Chromeleon 7

For each formulation, three individual SODFs were dissolved in 100 mL of distilled water. Samples of the solutions were then diluted and the drug concentration was determined by ultra-high performance liquid chromatography (UHPLC, Thermofisher Scientific, Waltham, MA, USA) using a UHPLC-DAD system. It consisted of a Thermo Scientific™ Dionex™ UltiMate™ 3000 BioRS equipped with a WPS-3000TBRS autosampler and a TCC-3000RS column compartment set at 35 °C. The system was operated using Chromeleon 7 software (Thermofisher Scientific, Waltham, MA, USA). An Accucore C18 column (2.6 µm, 100 × 2.1 mm²) combined with a security guard ultra-cartridge (Phenomenex Inc., Torrance, CA, USA) was used. An isocratic binary solvent system was utilized, consisting of water/formic acid (1%, v/v) as solvent A and acetonitrile/formic acid (1%, v/v) as solvent B (90%A, 10%B). The flow rate of the mobile phase was 1.5 mL/minute, and the injection volume was 50 μL. Quantitative analysis of paracetamol in the SODFs was carried out using an external standard method. The calibration curve was constructed using 5 different standard levels in the concentration range 1–20 mg/L. The peak of paracetamol was monitored at 244 nm.

Eight different chiral columns were selected, and the chiral column types were shown in Table 2. 2-Pentanol racemate was determined using a Thermo Fisher gas chromatograph (Trace 1300, Thermo Fisher, Waltham, Massachusetts, USA), and the best column was selected according to the separation resolution. Take 5 µL 2-pentanol racemate with anhydrous ethanol constant volume to 10 mL, filtered through a 0.22 µm nylon syringe filter membrane into the injection vial, and stored in a refrigerator at 4 °C until GC analysis. The 2-pentanol racemate was injected into the injection port in split mode (injector temperature 250 °C, split ratio: 20:1). The separation was carried out on eight different chiral columns under the same chromatographic conditions. GC conditions: column oven of 40 °C, held for 1 min, increased to 120 °C at a rate of 2 °C/min, and finally increased to 210 °C at 3 °C/min and held for 1 min. The carrier gas was nitrogen (99.999%) at a constant flow rate of 1 mL/min. Chromatograms were analyzed using Chromeleon 7 software (Thermo Fisher, Waltham, Massachusetts, USA).

SEC was used to assess the monomer and HMWI content and product losses. Experiments were carried out on a Dionex UltiMate 3000 HPLC system (Thermo Fisher Scientific, Waltham). Isocratic elution was carried out with a 50 mM sodium phosphate buffer with 150 mM NaCl at pH 7.0. 10 μl of 0.22 μm filtered sample were applied to a TSKgel® G3000SWXL HPLC column (5 μm; 7.8 mm × 300 mm) with a TSKgel® SWXL guard column (7 μm; 6.0 × 40 mm) (both Tosoh, Japan). Chromeleon™ 7 software (Thermo Scientific) was used to monitor the signals at 215 nm (for HMWI) and 280 nm (for monomer content and product loss). The starting material is referred to as T₀ material. Monomer content is based on the ratio of product peak area (monomer, detected at 280 nm) to the sum of all peak areas. HMWI are calculated by taking the sum of all peaks eluting before the main antibody peak (at 215 nm) and dividing it by the sum of main peak and earlier eluting species. Concentration in relation to T₀ is the ratio between the monomer peak at 280 nm of the respective sample with the T₀ sample of the respective capture method (protein A or precipitation). Average values and standard deviations are based on triplicate measurements. Injection volume and dilutions were kept constant for all samples.

Relevant samples were analyzed by HPLC‐SEC‐MALS in order to determine particle composition and estimate purity. All experiments were performed using the HPLC system mentioned in 2.11 with the Chromeleon 7 software (Thermo Fisher Scientific) for method programming, control and data acquisition. A TSKgel G5000PWXL column (300.0 × 7.8 mm i.d.) combined with a TSKgel PWXL guard column (40.0  × 6.0 mm i.d.) (Tosoh Bioscience, Stuttgart, Germany) was used. A volume of 50 µL of each sample was injected. The flow rate was 0.3 mL/min. Isocratic elution was performed with 50 mM HEPES, 100 mM NaCl, pH 7.2. UV signals at 280 and 260 nm were recorded by the Chromeleon software and LS signal was acquired with a DAWN HELEOS 18‐angle detector (Wyatt) with the Astra Software 5.3.4 (Wyatt). Data evaluation was performed in Astra 6.1.2.

At‐line MALS measurements for the determination of the LS intensity were performed using an Ultimate 3000 HPLC system (Thermo Fisher) with a quaternary LPG‐3400SD pump, a WPS‐3000TSL autosampler, and a DAD 3000 UV‐detector. Mobile phase consisted of 50 mM HEPES, 100 mM NaCl, pH 7.2. A sample volume of 50 µL was injected in bypass mode using a flow rate of 0.3 mL/min. All samples were measured in duplicates. MALS signals were acquired by the DAWN HELEOS 18‐angle detector (Wyatt, Santa Barbara, CA, USA). For HPLC programming, Chromeleon 7 software (Thermo Fisher) was used. MALS data collection and analysis were performed with ASTRA software, version 6.1.2 (Wyatt).

Methanol in samples producing lactic acid were quantified via UPLC (UPLC Ultimate 3000) equipped with a Rezex ROA UPLC column (Rezex ROA-Organic Acid H+ (8%): 300 × 7.8 mm; Phenomenex) coupled to a refractive index detector (RefractoMax 521, Thermo Fisher Scientific). As the solvent, 2.5 mM sulfuric acid in ultrapure water was used. To separate the metabolites, a flow rate of 600 μl min⁻¹ was used. Peaks were identified and the peak areas were quantified using Chromeleon 7 software (Thermo Fisher Scientific) on the basis of an external standard curve of pure methanol (Sigma-Aldrich).

To estimate product purity, size exclusion chromatography was performed. For HPLC analytics, we used a Dionex UltiMate 3000 HPLC system equipped with a diode array detector (Thermo Fisher Scientific). The running buffer was a 50 mM potassium phosphate buffer with 150 mM NaCl, pH 7.0 (Merck KGaA). The buffer was filtered through 0.22‐μm filters (Merck KGaA) and degassed. We applied 20 μl of the filtered sample to a TSKgel® G3000SWXL HPLC column (5 μm, 7.8 × 300 mm²) in combination with a TSKgel SWXL Guard column (7 μm, 6.0 × 40 mm²; Tosoh). The absorbance at 280 nm was recorded, and the results were evaluated with the Chromeleon™ 7 software (Thermo Fisher Scientific). The antibody purity was calculated as the ratio of the monomer peak area (retention time: 21.2 min) to the sum of all peak areas, based on the 280 nm signal.