analytical methods & Validation

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					          Understanding and
          Implementing Efficient
          Analytical Methods
          Development and
          Jay Breaux , Kevin Jones, and Pierre Boulas

                                                              o ensure compliance with
Analytical methods                                            quality and safety standards,
development and validation                                    the United States, Europe,
play important roles in the                             Japan, and other countries have
discovery, development, and                             published compendia, or pharma-
manufacture of                                          copeias, that describe official test
pharmaceuticals. The official                           methods for many marketed drug
test methods that result from                           products. For example, compendial
these processes are used by                             analytical methods found in United
                                                        States Pharmacopeia 25 (USP 25)
quality control laboratories
                                                        are legally recognized analytical
to ensure the identity, purity,
                                                        procedures under section 501 (b)
potency, and performance of                             of the Federal Food, Drug, and
drug products.                                          Cosmetic Act. For these compendial
                                                        methods, USP provides regulatory
          Jay Breaux, PhD, is senior                    guidance for method validation (1).
          director, Analytical Services                 In addition, validation of
          (Chapel Hill, NC), Jay.Breaux@                analytical methods is covered by
; Kevin Jones is
          director, Technical Specialists
                                                        the United States Code of Federal
          Group (North Brunswick, NJ),                  Regulations (CFR). Specific refer-
; and                  ences are 21 CFR 211.165 (e) and
          Pierre Boulas, PhD,                           21 CFR 211.194 (a).
          is senior manager, Analytical                    Method validation is defined as
          Services (Wilmington, NC),
, all
                                                        the process of proving (through sci-
          at AAI International.                         entific studies) that an analytical
                                                        method is acceptable for its intended
6   Pharmaceutical Technology   Analytical Chemistry & Testing 2003      www.phar
use. Recent guidelines for methods                         In recent years, a great deal of
development and validation for new                      effort has been devoted to the
noncompendial test methods are                          harmonization of pharmaceutical
provided by the FDA draft docu-                         regulatory requirements in the
ment, “Analytical Procedures and                        United States, Europe, and Japan.
Methods Validation: Chemistry,                          As part of this initiative, the Inter-
Manufacturing, and Controls                             national Conference on Harmo-
Documentation” (2). This recent                         nization (ICH) has issued guide-
document applies to the method                          lines for analytical method
development and validation process                      validation. The recent FDA methods
for products included in investiga-                     validation draft guidance document
tional new drug (IND), new drug                         as well as USP both refer to ICH
application (NDA) and abbreviated                       guidelines (2). Analytical guidance
new drug application (ANDA)                             documents recently published by
submissions. Therefore, expecta-                        the ICH are the following:
tions from regulatory agencies                          ● stability testing (Q1)

for method development and                              ● validation of analytical procedures

validation are clear.                                     (Q2)
8   Pharmaceutical Technology   Analytical Chemistry & Testing 2003      www.phar
● impurities in drug substances and        veloped. However, the following
  products (Q3)                            steps are common to most types of
● specifications for new drug sub-         projects:
  stances and products (Q6).               ● method development plan

   Additional regulatory guidance            definition
can be found on the FDA Web site           ● background information and on             gathering
the ICH Web site              ● laboratory method development

These sites ensure access to current       ● generation of test procedure

methods development and valida-            ● methods validation protocol

tion guidelines.                             definition
   The methods validation docu-            ● laboratory methods validation

mentation requirements for IND             ● validated test method generation

and NDA submissions are outlined           ● validation report.

in the chemistry, manufacturing               A well-developed method should
and controls (CMC) guidance                be easy to validate. A method should
document (2). The current trend            be developed with the goal to rapidly
continues to be in the direction of        test preclinical samples, formulation
phase-dependent methods develop-           prototypes, and commercial sam-
ment and validation. Nonvalidated          ples. As the methods development
screening methods are used to              and validation processes advance,
monitor the synthesis of active            the information gathered is cap-
ingredients or to confirm their            tured in the design and subsequent
identity during discovery and pre-         improvement of the method. Ideally,
clinical research. Analytical meth-        the validation protocol should be
ods are progressively optimized and        written only following a thorough
a preliminary validation package is        understanding of the method’s
furnished as part of the IND appli-        capabilities and intended use. The
cation before Phase I safety trials        validation protocol will list the ac-
are initiated. All analytical meth-        ceptance criteria that the method
ods should be fully optimized and          can meet. Any failure to meet the
validation completed before the            criteria will require that a formal
NDA is submitted at the end of             investigation be conducted.
Phase III studies.                            The required validation parame-
   Method validation is a continu-         ters, also termed analytical perform-
ous process. The goal is to ensure         ance characteristics, depend upon
confidence in the analytical data          the type of analytical method. Phar-
throughout product development.            maceutical analytical methods are
                                           categorized into five general types
The method development and                 (3):
validation processes                       ● identification tests

The steps of methods development           ● potency assays

and method validation depend               ● impurity tests: quantitative

upon the type of method being de-          ● impurity tests: limit

                             Pharmaceutical Technology   Analytical Chemistry & Testing 2003   9
Analytical Chemistry & Testing

● specific tests.                                       submitted as parts of the NDA or
   The first four tests are universal                   ANDA.
tests, but the specific tests such as
particle-size analysis and X ray                        Advances in technology
diffraction are used to control                         and equipment
specific properties of the active                       Recent progress in methods devel-
pharmaceutical ingredient (API) or                      opment has been largely a result of
the drug product.                                       improvements in analytical instru-
   Validation requirements depend                       mentation. This is especially true
upon the type of test method,                           for chromatographs and detectors.
including                                               Isocratic and gradient reverse-phase
● specificity: ability to measure de-                   HPLC have evolved as the
  sired analyte in a complex mix-                       primary techniques for the analysis
  ture                                                  of nonvolatile APIs and impurities.
● accuracy: agreement between                              The HPLC detector of choice for
  measured and real value                               many types of methods develop-
● linearity: proportionality of meas-                   ment is the photodiode array (PDA)
  ured value to concentration                           detector because it can be used for
● precision: agreement between a                        both quantitative and qualitative
  series of measurements                                analysis. The use of a PDA detector
● range: concentration interval                         to determine peak purity of the
  where method is precise, accurate,                    active ingredient in stressed samples
  and linear                                            greatly facilitates the development
● detection limit: lowest amount of                     of stability-indicating assays.
  analyte that can be detected                             The emphasis on the identifica-
● quantitation limit: lowest amount                     tion of trace impurities and
  of analyte that can be measured                       degradants has led to the increased
● robustness: reproducibility under                     use of hyphenated techniques such
  normal but variable laboratory                        as liquid chromatography–mass
  conditions.                                           spectrometry (LC–MS) and
   Only specificity is needed for an                    liquid chromatography–nuclear
identification test. However, the full                  magnetic resonance spectroscopy
range of specificity, accuracy, linear-                 (LC–NMR). This trend will con-
ity, range, limit of detection (LOD),                   tinue with the need to better define
limit of quantitation (LOQ),                            degradation pathways.
precision, and robustness testing is                       The ultraviolet (UV) absorbance
needed for more-complex methods                         detector remains the most common
such as quantitative impurity                           HPLC detector for potency and
methods.                                                impurity analysis. Once specificity
   The validated test method is                         has been demonstrated, the PDA
included in the validation report                       detector is replaced with a variable
that summarizes the results of the                      wavelength detector and the HPLC
validation studies. Both the valida-                    effluent is monitored at fixed wave-
tion report and test method are                         lengths. Stability-indicating and
10    Pharmaceutical Technology   Analytical Chemistry & Testing 2003    www.phar
Analytical Chemistry & Testing

impurity methods often are required                    mentation is beyond the scope of
to measure analytes within a wide                      this article. However, several meth-
concentration range. For example,                      ods should be noted. Advances in
process impurities and/or degrada-                     the use of nondestructive infrared
tion products may be present at                        (IR) and near-infrared spectroscopy
levels of 0.1%, and the main active                    (near IR) and NMR techniques are
ingredient typically is present at the                 particularly promising for methods
nominal concentration (100%).                          development scientists.
This amount is well within the
linear range of a fixed wavelength                     Issues and challenges
detector but not within the linear                     For a methods development and
range for LC–MS detectors.                             validation program to be
   Recent FDA and ICH guidance                         successful, a holistic approach is
about chiral drug products and                         recommended. A common chal-
impurities has posed new challenges                    lenge encountered during methods
for methods development scientists                     development and validation is that
(3). However, recent advances in the                   methods are typically developed by
use of chiral HPLC columns has                         the R&D department, whereas vali-
greatly facilitated progress in this                   dation is typically the responsibility
area. The advances are primarily a                     of a validation group. It’s important
result of the introduction of chiral                   that the R&D and validation groups
stationary phases (CSPs) prepared                      work as one team.
by reacting amylose or cellulose de-                      Various groups also may be re-
rivatives with silica. The new CSPs                    sponsible for ensuring the suitabil-
allow trace levels of enantiomeric                     ity of the methods to support early
impurities to be measured.                             clinical phases and commercial
   Gas chromatography remains the                      manufacturing. The transfer of
method of choice for the analysis of                   analytical methods from one group
volatile compounds. Gas chro-                          to another then becomes an impor-
matography with mass spectrome-                        tant step for ensuring that the
try detection (GC–MS) is increas-                      proper validation is in place to jus-
ingly being used to identify                           tify its intended use.
impurities and to determine active                        Because the method will be run
ingredient peak purity in stressed                     by several groups during its pro-
samples.                                               gression from development to vali-
   Advances in laboratory robotics                     dation, the method must be robust.
and automation also are beginning                      This means the method should
to be applied to methods develop-                      provide reliable data, both on a
ment and validation. Development                       wide range of equipment and in the
teams are using laboratory robotics                    hands of several chemists. A com-
to develop automated methods for                       mon weakness in development and
high-volume tests.                                     validation of methods is that the
   An in-depth review of all the                       methods are not robust enough. If
recent advances in analytical instru-                  robustness is not built into methods
12   Pharmaceutical Technology   Analytical Chemistry & Testing 2003    www.phar
early in development, then the                the fact that, in some cases, the tools
result most likely will be loss of effi-      used to characterize reference stan-
ciency during routine QC testing              dard materials are being developed
and a lengthy and complicated vali-           and validated at the same time as
dation process as well.                       the reference standard itself.
   Another challenge encountered
early in the development of meth-             Conclusion
ods intended to support stability             The efficient development and vali-
studies is ensuring that the method           dation of analytical methods are a
is stability indicating. This process is      critical elements in the development
typically achieved by conducting              of pharmaceuticals. Success in these
forced-degradation studies. The               areas can be attributed to several
design and execution of these stud-           important factors, which in turn
ies requires thorough knowledge of            will contribute to regulatory com-
the product being tested as well as a         pliance. Experience is one of these
good understanding of the analysis            factors––both the experience level
technique.                                    of the individual scientists and the
   As mentioned previously, new               collective experience level of the
regulatory guidelines are being pub-          development and validation depart-
lished governing the expectations of          ment. A strong mentoring and
regulatory agencies throughout the            training program is another impor-
world for methods development and             tant factor for ensuring successful
validation. Another challenge is that         methods development and valida-
many pharmaceutical companies                 tion. Companies must maintain an
must upgrade methods to meet cur-             appropriate level of expertise in this
rent regulatory standards. From a             important dimension of developing
simple method improvement to a                safe and effective drugs.
complete redevelopment and subse-
quent cross-over to an older                  References
method, the upgrade of analytical               1. USP 25–NF 20 (United States Phar-
methods can be a daunting task. For                macopeial Convention, Rockville,
                                                   MD, 2002), p. 2256.
this reason, one must be alert to cur-          2. FDA, “Analytical Procedures and
rent trends in regulatory guidelines               Methods Validation: Chemistry,
and to adopt a proactive approach                  Manufacturing, and Controls,”
to changes that may affect develop-                Federal Register (Notices) 65 (169),
ment and validation programs.                      52,776–52,777 (30 August 2000).
                                                3. “International Conference on Harmo-
   Finally, one of the key require-                nization; Draft Guidance on Specifi-
ments for methods validation                       cations: Test Procedures and Accep-
(which is also one of the key chal-                tance Criteria for New Drug
lenges), is that only well-character-              Substances and Products: Chemical
ized reference materials with well-                Substances,” Federal Register (Notices)
                                                   65 (251), 83041–83063 (29 December
documented purities should be                      2000). P T
used during method validation
activities. The challenge stems from
                              Pharmaceutical Technology   Analytical Chemistry & Testing 2003   13