Page 104 - Tyrosine-Based Bioconjugations - Jorick Bruins

Chapter 6
Figure 2. Comparison between inserted tags in trastuzumab.
Having the various mutants in hand, SPOCQ was attempted on both Tras-Y1 and Tras-Y2 antibodies in PBS (50 mM phosphate, 150 mM NaCl) at pH 5.5 or pH 7.1, and at 4 °C or 37 °C. The higher temperature and pH would generally increase the reaction rate of the oxidation by mushroom tyrosinase,8, 22 although more side reactions might occur due to an increase in non- selective Michael additions of nucleophilic amino acid side-chains.23 The antibodies were modified at 3 mg/mL with 6 eq. of BCN-lissamine with overnight incubation, after which SDS- PAGE and HPLC analyses were performed.
A few interesting observations can be made, the first of which is that Tras-Y1 seems to outperform Tras-Y2 based on a higher fluorescence intensity, which is most clearly visible at pH 7.1 (Figure 3A and 3B, lanes I and L). Furthermore, SPOCQ at pH 5.5 seems to lead to lower yield than at pH 7.1, which is in contrast with SPOCQ on termini where a shift in pH generally only results in different rates of oxidation by mushroom tyrosinase. In those cases, a lower pH resulted in decreased non-specific Michael addition by nucleophilic amino acids, and therefore a better conjugation yield.23 A case can be made for the oxidation not going to completion due to the inaccessibility of the tyrosine by mushroom tyrosinase, but then one would expect a difference in conversion rate between 4 °C and 37 °C as well, which is not the case (Figure 3A and 3B, lane C vs lane F). A control experiment with mushroom tyrosinase in the absence of BCN- lissamine for Tras-Y1 at pH 7.1 (Figure 3A, lanes H and K), indicated that the antibody seems to fragment into lower molecular weight fragments. In contrast, a similar control experiments with terminal tyrosine-bearing antibodies, generally induced cross coupling between light and heavy chains, resulting in higher molecular weight conjugates.
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