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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: 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.


Protein Retention behaviour on a Toyopearl AF-Formyl dead column under different pH conditions (0.8 M NaCl).
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Related In: Results  -  Collection


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Fig1: Protein Retention behaviour on a Toyopearl AF-Formyl dead column under different pH conditions (0.8 M NaCl).

Mentions: Figure 1 shows that catalase and BSA have a retention behaviour that is independent of pH. The pH independent behaviour of these proteins is consistent with the hypothesis previously advanced that 0.8 M NaCl is able to shield the protein-surface electrostatic interactions (23). However, for mAbs, and a lesser extent lysozyme, there is a clear dependency of retention volume with pH. This observation is very significant. Firstly, it implies that electrostatic interactions between the proteins were not fully shielded by the 0.8 M NaCl of electrolyte present in the protein solutions. Whilst secondly, it shows that dead volume measurements can be both pH and protein-column dependent.Fig. 1


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

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

Protein Retention behaviour on a Toyopearl AF-Formyl dead column under different pH conditions (0.8 M NaCl).
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig1: Protein Retention behaviour on a Toyopearl AF-Formyl dead column under different pH conditions (0.8 M NaCl).
Mentions: Figure 1 shows that catalase and BSA have a retention behaviour that is independent of pH. The pH independent behaviour of these proteins is consistent with the hypothesis previously advanced that 0.8 M NaCl is able to shield the protein-surface electrostatic interactions (23). However, for mAbs, and a lesser extent lysozyme, there is a clear dependency of retention volume with pH. This observation is very significant. Firstly, it implies that electrostatic interactions between the proteins were not fully shielded by the 0.8 M NaCl of electrolyte present in the protein solutions. Whilst secondly, it shows that dead volume measurements can be both pH and protein-column dependent.Fig. 1

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.