Trace level analysis of leached Protein A in bioprocess samples without interference from the large excess of rhMAb IgG

Analytical Operations, Process Research and Development, Genentech, Inc. 1 DNA Way, South San Francisco, CA 94080, United States.
Journal of Immunological Methods (Impact Factor: 1.82). 12/2008; 341(1-2):59-67. DOI: 10.1016/j.jim.2008.10.015
Source: PubMed


Resins containing immobilized Staphylococcal Protein A (PA) are widely used in the commercial purification of recombinant human monoclonal antibody (rhuMAb IgG) biotherapeutics. Therefore, a sensitive assay for leached PA is needed to ensure that PA is not present at unacceptable levels as an impurity in the final product. PA impurities are measured by an ELISA using chicken anti-PA antibodies. However, PA in the presence of IgG product forms a PA/IgG complex that interferes in the assay. In this report a multi-product PA ELISA is described, wherein the PA/IgG complex is dissociated by heating in the presence of detergents and chelators prior to the ELISA. The dissociation facilitates the accessibility of the anti-PA antibodies to bind to PA in the immunoassay. Heat is provided by a novel microwave technology which allows brief heating time and high sample throughput using a microtiter plate for sample heating. Thus, broadly applicable dissociation conditions, suitable for all 21 rhMab IgGs tested to date were identified. This approach streamlines the measurement of leached PA, allows higher sample testing throughput, facilitates application across multiple products, and facilitates assay automation. Data comparing in-process samples tested with both the former product-specific ELISA and this new multi-product assay are shown.

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Available from: Judith Zhu-Shimoni, Jun 26, 2014
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    • "However in most cases, standard techniques such as sodium dodecyl sulfate polyacrylamide gel electrophoresis (or SDS-PAGE) are considered sensitive enough to detect the presence of leached protein-A which is co-eluted along with a huge excess of mAb and is therefore likely to be present in a complexed form with one or several mAb molecules once the pH of the eluate from the protein-A column is neutralized. To measure the exact concentration of protein-A, such complexes need to be first dissociated by boiling in the presence of SDS [28] or by employing microwave [29]. Leached protein- A and its fragments could be removed using anion exchange chromatography [27] [30], cation exchange chromatography [10] [30], and hydroxyapatite chromatography [18]. "
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    • "Further problems include leakage of protein A or its degradation by feedstock proteases that decrease the adsorption capacity. Moreover, protein A is a bacterial toxin which can cause immunogenic responses and therefore it has to be subsequently removed from the purified antibody solution (Huse at al., 2002; Ghose et al., 2006; Guerrier et al., 2001; Zhu-Shimoni et al., 2009). Although the stability of affinity adsorbents has recently been improved by using recombinant protein A with changed sequences for cleavage protection and a direct attachment of the ligand to an adsorbent matrix (Hober et al., 2007; Carter-Franklin et al., 2007; Hahn et al., 2006; Roque et al., 2007), its essential drawback remains, costs several times higher compared to other chromatographic adsorbents. "
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