Chromatography Forum

HPLC: Waters Alliance 2695
Column: Poros A/20 2.1x 30mm
Mobile Phase A= PBS pH 7.4
Mobile Phase B= PBS pH 2.6
Inj vol= 50ul
Flow Rate = 2ml/min
Abs=280nm


We are trying to meet acceptance criteria of the lowest cal std (0.05mg/ml) +/- 15% and continually fail at around 20%. The rest of this 7 point curve is fine. I am limited with the amount of variables I can change since the method is set in stone.
Any suggestions? The cal curve goes from 0.05mg/ml to 2.0 mg/ml.


thanks

Im trying to analyze Protein A

Any carryover in blanks/washes?

Is it failing high or low? Low probably means you have a problem of sample adsorption somewhere. High probably means contamination or carryover. Is the result dependent on the order of the calibrators? What about your buffer blank? Is the integration good?

Im trying to analyze Protein A

What are you doing? It looks like you are using the "Poros protein A" affinity column. I assume you are analysing IgG. Your conditions seem similar to what I have seen published for this column.

How is your standard curve?
Is the curve linear to your low end standards?
How large a peak area are you getting for the low end standards?
Is this below the LOQ for your system?

I run 3 blanks of mobile phase at the start of the sequence, they are all <.05mg/ml peak areas. There are no other blanks run on this assay. I always fail high at about 19%. I believe this is due to the small fronting shoulder that is seen on every injection. This is atypical. The waters alliance should be able to handle a 50ul injection, the agilents handle it fine for the same assay/same column.( no fronting shoulders are observed) I inject each standard once and a theoretical %RSD is calculated on each curve point. The .05 mg/ml always fails high at ~19%
Any ideas?

Personally I would add more blanks and check out carryover. Add a blank after your highest standard and before your samples, and also add one after your samples and before your second curve if you use one.

Also, you said that blanks are <0.05. But what is <0.05? i.e. if your blanks are coming out at 0.04 then that is not good.

Your blanks should be <20% peak area of your lowest standard. So in your case blanks should back calculate at <0.01.

Also, for the peak fronting, try injecting half the volume of samples that are double the concentration. If the fronting disappears it is a sample volume thing. If it's still there, then there's another cause!

Some questions for clarification:

Is the +/- 15% acceptance ctiteria for the recovery?

Do you use the same limit for all standard concentrations in the calibration curve?

Is the acceptance criteria met on the Agilent system where the peak doesn't show the fronting?

Comments:

Perhaps +/- 15% is to tight a limit; unless you are meeting that on the Agilent system.

If you do meet the acceptance criteria on the Agilent, then you may have an instrument problem with the Waters system or a method robustness problem. If you can't change the +/- 15% limit, then you may have to accept that this method cannot be run on certain systems (may not be limited to just the Waters system). That may not be a problem depending on your instrument availablility.

Regards,
Dan

The bigger issue here is retention time and peak shape inconsistencies. Overlaid calibration points on the Agilent systen yields perfect symmetry, whereas the Waters systems yields shifts in retention time.
See enclosed link to overlaid Chromatograms. (The red is the 2.0mg/ml, highest standard)
Can anyone make any sense of this?

http://tinypic.com/view.php?pic=48xuutj

If you say, that on the Agilent system it's working well with the same column and injection volumes than I would take a closer look on the differences of both systems.

In front of my mind is the similarity of the dwell or gradient delay volume if it's a gradient method. Maybe you have to make a correction for the different volumes. The Waters 2695 is a quarternary low-pressure gradient system, isn't it?
If it's the quarternary system, what about the gradient proportioning valve? Is it working correct? I would check some other combination of the reservoirs (C&D, A&C, B&D).

Second I would measure the real %RSD of multiple injections (maybe 3 or 6) of the same concentration. Is the reproducability for theses injections ok?
Are these %RSDs in the range you have calculated from the calibration or somewhat different?

What is your elution ?
It looks like this is a step bump with the low pH phosphate buffer.

I have done alot of process validation for IgG purification with Protein A.
With a large elution peak, the protein actually elutes in front of the low pH salt front. With a smaller peak, it is not "pushed" in front of the elution front. If you had pH and conductivity probes, that would explain much of what is happening (typical for process chromatography - not analytical).

This would explain the difference in retention time between larger peaks and small peaks.

The difference with your 2 HPLC systems is due to hold up volume and different mix chambers.

Your elution is a sudden pH shift with a step bump, but due to the mix chamber in system - who knows what is happening? Also remember that phosphate has almost no buffering capacity between pH 6.5 and 2.5.

You can get more reproducable peaks by washing with a lower pH before elution (such as a weak acetate at pH 4 or 5). Then when you hit it with the sudden low pH elution buffer - it is not "fighting it" as much.

I suspect that some of the peaks for your blanks and low concentration std's is just "buffer fronts" and not true protein elution peaks. Although you should check to see if you fully strip the column between samples or if there is carry over.

I am also surprised you are so concerned about %RSD of replicates for your standard curve, but no mention of "r squared" or other linearity for your curve.

Yes I am running a gradient method. How do I test the gradient proportioning valve to see if it is working correctly?

I replaced the check valves and have no improvements on the erratic retention time. Should I try running lines C and D instead of A and B?

Also, there is a constant r"ringing" sound when the flow is increased past 1.8ml/min. Im running a 2695 quaternary pump. Is the ringing an inndicator of stress? My method calls for 2ml/min. Its as if the gradient proportioning valve is switching the solvents at the wrong time?????
thank for your input

Find your operator's manual, and read the part about Operational Qualification. It should describe a series of tests to assess the performance of the pump. To make a long story short: first establish the flow rate by pumping water and weighing it. Second test the proportioning valve by running steps of water versus 0.1% acetone and collecting UV data at 270nm; compare the height of the steps to the theoretical. There are factory specifications for the results.

If you can't find any help within your organization, this topic has been discussed before in the forum, and you can search for it.

It looks like your problems could be both hardware and assay protocol.

That Waters pump is rated for 5 ml/min , if the pump is complaining - then you could have mechanical problems.
Just to guess - are you starving the pump inlet with clogged solvent filters or are the buffer reservoirs below the level of the pump?

Concerning your assay -Step bumps are recomended by Applied Biosystems and also every procedure I have seen using this type of column.

I see no reason to use a gradient elution, especially with phosphate buffers!
When you are between pH 3 and 5 you have NO BUFFERING capacity and your eluted protein is probably the strongest buffer.
Think of the theory of what would this do to the peak shape???

A guide for this column can be found at :
http://docs.appliedbiosystems.com/pebio ... 003401.pdf
This discusses a problem with "refractive index changes with phosphate elution buffer" causing distorted peaks with your low concentration standards and blanks.

I also have some other literature on validation of an assay for MAb's using this column. Unfortunatly I can not find a current web link for the paper. Send me your email and I will forward it to you.


I appoligize if I sound critical, it is just that you have hit on a topic that I have spent too many years working on.
I do understand how frustrating it is when you are given a protocol that is "set in stone".
If you can't reproduce it ... it makes you look bad.
Also this might be a low prority for your time, you just have to get this done and move on to something else.


It is one of my faults that I have a low tolerance for "Bad Science" repeated forever , because "that is how it has always been done" !!

(Been there - Done that .... I have not always made friends in the process.)

Good Luck and Have FUN !

thanks rande and everyone else that has added insight.
Email me and I will discuss my gradient, its just affinity chromatography, bind then elute.
jtoshea2005@yahoo.com