Single-step Purification Research Articles

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

Enzymatische Peptid‐ und Proteinbromierung: Der BromoTrp Tag

AbstractBioorthogonale Reaktionen von Proteinen und ungeschützten Peptiden sind von hohem Wert für die chemische Biologie. Die Kombination aus enzymatischer Halogenierung und übergangsmetallkatalysierter Kreuzkupplung bietet einen praktikablen Ansatz zur Modifizierung von Proteinen und ungeschützten Peptiden. Durch semirationales Protein Engineering, wurden Varianten der Tryptophan 6‐Halogenase Thal identifiziert, welche die Bromierung von Peptiden mit einem C‐terminalen Tryptophanrest ermöglichen. Das Substratspektrum wurde anhand von Di‐, Tri‐ und Tetrapeptid‐Bibliotheken charakterisiert, und ein optimierter Peptid‐Tag identifiziert, dem wir den Namen BromoTrp‐Tag gegeben haben. Dieser Tag wurde in drei Modellproteine eingeführt. Deren präparative, posttranslationale Bromierung mit nur einem Kultivierungs‐ und Aufreinigungsschritt wurde durch das bromierende E. coli Koexpressionssystem Brocoli ermöglicht. Pd‐katalysierte Suzuki–Miyaura Kreuzkupplung war mittels Pd‐Nanopartikeln als Katalysator bei 37 °C möglich, was das vielversprechende Potential dieser Strategie zum Zweck bioorthogonaler Funktionalisierung und Konjugation demonstriert.

Enzymatic Peptide and Protein Bromination: The BromoTrp Tag.

Bio-orthogonal reactions for modification of proteins and unprotected peptides are of high value in chemical biology. The combination of enzymatic halogenation with transition metal-catalyzed cross-coupling provides a feasible approach for the modification of proteins and unprotected peptides. By a semirational protein engineering approach, variants of the tryptophan 6-halogenase Thal were identified that enable efficient bromination of peptides with a C-terminal tryptophan residue. The substrate scope was explored using di-, tri-, and tetrapeptide arrays, leading to the identification of an optimized peptide tag we named BromoTrp tag. This tag was introduced into three model proteins. Preparative scale post-translational bromination was possible with only a single cultivation and purification step using the brominating E. coli coexpression system Brocoli. Palladium-catalyzed Suzuki-Miyaura cross-coupling of the bromoarene was achieved with Pd nanoparticle catalysts at 37 °C, highlighting the rich potential of this strategy for bio-orthogonal functionalization and conjugation.

Single injection by LC-ESI-MS/MS for simultaneous determination of organophosphate tri- and di-esters in plant tissue based on ultrasonic-assisted sequential extraction and single-step purification

A novel methodology based on ultrasonic-assisted sequential extraction, dispersive-SPE purification, and single-injection on liquid chromatography-tandem mass spectrometry (LC-MS/MS) is proposed, for the first time, to simultaneously measure 14 tri-OPEs and 9 di-OPEs in plant tissues. The samples were successively ultrasonicated with a mixture of hexane:dichloromethane (1:1, v/v) and 8% acetic acid in acetonitrile for extracting tri- and di-OPEs purified with graphitized carbon black and quantitated on LC-MS/MS at the same time. The recoveries of targeted tri- and di-OPEs in the matrix spike ranged from 66% to 120% and 71% to 110% respectively. The proposed method was validated by processing eight types of common vegetables including spinach (Spinacia oleracea L.), lettuce (Lactuca sativa), carrot (Daucus carota var. sativa Hoffm.), sweet potato (Solanum tuberosum L.), cucumber (Cucumis sativus L.), tomato (Solanum lycopersicum L.), green beans (Phaseolus vulgaris), and cowpeas (Vigna unguiculata), with the recoveries of surrogates ranging from 84% to 98%.

A Convenient Self-Removing Affinity Tag Method for the Simple Purification of Tagless Recombinant Proteins.

In this work, we describe a novel self-cleaving affinity tag technology based on a highly modified split-intein cleaving element. In this system, which has recently been commercialized by Protein Capture Science, LLC under the name iCapTagTM , the N-terminal segment of an engineered split intein is covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong binding between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex. After unwanted impurities are washed from the resin, cleavage of the target protein is initiated by a shift of the buffer pH from 8.5 to 6.2. As a result, the highly purified tagless target protein is released from the column in the elution step. Alternately, the resin beads can be added directly to cell culture broth or lysate, allowing capture, purification and cleavage of the tagless target protein using a column-free format. These methods result in highly pure tagless target protein in a single step, and can thereby accelerate characterization and functional studies. In this work we demonstrate the single step purification of streptokinase, a fibrinolytic agent, and an engineered recombinant human hemoglobin 1.1 (rHb1.1). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Expression of high-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Basic Protocol 2: Purification of high-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform Alternate Protocol 1: Expression of low-titer protein tagged with the Nostoc punctiforme (Npu) DnaE split-intein on the N-terminus Alternate Protocol 2: Purification of low-titer protein using the Nostoc punctiforme (Npu) DnaE split-intein purification platform.

Enhancement in T1 lipase purification recovery using the novel construct pGEX4T1/His-T1

The Geobacillus zalihae strain T1 produces a thermostable T1 lipase that could be used for industrial purposes. Previously, the GST-T1 lipase was purified through two chromatographic steps: affinity and ion exchange (IEX) but the recovery yield was only 33%. To improve the recovery yield to over 80%, the GST tag from the pGEX system was replaced with a poly-histidine at the N-terminal of the T1 lipase sequence. The novel construct of pGEX/His-T1 lipase was developed by site-directed mutagenesis, where the XbaI restriction site was introduced upstream of the GST tag, allowing the removal of tag via double digestion using XbaI and EcoRI (existing cutting site in the pGEX system). Fragment of 6 × His-T1 lipase fusion was synthesized, cloned into the pGEX4T1 system, and expressed in Escherichia coli BL21 (DE3) pLysS, resulting in lipase-specific activity at 236 U/mg. The single purification step of His-T1 lipase was successfully achieved using nickel Sepharose 6FF with an optimized concentration of 5 mM imidazole for binding, yielding the recovery of 98%, 1,353 U/mg lipase activity, and a 5.7-fold increase in purification fold. His-T1 lipase was characterized and was found to be stable at pH 5–9, active at 70 °C, and optimal at pH 9.

Merging Solid-Phase Peptide Synthesis and Automated Glycan Assembly to Prepare Lipid-Peptide-Glycan Chimeras.

Biomaterials with improved biological features can be obtained by conjugating glycans to nanostructured peptides. Creating peptide-glycan chimeras requires superb chemoselectivity. We expedite access to such chimeras by merging peptide and glycan solid-phase syntheses employing a bifunctional monosaccharide. The concept was explored in the context of the on-resin generation of a model α(1→6)tetramannoside linked to peptides, lipids, steroids, and adamantane. Chimeras containing a β(1→6)tetraglucoside and self-assembling peptides such as FF, FFKLVFF, and the amphiphile palmitoyl-VVVAAAKKK were prepared in a fully automated manner. The robust synthetic protocol requires a single purification step to obtain overall yields of about 20 %. The β(1→6)tetraglucoside FFKLVFF chimera produces micelles rather than nanofibers formed by the peptide alone as judged by microscopy and circular dichroism. The peptide amphiphile-glycan chimera forms a disperse fiber network, creating opportunities for new glycan-based nanomaterials.

Enhanced production and single step purification of biologically active recombinant anti-IL6 scFv from Escherichia coli inclusion bodies

Interleukin 6 (IL-6) is a proinflammatory cytokine that plays a critical role in immune responses and inflammation. Overexpression of IL-6 serves as a biomarker for various diseases. Therefore, it is vital to develop IL-6-specific antibodies for the diagnosis and treatment of inflammatory diseases. Alternatively, high-yield production of single-chain variable fragments (scFv) in prokaryotes reduces the time and cost required while attaining a similar specificity. However, overexpression of eukaryotic proteins in bacterial expression systems often leads to the formation of inclusion bodies. Here, a codon-optimized anti-IL6 scFv was expressed in the E. coli SHuffle strain, and the effects of inclusion body solubilisation methods on the product yield were investigated. A high concentration (23 mg/L) of biologically active and pure (>95 %) scFv was obtained without any need for a refolding step following solubilisation with a non-denaturing solubilisation agent, N-lauroyl sarcosine. Activity assays showed that anti-IL6 scFv could specifically bind to its antigen. Overall, we present reproducible and efficient production of anti-IL6 scFv in E. coli by using a one-step purification method. While the biologically active and pure antibody fragment can be used for diagnostic studies of the IL-6 cytokine, the developed method can be applied to other recombinant proteins expressed in E. coli.

Single-step ethylene purification from ternary C2 hydrocarbon mixtures in a scalable metal–organic framework

Single-step purification of the ternary C2 hydrocarbon mixture to produce ethylene (C2H4) directly using adsorption-based technologies is desirable for reducing large energy consumption. However, it is challenging to develop an appropriate adsorbent, having preferential adsorption characteristics toward ethane (C2H6) and acetylene (C2H2) rather than C2H4 coupled with high adsorption capacity owing to their similar physicochemical properties. Herein, we present a highly stable, cheap, and scalable CAU-23 adsorbent, which enables single-step production of high-purity C2H4 (>99.9%) from the ternary C2 hydrocarbon mixture. CAU-23 exhibited higher uptake capacity of C2H6 (4.0 mmol g−1) and C2H2 (4.7 mmol g−1) compared to that of C2H4 (3.8 mmol g−1). Further, it showed the reasonable selectivity for both C2H6/C2H4 (1.54) and C2H2/C2H4 (1.5) with an equimolar binary mixture, indicating that the separation performance of CAU-23 is comparable to the benchmark porous materials for separating C2 ternary gas mixture. The breakthrough experiments demonstrated its capability to produce high-purity C2H4 (>99.9%) with various C2H6/C2H4/C2H2 compositions at 298 K and 1 bar with high recyclability. The origin of the separation performance was further explored by using computational simulations of the grand canonical Monte Carlo (GCMC) and density functional theory (DFT).

Efficient adeno-associated virus serotype 5 capture with affinity functionalized nanofiber adsorbents.

Adeno-associated viruses (AAVs) are one of the most promising tools for gene therapy applications. These vectors are purified using affinity and ion exchange chromatography, typically using packed beds of resin adsorbents. This leads to diffusion and pressure drop limitations that affect process productivity. Due to their high surface area and porosity, electrospun nanofiber adsorbents offer mass transfer and flow rate advantages over conventional chromatographic media. The present work investigated the use of affinity cellulose-based nanofiber adsorbents for adeno-associated virus serotype 5 (AAV5) capture, evaluating dynamic binding capacity, pressure drop, and AAV5 recovery at residence times (RT) less than 5s. The dynamic binding capacity was found to be residence time-dependent, but nevertheless higher than 1.0 × 1014TP mL-1 (RT = 1.6s), with a pressure drop variation of 0.14MPa obtained after loading more than 2,000 column volumes of clarified AAV5 feedstock. The single affinity chromatography purification step using these new affinity adsorbents resulted in 80% virus recovery, with the removal of impurities comparable to that of bead-based affinity adsorbents. The high binding capacity, virus recovery and reduced pressure drop observed at residence times in the sub-minute range can potentially eliminate the need for prior concentration steps, thereby reducing the overall number of unit operations, process time and costs.

Solid-Phase Synthesis of s-Tetrazines.

An efficient synthesis of s-tetrazines by solid-phase methods is described. This synthesis route was compatible with different solid-phase resins and linkers and did not require metal catalysts or high temperatures. Monosubstituted tetrazines were routinely synthesized using thiol-promoted chemistry, using dichloromethane as a carbon source, while disubstituted unsymmetrical aryl or alkyl tetrazines were synthesized using readily available nitriles. This efficient approach enabled the synthesis of s-tetrazines in high yields (70-94%), eliminating the classical solution-phase problems of mixtures of symmetrical and unsymmetrical tetrazines, with only a single final purification step required, and paves the way to the rapid synthesis of s-tetrazines with various applications in bioorthogonal chemistry and beyond.

Calcium-dependent affinity ligands for the purification of antibody fragments at neutral pH

The emerging formats of antibody fragments for biotherapeutics suffer from inadequate purification methods, delaying the advances of innovative therapies. One of the top therapeutic candidates, the single-chain variable fragment (scFv), requires the development of individual purification protocols dependent on the type of scFv. The available approaches that are based on selective affinity chromatography but do not involve the use of a purification tag, such as Protein L and Protein A chromatography, require acidic elution buffers. These elution conditions can cause the formation of aggregates and thereby greatly compromise the yield, which can be a major problem for scFvs that are generally unstable molecules. Due to the costly and time-consuming production of biological drugs, like antibody fragments, we have engineered novel purification ligands that elute the scFvs in a calcium-dependent manner. The developed ligands are equipped with new, selective binding surfaces and were shown to efficiently elute all captured scFv at neutral pH with the use of a calcium chelator. Further, two of three ligands were proven not to bind to the CDRs of the scFv, indicating potential for use as generic affinity ligands to a range of different scFvs. Multimerization and optimization of the most promising ligand led to a 3-fold increase in binding capacity for the hexamer compared to the monomer, in addition to highly selective and efficient purification of a scFv with >95% purity in a single purification step. This calcium-dependent ligand could revolutionize the scFv industry, greatly facilitating the purification procedure and improving the quality of the final product.

A Simple and Cost-Efficient Platform for a Novel Porcine Circovirus Type 2d (PCV2d) Vaccine Manufacturing.

Porcine circovirus type 2d (PCV2d) is becoming the predominant PCV genotype and considerably affects the global pig industry. Nevertheless, currently, no commercial PCV2d vaccine is available. Preventing and controlling the disease caused by PCV2d is therefore based on other genotype-based vaccines. However, their production platforms are laborious, limited in expression level, and relatively expensive for veterinary applications. To address these challenges, we have developed a simple and cost-efficient platform for a novel PCV2d vaccine production process, using fed-batch E. coli fermentation followed by cell disruption and filtration, and a single purification step via cation exchange chromatography. The process was developed at bench scale and then pilot scale, where the PCV2d subunit protein yield was approximately 0.93 g/L fermentation volume in a short production time. Moreover, we have successfully implemented this production process at two different sites, in Southeast Asia and Europe. This demonstrates transferability and the high potential for successful industrial production.

Single-Step Non-Chromatographic Purification of Recombinant Mammalian Proteins Using a Split Intein ELP Tag System.

Glycoprotein therapeutics are currently used by large patient populations and generate significant revenue for the biopharmaceutical industry. These therapeutic proteins are currently purified at industrial scale using individualized processes involving multiple chromatographic steps. In the absence of a viable affinity platform method, the required chromatographic steps are difficult to develop and inevitably lead to significant yield losses. Further, during preclinical development, there is a need for reliable platform technologies capable of performing high-throughput screening for biologic candidates. Although affinity tags can provide a solution to some of these challenges, they require specific affinity resins, and the tag itself can interfere with the target protein characteristics. Fusion protein systems consisting of elastin-like polypeptide (ELP) and self-cleaving split inteins such as Npu DnaE can serve as potential non-chromatographic platform technologies for the single-step purification of tagless glycoproteins expressed in mammalian cells. In this chapter, we demonstrate the use of this technology to obtain highly purified anti-ErbB2 ML39 single-chain variable fragment (scFv) expressed from Expi293F suspension cells.

Production of active Exendin-4 in Nicotiana benthamiana and its application in treatment of type-2 diabetics.

GLP-1 (Glucagon-like peptide-1) is a peptide that stimulates insulin secretion from the β-cell for glycemic control of the plasma blood glucose level. Its mimetic exenatide (synthetic Exendin-4) with a longer half-life of approximately 3.3-4 h is widely used in clinical application to treat diabetes. Currently, exenatide is chemically synthesized. In this study, we report that the GLP-1 analogue recombinant Exendin-4 (Exdn-4) can be produced at a high level in Nicotiana benthamiana, with an estimated yield of 50.0 µg/g fresh biomass. For high-level expression, we generated a recombinant gene, B:GB1:ddCBD1m:8xHis : Exendin-4 (BGC : Exdn-4), for the production of Exendin-4 using various domains such as the BiP signal peptide, the GB1 domain (B1 domain of streptococcal G protein), a double cellulose binding domain 1 (CBD1), and 8 His residues (8xHis) to the N-terminus of Exendin-4. GB1 was used to increase the expression, whereas double CBD1 and 8xHis were included as affinity tags for easy purification using MCC beads and Ni2+-NTA resin, respectively. BGC : Exdn-4 was purified by single-step purification to near homogeneity using both Ni2+-NTA resin and microcrystalline cellulose (MCC) beads. Moreover, Exdn-4 without any extra residues was produced from BGC : Exdn-4 bound onto MCC beads by treating with enterokinase. Plant-produced Exdn-4 (Exendin-4) was as effective as chemically synthesized Exendin-4 in glucose-induced insulin secretion (GIIS) from mouse MIN6m9 cells a pancreatic beta cell line.

Affinity-based and in a single step purification of recombinant horseradish peroxidase A2A isoenzyme produced by Pichia pastoris.

Horseradish peroxidase (HRP) is an oxidoreductase enzyme and oxidizes various inorganic and organic compounds. It has wide application areas such as immunological tests, probe-based test techniques, removal of phenolic pollutants from wastewater and organic synthesis. HRP is found in the root of the horseradish plant as a mixture of different isoenzymes, and it is very difficult to separate these enzymes from each other. In this regard, recombinant production is a very advantageous method in terms of producing the desired isoenzyme. This study was performed to produce HRP A2A isoenzyme extracellularly in Pichia pastoris and to purify this enzyme in a single step using a 3-amino-4-chloro benzohydrazide affinity column. First, codon-optimized HRP A2A gene was amplified and inserted into pPICZαC. So, obtained pPICZαC-HRPA2A was cloned in E. coli cells. Then, P. pastoris X-33 cells were transformed with linearized recombinant DNA and a yeast clone was cultivated for extracellular recombinant HRP A2A (rHRP A2A) enzyme production. Then, the purification of this enzyme was performed in a single step by affinity chromatography. The molecular mass of purified rHRP A2A enzyme was found to be about 40kDa. According to characterization studies of the purified enzyme, the optimum pH and ionic strength for the rHRP A2A isoenzyme were determined to be 6.0 and 0.04M, respectively, and o-dianisidine had the highest specificity with the lowest Km and Vmax values. Thus, this is an economical procedure to purify HRP A2A isoenzyme without time-consuming and laborious isolation from an isoenzyme mixture.

Landfill gas upgrading using amine-functionalized silica sorbents

ABSTRACT Separation of acid gases such as CO2 from biogas is a key step for its purification before biomethane is used in various applications such as injection into a pipeline or use as a vehicle fuel. In this study, the single step purification of real biogas (locally collected landfill gas) was demonstrated using (3-Aminopropyl) triethoxysilane (APTES)-grafted SBA-15. The sorbent was optimized between amine amount and accessibility to adsorption sites. In studies using a surrogate mixture of CH4/CO2 the sorbent recorded an adsorption capacity of 0.85 mmolCO2/gads under dry conditions and this decreased to 0.72 mmolCO2/gads when the surrogate biogas was saturated with steam. Even though the capacity was further decreased to 0.55 mmolCO2/gads using real landfill gas, it remained fairly stable over 5 cycles despite the presence of impurities, namely hydrogen sulfide (68 ppm). In all cases, the capacity was regenerated with a thermal treatment in inert gas at 100°C. The results suggested possibilities for a single step purification unit to remove acid gasses and water and provide potential for a streamlined operation for the separation of carbon dioxide from biogas. The enhanced economics should propel the move toward a green energy future.

Single-Step Protocol for Isolating the Recombinant Extracellular Domain of the Luteinizing Hormone Receptor from the Ovis aries Testis.

The luteinizing hormone receptor (LHR) is a glycoprotein member of the G protein-coupled receptors superfamily. It participates in corpus luteum formation and ovulation in females and acts in testosterone synthesis and spermatogenesis in males. In this study, we extracted RNA from sheep testicles and synthetized the cDNA to amplify the gene lhr-bed. This gene consists of 762 bp and encodes 273 amino acids of the extracellular domain of LHR. The lhr-bed was cloned into pJET1.2/blunt, then subcloned into pCOLD II, and finally, transformed in E. coli BL21 (DE3) cells. Because the induced rLHR-Bed protein was found in the insoluble fraction, we followed a modified purification protocol involving induction at 25 °C, subjection to denaturing conditions, and on-column refolding to increase solubility. We confirmed rLHR-Bed expression by means of Western blot and mass spectrometry analysis. It is currently known that the structure stem-loop 5'UTR on pCOLD II vector is stable at 15 °C. We predicted and obtained RNAfold stability at 25 °C. We successfully obtained the recombinant LHR extracellular domain, with protein yields of 0.2 mg/L, and purity levels of approximately 90%, by means of a single chromatographic purification step. The method described here may be used to obtain large quantities of rLHR-Bed in the future.

Single-step rapid chromatographic purification and characterization of clinical stage oncolytic VSV-GP.

Purification of viruses, especially for therapeutic purposes, is a tedious and challenging task. The challenges arise due to the size and surface complexity of the virus particles. VSV-GP is a promising oncolytic virus, which has been approved for phase I clinical trials by the Food and Drug Administration (FDA) of United States and Paul Ehrlich Institute (PEI) of Germany. The virus particles of VSV-GP are larger in size than vectors commonly used for gene therapy (e.g., adenovirus, adeno-associated virus, etc.). The current established proprietary clinical-grade manufacturing process for the purification of VSV-GP encompasses several chromatographic and non-chromatographic steps. In this study, we describe a new single-step purification process for the purification of VSV-GP virus, using cation exchange convective flow column with relatively higher yields. The purified virus was characterized for its quality attributes using TCID50 assay (for viral infectivity), host cell protein contaminant ELISA, SDS-PAGE, size exclusion chromatography (SEC), and cryo-electron microscopy. Furthermore, the purified viral therapeutic material was tested in vivo for its efficacy and safety. All these characterization methods demonstrated a therapeutic virus preparation of high purity and yield, which can be readily used for various studies.

Development and evaluation of a semi-automatic single-step clean-up apparatus for rapid analysis of 18 antibiotics in fish samples

Here we report a novel semi-automatic pre-treatment apparatus to fulfill a single-step clean-up aim for rapid quantification of 18 antibiotics in fish samples. Trimethoprim and 17 sulfonamides were extracted from fish samples by acetonitrile solution based on ultrasonic-assisted extraction. Cheap analytical filtration syringe (CAFS) clean-up column was used to fulfill single-step clean-up of the extract which was set on the developed semi-automatic clean-up apparatus. This semi-automatic clean-up apparatus is quite convenient for single-step purification by precisely controlling the flow rate of the extracts, in which up to 10 samples can be purified in parallel at one time. The time needed to clean sample was only about 2 min per 10 samples. Type of compatible purification column ranged from 1 mL (inner diameter × length, 5.6 × 55 mm) to 12 mL (inner diameter × length, 15.6 × 83 mm). The apparatus also did not need additional gas source such as nitrogen or air. After purification, the analytes were simultaneously separated and quantified by high-performance liquid chromatography/quadrupole-linear ion trap tandem mass spectrometry (HPLC-QTRAP-MS/MS). The limits of detections ranged from 0.5 to 1.0 μg kg −1 , and the limits of quantifications ranged from 1.0 to 5.0 μg kg −1 . All the recoveries ranged from 81.39% to 116.01%. Intra-day and inter-day relative standard deviations (RSDs) were lower than 14% for all analytes. • A semi-automatic apparatus was developed to fulfill single-step clean-up of the extract. • Up to 10 samples could be purified in parallel at one time. • The flow rate of the extract could be precisely controlled. • The time needed to clean sample was only about 2 min per 10 samples. • The novel apparatus was applied for rapid clean-up of 18 antibiotics in fish.