Method Development HPLC_01

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Development and Use of OrthogonalMethods for Impurity Profiling of

Pharmaceuticals by HPLC

Henrik T. Rasmussen, Fengmei Zheng, Dora Visky,

Rhonda Jackson, Analytical Development

5th Annual Forced Degradation Studies Conference

February 25-27, 2008



Overview

• Requirements for Pharmaceutical Separations

• Strategy for Method Development

• Case Study – Method Development

• Case Study – Value of Orthogonal Methods

• HILIC as an Orthogonal Approach

• Conclusions



Pharmaceutical Development

EARLY PHASE LATE PHASE

Controlled ManufacturingSynthesis optimization

Formulation optimization

Limited Drug Chemistry



Evaluating a New NCE

• What is it?

– NMR, IR, MS, XRD (polymorph)

• What else is it? – Organic impurities (including enantiomers), residual

solvents, moisture, heavy metals, other…

• What will it become? – Degradation products

• What else will be in the next batch?

– …and will our methods be able to measure it?



Terminology

• Reporting Threshold – A limit above which an

impurity or degradation product should be reported.

• Identification Threshold – A limit above which animpurity or degradation product should be identified.

• Qualification Threshold – A limit above which the

biological safety of an impurity or degradation productshould be assessed.



ThresholdsMaximum daily dose Reporting Threshold Identification Threshold Qualification Threshold

Drug Substance Impurities< 2g/day 0.05% 0.10% or 1.0 mg per day* 0.15% or 1.0 mg/day*

> 2g/day 0.03% 0.05% 0.05%

Drug Product Degradation Products

< 1g 0.10%> 1g 0.05%

<1mg 1.0% or 5 ug*

1 mg - 10 mg 0.5% or 20 ug*

>10mg - 2 g 0.2% or 2 mg*

>2g 0.10%<10mg 1.0% or 50 ug*

10 mg -100 mg 0.5% or 200 ug*

>100 mg - 2 g 0.2% or 3 mg*

>2 g 0.15%

* whichever is lower

% are wt/wt relative to the API



HPLC Method Development

s

Pro-Active

Method

Development -Method Validation IND/CTA

methods

Supportive methods

Final

Method

Development

andValidation

Secondary

methods



Key Concerns During HPLC Method Development

0 2 4 6 8 10 12 14Time (min)

1 0 . 9

1 7

2 1 . 0

5 0

3 4 . 6

0 0

4 5 . 6

6 7

5 9 . 8

3 3

34 1 2

co-elution? non-elution?

solvent front/no retention? impurities not separated from API?



Proactive Method Development: Experiments

• Samples – All available DS batches

– API Precursors (knowledge of drug synthesis)

– Stressed DS (acid, base, temperature, humidity, oxygen, light)

– Stressed DP (or DS/Excipient mix) if available

– Excipients

• Conditions – Broad gradients at low and high pH (5 -95% strong solvent) on multiple

orthogonal HPLC Columns (Lab-Pro Valve). HILIC and NP

Chromatography

– PDA, MS (and NMR) detection

– Other separation methods such as GC, SFC and CE may also be evaluated



Stress ConditionsTime Pulls Degradation Type

Solution/suspension at 50oC 0.1N HCl, 0.1N NaOH, water 0.5, 1, 3 days Hydrolytic

pH 2, 4, 6, 8, 10 3, 7,14 days Hydrolytic

0.5% Methocel 3, 7,14 days Hydrolytic

PEG-400 3, 7,14 days Oxidation

20% HPbCD 3, 7,14 days Hydrolytic

AIBN 0.5, 1, 3 days OxidationFe (III)/Cu (II) 0.5, 1 days Oxidation

Solution/suspension at room temp H2O2 0.5, 1, 3, 7 days Oxidation

Solid at 80oC solid state @ ambient humidity 0, 7, 14 days Thermolytic

solid state @ 75% RH 0, 7, 14 days Hydrolytic, Thermolytic

Excipient blends 1-4, @ 75% RH 7, 14 daysLight Solid - Xenon lamp, 765 W/m2 1xICH, 2xICH Photolytic

Solid - cool white light 1, 2, 7 days Photolytic

DS in water - cool white light 1, 2 days Photolytic

Sample solvent - cool white light 1, 2 days Photolytic

Conditions



Automated Forced Decomposition Studies

• Fermier, A. M.; Armstrong, B. L.; Oyler, A. R.; Weber, J. V.;

Nalasco, J., Automated Degradation Instrument. US Patent

Application Serial No. 09/816,787 ; 2001.

• Fermier, A. M.; Oyler, A. R.; Armstrong, B. L.; Weber, B. A.:

Rodriguez, R. L.; Weber, J. V.; Nalasco, J.A.; Automation of

Chemical Reaction Kinetics and Product Distribution Studies in

Pharmaceutical Development. JALA, 7(1) (2002) 68.

• Jackson, Rhonda A. “A Forced Degradation Strategy: How

Johnson & Johnson PRD Approaches Degradation” Am. Pharm.

Rev., 10 (5), (2007) 59.



HPLC Conditions

Orthogonal Screening Method DescriptionTime (min) %Water %Acetonitrile

% Modifier

a Flow Rate (ml/min)

0 85 10 5 1.0

40 10 85 5 1.0

45 10 85 5 1.0

45.10 85 10 5 1.060 85 10 5 1.0

Injection Volume 5 µL

Detection 280 nm (compound dependent); DAD (190 – 400 nm),

Column Temperature Ambient

Sample Temperature 5oCaModifier stock solutions are prepared at a concentration 20 times higher than the desired mobile phase

concentration since mobile phases are prepared at time of use with the HPLC quaternary pump.



Mobile Phase Modifiers

Modifier Mobile PhaseConcentration

Approximate pH

Trifluoroacetic Acid (TFA) 0.05% 2

Formic Acid 0.1% 2.8

Ammonium Acetate + Acetic Acid 8 mM + 0.1% 4

Ammonium Acetate 8 mM 7

Ammonium Acetate + Ammonium

Hydroxide

8 mM + 0.05% 10.2

Ammonium Hydroxide 0.05% 10.8



HPLC Columns

Orthogonal Screening – Columns

Stationary Phasea Column pH Range

bManufacturer Part Number

C18 – Twin Technology Gemini C18, 5µm, 110A, 4.6 x 150 mm 1-12 Phenomenex 00F-4435-E0

Phenyl with Hexyl (C6) linker,

endcappedLuna Phenyl-Hexyl, 3µm, 4.6 x 150 mm 1.5-10 Phenomenex 00F-4256-E0

C18-20% C loading Discovery HS-C18, 3µm, 4.6 x 150 mm 2-8 Supelco 569252-UC18 – polar embedded, hybrid

particle with Shield TechnologyXTerra RP18, 3.5µm, 4.6 x 150 mm 1-12 Waters 186000442

C18– silica Sunfire C18, 3.5µm, 4.6 x 150 mm 2.8 Waters 186002554

Pentafluorophenyl Curosil PFP, 3µm, 4.6 x 150 mm 2-7.5 Phenomenex 00F-4122-E0aOther columns could be selected based on the compound properties.

bColumns were screened only against mobiles phases within their compatible pH range.

C l S l ti it Ch t



Column Selectivity Chart

SunFire ™ C18

YMC-Pack™ PolymerC18™

Hypersil® CPS Cyano

YMC-Pack™ CN

Waters Spherisorb® S5 P

Hypersil® BDS Phenyl

Nova-Pak® Phenyl

YMC-Pack™

Phenyl

Hypersil® Phenyl

Inertsil® Ph-3

YMC-Pack™ Pro C4™

YMCbasic™

Symmetry® C8YMC-Pack™ Pro C8™

Nova-Pak®

C8

XTerra®

MS C18 Symmetry® C18

YMC-Pack™

Pro C18™

Inertsil® ODS-3

YMC-Pack™ ODS-A™

Nova-Pak®

C18

YMC J'sphere™

ODS–L80 Nucleosil® C18

Waters Spherisorb® ODS2

Waters Spherisorb® ODS1

Resolve® C18

µBondapak™ C18

YMC-Pack™ ODS–AQ™

YMC J'sphere™ ODS–H80YMC J'sphere™ ODS–M80

Inertsil® CN-3

Waters Spherisorb® S5CN

Nova-Pak® CN HP

SymmetryShield™ RP8

SymmetryShield™ RP18

XTerra® RP8

XTerra® RP18

-0.6

-0.3

0

0.3

0.6

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3

3.3

3.6

-1.5 -0.5 0.5 1.5 2.5 3.5

( l n

[ ] a m i t r i p t

y l i n e / a c e n a p

h t h e n e )

XTerra® MS C8

Luna ®

C18(2)

ACQUITY UPLC™

BEH C18

XTerra ®Phenyl Luna ™

Phenyl Hexyl

ChromolithTM

RP-18

Atlantis® dC18

Zorbax® XDB C18 ACT Ace® C18

Zorbax® SB C18

SunFire C8

Luna®

C8 (2)

ACQUI Y UPLC™

Shield RP18

ACQUITY UPLC™

BEH C8

ACQUITY UPLC™

BEH Phenyl

Diagram courtesy of Waters Corp.

ln [k] acenapthene

™

T



Case Study: Method Development

H. T. Rasmussen, K. A. Swinney and S. Gaiki, “HPLC Method

Development in Early Phase Pharmaceutical Development” in HPLC

Method Development for Pharmaceuticals, Volume 8 (Separation Science

and Technology), S. Ahuja and H. Rasmussen, eds., (Academic Press, Amsterdam, The Netherlands, 2007). Chapter 12.

1 Run all samples by one condition to identify samples of



1. Run all samples by one condition to identify samples of

interestChromatograms of significant forced degraded samples.

A

U

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

0 . 0 9 0

0 . 1 0 0

0 . 1 1 0

0 . 1 2 0

0 . 1 3 0

0 . 1 4 0

0 . 1 5 0

M i n u te s0 .0 0 5 .0 0 1 0 . 0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 30 .0 0 3 5 .0 0

A: drug substance in water stressed under fluorescent light for 7 days

B: solid stressed under fluorescent light for 7 days

C: drug substance stressed with 0.3% H2O2 for 7 days at Ambient

D: drug substance stressed with 0.1N NaOH for 7 days at 70oC

E: standard (0.175 mg/mL)

C

B

A

E

D

Oxidation degradant

Base degradants

2 Co-eluting light degradantsLight degradant

2 Co-eluting light

degradants

Process impurities



2. Identify Screening conditions that provide optimal separation*

Orthogonal Screening Results: XTerra RP18 Column with 8mM Ammonium Acetate + 0.1%

Acetic Acid modifier.

A

U

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

0 . 0 9 0

0 . 1 0 0

0 . 1 1 0

0 . 1 2 0

0 . 1 3 0

0 . 1 4 0

0 . 1 5 0

M i n u te s

0 .0 0 5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 . 0 0

A: drug substance in water stressed under fluorescent light for 7 days

B: drug substance stressed with 0.3% H2O2 for 5 days at Ambient

C: drug substance stressed with 0.1N NaOH for 5 days at 70oC

Oxidation degradant

Base degradants

A

B

C

Light degradantsLight degradant



*Identifying candidate conditions

• Export data set to Microsoft Excel and:

– Sort by Area% of largest peak – to identify meaningful stressconditions (2-5% degradation)

– Sort by number of peaks to identify candidate chromatographicconditions



3. Identify an orthogonal method from the screening module

Orthogonal Screening Results: Sunfire C18 Column with 0.1% Formic Acid modifier

A

U

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

0 . 0 9 0

0 . 1 0 0

0 . 1 1 0

0 . 1 2 0

0 . 1 3 0

0 . 1 4 0

0 . 1 5 0

M i n u te s

0 .0 0 5 .0 0 10 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0

A: drug substance in water stressed under fluorescent light for 7 daysB: drug substance stressed with 0.3% H2O2 for 5 days at Ambient

C: drug substance stressed with 0.1N NaOH for 5 days at 70oC

Base degradants

Light degradant

Oxidation degradant

2 Co-eluting light degradants



Orthogonality

Orthogonality Plot for Candidate and Secondary Methods

K' for Candidate Method

6 8 10 12 14 16

K ' f o r S e c o n d a r y M e t h o d

2

4

6

8

10

12

14

R²=0.40

4 Run Samples from each stress condition by candidate



4. Run Samples from each stress condition by candidate

method (to assure nothing was missed)Candidate Method: Xterra RP18 Column (4.6×150 mm) with mobile phase additive

8 mM Ammonium Acetate + 0.1% Acetic Acid – Chromatograms of representativeforced degraded samples.

A

U

- 0 . 0 0 2

0 . 0 0 0

0 . 0 0 2

0 . 0 0 4

0 . 0 0 6

0 . 0 0 8

0 . 0 1 0

0 . 0 1 2

0 . 0 1 4

0 . 0 1 6

0 . 0 1 8

0 . 0 2 0

0 . 0 2 2

0 . 0 2 4

0 . 0 2 6

0 . 0 2 8

0 . 0 3 0

0 . 0 3 2

0 . 0 3 4

0 . 0 3 6

0 . 0 3 8

0 . 0 4 0

M i n u te s0 .0 0 5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0 4 0 .0 0

A: solid stressed under intense UV & Visible light – 1X ICH

B: drug substance stressed with 0.3% H2O2 – 3days

C: solid at elevated temperature (60oC) – 3 days

D: solid at elevated temperature and humidity (60oC/75% RH) – 3 days

E: drug substance stressed with 0.1N HCl at 70oC – 3days

F: drug substance stressed with 0.1N NaOH at 70oC – 3days

A

B

C

D

E

F

Light degradantsLight degradant

Oxidation degradant

Base degradants

5 R S l f h t diti b th l



5. Run Samples from each stress condition by orthogonal

method (to assure nothing was missed)Secondary Method: Sunfire C18 Column (4.6×150 mm) with mobile phase additive 0.1% Formic Acid

- Chromatograms of representative forced degraded samples.

A

U

0 . 0 0 0

0 . 0 1 0

0 . 0 2 0

0 . 0 3 0

0 . 0 4 0

0 . 0 5 0

0 . 0 6 0

0 . 0 7 0

0 . 0 8 0

0 . 0 9 0

0 . 1 0 0

0 . 1 1 0

0 . 1 2 0

0 . 1 3 0

0 . 1 4 0

0 . 1 5 0

0 . 1 6 0

0 . 1 7 0

0 . 1 8 0

M i n ute s0 .0 0 5 .0 0 1 0 .0 0 1 5 .0 0 2 0 .0 0 2 5 .0 0 3 0 .0 0 3 5 .0 0

A: solid stressed under intense UV & Visible light – 1X ICHB: drug substance stressed with 0.3% H2O2 – 3days

C: solid at elevated temperature (60oC) – 3 days

D: solid at elevated temperature and humidity (60oC/75% RH) – 3 days

E: drug substance stressed with 0.1N HCl at 70oC – 3days

F: drug substance stressed with 0.1N NaOH at 70oC – 3days

Oxidation degradant

A

B

C

D

E

FBase degradants

2 co-eluting light degradants

Light degradant



6. Optimize the candidate method (run time,resolution, temperature, etc. via DryLab) wherenecessary.

7. Use candidate method for release and stabilityactivities.

8. Use orthogonal method (in addition to candidatemethod) for evaluation of pivotal stability samplesand Drug substances synthesized via new routes(to assure candidate method is still specific).

9. Develop final methods based on batch histories

generated using both methods.



Use of Orthogonal Methods DuringPharmaceutical Development:

Case Studies

Chromatograms of two Drug Substance batches



g g(primary method)

1st DS batch

New DS batch

API

A U

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00

Impurity A Impurity B

Impurity C, 0.1%

• Are there any impurities missing?

O th l R lt



Orthogonal Results

1st DS batch

New DS batch

API

A U

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes

6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

Impurity A1

Impurity B

Impurity A2

Dimer1, 0.4%

Dimer2, 0.1%Impurity C

• Two isomers of impurity A were separated.

• Two new impurities, dimer1 and dimer2, were detected which were not

detected in the primary method

Chromatograms of two Drug Substance batches



g g

(primary method)

A U

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

0.045

0.050

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

1st GMP DS batch

New DS batch

Impurity A, 0.08 w/w%

API

Impurity A, 0.40 w/w%

• Problem: Impurity A exceeds the Tox. Qualification Threshold.

• Are there any impurities missing?

Orthogonal Results:



Orthogonal Results:

A U

-0.010

-0.005

0.000

0.005

0.010

0.015

0.020

0.025

0.030

0.035

0.040

Minutes

0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00 30.00

1st GMP DS batch

New DS batch

API

Impurity A, 0.21 w/w%Isomer of the API, 0.14 w/w%

Impurity B, 0.18 w/w%

• Impurity A is 2 compounds below the Qualification Threshold.

• An isomer of the API is separated from API.

H d hili I i Ch h



Hydrophilic Interaction Chromatography

• Potentially useful as alternative orthogonal approach due to

differences in retention mechanism

– Polar column

– Aqueous/organic mobile phase (water is the “strong” solvent) – Salt type, concentration and pH affect retention

– The same sample preparations used for RP-HPLC can be used for

HILIC.

X. Wang, W. Li* and H. T. Rasmussen, J. Chromatogr., 1083 (2005) 58.

HILIC C St d



HILIC – Case Study

Compound Identification Functional Groups

Comp 1 Aromatic, -NH2, -OHComp 2 (API) Aromatic, -NH2, -O-CONH2

Comp 3 Aromatic, -NH-CONH2, -OH

Comp 4 Aromatic, -O-CONH-

HILIC Case Study



HILIC Case Study

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

C o m p 1

C

o m p 2

C o m p 3

C o m p 4

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

Minutes

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0 20.0

C o m p 4

C o m p 3

C o m p 1

C o m p 2

a

b

• (a) RP-HPLC: RP-HPLC conditions: column 100× 4.6mm 5µm Xterra MS C18; gradient elution with A: 0.1% phosphoric acid and B: acetonitrile (2-25%B in 20 minutes); column temperature 35°C; flow rate 1mL/min; UVdetection 215 nm.

• (b) HILIC: HILIC conditions: column 250× 4.6mm 5µm YMC-pack Diol-120 NP; mobile phase 10 mM NH4Cl in

acetonitrile-water (95:5, v/v); column temperature 30°C; flow rate 1.5ml/min; UV detection 215 nm.

C l i



Conclusions

• Orthogonal methods are necessary for ongoing

assessment of method specificity.

• A generic screening approach may be used to obtainorthogonal methods.

• Alternative types of HPLC such as HILIC additionally

provide selectivity differences vs. conventional RP-

HPLC.



Acknowledgements

THANKS TO ALL THE CONTRIBUTORS:

A successful filing is driven by pro-activecommunication amongst scientists working

within and across groups as an effective team.



Questions?

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