A novel anion-exchange resin suitable for both discovery research and clinical manufacturing purposes

Strong ion-exchange protein chromatography is one of the most powerful and most common steps for protein purification in both discovery research and manufacturing. However, the demands on protein purification of early drug discovery and later stage manufacturing are quite different. In order to shorten the time of developing a purification process for new protein drug candidates, there is a need for a strong ion-exchange resin that will be optimum for both stages. This article details a novel anion-exchange resin suitable for research, as well as for clinical manufacturing. In this study, a novel Q resin anion-exchange prototype was evaluated and compared to the GE Healthcare Q Sepharose? Fast Flow (QFF) and Q Sepharose? High Performance (QHP) resins. This study specifically focused on the following: resolution, dynamic binding capacity, flow rate, back pressure, and scale up. The evaluation was performed in both small- and large-scale experiments. From all the comparable data, the prototype resin is adaptable for both discovery research and manufacturing. Its wide-range operation suitability could potentially shorten the time required to develop conventional purification protocols for clinical manufacturing.     

Improved purification of recombinant adenoviral vector by metal affinity membrane chromatography

The increasing importance of adenoviral vectors for gene therapy clinical trials necessitates the development of processes suitable for large-scale and commercial production of adenovirus. Here, we evaluated a novel purification process combining an anion-exchange chromatography and an immobilized metal affinity membrane chromatography for the purification of recombinant adenovirus. Adenovirus was initially purified from clarified infectious lysate by anion-exchange chromatography using Q Sepharose XL resin and further polished using a Sartobind IDA membrane unit charged with Zn²⁺ ions as affinity ligands. The metal affinity membrane chromatography efficiently removed residual host cell impurities that co-eluted with adenovirus during the previous anion-exchange chromatography step. The metal affinity membrane chromatography also separated defective adenovirus particles from the infectious adenovirus fraction. Furthermore, the metal affinity membrane chromatography showed an improved yield, when compared with a conventional bead-based metal affinity chromatography. The purity and specific activity of the adenovirus prepared using this two-step chromatography was comparable to those of adenovirus produced by the conventional CsCl density centrifugation. Therefore, our data provide an improved method for the purification of adenoviral vectors for clinical applications.     

Purification of a full-length recombinant glucocorticoid receptor

We described a novel purification method for a recombinant glucocorticoid receptor (GR) in detail. The purification procedure consists of sequential chromatographies using common ion-exchange columns (Mono Q and Mono S). This procedure is based upon a new finding that the activated GR binds both to a Mono Q column and to a Mono S column at the same pH. The entire chromatographies took about 3 h and GR represented 97% of the purified protein sample. This purification protocol will be applicable to the purification of native GR, point-mutated recombinant GR and other nuclear receptors.     

Application of mixed modal resin for purification of a fibrinolytic enzyme

Recent advances in purification technologies for therapeutic molecules have stirred the research consortium. Mixed mode chromatography, having multiple interactions with the solute molecule, has drawn significant attention due to its overall advantage over traditional ion-exchange and reverse-phase chromatography. Capto adhere, a mixed mode chromatography resin with strong anion-exchange and reverse-phase interaction with solutes, was explored for purification of fibrinolytic enzyme from Bacillus sphaericus MTCC 3672. Static and dynamic resin binding study revealed that 30°C temperature, pH 8, and 0.5 mL/min flow rate were optimum for maximum binding of fibrinolytic enzyme. Maximum static dynamic binding and breakthrough capacities for Capto adhere were 249 and 196 U/mL of resin, respectively. Final purification with Sephadex G 100 gel chromatography resulted in 38-fold purity of fibrinolytic enzyme with 39% enzyme recovery. Purified enzyme was further characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis to homogeneity, and molecular mass was found to be around 55–70 kD. Like most of the serine alkaline proteases, purified fibrinolytic enzyme was stable in a temperature range of 25–40°C and pH range of 7–9. Offshoots of our research findings have revealed a broad application area of mixed mode chromatography.     

Investigation of Factors Affecting the Bioregeneration Process for Perchlorate-Laden Gel-Type Anion-Exchange Resin

Ion exchange is the most common process for perchlorate removal from waters. Selective ion-exchange resins are widely used for perchlorate removal from waters, but are incinerated after one-time use, making the ion-exchange process incomplete for perchlorate removal. As perchlorate ions are readily biodegradable, direct contact of spent ion-exchange resins with perchlorate-reducing bacteria for its regeneration has been studied recently. In this research, some factors affecting the bioregeneration of perchlorate-laden gel-type anion-exchange resin were investigated. Bioregeneration is a sustainable process when compared to one-time use of resin and disposal by incineration. Batch bioregeneration experiments were performed to determine (a) the effect of initial perchlorate load in the resin, (b) the effect of microbial concentration, and (c) the effect of nitrate load on the degradation of perchlorate in the resin bead. The results of the bioregeneration tests suggested that the bioregeneration process may be controlled by both kinetics and diffusion. Higher perchlorate load in the resin had a positive effect on perchlorate degradation rates, whereas varying microbial concentration did not have a significant effect on perchlorate degradation in gel-type resin. The presence of nitrate suppressed perchlorate degradation initially, but once all nitrate was utilized, perchlorate degradation took place.     

Use of anion-exchange high-performance liquid chromatography for the study of smooth muscle myosin light-chain kinase and its catalytic domain

Avian myosin light-chain kinase from smooth muscle of the gizzard and its catalytic domain, derived from the intact enzyme by trypsin digestion, was purified within 30–40 min by both analytical and preparative anion-exchange high-performance liquid chromatography. The proteins obtained were more than 95% pure and retained their biological activity. The high-performance anion-exchange chromatography protocols represent a significant decrease in purification time when compared with conventional ion-exchange chromatography.     

Biodiesel production using anionic ion-exchange resin as heterogeneous catalyst

The transesterification reactions of triolein with ethanol using various ion-exchange resin catalysts were conducted to produce ethyl oleate as a biodiesel. The anion-exchange resins exhibited much higher catalytic activities than the cation-exchange resin. The anion-exchange resin with a lower cross-linking density and a smaller particle size gave a high reaction rate as well as a high conversion. By combining the three-step regeneration method, the resin could be repeatedly used for the batch transesterification without any loss in the catalytic activity. A continuous transesterification reaction was carried out using an expanded bed reactor packed with the most active resin. The reactor system permitted the continuous production of ethyl oleate with a high conversion.     

Study of support materials for sol-gel immobilized lipase

Abstract

A variety of support materials for sol-gel immobilized lipase were considered based on their ability to provide superior sol-gel adhesion, load protein, and synthesize methyl oleate. A standard approach was developed to formulate the supported lipase sol-gels and to allow comparison of the resulting hybrid materials. These supported sol-gels are proposed as an alternative immobilization regime to overcome some challenges associated with enzymatic biodiesel production such as enzyme stability and cost. The support materials considered were 6–12 mesh silica gel, Celite^® R633, Celite^® R632, Celite^® R647, anion-exchange resin AG3-X4, and Quartzel^® felt. Each support material exhibited unique properties that would be beneficial for this application including: Quartzel^® felt had the highest initial sol-gel capacity (62.5 mL/g) and sol-gel adhesion (1100 mg sol-gel/g material); silica gel had the most uniform coating of deposited sol-gel; the anion-exchange resin AG3-X4 supported sol-gel had the highest protein loading (1060 μg lipase/g) and reaction rate [1.25 mM/(min g-material)]; the Celite^® support series were the most thermally stable and had the lowest water content; and the Celite^® R632 supported lipase sol-gels had the highest 6 h biodiesel conversion per gram of supported material (68%) and enzymatic activity [9.4 mmol/(min g-lipase)]. The supported sol-gels with the highest enzymatic properties (conversion, activity, and reaction rate) were those supported on Celite^® R632, anion-exchange resin AG3-X4, and Quartzel^®. These supported sol-gels had superior performance in comparison with the unsupported sol-gels. Based on this study, the lipase sol-gel support material with the most potential for biodiesel production is Celite^® R632.     

High-performance liquid chromatography of proteins: Purification of the acidic isozyme of adenylosuccinate synthetase from rat liver

High-performance ion-exchange liquid chromatography was utilized for the purification of the acidic isozyme of adenylosuccinate synthetase from rat liver. Initial steps in the purification included ammonium sulfate fractionation and DEAE-cellulose and agarose-GTP affinity columns. The final steps were done on a SynChropak AX-300 anion-exchange support. The enzyme was purified 3000-fold with an overall yield of 10%. The enzyme preparation exhibited only one protein band on gel electrophoresis.     

Isolation of U3 snoRNP from CHO cells: a novel 55 kDa protein binds to the central part of U3 snoRNA.

U3 snoRNP, the most abundant of the small nucleolar ribonucleoprotein particles (snoRNPs), has previously been demonstrated to participate in pre-rRNA maturation. Here we report the purification of U3 snoRNP from CHO cells using anti-m3G-immunoaffinity and mono Q anion-exchange chromatography. Isolated U3 snoRNPs contain three novel proteins, of 15, 50 and 55 kDa respectively. These proteins may represent core U3 snoRNP proteins whose binding mediates the association of other proteins, such as fibrillarin, that are lost during purification. Using a rabbit antiserum raised against the 55 kDa protein, and an in vitro reconstitution assay, we have localised the 55 kDa protein binding site on the U3 snoRNA. Stable binding of the 55 kDa protein requires sequences located between nucleotides 97 and 204 of the human U3 snoRNA, including the evolutionarily conserved B and C sequence motifs.     

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.     

High-throughput screening of chromatographic separations: I. Method development and column modeling

The development of purification processes for protein biopharmaceuticals is challenging due to compressed development timelines, long experimental times, and the need to survey a large parameter space. Typical methods for development of a chromatography step evaluate several dozen chromatographic column runs to optimize the conditions. An efficient batch-binding method of screening chromatographic purification conditions in a 96-well format with a robotic liquid-handling system is described and evaluated. The system dispenses slurries of chromatographic resins into filter plates, which are then equilibrated, loaded with protein, washed and eluted. This paper evaluates factors influencing the performance of this high-throughput screening technique, including the reproducibility of the aliquotted resin volume, the contact time of the solution and resin during mixing, and the volume of liquid carried over in the resin bed after centrifugal evacuation. These factors led to the optimization of a batch-binding technique utilizing either 50 or 100 microL of resin in each well, the selection of an industrially relevant incubation time of 20 min, and the quantitation of the hold-up volume, which was as much as one quarter of the total volume added to each well. The results from the batch-binding method compared favorably to chromatographic column separation steps for a cGMP protein purification process utilizing both hydrophobic interaction and anion-exchange steps. These high-throughput screening tools can be combined with additional studies on the kinetics and thermodynamics of protein-resin interactions to provide fundamental information which is useful for defining and optimizing chromatographic separations steps.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein–protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Isolation and purification of plastocyanin from spinach stored frozen using hydrophobic interaction and ion-exchange chromatography

A new simple three-day procedure for preparative isolation and purification of plastocyanin from spinach stored in the frozen state is described. This procedure is based on batch adsorption on ion-exchange resin, ammonium sulphate precipitation, and purification on a Phenyl-Sepharose hydrophobic interaction column and a single Q Sepharose High Performance ion-exchange column. Approximately 100 mg of plastocyanin with an absorbance ratio A₂₇₈/A₅₉₇ of 1.10±0.02 in the oxidized state was typically obtained from 12 kg of spinach leaves. The purified spinach plastocyanin is shown to be homogeneous to the resolution of free solution capillary electrophoresis.Abbreviations MES 2(N-morpholino)ethanesulfonic acid - Tris Tris(hydroxymethyl)aminomethane - FSCE free solution capillary electrophoresis     

Determination of gliclazide in human serum by high-performance liquid chromatography using an anion-exchange resin

A method for the routine clinical examination of serum gliclazide by high-performance liquid chromatography (HPLC) on a column packed with a macroporous anion-exchange resin, Diaion CDR-10, was developed. The elution was performed with acetonitrile—methyl alcohol—1.2 M ammonium perchlorate (4:3:7, v/v/v) at a flow-rate of 0.4 ml/min. The retention time of gliclazide was 15 min. It seems that the retention mechanism of gliclazide under the HPLC conditions described is not only ion-exchange mode but reversed-phase mode between the anion-exchange resin and the mobile phase. The detection limit of gliclazide was 0.2 μg/ml in plasma. The coefficient of variation for the within-day assay was 5.0% (0.2 μg/ml, n=8). The decay curve of serum gliclazide in diabetic patients was determined.     

The suitability of DEAE-Cl active groups on customized poly(GMA-co-EDMA) continuous stationary phase for fast enzyme-free isolation of plasmid DNA

The creation of a commercially viable and a large-scale purification process for plasmid DNA (pDNA) production requires a whole-systems continuous or semi-continuous purification strategy employing optimised stationary adsorption phase(s) without the use of expensive and toxic chemicals, avian/bovine-derived enzymes and several built-in unit processes, thus affecting overall plasmid recovery, processing time and economics. Continuous stationary phases are known to offer fast separation due to their large pore diameter making large molecule pDNA easily accessible with limited mass transfer resistance even at high flow rates. A monolithic stationary sorbent was synthesised via free radical liquid porogenic polymerisation of ethylene glycol dimethacrylate (EDMA) and glycidyl methacrylate (GMA) with surface and pore characteristics tailored specifically for plasmid binding, retention and elution. The polymer was functionalised with an amine active group for anion-exchange purification of pDNA from cleared lysate obtained from E. coli DH5alpha-pUC19 pellets in RNase/protease-free process. Characterization of the resin showed a unique porous material with 70% of the pores sizes above 300 nm. The final product isolated from anion-exchange purification in only 5 min was pure and homogenous supercoiled pDNA with no gDNA, RNA and protein contamination as confirmed with DNA electrophoresis, restriction analysis and SDS page. The resin showed a maximum binding capacity of 15.2 mg/mL and this capacity persisted after several applications of the resin. This technique is cGMP compatible and commercially viable for rapid isolation of pDNA.     

NMR techniques for characterization of ligand binding: utility for lead generation and optimization in drug discovery.

Over the last ten years, nmr spectroscopy has evolved into an important discipline in drug discovery. Initially, nmr was most useful as a technique to provide structural information regarding protein drug targets and target-ligand interactions. More recently, it has been shown that nmr may be used as an alternative method for identification of small molecule ligands that bind to protein drug targets. High throughput implementation of these experiments to screen small molecule libraries may lead to identification of potent and novel lead compounds. In this review, we will use examples from our own research to illustrate how nmr experiments to characterize ligand binding may be used to both screen for novel compounds during the process of lead generation, as well as provide structural information useful for lead optimization during the latter stages of a discovery program.     

Characterization of the constitutive and wound-inducible monoterpene cyclases of grand fir (Abies grandis)

Monoterpene cyclase activity is greatly increased in grand fir (Abies grandis) sapling stems in response to wounding and the composition of the cyclic olefin mixture generated differs from that produced constitutively as determined by radio gas-liquid chromatography. Cell-free extracts from wounded stems and from non-wounded controls were systematically compared for monoterpene cyclase activities following partial purification and separation of these enzymes by anion-exchange chromatography (Mono Q FPLC) and native PAGE. The increase in monoterpene cyclase activity following wounding represents both the apparent enhancement of constitutive cyclase activities and the appearance of novel cyclization enzymes that are absent in nonwounded controls. A pinene cyclase was shown to be the major wound-inducible enzyme directly responsible for oleoresin monoterpene formation and was tentatively identified as a 62-kDa protein by SDS-PAGE.     

Discovery of regulatory molecular events and biomarkers using 2D capillary chromatography and mass spectrometry

An important component of proteomic research is the high-throughput discovery of novel proteins and protein-protein interactions that control molecular events that contribute to critical cellular functions and human disease. The interactions of proteins are essential for cellular functions. Identifying perturbation of normal cellular protein interactions is vital for understanding the disease process and intervening to control the disease. A second area of proteomics research is the discovery of proteins that will serve as biomarkers for the early detection, diagnosis and drug treatment response for specific diseases. These studies have been referred to as clinical proteomics. To discover biomarkers, proteomics research employs the quantitative comparison of peptide and protein expression in body fluids and tissues from diseased individuals (case) versus normal individuals (control). Methods that couple 2D capillary liquid chromatography (LC) and tandem mass spectrometry (MS/MS) analysis have greatly facilitated this discovery science. Coupling 2D-LC/MS/MS analysis with automated genome-assisted spectra interpretation allows a direct, high-throughput and high-sensitivity identification of thousands of individual proteins from complex biological samples. The systematic comparison of experimental conditions and controls allows protein function or disease states to be modeled. This review discusses the different purification and quantification strategies that have been developed and used in combination with 2D-LC/MS/MS and computational analysis to examine regulatory protein networks and clinical samples.     

Rapid purification of native C protein from nuclear ribonucleoprotein particles

A rapid three step procedure is described for the purification of C protein from HeLa 40 S hnRNP particles. The procedure takes advantage of the salt resistant RNA binding of C protein, the size of the C protein-RNA complex, and the strong binding of C protein to an anion-exchange resin. Typically 120 micrograms of C protein is obtained from 4.0 X 10(9) cells with greater than 95% electrophoretic purity. Proteins C1 and C2 copurify in the ratio of 3.5 Cl to 1 C2. The purified C protein participates in hnRNP particle reconstitution and on this basis is judged to be native. The purified C protein binds to a gel filtration matrix at 0.5 M NaCl but at higher salt concentrations it elutes before the marker protein, apoferritin (Mr = 443,000). An abbreviated two step purification procedure utilizing anion-exchange chromatography is also described. This procedure results in relatively pure C protein, as well as a useful separation of the other hnRNP proteins.