5.3.4.2 HCPs

Biopharmaceutical tools

limits established that reflect a level of purity that can be achieved reasonably and consistent.”

5.3.4.2 HCPs HCPs come from the host organism and constitute a major purification problem due to variability structure and surface properties. The amount released into the culture medium depends on the expression system used, the culture conditions, and whether the cells are disrupted or not in order to intracellularly extract expressed target protein. Notice that foreign cellular proteins can be introduced by means of recombinantly derived raw materials (e.g., enzymes) used in the downstream process. The range of preventive actions includes use of expression systems with direct expression of the target protein to the culture medium, gentle handling of intact cells, and purification procedures such as chromatography, filtration, precipitation, and crystallization. If milk from transgenic animals is used as the product source, the presence of the animal equivalent of the target protein should be of concern (as it might copurify with the target protein). Protein impurities should be considered on a case-by-case basis and be relegated to process validation rather than final product testing. Due to molecular diversity, no specific purification method can be recommended, as the separation depends on the difference in affinity (selectivity) between the HCP and the target protein for chosen chromatographic media and operating conditions. The use of a combination of different chromatographic principles is recommended. Most proteins will be removed (CIP) by means of 0.1–1 M NaOH (make sure that equipment, filters, and chromatographic media are not affected by NaOH). Several analytical methods have been used to monitor HCP including SDS-PAGE, 2DE, WB, and immunoassays. SDS-PAGE separates molecules according to the molecular weight of the protein. The method, which is semiquantitative, has a sensitivity of 100 pg/band (silver staining). 2-DE (IEF in combination with SDS-PAGE) provides the most powerful separation of protein mixtures. The method, which is semiquantitative, provides the widest window of the methods used with a sensitivity of 100 pg/spot. The WB method provides information about immunological identity. The sensitivity is 0.1–1 ng/band and the method is qualitative. Immunoassays are widely used for HCP analysis in drug substances and drug products. The method depends on the nature and the quality of antibodies used, and not all proteins (regardless of quantity) are detected. The method also provides the highest sensitivity (<0.1 ng/mL). Two types of assays have been used to detect HCPs: generic and specific assays. In generic assays, the HCPs typical for a given expression system are detected. If the assays are based on competently produced antibodies raised against cell lysates, the quality may be adequate or better than those produced by individual companies. Generic assays are typically used during process development, as process specific assays by definition are not available before the process has been locked. However, it is also recommended to use generic HCP assays for lot release for several reasons. Firstly, the protein 231