56 Chapter 3 FVIII. A stable isotopic labeled peptide GELNEHLGLLGPYIR[13C6, 15N4] was synthesized and was used as internal standard to correct for MS ionization variability. Sample handling procedure was based on our previous work where we have compared various denaturation conditions for simultaneous quantification of adalimumab and infliximab [30]. Using the 80 °C denaturation procedure we found that most signature peptides provided similar digestion efficiency as the commercial Smart Digest kit and was superior to denaturation and reduction with DTT at 60 °C. Finally, remaining steps in the method were evaluated and optimized to ensure a sensitive, repeatable and accurate result. Camelid nanobody amount and signature peptide signal intensity The optimum amount of biotinylated camelid nanobody needed for efficient purification of activated FVIII light chain was investigated. Camelid nanobody consist of an antibody variable domain fragment with a molecular mass of 13kDa targeting the light chain of FVIII. This is an important advantage over traditional full length antibodies derived from other mammals which are 10 times bigger. More camelid nanobody can fit in each streptavidin coated well thus providing increased binding sites for factor FVIII. The saturation curve in Figure 2 showed there was no significant (p = 0.075) change in signal intensity between 100 and 500 ng camelid nanobody. Therefore, 200 ng (15 pmol) camelid nanobody per well was used for all experimentations. Figure 2. On the x-axis, coated amount of biotinylated camelid anti FVIII nanobodies are plotted versus signal intensity of the signature peptide of the highest standard (500 ng/µL) on the y-axis, error bars represent SD with n = 3. Binding time and signature peptide signal intensity Another important parameter is the time required for the dissociation of VWF, the conversion of FVIII to FVIIIa and the binding of FVIIIa light chain to the 96 well plate coated camelid nanobodies. This experiment was performed with human derived FVIII plasma, since octocog alfa in the standard solution is not pre-conjugated with VWF. At 24 hours the signal intensity was significantly higher (p = 0.0004) than the signal intensity obtained at 5 hours (Figure 3). However, the difference in signal intensity between 1 and
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