1. Stability Indicating Assays for Dose Formulation Stability Testing Teresa Fuller GSK

2. Stability Indicating Assays for Dose Formulation Stability Testing –Stability Studies for Dose Formulations –What the white paper says –What is a stability indicating assay? –So do we need a stability-indicating assay? –Closing thoughts

3. Stability Studies for Formulations –Performed for every formulation to be used on a GLP study –Test the formulation for concentration on day 0 –Test the formulation for concentration on further timepoints to assess stability, eg. day 1, day 7 and day 14 –Based on concentration results over time, assess stability

4. What the white paper says “The analytical method should be suitable for the intended purpose. It is conceivable to have a rapid analytical method for test article only, which would not be suitable as a stability-indicating assay where degradents are separated from the test article or a more elaborate method which may have the additional advantage of identifying degradents. Both types of assays are acceptable and would require the appropriate level of validation for their intended purposes.”

5. What is a Stability Indicating Assay? –According to the US-FDA stability guideline of 1998 ‘validated quantitative analytical methods that can detect the changes with time in the chemical, physical, or microbiological properties of the drug substance and drug product, and that are specific so that the contents of active ingredient, degradation products, and other components of interest can be accurately measured without interference.’ –But this applies to drug substance and drug product not dose formulations! –For dose formulations we do not necessarily need to quantify the degradents –So what should the formulation analytical method look like?

6. Isocratic UPLC/UV –Chromatography column, %organic solvent and wavelength chosen for analyte of interest –K’ of at least 3 [K’ = (V t – V 0 )/V 0 ] –Eg. HPLC System Waters Acquity UPLC with TUV Detector using High Sensitivity Flow cell Typical Injection Volume1 µL Flow Rate1 mL/min Wavelength245 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% Trifluoroacetic Acid Mobile Phase BMethanol Isocratic Composition A:B69:31

7. Isocratic UPLC/UV Reference standard Stability sample day 7, ambient PFL 0.02 mg/mL in water

8. Generic UPLC-UV Gradient HPLC SystemWaters Acquity UPLC Typical Injection Volume0.5 – 5 uL Flow Rate0.6 mL/min WavelengthAnalyte specific Analytical Column100 x 2.1 mm i.d. Waters BEH C18 1.7 µm Column Temperature50 ºC Run Time12 min Mobile Phase A0.1% Trifluoroacetic Acid in water Mobile Phase B0.1% Trifluoroacetic Acid in acetonitrile Gradient profile Time%B 03 10100 10.5100 113

9. Generic UPLC-UV Gradient

10. Isocratic and Gradient Compared IsocraticGradient Run Time1-2 mins12 mins Method development1 day to several weeksMinimal Visible degradentsSignificantly different degradents probably not visible Most degradents and impurities likely to be visible Stability-indicatingpossibly

11. How can we assess how stability indicating the assay is? –Forced degradation –Store sample at 30°C, 65% RH or 40°C, 75% RH or 50°C, ambient RH –UV light exposure –Oxidation, eg. By addition of hydrogen peroxide –Acid/alkali hydrolysis –Compare forced samples to a fresh sample –If any additional peaks are seen in the forced samples but these are well resolved from the analyte then assume assay is adequate

12. How can we assess how stability indicating the assay is? –Peak purity analysis –Acquire UV spectrum at several points (at least 3) across the chromatographic peak –Compare spectra to look for differences which may indicate underlying peaks co-eluting with the analyte

13. How can we assess how stability indicating the assay is? –Mass spectral peak purity –Acquire mass spectrum at several points across the peak, similarly to UV peak purity

14. The ideal stability indicating method? –UPLC-MS/MS –Use specific parent and daughter ions to monitor the analyte Analyte Precursor ion (m/z) Product Ion (m/z) GSK123456539290 Product 290

15. Pros and Cons TechniqueProsCons Forced Degradation No additional equipment reqd Time Degradation may not be the same in real samples UV Peak purityQuickDegradation products may have similar spectrum Needs PDA detector Mass specSpecificExpensive equipment Mass spectral comparison difficult

16. So do we need a stability-indicating assay? –White paper says we don’t! –Unstable formulation considered to be greater than 10% drop from t0 –Complex oral and iv formulations are likely to have had previous development performed by a Product Development group –storage stability already tested to some degree –We need some confidence in our assay

17. Closing Thoughts –Several factors to consider –Balancing act between having confidence in the method and minimising method development time and effort –Is a gradient better than isocratic? –Is MS detection better than UV as it becomes more affordable?

18. Acknowledgements –Emma Simmonds, GSK PD –Tammy Clegg, GSK DMPK –Louise Kay, GSK DMPK

19. Useful Reference –Review of Development of Validated Stability Indicating Assay Methods –Journal of Pharmaceutical and Biomedical Analysis 28 (2002) 1011–1040