Mabthera

For rituximab, the buffer of 2 mL formulation solution of Mabthera (Mabthera, Roche; exp. year: 2013, 2 mL, 10 mg/mL) was exchanged to 0.25 M ammonium acetate buffer (pH 4) with Amicon Ultra-15 Centrifugal Filter Units (Cutoff: 30 kDa, Merck) and the sample was incubated for 138 h at 40 °C. Afterward, the buffer was changed to ddH₂O overnight with a Spectra/Por dialysis membrane and lyophilized. For NMR measurements the sample was dissolved in 500 μL of a 7 M urea-d₄ (98 atom%D, ARMAR Chemicals) solution in D₂O (100 at.% D, ARMAR Chemicals) resulting in a concentration of 30 to 40 mg/mL mAb. Urea solutions were freshly prepared to minimize the formation of isocyanic acid which leads to carbamylation of lysine residues. For reducing the disulfide bonds, approx. 1–1.6 mg of tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (Sigma-Aldrich) was added to the sample corresponding approximately to 11 mmol L⁻¹ followed by incubation at 60 °C for 15 min. The pH was adjusted to 2.3 or 7.4 by adding DCl or NaOD (ARMAR Chemicals), respectively.
1 to 2 mg of each reference peptide (Table 2) was dissolved at the same conditions as full-length rituximab but without TCEP treatment. Afterward, the pH was adjusted to 2.3 or 7.4 by adding DCl or NaOD (ARMAR Chemicals), respectively.

Rituximab was obtained in solution (Mabthera^®, 10 mg/mL, Roche Pharma AG, Grenzach-Wyhlen, Germany) and a PD-10 (GE Healthcare, Vienna, Austria) size exclusion column was used for buffer exchange according to manufacturer’s protocol to give RTX in 0.1 M NaHCO₃ solution (7 mg/mL). For the conjugation of trans-cyclooctene (TCO), 2 mL of the RTX solution were mixed with 20 molar equivalent of TCO-NHS ester dissolved in DMSO and the reaction was stirred for 30 min at ambient temperature followed by incubation overnight at 4 °C under light exclusion. Subsequently, the modified antibody (RTX-TCO) was purified using size exclusion chromatography (PD-10) to give 10 mg of RTX-TCO dissolved in PBS. The antibody was treated following the same procedure as described above without adding TCO-NHS ester, in order to obtain a non-modified RTX counterpart as negative control.

All chemicals and solvents were purchased as reagent grade from commercial sources unless otherwise stated. trans-Cyclooctene-NHS ester and tetrazine-PEG₅-NHS ester were bought from Click Chemistry Tools (Scottsdale, AZ, USA). Rituximab (MabThera^®, Roche Pharma AG, Grenzach-Wyhlen, Germany) was of pharmaceutical grade and was a kind gift from the University Hospital of Innsbruck.