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


METHODS
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.


CONCLUSIONS
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.
Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK.
S H M
SH
Hedberg
Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK.
J Y Y
JY
Heng
Surfaces and Particle Engineering Laboratory, Department of Chemical Engineering, Imperial College London, London, UK. d.r.williams@imperial.ac.uk.
D R
DR
Williams
R&D Group, FUJIFILM Diosynth Biotechnologies, Billingham, UK.
J M
JM
Liddell
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D002417:Cattle; D002645:Chickens; D002845:Chromatography / Q000379:methods*; D009113:Muramidase / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D012996:Solutions / Q000737:chemistry
D000911:Antibodies, Monoclonal; D012996:Solutions; D012710:Serum Albumin, Bovine; D009113:Muramidase
dead volume; mAbs; protein-protein interactions; second virial coefficient; self-interaction chromatography