Measuring protein mobility using modern microelectrophoresis

Corbett, Jason C.W.; Jack, Robert O.

doi:10.1016/j.colsurfa.2010.10.029

Cited by 16 publications

(28 citation statements)

References 18 publications

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“…Method parameters were first optimized using bovine serum albumin (BSA) to obtain values that have been previously reported. 43 Based on the data from BSA, a concentration of 10 mg/ml was selected for experiments using formulated mAbs. For samples that contained high salt (e.g., G1-6 and G1-7), to overcome saturation of the current due to high ionic strength, dilutions were performed in 10 mM histidine buffer, pH 6.0.…”

Section: Zeta Potential and K D Measurementsmentioning

confidence: 99%

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

Thiagarajan

Semple

James

et al. 2016

mAbs

104

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With the rapid growth of biopharmaceutical product development, knowledge of therapeutic protein stability has become increasingly important. We evaluated assays that measure solution-mediated interactions and key molecular characteristics of 9 formulated monoclonal antibody (mAb) therapeutics, to predict their stability behavior. Colloidal interactions, self-association propensity and conformational stability were measured using effective surface charge via zeta potential, diffusion interaction parameter (k D ) and differential scanning calorimetry (DSC), respectively. The molecular features of all 9 mAbs were compared to their stability at accelerated (25 C and 40 C) and long-term storage conditions (2-8 C) as measured by size exclusion chromatography. At accelerated storage conditions, the majority of the mAbs in this study degraded via fragmentation rather than aggregation. Our results show that colloidal stability, self-association propensity and conformational characteristics (exposed tryptophan) provide reasonable prediction of accelerated stability, with limited predictive value at 2-8 C stability. While no correlations to stability behavior were observed with onset-of-melting temperatures or domain unfolding temperatures, by DSC, melting of the Fab domain with the C H 2 domain suggests lower stability at stressed conditions. The relevance of identifying appropriate biophysical assays based on the primary degradation pathways is discussed.

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Section: Zeta Potential and K D Measurementsmentioning

confidence: 99%

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

Thiagarajan

Semple

James

et al. 2016

mAbs

104

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“…The pH of the suspensions was monitored using the Metrohm 827 pH lab (Metrohm, http://repository.uwc.ac.za Switzerland) equipped with a Primatrode pH electrode. A syringe was used to fill a semidisposable capillary cell with the sample which was then immersed into a temperature controlled block holder to avoid thermal gradients in the absence of the applied electric field [23]. Electrophoretic mobility of particles was measured by applying a fixed voltage of 100 V cm -1 .…”

Section: 2mentioning

confidence: 99%

Optimisation of electrophoretic deposition parameters for gas diffusion electrodes in high temperature polymer electrolyte membrane fuel cells

Jao

Pasupathi

Pollet

2013

Journal of Power Sources

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Felix, C. et al. (2013). Optimisation of electrophoretic deposition parameters for gas diffusion electrodes in high temperature polymer electrolyte membrane fuel cells. AbstractElectrophoretic deposition (EPD) method was used to fabricate gas diffusion electrodes (GDEs) for high temperature polymer electrolyte membrane fuel cells (HT PEMFC). Parameters related to the catalyst suspension and the EPD process were studied. Optimum suspension conditions are obtained when the catalyst particles are coated with Nafion ® ionomer and the pH is adjusted to an alkaline range of about 8 e10. These suspensions yield good stability with sufficient conductivity to form highly porous catalyst layers on top of the gas diffusion layers (GDLs). GDEs were fabricated by applying various electric field strengths of which 100 V cm -1 yields the best membrane electrode assembly (MEA) performance. Compared to an MEA fabricated by the traditional hand sprayed (HS) method, the EPD MEA shows superior performance with a peak power increase of about 73% at similar platinum (Pt) loadings. Electrochemical Impedance Spectroscopy (EIS) analysis shows lower charge transfer resistance for the MEA fabricated via the EPD method compared to the HS MEA. The EPD GDE exhibits a greater total pore area (22.46 m 2 g -1 ) compared to the HS GDE (13.43 m 2 g -1 ) as well as better dispersion of the Pt particles within the catalyst layer (CL).

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“…The procedures used follow closely the work of Corbett and Jack [5] -and only a brief description is given here for brevity.…”

Section: Experimental Settingsmentioning

confidence: 99%

“…Unless otherwise stated, a 13.2 V/cm field strength was used with a 15 s integration time per measurement. A 10-Hz field reversal frequency was also used throughout, appropriate for the size of the capillary to remove any dependency of the measured mobility on electrophoresis at the cell wall [5,8]. The remaining systematic uncertainty in the final mobility value, due to these effects, was limited to o5%.…”

Section: Experimental Settingsmentioning

confidence: 99%

“…This is typically the case when the electrodes are close together and close to the detection volume. The mobility of proteins and their aggregates have been shown to be significantly different [5], which highlights the importance of preventing their creation and/or detecting their presence. Furthermore, the volumes typically associated with LDE, which are of the orders of 1-10 mL or so, are problematic due to the high sample cost and the iterative nature of LDE measurement optimisation.…”

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confidence: 99%

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Advances in the measurement of protein mobility using laser Doppler electrophoresis – the diffusion barrier technique

Corbett¹,

Connah²,

Mattison³

2011

Electrophoresis

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A new technique for the measurement of protein mobility using laser Doppler electrophoresis (LDE) is introduced and characterised. The diffusion barrier approach loads a tiny protein sample volume into a much larger volume of dispersant, which contains the electrodes; the LDE measurement is then recorded before the sample can diffuse to the electrodes. We demonstrate that sample volumes are reduced by up to two orders of magnitude to volumes typically associated with separation techniques (∼50 μL), no reduction in measurement sensitivity occurs, samples can be retrieved usefully intact, post-measurement and typical measurement times are of the order of minutes. Measurements of BSA mobility up to 75°C and 1 M buffer concentration and lysozyme at a concentration as low as 0.5 mg/mL are demonstrated using the technique with good agreement with literature values.

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Measuring protein mobility using modern microelectrophoresis

Cited by 16 publications

References 18 publications

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

A comparison of biophysical characterization techniques in predicting monoclonal antibody stability

Optimisation of electrophoretic deposition parameters for gas diffusion electrodes in high temperature polymer electrolyte membrane fuel cells

Advances in the measurement of protein mobility using laser Doppler electrophoresis – the diffusion barrier technique

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