(herein referred to as VP3 prime) was detected in all runs. MS deconvolution revealed this peak to contain a mixture of mostly deamidated VP3 proteoforms, as later discussed in Section 3.3. As column temperature increased, VP retention decreased resulting in VPs eluting earlier in the gradient as expected. More crucially, separation of VP1 and VP2 improved as column temperature decreased, with significant VP1 and VP2 peak overlap occurring when separation was performed at 60°C and near baseline separation achieved when separation was performed at 25°C. Determination of VP ratios is important as studies have shown that different VP stoichiometry during manufacturing can impact product potency . VP ratios were calculated using two methods, one fluorescence (FLR) based, and one MS based. For the FLR based method, VP ratios were calculated using the integrated VP peak areas from the FLR traces. With the MS based method, VP stoichiometry was determined using the area under the extracted ion chromatograms (XICs) of the proteoforms identified in Section 3.3. As Table illustrates, column temperature had a direct influence on VP stoichiometry determination in the FLR method. Improved full-length VP separation, particularly between VP1 and VP2, at lower temperatures enabled improved peak integration. This resulted in the detected copy numbers of VP1 increasing and the detected copy numbers of VP2 decreasing as column temperature was lowered, despite the copy numbers of VP3 only minimally changing. It has been suggested that varying ratios of VP1 to VP2 could have an impact on AAV potency , so the discrepancies seen at the different column temperatures illustrate a limitation to using FLR based quantitation methods. *The average areas were calculated using FLR traces with values calculated as an average of the three runs performed at each column temperature. Given that an AAV capsid contains a total of 60 VP copies, the number of VP copies per VP was determined by dividing the total number of VPs by the summed integrated area of VP1, VP2, VP3, and VP3 Prime and then multiplying the result by the area of each respective VP. VP ratios were calculated based on the theoretical ratio of VP1:VP2:VP3 being 1:1:10 as described in the literature. The summed total of 12 theoretical copies was divided by the summed integrated area of VP1, VP2, VP3, and VP3 Prime and then multiplied by the area of each respective VP.