The published assays available for capsular

polysaccharides typically quantify a specific subunit of the repeating structure. Hence, each capsular polysaccharide or subset of serotypes tends to have a custom method for polysaccharide quantification. Many of these assays involve complex colorimetric procedures but research groups have found alternative approaches for measuring polysaccharide quantity [16], [17] and [18]. Several authors have recognized the analytical bottleneck posed by sugar quantitation and devised high throughput methods. Methods based on anthrone have been developed and further scaled-down Bcl-2 inhibitor to microplates [17], [18] and [19]. This assay’s limitations include reagent instability, poor reactivity with pentoses and methylated sugars, interference by process substance such as phenol, and issues with consistency

Pictilisib in vitro [20] and [21]. Refractive index has been used in conjunction with HPLC for many years to estimate sugar content. However, without the added purification and normalization provided by chromatography, this approach is exceedingly sensitive to background interference. Other methods involving phenol, 1-napthosulfonate, or aniline phthalate/trichloroacetic acid have been proposed but suffer from toxicity, interference, and limited reactivity with ketoses, respectively [20]. The phenol sulphuric acid method (PHS) is perhaps the most promising assay for integration with high throughput screening. This method is based on a colorimetric product formed when phenol, sulphuric acid, and sugar are reacted and was first described by Dubois et al. in 1951 [22]. This assay is broadly applicable and measures hexoses and pentoses in a variety of oligosaccharides, making it useful for quantifying neutral sugars [20] and [23]. The broad carbohydrate specificity of this assay underlies

its attractiveness but the measurement may be confounded by the reaction of heterogeneous carbohydrate-containing substances, such as glycoproteins. In one modification on the original method, Oxymatrine the PHS procedure was refined by reversing the sequence of reagent addition to improve sensitivity for glycated proteins and uniformity with respect to sugar type [24]. Saha et al. removed the heating step and reduced volumes to 2.5 mL total per sample [25]. Subsequent efforts have focused on reducing the volume further and/or improving throughput but have required cumbersome heating and/or specialized pipetting not amenable to automation [25], [26], [27] and [28]. To further optimize and minimize interference, procedures for cleaning up protein interference have been described [29] and [30]. However, none of the described methods minimize sample utilization nor are microplate-based, while concurrently simplifying the heating procedures sufficiently for transfer to a robot for automation. Rapid impurity measurements are critical for the development of purification processes from biological feedstreams.