diversity of molecules and the resulting analytical challenges chaotic 24 april 2013

Diversity of molecules and the resulting analytical challenges

CHAOTIC 24 April 2013

Overview

Structure of Dose Analysis at GSK

Techniques at our Disposal

Molecule Diversity

Case Studies

Summary

Structure of Dose Analysis at GSK

Multi discipline team within Bioanalytical Science and Toxicokinetics

– Dose concentration analysis

– Bioanalysis

– Toxicokinetics

Purely analytical and interpretation group

Techniques at our Disposal

HPLC- UV

UHPLC-UV

HPLC or UHPLC – CAD (Charged aerosol detector)

A280

LC-MS/MS

Molecular diversity

Traditional Small molecules

Bio-pharm molecules

Peptides

Intermediates in the manufacturing process– Analysis for genetic toxicolgy screens

Natural products

Case Studies - Small Molecules, Compound A

Method Validated with out issue

All samples and standards diluted 1:1 with water prior to injection to improve peak shape

Typical Injection Volume 3 µL

Flow Rate 1 mL/min

Wavelength 311 nm

Analytical Column 50 x 2.1 mm i.d. Waters BEH C18 1.7 µm

Column Temperature 60 ºC

Run Time 1 min

Typical Retention Time 0.8 min

Mobile Phase A 0.05% TFA

Mobile Phase B Acetonitrile

Isocratic Composition A:B 60:40

Diluent Dimethyl Sulphoxide (DMSO)

Case Studies - Small Molecules Compound A

Consistently High results for formulations in 1.14% (w/v) 10M aqueous sodium hydroxide containing 25mM sodium bicarbonate and up to 1M equivalent D-Mannitol.

Why? Was it the formulation or the analysis?

Investigations into the analytical method suggested it was working as it should

On one set of samples for analysis, wrong set of vials injected (undiluted with water)

Standard peak area considerably higher

Samples with in Specification. (although poor peak shape)

Injection of correct samples gave high results

Case Study - Small Molecules, Compound A

Conclusion -– Standards falling out of solution on the addition of

water.– Samples remain in solution due to presence of

solubilising agent in Vehicle– Not picked up on vehicle interference test in validation

due to difference on the way sample prepared

Assay revalidated using a different diluent not requiring the addition of water

Case Study - Bio-Pharm, Compound B

Protein based molecule

Analysis by Absorbance at 280nm on UV-Vis spectrophotometer

Supplied with solution with known concentration and extinction co-efficient

Response from vehicle greater than acceptable, standard approach

All dilutions to be done in vehicle, and response of blank vehicle to be measured at each analysis point and subtracted from sample response

Case Study - Bio-Pharm, Compound B

In general assay of this type give sample results within ±2% of nominal concentration

This compound ≈ 108% of nominal, although within specification this raised questions as to the accuracy of the method

All investigative work under taken indicated this to true result

Subsequent re-analysis of Stock by Bio-pharm group, found concentration to have changed and as such possible cause of high results.

Case Study – Peptides, Compound C

Molecular Weight 2284 amu

Reasonable solubility in organic/aqueous diluent (9mg/mL)

Range of pKa

Low λmax 220nm (relatively insensitive)

Case Study – Peptides Compound C

Uncommonly large variation in retention time across systems

pH requires close attention

Use of Water or 0.9% (w/v) Aqueous Sodium Chloride (vehicle) as diluent not suitable due to reduced UV response

Requires the presence organic in diluent and acidic pH

Assay validated to within usual parameters

Case Study - Intermediates in the Manufacturing Process, Compound D

Compound D has poor UV chromaphore

HPLC-UV analysis unsuitable for levels required

Ionisation not sufficient for CAD detector

LC-MS/MS method validated in vehicle -10% DMSO in 0.9% (w/v) Aqueous Sodium Chloride

– On edge of acceptance criteria for accuracy and precision at LLQ

– 2 initial runs failed to meet acceptance du to variablility attributed to sample preparation


Change of vehicle to 1% Aq. Methylcellulose

Validation attempted with the dilution (x10) as initial validation

Validation runs fail on several occasions due to front and back calibration lines have different response and high variation of QCs


Logical conclusion, due to change in vehicle therefore diluted further (x1000)

No change in out come

It appeared that the run performance of assay benefitted from allowing the mass spectrometer to settle for an extended period prior to run.

Initially looks promising during tests, however validation runs continue to fail due to increased variability

Chromatography investigated to assess ion suppression

Appearance of second peak on leading edge observed


Second Peak


Sample sent for NMR analysis

Confirmed the presence of 2 anomeric forms

Confirmation received from chemist that;

– that in solution 2 anomers are formed

– the anomers have differing MS responses due to one anomer being ionised more readily than the other

– The rate of interconversion was specific to media, pH and temperature

Consequently there was no realistic way to resolve the issues

Acceptance criteria widened for accuracy and precision to 20%


TertiaryButylamine

No UV chromaphore

MW 73 amu

Simple structure, volatile

Classic method of analysis GC-MS, not available to us.

CH3

CH3

CH3

NH2

Intermediates in the Manufacturing Process Compound E

Attempted derivitisastion to allow LC-MS/MS analysis failed

Analysis not possible without derivitisation due to;– no retention on reverse phase chromatography– poor ionisation in MS source– no fragmentation (although possible to use SIM)

No way of telling if the all the all the TBA had been derivitised

Possibility of using accurate mass instrument (Waters Synapt G2-S, quantitative ToF) but not GLP validated

Contracted out for analysis by GC-MS

Case Study - Natural products, Compound F

Mono/Oligosaccharides

UV not suitable CAD Detection required

What peaks to monitor?

Soluble Insoluble


Validated HPLC ConditionsHPLC System Agilent 1100Typical Injection Volume 5 L

Flow Rate 1 mL/minAnalytical Column

Waters XBridge Amide, 3.5 m, 150 x 4.6 mm

Column Temperature 45C

Run Time 50 minutesTypical Retention Time

Approximately 24.5 minutes (Peak 1) Approximately 29.2 minutes (Peak 2)

Mobile Phase A (90:10) Acetonitrile:Water containing 0.02% (v/v) NH4OH

Mobile Phase B (50:50) Acetonitrile:Water containing 0.02% (v/v) NH4OH

Time (mins) %A %B

0.0 95 5

2 95 5

42 25 75

42.1 95 5

Gradient profile


HPLC-CAD Chromatogram


For quantification, sum of 2, 3-chain sugars

Sum of areas means transfer to excel and 100% check of data

Poor sensitivity reference standard at 15mg/mL

23 days stability generated @ room teperature


Anthocyanin analysis

Warned about lack of stability

Diluted in 10% phosphoric acid to increase stability

Unstable in light (>40% loss in 12 hours on the bench) confirmed even with acid

All work done in amber glassware


Validated HPLC ConditionsHPLC System Agilent 1100Typical Injection Volume 10 L

Flow Rate 0.8 mL/minAnalytical Column

250 x 4.6 mm i.d. LiChrosorb RP 18.5 µm

Column Temperature 35C

Run Time 25 minutes

Typical Retention Time

Peak 1: ~10.79 minPeak 2: ~11.17 minPeak 3: ~13.24 minPeak 4: ~14.39 min

Mobile Phase A 10% Formic Acid (FA)

Mobile Phase B (75:15:10) Water:Acetonitrile:Formic Acid

Time (mins) %A %B

0.0 70 30

20 70 30

20.1 0 100

25 70 30

Gradientt profile


HPLC-UC Chromatogram of Anthocyanins


For quantification, sum 4 separate anthocyanins

Sum of areas means transfer to excel and 100% check of data

6 Days stability generated in acidic conditions, protected from light

Summary

Dose analysis set up slightly different at GSK with a pure analytical that also performs other analytical tasks

Dose analysis has to develop and validate method suitable for quantification of highly varied compounds

Ready access to a variety techniques

Still some things are beyond our capability

This set up allows the dose analysis group to be true analytical experts and better placed to deal with diversity

Acknowledgements

Amanda Foster

Tammy Clegg

Aida Merchan

Connie Parker

Mark Mullin (RTP)

Back Up

CAD

diversity of molecules and the resulting analytical challenges chaotic 24 april 2013

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