Assays for Controlling Host-Cell Impurities in Biopharmaceuticals

the manufacture of biopharmaceuticals is purification of the drug substance. The downstream process must remove all contaminants, including host cell material such as DNA and cellular protein. Trace amounts of host-cell DNA and proteins can be copurified along with the drug substance. Such contaminants are obviously undesirable because of possible consequences if they are injected into patients along with it. They could potentially cause allergic reactions (proteins) or even transfection of cells (DNA) resulting in tumors. Because of those potential negative effects, regulatory authorities have released several guidance documents about what levels of impurities are acceptable. Residual DNA in final bulk products should be generally lower than 100 pg per therapeutic dose (1). Other references suggest that higher levels may be acceptable (2). The 100pg limit requires a purification process that is very effective and robust in removing DNA — as well as analytical methods that are extremely sensitive and reliable to prove it. Host-cell proteins in the drug substance should be “below detectable levels using a highly sensitive analytical method” (1). As a rule, that amount should not exceed a level of about 100 ppm. But no exact limit is set for proteins; therefore, the specification for proteins must be determined case by case. According to the European Agency for the Evaluation of Medicinal Products, EMEA (3), two different strategies can be used to ensure that a drug substance is within the allowable limits of contamination with DNA or hostcell proteins. You can either validate that a process removes sufficient amounts of such contaminants or perform routine final product testing to determine whether they are present. With the validation approach, known amounts of DNA are spiked into a downstream process. The various steps within that process are then examined to determine their capability to remove the contamination. This approach is most commonly used for DNA. Robustness and consistency in DNA removal also must be shown by measuring the amounts contaminating several batches of product. Routine testing determines host-cell DNA and proteins in each product batch as part of the lotrelease data. No matter which approach is used, the assays and methods involved in determination of residual DNA and proteins must fulfill ICH validation requirements (4, 5). Obviously those techniques must be sensitive enough to determine very low levels of contamination (e.g., in the ppm range). Here we describe some common analytical methods used.