Lyophilization does not affect the activity or secondary structure of cat allergen vaccines

INTRODUCTION: Licensed allergen vaccines are manufactured and sold as aqueous solutions, glycerinated solutions, and lyophilized powders.Lyophilized proteins are stable for longer than proteins in solution, and the use of lyophilized reference standards can lower the costs and increase the consistency associated with the maintenance of references.However, a previous study (J Mol Bio12001 310:169-79) has suggested that lyophilization may affect the secondary structure of proteins.We examined the effect of lyophilization on SDS-PAGE, specific antibody binding, and secondary structure of cat allergen vaccines.METHODS: Licensed commercial glycerinated cat hair and pelt extracts were dialyzed to remove glycerin and lyophilized using a Genesis Model 25EL lyophilizer (Virtis).Samples were frozen at -40,,aC and lyophilization was initiated at 100 millitorr.After about 12 hr, the chamber temperature is allowed to rise to 20,,aC for about 8 hours or until the vacuum and temperature are stable, with a final pressure of 10 millitorr when stoppered.Lyophilized, aqueous or glycerinated products were stored at 4-8,,aC.SDS-PAGE was performed using Novex 4-12% gradient gels.Fel d 1 concentrations were determined by radial immunodiffusion assay (RID) using sheep anti-Fel d 1 serum.Circular dichroism (CD) measurements were made using a Jasco 710 spectropolarimeter.For the SDS-PAGE and RID studies, the extracts were dialyzed into water, lyophilized and reconstituted in PBS/50% glycerin.For the CD studies, the extracts were dialyzed into 10 mM phosphate buffer, lyophilized and reconstituted in distilled water.RESULTS: SDS-PAGE analysis indicated that the putative Fel d 1 and albumin bands appear to be unchanged, but a single 28 kD band disappeared with lyophilization.RID analysis demonstrated no changes in Fel d 1 content.CD analysis indicated that cat hair extracts had random coil conformation before and after lyophilization.Cat pelt extracts contained approximately 40% alpha-helix; this too was unchanged by lyophilization.CONCLUSIONS: The removal of glycerol and lyophilization of cat hair and cat pelt allergenic extracts has no measurable effect upon the potency or the secondary structure of the cat allergen extracts.There was the loss of a single 28 kD band of uncertain significance.