Integration of scale-down experimentation and general rate modelling to predict manufacturing scale chromatographic separations

Chromatography is an essential downstream processing step in the production of biopharmaceuticals. Here we present an approach to chromatography scale-up using scale-down experimentation integrated with general rate modelling. This type of modelling takes account all contributions to the mass transfer kinetics providing process understanding. The model is calibrated using a 2.5 cm height, 1 ml column and used to predict chromatograms for 20 cm height columns from 40 ml to 160 L volume. Simulations were found to be in good agreement with experimental results. The envisaged approach could potentially reduce the number of experiments, shorten development time and reduce costs.     

Application of a chromatography model with linear gradient elution experimental data to the rapid scale-up in ion-exchange process chromatography of proteins

We applied the model described in our previous paper to the rapid scale-up in the ion exchange chromatography of proteins, in which linear flow velocity, column length and gradient slope were changed. We carried out linear gradient elution experiments, and obtained data for the peak salt concentration and peak width. From these data, the plate height (HETP) was calculated as a function of the mobile phase velocity and iso-resolution curve (the separation time and elution volume relationship for the same resolution) was calculated. The scale-up chromatography conditions were determined by the iso-resolution curve. The scale-up of the linear gradient elution from 5 to 100mL and 2.5L column sizes was performed both by the separation of beta-lactoglobulin A and beta-lactoglobulin B with anion-exchange chromatography and by the purification of a recombinant protein with cation-exchange chromatography. Resolution, recovery and purity were examined in order to verify the proposed method.     

Economic considerations important in the scale-up of an ovalbumin separation from hen egg-white on the anion-exchange cellulose DE92.

The scale-up of the separation of hen egg-white proteins has been investigated using Whatman DE92 anion-exchange cellulose. Having developed suitable chromatographic conditions, a maximum binding capacity of 100 mg protein/ml packed DE92 was determined in a 25-ml column. The process was scaled up 1000-fold and the influence of batch and column techniques on the chromatographic step assessed. Data indicate column processes to be more efficient than batch in the adsorptive stage.     

Preparative Hydrophobic Interaction Chromatography of Proteins Using Ether Based Chemically Bonded Phases

Abstract

This paper examines the use of 15–20 micron wide-pore silica-based ether bonded phases for the preparative hydrophobic interaction chromatography of proteins. In particular, silyl ethers are immobilized on large particle silica in an analogous manner to previously developed ether bonded 5 um analytical supports. The preparative supports are reproducibly prepared and exhibit constant chromatographic retention for at least five months of continual use. Preparative columns can be operated for protein chromatography with peak shapes and capacity as predicted by the Snyder gradient elution model. Moreover, similar retention times are obtained relative to those on the 5 um analytical columns, enabling the direct transition and scale-up of separation. Gradient optimization is seen to directly parallel that performed on 5 um bonded ether analytical columns. Acceptable chromatographic resolution was obtained with sample capacity of >15 mg protein/ml column volume using a repetitive injection technique. A column clean-up strategy is examined for rapid and safe removal of contaminants. An illustrative example of use of the bonded ether preparative columns is made by application to soybean trypsin inhibitor purification. Initial results are presented on a column-switching method for the analytical monitoring of preparative separation.     

甲壳素色谱柱富集—快速吸光光度法测定环境试样中痕量铁

研究了甲壳素色谱柱富集Fe(Ⅱ)-磺基水杨酸-十八烷基二甲基氨基乙酸的红紫色缔合物,用磺基水杨酸洗脱后,在分光光度计上直接测定洗脱液的吸光度,方法快速,简单、富集倍数高,用于环境水样中痕量铁的测定,结果令人满意。     

Purification and characterization of Taxol and 10-Deacetyl baccatin III from the bark,needles, and endophytes of Taxus baccata by preparative high-performance liquid chromatography,ultra-high-performance liquid chromatography-mass spectrometry,and nuclear magnetic resonance

Taxol and 10-Deacetyl baccatin III are major taxanes in the bark, needles, and endophytes of Taxus baccata. The current study aimed to develop a process for their separation from different matrices. Crude taxoid was prepared by extraction of samples with methanol, followed by partitioning with dichloromethane and precipitation with hexane. Analytical high-performance liquid chromatography involved isocratic elution on C18 column (4.6 × 250 mm, 5 μm) with methanol-water (70:30 v/v) at a flow rate of 1 ml/min. Injection volume was 20 μl and detection was carried out at 227 nm. The content of Taxol and 10-Deacetyl baccatin III in bark, needles and endophytic culture broth was 11.19 and 1.75 μg/mg; 11.19 and 1.75 μg/mg; and 2.80 and 0.22 μg/L, respectively. Preparative high-performance liquid chromatography was done on C18 column (10 × 250 mm, 5 μm) at a flow rate of 10 ml/min. About 20 g crude taxoid was processed in < 3 h with a recovery of about 90% for both the analytes. The purity of recovered Taxol and 10-Deacetyl baccatin III determined by ultra-high-performance liquid chromatography-mass spectrometry was found to be 95.78 ± 3.63% and 99.72 ± 0.18%, respectively. The structure of recovered Taxol was confirmed by nuclear magnetic resonance. The method can find use in biotransformation studies.     

Bengal gram seed husk as an adsorbent for the removal of dyes from aqueous solutions – Column studies

A continuous fixed bed (column) study was carried out by using seed husk of Bengal gram (Cicer arietinum) (SHBG) as a biosorbent for the removal of direct dye, Congo red (CR) from aqueous solutions. The effects of important factors, such as the value of initial pH, the flow rate, the influent concentration of CR, bed depth, particle size of SHBG, foreign ions and regeneration of CR were studied. The effect of similar type of direct dyes like direct turquoise blue 86 (DTB) and direct black 38 (DB) on the adsorption of CR in column containing SHBG is also studied by keeping other parameters constant. The studies confirmed that the breakthrough curves were dependent on flow rate, initial dye concentration, size of SHBG, initial pH of solution of CR and bed depth. The bed depth service time analysis (BDST) model was applied at different bed depths to predict the breakthrough curves. The model is found suitable for describing the biosorption process of the dynamic behaviour of the SHBG column and the data were in good agreement with BDST model. When the flow rate was 0.67 mL/min and the influent concentration of CR was mg L⁻¹, the column capacity was 6.572 mg g⁻¹. The removal capacity of SHBG was more in case of CR (6.572 mg g⁻¹) compared to other similar direct dyes of DTB (1.984 mg g⁻¹) and DB (1.612 mg g⁻¹). The removal of CR was enhanced in the presence of foreign ion potassium (8.308 mg g⁻¹) and decreased in the presence of calcium (5.58 mg g⁻¹). 120 ml of acetone is required for the completion of regeneration of the column and the total amount of CR recovered in this case. All the results suggested SHBG as a potential adsorbent for removal of CR from aqueous solution so that the rate of bio-sorption process is rapid.     

Continuous chromatographic protein refolding

Column-based protein refolding requires a continuous processing capability if reasonable quantities of protein are to be produced. A popular column-based method, size-exclusion chromatography (SEC) refolding, employs size-exclusion matrices to separate unfolded protein from denaturant, thus refolding the protein. In this work, we conduct a comparison of SEC refolding with refolding by batch dilution, using lysozyme as a model protein. Lysozyme refolding yield was found to be extremely sensitive to the chemical composition of the refolding buffer and particularly the concentration of dithiothreitol (DTT) introduced from the denatured protein mixture. SEC refolding was not adversely affected by DTT carry-over as small contaminants in the denatured solution are separated from protein during the refolding operation. We also find that, contrary to previous reports, size-exclusion refolding on batch columns leads to refolding yields slightly better than batch dilution refolding yields at low protein concentrations but this advantage disappears at higher protein concentrations. As batch-mode chromatography would be the limiting step in a column based refolding downstream process, the batch column refolding method was translated to a continuously operating chromatography system (preparative continuous annular chromatography, P-CAC). It was shown that the P-CAC elution profile is similar to that of a stationary column, making scale-up and translation to P-CAC relatively simple. Moreover, it was shown that high refolding yields (72%) at high protein concentration (>1 mg ml(-1)) could be obtained.     

Strategies for Enhancing Laccase Yield from <Emphasis Type="Italic">Streptomyces psammoticus</Emphasis> and Its Role in Mediator-based Decolorization of Azo Dyes

Enhanced production of laccases from Streptomyces psammoticus in solid-state fermentation was carried out using two different strategies: laccase inducers and scale-up process. Laccase yield was enhanced by a wide range of aromatic inducers. The best inducer was pyrogallol, which yielded 116 U/g as compared to the control (55.4 U/g). Scale-up studies in packed bed bioreactor was performed at different aeration rates. Aeration at 1.5 vvm was identified as the optimum condition for laccase production (75.4 U/g) in the column bioreactor. The enzyme yield was enhanced further by combining the best conditions from the first two experiments. Fermentation was carried out in bioreactors in the presence of 1 mM pyrogallol, which resulted in 3.9-fold increase in laccase yield (215.6 U/g). The role of laccase in azo dye decolorization was evaluated in the presence of four different laccase mediators, at different concentrations. 1-Hydroxybenzotriazole (HOBT) proved to be the best mediator for S. psammoticus laccase and decolorized the azo dyes efficiently. Acid orange, Methyl orange, and Bismarck brown were decolorized at the rates of 86%, 71%, and 75% respectively, by HOBT.     

A sensitive dispersive micro solid‐phase extraction coupled with high performance liquid chromatography for determination of three flavonoids in complex matrics by using crab shell as a sorbent

A sensitive dispersive micro solid‐phase extraction coupled with HPLC has been developed for preconcentration and determination of three flavonoids (quercetin, kaempferol, and isorhamnetin) in complex matrix samples. Parameters that affect extraction efficiency have been optimized. The optimal extraction conditions are using 2 μg/mL of crab shell as the sorbent, extraction for 2 min at pH 7, and then eluting with 100 μL of methanol. As a result, the method shows good linearity (R > 0.9994), low LODs (even 0.08 ng/ml) and satisfactory recovery in real honey and rat urine samples. As an eco‐friendly biomaterial, crab shell powder is used as sorbent in pretreatment of flavonoids, and its adsorption mechanism has been investigated for the first time. Compared with the other reported methods, the proposed strategy is time‐saving, eco‐friendly, and highly sensitive using HPLC (even achieving MS grade sensitivity).     

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.     

Simple, non-moving modulation interface for comprehensive two-dimensional gas chromatography.

A simple, non-moving dual-stage CO2 jet modulator is described, which cools two short sections of the front end of the second-dimension column of a comprehensive two-dimensional gas chromatograph. A stream of expanding CO2 is sprayed directly onto this capillary column to trap small fractions eluting from the first-dimension column. Remobilization of the trapped analytes is performed by direct heating by the GC oven air. Installation, maintenance and control of the modulator is simple. Focusing and remobilization of the fractions is a very efficient process, as the bandwidths of the re-injected pulses are less than 10 ms. As a result, alkane peaks eluting from the second-dimension column have peakwidths at the baseline of only 120 ms.     

Automated sample clean-up and fractionation of organochlorine pesticides and polychlorinated biphenyls in human milk using NP-HPLC with column-switching

A method has been developed for the automated sample pretreatment of organochlorine pesticides (OCP's) and polychlorinated biphenyls (PCB's) in extracts of human milk. This work was part of a regular monitoring program presently carried out at our institute. In this program several hundreds of human milk samples have to be analyzed for the occurrence of PCB's and OCP's. With a normal bore straight phase HPLC system, utilizing column switching we are able to separate the fat from the compounds of interest and, moreover, complete separation of the PCB fraction from the OCP's can be achieved. Under the conditions used to separate the PCB fraction from intefering OCP's column-switching is essential since the retention times for the OCP's vary from 4 minutes for hexachlorobenzene (HCB) to more than 2 hours for dieldrin. 1 ml of an extract containing 45 mg of fat is injected on the first (pre)column, the fat is retained on this column and the early eluting HCB, the PCB fraction, and the DDT complex are transferred to the second (analytical) column. Compounds eluting later than p,p′-DDT are collected directly from the precolumn. Meanwhile, the PCB fraction is separated from the rest of the OCP's on the analytical column. Contrary to conventional gravity-controlled chromatography or solid phase extraction the clean-up process can be monitored on-line by UV-detection, thus rendering a fast and reliable optimization of the system. The OCP-fractions collected from the LC are pooled before they are transferred to a high resolution gas chromatograph equipped with a large volume option. The PCB-fraction is injected directly in a HRGC equipped with a concurrent solvent evaporation injection device. The limits of detection for the OCP's (HCB, α-,β- and γ-HCH, β-Hepo (heptachlorepoxide), dieldrin, p,p′-DDE, o,p′-DDT p,p′-DDT and TDE) and the PCB's investigated are at sub-ppb level (fat basis); the recoveries vary from 80 to 100%.     

Continuous separation of maslinic and oleanolic acids from olive pulp by high‐speed countercurrent chromatography with elution‐extrusion mode

In this work, a continuous high‐speed countercurrent chromatography method has been developed on the basis of elution‐extrusion mode and this method was successfully applied to the separation of maslinic and oleanolic acid from the extract of olive pulp. In the process of ‘elution’, the sample solution was continuously loaded into the column and the maslinic acid was steadily eluted out in this step while highly retained oleanlic acid always stayed in the column. In the process of ‘extrusion’, the oleanlic acid was pushed out of the column with the stationary phase. In this way, we achieved a large sample loading. A total of 120 mL sample solution (about 89.55% of the column volume) which contains 600 mg olive pulp extract was pumped in the apparatus by a constant‐flow pump and the maslinic and oleanolic acids were largely separated within 120 min. Both of these two compounds presented high yields and high purities (271.6 mg for maslinic acid with 86.7% and 83.9 mg oleanolic acids with 83.4%).     

Preparation and characterization of molecularly‐imprinted polymers for extraction of sanshool acid amide compounds followed by their separation from pepper oil resin derived from Chinese prickly ash (Zanthoxylum bungeanum)

Molecularly imprinted polymers were prepared using the molecular structure analogs of sanshool as template molecule, 2‐vinylpyridine and β‐cyclodextrin as double functional monomers, ethylene dimethacrylate as cross linker, and azobisisobutyronitrile as initiator. The structural characteristics of the polymers were determined by Fourier‐transform infrared spectroscopy and scanning electron microscopy. Dynamic adsorption and isothermal adsorption were also investigated. The molecularly imprinted polymers were used to prepare a molecularly imprinted solid‐phase extraction column in order to separate acid amide components from pepper oil resin derived from Chinese prickly ash (Zanthoxylum bungeanum). After eluting, the percentage of acid amide components was enhanced to 92.40 ± 1.41% compared with 23.34 ± 1.21% in the initial pepper oil resin, indicating good properties of purification of molecularly imprinted polymers and potential industrial application.     

Extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance using supercritical fluid extraction followed by supercritical fluid chromatography

In this paper, an off-line combination method of supercritical fluid extraction and supercritical fluid chromatography was developed for the selective extraction and isolation of diphenylheptanes and flavonoids from Alpinia officinarum Hance. The enrichment of target components was successfully achieved using supercritical fluid extraction with the following conditions (8% ethanol as co-solvent at 45°C and 30 MPa for 30 min). Taking full advantage of the complementarity of supercritical fluid chromatography stationary phases, a two-step preparative supercritical fluid chromatography strategy was constructed. The extract was firstly divided into seven fractions on a Diol column (250 × 20 mm internal diameter, 10 μm) within 8 min by gradient elution increasing from 5% to 20% modifier (methanol) at 55 ml/min and 15 MPa. Then the seven fractions were separated by using a 1-AA or a DEA column (250 × 19 mm internal diameter, 5 μm) at 50 ml/min and 13.5 MPa. This two-step strategy showed superior separation ability for structural analogs. As a result, seven compounds, including four diphenylheptanes and three flavonoids with high purity, were successfully obtained. The developed method is also helpful for the extraction and isolation of other structural analogs of traditional Chinese medicines.     

Hydrodynamic Radius Characterization of a Vinyl-Type Polynorbornene by Size-Exclusion Chromatography with a Static and Dynamic Laser Light Scattering Detector

Both absolute molecular weight and molecular sizes (radius of gyration and hydrodynamic radius) of a vinyl-type polynorbornene eluting from size-exclusion chromatography columns were determined by combined with a static and dynamic laser light scattering detector. The hydrodynamic radius of polymer fraction eluting from size-exclusion chromatography columns was obtained from dynamic laser light scattering measurements at only a single angle of 90° by introducing a correction factor. According to the scaling relationship between molecular sizes and molecular weight and the ratio between radius of gyration and hydrodynamic radius, the vinyl-type polynorbornene took a random coil conformation in 1,2,4-trichlorobenzene at 150 °C.     

Solid-phase extraction of bismuth, lead and nickel from seawater using silica gel modified with 3-aminopropyltriethoxysilane filled in a syringe prior to their determination by graphite furnace atomic absorption spectrometry

In this study, the use of syringe filled with sorbent for the separation and enrichment of bismuth, lead and nickel prior to their analysis by graphite furnace atomic absorption spectrometry was described to substitute for batch and column techniques. The method proposed in this paper was compared with column technique with respect to easiness, fastness, simplicity, recovery and risk of contamination. The syringe was filled with 0.5 g of sorbent and in order to retain the analyte elements, 5 ml of sample solution (pH≥5) was drawn into the syringe to 15 s and discharged again in 15 s. Then, 2.0 M of HCl, as the eluent, was drawn into the syringe and ejected back to desorb the analyte elements. At optimum conditions, the recoveries of Bi, Pb and Ni were 95-99% with relative standard deviations (RSDs) of around ±2%. Detection limit (δ) was 0.5 μg l⁻¹ for Bi, Pb and Ni, respectively. The elements could be concentrated by drawing and discharging several portions of sample successively but eluting only one time. Bi, Pb and Ni added to a seawater sample were quantitatively recovered (>95%) with low RSD values of around ±2-3%. The risk of contamination is less than that with the column technique. In addition, it is much faster, simpler, easier, more practical and handy compared with column technique.     

A protocol for fabrication of polymer monolithic capillary columns and tuning the morphology targeting high-resolution bioanalysis in gradient-elution liquid chromatography

Polymer monolithic stationary phases are designed as a continuous interconnected globular material perfused by macropores. Like packed column, where separation efficiency is related to particle diameter, the efficiency of monoliths can be enhanced by tuning the size of both the microglobules and macropores. This protocol described the synthesis of poly(styrene-co-divinylbenzene) monolithic stationary phases in capillary column formats. Moreover, guidelines are provided to tune the macropore structure targeting high-throughput and high-resolution monolith chromatography. The versatility of these columns is exemplified by their ability to separate tryptic digests, intact proteins, and oligonucleotides under a variety of chromatographic conditions. The repeatability of the presented column fabrication process is demonstrated by the successful creation of 12 columns in three different column batches, as evidenced by the consistency of retention times (coefficients of variance [c.v.] = 0.9%), peak widths (c.v. = 4.7%), and column pressures (c.v. = 3.1%) across the batches.     

Pressurized gradient capillary electrochromatographic separation of eighteen amino acid derivatives

A pressurized gradient capillary electrochromatography (pCEC) instrument was developed to separate 18 amino acid derivatives. A reversed-phase C18 column (3 microm, 130 mm x 75 microm I.D.) and an acetate buffer (50 mmol/l NaAc, pH 6.4) with an ion-pair reagent (1% N,N-dimethylformamide) were used to separate derivatized amino acids from a standard solution (2 microg/ml), and the wavelength of the UV-Vis detector was 360 nm. The pressure on the capillary column was kept at approx. 70 Pa and 3 kV positive voltage was added on the outlet end of column. The effect of voltage on the eluting order of amino acids and the resolution of separation were studied, and it was found that when the voltage was higher than 3 kV, the adsorption of amino acids in the porous C18 column occurred. The effect of salt concentration, injection volume, and column length on the separation of amino acids was determined. The amino acid sample was separated by pCEC, and RSDs of the migration times of each amino acid were all less than 2.5%.