And we used anti-HCP antibodies to Chinese hamster ovary (CHO) proteins purchased from Cygnus Technologies (3G-0016-PA) as the primary antibody and HRP-conjugated bovine antigoat antibody purchased from Santa Cruz Biotechnology as the secondary antibody. NuPage gels and Gibco cell-culture media were purchased from Life Technologies (now Thermo Scientific), and ultrafiltration protein concentration/buffer exchange cartridges were purchased from Sartorius. We purchased the following materials from Bio-Rad Laboratories: a ReadyPrep 2D cleanup kit and ReadyPrep rehydration/sample buffer, ReadyStrip immobilized pH gradient (IPG) strips, Criterion gels, Trans-Blot Turbo PVDF transfer packs, Bio-Safe Coomassie stain, Oriole fluorescent gel stain, SYPRO Ruby protein blot stain, blotting grade-blocker, and Clarity Western enhanced chemiluminescence (ECL) substrate. Further, we apply these enhancements as a model example to the evaluation of a commercially available anti-HCP antibody reagent. To increase reliability of anti-HCP antibody evaluations and facilitate downstream decisions for effective HCP impurity profiling, we present here enhancements to the standard 2DE and WB workflow. That complicates downstream decisions for effective HCP impurity profiling of purified biologics, including identification of potential protein impurities “missed” by anti-HCP antibodies and development of orthogonal methods to monitor those impurities. Although it is widely used, the laborious nature of this workflow - and experimental variation inherent to comparisons between replicate 2DE analyses - can affect overall reliability of final results. Finally, images from 2DE and WB membranes are overlaid to obtain a match rate for determining the percentage of HCPs that the anti-HCP antibodies can detect. Third, proteins from replicate 2DE gels are transferred to a solid support membrane such as nitrocellulose or polyvinylidene fluoride (PVDF) for WB, with the polyclonal anti-HCP antibodies being evaluated. Second, one post-2DE gel is processed for total protein detection by a sensitive protein stain. First, 2DE of host-cell protein preparations is performed in replicate gels. The standard workflow for this evaluation comprises four steps. KEYWORDS: HOST-CELL PROTEINS, ASSAY DEVELOPMENT, REPLICATES, QUALIFICATIONĪmong multiple technical approaches for evaluating the antibody reagent, a combination of two-dimensional electrophoresis (2DE) and Western blotting (WB) is the current “gold standard” for visualizing total HCP populations and assessing the percent immunocoverage of an anti-HCP polyclonal antibody reagent ( 5, 6). WHO SHOULD READ: ANALYTICAl, QA/QC, PROCESS DEVELOPMENT So that reagent must be validated to be suitably specific for its intended use by testing for the percentage of immunodetection it offers for the total population of HCPs before it is used in the HCP ELISA. The accuracy of this immunoassay depends on the antibody reagent’s ability to detect essentially all potential HCP impurities. An HCP ELISA uses a polyclonal “antibody reagent” (anti-HCP antibodies) grown in animals against HCP preparations from a product expression system. One of the most widely used methods for monitoring HCPs is the enzyme-linked immunosorbent assay (ELISA) due to its high sensitivity (low ppm) and potential for broad specificity for the population of HCPs ( 1, 2, 3, 4).
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