AChE-Fc was captured by the aromatic complexes (Step 1); unbound or weakly bound protein impurities were removed with 20 mM NaCl (Step 2); and AChE-Fc was then extracted at pH 7 (Step 3) using 100 mM Na citrate buffer in 250 mM NaCl. Purified AChE-Fc was not aggregated (as determined by dynamic light scattering (DLS) and Native PAGE). However, full enzymatic activity was only preserved with the [(batho)₃:Zn²⁺] complex. Interaction between AChE-Fc and [(batho)₃:Zn²⁺] led to ~83–88% overall protein yield. Thirty-fold process upscaling by volume required only proportional increase in the amounts of [(batho)₃:Zn²⁺] and PEG-6000. Efficient (95–97%) chelator recycling was achieved by recrystallization. Chelator leaching into purified AchE-Fc was estimated to be ~0.3% relative to the total amount used. Taken together, this novel procedure has the potential to provide an economical and practical avenue for the industrial purification of Fc-fusion proteins. Full article

Olive oil extraction by two-phase systems generates a by-product called “alperujo” which presents several difficulties for its valorization. The present work evaluated an industrial approach, based on the application of thermal treatments to alperujo followed by solid/liquid separation using standard two-phase olive oil mill equipment. Treatments consisted of the thermo-malaxation of alperujo at 70 °C for 45 or 90 min, with or without acid addition, followed by solid/liquid separation in an industrial decanter. The solid was characterized concerning subsequent use for composting, while total and hydrophilic phenolics were analyzed in liquid for their recovery. Additionally, a laboratory-scale trial to compare phenolic purification by ethylic acetate extraction with chromatographic procedures was also included. The static respiration index showed that solid fractions presented higher susceptibility to biodegradation processes than raw alperujo. The phenolic content of treated liquid fractions was higher than in raw alperujo. Total phenolics were maximum at the longest exposure time without acid addition, while hydrophilic phenolics were highest at the shortest exposure time in acidified samples. The use of non-ionic resins seemed attractive for obtaining highly concentrated phenolic fractions. The proposed thermal treatments can be applied in olive oil industries, allowing in situ pomace valorization and the recovery of phenolic-enriched liquid fractions. Full article

Lepidium draba (hoary cress) is a perennial plant belonging to the Brassicaceae family that produces two dominant glucosinolates (GLSs): glucoraphanin (GRN) and sinalbin (SBN). They represent the stored form, which is converted upon the myrosinase (Myr) hydrolysis activity to active compounds, mainly isothiocyanates (ITCs) such as sulforaphane (SFN) or p-hydroxybenzyl isothiocyanate (pHBITC). Research on ITCs that have proven anticancer, antimicrobial, and chemoprotective properties is usually conducted with pure commercially available compounds. However, these are chemically reactive, making it difficult to use them directly for preventive purposes in dietary supplements. Efforts are currently being made to prepare dietary supplements enriched with GLS and/or Myr. In this study, we report a simple but efficient chromatographic procedure for the isolation and purification of GLSs from MeOH extract from hoary cress based on a combination of ion exchange and gel permeation chromatography on DEAE-Sephadex A-25 and Sephadex LH-20. To obtain the Myr required for efficient hydrolysis of GLSs into antibacterial ITCs, we developed a rapid method for its extraction from the seeds of Lepidium sativum (garden cress). The yields of GLSs were 22.9 ± 1.2 mg GRN (purity 96%) and 10.4 ± 1.1 mg SBN (purity 92%) from 1 g of dry plant material. Both purified GLSs were used as substrates for the Myr. Analysis of the composition of hydrolysis products (HPs) revealed differences in their hydrolysis rates and in the degree of conversion from GLSs to individual ITCs catalyzed by Myr. When GRNs were cleaved, SFNs were formed in an equimolar ratio, but the formation of pHBITCs was only half that of cleaved SBNs. The decrease in pHBITC content is due to its instability compared to SFN. While SFN is stable in aqueous media during the measurement, pHBITC undergoes non-enzymatic hydrolysis to p-hydroxybenzyl alcohol and thiocyanate ions. Testing of the antimicrobial effects of the HPs formed from GRN by Myr under premix or in situ conditions showed inhibition of the growth of model prokaryotic and eukaryotic microorganisms. This observation could serve as the jumping-off point for the design of a two-component mixture, based on purified GLSs and Myr that is, usable in food or the pharmaceutical industry in the future. Full article

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM^® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5–7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3–7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H₂O at room temperature. Full article

Many non-traditional isotopes, such as chlorine, magnesium, calcium, etc., are widely used as groundwater tracers. A new sample processing protocol of purification and concentration for isotopic analysis is presented to overcome many of the major drawbacks of existing methods. Contemporary sample preparation often requires several laborious off-line procedures in a ultra clean laboratory prior to instrumental determination; additionally, interference ions in real samples are difficult to completely remove, especially when the concentration of those ions is equal to that of the target ions. The new protocol includes the following steps: (i) one-step purification using a newly developed isotopic preparative chromatograph (IPC) with a background suppressed mode to obtain extremely pure components that only have target ions and H₂O; (ii) enrichment of the collected pure solution from the previous step using a newly developed ultra clean concentrator filled with high purity nitrogen; (iii) transforming the enriched target ion into suitable speciation inside the ultra clean concentrator; (iv) finally, sending the enriched solutions to a multi-collector inductively coupled-plasma mass-spectrometer (MC-ICP-MS) or thermal ionization mass spectrometer (TIMS). The present method was validated using certified reference materials and real samples for both chlorine and magnesium; the precision of chlorine ratio value was generally below 0.22‰ and that of Mg was below 0.12‰. This processing protocol provides a potential method for isotope sample preparation and analysis in a small number of geological samples with low concentrations of many other elements or compounds such as nitrate, sulfate, lithium, calcium, strontium, etc. Full article

Several chromatographic approaches have been established over the last decades for the production of pharmaceutically relevant viruses. Due to the large size of these products compared to other biopharmaceuticals, e.g., proteins, convective flow media have proven to be superior to bead-based resins in terms of process productivity and column capacity. One representative of such convective flow materials is membranes, which can be modified to suit the particular operating principle and are also suitable for economical single-use applications. Among the different membrane variants, affinity surfaces allow for the most selective separation of the target molecule from other components in the feed solution, especially from host cell-derived DNA and proteins. A successful membrane affinity chromatography, however, requires the identification and implementation of ligands, which can be applied economically while at the same time being stable during the process and non-toxic in the case of any leaching. This review summarizes the current evaluation of membrane-based affinity purifications for viruses and virus-like particles, including traditional resin and monolith approaches and the advantages of membrane applications. An overview of potential affinity ligands is given, as well as considerations of suitable affinity platform technologies, e.g., for different virus serotypes, including a description of processes using pseudo-affinity matrices, such as sulfated cellulose membrane adsorbers. Full article

Sesquiterpene lactone (SL) subtypes including hirsutinolide and cadinanolide have a controversial genesis. Metabolites of these classes are either described as natural products or as artifacts produced via the influence of solvents, chromatographic mobile phases, and adsorbents used in phytochemical studies. Based on this divergence, and to better understand the sensibility of these metabolites, different pH conditions were used to prepare semisynthetic SLs and evaluate the anti-inflammatory and antiproliferative activities. Therefore, glaucolide B (1) was treated with various Brønsted–Lowry and Lewis acids and bases—the same approach was applied to some of its derivatives—allowing us to obtain 14 semisynthetic SL derivatives, 10 of which are hereby reported for the first time. Hirsutinolide derivatives 7a (CC₅₀ = 5.0 µM; SI = 2.5) and 7b (CC₅₀ = 11.2 µM; SI = 2.5) and the germacranolide derivative 8a (CC₅₀ = 3.1 µM; SI = 3.0) revealed significant cytotoxic activity and selectivity against human melanoma SK-MEL-28 cells when compared with that against non-tumoral HUVEC cells. Additionally, compounds 7a and 7c.1 showed strongly reduced interleukin-6 (IL-6) and nitrite (NOx) release in pre-treated M1 macrophages J774A.1 when stimulated with lipopolysaccharide. Despite the fact that hirsutinolide and cadinanolide SLs may be produced via plant metabolism, this study shows that acidic and alkaline extraction and solid-phase purification processes can promote their formation. Full article

Ochna kibbiensis (Family: Ochnaceae) has been employed in ethnomedicine for the treatment of malaria and inflammation, among others. The aim of this study was to isolate and characterize prophylactic antimalarial agents from the leaves of O. kibbiensis against Plasmodium berghei, in vivo and in silico. The median lethal dose (LD₅₀) of the methanol extract and its fractions (hexane, dichloromethane, ethylacetate and butanol) was determined according to Lorke’s method while the antimalarial effect of the extract and its fractions was investigated according to the method described by Peters prophylactic test using Chloroquine-sensitive Plasmodium berghei (NK65). All the extract/fractions exhibited LD₅₀ values ≥ 5000 mg/kg with the exception of the n-butanol fraction (1702.94 mg/kg), which indicate that the plant is non-toxic. Dichloromethane fraction exhibited a significant (p < 0.05) and dose-dependent prophylactic effect with 47.62, 85.12, and 100.0% prophylaxis (at 500, 250, and 125 mg/kg), while the least effect was observed by the butanol fraction with a percentage prophylaxis of 64.29 and 76.19, respectively; the standard drug, pyrimethamine, had 95.24% prophylaxis. Based on the results obtained, dichloromethane fraction of O. kibbiensis was subjected to chromatographic purification, which led to the isolation of a mixture of two compounds identified as stigmasterol and β-sitosterol by analysis of the NMR spectral data and comparison with existing literature; the compounds exhibited good binding affinities (−5.129 and −4.889 kcal/mol) against pfLDH and a favorable ADMET profile. In conclusion, the leaves of O. kibbiensis have demonstrated a significant prophylactic antimalarial activity and the two known steroids (stigmasterol and β-sitosterol) were isolated from the dichloromethane fraction for the first time. Full article

Growing medical, engineering, biochemical, and biological interest has led to a steady pace of research and development into polymeric monolithic structures with densely interconnected pores for purifying bio compounds. Cryogels, which are generated by freezing a reactive polymerization mixture, are highlighted due to their versatility and low relative cost as macroporous, polymeric, monolithic adsorbents. The conversion of cryogels into affinity adsorbents is one possible alternative to their optimal application. Some of the most often utilized supports for immobilizing particular ligands are monolithic columns manufactured with epoxy radicals on their surfaces. The purification of biomolecules with a high degree of specificity, such as lectins and glycoproteins with an affinity for glycosylated groups, has garnered interest in the use of fixed non-traditional beds functionalized with ligands of particular interest. The interaction is both robust enough to permit the adsorption of glycoproteins and reversible enough to permit the dissociation of molecules in response to changes in the solution’s pH. When compared to other protein A-based approaches, this one has been shown to be more advantageous than its counterparts in terms of specificity, ease of use, and cost-effectiveness. Information on polymeric, macroporous, monolithic adsorbents used in the affinity chromatographic purification of lectins has been published and explored. Full article

Protein deamidation can severely alter the physicochemical characteristics and biological functions of protein therapeutics. Cobratide is a non-addictive analgesic with wide clinical acceptance. However, the Asn residue at position 48 from the N-terminus of the cobratide amino acid sequence (N48) tends to degrade during purification, storage, and transport. This characteristic could severely affect the drug safety and clinical efficacy of cobratide. Traditional methods for quantitating deamidation reported in previous research are characterised by low efficiency and accuracy; the quality control of cobratide via this method is limited. Herein, we developed an improved ¹⁸O-labelling method based on the detection of a unique peptide (i.e., the protein fragment of cobratide containing the N48 deamidation hotspot after enzymolysis) using an Orbitrap high-resolution mass spectrometer to quantify deamidated cobratide. The limits of detection and quantification of this method reached 0.02 and 0.025 μM, respectively, and inter- and intra-day precision values of the method were <3%. The accuracy of the ¹⁸O-labelling strategy was validated by using samples containing synthesised peptides with a known ratio of deamidation impurities and also by comparing the final total deamidation results with our previously developed capillary electrophoresis method. The recoveries for deamidation (Asp), deamidation isomerisation (iso-Asp), and total deamidation were 101.52 ± 1.17, 102.42 ± 1.82, and 103.55 ± 1.07, respectively. The robustness of the method was confirmed by verifying the chromatographic parameters. Our results demonstrate the applicability of the ¹⁸O-labelling strategy for detecting protein deamidation and lay a robust foundation for protein therapeutics studies and drug quality consistency evaluations. Full article

The downstream processing of natural active molecules remains the most significant cost in the production pipeline. This considerable cost is largely attributed to rigorous chromatographic purification protocols. In an ongoing effort to abate the dependence on chromatography in downstream processing, alternative affinity matrices in the form of magnetic particles (e.g., iron oxide) have emerged as viable candidates. Nevertheless, biotechnological applications of iron oxide particles are still confined to the research level or for low-throughput clinical applications. Herein, we describe an efficient, quick, and environmentally friendly method for the isolation of astaxanthin and lutein, two carotenoids with very similar chemical structure, from extracts of the microalga Haematococcus pluvialis. The technology proposed, named Selective Magnetic Separation (SMS), is based on the use of magnetic materials carrying affinity ligands that bind carotenoids and is applied as second step of purification. The method, thanks to functionalized magnetic nanoparticles, reduces the use of organic or toxic solvents. In the present work, we examined the most efficient binding conditions such as temperature, magnetic nanoparticles concentration, and elution time, as well as their effects on carotenoids recovery, with the aim to improve the non-covalent binding between the ligand (amines) and astaxanthin/lutein. Our initial results clearly showed that it is possible to use magnetic separation as an alternative to chromatography to isolate important and valuable compounds. Full article

Cyclotron-produced radiometals must be separated from the irradiated target and purified from other metal impurities, which could interfere with the radiolabeling process. We compared different chromatographic and colorimetric methods to determine the amount of transition metals in radioactive samples. Besides commercially available colorimetric tests, 4-(2-pyridylazo)resorcinol and xylenol orange were used as a non-selective metal reagents, forming water-soluble chelates with most of the transition metals immediately. We compared the applicability of pre- and post-column derivatization, as well as colorimetric determination without separation. The studied chromatographic and colorimetric analyses are not suitable to completely replace atomic spectroscopic techniques for the determination of metal contaminants in radioactive samples, but they may play an important role in the development of methods for the purification of radiometals and in their routine quality control. Full article