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Quality-by-Design in Method Development
Dr. Daniel Rathmann Head of pharmaceutical development
Agenda
Chromicent – who we are
Traditional vs. Systematic strategies for method
development
QbD – a new approach in development
Case study : Quality by Design in analytical method development
Case study : Fast UPLC method development and method transfer to HPLC (for business needs)
Who we are ?
Company Pharmaceutical Service Provider offering … Method Development in a Quality-by-Design
framework in compliance with ICH Q8 Method Validation in compliance with ICH Q2 Forced degradation studies and impurity
profiling in compliance with ICH Q1 Cleaning Validation Extractables and Leachables Method Transfer Consulting and Training Auditing (GMP)
Who we are ?
Company Founded in 2013, based in Berlin Adlershof
GMP approved by local drug authority
Allowance to handle controlled substances and
narcotics Reference customer and training
facility for Waters Corp.
Co-operation partner of WADA in development of methods
Who we are ?
Analytical dept. HPLC (Alliance) with PDA-, UV-, FL-, RI-,
Conductivity, Electrochemical and Charged Aerosol Detection
UPLC (Acquity classic, H-Class, I-Class) with PDA-, FL-, ELS-, single-MS and tandem-MS-Detection
SFC (Acquity UPC² ) with PDA-, ELSD and tandem-MS-Detection
Ion chromatograph with Conductivity Detection
Prep LC system with fraction collector
Traditional vs. Systematic strategies in
method development
• Mainly by trial and error • Varying one-factor-at-a-time (OFAT) • Problems: - additional or missing peaks
- changes in selectivity - decreasing resolution of the critical peak pair
• Trying to test “quality“ into the method – this is however the wrong way • Results: - time consuming approach
- no understanding of the influence of key factors
Traditional strategy for HPLC method development (I)
One factor at a time - OFAT
X
Y
One factor at a time - OFAT
X
Y
One factor at a time - OFAT
X
Y
• System to system variations • Day to day variation • Column quality changes from batch to batch • Peaks move with pH, temperature and %B
0 10 20 30 40
Problems with non-robust methods
Courtesy of Imre Molnár, Molnár-Institute, Berlin, Germany
• System to system variations • Day to day variation • Column quality changes from batch to batch • Peaks move with pH, temperature and %B
0 10 20 30 40
0 10 20 30 40
Problems with non-robust methods
Courtesy of Imre Molnár, Molnár-Institute, Berlin, Germany
• System to system variations • Day to day variation • Column quality changes from batch to batch • Peaks move with pH, temperature and %B
0 10 20 30 40
0 10 20 30 40
0 10 20 30
Problems with non-robust methods
Courtesy of Imre Molnár, Molnár-Institute, Berlin, Germany
• Chromatographers tried to adapt already validated methods • published in pharmacopoeias (USP, EP, JP) or in literature
Traditional strategy for HPLC method development (II)
Example for adapting an „old“ method
• Official HPLC method for ebastine published in E.P. • Run time (unbelievable) 160 min • Peak width for ebastine (API) = 8 min • Retention factors k* = 0.25 – 70 (recommended 2 – 20)
0 20 40 60 80 100 120 140 160Time (min)
Impu
rity
AIm
purit
y B
Impu
rity
DIm
purit
y C
Impu
rity
F
Impu
rity
G
Eba
stin
Impu
rity
E
• Using experimental design plans as an efficient and fast tool for method development.
• In a full or fractional factorial design a couple of experiments are carried out in which one or more factors are changed at the same time.
• Typical examples are Plackett-Burman design • Software packages (e.g. Fusion AE, DesignExpert)
Systematic LC method development I.) statistical software tools
• A very smart and computer-assisted way of developing a chromatographic method is by using software modeling packages (e.g. DryLab, ChromSword, ACD/LC simulator).
Systematic LC method development II.) modeling software tools
Case study 1: Quality-by-Design
in analytical method development
Initial situation
• Official HPLC method for ebastine published in E.P. • Run time (unbelievable) 160 min • Peak width for ebastine (API) = 8 min • Retention factors k = 0.25 – 70 • Who developed this method ….???
0 20 40 60 80 100 120 140 160Time (min)
Impu
rity
DIm
purit
y C
Impu
rity
F
Impu
rity
G
Eba
stin
Impu
rity
EImpu
rity
B
Impu
rity
A
„Our“ method development strategy by using a Quality-by-Design approach
Quality-by-Design Key components defined by ICHQ8
Analytical method development strategy
Quality target product profile Define method goals / ATP
Critical quality attributes Risk assessment … Critical quality attributes … Linking CQA to CPP
Risk assessment Design of Experiments (DoE) … Screening for stationary and mobile phase
… Optimization
Design space Design space … Select working point and verification
… Method validation and robustness testing
Control strategy Control strategy … system suitability test
Continuous Improvements Continuous Improvements
Step 1: Define method goal / ATP
• Adequate baseline separation of all components (Rs > 2.0) • Minimum analysis time (< 10 min). • k*-values for ebastine and impurities should be between 2 and 10 • Impurities: 0.05% lvl • Visualize a design space, in which the method is robust
Step 2: Risk assessment
Identified influencial parameters • Column (chemistry) • gradient time tG, start and end • temperature T • ternary composition of the eluent • pH of the eluent
Step 3a: Design of experiments – screening for the selection of column
Step 3a: Design of experiments – screening for the selection of column
Design of screening experiments
Four columns Linear gradients of 10 to 90% methanol acetonitrile 2-propanol
Column Mobile phase (Eluent B)
Critical Resolution Rs (crit peak pair)
Acquity UPLC BEH C18
Methanol 1.64 (A,D)
Acetonitrile 1.94 (C,D)
2-propanol 1.88 (C,D)
Acquity UPLC HSS T3
Methanol < 1.5 (C,D)
Acetonitrile < 1.5 (C,D)
2-propanol 1.57 (C,D)
Acquity UPLC BEH Phenyl
Methanol < 1.5 (C,D)
Acetonitrile < 1.5 (C,D)
2-propanol < 1.5 (C,D)
Acquity UPLC HSS C18 SB
Methanol < 1.5 (C,D)
Acetonitrile < 1.5 (C,D, B )
2-propanol < 1.5 (C,D, B)
Step 3b: Design of experiments – Optimization phase
Run experiments on a WATERS Acquity UPLC H-class system
WATERS Acquity UPLC H-class system • Solvent Manager with SSV for up to 9 solvents • Sample Manager FTN • Column Manager for up to 4 columns and different temp. zones • PDA-detector •QDa single-MS-detector •Empower 3
Step 4: Design space (Analyse and process data and build models) 2D-modell tG/T of 100%ACN
tG [min]
T [°
C]
Col
or c
ode:
R
esol
utio
n R
s
2D-modell tG/T of 30% iPrOH in ACN
tG [min]
T [°
C]
Col
or c
ode:
R
esol
utio
n R
s
2D-modell tG/T of 60% iPrOH in ACN
tG [min]
T [°
C]
Col
or c
ode:
R
esol
utio
n R
s
3D-modell tG/T of 0-60% iPrOH in ACN
Select working point
Investigation of influence of pH
tG [min]
pH
Col
or c
ode:
R
esol
utio
n R
s
Verification of model with real experiment
Verification of model with real experiment
R² = 0,9996
0,0
0,5
1,0
1,5
2,0
2,5
3,0
0,0 0,5 1,0 1,5 2,0 2,5 3,0
expe
rimen
tal r
eten
tion
time
[min
]
DryLab predicted retention time [min]
• Variation of chromatographic parameters • tG (3 min ± 0.3 min) • T (60°C ± 6°C) • tC (50% ± 5% ACN in PrOH ) • flow rate (0.5 mL/min ± 0.05 mL/min) • %start (30% ± 2%) • %end (90% ± 2%) of the gradient • Full factorial design: 3 levels (+1, 0, -1) = 36 = 729 experiments
Method robustness
• Variation of chromatographic parameters by using the Robustness Module of the DryLab 4.0 (in silico)
• tG (3 min ± 0.3 min) • T (60°C ± 6°C) • tC (50% ± 5% ACN in PrOH ) • flow rate (0.5 mL/min ± 0.05 mL/min) • %start (30% ± 2%) • %end (90% ± 2%) of the gradient • Full factorial design: 3 levels (+1, 0, -1) = 36 = 729 experiments
Method robustness
Method robustness
729 experiments with Rs > 2.0
Step 5: Control strategy
• Based on the validation data and the robustness of the method, the risk assessment indicates that there is extensive knowledge gained about the performance of the method, so that a suitable system suitability test may be the only control element needed in the method control strategy.
• Therefore, the resolution of the critical peak pair impurity C and D, which
shows the lowest resolution of all impurity peaks was chosen as a system suitability test parameter and should be not less than 2.0.
Method validation
• Specificity • Linearity, LoD, LoQ • Coefficient of correlation > 0.999 • Accuracy and Precision (Repeatability) • RSD < 5.0 %, Recovery rate between 98.0 – 102.0 % • Precision - Intermediate Precision • RSD < 5.0 %, Mean-t-test must comply • Precision - System Precision • RSD < 2.0 %
Finale UPLC method for ebastine developed with DryLab
1,0 2,0Time (min)
Imp.
CIm
p. D
Imp.
A
Imp.
B
Imp.
F
Imp.
G
Imp.
E
Comparison finale UPLC method vs. official HPLC method
1,0 2,0Time (min)
Imp.
CIm
p. D
Imp.
A
Imp.
B
Imp.
F
Imp.
G
Imp.
E
0 20 40 60 80 100 120 140 160Time (min)
Impu
rity
AIm
purit
y B
Impu
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DIm
purit
y C
Impu
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F
Impu
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G
Ebas
tin
Impu
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E
Conclusion
All previous defined method goals were met:
Baseline separation of the components of interest (Rs >>2.0) k*-values for ebastine and impurities are between 2.2 and 4.3 The design space – an area in which the method is robust – is
defined and visualized Analysis time is only 4 min, which is a impressive 40-fold
increase in productivity in comparison to the method published in the E.P. monograph and allowed purity testing of more than 360 samples per day.
Step 6: Continuous Improvement
HPLC <-> UPLC <-> UPC²
Case study 2:
Fast UPLC method development and method
transfer to HPLC (for business needs)
Case study Omeprazole
0 10 20 30 40time (min)
Imp.
AIm
p. I
Imp.
E
Imp.
F+G
Imp.
BIm
p. D
Imp.
H
Imp.
C
4 6 8 10time (min)
Imp.
AIm
p. I
Imp.
E
Imp.
F+G
Imp.
BIm
p. D
Development of a new UPLC method for omeprazole
pH: 7.5, 8.0, 8.5, 9.0 Solvents: methanol, acetonitril Gradient time: 4, 10 min Temperature: 30, 60 °C
Design of Experiments: (Parameters and values)
Methanol as solvent
Design space Robust region
Acetonitrile as solvent
Design space Robust region
Conditions of the working point for new UPLC method Parameters and values
pH: 8.5 Solvent: acetonitrile Gradient time: 4 min Temperature: 35 °C Flow rate: 0.7ml/min Column: BEH C18, 50x2.1mm; 1.7µm Gradient: 10%-60% acetonitrile
New UPLC method for omeprazole
predicted chromatogram
1.0 2.0 3.0 4.0time (min)
1.064
Imp. A 1.4
50 Imp
. I
1.710
Imp. E
1.972
Imp. D
2.173
Imp. B
2.683
Imp. H
2.964
Imp. C
3.685
Imp. F
3.817
Imp. G
1.0 2.0 3.0 4.0time (min)
1.144 Im
p. A
1.479 Im
p. I
1.743 Im
p. E
2.002 Im
p. D
2.210 Im
p. B
2.718 Im
p. H
2.988 Im
p. C
3.711 Im
p. F3.84
0 Imp. G
experimental chromatogram
Robustnes testing UPLC Frequency distribution of the Rs,crit -values for all 729 experiments
of the robustness study on the UPLC system.
The six parameters tG (4 min ± 0.1 min), T (35°C ± 2°C), pH (8.75 ± 0.1), flow rate (0.7 mL/min ± 0.05 mL/min) and the %Bstart (10% ± 1%) and %Bend (60% ± 1%)
of the gradient were varied at 3 levels (+1, 0, -1).
Transfer from UPLC to HPLC for business reasons
The transfer was calculated by DryLab by changing the values of the parameters
UPLC HPLC Column: BEH C18, 50x2.1mm; 1.7µm 50x4.6mm; 2.5µm Dwell volume: 0.4 ml UPLC 1.0 ml HPLC
Transfered HPLC method
1.0 2.0 3.0 4.0 5.0 6.0time (min)
1.50
6 Im
p. A
2.09
8 Im
p. I
2.54
3 Im
p. E
3.00
6 Im
p. D
3.35
1 Im
p. B
4.24
0 Im
p. H
4.73
1 Im
p. C
5.99
6 Im
p. F
6.22
6 Im
p. G
1.0 2.0 3.0 4.0 5.0 6.0time (min)
1.63
2 Im
p. A 2.
090
Imp.
I
2.49
0 Im
p. E
2.97
9 Im
p. D
3.26
0 Im
p. B
4.13
0 Im
p. H
4.59
1 Im
p. C
5.84
0 Im
p. F
6.06
7 Im
p. G
predicted chromatogramm
experimental chromatogramm
pH: 8.5 Solvent: acetonitrile Gradient time: 7 min Temperature: 35 °C Flow rate: 1.9 ml/min Column: BEH C18, 50x4.6mm; 2.5µm Gradient: 10%-60% acetonitrile
Robustness testing HPLC
Frequency of the distribution of the resolution values Rs,crit for all 729 experiments of the robustness study after the transfer to the HPLC system.
The six parameters tG (7 min ± 0.1 min), T (35°C ± 2°C), pH (8.75 ± 0.1),
flow rate (1.9 mL/min ± 0.1 mL/min) and the %Bstart (10% ± 1%) and %Bend (60% ± 1%) of the gradient were varied at 3 levels (+1, 0, -1).
working point verification point 1 verification point 2 verification point 3 verification point 4 Flow rate [mL/min] 0.70 0.70 0.75 0.70 0.65
tG [min] 4.0 3.9 4.1 4.0 3.9 Temp [°C] 35 37 33 33 35
pH 8.75 8.75 8.75 9.00 9.00 %start 10 9 10 11 10 %end 60 60 61 60 61
Retention time [min] Pred. Exp. Pred. Exp. Pred. Exp. Pred. Exp. Pred. Exp. Imp. A 1,06 1,14 1,13 1,18 1,04 1,09 0,96 1,08 1,09 1,15 Imp. I 1,45 1,48 1,50 1,52 1,37 1,41 1,30 1,32 1,43 1,46 Imp. E 1,71 1,74 1,75 1,77 1,65 1,68 1,57 1,59 1,69 1,73 Imp. D 1,97 2,00 2,01 2,02 1,91 1,93 1,79 1,83 1,90 1,91 Imp. B 2,17 2,21 2,20 2,21 2,11 2,14 2,06 2,08 2,15 2,18 Omeprazole 2,26 2,29 2,28 2,29 2,20 2,22 2,15 2,18 2,24 2,27 Imp. H 2,68 2,72 2,68 2,70 2,62 2,65 2,58 2,62 2,65 2,68 Imp. C 2,96 2,99 2,95 2,96 2,90 2,92 2,91 2,93 2,96 2,98 Imp. F 3,68 3,71 3,64 3,65 3,62 3,65 3,66 3,67 3,67 3,69 Imp. G 3,82 3,84 3,76 3,77 3,75 3,78 3,79 3,81 3,80 3,82
Verification of the UPLC method
verification point 5 verification point 6 correlation between exp. vs. pred. RT Flow rate [mL/min] 0.65 0.75
tG [min] 4.1 4.0 Temp [°C] 35 37
pH 8.50 8.50 %start 11 9 %end 59 59
Retention time [min] Pred. Exp. Pred. Exp. Imp. A 1,07 1,21 1,10 1,19 Imp. I 1,54 1,61 1,53 1,62 Imp. E 1,81 1,86 1,77 1,85 Imp. D 2,14 2,18 2,07 2,16 Imp. B 2,30 2,32 2,22 2,30 Omeprazole 2,38 2,40 2,30 2,38 Imp. H 2,84 2,85 2,72 2,80 Imp. C 3,11 3,10 2,96 3,04 Imp. F 3,88 3,84 3,66 3,71 Imp. G 4,02 3,97 3,79 3,84
working point verification point 1 verification point 2 verification point 3 verification point 4 Flow rate [mL/min] 1.9 1.9 2.0 1.9 1.8
tG [min] 7.0 6.8 7.2 7.0 6.8 Temp [°C] 35 37 33 33 35
pH 8.75 8.75 8.75 9.00 9.00 %start 10 9 10 11 10 %end 60 61 61 60 61
Retention time [min] Pred. Exp. Pred. Exp. Pred. Exp. Pred. Exp. Pred. Exp. Imp. A 1,51 1,64 1,59 1,71 1,52 1,61 1,37 1,49 1,51 1,69 Imp. I 2,10 2,09 2,18 2,19 2,05 2,04 1,84 1,83 1,99 2,02 Imp. E 2,54 2,49 2,61 2,57 2,50 2,44 2,31 2,25 2,43 2,42 Imp. D 3,01 2,98 3,06 3,05 2,95 2,92 2,69 2,65 2,81 2,81 Imp. B 3,35 3,26 3,38 3,31 3,31 3,21 3,15 3,06 3,24 3,19 Omeprazole 3,50 3,40 3,52 3,44 3,45 3,35 3,31 3,21 3,39 3,32 Imp. H 4,24 4,13 4,23 4,14 4,20 4,09 4,06 3,94 4,10 4,03 Imp. C 4,73 4,59 4,69 4,58 4,70 4,55 4,64 4,51 4,65 4,55 Imp. F 6,00 5,84 5,90 5,77 5,97 5,81 5,95 5,77 5,89 5,78 Imp. G 6,23 6,08 6,12 5,99 6,20 6,04 6,18 6,03 6,10 6,00
Verification of the HPLC method
verification point 5 verification point 6 correlation between exp. vs. pred. RT
Flow rate [mL/min] 1.8 2.0
tG [min] 7.2 6.8 Temp [°C] 35 37
pH 8.50 8.50 %start 11 9 %end 59 59
Retention time [min] Pred. Exp. Pred. Exp. Imp. A 1,52 1,56 1,59 1,67 Imp. I 2,21 2,19 2,26 2,26 Imp. E 2,66 2,55 2,66 2,58 Imp. D 3,24 3,20 3,18 3,17 Imp. B 3,51 3,36 3,43 3,31 Omeprazole 3,66 3,50 3,56 3,44 Imp. H 4,46 4,28 4,28 4,15 Imp. C 4,92 4,72 4,70 4,54 Imp. F 6,27 6,05 5,90 5,73 Imp. G 6,52 6,29 6,12 5,95
Implementation of both methods
Chromatographic
parameter
UPLC condition HPLC condition
Column ACQUITY BEH C18; 2.1 x 50 mm, 1.7 µm XBridge BEH C18; 4.6 x 50 mm, 2.5 µm
Eluent A 10mM ammoniumbicarbonat buffer pH 8.75 (± 0.1 pH units)
Eluent B acetonitrile
Gradient linear increase from 10% (±1%) to 60%
(±1%) of eluent B in 4.0 min (±0.05 min),
followed by re-equilibration
linear increase from 10% (±1%) to 60%
(±1%) of eluent B in 7.0 min (±0.5 min),
followed by re-equilibration
Stop time 5 min 8 min
Flow rate 0.70 mL/min (±0.05 mL/min) 1.90 mL/min (±0.05 mL/min)
Column temp. 35°C (±2°C)
Injection volume 2 µL 20 µL
Detection UV @ 303 nm
With this approach it is possible to switch between HPLC and UPLC instruments
1.0 2.0 3.0 4.0time (min)
1.144 Im
p. A
1.479 Im
p. I
1.743 Im
p. E
2.002 Im
p. D
2.210 Im
p. B
2.718 Im
p. H
2.988 Im
p. C
3.711 Im
p. F3.84
0 Imp. G
1.0 2.0 3.0 4.0 5.0 6.0time (min)
1.632 Im
p. A 2.090 Im
p. I
2.490 Im
p. E
2.979 Im
p. D
3.260 Im
p. B
4.130 Im
p. H
4.591 Im
p. C
5.840 Im
p. F6.06
7 Imp. G
UPLC Method
HPLC Method
0 10 20 30 40time (min)
Imp.
AIm
p. I
Imp.
E
Imp.
F+G
Imp.
BIm
p. D
Imp.
H
Imp.
CPh.Eu. Method
0,0%
10,0%
20,0%
30,0%
40,0%
50,0%
60,0%
70,0%
80,0%
90,0%
100,0%
100,0%
7,3% (14x)
purit
y te
stin
g in
h
comparison of 73 adapted methods vs. Chromicent developed methods
Time & eluent adapted method time new method Eluent new method
3,3% (30x)
73 developed analytical methods: 58 methods for pharma 05 methods for chemistry 04 methods for food 04 methods for clinic / doping 02 methods for renewable energies
