1. Diversity of molecules and the resulting analytical challenges CHAOTIC 24 April 2013

2. Overview Structure of Dose Analysis at GSK Techniques at our Disposal Molecule Diversity Case Studies Summary

3. Structure of Dose Analysis at GSK Multi discipline team within Bioanalytical Science and Toxicokinetics –Dose concentration analysis –Bioanalysis –Toxicokinetics Purely analytical and interpretation group

4. Techniques at our Disposal HPLC- UV UHPLC-UV HPLC or UHPLC – CAD (Charged aerosol detector) A280 LC-MS/MS

5. Molecular diversity Traditional Small molecules Bio-pharm molecules Peptides Intermediates in the manufacturing process –Analysis for genetic toxicolgy screens Natural products

6. 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 Volume3 µL Flow Rate1 mL/min Wavelength311 nm Analytical Column50 x 2.1 mm i.d. Waters BEH C18 1.7 µm Column Temperature60 ºC Run Time1 min Typical Retention Time0.8 min Mobile Phase A0.05% TFA Mobile Phase BAcetonitrile Isocratic Composition A:B60:40 DiluentDimethyl Sulphoxide (DMSO)

7. 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

8. 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

9. 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

10. 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.

11. Case Study – Peptides, Compound C Molecular Weight 2284 amu Reasonable solubility in organic/aqueous diluent (9mg/mL) Range of pKa Low λ max 220nm (relatively insensitive)

12. 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

13. 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

14. Case Study - Intermediates in the Manufacturing Process, Compound D 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

15. Case Study - Intermediates in the Manufacturing Process, Compound D 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

16. Case Study - Intermediates in the Manufacturing Process, Compound D Second Peak

17. Case Study - Intermediates in the Manufacturing Process, Compound D 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%

18. Case Study - Intermediates in the Manufacturing Process, Compound D TertiaryButylamine No UV chromaphore MW 73 amu Simple structure, volatile Classic method of analysis GC-MS, not available to us.

19. 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

20. Case Study - Natural products, Compound F Mono/Oligosaccharides UV not suitable CAD Detection required What peaks to monitor? Soluble Insoluble

21. Case Study - Natural products, Compound F Validated HPLC Conditions HPLC SystemAgilent 1100 Typical Injection Volume 5  L Flow Rate1 mL/min Analytical Column Waters XBridge Amide, 3.5  m, 150 x 4.6 mm Column Temperature 45  C Run Time50 minutes Typical 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) NH 4 OH Mobile Phase B (50:50) Acetonitrile:Water containing 0.02% (v/v) NH 4 OH Time (mins) %A%B 0.0955 2 5 422575 42.1955 Gradient profile

22. Case Study - Natural products, Compound F HPLC-CAD Chromatogram

23. Case Study - Natural products, Compound F 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

24. Case Study - Natural products, Compound F 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

25. Case Study - Natural products, Compound F Validated HPLC Conditions HPLC SystemAgilent 1100 Typical Injection Volume 10  L Flow Rate0.8 mL/min Analytical Column 250 x 4.6 mm i.d. LiChrosorb RP 18.5 µm Column Temperature 35  C Run Time25 minutes Typical Retention Time Peak 1: ~10.79 min Peak 2: ~11.17 min Peak 3: ~13.24 min Peak 4: ~14.39 min Mobile Phase A10% Formic Acid (FA) Mobile Phase B (75:15:10) Water:Acetonitrile:Formic Acid Time (mins) %A%B 0.07030 207030 20.10100 257030 Gradientt profile

26. Case Study - Natural products, Compound F HPLC-UC Chromatogram of Anthocyanins

27. Case Study - Natural products, Compound F 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

28. 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

29. Acknowledgements Amanda Foster Tammy Clegg Aida Merchan Connie Parker Mark Mullin (RTP)

30. Back Up CAD