SPR pollution - baseline deviation

Dear alll,

I encounters some issues on our T200 device. I think something is sticking on the IFC and I cannot manage to get rid off it. Here are the characteristics of the runs:

Immobilization : Neutravidin on CM5 chip
Fc1 = reference
Fc2 = carbonic anhydrase
Fc3 & Fc 4 = protein of interest at 4000 & 8000 RUs

Buffer: HBS-P + 2% DMSO
Run is launched after 10 startups in running buffers.

When I inject furosemide (binder of carbonic anhydrase), I get a proper sensorgram with return to baseline to 0 RU.
When I inject the reference binder of my protein of interest, ,No return to baseline is observed even if it is a well-known reference that dissociate very quickly and that I injected several times before perfectly.

It's been several days I have this phenomenon. I tried different cleaning steps:
- desorb, desorb & sanitize, superclean
- a lot of extra wash with buffer or 50 %DMSO between runs
- a lot of wash on maintenance chip with H2O over the week end etc...

I don't know what is going on. Do you have any advice or ideas?

Thank you all for your help !

Hi and welcome to the forum.
The measurement in the SPR instrument is on the sensor surface alone. Apparently some part of the analyte is sticking to the sensor surface or immobilized ligand. When something is sticking to the IFC generally the actual analyte concentration will be lower at the sensor surface and after the sample plug has passed will leach again into the buffer giving a non-zero dissociation. This results in drift (sometimes at the association) in the dissociation. Therefore cleaning the instrument will not lower the baseline after analyte injection and the first injections after cleaning can suffer from extra adsorption of the analyte.
I would check the reference analyte on purity and aggregation. Also, does the reference analyte has the same problem on the CAII channel? Further is the residual baseline higher at channel 4 (8000 RU ligand) and is it higher at higher ref. analyte concentrations? This could point to sticking to the ligand or the analyte purity.
I hope you can solve your issue.

Kind regards
Arnoud

Hi Arnoud,

Thank you for your answer,

The reference analyte has the same problem on the CAII channel but only with a drift of 3 RUs.
I see your point, but this reference analyte was injected a lot of time months ago (it is our control that we put at the beginning, middle and end of the run) and it went back perfectly to baseline.

Could there be a problem with DMSO? (more micro aggregates because of its tendancy to be hygroscopic) I can try to change it.

Thank you for your kind help !

Clemence

Hi Arnoud,

After changing a lot of things without conclusive change in my problems. I tried to go back to SA immobilization (we did it several time with this reference).
And with SA, the return to baseline is obvious for the reference in dose-response or monodose (see picture).

Do you have an explanation for this sticking on NA chip ?
I have to mentionned that I tried:
- new batch of neutravidine
- new CM5 chip lot number

And the effect on NA immobilization remains the same.

Thank you for your help

Hi Clemence,

I thought that the Neutravin was engineered to have less non-specific binding compared to Streptavidin. Somehow it seems that you are unlucky in the NA-analyte combination this time. You can try to raise the detergent or salt concentration of the running buffer to diminish the non-specific binding. Sometime a slight altering (± 0.2 points) of the buffer pH can also help.

Kind regards
Arnoud