Assay qualification by standard FDA and ICH guidelines, including dilution linearity, spike and recovery analysis, interference studies, precision studies, and determination of analytical sensitivity.
Utilization of sensitive and specific orthogonal methods such as Antibody Affinity Extraction™ (AAE™), mass spectrometry and 2D PAGE to demonstrate antibody coverage and identify host cell protein impurities that persist through purification.
Together with data from ELISA, the data from these orthogonal methods provide a comprehensive analysis of HCP content that will meet regulatory expectations.
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Dilution linearity should be the first step in assay qualification. Dilution linearity study establishes freedom of sample matrix interference and demonstrates the important condition of antibody excess for the array of impurities in your samples. This analysis is critical for HCP assays as very high concentrations of certain HCPs may approach saturation of the antibody against that HCP indicating there is a risk of significant under-quantitation for that HCP.
Spike recovery analysis demonstrates that the assay can recover added HCP or other impurity or contaminant spiked into that sample matrix; usually performed for final product or in-process samples.
For more information, watch the video, "Dilution Linearity and Spike Recovery".
Anti-HCP Antibodies are the critical reagents for HCP ELISA. HCP coverage analysis is required for assay qualification as it assesses the ability of the antibodies to recognize a wide range of HCPs in the calibration standard and those present in in-process and drug substance samples. Antibody Affinity Extraction™ (AAE™) is a new, state-of-the art method for determination of antibody coverage to complex mixtures of HCP antigens. AAE overcomes most of the limitations of 2D WB while enabling the determination of coverage to the most relevant host cell proteins (HCPs)--those that co-purify with your product. Traditional attempts to determine anti-HCP coverage by comparing a non-specifically stained 2D PAGE to WB provide very little predictive value in determining how the ELISA using that antibody, will quantitatively react to the more limited array of HCPs in downstream samples. We conduct AAE for antibody coverage analysis and AAE for Mass Spectrometry identification of individual HCP.
Benefits of AAE as an orthogonal method for HCP analysis:
2D PAGE analysis provides very good fractionation and resolution of complex samples such as harvest material that can contain up to thousands of proteins. The detection methods of silver stain or fluorescent labeling can yield sensitivities as low as 1 ng for some individual proteins. However, the sensitivity of 2D PAGE is limited for downstream samples which contain reduced levels of HCPs and high concentration of drug substance. In addition, 2D PAGE is not specific in discriminating HCPs from product heterogeneity and other non-HCP contaminates. Antibody coverage analysis by AAE with 2D PAGE yields percentage HCP coverage as required for HCP assay qualification.
AAE combined with mass spectrometry (AAE-MS™) is a powerful method to assess antibody coverage to HCPs in a given process. The advantage of AAE-MS approach is that in addition to percentage coverage, it identifies HCPs in the harvest material and HCPs reactive with the antibody and yields protein MW and pI information.
The major factor limiting the application of MS in the detection of downstream HCPs is sensitivity and specificity. In most well purified products HCP content will be composed of numerous individual HCPs that in total are less than 100 ppm. MS discrimination of the drug substances at mg/mL from individual HCP at low ppm levels is problematic. We combine MS analysis with AAE as a sample preparation step to deplete drug substance and concentrate HCPs, making mass spectrometry a powerful method for detection and identification of HCPs in final DS. Taken together, this data enables more accurate assay qualification.