Chromatography Forum

Hello -

In the past 12 months, my company has changed its focus from general protein formulation to mAb formulation. Our analytical lab is now running mostly SEC methods for integrity (aggregation), and most of them are very similar: isocratic, with a salt buffer (various salts, mostly between 50 - 200 mM) at a pH between 6.5 and 7.5. We've used mostly Toso columns (SW3000xl, SW2000xl) and a Bio-Rad column (BioSil). We always use guard columns, too.

Most of our samples are early-stage feasibility, so there may be strange salts or polymers or metals (all residual, < 3%) in the samples, but we do centrifuge and filter everything before placing on the HPLC.

We run a Gel Filtration standard with each run to check on the column's performance. We're finding that our columns are not lasting long at all - in most cases, not even 100 injections - before they have to be "regenerated" using the manufacturer's recommended procedures. After a couple regenerations, the regeneration process isn't very effective anymore.

Also, we've found that our HPLC systems (Waters Alliance) are requiring more frequent maintenance calls for things such as pressure fluctuations and shifting RTs. We pump hot water through the systems after each run to try to wash residual salt out of the system, and this has helped somewhat.

I guess my general question is this: Why are our columns lasting only a few injections? Are we doing something wrong? Is this the typical performance people see?

Thank you for reading,

Eric Moore

Have you considered installing a filter between the injection valve and the pre-column? I've never worked with SEC or proteins before, so I can't recommend a size, but I believe they make in-line glass fiber filters specifically designed for the removal of particulates from environmental or biological samples.

Perhaps someone can offer some better advice than that, but this phenomenex catalog I'm looking at has some pre-column filters that are fairly cheap (and I'm sure you could probably find better and/or cheaper ones if you dug around a bit). They have some peek ones that replace a finger-tight fitting, or stainless/peek assemblies with replaceable filters inside.

If you search this forum you might find the problem with a Tosoh column which I mentioned several times. In short: It changed to the worse, and partially irreversibly, when TFA and other perfluoro-acids were used in the mobile phase. Its performance was very touchy regarding other mobile phases as well, but reversible (we also did Mab + Fab at diff pH, different org. modifyers, different salts and their concentrations).
For the case that you didn´t use TFA, etc., my guess would be that your Mab are of too low in purity to put on these columns.

Thank you, everyone.

We do use a precolumn filter, so I think we're covered there. However, the mAb purity might be an issue - a lot of these are non-therapeutic mAbs, and so aren't manufactured to be ultra-clean (so to speak.)

I'll search the forum for the Tosoh info HW mentioned.

Keep the ideas coming!

Eric

What is the observation with the standard that tells you that the column has deteriorated? What is the standard, and what is the test method?

Uwe -

The observation that the column is failing is based on injecting a GF standard that we purchase (run on the same method we're using, as described, generally, in my first post.) It contains Vitamin B12, Myoglobulin, Ovalbumin, and a few other guys whose names I forget. We begin to see plate counts deteriorate (from ~9500 to ~4500 or lower) and decreased resolution - both caused by altered peak shape (shorter, fatter, mishapen peaks, sometimes with a shoulder.) These observed changes in the GF std seem to correlate to diminished ability of the column to pick up higher molecular weight peaks in our test subjects - control samples that have always been 99.1% monomer begin to show up as 99.8% monomer.

Eric

Hi, all -

As a follow up - after discussions with the manufacturer, it looks like some of our more recent problems were due to a bad column lot. Of course, this only muddies the water in terms of trying to figure out what we're doing right and wrong!

Eric

This is very interesting and unusual, a manufacturer who suggests that a bad lot could be the cause of a column that works fine, then deteriorates?

On the other hand, I don´t see how a prefilter and guard column will take care of a lot of gunk in your sample (if that´s the case. . . . . we don´t know what the prior treatment of the samples was, exactly).

A column deterioration from "bad column lots" is not impossible, but does not explain anything.

Was the deterioration uniform for all the standards that you have injected? For example, if fronting would have appeared, was this the same for all the peaks independent of the MW, or where there differences between the different standards?

This is very interesting and unusual, a manufacturer who suggests that a bad lot could be the cause of a column that works fine, then deteriorates?

I guess "bad column lot" was a little too general - what happened was that one column got >400 injections and was still going strong. Two other columns from the same lot had about 30 injections then drastically deteriorated (resolution and plate count dropped, as described above.) The manufacturer told us to send back the 2 columns that didn't work. They felt, based on our discussions, that the columns were bad. (Obviously, there are more details than I can put into this post: sample types, sample conditions, etc, also led to their analysis that the columns were suspect.)

Was the deterioration uniform for all the standards that you have injected? For example, if fronting would have appeared, was this the same for all the peaks independent of the MW, or where there differences between the different standards?

The deterioration is always the same. The lower molecular weight peaks begin to change.

Thanks, all.

Eric

Dear Forum,

We are having a similar problem - only the manufacturer has not responded to our queries, which is not at all helpful with our troubleshooting....

Our 1st BioSep columns lasted well over 3 years, and thousands of injections. But our newly-purchased BioSep columns have deteriorated (as shown by peak tailing) after only ~ 2 months and < 300 injections!

We're thinking of changing brands even though it's a lot of work to re-validate the method...

Can anyone share their experience with the different brands of SEC columns
- Phenomenex's BioSep,
- Tosoh's TSK, and
- Agilent's Zorbax?
Which is the best and most robust ? We're analyzing mAbs and other recombinant proteins

Zorbax and TSK are good. In the same category is Waters. Finally, one of the finest brands in SEC context is: Sephadex.
Just google them and you’ll find a wealth of info. about each of the brands.

Best Regards

Thanks for the tips.

Does anyone have any experience with the Phenomenex SEC columns?

How do they compare with the rest in terms of resolution and stability?

From the standard protein chromatogram that every brand shows, the peak shapes look roughly similar, for similar particle sizes. I can only see a discernible difference in resolution once the particle size starts to drop.