Page 117 - Tyrosine-Based Bioconjugations - Jorick Bruins

7.2. SPOCQ on proteins – Critical process parameters
As described in this thesis, reaction conditions for SPOCQ have been optimized to enable >95% labeling of antibodies in a reliable fashion. While specific conditions were already included in the supporting information of chapters 3 and 4, a detailed explanation of each process parameter and its impact on SPOCQ has not been provided yet. As concluded in chapter 5, the rate-limiting step of SPOCQ is the enzymatic oxidation of tyrosine, which in this thesis was performed by mushroom tyrosinase (mTyr). Thus, for faster conjugations, the rate-limiting oxidation rate of tyrosine must be accelerated. At the same time, the relative rate of intermolecular cycloaddition with strained alkynes or alkenes versus non-specific side-reactions is critical for product purity: the in situ generated quinone has only a limited lifetime before undergoing Michael addition by nucleophilic amino acids, hence cycloaddition by either BCN, TCO or cpTCO must be rapid. Here, the impact of various process parameters on enzymatic oxidation and cycloaddition chemistry are discussed in detail.
pH: Over the course of this research, it was found that the pH of the reaction is of paramount importance to the reaction rate and conversion rate of SPOCQ (chapter 3, Figure S10 and S11). At lower pH, reaction rate of Michael addition by nucleophilic amino acids is mitigated due to protonation of amine and thiol residues. Unfortunately, oxidation rate by tyrosinase is also directly correlated to pH, with mushroom tyrosinase having barely any activity below pH 5.0. Generally, a pH of 5.5 is optimal for selective antibody modification, yielding clean conjugates without hampering the rate of tyrosine oxidation.
Buffer type: While buffer seems to have little influence on SPOCQ, it is noted that tris(hydroxymethyl)aminomethane (present in Tris buffer) is also a nucleophilic agent that is able to perform Michael addition to quinones and therefore incompatible with SPOCQ. Generally, any non-nucleophilic buffer capable of maintaining the desired pH of the reaction is suitable. Low chloride concentration is preferred, because mushroom tyrosinase is inhibited during storage over time by chloride ions.
Temperature: Increase in temperature increases the rate of oxidation by mushroom tyrosinase. However, it also leads to increased non-selective Michael additions. Generally, performing the reaction at 4 °C is optimal (chapter 3, Figure S10).
Protein concentration & tag stoichiometry: In case of C-terminally labeled antibodies, 5 mg/mL antibody and 3-5 eq. of BCN, TCO or cpTCO-bearing label results in optimal conversion. However, for N-terminal labeling of antibodies on the light chain, it was found that a significantly higher protein concentration is necessary to prevent non-selective Michael additions. This is most likely due to the tag being near the N-terminus of the heavy chain, as we found the main side-product to be 75 kDa in weight (chapter 3, Figure S10). Generally, a reaction consisting of 5 mg/mL mAb,
Summary and General Discussion
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