Stability Indicating Assays for Dose Formulation Stability Testing

Stability Indicating Assays for Dose Formulation Stability Testing Teresa Fuller GSK

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

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

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. ”

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?

Isocratic UPLC/UV – Chromatography column, %organic solvent and wavelength chosen for analyte of interest – K’ of at least 3 [K’ = (Vt – V 0)/V 0] – Eg. HPLC System Waters Acquity UPLC with TUV Detector using High Sensitivity Flow cell Typical Injection Volume 1 µL Flow Rate 1 m. L/min Wavelength 245 nm Analytical Column 50 x 2. 1 mm i. d. Waters BEH C 18 1. 7 µm Column Temperature 60 ºC Run Time 1 min Typical Retention Time 0. 8 min Mobile Phase A 0. 05% Trifluoroacetic Acid Mobile Phase B Methanol Isocratic Composition A: B 69: 31

Isocratic UPLC/UV Reference standard Stability sample day 7, ambient PFL 0. 02 mg/m. L in water

Generic UPLC-UV Gradient HPLC System Waters Acquity UPLC Typical Injection Volume 0. 5 – 5 u. L Flow Rate 0. 6 m. L/min Wavelength Analyte specific Analytical Column 100 x 2. 1 mm i. d. Waters BEH C 18 1. 7 µm Column Temperature 50 ºC Run Time 12 min Mobile Phase A 0. 1% Trifluoroacetic Acid in water Mobile Phase B 0. 1% Trifluoroacetic Acid in acetonitrile Gradient profile Time %B 0 3 10 10. 5 100 11 3

Generic UPLC-UV Gradient

Isocratic and Gradient Compared Isocratic Gradient Run Time 1 -2 mins 12 mins Method development 1 day to several weeks Minimal Visible degradents Significantly different degradents probably not visible Most degradents and impurities likely to be visible Stability-indicating possibly

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

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

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

The ideal stability indicating method? – UPLC-MS/MS – Use specific parent and daughter ions to monitor the analyte Analyte Precursor ion (m/z) 539 GSK 123456 Product 290 Product Ion (m/z) 290

Pros and Cons Technique Pros Cons Forced Degradation • No additional equipment • Time reqd • Degradation may not be the same in real samples UV Peak purity • Quick • Degradation products may have similar spectrum • Needs PDA detector Mass spec • Specific • Expensive equipment • Mass spectral comparison difficult

So do we need a stability-indicating assay? – White paper says we don’t! – Unstable formulation considered to be greater than 10% drop from t 0 – 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

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?

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

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