Acquity beh300 c4 column

The reversed-phase chromatography was performed on an Agilent 1290 UHPLC system using an ACQUITY BEH 300 C4 column (1.7 µm, 2.1 × 100 mM) from Waters. The column was conditioned in 0.1% trifluoroacetic acid (TFA) in Milli-Q water at 65°C, 0.4 mL/min, and the antibody fragments were eluted in a gradient of 0.1% TFA in 60% acetonitrile/40% isopropanol. Details on the gradient: 0–3 min 5% B, 3–6 min 5–15% B, 6–36 min 15–45% B, 36–37 min 45–80% B, 37–42 min 80% B, 42–42.1 min 80–5% B, 42.1–55 min 5% B. Detection was at 280 nm.

Oxidized forms of filgrastim, to be used as standards for RP-UPLC, were obtained according to the Ph. Eur. monograph for filgrastim concentrated solution (28 ). Briefly, an aliquot of 100 μl of 0.5 mg/ml filgrastim CRS was treated with 3 μl of 30% hydrogen peroxide (Merck) and incubated at 25°C for 15 min before adding 0.8 mg of L-methionine (Sigma-Aldrich). Reduced forms were produced by adding 0.125 mg dithiothreitol to 100 μl of 0.5 mg/ml filgrastim CRS and incubated at 35°C for 60 min.
RP-UPLC was performed on an Acquity Ultra Performance LC system (Waters Corporation) where a UPLC Acquity BEH300 C4 column (1.7 μm, 2.1 × 50 mm) was installed. The column was equilibrated at 60°C with 85% mobile phase A (0.1% trifluoroacetic acid [TFA, Sigma-Aldrich] in Milli-Q water) / 15% mobile phase B (0.1% TFA in 90% HPLC grade acetonitrile [Biosolve, Valkenswaard, The Netherlands]) until a stable baseline was reached. Separation was achieved by applying the following linear gradients at a flow rate of 0.25 ml/min: 15% B (0–0.5 min), 15% B-75% B (0.5–10.5 min), 75% B-15% B (10.5–11 min) and 15% B (11–11.5 min). While being maintained at 20°C, 1.5 μg of each filgrastim product was injected in triplicate. Detection took place with an Acquity™ PDA detector (Waters Corporation) at 215 nm.