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  1. Host Cell Protein Analysis-related Questions:
    1. What is Host Cell Protein (HCP)?
      Host Cell Proteins are proteins and their modified forms derived from the hosts of biologics expression and co-exist during the biologics purification process.
    2. How to express the level of HCPs?
      HCPs are typically expressed as ng/mg or parts per million (ppm).
    3. What are the common technologies for HCP analysis?
      Common technologies used for HCP analysis include ELISA (Enzyme-linked Immunosorbent Assay), one-dimensional or two-dimensional gel electrophoresis, Western Blot and Mass Spectrometry.
    4. What guidelines are related to HCP analysis?
      ICH Q6B section 2.3.1; 4.1.3 and 6.2.1 have detailed descriptions on impurity analysis including HCP analysis.
    5. How the methods are developed?
      HCP methods are developed based on the performance of the anti-HCP antibodies including antibody selectivity, sensitivity. Many formats have been developed for HCP analysis, the most commonly used format is ELISA.
    6. How the methods are validated?
      HCP method validation especially the ELISA-based method validation is the result of extensive study of the anti-HCP reagent capability and their performance in a specific biologics product, therefore method validation is product-specific. ICH Q2 is the most cited guideline for analytical method validation.
    7. What are the most important parameters when validating a HCP ELISA?
      The most important parameters in HCP method validation are quantitation limit (QL), Accuracy, Precision and range.
    8. How much time needs to budget for a HCP method validation?
      Typically 3-6 months will be needed for an ICH method validation.
    9. How the HCP immunogens are prepared?
      HCP immunogens are prepared using a null cell line, it should be the same cell line that is used for the biologics production without the expression of the biologics.
    10. How the HCP antibodies are prepared?
      HCP antibodies are raised in rabbits or other animals that could provide a relatively large pool of antiserum because the biologics under development could have a long development and market life.
    11. What format of ELISA needs to be used?
      Sandwich ELISA is commonly used for HCP quantitation because the impurities only exist in parts per million level, enrichment step is necessary to achieve the required sensitivity.
    12. What are the limitations of HCP ELISA?
      The current sandwich ELISA can only detect HCPs producing two high affinity antibodies during the HCP antibody production, HCPs with low or no immunogenicity in the host animals will not be detected with this assay format. It is also possible that HCPs with relatively small molecular size will not be detected because they are cleared quickly in the animal host during antibody production.
    13. What is the value of gel electrophoresis?
      The value of gel electrophoresis is to complement the limitations of ELISA for HCP analysis, i.e. detection of HCPs that are weak or not immunogenic, small molecular size HCPs or HCPs that could be associated with biologics and not detected by the ELISA.
    14. What is the value of HCP Western Blot?
      HCP Western Blot could detect HCPs with one or more high affinity antibodies (ELISA only detects HCPs with two high affinity antibodies), therefore provide a semi-quantitative HCP measurement that sometimes could also be more sensitive than ELISA.
    15. What is the value of a bioinformatics analysis?
      Bioinformatices could be used to analyze the function of the HCPs identified, this will provide the basis for a rick assessment. If the HCPs or some of their domains identified share high homology with important regulatory proteins in humans, more efforts are necessary to further evaluate their risk or reduce their levels in the biologics.
    16. How to do a bridging study when switching testing reagents?
      Because HCP antibodies are polyclonal and multiple batches are typically produced during the development and marketing of a biologics, a bridging study is necessary to ensure the consistency of the reagents. All the reagents and methods should be developed under cGMP, and any future production of any components in the ELISA should be evaluated with the HCP reference and biologics sample retains to make sure that the reporting values are consistent after the switch of reagent(s), adjustment of certain reagents will be needed if there is any significant value changes during the bridging studies.
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