Simple method for determining the amount of ion-exchange groups on chromatographic supports

Lendero, Nika; Vidič, Jana; Brne, Peter; Podgornik, Aleš; Štrancar, Aleš

doi:10.1016/j.chroma.2004.10.072

Cited by 48 publications

(29 citation statements)

References 5 publications

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“…It is based on a different response of the particular group on the step change between the buffer ionic strengths. From the conceptual point of view, this novel method is similar to the method for determining the ligand density, described elsewhere in detail [15] and briefly later in this article. The groups can be distinguished due to differences in pH profiles measured after a step change.…”

Section: Resultsmentioning

confidence: 97%

“…Recently, a simple and fast method for determining ionic capacity has been introduced [15]. This method is based on the formation of the pH transient during the step change between high and low ionic strength buffer, which can be monitored with a change in UV absorbance (details are described elsewhere) [15]. Similar to the method for identifying the active group type, biological buffers of the same pH value are used, as illustrated in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…In this case also part of the active groups is no longer accessible which results in a decrease in ionic capacity. Recently, a simple and fast method for determining ionic capacity has been introduced [15]. This method is based on the formation of the pH transient during the step change between high and low ionic strength buffer, which can be monitored with a change in UV absorbance (details are described elsewhere) [15].…”

Section: Resultsmentioning

confidence: 99%

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Noninvasive Methods for Characterization of Large‐Volume Monolithic Chromatographic Columns

et al. 2005

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Chromatographic monoliths exhibit several properties like flow-unaffected resolution and dynamic binding capacity, low pressure drop and high capacity for extremely large molecules, which are advantageous to the purification of large biomolecules such as proteins, DNA or even viruses. Recently, large-volume monolithic columns were introduced enabling the monoliths to be incorporated into industrial downstream processes. Due to the monolithic structure, however, conventional methods for resin characterization cannot be applied to monoliths, which limit their usage under cGMP conditions. In this article, a methodology of how to overcome this problem and to determine the most important parameters is presented. Several fast and noninvasive methods for monolith characterization are introduced. Their usage enables accurate determination of monolith pore size distribution and specific surface area, uniformity of the monolith as well as type and density of the active groups before or during monolith usage.

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Section: Resultsmentioning

confidence: 97%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Noninvasive Methods for Characterization of Large‐Volume Monolithic Chromatographic Columns

et al. 2005

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“…The time interval ∆t (pH) was determined between the switching point of the mobile phase and the point at which 50% of the maximum pH value was achieved. The phosphate capacity was calculated using Equation (3), where the volumetric flow rate (ϕv), concentration of elution buffer solution (C2), and column volume (Vc), as well as time interval (∆t) are used in the expression [50,51]. Q Sepharose FF beads were utilized as a control ion exchange sample.…”

Section: Determination Of the Ionic Capacitymentioning

confidence: 99%

Chromatographic Characterization and Process Performance of Column-Packed Anion Exchange Fibrous Adsorbents for High Throughput and High Capacity Bioseparations

et al. 2015

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Fibrous materials are prominent among novel chromatographic supports for the separation and purification of biomolecules. In this work, strong anion exchange, quaternary ammonium (Q) functional fibrous adsorbents were evaluated with regards to their physical and functional characteristics. A column packed with Q fibrous adsorbent illustrated the good column packing efficiency of theoretical plate height (H) values and higher permeability coefficients (>0.9 × 10 −7 cm 2 ) than commercial adsorbents. For pulse experiments with acetone and lactoferrin as tracers under nonbinding conditions, the total porosity (for acetone) and the interstitial porosity (for lactoferrin) measured 0.97 and 0.47, respectively. The total ionic capacity of the chemically-functionalized Q fiber was 0.51 mmol/mL. The results indicated that the Q fiber had a static binding capacity of 140 mg/mL and a dynamic binding capacity (DBC) of 76 mg/mL for bovine serum albumin (BSA) and showed a linearly-scalable factor (~110 mL) for a column volume with high capacity and high throughput. Furthermore, this adsorptive material had the ability to bind OPEN ACCESSProcesses 2015, 3 205 the high molecular weight protein, thyroglobulin, with a capacity of 6 mg/mL. This work demonstrated the column-packed Q fibrous adsorption system as a potential chromatography support that exhibits high capacity at higher flow rates.

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“…The maximum product binding capacity is important to enable economic processing (13)(14)(15). The experimental conditions affecting the observed capacity of an ion exchange resin are: pH, ionic strength of the buffer, nature of the counter-ion, flow rate, and temperature (2,7,8,(16)(17)(18)(19)(20). Flow rate is important for dynamic binding capacity.…”

Section: Introductionmentioning

confidence: 99%

Optimization of Dynamic Binding Capacity of Anion Exchange Chromatography Media for Recombinant Erythropoietin Purification

et al. 2014

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Background:The dynamic binding capacity (DBC) of a chromatography matrix in protein purification is the amount of the total protein absorbed into the matrix, before occurrence of a significant break in the breakthrough curve. Optimization of the process criteria for maximum DBC avoids extra process scale-up and reduces the processing time, costs and protein loss. Taguchi method is a simple useful tool in experimental design to estimate the optimal condition with minimum experiments. Objectives: In this research, linear flow rate, pH and protein concentration of the feed were checked according to an L9 orthogonal Taguchi array, to estimate the best conditions for maximum DBC of Q-sepharose fast flow (QSFF) resin in recombinant human erythropoietin purification process. Materials and Methods: A crud sample containing human recombinant erythropoietin was harvested from a cell culture of Chinese hamster ovary (CHO) cell line. Desalted harvests with different total protein concentrations (30, 40 and 50 µg.mL -1 ) and pH values (5, 6 and 7) were loaded into a packed column of QSFF with different linear flow rates (60, 120 and 280 cm.h -1 ) up to 10% of the breakthrough curve. The total protein loading to the column was checked by UV absorbance and Lowry method, and erythropoietin concentration was measured by ELISA. Analysis of variance (ANOVA) was applied to determine the optimum condition. Results: Finally, total protein concentration of 50 µg.mL -1 , pH of 5 and flow rate of 120 cm.h -1 , were anticipated as the optimal process conditions with 5.85 mg.mL -1 of resin as the dynamic binding capacity. Conclusions:Experiments with anticipated optimal criteria were performed three times and no significant difference was observed (p = 0.136, and 6.06 mg/mL as the average dynamic binding capacity).

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Simple method for determining the amount of ion-exchange groups on chromatographic supports

Cited by 48 publications

References 5 publications

Noninvasive Methods for Characterization of Large‐Volume Monolithic Chromatographic Columns

Noninvasive Methods for Characterization of Large‐Volume Monolithic Chromatographic Columns

Chromatographic Characterization and Process Performance of Column-Packed Anion Exchange Fibrous Adsorbents for High Throughput and High Capacity Bioseparations

Optimization of Dynamic Binding Capacity of Anion Exchange Chromatography Media for Recombinant Erythropoietin Purification

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