Chromatography Forum

Dear All,

I would like to separate three insulins Humulin (human insulin), Humalog and Lantus. I run Google search but could not find method for the separation of all three insulin. Humulin and Humalog are almost identical. Teh difference is very small. Humalog is identical to human insulin except for the transposition of proline and lysine at positions 28 and 29 on the B chain. Is it a big difference for HPLC separation. I tried two columns for proteins and obtained no separation. After a week of trying I decided to seek help here.

Thank you in advance. ???

Are you trying to assay, or do you need to recover the proteins or measure biological activity?

This might be tough to do. I would first search the literature thorroughly to see if anybody has done this already. If you don't find anything, here is how I would approach it.

First, I would look for a very flat gradient (very small change in % organic) around the elution of the humalog/humulin. Then I would change the pH by small increments around pH 2 to see if there are subtle differences that can be observed between the two. I would probably use a phosphate buffer instead of the usual TFA. One I see a subtle difference, I would finally decrease the flow rate while increasing the gradient run time to see if the higher resolving power at the slower flow rate will finally do the trick.

All of this is a lot of work, but it appears to be a tough problem.

I am trying to assay all three insulins in a mixture (Humulin, Humalog, Lantus). Right now it is just a mixture of standards. I tried two columns from different manufacturers and various gradients (slow, fast, steep, shallow, low pH, high pH, different concentrations of buffer, etc.) So far I screened over 50 different mobile phases and gradients with no luck. I did Google and SciFinder searches and found no method for the analysis of all three (without breaking insulins apart and looking at fragmentation). Does the method even exist?..

regards

Amersham´s (GE now?) "Guide to radioiodination techniques" mentiones a probably even more difficult separation, namely that of the four iodine isomers formed during iodination of insulin. This booklet is referenzed badly, but it could be the following: Frank, et al, J Chromatogr, 266, 239 (1983). If this is the correct ref it might help.

The way I read your post, all of the conditions you tried were reversed-phase. Evidently, all three are chromatographically similar by reversed-phase. What about ion-exchange or HIC?

Nalizer,

Would you please provide me with your email address, we have a pretty good lead on insulins and would like to send you some chromatograms.

TIA

Nalizer:

How about giving us some details on what you tried and how far you got?

HW Muller,

If you are interested we can try to separate your peptides/proteins if you provide us with sample (no obligations from you). If anybody would like to, I can also send you insulin application for the separation of Humalog and Humulin

SIELC_Tech, send me your columns and I will give them a nice critical spin, what I just did today is label an antibody with 90-Y (~2.6 day halflife, hard beta, 1.3GBq), problem: the 90-Y has a tendency to come off, if it does it even sticks to teflon.... No way to send this stuff around. Could you guarantee that, if it ever came out of your columns, that we can infuse that into humans? (Well there are some analytical aspects here also).