Micro scale self-interaction chromatography of proteins: A mAb case-study

Hedberg, Sarah; Heng, Jerry Y. Y.; Liddell, John

doi:10.1016/j.chroma.2015.12.034

Cited by 11 publications

(5 citation statements)

References 23 publications

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“…The mobile phase flow rate was very low; 0.05 mL/min. The lower flow rates and protein concentration in the microcolumn gave consistent results with regular flow rate and higher protein concentration in a 1–2 mL laboratory scale column . All injections were performed in triplicate on two different protein immobilized columns to ensure reproducibility of results.…”

Section: Experimental Proceduresmentioning

confidence: 70%

“…Such a 2 day SIC screen will result in an over 90% saving in time compared to the equivalent SEC study. SIC also enables the use of microcolumns that can be used on conventional chromatography instruments, allowing savings in terms of the amount of proteins used in addition to time . Similarly, the total amount of mAb used for SIC was just over 6 mg (1 column and 30+ conditions).…”

Section: Resultsmentioning

confidence: 99%

“…A number of thermodynamic-based solubility models has been presented and directly verified with experimental data, − while other papers have taken an experimentally led approach and established a correlation between B 22 and solubility. , The use of B 22 to determine the phase behavior and crystallization conditions has also been widely reported for different proteins with varying levels of success, , and similarly, it was shown to quickly screen formulation conditions . Improvements in SIC include operational speed, the application of microfluidic chips, and microcolumns with low levels of protein. Despite reports of success, conflicting research findings have been found that show no correlation between B 22 and aggregation.…”

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confidence: 99%

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Mapping the mAb Aggregation Propensity Using Self-Interaction Chromatography as a Screening Tool

et al. 2018

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The osmotic second virial coefficient ( B), which describes protein-protein molecular interactions in solution, was determined using self-interaction chromatography (SIC) for an IgG1-type mAb across a wide range of solution conditions. These data were compared with its time dependent aggregation behavior, as determined using size-exclusion chromatography (SEC), and its temperature dependent aggregation behavior using dynamic light scattering (DLS) over a four-week period (SEC) or overnight (DLS). DLS and SEC gave consistent data on aggregation behavior, which correlated well with experimental B trends across the wide pH (4-9) and NaCl concentration (0-1.0 M) ranges studied. The IgG aggregated at pH 4 for 0.5-1.0 M NaCl concentrations and for 0 M NaCl concentrations at pH 8. Best stability against aggregation was exhibited for the pH range from 5 to 8 at 0.8-1.0 M NaCl. SIC data were able to be classified within the one-day solution conditions for aggregation, which were not identified for 2-3 weeks in the accelerated SEC stability study. The ability of SIC to provide such data rapidly reflects the fundamentally thermodynamic nature of this parameter and of the aggregation process itself. Proteins with attractive protein-protein interactions and negative B coefficients in the range -3 to -6 clearly exhibit aggregation behavior, while B values in the range 0 to 2 showed good stability toward aggregation. SIC allows the rapid screening of solution conditions for which mAbs will exhibit stability to aggregation while requiring 90% less time and material compared with that required for a conventional SEC aggregation study.

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Section: Experimental Proceduresmentioning

confidence: 70%

Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Mapping the mAb Aggregation Propensity Using Self-Interaction Chromatography as a Screening Tool

et al. 2018

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“…All the proteins used in this paper have different sizes from 14.3 kDa (lysozyme) to around 250 kDa (catalase), which means that for the same amount in mg immobilised more protein molecules of the smaller proteins are bound to the resin. Based on the cross-sectional surface area, more mass of the larger proteins is needed in order to achieve the same surface coverage as the smaller proteins (Hedberg et al 2016). In addition the kinetics of the reactions are faster for the smaller proteins in terms of more protein molecules bound in a shorter time frame.…”

Section: Resultsmentioning

confidence: 99%

“…The protein immobilisation procedure has long been considered a bottleneck of SIC (Bajaj et al 2007), due to the experiments being non-automated as well as requiring relatively substantial amounts of proteins. However, studies has shown that the immobilisation requirements can be reduced with cross-interaction chromatography (Jacobs et al 2010; Hedberg et al 2018b) or can be done with minimal amounts of protein using both microfluidic chips and micro columns (García et al 2003; Deshpande et al 2009; Martin and Lenhoff 2011; Hedberg et al 2016).…”

Section: Introductionmentioning

confidence: 99%

Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised on affinity resins

2019

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Protein adsorption on solid state media is important for the industrial affinity chromatography of biotherapeutics and for preparing materials for self-interaction chromatography where fundamental protein solution thermodynamic properties are measured. The adsorption of three model proteins (lysozyme, catalase and BSA) and two antibodies (a monoclonal and a polyclonal antibody) have been investigated on commercial affinity chromatography media with different surface functionalities (Formyl, Tresyl and Amino). Both the extent of protein immobilised (mg protein/ml media) and the reaction kinetics are reported for a range of reaction conditions, including pH, differing buffers as well as the presence of secondary reactants (glutaraldehyde, sodium cyanoborohydride, EDC and NHS). Compared to the reaction conditions recommended by manufacturers as well as those reported in previous published work, significant increases in the extent of protein immobilisation and reaction kinetics are reported here. The addition of glutaraldehyde or sodium cyanoborohydride was found to be especially effective even when not directly needed for the adsorption to happen. For mAb and pIgG, immobilisation levels of 50 and 31 mg of protein/ml of resin respectively were achieved, which are 100% or more than previously reported. Enhanced levels were achieved for lysozyme of 120 mg/ml with very rapid reaction kinetics (< 1 h) with sodium cyanoborohydride. It can be concluded that specific chromatography resins with Tresyl activated support offered enhanced levels of protein immobilisation due to their ability to react to form amine or thio-ether linkages with proteins. Additionally, glutaraldehyde can result in higher immobilisation levels whilst it can also accelerate immobilisation reaction kinetics.

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Molecular Weight Determination by Luminescent Chemo–enzymatics

Pires¹,

Moro

Lourenço

et al. 2016

ChemistrySelect

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A novel methodology for the determination of the number‐average molecular weight (Mw) of linear, branched and crosslinked polymers is disclosed. The method relies on chemo‐enzymatic‐triggered fluorescence of dormant CdTe polymer‐caged quantum dots synthesised in supercritical carbon dioxide. Polyurea (PURE) dendrimers (G1, G4, G5 and G6) are presented as a case study using the enzyme urease but the protocol is applicable to other synthetic and natural polymers. Simple, versatile and accurate, luminescent chemo‐enzymatics is a low cost alternative to conventional techniques (e. g. GPC/SEC, MALDI‐TOF).

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Micro scale self-interaction chromatography of proteins: A mAb case-study

Cited by 11 publications

References 23 publications

Mapping the mAb Aggregation Propensity Using Self-Interaction Chromatography as a Screening Tool

Mapping the mAb Aggregation Propensity Using Self-Interaction Chromatography as a Screening Tool

Improved adsorption reactions, kinetics and stability for model and therapeutic proteins immobilised on affinity resins

Molecular Weight Determination by Luminescent Chemo–enzymatics

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