Purification with Hydroxyapatite
Crude preparations, whether generated from serum, bacterial cells, or culture media, are a complex brew of salts, ancillary proteins, endotoxins, and aggregates, which present special challenges to the protein purification specialist. Affinity chromatography with staphylococcal Protein A is widely used for both commercial and research levels of purification.
The widespread use of protein A as a purification ligand has driven much research into its molecular structure as companies seek to improve its performance. Sanchayita Ghose, Ph.D., and his collaborators at Amgen (www.amgen.com), have investigated elution pH differences during Protein A chromatography among several IgG1s, IgG2s, antibody fragments, and Fc-fusion molecules.
Variable region interactions determine elution pH for various antibody subclasses with traditional protein A chromatographic materials. The Amgen group have shown that a chopped down, engineered Protein A will respond to a single elution pH for a range of antibodies, mitigating problems associated with low pH induced aggregation.
The strong interaction between protein A and members of the IgG subclass ensures a powerful and specific binding, but elution of the antibody molecules can present difficulties. Protein A binds to the Fc region at the juncture of the Cd2 and Cd3 domains through hydrophobic interactions, hydrogen bonding, and salt bridges.
At low pH complementary histidine groups facing one another take on positive charges, resulting in an electrostatic repulsion. While this force is powerful enough to allow elution from a Protein A-packed column, these conditions can be harsh and cause aggregate formation and denaturation of the antibody molecules. Circumventing this molecular challenge has driven a number of independent companies to develop strategies before and after elution from Protein A.
Scientists at Bio-Rad Laboratories (www.bio-rad.com) have optimized the use of ceramic hydroxyapatite, according to Pete Gagnon, process applications and new technologies R&D manager, process chromatography division of the company. Gagnon adapted this material for antibody purification. Ceramic calcium hydroxyapatite, Ca10(PO4)6(OH)2, can be especially useful in removing aggregate contaminants. Protein aggregates are chronic problems in the case of proteins expressed in E. coli. Formed in inclusions bodies, they can prove to be highly persistent in bacterial protein expression systems.
Calcium participates in metal affinity interactions, while the phosphate portion is active in cation exchange exclusion reactions, Gagnon explains. CHT ceramic hydroxyapatite has the added quality of being stable down to pH 6.5 in the presence of 5 mM phosphate.
CHT ceramic hydroxyapatite fractionation of contaminants on linear sodium chloride gradients allowed separation of Protein A-purified human IgG1 from both large and small aggregates, as well as DNA, endotoxins, and lipopolysaccharides.
Aggregate removal was very efficient, greater than 99%. Leached protein Aremoval was greater than 90%, while DNA removal was reduced by three logs, and endotoxins was brought down by greater than four logs. Gagnon and his colleagues optimized the process for scale-up by adjusting the slope and amplitude of the salt gradient.
When CHT ceramic hydroxyapatite columns are eluted with a sodium chloride gradient at a low concentration of phosphate, we simultaneously achieve reductions in the levels of aggregates, leached protein A, DNA, and endotoxin, says Gagnon. The method is easily integrated into a two step platform with Protein A, or with an additional step to exploit alternative fractionation mechanisms.