Determination of residual trifluoroacetate in protein purification buffers and peptide preparations by ion chromatography

Trifluoroacetate (TFA) is commonly used in a variety of pharmaceutical applications. Because of its toxic nature, it is important to reliably measure the effective removal of TFA. We developed an ion chromatography (IC) method to determine the concentration of residual TFA in samples found in the pharmaceutical industry. A high-capacity anion-exchange column was used to separate trace trifluoroacetate from an excess of chloride, phosphate, and other anions without the need for sample preparation. TFA was detected by suppressed conductivity. A method with four KOH eluent step changes was optimized and reproducibly executed using automated generation of the KOH eluent. We used this method to determine TFA in the following samples: a phosphate-buffered saline (PBS), an acetate-buffered saline containing protein, and a commercial peptide. The method detection limits for TFA in these samples were all less than 90 ng/ml.     

An indirect competitive enzyme-linked immunosorbent assay for determination of norfloxacin in waters using a specific polyclonal antibody

A specific polyclonal anti-norfloxacin antibody was obtained, and a sensitive indirect competitive enzyme-linked immunosorbent assay (icELISA) was developed for determining trace amounts of norfloxacin in various waters. Good linearity was achieved in the range from 0.1 to 10 μg L⁻¹. The average IC₅₀ value was determined to be 2.2 μg L⁻¹ and the limit of detection was 0.016 μg L⁻¹ at a signal-to-noise ratio of 3 in phosphate-buffered saline buffer. Recoveries of norfloxacin at various spiking levels ranged from 74 to 105% in groundwater, surface water, treated and untreated wastewater samples, with relative standard deviations of 3–5%. The assay was applied for determining norfloxacin in municipal wastewater, surface water, and groundwater collected in a metropolis of China. Raw wastewater samples were only submitted to filtration and pH adjustment while the other water samples were pre-concentrated by solid phase extraction prior to the icELISA assay. Good agreement of the results obtained by the icELISA and liquid chromatography tandem mass spectrometry further confirmed the reliability and accuracy of the icELISA for rapid detection of norfloxacin in waters.     

Purification and quantification of recombinant Epstein-Barr viral glycoproteins gp350/220 from Chinese hamster ovary cells.

Truncated Epstein-Barr virus (EBV) membrane antigen gp350/220 (EBV-MA) lacking the membrane anchor was expressed and secreted into the medium of recombinant Chinese hamster ovary cells that had been cultured in Plasmapur hollow-fibre modules using defined serum-free medium. The EBV-MA in the medium was concentrated by 70% (w/v) ammonium sulphate precipitation and subsequently purified by immunoaffinity chromatography using an anti-EBV-MA (EBV.0T6) monoclonal antibody (mAb) column. Adsorbed antigen was eluted with 3 M MgCl2 in phosphate-buffered saline, concentrated by Mono Q anion-exchange chromatography and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, silver staining and Western blotting using EBV-positive serum and anti-EBV-MA specific mAbs. Monospecific polyclonal rabbit antibodies against the purified EBV-MA were raised and purified by protein G affinity chromatography. For the measurement of EBV-MA antigen levels a sandwich enzyme-linked immunosorbent assay using rabbit polyclonal antibodies and a horseradish peroxidase-conjugated anti-MA mAb was developed having a detection level of 10 ng/ml.     

Immobilized artificial membrane chromatography with mass spectrometric detection: a rapid method for screening drug-membrane interactions.

A high-performance liquid chromatography (HPLC)/electrospray ionization mass spectrometry method for measuring drug-membrane interactions was developed using immobilized artificial membrane (IAM) fast-screening mini-columns. The HPLC mobile phase consisted of phosphate-buffered saline (i.e., 5.0 mM phosphate buffer at pH 7.4, 1.35 mM KCl, and 68.5 mM NaCl) and acetonitrile. This method facilitated the measurement of IAM retention time of over ten compounds in one experiment, significantly reducing analysis time compared with the earlier IAM-HPLC method. The particular electrospray source used demonstrated the ability to tolerate the high salt-containing nonvolatile buffer used for retention time measurement.     

Rapid mastitis detection assay on porous nitrocellulose membrane slides

We have developed a rapid mastitis detection test based on the immobilization of tag-specific antibody molecules, the binding of double-tagged amplicons, and as a secondary signal a conjugate of black carbon nanoparticles having molecules of a fusion protein of neutrAvidin and alkaline phosphatase at their surface. The antibodies were inkjet printed onto three different nitrocellulose membrane slides, Unisart (Sartorius), FAST (GE Whatman), and Oncyte-Avid (Grace-Biolabs), and the final assay signals on these slides were compared. The blackness of the spots was determined by flatbed scanning and assessment of the pixel gray volume using TotalLab image analysis software. The black spots could be easily read by the naked eye. We successfully demonstrated the detection of specific amplicons from mastitis-causing pathogens in less than 3 h. Using a similar protocol, we also showed that it was possible to detect specific amplicons from four different mastitis-causing pathogens (six strains) on the same pad. The influence of two different printing buffers, phosphate-buffered saline (pH 7.4) and carbonate buffer (pH 9.6), on the functionality of the primary antibodies was also compared.     

Exploration of overloaded cation exchange chromatography for monoclonal antibody purification

Cation exchange chromatography using conventional resins, having either diffusive or perfusive flow paths, operated in bind-elute mode has been commonly employed in monoclonal antibody (MAb) purification processes. In this study, the performance of diffusive and perfusive cation exchange resins (SP-Sepharose FF (SPSFF) and Poros 50HS) and a convective cation exchange membrane (Mustang S) and monolith (SO(3) Monolith) were compared. All matrices were utilized in an isocratic state under typical binding conditions with an antibody load of up to 1000 g/L of chromatographic matrix. The dynamic binding capacity of the cation exchange resins is typically below 100 g/L resin, so they were loaded beyond the point of anticipated MAb break through. All of the matrices performed similarly in that they effectively retained host cell protein and DNA during the loading and wash steps, while antibody flowed through each matrix after its dynamic binding capacity was reached. The matrices differed, though, in that conventional diffusive and perfusive chromatographic resins (SPSFF and Poros 50HS) demonstrated a higher binding capacity for high molecular weight species (HMW) than convective flow matrices (membrane and monolith); Poros 50HS displayed the highest HMW binding capacity. Further exploration of the conventional chromatographic resins in an isocratic overloaded mode demonstrated that the impurity binding capacity was well maintained on Poros 50HS, but not on SPSFF, when the operating flow rate was as high as 36 column volumes per hour. Host cell protein and HMW removal by Poros 50HS was affected by altering the loading conductivity. A higher percentage of host cell protein removal was achieved at a low conductivity of 3 mS/cm. HMW binding capacity was optimized at 5 mS/cm. Our data from runs on Poros 50HS resin also showed that leached protein A and cell culture additive such as gentamicin were able to be removed under the isocratic overloaded condition. Lastly, a MAb purification process employing protein A affinity chromatography, isocratic overloaded cation exchange chromatography using Poros 50HS and anion exchange chromatography using QSFF in flow through mode was compared with the MAb's commercial manufacturing process, which consisted of protein A affinity chromatography, cation exchange chromatography using SPSFF in bind-elute mode and anion exchange chromatography using QSFF in flow through mode. Comparable step yield and impurity clearance were obtained by the two processes.     

Hydrophobic charge‐induction resin with 5‐aminobenzimidazol as the functional ligand: preparation,protein adsorption and immunoglobulin G purification

A new hydrophobic charge‐induction chromatography resin was prepared with 5‐aminobenzimidazol as functional ligand and polyacrylic ester beads as matrix. Adsorption isotherms and adsorption in columns were investigated using human immunoglobulin G and bovine serum albumin as model proteins, and the influence of pH and NaCl concentration was discussed. Results showed that the ligand density was 195 μmol/mL gel, and protein selectivity can be improved by controlling pH and salt addition. An optimized purification process (sample loading at pH 8.0 with 0.2 M NaCl and elution at pH 5.0) was performed to purify human immunoglobulin G from bovine serum albumin containing feedstock, which resulted in human immunoglobulin G purity of 99.7% and recovery of 94.6%. A similar process was applied for the purification of monoclonal antibody from cell culture supernatant, which showed antibody purity of 94.9% and recovery of 92.5%. The results indicated that the new resin developed had comparable performance as Protein A chromatography and would be suitable for antibody purification from complex feedstock.     

Determination of vegetal proteins in milk powder by enzyme-linked immunosorbent assay: interlaboratory study

Eight laboratories participated in a collaborative study to evaluate an enzyme-linked immunosorbent assay (ELISA) to determine soy, pea, and wheat proteins in pasteurized or ultra-high temperature (UHT) milk powders. To perform this assay, polyclonal antibodies for soy, pea, and wheat proteins were obtained from rabbit sera. Collaborators received calibration standards composed of milk powder containing 0-8% (w/w) vegetal protein in total protein and blind test samples containing approximately 1, 2, and 5% (w/w) vegetal protein. An indirect competitive ELISA was performed with a kit prepared by a participating laboratory; the kit contained plates coated with soy, pea, or wheat proteins, the corresponding specific antisera, enzyme-labeled second antibody, and substrate solution. Test samples and calibrants were extracted with phosphate-buffered saline, pH 7.4, containing 0.05% Tween and assayed with the ELISA kits. The degree of adulteration was affected by the type of heat treatment applied to the samples. The estimated percentage of vegetal protein addition was close to the theoretical value for pasteurized samples but much lower for UHT samples. For pasteurized samples, intralaboratory relative standard deviations ranged from 5 to 22% and interlaboratory relative standard deviations ranged from 14 to 34%.     

Automated high-performance liquid chromatographic determination of chloramphenicol in milk and swine muscle tissue using on-line immunoaffinity sample clean-up.

An on-line high-performance liquid immunoaffinity chromatographic (HPLIAC) system for the direct determination of chloramphenicol in milk and swine muscle tissue is described. The system consisted of a dual-column system in which an HPLIAC column was directly coupled to an RP-8 high-performance liquid chromatographic column. Skimmed and deproteinated milk or aqueous muscle tissue extract was directly injected into the HPLIAC column. After a washing step with phosphate-buffered saline, chloramphenicol was desorbed by a glycine-NaCl buffer (pH 2.8) and directly concentrated on the RP-8 column. Next, chromatography was carried out using acetonitrile-sodium acetate buffer as the mobile phase. Chloramphenicol was detected at 280 nm. Mean recoveries from spiked raw milk were 70 +/- 2% (1-50 micrograms/kg) and from spiked swine muscle tissue 64 +/- 2% (10-50 micrograms/kg). The calibration curves were linear in the range 1-200 micrograms/kg spiking levels. Limits of determination were 1 microgram/kg for milk and 10 micrograms/kg for muscle tissue.     

Characterization of N-terminal formaldehyde adducts to hemoglobin

A procedure to prepare and purify adducts of formaldehyde (FA) to the N-terminus of peptides was developed. FA-VHLTPEEK and FA-VLSPADK were produced with purities >95% upon incubation of the peptides with FA in phosphate-buffered saline (PBS) at a pH level of 7.4. The peptides were purified by preparative liquid chromatography and were characterized by their retention times in liquid chromatography, their fragmentation patterns obtained by tandem mass spectrometry, and their accurate mass and nuclear magnetic resonance measurements. This is the first time an imidazolidone-type structure has been reported for FA adducts. The same peptides were identified in tryptic digests of human hemoglobin incubated with FA at physiological conditions and in human hemoglobin specimens. These peptides are suitable for use as calibrators for the quantitative assessment of internal exposure to FA.     

Comparison of ceramic hydroxy- and fluoroapatite versus Protein A/G-based resins in the isolation of a recombinant human antibody from cell culture supernatant

A recombinant human antibody (IgG(1)-subtype) was produced in Chinese Hamster Ovary (CHO) cells. Alternatives to the established isolation by Protein A affinity chromatography were investigated. Neither an alternative elution agent (Arginine) nor an alternative affinity ligand (Protein G) resulted in an improvement in yield and/or purity. Subsequently, apatite stationary phases including a novel ceramic fluoroapatite material were tested. By applying a double gradient (first 0 to 1M NaCl, then 0.01 to 0.4M phosphate) the culture supernatant was separated into three fractions: the flow through, which contained no active antibody, the NaCl-eluate, which contained the antibody and no other discernible protein contaminants, and a fraction that eluted in the phosphate gradient and contained several proteins, but no active antibody. In case of the hydroxyapatite, retention of the antibody decreased and yield increased when the pH was raised from 6.0 to 8.2 (isoelectric point (pI) of the antibody: 8.3), to reach a yield of 71% at pH of 8.2. In case of the fluoroapatite, retention was also found to increase with increasing mobile phase pH, but the yields went through a maximum (of ca. 90%) at a mobile phase pH of 7.0. No traces of contaminants were seen in the corresponding gel. This is the first time that yields of 90% and such high purities have been reported as the result of a single chromatographic step for the antibody in question with either (Protein A) affinity or apatite chromatography.     

Determination of neomycin in animal tissues by liquid chromatography

Tissue samples are digested under hot alkaline conditions after initial conditioning at room temperature with phosphate-buffered saline. The cooled digest is deproteinated with concentrated perchloric acid. After centrifugation and pH adjustment, the clear supernatant is applied to an ion-exchange cartridge, and after the cartridge is washed, the neomycin is eluted with dilute perchloric acid. This eluate is derivatized with 9-fluorenylmethyl chloroformate prior to liquid chromatography using a wide-pore spherical silica C4 column and fluorescence detection. Recovery and repeatability are calculated from tissue extract standard calibration curves produced from the same assay. Recoveries ranged from 80 to 120% for fortifications of 0.25-1.00 mg/kg for muscle tissue and from 80 to 100% for fortifications of 0.50-10.0 mg/kg for kidney tissue. Limits of quantitation were 0.25 and 0.50 mg/kg, respectively, for muscle and kidney tissues. Limits of detection were 0.125 and 0.20 mg/kg, respectively, for muscle and kidney tissues.     

Mathematical model using non-uniform flow distribution for dynamic protein breakthrough with membrane adsorption media

A mathematical model has been investigated to predict protein breakthrough during membrane adsorption/chromatography operations. The new model incorporates a non-uniform boundary condition at the column inlet to help describe the deviation from plug flow within real membrane adsorption devices. The model provides estimated breakthrough profiles of a binding protein while explicitly accounting for non-uniform flow at the inlet of the separation operation by modeling the flow distribution by a polynomial. We have explored experimental breakthrough curves produced using commercial membrane adsorption devices, as well as novel adsorption media of nanolayered nanofiber membranes, and compare them to model predictions. Further, the impact of using various simplifying assumptions is considered, which can have a dramatic effect on the accuracy and predictive ability of the proposed models. The new model, using only simple batch equilibrium and kinetic uptake rate data, along with membrane properties, is able to accurately predict the non-uniform and unsymmetrical shape for protein breakthrough during operation of membrane adsorption/chromatography devices.     

金属-有机骨架MIL-101(Cr)掺杂聚合物整体柱的制备及其用于酚类化合物的在线固相萃取

制备了金属-有机骨架（MOF）MIL-101（Cr）掺杂聚合物整体柱，建立了在线固相萃取-高效液相色谱检测水中4-硝基苯酚、2-硝基苯酚、3-甲基苯酚和2，4-二氯苯酚的方法。考察了样品溶液pH值、上样时间、上样流速和解吸时间对酚类化合物萃取效果的影响。在最优萃取条件下，采用制备的整体柱检测水中酚类化合物，其富集因子高，线性范围宽，精密度好，检出限低，适用于水中酚类化合物的检测。制备掺杂聚合物整体柱是促进和拓宽MOFs在线固相萃取应用的有效方法之一。     

A Model On-line Flow Injection Fluorescence Immunoassay Using a Protein a Immunoreactor and Lucifer Yellow

Abstract

A heterogeneous on-line fluorescence immunoassay has been developed for a model analyte (transferrin) using a flow injection analysis system containing a controlled pore glass protein A immunoreactor. Lucifer yellow VS (LYVS) a 4-aminonaphthalimide with a large stokes shift and pH independence was the fluorophore. For each assay the antibody-protein A reaction takes place at near neutral pH, and the complexes are eluted at acid pH. Transferrin levels in human serum has been determined by this method, and good within assay variations have been achieved.     

Photogenerated polyelectrolyte bilayers from an aqueous-processible photoresist for multicomponent protein patterning

A novel photoresist (PR) that can be processed under mild aqueous conditions was synthesized and used to create photogenerated polyelectrolyte bilayers. Thin films of the PR cast on polycation-coated substrates were exposed to UV irradiation to generate carboxylate groups in the photoresist. The bulk of the UV-exposed PR film was dissolved by rinsing with pH 7.4 phosphate-buffered saline, but a polyelectrolyte bilayer formed in situ at the PR/polycation interface on exposure remained bound to the substrate. The UV-exposed photoresist also exhibited pH-dependent solubility; it was soluble in water above pH 6.6, but insoluble at lower pHs. Using these unique properties, two-component protein patterning was achieved using biotinylated PR films under conditions that avoid exposing the proteins to conditions outside the narrow range of physiological pH, ionic strength, and temperature where their stability is greatest.     

Water-soluble red-emitting distyryl-borondipyrromethene (BODIPY) dyes for biolabeling

A series of water-soluble red-emitting distyryl-borondipyrromethene (BODIPY) dyes were designed and synthesized by using three complementary approaches aimed at introducing water-solubilizing groups on opposite faces of the fluorescent core to reduce or completely suppress self-aggregation. An additional carboxylic acid functional group was introduced at the pseudo-meso position of the BODIPY scaffold for conjugation to amine-containing biomolecules/biopolymers. The optical properties of these dyes were evaluated under simulated physiological conditions (i.e., phosphate-buffered saline (PBS), pH 7.5) or in pure water. The emission wavelength (λ(max)) of these labels was found in the 640-660 nm range with quantum yields from modest to unprecedentedly high values (4 to 38%). The bioconjugation of these distyryl-BODIPY dyes with bovine serum albumin (BSA) and the monoclonal antibody (mAb) 12A5 was successfully performed under mild aqueous conditions.     

Diffusion of small molecules inside a peptide hydrogel

A set of four phenylalanine analogues experiences diffusion retardation when transferred from phosphate-buffered saline into a peptide hydrogel of the same pH and ionic strength. The extent of retardation increases linearly with logP(oct), their lipophilicity.     

Detection of hazelnut in foods using ELISA: challenges related to the detectability in processed foodstuffs

Hazelnuts are widely used nowadays, and can pose a serious threat to allergic consumers due to cross-contamination that may occur during processing. This might lead to the presence of hidden hazelnut in foods. Therefore, reliable tests are needed to detect hazelnut, especially in processed foods. A hazelnut-specific indirect competitive ELISA based on polyclonal chicken antibodies was developed. The polyclonal antibodies were raised against modified hazelnut proteins in order to improve the detectability of hazelnut proteins in processed foods. The assay showed a detection limit of 1.36 microg hazelnut protein/mL of 5 mM urea in phosphate-buffered saline buffer (pH 7.4). Limited cross-reactivity with walnut and pecan nut was observed; no cross-reactivity was observed with other food ingredients. Blank cookies spiked before analysis showed recoveries of 73-107%. However, cookies spiked before baking showed that the detectability was severely decreased. Addition of lactose to the cookies, which led to more severe modification through the Maillard reaction, led to an increase in the detectability. These results indicate that using antibodies developed toward allergens modified through food processing-simulating reactions is a better approach for detection.