Abstract
Biologics manufacturing requires the clearance of Host Cell Proteins (HCPs) from recombinant therapeutic protein to acceptable low levels to ensure product purity and patient safety. To ensure adequate removal, a highly sensitive method, commonly in the form of Enzyme-Linked Immunosorbent Assay (ELISA), is necessary to quantify the HCPs amount in process intermediates and drug substance. We report the development of a chemiluminescent detection based ELISA (luminescent ELISA) in lieu of previously used colorimetric method (colorimetric ELISA) to improve assay sensitivity for the quantification of Chinese Hamster Ovary (CHO) HCPs in a monoclonal antibody product (mAb-A). For luminescent ELISA, Pierce Supersignal ELISA Femto was chosen as the substrate to replace colorimetric substrate TMB. The assay performance of luminescent and colorimetric ELISA was directly compared side-by-side. Our data show that luminescent ELISA has better signal/background ratio, broader linear range over logarithmic scales, and better linearity within the same linear range than colorimetric ELISA. Luminescent ELISA also demonstrates better lowend linearity, greater accuracy and precision. In addition, the Limit of Detection (LOD) and Limit of Quantification (LOQ) are significantly improved with luminescent ELISA as compared to colorimetric ELISA. In summary, luminescent ELISA is a more sensitive method and demonstrates superiority over colorimetric method for CHO HCP quantification.
Highlights
Monoclonal antibodies have become a significant focus of the pharmaceutical industry due to their high specificity and their ability to engage a wide variety of targets [1,2]
enzyme-linked immunosorbent assay (ELISA) was directly compared by a variety of assay parameters such as signal/noise ratio, linear range and linearity over logarithmic scales, accuracy and precision, lower limit of detection (LLOD) and quantification (LLOQ)
Assay performance comparison for colorimetric ELISA and luminescent ELISA in Corning Costar clear 96-well plate using conditions optimized for colorimetric ELISA
Summary
Monoclonal antibodies (mAbs) have become a significant focus of the pharmaceutical industry due to their high specificity and their ability to engage a wide variety of targets [1,2]. Due to its high specificity and sensivity, enzyme-linked immunosorbent assay (ELISA) is the most commonly accepted method by regulators for HCPs quantification [10] Alternative immunospecific methods such as a quantitative slot blot assay [11] and solid-phase proximity ligation assay [12] as well as non-specific methods including mass spectrometry (MS) and 2D liquid chromatography (LC)-MS are being developed or explored [8,10]. None of these methods are robust enough or can achieve the same level of sensitivity as ELISA, which remains the gold standard for HCP quantification. Engineered CHO cell line is used to manufacture mAb-A and a process-specific HCP ELISA using proprietary antibodies raised against the null CHO cells has been developed in-house for Phase III mAb-A to measure HCP components in the drug substance (DS)
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