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Comparison of Assay Methods for the Detection of Residual Protein A in Biological Therapeutics

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Peter J. Brescia 1, Peter Banks 1, M. Cortes-Caminero 2
1 BioTek Instruments, Inc., Winooski, VT
2 Enzo Life Sciences, Farmingdale, NY

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There continues to be a focus on the development of recombinant human monoclonal antibodies (rhuMAb IgGs) for therapeutic use with dozens reaching the market in the last decade. Therapeutic proteins of the scale needed for treatment of even a small population require industrial scale production using a variety of bioprocessing methods including recombinant cell line expression systems, chromatographic purification methods, and stringent purity assessment. Purity requirements include minimizing the concentration of host cell proteins and DNA ranging in the parts per million or lower relative to the product. Additionally, the formulation must be sterile insuring no viable microorganisms exist in the final product and void of any residual contaminants from the purification process itself.

A recombinant human monoclonal antibody is commonly produced in a mammalian cell line such as Chinese Hamster Ovary (CHO) cells during large scale manufacturing. Purification typically relies on the use of a three-column chromatography process to meet the stringent purification requirements: 1) Protein A affinity chromatography, 2) Cation exchange (CEX), and 3) Anion Exchange (AEX). Additionally, a viral filtration (VF) step is generally used during the final stages of production. Resin with immobilized Staphylococcal Protein A (PA) has a high affinity for the crystallizable fragment (Fc) region present in rhuMAb IgGs allowing capture from the culture media or crude cell lysate of the host cell line. While these resins provide a high capacity and selectivity for the target protein, trace amounts of the PA ligand has been found contaminating the antibody product. Residual PA contamination of a biotherapeutic may result in immunogenic consequences as well as toxicological and/or mitogenic effects. Therefore, reliable, robust methods for the detection and quantification of trace amounts of PA are necessary and mandated by the FDA.

Here we demonstrate automation of a HTS compatible homogenous proximity assay and conventional ELISA method for the detection of residual PA in biological therapeutics. The demonstration includes evaluation of the lower detection limit (LDL) as well as screening results for detection of residual PA in a panel of ten (10) human IgG antibodies including the active therapeutic components of Herceptin®, Rituxan®, and Erbitux®along with the antibodies trastuzumab, rituximab, and cetuximab, respectively.

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