Skip to Content
Merck
  • Scale-up protein separation on stainless steel wide bore toroidal columns in the type-J counter-current chromatography.

Scale-up protein separation on stainless steel wide bore toroidal columns in the type-J counter-current chromatography.

Journal of chromatography. A (2015-03-31)
Yue Hugh Guan, Peter Hewitson, Remco N A M van den Heuvel, Yan Zhao, Rick P G Siebers, Ying-Ping Zhuang, Ian Sutherland
ABSTRACT

Manufacturing high-value added biotech biopharmaceutical products (e.g. therapeutic proteins) requires quick-to-develop, GMP-compliant, easy-to-scale and cost effective preparatory chromatography technologies. In this work, we describe the construction and testing of a set of 5-mm inner diameter stainless steel toroidal columns for use on commercially available preparatory scale synchronous J-type counter-current chromatography (CCC) machinery. We used a 20.2m long column with an aqueous two-phase system containing 14% (w/w) PEG1000 and 14% (w/w) potassium phosphate at pH 7, and tested a sample loading of 5% column volume and a mobile phase flow rate of 20ml/min. We then satisfactorily demonstrated the potential for a weekly protein separation and preparation throughput of ca. 11g based on a normal weekly routine for separating a pair of model proteins by making five stacked injections on a single portion of stationary phase with no stripping. Compared to our previous 1.6mm bore PTFE toroidal column, the present columns enlarged the nominal column processing throughput by nearly 10. For an ideal model protein injection modality, we observed a scaling up factor of at least 21. The 2 scales of protein separation and purification steps were realized on the same commercial CCC device.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Myoglobin from equine skeletal muscle, 95-100%, essentially salt-free, lyophilized powder
Sigma-Aldrich
Poly(ethylene glycol), average Mn 950-1,050
Sigma-Aldrich
Lysozyme from chicken egg white, powder or granules, ≥90 %, ≥39,000 units/mg protein
Sigma-Aldrich
Potassium phosphate dibasic, ACS reagent, ≥98%
Sigma-Aldrich
Potassium phosphate monobasic, ACS reagent, ≥99.0%