Chromatographic Method Development in

an Analytical Quality by Design (AQbD)

Framework
James Morgado*, Kimber Barnett, David
Fortin, Pedro Daddario, Pankaj Aggarwal,
Jian Wang

COSMOS Conference Presentation

August 19, 2015
Abstract

• Analytical methods provide critical data in support of the understanding and control of pharmaceutical materials. There
are a multitude of approaches to methods development from utilization of the “favorite” column, stationary phase or
vendor to systematic method screens employing software optimization and nuances in between. The demonstration
that the method is suitable for its intended purpose is shown from the method development (ensuring the method
performs as required) through method validation and subsequent use. The method needs (e.g., purpose, specificity,
sensitivity, cycle time, accuracy, precision) must be well understood, and these requirements used to build a method
that meets the needs of the program.
• Quality by Design (QbD) is well established in the development and manufacture of pharmaceutical drug substance
and drug product processes. The aim of QbD is to design a quality product that consistently delivers the intended
performance. These same principles and concepts have been applied to the development of methods and termed
Analytical QbD (AQbD). The outcome of AQbD is the design of a quality, robust method that consistently delivers the
intended performance. The knowledge obtained during method development, optimization and performance verification
help justify the establishment of the method operable design region (MODR), the “design space” of the analytical
method. As a result of AQbD methods development, enhanced method understanding and robustness will result in
fewer method failures and transfer issues over the lifecycle of the method.
• This presentation will discuss AQbD concepts, a typical reversed-phase LC workflow and a case
study. The chromatographic method development occurs in a series of experiments meant to roughly
understand robustness and select draft conditions. Based upon the project specific method
development knowledge gained and experience, a risk assessment is performed to identify risk factors
that should be experimentally evaluated in a design of experiment (DOE). An assessment of the DOE
data provides a method operable design region and center point/control strategy (defined method
parameter set points and operating space) for the method.

2
Outline

• What is AQbD
– Concept
– Workflow
– Benefits
• Case Study
– RP LC Method development workflow
• Risk Assessments
• DOE
• MODR
• Conclusions

3
What is (Analytical) Quality by Design?

• The aim of Process QbD is to design a quality product

that consistently delivers the intended performance.
– International Conference on Harmonization Q8 – Q10, Q11 (Draft)

• The outcome of Analytical (AQbD) is the design of a

quality and robust analytical method that consistently
delivers the intended performance.
• The knowledge obtained during method development,
optimization (including risk assessments and design of
experiments) and performance verification provide
enhanced method understanding…

4
Quality by Design Elements (Parallels)
The same principles that are applied to Manufacturing Processes (ICH Q8-11)
can be applied to Analytical Methods

Processes Element Analytical Methods

Target Product Profile Establish Criteria Analytical Target Profile
(ATP)
Process Design Design supported by sound Method Design
science
Risk Assessment Identify Risks Risk Assessment
Process Design Space Demonstrate Robustness Method Operable
Design Region (MODR)
Control Strategy Establish Appropriate Control Strategy
Controls
Monitor Process Monitor Performance Monitor Method
Performance Performance
Continual Improvement Evaluate innovative Continual Improvement
approaches
5
 Why QbD for Analytical Methods?

Facilitates a framework to…

• Understand, identify, reduce and control, sources of analytical
method variability
• Enhance method understanding improves robustness and
ruggedness
• QbD concepts offer an opportunity to enhance current practices

The concepts are not new…

• Activities are better connected throughout the method lifecycle
• We’re applying the concepts in a rigorous, consistent, and
harmonized manner to provide accurate, precise, and reliable
results

6
Benefits of AQbD

• Ensures high quality and rugged methods

• Understand method “Operational Boundaries”
• Normal Operating Conditions set for “ensured method robustness”
• Risk assessments to identify and rank risks to method robustness
• DOEs to understand the relationship with and de-risk experimental
conditions in a systematic approach
• Ensures more consistent methods
• Enables transfer of analytical methods group to group and site to site
• Reduces inventory of columns via standardization…though priority is
a robust/rugged analytical method
• Reduces method variability
• Leads to a better understanding of the contributions to variability of
analytical measurements.
• Enables better understanding (and control) of process variability as
σ2 (measured) is a  (σ2 (process) + σ2 (method))

7
AQbD Workflow (Method Design, Evaluation, Control)

Verify MODR &

Define Objectives Method Selection Develop Method Knowledge
Establish Control
(Method Design) (Method Design) Understanding Management
Strategy

Develop
Analytical Target MODR and Identify Quality
Profile Control Strategy Attributes
(Risk Mitigation)

Method
Understanding Quality
Perform Risk Assessment
Experimental Strategy Identify and Prioritize
MethodParameters

Risk Assessment ID Experiments

Prioritize Understand
Experiments CQA = f(CPP)

8
Method Design: The Analytical Target Profile

• Describes the needed performance of the method in terms of

sources of “Uncertainty”
– Expressed in terms of accuracy and precision jointly,
through a probability statement of measurements meeting
pre-defined criteria

• Can be structured so that the ATP is not linked to a specific

technique
– Linked to the result generated
– The method is just a tool to generate a result(s)
– More than one technique may satisfy ATP requirements
• (e.g., HPLC, CE, IC, SFC, etc…)

9
Method Design: Analytical Target Profile

• ATP describes measurement requirements

(method performance criteria)
– based on a probability of reportable result being within a given
range from the “true value”

The procedure must be able to accurately quantify [drug] in film

coated tablets over the range of 70 – 130% of the nominal
concentration with accuracy and precision such that
measurements fall within ± 3.0% of the true value with 95%
probability

10
Method Design: Traditional Method (Precision/ Bias)
Validation Criteria Graphical Representation

Low bias, high

variability
•At boundaries: no trade-off
between method bias and
precision

•It is possible to accept a

method with both high bias
(low accuracy) and high
variability (low precision)

•Does not consider TOTAL

Criteria method variability
Bias (Accuracy): NMT 3.0%
Variability (Precision): NMT 2.0%
11
Method Design: The ATP Equation defines a joint
(Precision/ Bias) Probability Contour
ATP plot: measurement ± 3.0% of the true value with ≥95% probability

• A contour line is a
Traditional Criteria
curve connecting
points of a function of
two variables, where
the function has the
ATP Criteria same value.

12
Outline

• What is AQbD
– Concept
– Workflow
– Benefits
• Case Study
– RP LC Method development workflow
• Risk Assessments
• DOE
• MODR
• Conclusions

13
AQbD Method Development Workflow

14
Wave 0: Log D Plot of KPSS (Informational Assessment)

Compound Log D vs. pH

5 Compound 1

4 Compound 2

Compound 3
3
Compound 4 (API)
2 Compound 5
Log D

1 Compound 6

Compound 7
0
0 2 4 6 8 10 12 14 Compound 8
-1 Compound 9

-2 Compound 10

Compound 11
-3
pH

From this plot, it is observed that an area of stable Log D values (minimal change with pH) is found
between approximately pH 6 and 11, and should produce robust chromatographic conditions (stable
retention with minor changes in mobile phase pH). Also, Log D values vary under low and high pH
values (may result in non-robust chromatographic conditions) and compound 6 may not be retained
under low pH conditions.

15
Wave 1 Screen: What and Why ?

• Rationale for the selection of UPLC Columns, pH, and

Organic Conditions?
• Analytes • Organic solvents
– 44 compounds (8 mixtures) – Acetonitrile
– Range of neutrals, acids, zwitterions and – Methanol
bases of varying polarity

• Buffers • Columns
– 0.1% TFA (pH unadjusted, approx. 2.0)
– Waters BEH
– 0.1% HCOOH (pH unadjusted, approx. 2.7)
• C8, C18, RP C18 and Phenyl
– 10 mM NH4CH3COO (pH unadjusted, approx.
pH 6.8) – Zorbax
– 0.1% NH4OH (pH unadjusted, approx. pH 10.6) • Not tested with methanol
10 mM NH4HCO3 pH 10.0 • Stablebond C8, C18, CN
• Extend C18
– Thermo
• Gold C18, PFP, Aq C18

16
Wave 1 Screen: How ?

• Principle Component Analysis (PCA) used to Identify Orthogonal

Selectivity Conditions

17
Wave 1 Screen: Column, pH, Organic

0.1% TFA 0.1% formic acid 10 mM NH4OAc 0.1% NH4OH

pH 2 pH 2.6 pH 6.8 pH 10.5
Column
ACN MeOH ACN MeOH ACN MeOH ACN MeOH
100mm x 2.1mm
ACQUITY UPLC BEH C18 (1.7 um) X X X X X X X X
ACQUITY BEH Shield RP18 (1.7 um) X - X - X - X -
ACQUITY CSH Phenyl-Hexyl (1.7 um) X X X X X X X X
ACQUITY UPLC HSS C18 (1.8 um) X - X - X - - -

• Upon completion of wave 1 screening, in most cases promising (e.g., good peak shape, selectivity for analytes)
separation condition are identified. Note that baseline separation of the eleven components is achieved in
approximately eight minutes and the (neutral) pH is included in the area of pH robustness identified in wave 0.

Selected Wave 1 condition:

Column Name ACQUITY UPLC BEH C18
Flow Rate 0.4 ml/min
Gradient 95-5B (15 min)
Organic ACN
Aqueous 10 mM Ammonium Acetate
Temperature 45 °C

18
Wave 2: Narrow pH Screen

• Wave 2 is a multi-5-point experiment that fits the retention data to pH (determined

from wave 1) using a 2nd order quadratic function which accounts for potential
curvature within the narrow pH range.
Quadratic expression: ln (k’)= a+ bX + cX2

• Computational tools (e.g., ACD LC simulator) are used to aid visualization

and optimization of the mobile phase pH.
10 mM NH4OAc / Acetonitrile
ACQUITY UPLC BEH C18 (1.7 um) pH 5.5 pH 6.0 pH 6.5 pH 7.0 pH 7.5

• Maximum resolution is predicted at pH 6.5.

• A pH 6.5 will be fixed for wave 3.
• The simulation also provides an assessment
of robustness (resolution ≥ 1.5) from
approximately pH 5.8 to 7.0.

19
Wave 3: Temperature, Gradient Optimization (Why ?)

• Complex molecules do not always conform to the simplistic 1st order

linear relationships between retention and temperature.

• Optimize the gradient and temperature so that critical pair resolution,

run time, and method robustness can be further established.

Linear behaviour for simple molecule

ln(k’)= b. (1/T) + a

Higher order behaviour - complex molecule

Polynomial expression
ln (k’)= a+ b.(1/T)-1 +c.(1/T)-2

20
 Wave 3: Temperature, Gradient Optimization

• Upon completion of wave 2, mobile phase pH, column and organic

modifier are fixed.
• The main goal of this experiment is to optimize gradient and
temperature so that critical pair resolution, run time, and method
robustness are further defined.

Stationary Phase: Temperature

ACQUITY UPLC BEH C18 30, 45, 60 ° C 45
2.1 x 100mm (1.7 um)
Gradient
10mM Ammonium Acetate 5 – 95 % Acetonitrile
Gradient
pH 6.50 15 minutes time
45 minutes (min)
A six-point experiment was carried out where gradient slope
steepness (15 and 45 minute gradient time from 5% to 95% 15
organic) and temperature (30°C, 45°C and 60°C) are evaluated.
Data from this experiment is subsequently imported into an
optimization program (e.g., DryLab, ACD LC simulator) for 30 45 60
simulation of optimum and robust conditions. Temperature (C)

21
 Wave 3: Temperature, Gradient 2-D Optimization

This fine tuning of the conditions allows for a good simulation of selectivity, enabling optimization of run
time with gradient and temperature conditions. The optimized conditions are depicted below.
ACQUITY UPLC BEH C18
60
Column
5.37 (2.1  100 mm, 1.7 m)
5.15
55 4.93 Mobile Phase 10 mM NH4OAc pH 6.5
4.70
4.48
Organic Phase Acetonitrile
50 4.25 Injection Vol 2 L
4.03
3.81 Column Temp 32C
Column Temperature, °C

45 3.58
3.36
Flow Rate 0.4 mL/minute
40
3.13 Detection UV @ 260 nm and ES+ MS
2.91
2.69 Assay Conc ca. 0.1 mg/mL
2.46
35
2.24
Sample Diluent (80/20) 0.1N HCl/ACN
2.02 Run Time 10 minutes
1.79
30
1.57 Elution Mode Gradient
1.34
1.12
Gradient Time (Min) % Aq % ACN
25
0.90 Timepoint 0 0.0 82 18
0.67
20 0.45 Timepoint 1 1.0 82 18
0.22
25 30 35 40 45 50 55 60 65 70 75 80 85 0.00
Timepoint 2 8.0 43 57
Solvent B, % Timepoint 3 8.1 82 18
Timepoint 4 10 82 18
Constraints:
• Resolution ≥ 2.0
• Runtime  10 min

22
Wave 3: Temperature, Gradient Optimization

A verification run is carried out to ensure that the in-silico results can be replicated experimentally.
The figure below shows the simulated and experimentally verified results. Note the accuracy of the
simulation with the experimental results. With the completion of the three wave work flow, the
method development aspect is complete in a matter of a few days.

Initial
Wave 3 simulation of
optimum conditions

Experimental verification

23
Outline

• What is AQbD
– Concept
– Workflow
– Benefits
• Case Study
– RP LC Method development workflow
• Risk Assessments
• DOE
• MODR
• Conclusions

24
Develop Method Understanding:
Risk Assessments: Quality Risk Management
Method
Understanding

Risk Identification
List method parameters that could potentially affect the
method

Examples:
Material Properties: Formulation composition, Solubility,
Tablet hardness

Extraction: Diluent, Extraction type, Shaker speed

Mobile Phase: Reagent purity, solvent grade

Injector: Strong/weak needle wash, Volume

Separation: Column temperature, Flow rate, Ionic

strength, pH

Detector: Wavelength, Data rate, Band width

25
Develop Method Understanding:
Risk Assessments: Quality Risk Management
Method
Understanding

Risk Analysis
Qualitative or quantitative process to estimate
risk associated with method parameters
Failure mode and effects analysis
QRM Tools:
- Process Flow Diagrams
- Cause & Effect Matrix
- FMEA
- Ishikawa Diagram

Ishikawa Diagram
Process Flow Diagrams

26
Develop Method Understanding:
Risk Assessments: Quality Risk Management
Method
Understanding

Risk Evaluation - Weightings

Compare the identified and analyzed risk against
given risk criteria.

Agree which method parameters need to be further

evaluated experimentally, or controlled through
specific method instructions (or other means). (e.g.,
Comprehensive Risk Assessment…below)

27
Develop Method Understanding:
Risk Assessments: DoE Implementation
Method
Assessment of experimental results via DOE or One Off experiments Understanding

Risk Evaluation - DOE

Compare the identified and analyzed risk against given
risk criteria.
Agree which method factors need to be further
evaluated experimentally, or controlled through specific
method instructions (or other means). Response
results evaluated against the ATP…

% Organic Parameters (Factors): Units Low Target High

- +
1 Initial Organic Content % 13 18 23
+ + 2 Initial Hold Time min 0.5 1.0 1.5
3 Final Organic Content % 52 57 62
Buffer Buffer
Strength Strength
4 Gradient Time min 7.5 8.0 8.5
- - 5 Mobile Phase pH N/A 6.0 6.5 7.0
+ +

Temp
6 Column Temperature °C 27 32 37
Temp
7 Buffer Concentration mM 10
- -
- pH + - pH +
8 Flow Rate mL/min 0.4

28
Outline

• What is AQbD
– Concept
– Workflow
– Benefits
• Case Study
– RP LC Method development workflow
• Risk Assessments
• DOE
• MODR
• Conclusions

29
Design of Experiments (DOE Models)

• The objectives of the design were the clear determination

of Main Effects and 2-Factor Interactions
• Linear models were generated for all Retention Time,
Resolution, and critical Tailing Responses

DoE Model: 26-1 (2-level Fractionated Factorial , 6- Factors, 36 Design

Points, and 4 Center points, 40 - Runs)

Y   0  1  x1   2  x2  3  x3   4  x1  x2  5  x1  x3   6  x2  x3   7  x1  x2  x3

A B C D
Where,…Y = Response (e.g., Retention Time, Resolution, Tailing,...etc.); A = Intercept; B = Main Effect Terms; C =2-Factor
Interaction Terms; D = 3-Factor Interaction Terms

30
Design of Experiments (Optimization Strategy)

DOE Optimization

Statistical analysis software was used to evaluate the multi-response analysis through the use of
numeric and graphical optimizations tools by setting response acceptance limits and evaluating the
resultant interactions plots. The results of the numeric optimization are presented as a desirability
interaction plot, the numeric limits were set to maximize all resolution responses for the respective
models. The set-point from the end of wave 3 is designated as a red dot. The plot indicates that
optimal resolution results for all pairs would be obtained if the center point was shifted to the lower
left quadrant.
Design-Expert® Software Desirability
23.00

0.438
Desirability 0.452
Design Points 22.00
0.463
0.468 0.473
1 0.479
Optimization is often 21.00 0.485
0
achieved iteratively with 20.00

respect to the combination

A: G1%Organic
0.491
X1 = B: G1Time
19.00

of factors X2 = A: G1%Organic
0.497
18.00 4
Actual Factors
0.506
C: G2%Organic = 57.00
D: G2Time = 8.00 17.00
0.514
E: pH = 6.50
0.520 0.485
F: Temp = 32.00 16.00
0.525
0.479
0.473
0.531
15.00 0.468
0.463
0.452
0.540
14.00 0.438

0.418

13.00

0.50 0.75 1.00 1.25 1.50

B: G1Time
31
 DoE Data Analysis (Optimization and Simulation)

Simultaneous multi-dimensional projection and optimization of response

data from DoE study…further examples of optimization approaches…
(1) 3-D PCA: PLS-DA Simulation (2) 2-D Factorial Analysis
Approach for all resolutions… Desirability Approach
Minimum Res < 1.5
2.5 1.5 < Minimum Res < 2.0 Optimal
2.0 < Minimum Res < 2.5
Design-Expert® Software direction
2
Optimal 2.5 < Minimum Res
Factor Coding: Actual
Desirability 51.00
Desirability
1.5 1.000
Score #3 (Explained variance=9%)

direction
Prediction 0.579

0.000
1
50.00
X1 = D: Buffer pH
0.5 X2 = E: Column Temp

Actual Factors

E: Column Temp
0 A: Gradient Hold Time = 1.00 49.00
B: Gradient Organic T=1 = 3.00 0.500
C: Gradient Organic T=2 = 72.00
-0.5
0.200
48.00 0.100
0.400
-1

-1.5
47.00 0.300

-2

-2 46.00

-1

0 45.00
0.200

5.80 5.90 6.00 6.10 6.20

1 -2
-1 -1.5
0 -0.5
2 0.5
1.5 1
Score #1 (Explained variance=46%) D: Buffer pH

Method Condition Min Res=1.46

Score #2 (Explained variance=28%)

32
Outline

• What is AQbD
– Concept
– Workflow
– Benefits
• Case Study
– RP LC Method development workflow
• Risk Assessments
• DOE
• MODR
• Conclusions

33
Method Operable Design Region

 A description of the multidimensional combinations and interaction

of method parameters that have been demonstrated to meet
suitability requirements and conversely the Analytical Target Profile
(not always a discrete range)
 A region over which changes to Normal Operating Conditions
(NOC) can be made without risk to method performance.

Unevaluated Space
Knowledge Space

MODR

NOC

34
 MODR

Experimental DOE Chromatographic Conditions and Method Operable Design Region (MODR)
Exploration of the multivariate design space through an iterative approach indicates that the center point
(target) conditions obtained from end of Wave 3 experiments should be relocated….
(Old) (Revised) MODR
Method Parameters (Factors): Units
Target Target Low High The final MODR is defined
1 Initial Organic Content % 18 16 13 19 by the actual linear
2 Initial Hold Time min 1.0 0.75 0.5 1.5 equations for the critical
3 Final Organic Content % 57 55 52 62
responses set to their
respective acceptance limit,
4 Ramp Time min 8.0 8.0 7.5 8.5
with respect to the method
5 Aqueous Mobile Phase pH pH 6.5 6.5 6.0 7.0 factor input values
6 Column Temperature °C 32 35 27 37
7 Buffer Concentration mM 10 10
8 Flow Rate mL/min 0.4 0.4
Initial

Experimental results of the final

chromatographic conditions

9
Wave 3

7 11
1 2 3 5 6 8 10

3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0
Time (min)

35
Verify MODR & Establish Control Strategy
Method Evaluation and Verification Strategy

Experimental Verification (Example)

Example of Parameters confirmed at boundary points of the MODR
•Precision (API and Degradants): (Injection Repeatability)
•Accuracy (API): (Accuracy Determined by Wet Spike in Placebo versus an Ext. Standard))
•Repeatability (API): (Accuracy Precision across the design space)
•Accuracy (Specified Degradant): (Accuracy at 0.05% and 0.2% Determined by Wet Spike Assay
in the presence of Placebo determined by Area % vs. Theoretical)
•Repeatability (Specified Degradant): (Accuracy Precision across the design space)

Multivariate method factor combinations identified as critical boundary points

Factor Name Units Point 1 Point 2 (Nominal) Point 3
A Temperature °C 37 40 43
B Flow Rate mL/min 0.90 1.0 1.10
C Organic Modifier %B 62 64 66

36
Conclusions

• AQbD: holistic view towards methods development

– Science and Risk based concepts
• Principles incorporates:
– Method needs (Analytical Target Profile)
– Systematic Method Development
– Systematic sample preparation
– Risk assessments
– Multivariate (Univariate as needed) Design of Experiments
– MODR ----> Method Verification
• Aids development of consistent and rugged methods
– Reduced resources over method lifecycle as compared to traditional approaches.
• Improves method transferability
• Provides understanding of method robustness/operational boundaries that
could present risk to meeting the ATP

37
Acknowledgements

• Members of the AQbD Methods Development Group

– Jian Wang, Jeff Harwood, Dave Fortin, Kimber Barnett, Pankaj Aggarwal, Pedro
Daddario

• Members of the AQbD Core Strategy Team

– Kimber Barnett, Timothy Graul, Brent Harrington, Loren Wrisley, Neil Clayton,
Melissa Hanna-Brown, Chuck Melucci, Steve Chestnut, Janice Ensing, and Mike
Cohen

• Dave De Antonis (VP Analytical R&D)

• Audience

38
Questions?

39