2.  What was the issue?  Why does it happen?  How does it happen?  Influencing factors  Filtration effects  Remedial action  Conclusion

4.  Issue  Stability study conducted at GSK  Low Day 0 analytical results during Phase 1  20% reduction in nominal conc. over time (Days 0 – 7)  TM; Compound A  Vehicle; Water  Hypothesis  Low results possibly due to TM adhesion to scintillation vials  Issue experienced during previous stability studies

6.  Scintillation vials are made of Borosilicate glass  Consists of BoronOxide and Silica  More thermo stable & less dense compared to common glass (prepared from soda lime)  They contain Silanol (Si-O-H) functional groups on their surface  Susceptible to deprotonation;  During interaction with aqueous solutions (e.g. Water), deprotonation of scintillation vial surface occurs  Removal of H+ proton from Silanol group; SiO-H → SiO- + H+  Produces electro-negative (-) charge on the inner walls of scintillation vial  Creates an attractive force for electro-positively (+) charged molecules  i.e. Acidic functional groups present in Compound A  Resulting in initiation of adsorption of Compound A

8.  Adsorption: Adhesion of atoms / ions / molecules from a dissolved solid to a surface  In our formulation;  Acidic Carbonyl (C=O) group of Compound A undergoes nucleophilic chemical reaction  Nucleophile (Nu-) is the deprotonated scintillation vial surface  Chemical bond between (C=O) and (Nu-)  Results in hydrolysis of Compound A  Consequently, denaturing the chemical structure of Compound A  Reduction of Compound A (active material) within the formulation  Verified by low analytical results in stability study

9.  pH  Adsorption may affect the formulation pH  Measured pH values within stability study: 2.5 – 5.5 (at 10 – 0.01 mg/mL respectively)  Results typically on the low side – Indicative of acidic nature of Compound A  Temperature  Adsorption becomes more profound when interaction between adsorbant (scintillation vial) and adsorbate (Compound A) occurs across thermo stable surface  Scintillation vials are known to be good at retaining heat (high thermal stability)  Provide a perfect medium for this interaction  Concentration  Adsorption may occur irrespective of formulation conc.  This suggests it is not relative (or progressive) to formulation conc.  In most cases, only 1-2% of TM adheres to scintillation vials (across all conc.)  However, at lower conc. there is greater possibility of TM to be hydrolysed  Due to larger surface area of nucleophilic sites in proportion to acidic molecules within formulation  Therefore, adsorption more evident at lower conc. (0.01 mg/mL), compared to higher conc. (10 mg/mL)  Consistent with results obtained throughout stability study

10.  Adsorption may also explain low analytical results obtained from formulation filtration via membrane filters  Millex-GV PVDF membrane filters used at GSK  Consist of open colloidal structures  Hydrophilic  Known to cause less adhesion compared to Nylon / PTFE membrane filters  Therefore, any TM content reduction may not be attributed to hydrolysis

11.  Adsorption observed using PVDF membrane filters may be related to electrostatic interaction between TM and Filter  Opposite electro charges between the TM (+) and Filter (-) attract via Van Der Waal’s forces  VDW Force: Attraction between opposite electro charges in atoms / molecules / surfaces  Typically weak bonding  However, VDW force of adhesion is dependent upon the surface of interaction  Fewer surface asperities (unevenness) result in a larger area of contact between the particles and surface (i.e. TM + Filter)  Consequently increasing the force of attraction  Open colloidal structure of PVDF filters provide an ideal medium for VDW interaction

13.  Stability study repeated – Phase 2  Samples for analytical submission placed into common glassware (made of soda lime) instead of scintillation vials  Non silanised  Do not act as an initiator for TM adsorption  New time-points of analysis implemented; Days 0, 1 & 7  Analytical results;  Within the acceptable limits of ±10% of nominal conc. Day 0  No significant reduction in nominal conc. over time  Adsorptive effects using membrane filters reduced by;  Saturating filter with small volume of formulation (1-2 mL) prior to sampling  Filter adsorptive sites gradually occupied by the formulation mix  Therefore, additional filtration will produce minimal adsorptive effects

15.  Scintillation vials contain Silanol functional groups  Undergo deprotonation when in contact with aqueous solutions  Creates a electro-negative (-) charge inside container  Attracts electro-positively (+) charged TMs  Susceptible TMs undergo hydrolysis via nucleophilic reaction, causing adsorption  Results in low TM % nominal conc.  Degree of adsorption influenced by formulation conc.  More apparent at lower levels compared to higher levels  Due to greater possibility of TM undergoing hydrolysis  Issue overcome by replacing scintillation vials with common glassware (made of soda lime)  Adhesion of TM in formulation also influenced by membrane filters  Via Van Der Waal’s interaction  Attraction between opposite electro charges in TM and PVDF Filter  Minimised by passing 1-2 mL of formulation through filter prior to sampling  Saturates filter membrane adsorptive sites