Approaches for Developing Residual Impurity Methods
The first step is to determine how to handle the sample and may involve removing the protein. Acetonitrile or sodium chloride
can be added to precipitate out the protein, and then the supernatant can be obtained by centrifugation or filtration. Care
must be taken to ensure that the residual impurity of interest is not co-precipitated or removed with the protein through
The next step may involve further sample preparation including extraction, distillation, and cleanup. Extraction approaches
may include liquid–liquid extraction with appropriate solvent or solid phase extraction. Some methods may require derivatization,
which is an approach that modifies the impurity of interest to make it more amenable to a specific detector. These steps must
be evaluated through validation.
Next, the determinative approach must be investigated. For example, a volatile organic compound will most likely be best suited
for GC, whereas a nonvolatile compound by HPLC. Also, the detector must be chosen based on the analyte of interest, the sample
matrix, the sensitivity, and the selectivity required. As mentioned above, HPLC with charged aerosol detection may be a good
approach for compounds that do not respond to UV. Also, if the compound can be ionized, MS/MS is usually a good approach because
of selectivity and sensitivity.
After these conditions are established, the method is evaluated for potential interferences and limit of detection and limit
of quantitation within its particular matrix. Also, the method must be tested to ensure acceptable levels of precision, accuracy,
and linearity for the intended application. It then can be used as a qualified method, or a protocol could be drafted to perform
a formal method validation.
Monitoring of residual impurities seen in bioprocessing can be quite challenging. Because of the range of potential impurities,
many different analytical approaches may need to be used. After developed, these methods must be qualified or validated for
the intended use.
Jon S. Kauffman, PhD, is the director of method development & validation and biopharmaceutical services at Lancaster Laboratories,
Lancaster, PA, JKauffman@lancasterlabs.com
1. US Food and Drug Administration. Guidance for industry. Genotoxic and carcinogenic impurities in drug substances and products.
Rockville, MD; 2008 Dec.
Available from: http://www.fda.gov/downloads/Drugs/GuidanceComplianceRegulatoryInformation/Guidances/ucm079235.pdf
2. European Commission. EMEA Guideline on the limits of genotoxic impurities. Brussels, Belgium; 2006 Jun. Available from: