**5. Performance characteristics**

In order to produce a reliable assessment of method performance, all necessary performance characteristics should be evaluated in carefully designed experiments. ICH guideline Q2R1 specifies which performance characteristics should be evaluated for validation. However, interpretation of the table for protein products is not straightforward. This is due in part to the fact that ICH Q2R1, Q6B and the industry used different nomenclature to describe the type of methods. The table below details the performance characteristics that should be assessed during qualification, and subsequently during the confirmatory validation experiments for protein products.

Analytical Method Validation for Biopharmaceuticals 123

Nearest Reporting Interval

due to the inability of bioassays to provide precision that would justify such a reporting interval. Therefore, bioassays with a standard deviation of 11.8 would result in a reporting interval of 1. Similarly, an HPLC assay with a standard deviation of 1.3 for the main peak would result in a reporting interval of 0.1. Reporting intervals for impurities (minor peaks) need to be consistent with reporting intervals for the main peak. In general, STD of equal or less than 2 (in units reported by the method) is required to ensure a reporting interval of one decimal place. The argument can be raised that for low level, minor analytes (for example, the dimer in SEC present at 1%), the requirement for STD to be at or below 2% is too generous. This will result in an RSD of 200% for the peak. In such an instance, this would indicate that the minor peak is well below the detection level, because theoretically the RSD at the LOD

level should not exceed 33% (Long and Winefordner 1983; Hayashi and Matsuda 1995)

obtained during qualification for protein products.

Horwitz and Albert 1997; Horwitz and Albert 1997)

where, C is the concentration of the analyte in mg/g.

fully suitable for the intended application.

concentration method), will be 16 and 2.8 %, respectively.

Standard Deviation (in reported units)

**Table 2.** Recommended Nearest Reporting Results Based on Standard Deviation

The table below proposes the nearest reporting intervals based on standard deviations

≤2.0 0.1 ≤20 1

Method precision is closely linked to the concentration of the analyte. The best-known relationship between analyte concentration and RSD is the Horwitz equation (Horwitz 1982;

1-0.5 ) logC( RSD = 2

Based on the Horwitz equation, the precision of the measurement, expressed as RSD,

The Horwitz relationship can provide good guidance for method precision targets during method development and qualification. Intermediate precision obtained during these studies should meet the variability derived from the Horwitz equation for each individual analyte. If, during execution of the qualification experiments, the precision of the measurements exceeds values derived from the Horwitz equation, this may indicate that the assay may need to be redeveloped, or that the technology utilized in the assay may not be

Typically, proteins are available for analysis as solutions, with concentrations ranging widely from 1 g/ml (e.g., a growth factor) to 100 mg/ml or higher (e.g., a monoclonal antibody). In such cases, expectations for the RSD of measurements of the main protein analyte in these solutions, based on the Horwitz relationship (e.g., using protein

doubles for each decrease of analyte concentration of two orders of magnitude.


\*In some cases may be required by USP <1225>.

**Table 1.** Performance Characteristics that need to be Evaluated During Qualification/Validation by Method Type
