Application 1. Direct Capture of Albumin from Undiluted Plasma
The objective is to evaluate HyperCel STAR AX resin as the first step in a two-step purification sequence to capture human serum albumin (HSA) from undiluted plasma (conductivity 11 mS/cm). HyperCel STAR AX resin was used as a first capture step, followed by an orthogonal cation exchange step performed on S HyperCel resin, without pH or conductivity adjustment of the plasma. Neat undiluted plasma (pH 7.6, 11mS/cm), was loaded with DBC of 30 mg/mL.
Design of Experiments (DoE) in 96-well filter plates was performed to determine optimal conditions to achieve the best yield/purity ratio.
These conditions were then transferred to column chromatography on PRC prepacked columns.
Conditions determined in 96-well plates were applied to chromatography of undiluted plasma on HyperCel STAR AX resin in a 1 mL PRC prepacked column. Chromatogram and SDS-PAGE analysis of fractions confirmed that most contaminants were eliminated by high conductivity wash, leading to a 99% pure HSA fraction in a single step, with 90% yield.
In addition, HyperCel STAR AX resin used at capture step with undiluted plasma had a DBC >30 mg/mL, more than 2-fold higher than that of a conventional DEAE agarose resin tested in these conditions.
HSA was eluted by a simple decrease of pH (4.0), without addition of salt, allowing a direct load on an orthogonal cation exchange column (S HyperCel resin).
This last polishing step on S HyperCel resin led to a purified fraction of HSA eluted at pH 7.0 with purity >99%, and a capacity around 65 mg/mL.
|Capture on HyperCel STAR AX resin
||> 30 mg/mL
|Polishing on S HyperCel resin
||pH 4.0 eluate from HyperCel STAR AX resin
Application 2. Early Removal of CHOPs before Protein A Capture of a Monoclonal Antibody (MAb)
The objective of this study was to evaluate the impact of a pre-purification step using HyperCel STAR AX resinbefore conventional Protein A resin capture of a monoclonal antibody (MAb) from mammalian cell culture feedstock.
The content of contaminating CHOPs (Chinese Hamster Ovary Proteins) was compared using a commercial ELISA assay for the two purification schemes.
Data shows that using HyperCel STAR AX resin prior to Protein A results in a better CHOP reduction (>8-fold). Due to this synergistic effect, starting from an initial Host Cell Protein (HCP) content of 413,000 ppm, the final CHOP level is reduced to ∼400 ppm (3-Log reduction), with a MAb recovery of 90%.
A pre-purification step can impact positively process economics by extending the lifetime of an expensive Protein A column used as standard step for MAb capture.