Chromeleon 6

Catecholamines were measured by HPLC with electrochemical detection as described previously [27 (link)–29 (link)]. Striatal tissue was homogenized in 50 μl of 0.1 M perchloric acid per mg of striatal tissue. Cell debris was pelleted by centrifugation (17,000 x g for 20 min at 4°C). 20 μl of supernatant was injected onto a C18 reverse-phase column (Prontosil 120-3-C18, Thermo Fisher). The mobile phase consisted of 85 mM sodium acetate, 35 mM citric acid, 0.5 mM octane sulfonic acid, 0.15 mM EDTA solution and 10% methanol (pH 4.3). Flow rate was 0.8 ml/min. Electrochemical detection of dopamine, DOPAC (3,4-dihydroxyphenylacetic acid) and homovanillic acid concentrations was carried out at 800 mV. Calibration was achieved by comparison with external standards run after every third sample and Chromeleon 6.80 software (Thermo Fisher Scientific). Values are represented as ng catecholamine per mg wet weight tissue.

The chromatographic analyses were performed using a liquid chromatograph from Thermo Fisher Scientific (Sunnyvale, CA, USA), an Ultimate 3000 equipped with an LPG-3400SD pump, an ACC-3000 autosampler, a 5 μL loop, a VWD-3400RS UV detector. Data processing was performed using Chromeleon 6.80 software (Thermo Fisher Scientific, Sunnyvale, CA, USA). The enantioselective separation was performed using a Supelco CHIROBIOTIC T column (150 mm × 2.1 mm × 5 µm) at 25 °C. The mobile phase was composed of 65% of methanol and 35% water containing 0.04% of formic acid. The flow rate was set at 0.2 mL/min. The detection wavelength was set at 207 nm. The injected sample volume was 5 µL.

GDP profiling was performed as previously reported [17 (link)]. Briefly, α-dicarbonyls were derivatized with o-phenylenediamine yielding their respective quinoxaline derivatives. The pH of the double-chamber bag model was adjusted to 5.5 prior to derivatization. A Thermo Fisher UltiMate 3000RS liquid chromatography system consisting of a pump with degasser, autosampler, column compartment, and diode array detector equipped with an ACQUITY UPLC® BEH phenyl column (1.7 μm particle size; 2.1 × 100 mm, Waters, Eschborn, Germany) was used for the chromatographic separation of the quinoxalines. The system was controlled by Chromeleon 6.80 (Thermo Fisher Scientific, Dreieich, Germany). The quinoxaline derivatives were analyzed between 120 and 650 min after adding the derivatizing reagent.

A Waters 2695 Separation module (Waters GmbH, Eschborn, Germany) equipped with a Waters 2487 Dual λ Absorbance Detector (Waters GmbH, Eschborn, Germany) at 214 and 280 nm was used. Isocratic elution with a 25 mM sodium phosphate running buffer containing 200 mM sodium chloride (pH 7.0) was performed.
For mAb1-formulations (F1–F3), 10 µL of a reconstituted solution corresponding to a loading of 50 µg protein were loaded on a Tosoh TSKgel G3000SWxl, 7.8 × 300 mm, 5 µm column (Tosoh Bioscience, Griesheim, Germany) and separated with a flow rate of 0.7 mL/min.
For mAb2 (F4), samples were diluted with 10 mM histidine buffer (pH 5.5) to 1 g/L protein concentration, and 25 µL was injected, corresponding to a load of 25 µg protein. A YMC-Pack Diol-300, 300 × 8.0 mm, 5 µm column (YMC Europe GmbH, Dinslaken, Germany) with a flow rate of 0.8 mL/min was used for separation. Samples were measured in triplicates with three individual injections. Data integration of relative areas at 280 nm was performed using Chromeleon 6.80 (Thermo Scientific, Wilmington, DE, USA), provided that every peak eluting before the monomer corresponded to high molecular weight (HMW) species. No peaks could be detected after the monomer. For verification of equipment performance, an internal standard of thawed mAb formulation was injected at the beginning and end of a sequence.

The thawed filtrates were derivatized and analyzed by UHPLC-DAD, as described before [53 (link)]. Following this method, 80 µL of sample containing α--dicarbonyls were reacted with 20 µL of derivatizing reagent containing o-PD and 2,3-dimethylquinoxaline, as the internal standard. After at least 2 h, the formed quinoxalines were subsequently separated, applying a multi-step gradient, consisting of ammonium formate buffer (pH 2.8) and methanol on an ACQUITY UPLC^® BEH phenyl column (2.1 × 100 mm, 1.7 µm particle size; Waters, Eschborn, Germany) equipped with a pre-column of the same material, detected by DAD at 316 nm (3-DG_Qx and 3-DGal_Qx) and 335 nm (3,4-DGE_Qx), and quantified via external calibrations. The applied UHPLC-DAD system (Ultimate 3000RS, Thermo Fisher Scientific, Dreieich, Germany) comprised a pump with degasser, autosampler, column oven, and DAD. The software, Chromeleon 6.80 (Thermo Fisher Scientific, Dreieich, Germany), was used for system control and data analysis. The areas of the two 3,4-DGE–GSH monoadducts were summed up and quantified via the 3-DG calibration curve because of their similar absorption behavior.

The synthesis of 1a was done following the previously described methods for the synthesis of SAB^{59 (link), 60 (link)}. Briefly, to 10 µL of acetic acid were added 25 µL of 2 mg/mL of N-chlorosuccinimide in methanol and 1,1 mg of ethyl 3-(tri-n-butylstannyl) benzoate (1c) in 25 µL of methanol in an HPLC vial. Then 50 µL of ²¹¹At in chloroform were added (roughly corresponding to 5–10 MBq of activity). After 20 min incubation, 1a was purified by HPLC using a Dionex Ultimate3000 HPLC device with an Interchrom C18 column piloted by the Chromeleon 6.80 software (ThermoFisher Scientific Inc.). It was coupled with a dual-flow cell gamma detection system^{42 (link)} using a γ-ray detector (raytest GABI Star) piloted by the Gina software (raytest Isotopenmeßgeräte GmbH).

The Element 2 High-Resolution ICP-SFMS (Thermo Fisher Scientific, Bremen, Germany) was used in low resolution mode for the determination of Ag, Pt, Pd and Au. In order to achieve the minimum oxide rates, calculated from the UO/U ratio, a membrane desolvation system (Apex Ω, ESI Elemental Scientific Inc., Omaha, NE, USA) consisting of a heated quartz cyclonic spray chamber and an EPTFE fluoropolymer membrane was used. The eluted analytes were introduced into the membrane desolvation unit through a microflow nebulizer (PFA MicroFlow Nebulizer, ESI). The Element 2 was equipped with a sapphire injector and nickel sampler and skimmer cones.
Before each measurement sequence, the tuning parameters were optimized in order to achieve the best sensitivity (approximately 4,000,000 cps for 1-µg L⁻¹ In), lowest oxidation rate (<0.05% for UO/U) and high signal stability. The operation parameters are briefly summarized in Table 7, and the following isotopes were measured as transient signals: ⁸⁸Sr, ⁹⁵Mo, ¹⁰⁶Pd, ¹⁰⁸Pd, ¹⁰⁷Ag, ¹⁰⁹Ag, ¹¹¹Cd, ¹¹⁵In (in the elution acid to monitor the signal stability), ¹⁹⁵Pt, ¹⁹⁶Pt and ¹⁹⁷Au.
Data acquisition was performed in E-scan mode (495 runs * 1 passes, 10% mass window, 300 samples/peak and 1-ms sample time).
For integration of the measured transient signals, Chromeleon 6.80 (Thermo Fisher Scientific) was used.