Wednesday, February 18, 2015

Detection of Residual Protein A in Biological Therapeutics

Over the past 10 years the pharmaceutical community has witnessed an explosion in growth of biotherapeutics. An increased focus on developing and marketing antibody based products resulted in a product shift to the point that in 2013 antibody based therapies represented one-third of all biotherapeutics and nearly half of all revenue in the product category. In fact, 2014 saw a new approval record set with nine new antibody products entering the market and many more in the pipeline or under review for approval in 2015.

Treatment of even a small population will require industrial scale production of therapeutic proteins, 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 Anion Exchange (AEX); a viral filtration (VF) step is also 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 to leach from the column 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 in the US by the FDA.

Recently we demonstrated automation of a HTS compatible homogenous proximity assay for the detection of residual PA in biological therapeutics. The application includes screening results for detection of residual PA in a panel of ten (10) human IgG antibodies including samples of the  biologically active antibody components in the therapeutic products Herceptin®, Rituxan®, and Erbitux®.

D Ekers. (2015, Jan. 29) Antibodies: $75 million in sales and no slowing down in sight. [Web log comment] Retrieved from
Mehta, A., et al. 2007. Purifying Therapeutic Monoclonal Antibodies. CEP. SBE Special Section: Bioprocessing. S14-S20.
Zhu-Shimoni, J.; Gunawan, F.; Thomas, A.; Vanderlaan, M.; Stults, J. 2009. Trace level analysis of leached Protein A in bioprocess samples without interference from the large excess of rhMAb IgG. J. Immunol. Methods, 341 (1-2), pp. 59–67.

By: BioTek Instruments, Peter J. Brescia Jr., MSc, MBA

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