Surface Neutralization System - Pharmaceutical Technology

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Surface Neutralization System
A new, robust method for protein elution from ceramic hydroxyapatite.

Pharmaceutical Technology
Volume 35, Issue 9, pp. 66-72

Figure 5: Calcium and pH measurements during surface neutralization. The purple curve is on-line pH measurements, the red curve is on-line conductivity.
Off-line measurements are presented again in Figure 5, this time using a PIPES-based SNS solution. In this experiment pH and calcium values were measured during a protein-free cycle for a 3.2 20 cm column of CHT. The equilibration buffer was 10 mM sodium phosphate, pH 6.8; the load buffer contained 20 mM Tris-Arginine, pH 7.4; the SNS solution was 25 mM Tris, 25 mM NaCl, 5 mM sodium phosphate; and the elution buffer was 10 mM sodium phosphate, 0.55 M NaCl, and 10 ppm (0.25 mM) calcium chloride, pH 6.8. The purple curve represents on-line pH measurements, while the red curve represents on-line conductivity. Again, the SNS solution maintained a stable elution pH of 6.5, and the calcium in the elution buffer was sufficient to prevent calcium leaching from CHT; in contrast, the calcium present in the pre-elution effluents resulted from slight dissolution of the CHT itself because these buffers did not contain added calcium chloride.

To demonstrate the robustness of SNS at commercial scales, this protocol was repeated on a 20 20 cm column packed with CHT. At commercial scale, the primary endpoint for CHT use is unacceptable backpressure. Under control conditions where the SNS step was omitted, approximately 10 cycles were obtained before the backpressure in the column rose to > 3 bar. However, the addition of the SNS buffer allowed for at least 46 cycles before a significant increase in pressure. Not only could early failure be easily demonstrated as a negative control, but the clear protective effect of SNS was also shown. This is a crucial validation of the usefulness of SNS technology for manufacturing processes.

The effectiveness of SNS in stabilizing CHT, as well as a lack of general effect on monoclonal IgG elution, is thus demonstrated. Monoclonal antibodies, however, provide more precise ways to examine specific chromatographic effects. Accordingly, further work was performed with two available monoclonal antibodies to determine whether SNS cycling would affect CHT itself or whether SNS would alter antibody elution or impurity clearances.

Figure 6: Protein elution using NaCl step gradient with and without surface neutralizaton system (SNS) step: (A) monomer yield without SNS step; (B) monomer yield with SNS step; and (C) aggregate analysis of B by size exclusion chromatography.
A 1.1 21.7 cm column of either fresh CHT (see Figure 6A) or CHT from the 20 20 cm SNS experiment described above (see Figure 6B) was equilibrated with 10 mM sodium phosphate, pH 6.8 and loaded with ~10 mg mAb G/mL bed volume. The column was then washed either with 8 CV of SNS (25 mM Tris, 25 mM NaCl, 5 mM sodium phosphate, pH 7.75) or equilibration buffer before elution with 10 mM sodium phosphate, 330 mM NaCl, 10 ppm calcium (0.25 mM), pH 6.8. In both cases, the peak elution shape remains the same, even though the pH drop seen in Figure 6A is eliminated in Figure 6B. The yields were 83% and 86%, within historical values (80–88%). The similar peak shapes indicate that prior exposure to SNS does not alter the ability of CHT to bind and elute this monoclonal antibody. Figure 6C shows HPLC-SEC chromatograms of the load and eluate pools taken from experiment 6B. Even after almost 50 cycles of exposure to SNS, CHT still completely retained its ability to remove aggregates to below the limit of detection (0.03%).

Figure 7: Surface neutralizaton system step with protein eluted using NaCl linear gradient.
Peak retention times provide a more precise comparison of protein-matrix interactions. The previous experiment was repeated, except that the elution was a gradient from 10 mM sodium phosphate, pH 6.8 to 10 mM sodium phosphate, pH 6.8 with 1.5 M NaCl over 20 CV. Again, the elution time of the antibody peak was unaltered even after exposure of CHT to multiple SNS cycles (see Figure 7). This is a precise demonstration that CHT-protein interactions are not affected by prolonged cycling with SNS solutions.


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