15


ligand on epoxy-modified columns were performed at a Merck-Hitachi L-6200 HPLC Pump (Darmstadt, Germany). The following chromatographic studies were performed on an Ultimate 3000 HPLC system consisting of LPG-3400RS HPLC pump, WPS-3000TRS autosampler, TCC-3000RS column oven compartment and VWD-3000 UV detector from Dionex (Germering, Germany).


Preparation of rSPA Silica Monoliths


Figure 2: Analysis of 50 injections of cetuximab (1 mg/mL) on immobilised rSPA silica monolith. Only first and every 10th chromatogram is shown. Chromatographic conditions: Stepwise gradient: 100 mM sodium phosphate pH 7.4/100 mM sodium phosphate pH 2.5; 0.25 min 100/0, 0.25-0.26 min 0/100, 1.00 min 0/100, 1.00-1.01 100/0, 1.01-2.50 min 100/0; flow rate: 2.0 mL/min; detection: 280 nm; injection: 10 µL; temperature: 25°C


characteristics resulting in much lower column backpressure compared to columns packed with particles, and maintaining column performance even at higher flow rates [11-13]. Due to the hydrodynamic volume of mAbs (mostly 5-6 nm), the alteration of mesopore development up to 30 nm is necessary allowing them to enter the pores for improved chromatographic separation and to exclude separation effects by size. Recently, the development of silica monoliths containing larger mesopores and its application for the analysis of biomolecules has been reported [14].


Another continuously growing sector in chromatography is the coupling of desired ligands for specific interactions with certain analytes e.g. antibodies, aptamers or enzymes [15-20]. The majority of described protein immobilisations were performed on polymeric particles or monoliths and silica particles. In the last decade, silica-based monoliths were also used for the immobilisation of several ligands to enable their use in the applications of affinity chromatography, for chiral separations or as on-column bioreactors [21-26]. The development of wide pore silica monoliths enhanced the column properties for the immobilisation and analysis of larger molecules. In this study, recombinant staphylococcal protein A (rSPA) is immobilised onto a silica monolith containing larger mesopores suitable for the separation of mAbs. The analysis performed on the monolithic column is characterised by different parameters including detector response linearity, reproducibility and long- term stability.


Experimental Chemicals


Sodium phosphate dehydrate (≥99.5%), ammonium sulphate (≥99.5%), sulphuric acid (w=98%), ortho-phosphoric acid (w=85%) and sodium hydroxide (w=50%) for analysis were purchased from Merck (Darmstadt, Germany). Pure water was obtained from Milli-Q system from Merck (Darmstadt, Germany). Native recombinant staphylococcal protein A (rSPA) ligand was purchased from Repligen (Waltham, MA, USA) and bovine serum albumin (BSA) was obtained from VWR (Darmstadt, Germany). Pure cetuximab stock solution was a research sample from Merck (Darmstadt, Germany) and gammanorm IgG was obtained from Octapharma (Heidelberg, Germany).


Epoxy-modified widepore silica monolith (25 mm x 4.6 mm) columns (Chromolith® WP 300 Epoxy) were prepared as research samples at Merck (Darmstadt, Germany).


Apparatus


All modification and cleaning steps necessary for the immobilisation of rSPA


Wide pore silica monoliths were prepared according to the sol-gel process [27, 28] possessing a macropore size of 2 µm. Thermal treatment under alkaline conditions allowed mesopore formation to a size of 30 nm. All silica monoliths were cladded with solvent-resistant polymer (polyether ether ketone [PEEK]) housings. The silica monoliths were chemically derivatised to epoxy-modified monoliths following a procedure described elsewhere [22].


The immobilisation of rSPA on wide pore epoxy-modified monoliths was done according to a dynamic process circulating ligand solution through the column. The rSPA ligand solution was dissolved in 6 mL immobilisation buffer (50 mM sodium phosphate + 1.9 M ammonium sulphate pH8.0) resulting in a rSPA concentration of 2 mg/mL. Before immobilisation, columns were equilibrated with 50 mL immobilisation buffer. The protein solution was circulated at a flow rate of 0.2 mL/min for 4 hours. Finally, rSPA silica monolith was washed with 50 mL of 100 mM sodium phosphate buffer pH7.4 and remaining epoxy functions were hydrolysed with 150 mM phosphoric acid pH 1.5.


Results and Discussion General Column Evaluation


Before the immobilisation process, the silica monoliths were characterised by mercury intrusion porosimetry and nitrogen


Table 1: Chromatographic data of separated IgG by immobilised rSPA silica monolith from different batches. Chromatographic conditions: Stepwise gradient: 100 mM sodium phosphate pH 7.4/100 mM sodium phosphate pH 2.5; 0.25 min 100/0, 0.25-0.26 min 0/100, 1.00 min 0/100, 1.00-1.01 100/0, 1.01-2.50 min 100/0; flow rate: 2.0 mL/min; detection: 280 nm; injection: 10 µL; temperature: 25°C


Batch No.


1 (n=6) 2 (n=6) 3 (n=6)


Retention time [min]


1.15 ± 0.01 1.16 ± 0.00 1.15 ± 0.00


Total (n=18) 1.15 ± 0.00


Peak Symmetry (USP)


1.57 ± 0.15 1.53 ± 0.12 1.52 ± 0.06 1.54 ± 0.11


Peak Width (10%) [min]


Column back pressure [bar]


0.037 ± 0.003 10 ± 0 0.035 ± 0.003 10 ± 0 0.035 ± 0.003 10 ± 0 0.036 ± 0.003 10 ± 0


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