Development of rapid chromatographic technologies for complex biofermentation sample analysis.

The recent growth in the biopharmaceutical industry is remarkable due to the introduction of many new therapeutic proteins for the treatment of different diseases. The production processes of the biotherapeutics are complicated and have to be maintained under strict and regulated conditions. Therefore, the development of rapid, sensitive and cost-effective analytical assays is highly demanded for monitoring the biofermentation processes and the key parameters that could affect the final product quality and production consistency. The overall aim of this research is to develop sensitive and selective analytical methods for the determination of raw material components in highly complex samples supplied from the biopharma industry based on solid phase extraction (SPE) and rapid resolution liquid chromatography (RRLC). This development includes the use of narrow-bore columns packed with sub-2 μm silica particles or made of monolithic materials. The developed methods were applied for the qualitative and quantitative analysis of common monosaccharides, including sialic acid, and cysteine/cystine ratio in a range of biopharmaceutical production samples such as raw material yeast extract powders, fermentation feedstocks, chemically defined media and in-process fermentation broth samples in which they were taken from different lots in order to estimate lot-to-lot variability. For evaluation purposes, standard analytical performance criteria were examined for all the developed methods. In addition, a novel solid phase microextraction in a pipette tip for selective enrichment of galactosylated proteins is presented. The extraction device is fabricated by in-situ photopolymerisation of ethylene dimethacrylate porous polymer monolith within the confines of 20 μL polypropylene pipette tip. Then the surface of the monolith was significantly enhanced by immobilising gold nano-particles (AuNPs) which was functionalised with Erythrina cristagalli lectin (ECL) afterwards. The ECLmodified tip was successfully applied for the enrichment of galactosylated proteins versus non-galactosylated proteins from different sample matrices including Escherichia coli cell lysate. Reversed-phase capillary LC was used to validate the efficiency and selectivity of the developed extraction device which resulted in an increase in extraction recovery of ~95% due to the AuNPs enhanced surface area

AuNP-Agglomerated monoliths in pipette tips for lectin affinity extraction of glycoproteins

• To in situ fabricate ethylene dimethacrylate porous polymer monoliths within the confines of a commercial 20 μL polypropylene pipette tips. • To enhance the monolith surface area by immobilising AuNPs and then functionalise the AuNPs with ECL lectin for selective extraction of galactosylated proteins from complex media

A Simple and Highly Structured Procaine Hydrochloride as Fluorescent Quenching Chemosensor for Trace Determination of Mercury Species in Water

An ultrasensitive, simple and highly selective spectrofluorometric strategy for quantifying traces of mercury(II) in environmental water has been established using the fluorescent probe procaine hydrochloride (PQ+.Cl−). The procedure was based upon the formation of the ternary ion associate complex [(PQ+)2.(HgI4)2−] between PQ+.Cl− and mercury(II) in iodide media at pH 9.0–10.0 with its subsequent extraction onto dichloromethane accompanied by a change in fluorescence intensity at λex/em = 268/333 nm. The developed strategy exhibited a linear range of 1–114 μg L−1 with lower limit of detection (LOD) and quantification (LOQ) of mercury(II) 1.3 and 3.98 nM, respectively. Intra and inter-day laboratory accuracy and precision for trace analysis of mercury(II) in water were performed. Complexed mercury(II) in environmental water, chemical speciation and successful literature comparison was performed. The proposed system offered excellent selectivity towards mercury(II) ions examined in the presence of competent ions in excess, relevant to real water samples. The method was applied for analysis of mercury(II) in tap water samples. Statistical comparison (Student’s t and F tests) of the proposed method with the reference ICP-OES method revealed no significant differences in the accuracy and precision

Chromatographic Separation, Total Determination and Chemical Speciation of Mercury in Environmental Water Samples Using 4-(2-Thiazolylazo) Resorcinol-Based Polyurethane Foam Sorbent-Packed Column

A simple method has been developed for quantitative retention of traces of mercury(II) ions from aqueous media using polyurethane foams (PUFs) loaded with 4-(2-thiazolylazo) resorcinol (TAR). The kinetics and thermodynamics of the sorption of mercury(II) ions onto PUFs were studied. The sorption of mercury(II) ions onto PUF follows a first-order rate equation with k = 0.176 ± 0.010 min−1. The negative values of ΔH and ΔS may be interpreted as the exothermic chemisorption process and indicative of a faster chemisorption onto the active sites of the sorbent. The sorption data followed Langmuir, Freundlich and Dubinin-Radushkevich (D–R) isotherm models. The D-R parameters β, KDR and E were 0.329 mol2 kJ−2, 0.001 μmol g−1 and 1.23 ± 0.07 kJ/mol for the TAR-loaded PUFs, respectively. An acceptable retention and recovery (99.6 ± 1.1%) of mercury(II) ions in water at ≤10 ppb by the TAR-treated PUFs packed columns were achieved. A retention mechanism, involving absorption related to “solvent extraction” and an “added component” for surface adsorption, was suggested for the retention of mercury(II) ions by the used solid phase extractor. The performance of TAR-immobilized PUFs packed column in terms of the number (N), the height equivalent to a theoretical plate (HETP), the breakthrough and critical capacities of mercury(II) ion uptake by the sorbent packed column were found to be 50.0 ± 1.0, 1.01 ± 0.02 mm, 8.75 and 13.75 mg/g, respectively, at 5 mL/min flow rate

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Development of a rapid and sensitive method for determination of cysteine/cystine ratio in chemically defined media

Two rapid, sensitive and quantitative methods for the determination of the cysteine and cystine ratio in complex defined media feedstock using monolithic reversed-phase liquid chromatography (RPLC) and RPLC-MS are presented. Cysteine is pre-derivatised with purified 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) and separated from other derivatisation products on a narrow-bore 50 mm x 2 mm I.D. monolithic C-18 column with UV detection at 355 nm. For reversed-phase LC (RPLC) the separation is carried out isocratically using a mobile phase of 50 mM trichloroacetic acid (TCA) adjusted to pH 2.5 with lithium hydroxide (LiOH) and acetonitrile (83:14) pumped at 1.5 mL/min with an elevated column temperature. For RPLC-MS an ammonium acetate and acetonitrile gradient method was developed with a reduced flow rate of 0.3 mL/min. The treatment of the samples consisted of dividing them into two aliquots, the first aliquot is analysed for cysteine and the second aliquot is analysed for cystine after its quantitative reduction to cysteine using tris(2-carboxyethyl)phosphine (TCEP). Both methods are linear, with R-2 &gt; 0.999 for 0.25-500 mu M for cysteine and 0.25-250 mu M for cystine using the LC-UV method, sensitive, with detection limit of 36 nM for cysteine, and precise, with &lt;= 1.1% RSD for both retention time and peak area (n = 6). Samples (n = 31) of an industry standard and supplied chemically defined media feedstock were analysed, finding cysteine ranging from 1.56 to 2.26 mu g/mL and cystine from 1062.02 to 1348.13 mu g/mL (C) 2010 Elsevier B.V. All rights reserved.</p

AuNP-Agglomerated monoliths in pipette tips for lectin affinity extraction of glycoproteins

• To in situ fabricate ethylene dimethacrylate porous polymer monoliths within the confines of a commercial 20 μL polypropylene pipette tips. • To enhance the monolith surface area by immobilising AuNPs and then functionalise the AuNPs with ECL lectin for selective extraction of galactosylated proteins from complex media

Pipette-tip selective extraction of glycoproteins with lectin modified gold nano-particles on a polymer monolithic phase

The selective extraction of specific proteins (non glycosylated, glycosylated or different glycoforms) from complex sample matrices utilising selective solid phase extration (SPE) is of significant interest within the fields of proteomics and glycoproteomics. Polymer monoliths have proven to be an excellent solid support for SPE applications in bio-analysis due to their excellent mass transfer characteristics for large biomolecules. Although biorecognition molecules such as lectins can be covalently immobilised directly onto the 10 monolith surface using a variety of chemistries, the relatively low surface area of these monoliths results in a correspondingly low sample capacity. Recently, we have described the covalent attachment of 20 nm gold nanoparticles (AuNPs) upon a polymer monolith with excellent surface coverage leading to a significant increase in surface area. In this work therefore wedescribe the in-situ preparation of an ethylene dimethacrylate porous polymer monolith within the confines of 20 μL polypropylene pipette tips, onto which methacrylate anchor sites were previously grafted to ensure intimate monolith/wall contact. Then AuNPs were immobilised onto the monolith pore 15 surface utilising azlactone cemistry. Field emission scanning electron microscopy was used to verify the high surface coverage of AuNPs. Erythrina cristagalli lectin (ECL) was immobilised upon the attached AuNPs via a biofunctional linker. The ECL-modified tip was successfully applied for the enrichment of galactosylated protein (desialated transferrin) versus a non-galactosylated protein (ribonuclease B) due to the specificity of ECL. Reversed phase capillary HPLC was used to validate the efficiency and selectivity of the developed micro-extraction phase which resulted in an increase in extraction recovery of ~95 % in the presence of AuNPs. The 20 specificity of the ECL-modified tip was further studied by using more complex mixture of proteins which included non-glycosylated proteins and glycosylated proteins with different terminal sugar structure. Finally, the lectin affinity extraction device was tested with a real sample (E. coli cell lysate) spiked with target galactosylated glycoproteins