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With the increasing global demand for precise and efficient pharmaceuticals and the biopharma industry moving towards Industry 4.0, the need for advanced process integration, automation, and modeling has increased as well. In this work, a method for automatic modeling and calibration of an integrated preparative chromatographic system for pharmaceutical development and production is presented. Based on a user-defined system description, a system model was automatically generated and then calibrated using a sequence of experiments. The system description and model was implemented in the Python-based preparative chromatography control software Orbit. [ABSTRACT FROM AUTHOR]

[Display omitted] • PBDEs were measured in various materials from seating furniture wastes. • A single MAE-GC/MS process was adapted for both plastics, foams and textiles. • From 100 samples the sum of 8 PBDEs did not exceed the tolerated threshold. • BDE-209 was identified at higher contents mainly in hard plastics. • No direct correlation could be found between Br levels measured by XRF and PBDEs. Polybrominated diphenyl ethers (PBDEs) are brominated flame retardants that are used in polymeric materials. Due to their adverse health effects, the use of recycled wastes has been forbidden if the total PBDE content exceeds 0.1% (w/w). The objective was to estimate the proportion of PBDEs in professional seating furnishing wastes to identify the materials in which the content of PBDEs (and particularly BDE-209) could exceed the limit to eliminate them from recycling. An analytical process (microwave extraction followed by purification and chromatographic analysis) was adapted to assess with a unique methodology the amounts of eight PBDEs in materials that result from various seating wastes, such as hard plastics, foams and accompanying textiles. X-ray fluorescence (XRF) was used to rapidly predict critical PBDE concentrations via Br. From 100 samples, the total PBDE content did not exceed the current tolerated threshold. The examined materials contained only trace levels of former PBDE formulations, and BDE-209 was identified at higher amounts, mainly in hard plastics, but these amounts were less than 312 mg kg−1. Since XRF was not reliable for quantitative measurements and was not specific, no direct correlation could be identified between Br and PBDE levels. Br was strongly associated with As in all the materials, but the presence of PBDEs was not clearly associated with the presence of other metals that are used in flame retardants. [ABSTRACT FROM AUTHOR]

The extraction condition of curcumin from Curcuma longa L was optimized through four factors and three levels orthogonal experiment based on the results of single factor tests. Under the optimal conditions: the concentration of ethanol 80%, extraction temperature 70°C, the ratio of liquid to material 20, and extraction time 3 h, a crude extract with the yield of curcumin 56.8 mg/g could be obtained. The isolation and purification of curcuminoids from the crude extract was performed on high performance counter current chromatography employing an optimized solvent system n‐hexane/ethyl acetate/methanol/water (2/3/3/1, v/v/v/v). From 97 mg crude sample (in which the purity of curmumin was 68.56%), 67 mg curmumin, 18 mg demethoxycurcumin, and 9.7 mg bisdemethoxycurcumin with a high‐performance liquid chromatography purity of 98.26, 97.39, and 98.67%, respectively, were obtained within 70 min. The antioxidant activities and cytotoxicity of purified curcumin was comparable to that of the commercial product, indicating that the biological activity of curcumin could be maintained by this method. [ABSTRACT FROM AUTHOR]

Pulmonary surfactant preparations extracted from natural sources have been used to treat millions of newborn babies with respiratory distress syndrome (RDS) and can possibly also be used to treat other lung diseases. Due to costly production and limited supply of animal-derived surfactants, synthetic alternatives are attractive. The water insolubility and aggregation-prone nature of the proteins present in animal-derived surfactant preparations have complicated development of artificial surfactant. A non-aggregating analog of lung surfactant protein C, SP-C33Leu is used in synthetic surfactant and we recently described an efficient method to produce rSP-C33Leu in bacteria. Here rSP-C33Leu obtained by salt precipitation of bacterial extracts was purified by two-step liquid gel chromatography and analyzed using mass spectrometry and RP-HPLC, showing that it is void of modifications and adducts. Premature New Zealand White rabbit fetuses instilled with 200mg/kg of 2% of rSP-C33Leu in phospholipids and ventilated with a positive end expiratory pressure showed increased tidal volumes and lung gas volumes compared to animals treated with phospholipids only. This shows that rSP-C33Leu can be purified from bacterial lipids and that rSP-C33Leu surfactant is active against experimental RDS. [ABSTRACT FROM AUTHOR]

Highlights • First report on the main expanded-bed chromatography parameters to purify peroxidases. • Cationic adsorbent was used to purify rice bran peroxidase. • Purification efficiency was increased by adding CaCl 2 in buffer during elution step. • Rice bran peroxidase was purified in 14.1-fold using a single technique. Abstract Peroxidases catalyze the oxidation-reduction reactions of organic substrates and present several applications in the biotechnological, clinical, environmental, and industrial areas. Among the chromatographic strategies applied to peroxidase purification to allow future uses, expanded-bed adsorption (EBA) stands out due to the large volumes of purified enzyme and potential for scale-up. In this paper, the main EBA parameters were evaluated in the purification of rice bran peroxidases. Sodium acetate buffer (pH 4.5) and expansion degree of 2.5 were the most favorable conditions to the enzyme adsorption onto Streamline® SP resin, showing an enzymatic activity at equilibrium on the solid phase (q*) of 2.68 U/mL resin, a partition coefficient (f) of 38.35 and a dynamic binding capacity (Q 10%) of 0.19 U/mL resin. Rice bran peroxidase was purified in 14.1-fold (± 1.3) with 49.8% (± 2.5) of recovery by using 25 mmol/L acetate buffer pH 5.5 in the washing step, and elution combining stepwise (0.15 mol/L NaCl) and linear gradient (0.15–1 mol/L NaCl), using acetate buffer pH 5.5 with 1 mmol/L of CaCl 2. Based on the results obtained, EBA showed to be a viable single-step strategy for peroxidase purification. [ABSTRACT FROM AUTHOR]

Modern technological approaches to ensuring the specific safety of human immunoglobulin and albumin preparations are analyzed. Introduction into human immunoglobulin preparation technology of donor screening for group- and rhesus-affiliations and chromatographic purification steps to reduce the contents of anti-A and anti-B hemagglutinins and anti-D antibodies are shown to be promising. Technological capabilities for reducing the anticomplementary activity and thrombogenic potential of immunoglobulin preparations and decreasing the contents of prekallikrein activator in human i.v. immunoglobulin and albumin preparations are discussed. [ABSTRACT FROM AUTHOR]

A chromatographic procedure for Rb, K, Ba and Sr one-by-one separation from geological materials has been developed by using a single column with packing Sr-spec resin, followed by high-precision Rb isotope measurement by multi-collector inductively coupled plasma mass spectrometry (MC-ICP-MS). The main matrix elements (Ti, Al, Mg, Fe, Mn, Na and Ca) were removed with 4.4 mL of 3 M HNO3 first; Rb and K were then sequentially eluted in different volumes with the same acid. After that, Ba was eluted with 8 M HNO3, and finally Sr was eluted with Milli-Q water. This procedure enables us to collect pure Rb, K, Ba and Sr one by one in a single column with recovery close to 100%. The purified Rb was measured by using a standard-sample bracketing method via MC-ICP-MS. The short-term precision for δ87Rb was better than ±0.03‰ (2SD, for 15 hours), and the long-term (more than 12 months) external precision was better than ±0.06‰ (2SD) based on repeated analysis of Rb standard solution NIST SRM 984. Matrix effects on Rb isotope measurement by MC-ICP-MS were tested by doping pure Rb standard with various amounts of matrix elements and were found to be insignificant when Na/Rb ＜ 2, Ca/Rb ＜ 4 and K/Rb ＜ 25. We measured a set of geological reference standards by this method and found significant Rb isotopic variation. Our study suggests that Rb isotope geochemistry may be a promising tracer of various cosmochemical and geological processes. [ABSTRACT FROM AUTHOR]

A new protease was isolated from the intestine of small red scorpionfish. After ammonium sulfate precipitation, the enzyme was purified to homogeneity by a two-step chromatography with 6.69% recovery and fivefold increase in specific activity. The molecular weight of the protease was about 24 kDa as estimated by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme optimum pH and temperature were pH 10.0 and 40°C, respectively. The protease was stable at temperatures below 40°C and over a broad pH range (8.0-11.0). The Km and Kcat values were 0.36 mmol L-1 and 1.61 s-1, respectively. Interestingly, the enzyme presented specificity for casein, egg albumin, bovine serum albumin, and gelatin with obtained degrees of hydrolysis ranging from 3.14% to 6.58%, after 2 h of hydrolysis. Moreover, gluten hydrolysate analysis confirmed protein hydrolysis and solubilization. These results suggested the potential use of the protease in the preparation of food protein hydrolysates with interesting properties. [ABSTRACT FROM AUTHOR]

This instalment in our series on ultrahigh-pressure liquid chromatography (UHPLC) highlights its benefits in fast analysis, high-resolution separations, high performance liquid chromatography (HPLC) method development, reduced solvent and sample usage, and enhanced sensitivity and precision performance. [ABSTRACT FROM AUTHOR]

Dual flow chromatography (DFC) separations are performed with back and forth flow for rapid method development, design of experiments (DOE), quality-by-design (QbD), or high-throughput chromatographic purification. Although different than conventional unidirectional flow through chromatography, chromatographic principles still control the separations. Selectivity coefficients and Langmuir adsorption isotherms control the separation chemistry properties of the column and dictate the mobile phase conditions needed to achieve separation. However, the kinetic rates of diffusion and interaction of mobile phase molecules with the stationary phase, column channeling, and other column properties are not germane to the practice of DFC. Chromatographic conditions developed with DFC can be scaled to any size, including laboratory and industrial preparative columns. One practical use of DFC is in the pharmaceutical industry where multidimensional method development and high-throughput, parallel processing has been used to improve R&D productivity and decrease the time to manufacturing of biopharmaceutical drugs. [ABSTRACT FROM AUTHOR]

In this study we report the purification of laccase produced by Trichoderma harzianum strain HZN10 (using wheat bran under solid state fermentation) and its application in decolorization of synthetic dyes. Extracellular laccase was purified to homogeneity by DEAE-Sepharose and Sephadex G-100 chromatography with specific activity of 162.5 U/mg and 25-fold purification. Purified laccase was immobilized in various entrapments like calcium alginate, copper alginate, calcium alginate–chitosan beads and sol–gel matrix. Optimization results revealed that the laccase immobilized in sol–gel was optimally active in wide pH range (4.0–7.0) and thermo-stable (50–70 °C) than free enzyme which was optimum at 50 °C and pH 6.0. Kinetic analysis showed K m of 0.5 mM and 2.0 mM and V max of 285 U/mg and 500 U/mg by free laccase and sol–gel immobilized laccase respectively with 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) [ABTS] substrate. Free and immobilized laccase was employed for decolorization of three different synthetic dyes (malachite green, methylene blue and congo red). High performance liquid chromatography (HPLC) analysis results revealed that approximately 100% of malachite green, 90% of methylene blue and 60% of congo red dyes at initial concentration of 200 mg/L were decolorized within 16, 18 and 20 h, respectively by laccase immobilized in sol–gel matrix in the presence of 1-hydroxybenzotriazole (HBT) mediator. During the decolorization all three synthetic dyes showed various peaks on HPLC chromatogram indicating different by-products formation. Finally, phytotoxicity analysis results revealed that the by-products of synthetic dyes (formed during decolorization) showed less toxicity against Phaseolus mungo compared to untreated synthetic dyes. [ABSTRACT FROM AUTHOR]

Various high-throughput systems and strategies are employed by the biopharmaceutical industry for early to late-stage process development for biologics manufacturing. The associated increases to experiment productivity and reduction in material consumption makes high throughput tools integral for bioprocess development. While these high-throughput systems have been successfully leveraged to generate high quality data representative of manufacturing scale processes, their data interpretation often requires complex data transformation and time-intensive system characterization. With respect to high throughput purification development, RoboColumns by Repligen operated on Tecan automated liquid handling systems offer superior performance scalability, but lack an optimized liquid delivery system that is representative of preparative chromatography. Particularly, stock Tecan liquid handling systems lack the capability to provide high-capacity continuous liquid flow and ideal linear gradient chromatography conditions. These limitations impact protein chromatography performance and hinder the application of high-throughput gradient elution experiments. In this work, we describe a Tecan Freedom EVO high-throughput purification tool that provides more continuous liquid delivery enabling continuous gradient elution capability for RoboColumn experiments as demonstrated by generation of highly linear conductivity gradients. Results demonstrate that the tool can provide RoboColumn performance and product quality data that is in agreement with larger, bench scale chromatography formats for two model purification methods. The described gradient purification method also provides more consistent performance between RoboColumns and larger column formats compared to step elution methods using the same optimized Tecan system. Lastly, new insights into the impact of discontinuous flow on RoboColumn elution performance are introduced, which may help further improve application of these data towards bioprocess development. [ABSTRACT FROM AUTHOR]

Given recent advances in automated oligosaccharide synthesis, analytical techniques that can be coupled to a synthetic framework are needed to not just identify but also purify to homogeneity protected carbohydrate compounds at levels of ≥99.5% purity. Herein, an alternate-pump recycling high-performance liquid chromatography (R-HPLC) method has been developed to allow purification of protected carbohydrates at levels of ≥99.5% purity. [ABSTRACT FROM AUTHOR]

In the present study, the feasibility of immunoaffinity chromatography (IAC) as a purification technology for the analysis of bioactive components in Traditional Chinese Medicine (TCM) was evaluated. IAC was used to analyze hesperidin (HP) and narirutin (NR) in TCM preparations containing Citri reticulatae Pericarpium (CRP, Chenpi in Chinese). An IAC column for the specific extraction and enrichment of HP and NR from TCM preparations containing CRP was developed and characterized. After HP reacted with carbonyl diimidazole and coupled to protein, it was used to immune mice for the generation of antibody. Through cell fusion, cloning and screening, monoclonal antibody was obtained. The IAC column was constructed by covalently coupling specific monoclonal antibody against HP and NR to CNBr-activated Sepharose 4B and packed into a common solid phase extraction cartridge. The extraction conditions including loading, washing and eluting, as well as flow rate for the extraction of HP and NR were optimized. Under optimal conditions, the maximum capacity, extraction recovery rate and stability of IAC column was also characterized. Results revealed that the maximum capacity of IAC column for HP and NR was approximately 16 μg and the relative binding capacity per 1 mL of the column volume was 27 μg. The extraction recovery rate of IAC column for HP and NR at three spiked levels was in the range of 94.05–109.15%. After the repeated application for 5 times, no significant loss of specific recognition was observed. Using high performance liquid chromatography (HPLC) as an effective analytic tool, HP and NR could be successfully separated via IAC column without the inference from impurities, suggesting that the extraction of HP and NR using the prepared IAC column is feasible. The application of IAC can solve the problem of quantitative analysis due to severe interference or low content. Furthermore, pretreatment methods in different matrixes can be unified. The IAC purification procedure can be used as an alternative effective analytical method for the pretreatment of bioactive components in TCM. [ABSTRACT FROM AUTHOR]

Chromatographic purification of chromium (Cr), which is required for high-precision isotope analysis, is complicated by the presence of multiple Cr-species with different effective charges in the acid digested sample aliquots. The differing ion exchange selectivity and sluggish reaction rates of these species can result in incomplete Cr recovery during chromatographic purification. Because of large mass-dependent inter-species isotope fractionation, incomplete recovery can affect the accuracy of high-precision Cr isotope analysis. Here, we demonstrate widely differing cation distribution coefficients of Cr(III)-species (Cr 3+ , CrCl 2+ and CrCl 2 + ) with equilibrium mass-dependent isotope fractionation spanning a range of ∼1‰/amu and consistent with theory. The heaviest isotopes partition into Cr 3+ , intermediates in CrCl 2+ and the lightest in CrCl 2 + /CrCl 3 °. Thus, for a typical reported loss of ∼25% Cr (in the form of Cr 3+ ) through chromatographic purification, this translates into 185 ppm/amu offset in the stable Cr isotope ratio of the residual sample. Depending on the validity of the mass-bias correction during isotope analysis, this further results in artificial mass-independent effects in the mass-bias corrected 53 Cr/ 52 Cr (μ 53 Cr* of 5.2 ppm) and 54 Cr/ 52 Cr (μ 54 Cr* of 13.5 ppm) components used to infer chronometric and nucleosynthetic information in meteorites. To mitigate these fractionation effects, we developed strategic chemical sample pre-treatment procedures that ensure high and reproducible Cr recovery. This is achieved either through 1) effective promotion of Cr 3+ by >5 days exposure to HNO 3 H 2 O 2 solutions at room temperature, resulting in >∼98% Cr recovery for most types of sample matrices tested using a cationic chromatographic retention strategy, or 2) formation of Cr(III)-Cl complexes through exposure to concentrated HCl at high temperature (>120 °C) for several hours, resulting in >97.5% Cr recovery using a chromatographic elution strategy that takes advantage of the slow reaction kinetics of de-chlorination of Cr in dilute HCl at room temperature. These procedures significantly improve cation chromatographic purification of Cr over previous methods and allow for high-purity Cr isotope analysis with a total recovery of >95%. [ABSTRACT FROM AUTHOR]

A novel method for Fmoc/ tBu solution-phase peptide synthesis and the development of a new benzyl-type group-assisted purification (GAP) protecting group is reported. This GAP protecting group is utilized in place of a polymer support, facilitating C→N Fmoc peptide synthesis without chromatography or recrystallization. The GAP group can be added and removed in high yield, and was used to synthesize over 1 gram of the immunostimulant, thymopentin, in high overall yield (83 %) and purity (99 %). [ABSTRACT FROM AUTHOR]

The interest of pharmaceutical companies for complementary high-performance chromatographic tools to assess a product's purity or enhance this purity is on the rise. The high-throughput capability and economic benefits of supercritical fluid chromatography, but also the 'green' aspect of CO2 as the principal solvent, render supercritical fluid chromatography very attractive for a wide range of pharmaceutical applications. The recent reintroduction of new robust instruments dedicated to supercritical fluid chromatography and the progress in stationary phase technology have also greatly benefited supercritical fluid chromatography. Additionally, it was shown several times that supercritical fluid chromatography could be orthogonal to reversed-phase high-performance liquid chromatography and could efficiently compete with it. Supercritical fluid chromatography is an adequate tool for small molecules of pharmaceutical interest: synthetic intermediates, active pharmaceutical ingredients, impurities, or degradation products. In this review, we first discuss about general chromatographic conditions for supercritical fluid chromatography analysis to better suit compounds of pharmaceutical interest. We also discuss about the use of achiral and chiral supercritical fluid chromatography for analytical purposes and the recent applications in these areas. The use of preparative supercritical fluid chromatography by pharmaceutical companies is also covered. [ABSTRACT FROM AUTHOR]

The total alkaloids from Nitraria sibirica leaves have been confirmed to exhibit significant protective effects against inflammatory renal injury, hypertension and albuminuria in angiotensin II-salt hypertension. In the present study, a separation method of pH-zone-refining counter-current chromatography was established for separation of the alkaloids from N. sibirica . The separation was performed with a solvent system of MtBE- n -BuOH-H 2 O (2:2:5, v / v ) at a flow rate of 2.0 mL/min. And 15 mM triethylamine (TEA) was added to the upper organic phase, while 10 mM hydrochloric acid was added to the lower aqueous phase. As a result, a new alkaloid, schobemine (5.6 mg), and a known alkaloid, nitraramine (5.0 mg), together with fractions A and B were obtained from the total alkaloids of N. sibirica . The fractions A and B were further purified by means of pH-zone-refining counter-current chromatography with solvent systems of n -hexane- n -BuOH-H 2 O (1.5:3.5:5, v / v ) and (2:3:5, v / v ), respectively. TEA (10 mM) was added to the upper phase, and 10 mM of HCl was added to the lower phase in above two solvent systems, respectively. As a result, a known alkaloid, schoberidine (5.0 mg), and a new alkaloid, schoberimine (3.0 mg) were obtained from fractions A and B, respectively. The purities of the compounds were measured by HPLC–ELSD, and their structures were identified by ESI–MS, 1D and 2D NMR. [ABSTRACT FROM AUTHOR]

This study was carried out to investigate the purification and biochemical characterization of a new extracellular alkaliphilic and thermostable pectate lyase (Pel-20) isolated from Actinomadura keratinilytica strain Cpt20. Pure protein was obtained after sequential chromatographies on a fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC) columns. Matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis revealed that the purified enzyme was a monomer with a molecular mass of 34125.11-Da. The enzyme had an NH 2 -terminal sequence of GFATNQGGTTGGAGGTLS, thus, sharing high homology with actinomycetes pectate lyase family. The results showed that this enzyme was completely inhibited by EDTA, which supports its belonging to the pectate lyase superfamily. It showed optimum activity at pH 10.5 and 70 °C. The thermoactivity and thermostability of Pel-20 were enhanced in the presence of 1 mM Ca 2+ . Its half-life times at 70, 80, 90, and 100 °C were 18, 12, 7, and 2 h, respectively. Its kinetic parameters, K m and V max values were 0.45 mM and 21,700 U/mg, respectively. Low-esterified pectin was the optimum substrate for the Pel-20. However, higher-esterified pectin was also weakly cleaved. Overall, the alkaliphilicity and thermostability properties of Pel-20 make it a potential candidate for future application in industrial bioprocesses. [ABSTRACT FROM AUTHOR]

This article presents the use of caprylic acid (CA) to precipitate impurities from the protein A capture column elution pool for the purification of monoclonal antibodies (mAbs) with the objective of developing a two chromatography step antibody purification process. A CA-induced impurity precipitation in the protein A column elution pool was evaluated as an alternative method to polishing chromatography techniques for use in the purification of mAbs. Parameters including pH, CA concentrations, mixing time, mAb concentrations, buffer systems, and incubation temperatures were evaluated on their impacts on the impurity removal, high-molecular weight (HMW) formation and precipitation step yield. Both pH and CA concentration, but not mAb concentrations and buffer systems, are key parameters that can affect host-cell proteins (HCPs) clearance, HMW species, and yield. CA precipitation removes HCPs and some HMW species to the acceptable levels under the optimal conditions. The CA precipitation process is robust at 15-25°C. For all five mAbs tested in this study, the optimal CA concentration range is 0.5-1.0%, while the pH range is from 5.0 to 6.0. A purification process using two chromatography steps (protein A capture column and ion exchange polishing column) in combination with CA-based impurity precipitation step can be used as a robust downstream process for mAb molecules with a broad range of isoelectric points. Residual CA can be effectively removed by the subsequent polishing cation exchange chromatography. [ABSTRACT FROM AUTHOR]

An improved and efficient synthesis of (+)-cloprostenol has been accomplished in nine steps and 26% overall yield from commercially available (-)-Corey lactone 4-phenylbenzoate alcohol 1. The present route avoids tedious purifications and requires only one column chromatography operation, which reduces the generation of waste and is suitable for . Chirality 27:392-396, 2015. © 2015 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Objective: The purpose of the present study was to purify and characterize an esterase from a thermophilic bacterium Thermus sp. NCCB 100425T and to check its suitability for industrial applications. Methods: Thermus sp. NCCB 100425T esterase was purified by using ammonium sulphate precipitation and hydrophobic interaction chromatography and then characterized biochemically. Results: The purity of the enzyme was observed as a single band on native- and SDS- PAGE. In the presence of p-nitrophenyl acetate (pNPA) as a substrate, the optimum pH and temperature of the enzyme were found to be 7.5 and 60°C, respectively. Km and Vmax values are calculated as 18.32 mM and 96.15 U/mg protein, respectively, with pNPA. pH stability was investigated in the range of pH 4.0-9.0 at 4°C and 60°C. After 7 days incubation, activity of pure enzyme was retained 85-90% for all pH at 4°C and 59±5.2% for pH 8.0 at 60°C. It was determined that approximately 80% of enzyme activity was retained between 30-60°C after 7 days incubation. In the presence of 10% ethanol and DMSO, the enzyme activity was retained 96±2.7% and 78±2.5%, respectively. Additionally, it was detected that some metal ions affect the enzyme activity at different ratios. Conclusion: It is clear that Thermus sp. NCCB 100425T esterase might have advantages for industrial and/or clinical applications in terms of especially its high thermal- and pH-stability. [ABSTRACT FROM AUTHOR]

Immunoglobulins G (IgG) against hepatitis B core antigen (HBcAg) was successfully purified using a purification scheme comprising ammonium sulphate precipitation and SepFast™ MM AH-1 column chromatography. Ammonium sulphate precipitation performed at 40% saturation was optimum in terms of the recovered polyclonal IgG concentration (7.8 mg/ml) and the removal of albumin (72%). The yield, purity and purification factor achieved from this simple purification method were 99%, 94% and 7.8, respectively. The IgG recovered from ammonium sulphate precipitation was subjected to SepFast™ MM AH-1 column chromatography and the purity of IgG was further increased to 98%, corresponding to a purification factor of 8.1. Protein aggregation was also reduced significantly in the purified IgG sample. Furthermore, the salt content in the purified sample was reduced by 75% and therefore the need of desalting final product was eliminated. Enzyme-linked immunosorbent assay (ELISA) showed that the antigenicity of anti-HBcAg IgG obtained after these purification processes was maintained. [ABSTRACT FROM AUTHOR]

Herein, we report a short, three-step synthesis of d-[1-14C]-serine (4) in high enantiomeric purity. Starting from [14C]-KCN and 2-(benzyloxy)acetaldehyde, Strecker reaction using ( R)-1-phenylethylamine as the chiral auxiliary gave two diastereomeric aminonitriles 1 and 2 in the ratio of 4:3, which were conveniently separated and purified chromatographically. Following hydrolysis and subsequent hydrogenolysis, the purified major diastereomer 1, was smoothly converted to d-[1-14C]-serine (4) in an enantiomeric excess of >99%, thus circumventing time intensive chiral HPLC enantiomeric resolution. [ABSTRACT FROM AUTHOR]

Monoclonal antibodies (mAbs) are one of the fastest growing drug molecules targeting the treatment of diseases ranging from arthritis, immune disorders, and infectious diseases to cancer. Due to its unique application principle, antibodies are commonly produced in large quantities. Plants, such as Nicotiana benthamiana , offer a unique production platform for bio-therapeutics due to their ability to produce large amounts of biomolecules in a relatively quick manner. However, purification of a target protein from plant is an arduous task due to the presence of toxic compounds in ground plant tissue and the large quantities of plant tissues to be processed. Here, a process was developed prior to the chromatographic purification of a mAb against Ebola GP1 protein expressed in N. benthamiana. The process includes a diafiltration step and a charged polyelectrolyte precipitation. The diafiltration step significantly improved the precipitation efficiency, reducing the usage of polyelectrolyte by more than 2000 fold while improving the native plant protein removed from 60% to 80%. The mAb can then be purified to near homogeneity judging from SDS-PAGE by either Protein A affinity chromatography or a tandem of hydrophobic interaction chromatography and a hydrophobic charge induction chromatography. The purified mAbs were shown to retain their binding specificity to irradiated Ebola virus. [ABSTRACT FROM AUTHOR]

ABSTRACT The concept of Process Analytical Technology ( PAT) introduced by the US FDA has received a lot of attention lately. The use of such a scheme ensures robust process performance over the entire life cycle of the product and consistent product quality at the end of the manufacturing process. Successful implementation of PAT requires use of high resolution, orthogonal analytical tools that are able to measure the critical quality attributes ( CQA) of the raw materials and in-process materials with the aim of ensuring final product quality. This article presents a review of PAT applications for monitoring commonly used downstream biotech unit operations. Focus of the review will be on recent advancements (last 5 years). Analytical tools that have been used in these approaches have also been discussed with reference to their speed of analysis, robustness and sensitivity. Also discussed is their potential to replace the traditional high performance liquid chromatography ( HPLC) which continues to be the gold standard for analysis of biotech therapeutics. © 2014 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

A methodology to purify a Ho solution has been developed by a combination of activity and mass concentration measurements in order to further determine the Ho half-life. The isobaric interference at m/ q ≃ 166 requires Ho purification from non-natural Er with a high purification degree due to the large amount of Ho as opposed to Er. The Ho/Er separation was achieved using high-performance liquid chromatography on a semi-preparative column followed by purification on gravitational chromatography. The efficiency of the separation was evaluated after precise determination of the Er isotopic composition. The purification methodology enabled to separate Ho from Er. [ABSTRACT FROM AUTHOR]

Highlights: [•] We develop a microfluidic chip to purify and enrich virus by hydroxyapatite chromatography. [•] We examine purification and enrichment of the virus. [•] The viruses were isolated from the mixture of virus and protein. [•] The virus concentration was increased by hydroxyapatite chromatography. [ABSTRACT FROM AUTHOR]

Magnetic poly(2-hydroxyethyl methacrylate-co-N-methacryloyl–L-phenyl alanine), p(HEMA-co-MAPA) nanoparticles were produced by emulsion polymerization technique. The nanoparticles were characterized by zetasizer, atomic force microscopy (AFM), transmission electron microscopy (TEM), electron spin resonance spectroscopy (ESR), and Fourier transformed infrared spectroscopy (FTIR) techniques. Experimental conditions were optimized for DNA adsorption in aqueous media. The effects of several factors (i.e., temperature, pH, equilibrium concentration of DNA, salt type, and ionic strength) on adsorption capacity were examined in batch studies. The maximum DNA adsorption capacity was 211.3 mg/g for nanoparticles consisting of magnetite. The adsorption isotherms and kinetics were also examined. The Langmuir model and pseudo second-order kinetics are the best fitted to the data. Following the ten adsorption-desorption cycles, the decrease in adsorption capacity was only 12%. Finally, plasmid DNA was purified fromE.colilysate by magnetite nanoparticles consisting of MAPA. The purity and molecular mass of purified plasmid DNA were determined by the use of agarose gel electrophoresis resulting in 7000 base pairs for molecular mass and OC pDNA form following elution. [ABSTRACT FROM AUTHOR]

Flavonoids are the main chemical constituents ofSmilax chinaL. and thought to be responsible for the anti-inflammatory activity ofSmilax chinaL. In this study, a suitable HSCCC method was developed to separate the three main flavonoids in a one-separation from the anti-chronic pelvic inflammation disease (CPID) effective fraction ofSmilax chinaL. with a two-phase solvent system composed of n-hexane-ethyl acetate-methanol-water (1:10:1:10, v/v/v/v). 42 milligrams of astilbin, 10 mg neoisoastilbin, and 6 mg quercetin-3-O-α-L-rhamnoside were separated from 200 mg of the anti-CPID effective fraction ofSmilax chinaL. with purity of 98.3%, 98.7%, 98.5%, respectively, by HPLC analysis. Orthogonal test L9(34) was also applied to investigate the optimum extracting conditions of the three flavonoids. HSCCC was successfully used for the isolation and purification of the three flavonoids from the anti-CPID effective fraction ofSmilax chinaL. and the neoisoastilbin was first separated fromSmilax chinaL. [ABSTRACT FROM AUTHOR]

• Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving. • High throughput (HTP) chromatography significantly speed up AAV process development. • HTP chromatography using resin slurry plate and mini-column for AAV purification. • Demonstrated performance comparability between HTP and bench-scale chromatography. Recombinant adeno-associated virus (AAV) has been broadly used as a delivery tool for gene therapy applications. The development of a robust purification process is essential for delivering high purity and quality AAV products to clinic. The short clinical timelines and material limitations of early-stage development pose unique challenges to developing robust and scalable downstream purification processes. One approach to overcome these limitations is to leverage high throughput (HTP) strategies and automation technologies for purification process development, an approach that is well established in protein biologics and other areas. However, due to the unique challenges related to viral vector purification, implementing HTP approaches for gene therapy process development has not been explored extensively. In this paper, we established a HTP chromatography platform and demonstrated its capability to facilitate gene therapy purification process development using both mini-columns and self-packed resin plates. The end-to-end development workflow for AAV HTP purification is detailed in this work with the expectation of serving as an introductory for the AAV purification development field. Comparable process performance was confirmed between a bench-scale chromatography process and an HTP chromatography format. Slightly lower recovery was observed using the HTP format (62% vs 75%), as well as %full capsid enrichment (71% vs. 82%). Comparable impurity clearance capability was demonstrated between the two different systems as well. It was concluded that the established HTP chromatography formats can serve as a surrogate to bench-scale chromatography development to reduce material needs and development timelines for AAV purification development. [ABSTRACT FROM AUTHOR]

Preparative and process chromatography is a versatile unit operation for the capture, purification, and polishing of a broad variety of molecules, especially very similar and complex compounds such as sugars, isomers, enantiomers, diastereomers, plant extracts, and metal ions such as rare earth elements. Another steadily growing field of application is biochromatography, with a diversity of complex compounds such as peptides, proteins, mAbs, fragments, VLPs, and even mRNA vaccines. Aside from molecular diversity, separation mechanisms range from selective affinity ligands, hydrophobic interaction, ion exchange, and mixed modes. Biochromatography is utilized on a scale of a few kilograms to 100,000 tons annually at about 20 to 250 cm in column diameter. Hence, a versatile and fast tool is needed for process design as well as operation optimization and process control. Existing process modeling approaches have the obstacle of sophisticated laboratory scale experimental setups for model parameter determination and model validation. For a broader application in daily project work, the approach has to be faster and require less effort for non-chromatography experts. Through the extensive advances in the field of artificial intelligence, new methods have emerged to address this need. This paper proposes an artificial neural network-based approach which enables the identification of competitive Langmuir-isotherm parameters of arbitrary three-component mixtures on a previously specified column. This is realized by training an ANN with simulated chromatograms varying in isotherm parameters. In contrast to traditional parameter estimation techniques, the estimation time is reduced to milliseconds, and the need for expert or prior knowledge to obtain feasible estimates is reduced. [ABSTRACT FROM AUTHOR]

Separation from bed, not board: Simulated moving bed chromatography, a continuous separation method, enables the nearly quantitative recovery of sugar products and ionic liquid solvent from chemical hydrolysates of biomass. The ensuing sugars support microbial growth, and the residual lignin from the process is intact. [ABSTRACT FROM AUTHOR]

Forsythoside A and suspensaside A were isolated and purified fromForsythia suspensaby one step of high-speed counter-current chromatography for the first time using a solvent system of ethyl acetate-n-butanol-methanol-water (4:0.5:0.5:5,v/v). The purities of the two compounds were 98.19% and 98.05%, respectively, as determined by HPLC. Their structures were identified by MS, UV, and NMR analysis. Such a simple and effective method was fairly useful to prepare pure compounds as reference substances for related study onForsythia suspensa. [ABSTRACT FROM AUTHOR]

Abstract: Recently, nanofiltration techniques have been introduced by pharmaceutical industries in the downstream processes for peptides, to perform concentration, purification and salt/solvent exchange, and have been demonstrated as being suitable for integration with conventional chromatographic purification techniques, providing savings in terms of time and costs. Design of Experiments (DoE) methods have been extensively applied in the process design of the conventional techniques, under the so-called Quality by Design (QbD) concept, but applications of DoE methods to nanofiltration are still few. In this study, the nanofiltration of a model peptide, conventionally named PEP1, in trifluoroacetic acid/acetonitrile/water mixtures is studied by DoE. Statistical models for solvent flux, peptide and trifluoroacetic acid rejections are obtained by statistical Analysis of Variance and the best operating conditions for concentration are found by numerical optimization of the statistical models. The statistical models from DoE are included in the mathematical framework of the diafiltration process, to calculate the evolution of peptide, counter-ion and solvent concentrations over time, compare constant volume vs. variable volume diafiltration modes, and select the best process in terms of operating time and solvent consumption. This work demonstrates that empirical DoE models can provide phenomenological understanding of the transport mechanism through nanofiltration membranes for a specific solute of interest and successfully support the process modelling for concentration and diafiltration, providing a methodology to select the most appropriate filtration technique for a given separation problem. [Copyright &y& Elsevier]

Highlights: [•] Grapefruit coumarins were fractionated by solvent partitioning. [•] C-18 column facilitated purification of minor coumarins than Silica. [•] Nine compounds were purified using rapid hyphenated chromatography. [•] Structure elucidation of coumarins was confirmed by spectral analysis. [ABSTRACT FROM AUTHOR]

Several methods have been demonstrated in the recovery and purification of cutinase; however, hydrophobic interaction membrane chromatography (HIMC) has yet to be implemented for this purpose. In this study, two factors, that is, buffer pH and ammonium sulphate concentration for HIMC operation, were optimized using response surface methodology (RSM), aiming to obtain the highest cutinase purification factor and recovery possible. The experimental design used was a central composite design (CCD) comprised of 13 experimental runs. The analysis of variance (ANOVA) indicated that a quadratic model best represented the system. Based on theR2values, it can be concluded that around 90% of the variability in the system can be explained by the fitted models. The optimized HIMC conditions were buffer pH 6.0 and 1.3 M ammonium sulphate, which was expected to provide 27-fold cutinase purification factor and 85% recovery. However, the actual values deviated from the predicted values with 13% and 18% error, respectively. The statistical analysis showed that only the ammonium sulphate concentration had a great influence on cutinase purification factor. Meanwhile, for cutinase recovery, both parameters were important in determining the optimal condition for cutinase adsorption and desorption to the chromatographic media. [ABSTRACT FROM AUTHOR]

Background The phycobiliprotein C-phycocyanin (C- PC) is used in cosmetics, diagnostics and foods and also as a nutraceutical or biopharmaceutical. It is produced in the cyanobacterium Arthrospira platensis grown phototrophically in open cultures. C- PC may alternatively be produced heterotrophically in the unicellular rhodophyte Galdieria sulphuraria at higher productivities and under improved hygienic standards if it can be purified as efficiently as C- PC from A. platensis. Results Ammonium sulfate fractionation, aqueous two-phase extraction, tangential flow ultrafiltration and anion exchange chromatography were evaluated with respect to the purification of C- PC from G. sulphuraria extracts. Galdieria sulphuraria C- PC showed similar properties to those described for cyanobacterial C- PC with respect to separation by all methodologies. The presence of micelles in G. sulphuraria extracts influenced the different procedures. Only chromatography was able to separate C- PC from a second phycobiliprotein, allophycocyanin. Conclusion C- PC from heterotrophic G. sulphuraria shows similar properties to cyanobacterial C- PC and can be purified to the same standards, despite initial C- PC concentrations being low and impurity concentrations high in G. sulphuraria extracts. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Soybean oil 2-monoacylglycerol (2-MAG) was synthesized via the Novozym 435-catalyzed ethanolysis of triacylglycerols and purified by conventional liquid-liquid extraction. The 2-MAG was subjected to incubation at 20 and 40 °C in the presence of five solid commercial support materials, Lewatit, Silica Gel 60, Alumina-Neutral Brockman Activity 1, Amberlyst-15, and SBA-15, to determine their effects on acyl migration rates. Lewatit and SBA-15 did not catalyze acyl migration rates after 144 h, while silica gel slightly increased migration rates. The more polar alumina and the cationic Amberlyst 15 significantly increased migration rates. Flash chromatography purification of 2-MAG using silica gel as the immobile phase developed with an acetone/hexane binary gradient proved to be a comparable purification method to liquid-liquid extraction, resulting in 60 % 2-MAG yield, no residual triacylglycerol, diacylglycerol, or glycerol co-products, and a 95 mol% 2-MAG purity vs. 1-MAG, demonstrating that flash chromatography did not catalyze acyl migration. [ABSTRACT FROM AUTHOR]

Highlights: [•] Retention behavior of monoclonal IgG1 was investigated on IEC and HIC media. [•] Dynamics of both processes was dominated by kinetic effects. [•] A short-cut method was proposed for mathematical modeling. [•] A dynamic model was used for optimization of the integrated process IEC/HIC. [ABSTRACT FROM AUTHOR]

Abstract: One of the caveats of the Mitsunobu reaction is the often difficult and laborious purification of the reaction mixture. Herein, we show that azodicarbonyl dimorpholide (ADDM) is an effective and versatile Mitsunobu reagent, which, along with its reduced form ADDM-H2, may be removed from the reaction mixture by a simple aqueous extraction. Furthermore, we demonstrate that the isolated ADDM-H2 may be re-oxidized with silver(I) oxide to regenerate ADDM. Finally, a chromatography-free Mitsunobu reaction is presented. [Copyright &y& Elsevier]

An effective high–speed counter–current chromatography (HSCCC) method was established for semi-preparative isolation and purification of bioactive chemical constituents from the Tibetan medicinal plantPedicularis longifloravar.tubiformis(Klotzsch) P. C. Tsoong. With a two-phase solvent system composed of chloroform–methanol–water (8:4:5, v/v), 40 mg of extract was separated to yield luteolin (12.5 mg), apigenin (9.6 mg), and chrysoeriol (4.8 mg), with purities of 99.3, 98.2, and 98.6%, respectively, as determined by high-performance liquid chromatography (HPLC). The chemical structures of these three components were identified by1H NMR and13C NMR. [ABSTRACT FROM AUTHOR]

A facile and efficient one-pot procedure for the preparation of functionalized dihydro-lH-indol-4(5H)-ones by a catalyst-free, three-component reaction of 1,3-dicarbonyl compounds, arylglyoxal monohydrate and enaminones under mild conditions in excellent yield is reported. This synthesis was confirmed to follow the group-assisted-purification (GAP) 0 chemistry process, which can avoid traditional purifications, chromatography, and recrystallization. [ABSTRACT FROM AUTHOR]

The detrimental effect of heterogeneity on the efficiency of particle packed columns has been a long standing issue in chromatographic separations. This heterogeneity arises from the difference in packing density between the wall and central regions of the chromatographic bed and imparts radial variance in mobile phase velocity through the column. In conjunction with the uneven distribution of the sample band through frits and distributors at the head of the column, the resultant effect is a decrease in efficiency due to the formation of a parabolic-“like” sample band. We introduce the concept of “Active Flow Management” to counteract the effects of column heterogeneity via the separation of the flow eluting from the center of the column from that of the flow eluting near the peripheral wall region. The flow streams from the two aforementioned regions are separated by the use of a specialized column outlet fitting. By varying the ratio of flow eluting from the center to the peripheral region it was possible to increase the efficiency of a 100 × 21 mm column by up to 57% compared to a column operated in the conventional manner. [ABSTRACT FROM AUTHOR]

Abstract: Hydrophobic charge-induction chromatography (HCIC) is a novel downstream bioprocessing technology for antibody purification and it has several advantages over traditional purification processes. However, its separation selectivity still needs to be improved. In this work, sodium caprylate (NaCA) was used as the selective modifier to improve IgG purification from serum albumin containing feedstock with a typical HCIC resin, MEP HyperCel. The effects of NaCA on the adsorption equilibrium of bovine serum immunoglobulin G (IgG) and bovine serum albumin (BSA), as well as the dynamic binding and displacement behaviors were investigated. The binding and elution behaviors of these two proteins in the column were studied. It was found that adding 50–75mM NaCA in the liquid phase could effectively reduce the adsorption of BSA on the MEP resin, but the same treatment has little influence on the adsorption of IgG. Moreover, the mechanism of the competitive binding between caprylate and MEP ligands on the surface of BSA is discussed. It was found that by controlling NaCA addition in the loading or washing buffer, the process efficiency of IgG purification from BSA containing feedstock can be improved, and the purity of IgG could reach to over 98%. The results indicated that caprylate could be a promising albumin-selective modifier to improve the separation efficiency of antibodies with the HCIC process. [Copyright &y& Elsevier]

A successful method of preparative high-speed counter-current chromatography (HSCCC) was established for direct isolation and purification of hydrolysis products ofEpimedium koreanumNakai to obtain the secondary glycosides and aglycons of flavonoids inE. koreanum. The two-phase solvent systems of HSCCC composed n-hexane-ethyl acetate-methanol-water at the ratios of 6:3.5:6:5 (v/v) and 6:3.5:8:5 (v/v) were used in a stepwise elution. Totally, 60.1 mg of FractionIthat would be isolated and purified by preparative HPLC, 9.4 mg of icartin (4), 3.5 mg of desmethylicartin (5), 4.7 mg of 3′′-ethyoxyl-icartin (6), 7.3 mg of anhydroicaritin (7), and 10.1 mg of β-anhydroicaritin (8) were obtained in one-step separation from 100 mg of the crude materials with purities of 97.7%, 96.2%, 99.2%, 99.5%, and 91.0%, respectively. And FractionIof HSCCC was purified by the preparative HPLC to obtain 12.7 mg of pletypetaloside (I-1), 6.8 mg of icariside I (I-2) and 16.3 mg of 3′′-ethyoxyl-pletypetaloside (I-3) with purities of 90.8%, 93.4%, and 99.2%. All of the chemical structures were identified on the basis of widely spectral technology including 2D-NMR. Among them, compoundsI-3and6are novel. [ABSTRACT FROM AUTHOR]

Abstract: Molecular imprinted polymer (MIP) techniques have been increasingly used in a variety of fields including chromatography, sample pretreatment, purification, sensors, drug delivery, and catalysts, etc. MIP is a specific artificial receptor that shows favored affinity to the template molecule. The cavities of the template are produced by carrying out polymerization of a reaction mixture followed by eliminating the template molecules by washing. Various forms of MIP materials have been prepared for diverse applications including irregularly ground particles, regular spherical particles, nanoparticles, monoliths in a stainless steel or capillary column, open tubular layers in capillaries, membranes, surface attached thin layers, and composites, etc. When an enantiomer is used as the template, then the resulting MIP can show capability of enantiomeric recognition between the pair of enantiomers. In this review, progresses in applications of enantio-selective recognition by MIPs will be critically reviewed for the recent period since 2007. [Copyright &y& Elsevier]

Perchloric acid was found to be a suitable medium for the quantitative leaching of Sr(II) from homogeneous and calcined (Th,Sr)O particularly with respect to the contamination from Th(IV). Sr is a cause of major concern to the environment due to its long half life (28.6 years), significant abundance in large inventory of spent nuclear fuels (~350 thousand tons) awaiting geological disposal and its chemical similarity to Ca(II), an essential element for the living beings. Application of Sr as a parent radionuclide for Y (used in therapy radiopharmaceuticals) is possible provided it can be made available at desired high purity. In this context, the distribution coefficients of Sr(II), Th(IV), Zr(IV), Y(III), Eu(III) and Cs(I) were determined using Sr selective crown ether 4,4′(5′)-di -tert-butyl -dicyclohexano -18 -crown -6 by solid-liquid extraction in perchloric acid medium. Feasibility of employing extraction chromatography using Sr selective resin for the recovery and purification of Sr(II) from leached perchloric acid medium was explored. Perchloric acid medium is better than nitric acid medium for the uptake of Sr by Sr selective chromatographic resin under varying loading conditions of Sr(II). Similarly pH 2 solution appears better eluent of Sr(II) than distilled water. Present work offers a novel approach for setting up a Sr-Y generator. [ABSTRACT FROM AUTHOR]