Noninvasive method for determination of immobilized protein A

Mravljak, Rok; Stantič, Metka; Bizjak, Ožbej; Podgornik, Aleš

doi:10.1016/j.chroma.2022.462976

Cited by 5 publications

(14 citation statements)

References 46 publications

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“…Due to the importance of protein immobilization, a variety of different methods have been developed to determine the amount of immobilized protein. ,− ,− Nevertheless, there is a lack of a direct method that can be implemented in a noninvasive manner, allowing inspection of a particular sample without affecting its properties. As recently demonstrated, the pH transition method, which allows determination of the amount of ionizable groups on the matrix, ,,,, can also be used for the detection of immobilized protein A. Therefore, we investigated whether a modified method could be applied to a specific immobilized protein and whether its amount could be determined accurately.…”

Section: Resultsmentioning

confidence: 99%

“…The method is based on a pH transition phenomenon that occurs when two mobile phases of the same pH but different ionic strengths are exchanged with a matrix bearing ion-exchange functionalities. It has been demonstrated that the duration and/or amplitude of the pH excursion that occurs during such a shift depends linearly on the amount of ion-exchange groups on the matrix. − Recently, this approach was modified to allow the detection of immobilized protein A, a ligand commonly used for monoclonal antibody purification . Here, we demonstrate that this technique can be applied to various immobilized proteins and allows determination of their amount in flow-through mode without any pretreatment.…”

Section: Introductionmentioning

confidence: 82%

“…Therefore, one would expect pH transition profiles similar to those of anion exchange groups, for which a pH increase with the increase in the buffer ionic strength and vice versa has been reported. 40 In fact, such a trend was correctly predicted by a mathematical model. Furthermore, the higher the amount of immobilized protein A, the more pronounced the pH shift.…”

Section: ■ Introductionmentioning

confidence: 89%

“…Additional reason to evaluate the chromatographic peak shape is method sensitivity, which was demonstrated to be higher as for flat-top peaks. 40 Evaluation of pH Profiles for Estimation of Immobilized Protein Amount. As discussed in our previous work, 40 the magnitude of the pH peak can be correlated with the amount of immobilized protein by determining either the full width at half-maximum (fwhm) or the height (ΔpH) of the peak for the flat-top or chromatographic peak shape, respectively.…”

Section: ■ Introductionmentioning

confidence: 99%

“…40 Evaluation of pH Profiles for Estimation of Immobilized Protein Amount. As discussed in our previous work, 40 the magnitude of the pH peak can be correlated with the amount of immobilized protein by determining either the full width at half-maximum (fwhm) or the height (ΔpH) of the peak for the flat-top or chromatographic peak shape, respectively. Since chromatographic peaks were predominant in our experiments, a linear correlation between the height of the pH transition peak (ΔpH) and the amount of immobilized protein was investigated.…”

Section: ■ Introductionmentioning

confidence: 99%

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Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein

2023

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Protein immobilization is of utmost importance in many areas, where various proteins are used for selective detection of target compounds. Despite the importance given to determine the amount of immobilized protein, there is no simple method that allows direct, noninvasive detection. In this work, a method based on pH transition, occurring during change of solution ionic strength, was developed. The method utilized the ionic character of the immobilized protein while implementing biologically compatible buffers. Five different proteins, namely, glucose oxidase, horseradish peroxidase, bovine serum albumin, lysozyme, and protein A, were immobilized in different amounts on a porous polymeric matrix, and their pH transition was measured using lactate buffer of various concentrations and pH values. A linear correlation was found between the amount of immobilized protein and the amplitude of the pH transition, allowing the detection down to 2 nmol of immobilized protein. By changing the buffer concentration and pH, the sensitivity of the method could be tailored. Criteria based on the symmetry of the pH transition peak have been developed to determine if a particular measurement is within a linear range. In addition, a mathematical model was developed enabling prediction of pH transition profiles based solely on the protein amino acid sequence, the buffer pK a value(s), and the amount of immobilized protein.Hence, it can be used to design pH transition method experiments to achieve the required sensitivity for a target sample. Since the proposed method is noninvasive, it can be routinely applied during optimization of the immobilization protocol, for quality control, and also as an in-process monitoring tool.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 82%

Section: ■ Introductionmentioning

confidence: 89%

Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein

2023

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Purification of monoclonal antibodies using novel 3D printed ordered stationary phases

Conti,

Boland,

Heeran

et al. 2024

Journal of Chromatography A

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Mechanism of Interconnected Pore Formation in High Internal Phase Emulsion-Templated Polymer

Zhang,

Chen,

You

et al. 2024

ACS Macro Lett.

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High internal phase emulsion-templated polymer, named polyHIPE, has received widespread attention due to its great potential applications in many fields, such as separation, adsorption, heterogeneous catalysis, and sound absorption. The broad applicability is largely dependent on its adjustable opening structure. However, the question of why polyHIPE has an interconnected pore network structure is still to be discussed. Herein, different types (w/o, o/w, and o/o) of HIPEs are prepared and subsequently detected with laser scanning confocal microscopy (LSCM), and the polyHIPEs obtained by curing the HIPEs are characterized by SEM. The observations suggest that the interconnected pore formation is primarily due to the presence of the surfactant-rich phase in the film between the neighboring droplets in HIPE. The interconnected pores are generated by removal of the surfactant-rich domains in the postcuring procedure, and their sizes would be enlarged if the solubility of the surfactant in the continuous phase decreases in the curing stage.

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Noninvasive method for determination of immobilized protein A

Cited by 5 publications

References 46 publications

Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein

Rapid, Direct, Noninvasive Method to Determine the Amount of Immobilized Protein

Purification of monoclonal antibodies using novel 3D printed ordered stationary phases

Mechanism of Interconnected Pore Formation in High Internal Phase Emulsion-Templated Polymer

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