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

Materials and methods

CHT (type I 40 m), Unosphere Supra cartridges, Bio-Scale Mini CHT columns, Bio-Sil 250 HPLC-SEC columns, and the Biologic Duoflow chromatographic workstation are all products of Bio-Rad Laboratories. Human polyclonal IgG antibody was purchased from Sigma-Aldrich. Chinese hamster ovary (CHO) ELISA immunodetection and Picogreen DNA analysis kits were purchased from Cygnus Technology and Invitrogen, respectively. All other chemicals were obtained commercially. Monoclonal antibodies (mAb) R and G were kindly provided by external collaborators.

SNS is implemented by adding a special buffer solution immediately before elution. All other process buffers as originally developed in a given process can remain the same. The addition of low levels of calcium significantly enhances CHT stability by the same common-ion effect as phosphate stabilization, and should be included in all steps up to and including elution (9, 10). The exact composition of each buffer used for this study is detailed below. SNS solutions include a stabilizing buffer (25–50 mM, such as Tris, arginine, lysine, histidine, PIPES, HEPES, ACES MOPS, and MOPSO), 25 mM NaCl, and 5 mM sodium phosphate, pH 7.5–8.4. The stabilizing buffer maintains the slight alkalinity of the solution; the low amount of NaCl slowly removes protons from the surface of CHT without desorbing target proteins; and phosphate is added to improve CHT robustness through the common ion effect. Application of six column volumes (CV) is sufficient to neutralize the CHT surface charge; in the experiments that followed, volumes of at least 6 CV were employed.

MAb G was loaded at approximately 20 mg/mL onto a 3.2 20 cm CHT column equilibrated in 5 mM sodium phosphate, pH 6.5. The column was then washed with 3 CV of this buffer prior to the application of 8 CV of SNS solution (50 mM Tris, 25 mM NaCl, 5 mM sodium phosphate, pH 8.41). Elution was then carried out using 5 mM phosphate, 330 mM NaCl, 12 ppm calcium (added as CaCl2), pH 6.5, followed by regeneration with 0.5 M sodium phosphate, pH 6.5. Note that all ppm measurements in this study were of calcium, so that 40 ppm calcium = 1 mM calcium (formula weight 40.08).

Results and discussion


Figure 3: Behavior of monoclonal antibody G during surface neutralizaton.
The ultraviolet (UV) profile demonstrates that all applied IgG remained bound to the CHT during the SNS step (see Figure 3). The pH trace shows no acidic excursion during the elution step; this contrasts with a drop to pH ~5.5 in the absence of SNS. The stable pH during elution, as measured by the online probe, demonstrates that all protons have been exchanged for sodium ions on the surface of CHT.


Figure 4: Fifty cycles with PIPES-buffered surface neutralizaton: (A) visual measurement of fines after 50 cycles; and (B) pH and calcium measurements.
The protective effects of SNS on CHT are shown in Figure 4. In Figure 4A, a 3.2 20 cm column of CHT was cycled 50 times in the absence of protein as above, but with an SNS solution that was 10 CV of 25 mM PIPES, pH 7.75, 25 mM NaCl, and 5 mM sodium phosphate. The picture indicates an absence of fines or broken particles after cycling; the presence of such species indicates reduced CHT robustness (9, 10). The ability of SNS to suppress an acidic excursion is further substantiated in Figure 4B, where the on-line pH trace is shown with concurrent off-line measurements. For comparison, the pH drop observed without the SNS step included is shown with a blue dotted line. Note that the pH of the eluant never drops below pH 6.5, and the output calcium content is near or lower than the input calcium level in the elution buffer. The input buffer calcium is thus sufficient to prevent virtually all calcium leaching into the effluent stream. In the absence of added buffer calcium, detectable calcium in the effluent represents loss of CHT into the solvent phase.


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