16 February / March 2017


ranging from 0.0125 mg/ml to 10 mg/mL. The 15 used calibration standards revealed a broad analysis range from 1.25-200 µg of injected cetuximab for the immobilised rSPA silica monolith. The retention time of eluted cetuximab concentrations varied by only 0.1% and was confirmed by an overlay of all calibration chromatograms as shown in Figure 3. The correlation coefficient was higher than 0.999 supporting the hypothesis of a high column capacity.


Flow rate


Figure 3: Calibration curve of Cetuximab on rSPA silica monolith ranging from 1.25 µg to 200 µg. 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: 20 µL; temperature: 25°C


adsorption/desorption (BET). They consisted of macropores of 1.83 µm and mesopores of 28.6 nm resulting a total surface area of 120 m2


/g silica. The column was immobilised


with rSPa ligand circulating through the column for four hours. The final column possessed a dynamic binding capacity of 2.75 (± 0.11) mg for monoclonal IgG.


The rSPA modified silica monoliths were used for separation of monoclonal antibodies from their related impurities. The separation of cetuximab from BSA impurity was performed in around one minute and a typical chromatogram is shown in Figure 1. The unbound BSA eluted first followed by cetuximab which was released due to the pH shift to pH2.5. The rSPA silica monoliths provided very sharp peaks with peak widths < 0.040 minutes in a reasonable run time which was supported by the hydrophilic column surface reducing undesirable backbone interactions with the analytes.


Another important criteria for analytical protein A columns is column performance reproducibility. Therefore, cetuximab sample was injected 50 times and all chromatographic parameters were evaluated especially with respect to their relative standard deviation (RSD). An overlay of all 50 chromatograms is shown in Figure 2 indicating a robust and reproducible column performance. The RSD of the retention time of eluted cetuximab was < 0.1% with the peak width being equal for every run. The peak area RSD was 0.4% supporting the previous data and assumptions of a robust column modification and reproducible column performance.


Furthermore, different batches were synthesised under exactly same conditions and six columns of each batch were immobilised with rSPA ligand, respectively. The summary of chromatographic data of each batch are shown in Table 1. The data revealed only slight differences between columns from the same batch as well as minor differences between different batches. Those results demonstrate that the immobilisation process via epoxide functions is very robust and reproducible.


Column Linear Range


For titre determination, linear range of the analytical column is a key property for its application. In antibody production, the used bioreactors contain antibody concentrations between 0.5-7 g/L, which is the minimum requirement for an analytical protein A column. The rSPA modified monolith was evaluated by analysing different concentrations of pure cetuximab


Since monolithic silica columns enable a low column backpressure due to their high flow through pores, the separation capability of immobilised rSPA silica monoliths was evaluated at higher flow rates. The comparison of column performance was done with pure cetuximab spiked with BSA. It was expected that retention time and peak width of eluted cetuximab would decrease with increasing flow rate.


In Table 2, the most important data were shown for flow rate comparison. It was visible that cetuximab column binding was not effected by flow rate and the relative peak area of eluted cetuximab was constant. Additionally, column backpressure increased linearly (R2


= 0.998) enabling high flow rates and even shorter run times with constant column performance and without any loss in binding efficiency. The high-speed separation of monoclonal antibodies at high flow rates was enhanced by the high mass transfer properties of the macropores. The flow rate data demonstrated the large time savings and high separation efficiencies obtained by using silica monoliths.


Stability


The examination of column long-term stability was performed with IgG as a control. The stability of the monolithic structure, column modification and protein


Table 2: Chromatographic data of separated IgG (1 mg/mL) and BSA (1 mg/mL) by immobilised rSPA silica monolith at different flow rates. Chromatographic conditions: Stepwise gradient: 100 mM sodium phosphate pH 7.4/100 mM sodium phosphate pH 2.5; same gradient was used as shown in Figure 2 and was adjusted based on used flow rate; flow rate: 1.0-5.0 mL/min; detection: 280 nm; injection: 10 µL; temperature: 25°C


Flow rate [mL/min]


1.0 2.0 3.0 4.0 5.0


Retention time [min]


2.31 1.16 0.77 0.58 0.47


Peak Width (10%) [min]


0.058 0.032 0.024 0.021 0.020


Unbound area


39% 39% 39% 39% 39%


Bound area Column back pressure [bar]


61% 61% 61% 61% 61%


3 6


10 13 17


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