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

• 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

0 10 20 30 40

0 10 20 30

• 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)

• 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)

„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

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

Acetonitrile < 1.5 (C,D)

2-propanol < 1.5 (C,D)

Acquity UPLC HSS C18 SB

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]

2D-modell tG/T of 30% iPrOH in ACN

tG [min]

2D-modell tG/T of 60% iPrOH in ACN

tG [min]

3D-modell tG/T of 0-60% iPrOH in ACN

Select working point

Investigation of influence of pH

tG [min]

Verification of model with real experiment

R² = 0,9996

0,0 0,5 1,0 1,5 2,0 2,5 3,0

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

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)

Comparison finale UPLC method vs. official HPLC method

1,0 2,0Time (min)

0 20 40 60 80 100 120 140 160Time (min)

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)

4 6 8 10time (min)

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)

Imp. A 1.4

50 Imp

Imp. E

Imp. D

Imp. B

Imp. H

Imp. C

Imp. F

Imp. G

1.0 2.0 3.0 4.0time (min)

1.144 Im

1.479 Im

1.743 Im

2.002 Im

2.210 Im

2.718 Im

2.988 Im

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)

p. A 2.

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

1.479 Im

1.743 Im

2.002 Im

2.210 Im

2.718 Im

2.988 Im

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

2.490 Im

2.979 Im

3.260 Im

4.130 Im

4.591 Im

5.840 Im

p. F6.06

7 Imp. G

UPLC Method

HPLC Method

0 10 20 30 40time (min)

CPh.Eu. Method

100,0%

7,3% (14x)

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

Thank you very much for your interest !

info@chromicent.de

Quality-by-Design in Method Development · 2017-11-29 · „Our“ method development strategy by...

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