Detection and quantitation of cyclodextrins
View Patent ↗The invention relates to the detection and quantitation of cyclodextrins and cyclodextrin derivatives in solutions comprising a protein. The invention further relates to methods of evaluating pharmaceutical preparations for the presence of residual cyclodextrins.
1. A method for recovering a cyclodextrin or cyclodextrin derivative from a solution comprising a protein recombinantly expressed in the presence of the cyclodextrin or cyclodextrin derivative, the method comprising loading the solution on a size exclusion chromatography (SEC) column, wherein the solution does not undergo a step of extraction prior to being loaded on the SEC column; separating the cyclodextrin or cyclodextrin derivative from the recombinantly expressed protein by SEC; contacting the cyclodextrin or cyclodextrin derivative with an agent that forms a detectable inclusion complex with the separated cyclodextrin or cyclodextrin derivative; and measuring the amount of a signal from the inclusion complex, wherein the signal is indicative of the quantity of the cyclodextrin or cyclodextrin derivative recovered from the solution.
2. The method of claim 1 , wherein the cyclodextrin or cyclodextrin derivative is methyl-β-cyclodextrin.
3. The method of claim 1 , wherein the recombinantly expressed protein is an antibody.
4. The method of claim 1 , wherein the solution is a pharmaceutical preparation.
5. The method of claim 1 , wherein the solution comprises less than 20 μg/mL of the cyclodextrin or cyclodextrin derivative.
6. The method of claim 1 , wherein the agent is a fluorescent compound.
7. The method of claim 6 , wherein the agent is 1-naphthol.
8. The method of claim 1 , wherein the method has a cyclodextrin or a cyclodextrin derivative limit of detection of less than 10 μg/mL.
9. The method of claim 8 , wherein the method has a cyclodextrin or a cyclodextrin derivative limit of detection of less than 2 μg/mL.
10. The method of claim 1 , wherein the SEC column has a separation range of about 5-20 kDa to about 75-300 kDa.
11. The method of claim 1 , wherein the SEC column has a particle size of about 2 to about 6 microns.
12. The method of claim 11 , wherein the SEC column has a particle size of about 4 microns.
13. The method of claim 1 , wherein at least 97%±5% of the cyclodextrin or cyclodextrin derivative is recovered.
14. The method of claim 1 , wherein the separation uses a mobile phase comprising a salt at a concentration of about 50 mM to about 300 mM.
15. The method of claim 14 , wherein the solution and the mobile phase have a pH of about 2.0 to about 8.0.
16. The method of claim 15 , wherein the solution and the mobile phase have a pH of about 5.0 to about 7.5.
17. The method of claim 14 , wherein the agent is present in the mobile phase during the separating step.
18. The method of claim 1 , wherein the recombinantly expressed protein is at least 90% pure.
19. The method of claim 18 , wherein the recombinantly expressed protein is at least 95% pure.
20. The method of claim 19 , wherein the recombinantly expressed protein is at least 98% pure.