Assay Qualification

Assay qualification by standard FDA and ICH guidelines, including dilution linearity, spike and recovery, precision studies, interference studies, and determination of analytical sensitivity.

Application of sensitive and specific orthogonal methods (mass spectrometry, 2D PAGE, HPLC, and Cygnus' proprietary antibody affinity extraction method) 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 & Spike Recovery

Dilution linearity study to establish freedom of sample matrix interference and demonstrate 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 to demonstrate 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.

Orthogonal Coverage Analysis by AAE

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:

  • Coverage analysis is more predictive of the performance of the antibody in the HCP ELISA 
  • Facilitates identification of individual downstream HCPs 
  • Trusted and accepted by regulatory agencies around the world (FDA, EMA, CFDA) 
  • Included in the USP Chapter 1132 on HCP Analysis 
  • Can be used as a single Liquid Chromatography step to improve sensitivity of Mass Spectrometry

Orthogonal Coverage Analysis by 2D PAGE

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. When used together with the AAE, 2D PAGE allows identification of individual HCPs that co-purify with drug substance.

Orthogonal Coverage Analysis by Mass Spectrometry

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 remove drug substance and concentrate HCPs, making mass spectrometry a powerful method for detection and identification of downstream HCPs.


Frequently Asked Questions

  • How do I Perform Spike & Recovery Studies?
  • How do I Qualify HCP & Bioprocess Impurity Assays?
  • Why is Dilution Linearity important?

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lf you have questions or would like a quote, a Cygnus Technologies expert is happy to help!

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