Method Transfer Crossing Multiple Borders

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Method Transfer: Crossing Multiple Borders

Xiande (Andy) Wang, Ph.D.

Analytical Research and Development Cordis Corporation,

A Johnson & Johnson Company,Warren, NJ 07059

IVT Annual Method Validation 2010

Outline

1. Lifecycle of Analytical Methods

2. Border I: Between Methods

– Case study: combination of 3 methods into one

3. Border Between Instrumentation/Technique

– Case study: validation of an HPLC and UPLC method side by side

4. Border Between Groups

– Case study: Troubleshooting cross functions during method transfer

5. Conclusions

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Life Cycle of an Analytical method

Method Development

Method validation

Method transfer

Life Cycle of Analytical methods

Development

validationtransfer

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Life Cycle of Analytical Methods - Examples

Development

validationtransfer

Life Cycle of Analytical Methods - Examples

Development

validationtransfer

HPLC

UPLC HPLC

UPLC

HPLCUPLC

HPLC

HPLC

HPLC

UPLC

UPLC

UPLC UPLC

UPLC

UPLCHPLC

HPLC

HPLC

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Borders to Cross in Lifecycle of Method

Development

validationtransfer

HPLC

UPLC HPLC

UPLC

HPLCUPLC

HPLC

HPLC

HPLC

UPLC

UPLC

UPLC UPLC

UPLC

UPLCHPLC

HPLC

HPLC

Border II

Border I

Border III

Border I: Between Methods

UPLC

UPLC

UPLCHPLC

HPLC

HPLC

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Questions Around Border I

• Can the method be used interchangeably?

• Methods require the same instrumentation, column, reagents, materials?

• Solution (standard, sample, mobile phase) storage and stability

• Timing of validation, transfer; effective date, versions

• Will methods be run in the same or different lab?

• Sample shipping, storage at different environment

• Consistency in any other areas

How is HPLC Assay Method Developed?Method Objectives

– Stability indicating• Peak purity

• Resolution of all species

• LC-MS compatible

• Elution of all species/compounds

– Long gradient method

– Orthogonal

– Robust

– Sensitivity

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11

Systematic HPLC Assay Method Development

Samples are stressed under

different conditions

Stressed samples are analyzed with a generic method

Representative stressed samples are

chosen

Method screening is conducted with selected samples

A primary method is identified

The method is optimized

Stressed samples are analyzed with

optimized method

The primary method is ready for further optimization /

validation

12

General guidelines for HPLC Column Selection

• Select high-purity silica-based columns• C18 and C8– Hydrophobic, retentive and stable• Phenyl – medium polarity components; unique selectivity for

aromatics• Hydrophilic end-capped phases (retentive for water soluble

compounds)• Polar-embedded phases (amide, carbamate, ether, sulfonamide)

– Less tailing for basic analytes– “Orthogonal” to C8/C18, No phase collapse

• Explore selectivity differences between C18, polar-embedded or phenyl bonded phases – Consult column selectivity chart – For low pH Applications, select column resistant to hydrolytic cleavage

(e.g., StableBond, X-Bridge C18)

– For high-pH application, select columns stable at high pH (e.g., Gemini, X-Bridge, Extend, Luna)

M.W. Dong, Modern HPLC for Practicing Scientists, Wiley, 2006, Chap. 3.

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13

• Waters: Symmetry, SunFire, XTerra *, ACQUITY*, X-Bridge*, Atlantis, NovaPak, m-Bondapak, Spherisorb

• Agilent: Zorbax StableBond, Eclipse XDB, Extend C18*, Bonus

• Phenomenex: Luna*, Prodigy, Synergi*, Gemini*

• Supelco: Discovery, Ascentis, Supelcosil

• Varian: Inertsil, Polaris*

• Thermo: HyPURITY, Hypersil, Prism, Hypersil Gold *

• MacMod: ProntoSIL, ACE (Adv. Chrom. Tech.)

• YMC: YMCbasic, Pack Pro

• Eka Chemicals: Kromasil

• GL Sciences: Inertsil

• Macherey Nagel: Nucleosil

• Merck KGaA: Chromolith (Monolith)

• Bischoff: ProntoSIL*

• Grace: Vydac, Platinum (Alltech)

• Dionex: Acclaim, Acclaim PA, Acclaim PA2*

Columns based on high-purity silica are underlined. Hybrid particles are in bold. Phases stable in high pH are italicized and marked with *.

Some Popular HPLC Columns

M.W. Dong, Modern HPLC for Practicing Scientists, Wiley, 2006, Chap. 3.

HPLC Assay Method Development: Column Screening

E. F. Hewitt, P. Lukulay, and S. Galushko, J Chromatography A, 1107, 79.

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15

HPLC Column Screen Set: An Example

Orthogonal Screening – Columns Stationary Phase Column pH Rangea Manufacturer Part Number

C18 – Twin Technology Gemini C18, 5 μm, 110A, 4.6 x 150 mm 1-12 Phenomenex 00F-4435-E0 Phenyl with Hexyl (C6) linker,

endcapped Luna Phenyl-Hexyl, 3 μm, 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-U C18 – polar embedded, hybrid

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

C18– silica Sunfire C18, 3.5 μm, 4.6 x 150 mm 2.8 Waters 186002554 Pentafluorophenyl Curosil PFP, 3 μm, 4.6 x 150 mm 2-7.5 Phenomenex 00F-4122-E0

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

Slide courtesy of H. Rasmussen et al

16

HPLC Screening Conditions: An Example

Orthogonal Screening Method Description Time (min) %Water %Acetonitrile % Modifiera 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 1.0 60 85 10 5 1.0

Injection Volume 5 μL Detection 280 nm; DAD (190 – 400 nm)

Column Temperature Ambient Sample Temperature 5oC

aModifier 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.

Modifier Mobile Phase Concentration

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

Slide courtesy of H. Rasmussen et al

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Summary of HPLC Assay Method Development

• There is a systematic approach for assay method development.

• Column screening is an effective tool.

• It will enhance efficiency by narrowing down the list of columns for screening/selection.

• It is critical to seek input from other labs (QA, QC) during method development.

• It’s important to look at the big picture across methods.

What is Important in Developing HPLC CU Method?Method Objectives

– Short

– Specific

– Isocratic preferred

– Robust

– Compatible with assay method

– Can be used for dissolution testing

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19

Systematic HPLC CU Method Development

samples are analyzed with a

generic method

Specificity, retention, peak shape etc.

acceptable

Method screening is conducted

A primary method is identified

The method is optimized

The primary method is ready for further optimization / validation

Yes

No

HPLC CU Method Development: Column Screening

E. F. Hewitt, P. Lukulay, and S. Galushko, J Chromatography A, 1107, 79.

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What is Important in Developing HPLC Method for Dissolution Testing?

– Short

– Specific

– Isocratic preferred

– Robust

– Compatible with CU method

– Sensitive

How to Minimize Border I (Between Methods)

• Use same materials/chemicals: grade, vendor

• Adopt similar standard/sample solution: procedure of preparation, concentration, pH, storage, expiry

• Use same column: vendor, stationary phase, dimension, particle size; alternative column

• Use same Instrumentation

• Be consistent in write-up: same product description, formulation number, etc.

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Case Study: Combination of 3 methods into 1

Method number

Method title

N1 Identification, Assay and Content Uniformity Determination of … by RP-HPLC-UV

O1 Identification and Assay of … by RP-HPLC-UV

O2 Determination of Content Uniformity of … by RP-HPLC-UV

O3 Identification of … by UV Spectroscopy

Method O1: Identification and Assay of … by RP-HPLC-UV

Parameter Value

HPLC Column Agilent, Zorbax Eclipse XDB-C18, 150 mm x 4.6 mm x 3.5 µm

Flow Rate 1.0 ± 0.1 mL/min Injection Volume 25 µL

Column Temperature 40 ± 2 °C

Detection Wavelength

278 nm.Note: If using an Agilent DAD detector, set the bandwidth to 4

nm and the reference wavelength off.

Mobile Phases A: 80:20% (v/v) 0.02% formic acid:THF

B: 75:20:5:0.02% (v/v/v/v), acetonitrile: THF : water: formic acid

Gradient Program

(Linear Gradient Profile)

Time (minutes) % Mobile Phase A % Mobile Phase B

0.0 55 45 3 55 45 7 42 58 13 10 90 15 55 45

AU

0.000

0.005

0.010

0.015

0.020

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00 10.00 11.00 12.00 13.00 14.00 15.00

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Method O2: Determination of Content Uniformity of and Dissolution … by RP-HPLC-UV

Parameter Value

Column

Phenomenex Luna C18 (2), 4.6 x 50 mm, 3 μm or Phenomenex Gemini C18, 4.6 x 50 mm, 3 μm

HPLC Column Column

Temperature 35oC ± 2oC

Autosampler Temperature

Ambient (20 to 25 °C if temperature control is

available)

Mobile Phase 55:45, 0.02% v/v Formic Acid:THF

Flow Rate 1.2 mL/minute

Detector

278 nmNote: If using Agilent PDA

detector, set bandwidth to 4 nm and reference wavelength off.

Injection Volume 25 μL Run Time 10 minutes

Method O3: Identification of … by UV Spectroscopy

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Method N1: Identification, Assay and Content Uniformity Determination of … by RP-HPLC-UV

Parameter Value

HPLC Column Agilent Zorbax Eclipse XDB-C18, 100 mm x 4.6 mm, 3.5 µm

Flow Rate 1.0 mL/min Injection Volume 20 µL

Column Temperature 45 oC ± 2°C


Assay Identification278 nm

Note: If using an Agilent DAD detector, set the bandwidth to 4 nm and

the reference wavelength off.

200 to 400 nm

Mobile Phases A: (20:80)THF: Formate Buffer B: (75:20:5)ACN:THF:Formate Buffer

Gradient Program (Linear

Gradient)

Time (min) % Mobile Phase A

% Mobile Phase B

0.0 47 53 3.0 47 53 5.0 20 80 6.0 2 98 7.5 2 98 7.6 47 53 10 47 53

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

2.18

1

2.57

7

3.05

93.

390

3.59

5

RAPA

MYC

IN -

4.05

5

4.97

9

BHT

- 7.1

96

AU

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

nm200.00 220.00 240.00 260.00 280.00 300.00 320.00 340.00 360.00 380.00 400.00

208.0

269.0

279.0

291.0

349.0

Benefits of a Combined Method

Benefit Details

Shortened project timelines

One, instead of three, set of test methods, validation protocols, validation reports

Enhancedthroughput

Run time cut from 18 to 10min; one instrument/set-up for both assay/CU; eliminate requirement of a UV spectrometer.

Reduce cost One , instead of two, set of HPLC column is neededLess reference standard, organic solvent Less sample shipment cost

Eliminated causes for bias between assay and CU

When two methods are used, possible bias could come from different instrument set up, different way of peak integration, different separation capacity, etc. One method eliminate all these possible causes.

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Benefits of Elimination of Border I

Assay/CU/dissolution

Assay/CU/dissolutionAssay/CU/dissolution

• Same columns

• Same reagents, vendor, expiry dates

• Same materials such as glassware, HPLC vials, etc.

• No discrepancy (bias) of results due to integration, separation capacity, etc.

• Same solution (standard, sample, mobile phase)storage and stability

• Transfer at the same time

• Same method, same version

• One HPLC system needed

• Minimize sample shipping, storage at different environment

Summary of Strategy to Minimize/Eliminate Border I

• Assay, CU, and dissolution (HPLC) methods have different objectives and hence the strategy for method development may be different.

• However, it is important to keep the other method in mind during method development.

• Column screening is an effective tool for assay, CU or dissolution methods.

• It is effective to narrow down the list of columns for screening/selection.

• It is critical to seek input from other labs (QA, QC) during method development.

• It’s important to leverage the knowledge, as much as possible, of one method and apply it to another.

• It offers a lot of benefit to use similar or the same procedures, instrumentation and materials for all methods, and transfer them at the same time.

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Border II: HPLC vs. UPLC

UPLC

UPLC

UPLCHPLC

HPLC

HPLC

UPLC vs. HPLC: Example of Assay Method

Ref: L. Pereira, Poster at Pittcon 2007, Chicago, Illinois, February 2007.

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UPLC vs. HPLC: Example of CU Method

http://www.waters.com/waters

Issues Around Border II (HPLC vs. UPLC)

• Availability of instruments at all sites

• Different UHPLC systems have different design, not a direct transfer

• Training/knowledge and technical challenges on the new instruments

• Limited availability and technical challenges of columns

• Project history

• Cost

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How to Minimize Border II (HPLV vs. UPLC)

• Use the same stationary phase with different dimension and particle size

• Same mobile phase but slight different gradient

• Same protocol template

• Use the same solutions and validate the methods side by side

Case study: Validation of HPLC and UPLC Assay Method Side by SideChromatograms of the Final Methods

HPLC

UPLC

AU

-0.004

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

0.014

0.016

0.018

0.020

0.022

0.024

0.026

0.028

Minutes1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 9.00

2.18

1

2.57

7

3.05

9

3.39

03.

595

RA

PAM

YC

IN -

4.05

5

4.97

9

BHT

- 7.

196

AU

-0.002

0.000

0.002

0.004

0.006

0.008

0.010

0.012

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00

RAPA

MYC

IN -

2.35

8

BHT

- 4.2

02

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Case study: Validation of HPLC and UPLC Assay Method Side by SideFinal Method Parameters

HPLC UPLCParameter Value

HPLC Column Agilent Zorbax Eclipse XDB-C18, 100 mm x 4.6 mm, 3.5 µm

Flow Rate 1.0 mL/min Injection Volume 20 µL

Column Temperature 45 oC ± 2°C


Assay Identification278 nm

Note: If using an Agilent DAD detector, set the bandwidth to 4 nm and


200 to 400 nm

Mobile Phases A: (20:80)THF: Formate Buffer B: (75:20:5)ACN:THF:Formate Buffer


Gradient)


% Mobile Phase B

0.0 47 53 3.0 47 53 5.0 20 80 6.0 2 98 7.5 2 98 7.6 47 53 10 47 53

Parameter Value UPLC

Column AgilentZorbax Eclipse XDB-C18, 100

mm x 3.0 mm, 1.8 µm Flow Rate 0.7 mL/min Injection Volume 5 µL

Autosampler Temperature

10 oC

Column Temperature 45 oC ± 2 °C


Assay Sampling Rate

278 nm Note: If using an Agilent

DAD detector, set the bandwidth to 4 nm and


20 (points/sec)

Mobile Phases

A: (20:80)THF: Formate Buffer B: (75:20:5)ACN:THF:Formate

Buffer


Gradient)


% Mobile Phase B

0.0 47 53 2.0 47 53 3.5 20 80 4.2 2 98 5.2 2 98 5.3 47 53 6.5 47 53

Run Time 6.5 minutes (Retention time is ~2.5 min for sirolimus, ~4.3 min for BHT.

Seal Wash Acetonitrile

Weak Wash Acetonitrile/water: 50/50, 600 µL volume

Case study: Validation of HPLC and UPLC Assay Method Side by SideAccuracy (spiked recovery)

~Level Actual

Concentration (μg/mL)

Individual % Recovery

Mean % Recovery

% RSD

40% 67.4 99.9

99.9 0.1 99.7 99.9

100% 151.7 99.5

99.6 0.2 99.8 99.5

160% 242.7 100.6

100.5 0.2 100.6 100.3

~Level Actual


Individual % Recovery

Mean % Recovery

% RSD

40% 67.4 100.9

101.0 0.1 101.0 101.1

100% 151.7 99.4

99.6 0.2 99.6 99.8

160% 242.7 99.3

99.0 0.2 98.9 98.9

HPLC

UPLC

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Validation of HPLC and UPLC CU Method Side by Side:Accuracy (spiked recovery)

HPLC

UPLC

~Level Actual


Individual %

Recovery

Mean % Recovery % RSD

40% 12.1 100.6

100.4 0.4 100.7 100.0

100% 30.3 98.9

98.9 0.1 98.8 99.0

160% 48.5 99.6

99.8 0.2 100.0 99.6

~Level Actual


Individual %

Recovery

Mean % Recovery % RSD

40% 12.1 100.9

101.2 0.3 101.6 101.3

100% 30.3 100.4

100.0 0.4 100.0 99.5

160% 48.5 100.4

101.3 0.9 102.2 101.5

Validation of HPLC and UPLC Assay Method Side by Side:Intermediate Precision

HPLC

UPLC

Determination %LC Analyst 1 %LC Analyst 2

1 103.1 103.42 101.9 102.63 101.4 103.04 101.6 103.05 102.4 103.46 102.2 104.0

Mean (n=6) 102.1 103.2%RSD (n=12) 0.8

Determination %LC Analyst 1 %LC Analyst 2

1 103.8 101.32 101.8 102.13 100.9 102.34 100.6 102.25 101.8 102.76 102.2 102.9

Mean (n=6) 101.9 102.2%RSD (n=12) 0.5

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Validation of HPLC and UPLC CU Method Side by Side:Intermediate Precision

HPLC

UPLC

Determination %LC Analyst 1 %LC Analyst 21 101.5 102.72 102.0 104.23 101.0 102.34 103.4 102.35 105.1 102.36 101.3 101.77 103.1 102.68 103.1 102.79 102.1 102.7

10 102.9 102.0Mean (n=10) 102.6 102.6

%RSD (n=20) 0.9

Determination %LC Analyst 1 %LC Analyst 21 102.0 103.42 101.6 103.73 101.3 103.44 100.4 102.75 102.6 102.26 101.0 101.77 101.7 103.68 100.8 102.69 101.6 102.9

10 101.4 102.2Mean (n=10) 101.4 102.7

%RSD (n=20) 0.9

Validation of HPLC and UPLC Assay Method Side by Side:Linearity

HPLC

UPLC

R=0.9997

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Validation of HPLC and UPLC CU Method Side by Side:Linearity

HPLC

UPLC

R=0.9997

R=1.0000

Validation of HPLC and UPLC CU Method Side by Side:Method Equivalency

HPLC UPLC

Determi-nation

%LC with new

method

%LC old CU

method1 98.4 98.72 99.2 99.63 98.7 99.94 100.1 100.35 100.0 99.96 100.4 100.67 100.4 100.28 100.1 100.59 100.7 100.210 99.9 100.4

Mean (n=10) 99.8 100.0

%RSD (n=10) 0.8 0.6

Absolute Difference

(%)0.2

Determi-nation %LC with

new method

%LC with old

CU method

1 102.0 101.32 101.6 100.63 101.3 100.84 100.4 100.45 102.6 102.26 101.0 100.77 101.7 101.18 100.8 100.69 101.6 101.410 101.4 101.0

Mean (n=10)

101.4 101.0

%RSD 0.6 0.5Absolute

difference (%)

0.4

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Summary of Case Study to Minimize Border II (HPLC vs. UPLC)

• Use the same stationary phase with different dimension and particle size

• Same mobile phase but slight different gradient

• Same protocol template

• Use the same solutions and validate the method side by side

• The knowledge of both methods were exchanged during the process

• HPLC and UPLC method proved to be equivalent and can be used interchangeably

Border III. Between Functions

Development

validationtransfer

HPLC

UPLC HPLC

UPLC

HPLCUPLC

HPLC

HPLC

HPLC

UPLC

UPLC

UPLC UPLC

UPLC

UPLCHPLC

HPLC

HPLC

Border II

Border I

Border III

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III-a: Method Development vs. Validation

• Often reside in the same functional group

• Natural redundancy in terms of robustness, forced degradation, alternative columns

Method Development

Method validation

Method transfer

What Is Analytical Method Transfer

• Protocol driven study with pre-defined acceptance criteria

• Transfer of validated analytical procedures to a new laboratory

• Verification of a method’s suitability for its intended use

• Demonstration of a laboratory’s proficiency in running a particular method

• No official guidelines

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Options for Method Transfer

• Comparative testing:A set of samples are tested in both labs and resulting data are compared with predetermined acceptance criteria.

• Co-validation between two labs:The receiving laboratory is involved in method validation but have to identify which validation parameters are to be generated or challenged by the two labs.

• Complete or partial method validation:A repeat of method validation either completely or partially.

• Transfer waiver (omission of formal validation):Needs justification as to why method transfer was not needed. For example, lab is already testing the product.

Border III-b: Method Transfer and Validation

• Method transfers are closely related to validation

• Method transfer is more challenging because multiple laboratories and companies are involved– Different approaches to Validation and Transfer– Different expectations of what is an acceptable

validation– Different instruments and facilities

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Method Transfer and Validation

Method transfer• Can be part of the validation

• Protocol driven study with pre-defined acceptance criteria

• Transfer of validated analytical procedures to a new laboratory

• Verification of a method’s suitability for its intended use in a new laboratory


• No official guidelines

Method validation• Protocol driven study with pre-defined

acceptance criteria

• Validation of analytical procedures in a laboratory

• Verification of a method’s suitability for its intended use


• http://www.ich.org/LOB/media/MEDIA417.pdf; http://www.fda.gov/cder/guidance/2396dft.pdf

Objectives of Method Transfer

• Maintain the validated state of the method and meet all regulatory requirements

• Minimize surprises!

– Open and responsive communication

– Pre-determined expectations

– Clearly documented and communicated technical details

– Pre-transfer evaluation by experienced technical staff at receiving site

– Technical contact available for troubleshooting at transferring site

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Typical Method Transfer Steps

• Discussions Initiated

• Review of Method and Validation

• Laboratory Evaluation

• Protocol (Transfer or validation) Written

• Protocol Approved

• Experimental evidence from a transfer study generated

• Report (Transfer or validation) Written

• Report Approved

• Transfer Complete

Preparation for a Method Transfer

• Method – Details about method– Specific instrument

• Method development history report• Training/discussion on the method • Materials

– Reference Standard– Samples for Evaluation– Difficult to purchase supplies

• Specifications• Technical Contact• Details about product

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Prior to Formal Method Transfer

• Receiving laboratory should perform the method– Helps to determine where there are differences

and gaps in documentation• Lack of detailed test method instructions

– Assay Conditions– Calculations– System Suitability

– Differences with instrumentation or reagents

Prior to Formal Method Transfer

• Training of Personnel– Review of relevant SOPs

– Observation of test procedure

– Performing test procedure

• Helpful to include development, qualification and validation reports to recipient laboratory

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Method Validation and Transfer

• Method transfers are closely related to validation

• Method transfer is usually part of validation

Method Development

Method validation

Method transfer

Border III-c: Method Transfer and Method Development:Before or During Method Development

• Define goals of end method

• Dynamic platform of communication

• Exchange of knowledge

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Border III-c: Method Transfer and Development:

• Is the method inadequate by today’s scientific standard or regulatory requirement?

• Is sufficient data available to permit simplification of the method?

• Does monitoring of laboratory deviation suggest a need for method improvement ?

• Do newer method for similar products significantly outperform?

• Is the volume of testing justify further method optimization or automation?

After method Transfer

Border III-c: Method Transfer and Development:

– Concerns?

– Observations?

– Investigations/troubleshooting: Must involve multiple labs

During Method Transfer

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Case Study: Extraneous peaks associated with HPLC vials

AU

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00

3.10

8

3.67

0

RA

PA

MY

CIN

- 4.

133

Polypropylene

Deactivated glass vials

Case Study: Extraneous peaks associated with Glass Pipettes

AU

-0.002

-0.001

0.000

0.001

0.002

0.003

0.004

0.005

0.006

0.007

0.008

0.009

0.010

0.011

Minutes0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00

Unk

now

n1 -

1.69

0

Unk

now

n2 -

2.52

6

Unk

now

n3 -

2.97

7

Unk

now

n4 -

3.49

6

Rap

amyc

in -

3.94

7

Above: chromatograms of two different lots of glass pipettes .Plastic transfer pipettes, or no pipettes, were recommended.

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Case Study: Extraneous peaks associated with Extraction Vials

Extraction Vial Volume

(mL)

Number per case

Price ( $/case)

Price ($/vial) Comments

Nalgene-Teflon 10 10 224.70 22.47 Re usable. VWR Cat# 210030 10 333.30 33.33 Re usable. VWR Cat# 2100

BD Falcon-Poly Propylene, snap cap

5 500 160.00 0.32 VWR Cat # 60819-706

14 500 195.30 0.39 VWR Cat # 60819-740

BD Falcon-Poly Propylene, screw cap

15 500 220.89 0.44 VWR Cat# 21008-918

Starplex-Polypropylene

screw cap

5 1500 186.18 0.12 only 2 sizes available VWR14216-262

10 1000 135.96 0.14 only 2 sizes available VWR14216-266

15 500 192.00 0.38 Sigma Aldrich Cat# Z7204NUNC Poly propylene snap cap

Supelco pre-cleaned clear glass

7 100 92.50 0.93 Pre-cleaned* Sigma Aldrich27341



Supelco Silanized Clear Glass 4 1000 267.00 0.27

Larger sizes are available urequest, caps not included. Sigma Aldrich Cat# 27114

Kimble-glass 8 144 152.49 1.06 Currently being used VWR

66009-984

16 144 193.44 1.34 Currently being used VWR66009-986

Treatment to Glass Extraction Vials

• Rinse with 0.02% formic acid in acetonitrile

• Rinse with acetonitrile

• Wash with a regular washing cycle for other glassware

• Soak with 0.02% acid (formic, acetic or nitric) in water and rinse with DI water

• Soak/sonicate in DI water

• Details are critical to ensure accurate comparison cross labs.

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Standard Solution in Unwashed Glass Vials

0.00

0.50

1.00

1.50

2.00

2.50

3.00

3.50

0 1 2 3 4 5 6 7 8

Tota

l Im

purit

y A

rea%

Day

Standard Solution in Pre-cleaned Glass Vials

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 1 2 3 4 5 6 7 8

Tota

l Im

purit

y A

rea

%

Day

2ml

3ml

4ml

5ml

6ml

7ml

9ml

10ml

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Standard Solution in Silanized Glass Vials

Standard Solutions in Glass Vials Soaked and Sonicated in DI Water

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Standard Solution in Glass Vials Rinsed with MeCN 3 Times

0

0.5

1

1.5

2

2.5

3

0 1 2 3 4 5 6 7 8

Tota

l Im

purit

y ar

ea %

Day

Direct pour

8ml- std soln-1

8ml- std soln-2

8ml- std soln-3

8ml- std soln-4

8ml- std soln-5

Standard Solution in Glass Vials Rinsed with 0.02% Formic Acid in MeCN 3 Times

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1

0 1 2 3 4 5 6 7 8

Tota

l Im

purit

y A

rea%

Day

Direct pour

8ml- std soln-1

8ml- std soln-2

8ml- std soln-3

8ml- std soln-4

8ml- std soln-5

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Standard Solutions in Glass Vials Treated with 0.02% Acid (Nitric, Acetic or formic)

Standard Solution in Washed Glass Vials

0

0.02

0.04

0.06

0.08

0.1

0 1 2 3 4 5 6 7

To

tal i

mp

uri

ty %

Day

Standard in Washed Extraction Vial

2

3

4

5

6

7

9

10

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Standard Solution in BD Falcon Polypropylene Vials

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0 1 2 3 4 5 6 7

Tota

l Im

puri

ty a

rea%

Day

small-1

small-2

small-3

small-4

small-5

small-6

small-7

small-8

small-9

Summary to Case Study: Extraneous peaks Associate with Glassware

• Pay attention to the glass grade, vendor and treatment.

• Glass vials vary within the same lot/box.

• Rinse with 0.02% acid (formic, acetic, nitric) acid in water is effective for this method.

• Wash the glass vials with acidic detergent is effective.

• Use of polypropylene vials eliminates the problem.

• It is critical for multiple labs to be involved, to carry our experiments and share data with details.

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Method transfer vs. method development

• Start from the beginning of the lifecycle (define goals, communicate limitations)

• Maintain a dynamic and continuous process

• Build strong partnership and co-ownership through regular meetings, visits, design and execute experiments together

• Transfer knowledge, not just method

Method Transfer vs. validation Vs. Development

Method Development

Method validation

Method transfer

• Dynamic platform of communication

• Design and execute experiments (AMERT, Investigations)

• Share ownership• Exchange of knowledge

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Conclusions

• Analytical method lifecycle is a dynamic and continuous process

• Transfer of knowledge, instead of method, is desired in every stage of the life cycle

• Scientists/managers need to zoom in and zoom out to consider needs of other projects, methods or labs.

• It enhances efficiency to eliminate borders between methods, techniques and functional groups, as much as possible

• Strong partnership and co-ownership is the key to successful methods

Method Transfer Crossing Multiple Borders

Health & Medicine

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