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


Table I: Effect of repeated use of surface neutralizaton system on binding capacity.
In further robustness studies, columns (1.1 21.7 cm) of either fresh CHT or CHT from the 20 20 cm experiment described above were equilibrated in 10 mM sodium phosphate, pH 6.5 and then loaded with a 1 mg/mL solution of human polyclonal IgG. The 5% and 10% breakthrough points were established based on UV absorbance, using the absorbance of pure load material as the UV value for 100% breakthrough. The data demonstrate that repeated exposure to SNS does not demonstrably alter the binding capacity of the monoclonal antibody when two loading conditions were chosen (see Table I).


Table II: Effect of surface neutralizaton system (SNS) on process impurity clearance.
Finally, for SNS to be commercially viable it should not alter the ability of CHT to clear other process impurities. To study this, approximately 10 mg of mAb R was purified over a UnoSphere Supra cartridge using an eluant of 0.1 M glycine, pH 3.5, which was then neutralized to pH 6.8. The solution was then applied to a 1 mL Bio-Scale Mini CHT column equilibrated in 10 mM sodium phosphate and 10 mM NaCl, pH 6.8. The column was washed with 25 CV of equilibration buffer followed by 0 (control) or 10 (SNS) CV of 25 mM Tris, 25 mM NaCl, 5 mM sodium phosphate, pH 7.75. The column was then developed with 25 CV of a linear gradient of 10 mM sodium phosphate and 10 mM NaCl, pH 6.8, down to 10 mM sodium phosphate and 1 M NaCl, pH 6.8. Antibody eluate pools were then analyzed for CHO host-cell protein and DNA. SNS did not alter the clearance of either type of impurity within the reproducibility limits of the assay (see Table II).

Conclusion

The results of these experiments demonstrate several points. The application of a simple, inexpensive buffer immediately before elution of a protein from CHT mitigated uncontrolled pH excursions and dramatically extended the number of cycles that could be obtained prior to a significant backpressure increase. This result stemmed from the replacement of protons on the surface of the matrix with sodium ions. The positive effects of SNS were demonstrated visually and by pH and calcium measurements, and reproduced at a column scale (20 20 cm) that has been shown to produce early column backpressure failure in negative control experiments.

The insertion of the SNS step did not alter basic properties of CHT, such as binding capacity, target protein interaction, aggregate clearance, or host cell protein and DNA removal. Thus, the highly desirable properties of CHT remain unaltered as its robustness was significantly increased. Also, the performance of CHT on a per-cycle basis seems to be equivalent with or without SNS. Taken together, these data suggest the desirability of this technology for development-phase processes, and also suggest that it is a reasonably straightforward regulatory pathway for post-licensing modification of an existing process.

The addition of calcium independently supports CHT robustness, and is expected to act through the common-ion effect in the same way as phosphate (10–12). Unpublished studies demonstrate this as well. The presence of calcium in effluents from calcium-free buffers demonstrates that CHT is leaching this ion into solution. By adding calcium at appropriate concentrations to various buffers, this leaching can be dramatically diminished. The simple method for determining how much calcium to add, as well as further experimentation on the positive effects of calcium on CHT robustness, will be discussed in the future.

Mark A. Snyder* is manager, process R&D applications, Daniel Yoshikawa is product manager, and Larry J. Cummings is a consulting scientist, all at Bio-Rad Laboratories, 6000 James Watson Dr., Hercules, CA 94547.
, tel. 510.741.4675.

*To whom all correspondence should be addressed.

Submitted: Apr. 14, 2011 Accepted: May 31, 2011.


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