Comparative study of recent wide-pore materials of different stationary phase morphology, applied for the reversed-phase analysis of recombinant monoclonal antibodies.
Various recent wide-pore reversed-phase stationary phases were studied for the analysis of intact monoclonal antibodies (mAbs) of 150 kDa and their fragments possessing sizes between 25 and 50 kDa. Different types of column technology were evaluated, namely, a prototype silica-based inorganic monolith containing mesopores of ~250 Å and macropores of ~ 1.1 μm, a column packed with 3.6 μm wide-pore core-shell particles possessing a wide pore size distribution with an average around 200 Å and a column packed with fully porous 1.7 μm particles having pore size of ~300 Å. The performance of these wide-pore materials was compared with that of a poly(styrene-divinyl benzene) organic monolithic column, with a macropore size of approximately 1 μm but without mesopores (stagnant pores). A systematic investigation was carried out using model IgG1 and IgG2 mAbs, namely rituximab, panitumumab, and bevacizumab. Firstly, the recoveries of intact and reduced mAbs were compared on the two monolithic phases, and it appeared that adsorption was less pronounced on the organic monolith, probably due to the difference in chemistry (C18 versus phenyl) and the absence of mesopores (stagnant zones). Secondly, the kinetic performance was investigated in gradient elution mode for all columns. For this purpose, peak capacities per meter as well as peak capacities per time unit and per pressure unit (PPT) were calculated at various flow rates, to compare performance of columns with different dimensions. In terms of peak capacity per meter, the core-shell 3.6 μm and fully porous 1.7 μm columns outperformed the two monolithic phases, at a temperature of 60 °C. However, when considering the PPT values, the core-shell 3.6 μm column remained the best phase while the prototype silica-based monoliths became very interesting, mostly due to a very high permeability compared with the organic monolith. Therefore, these core-shell and silica-based monolith provided the fastest achievable separation. Finally, at the maximal working temperature of each column, the core-shell 3.6 μm column was far better than the other one, because it is the only one stable up to 90 °C. Lastly, the loading capacity was also measured on these four different phases. It appeared that the organic monolith was the less interesting and rapidly overloaded, due to the absence of mesopores. On the other hand, the loading capacity of prototype silica-based monolith was indeed reasonable.
School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, Geneva 4, Switzerland. szabolcs.fekete@unige.ch
Szabolcs
S
Fekete
Jean-Luc
JL
Veuthey
Sebastiaan
S
Eeltink
Davy
D
Guillarme
D000327:Adsorption; D000911:Antibodies, Monoclonal / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D056148:Chromatography, Reverse-Phase / Q000295:instrumentation* / Q000379:methods; D007700:Kinetics; D010316:Particle Size; D016062:Porosity; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012117:Resins, Synthetic / Q000737:chemistry*
D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D012117:Resins, Synthetic