Issue link: https://resources.nanotempertech.com/i/1050512
Methods Thermal unfolding by nanoDSF. Samples 014-01 to 014-16 and the reference molecule were measured as triplicates in thermal unfolding experiments with a heating rate of 1 °C/min in standard treated capillaries. The reference was previously diluted in sample buffer to reach a final concentration of 2 mg/ml, matching the concentration range of the other samples. 10 µl of sample were required for one thermal unfolding profile. For calculation of the overlap index, the first derivative data of the F350/F330 fluorescence ratio unfolding curves were used. Averages of the triplicate measurements were calculated and subtracted from the 1 st derivative value of the reference molecule for each scan. Subsequently, absolute values were added to yield the 1 st derivative overlap index for each sample. Protein charge variant profiling. Protein charge variants were profiled using a strong anion exchange column with a salt gradient at pH 7 and detected. N-linked glycans were released from the protein by PNGase F digest and then labelled by 2-AB. N-glycan profiling. N-linked glycan profiles were acquired by separation of labeled glycans on an amide column run in HILIC mode with fluorescent detection. Sialic acid quantification. Sialic acids were released by acidic hydrolysis followed by DMB-labelling. Total sialic acid was then quantified by fluorescent detection of labelled sialic acid species separated on a reversed phase chromatography column following normalization with internal standards. Acknowledgements Authors thank Silvia Verzini and Jenny Heßler from Downstream Processing Department at UGA Biopharma for purification of samples for the analyses. References Berkowitz SA, Engen JR, Mazzeo JR, Jones GB (2012) Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars. Nature reviews Drug discovery 11: 527- 540 Bui LA, Hurst S, Finch GL, Ingram B, Jacobs IA, Kirchhoff CF, Ng CK, Ryan AM (2015) Key considerations in the preclinical development of biosimilars. Drug discovery today 20 Suppl 1: 3-15 Cai XY, Gouty D, Baughman S, Ramakrishnan M, Cullen C (2011) Recommendations and requirements for the design of bioanalytical testing used in comparability studies for biosimilar drug development. Bioanalysis 3: 535-540 van Aerts LA, De Smet K, Reichmann G, van der Laan JW, Schneider CK (2014) Biosimilars entering the clinic without animal studies. A paradigm shift in the European Union. mAbs 6: 1155-1162