High-performance liquid chromatography using spherical aggregates of hydroxyapatite micro-crystals as adsorbent

High-performance liquid chromatography (HPLC) using spherical aggregates of hydroxyapatite (HA) microcrystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. In comparison with previously developed plate-like HA packed columns for HPLC, spherical HA packed columns show considerably high chromatographic resolutions in spite of extremely reduced column lengths of 0.5-3 cm. The pressure generated by the latter columns is much higher than that generated by the former, however.     

Strontium-phosphate hydroxyapatite high-performance liquid chromatography

High-performance liquid chromatography using spherical aggregates of strontium-phosphate hydroxyapatite(SrHA) micro-crystals as adsorbent has been developed; preliminary performance tests were carried out by using several types of protein. It can be deduced that, in parallel with the case of usual calcium-phosphate hydroxyapatite(CaHA), with SrHA also, two types of effective surface, vector a (or vector b) and vector c surfaces, appear on the crystal: the same protein molecular generally shows slightly different chromatographic behaviors between the CaHA and the SrHA packed column. Combining the SrHA and the CaHA packed column would lead to an efficient fractionation of a particular molecule from an assembly of molecules with subtle structural differences from one another.     

Molecular-size determination of xanthan polysaccharide

Hydrodynamic chromatographic separations of xanthan polysaccharide of ultrahigh molecular weight have been obtained by using columns packed with 30-μm, non-porous spheres. From calibration curves of the elution volume versus particle size for spherical, polystyrene latexes, it was found that xanthan is eluted at the same volume as a 0.153-μm diameter sphere. Extremely dilute samples of xanthan (70 p.p.m.) were injected to preclude self-association and aggregate formation. Detection at these low concentrations was accomplished by tagging the xanthan with a fluorescein derivative and using a flow-through fluorometer detector. Flow rates of 1 mL/min yielded run times of ～7 min. Comparison of the accepted molecular conformation of xanthan—a rigid rod-like molecule—with the apparent molecular volume from the spherical-latex calibration indicates that the xanthan molecules are substantially oriented by the flow field in the chromatography column.     

Pressure-flow relationships for packed beds of compressible chromatography media at laboratory and production scale

Pressure drop across chromatography beds employing soft or semirigid media can be a significant problem in the operation of large-scale preparative chromatography columns. The shape or aspect ratio (length/diameter) of a packed bed has a significant effect on column pressure drop due to wall effects, which can result in unexpectedly high pressures in manufacturing. Two types of agarose-based media were packed in chromatography columns at various column aspect ratios, during which pressure drop, bed height, and flow rate were carefully monitored. Compression of the packed beds with increasing flow velocities was observed. An empirical model was developed to correlate pressure drop with the aspect ratio of the packed beds and the superficial velocity. Modeling employed the Blake-Kozeny equation in which empirical relationships were used to predict bed porosity as a function of aspect ratio and flow velocity. Model predictions were in good agreement with observed pressure drops of industrial scale chromatography columns. A protocol was developed to predict compression in industrial chromatography applications by a few laboratory experiments. The protocol is shown to be useful in the development of chromatographic methods and sizing of preparative columns.     

Transport and binding characterization of a novel hybrid particle impregnated membrane material for bioseparations

The transport and binding properties of a novel hybrid particle-nonwoven membrane medium are described. In this construct, a polymeric chromatographic resin is entrapped between two layers of a nonwoven polypropylene membrane. The membrane-supported resin medium offers the advantage of increased interstitial pore diameter to allow passage of cells and other debris in the feed, while providing sufficiently high surface area for product capture within the resin particles. Columns packed with PIM displayed excellent flow distribution and had interstitial porosities of 0.48 ± 0.01, 25-60% larger than those typical of a packed bed. These columns were able to pass over 95% of E. coli cells and human red blood cell concentrate in 30 column volumes while maintaining a pressure drop significantly lower than that of a packed bed with a similar amount of resin. The dynamic binding capacity of bovine serum albumin (BSA) to the chromatographic resin entrapped in the PIM packed column was essentially the same as that observed with the same volume of resin in a packed bed. The General Rate (GR) model of chromatography was used to analyze experiments indicating the breakthrough behavior of the PIM columns is predictable, and very similar to those of a normal packed bed. These results suggest that PIM constructs can be designed to process viscous mobile phases containing particulates while retaining the desirable binding characteristics of the embedded chromatographic resin and could find uses in adsorption separation processes from complex feed streams such as whole blood, cell culture, and food processing.     

Chromatography of nucleic acids, proteins and certain phages on granulated hydroxyapatite]

A modification of the classical method of hydroxyapatite synthesis is proposed. The essence of the modification is hydroxyapatite synthesis in the presence of an additional component silicic acid particles. The subsequent steps of the method are modified so, as to retain the intactness of crystals at all the stages of preparation and use of the adsorbent. The final product consists of large spherical agregates (200-250 mu in diameter) and contains about 1% of tightly bound silicic acid. It slightly differs from usual hydroxyapatite in its chromatographic properties. Granulated hydroxyapatite obtained has a high specific capacity and can be repeatedly used in experiments (up to 50 chromatographic cycles). Native high-polymeric T2 phage DNA was practically quantitatively eluated from the column. Conditions for chromatography of some proteins (lysozyme, RNase, DNase) are described. Fractionation and purification of T2 and T3 bacteriophages and TMV are carried out by means of chromatography on granulated hydroxyapatite.     

Slalom chromatography: size-dependent separation of DNA molecules by a hydrodynamic phenomenon

Slalom chromatography, a size-dependent DNA fractionation method based on a new principle [Hirabayashi, J., & Kasai, K. (1989) Anal. Biochem. 178, 336-341], was systematically studied in detail. In this method, larger DNA fragments are eluted much later than smaller ones from columns packed with spherical microbeads. Elution of a series of DNA fragments was systematically examined by using columns packed with polymer-based packings of different diameter and different pore size for high-performance gel permeation chromatography. Packings of smaller diameter proved to be superior for resolving the smaller size range of DNA, while the reverse was the case for larger DNAs. Application of a faster flow rate led to larger retardation of every DNA fragment, while at the lowest flow rate applied (0.067 cm/min), all the fragments were eluted almost at the void volume. When the column temperature was lowered, retardation of DNA became larger. On the other hand, differences in the chemical nature and the pore size of packings, or in the hydrophobicity of the eluting solvent, had little effect on DNA retardation. Size-dependent fractionation of DNA was also achieved even on columns packed with nonporous packings having anionic groups (cation exchangers). In conclusion, these results confirmed the previous conclusion that slalom chromatography is not based on an adsorption or equilibrium phenomenon but should be attributed to a hydrodynamic phenomenon.     

Pre-erythrocytic development and associated host responses to Haemoproteus meleagridis (Haemosporina: Haemoproteidae) in experimentally infected domestic turkeys

Two generations of pre-erythrocytic schizogony occurred in skeletal and cardiac muscle of domestic turkeys infected with sporozoites of Haemoproteus meleagridis. First generation schizonts reached maturity approximately five days post-inoculation (DPI) and developed in capillary endothelial cells and myofibroblasts. The schizonts ranged from 12 to 20 microns in diameter and produced long (5-6 microns), slender merozoites. Early second generation schizonts were first detected in capillary endothelial cells between 5 and 8 DPI. They were cylindrical and ranged in size from 5 to 8 microns in diameter and up to 28 microns in length. Second generation schizonts which reached maturity by 17 DPI were surrounded by a thick, hyaline wall and were packed with numerous spherical merozoites less than 1 micron in diameter. Mature megaloschizonts were fusiform, ranged from 30 to 113 microns in diameter, and extended as much as 465 microns along the long axis of muscle fibers. Merozoites developed as buds from cytomeres that formed between 8 and 14 DPI. Infected turkeys developed a moderate to severe myositis within 5 DPI and were lame in one or both legs. The myositis was associated with the necrosis of scattered groups of muscle fibers. Muscle fibers surrounding mature megaloschizonts were swollen and hyaline. Megaloschizonts were surrounded occasionally by fibroblasts and infiltrates of mononuclear cells. The morphology and site of development of mature megaloschizonts of Haemoproteus meleagridis are contrasted with those of other avian haemosporidians.     

Heterogeneity of tropocollagen as investigated by chromatography on hydroxyapatite columns

The chromatography of native acid-soluble tropocollagen from calf skin on hydroxyapatite columns has been investigated. Resolution of a number of chromatographic peaks has been obtained by using shallow slopes of the eluting phosphate gradient. The results obtained suggest a heterogeneity of tropocollagen molecules which might be due to different distributions of absorbing sites on the molecules. The results obtained have also been used to study the mechanism of chromatography of proteins on hydroxyapatite and the resolving power of the columns.     

Isocratic separation of DNA bases and deoxyribonucleosides by high-performance liquid chromatography on anex OSTION.

A high-performance liquid chromatographic method has been developed for the resoiusion of DNA bases and deoxyribonucleosides in mixture. The isocratic separation based on ion exelusion was achieved in column of 250-mm length and 2 mm i.d. packed with the spherical, totally porous anex OSTION LGAT 0800 of particle size 10–12 μm. The mobile phase consists of 5 mm ammonium formate pH 4.5 and complete resolution of an eight-component system is possible at a flow rate of 2 ml/h in 100 min. Rapid separation of the components, with exception of Cyt-dCyd, is reached at flow rate of 20 ml/h in 10 min.     

Electronic particle counting for evaluating the quality of air in operating theatres: a potential basis for standards?

Airborne particle counting in eight size ranges (0.5- greater than 20 microns), by computerized electronic equipment, was compared with the numbers of bacteria-carrying particles (BCP) assessed by slit sampling in ultra-clean and turbulently ventilated operating theatres. In the ultra-clean theatre the number of particles of 5-7 microns size range correlated with BCP while peaks in the numbers of particles less than 3 microns and greater than 15 microns corresponded with activity. Comparative relationships also occurred in the turbulently ventilated theatre but the use of this equipment in that environment cannot yet replace counts of airborne bacteria. We consider that electronic particle counting in the 0-20 microns size range may be used to judge the performance of a clean air operating theatre distribution system, including efficiency and integrity of the filter/seal systems and the presence or absence of entrainment of bacteria and other particles. The sampling techniques and analysis of particle concentration results described here may be a suitable basis for standards.     

Analysis of Carnosine, Homocarnosine, and Other Histidyl Derivatives in Rat Brain

Isocratic reverse-phase analytical HPLC has been used to examine naturally occurring imidazoles of rat brain. Elution of brain extracts with a phosphate buffer mobile phase from columns packed with Hypersil ODS (5 microns) resulted in good separation of the well-documented brain imidazole-containing dipeptides carnosine and homocarnosine. Measured concentrations corresponded to published values. Several further peaks observed had properties consistent with those of N-acetyl derivatives of compounds related to carnosine and homocarnosine. N-Acetyl forms not commercially available were prepared and their identities verified by nuclear magnetic resonance spectroscopy. A number of these had chromatographic properties identical to those of compounds in brain extracts. Fractions corresponding to some of the peaks were examined using staining systems specific for certain chemical features and compared with results obtained for commercial or synthetic standards. The results of these tests supported the chromatographic data. Thus, chromatographic and microchemical evidence is presented for the existence of N-acetyl forms of histidine, 1-methylhistidine, carnosine, anserine, and homocarnosine in rat brain.     

Efficiency of particle retention and clearance rate in the polychaete Sabellaria alveolata L

The development of Sabellaria alveolata, a gregarious reef-building polychaete species, is maximal in Mont-Saint-Michel Bay (France), where trophic capacity is now threatened by increasing shellfish farming. As no data are available concerning the ecophysiological response of this species, the purpose of the present study was to obtain clearance rate and retention efficiency values to provide a first order of magnitude for the trophic role of this species. Data were obtained using a flow-through system with novel troughs suitable for 225 cm2 reef blocks containing a mean number of 940 +/- 102 (S.E.) individuals. The experimental diet used consisted of a mixture of two live microalgae, Skeletonema costatum (3800 cell ml-1) and Isochrysis galbana (23,700 cell ml-1), chosen to cover a broad size range (2 to 16 microns equivalent spherical diameter, ESD), as determined by a particle counter. On the basis of a mean clearance rate of 0.7 lh-1 obtained with reef blocks, the mean rate for an individual was estimated at 7.5 x 10(-4) L h-1. Particles larger than 6 microns ESD were cleared with 100% efficiency, but S. alveolata was unable to retain particles smaller than 2 microns ESD. The results are compared with data obtained for other polychaete species, and clearance rate values are extrapolated to an entire reef.     

Purification of a functional gene therapy vector derived from Moloney murine leukaemia virus using membrane filtration and ceramic hydroxyapatite chromatography

The ability of membrane ultra- and diafiltration and two chromatography media, Matrex Cellufine Sulfate (Millipore) and Macro-Prep ceramic hydroxyapatite (Bio-Rad), to adsorb, elute, and purify gene therapy vectors based on Moloney murine leukaemia virus (MoMuLV) carrying the 4070A amphotropic envelope protein was studied. Membrane ultra- and diafiltration provided virus concentration up to 160-fold with an average recovery of infectious viruses of 77 +/- 14%. In batch experiments, Macro-Prep ceramic hydroxyapatite (type 2, particle size 40 microm) proved superior to Matrex Cellufine Sulfate for MoMuLV vector particle adsorption. Furthermore, functional vector particles could be eluted using phosphate buffer pH 6.8 (highest titres from >or=300 mM phosphate) from the Macro-Prep adsorbent, with higher specific titres (cfu/mg protein) than the starting material. Similar results were obtained when this ceramic hydroxyapatite was packed into a column and used in a liquid chromatography system. Recovery of transduction-competent virus was between 18 and 31% for column experiments and 32 and 46% for batch experiments.     

Performance and characterization of a nanophased porous hydroxyapatite for protein chromatography

Nanophased porous hydroxyapatite beads with particle diameters of 25 microm and 30 microm intended for use in protein and biomolecule separation are characterized with respect to chromatographic characteristics. These particles were produced from a hydroxyapatite gel by a controlled spray process yielding microspheres containing hydroxyapatite nanocrystals. By calcification of the microspheres, nanophased porous hydroxyapatite beads were obtained. As a reference material, ceramic hydroxyapatite Types I and II with a particle diameter of 40 microm was chosen. SEM pictures show that the surface of the nanophased hydroxyapatite is very rough compared to ceramic hydroxyapatite Types I and Type II. The calcium-to-phosphorous ratio of this nanophased hydroxyapatite is 1.6, which is slightly below the theoretical ratio of 1.67 of pure hydroxyapatite. The porosity is greater than 60%. An IgG binding capacity of 60.7 mg/ml for Bio-Rad Type I and 36.0 mg/ml for Type II, 42.0 mg/ml for the nanophased material with 25 microm and 19.7 mg/ml for the nanophased material with 30 microm were observed. The nanophased material with 30 microm had the lowest mass transfer resistancy as indicated by the dependency of the dynamic binding capacity on velocity. It is assumed that the mass transport properties are characterized by a low particle diffusion resistancy or by slight intraparticle convection. The material also showed high selectivity for IgG. When culture supernatant with 5% FCS containing 3 mg/ml was loaded, pure IgG could be eluted by linear gradient with increasing sodium phosphate concentration. This nanophased material comprises a novel stationary phase for IgG separation.     

HPLC separation of some purine and pyrimidine derivatives on Chromolith Performance Si monolithic column

The chromatographic behavior of some purines and pyrimidines on a monolithic Chromolith Performance Si column under normal-phase high-performance liquid chromatography mode has been studied. Column pressure, column efficiency and selectivity of Chromolith Performance Si column were compared to those of conventional spherical 5 microm silica packed columns Econosphere Silica and Zorbax Rx-SIL. The investigation has shown that application of Chromolith Performance Si column for analysis of polar solutes can reduce the separation time without sacrificing column efficiency and selectivity. Improvement of the monolithic silica column efficiency for polar solutes is observed when ternary mobile phases (mixtures of hexane-isopropanol with ethylene glycol, water or acetonitrile) are applied.     

Characterization of the swelling of a size-exclusion gel.

The swelling of a dextran gel, Sephadex G-75, was observed in an aqueous environment at room temperature by a noninvasive technique that uses light microscopy coupled to an image analysis system via a video camera. The rate of swelling was found to follow the Tanaka and Fillmore theory, from which the overall gel diffusion coefficient was estimated as 6.3 x 10(-7) cm2/s. In addition to giving a quantitative measure of gel swelling that could be useful in the mechanical design of liquid chromatography columns, this approach provides data on wet particle size and particle size range, which is needed for the modeling of diffusional and mass transfer effects in size-exclusion chromatography. In this context, key observations are that the gel particles are nearly spherical with an elliptical shape factor of 0.98 (perfect sphere = 1) and that there is little difference between sizes of particles obtained in water, 50 mM Tris-glycine buffer (pH 10.2), and buffer containing 1 mg/mL protein. The diameter of the dry material ranged from 20 to 100 microns, while the hydrated particles had diameters of 40-350 microns. The rate of swelling is rapid, with 50% swelling occurring in about 10 s and swelling to 99% of the final wet particle size being obtained in less than 90 s.     

Isolation of a recombinant antibody from cell culture supernatant: continuous annular versus batch and expanded-bed chromatography

Annular chromatography represents a crossflow approach to chromatographic separations, that allows the continuous separation of multicomponent mixtures. The potential of the method for continuous bioseparation has been discussed for some time, however, we demonstrate for the first time the processing of a complex feed (cell culture supernatant) taken from an actual (bio)process. Moreover, while previously published applications of annular chromatography concentrated on noninteractive (gel filtration) or nonspecific (ion exchange) chromatography, we show the possibility of continuous annular affinity chromatography. In particular, a commercially available preparative continuous annular chromatography (P-CAC) system was used to purify a recombinant antibody (human IgG(1)-kappa) from CHO cell culture supernatants by (pseudo)affinity chromatography on hydroxyapatite (HA) and rProtein A. Methods developed using small (2 mL) batch columns could be directly transferred to the P-CAC, where they yielded similar results in terms of final product quality. Yields were between 87% and 92% in the case of HA and between 77% and 82% in the case of rProtein A chromatography. DNA removal was nearly quantitative in all cases. Concomitantly, the antibody fraction of the total protein content was raised by one order of magnitude in HA and by a factor of 50 by rProtein A chromatography. In addition, a novel HA material (particle diameter -120 microm) was investigated, which was compatible with expanded-bed applications. However, the final purity of the antibody thus obtained and also the yields (<70%) were less than satisfactory.     

Separation and automated analysis of phosphorylated metabolic intermediates

A chromatographic procedure has been described using a new automated detection system that allows separation at the 10 nmole level of all the glycolytic intermediates as well as AMP, ADP, and ATP in pure samples and samples of skeletal muscle and blood. Separations are carried out on 3 × 500 mm columns packed with modern anion exchange resins of closely sized, fine particles and a linear gradient of ammonium chloride containing borate in order to complex the sugar phosphates. Pressures are moderate, and elutions are complete within 5–8 hr with excellent reproducibility and recovery of each compound. Screening runs can be made in only 90 min with shorter columns and with some sacrifice in resolution for certain compounds.     

Mathematical Modeling of Size Exclusion Chromatography

A mathematical model of the size exclusion chromatography (SEC) process in chromatographic columns has been developed. It considers the following three mass transfer processes in the SEC column: axial dispersion in the bulk‐fluid phase, interfacial film mass‐transfer between the stationary and mobile phases, and diffusion of solutes within the macro pores of the packing particles. Differential equations of the process model were solved by the finite difference method. Characteristics of the column and the packing particles (bed void volume fraction, particle porosity, accessible particle porosity) were obtained experimentally, as well as retention times of different molecules with known molecular weights. Experiments were performed with two different columns containing two different packing materials, Superdex 75 HR 10/30 and BioSep SEC S2000, respectively. The model has been validated by comparing theoretical and experimental retention times for the different columns.