Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6®‐derived recombinant antibody

Membrane chromatography has already proven to be a powerful alternative to polishing columns in flow‐through mode for contaminant removal. As flow‐through utilization has expanded, membrane chromatography applications have included the capturing of large molecules, including proteins such as IgGs. Such bind‐and‐elute applications imply the demand for high binding capacity and larger membrane surface areas as compared to flow‐through applications. Given these considerations, a new Sartobind Phenyl? membrane adsorber was developed for large‐scale purification of biomolecules based on hydrophobic interaction chromatography (HIC) principles. The new hydrophobic membrane adsorber combines the advantages of membrane chromatography—virtually no diffusion limitation and shorter processing time—with high binding capacity for proteins comparable to that of conventional HIC resins as well as excellent resolution. Results from these studies confirmed the capability of HIC membrane adsorber to purify therapeutic proteins with high dynamic binding capacities in the range of 20 mg‐MAb/cm³‐membrane and excellent impurity reduction. In addition the HIC phenyl membrane adsorber can operate at five‐ to ten‐fold lower residence time when compared to column chromatography. A bind/elute purification step using the HIC membrane adsorber was developed for a recombinant monoclonal antibody produced using the PER.C6® cell line. Loading and elution conditions were optimized using statistical design of experiments. Scale‐up is further discussed, and the performance of the membrane adsorber is compared to a traditional HIC resin used in column chromatography. Biotechnol. Bioeng. 2010; 105: 296–305. © 2009 Wiley Periodicals, Inc.     

Paper-PEG-based membranes for hydrophobic interaction chromatography: purification of monoclonal antibody

This article discusses the preparation of novel Paper-PEG interpenetrating polymer network-based membranes as inexpensive alternative to currently available adsorptive membranes. The Paper-PEG membranes were developed for carrying out hydrophobic interaction membrane chromatography (HIMC). PEG is normally very hydrophilic but can undergo phase separation and become hydrophobic in the presence of high antichaotropic salt concentrations. Two variants of the Paper-PEG membranes, Paper-PEG 1 and Paper-PEG 2 were prepared by grafting different amounts of the polymer on filter paper and these were tested for their hydraulic properties and antibody binding capacity. The better of the two membranes (Paper-PEG 1) was then used for purifying the monoclonal antibody hIgG1-CD4 from simulated mammalian cell culture supernatant. The processing conditions required for purification were systematically optimized. The dynamic antibody binding capacity of the Paper-PEG 1 membrane was about 9 mg/mL of bed volume. A single step membrane chromatographic process using Paper-PEG 1 membrane gave high monoclonal antibody purity and recovery. The hydraulic permeability of the paper-based membrane was high and was maintained even after many runs, indicating that membrane fouling was negligible and the membrane was largely incompressible.     

Nanotubular structures developed from whey‐based α‐lactalbumin fractions for food applications

Whey proteins have high nutritional value providing use in dietary purposes and improvement of technological properties in processed foods. Functionality of the whey‐based α‐lactalbumin (α‐La) may be increased when assembled in the form of nanotubes, promising novel potential applications subject to investigation. The purpose of this study was to extract highly pure α‐La from whey protein isolate (WPI) and whey powder (WP) and to construct protein nanotubes from them for industrial applications. For protein fractionation, WPI was directly fed to chromatography, however, WP was first subjected to membrane filtration and the retentate fraction, whey protein concentrate (WPC), was obtained and then used for chromatographic separation. α‐La and, additionally β‐Lg, were purified at the same batches with the purities in the range of 95%–99%. After enzymatic hydrolysis, WPI‐based α‐La produced chain‐like and long nanotubules with ～20 nm width while WPC‐based α‐La produced thinner, miscellaneous, and fibril‐like nanostructures by self‐assembly. Raman and FT‐IR spectroscopies revealed that α‐La fractions, obtained from both sources and the nanostructures, developed using both fractions have some structural differences due to conformation of secondary structure elements. Nanotube formation induced gelation and nanotubular gel network entrapped a colorant uniformly with a transparent appearance. Dairy‐based α‐La protein nanotubules could be served as alternative gelling agents and the carriers of natural colorants in various food processes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1301–1310, 2014     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Antibody purification from CHO cell supernatant using new multimodal membranes

This contribution describes strategies to purify monoclonal antibodies from Chinese hamster ovary (CHO) cell culture supernatant using newly designed multimodal membranes (MMMs). The MMMs were used for the capture step purification of human IgG₁ following a size‐exclusion desalting column to remove chaotropic salts that interfere with IgG binding. The MMM column attained higher dynamic binding capacity than a Protein A resin column at an equivalent residence time of 1 min. The two‐step MMM chromatography process achieved high selectivity for capturing hIgG₁ from the CHO cell culture supernatant, though the desalting step resulted in product dilution. Product purity and host cell protein (HCP) level in the elution pool were analyzed and compared to results from a commercial Protein A column. The product purity was >98% and HCP levels were <20 ppm for both purification methods. In addition, hIgG₁ could be eluted from the MMM chromatography column at neutral pH, which is important for limiting the formation of aggregates; although slow elution dilutes the product. Overall, this paper shows that MMMs are highly effective for capture step purification of proteins and should be considered when Protein A cannot be used, e.g., for pH sensitive mAbs or proteins lacking an Fc binding domain. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:658–665, 2017     

Separation of human plasma proteins HSA and HIgG using high-capacity macroporous gel-filled membranes

This paper discusses the separation of human serum albumin (HSA), the most abundant protein present in plasma from human immunoglobulin G (HIgG) by membrane chromatography using a novel macroporous gel-filled membrane (designated Q Type 2). The membrane was prepared by anchoring a quaternary ammonium salt macroporous gel within the pores of a non-woven, polypropylene fabric. Factors affecting HSA binding were examined and operating conditions suitable for separating it from human plasma were identified. At an optimized condition, the HSA binding capacity of this novel membrane under saturating conditions was in the range of 290–300 mg/ml. This was not only significantly higher than binding capacities reported for other chromatographic membranes, but also higher than binding capacities of conventional gel based chromatographic media. The protein binding capacity was also largely insensitive to the superficial velocity, indicating the dominance of convective protein transport to and from the binding sites. The suitability of using this membrane for plasma fractionation was demonstrated by the separation of a simulated feed solution consisting of HSA and HIgG.     

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β‐barrel membrane proteins using Opa₆₀ from Neisseria gonorrhoeae as a model system. Opa₆₀ is an eight‐stranded β‐barrel with long extracellular loops (～63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa₆₀ in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β‐barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β‐barrel. The removal of the loop resonances significantly improved the assignment of the Opa₆₀ β‐barrel region (97% of the resonances corresponding to the β‐barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full‐length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β‐barrel membrane proteins.     

Zonal rate model for stacked membrane chromatography part II: characterizing ion-exchange membrane chromatography under protein retention conditions

The Zonal Rate Model (ZRM) has previously been shown to accurately account for contributions to elution band broadening, including external flow nonidealities and radial concentration gradients, in ion-exchange membrane (IEXM) chromatography systems operated under nonbinding conditions. Here, we extend the ZRM to analyze and model the behavior of retained proteins by introducing terms for intra-column mass transfer resistances and intrinsic binding kinetics. Breakthrough curve (BTC) data from a scaled-down anion-exchange membrane chromatography module using ovalbumin as a model protein were collected at flow rates ranging from 1.5 to 20 mL min(-1). Through its careful accounting of transport nonidealities within and external to the membrane stack, the ZRM is shown to provide a useful framework for characterizing putative protein binding mechanisms and models, for predicting BTCs and complex elution behavior, including the common observation that the dynamic binding capacity can increase with linear velocity in IEXM systems, and for simulating and scaling separations using IEXM chromatography. Global fitting of model parameters is used to evaluate the performance of the Langmuir, bi-Langmuir, steric mass action (SMA), and spreading-type protein binding models in either correlating or fundamentally describing BTC data. When combined with the ZRM, the bi-Langmuir, and SMA models match the chromatography data, but require physically unrealistic regressed model parameters to do so. In contrast, for this system a spreading-type model is shown to accurately predict column performance while also providing a realistic fundamental explanation for observed trends, including an observed increase in dynamic binding capacity with flow rate.     

Characterization of cryogel monoliths for extraction of minor proteins from milk by cation exchange

Extraction and purification of high‐value minor proteins directly from milk without pre‐treatment is a challenge for the dairy industry. Pre‐treatment of milk before extraction of proteins by conventional packed‐bed chromatography is usually necessary to prevent column blockage but it requires several steps that result in significant loss of yield and activity for many minor proteins. In this paper, we demonstrate that it is possible to pass 40–50 column volumes of various milk samples (raw whole milk, homogenized milk, skim milk and acid whey) through a 5 mL cryogel chromatographic column at 550 cm/h without exceeding its pressure limits if the processing temperature is maintained above 35°C. The dynamic binding capacity obtained for the cryogel matrix (2.1 mg/mL) was similar to that of the binding capacity (2.01 mg/mL) at equilibrium with 0.1 mg/mL of lactoferrin in the feed samples. The cryogel column selectively binds lactoferrin and lactoperoxidase with only minor leakage in flowthrough fractions. Lactoferrin was recovered from elution fractions with a yield of over 85% and a purity of more than 90%. These results, together with the ease of manufacture, low cost and versatile surface chemistry of cryogels suggest that they may be a good alternative to packed‐bed chromatography for direct capture of proteins from milk. Biotechnol. Bioeng. 2009;103: 1155–1163. © 2009 Wiley Periodicals, Inc.     

β‐Methylphenylalanine exerts neuroprotective effects in a Parkinson's disease model by protecting against tyrosine hydroxylase depletion

We evaluated the neuroprotective effects of β‐methylphenylalanine in an experimental model of rotenone‐induced Parkinson's disease (PD) in SH‐SY5Y cells and rats. Cells were pre‐treated with rotenone (2.5 µg/mL) for 24 hours followed by β‐methylphenylalanine (1, 10 and 100 mg/L) for 72 hours. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), mitochondrial fragmentation, apoptosis, and mRNA and protein levels of tyrosine hydroxylase were determined. In a rat model of PD, dopamine (DA) and 3,4‐dihydroxyphenylacetic acid (DOPAC) levels, bradykinesia and tyrosine hydroxylase expression were determined. In rotenone–pre‐treated cells, β‐methylphenylalanine significantly increased cell viability and MMP, whereas ROS levels, apoptosis and fragmented mitochondria were reduced. β‐Methylphenylalanine significantly increased the mRNA and protein levels of tyrosine hydroxylase in SH‐SY5Y cells. In the rotenone‐induced rat model of PD, oral administration of β‐methylphenylalanine recovered DA and DOPAC levels and bradykinesia. β‐Methylphenylalanine significantly increased the protein expression of tyrosine hydroxylase in the striatum and substantia nigra of rats. In addition, in silico molecular docking confirmed binding between tyrosine hydroxylase and β‐methylphenylalanine. Our experimental results show neuroprotective effects of β‐methylphenylalanine via the recovery of mitochondrial damage and protection against the depletion of tyrosine hydroxylase. We propose that β‐methylphenylalanine may be useful in the treatment of PD.     

Fractionation of whey proteins with high-capacity superparamagnetic ion-exchangers

In this study we describe the design, preparation and testing of superparamagnetic anion-exchangers, and their use together with cation-exchangers in the fractionation of bovine whey proteins as a model study for high-gradient magnetic fishing. Adsorbents prepared by attachment of trimethyl amine to particles activated in sequential reactions with allyl bromide and N-bromosuccinimide yielded a maximum bovine serum albumin binding capacity of 156 mg g(-1) combined with a dissociation constant of 0.60 microM, whereas ion-exchangers created by linking polyethylene imine through superficial aldehydes bound up to 337 mg g(-1) with a dissociation constant of 0.042 microM. The latter anion-exchanger was selected for studies of whey protein fractionation. In these, crude bovine whey was treated with a superparamagnetic cation-exchanger to adsorb basic protein species, and the supernatant arising from this treatment was then contacted with the anion-exchanger. For both adsorbent classes of ion-exchanger, desorption selectivity was subsequently studied by sequentially increasing the concentration of NaCl in the elution buffer. In the initial cation-exchange step quantitative removal of lactoferrin (LF) and lactoperoxidase (LPO) was achieved with some simultaneous binding of immunoglobulins (Ig). The immunoglobulins were separated from the other two proteins by desorbing with a low concentration of NaCl (< or = 0.4 M), whereas lactoferrin and lactoperoxidase were co-eluted in significantly purer form, e.g. lactoperoxidase was purified 28-fold over the starting material, when the NaCl concentration was increased to 0.4-1 M. The anion-exchanger adsorbed beta-lactoglobulin (beta-LG) selectively allowing separation from the remaining protein.     

Development of rigid bidisperse porous microspheres for high-speed protein chromatography

Development of a high-performance stationary phase is an essential demand for high-speed separation of proteins by liquid chromatography. Based on a novel porogenic mode, that is, using superfine granules of calcium carbonate as solid porogen and a mixture of cyclohexanol and dodecanol as liquid porogen, a rigid spherical biporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension-polymerization. The epoxide groups of the matrix were modified with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydeoxypropy functionalities that are required for ion exchange chromatography. Results from scanning electron microscopy and mercury intrusion porosimetry measurements revealed that the matrix contained two families of pores, that is, micropores (10-90 nm) and macropores (180-4000 nm). Furthermore, the biporous medium possesses specific surface area as high as 91.3 m(2)/g. Because of the presence of the macropores that provided convective flow channels for the mobile phase, the dynamic adsorption capacity was found to be as high as 54.6 mg/g wet bead at 300 cm/h, approximately 63.2% of its static capacity. In addition, the column efficiency and dynamic binding capacity decreased only slightly with mobile-phase flow rate in the range of 300-3000 cm/h. These properties made the packed bed with the bidisperse porous matrix suitable for high-speed protein chromatography.     

Hydrolysis of lactose in whey permeate by immobilized β-galactosidase from Penicillium notatum

A new low-cost β-galactosidase (lactase) preparation for whey permeate saccharification was developed and characterized. A biocatalyst with a lactase activity of 10 U/mg, a low transgalactosylase activity and a protein content of 0.22 mg protein/mg was obtained from a fermenter culture of the fungus Penicillium notatum. Factors influencing the enzymatic hydrolysis of lactose, such as reaction time, pH, temperature and enzyme and substrate concentration were standardized to maximize sugar yield from whey permeate. Thus, a 98.1% conversion of 5% lactose in whey permeate to sweet (glucose-galactose) syrup was reached in 48 h using 650 β-galactosidase units/g hydrolyzed substrate. After the immobilization of the acid β-galactosidase from Penicillium notatum on silanized porous glass modified by glutaraldehyde binding, more than 90% of the activity was retained. The marked shifts in the pH value (from 4.0 to 5.0) and optimum temperatures (from 50°C to 60°C) of the solid-phase enzyme were observed and discussed. The immobilized preparation showed high catalytic activity and stability at wider pH and temperature ranges than those of the free enzyme, and under the best operating conditions (lactose, 5%; β-galactosidase, 610–650 U/g lactose; pH 5.0; temperature 55°C), a high efficiency of lactose saccharification (84–88%) in whey permeate was achieved when lactolysis was performed both in a batch process and in a recycling packed-bed bioreactor. It seems that the promising results obtained during the assays performed on a laboratory scale make this immobilizate a new and very viable preparation of β-galactosidase for application in the processing of whey and whey permeates.     

Influence of different spacer arms on Mimetic Ligand™ A2P and B14 membranes for human IgG purification

Microporous membranes are an attractive alternative to circumvent the typical drawbacks associated to bead-based chromatography. In particular, the present work intends to evaluate different affinity membranes for antibody capture, to be used as an alternative to Protein A resins. To this aim, two Mimetic Ligands? A2P and B14, were coupled onto different epoxide and azide group activated membrane supports using different spacer arms and immobilization chemistries. The spacer chemistries investigated were 1,2-diaminoethane (2LP), 3,6-dioxa-1,8-octanedithiol (DES) and [1,2,3] triazole (TRZ). These new mimetic membrane materials were investigated by static and by dynamic binding capacity studies, using pure polyclonal human immunoglobulin G (IgG) solutions as well as a real cell culture supernatant containing monoclonal IgG(1). The best results were obtained by combining the new B14 ligand with a TRZ-spacer and an improved Epoxy 2 membrane support material. The new B14-TRZ-Epoxy 2 membrane adsorbent provided binding capacities of approximately 3.1mg/mL, besides (i) a good selectivity towards IgG, (ii) high IgG recoveries of above 90%, (iii) a high Pluronic-F68 tolerance and (iv) no B14-ligand leakage under harsh cleaning-in-place conditions (0.6M sodium hydroxide). Furthermore, foreseeable improvements in binding capacity will promote the implementation of membrane adsorbers in antibody manufacturing.     

Advective hydrogel membrane chromatography for monoclonal antibody purification in bioprocessing

Protein A chromatography is widely employed for the capture and purification of monoclonal antibodies (mAbs). Because of the high cost of protein A resins, there is a significant economic driving force to seek new downstream processing strategies. Membrane chromatography has emerged as a promising alternative to conventional resin based column chromatography. However, to date, the application has been limited to mostly ion exchange flow through (FT) mode. Recently, significant advances in Natrix hydrogel membrane has resulted in increased dynamic binding capacities for proteins, which makes membrane chromatography much more attractive for bind/elute operations. The dominantly advective mass transport property of the hydrogel membrane has also enabled Natrix membrane to be run at faster volumetric flow rates with high dynamic binding capacities. In this work, the potential of using Natrix weak cation exchange membrane as a mAb capture step is assessed. A series of cycle studies was also performed in the pilot scale device (> 30 cycles) with good reproducibility in terms of yield and product purities, suggesting potential for improved manufacturing flexibility and productivity. In addition, anion exchange (AEX) hydrogel membranes were also evaluated with multiple mAb programs in FT mode. Significantly higher binding capacity for impurities (support mAb loads up to 10Kg/L) and 40X faster processing speed were observed compared with traditional AEX column chromatography. A proposed protein A free mAb purification process platform could meet the demand of a downstream purification process with high purity, yield, and throughput. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:974–982, 2015     

The role of polymer nanolayer architecture on the separation performance of anion-exchange membrane adsorbers: I. Protein separations

This contribution describes the preparation of strong anion-exchange membranes with higher protein binding capacities than the best commercial resins. Quaternary amine (Q-type) anion-exchange membranes were prepared by grafting polyelectrolyte nanolayers from the surfaces of macroporous membrane supports. A focus of this study was to better understand the role of polymer nanolayer architecture on protein binding. Membranes were prepared with different polymer chain graft densities using a newly developed surface-initiated polymerization protocol designed to provide uniform and variable chain spacing. Bovine serum albumin and immunoglobulin G were used to measure binding capacities of proteins with different size. Dynamic binding capacities of IgG were measured to evaluate the impact of polymer chain density on the accessibility of large size protein to binding sites within the polyelectrolyte nanolayer under flow conditions. The dynamic binding capacity of IgG increased nearly linearly with increasing polymer chain density, which suggests that the spacing between polymer chains is sufficient for IgG to access binding sites all along the grafted polymer chains. Furthermore, the high dynamic binding capacity of IgG (>130 mg/mL) was independent of linear flow velocity, which suggests that the mass transfer of IgG molecules to the binding sites occurs primarily via convection. Overall, this research provides clear evidence that the dynamic binding capacities of large biologics can be higher for well-designed macroporous membrane adsorbers than commercial membrane or resin ion-exchange products. Specifically, using controlled polymerization leads to anion-exchange membrane adsorbers with high binding capacities that are independent of flow rate, enabling high throughput. Results of this work should help to accelerate the broader implementation of membrane adsorbers in bioprocess purification steps.     

肝素纯化工艺中离子交换树脂的选择与应用研究

研究比较了5种树脂对肝素的吸附能力,从中选出S5428阴离子交换树脂来纯化肝素。通过对各因素的研究,确定了树脂对肝素的静态、动态吸附以及解吸的最佳条件。结果表明:静态吸附的温度45℃,pH 8.0的条件下吸附2 h,肝素的吸附率为90.5%;层析柱的动态吸附温度45℃,肝素溶液进样浓度1.0 mg/mL,进样速度1.5 mL/min,树脂柱能处理1.5 BV肝素液而不发生泄露,吸附量为3.05 mg/mL,达到饱和吸附时可处理4BV的料液,吸附量为9.18 mg/mL;采用2.0 mol/L NaCl洗脱,洗脱流速1.5 mL/min,肝素解吸率可达95.8%,肝素效价可达150 U/mg,效价回收率98%。     

Improved β‐Glucanase Production by a Recombinant Escherichia coli Strain using Zinc‐Ion Supplemented Medium

In order to investigate the suitability of different metal chelates for affinity chromatography, an expression vector was constructed. It contained a hybrid β‐glucanase as a model protein fused with a His₆‐tag and a secretion cassette providing the ability to secrete β‐glucanase into the culture medium. Supplementation of zinc to the medium led to a rapidly increased expression and release of the target protein into the cultivation medium. Results in respect to the supplementation of the commonly used Terrific Broth “TB‐medium” with different metal ions are reported with special emphasis on the influence of zinc ions. A concentration of zinc ions in the order of about 0.175 mM led to optimal results. Batch cultivation under well‐controlled conditions showed that the growth behavior did not change significantly by adding zinc ions. Growth in a stirred tank bioreactor was much faster in unsupplemented TB‐medium compared to shake flask experiments leading to a much higher biomass concentration (15 g/L instead of 3 g/L). The secretion of β‐glucanase under theses conditions started at the transition into the stationary phase and increased to yield an extracellular activity of 1350 U/mL at the end of the fermentation process. An even higher yield of extracellular β‐glucanase (2800 U/mL) was reached when the fermentation was carried out with TB‐medium supplemented with 0.175 mM ZnSO₄.     

Preparative Enantioseparation of β‐Substituted‐2‐Phenylpropionic Acids by Countercurrent Chromatography With Substituted β‐Cyclodextrin as Chiral Selectors

Preparative enantioseparation of four β‐substituted‐2‐phenylpropionic acids was performed by countercurrent chromatography with substituted β‐cyclodextrin as chiral selectors. The two‐phase solvent system was composed of n‐hexane‐ethyl acetate‐0.10 mol L‐1 of phosphate buffer solution at pH 2.67 containing 0.10 mol L^‐1 of hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) or sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD). The influence factors, including the type of substituted β‐cyclodextrin, composition of organic phase, concentration of chiral selector, pH value of the aqueous phase, and equilibrium temperature were optimized by enantioselective liquid–liquid extraction. Under the optimum separation conditions, 100 mg of 2‐phenylbutyric acid, 100 mg of tropic acid, and 50 mg of 2,3‐diphenylpropionic acid were successfully enantioseparated by high‐speed countercurrent chromatography, and the recovery of the (±)‐enantiomers was in the range of 90–91% for (±)‐2‐phenylbutyric acid, 91–92% for (±)‐tropic acid, 85–87% for (±)‐2,3‐diphenylpropionic acid with purity of over 97%, 96%, and 98%, respectively. The formation of 1:1 stoichiometric inclusion complex of β‐substituted‐2‐phenylpropionic acids with HP‐β‐CD was determined by UV spectrophotometry and the inclusion constants were calculated by a modified Benesi‐Hildebrand equation. The results showed that different enantioselectivities among different racemates were mainly caused by different enantiorecognition between each enantiomer and HP‐β‐CD, while it might be partially caused by different inclusion capacity between racemic solutes and HP‐β‐CD. Chirality 27:795–801, 2015. © 2015 Wiley Periodicals, Inc.     

Enantiomeric separation of β‐blockers and tryptophan using heparin as stationary and pseudostationary phases in capillary electrophoresis

The separation methods of the enantiomers of two β‐blockers and tryptophan were studied using capillary electrochromatography with heparin covalently as well as non‐covalently, bonded onto the capillary inner wall as stationary phase and electrokinetic chromatography with heparin as pseudostationary phase. In the case of heparin, used as a stationary phase, the method was unable to resolve enantiomers in both cases β‐blockers and tryptophan. On the other hand, when heparin was used as a pseudostationary phase, the resolution of the enantiomers was obtained only with 3‐aminopropyltriethoxysilane which were immobilised onto the inner phase of the capillary. The results of this study let us infer that the electrostatic, hydrophobic, and steric interactions were involved in the separation mechanisms. The separation was achieved in less than 10 minutes under the optimized conditions: 30 mM phosphate buffer (pH 2.5) with the adding of 15 mg/mL of heparin at 15°C and 10 kV. The usefulness of heparin as a chiral selector both in electrokinetic chromatography using 3‐aminopropyltriethoxysilane attached to the capillary was demonstrated for the first time. The developed method was powerful, sensitive, and fast, and it could be considered an important alternative to conventional methods used for chiral separation.