ISSN:0006-3592    

DOI:10.1002/bit.260480502

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:0006-3592    

DOI:10.1002/bit.260480503

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractHighly selective ligand‐exchange absorbents have been prepared by template polymerization, a process in which the target molecule serves as a template for assembly of specific recognition sites. In an effort to develop materials suitable for chromatographic separations, thin coatings of the selctive templated polymers have been grafted to two reactive macroporous supports, poly(trimethylolpropane trimethacrylate) (TRIM), and propylmethacrylate‐derivatized silica beads. The precursor polymer prepared from the trifunctioal TRIM monomer is macroporous and highly crosslinked, providing a stable structure for surface grafting. The TRIM precursor polymer and various surface‐grafted copolymers have been characterized by scanning electron microscopy (SEM) and IR,13C NMR, and XPS spectroscopic techniques. Composite adsorbents have also been prepared using propylmethacrylate‐modified silica particles. While equilibrium rebinding selectivites for both types of surface‐templated materials are similar to those reported previously for bulk‐polymerized template polymers, the composite materials are far better suited to chromatographic separatios. Highly similarbis‐imidazole substrates can be separated by ligand‐exchange chromatography on these new templated adsorbents. © 1995 John

ISSN:0006-3592    

DOI:10.1002/bit.260480504

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractStudies carried out using engineered proteins clearly demonstrate that adsorption to derivatized surfaces involves multiple interactions between functional groups on the protein and complementary sites distributed on the surface. The fact that adsorption involves multipoint interactions has important implications for the design of separations processes and for the interpretation of heterogeneity in biological recognition phenomena. Increasing the density of surface metal sites (immobilized copper ions) is found to be functionally equivalent to increasing the number of metal‐coordinating groups on the protein (histidines and deporotonated amines), m in that both processes increase the likelihood of simultaneous interactions between the protein and the surface. A consequence of multiple‐site interactions is a significant in crease in protein binding affinity that depends on the arrangement of surface sites. A protein will show the highest affinity for arrangements of surface sites which best match its own pattern of functioal groups and will show lower affinity for less optimal arrangements, resulting in binding that is inherently heterogeneous. We have found that reversible protein adsorption in immobilized metal affinity chromatography (IMAC) is described by the Temikin model, which characterizes binding heterogeneity by a uniform distribution of binding energies over the population of surface binding sites. © 1995 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260480505

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractA method for determining the plate height HETP from the elution curve obtained by the linear gradient elution (LGE) ion‐exchange chromatography (IEC) of proteins is presented. The method was developed on the basis of the numerical solutions of a chromatography model which considers the zone sharpening and the distribution coefficient as a function of the salt concentration. The plate height HETP is determined from the peak width and the salt concentration at which the peak is eluted in LGE. The method was applied to the experimental results with various ion‐exchange chromatography media. A calculation example based onthe present method is presented to show how the chromatographic and operating parameters should be tuned to obtain a desired resolution. A simplified calculation procedure for the peak profile is also described. © 1995 John Wiley&Sons,

ISSN:0006-3592    

DOI:10.1002/bit.260480506

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractAlthough the ability to carry out simultaneous concentration and purification in a single displacement step has significant advantages for downstream processing of pharmaceuticals, a major impediment to the implementation of displacement chromatography has been the lack of suitable displacer compounds. An important recent advance in the state of the art of displacement chromatography has been the discovery that low‐molecular‐weight dendritic polymers can be successfully employed as displacers for protein purification in ion‐exchange systems. In this article, protected amino acid esters (based on arginine and lysine) are shown to be useful displacers for protein purification in cation‐exchange systems. A dynamic affinity plot is employed to evaluate the affinity of these low‐molecular‐weight compounds under dis‐placement conditions. In contrast to large polyelectroyte displacers, the efficacy of these low‐molecular‐weight displacers was shown to be dependent on both the initial carrier salt concentration and the displacer concentration. In addition to the funcamental interest generated by low‐molecular‐weight displacers, it is likely that these displacers will have significant operatioal advantages as compared with large polyelectrolyte displacers. © 199

ISSN:0006-3592    

DOI:10.1002/bit.260480507

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe continuous separation of proteins was performed in a countercurrent gradient chromatography (CGC) system. A magnetically stabilized fluidized bed (MSFB) was used to establish true countercurrent contact of a solid resin with a liquid buffer. STable pH gradients were formed in the system in less than 10 min and remained stable throughout the course of the separation experiment (>2 h). The shape of the pH gradient, which ultimately controls the resolution and purity of the separation, can be controlled by making simple adjustments in the interstitial velocities of the liquid and solid phases. We have performed the separation of myoglobin and human serum albumin (HSA) using this device and achieved concentration factors of 1.75 for myoglobin and 1.2 for HSA. A mathematical model that has no adjustable parameters has been developed that predicts the focusing behaviour and capabilities of the CGC system. Using the model, we have estimated the optimum phase velocities, particle diameters, and equilibrium parameters necessary for achieving high purity and high concentrations. © 1995 John Wiley&Sons, Inc

ISSN:0006-3592    

DOI:10.1002/bit.260480508

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractA continuous rod of porous poly(glycidy1 methacrylate‐co‐ethylene dimethacrylate) has been prepared by a free radical polymerization within the confines of a 16‐mm‐i.d. glass column. The epoxide groups of the rod have been modified in situ by their reaction with diethylamine to afford the ionizable weak base 1‐N,N‐diethylamino‐2‐hydroxypropyl functionalities that are required for the ion‐exchange chromatographic mode. The bimodal pore size distribution curve typical for other molded separation media also prevail for the preparative‐size rod. The column has been used successfully for the chromatographic separation of a mixture of standard proteins and yeast enzymes. The column exhibits a dynamic capacity that exceeds 420 mg of bovine serum albumin at a flow velocity of 60 cm/h. © 1995

ISSN:0006-3592    

DOI:10.1002/bit.260480509

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractThe recombinant production of proteins leads to inclusion bodies which contain aggregated proteins in active, partially active, and inactive conformational states. These aggregated proteins must be extracted from the inclusion bodies, unfolded, and carefully refolded to the active and the stable conformational state. Mechanistic models for protein refolding are briefly presented. Different strategies and protocols are presented that lead to the active and stable protein conformational state. The techniques presented include chaperonin‐assisted refolding, amino acid substitution, polyethylene glycolassisted refolding, protein refolding in reverse micelles, and antibody‐assisted refolding of proteins. The techniques presented together provide a reasonable framework of the state‐of‐the‐art and may be carefully applied to the bioseparation of other proteins and biological macromolecules of interest. © 1995 John Wiley

ISSN:0006-3592    

DOI:10.1002/bit.260480510

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractOxidation is one of the major chemical degradation pathways for protein pharmaceuticals. Methionine, cysteine, histidine, tryptophan, and tyrosine are the amino acid residues most susceptible to oxidation due to their high reactivity with various reactive oxygen species. Oxidation during protein processing and storage can be induced by contaminating oxidants, catalyzed by the presence of transition metal ions and induced by light. Oxidative modification depends on the structural features of the proteins as well as the particular oxidation mechanisms inherent in various oxidative species, and may also be influenced by pH, temperature, and buffer composition. Protein oxidation may result in loss of biological activity and other undesirable pharmaceutical consequences. Strategies to stabilize proteins against oxidation can be classified into intrinsic methods (site‐directed mutagenesis and chemical modification), physical methods (solid vs. liquid formulations) and use of chemical additives. The optimum choice of chemical additives needs to be evaluated on the basis of the specific oxidation mechanism. Oxidation induced by the presence of oxidants in the system is referred to as a non‐site‐specific mechanism. Under such conditions, oxidation can be effectively inhibited by the appropriate addition of antioxidants or free radical scavengers. metal‐catalyzed oxidation is a site‐specific process, in which the addition of antioxidants may accelerate the oxidation reaction. Careful screening of chelating agents has been shown to be an alternative method for preventing metal‐catalyzed oxidation. © 1995 John Wil

ISSN:0006-3592    

DOI:10.1002/bit.260480511

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY