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

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

Bottom Line: Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency.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.

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.


B2 values for different NaCl concentrations depending on the dead volume estimates used (20 mM sodium acetate pH 4.5).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4628098&req=5

Fig8: B2 values for different NaCl concentrations depending on the dead volume estimates used (20 mM sodium acetate pH 4.5).

Mentions: The importance of using accurate and robust estimates for the dead volume in determining B2 values cannot be over emphasised. Erroneous dead volumes lead directly to inaccurate estimates of B2 values. The dead volume estimates from Table I are shown in Fig. 8 where B2 of the mAb is determined as a function of increasing NaCl concentrations. The B2 estimates in this experiment are decreasing for increasing NaCl concentrations because of increasing charge shielding and therefore subsequent loss of electrostatic stability. However, the actual B2 values estimated vary substantially depending upon the dead volume used. Figure 8 provides three different estimated B2 values based on the three dead volumes determined in this study, where the second and third dead volumes are very similar (Table I).Fig. 8


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

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

B2 values for different NaCl concentrations depending on the dead volume estimates used (20 mM sodium acetate pH 4.5).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig8: B2 values for different NaCl concentrations depending on the dead volume estimates used (20 mM sodium acetate pH 4.5).
Mentions: The importance of using accurate and robust estimates for the dead volume in determining B2 values cannot be over emphasised. Erroneous dead volumes lead directly to inaccurate estimates of B2 values. The dead volume estimates from Table I are shown in Fig. 8 where B2 of the mAb is determined as a function of increasing NaCl concentrations. The B2 estimates in this experiment are decreasing for increasing NaCl concentrations because of increasing charge shielding and therefore subsequent loss of electrostatic stability. However, the actual B2 values estimated vary substantially depending upon the dead volume used. Figure 8 provides three different estimated B2 values based on the three dead volumes determined in this study, where the second and third dead volumes are very similar (Table I).Fig. 8

Bottom Line: Toyopearl AF-Amino packed dead columns showed no such problems and acted as suitable dead columns without any solution condition dependency.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.

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.