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Self-Interaction Chromatography of mAbs: Accurate Measurement of Dead Volumes.

Hedberg SH, Heng JY, Williams DR, Liddell JM - Pharm. Res. (2015)

Bottom Line: Measurement of the second virial coefficient B22 for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour.SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements.It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns.

View Article: PubMed Central - PubMed

Affiliation: Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK.

ABSTRACT

Purpose: Measurement of the second virial coefficient B22 for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour. In common with all physicochemical chromatographic methods, measuring the dead volume of the SIC packed column is crucial for accurate retention data; this paper examines best practise for dead volume determination.

Method: SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements.

Results: It was observed that lysozyme and mAb interacted specifically with Toyopearl AF-Formyl dead columns depending upon pH and [NaCl], invalidating their dead volume usage. Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency. Dead volume determinations using dextran MW standards with protein immobilised SIC columns provided dead volume estimates close to those obtained using Toyopearl AF-Amino dead columns.

Conclusion: It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns. Two other methods were shown to provide good estimates for the dead volume.

No MeSH data available.


Related in: MedlinePlus

Retention ratios for lysozyme injections on a Toyopearl AF-Formyl column (a) and on a Toyopearl AF-Amino column (b) as a function of NaCl concentrations (20 mM sodium acetate pH 4.5).
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Fig6: Retention ratios for lysozyme injections on a Toyopearl AF-Formyl column (a) and on a Toyopearl AF-Amino column (b) as a function of NaCl concentrations (20 mM sodium acetate pH 4.5).

Mentions: Figure 6a displays the effects of NaCl concentration for lysozyme on a Toyopearl AF-Formyl column. Similarly to Fig. 5a, at a NaCl concentration of 0.2 M or less the interactions are increasing again, possibly due to interactions with the matrix.Fig. 6


Self-Interaction Chromatography of mAbs: Accurate Measurement of Dead Volumes.

Hedberg SH, Heng JY, Williams DR, Liddell JM - Pharm. Res. (2015)

Retention ratios for lysozyme injections on a Toyopearl AF-Formyl column (a) and on a Toyopearl AF-Amino column (b) as a function of NaCl concentrations (20 mM sodium acetate pH 4.5).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4628098&req=5

Fig6: Retention ratios for lysozyme injections on a Toyopearl AF-Formyl column (a) and on a Toyopearl AF-Amino column (b) as a function of NaCl concentrations (20 mM sodium acetate pH 4.5).
Mentions: Figure 6a displays the effects of NaCl concentration for lysozyme on a Toyopearl AF-Formyl column. Similarly to Fig. 5a, at a NaCl concentration of 0.2 M or less the interactions are increasing again, possibly due to interactions with the matrix.Fig. 6

Bottom Line: Measurement of the second virial coefficient B22 for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour.SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements.It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns.

View Article: PubMed Central - PubMed

Affiliation: Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK.

ABSTRACT

Purpose: Measurement of the second virial coefficient B22 for proteins using self-interaction chromatography (SIC) is becoming an increasingly important technique for studying their solution behaviour. In common with all physicochemical chromatographic methods, measuring the dead volume of the SIC packed column is crucial for accurate retention data; this paper examines best practise for dead volume determination.

Method: SIC type experiments using catalase, BSA, lysozyme and a mAb as model systems are reported, as well as a number of dead column measurements.

Results: It was observed that lysozyme and mAb interacted specifically with Toyopearl AF-Formyl dead columns depending upon pH and [NaCl], invalidating their dead volume usage. Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency. Dead volume determinations using dextran MW standards with protein immobilised SIC columns provided dead volume estimates close to those obtained using Toyopearl AF-Amino dead columns.

Conclusion: It is concluded that specific interactions between proteins, including mAbs, and select SIC support phases can compromise the use of some standard approaches for estimating the dead volume of SIC columns. Two other methods were shown to provide good estimates for the dead volume.

No MeSH data available.


Related in: MedlinePlus