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Analytical methods validation in-process control methods for the manufacture of active pharmaceutical ingredients

Analytical methods validation in-process control methods for the manufacture of active pharmaceutical ingredientsIn-process methods are key components of quality control in a chemical manufacturing plant. These methods ensure that a production reaction step conducted by trained operators within the entire validated process will produce a quality chemical entity in the expected yields. The presence of impurities and related compounds (derived from the reaction or secondary reactions) is a critical parameter that determines a synthetic material's quality.

Chemical processing differs from product manufacturing. For example, the manufacture of a finished product typically involves a molecular entity that is stable under normal conditions and can be stored for prolonged periods without losing its physical and chemical characteristics. Most chemical reactions, however, require very tight controls and close monitoring of their progress because any of several potential result paths may be followed if conditions are not monitored closely. Other factors such as temperature and pressure are critical parameters for the successful completion of the chemical conversion process.

Each chemical reaction is unique. Consider the combination of reactants and the resulting end products, for example. One must examine conditions such as temperature, light, heat, environment, and the reaction vessel's surface. In addition, whether the reaction is chemical or biological is an important factor. Therefore, each process must be analyzed separately and classified according to the International Conference on Harmonization (ICH) Q7A guidance. This task is important because a reaction step may generate an impurity that may be carried over to the active pharmaceutical ingredient (API), regardless of how far apart that process may be from the API.

ICH's Q7A guidance briefly mentions analytical methods validation and does not discuss the in-process control methods for each reaction. The guidance does indicate, however, that as the process gets closer to the manufacture of the key intermediate and the API, the current good manufacturing practices (CGMP) requirements become more demanding.

This article outlines a plan for classifying and validating in-process testing methods and is intended as a foundation for assessing the parameters and acceptance criteria needed for validation.

Matrix and interferences

To characterize a reaction by means of an analytical method, it may be necessary to prepare a matrix, which functions similarly to a placebo for a finished product. A matrix is the combination of the reactants without the main component or precursor being converted. Because of the nature of some reactions, the combination of reagents may not be possible. On the other hand, adducts or complexes can be formed, which would not otherwise be formed in the presence of a component being converted into a product. The preparation of the matrix must be judged by the scientists working with the reaction process.

Classification of methods

For monitoring purposes, analytical methods can be classified according to the manufacturing step in which they are applied. The document indicates that the GMP requirements become more stringent as the synthesis steps approach the API. Beginning with the introduction of the starting material into the process, manufacturing processes can be divided into three classes, which reflect the practices established in the ICH Q7A guidance.

* intermediates production (e.g., alkylation, hydrogenation)

* isolation and purification (e.g., washing, crystallization)

* physical processing and packaging (e.g., micronization).

For the purposes of this article, intermediates production is subdivided into intermediates and key intermediates production. The classification of the methods (e.g., in-process controls and intermediate-release methods) is determined by how far the stage or step is removed from the API. Figure 1 represents the application of the ICH guidance to these classes.


The ICH Q2B guidance enables chromatographic resolution to be used as an indicator of specificity for critical separations, which means that peak purity is not necessary. Furthermore, peak purity should not be a consideration because samples are submitted only to confirm the disappearance of the starting material and the formation of the desired adduct. No peak will be as pure as required when the analysis is a crude reaction mixture.

Solution and standard stability should be included as part of some of the studies. The length of the stability study is defined by the process requirements.

Class 1. The Class 1 classification is exclusive to methods used for in-process control (and monitoring) of intermediate steps during an API manufacturing process. The classification pertains to reactions that are at least two steps from the processing of the key intermediate. Because the formation or source of impurities should be known and each impurity identified, it is possible that in some instances, the classification becomes Class 2 several steps before the key intermediate production.

Class 2. Class 2 is exclusive to methods used for in-process control (and monitoring) of intermediate steps during an API manufacturing process. The classification pertains to those reactions that precede the formation of the key intermediate.

Class 3. This classification includes methods used as intermediate-release methods when the product formed is an intermediate that will be used further after isolation or supplied as a starting material for another synthesis. This classification pertains to the key intermediate or isolated entity that eventually will be converted to an API.

An example of how processes would be classified is shown in Figure 2. Substance D is the key intermediate, one step before the API formation. Substance E represents the final API molecule before purification. Substance B, for example, could be subject to intermediate-release method testing if the material is isolated and the starting material is used in a parallel synthesis. In this case, the purification step does not involve any chemical conversion and the API is structurally identical to Substance E. The purification step can be recrystallization, micronization, or any other physical manipulation of the active that does not involve a chemical conversion or change in chemical structure. In addition, a reaction sequence may involve the isolation of an intermediate that is several steps away from the formation of the key intermediate. The intermediate would be classified as an intermediate-release method or Class 3.


Validation of methods. The suitability of all methods used as in-process control methods and as intermediate-release methods should be verified and documented under actual conditions of use. Each category has a recommended suitability procedure defined. The degree of analytical validation performed must reflect the purpose and stage of the API production process. All analytical equipment must be qualified before it is used for method validation. Complete records must be maintained for any and all equipment modifications made to validate analytical methods.

The validation process may require that intermediates be characterized, isolated, and used as reference markers for establishing the relative retention times. The preparation of a matrix or reaction mixture without the active may help establish unknown peaks and potential interferences. The matrix must be treated according to the procedures established for in-process control monitoring methods.

If an intermediate is not isolated, but reacted in situ to a later step, isolation may not be necessary for its characterization if it is an unstable entity. Should it be a stable molecule, however, its isolation and characterization may be necessary.

Chromatographic methods

Chromatographic methods are validated according to their classification, as discussed previously. The method validation protocol should include a discussion of the method's classification and the justification for the classification. The validation described for each classification is for quantitative chromatographic methods. Chromatographic identification and semiquantitative techniques such as thin-layer methods must be validated (described later in this article). These methods require the determination of accuracy in their semiquantitative level. Table I summarizes the requirements of each classification.

Class 1. Description. Class 1 methods are limit tests and must be validated accordingly. This validation should include a demonstration of the method's detection limit specificity and determination because these steps are far removed from the formation of the key intermediate and API. Therefore, one must be able to identify the peak of interest, properly resolved from the starting materials (reactants). The method should confirm the disappearance of the reactants or the formation of the adduct.