Publications (2)8.14 Total impact
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Article: Cation exchange surface-mediated denaturation of an aglycosylated immunoglobulin (IgG1).
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ABSTRACT: Cation exchange chromatography of an aglycosylated IgG1 resulted in two distinct peaks during gradient elution. The early eluting peak contained <1% high molecular weight (HMW) species, while the later peak contained 23% HMW species. Analysis by hydrogen-deuterium exchange and Fourier transform infrared spectroscopy (FTIR) indicated that aggregate formation and generation of the second peak were caused by antibody denaturation on the resin surface. Denaturation and HMW generation was increased by the use of strong cation exchange media, by increasing antibody residence time on the exchanger, or increasing temperature. Denaturation and HMW generation was reduced by increasing pH or ionic strength, by the use of preferentially excluded solutes such as citrate or glycine and controlled entirely by addition of 125 mM arginine to the process buffers. This leads to the hypothesis that denaturation and HMW generation of this antibody can be managed by reducing the strength of binding, by increasing its conformational stability, or by suppressing non-native protein-protein interactions. The glycosylated version of this antibody exhibited less than 2% denatured form, suggesting that glycosylation contributes significantly to the stability of this antibody. These findings may be helpful in managing aggregation in other antibodies, and particularly useful in developing purification processes for aglycosylated antibodies.Journal of chromatography. A 06/2012; 1251:101-10. · 4.19 Impact Factor -
Article: Cation exchange chromatography provides effective retrovirus clearance for antibody purification processes.
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ABSTRACT: One measure taken to ensure safety of biotherapeutics produced in mammalian cells is to demonstrate the clearance of potential viral contaminants by downstream purification processes. This paper provides evidence that cation exchange chromatography (CEX), a widely used polishing step for monoclonal antibody (mAb) production, can effectively and reproducibly remove xMuLV, a retrovirus used as a model of non-infectious retrovirus-like particles found in Chinese hamster ovary cells. The dominant mechanism for xMuLV clearance by the strong cation exchanger, Fractogel SO ₃⁻, is by retention of the virus via adsorption instead of inactivation. Experimental data defining the design space for effective xMuLV removal by Fractogel SO ₃⁻ with respect to operational pH, elution ionic strength, loading, and load/equilibration buffer ionic strength are provided. Additionally, xMuLV is able to bind to other CEX resins, such as Fractogel COO⁻ and SP Sepharose Fast Flow, suggesting that this phenomenon is not restricted to one type of CEX resin. Taken together, the data indicate that CEX chromatography can be a robust and reproducible removal step for the model retrovirus xMuLV.Biotechnology and Bioengineering 08/2011; 109(1):157-65. · 3.95 Impact Factor
