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?(Fig.5).5). therapeutic formulations, and Sulfacarbamide subjected to sedimentation velocity (SV) analytical ultracentrifugation (AUC). Additionally, Advate and Kogenate were concentrated and subjected to buffer exchange by ultrafiltration to remove viscous cosolvents for the purpose of measuring gene 15. Additionally, observational studies have indicated that, among the recombinant full\length FVIII products, Kogenate and Helixate are more immunogenic than Advate 4, 16, 17. The problems associated with interpreting these studies have been discussed extensively 18, 19, 20. Protein aggregates in biopharmaceuticals constitute a risk factor for the development of anti\drug antibodies 21. Aggregation is usually defined as self\association of a protein to form stable covalent or non\covalent complexes. Sedimentation velocity (SV) analytical ultracentrifugation (AUC) has emerged as a powerful method for detecting protein aggregates in biopharmaceuticals 22. In contrast to size exclusion chromatography (SEC), gel electrophoresis, and other fractionation\dependent techniques, SV AUC is usually a matrix\free method that does not disturb the self\association process, and provides size and conformation information about the protein and its aggregates. Additionally, analysis can be performed in the product formulation buffer. The most widely used method for measuring protein aggregates by SV AUC is at 4 C to ~ 0.05 mL; this was followed by the addition of 3.9 mL of HBS/Ca2+, and further concentration to ~ 0.05 mL. After the centrifugation/filtration step had been repeated three times, the sample was collected in 0.33 mL of HBS/Ca2+. Wilate (870 IU of VWF ristocetin cofactor activity per vial; 940 IU of FVIII per vial) was purchased from Octapharma (Vienna, Austria), and reconstituted to its therapeutic formulation by adding Sterile Water for Injection. Human plasma\derived VWF was purified as explained previously 26. Polysorbate 80 (10% Tween\80 Surfact\Amps Detergent Answer) was purchased from Thermo Scientific (Waltham, MA, USA). Anotop 10 0.02\m syringe filters were purchased from Sigma Aldrich (St Louis, MO, USA). Amicon Ultra\15 Ultracel\30K centrifugal filters were purchased from Sulfacarbamide Merck Millipore (Billerica, MA, USA). UV absorbance spectroscopy UV absorbance scans of formulated Advate, Helixate and Kogenate and of polysorbate 80 were performed in a Beckman DU650 spectrophotometer (Indianapolis, IN, USA) in a 1\cm\pathlength quartz cell blanked against water. AUC SV experiments were performed at 105 000 on Advate, Helixate, Kogenate, and Wilate, or at 42 000 on purified VWF at 20 C in a Beckman Coulter ProteomeLab XLI analytical ultracentrifuge. Scanning Sulfacarbamide was performed at 280 nm in an CCR3 An\60 rotor equipped with 12\mm\pathlength double\sector cells and sapphire windows. For formulated Advate, Helixate, Kogenate, Wilate, and VWF, water was used in the reference sector. For buffer\exchanged samples, ultrafiltration buffer was used in the reference sector. Sample and reference buffer volumes were 0.40 mL each. Scans were initiated in continuous mode with a radial spacing of 0.003 cm after reaching the target rotor velocity, and were acquired at intervals of 3 min. Data were analyzed with sedfit, version 15.01c ( by use of the continuous and are the signal of the integrated SEC peak and the extinction coefficient of HC species is Sulfacarbamide given by is the molecular excess weight and is the mole portion. The excess Sulfacarbamide weight\average molecular excess weight is usually yielded a excess weight\average molecular excess weight for full\length FVIII of 246 kDa. The partial specific volume, , of a glycoprotein was estimated by using and are the excess weight fractions and partial specific volumes of the polypeptide and glycan. The partial specific volumes of the HC1C817CLC, HC1C1115CLC and HC1C1313CLC heterodimers estimated with sednterp are, identically, 0.733 mL g?1. A partial specific volume of glycoprotein glycans of 0.63 mL g?1 was used 36. With these values and the fractional glycan content of the FVIII heterodimers, a excess weight\average partial specific volume of 0.719 mL gC1 was obtained for full\length FVIII. SEC SEC was performed on 0.1\mL samples with a Superdex 200 Increase 10/300GL column (GE Healthcare Life Sciences, Marlborough, MA, USA) at 0.75.