20/12/2017 Design of Experiments for Analytical Method Development and Validation

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Design of Experiments for Analytical Method

Development and Validation
Design of experiment is a powerful development tool for method characterization and
method validation.
Mar 01, 2014
By Thomas A. Little, PhD [1]
BioPharm International
Volume 27, Issue 3

Design of experiments (DOE) is a

well-proven characterization RELATED CONTENT
approach within product and process
development and a key aspect of
quality by design. Recently, more Understanding and Modeling Product and
attention has been placed on Process Variation [2]
applying DOE to analytical methods.
DOE for analytical methods has three Performing a Protein Purity Analysis
major applications: method
Comparability Study [3]
development for new methods or
those that need improvement,
method validation, and quantitation of Analytical Best Practices [4]
the influence of analytical methods
on product and process acceptance and out-of-specification (OOS) rates. Method
development seeks to understand where critical process parameters are in the
analytical method and to minimize their influence on accuracy and precision. DOE for
method validation seeks to validate the analytical method for a range of concentrations
so that changes in formulation or concentration will not require additional validation as
they are changes within a characterized design space. Once methods have been
developed, qualified, and validated, the impact they have on OOS rates and process
capability needs to be quantified and evaluated to determine their fitness for use.

A systematic approach for using DOE for analytical method development and
validation is discussed in this paper and was written in line with the International
Conference of Harmonization (ICH) Q2(R1), Q8(R2), and Q9 guidelines (1-3). A
quantitative understanding of the factors that influence resolution, linearity, precision,
and accuracy is integral to applying DOE to method development.

Textbook approaches to DOE generally suggest a sequential approach to DOE:

screening studies, characterization studies, and optimization of the method or process.
This approach applied to analytical methods is often not practical as 10-20 methods
are often used for drug substance and drug-product evaluation and the amount of time
and materials needed to follow the three steps (i.e., screen, characterize, and
optimize) would consume unreasonable amounts of resources. The sequence
generally recommended by the author for method development is understanding the
purpose of the study, perform risk assessments to screen out factors that may or may
not have an influence on the analytical method (screening variables by logic and an
examination of their scientific potential for influence), and characterization studies to
quantify and minimize their influence on precision, accuracy, and linearity.

Assays and measurement systems must be viewed as a process (see Figure 1). The
measurement process is made up of methods, standards, software, materials,
chemistry, reagents, analysts, sample preparation methods, environmental conditions,
and instrumentation/equipment. Quality risk management and statistical data analysis
techniques should be used to examine the process of measurement and identify
factors that may influence precision, accuracy, linearity, signal to noise, limits of
detection and quantification, and/or any other assay attributes to achieve optimal
assay results.

Figure 1: Measurement process elements.

DOE for Method Development

Design of experiments can be applied to many aspects of method development;
however, the following will provide the typical steps for designing and analyzing
experiments for analytical methods.

• Define the purpose (e.g., repeatability, intermediate precision, accuracy, LOD/LOQ

linearity, resolution).
• Define the range of concentrations the method will be used to measure and the
solution matrix it will be measured in.
• Develop/define the reference standards for bias and accuracy studies.

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20/12/2017 Design of Experiments for Analytical Method Development and Validation
• Define the steps in the method and any associated documentation.
• Determine the responses that are aligned to the purpose of the study.
• Complete a risk assessment of all materials, equipment, analysts, and method
components aligned to the purpose of the study and the key responses that will be
quantified.
• Design the experimental matrix and sampling plan.
• Identify the error control plan and run the study.
• Analyze the study and determine settings and processing conditions that improve
method precision and minimize bias errors. Document the design space of the method
and associated limits of key factors.
• Run confirmation tests to confirm settings improve precision, linearity, and bias.
Evaluate the impact of the method on product acceptance rates and process
capability.

Identify the Purpose of the Method Experiment

The purpose of the analytical method experiment should be clear (i.e., repeatability,
intermediate precision, linearity, resolution). The structure of the study, the sampling
plan, and ranges used in the study all depend on the purpose of the study. Designing
a study for accuracy determination is very different from a study that is designed to
explore and improve precision. Accuracy, for example, does not require sample
replicates to estimate the mean change in the response. Precision, however, requires
replicates and duplicates to evaluation variation in the sample preparation and in other
aspects of the method. The purpose of the study should drive the study design.

Define the Range of Concentrations to be Evaluated

Define the range of concentrations used to measure and the solution matrix it will be
measured in. Ranges of the concentration will generate the characterized design
space so they should be selected carefully as it will put restrictions on how the method
may be used in the future (see Figure 2). Normally five concentrations should be
evaluated per ICH Q2R1.

Figure 2: Concentration ranges and other constituents.

Define all Reference Standards Used in the Study
Develop/define the reference standards for bias and accuracy studies. Without a well-
characterized reference, standard bias/accuracy cannot be determined for the method.
Care should be made in selecting, storing, and using reference materials. Stability of
the reference is a key consideration and accounting for degradation when replacing
standards is critical.

Identify all Steps in the Analytical Method

Lay out the flow or sequence used in the analytical method. Define the steps in the
method (e.g., standard operating procedures [SOPs], procedures, or work
instructions), all chemistries, reagents, plates, and materials used in the method and
all instruments/sensors and equipment. Identify any steps in the process, materials,
analyst techniques, or equipment that may influence bias or precision.

Determine the Reponses

Determine the responses that are aligned to the purpose of the study. Raw data and
statistical measures such as bias, intermediate precision, signal to noise ratio, and CV
are all responses and should be considered as independent results from the method.
Make sure the data table is set up so that it can collect the raw data, the statistics can
be easily generated from that raw data, and there is a direct link from the statistics to
the data.

Perform a Risk Assessment

A risk assessment of the analytical method is used to identify areas/steps in the
method that may influence precision, accuracy, linearity, selectivity, signal to noise,
etc. Specifically, the risk question is: Where do we need characterization and

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20/12/2017 Design of Experiments for Analytical Method Development and Validation
development for this assay? Complete a risk assessment of all materials, equipment,
analysts, and method components aligned to the purpose of the study and the key
responses. The outcome of the risk assessment is a small set (3 to 8) of risk-ranked
factors that may influence the reportable result of the assay. There are many kinds of
factors, so factor identification and how to treat the factor in the analysis are crucial to
designing valid experiments. There are controllable factors: continuous, discrete
numeric, categorical, and mixture. There are uncontrollable factors: covariate and
uncontrolled. In addition, there are factors used in error control: blocking and
constants (see Figure 3).

Figure 3: Analytical method risk assessment example.

Design the Experimental Matrix and Sampling Plan

For small studies using two or three factors, a full factorial type design may be
appropriate. When the number of factors rises above three, a D-optimal type custom
DOE design should be used to more efficiently explore the design space and
determine factors that impact the method. There are many good software programs
today that help the user define statistically valid experiments and can be customized to
meet the user’s needs.

The experimental matrix is one consideration and the sampling plan is another.
Replicates and duplicates are essential to quantification of factor influence on
precision. Replicates are complete repeats of the method including repeats of the
sample preparation, duplicates are single sample preparations but with multiple
measurements or injections using the final chemistry and instrumentation. Replicates
provide total method variation and duplicates provide instrument, plate, and chemistry
precision independent of sample preparation errors. If the experiment is designed
properly many of the requirements for method validation (Figure 4) can be directly met
from the outcomes of the method DOE.

Figure 4: Method validation quick reference guide.

Identify the Error Control Plan

Make sure to measure and record uncontrolled factors during the study. Analyst name,
equipment ID, out time, hold times, ambient temperature, temperature at the beginning
and end of an operation, transfer times, pH, and incubation time may hold valuable
information on factors that impact the method. What factors will be restricted or held
constant during the study? Do you need to block for batch, lot, sample prep, or
instruments that may have an influence on the reportable result?

Analyze the DOE and Determine the Settings and Design Space
Use a good multiple regression/analysis of covariance (ANCOVA) software package
that allows the DOE factors and any uncontrolled variables to be correctly evaluated.
Analyze the study and determine settings and processing conditions that improve
method precision and minimize bias errors (see Figure 5). When using statistics from
the method (e.g., CV, mean, standard deviation), rather than raw data, make sure to
weigh the analysis by the number of replicates or duplicates to assure statistical tests
and confidence intervals are meaningful. Determine the design space and allowable
ranges for all key factors that influence the method.

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Figure 5: DOE design space for method precision and bias.

Verify the Model and Determine the Impact of the Method on Specifications and
Capability
Run confirmation tests to confirm settings improve precision, linearity, and bias.
Evaluate the impact of the method on product acceptance rates and process
capability. Using an accuracy-to-precision (ATP) model (4), it is possible to visualize
the relationship of precision and accuracy on product acceptance rates. The ATP
model shows how changes in precision and accuracy impact product acceptance rates
and the assay error design space relative to product acceptance specification limits.
The attention paid to method development, validation, and control will greatly improve
the quality of drug development, patient safety, and predictable, consistent outcomes
(see Figure 6).

Figure 6: Accuracy to precision modeling.

Summary
Design of experiment is a powerful and underutilized development tool for method
characterization and method validation. Analytical professionals need to be
comfortable using it to characterize and optimize the analytical method. If used
properly and during development, DOE will provide significant improvements in
precision and a reduction in bias errors. It will further help to avoid costly and time

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consuming validation studies as concentrations are modified in formulations and
dosing schemes are changed for drug product and drug substance.

References
1. ICH, Q2(R1) Validation of Analytical Procedures: Text and Methodology (ICH,
2005).
2. ICH, Q8(R2) Pharmaceutical Development (ICH, 2009)
3. ICH, Q9 Quality Risk Management (ICH, 2006).
4. T.A. Little, Assay Development and Method Validation (2014).

About the Author

Thomas A. Little is president of Thomas A. Little Consulting, [email protected]. [5]

© 2017 UBM. All rights reserved.

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