The impurity clearance and mapping study is to assess the capacity of the biological manufacturing process steps (especially downstream purification steps) to clear impurities, including elemental impurities, process-related impurities and product-related impurities. In particular, for Process Related Impurities (PRI), data will be collected from batch record sampling points and supplemental sampling plan to evaluate the removal of impurities such as Host cell protein (HCP), DNA, Residual Protein-A, high molecular weight (HMW), antifoam (Simethicone Emulsion), detergents (e.g., Triton X-100), growth factors (e.g., MTX, IGF, etc.), and other impurities that are process or product specific.

Data source should also include development reports, process characterization reports, and Process Performance Qualification (PPQ) study reports. Appropriate statistical analysis and Logarithmic Reduction Value (LRV, min., max., and average values) for impurities are calculated when applicable. The LRV calculation is given below, at end of the study, addition of each step LRV will be used to generate overall LRV, usually LRV ≥ 4 is acceptable value to show the steps can effectively remove related impurities.

The testing results should support sufficient impurity removal during manufacturing process, and also demonstrate that the downstream purification steps are effective and consistent in controlling the purity profile. Examples of testing sampling points during manufacturing are listed below

Characterization studies of impurity clearance studies using simulate impurities can be conducted in development labs (e.g., PD or MS&T labs), they include spike the load material with impurity at levels higher than those observed in manufacturing, and the load materials was then processed through filter and columns to evaluate removal effectiveness. The following conditions should be considered during studies:

The test columns need to be packed with the appropriate resins and pass Height Equivalent to a Theoretical Plate (HETP) and asymmetry criteria to demonstrate suitability for testing. Columns are loaded to maximum capacity to challenge removal.

Buffers should be obtained from manufacturing or prepared at laboratory scale using same raw materials as manufacturing. Load materials should be obtained from manufacturing if possible, frozen storage and thawed materials are also permit in consideration of timelines and project priorities.

Simulate impurities, such as Simethicone, genomic DNA standard reagent, 5% Triton X-100 spike, lab generated HMW spike, protein A ligand, etc., can be used for the study. Spike of impurities can be done at different concentration (e.g. 10, 100 or 1000ppm of protein A ligand) or worst-case concentration to evaluate difference. 

Equipment such as balance, conductivity/pH meter, spectrophotometer need to remain within calibration during study

Analytical methods should be qualified and suitable for intended use.

Process parameters must meet acceptance criteria that are defined in the protocols.  

The experiment runs are duplicated or tripled to confirm the results, if possible.