Mabpac sec 1

Synechocystis cell lysates were fractionated by SEC and IEX on an Ultimate 3000 HPLC system (ThermoFisher Scientific, Bremen, Germany). For SEC, the lysates were injected (350 μl per injection) onto MAbPac SEC-1 (5 μm, 300 mm × 4.0 mm; ThermoFisher Scientific) or Superose 6 10/300GL column (GE Life Sciences). There were 24 fractions collected by using MAbPac SEC-1, with a flow rate of 0.2 ml/min, and 45 fractions collected by using Superose 6 10/300GL column, with a flow rate of 0.3 ml/min. Protein standards (thyroglobulin, BSA, Albumin egg, and myoglobin) were analyzed with the same method to obtain the approximate MW range across fractions. For IEX, the ion-exchange column (12 μm, 200 mm × 4.6 mm, 1500 Å; Columnex, San Diego, CA) was used, and a 110 min salt gradient (0.12–1.2 M NaCl) was used to collect 43 fractions. The elution buffer A containing 10 mM Tris-HCl (pH 7.6), 0.5 mM DTT, and 5% glycerin, while elution buffer B with additional 1.2 M NaCl. For SDGC, lysates were loaded onto a 12 ml 15%–70% (w/v) linear sucrose gradient, which were then centrifuged at 160,000 g at 4 °C for 16 h in a Beckman MLS-50 rotor (Beckman-Coulter, CA), and 24 fractions were collected. In total, 181 fractions were collected.

When performing the HPLC‐SEC analysis, the solution of antibodies was centrifuged at 16 000 × g for 10 min, and only the first third of the solution was aspirated to prepare the test samples to avoid large protein aggregates. The antibody purity was detected using an Agilent 1260 HPLC system, and the absorbance at 280 nm was monitored. The system was formulated with a MabPac SEC‐1 (Thermo) size exclusion column, 4 × 300 mm, 5 μm, and 300 Å. The mobile phase contained 25.30 mm of NaH₂PO₄, 30.50 mm of Na₂HPO₄, and 300 mm of NaCl at a pH of 6.8. Samples with a concentration of 1 mg·mL⁻¹ and a volume of 10 μL were run in the mobile phase for ~ 20 min. The flow rate was 0.2 mL·min⁻¹, the maximum column pressure was 55 bar, and the column temperature was 25 °C. The percentage of monomers was calculated using the area normalization method, and the system software was lc1260 (Agilent Technologies, Palo Alto, CA, USA).

SEC-HPLC analysis was conducted to evaluate the purity, aggregation, and degradation of antibodies. Samples were analyzed with an Agilent 1260 HPLC system (Agilent) on a Thermo MAbPac SEC-1, 5 μm, (7.8 mm × 300 mm) column (P/N 088460). Mobile-phase condition was 50 mM sodium phosphate, 300 mM sodium chloride, pH 6.8. SEC chromatograms were observed at 280-nm absorbance at 25 °C, with a flow rate of 0.7 ml/min and sample injection volume of 15 μl.