Cross-Validation of Bioanalytical Methods: When, Why and How?

Pharma IQ

Bioequivalence studies are extremely important for the pharmaceutical industry to ensure that generic drugs are up to standard, and make their way to the market in a timely manner.

Around two-thirds of clinical studies undertaken worldwide are to determine bioequivalence. Therefore changes in the guidelines which govern such research could have large ramifications on those working in the field of drug development.

Across the industry it is becoming accepted that bioequivalence studies should be included earlier within the development process, yet there is still a lack of information and guidance on how this can be achieved.

The United States Food and Drugs Administration (FDA) recently released new pharmaceutical industry guidance on the bioequivalence recommendations for specific products, which details how the organisation will make information on how to design specific bioequivalence studies available to members of the public.

Streamlined Process

Previously bodies that wished to carry out bioequivalence studies had to contact the FDA for the specific guidance and wait for a reply, which proved time consuming.

In the guidance, the organisation said it believes "making this information available on the Internet will streamline the guidance process, making it more efficient than the previous process."

General guidance on bioequivalence studies has been available to the pharmaceutical industry since 2000.

The changes will assist members of the pharmaceutical industry filling out abbreviated new drug applications (ANDA), who are required to prove that their product is bioequivalent to the reference listed drug. However, the FDA will also not be required to publish information on a specific bioequivalence study before approving ANDAs.


In recent years the FDA has been working on streamlining the process for getting generic drugs to market, publishing the Critical Path Opportunities for Generic Drugs in 2007.

The report identified a number of issues associated with bioequivalence studies, including the problems with bioequivalence studies for drugs which cannot be assessed based on blood plasma levels, such as asthma inhalers, nasal sprays, and topical skin applications.

European Guidance for the Pharmaceutical Industry

Further guidance has also been issued for pharmaceutical industry companies working within Europe by the European Medicines Agency (EMEA).

Specific guidelines relating to bioequivalence investigations were issued in late January 2009, which created requirements for the design, conduct and evaluation of such studies. However, the issue of creating bioequivalence studies for drugs which cannot be tested using generic blood plasma concentrations was not addressed.

The comprehensive guidelines for the pharmaceutical industry addressed the number and nature of test subjects, required doses, selection of the reference product and the standardisation of the subjects and test conditions.

More recently the EMEA published guidelines on the validation of bioanalytical methods for the pharmaceutical industry. A consultation on the guidance was closed on May 31st 2010, before the final document was published in June.

Under the guidelines there is a requirement for bioanalytic methods to be verified and guidance for when cross-validation or partial validation could be used as an alternative to complete analytical validation.

"A complete method validation should be performed for any analytical method whether new or based upon literature," the guidance stated.

"The reference standards used for the analytical validation and analysis should be obtained from an authentic and traceable source," it added.

Partial validation will be applicable when "minor changes are made to an analytical method that has already been validated," which can include the movement of the study to another laboratory or a change of equipment.

Cross-validation will occur when data is obtained from separate study sites and should be carried out prior to the analysis.

The guidance outlined: "The outcome of the cross-validation is critical in determining whether the obtained data are reliable and whether they can be compared and used."