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
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.
[FIGURE 1 OMITTED]
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
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.
[FIGURE 2 OMITTED]
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
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 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