The ICH Q2(R1) guideline “Validation of analytical procedures: text and methodology” is the most important guideline used for analytical method validation. According to GMP, each method which is used for release or stability testing of drugs in a quality control laboratory of a pharmaceutical company needs to be validated or in case of compendial methods verified before being allowed to be used for routine analysis. Validation and verification are the proof that the method is suitable for its intended use. For the methods not mentioned in any pharmacopoeia i.e. in-house developed analytical methods = non-compendial methods, the ICH guideline provides information about the parameters used for validation, which differ depending on the type of method. Therefore, the methods need to be clustered.
Thus, the analytical methods are mainly categorized into 3 major types (identification tests, impurity tests and assays). This subdivision is also reflected in the definition of pharmaceutical quality by the German Medicines Act (AMG) in section 4: "Quality is the nature of a medicinal product, determined by identity, content, purity and other chemical, physical and biological properties or by the manufacturing procedure". Put simply, it provides answers regarding the following questions:
- Does it contain what is declared? (--> identity)
- Does it exclusively contain what is declared? (--> purity)
- Does it contain as much as declared? (--> content)
1. Identification tests
As the name suggests, identification tests are performed to characterize the identity of an analyte in a given sample. It is a requirement of the authorities to proof that in the drug you'd like to sell, really the drug substance is inside which is promised to possess the function of healing. It is often done by comparing the property of an analyte to that of a reference standard. For identification tests specificity (sometimes also termed as “selectivity” although defined differently, see also our article about that topic) is important. The identification parameter “specificity” essentially requires the method to discriminate the analyte from structurally similar molecules.
E.g. a capillary isoelectric focussing (cIEF) method to identify a known monoclonal antibody charge variant among a pool of other charge variants is an example of an identification test. Other examples are e.g. peptide mapping as you get a specific cleavage pattern just belonging to your protein of interest or somehow “old-fashioned” western blotting as you use specific antibodies just able to bind to your target protein. More modern, although following the same principle, is an immunofluorescence detection method to determine the identity of viral live vaccines. And while we are on the subject of viral vaccines, i.e. pharmaceuticals containing nucleic acids, PCR is also a suitable identity test, since specific primers ensure that only a defined gene sequence typical of this virus is amplified.
Much simpler identification methods are of course used in a pharmacy. Therefore, in the European Pharmacopoeia (Ph. Eur.) easy identification methods such as e.g. color reactions are listed for most active pharmaceutical ingredients.
2. Impurity tests
The impurity tests are performed to accurately define the purity profile of the sample to show that all impurities present in the drug are below acceptable limits. Hence to proof its harmlessness for the patient. All drugs have to be shown to be as safe as possible. It goes without saying that it must be clarified in advance which impurities / degradation products are to be expected and which analytical method is suitable for the specific detection of these substances in addition to the other ingredients (as also mentioned by the Aide-mémoire AiM 07123101 of the ZLG). As per the requirements, the method could be either a quantitative or a limit test for the determination of impurities in the sample. In any case, it should reflect the purity of the analyte in the sample. Some more validation characteristics are required for quantitative tests than for limit tests. Applying a quantitative method, you’ll get a result with “real”, scalable value and you know exactly how much (which quantity) of the substance you’ve determined is inside the sample (like in this example). Whereas when you apply a limit test, you just get a result like “nothing can be seen” when it is still below the limit or “something is inside” being above the limit, but you don’t know the amount. Colorimetric / photometric methods with a color changeover after reaching an appropriate limit are examples of limit tests. Another example is a HPLC/MS method to detect toxic compounds in a sample (check this publication). Limit tests are used to detect frequently occurring contaminants that are only tolerable in low concentrations but inevitable, e.g. substances for which there are also limit values in the Hazardous Substances Ordinance (German: Gefahrstoffverordnung). In the European Pharmacopoeia (Ph. Eur.) there are limit tests for e.g. methanol, formaldehyde, and arsenic.
Assays are usually performed for the quantification of the analyte in a sample. They could either asses the content of the analyte or the potency of it. For both aspects appropriate methods are required by the authorities. This is due to the fact, that it must be shown that the claimed amount of active pharmaceutical ingredient is indeed inside the drug product and that the active pharmaceutical ingredient is indeed “active”. In other words, assays essentially measure either how much of the analyte (in this case the active pharmaceutical ingredient) is present in the sample or its declared potency.
E.g. a photometric method for the determination of Fluvastatin-Natrium or a TLC-densitometric procedure for the estimation of clobetasol propionate in topical solutions are examples for methods to determine the content. In case of protein drugs, the content is often determined by a simple UV 280 nm measurement. A method determining an enzyme’s activity like a clot lysis assay for tissue plasminogen activator (tPA) is an example of a potency test. For drugs like antibodies to treat cancer by binding to specific cell receptors and inducing cell death, a specific cell culture assay (bioassay) is used to demonstrate potency.
Sometimes, however, content and potency can’t be distinguished so easily. This is the case, for example, with live vaccines. In case of live viral vaccines (e.g. against rotavirus), the amount of infectious virus particles in the vial can be determined using a PFU virus titration or in case of bacterial live vaccines (e.g. against tuberculosis bacteria) by determining the bacterial plate count. This clearly sounds like content determinations. However, since the potency depends on the number of viruses or bacteria present and the dose was previously determined during clinical studies, the viral plaques that have formed or the colonies that have grown not exclusively indicate their amount (= content), but due to their "liveliness" the potency of the vaccine.
Maybe you've already wondered why there is sometimes information above 100% in content determinations. Can any content be higher than 100%? Of course not in reality, but even the best analysis method can't be so specific that it only detects the analyte of interest. Impurities that can react in the same way are always also compulsorily detected. It is therefore important to quantify the impurities as well (with a different method).
In summary, these tests serve to guarantee the pharmaceutical quality by evaluating
- the product-specific (physicochemical) characteristics (--> identity)
- degradation and by-products or other impurities (--> purity) and
- the amount of the active ingredient present (--> content).