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In this manuscript we describe the two-step purification of a mono-PEGylated anti-epidermal growth factor receptor (EGFR) single-chain Fv. A weak cation exchanger was used for capture. Elution using arginine suppressed protein aggregation and allowed a very good resolution with purity and product-recovery was above 90%. Free PEG was removed completely. The use of hydrophobic interaction chromatography (HIC) increased purity to 98%. Increasing the size of PEG from 5 to 30 kDa increased retention on HIC and reduced it on cation exchangers. Bioactivity of PEGylated scFv was confirmed by (125)I based cell tests. Proteins modified with 5 kDa PEG showed higher bioactivity than proteins modified with larger PEGs. The combination of cation exchange and HIC provides a rational and effective basis for PEGylated scFv purification., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Dynamic binding capacity (DBC) measurements of cation-exchange resins were performed with two human monoclonal antibodies. DBC showed a pH dependent maximum, which was shifted to lower pH values with increasing buffer concentrations and increasing salting-out effect of the buffer anion according to the Hofmeister series. As this downshift correlates well with zeta potential values, a measurement of the latter allows the determination of the pH value for maximum DBC under a given set of conditions. Thus, the use of zeta potential values can accelerate the purification process development and helps to understand the protein adsorption mechanism.