300sb c8

The mobile phase A contained 2% IPA, 98% Water with 0.1% TFA. The mobile phase B contained 70% IPA, 20% ACN, 10% Water with 0.1% TFA. The sample was diluted in formulation buffer without surfactant at 1 mg/mL. Detection was UV absorbance at 280 nm.
For column assessment, the same separation condition was used for the UPLC column (ACUITY Protein BEH C4) and SPP column (HALO Protein C4 400 Å and 1000 Å). The gradient was 25%–35% B in 8 min under a flow rate of 0.6 mL/min at 75 °C. The total run time including conditioning and washing is 12 min and the injection quantity was 2.5 μL at 1 mg/mL protein. For the reference standard HPLC column (ZORBAX 300SB-C8), the gradient elution employed 25%–40% B in 20 min (0.75% B/min.) at the flow rate of 0.6 mL/min and column temperature of 75 °C.
An improved separation method for the HALO Protein C4 column used the gradient of 25% to 33% B in 7.5 min to (1.07% B/min.) achieve better resolution and throughput. All the other conditions such as flow rate, temperature and sample loading amount were varied during the optimization process to explore effects on separation qualities.
For the redox enriched samples and tagged samples analysis, the final optimized conditions were used, as described in the figures.

UV-VIS spectrophotometric assays were performed on a BioTek Synergy HT Microplate Reader. LC-ESI-MS analysis was carried out using a Waters single quadrupole mass spectrometer and Acquity H Class UPLC with photodiode array detector. LC-ESI-MS samples were run on a 10 min gradient, eluting with increasing acetonitrile containing 0.1% formic acid over a C₈ column (Zorbax 300SB-C₈).