Does RP-HPLC of polypeptides affect biological activity?

Biological activity of proteins depends on tertiary structure and permanent disruption of tertiary structure usually reduces biological activity. RP-HPLC may disrupt protein tertiary structure because of hydrophobic solvents used for elution or because of the interaction of the protein with the hydrophobic surface of the material. The amount of biological activity lost depends on the stability of the protein and on the elution conditions used. The loss of biological activity can be minimized by proper post-chromatographic treatment. Small peptides and very stable proteins are less likely to lose biological activity than large enzymes. Some specific points to keep in mind are:

	Denaturation of proteins on hydrophobic surfaces is kinetically slow. Reducing the residence time of the protein in the column generally reduces the loss of biological activity.
	Some solvents are less likely to cause a loss of biological activity than others. Isopropanol is the best solvent for retaining biological activity. Ethanol and methanol are slightly worse and acetonitrile causes the greatest loss of biological activity.
	Stabilizing factors, such as enzyme cofactors, added to the chromatographic eluent, stabilize proteins and reduce the loss of biological activity.
	The most important factor in maintaining or regaining biological activity is post-column sample treatment. Dissolution of a collected protein in a stabilizing buffer often allows the protein to re-fold. An example is HIV protease (Table 1).
Examples of biological activity after RP-HPLC
	Trypsin. Reversed phase chromatography has been used to purify trypsin for use in protein digestion.
	Poliovirus proteins. Poliovirus proteins purified by reverse phase chromatography were able to induce production of specific antibodies in rabbits, indicating a retention of biological activity.

Dissolution
After lyophilization, dissolve residue at 5-15 mg/ml in 50
mM sodium acetate, pH 5.5, containing 8 M urea, 1mM
EDTA and 2.5 mM dithiothreitol.
Refolding
Dilute with 9 volumes of 50 mM acetate, pH 5.5,
containing 1 mM EDTA and 2.5 mM dithiothreitol, 10%
glycerol, 5% ethylene glycol and 0.2% Non-idet P-40 at
4⁰C.
Result
Specific activity = 1.0+-0.1 µmol substrate hydrolyzed
per minute per mg of protein- compared to specific
activity of 1.2 for enzyme expressed in E. coli.

Table 1

Procedure used
to regain
biological activity
of HIV protease
after reversed-
phase chromatography

	Pollen Allergens. The main protein allergen of *Parietaria judaica* retained IgE-binding activity even after RP-HPLC purification because it eluted at, low acetonitrile concentration.
	HIV protease. HIV protease regained most of its biological activity after reverse phase chromatography and post chromatographic treatment to allow refolding (Table 1).

Perhaps the most compelling evidence that biological activity is not inevitably lost during reversed-phase chromatography is the fact that several commercial bio-therapeutics use reversed-phase chromatography in the purification of the marketed product.

	Erythropoetin may be purified using reversed-phase chromatography as an integral part of the purification process.
	Leukine, a marketed polypeptide therapeutic, uses reversed-phase HPLC as an integral part of its purification procedure.
	Human recombinant insulin purification uses reversed-phase chromatography in its production.

Conclusion
While the conditions of reversed-phase chromatography may cause some loss of tertiary structure and biological activity, in most cases this loss of biological activity may be moderated or eliminated by use of optimum chromatographic conditions or by post-chromatographic treatment.