Using Fusion QbD as an Analytical Quality by Design Software for Method Development

©2019 Waters Corporation 1COMPANY CONFIDENTIAL©2019 Waters Corporation COMPANY CONFIDENTIAL
Using Fusion QbD® as an
Analytical Quality by Design
Software for Method Development
Pittcon
20-March-2019
Oral Session: 01:30-2:30
Fadi Alkhateeb, Senior Scientist
Waters Corporation, Milford MA

©2019 Waters Corporation 2COMPANY CONFIDENTIAL
• QbD is a systematic approach to development …
–That includes defining a design space, control strategy and continual improvement to
increase method robustness and understanding
• … that begins with predefined objectives…
–Defining criteria (performance goals) to be used to measure the quality of the method
• …sound science and quality risk management…
–Statistical treatment of data to provide quantitative values to goals and limits of
performance goals
• QbD approach is implemented with an experimental design
*ICH: International Conference on Harmonization
Quality by Design (QbD)
A systematic approach to development that begins with predefined objectives and
emphasizes product and process understanding and process control, based on sound science
and quality risk management.
ICH* Q8(R2) Guidelines
Some Definitions

Design of Experiment (DoE)
Formal Experimental Design: A structured, organized method for determining the
relationship between factors affecting a process and the output of that process.
Also known as “Design of Experiments”.
ICH Q8(R2) Guidelines
• A structured and organized method of gathering empirical
knowledge
• DoE are represented as a multi-dimensional space (matrix)
• Screening parameters (study variables) are the dimensions of
this space
• Number of experiments is optimized using an appropriate study
design (full factorial design & partial factorial design)
X
(Temperature)
Y (Gradient Time)
Z (pH)
Best Method
Some Definitions

Chromatographic
Parameters
Numerical
(e.g. Temperature, Flow
Rate, pH, Injection Volume,
etc.
Categorical
( e.g. Stationary Phase,
Mobile Phase, Additive
Type, etc.
Fusion QbD – LC Method Development Module
Numerical factors are factors with
levels which are represented by
numbers on a continuous scale.
Categorical factor levels represent
disjoint (discontinuous) systems

DOE = Statistical Sampling (Efficient Multifactor Studies)
Consider two variables – five study levels each:
25 Methods
Gradient
Time
(min)
Oven Temp.
(°C)
Full Factorial Approach =
All Possible Combinations
25 30 35 40 45
12
10
8
6
4
13 Methods
Oven Temp.
(°C)
Gradient
Time
(min)
Design of Experiments =
Statistical Sampling
25 30 35 40
12
10
8
6
4

DOE – Adding a 3rd and 4th Factor to the Study
3rd Variable – pH
Oven
Temp
∆tG
pH
Oven
Temp
∆tG
pH
C18 Phenyl C8
Oven
Temp
∆tG
pH
Oven
Temp
∆tG
pH
4th Variable – Column Type

Fusion QbD – Supports ALL your Waters LC Systems

• Select preliminary study variables such as
injection volume, λ Max, initial hold time
• Select “major” factors & study ranges
Columns, Organic solvents, pH
• Identify method conditions for acceptable
separation
• Data are imported to Fusion
• Analyze and process the imported results to
create a design space
• Fine tune “minor” factors, Column temperatures
Gradient slopes Gradient times Flow rates
• Identify method for optimum performance &
robustness
Optimization
Importing Data to
Fusion and Developing
Knowledge
Space
Initial Sample Workup
Selecting
Chromatographic
Parameters/Screening
Fusion QbD Method Development: Steps

Method Development for Symbicort® Using Fusion Software
Budesonide, Mwt= 430.53 g/mol
(A pair of epimers) Formoterol, Mwt= 344.41 g/mol
Budesonide Related
Compound L,
Mwt= 428.22 g/mol
Budesonide Related
Compound E,
Mwt= 428.22 g/mol
Budesonide Related
Compound G,
Mwt= 432.25 g/mol
( A pair of epimers)

QbD Method Development: Steps
Select preliminary chromatographic parameters
Variables
• Injection volume
• Wavelength
• Two Columns max
Constants
• Initial hold time
• Gradient Time
• Temperature

• Select preliminary study variables such as injection
volume, λ Max, initial hold time
Method Development of Budesonide/Formoterole
and their Related Compounds, ongoing project

Only one gradient slope can be
screened in a screening experiment.
In our experiment the one slope was
10-95% organic. Gradient time was
made variable as a numerical factor.
𝐺𝑟𝑎𝑑𝑖𝑒𝑛𝑡 𝑆𝑙𝑜𝑝𝑒 = 𝑉° × ∆∅ ×
1
𝐹 × 𝑡𝑔
Selecting
Chromatographic
• Creating a Design

Selecting
Chromatographic
• Creating a Design

Selecting
Chromatographic
• Exporting methods to Empower

Fusion Creates all methods and method
sets for Empower (time saving!)
The created Sample Set can be
modified/customized
It also creates all methods and method
sets for equilibration/conditioning.
Selecting
Chromatographic
• Exporting methods to Empower

1. Formoterol
2. Budesonide Related E
3. Budesonide Related L
4. Budesonide Epimer B
5. Budesonide Epimer A
6. Budesonide Related G (Epimer 1)
1
23
4
5
6
7

Importing Data to
Knowledge
Space

Importing Data to
Knowledge
Space
• Execute Search

Importing Data to
Knowledge
Space
• Best Overall Answer
Shaded regions indicates pH and Gradient Time combinations that do not meet performance goals.
APR

Second Screening
Empirical Titration Curves
Developed using an H-Class
High pH
ACQUITY H-Class PLUS

1. Formoterol
1
23
4
5
6
7

Second Screening
Creating design space and finding the
“Acceptable Performance Region”
Acceptable Performance
Region (APR)

• Fine tune “minor” factors, Column temperatures
Gradient slopes Gradient times Flow rates
• Identify method for optimum performance &
robustness
Optimization

Optimization
1
2
3 5
6
741. Formoterol

Optimization

Tracking Peaks by UV spectra is very difficult
 Chemically related compounds, such as process impurities, and degradation products often have
similar UV Spectra
 Coeluting peaks have spectra that are a blend of the individual peaks
– Difficult to interpret
– Cannot determine which components are coeluting by UV Spectra alone
– For related species, UV tracking requires separate injections using pure standards, which
decreases efficiency of laboratory's workflow
Empower MS Tracking
 Empower MS Peak tracking uses the mass-to-charge (m/z) ratio to automatically track each peak in a
sample injection
 Using mass spectra data for peak tracking enables analytical laboratory to correctly monitor peak
retention during method development experiments
Peak Tracking
MS Peak Tracking for Method Development

MS Peak Tracking for Method Development:
Empower 3 MS Peak Tracking
 Specify Spectra for assigned mass
 The Assigned Mass Precision past the decimal point
- Default values of 0 or 1 are suitable for a quadrupole mass
detector

Spectra for Assigned Mass
Spectra for
Assigned
Mass
Description
None Peak tracking processing is disabled. As a result,
the Assigned Mass Precision field is disabled.
Apex Peak tracking is enabled and the Base Peak value
obtained from the peak’s apex spectra is copied into
the peak’s Assigned Mass Value field. Enter a value
in the Assigned Mass Precision field to specify how
many digits to display after the decimal when
formatting the Assigned Mass Value field as a string
in the Assigned Mass field.
Combined Peak tracking is enabled and the Base Peak
(Combined) value obtained from the peak’s
combined spectra is copied into the peak’s Assigned
Mass Value field. The combined spectrum is the
average mass spectrum across the peak centered
on the apex. Enter a value in the Assigned Mass
Precision field to specify how many digits to display
after the decimal when formatting the Assigned
Mass Value field as a string in the Assigned Mass
field.
% Peak height
Combined
spectra
Peak height

 Peak table displays assigned mass
(m/z) to each peak
 Fields in software
– Assigned Mass
– Assigned Mass Value (m/z)
– Notes
CSH C18
Process chromatographic
data
Review Data Window

Peaks with the Same Assigned Mass
Review Data Window
2 peaks with the
same masses
AU
0.00
0.20
0.40
Minutes
1.00 1.50 2.00 2.50 3.00 3.50
1-286.1
2-300.1
3-342.1
4-316.0
5-182.0
6-202.0
7-202.0
8-224.1
9-258.0
Peak #7 - 2.483 - QDa 1: MS Scan
Leading Apex Trailing
202.0
196.0
202.0 196.0
202.0
2 coeluting peaks
Imp. C: 202.0 m/z
Imp. H: 196.0 m/z
Method development: CSH Phenyl Hexyl

Manually Modify Assign Mass
Review Data
Manually modify Assigned Mass for peak 7
Right click on the
table to modify
the mass
 Enter the correct mass, and a note explaining why the
change was made
 After you have made your changes, don’t forget to save
the new result

Manually Modified Mass
Review Data
Manually assigned mass
for peak 7
AU
0.00
0.20
0.40
Minutes
1.00 1.50 2.00 2.50 3.00 3.50
1-286.1
2-300.1
3-342.1
4-316.0
5-182.0
6-202.0
7-196.0
8-224.1
9-258.0

Facilitate Method Development: Column Screening
AU
0.00
0.15
0.30
AU
0.00
0.20
0.40
AU
0.00
0.15
0.30
AU
0.00
0.12
0.24
Minutes
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00
CSH C18
CORTECS C18+
CSH Phenyl Hexyl
HSS PFP

PeakTracker – Auto-imports all Required PDA & QDa Data
Used for samples with peaks
which Do Not Ionize
Used for samples with peaks
which have No UV Absorbance
Copyright © 2019 S-Matrix Corporation. All Rights Reserved.
Fusion AQbD MS Peak Tracking

PeakTracker – Utilization of Acquity PDA and QDa Data
Peak Tracking
Fingerprint
Data
UV Chromatogram
Total Ion Chromatogram (TIC)
PeakTracker can automatically address these complex separation
and tracking challenges:
• Auto-deconvolution of partially and completely co-eluted peaks,
• Two or more peaks with identical mass data.
• Non-ionizable and non-absorbing compounds.
Global Tracking Method (GTM)
PeakTracker automatically builds
a customizable GTM by scanning
all UV and TIC chromatograms to
identify all integrated peaks.

Peak Has No
UV Absorbance
Peak is Present
In the TIC
Just add Peak Names for non-
absorbing peaks to the
automatically imported MS data
for the peaks displayed in the
Global Tracking Method.
PeakTracker will automatically
track the peaks and add them
to the UV chromatograms for
visualization and modeling.
Mass and UV
Spectral
Analysis
Window in
Fusion QbD
PeakTracker – Peaks with No UV Absorbance

Peak Has
UV Absorbance
Peak Does Not
Ionize
PeakTracker – Peaks which Do Not Ionize
UV Spectra
Analysis Window
in Fusion QbD

PeakTracker – Auto & Manual Update of Results Data
Response Data
View Filter
Easy edit mode.
• Blue background for data auto-updated by
PeakTracker.
• Yellow background for missing data which
can be edited by user.
Blue Background
Yellow Background

PeakTracker – Auto-updates Results Data for Analysis
PeakTracker automatically associates compound names with all of the UV
results data computed by the CDS for all identified peaks in each experiment
chromatogram. It also automatically deconvolutes co-eluted peaks, and
updates missing UV results data for the co-eluted peaks.

• Using Fusion QbD as a tool for developing LC methods can be
advantageous as it:
–Automates the entire process of method development
–Saves time by determining the minimum number of
experiments needed for valid results
–Provides tools to visualize the impact of chromatographic
parameters
• Utilizing the QDa detector and Empower speed up the
method development process through efficient peak tracking.
• The new Fusion Peak Tracker feature automatically tracks
peaks across all experiment chromatograms.
• Fills in missing data for co-eluting peaks, and generates
correct results data sets for non-absorbing peaks for robust
method optimization.
Conclusions

Using Fusion QbD as an Analytical Quality by Design Software for Method Development

Using Fusion QbD as an Analytical Quality by Design Software for Method Development

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