|Possibilities of modern size-exclusion chromatography for therapeutic proteins; feasibility assessment for future mass spectrometry hyphenation
|(R)evolutions in Biopharmaceutical Analysis (KVCV)
|Dr Szabolcs Fekete
|Mr Alexandre Goyon
Prof Jean-Luc Veuthey
Dr Davy Guillarme
|University of Geneva
Abstract Information :
In the last few years, highly efficient UHP-SEC columns packed with sub-3 µm particles have been commercialized by several providers.
Besides the particle size reduction, the dimensions of modern SEC columns (150 x 4.6 mm) were also reduced, compared to regular SEC columns (300 x 6 or 300 x 8 mm) enabling to reduce analysis time. However, the contribution of the chromatographic system itself to peak variance can become a serious problem under UHP-SEC conditions and need to be evaluated.
The inertness of SEC stationary phases is another important feature as slight secondary (non-specific) interactions can drastically affect the observed amount of protein aggregates. The possible undesired interactions can be controlled by the addition of various mobile phase additives (salts, buffers, organic modifiers). To support the growth of mass spectrometry (MS) in the biopharmaceutical field and disclose the unambiguous identification of the separated species, volatile mobile phases have been recently suggested in SEC-MS for the analysis of reduced, denatured and intact monoclonal antibodies (mAbs). However by using such mobile phases, compromises have to be find between chromatographic efficiency and MS sensitivity.
The presentation discusses modern SEC column technology, the impact of stationary phase inertness and possibilities to perform direct UHP-SEC-MS experiments. The SEC chromatographic behavior of several commercial mAbs and mAb-related products covering a wide range of physico-chemical properties (molecular weights between 54 and 153 kDa, pI values comprised between 6.1 and 9.4) are illustrated. Possible MS hyphenation and multi-dimensional separations are discussed in details. Problems related to extra-column volume and band broadening in modern SEC are also shown. Finally, various applications and method development approach are presented.