Alkaline cation-exchange chromatography for the reduction of aggregate and a mis-formed disulfide variant in a bispecific antibody purification process.
Troii Hall, Joseph J Wilson, Tammy J Brownlee, James R Swartling, Sarah E Langan, Peter K Lambooy
Index: J. Chromatogr. B. Analyt. Technol. Biomed. Life Sci. 975 , 1-8, (2014)
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Abstract
During the purification development of a bispecific antibody, cation-exchange chromatography was screened for its ability to separate a prominently expressed (>12%) mis-formed disulfide bond variant, termed MAb-diabody, and aggregate from the product of interest. The influence of pH, product load (g of product per liter of resin) and linear velocity on the separations were evaluated for the strong cation-exchange resins SP Sepharose HP and POROS(®) HS50. Cation-exchange chromatography is commonly operated distant to the isoelectric point of a molecule, generally leading to acidic conditions for antibody purification. However, the results herein demonstrated improved removal of MAb-diabody with increasing pH, resulting in reduction of MAb-diabody content greater than 12-fold when operating near the alkaline pI of the product. This approach was successful over a range of linear velocities and g/L of resin loading. Aggregate removal was less affected by pH and was effectively reduced from 10.9% to less than 3% for each condition. Furthermore, this method was successfully scaled to a 60 cm diameter column using SP Sepharose HP resin.Copyright © 2014 Elsevier B.V. All rights reserved.
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