Varsha S. Joshi

A systematic approach for formation of sigmoidal gradient for analysis of biopharmaceutical proteins has been reviewed and discussed together with two case studies. The first application involves HPLC separation of the various product related variants of a microbial expressed biotherapeutic, granulocyte colony stimulating factor (GCSF). The second case study involves separation of the charge heterogeneity related variants for a monoclonal antibody (mAb) biotherapeutic product. In all cases, it is observed that the use of sigmoidal gradient successfully reduces the analysis time significantly (from 70 minutes to 15 minutes for GCSF and from 40 minutes to 4 minutes for the mAb) while retaining the selectivity and the resolution.

In this article, we discuss the role of the gradient in protein separations by reversed-phase and ionexchange high performance liquid chromatography (HPLC).

A discussion of the role of the gradient in separation by reversed-phase and ion-exchange HPLC, with data from two products to illustrate the key points

The use of a monolithic column reduces analysis time in an on-line biopharmaceutical HPLC analysis of granulocyte colony stimulating factor.