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A solid phase extraction, liquid chromatography and fluorescence (SPE-RPLC-FL) based protocol for the determination of free monosaccharides in highly complex raw material powders and formulated fermentation feedstocks and broths has been developed. Monosaccharides within sample extracts were derivatised pre-column with anthranilic acid and the derivatives separated using reversed-phase LC with fluorescence detection. Using a 2.1 mm × 50 mm 1.8 µm Zorbax Eclipse XDB-C18 column, a flow rate of 0.4 mL min−1 and an acetonitrile gradient in a sodium acetate buffer (pH 4.3; 50 mmol L−1) the baseline resolution of glucosamine, mannosamine, galactosamine, galactose, mannose, glucose, ribose, xylose, fucose and sialic acid within 20 minutes was achieved. Pre-column derivatisation involved combining a 30 mg mL−1 solution of anthranilic acid in a 1 : 1 ratio with an aqueous standard prior to injection. Standard analytical performance criteria were used for evaluation purposes, with the method found to exhibit LOD's as low as 10 fmol, and be linear and precise (%RSD < 2.2% (n = 7). The method was applied to the analysis of a range of highly complex biopharmaceutical production samples, including yeastolate powders, chemically defined media and in-process fermentation broth samples. Sample preparation involved passing an aqueous sample through a C18 solid phase extraction cartridge to trap hydrophobic peptides and vitamins, with recovery of all test sugars exceeding 90%. Finally, standard statistical analysis was performed on samples taken from different lots in order to estimate lot-to-lot variability.

Two rapid, sensitive and quantitative methods for the determination of the cysteine and cystine ratio in complex defined media feedstock using monolithic reversed-phase liquid chromatography (RPLC) and RPLC-MS are presented. Cysteine is pre-derivatised with purified 2-chloro-1-methylquinolinium tetrafluoroborate (CMQT) and separated from other derivatisation products on a narrow-bore 50mmx2mm I.D. monolithic C(18) column with UV detection at 355nm. For reversed-phase LC (RPLC) the separation is carried out isocratically using a mobile phase of 50mM trichloroacetic acid (TCA) adjusted to pH 2.5 with lithium hydroxide (LiOH) and acetonitrile (83:14) pumped at 1.5mL/min with an elevated column temperature. For RPLC-MS an ammonium acetate and acetonitrile gradient method was developed with a reduced flow rate of 0.3mL/min. The treatment of the samples consisted of dividing them into two aliquots, the first aliquot is analysed for cysteine and the second aliquot is analysed for cystine after its quantitative reduction to cysteine using tris(2-carboxyethyl)phosphine (TCEP). Both methods are linear, with R(2)>0.999 for 0.25-500microM for cysteine and 0.25-250microM for cystine using the LC-UV method, sensitive, with detection limit of 36nM for cysteine, and precise, with < or =1.1% RSD for both retention time and peak area (n=6). Samples (n=31) of an industry standard and supplied chemically defined media feedstock were analysed, finding cysteine ranging from 1.56 to 2.26microg/mL and cystine from 1062.02 to 1348.13microg/mL.

The in situ preparation of ethylene dimethacrylate porous polymer monoliths within 20 μL polypropylene pipette tips, bound via surface grafted methacrylate anchor sites, is reported. Gold nano-particles (AuNPs) were immobilised onto the monolith pore surface utilising azlactone chemistry and coverage verified using field emission scanning electron microscopy. Erythrina cristagalli lectin (ECL) was immobilised upon the attached AuNPs via a bio-functional linker. The ECL-modified tip was successfully applied for the enrichment of galactosylated protein (desialylated transferrin) versus a non-galactosylated protein (ribonuclease B) due to the specificity of ECL. Reversed-phase capillary HPLC was used to validate the efficiency and selectivity of the developed micro-extraction phase which resulted in an increase in extraction recovery of ∼95% due to the AuNP enhanced surface area. Further specificity of the ECL-modified tip was demonstrated with a complex mixture of non-glycosylated and glycosylated proteins with differing terminal sugar structures. Finally, the lectin affinity phase was applied to a galactosylated glycoproteins spiked Escherichia coli cell lysate to successfully demonstrate matrix tolerance.

This paper describes a rapid, sensitive and reproducible liquid chromatographic method specifically for the quantitative determination of total free sialic acid, employing thiobarbituric acid as pre-column tagging agent, following the oxidation of sialic acid with periodic acid. The derivatised sialic acid was separated from 2-deoxy-D-ribose and the other components utilising a short C18 monolithic column, with total run-times of under 90 s per sample. The method was successfully applied to quantify sialic acid in a range of complex samples, e.g. yeastolate powders, basal media and in-process samples supplied from the biopharmaceutical industry. The selective method was highly reproducible (%RSD for method (n = 6) =