RESUMEN
A continuously regenerated cationic impurity removal device (CR-CRD) has been fabricated and applied for ion chromatography (IC). The removal of cationic impurities is realized by electrodialytically replacing the cationic impurities with hydronium ions. The device is configured in a sandwich structure and the central eluent channel is respectively isolated from both electrodes by stacked cation exchange membranes and a bipolar membrane (BPM) plus stacked anion exchange membranes. The eluent channel is packed with cation exchange resins in hydronium form and their continuous regeneration can be achieved by electrodialysis. A desirable feature of the device is gas-free, and no degasser is required. It showed sufficient ability to remove cationic impurities, as indicated by > 99.9 % removal of 10 mL of 1 mM LiOH solution injected (â¼10 µmol) or continuous removal of 1 mM LiOH solution at the flow rate of 1 mL/min (1 µmol/min). A useful application was for sample pretreatment in nuclear power industry, by eliminating strong matrix interference of the sample containing LiOH (1 mM) and boric acid (2000 mg/L) with trace anion analysis.
Asunto(s)
Cationes , Cromatografía por Intercambio Iónico/métodos , Cromatografía por Intercambio Iónico/instrumentación , Cationes/química , Membranas Artificiales , Resinas de Intercambio de Catión/química , Diseño de EquipoRESUMEN
Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic anions and oxyhalides. Preparation of base coating included grafting highly polar N-vinylformamide to the ethylvinylbenzene-divinylbenzene (EVB-DVB) substrate surface in highly polar solvent (methanol) with subsequent hydrolysis of grafted amide polymer in basic media, which resulted in preparation of polymer chains with multiple primary amino groups. Those amino groups were used as attachment points for forming hyperbranched anion-exchange layers using 1,4-butanediol diglycidyl ether and primary mono- or diamine (methylamine or 1,3-diaminopropane, respectively). The effects of hyperbranching reaction cycles number on selectivity were evaluated which revealed that selectivity and capacity can be controlled independently for the covalently bonded stationary phases in contrast to electrostatically bonded phases. It was demonstrated that unlike for electrostatically bonded phases, the intentional increase of crosslink by using primary diamine instead of primary monoamine doesn't cause the shift of selectivity coefficients. It was also shown that crosslink distribution throughout the hyperbranched layer is an important factor determining selectivity of hyperbranched anion exchangers.
Asunto(s)
Cromatografía por Intercambio Iónico , Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , Aniones/química , Aminas/químicaRESUMEN
Polyallylamine (PAA) has been utilized as a salt tolerant anion exchange chromatography ligand in downstream processing of biopharmaceuticals. We have developed novel MMC resins based on PAA polymer ligand partially modified with hydrophobic butyl or phenyl group. The resulting hydrophobic modified PAA ligand reduced HCP level to 12% (21-23 ppm) under 6 mS/cm in a flow-through polishing step of mAb, while not modified PAA ligand showed only 79% (145 ppm). We also found that structure of hydrophobic groups in the ligand mainly influenced on mAb yield. That is 25% increase of phenyl group modification ratio reduces mAb yield from 95% to 90%. On the other hand, modification with butyl group kept mAb yield more than 95%. The optimized ligand structure displayed a wide operational conductivity range. Extended purification studies of mAb using the MMC resin in the flow-through polishing step were carried out under optimized pH and conductivity condition as determined in a DOE study. The study revealed that the MMC resin was effective for developing one-step flow-through polishing workflow for mAb purification. In addition, the MMC flow-through polishing step could be directly coupled with a specified CEX chromatography step to efficiently remove mAb aggregates from 2.3% to <1.0% to achieve a biopharmaceutical-grade quality and a high yield of mAb (>93%) with a high loading capacity around 1000 mg/mL-resin. This new MMC resin will be useful in future mAb manufacturing platforms comprising of a robust and cost-effective flow-through polishing step.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Resinas de Intercambio Iónico/química , Poliaminas/química , Animales , Anticuerpos Monoclonales/química , Células CHO , Cromatografía por Intercambio Iónico/instrumentación , Cricetinae , Cricetulus , Interacciones Hidrofóbicas e Hidrofílicas , LigandosRESUMEN
Two-dimensional separations provide a simple way to increase the resolution and peak capacity of complex protein separations. The feasibility of a recently developed instrumental approach for two-dimensional separations of proteins was evaluated. The approach is based on the general principle of two-dimensional gel electrophoresis. In the first dimension, semi-preparative strong anion exchange high-performance liquid chromatography is utilized and fractions are collected by means of a fraction collector. They are subsequently analyzed in the second dimension with microchip capillary electrophoresis sodium dodecyl sulfate. Microchip capillary electrophoresis provides the necessary speed (approximately 1 min/fraction) for short analysis. In this study, three different samples were investigated. Different constructs of soluble guanylyl cyclase were expressed in Sf9-cells using the baculovirus expression system. Cell lysates were analyzed and the resulting separations were compared. In our experimental setup, the soluble guanylyl cyclase was identified among hundreds of other proteins in these cell lysates, indicating its potential for screening, process control, or analysis. The results were validated by immunoblotting. Samples from Chinese hamster ovary cell culture before and after a purification step were investigated and approximately 9% less impurities could be observed. The separation patterns obtained for human plasma are closely similar to patterns obtained with two-dimensional gel electrophoresis and a total of 218 peaks could be observed. Overall, the approach was well applicable to all samples and, based on these results, further directions for improvements were identified. .
Asunto(s)
Cromatografía por Intercambio Iónico/instrumentación , Electroforesis Capilar/instrumentación , Proteínas/aislamiento & purificación , Animales , Aniones/química , Células CHO , Cromatografía Líquida de Alta Presión/instrumentación , Cricetulus , Humanos , Dodecil Sulfato de Sodio/químicaRESUMEN
We describe a quenching-free, 'online' ion exchange chromatography (oIEC) method for the quantitative analysis of enzymatic reactions in real-time. We show that separate quenching of the ongoing reaction performed conventionally is not required, since enzymatic reactions are interrupted upon immobilization of the reaction compounds by binding to the stationary phase of the ion exchange column. The reaction mix samples are directly injected into the column, thereby improving data consistency and allowing automation of the process. The method allows reliable and efficient acquisition of enzymatic progress curves by automatic loading of aliquots of an ongoing reaction at predefined timepoints. We demonstrate the applicability of this method for a variety of enzymatic reactions. SUBJECT: Enzymatic assays and analysis.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Pruebas de Enzimas/métodos , Cromatografía por Intercambio Iónico/instrumentación , Pruebas de Enzimas/instrumentación , Diseño de Equipo , Proteínas Fúngicas/metabolismo , Hexoquinasa/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/metabolismoRESUMEN
The development of ion chromatography has made remarkable progress in the past few decades, and it is now widely used for the analysis of common ions and organic compounds. Ion chromatography has many advantages, such as fast, high sensitivity, good selectivity and support for simultaneous analysis of multiple ionic compounds. In order to meet the high requirements of material analysis, new packing materials for ion chromatography with higher sensitivity and selectivity have been developed. In this paper, a lot of knowledge of ion chromatography is reviewed, and the development of ion chromatographic packings in recent years, especially in the last five years, is summarized.
Asunto(s)
Cromatografía por Intercambio Iónico , Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/tendencias , Ciencia de los MaterialesRESUMEN
A high-performance polyacid ion exchange (IEX) nanofiber membrane was used in membrane chromatography for the recovery of lysozyme from chicken egg white (CEW). The polyacid IEX nanofiber membrane (P-BrA) was prepared by the functionalization of polyacrylonitrile (PAN) nanofiber membrane with ethylene diamine (EDA) and bromoacetic acid (BrA). The adsorption performance of P-BrA was evaluated under various operating conditions using Pall filter holder. The results showed that optimal conditions of IEX membrane chromatography for lysozyme adsorption were 10% (w/v) of CEW, pH 9 and 0.1 mL/min. The purification factor and yield of lysozyme were 402 and 91%, respectively. The adsorption process was further scaled up to a larger loading volume, and the purification performance was found to be consistent. Furthermore, the regeneration of IEX nanofiber membrane was achieved under mild conditions. The adsorption process was repeated for five times and the adsorption capacity of adsorber was found to be unaffected.
Asunto(s)
Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , Clara de Huevo/química , Membranas Artificiales , Muramidasa/aislamiento & purificación , Acetatos/química , Resinas Acrílicas/química , Adsorción , Animales , Pollos , Etilenodiaminas/química , Concentración de Iones de Hidrógeno , Muramidasa/química , Nanofibras/químicaRESUMEN
Chromatographic separation serves as "a workhorse" for downstream process development and plays a key role in the removal of product-related, host-cell-related, and process-related impurities. Complex and poorly characterized raw materials and feed material, low feed concentration, product instability, and poor mechanistic understanding of the processes are some of the critical challenges that are faced during the development of a chromatographic step. Traditional process development is performed as a trial-and-error-based evaluation and often leads to a suboptimal process. A high-throughput process development (HTPD) platform involves the integration of miniaturization, automation, and parallelization and provides a systematic approach for time- and resource-efficient chromatographic process development. Creation of such platforms requires the integration of mechanistic knowledge of the process with various statistical tools for data analysis. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing the HTPD of chromatography steps. It describes the operation of a commercially available device (PreDictor™ plates from GE Healthcare). This device is available in 96-well format with 2 or 6 µL well size. We also discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.93). We think that this protocol will be of significant value to those involved in performing the high-throughput process development of the chromatography process.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Animales , Cromatografía por Intercambio Iónico/instrumentación , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Receptores de Factor Estimulante de Colonias de Granulocito/análisis , Receptores de Factor Estimulante de Colonias de Granulocito/aislamiento & purificaciónRESUMEN
Membrane chromatography is gradually emerging as an alternative to conventional column chromatography. It alleviates some of the major disadvantages associated with the latter, including high-pressure drop across the column bed and dependence on intraparticle diffusion for the transport of solute molecules to their binding sites within the pores of separation media. In the last decade, it has emerged as a method of choice for final polishing of biopharmaceuticals, in particular, monoclonal antibody products. The relevance of such a platform is high in view of the constraints with respect to time and resources that the biopharma industry faces today.This protocol describes the steps involved in performing HTPD of a membrane chromatography step. It describes the operation of a commercially available device (AcroPrep™ Advance filter plate with Mustang S membrane from Pall Corporation). This device is available in 96-well format with a 7 µL membrane in each well. We will discuss the challenges that one faces when performing such experiments as well as possible solutions to alleviate them. Besides describing the operation of the device, the protocol also presents an approach for statistical analysis of the data that are gathered from such a platform. A case study involving the use of the protocol for examining ion-exchange chromatography of the Granulocyte Colony Stimulating Factor (GCSF), a therapeutic product, is briefly discussed. This is intended to demonstrate the usefulness of this protocol in generating data that are representative of the data obtained at the traditional lab scale. The agreement in the data is indeed very significant (regression coefficient 0.9866). We think that this protocol will be of significant value to those involved in performing high-throughput process development of membrane chromatography.
Asunto(s)
Cromatografía/instrumentación , Membranas Artificiales , Animales , Cromatografía/métodos , Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , Factor Estimulante de Colonias de Granulocitos/análisis , Factor Estimulante de Colonias de Granulocitos/aislamiento & purificación , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , HumanosRESUMEN
High-throughput process development is more and more used in chromatography. Limitations are the tools provided by the manufacturers. Here, we describe a method to select ion exchange chromatographic media using a 96-well filter microplate.
Asunto(s)
Cromatografía por Intercambio Iónico/instrumentación , Animales , Tampones (Química) , Cromatografía por Intercambio Iónico/métodos , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Proteínas/análisis , Proteínas/aislamiento & purificaciónRESUMEN
A stirred fluidized bed (SFB) ion exchange chromatography was successfully applied in the direct recovery of recombinant enhanced green fluorescent protein (EGFP) from the unclarified Escherichia coli homogenate. Optimal conditions for both adsorption and elution processes were determined from the packed-bed adsorption systems conducted at a small scale using the clarified cell homogenate. The maximal adsorption capacity and dissociation constant for EGFP-adsorbent complex were found to be 6.3 mg/mL and 1.3 × 10-3 mg/mL, respectively. In an optimal elution of EGFP with 0.2 M of NaCl solution (pH 9) and at 200 cm/h, the recovery percent of the EGFP was approximately 93%. The performances of SFB chromatography for direct recovery of EGFP was also evaluated under different loading volumes (50-200 mL) of crude cell homogenate. The single-step purification of EGFP by SFB recorded in a high yield (95-98%) and a satisfactory purification factor (~3 folds) of EGFP from the cell homogenate at 200 rpm of rotating speed.
Asunto(s)
Cromatografía por Intercambio Iónico/métodos , Escherichia coli/química , Proteínas Fluorescentes Verdes/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificación , Adsorción , Cromatografía por Intercambio Iónico/instrumentación , Diseño de Equipo , EtanolaminasRESUMEN
Over the past several years, hyphenation of native (nondenaturing) liquid chromatography (nLC) methods, such as size exclusion chromatography (SEC), ion exchange chromatography (IEX), and hydrophobic interaction chromatography (HIC) with mass spectrometry (MS) have become increasingly popular to study the size, charge, and structural heterogeneity of protein drug products. Despite the availability of a wide variety of nLC-MS methods, an integrated platform that can accommodate different applications is still lacking. In this study, we described the development of a versatile, sensitive, and robust nLC-MS platform that can support various nLC-MS applications. In particular, the developed platform can tolerate a wide range of LC flow rates and high salt concentrations, which are critical for accommodating different nLC methods. In addition, a dopant-modified desolvation gas can be readily applied on this platform to achieve online charge-reduction native MS, which improves the characterization of both heterogeneous and labile biomolecules. Finally, we demonstrated that this nLC-MS platform is highly sensitive and robust and can be routinely applied in protein drug characterization.
Asunto(s)
Anticuerpos Monoclonales/análisis , Cromatografía Liquida/instrumentación , Espectrometría de Masas/instrumentación , Cromatografía en Gel/instrumentación , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía por Intercambio Iónico/instrumentación , Diseño de EquipoRESUMEN
BACKGROUND: The ADAMS™ HA-8180V is the 8th generation of a fully automated ion-exchange HPLC system from ARKRAY, and the first to be released onto the US market. We evaluated the HA-8180V, for routine hemoglobin A1c measurement in comparison with the Roche Cobas c501, the Tosoh G8 analyzer for normal hemoglobin, and with the Trinity analyzer for hemoglobin variants. METHODS: The analytical performance (linearity, precision, carryover, and sample stability) was assessed based on the Clinical and Laboratory Standards Institute (CLSI) and manufacturer guidelines. A comparison of the HA-8180V against two major analytical methods was performed for 100 whole blood samples. HA-8180V variant mode was also compared against the Trinity ultra2 A1c analyzer for 50 samples containing hemoglobin variants (HbC 14, HbS 14, HbD 12, and HbE 10). RESULTS: The within-run and total CVs were <0.01 and 0.75% at low HbA1c concentration and 0.46 and 0.63% at high HbA1c concentration, respectively. Linearity was shown in the concentration range 3.4-18.1% HbA1c, carryover was 0.00%, and stability values were excellent. Method comparison demonstrated a high concordance between methods. CONCLUSION: The eighth generation ADAMS HA-8180V A1c analyzer demonstrated high analytical performance adequate for routine clinical use.
Asunto(s)
Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía por Intercambio Iónico/instrumentación , Hemoglobina Glucada/análisis , Humanos , Reproducibilidad de los ResultadosRESUMEN
Multidimensional protein identification (MudPIT), developed in the Yates Laboratory 20 years ago, is regarded as a powerful tool for proteomics research. Due to its remarkable online separation advantages, MudPIT has been widely used to facilitate discoveries in the field of proteomics research. However, it has one major disadvantage: the process of eluting peptides during strong cation exchange introduces salts, of different concentrations, into the mass spectrometer. Considering the sensitivity of the new generation of high-resolution mass spectrometers, developing a new version of MudPIT that could eliminate the introduction of salts in the elute would be a significant advancement to current technology. Herein, we developed a new, clean version of MudPIT called parallel channels-multidimensional protein identification technology (PC-MudPIT) to overcome this issue. In this design, the original biphasic trapping column was replaced by two parallel analytical column channels. We successfully averted the salt contamination yet retained all the other advantages of MudPIT. A total of 8161 and 7359 protein groups were identified from A549 whole cell lysate using PC-MudPIT and classic MudPIT, respectively. Moreover, we discovered the additional advantage that, in online mode, PC-MudPIT can also be used for an enrichment process of phosphopeptide identification. We identified a total 11453 phosphopeptides using PC-MudPIT and 7729 phosphopeptides using offline TiO2 enrichment followed by classic MudPIT. These advances indicate the possibility of other innovative applications of PC-MudPIT technology in deep proteome exploration.
Asunto(s)
Cromatografía por Intercambio Iónico , Proteínas/análisis , Proteómica/métodos , Células A549 , Cromatografía Líquida de Alta Presión/instrumentación , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/instrumentación , Cromatografía por Intercambio Iónico/métodos , Diseño de Equipo , Humanos , Fosfopéptidos/análisis , Fosfopéptidos/química , Fosfopéptidos/clasificación , Proteínas/química , Proteínas/clasificación , Sales (Química) , Espectrometría de Masas en Tándem/métodosRESUMEN
A two-membrane electrodialytic carbonate eluent generator (EDG) for ion chromatography (IC) is described. It is in a sandwich-like configuration, in which the central eluent channel is spatially isolated from two outer regenerant channels by stacked cation exchange membranes (CEMs) and anion exchange membranes (AEMs). A platinum screen electrode is placed in each of two outer regenerant channels. The electrode at the CEMs side is set as an anode with respect to the electrode at the AEMs side being cathode. Potassium carbonate and/or bicarbonate solution is pumped into the regenerant channel as feed solution. The electromigration of carbonate and/or bicarbonate and potassium from feed solution respectively through AEMs and CEMs will form a gas-free eluent. With this configuration, ion transport behavior through AEMs was explored. The device demonstrated good reproducibility, as indicated by the relative standard deviation of retention time of less than 0.08%.
Asunto(s)
Carbonatos , Técnicas de Química Analítica , Cromatografía por Intercambio Iónico , Bicarbonatos/química , Carbonatos/química , Cationes/química , Técnicas de Química Analítica/instrumentación , Técnicas de Química Analítica/métodos , Cromatografía por Intercambio Iónico/instrumentación , Electrodos , Potasio/química , Reproducibilidad de los ResultadosRESUMEN
The analysis of complex mixtures of closely related species is quickly becoming a bottleneck in the development of new drug substances, reflecting the ever-increasing complexity of both fundamental biology and the therapeutics used to treat disease. Two-dimensional liquid chromatography (2D-LC) is emerging as a powerful tool to achieve substantial improvements in peak capacity and selectivity. However, 2D-LC suffers from several limitations, including the lack of automated multicolumn setups capable of combining multiple columns in both dimensions. Herein, we report an investigation into the development and implementation of a customized online comprehensive multicolumn 2D-LC-DAD-MS setup for screening and method development purposes, as well as analysis of multicomponent biopharmaceutical mixtures. In this study, excellent chromatographic performance in terms of selectivity, peak shape, and reproducibility were achieved by combining reversed-phase (RP), strong cation exchange (SCX), strong anion exchange (SAX), and size exclusion chromatography (SEC) using sub-2-µm columns in the first dimension in conjunction with several 3.0 mm × 50 mm RP columns packed with sub-3-µm fully porous particles in the second dimension. Multiple combinations of separation modes coupled to UV and MS detection are applied to the LC × LC analysis of a protein standard mixture, intended to be representative of protein drug substances. The results reported in this study demonstrate that our automated online multicolumn 2D-LC-DAD-MS workflow can be a powerful tool for comprehensive chromatographic column screening that enables the semi-automated development of 2D-LC methods, offering the ability to streamline full visualization of sample composition for an unknown complex mixture while maximizing chromatographic orthogonality. Graphical Abstract.
Asunto(s)
Cromatografía Liquida/instrumentación , Evaluación Preclínica de Medicamentos/instrumentación , Espectrometría de Masas/instrumentación , Cromatografía en Gel/instrumentación , Cromatografía por Intercambio Iónico/instrumentación , Cromatografía de Fase Inversa/instrumentación , Descubrimiento de Drogas/instrumentación , Diseño de Equipo , Preparaciones Farmacéuticas/análisis , Proteínas/análisis , Flujo de TrabajoRESUMEN
This rapid and efficient method to prepare highly purified recombinant adeno-associated viruses (rAAVs) is based on binding of negatively charged rAAV capsids to an anion-exchange resin that is pH dependent.
Asunto(s)
Centrifugación/métodos , Cromatografía por Intercambio Iónico/métodos , Dependovirus/aislamiento & purificación , Vectores Genéticos/aislamiento & purificación , Ácidos Triyodobenzoicos/química , Virión/genética , Aniones , Cromatografía por Intercambio Iónico/instrumentación , Dependovirus/genética , Vectores Genéticos/genética , Concentración de Iones de Hidrógeno , Recombinación Genética , Reproducibilidad de los Resultados , Temperatura , Empaquetamiento del Genoma ViralRESUMEN
Biomedical analytical methods often rely on indirect measurements, such as immunoassays, which can lack effective metrological traceability. In the nephelometric determination of ceruloplasmin (Cp), an important protein whose circulating level is altered in Wilson's disease (WD), the anti-Cp antibody used is not specific for the biologically active holoprotein so the assay can overestimate the concentration of Cp due to the presence of the apoprotein. By providing quantitation using elemental standards, the use of strong anion exchange chromatography (SAX) coupled to triple quadrupole inductively coupled plasma mass spectrometry (ICP-MS-MS) can overcome the drawbacks of methods for the measurement of metalloproteins reliant on immunoassays. In the current study, a SAX-ICP-MS-MS method for Cp quantification was designed and tested in samples of blood serum of WD patients and healthy controls. Using standards based on a copper-EDTA complex for calibration, the method provides relatively simple quantification of Cp with the limit of detection of 0.1 µg L-1 (limit of quantification 0.4 µg L-1). The method was also used to investigate the copper species separated by using a 30 kDa cut-off ultrafiltration device. The so-called "exchangeable" copper fraction is considered as an alternative clinical biomarker of WD. Using the designed speciation approach, it was shown that the ultrafiltration method can overestimate the "exchangeable" copper fraction due to a removal of copper from Cp. This was confirmed by comparing the enzymatic activity of the fractions. Thus, the specificity of the "exchangeable" copper test can be ensured only under strict maintenance of ultrafiltration conditions.
Asunto(s)
Investigación Biomédica , Cobre/sangre , Degeneración Hepatolenticular/sangre , Cromatografía por Intercambio Iónico/instrumentación , Degeneración Hepatolenticular/diagnóstico , Humanos , Espectrometría de Masas/instrumentaciónRESUMEN
Novel biopharmaceutical products, such as vaccines and viral vectors, play a significant role in the development of innovative therapeutic, prophylactic, and clinical applications. However, several challenges are posed when manufacturing these products. The diversity of cell lines and the different physical and chemical properties of these biologicals require the use of different production and processing technologies. Alternative purification strategies that can improve the purification yield, such as continuous chromatography, are regarded nowadays as enabling technologies to overcome some of the bottlenecks in biomanufacturing. This chapter offers a shortcut approach to implement a semi-continuous chromatography purification of hepatitis C virus-like particles produced in insect cells with recombinant baculovirus. Although the purification is based on ion exchange chromatography, the present methodology can be extended to other types of chromatography.
Asunto(s)
Productos Biológicos/aislamiento & purificación , Cromatografía por Intercambio Iónico/métodos , Hepacivirus/aislamiento & purificación , Vacunas de Partículas Similares a Virus/aislamiento & purificación , Animales , Baculoviridae/genética , Baculoviridae/metabolismo , Cromatografía por Intercambio Iónico/instrumentación , Vectores Genéticos , Células Sf9RESUMEN
It has been known that anion exchangers prepared by grafting poly(ethyleneimine) (PEI) onto Sepharose FF (PEI-Sepharose) at ionic capacities (IC) over 600â¯mmol/L show both high protein adsorption capacity and uptake kinetics, and charge reversal of PEI-Sepharose by modification with succinic anhydride can produce protein cation exchangers of high capacity and uptake rate. Previously, a Charge Reversal-then-Reduction procedure (route A) was studied for preparation of cation exchangers of different IC values from PEI-Sepharose. In this work, we proposed a new route, that is, Charge Reduction-then-Reversal route (route B), to develop cation exchangers of different IC values from PEI-Sepharose FF with an IC of 700â¯mmol/L (FF-PEI-L700) as the starting resin. The two kinds of cation exchangers (route A, PEI-L700-CRn; route B, PEI-Rm-Cn) are compared for lysozyme (Lys) adsorption and chromatography. The two modification routes result in the difference in the ligand structures that significantly affect protein adsorption equilibrium and kinetics. Route A introduces long electroneutral groups that hinder protein adsorption and reduce equilibrium capacity. Moreover, charge reversal by reaction with succinic anhydride could cause diamide formation, which reduces remaining carboxyl groups or the IC. In the charge-reduced FF-PEI-Rm resins of the lowest IC (394â¯mmol/L) prepared in route B, the diamide formation was little due to the lack of primary and secondary amine groups, so its charge reversal makes a higher-IC cation exchanger. This makes PEI-Rm-Cn show a higher IC (589â¯mmol/L) than PEI-L700-CRn (463â¯mmol/L) in which De/D0 jumps about four times. The differences in the adsorption equilibrium and kinetics make the two kinds of resins behave distinctly in dynamic adsorption and chromatography. Namely, PEI-Rm-Cn resins display obviously higher dynamic binding capacities than PEI-L700-CRn resins in the IC range studied. For instance, the DBC (at 10% breakthrough) of PEI-R590-C680 (192â¯mg/mL) is 33% higher than that of PEI-L700-CR680 (144â¯mg/mL). This is proved by the purification of Lys from chicken egg white solution, in which the PEI-R590-C680 column purified Lys with a recovery yield 14% higher than the PEI-L700-CR680 column. This research thus demonstrated that Charge Reduction-then-Reversal route is superior over Charge Reversal-then-Reduction route in fabricating a high-capacity cation exchanger from PEI-Sepharose.